Citation
Maternal recognition of pregnancy in the ewe

Material Information

Title:
Maternal recognition of pregnancy in the ewe the structurefunction relationship of ovine interferon tau
Creator:
Schalue, Tammie Kay
Publication Date:
Language:
English
Physical Description:
xv, 265 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Conceptus ( jstor )
Estrogens ( jstor )
Ewes ( jstor )
Interferons ( jstor )
Messenger RNA ( jstor )
Pregnancy ( jstor )
Prostaglandins ( jstor )
Receptors ( jstor )
Secretion ( jstor )
Sheep ( jstor )
Animal Science thesis, Ph. D ( lcsh )
Dissertations, Academic -- Animal Science -- UF ( lcsh )
Ewes -- Pregnancy ( lcsh )
Ewes -- Reproduction ( lcsh )
Interferon -- Physiological effect ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1997.
Bibliography:
Includes bibliographical references (leaves 235-264).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Tammie Kay Schalue.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
028066445 ( ALEPH )
37819069 ( OCLC )

Downloads

This item has the following downloads:


Full Text












MATERNAL RECOGNITION OF PREGNANCY IN THE EWE:
THE STRUCTURE/FUNCTION RELATIONSHIP OF OVINE INTERFERON TAU




















By

TAMMIE KAY SCHALUE



















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1997



































For

The Trees















ACKNOWLEDGMENTS


I would like to express my gratitude to Dr. F. W. Bazer and Dr. W. W. Thatcher for their invaluable guidance and support during the course of my program. I would also like to thank all the members of my committee, Dr. W.C. Buhi, Dr. H.M. Johnson and Dr. D.C. Sharp, for their support and their contributions to my Ph.D. program as a whole. Special thanks are extended to Dr. T.F. Ogle for conducting the steroid receptor assays, to Dr. Troy Ott for supplying the roIFNT, to Dr. Tom Spencer for supplying the estrogen probe and for his gracious support, and to Marie-Joelle Thatcher for her assistance with the PGFM assay.

This work would not have been possible without the

assistance and support of the other students, postdocs and technicians of the lab. I would also like to thank all the other faculty, postdocs and graduate students of the Animal and Dairy and Poultry Science Departments for their assistance and support.

Special consideration must also go to my family, for they have supported me throughout my life, as I chased my dreams.





iii
















TABLE OF CONTENTS



ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . 111

LIST OF FIGURES . . . . . . . . . . . . . .. . . . . viii

KEY TO ABBREVIATIONS . . . . . . . . . . . . . . . . . xi

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . xiv

CHAPTERS

1 GENERAL INTRODUCTION . . . . . . .... . . . . 1

2 REVIEW OF THE LITERATURE . . . . . . . . . . . . 6

Arachidonic Acid Metabolism And Prostaglandin
Synthesis . . . . . . . . . . . . . . . . . . 6
Interferon Receptors/Signal transduction . . . 8 Steroid Receptors . . . . . . . . . . . . . . . 13
Models For Steroid Receptors; Historical
Perspective . . . . . . . . . . . . . 13
Current Model For Steroid Receptors 15
Structure/Function Of The Steroid
Receptor . . . . . . . . . . . . . . . 16
Receptor Activation . . . . . . . . . . 18 Hormone Response Elements . . . . . . . 20
Estrous Cycle . . . . . . . . . . . . . . . . . 21
Luteolysis . . . . . . . . . . . . . . . . . . 29
Effects of Ovarian Steroids on
Prostaglandin-Fu Secretion . . . . . 32
Progesterone affects . . . . . . . .. . 32 Estrogen affects . . . . . . . . . . . . 35
Summary . . . . . . . . . . .. . . . . 38
Endometrial Oxytocin Receptor . . . . . . . . . 39 Maternal Recognition Of Pregnancy . . . . . . . 44
Ovine Trophoblast Protein-i . . . . . . 55
Ovine Trophoblast Protein-i is an
Interferon . . . . . . . . . . . . . . 57
Maternal Recognition Effects of Ovine
Interferon Tau and Type I Interferons 61
Effects on prostaglandin Synthesis . . . 64



iv









The Ovine Interferon Tau Receptor and Signal
Transduction . . . . . . . . . . . . . . . 68


3 THE EFFECTS OF RECOMBINANT OVINE INTERFERON TAU
AND SYNTHETIC PEPTIDES, CORRESPONDING TO
PORTIONS OF RECOMBINANT OVINE INTERFERON TAU,
ON OXYTOCIN-STIMULATED ENDOMETRIAL INOSITOL
PHOSPHATE METABOLISM AND ENDOMETRIAL OXYTOCIN
RECEPTOR CONCENTRATION. . . . . . . . . . . . 70

Introduction . ............ .... . 70
Materials and Methods . . . . . . . . . . . . . 74
Animals . . . . . . . . . . . . . . . . 74
Protein And Peptide Preparation . . . 74
Ovine conceptus secretory protein
preparation . . . . . . . . . . . 74
Recombinant interferon tau
preparation . . . . . . . . . . . 75
Serum protein preparation . . . . . 76 Synthetic peptide production . . 76
Experimental Design . . . . . . . . . . 77
Experiment 1 . . . . . . . . . . . 77
Experiment 2 . . . . . . . . . . . 78
Experiment 3 . . . . . . . . . . . 82
Experiment 4 . . . . .. . . . . . 85
Experiment 5 . . . .......... 91
Inositol Phosphate Metabolism . . . . . 92 Oxytocin-induced IP metabolism . . . . . 99 Endometrial Oxytocin Receptor Assay 103
Filter procedure . . . . . . . . 103 PEG assay . . . . . . . . . . . . 107
Statistical Analysis . . . . . . . . . 109
Results . . . . . . . . . . . . . . . . . . . 114
Inositol Phosphate Metabolism . . .. 114 Endometrial Oxytocin Receptor Assay 120
Discussion . . . . . . . . . . . . . . . . . 130

4 THE EFFECTS OF RECOMBINANT OVINE INTERFERON TAU
AND SYNTHETIC PEPTIDE DOMAINS OF INTERFERON TAU
ON OXYTOCIN-STIMULATED PROSTAGLANDIN-F
METABOLITE CONCENTRATIONS IN PLASMA OF EWES . 137

Introduction . . . ... ............... 137
Materials and Methods . . . . . . . . . . . . 139
Animals ............... 139
Protein And Peptide Preparation . . 139
Recombinant ovine interferon tau
preparation . . . . . . . . . . 139 Serum protein preparation . . . . 139 Synthetic peptide production . 139
Experimental Design . . . . . . . . . 140

v









Prostaglandin-F metabolite assay . . 140 Statistical Analysis . . . . . . . . . 141
Results . . . . . . . . . . . . . . . . . . . 142
Discussion . . . . . . . . . . . . . . . . . 149

5 THE EFFECTS OF OVINE CONCEPTUS SECRETORY
PROTEINS, RECOMBINANT OVINE INTERFERON TAU AND SYNTHETIC PEPTIDES CORRESPONDING TO PORTIONS OF INTERFERON TAU ON ENDOMETRIAL CONCENTRATIONS OF ESTROGEN AND PROGESTERONE RECEPTOR PROTEIN AND
mRNA . . . . . . . . . . . . . . . . . . . . 159

Introduction . . . . . . . . . . . . . . . . 159
Materials and Methods . . . . . . . . . . . . 163
Animals . ..... ............... 163
Protein And Peptide Preparation . . 164 Ovine conceptus secretory protein preparation . . . . . . . . . . 164 Recombinant ovine interferon tau preparation . . . . . . . . . . 164 Serum protein preparation . . .. 164 Synthetic peptide production . 164 Experimental Design . . . . . . . . . 164 Estrogen Receptor mRNA . . . . . . . . 165 Ribonucleic acid isolation . . 165 Northern blot procedure . . . . . 166 Slot blot procedure . . . . . . . 167 Hybridization . . . . . . . . . . 169 Estrogen Receptor Assay . . . . . . . 170 Progesterone Receptor Binding Assay 172 Statistical Analysis . . . . . . . . . 173
Results . . . . . . . . . . . . . . . . . . . 175
Estrogen Receptor mRNA . . . . . . . . 175 Estrogen Receptor Binding Assay . . 176 Progesterone Receptor Binding Assay 183
Discussion . . . . . . . . . . . . . . . . . 190

6 GENERAL DISCUSSION . . . . . . . . . . . . . . 198

Working Model of Maternal Recognition of
Pregnancy in the Ewe . . . . . . . . 198
Effects of oIFNT on IP metabolism and
OTr Concentrations . . . . . . . . . 207
Effects of oIFNT on Oxytocin-Stimulated
PGFM . . ..... ............... 208
Effects of oIFNT on Er Protein and mRNA 208
Summary . . . . . . . . . . . . . . . . . . 210


APPENDICES

A PROTOCOL FOR PGFM ASSAY . . . . . . . . . . 212

vi










B PROTOCOL FOR OXYTOCIN RECEPTOR ASSAY
(PEG PROCEDURE) . . . .. . . . . . . . . . 216

C PROTOCOL FOR BICINCHONINIC ACID PROTEIN
ASSAY . . . . . . . . . . . . . . . . . . 221

D PROTOCOL FOR OXYTOCIN RECEPTOR ASSAY
(FILTER PROCEDURE) . ..... . . . . . 224

E PROTOCOL FOR INOSITOL PHOSPHATE ASSAY . . . 229 REFERENCE LIST . . . . . . . . . . . . . . . . . . . 235

BIOGRAPHICAL SKETCH .. . . . . . . . . .. . . . . . 265









































vii















LIST OF FIGURES


Figure pane

3.1 - Design for Experiment 2 ......................... 80

3.2 - Design for Experiment 3 .......................... 84

3.3 - Design for Experiment 4 ...................... 87

3.4 - Detailed of treatment period for Experiment 4 ... 89 3.5 - Design for Experiment 5 ......................... 94

3.6 - Schematic of the AVP/OT-stimulated IP assay ..... 96

3.7 - Schematic of the OT-stimulated IP assay .......... 102

3.8 - Validation of the OTr assay (filter procedure) .. 106

3.9 - Time and temperature validation for the OTr
assay (PEG procedure) .......................... 111

3.10 - Optimal protein concentration validation for
the OTr assay (PEG procedure) .................. 113

3.11 - Mean inositol phosphate metabolism in endometrium
treated with oxytocin, AVP, and/or antagonists
for each of the neurophysin receptors ............ 116

3.12 - Mean inositol phosphate metabolism in endometrium
from ewes treated with SP, oCSP, SP + CT,
SP + NT, oCSP + CT or oCSP + NT ................. 119

3.13 - Mean inositol phosphate metabolism in endometrium
from ewes treated with SP, roIFNT, or synthetic
peptides corresponding to portions of oIFNT ..... 123

3.14 - Mean endometrial OTr concentration in ewes treated
with SP, oCSP, SP + CT, SP + NT, oCSP + CT
or oCSP + NT ................................... 125

3.15 - Mean endometrial OTr concentration in ewes treated
with SP, roIFNT, or synthetic peptides
corresponding to portions of oIFNT ............... 127

viii








3.16 - Mean endometrial OTr concentration in ewes treated
with SP, NT or roIFNT .......................... .. 129

3.17 - Mean endometrial OTr concentration in ewes treated
with SP or NT for 16, 17 or 18 days ............. 132

4.1 - Individual SP-treated ewe PGFM response following
oxytocin challenge ............................... 144

4.2 - Individual NT-treated ewes PGFM response following
oxytocin challenge ................................... 146

4.3 - Individual roIFNT-treated ewes PGFM response
following oxytocin challenge ..................... 148

4.4 - Mean PGFM response following oxytocin challenge in
ewes treated with SP, NT, or roIFNT .............. 151

4.5 - Mean of PGFM response following oxytocin challenge
in ewes treated with SP, NT, or roIFNT ............ 153

4.6 - Mean treatment*day effects on PGFM response
following oxytocin challenge on Day 13 and 15 in ewes treated with SP, NT, or roIFNT ................... 155

4.7 - Mean treatment*day*time effects on PGFM response
following oxytocin challenge in ewes treated with
SP, NT, roIFNT ................................... 157

5.1 - Mean endometrial Er mRNA concentration in ewes
treated with SP, oCSP, SP + CT, SP + NT, oCSP + CT
or oCSP + NT ..................................... 178

5.2 - Mean endometrial Er mRNA concentration in ewes
treated with SP, roIFNT, or synthetic peptides
corresponding to portions of oIFNT ............... 180

5.3 - Mean endometrial Er mRNA concentration in ewes
treated with SP, NT, or roIFNT ................... 182

5.4 - Mean endometrial Er mRNA concentration in ewes
treated with SP or NT on Days 16, 17 or 18 ....... 185

5.5 - Mean endometrial Er protein concentration in ewes
treated with SP, NT or roIFNT .................... 187

5.6 - Mean endometrial Er protein concentration in ewes
treated with SP or NT on Days 16, 17 or 18 ....... 189

5.7 - Mean endometrial Pr protein concentration in ewes
treated with SP, NT or roIFNT .................... 192


ix








5.8 - Mean endometrial Pr protein concentration in ewes
treated with SP or NT on Days 16, 17 or 18 ....... 194

6.1 - Schematic diagram of the regulation of hormone
receptor expression during late diestrus ......... 201

6.2 - Schematic diagram of the regulation of hormone
receptor expression during establishment
of pregnancy ...................................... 203

6.3 - Schematic diagram of the regulation of hormone
receptor expression during establishment of
pregnancy involving the mechanism of oIFNT
action ........................................... 205








































x















KEY TO ABBREVIATIONS


aa ... Amino Acid Ala ... Alanine ANOVA... Analysis of Variance Arg ... Arginine AVP ... Arginine Vasopressin CT ... Carboxyl-terminus of oIFNT; aa 139-172 Cys ... Cysteine DNA ... Deoxyribonucleic Acid GLM ... General Linear Models Gln ... Glutamine Glu ... Glutamic Acid HSP ... Heat Shock Protein IFN ... Interferon IFNr ... Type I Interferon Receptor IFNa ... Interferon alpha IFNS ... Interferon beta IFNw ... Interferon omega IRF-2 .. Interferon Regulatory Factor-2 IRF-E .. Interferon Regulatory Factor Element ISGF3 .. IFN Stimulated Gene Factor-3 Complex ISGF3a . IFN Stimulated Gene Factor-3a ISGF37 IFN Stimulated Gene Factor-37 xi









ISRE ... IFN Stimulated Response Element IP ... Inositol Phosphate JAK ... Janus Kinase KRB ... Kreb's Ringer Bicarbonate Leu ... Leucine mRNA ... Messenger Ribonucleic Acid NT ... Amino-terminus of oIFNT; aa 1-37 oCSP ... Ovine Conceptus Secretory Protein oIFNT .. Ovine Trophoblast Interferon OT ... Oxytocin oTP-1 .. Ovine Trophoblast Protein-i OTr ... Oxytocin Receptor PEG ... Polyethyleneglycol Pep 2... aa 34-64 of oIFNT Pep 3... aa 62-92 of oIFNT Pep 4... aa 90-122 of oIFNT Pep 5... aa 119-150 of oIFNT PGFu2 .. Prostaglandin-F2 PGFM ... 13,14-dihydro-15-keto Prostaglandin-F2u Pro ... Proline RNA ... Ribonucleic Acid rbIFN . Recombinant Bovine Interferon-alpha roIFNT . Recombinant Ovine Trophoblast Interferon SAS ... Statistical Analysis System SEM ... Standard Error of the Mean Ser ... Serine xii









Statla. . Signal Transducers and Activators of Transcription-la Statl. . Signal Transducers and Activators of Transcription-1l Stat2 . Signal Transducers and Activators of Transcription-2 SP ... Serum Protein Tyk2 ... Tyrosine Kinase Tyr ... Tyrosine Trp ... Tryptophan










































xiii















Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

MATERNAL RECOGNITION OF PREGNANCY IN THE EWE:
THE STRUCTURE/FUNCTION RELATIONSHIP OF OVINE INTERFERON TAU By

Tammie Kay Schalue

August, 1997

Chairperson: Dr. F. W. BAZER Co-Chairperson: Dr. W. W. THATCHER Major Department: Animal Science

In the ewe, pregnancy success is dependent on the

conceptus secreting the pregnancy recognition factor, oIFNT, during the maternal recognition of pregnancy period. oIFNT is secreted by the trophoblast cells of the developing conceptus. Binding to specific endometrial receptors initiates a series of events which block the release of luteolytic PGFa. Our current working hypothesis is that oIFNT accomplishes this attenuation of pulsatile PGF2u secretion by the endometrium, through preventing expression of the Er gene, and subsequent up-regulation of expression of the OTr gene that normally occurs in non-pregnant ewes. The NT portion of the oIFNT has been shown, through competitive binding studies, to act through a specific domain of the Type I IFN receptor while the CT is believed



xiv








to act through a more common domain of that receptor. The present studies were conducted to determine if the NT, or the other portions of oIFNT, has pregnancy recognition properties when injected into the uterine lumen of cyclic ewes. Intrauterine injections were either one of six overlapping synthetic oIFNT peptides (NT was the peptide most closely examined), ovine conceptus protein or roIFNT. The NT peptide was nearly as effective as roIFNT in suppressing expression of endometrial Er and OTr, blocking the metabolism of inositol phosphate in endometrial tissues after an oxytocin challenge test in vitro and PGFM response to an oxytocin challenge test in vivo. NT had no effect on endometrial Pr expression. Collectively, these results support the hypothesis that oIFNT suppresses expression of endometrial Er and OTr, thereby preventing the pulsatile release of PGFz2 without affecting expression of Pr. Also, these results indicate that the NT peptide is as effective as oIFNT in producing a pregnancy recognition response. This finding supports the hypothesis that the NT of oIFNT is the most likely portion of oIFNT responsible for the antiluteolytic properties of oIFNT which distinguish it from other Type I IFNs.










xv















CHAPTER 1
GENERAL INTRODUCTION





The "term maternal recognition of pregnancy" was first used in 1969 by R.V. Short. Identification of the maternal pregnancy recognition factor as an IFN came about as the result of molecular cloning and amino acid sequencing (Stewart et al., 1987; Imakawa et al., 1987, 1989; Charpigny et al., 1988). These reports identified the maternal pregnancy recognition factor, referred to currently as oIFNT, as a Type I IFN. Trophoblast IFNs are biologically similar to other Type I IFNs, and like the other IFNTs, oIFNT displays both antiviral and antiproliferative properties as does IFNa (Pontzer et al., 1988). Ovine IFNT binds to high affinity (Godkin et al., 1984a) Type I IFN receptors (Stewert et al., 1987) which are distributed throughout endometrial tissues of the ewe and their expression may be influenced by ovarian steroids (Knickerbocker and Niswender, 1989). Synthetic peptides corresponding to the amino- (Pontzer et al., 1991) and carboxyl-terminal peptides of oIFNT, as well as two internal peptides (aa 62-92 and aa 119-150) were found to inhibit oIFNT receptor binding and antiviral activity in a dose

1








2

dependent manner (Pontzer et al., 1994) indicating specific competition between these peptides and oIFNT. The NT had no effect on the antiviral activity of IFN. These findings indicated that the NT may act through a novel domain of the oIFNT receptor while the other peptides may act through a more common domain of the Type I IFN receptor. This could explain the unique actions of oIFNT.

Ovine IFNT-induced hormone action is initiated by the transduction of signal via activation of four cytosolic proteins which bind to interferon stimulated response elements (see Williams, 1991a; Darnell et al., 1994) and is believed to act in the same manner as all other Type I interferons. Ovine IFNT and other Type I IFNs increase endometrial protein production dramatically (Gross et al., 1988b; Sharif et al., 1989; Ashworth and Bazer, 1989; see Spencer et al., 1996) and affect expression of Er concentration within the endometrium during early pregnancy (Mirando et al., 1993; Ott et al., 1993b; Wathes and Hamon, 1993; Spencer et al., 1995a, 1995b) to allow for establishment of pregnancy.

In the cyclic ewe, the Er is abundant just before,

during and after estrus. Receptor numbers fall dramatically from Day 3, to a low on Days 10 to 14, followed by a dramatic increase beginning on Day 14, to peak at estrus (Koligian and Stormshak, 1976; Miller et al., 1977; Zelinski et al., 1980; Cherny et al., 1991; Ott et al., 1993b;








3

Spencer et al., 1996). Progesterone is inhibitory to Er formation in most species (Brenner et al., 1974; Hsueh et al., 1975 and 1976; Tseng and Gurpide, 1975; West et al., 1976; Bhakoo and Katzenellenbogen, 1977) including sheep (Koligian and Stormshak, 1977b; Zelinski et al., 1980; Cherny et al., 1991). Progesterone in the cyclic animal is clearly inhibitory to its own endometrial receptor, probably through a down-regulation mechanism (Milgrom et al., 1973; Leavitt et al., 1974; Vu Hai et al., 1977; Spencer et al., 1996). Progesterone's down-regulation of its own receptor initiates removal of the proposed progesterone block (McCracken et al., 1984), allowing Er up-regulation, and formation of OTr which bind oxytocin to initiate pulsatile release of PGF, from the uterus.

In the cyclic ewe luteolysis is initiated by pulsatile release of PGF2a produced by the uterus (McCracken et al., 1981; Hooper et al., 1986; Thornburn et al., 1973; Niswender and Nett, 1994). Oxytocin from the ovary, binding to its endometrial OTr, and PGF2a from the uterus binding to its ovarian receptor, act together in a positive feedback loop to generate the pulses of PGFz2 required for luteolysis (Flint and Sheldrick, 1983; Hooper et al., 1987; Niswender and Nett; 1994). Endometrial OTr begin to increase on Day 14, reach maximum values at estrus and decline by Day 5 of the subsequent cycle. This is coincidental with the decrease in plasma progesterone and the rise in estrogen








4

(Sheldrick and Flint, 1985; Wallace et al., 1991) during proestrus.

Intrauterine injections of conceptus homogenates

increase the interestrous interval in ewes (Rowson and Moor, 1967) and oCSPs elicit the same response (Godkin et al., 1984b; Vallet et al., 1988). Intrauterine injection of oCSPs also blocks cyclic uterine PGF2. responsiveness after oxytocin challenge (Fincher et al., 1986; Vallet et al., 1988; Mirando et al., 1990a). The lack of PGFu2 responsiveness (Fincher et al., 1986; Vallet et al., 1988) and IP hydrolysis (Mirando et al., 1990b) in the pregnant ewe is due to low OTr (Flint and Sheldrick, 1986) and Er (Findlay et al., 1982) on the luminal epithelium (Spencer et al., 1996). The proposed progesterone block (McCracken et al., 1984) to OTr formation was initially believed to be conceptus maintained by preventing the normal downregulation of the Pr on Days 12 to 14. Recent results of Spencer et al. (1995a,b) indicate that in the pregnant ewe oIFNT prevents luteolysis, not by stabilizing Pr and maintaining the progesterone block, but by preventing expression of the Er gene which, in turn, prevents expression of the endometrial OTr gene.

Present studies determined the function of oIFNT on factors associated with maternal recognition and examined the functional properties of specific domains of oIFNT responsible for maternal recognition of pregnancy effects.








5

The experimental designs and methods for the five individual animal experiments which comprise the present studies are discussed in Chapter 3 and are referred to throughout the remainder of this dissertation by experiment number.















CHAPTER 2
REVIEW OF THE LITERATURE


Arachidonic Acid Metabolism And Prostaglandin Synthesis


Arachidonic acid, a 20 carbon fatty acid, is supplied through the diet or by anabolic metabolism of linoleic acid (Ramwell, 1977). Most arachidonic acid is present within integral membrane components of cells in the form of phospholipids. Arachidic acid is found most prominently at the 2n position of phosphatidylcholine, phosphatidylethanolamine, and, to a lesser extent, phosphatidylinositol (MacDonald and Sprecher, 1991). Arachidonic acid, once mobilized from phospholipids, usually enters into one of three major arachidonic acid metabolic cascades; the cyclooxygenase, lipoxygenase, or epoxygenase pathways. The cyclooxygenase pathway produces prostaglandins and thromboxanes (Smith et al., 1991). The lipoxygenase pathway produces leukotrienes, hydroxy acids and lipoxens (Samuelsson, 1987; Smith et al., 1991). Epoxy acids and dihydroxy acids are formed via the epoxygenase pathway (Fitzpatrick and Murphy, 1988).

Arachidonic acid is mobilized from phospholipids in response to extracellular stimuli, in part, through the



6








7

actions of one of three phospholipases; phospholipase A,, A2, and C (Smith, 1986). Phospholipase A, cleaves acyl residues from phospholipids at the 3n (3 carbon) position of the glycerol backbone to produce a lysophospholipid from which arachidonic acid is then cleaved. Phospholipase A2 mobilizes arachidonic acid directly from the phospholipid at the 2n position of the glycerol backbone (Loeb and Gross, 1986; Kramer et al., 1989;). Phospholipase C, via receptormediated G protein stimulation, cleaves phosphatidylinositol into inositol 1,4,5-trisphosphate and diacylglycerol, each of which can act as a transduction signal within the cell. Arachidonic acid is subsequently cleaved from diacylglycerol at the 2n position by an inositol specific phospholipase C enzyme with both diacylglycerol lipase and monoacylglycerol lipase activities (see Berridge, 1984; Smith et al., 1991). Arachidonic acid mobilization for prostaglandin synthesis is primarily through the actions of phospholipase A2 and phospholipase C (Martin and Wysolmerski, 1987).

The prostaglandins -A, -D, -E, -F, and -I, are 20

carbon compounds produced via the cyclooxygenase pathway of arachidonic acid metabolism, through the actions of prostaglandin-H synthetase (Smith, 1989). Prostaglandin-H synthetase is an integral membrane protein found on the cytoplasmic side of cell membranes (Smith et al., 1991). Prostaglandin-H synthetase exhibits both cyclooxygenase and hydroperoxidase activities as separate sites on the








8

synthetase enzyme (Miyamoto et al., 1976; Pagels et al., 1983) Cyclooxygenase catalyzes prostaglandin-G2 formation from arachidonic acid and hydroperoxidase acts on the 15hydroperoxyl group of prostaglandin-G2 to form prostaglandin-H2. Both cyclooxygenase and hydroperoxidase activities require heme (Kartheim et al., 1987). Prostaglandin production is strongly regulated by the availability of substrate (arachidonic acid) and control of prostaglandin-H synthetase activity to produce prostaglandin-H2, the precursor of all prostaglandins and thromboxane.

Prostaglandin-D2, prostaglandin-E2, prostaglandin-I2, and thromboxane are produced from prostaglandin-H by nonoxidative rearrangements through the action of their respective synthetase enzymes. Prostaglandin-F2. can be formed by three separate mechanisms through prostaglandin-F synthetase enzyme actions. Prostaglandin-F2a can be formed from prostaglandin-H2 through endoperoxide reductase activity or reduction from prostaglandin-E2 by a 9-ketoreductase. An active metabolite of PGF2a, 9a,11f-PGF2a can be produced from prostaglandin-D, via 11-keto-reductase (Smith et al., 1991).


Interferon Receptors/Signal transduction


Interferons can be divided into two general types (for review, see Roberts et al., 1992). Type II interferon is








9

the product of a single gene and is known as IFNy. Type I IFN is composed of at least three distinct subtypes known as IFNa, IFNS and IFNw. Although these three subtypes differ markedly in amino acid sequence, they elicit their actions through a common receptor and exhibit similar biological properties. Although commonly thought to be derived primarily from T-lymphocytes and natural killer cells in the presence of other cells infected with virus, Type II IFN has been shown to be released from a variety of cells, including porcine trophoblast (Lefevre et al., 1990). Type-I IFNs can be induced by many cells within the body. Interferon-a, produced by immune cells, is the most prominent, while IFNT is produced exclusively by trophoblast cells. It is IFNT, a member of the Type-I IFN family which is involved in pregnancy recognition, therefore, this discussion on IFN receptors and signal transduction will be limited to the Type-I IFN family.

The Type-I IFN receptor is a transmembrane receptor

comprised of two subunits (a and S; Platanias et al., 1994) Coupling of IFN to its receptor sets into motion a series of phosphorylations of proteins within the cell which ultimately affect transcription of various proteins. How specificity is acquired is not understood at this time as apparently all Type-I IFNs bind to the same or similar receptors and initiate their responses through the same transduction signaling pathway (see, Johnson et al., 1994).








10

Signal transduction for IFNs is not through stimulation of CAMP, cGMP, or IP turnover (see Bazer, 1992; Bazer et al., 1993; Johnson et al., 1994). Rather, IFNs elicit their action on gene function through a complex signal transduction cascade involving activation of protein tyrosine kinases including those belonging to the Janus kinase (Jak) family, including Tyk2 and Jakl (for review, see Darnell et al., 1994). Ligand binding initiates the activation of Tyk2, which is associated with the cytoplasmic domain of the Type-I IFN receptor. Either ligand binding or activation of Tyk2 results in activation of Jakl. These two kinases, either separately or together, act to phosphorylate proteins within the cytoplasm which comprise the interferon stimulated gene factor-3 complex (see, Pfeiffer et al., 1994, Johnson et al., 1994). These subsequently translocate to the nucleus where they bind to specific interferon stimulated response elements on DNA sequences that direct IFN-induced transcriptional responses. interferon stimulated gene factor-3 is actually two complexes composed of four separate components which normally reside in a dissociated form in untreated cells. The interferon stimulated gene factor-3a complex forms within minutes following IFN treatment, and is composed of three proteins (113 kDa STAT2, 91 kDa STATla and 84 kDa STAT1I; Fu et al., 1992; David and Larner, 1992). It has been suggested that Jakl is the kinase responsible for activating STAT1 while








11

Tyk2 phosphorylates STAT2 (Silvennoinen et al., 1993; Darnell et al., 1994). Inhibitors of the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism increase activity of interferon stimulated gene factor-3a (Hannigan et al., 1991). This indicates that a factor produced from metabolism of arachidonic acid in the epoxygenase pathway may amplify the interferon stimulated gene factor-3a signal (Hannigan et al., 1991; Williams, 1991b). Once activation and binding of these three components occurs, interferon stimulated gene factor-3a binds a fourth DNA-binding protein (48 kDa interferon stimulated gene factor-3; Levy et al., 1990; see Marx, 1992), and the multimeric complex translocates to the nucleus and binds to the interferon stimulated response elements (Fu, 1992; Schindler et al., 1992; Larner et al., 1993).

Interferon-stimulated response elements are cis-acting DNA elements found upstream in IFN responsive genes which activate transcription when bound (see Williams, 1991b). Transcription is activated after binding of the activated interferon stimulated gene factor-3 complex, made up of interferon stimulated gene factor-3a and interferon stimulated gene factor-3, to interferon stimulated response elements.

The consensus sequence of interferon stimulated

response elements for Type I IFNs is reported to have both a








12

common motif (GGAAA) and a specific motif (TGAAACT). The specific motif is reportedly separated from the common motif by 1 residue (3') (see Williams, 1991a; Kerr and Stark, 1991). Once the interferon stimulated response element is bound, RNA polymerase II initiates transcription.

Other members of the Type I IFN regulatory factor

family also play a major role in IFN action. The interferon regulatory factor-i gene contains an interferon stimulated response element, which when bound by the activated interferon stimulated gene factor-3 complex, is upregulated, resulting in an increase in the positive transcription factor interferon regulatory factor-1. The interferon regulatory factor-i protein, in contrast to the interferon stimulated gene factor-3 complex, can in turn bind to an interferon regulatory factor element, which is often contained within a larger interferon stimulated response element, and increase gene transcription. One such gene which contains a interferon regulatory factor element is the interferon regulatory factor-2 gene, and binding to this interferon regulatory factor element by interferon regulatory factor-i results in an increase in the production of the transcription factor interferon regulatory factor-2. interferon regulatory factor-i and interferon regulatory factor-2 are structurally similar DNA-binding factors which have contrasting roles in IFN action. interferon regulatory factor-i serves as a transcriptional activator of IFN and








13

IFN-inducible genes, while interferon regulatory factor-2 represses interferon regulatory factor-i action by competing for the same cis elements and displacing interferon regulatory factor-i (Harada et al., 1994). Thus, this "yinyang interaction", as described by Spencer et al. (1996), modulates IFN action by regulating the induction and repression of Type I IFN-responsive gene expression.



Steroid Receptors


Models For Steroid Receptors; Historical Perspective


Mueller et al. (1958) was the first to propose a

mechanism of action for a steroid. This report led to a search for the compartment in which the steroid receptor was located. Toft and Gorski (see Gorski et al., 1968) used sucrose gradient centrifugation to separate the cytosolic and nuclear fractions of cells treated with tritiated estradiol. They found most of the radioactivity in the nuclear fraction. Only after an excess of estradiol was added was there detectable radioactivity in the cytosol. However, previous studies indicated the presence of unbound receptor in the cytosol. This led to studies which examined the effects of time and temperature, during incubation with estradiol, on location of the Er. In these studies whole uteri were incubated at OOC and 370C. After 1 h at 370C there was more estrogen in the nuclear fraction than in the








14

cytosolic fraction. However, at 00C most estradiol was in the cytosol. Incubation of uteri for 1 min at 00C with labeled estradiol, followed by transfer of the tissues to fresh medium without estradiol and incubation at 370C, resulted in the appearance of estradiol in the nuclear fraction. Because formation of the nuclear fragment (9.5s) resulted in loss of the estrogen-bound cytosolic fragment (4-6s) the question was whether the cytosolic fragment (46s) was extracellular in origin and whether estrogen from the 9.5s fraction was from the cytosol fragment. This was answered by experiments utilizing cell-free preparations of cytosol incubated with labeled estradiol. The cell-free cultures prepared from uteri incubated at 00C with labeled estrogen, followed by incubation at 370C without estrogen had very little estrogen in the cytosolic fraction. However, if the same experiment was conducted without estrogen during the 00C incubation the estrogen was found in the cytosol. These results led the authors to propose that estrogen moved from the cytosol to the nucleus and, in the process, the 9.5s fragment was lost. Autoradiographic studies by Jensen et al. (1968) showed that uterine tissues incubated at 370C with labeled estradiol had the majority of the label within the nucleus. However, when incubations were conducted at 20C the majority of the label was located within the cytosol. These findings were supported by earlier immunocytochemical studies.








15

This led to, a revision of the proposal for the

translocation model for the steroid receptors (Gorski et al., 1968). In this model, estrogen diffuses into cells from the blood and binds to a cytoplasmic receptor. The binding of estrogen induces a conformational change in the receptor which allows movement of the ligand-receptor complex from the cytosol to the nucleus. This conformational rearrangement changes the shape of the receptor and causes the release of one or more proposed subunits attached to the receptor in the inactive state. It was this combination of the loss of the subunits and the conformational change that caused the proposed change from the 9.5s cytosolic form to the 4-6s nuclear form. Once in the nucleus, it was believed that receptor-initiated events resulted in increased cellular protein production by estrogen-treated uterine tissues. Current Model For Steroid Receptors


In recent years, improved separation techniques,

utilizing a cytochalasin enucleation procedure, allowed more complete separation of the cytosolic and nuclear fractions into cytoplasts and nucleoplasts, respectively. With this procedure, Welshons et al. (1984, 1985) conclusively established that Er, Pr and Gr are located within the nucleus. Techniques, using frozen tissue for immunocytochemical detection of steroid receptor by








16

monoclonal antibody binding, also indicated that the unoccupied receptor for estrogen was in the nucleus (King and Greene, 1984). In light of these findings, Gorksi et al. (1986) proposed the current model for steroid receptor action. This model is very similar to that previously described except that the unoccupied receptor is found only in the nuclear compartment. The steroid diffuses into the nucleus, binds to the specific receptor which then undergoes conformational changes, and the receptor-ligand complex binds to the steroid response element in the promotor/enhancer region of DNA. Structure/Function Of The Steroid Receptor


Steroid receptors belong to a super-family of nuclear receptors that include receptors for all steroids, retinoic acid, thyroid hormone, vitamin D, and several other receptors which are termed orphan receptors. Orphan receptors are not fully characterized but are apparently involved in proper development in Drosophila. There is a high degree of homology among the steroid receptors, and all members of this super-family have common functional domains which account for their high homology. These domains consist of a DNA binding domain, a ligand binding domain, a dimerization domain, a heat shock protein 90 binding domain, a nuclear localization domain and a transactivation domain








17

(for reviews see Carson-Jurica et al., 1990; Wahli and Martinez, 1991; Freedman, 1992; and Orit et al., 1992).

The DNA binding domain, probably the most studied, is noted for many conserved cysteines. Eight of the cysteines are arranged into zinc finger complexes where four cysteines in each of the two fingers bind a zinc ion. The DNA binding domain of the steroid super-family is divided into two separate subfamilies based on differences in the zinc fingers which are involved in DNA sequence recognition and receptor dimerization. These subfamilies are the glucocorticoid/progesterone subfamily, which includes androgen receptors and mineralocorticoid receptors, and the estrogen subfamily, which includes retinoic acid, thyroid hormone, vitamin D, and the orphan receptors

The DNA binding domain of the glucocorticoid receptor was the first to be characterized. Subsequent studies indicated that only a portion of this domain was required for DNA binding and that the DNA binding domain also possessed hormone-dependent transcriptional activity (Hollenberg et al., 1987). As stated previously, this domain has two zinc finger structures similar to those originally reported in transcription factor IIIA. Cloning and sequencing of the steroid receptor zinc finger region led to the following consensus sequence: Cys-X2-Cys-X3-CysX2-Cys-Xl5-Cys-Xs-Cys-X,-Cys-X4-Cys (see Freedman, 1992).








18

The ligand binding domain (220 - 250 amino acids) located near the carboxyl terminal includes domains for dimerization, transactivation and heat shock protein 90 binding. The N-terminal domain is highly variable in length and exhibits the conserved least sequence homology of all the domains. It is, therefore, sometimes referred to as the immunologic domain to which antibodies are generated to distinguish between the various receptors within this superfamily. This domain also contains an area involved in transactivation.

Receptor Activation

Prior to ligand binding the steroid receptor is held in an inactive state by inhibitory proteins; heat shock protein 90 is the primary protein responsible for this inhibition. Salt treatment of steroid receptors removes heat shock protein 90 and causes activation of the receptor. The heat shock protein 90 binding occurs in the carboxyl terminal region of the receptor; the same area to which the ligand binding domain has been mapped. Apparently there are two molecules of heat shock protein attached to each receptor molecule. Studies which utilized fragments of the glucocorticoid receptor indicated that heat shock protein 90 may be involved in maintaining conformation of the receptor, such that the ligand is unable to bind. When the heat shock protein was removed, ligand binding was blocked (Bresnick et al., 1989) possibly due to protein unfolding characteristics








19

reported for other heat shock proteins (Pelham, 1988). The two heat shock protein 90 molecules are lost as a result of a conformational changes induced by ligand binding. Conformational change causes movement of the hinge region, loss of the heat shock protein 90 molecules and exposure of the DNA binding domain to allow binding to hormone response elements. This also allows formation of a dimmer of two steroid receptor molecules which is necessary for binding to the hormone response element (homodimer formation may not be necessary for binding of estrogen to its hormone response element; Furlow et al., 1993).

Phosphorylation of steroid receptors is also involved in activation, DNA binding, and activation of transcription. Hormone binding by steroid receptors is increased when tyrosine kinase is added to cultures of receptors. There are multiple sites for phosphorylation on most steroid receptors and hyperphosphorylation occurs after ligand binding. Serine is the primary amino acid phosphorylated, with minor amounts of threonine phosphorylated (this may be switched in the Er). Phosphorylation occurs in several domains, particularly the hormone binding and DNA binding domains and the hinge region. It has been proposed that phosphorylation must precede dimerization; however, this has not been proven. There are results which indicate that phosphorylation of serine/threonine in some domains inhibits normal steroid receptor function (see Orit et al., 1992).








20

Hormone Response Elements


Hormone response elements are the nucleotide sequences recognized by the steroid receptors. Hormone response elements are sequences which have palindromic structure. For steroid hormones receptors, hormone response elements are unequal halves with three non-conserved base pairs between each of the halves. Like the steroid receptor super-family, the hormone response elements can be separated into subfamilies with the glucocorticoid/progesterone subfamily in one group and the estrogen subfamily in another. The consensus sequence for the glucocorticoid/progesterone subfamily half palindrome hormone response element is TGTYCT, while it is TGACC for the estrogen subfamily. The difference between these two elements is at the third position, this is a T in the glucocorticoid/progesterone family and an A in the Er family. The sequence responsible for determination of progesterone or Er binding is in the area between Cys 3 and

4 and the amino acid residues just downstream from that point in the first zinc finger (Mader et al., 1989; Umesono and Evans, 1989). There are specific hormone response element recognition sites between Cys 5 and 6 located on the second finger as well. These may be important in dictating the orientation of the receptor dimers (Hard et al., 1990).

Steroid hormones control the activity of various genes in target cells through the action of ligand activated








21

transcriptional modulators which act by binding to the hormone response element on the DNA. This interaction with the DNA-bound receptor with basal transcriptional machinery and sequence specific transcription factors mediate the transcriptional effects of the steroid hormone.


Estrous Cycle


The estrous cycle in the ewe is 16 to 17 days in

length, but there are some minor variations in length, due to age and stress (Mckinzie and Terrill, 1937).

Estrous behavior occurs in most breeds of sheep during the fall and winter, while anestrus occurs during the spring and summer. This assures that birth of young occurs in the spring, when conditions for their survival are optimal. Unlike the estrous cycle, there is considerable variability in the duration of the breeding season among breeds. The breeding season is under photoperiodic control and those breeds originating from mild climates have much longer breeding seasons than those breeds which originated in more northern regions (Robinson, 1959).

Estrus is defined as the period of time when the ewe

willingly allows the ram to mount. The first day a ewe will allow a ram to mount is, by convention, designated Day 0 of the estrous cycle. Estrous behavior generally lasts 24 to 48 h; however, there is a considerable variation in duration. Differences in duration of estrous behavior can








22

be attributed to several factors. The most studied, however, are the effects of ovulation rate and the ram. Breeds which typically have high ovulation rates exhibit a longer period of estrus (Bindon et al., 1979). Continuous presence of a ram reduces length of estrus, as compared to that for ewes that experience intermittent exposure to a ram (Parsons, 1967). Regardless of the duration of estrus, ovulation occurs about 30 h after the onset of estrous behavior in response to the ovulatory surge of LH (Mckinzie and Terrill, 1937).

The two ovarian steroids primarily involved in the

estrous cycle are progesterone and estradiol. Progesterone is secreted by the CL, while estradiol is secreted by the follicles (Short et al., 1963). The CL is made up of two steroidogenic cell types distinguished by size and function. The large luteal cells are believed to be of granulosa cell origin and the small luteal cells appear to be of theca cell origin (Meidan et al., 1990). Large luteal cells secrete oxytocin (Rodgers et al., 1983) and spontaneously secrete large amounts of progesterone (Hulet and Shelton, 1980). Conversely, small luteal cells must be stimulated by LH to produce progesterone (Fitz et al., 1884). Small luteal cells have high numbers of LH receptors and very low numbers of PGFu2 receptors. In contrast, large luteal cells have abundant PGFu2 receptors and few LH receptors (Alila et al., 1988). The preponderance of PGFu2 receptors on large luteal








23

cells may explain why they are affected first during luteolysis, resulting in a rapid decrease in progesterone secretion at the onset of luteolysis (Branden et al., 1988). PGF2 activates the protein kinase C (PKC) system in large luteal cells, which leads to inhibition of progesterone synthesis. Although activation of the PKC system in small luteal cells also inhibits progesterone synthesis, it is not clear what hormone is involved in the activation of PKC in these cells (Niswender and Nett, 1994).

Progesterone levels are nearly undetectable from Day 0 to Day 3 and then increase gradually until Day 8. The levels remain constant, thereafter, ranging from 1.5 to 3 ng/ml (Bindon et al., 1979), until Day 15-16 when progesterone decreases rapidly in blood to below 1 ng/ml, and the next cycle commences (Stabenfeldt et al., 1969). While there is very little difference in temporal changes in progesterone secretion by CL, differences have been reported for maximal circulating concentrations of progesterone across breeds. Progesterone concentrations are generally higher at mid-cycle in those breeds with higher ovulation rates compared to breeds with lower ovulation rates (Bindon et al., 1979). Progesterone concentrations are also higher in the middle of the breeding season, compared to the beginning or the end of the season (Legan et al., 1985).

Progesterone levels begin to fall even before CL

regression becomes morphologically apparent and luteolysis








24

is initiated by PGF2, release from the non-gravid uterus. Pulsatile secretion of PGFu2 is first apparent about Day 15 of the cycle and continues to increase until estrus. The role of PGFu2 in luteolysis and the attenuation of its production will be discussed in more detail in the sections on luteolysis and maternal recognition of pregnancy.

Plasma estradiol concentrations begin to increase just after the initial decrease in progesterone is noted. Estrogen concentrations in the plasma continue to increase from basal levels of 1 pg/ml to highest levels of about 10 pg/ml (Baird et al., 1976) at the LH surge, after which time estrogen levels decline rapidly and remain at basal levels through the luteal phase except for small increases associated with follicular waves. Estradiol is produced by rapidly growing preovulatory follicles during proestrus (Days 15-16).

Both progesterone and estradiol are secreted in pulses. Progesterone, however, does not follow a pattern set by gonadotropins as does estradiol. Pulses of estradiol precede pulses of LH and both pulse frequency and amplitude of estradiol increase during the follicular phase (Baird, 1978).

LH is secreted episodically, in the ewe, and varies in both frequency and amplitude throughout the estrous cycle. The basal level of LH secretion (-1 ng/ml) is required for








25

CL function and steroidogenesis, while the LH surge initiates ovulation and CL formation (Goding et al., 1970).

The LH surge is characterized by a dramatic rise in LH pulse frequency a few hours prior to ovulation after which LH returns to basal levels. On average the entire ovulatory surge of LH occurs within 12 h, and coincides with the onset of estrus. This is followed by ovulation about 30 h later. The CL begins to produce progesterone by Day 3 and the recurring estrous cycles continue during the breeding season, until ewes become pregnant, or enter anestrus.

Changes in the secretion of LH during the normal cycle and ovulation are tightly controlled by changes in the generation of GnRH pulses (reviewed in Goodman, 1994). During the early luteal phase, tonic LH secretion decreases because the increasing amounts of progesterone, along with estradiol, suppress the activity of the GnRH neural oscillator and decrease LH pulse frequency. Tonic LH levels then remain low as long as progesterone levels are increased which attenuates GnRH pulse frequency. When progesterone levels fall following luteolysis, the frequency of the GnRH pulse generator increases, which may in turn stimulate GnRH receptor gene expression and increased expression of GnRH receptors (Turzillo et al., 1995). Increases in GnRH pulse generation then serve to increase the pulse frequency of LH and accounts for the rise in LH secretion during the follicular phase of the sheep.








26

Endometrial Steroid Receptors


It is clear that the uterine environment is dynamic and ever changing. The changes that occur throughout the estrous cycle with regard to both physiological and biochemical activities are controlled by both changing steroid hormone profiles and changes in expression of their receptors. While finite control of uterine steroid receptors is not fully understood, changes in Er and Pr dynamics throughout the estrous cycle and pregnancy are being examined.

In the cyclic ewe, the Er is generally abundant just before and after estrus (Wathes and Hamon, 1993). Numbers of Er fall dramatically from Day 3, to a low on Days 10 to 14, followed by a dramatic increase beginning on Day 14, to peak values at estrus (Koligian and Stormshak, 1976; Miller et al., 1977; Zelinski et al., 1980; Cherny et al., 1991; Ott et al., 1993b). Steady state levels of endometrial Er mRNA are highest on Day 1 of cyclic ewes, decline between Days 1 and 6, and increase between Days 11 and 15 (Spencer and Bazer, 1995). Myometrial Er mRNA in these animals was also highest on Day 1, but decreased to Day 6 and remained low thereafter. Cherny et al. (1991) reported that endometrial Er regulation was not homogenous throughout endometrial tissue, but varied by tissue type (caruncular or intracaruncular), as well as cell type (epithelial, stromal or glandular), and that steroidogenic control within tissues








27

was also variable. Spencer and Bazer (1995) have further demonstrated, using in situ hybridization and immunocytochemical approaches, that distinct and tissue- and cell-specific alterations in uterine Er and Pr mRNA and protein expression during the estrous cycle of the ewe generally paralleled the overall changes noted in steady state levels of Er and Pr mRNAs. In the endometrium, Pr mRNA and protein expression disappeared from the luminal and shallow glandular epithelium between Days 6 and 13, whereas Er mRNA and protein expression was low on Days 6 and 11 and increased between Days 11 and 15 in the luminal and shallow glandular epithelium. Er mRNA and protein were consistently present at low levels in the stroma and deep glandular epithelium. Progesterone is inhibitory to Er formation in several species (Brenner et al., 1974; Hsueh et al., 1975 and 1976; Tseng and Gurpide, 1975; West et al., 1976; Bhakoo and Katzenellenbogen, 1977; Spencer et al., 1995a; 1995b) including sheep (Koligian and Stormshak, 1977b; Zelinski et al., 1980; Cherny et al., 1991; Spencer et al., 1995a; 1995b). Estrogen, on the other hand, is stimulatory to it's own receptor formation (Anderson et al., 1975; Bhakoo and Katzenellenbogen, 1977; Zelinski et al., 1980; Cherny et al., 1991). Control of Er, however, is not a simple matter and probably involves several factors which affect each of the uterine cell populations in various ways (Cherny et al., 1991).








28

The Pr is most prevalent in ovine endometrium around estrus (Miller et al., 1977; Zelinski et al., 1980; Ott et al., 1993b) with the highest concentrations of receptors reported on Day 2 (Miller et al., 1977) and the lowest from Days 10 to 14. In cyclic ewes, endometrial Pr mRNA levels are highest on Day 1, decrease between Days 1 and 11, and then increase between Days 13 and 15. Myometrial Pr mRNA levels are highest on Day 1 and decline thereafter (Spencer and Bazer, 1995). Estrogen appears to be the primary stimulus for Pr formation (Milgrom et al., 1973; Leavitt et al., 1974; Zelinski et al., 1980; Aronica and Katzenellenbogen, 1991). Progesterone, however, can exert both positive and negative influences on uterine Pr expression. Progesterone in cyclic animals is clearly inhibitory to endometrial Pr expression, probably through down-regulation of Pr (Milgrom et al., 1973; Leavitt et al., 1974; Vu Hai et al., 1977). Progesterone down-regulation of its own receptor removes the progesterone block (McCracken et al., 1984), allowing Er up-regulation, and formation of OTr which, when bound by oxytocin, initiates pulsatile release of PGF2,. In pregnant ewes it had been assumed that Pr are maintained in the presence of high progesterone for extended periods (Ogle et al., 1989, 1990; Ott et al., 1993b; Mirando et al., 1993). In the pregnant ewe this was suggested to be achieved by stabilization of the Pr by a product of the conceptus (Ott et al., 1993b). However,








29

others have shown that continuous exposure of the endometrium to progesterone down-regulates endometrial Pr mRNA and protein abundance in the luminal epithelium, shallow glandular epithelium, and stroma (Wathes and Hamon, 1993; Spencer and Bazer, 1995; Spencer et al., 1995b). The mechanism responsible for this is currently poorly understood but may involve Pr-mediated decreases in Pr gene transcription (Alexander et al., 1989; Read et al., 1988). Results from studies performed by Spencer et al. (1995b) have shown that negative regulation of the Pr gene in the endometrial epithelium occurs in both cyclic and pregnant ewes, because Pr mRNA abundance and immunoreactive Pr protein declined in the endometrial luminal epithelium and shallow glandular epithelium after Day 6. Thus, the current hypotheses is that pregnancy does not stabilize or upregulate Pr gene expression in the endometrium.


Luteolysis


Luteolysis is initiated in ruminants in response to the pulsatile release of by PGFu2 produced by the uterus (McCracken et al., 1981; Hooper et al., 1986). PGFu is released from the uterus in the nonpregnant ewe in a series of 5-8 episodes (Thornburn et al., 1973) with 6-8 h between each series. McCracken et al. (1984) have shown that the CL must be exposed to approximately 5 pulses of PGF over a 25hour period to undergo complete luteolysis. These episodes








30

begin just prior to the onset of luteal regression. At this time the progesterone level has not begun to decline (Zarco et al., 1988b) but Pr are very low which allows expression of Er and OTr. The pulsatile secretion of PGF2a is initiated by the secretion of oxytocin from the posterior pituitary and is escalated by oxytocin of luteal origin. The result is a synchronous pulsatile release of oxytocin from the CL on each ovary and from the posterior pituitary in ewes (Hooper et al., 1986). Oxytocin from the CL and PGFz from the uterus act together in a positive feedback loop to generate pulses of PGF2a required for luteolysis.

Oxytocin is released from the posterior pituitary

(Hooper et al., 1986) as well as the CL where it is produced by large luteal cells (Rodgers et al., 1983; Wathes and Denning-Kendal, 1992) and is secreted into the ovarian vein (Wathes and Swann, 1982; Flint and Sheldirck, 1982; Flint and Sheldrick, 1986). In large luteal cells, the oxytocin gene is transcribed on Days 0-4 (Jones and Flint, 1986). Oxytocin mRNA is translated into oxytocin from Days 4-7. Stores of oxytocin and its neurophysin in luteal cells are highest on Days 10-12 (Silvia et al., 1991; Rhodes and Nathanielsz, 1990). Flint and Sheldrick (1983) reported that the rise and fall of oxytocin followed that of progesterone, with the lowest levels noted at the time of ovulation. However, there is no indication that progesterone directly affects the synthesis of oxytocin.








31

There is considerable evidence, however, for the association of oxytocin and PGF,,, in the formation of a positive feedback loop to increase pulsatile release of luteolytic PGF2a.

As stated earlier, there is a positive feedback loop between oxytocin and PGF,. That is, PGFu can stimulate luteal oxytocin secretion and oxytocin can stimulate endometrial PGF, secretion. There is evidence that the initiation of this loop is with PGFu secretion from the uterus due to the fact that PGFu2 release occurs prior to pulsatile release of luteal oxytocin (Moore et al., 1986). While it is possible that the uterus has an endogenous clock, possibly estrogen, that triggers the release of the initial PGFu secretion, pituitary oxytocin is the more accepted mediator of the initial uterine PGFu2 secretion which in turn begins the PGF2,/oxytocin feedback loop (see Silvia et al., 1991; McCracken et al., 1991).

Once the PGF2/oxytocin loop is established, it serves to increase PGF, concentrations in the ovarian pedicle. Increasing concentrations of PGF2, binding to their receptors on the large luteal cells, cause a reduction in viable cell numbers in vitro. Several different mechanisms have been put forth to explain the luteolytic effects of PGF2 ,(see Niswender and Nett, 1994; Spencer and Bazer, 1995). These mechanisms include: 1) a rapid decrease in luteal blood flow; 2) a reduction in the number of LH








32

receptors and/or an uncoupling of the LH receptor from adenylate cyclase; 3) activation of protein kinase C; 4) influx of high levels of calcium; and 5) a cytotoxic effect (Silvia et al., 1984a). Luteolysis does not require withdrawal of basal LH support, but PGF may activate protein kinase C in large luteal cells to inhibit progesterone production (Wiltbank et al., 1991) and cause luteolysis (McGuire et al., 1991). Treatment of large luteal cells with PGF increases intracellular calcium (Wiltbank et al., 1989), which appears to mediate the cytotoxic effects of PGFu2 probably through typical apoptotic changes (Sawyer et al., 1990) as well as decrease expression of mRNA for 3fhydroxysteroid dehydrogenase (Hawkins et al., 1993). Effects of Ovarian Steroids on Prostaqlandin-F2,, Secretion


There is a great deal of information on the effects of steroids on PGFu2 formation and/or luteolysis. It is obvious that progesterone and estrogen affect PGF2. production and secretion, and are, therefore, important steroids affecting luteolysis. Progesterone affects

One of the most compelling studies to link progesterone with normal cyclical estrus activity in ewes was that of French and Spennetta (1981) who showed that immunization of ewes against progesterone resulted in erratic estrous cycles. Progesterone initiates PGF,, secretion in








33

ovariectomized ewes after 7 days of treatment, but prior to progesterone treatment, PGFu2 secretion was minimal (Scaramuzzi et al., 1977). Prostaglandin-F2u secretion can also be induced early in the cycle (up to 32 h post-estrus) by progesterone treatment (Ginther, 1969; Ottobre et al., 1980). Administration of exogenous progesterone to cyclic ewes during metestrus decreases interestrous intervals, while administration of the Pr antagonist RU486 during the early luteal phase delays the onset of endometrial PGF production and luteolysis (Morgan et al., 1993). Apparently the uterus requires the influence of progesterone for a period of 10 to 12 days to produce luteolytic PGF2 in a normal cyclic manner (Vallet et al., 1990).

Progesterone is also necessary to elicit physiological changes in uterine responsiveness noted throughout the cycle. Humanics and Silvia (1988) utilized ovariectomized ewes to show this effect. Ewes were first pretreated with a hormonal regime that would mimic that of the steroid pattern noted in cyclic animals 6 days prior to estrus. Ewes were then treated with progesterone for 15 days and responsiveness to oxytocin (as determined by PGFM response) was measured on Days 5, 10 and 15 of treatment. They found that there was no PGFM response until Day 15. Vallet et al. (1990) used a similar model in which a subset of ewes received no steroid pretreatment and then progesterone alone








34

for 12 days to cause the uterus to become responsive to oxytocin challenge.

There are several ways that progesterone changes the endometrial milieu that could explain changes in uterine responsiveness to oxytocin. Brinsfield and Hawk (1973) reported that the accumulation of lipid droplets in uterine epithelium of ewes were induced by progesterone. In rats progesterone induces both phospholipid and triglyceride accumulation (Mamimekalai et al., 1979 ;Boshier et al., 1981). Progesterone has been shown to affect several factors associated with PGF2 metabolism. Raw and Silvia (1991) have shown that progesterone treatment of ovariectomized ewes for 16 days resulted in increased phospholipase-C activity. Phospholipase-C activity has been found to increase during the period of luteolysis in the ewe as well (Silvia and Raw, 1993). While there is no doubt that progesterone has an effect on phospholipase-C activity, Silvia and Raw (1993) suggest that control of PGF22 metabolism may occur later in the synthesis pathway.

Progesterone has an effect on the prostaglandin

synthetase enzyme in cattle, and ovariectomized ewes treated with progesterone also respond with elevated uterine endometrial prostaglandin-H synthetase activity (Raw et al., 1988). Eggleston et al. (1990) reported that prostaglandinH synthetase mRNA increased in intact ewes treated with progesterone early in the cycle. There was also an








35

increased incidence of early luteolysis in these ewes. An increase in PGFu2 release also occurs when progesterone is removed at a time corresponding to normal progesterone decline in cyclic ewes (Leavitt et al., 1985). Therefore, progesterone may stimulate PGF2a secretion early in the cycle, but exerts an inhibitory effect late in the cycle (see Silvia et al., 1991).

Estrogen affects

Ford et al. (1975) showed that pharmacological levels of estradiol (two injections given 12 h apart) resulted in an increase in PGF2a within 12 h after the second injection. This effect was the same for both control and progesterone primed ewes. McCracken (1980) used ovariectomized ewes, and Sharma and Fitizpatrick (1974) used anestrus ewes, to elicit a PGFu2 response to estradiol in as little as 6 h. Results of these studies indicate that in ovariectomized ewes, at least, there is no requirement for progesterone in PGF2G secretion. However, in studies in which physiological levels of estradiol were administered to ovariectomized ewes, estradiol alone was less effective in eliciting a PGFu2 response (Homantics and Silvia, 1988). In studies of intact ewes, estrogen was also ineffective in eliciting a PGFu2 response when administered alone. In a study in which the uterus was auto-transplanted to the neck of the ewe, Scaramuzzi et al. (1977) found that progesterone pretreatment was required to get a full PGF2,, response. In








36

ewes which received estradiol alone there was a slight, but nonsignificant, increase in the PGFu2 response. Endometrium from ewes treated with estradiol for several days in vivo were also unable to respond with PGF2a secretion in vitro (Findly et al., 1981; Raw and Silvia, 1991).

Effects of estrogens on PGFu2 release are much greater when estrogen is administered in conjunction with progesterone or in a regime that mimics the steroidogenic patterns of the normal estrous cycle. In cyclic ewes, estradiol injections on Days 11 and 12 (in one study), or Day 10 (in another), were able to induce luteal regression (Stormshak, 1969; Cook et al., 1974). In a similar study, Hawk and Bolt (1970) treated a total of 94 ewes with estradiol on two successive days with groups beginning treatment on Days 1 through 11. They found that estradiol had no effect on CL weights until Day 10 of the cycle. If ewes were not treated with estradiol until Days 11 and 12 CL weights were decreased by Day 15. Secretion of PGFz2 is also enhanced when ewes are treated with estrogen and progesterone (Ford et at., 1975). Hixon and Flint (1987) showed that inositol phosphate metabolism as well as PGF2. secretion increased when estradiol was administered at midcycle (Days 9 and 10). Barcikowski et al. (1974) concluded that the PGF2, response was due to a direct effect of estrogen on the uterus when physiological quantities of estradiol were infused either into the uterine artery or








37

systemically. Only those ewes which received treatment locally (at the uterus), and late in the cycle responded with elevated secretion of PGF2,. Ovariectomized ewes treated with progesterone for at least 5 days also responded with greater PGF2, secretion than ewes treated with estrogen alone (Scaramuzzi et al., 1977). As discussed earlier Vallet et al. (1990) reported that progesterone alone will elicit a PGF2a response when ewes are challenged with oxytocin. This study, however, showed that a combined treatment of progesterone and estrogen resulted in the highest response to oxytocin challenge.

Ovariectomized ewes, which have low basal

concentrations of both progesterone and estradiol, have also been used to study the factors which control oxytocininduced PGFu2 release (Beard and Lamming, 1994; Beard et al., 1994). In these ewes, expression of the endometrial OTr was constitutively present in high levels in luminal and superficial glandular epithelium, and treatment with progesterone initially caused a complete loss of OTr expression (Wathes and Lamming, 1995; Stevenson et al., 1994; Sheldrick and Flint, 1985). However, chronic progesterone treatment for 12 days resulted in an increase in endometrial phospholipid stores, OTr expression, prostaglandin synthase activity, and oxytocin-induced pulsatile release of PGF (Salamonsen, 1992; Spencer et al., 1995b; Vallet et al., 1990). The OTr that develop as








38

progesterone down-regulates Pr are localized exclusively to luminal and superficial glandular epithelium, which is essentially identical to the spatial expression of OTr during luteolysis (Wathes and Lamming, 1995).

Estradiol, like progesterone, affects the enzymes

involved in the synthesis of PGFa. Estrogen treatment on Days 9 and 10 of the cycle results in increased inositol phosphate metabolism (an indication of phospholipase-C activity) within 12 h, over that of controls. However, in ovariectomized ewes which received physiological doses of steroids for 16 days, there was a decrease in phospholipaseC activity in ewes treated with estrogen alone, or estrogen plus progesterone (Raw and Silvia, 1991). Prostaglandin synthetase mRNA was decreased in ovariectomized ewes treated with estrogen alone, however, prostaglandin synthetase mRNA was not different from controls when ewes were treated with progesterone and estrogen (Salamonsen et al., 1991). In this study, prostaglandin synthetase mRNA was decreased, but there was no difference in the levels of immunoreactive prostaglandin synthetase in luminal and glandular epithelial cells of ewes that received progesterone or progesterone plus estrogen.

Summary

The results of these studies clearly indicate that there is an effect of the ovarian steroids on PGF2. synthesis and secretion. While this effect is apparent








39

after progesterone treatment alone, estrogen appears to facilitate the complete response in the normal, cyclic ewe.


Endometrial Oxytocin Receptor


The release of PGF2 is initiated by oxytocin after

release from large luteal cells (Rodgers et al., 1983), and binding to its endometrial receptor (Flint and Sheldrick, 1983; Roberts et al., 1976). The appearance of OTr on endometrial epithelium (surface primarily) about Day 14 of the cycle is believed to set up the oxytocin/PGF2u feedback loop which initiates and insures luteolysis. Pulses of PGF22 have been shown to coincide with pulses of plasma oxytocin (Flint and Sheldrick, 1983; Hooper et al., 1987). The binding of oxytocin initiates the phosphoinositidephospholipase-C transduction signal, one action of which is to mobilize arachidonic acid, which results in the formation of PGF2,.

Endometrial OTr concentrations rise rapidly 48 h prior to estrus (Sheldrick and Flint, 1985). Endometrial OTr begin to increase on Day 14, reach maximum values at estrus and decline by Day 5 of the subsequent cycle. This is coincidental with the decrease in plasma progesterone, Pr, the rise in estrogen and Er (Sheldrick and Flint, 1985; Wallace et al., 1991) during proestrus. Through the use of receptor binding studies it was shown that endometrial OTr are more concentrated in the intercaruncular than in the








40

caruncular tissue of ewes in estrus, while Days 13 through 15 there were more receptors located in caruncular tissues (Shledrick and Flint, 1985). Autoradiographic studies have shown that at the time of luteolysis, endometrial OTr are found only on the luminal epithelial cells (Ayad et al., 1991a, 1991b; Wallace et al., 1991). The appearance of receptors in stromal tissues and glandular epithelial cells occurs after luteolysis. It is proposed that the apparent differential regulation of these receptor populations may be important in the process of luteolysis (or lack of luteolysis in the pregnant ewe).

The effect of oxytocin on its own receptor was examined by Flint and Sheldrick (primarily for therapeutic purposes; 1985). In this study, systemic infusion of oxytocin resulted in a lengthening of the interestrous interval. Upon examination there was no apparent lysis of luteal tissues. It was proposed by others that the action of oxytocin was to down-regulate its own receptor and that the increase in endometrial OTr at the time of luteolysis was due to removal of this down-regulation by decreasing concentrations of oxytocin in plasma. However, since plasma oxytocin concentrations decrease prior to this time, this seems unlikely. Down-regulation was also proposed as a possible explanation for the lack of OTr in pregnant ewes in response to oxytocin produced by the conceptus (Lacroix et al., 1988). This does not appear likely since ovine








41

trophoblast cells do not express oxytocin (Parkinson et al., 1991) and intrauterine infusions of oxytocin have no effect on cycle length or endometrial OTr expression (Parkinson et al., 1991; Ayad et al., 1993). It might be argued that uterine proteases denatured the infused oxytocin and this was the reason for the difference in cycle extension noted between these studies. However, OTr expression has been shown to be absent on the luminal epithelium if oxytocin was infused into the uterine lumen (Ayad et al., 1993). Thus, OTr may be localized to the basolateral domain of the epithelial cells so that intraluminal oxytocin may not have access to those receptors. Since the conceptus does not express oxytocin (Parkinson et al., 1991), down regulation does not appear to be a controlling factor in OTr concentration at the time of maternal recognition of pregnancy. The fact that endometrial OTr concentrations are highest when plasma oxytocin concentrations are low (Flint and Sheldrick, 1985) indicates that oxytocin effects are exerted at low receptor occupancy and suggests that control of uterine responsiveness to oxytocin is through control of OTr gene expression, rather than control of oxytocin secretion.

Oxytocin receptors are clearly under steroid hormone control. Uterine OTr increase (in rodents) in response to estrogen (Soloff, 1975). As indicated by PGF, secretion, prolonged progesterone treatment followed by estrogen








42

stimulation resulted in maximum OTr formation (McCracken et al., 1981). It was proposed (using a hamster model) that estrogen stimulates endometrial OTr formation and that progesterone exerts inhibitory actions through progesteroneinduced inhibitors which block estrogen binding to its receptor (Okulicz et al., 1981). These findings led McCracken et al. (1984) to propose that there was a progesterone block to uterine OTr formation during Days 5 to 14 of the cycle in ewes. At the end of this period the endometrium became refractory to the progesterone block, probably because of down-regulation of Pr by progesterone. Removal of the progesterone block, along with the stimulatory influence of rising estrogen, caused upregulation of endometrial OTr. Vallet et al. (1990) found that endometrial OTr were high in ovariectomized ewes, and treatment with progesterone initially caused a complete loss of OTr expression (Wathes and Lamming, 1995; Vallet et al., 1990; Lau et al., 1992) which indicates that the role of ovarian steroids, in control of endometrial OTr, is inhibitory. Interestingly, these receptors were apparently uncoupled from PGF2a synthesis as noted by the lack of PGFM response after challenge with exogenous oxytocin. Since progesterone increases phospholipid stores (Boshier et al., 1987) and prostaglandin synthase activity is necessary for conversion of arachidonic acid to PGFu2 (Eggleston et al., 1990) this "uncoupling" phenomenon was apparently related








43

to a reduction in the machinery necessary for the increased PGF synthesis. Progesterone treatment alone, for a period of 12 days, was sufficient to increase endometrial phospholipid stores, OTr expression, prostaglandin synthase activity, and oxytocin-induced pulsatile release of PGF2, (Salmonsen, 1992; Spencer et al., 1995b; Vallet et al., 1990). Treatment with progesterone for 5 days was not sufficient to stimulate endometrial OTr formation. A regime meant to closely mimic a normal estrous cycle (progesterone pretreatment, estradiol, progesterone for 12 days, and estradiol on Days 11 and 12 of progesterone treatment), resulted in only a slight increase in OTr number over that of progesterone alone. Estradiol alone was inhibitory to receptor formation. It was proposed by these authors that formation of endometrial OTr was under the inhibitory control of progesterone alone. Also, estrogen's action may be biphasic, in that estrogen may be stimulatory on Days 1 through 2 then inhibitory on Days 5 through 7. Similar findings were reported in studies utilizing ovariectomized ewes (Lamming et al., 1991; Zang et al., 1992; Lau et al., 1992; Lau et al., 1993) and uterine explant tissues (Sheldrick and Flick-Smith, 1993). The overall effects of estrogen on the timing, magnitude and pattern of PGF2. response to oxytocin may be mediated through increases in OTr gene expression (Beard and Lamming, 1994; McCrackin et al., 1984), enhanced coupling of OTr to its second messenger








44

signal transduction system (Bouvier et al., 1991) and increased activity of the machinery which drives prostaglandin synthase activity (Eggleston et al., 1990; Huslig et al., 1997).


Maternal Recognition Of Pregnancy


The term "maternal recognition of pregnancy" was first used in 1969 in a review published by R.V. Short. However, it had been known for some time that the conceptus in some way affected CL life-span. This was first alluded to in 1945 by Casida and Warwick who found that CL were maintained in pregnant ewes and that the conceptus was dependent on the CL for continued development until Day 55 of gestation. It was not until embryo transfer studies showed that synchronous transfers (Day 12 embryo into a Day 12 uterus) were successful, but embryo transfers on Day 13 were not, that the conceptus was directly implicated as having antiluteolytic effects. To determine that the Day 13 conceptus was not affected by the transfer procedure and that this was the reason there was not cycle extension in these ewes, Day 13 conceptuses were transferred to Day 12 cyclic sheep and the result was extension of the cycle. These results showed there was some affect of the conceptus that had to occur on Day 12 to prevent luteal regression in the pregnant ewe (Moor and Rowson, 1964; 1966a). A similar








45

study also detected the antiluteolytic effect of the ovine conceptus (Niswender and Dziuk, 1966).

To further examine the antiluteolytic effect of the conceptus, Moor and Rowson (1966b) removed conceptuses on Days 5 through 15 of pregnancy. They found that removal of the conceptus on Day 12 or before resulted in a cycle length that was normal (-17 days). However, if the conceptus was removed on Day 13 or after there was an extension in the life-span of the CL on average to Day 25. This study clearly indicated that the conceptus was affecting CL lifespan, and that this effect occurred on Day 12 of pregnancy. Since it was known that the uterus-initiated CL regression (see Luteolysis section) and that this was a local affect, Moor and Rowson (1966c) questioned whether the effect of the conceptus was also local. The authors found when conceptuses were transferred to an isolated ipsilateral horn the CL was maintained, but when the transfer was to an isolated contralateral horn the CL regressed. However, if embryo transfers to the contralateral horn coincided with removal of the ipsilateral horn the CL was maintained. These studies indicated that the conceptus exerts a local unilateral antiluteolytic effect. This was further indicated by the fact that embryo transfers to one isolated horn of a uterus in ewes with a CL on each ovary resulted in maintenance of the ipsilateral CL, but not the contralateral CL. Taken together, these studies clearly indicate that the








46

conceptus overcomes the local luteolytic effect of the uterus.

The next question was how does the conceptus exert its antiluteolytic effect. To examine this, Day 14 and Day 15 conceptus were collected, homogenized and frozen/thawed or heat treated. These homogenates were infused into the uterine lumen on Day 12 or daily for the treatment period. Infusions of conceptus homogenates on Day 12 alone or of heat-treated conceptus homogenates were not effective in extending the estrous cycle. Repeated daily infusions of frozen/thawed conceptus homogenates extended the cycle, on average to 22 days. Infusion of Day 25 sheep conceptuses homogenates or Day 14 pig homogenates had no effect on cycle length (Rowson and Moor; 1967). These studies indicated that the antiluteolytic effect was apparently species specific and time dependent. Furthermore, the fact that the antiluteolytic effect was present in frozen/thawed, but not heat-treated conceptus homogenates led the authors to suggest that the responsible factor was chemical in nature and heat labile. Similar studies indicated that there was an antiluteolytic effect of intrauterine infusion of bovine conceptus homogenates in the cow (Northly and French, 1980).

McCracken's report (1971) that PGF2a is released into

the uterine vein by endometrial tissues and that the timing of this release coincides with CL regression, initiated a great deal of interest in the effect of the conceptus on








47

PGF2a release. Moore and Watkins (1982) found that on Days 12 and 13, cyclic ewes responded with a pulse of PGFM for each pulse of oxytocin neurophysin and that this effect was absent in pregnant ewes. Pratt et al. (1977) reported that when PGF2a was injected into the largest follicle on the ovary bearing the CL of cyclic and pregnant ewes the cyclic ewes had a 1.5 day shorter interestrous interval, while none of the pregnant ewes returned to estrus. In this same report, intrauterine infusions of prostaglandin-E into pregnant ewes resulted in longer cycle lengths than for control ewes. Silvia and Niswender (1984) found that less PGF2a (4 mg/58 kg body weight as compared to 10 mg/58 kg) was required to cause luteolysis in cyclic compared to pregnant ewes. In further studies by these same authors, the protective properties the conceptus bestowed on the CL, in the presence of PGF2 challenge, did not occur until Day 13 and was lost by Day 26. Similar results were found when ewes were challenged with oxytocin (Fairclough et al., 1984; Silvia et al., 1992). Estradiol treatment also induces luteolysis in cyclic, but not pregnant ewes (Kittok and Britt, 1977), indicating an affect of estradiol in PGF2. production which the conceptus is able to block.

McCracken (1984) first proposed that estradiol-induced formation of endometrial OTr, which bound oxytocin, of pituitary or luteal origin, to induce pulsatile release of PGF2a. It was this process that began the feedback loop








48

which resulted in luteolysis (see Luteolysis section). Sheldrick and Flint (1985) reported that in pregnant ewes, the increase in OTr numbers detected in proestrus of cyclic ewes was attenuated. Oxytocin receptor expression is high from Day 14 of one cycle to Day 2 of the next cycle, but is almost completely absent in caruncular and intracaruncular endometrium of pregnant ewes (Flint and Sheldrick, 1986). Similar differences were found between cyclic and pregnant cows on Day 17 (Jenner et al., 1991). Autoradiological studies of endometrial receptors determined that labeled oxytocin was concentrated in luminal epithelium, glandular epithelia and caruncular stroma of Day 15 cyclic ewes. However, in pregnant ewes there was no labeling of endometrial tissues (Ayad et al., 1993).

Intrauterine injections of conceptus homogenates

increase the interestrous interval in ewes (Rowson and Moor, 1967). Since the conceptus was directly affecting the maternal environment it was believed that this effect was through a secretory product. To determine if conceptusconditioned culture media contained a pregnancy recognition factor, culture medium from incubations of Day 15 and 16 conceptuses were injected into the uterine lumen of cyclic ewes from Days 12 to 18 of the estrus cycle (Godkin et al., 1984b). The CL life-span was prolonged in all ewes treated with oCSP based on maintenance of progesterone secretion and CL which had previously been marked with India ink. One








49

treated ewe maintained a functional CL until Day 52 when the project was terminated. In comparison, all the ewes treated with SP, as controls, ovulated by Day 25 when CL were checked at hysterectomy, and progesterone secretion had fallen by Day 19. This was the first study to conclusively show that oCSP could prolong CL life-span. Similar findings were reported by Vallet et al. (1988).

To determine the effect of oCSPs on uterine PGFu

responsiveness after estradiol or oxytocin challenge, oCSP or plasma proteins were infused into the uterine lumen of ewes from Day 12 to Day 14 of the cycle (Fincher et al., 1986). On Day 14 all ewes were challenged with estradiol. Jugular blood samples were drawn hourly over a 10 hour period and assayed for PGFM. On Day 15 one-half of the ewes from each treatment group were challenged with oxytocin or saline. Blood was drawn for PGFM analysis. The PGFM response was lower in ewes which received oCSP, compared to SP-treated controls, after estradiol and oxytocin challenge. These results indicated that the factor in oCSP which affects the endometrium is a secretory protein produced by the conceptus and that it can induce changes in the uterine environment which prevent normal cyclic responsiveness to estradiol and oxytocin. Vallet et al. (1988) and Mirando et al. (1990a) also reported that PGF2a secretion after challenges with estradiol or oxytocin, was reduced significantly in oCSP-treated ewes.








50

The lack of PGFz2 responsiveness has been shown in the pregnant animal to be due to low oxytocin (Flint and Sheldrick, 1986) and estrogen (Findlay et al., 1982) receptors on the luminal epithelium. An indirect measure of the OTr can be obtained by measurement of IP which is a factor in the signal transduction pathway stimulated by activation of the OTr (Flint et al., 1986; Hixon et al., 1987). Mirando et al. (1990a, 1990b) reported that in Day 16 pregnant ewes there was an attenuation of IP hydrolysis within endometrial tissues after in vitro stimulation with oxytocin, while in Day 16 cyclic ewes there was an increase in IP hydrolysis. The pregnant ewes also had high plasma progesterone concentrations (Mirando et al., 1990a). Intrauterine injections of oCSP on Days 11 through 15 also resulted in lower IP hydrolysis after in vitro stimulation of endometrial tissues with oxytocin as compared to SPtreated controls (Mirando et al., 1990a, 1990b).

Maintenance of plasma progesterone concentrations noted in pregnant ewes may be important in sustaining the proposed progesterone block to OTr formation (McCracken et al., 1984). Ott et al. (1992) utilized an ovariectomized ewe model to indirectly (i.e. PGFM response to oxytocin challenge and IP hydrolysis) examine the interaction of oCSP and progesterone on OTr formation. Ovariectomized ewes received intrauterine injections of SP or oCSP from Day 11 through 14 (ewes were ovariectomized on Day 4 of the cycle)








51

and daily progesterone injections (im) were administered from Day 4 through Day 10 or Day 4 through Day 15. The oxytocin-induced PGFM response was completely blocked in oCSP-treated ewes which received progesterone until Day 15. Endometrial tissue of these ewes was not responsive to oxytocin-induced IP hydrolysis in vitro, while there was a doubling of the rate of IP hydrolysis in ewes treated with SP and progesterone. However, oCSP did not block endometrial responsiveness (either PGFM or IP hydrolysis) if progesterone treatment was stopped on Day 10. Interestingly, oxytocin-induced PGFM increases and IP hydrolysis were also blocked by treatment with progesterone alone until Day 15 (i.e., without intrauterine injection of oCSP). This supports McCracken's proposal that progesterone acts to block the increase in OTr formation until the time of luteolysis, but this does not support McCracken's proposed requirement for estrogen.

There is some evidence that ovine conceptuses secrete a factor (not oIFNT) that directly affects the luteal cells to attenuate PGF2,-induced luteolytic effects. Wiltbank et al. (1992) found that in vitro cultures of separated large and small luteal cells were affected by oCSP. When large cells were cultured with oCSP and PGFu2 the normal anti-steroidal effect of PGF,2 alone was blocked. There was no effect of oCSP on progesterone secretion by large luteal cells. However, oCSP increased progesterone secretion from small








52

luteal cells regardless of whether the small luteal cells were stimulated by LH or not. The anti-PGF2 factor in oCSP, while not fully characterized is apparently a protein as the protective action was lost when oCSP was heated. The anti-PGFu effect was shown not to be oIFNT when progesterone secretion was not decreased in the presence of PGFu2 and oCSP with oIFNT removed. While prostaglandin-E has been reported to possess luteoprotective properties (Henderson et al., 1977), prostaglandin-E was not considered the anti-PGFu factor since their dialysis procedure should have removed all prostaglandin-E. Also, there was no increase in progesterone secretion by the large luteal cells as noted when prostaglandin-E is added to large luteal cell cultures (Fitz et al., 1984). The anti-PGFu protein in oCSP apparently does not competitively inhibit binding of PGFu2 to its receptor as there was not a decrease in binding of 3H-PGF2. in the presence of oCSP. Therefore, it was proposed that the inhibitory action of this putative protein factor was at the post-receptor level, possibly at the level of the second messenger system. This study indicated that a factor in oCSP is capable of protecting progesterone production by luteal cells in the presence of PGF2a, and may explain why progesterone secretion is not decreased in pregnant ewes with higher basal levels of PGF2a (Ellinwood et al., 1979; Fincher et al., 1986; Zarco et al., 1988a; Vallet et al., 1989a; Burgess et al., 1990) and why








53

luteolysis in pregnant ewes requires higher doses of exogenous PGF22 (Inskeep et al., 1975; Silvia et al., 1984b; Silvia et al., 1986). However, this factor is not oIFNT, shown to be the only protein in oCSP to act on endometrial tissues to prevent pulsatile secretion of PGFa, thus maintaining pregnancy (Vallet et al., 1988). A supportive role of this, to-date unknown protein, to oIFNT in the maintenance of pregnancy should not be ruled out.

Since intrauterine injection of oCSP attenuates

oxytocin-induced PGF22 secretion (Fincher et al., 1986; Vallet et al., 1988) and IP hydrolysis (Mirnado et al., 1990b) by endometrium, it has been proposed that OTr formation is blocked in pregnant ewes by conceptus-mediated events (Flint et al., 1994, 1995; Flint, 1995). Vallet and Lamming (1991) reported that intrauterine injection of oCSP blocked oxytocin-induced PGF2 secretion and endometrial OTr formation. This was the first direct measure which confirmed that conceptus secretory products had an effect on OTr formation.

The proposed progesterone block to OTr formation was suggested to be maintained by the conceptus to prevent normal down regulation of the Pr on Days 12 to 14 (McCracken et al., 1984). In the pregnant ewe, Pr concentrations in endometrial tissues does not change from Day 10 to Day 16 (Ott et al., 1993b). However, during this time Pr mRNA actually decreased by 50 percent. Estrogen receptor protein








54

and mRNA decreased in pregnant ewes from Day 10 to Day 16. Mirando et al., (1993) reported that intrauterine injection of oCSP decreased Er protein and mRNA, as well as OTr in endometrial tissues. These results support an earlier hypothesis proposed by Ott et al. (1993) that Pr are stabilized in pregnant ewes and that Er formation is blocked. This in turn prevents the formation of endometrial OTr and, therefore, release of luteolytic pulses of PGFz2 in response to oxytocin by endometrium. Progesterone dependence has also been reported for uterine responsiveness to oxytocin (as measured by IP metabolism; Vallet et al., 1989b; Ott et al., 1992). However, as indicated previously, others have shown that continuous exposure of the endometrium to progesterone down-regulates endometrial Pr mRNA and protein abundance in the luminal epithelium, shallow glandular epithelium, and stroma (Wathes and Hamon, 1993; Spencer and Bazer, 1995). The mechanism responsible for this is currently poorly understood, but may involve Prmediated decreases in Pr gene transcription (Alexander et al., 1989; Read et al., 1988). Results from studies performed by Spencer et al. (1995) have shown that negative regulation of the Pr gene in the endometrial epithelium occurs in both cyclic and pregnant ewes, because Pr mRNA abundance and immunoreactive Pr protein declined in the endometrial luminal epithelium and shallow glandular epithelium after Day 6. Thus, results of this study were









55

used to modify the hypothesis to indicate that pregnancy does not stabilize or up-regulate Pr gene expression in the endometrium.


Ovine Trophoblast Protein-i


To determine whether ovine conceptuses produce a pregnancy recognition factor, ovine conceptuses were collected on Days 13 through 21 and cultured in the presence of 3H-leucine for 24 h (Godkin et al., 1982). Twodimensional polyacrylamide gel electrophoresis of the dialyzed medium reveled one major protein product with three isoelectric species. The isoelectric species had pI's of 5.5 to 5.7 and an estimated molecular weight of 17,000 to 20,000. This conceptuses product was initially referred to as protein X (Wilson et al., 1979; Godkin et al., 1982; 1984a) Protein X production could be detected by gel filtration chromatography 2D PAGE between Days 13 and 21 of pregnancy (Wilson et al., 1979; Godkin et al., 1982). In situ hybridization studies later showed that oTP-1 mRNA could be detected on Day 12, but full scale production of oTP-1 was not up-regulated until Day 13 (Hansen et al., 1985; Farin et al., 1990). Because this protein was produced transiently by the conceptus during the period of maternal recognition it was proposed to be the pregnancy recognition factor (Godkin et al., 1982). This same protein








56

had been partially characterized by Martal and coworkers in 1979 and named Trophoblastin.

Godkin et al. (1984a) further characterized the actions of oTP-1 in a series of experiments which showed that oTP-1 in Day 16 pregnant sheep was localized within the uterus. Ovine trophoblast protein-i was associated with the trophectoderm cells of the blastocyst and with the surface and upper glandular epithelium of the uterus. Uterine infusion of '51-oTP-1 into Day 12 cyclic ewes indicated that oTP-1 was retained in the uterine tissues with little reaching the vasculature draining the uterus. When tissues from the CL and other ovarian structures were examined no oTP-1 was found. This indicated that the action of oTP-1 is local, at the level of the endometrium. Ovine trophoblast protein-i did not stimulate progesterone production by dispersed luteal cells from Day 12 cycling ewes. However, there was an oTP-1-induced increase in protein production in vitro from uterine tissue acquired from ewes on Day 12 of their cycle. In competition assays, oTP-1 did not compete with oPRL in rabbit mammary cell cultures. Ovine trophoblast protein-i also did not compete with hCG or bLH in sheep luteal cell cultures. As a result of these studies, the authors suggested three possible functions for oTP-1: first; (1) induce the uterus to produce proteins to meet the nutritional requirements of the conceptus until attachment occurs; (2) induce endocrinological changes









57

within uterine tissues which control the synthesis, release, or sequestration of PGF2a; or (3) induce secretion of particular proteins from the endometrium which would act in a luteotropic fashion at the level of the ovary. The second and third functions have been shown to be incorrect (for oTP-1). For this reason oTP-1 is considered to be an antiluteolytic hormone and not a luteotropic hormone.

A protein similar to oTP-1 is produced by the bovine and caprine conceptuses, and termed bTP-1 and cTP-1, respectively. The bTP-1 is the major protein produced by the bovine conceptus during the period of maternal recognition of pregnancy (Day 16 to 24). Bovine trophoblast protein-1, like oTP-1, posses several molecular weight and isoelectric variants ranging from 20 to 26 kDa and pI of 4.5 to 6.5, respectively (Bartol et al., 1985). However, unlike oTP-1, bTP-1 is glycosylated. Caprine trophoblast protein-i has at least two isoforms with molecular weights of about 17,000 and pIs of 5.2-5.7. The cTP-1 is the major protein produced by the goat conceptus during the time of maternal recognition of pregnancy (Day 17) (Gnatek et al., 1989). Ovine Trophoblast Protein-i is an Interferon


The identification of oTP-1 as an IFN came about as

result of molecular cloning of cDNA and protein sequencing techniques (Imakawa et al., 1987; Stewart et al., 1987; Imakawa et al., 1989; Charpigny et al., 1988). These








58

reports identified oTP-1 (which has also been called Trophoblastin, Protein-X, oTP-1, oIFNa11, oTIFN-omega), as a Type I IFN. Ovine trophoblast protein-i is reportedly closest in homology to the omega IFNs (originally refereed to as alpha.l IFNs). Trophoblast interferons of different species (bTP-1, cTP-1 and oTP-1) are more closely related to one another than they are to the omega IFNs of their own species (i.e. oTP-1 and other ovine omega IFNs). The trophoblast interferons are apparently functionally related as well, as indicated by extension of the cycle in goats when sheep conceptuses were transferred to the goats uterus, prior to the period of maternal recognition or when roIFNT was injected into the uterine lumen (see Bazer et al., 1993). In cattle, the transfer of trophoblastic vesicles (Heyman et al., 1984) or intrauterine injection of roIFNT also results in extension of the cows estrous cycle (see Bazer et al., 1993). It has been proposed, for these reasons, that the nomenclature IFNT be used to refer to all the trophoblast interferons (ovine, oIFNT; bovine, bIFNT; and caprine, cIFNT) Genes for IFNs are believed to exist in all ruminants of the order Artiodactyla and are believed to have diverged from IFN-omega genes 30 to 65 million years ago (Roberts et al., 1992; Leaman et al., 1992). Several cDNA sequences have been published for oIFNT since the first reports; however, they all have common characteristics








59

(Stewart et al., 1989b; Klemann et al., 1990; Charlier et al., 1991).

All IFNTS are 172 amino acids in length. Trophoblast interferons, like other Type I IFNs, are intron-less. They are coded for by a 595 base pair open reading frame which codes for a 195 amino acid preprotein with a 23 amino acid signal sequence that is cleaved to produce the 172 amino acid mature protein. There are two disulfide bridges at highly conserved Cys residues. The first is between the Cys' residue and Cys9. The second is between Cys29 and Cys139. This last pair of Cys residues have been identified in all alpha, beta and omega IFNs and appears to be important for biological activity of the molecule (see Roberts et al., 1992). There are several other conserved residues found in all Type I IFNs, including the IFNTs. They are the four Cys just discussed, as well as Leu3, Leu30, Arg33, Phe38, Pro39, Glu50, Glu52, Ser73, Gln2, Leu6, Tyrl23, Tyr130, Leu131, Ala140, Trp141, and Val'" (Klemann et al., 1990).

Hydrophilicity-hydrophobicity plots of the different isoforms of Type I IFN are very similar in spite of the differences noted in the sequences as a whole. Secondary structures are also very similar. It is believed that there are five a-helices arranged in an anti-parallel manner with connected loop regions (see Roberts et al., 1992). This arrangement is similar to that reported for IFNS, interlukin-1, interlukin-4, growth hormone and granulocyte








60

macrophage-colony stimulating factor (see Bazer et al., 1993). Interestingly, a recent report indicates that granulocyte macrophage-colony stimulating factor may play a role in modulation of production of oIFNT (Imakawa et al., 1993)

The oIFNT molecule is not glycosylated; however, there is a site of potential glycosylation in some of the isoforms at Asn78 (Godkin et al., 1982; Anthony et al., 1988) This is in contrast to bIFNT and some isoforms of cIFNT which are glycosylated (Helmer et al., 1987; Helmer et al., 1988; Baumbach et al., 1990). Why oIFNT is not glycosylated and other IFNTs are is not currently known.

Trophoblast IFNs are biologically similar to other Type I IFNs. Ovine IFNT affords antiviral protection to cells cultured in the presence of virus just as IFNa (Pontzer et al., 1988). Ovine IFNT decreases proliferation of several cell lines as does IFNa (Pontzer et al., 1991). Incorporation of tritiated thymidine into lymphocytes following mitogen exposure is blocked by both oIFNT and IFNa (Newton et al., 1989). Finally, oIFNT up-regulates 2,5oligoadenylate synthetase in endometrial tissues (Mirando et al., 1991). The major difference between the IFNTs and other Type I IFNs is their apparent lack of cytotoxicity. Even in large concentrations, the IFNTs exert little or no cytotoxic effects. This lack of cytotoxicity has generated








61

considerable interest in IFNT for use as a therapeutic drug (see Bazer, 1991).

For future experiments in the area of maternal

recognition of pregnancy, and as a therapeutic drug, IFNT must be produced in larger quantities than those obtained from 30 hour cultures of a Day 16 conceptus (Godkin et al., 1982). For this reason, recombinant forms of oIFNT have been developed using synthetic oligonucleotides and cDNAs (Ott et al., 1991 and Martal et al., 1990, respectively). The roIFNT produced by Ott et al. (1991) was derived from a synthetic gene which was edited to include 17 unique restriction sites not found in the natural oIFNT sequence (Imakawa et al., 1989). While roIFNT was designed for expression in E. coli, yeast were used to overproduce the product. The use of yeast has allowed large amounts of roIFNT to be produced (Ott et al., 1991; Van Heeke et al., 1996). The restriction sites will allow for the easier production of mutants to investigate structure/function of oIFNT domains in the future. Biological activities for roIFNT and oIFNT have been shown to be identical (Ott et al. 1991).


Maternal Recognition Effects of Ovine Interferon Tau
and Type I Interferons


While it had been shown that the maternal pregnancy recognition factor produced by the ovine conceptus was contained within the milieu of oCSPs, and that oTP-1 (now








62

referred to as oIFNT) was the major protein produced by the conceptus during the pregnancy recognition period (Godkin et al., 1984b), studies with purified oIFNT were required to demonstrate that oIFNT is the maternal pregnancy recognition factor.

The ability of partially purified oIFNT to extend the interestrous interval was initially demonstrated by Godkin et al. (1984b). The purified oIFNT was obtained by pooling culture media from a large number of conceptus cultures and passing the media over DEAE cellulose ion-exchange and S-200 Sephacryl columns. The resultant purified product was injected into the uterine lumen via catheters surgically placed into the tip of each uterine horn on Days 12 through 21. Plasma progesterone was used to determine CL life-span. A decrease in plasma progesterone concentrations to below 1 ng/ml was used to indicate luteal regression. Progesterone levels in ewes treated with partially purified oIFNT remained elevated 4 days longer than controls. While this was significant it was not nearly as long as the cycle extension noted for ewes (from this same report) which received intrauterine injections of oCSP. The differences in the extension of cycle between ewes which received oCSP and those that received oIFNT could have been due to several factors. The purified oIFNT may have been degraded by uterine protease or the estimate of the amount used per infusion, calculated from conceptus production in culture








63

during a 24 hour period, may have been too low to elicit the same response as oCSP. The other major question was whether the difference between the two treatments could be due to some other factor which acted in concert with the oIFNT in the total oCSP to cause the longer cycle extension. To answer these questions, Vallet et al. (1988) prepared highly purified oIFNT. In addition, the oCSP remaining after oIFNT had been removed using an anti-oIFNT column several times to remove all traces of oIFNT, was used to treat ewes. Ewes were fitted with uterine catheters and injected (intrauterine) with either SP, oCSP, oIFNT, or oCSP minus oIFNT. There was no difference in the cycle length (19 days each; determined by fall in plasma progesterone) between ewes treated with SP and oCSP with oIFNT removed. From these findings, it is apparent that proteins other than oIFNT in oCSP are not involved in maternal recognition of pregnancy. Further, there was no difference in the intraestrous interval between the ewes which received oCSP or highly purified oIFNT (27 and 25 days, respectively). These results indicate that there is not a synergistic effect between other conceptus products and oIFNT in maternal recognition of pregnancy.

Intrauterine injections of recombinant forms of oIFNT also increase the interestrous interval in ewes. Martal et al. (1990) reported an extension of up to 64 days (confirmed by marked CL at slaughter) when large amounts of roIFNT were








64

administered (intrauterine; 340 [g/day). There was also an extension of the cycle in ewes which received levels of roIFNT comparable to that produced by Day 16 conceptus in culture (170 pg/day), as shown in more recent studies in which roIFNT was administered from Day 11.5 through Day 16 and resulted in an extension of the interestrous interval to that of about 31 days (OTT et al., 1993a). Recombinant forms of other Type I IFNs (rbIFNa) also extend the interestrous interval; however, larger doses were required (2 mg/day) (Stewart et al., 1989a; Parkinson et al., 1992). It is interesting to note that intramuscular injections of rbIFNa increased the lambing rate in treated ewes (Schalue et al., 1989, 1991; Nephew et al., 1990; Martinod et al., 1991). Although, it is not known how rbIFNa aided in pregnancy recognition it was proposed that it supplemented the activity of endogenous oIFNT to ensure recognition of pregnancy in ewes in which conceptuses may have been retarded in growth, or for some reason producing suboptimal levels of oIFNT.


Effects on prostaglandin Synthesis


Intrauterine injections of oIFNT affect endometrial

prostaglandin responsiveness to oxytocin and estrogen in a manner similar to that following intrauterine injections of oCSP in studies by Fincher et al. (1986). Vallet et al. (1988) reported that a challenge with estradiol on Day 14








65

resulted in a subsequent rise of plasma PGFM concentrations in ewes treated with intrauterine injections of SP as controls while there was no such rise in ewes which received oIFNT. These same ewes, when challenged with oxytocin on Day 15, responded with a lower PGFM response when treated with oIFNT. There was no effect of treatment on prostaglandin-E production. This indicates that the prostaglandin production is not shunted from production of PGF2a in cyclic ewes, to production of prostaglandin-E in pregnant ewes, to prevent luteolysis as proposed by McCracken et al. (1984).

Attenuation of the PGFu2 response to exogenous oxytocin in pregnant ewes is acquired over a period of several days. Endometrium collected from ewes on Day 15, after treatment with oIFNT or SP by intrauterine injection on Days 12 through 14 (Vallet et al., 1989a) was perifused with buffer plus oxytocin and the medium assayed for PGF2a content. There was a higher PGF,2 response to oxytocin for SP-treated than for oIFNT-treated ewes. This supported results from a previous in vivo study (Fincher et al., 1986). However, when Day 15 endometrium was obtained from ewes not treated with oIFNT in vivo, the in vitro PGFu2 response was higher for endometrium treated in vivo with oIFNT. This is exactly opposite from the effects reported with long term treatment with oIFNT. These results imply that oIFNT does not act directly to competitively interfere with binding of oxytocin








66

to its receptor or by other means to block the PGF2 response. Rather, oIFNT, prevents development of endometrial sensitivity to oxytocin-induced PGF2,, secretory response. It has been shown that Type I interferons increase arachidonic acid metabolism. If oIFNT has the same affect on uterine tissues the increase in PGF2, secretion reported in the short term perifusion experiments may be attributed to an increase in arachidonic acid metabolism within the tissues. However, several reports in the literature indicate that, in uterine tissues, this may not occur (Salamonsen et al., 1988) and that arachidonic acid mobilized by IFNa is shunted away from the cyclooxygenase pathway (Hannigan and Williams, 1991).

As mentioned previously, IFN produced by the bovine

conceptus is very similar to that of oIFNT. Bovine IFNa and other Type I IFNs, like oIFNT, extend CL life-span when administered at the time of maternal recognition in the cow (Thatcher et al., 1989; Helmer et al., 1989a: Plante et al., 1991). However, unlike the ewe, basal PGF2. is lower in both pregnant cows, and cows which receive IFN treatment, than in cyclic cows. It is not known why basal levels of PGF2, are lower in bIFNT-treated cows compared to controls, while there is no difference in basal levels of PGF2a noted in oIFNT-treated and control ewes. It may be that the cow blocks luteolysis by inhibition of PGF,, synthesis early in the PGF2/oxytocin feedback loop to prevent a rise in OTr.








67

There is evidence for this mode of action of bIFNT in cows (Shemesh et al., 1981; Basu and Kindahl, 1987; Gross et al., 1988a; Helmer et al., 1989b) The action of a prostaglandin inhibitor in sheep has not been well studied, but endogenous prostaglandin inhibitors have been detected in endometrium (Basu, 1989) and in allantoic fluid (Harper and Thornburn, 1984; Rice et al., 1987) of sheep. It is not known, however, if an inhibitor of PGFu2 synthesis plays a role in maternal recognition pregnancy in ewes. In the cow, bIFNT may induce synthesis of a prostaglandin inhibitor (DanetDesnoyers et al., 1993). Interferons affect arachidonic acid metabolism. Also, inhibitors of the cyclooxygenase or lipoxygenase pathways of arachidonic metabolism increase activities of the transduction signal for IFNa (Hannigan and Williams, 1991). Type I IFNs have been reported to attenuate prostaglandin secretion. Interferon-a was reported to attenuate prostaglandin production in human cells (Dore-Duffy et al., 1983; Browning and Ribolini, 1987) The prostaglandin affected in this study was prostaglandin-E which is produced via the same pathway (cyclooxygenase) as PGFa. Human IFNa incubated with endometrial cells from ovariectomized ewes, maintained on a steroid regime that mimics that of a normal estrous cycle, caused a decrease in PGFu2 secretion (Salamonsen et al., 1988; Salamonsen et al., 1989). Vallet et al. (1991) reported that intrauterine injection of rbIFNa on Days 12 through 14 was as effective








68

in blocking oxytocin-induced PGFu2 secretion from the uterus as was oCSP; however, this was believed to be through progesterone attenuation of the OTr.


The Ovine Interferon Tau Receptor and Signal Transduction


Ovine IFNT, like other Type I IFNs (alpha, beta, and omega) bind to a high affinity (Godkin et al., 1984a;) Type I IFN receptor (Stewert et al., 1987). Type I IFN receptors are distributed throughout endometrial tissues of the ewe and their expression may be influenced by ovarian steroids (Knickerbocker and Niswender, 1989). Type I receptors are also present in other tissues of the body (Knickerbocker and Niswender, 1989). Pontzer et al. (1990) reported that high concentrations of the NT of oIFNT attenuated antiviral effects of oIFNT, but not IFNa, in cell co-cultures. Conversely, a synthetic peptide corresponding to amino acids 139-172 (CT) blocked antiviral effects of both oIFNT and IFNa in cell co-cultures. The authors concluded that NT of oIFNT binds to a unique domain in the Type I IFN receptor while the CT binds to a domain common to Type I IFNs. This may explain the unique actions of oIFNT.

Ovine IFNT-induced hormone action is initiated by the

transduction of signal via activation of the JAK/STAT system (see Willians, 1991a) and is believed to act in the same manner as other Type I interferons (see Interferon Receptor/Signal Transduction section).








69

Ovine IFNT and other IFNT's increase endometrial

protein production dramatically (Gross et al., 1988b; Sharif et al., 1989; Ashworth and Bazer, 1989). Included in these is the enzyme 2',5'-oligoadenylate synthetase (Mirando et al., 1991; Short et al., 1991). Estrogen receptors are increased in endometrial adenocarcinoma cells by IFNa2b and in human breast cancer tissue and human endometrium. In rabbit endometrium Er expression is increased by IFNa. Progesterone receptors may be increased by IFNa2 in endometrial adenocarcinoma and by IFNa in AE-7 endometrial cancer cells (see Bazer et al., 1993). Full length Er and Pr genes for ruminants have not been cloned to determine the presence of an interferon stimulated response element in the 3'or 5' flanking region; however, analysis of partial clones of genomic DNA from human and rabbit Er and Pr indicate their presence (see Bazer et al., 1993). If an interferon stimulated response element(s) is present in the Er and Pr genes they may allow oIFNT to negatively regulate expression of Er and OTr within the endometrium during early pregnancy (Mirando et al., 1993; Ott et al., 1993b) to allow for establishment of pregnancy.
















CHAPTER 3
THE EFFECTS OF RECOMBINANT OVINE INTERFERON TAU AND
SYNTHETIC PEPTIDES, CORRESPONDING TO PORTIONS OF RECOMBINANT
OVINE INTERFERON TAU, ON OXYTOCIN-STIMULATED ENDOMETRIAL
INOSITOL PHOSPHATE METABOLISM AND ENDOMETRIAL OXYTOCIN RECEPTOR CONCENTRATION.


Introduction


The establishment of pregnancy in ewes requires that the conceptus, by Day 12 of pregnancy (Moor and Rowson, 1966a), activate a mechanism(s) (oIFNT; Godkin et al., 1982; Vallet et al., 1988) to prevent luteolysis. Luteolysis in ruminants is initiated by pulsatile secretion of PGFu2 by endometrial tissues (McCracken et al., 1981; Hooper et al., 1986). Pulsatile secretion of uterine PGF2a is thought to be responsible for luteolysis due to the fact that continuous infusion of PGF,,, or immunization against PGF,,, results in extension of the cycle (Scaramuzzi and Baird, 1976; Fairclough et al., 1981). During luteal regression, PGF2a is secreted from the endometrium in a series of five to eight, high amplitude, short duration episodes (Thornburn et al., 1973; Barcikowski et al., 1974; Flint and Sheldrick, 1983; Zarco et al., 1988b) with 6 to 8 h between each episode. McCracken et al. (1984) have shown that the CL must be exposed to approximately 5 pulses of PGF2, over a 25 70








71

h period to undergo complete luteolysis. The pulsatile secretion of PGF2a may be initiated by the secretion of oxytocin from the posterior pituitary and is escalated by oxytocin secreted by the CL (Flint et al., 1990). Oxytocin from the CL and PGF2, from the uterus act together in a positive feedback loop to generate the luteolytic pulses required for luteolysis (Flint and Sheldrick., 1986; Hooper et al., 1986; see Silvia et al., 1991 ;McCracken et al., 1991). However, the ability of the endometrium to secrete PGF2a in response to oxytocin does not develop until Day 13 to 14 of the cycle (Roberts et al., 1976; Roberts and McCracken, 1976; Fairclough et al., 1984; Silvia et al., 1991) when OTr concentrations increase (Sheldrick and Flint, 1985). It is the coupling of oxytocin to specific endometrial OTr sites that stimulates PGFu synthesis, through activation of the inositol phosphate/diacylglycerol signal transduction pathway (Flint et al., 1986; Silvia and Homanics, 1988).

During early pregnancy, pulsatile secretion of PGF2a

secretion by the uterus is attenuated or absent (Thornburn et al., 1973; Barcikowski et al., 1974; Moore and Watkins, 1982; Hooper et al., 1987; Zarco et al., 1988a) with a disruption of the oxytocin/PGF,2 positive feedback loop. The attenuation of the loop in the pregnant ewe is primarily due to the absence of expression of endometrial OTr and Er








72

(McCracken et al., 1984; Sheldrick and Flint, 1985, Spencer et al., 1995b).

Ovine IFNT is the antiluteolytic protein secreted by

the conceptus (Godkin et al., 1992; Vallet et al., 1988; see Bazer et al., 1991; Godkin et al., 1984a; 1984b; Bazer et al., 1995). Endometrial IP metabolism (Mirando et al., 1990 a,b; Ott et al., 1992), PGF2, secretion (Vallet et al., 1988; Mirando et al., 1990a; Ott et al., 1992) as well as endometrial OTr and Er expression (Vallet and Lamming, 1991; Mirando et al., 1993; Spencer et al., 1996) are inhibited by intrauterine injection of oIFNT. Intrauterine injection of roIFNT is as effective as oIFNT purified from conceptus culture medium in blocking luteolysis (Ott, 1992; Ott et al., 1993a). It is believed that oIFNT acts through its Type I IFN receptor, and activation of its transduction signal to prevent endometrial expression of Er and OTr. The Pr and progesterone are considered permissive to antiluteolytic effects of oIFNT, but the mechanism is not known (Spencer 1995).

Several questions have been raised as to whether or not the oxytocin-induced IP metabolism, previously reported from our laboratory, was due to stimulation through the OTr, or could it be through the AVP receptor. Experiment 1 was designed to answer this question, by determining if oxytocin-induced IP metabolism within endometrial tissues is mediated through the OTr or the AVP receptor. This was








73

accomplished by means of an IP metabolism assay that examined all possible combinations of OT, and AVP stimulation with all possible combinations of the receptor antagonist for the OTr and AVP receptor.

Other primary objectives of these experiments (as they relate to IP metabolism and endometrial OTr concentration), were to determine what effect treatment of cyclic ewes, with various synthetically produced peptides corresponding to overlapping segments of oIFNT, had on oxytocin-induced endometrial IP metabolism, and on endometrial OTr concentration. The NT and CT peptides were first examined (Experiment 2) due to the fact that they were the most extensively examined of the peptides (Pontzer et al., 1990, 1994). It has been proposed that the NT possesses the properties which makes oIFNT different from other IFNas, and that CT is the portion common to the IFNas. Experiment 3 examined the effect of the remaining peptides (2-5) on oxytocin-induced IP metabolism and endometrial OTr concentration. Experiments 4, also examined the effect of NT treatment on endometrial OTr concentration, but this experiment was specifically designed to determine what effect oxytocin challenge (in vivo) on Days 13 and 15 would have with regards to the main treatment of NT or roIFNT. The final experiment, Experiment 5, was designed to determine what effect NT has on endometrial OTr concentration through Day 18, which is two days longer than








74

oIFNT had been examined. This was to determine if NT has the ability to hold OTr concentration at a low level for an extended period of time.


Materials and Methods


Animals


Ewes of primarily Rambouillet breeding were checked

daily at 07:30 am for 20 min with vasectomized males of St. Croix or mixed Rambouillet breeding. Ewes which had previously exhibited at least two normal estrous cycles (16 to 17 days in length) were assigned to experimental groups. Ewes for experiments 1, 2, and 3 were housed at the Sheep Research Facility, University of Florida, Gainesville. Ewes for experiment 4 and 5 were housed at the Sheep Research Center, Texas A&M University, College Station. Protein And Peptide Preparation Ovine conceptus secretory protein preparation

Ovine conceptus secretory proteins were prepared as

previously reported by our laboratory (Vallet et al., 1988 and Mirando et al., 1990b). Briefly, ovine conceptuses were collected at laparotomy on Day 16 of pregnancy by flushing the uterus with 20 ml minimum essential medium (Earl's salts; Gibco/Life Technologies, Grand Island, New York). The conceptuses were cultured for 30 h in minimum essential medium as reported by Godkin et al. (1982). The resultant








75

oCSP-conditioned medium was collected, pooled and stored at -200C until used.

Medium containing oCSP was thawed, pooled and dialyzed (3500 Mr cutoff) at 40C against 4 liters of 0.9% NaCl (w/v), changed three times (4L each change). Ovine conceptus secretory proteins were concentrated to one-tenth the original volume using an Amicon ultrafilter (500 Mr cutoff; Amicon Co., Danvers, MA). The concentration of oIFNT in oCSP was determined by RIA (Vallet et al., 1988). Ovine conceptus secretory proteins were diluted with in 0.9% NaCl (w/v) to an oIFNT concentration of 25Ag/ml oIFNT. The concentration of total protein in oCSP was determined by the method of Lowry et al. (1951). The oCSP was diluted with SP in 0.9% NaCl (w/v) to a total protein concentration of 0.75 mg/ml, and stored at -200C in 2 ml aliquots until just prior to use when they were thawed under running water. Recombinant interferon tau preparation

Recombinant oIFNT, provided by Dr. Troy Ott, was

produced as described by Ott et al. (1991). Antiviral units of roIFNT were determined by antiviral assay using Madin Darby bovine kidney cells challenged with vesicular stomatitis virus (Pontzer et al., 1988). Protein concentration of roIFNT was determined by protein assay (Lowry et al., 1951). Recombinant oIFNT was diluted to 25 gg/ml for Experiment 3 and 50 Ag/ml for Experiments 4 and 5. The total protein concentration was brought up to 0.75 mg/ml








76

by the addition of SP in 0.9% NaCl (w/v). Aliquots of 2 ml each were stored in glass scintillation vials at -200C until just prior to use at which time they were thawed under running water.

Serum protein preparation

Blood was collected from the jugular vein of a pregnant ewe on Day 16 (07:00 am) and allowed to clot 1 h at room temperature and then overnight at 200C. Serum was collected and dialyzed (3500 Mr cutoff) at 40C against 4 L 0.9% NaCl (w/v), with three changes (4 L each change). Protein concentration was determined (Lowry et al., 1951) and diluted to a protein concentration of 0.75 mg/ml with 0.9% NaCl (w/v) and stored in glass scintillation vials at -200C in 2 ml aliquots until just prior to use when they were thawed under running water.

Synthetic peptide production

Synthetic peptides corresponding to the amino and

carboxyl-terminus of oIFNT were produce by Dr. Carol Pontzer in Dr. Howard M. Johnson's laboratory as described by Pontzer et al. (1990). Briefly, peptides were synthesized on a Biosearch 9500AT automated peptide synthesizer using fluorenylemthyloxycarbonyl chemistry. Peptides were cleaved from resins using trifluroacetic acid/ethanedithiol/thioanisole/anisole. Cleaved peptides were extracted in diethyl ether and ethyl acetate, dissolved in water and lyophilized. Reverse phase HPLC was used to








77

determine purity of the peptides. Peptides produced were: to the NT (aa 1-37); CT (aa 139-172): as well as four overlapping internal peptides: Pep 2 (aa 34-64); Pep 3 (aa 62-92); Pep 4 (aa 90-122); and Pep 5 (aa 119-150). All peptides were reconstituted in 0.9% NaCl (w/v) to 0.5 mg/ml. This concentration had previously been shown to block oIFNTinduced antiviral activity (Pontzer et al., 1990). Serum proteins were added to bring total protein concentration to 0.75 mg/ml in a 2 ml volume. Aliquots of 2 ml were stored in glass scintillation vials at -200C until just prior to use when they were thawed under water. Experimental Design


Experiment 1

To determine if oxytocin-induced IP metabolism within endometrial tissues is mediated through the OTr or the AVP receptor inositol phosphate metabolism was examined in endometrium from three ewes after in vitro stimulation of oxytocin or AVP. Ewes received no in vivo treatment. On Day 16 of their estrous cycle ewes, were anesthetized with halothane and ovariectomized-hysterectomized by mid-ventral laparotomy. Caruncular tissue was collected from the entire uterus into 100 mm petri dishes, maintained on ice and minced into fine pieces. Tissue (0.999 gm) was transferred to 16 separate 20 ml glass scintillation vials and placed on ice until the oxytocin/AVP IP assay was begun (- 10 min).








78

Experiment 2

Twenty-four ewes were randomly assigned in a 2 X 3 factorial arrangement to receive SP, SP+NT, SP+CT, oCSP, oCSP+NT or oCSP+CT (n=4/treatment; Fig. 3.1). On Day 6, ewes were anesthetized with halothane and the uterus exteriorized by laparotomy. The number and location of CL were recorded, and a catheter (8' in length; V6 tubing, Bolab, Lake Havasu City, Az) placed into each uterine horn (- 2 cm) via the oviduct at the utero-tubal junction (Vallet et al., 1988). Catheters were secured to the oviduct at the utero-tubal junction, and the uterine body at the external bifurcation, with suture on either side of a set of cuffs (-2 cm and 30 cm from one end; V10 Tubing, Bolab, Lake Havasu City, AZ) fused to the catheter with a drop of cyclohexanone (Fisher). Catheters were exteriorized through an incision in the flank, and attached to the skin by suturing cloth tape, which was wrapped around the tubing, to the skin. The catheters were flushed with sterile 0.9% saline to determine that there were no blockages and the ends sealed to prevent air from entering the tubing. The external portion of the tubing was stored, wrapped in betadine soaked 4 X 4 gauze, and placed in a pouch attached by suture to the skin at the point of exit (Vallet et al., 1988). Ewes were allowed to recover for 24 h. They were then returned to their respective group pens until where they were placed into individual pens located within group




























Figure 3.1: Experimental design for Experiment 2.









Twice Daily
Intrauterine Injections
Estrus Catheterization Hysterectomy


DayO 1 8 9 10 11 12 13 14 15 16 16pm

Treatments:
SP 1.5 mg/l uterine horn 2X daily
SP + 0.5 mg NT/ uterine horn 2X daily (aa 1-37)
SP + 0.5 mg CTI uterine horn 2X daily (aa 139-172)
oCSP (25 jtg olFNT) I uterine horn 2X daily
oCSP + 0.5 mg NT / uterine horn 2X daily oCSP + 0.5 mg CT I uterine horn 2X daily
Total proteinlintrauterine injection = 1.5 mg (Balanced with SP)
Total volumelintrauterine injection including saline flush = 3.1 ml
Tissues collected for IP, OTr (filter method), and Er mRNA

CD








81

pens until Day 11. This arrangement limited movement of the ewes during the treatment period while allowing them constant contact with the other ewes in their respective pens. Hay and water were available ad libitum and about 120 gm concentrate feed was provided each morning. Hay, water and food were withheld 12 h prior to surgery. On Day 12, treatments began and continued through the morning of Day 16. Each ewe received, per uterine horn, twice daily injections (06:00 and 18:00 h, respectively) of one of the following, according to group: 1.5 mg SP; 0.5 mg NT plus 1.0 mg SP; 0.5 mg CT plus 1.0 mg SP; 0.75 mg oCSP (containing 25 lg oIFNT by RIA) plus 0.75 mg SP; 0.75 mg oCSP plus 0.5 mg NT; or 0.75 mg oCSP plus 0.5 mg CT. All injections were balanced to a total protein concentration of 1.5 mg with SP and were adjusted to 2 ml in volume with 0.9% NaCl (w/v). Each injection also contained 50 mg ampicillin (Polyflex; Aveco Co. Inc., Fort Dodge, IA) in 0.1 ml 0.9% NaCl (w/v) which was mixed with the thawed treatment sample just prior to injection. Each catheter was flushed with 1 ml 0.9% NaCl (w/v) after injection, resulting in a total infused volume of 3.1 ml. The ends of the each catheter was resealed without introducing air and the catheters returned to the pouch in fresh 4 X 4 gauze sponge soaked in betadine. On Day 15, all food and water was removed from the ewes and on the morning of Day 16, after the last intrauterine injection, the ewes were ovariectomized-hysterectomized.









82

Endometrial tissue (-1.2 gm; primarily of caruncular origin) from the uterine horn ipsilateral to the CL was collected into ice-cold KRB for determination of IP metabolism. The remainder of the endometrium was collected into bags, snap frozen in liquid nitrogen, and stored at -800C for use in oxytocin receptor assays (filter method) and Er mRNA analysis (Chapter 5).

Experiment 3

Twenty-eight ewes were randomly assigned to one of

seven treatment groups to receive SP, roIFNT, NT, Pep 2, Pep 3, Pep 4, or Pep 5 (n=4/treatment; refer to the section on synthetic peptide preparation for amino acid determination of each peptide; Fig. 3.2). On Day 8 or Day 9 of the estrous cycle ewes were anesthetized and the uterus exteriorized by laparotomy, the number and location of CL noted and catheters placed as described in Experiment 2. Ewes were housed as described in Experiment 2 with the exception that individual crates were located in an adjoining pen in sight of ewes in their respective group pens. Intrauterine injections were administered as in Experiment 2 except that they began on Day 11. Treatments, per horn, consisted of 1.5 mg SP, 0.25 Ag roIFNT, 0.5 mg NT,

0.5 mg Pep 2, 0.5 mg Pep 3, 0.5 mg Pep 4, or 0.5mg Pep 5. The total protein concentration per treatment was brought to

1.5 mg with SP and the total volume adjusted to 2 ml with

0.9% NaCl (w/v). Each injection, also contained 50 mg



























Figure 3.2: Experimental design for Experiment 3.










Twice Daily
Estrus Catheterization Intrauterine Injections Hysterectomy



Day 0 1 8 9 10 11 12 13 14 15 16 16pm

Treatments:
rolFNT 100 ptgl ewe/ day
25 pg / uterine horn 2X daily NT (aa 1-37)
Pep 2 mg/ ewe/l day Pep 2 (aa 34-64)
500 ggl uterine horn 2X daily Pep 3 (aa 62-92) Pep 4 (aa 90-122)
SP 6 mg/l day Pep 5 (aa 119-150)
1.5 mg/ uterine horn 2X daily

O








85

ampicillin in 0.1 ml 0.9% NaCl (w/v) which was added with the sample just prior to injection. Each catheter was flushed with 1 ml of 0.9% NaCl (w/v) after injection, to clear the catheter, so the volume injected was 3.1 ml.

On Day 15 all food and water was removed from the ewes and on the morning of Day 16, after the last injection, the ewes were ovariectomized-hysterectomized. Endometrial tissue (-1.2 gm; primarily of caruncular origin) from the uterine horn ipsilateral to the CL was collected into icecold KRB for determination of IP metabolism. The remainder of the endometrium was placed in plastic bags, snap frozen in liquid nitrogen, and stored at -800C for use in oxytocin receptor assays (filter method) and Er mRNA analysis (Chapter 5).

Experiment 4

Twelve ewes were randomly assigned to receive intrauterine injection of either SP, roIFNT or NT (n=4/treatment; Fig. 3.3 and 3.4). On Day 8 or 9 of the estrous cycle ewes were anesthetized and the uterus exteriorized by laparotomy and catheters placed as described in Experiment 2. Ewes were allowed to recover for 24 h and then returned to a pen, separate from, but in sight of, ewes in their respective group pen until Day 10, when they were placed into individual pens located within sight of ewes in their group pens. Hay and water were available ad libitum. On Day 11 treatments began, and continued twice daily (06:00




Full Text

PAGE 1

MATERNAL RECOGNITION OF PREGNANCY IN THE EWE: THE STRUCTURE/FUNCTION RELATIONSHIP OF OVINE INTERFERON TAU By TAMMIE KAY SCHALUE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1997

PAGE 2

For The Trees

PAGE 3

ACKNOWLEDGMENTS I would like to express my gratitude to Dr. F. W. Bazer and Dr. W. W. Thatcher for their invaluable guidance and support during the course of my program. I would also like to thank all the members of my committee, Dr. W.C. Buhi , Dr. H.M. Johnson and Dr. D.C. Sharp, for their support and their contributions to my Ph.D. program as a whole. Special thanks are extended to Dr. T.F. Ogle for conducting the steroid receptor assays, to Dr. Troy Ott for supplying the roIFNt , to Dr. Tom Spencer for supplying the estrogen probe and for his gracious support, and to MarieJoelle Thatcher for her assistance with the PGFM assay. This work would not have been possible without the assistance and support of the other students, postdocs and technicians of the lab. I would also like to thank all the other faculty, postdocs and graduate students of the Animal and Dairy and Poultry Science Departments for their assistance and support. Special consideration must also go to my family, for they have supported me throughout my life, as I chased my dreams . iii

PAGE 4

TABLE OF CONTENTS ACKNOWLEDGMENTS iii LIST OF FIGURES viii KEY TO ABBREVIATIONS xi ABSTRACT xiv CHAPTERS 1 GENERAL INTRODUCTION 1 2 REVIEW OF THE LITERATURE 6 Arachidonic Acid Metabolism And Prostaglandin Synthesis 6 Interferon Receptors/Signal transduction ... 8 Steroid Receptors 13 Models For Steroid Receptors; Historical Perspective 13 Current Model For Steroid Receptors . . 15 Structure/Function Of The Steroid Receptor 16 Receptor Activation 18 Hormone Response Elements 20 Estrous Cycle 21 Luteolysis 29 Effects of Ovarian Steroids on Prostaglandin-F 2a Secretion 32 Progesterone affects 32 Estrogen affects 35 Summary 3 8 Endometrial Oxytocin Receptor 39 Maternal Recognition Of Pregnancy 44 Ovine Trophoblast Protein1 5 5 Ovine Trophoblast Protein1 is an Interferon 57 Maternal Recognition Effects of Ovine Interferon Tau and Type I Interferons 61 Effects on prostaglandin Synthesis ... 64 iv

PAGE 5

The Ovine Interferon Tau Receptor and Signal Transduction 68 3 THE EFFECTS OF RECOMBINANT OVINE INTERFERON TAU AND SYNTHETIC PEPTIDES, CORRESPONDING TO PORTIONS OF RECOMBINANT OVINE INTERFERON TAU, ON OXYTOCINSTIMULATED ENDOMETRIAL INOSITOL PHOSPHATE METABOLISM AND ENDOMETRIAL OXYTOCIN RECEPTOR CONCENTRATION 7 0 Introduction 70 Materials and Methods 74 Animals 74 Protein And Peptide Preparation .... 74 Ovine conceptus secretory protein preparation 74 Recombinant interferon tau preparation 7 5 Serum protein preparation 7 6 Synthetic peptide production ... 76 Experimental Design 77 Experiment 1 77 Experiment 2 78 Experiment 3 82 Experiment 4 85 Experiment 5 91 Inositol Phosphate Metabolism 92 Oxytocin-induced IP metabolism 99 Endometrial Oxytocin Receptor Assay . 10 3 Filter procedure 103 PEG assay 107 Statistical Analysis 109 Results 114 Inositol Phosphate Metabolism .... 114 Endometrial Oxytocin Receptor Assay . 120 Discussion 130 4 THE EFFECTS OF RECOMBINANT OVINE INTERFERON TAU AND SYNTHETIC PEPTIDE DOMAINS OF INTERFERON TAU ON OXYTOCINSTIMULATED PROSTAGLANDINF METABOLITE CONCENTRATIONS IN PLASMA OF EWES . 137 Introduction 137 Materials and Methods 139 Animals 139 Protein And Peptide Preparation . . . 139 Recombinant ovine interferon tau preparation 139 Serum protein preparation .... 139 Synthetic peptide production . . 139 Experimental Design 140 v

PAGE 6

Prostaglandin-F metabolite assay . . . 140 Statistical Analysis 141 Results 142 Discussion 149 5 THE EFFECTS OF OVINE CONCEPTUS SECRETORY PROTEINS , RECOMBINANT OVINE INTERFERON TAU AND SYNTHETIC PEPTIDES CORRESPONDING TO PORTIONS OF INTERFERON TAU ON ENDOMETRIAL CONCENTRATIONS OF ESTROGEN AND PROGESTERONE RECEPTOR PROTEIN AND raRNA 159 Introduction 159 Materials and Methods 163 Animals 163 Protein And Peptide Preparation . . . 164 Ovine conceptus secretory protein preparation 164 Recombinant ovine interferon tau preparation 164 Serum protein preparation .... 164 Synthetic peptide production . . 164 Experimental Design 164 Estrogen Receptor mRNA 165 Ribonucleic acid isolation . . . 165 Northern blot procedure 166 Slot blot procedure 167 Hybridization 169 Estrogen Receptor Assay 170 Progesterone Receptor Binding Assay . 172 Statistical Analysis 173 Results 175 Estrogen Receptor mRNA 17 5 Estrogen Receptor Binding Assay . . . 176 Progesterone Receptor Binding Assay . 183 Discussion 190 6 GENERAL DISCUSSION 198 Working Model of Maternal Recognition of Pregnancy in the Ewe 19 8 Effects of oIFNx on IP metabolism and OTr Concentrations 207 Effects of oIFNx on Oxytocin-Stimulated PGFM 208 Effects of oIFNx on Er Protein and mRNA 208 Summary 210 APPENDICES A PROTOCOL FOR PGFM ASSAY 212 vi

PAGE 7

B PROTOCOL FOR OXYTOCIN RECEPTOR ASSAY (PEG PROCEDURE) 216 C PROTOCOL FOR BICINCHONINIC ACID PROTEIN ASSAY 221 D PROTOCOL FOR OXYTOCIN RECEPTOR ASSAY (FILTER PROCEDURE) 224 E PROTOCOL FOR INOSITOL PHOSPHATE ASSAY . . . 229 REFERENCE LIST 235 BIOGRAPHICAL SKETCH 265 vii

PAGE 8

LIST OF FIGURES Figure Page 3.1 Design for Experiment 2 80 3.2 Design for Experiment 3 84 3.3 Design for Experiment 4 87 3.4 Detailed of treatment period for Experiment 4 ... 89 3.5 Design for Experiment 5 94 3.6 Schematic of the AVP/OTstimulated IP assay 96 3.7 Schematic of the OTstimulated IP assay 102 3.8 Validation of the OTr assay (filter procedure) .. 106 3.9 Time and temperature validation for the OTr assay (PEG procedure) Ill 3.10 Optimal protein concentration validation for the OTr assay (PEG procedure) 113 3.11 Mean inositol phosphate metabolism in endometrium treated with oxytocin, AVP, and/or antagonists for each of the neurophysin receptors 116 3.12 Mean inositol phosphate metabolism in endometrium from ewes treated with SP, oCSP, SP + CT, SP + NT, oCSP + CT or oCSP + NT 119 3.13 Mean inositol phosphate metabolism in endometrium from ewes treated with SP, roIFNx , or synthetic peptides corresponding to portions of oIFNx 123 3.14 Mean endometrial OTr concentration in ewes treated with SP, oCSP, SP + CT, SP + NT, oCSP + CT or oCSP + NT 125 3.15 Mean endometrial OTr concentration in ewes treated with SP, roIFNt, or synthetic peptides corresponding to portions of oIFNt 127 viii

PAGE 9

3.16 Mean endometrial OTr concentration in ewes treated with SP, NT or roIFNx 129 3.17 Mean endometrial OTr concentration in ewes treated with SP or NT for 16, 17 or 18 days 132 4.1 Individual SP-treated ewe PGFM response following oxytocin challenge 144 4.2 Individual NT-treated ewes PGFM response following oxytocin challenge 146 4.3 Individual roIFNx -treated ewes PGFM response following oxytocin challenge 148 4.4 Mean PGFM response following oxytocin challenge in ewes treated with SP, NT, or roIFNx 151 4.5 Mean of PGFM response following oxytocin challenge in ewes treated with SP, NT, or roIFNx 153 4.6 Mean treatment*day effects on PGFM response following oxytocin challenge on Day 13 and 15 in ewes treated with SP, NT, or roIFNx 155 4.7 Mean treatment*day* time effects on PGFM response following oxytocin challenge in ewes treated with SP, NT, roIFNx 157 5.1 Mean endometrial Er mRNA concentration in ewes treated with SP, oCSP, SP + CT, SP + NT, oCSP + CT or oCSP + NT 178 5.2 Mean endometrial Er mRNA concentration in ewes treated with SP, roIFNt, or synthetic peptides corresponding to portions of oIFNt 180 5.3 Mean endometrial Er mRNA concentration in ewes treated with SP, NT, or roIFNx 182 5.4 Mean endometrial Er mRNA concentration in ewes treated with SP or NT on Days 16, 17 or 18 185 5.5 Mean endometrial Er protein concentration in ewes treated with SP, NT or roIFNt 187 5.6 Mean endometrial Er protein concentration in ewes treated with SP or NT on Days 16, 17 or 18 189 5.7 Mean endometrial Pr protein concentration in ewes treated with SP, NT or roIFNx 192 ix

PAGE 10

5.8 Mean endometrial Pr protein concentration in ewes treated with SP or NT on Days 16, 17 or 18 194 6.1 Schematic diagram of the regulation of hormone receptor expression during late diestrus 201 6.2 Schematic diagram of the regulation of hormone receptor expression during establishment of pregnancy 203 6.3 Schematic diagram of the regulation of hormone receptor expression during establishment of pregnancy involving the mechanism of oIFNt action 205 x

PAGE 11

KEY TO ABBREVIATIONS aa Amino Acid Ala . . Alanine ANOVA. . Analysis of Variance Arg Arginine AVP . . Arginine Vasopressin CT Carboxylterminus of oIFNi; aa 139-172 Cys . Cysteine DNA . . . Deoxyribonucleic Acid GLM . . . General Linear Models Gin . . . Glutamine Glu . . . Glutamic Acid HSP . Heat Shock Protein IFN . . . Interferon IFNr . . . Type I Interferon Receptor IFNa . . . Interferon alpha IFNS . . . Interferon beta IFNco . . Interferon omega IRF-2 . . Interferon Regulatory Factor-2 IRF-E . Interferon Regulatory Factor Element ISGF3 . IFN Stimulated Gene Factor3 Complex ISGF3a IFN Stimulated Gene Factor3a ISGF37 IFN Stimulated Gene Factor-37 xi

PAGE 12

ISRE . . . IFN Stimulated Response Element IP ... Inositol Phosphate JAK . . . Janus Kinase KRB ... Kreb's Ringer Bicarbonate Leu . . . Leucine mRNA . . . Messenger Ribonucleic Acid NT ... Amino-terminus of oIFNt ; aa 1-37 oCSP . . . Ovine Conceptus Secretory Protein oIFNt . . Ovine Trophoblast Interferon OT ... Oxytocin oTP-1 .. Ovine Trophoblast Protein1 OTr . . . Oxytocin Receptor PEG . . . Polyethyleneglycol Pep 2... aa 34-64 of oIFNt Pep 3... aa 62-92 of oIFNt Pep 4... aa 90-122 of oIFNt Pep 5... aa 119-150 of oIFNt PGF 2a . . Prostaglandin-F 2a PGFM ... 13 , 14-dihydro-15-keto Prostaglandin-F 2a Pro . . . Proline RNA . . . Ribonucleic Acid rblFN . Recombinant Bovine Interf eron-alpha roIFNx . Recombinant Ovine Trophoblast Interferon SAS . . . Statistical Analysis System SEM . . . Standard Error of the Mean Ser . . . Serine xii

PAGE 13

csirrnal Transdurprs and. Q f a 1" 1 ft qt at 2 Si anal Transducers and Q IT . . Cpv-iirn P"rn1~^i n • O <3 -iLll LL r 1 U Tyk2 . . . Tyrosine Kinase Tyr . Tyrosine Trp . Tryptophan Activators of Transcriptionla Activators of Transcription113. Activators of Transcription-2 xiii

PAGE 14

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MATERNAL RECOGNITION OF PREGNANCY IN THE EWE: THE STRUCTURE/FUNCTION RELATIONSHIP OF OVINE INTERFERON TAU By Tammie Kay Schalue August, 1997 Chairperson: Dr. F. W. BAZER Co-Chairperson: Dr. W. W. THATCHER Major Department: Animal Science In the ewe, pregnancy success is dependent on the conceptus secreting the pregnancy recognition factor, oIFNx , during the maternal recognition of pregnancy period. oIFNx is secreted by the trophoblast cells of the developing conceptus. Binding to specific endometrial receptors initiates a series of events which block the release of luteolytic PGF 2a . Our current working hypothesis is that oIFNx accomplishes this attenuation of pulsatile PGF 2o! secretion by the endometrium, through preventing expression of the Er gene, and subsequent up-regulation of expression of the OTr gene that normally occurs in non-pregnant ewes. The NT portion of the oIFNt has been shown, through competitive binding studies, to act through a specific domain of the Type I IFN receptor while the CT is believed xiv

PAGE 15

to act through a more common domain of that receptor. The present studies were conducted to determine if the NT, or the other portions of oIFNt , has pregnancy recognition properties when injected into the uterine lumen of cyclic ewes. Intrauterine injections were either one of six overlapping synthetic oIFNt peptides (NT was the peptide most closely examined), ovine conceptus protein or roIFNt . The NT peptide was nearly as effective as roIFNi in suppressing expression of endometrial Er and OTr, blocking the metabolism of inositol phosphate in endometrial tissues after an oxytocin challenge test in vitro and PGFM response to an oxytocin challenge test in vivo. NT had no effect on endometrial Pr expression. Collectively, these results support the hypothesis that oIFNt suppresses expression of endometrial Er and OTr, thereby preventing the pulsatile release of PGF 2a without affecting expression of Pr. Also, these results indicate that the NT peptide is as effective as oIFNt in producing a pregnancy recognition response. This finding supports the hypothesis that the NT of oIFNt is the most likely portion of oIFNt responsible for the antiluteolytic properties of oIFNt which distinguish it from other Type I IFNs. xv

PAGE 16

CHAPTER 1 GENERAL INTRODUCTION The "term maternal recognition of pregnancy" was first used in 1969 by R.V. Short. Identification of the maternal pregnancy recognition factor as an IFN came about as the result of molecular cloning and amino acid sequencing (Stewart et al . , 1987; Imakawa et al., 1987, 1989; Charpigny et al., 1988). These reports identified the maternal pregnancy recognition factor, referred to currently as oIFNt , as a Type I IFN. Trophoblast IFNs are biologically similar to other Type I IFNs, and like the other IFNts, oIFNt displays both antiviral and antiproliferative properties as does IFNa (Pontzer et al . , 1988). Ovine IFNt binds to high affinity (Godkin et al. , 1984a) Type I IFN receptors (Stewert et al . , 1987) which are distributed throughout endometrial tissues of the ewe and their expression may be influenced by ovarian steroids (Knickerbocker and Niswender, 1989) . Synthetic peptides corresponding to the amino(Pontzer et al . , 1991) and carboxyl-terminal peptides of oIFNt , as well as two internal peptides (aa 62-92 and aa 119-150) were found to inhibit oIFNt receptor binding and antiviral activity in a dose

PAGE 17

dependent manner (Pontzer et al . , 1994) indicating specific competition between these peptides and oIFNt. The NT had no effect on the antiviral activity of IFN. These findings indicated that the NT may act through a novel domain of the oIFNt receptor while the other peptides may act through a more common domain of the Type I IFN receptor. This could explain the unique actions of oIFNt . Ovine IFNt -induced hormone action is initiated by the transduction of signal via activation of four cytosolic proteins which bind to interferon stimulated response elements (see Williams, 1991a; Darnell et al., 1994) and is believed to act in the same manner as all other Type I interferons. Ovine IFNt and other Type I IFNs increase endometrial protein production dramatically (Gross et al . , 1988b; Sharif et al . , 1989; Ashworth and Bazer, 1989; see Spencer et al., 1996) and affect expression of Er concentration within the endometrium during early pregnancy (Mirando et al . , 1993; Ott et al . , 1993b; Wathes and Hamon, 1993; Spencer et al . , 1995a, 1995b) to allow for establishment of pregnancy. In the cyclic ewe, the Er is abundant just before, during and after estrus. Receptor numbers fall dramatically from Day 3 , to a low on Days 10 to 14, followed by a dramatic increase beginning on Day 14, to peak at estrus (Koligian and Stormshak, 1976; Miller et al . , 1977; Zelinski et al., 1980; Cherny et al . , 1991; Ott et al . , 1993b;

PAGE 18

3 Spencer et al . , 1996). Progesterone is inhibitory to Er formation in most species (Brenner et al., 1974; Hsueh et al., 1975 and 1976; Tseng and Gurpide, 1975; West et al . , 1976; Bhakoo and Katzenellenbogen , 1977) including sheep (Koligian and Stormshak, 1977b; Zelinski et al., 1980; Cherny et al., 1991). Progesterone in the cyclic animal is clearly inhibitory to its own endometrial receptor, probably through a downregulation mechanism (Milgrom et al., 1973; Leavitt et al . , 1974; Vu Hai et al . , 1977; Spencer et al., 1996). Progesterone ' s downregulation of its own receptor initiates removal of the proposed progesterone block (McCracken et al . , 1984), allowing Er up-regulation , and formation of OTr which bind oxytocin to initiate pulsatile release of PGF^ from the uterus. In the cyclic ewe luteolysis is initiated by pulsatile release of PGF 2a produced by the uterus (McCracken et al . , 1981; Hooper et al . , 1986; Thornburn et al . , 1973; Niswender and Nett, 1994). Oxytocin from the ovary, binding to its endometrial OTr, and PGF 2a from the uterus binding to its ovarian receptor, act together in a positive feedback loop to generate the pulses of PGF 2a required for luteolysis (Flint and Sheldrick, 1983; Hooper et al . , 1987; Niswender and Nett; 1994) . Endometrial OTr begin to increase on Day 14, reach maximum values at estrus and decline by Day 5 of the subsequent cycle. This is coincidental with the decrease in plasma progesterone and the rise in estrogen

PAGE 19

4 (Sheldrick and Flint, 1985; Wallace et al . , 1991) during proestrus . Intrauterine injections of conceptus homogenates increase the interestrous interval in ewes (Rowson and Moor, 1967) and oCSPs elicit the same response (Godkin et al., 1984b; Vallet et al., 1988). Intrauterine injection of oCSPs also blocks cyclic uterine PGF 2a responsiveness after oxytocin challenge (Fincher et al . , 1986; Vallet et al., 1988; Mirando et al . , 1990a). The lack of PGF 2a responsiveness (Fincher et al., 1986; Vallet et al . , 1988) and IP hydrolysis (Mirando et al . , 1990b) in the pregnant ewe is due to low OTr (Flint and Sheldrick, 1986) and Er (Findlay et al., 1982) on the luminal epithelium (Spencer et al., 1996). The proposed progesterone block (McCracken et al., 1984) to OTr formation was initially believed to be conceptus maintained by preventing the normal downregulation of the Pr on Days 12 to 14. Recent results of Spencer et al. (1995a, b) indicate that in the pregnant ewe oIFNx prevents luteolysis, not by stabilizing Pr and maintaining the progesterone block, but by preventing expression of the Er gene which, in turn, prevents expression of the endometrial OTr gene. Present studies determined the function of oIFNt on factors associated with maternal recognition and examined the functional properties of specific domains of oIFNx responsible for maternal recognition of pregnancy effects.

PAGE 20

The experimental designs and methods for the five individual animal experiments which comprise the present studies are discussed in Chapter 3 and are referred to throughout the remainder of this dissertation by experiment number.

PAGE 21

CHAPTER 2 REVIEW OF THE LITERATURE Arachidonic Acid Metabolism And Prostaglandin Synthesis Arachidonic acid, a 20 carbon fatty acid, is supplied through the diet or by anabolic metabolism of linoleic acid (Ramwell, 1977). Most arachidonic acid is present within integral membrane components of cells in the form of phospholipids. Arachidic acid is found most prominently at the 2n position of phosphatidylcholine, phosphatidylethanolamine , and, to a lesser extent, phosphatidylinositol (MacDonald and Sprecher, 1991) . Arachidonic acid, once mobilized from phospholipids, usually enters into one of three major arachidonic acid metabolic cascades; the cyclooxygenase , lipoxygenase, or epoxygenase pathways. The cyclooxygenase pathway produces prostaglandins and thromboxanes (Smith et al . , 1991). The lipoxygenase pathway produces leukotrienes , hydroxy acids and lipoxens (Samuelsson, 1987; Smith et al., 1991). Epoxy acids and dihydroxy acids are formed via the epoxygenase pathway (Fitzpatrick and Murphy, 1988) . Arachidonic acid is mobilized from phospholipids in response to extracellular stimuli, in part, through the 6

PAGE 22

actions of one of three phospholipases ; phospholipase A 1( A 2 , and C (Smith, 1986) . Phospholipase A, cleaves acyl residues from phospholipids at the 3n (3 carbon) position of the glycerol backbone to produce a lysophospholipid from which arachidonic acid is then cleaved. Phospholipase A 2 mobilizes arachidonic acid directly from the phospholipid at the 2n position of the glycerol backbone (Loeb and Gross, 1986; Kramer et al . , 1989;). Phospholipase C, via receptormediated G protein stimulation, cleaves phosphatidylinositol into inositol 1 , 4 , 5 trisphosphate and diacylglycerol , each of which can act as a transduction signal within the cell. Arachidonic acid is subsequently cleaved from diacylglycerol at the 2n position by an inositol specific phospholipase C enzyme with both diacylglycerol lipase and monoacylglycerol lipase activities (see Berridge, 1984; Smith et al . , 1991). Arachidonic acid mobilization for prostaglandin synthesis is primarily through the actions of phospholipase A 2 and phospholipase C (Martin and Wysolmerski, 1987) . The prostaglandins -A, -D, -E, -F, and -I, are 20 carbon compounds produced via the cyclooxygenase pathway of arachidonic acid metabolism, through the actions of prostaglandin-H synthetase (Smith, 1989) . Prostaglandin-H synthetase is an integral membrane protein found on the cytoplasmic side of cell membranes (Smith et al., 1991). Prostaglandin-H synthetase exhibits both cyclooxygenase and hydroperoxidase activities as separate sites on the

PAGE 23

8 synthetase enzyme (Miyamoto et al . , 1976; Pagels et al . , 1983) Cyclooxygenase catalyzes prostaglandin^ formation from arachidonic acid and hydroperoxidase acts on the 15hydroperoxyl group of prostaglandin^ to form prostaglandin-H 2 . Both cyclooxygenase and hydroperoxidase activities require heme (Kartheim et al., 1987). Prostaglandin production is strongly regulated by the availability of substrate (arachidonic acid) and control of prostaglandin-H synthetase activity to produce prostaglandin-H 2 , the precursor of all prostaglandins and thromboxane . Prostaglandin-D 2 , prostaglandin-E 2 , prostaglandin-I 2 , and thromboxane are produced from prostaglandin-H by nonoxidative rearrangements through the action of their respective synthetase enzymes. Prostaglandin-F 2a can be formed by three separate mechanisms through prostaglandin-F synthetase enzyme actions. Prostaglandin-F 2a can be formed from prostaglandin-H 2 through endoperoxide reductase activity or reduction from prostaglandin-E 2 by a 9-ketoreductase. An active metabolite of PGF 2a , 9a,ll£-PGF 2a can be produced from prostaglandins, via 11-keto-reductase (Smith et al. , 1991) . Interferon Receptors/Signal transduction Interferons can be divided into two general types (for review, see Roberts et al., 1992). Type II interferon is

PAGE 24

9 the product of a single gene and is known as IFN7. Type I IFN is composed of at least three distinct subtypes known as iFNa, IFNS and IFNcj. Although these three subtypes differ markedly in amino acid sequence, they elicit their actions through a common receptor and exhibit similar biological properties. Although commonly thought to be derived primarily from T-lymphocytes and natural killer cells in the presence of other cells infected with virus, Type II IFN has been shown to be released from a variety of cells, including porcine trophoblast (Lefevre et al . , 1990). Type-I IFNs can be induced by many cells within the body. Interf eron-a, produced by immune cells, is the most prominent, while IFNt is produced exclusively by trophoblast cells. It is IFNt, a member of the Type-I IFN family which is involved in pregnancy recognition, therefore, this discussion on IFN receptors and signal transduction will be limited to the Type-I IFN family. The Type-I IFN receptor is a transmembrane receptor comprised of two subunits (a and 15; Platanias et al., 1994) Coupling of IFN to its receptor sets into motion a series of phosphorylations of proteins within the cell which ultimately affect transcription of various proteins. How specificity is acquired is not understood at this time as apparently all Type-I IFNs bind to the same or similar receptors and initiate their responses through the same transduction signaling pathway (see, Johnson et al . , 1994).

PAGE 25

10 Signal transduction for IFNs is not through stimulation of CAMP, cGMP, or IP turnover (see Bazer, 1992; Bazer et al . , 1993; Johnson et al . , 1994). Rather, IFNs elicit their action on gene function through a complex signal transduction cascade involving activation of protein tyrosine kinases including those belonging to the Janus kinase (Jak) family, including Tyk2 and Jakl (for review, see Darnell et al., 1994). Ligand binding initiates the activation of Tyk2 , which is associated with the cytoplasmic domain of the Type-I IFN receptor. Either ligand binding or activation of Tyk2 results in activation of Jakl. These two kinases, either separately or together, act to phosphorylate proteins within the cytoplasm which comprise the interferon stimulated gene factor-3 complex (see, Pfeiffer et al . , 1994, Johnson et al . , 1994). These subsequently translocate to the nucleus where they bind to specific interferon stimulated response elements on DNA sequences that direct IFN-induced transcriptional responses. interferon stimulated gene factor-3 is actually two complexes composed of four separate components which normally reside in a dissociated form in untreated cells. The interferon stimulated gene factor3a complex forms within minutes following IFN treatment, and is composed of three proteins (113 kDa STAT 2 , 91 kDa S TAT 1 a and 84 kDa STAT 113 ; Fu et al . , 1992; David and Larner, 1992). It has been suggested that Jakl is the kinase responsible for activating STAT1 while

PAGE 26

11 Tyk2 phosphorylates STAT 2 (Silvennoinen et al . , 1993; Darnell et al., 1994). Inhibitors of the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism increase activity of interferon stimulated gene factor3a (Hannigan et al . , 1991). This indicates that a factor produced from metabolism of arachidonic acid in the epoxygenase pathway may amplify the interferon stimulated gene factor-3a signal (Hannigan et al . , 1991; Williams, 1991b) . Once activation and binding of these three components occurs, interferon stimulated gene factor3a binds a fourth DNA-binding protein (48 kDa interferon stimulated gene factor-37; Levy et al . , 1990; see Marx, 1992) , and the multimeric complex translocates to the nucleus and binds to the interferon stimulated response elements (Fu, 1992; Schindler et al . , 1992; Larner et al . , 1993) . Interf eron-stimulated response elements are cis-acting DNA elements found upstream in IFN responsive genes which activate transcription when bound (see Williams, 1991b). Transcription is activated after binding of the activated interferon stimulated gene factor3 complex, made up of interferon stimulated gene factor-3a and interferon stimulated gene factor-37, to interferon stimulated response elements . The consensus sequence of interferon stimulated response elements for Type I IFNs is reported to have both a

PAGE 27

12 common motif (GGAAA) and a specific motif (TGAAACT) . The specific motif is reportedly separated from the common motif by 1 residue (3') (see Williams, 1991a; Kerr and Stark, 1991) . Once the interferon stimulated response element is bound, RNA polymerase II initiates transcription. Other members of the Type I IFN regulatory factor family also play a major role in IFN action. The interferon regulatory factor1 gene contains an interferon stimulated response element, which when bound by the activated interferon stimulated gene factor3 complex, is upregulated, resulting in an increase in the positive transcription factor interferon regulatory factor1. The interferon regulatory factor1 protein, in contrast to the interferon stimulated gene factor3 complex, can in turn bind to an interferon regulatory factor element, which is often contained within a larger interferon stimulated response element, and increase gene transcription. One such gene which contains a interferon regulatory factor element is the interferon regulatory factor-2 gene, and binding to this interferon regulatory factor element by interferon regulatory factor1 results in an increase in the production of the transcription factor interferon regulatory factor-2. interferon regulatory factor1 and interferon regulatory factor-2 are structurally similar DNA-binding factors which have contrasting roles in IFN action. interferon regulatory factor1 serves as a transcriptional activator of IFN and

PAGE 28

IFN-inducible genes, while interferon regulatory factor2 represses interferon regulatory factor1 action by competing for the same cis elements and displacing interferon regulatory factor-1 (Harada et al . , 1994). Thus, this "yinyang interaction", as described by Spencer et al. (1996), modulates IFN action by regulating the induction and repression of Type I IFN-responsive gene expression. Steroid Receptors Models For Steroid Receptors; Historical Perspective Mueller et al. (1958) was the first to propose a mechanism of action for a steroid. This report led to a search for the compartment in which the steroid receptor was located. Toft and Gorski (see Gorski et al., 1968) used sucrose gradient centrif ugation to separate the cytosolic and nuclear fractions of cells treated with tritiated estradiol. They found most of the radioactivity in the nuclear fraction. Only after an excess of estradiol was added was there detectable radioactivity in the cytosol. However, previous studies indicated the presence of unbound receptor in the cytosol. This led to studies which examined the effects of time and temperature, during incubation with estradiol, on location of the Er. In these studies whole uteri were incubated at 0°C and 37°C. After 1 h at 37°C there was more estrogen in the nuclear fraction than in the

PAGE 29

cytosolic fraction. However, at 0°C most estradiol was in the cytosol. Incubation of uteri for 1 min at 0°C with labeled estradiol, followed by transfer of the tissues to fresh medium without estradiol and incubation at 37 °C, resulted in the appearance of estradiol in the nuclear fraction. Because formation of the nuclear fragment (9.5s) resulted in loss of the estrogen-bound cytosolic fragment (4-6s) the question was whether the cytosolic fragment (46s) was extracellular in origin and whether estrogen from the 9.5s fraction was from the cytosol fragment. This was answered by experiments utilizing cellfree preparations of cytosol incubated with labeled estradiol. The cell-free cultures prepared from uteri incubated at 0°C with labeled estrogen, followed by incubation at 37°C without estrogen had very little estrogen in the cytosolic fraction. However, if the same experiment was conducted without estrogen during the 0°C incubation the estrogen was found in the cytosol. These results led the authors to propose that estrogen moved from the cytosol to the nucleus and, in the process, the 9.5s fragment was lost. Autoradiographic studies by Jensen et al . (1968) showed that uterine tissues incubated at 37°C with labeled estradiol had the majority of the label within the nucleus. However, when incubations were conducted at 2°C the majority of the label was located within the cytosol. These findings were supported by earlier immunocytochemical studies.

PAGE 30

This led to, a revision of the proposal for the translocation model for the steroid receptors (Gorski et al . , 1968). In this model, estrogen diffuses into cells from the blood and binds to a cytoplasmic receptor. The binding of estrogen induces a conformational change in the receptor which allows movement of the ligand-receptor complex from the cytosol to the nucleus. This conformational rearrangement changes the shape of the receptor and causes the release of one or more proposed subunits attached to the receptor in the inactive state. It was this combination of the loss of the subunits and the conformational change that caused the proposed change from the 9.5s cytosolic form to the 4-6s nuclear form. Once in the nucleus, it was believed that receptor-initiated events resulted in increased cellular protein production by estrogentreated uterine tissues. Current Model For Steroid Receptors In recent years, improved separation techniques, utilizing a cytochalasin enucleation procedure, allowed more complete separation of the cytosolic and nuclear fractions into cytoplasts and nucleoplasts , respectively. With this procedure, Welshons et al . (1984, 1985) conclusively established that Er, Pr and Gr are located within the nucleus. Techniques, using frozen tissue for immunocytochemical detection of steroid receptor by

PAGE 31

16 monoclonal antibody binding, also indicated that the unoccupied receptor for estrogen was in the nucleus (King and Greene, 1984). In light of these findings, Gorksi et al. (1986) proposed the current model for steroid receptor action. This model is very similar to that previously described except that the unoccupied receptor is found only in the nuclear compartment. The steroid diffuses into the nucleus, binds to the specific receptor which then undergoes conformational changes, and the receptor-ligand complex binds to the steroid response element in the promotor/enhancer region of DNA. Structure/Function Of The Steroid Receptor Steroid receptors belong to a superfamily of nuclear receptors that include receptors for all steroids, retinoic acid, thyroid hormone, vitamin D, and several other receptors which are termed orphan receptors. Orphan receptors are not fully characterized but are apparently involved in proper development in Drosophila. There is a high degree of homology among the steroid receptors, and all members of this superfamily have common functional domains which account for their high homology. These domains consist of a DNA binding domain, a ligand binding domain, a dimerization domain, a heat shock protein 90 binding domain, a nuclear localization domain and a transactivation domain

PAGE 32

17 (for reviews see CarsonJurica et al . , 1990; Wahli and Martinez, 1991; Freedman, 1992; and Orit et al., 1992). The DNA binding domain, probably the most studied, is noted for many conserved cysteines. Eight of the cysteines are arranged into zinc finger complexes where four cysteines in each of the two fingers bind a zinc ion. The DNA binding domain of the steroid superfamily is divided into two separate subfamilies based on differences in the zinc fingers which are involved in DNA sequence recognition and receptor dimerization . These subfamilies are the glucocorticoid/progesterone subfamily, which includes androgen receptors and mineralocorticoid receptors, and the estrogen subfamily, which includes retinoic acid, thyroid hormone, vitamin D, and the orphan receptors The DNA binding domain of the glucocorticoid receptor was the first to be characterized. Subsequent studies indicated that only a portion of this domain was required for DNA binding and that the DNA binding domain also possessed hormone -dependent transcriptional activity (Hollenberg et al., 1987). As stated previously, this domain has two zinc finger structures similar to those originally reported in transcription factor IIIA. Cloning and sequencing of the steroid receptor zinc finger region led to the following consensus sequence: Cys-X,-Cys-Xi 3 -CysX 2 -Cys-X 15 -Cys-X 5 -Cys-X,-Cys-X 4 -Cys (see Freedman, 1992) .

PAGE 33

18 The ligand binding domain (220 250 amino acids) located near the carboxyl terminal includes domains for dimerization , transactivation and heat shock protein 90 binding. The Nterminal domain is highly variable in length and exhibits the conserved least sequence homology of all the domains. It is, therefore, sometimes referred to as the immunologic domain to which antibodies are generated to distinguish between the various receptors within this superfamily. This domain also contains an area involved in transactivation. Receptor Activation Prior to ligand binding the steroid receptor is held in an inactive state by inhibitory proteins; heat shock protein 90 is the primary protein responsible for this inhibition. Salt treatment of steroid receptors removes heat shock protein 90 and causes activation of the receptor. The heat shock protein 90 binding occurs in the carboxyl terminal region of the receptor; the same area to which the ligand binding domain has been mapped. Apparently there are two molecules of heat shock protein attached to each receptor molecule. Studies which utilized fragments of the glucocorticoid receptor indicated that heat shock protein 90 may be involved in maintaining conformation of the receptor, such that the ligand is unable to bind. When the heat shock protein was removed, ligand binding was blocked (Bresnick et al., 1989) possibly due to protein unfolding characteristics

PAGE 34

19 reported for other heat shock proteins (Pelham, 1988) . The two heat shock protein 90 molecules are lost as a result of a conformational changes induced by ligand binding. Conformational change causes movement of the hinge region, loss of the heat shock protein 90 molecules and exposure of the DNA binding domain to allow binding to hormone response elements. This also allows formation of a dimmer of two steroid receptor molecules which is necessary for binding to the hormone response element (homodimer formation may not be necessary for binding of estrogen to its hormone response element; Furlow et al . , 1993). Phosphorylation of steroid receptors is also involved in activation, DNA binding, and activation of transcription. Hormone binding by steroid receptors is increased when tyrosine kinase is added to cultures of receptors. There are multiple sites for phosphorylation on most steroid receptors and hyperphosphorylation occurs after ligand binding. Serine is the primary amino acid phosphorylated, with minor amounts of threonine phosphorylated (this may be switched in the Er) . Phosphorylation occurs in several domains, particularly the hormone binding and DNA binding domains and the hinge region. It has been proposed that phosphorylation must precede dimerization ; however, this has not been proven. There are results which indicate that phosphorylation of serine/threonine in some domains inhibits normal steroid receptor function (see Orit et al . , 1992).

PAGE 35

20 Hormone Response Elements Hormone response elements are the nucleotide sequences recognized by the steroid receptors. Hormone response elements are sequences which have palindromic structure. For steroid hormones receptors, hormone response elements are unequal halves with three non-conserved base pairs between each of the halves. Like the steroid receptor superfamily , the hormone response elements can be separated into subfamilies with the glucocorticoid/progesterone subfamily in one group and the estrogen subfamily in another. The consensus sequence for the glucocorticoid/progesterone subfamily half palindrome hormone response element is TGTYCT , while it is TGACC for the estrogen subfamily. The difference between these two elements is at the third position, this is a T in the glucocorticoid/progesterone family and an A in the Er family. The sequence responsible for determination of progesterone or Er binding is in the area between Cys 3 and 4 and the amino acid residues just downstream from that point in the first zinc finger (Mader et al . , 1989; Umesono and Evans, 1989). There are specific hormone response element recognition sites between Cys 5 and 6 located on the second finger as well. These may be important in dictating the orientation of the receptor dimers (Hard et al., 1990). Steroid hormones control the activity of various genes in target cells through the action of ligand activated

PAGE 36

21 transcriptional modulators which act by binding to the hormone response element on the DNA. This interaction with the DNA-bound receptor with basal transcriptional machinery and sequence specific transcription factors mediate the transcriptional effects of the steroid hormone. Estrous Cycle The estrous cycle in the ewe is 16 to 17 days in length, but there are some minor variations in length, due to age and stress (Mckinzie and Terrill, 1937). Estrous behavior occurs in most breeds of sheep during the fall and winter, while anestrus occurs during the spring and summer. This assures that birth of young occurs in the spring, when conditions for their survival are optimal. Unlike the estrous cycle, there is considerable variability in the duration of the breeding season among breeds. The breeding season is under photoperiodic control and those breeds originating from mild climates have much longer breeding seasons than those breeds which originated in more northern regions (Robinson, 1959) . Estrus is defined as the period of time when the ewe willingly allows the ram to mount. The first day a ewe will allow a ram to mount is, by convention, designated Day 0 of the estrous cycle. Estrous behavior generally lasts 24 to 48 h; however, there is a considerable variation in duration. Differences in duration of estrous behavior can

PAGE 37

be attributed to several factors. The most studied, however, are the effects of ovulation rate and the ram. Breeds which typically have high ovulation rates exhibit a longer period of estrus (Bindon et al . , 1979). Continuous presence of a ram reduces length of estrus, as compared to that for ewes that experience intermittent exposure to a ram (Parsons, 1967). Regardless of the duration of estrus, ovulation occurs about 3 0 h after the onset of estrous behavior in response to the ovulatory surge of LH (Mckinzie and Terrill, 1937) . The two ovarian steroids primarily involved in the estrous cycle are progesterone and estradiol. Progesterone is secreted by the CL, while estradiol is secreted by the follicles (Short et al . , 1963). The CL is made up of two steroidogenic cell types distinguished by size and function. The large luteal cells are believed to be of granulosa cell origin and the small luteal cells appear to be of theca cell origin (Meidan et al . , 1990). Large luteal cells secrete oxytocin (Rodgers et al., 1983) and spontaneously secrete large amounts of progesterone (Hulet and Shelton, 1980) . Conversely, small luteal cells must be stimulated by LH to produce progesterone (Fitz et al . , 1884). Small luteal cells have high numbers of LH receptors and very low numbers of PGF 2a receptors. In contrast, large luteal cells have abundant PGF 2a receptors and few LH receptors (Alila et al . , 19 88) . The preponderance of PGF 2a receptors on large luteal

PAGE 38

23 cells may explain why they are affected first during luteolysis, resulting in a rapid decrease in progesterone secretion at the onset of luteolysis (Branden et al . , 1988). PGF 2a activates the protein kinase C (PKC) system in large luteal cells, which leads to inhibition of progesterone synthesis. Although activation of the PKC system in small luteal cells also inhibits progesterone synthesis, it is not clear what hormone is involved in the activation of PKC in these cells (Niswender and Nett, 1994) . Progesterone levels are nearly undetectable from Day 0 to Day 3 and then increase gradually until Day 8. The levels remain constant, thereafter, ranging from 1.5 to 3 ng/ml (Bindon et al . , 1979), until Day 15-16 when progesterone decreases rapidly in blood to below 1 ng/ml, and the next cycle commences (Stabenfeldt et al . , 1969). While there is very little difference in temporal changes in progesterone secretion by CL, differences have been reported for maximal circulating concentrations of progesterone across breeds. Progesterone concentrations are generally higher at mid-cycle in those breeds with higher ovulation rates compared to breeds with lower ovulation rates (Bindon et al . , 1979). Progesterone concentrations are also higher in the middle of the breeding season, compared to the beginning or the end of the season (Legan et al . , 1985). Progesterone levels begin to fall even before CL regression becomes morphologically apparent and luteolysis

PAGE 39

is initiated by PGF 2a release from the non-gravid uterus. Pulsatile secretion of PGF 2a is first apparent about Day 15 of the cycle and continues to increase until estrus. The role of PGF 2a in luteolysis and the attenuation of its production will be discussed in more detail in the sections on luteolysis and maternal recognition of pregnancy. Plasma estradiol concentrations begin to increase just after the initial decrease in progesterone is noted. Estrogen concentrations in the plasma continue to increase from basal levels of 1 pg/ml to highest levels of about 10 pg/ml (Baird et al . , 1976) at the LH surge, after which time estrogen levels decline rapidly and remain at basal levels through the luteal phase except for small increases associated with follicular waves. Estradiol is produced by rapidly growing preovulatory follicles during proestrus (Days 15-16) . Both progesterone and estradiol are secreted in pulses. Progesterone, however, does not follow a pattern set by gonadotropins as does estradiol. Pulses of estradiol precede pulses of LH and both pulse frequency and amplitude of estradiol increase during the follicular phase (Baird, 1978) . LH is secreted episodically, in the ewe, and varies in both frequency and amplitude throughout the estrous cycle. The basal level of LH secretion (~1 ng/ml) is required for

PAGE 40

CL function and steroidogenesis, while the LH surge initiates ovulation and CL formation (Goding et al . , 1970). The LH surge is characterized by a dramatic rise in LH pulse frequency a few hours prior to ovulation after which LH returns to basal levels. On average the entire ovulatory surge of LH occurs within 12 h, and coincides with the onset of estrus. This is followed by ovulation about 30 h later. The CL begins to produce progesterone by Day 3 and the recurring estrous cycles continue during the breeding season, until ewes become pregnant, or enter anestrus. Changes in the secretion of LH during the normal cycle and ovulation are tightly controlled by changes in the generation of GnRH pulses (reviewed in Goodman, 1994) . During the early luteal phase, tonic LH secretion decreases because the increasing amounts of progesterone, along with estradiol, suppress the activity of the GnRH neural oscillator and decrease LH pulse frequency. Tonic LH levels then remain low as long as progesterone levels are increased which attenuates GnRH pulse frequency. When progesterone levels fall following luteolysis, the frequency of the GnRH pulse generator increases, which may in turn stimulate GnRH receptor gene expression and increased expression of GnRH receptors (Turzillo et al., 1995). Increases in GnRH pulse generation then serve to increase the pulse frequency of LH and accounts for the rise in LH secretion during the follicular phase of the sheep.

PAGE 41

26 Endometrial Steroid Receptors It is clear that the uterine environment is dynamic and ever changing. The changes that occur throughout the estrous cycle with regard to both physiological and biochemical activities are controlled by both changing steroid hormone profiles and changes in expression of their receptors. While finite control of uterine steroid receptors is not fully understood, changes in Er and Pr dynamics throughout the estrous cycle and pregnancy are being examined. In the cyclic ewe, the Er is generally abundant just before and after estrus (Wathes and Hamon, 1993) . Numbers of Er fall dramatically from Day 3 , to a low on Days 10 to 14, followed by a dramatic increase beginning on Day 14, to peak values at estrus (Koligian and Stormshak, 1976; Miller et al., 1977; Zelinski et al . , 1980; Cherny et al . , 1991; Ott et al., 1993b). Steady state levels of endometrial Er mRNA are highest on Day 1 of cyclic ewes, decline between Days 1 and 6, and increase between Days 11 and 15 (Spencer and Bazer, 1995) . Myometrial Er mRNA in these animals was also highest on Day 1, but decreased to Day 6 and remained low thereafter. Cherny et al . (1991) reported that endometrial Er regulation was not homogenous throughout endometrial tissue, but varied by tissue type (caruncular or intracaruncular) , as well as cell type (epithelial, stromal or glandular) , and that steroidogenic control within tissues

PAGE 42

27 was also variable. Spencer and Bazer (1995) have further demonstrated, using in situ hybridization and immunocytochemical approaches, that distinct and tissueand cell-specific alterations in uterine Er and Pr mRNA and protein expression during the estrous cycle of the ewe generally paralleled the overall changes noted in steady state levels of Er and Pr mRNAs . In the endometrium, Pr mRNA and protein expression disappeared from the luminal and shallow glandular epithelium between Days 6 and 13, whereas Er mRNA and protein expression was low on Days 6 and 11 and increased between Days 11 and 15 in the luminal and shallow glandular epithelium. Er mRNA and protein were consistently present at low levels in the stroma and deep glandular epithelium. Progesterone is inhibitory to Er formation in several species (Brenner et al . , 1974; Hsueh et al . , 1975 and 1976; Tseng and Gurpide, 1975; West et al . , 1976; Bhakoo and Katzenellenbogen, 1977; Spencer et al., 1995a; 1995b) including sheep (Koligian and Stormshak, 1977b; Zelinski et al., 1980; Cherny et al., 1991; Spencer et al . , 1995a; 1995b). Estrogen, on the other hand, is stimulatory to it's own receptor formation (Anderson et al . , 1975; Bhakoo and Katzenellenbogen, 1977; Zelinski et al . , 1980; Cherny et al . , 1991). Control of Er, however, is not a simple matter and probably involves several factors which affect each of the uterine cell populations in various ways (Cherny et al., 1991) .

PAGE 43

28 The Pr is most prevalent in ovine endometrium around estrus (Miller et al., 1977; Zelinski et al . , 1980; Ott et al . , 1993b) with the highest concentrations of receptors reported on Day 2 (Miller et al . , 1977) and the lowest from Days 10 to 14. In cyclic ewes, endometrial Pr mRNA levels are highest on Day 1, decrease between Days 1 and 11, and then increase between Days 13 and 15. Myometrial Pr mRNA levels are highest on Day 1 and decline thereafter (Spencer and Bazer, 1995). Estrogen appears to be the primary stimulus for Pr formation (Milgrom et al . , 1973; Leavitt et al . , 197 4; Zelinski et al . , 1980; Aronica and Katzenellenbogen , 1991). Progesterone, however, can exert both positive and negative influences on uterine Pr expression. Progesterone in cyclic animals is clearly inhibitory to endometrial Pr expression, probably through down-regulation of Pr (Milgrom et al . , 1973; Leavitt et al . , 1974; Vu Hai et al . , 1977). Progesterone downregulation of its own receptor removes the progesterone block (McCracken et al . , 1984), allowing Er up-regulation, and formation of OTr which, when bound by oxytocin, initiates pulsatile release of PGF 2a . In pregnant ewes it had been assumed that Pr are maintained in the presence of high progesterone for extended periods (Ogle et al . , 1989, 1990; Ott et al., 1993b; Mirando et al . , 1993). In the pregnant ewe this was suggested to be achieved by stabilization of the Pr by a product of the conceptus (Ott et al . , 1993b). However,

PAGE 44

29 others have shown that continuous exposure of the endometrium to progesterone downregulates endometrial Pr mRNA and protein abundance in the luminal epithelium, shallow glandular epithelium, and stroma (Wathes and Hamon, 1993; Spencer and Bazer, 1995; Spencer et al . , 1995b). The mechanism responsible for this is currently poorly understood but may involve Pr-mediated decreases in Pr gene transcription (Alexander et al., 1989; Read et al., 1988). Results from studies performed by Spencer et al. (1995b) have shown that negative regulation of the Pr gene in the endometrial epithelium occurs in both cyclic and pregnant ewes, because Pr mRNA abundance and immunoreactive Pr protein declined in the endometrial luminal epithelium and shallow glandular epithelium after Day 6. Thus, the current hypotheses is that pregnancy does not stabilize or upregulate Pr gene expression in the endometrium. Luteolvsis Luteolysis is initiated in ruminants in response to the pulsatile release of by PGF 2ct produced by the uterus (McCracken et al . , 1981; Hooper et al., 1986). PGF 2a is released from the uterus in the nonpregnant ewe in a series of 5-8 episodes (Thornburn et al . , 1973) with 6-8 h between each series. McCracken et al. (1984) have shown that the CL must be exposed to approximately 5 pulses of PGF over a 25hour period to undergo complete luteolysis. These episodes

PAGE 45

30 begin just prior to the onset of luteal regression. At this time the progesterone level has not begun to decline (Zarco et al . , 1988b) but Pr are very low which allows expression of Er and OTr . The pulsatile secretion of PGF 2a is initiated by the secretion of oxytocin from the posterior pituitary and is escalated by oxytocin of luteal origin. The result is a synchronous pulsatile release of oxytocin from the CL on each ovary and from the posterior pituitary in ewes (Hooper et al., 1986). Oxytocin from the CL and PGF 2a from the uterus act together in a positive feedback loop to generate pulses of PGF 2a required for luteolysis. Oxytocin is released from the posterior pituitary (Hooper et al., 1986) as well as the CL where it is produced by large luteal cells (Rodgers et al . , 1983; Wathes and Denning-Kendal , 1992) and is secreted into the ovarian vein (Wathes and Swann, 1982; Flint and Sheldirck, 1982; Flint and Sheldrick, 1986). In large luteal cells, the oxytocin gene is transcribed on Days 0-4 (Jones and Flint, 1986) . Oxytocin mRNA is translated into oxytocin from Days 4-7. Stores of oxytocin and its neurophysin in luteal cells are highest on Days 10-12 (Silvia et al . , 1991; Rhodes and Nathanielsz, 1990). Flint and Sheldrick (1983) reported that the rise and fall of oxytocin followed that of progesterone, with the lowest levels noted at the time of ovulation. However, there is no indication that progesterone directly affects the synthesis of oxytocin.

PAGE 46

31 There is considerable evidence, however, for the association of oxytocin and PGF 2a , in the formation of a positive feedback loop to increase pulsatile release of luteolytic PGF 2a . As stated earlier, there is a positive feedback loop between oxytocin and PGF 2a . That is, PGF 2a can stimulate luteal oxytocin secretion and oxytocin can stimulate endometrial PGF 2a secretion. There is evidence that the initiation of this loop is with PGF 2a secretion from the uterus due to the fact that PGF 2a release occurs prior to pulsatile release of luteal oxytocin (Moore et al . , 1986). While it is possible that the uterus has an endogenous clock, possibly estrogen, that triggers the release of the initial PGF 2a secretion, pituitary oxytocin is the more accepted mediator of the initial uterine PGF 2a secretion which in turn begins the PGF 2a /oxytocin feedback loop (see Silvia et al., 1991; McCracken et al., 1991). Once the PGF 2a /oxytocin loop is established, it serves to increase PGF 2a concentrations in the ovarian pedicle. Increasing concentrations of PGF 2a binding to their receptors on the large luteal cells, cause a reduction in viable cell numbers in vitro. Several different mechanisms have been put forth to explain the luteolytic effects of PGF 2a (see Niswender and Nett, 1994; Spencer and Bazer, 1995). These mechanisms include: 1) a rapid decrease in luteal blood flow; 2) a reduction in the number of LH

PAGE 47

32 receptors and/or an uncoupling of the LH receptor from adenylate cyclase; 3) activation of protein kinase C; 4) influx of high levels of calcium; and 5) a cytotoxic effect (Silvia et al . , 1984a). Luteolysis does not require withdrawal of basal LH support, but PGF may activate protein kinase C in large luteal cells to inhibit progesterone production (Wiltbank et al . , 1991) and cause luteolysis (McGuire et al . , 1991). Treatment of large luteal cells with PGF increases intracellular calcium (Wiltbank et al . , 1989) , which appears to mediate the cytotoxic effects of PGF 2a probably through typical apoptotic changes (Sawyer et al . , 1990) as well as decrease expression of mRNA for 3/3hydroxysteroid dehydrogenase (Hawkins et al., 1993). Effects of Ovarian Steroids on Prostaglandin-F ^ Secretion There is a great deal of information on the effects of steroids on PGF 2a formation and/or luteolysis. It is obvious that progesterone and estrogen affect PGF 2a production and secretion, and are, therefore, important steroids affecting luteolysis. Progesterone affects One of the most compelling studies to link progesterone with normal cyclical estrus activity in ewes was that of French and Spennetta (1981) who showed that immunization of ewes against progesterone resulted in erratic estrous cycles. Progesterone initiates PGF 2a secretion in

PAGE 48

33 ovariectomized ewes after 7 days of treatment, but prior to progesterone treatment, PGF 2a secretion was minimal (Scaramuzzi et al . , 1977). Prostaglandin-F 2a secretion can also be induced early in the cycle (up to 32 h post-estrus) by progesterone treatment (Ginther, 1969; Ottobre et al . , 1980) . Administration of exogenous progesterone to cyclic ewes during metestrus decreases interestrous intervals, while administration of the Pr antagonist RU486 during the early luteal phase delays the onset of endometrial PGF production and luteolysis (Morgan et al., 1993). Apparently the uterus requires the influence of progesterone for a period of 10 to 12 days to produce luteolytic PGF 2a in a normal cyclic manner (Vallet et al., 1990). Progesterone is also necessary to elicit physiological changes in uterine responsiveness noted throughout the cycle. Humanics and Silvia (1988) utilized ovariectomized ewes to show this effect. Ewes were first pretreated with a hormonal regime that would mimic that of the steroid pattern noted in cyclic animals 6 days prior to estrus. Ewes were then treated with progesterone for 15 days and responsiveness to oxytocin (as determined by PGFM response) was measured on Days 5, 10 and 15 of treatment. They found that there was no PGFM response until Day 15. Vallet et al . (1990) used a similar model in which a subset of ewes received no steroid pretreatment and then progesterone alone

PAGE 49

34 for 12 days to cause the uterus to become responsive to oxytocin challenge. There are several ways that progesterone changes the endometrial milieu that could explain changes in uterine responsiveness to oxytocin. Brinsfield and Hawk (1973) reported that the accumulation of lipid droplets in uterine epithelium of ewes were induced by progesterone. In rats progesterone induces both phospholipid and triglyceride accumulation (Mamimekalai et al . , 1979 ;Boshier et al . , 1981) . Progesterone has been shown to affect several factors associated with PGF 2a metabolism. Raw and Silvia (1991) have shown that progesterone treatment of ovariectomized ewes for 16 days resulted in increased phospholipase-C activity. Phospholipase-C activity has been found to increase during the period of luteolysis in the ewe as well (Silvia and Raw, 1993) . While there is no doubt that progesterone has an effect on phospholipase-C activity, Silvia and Raw (1993) suggest that control of PGF 2a metabolism may occur later in the synthesis pathway. Progesterone has an effect on the prostaglandin synthetase enzyme in cattle, and ovariectomized ewes treated with progesterone also respond with elevated uterine endometrial prostaglandin-H synthetase activity (Raw et al., 1988). Eggleston et al . (1990) reported that prostaglandinH synthetase mRNA increased in intact ewes treated with progesterone early in the cycle. There was also an

PAGE 50

35 increased incidence of early luteolysis in these ewes. An increase in PGF 2a release also occurs when progesterone is removed at a time corresponding to normal progesterone decline in cyclic ewes (Leavitt et al., 1985). Therefore, progesterone may stimulate PGF 2a secretion early in the cycle, but exerts an inhibitory effect late in the cycle (see Silvia et al . , 1991). Estrogen affects Ford et al. (197 5) showed that pharmacological levels of estradiol (two injections given 12 h apart) resulted in an increase in PGF 2a within 12 h after the second injection. This effect was the same for both control and progesterone primed ewes. McCracken (1980) used ovariectomized ewes, and Sharma and Fitizpatrick (1974) used anestrus ewes, to elicit a PGF 2a response to estradiol in as little as 6 h. Results of these studies indicate that in ovariectomized ewes, at least, there is no requirement for progesterone in PGF 2a secretion. However, in studies in which physiological levels of estradiol were administered to ovariectomized ewes, estradiol alone was less effective in eliciting a PGF 2a response (Homantics and Silvia, 1988) . In studies of intact ewes, estrogen was also ineffective in eliciting a PGF 2a response when administered alone. In a study in which the uterus was autotransplanted to the neck of the ewe, Scaramuzzi et al . (1977) found that progesterone pretreatment was required to get a full PGF 2a response. In

PAGE 51

36 ewes which received estradiol alone there was a slight, but nonsignificant, increase in the PGF 2a response. Endometrium from ewes treated with estradiol for several days in vivo were also unable to respond with PGF 2a secretion in vitro (Findly et al . , 1981; Raw and Silvia, 1991). Effects of estrogens on PGF 2a release are much greater when estrogen is administered in conjunction with progesterone or in a regime that mimics the steroidogenic patterns of the normal estrous cycle. In cyclic ewes, estradiol injections on Days 11 and 12 (in one study) , or Day 10 (in another) , were able to induce luteal regression (Stormshak, 1969; Cook et al . , 1974). In a similar study, Hawk and Bolt (1970) treated a total of 94 ewes with estradiol on two successive days with groups beginning treatment on Days 1 through 11. They found that estradiol had no effect on CL weights until Day 10 of the cycle. If ewes were not treated with estradiol until Days 11 and 12 CL weights were decreased by Day 15. Secretion of PGF 2a is also enhanced when ewes are treated with estrogen and progesterone (Ford et at., 1975). Hixon and Flint (1987) showed that inositol phosphate metabolism as well as PGF 2a secretion increased when estradiol was administered at midcycle (Days 9 and 10). Barcikowski et al. (1974) concluded that the PGF 2a response was due to a direct effect of estrogen on the uterus when physiological quantities of estradiol were infused either into the uterine artery or

PAGE 52

37 systemically . Only those ewes which received treatment locally (at the uterus) , and late in the cycle responded with elevated secretion of PGF 2a . Ovariectomized ewes treated with progesterone for at least 5 days also responded with greater PGF 2a secretion than ewes treated with estrogen alone (Scaramuzzi et al . , 1977). As discussed earlier Vallet et al. (1990) reported that progesterone alone will elicit a PGF 2a response when ewes are challenged with oxytocin. This study, however, showed that a combined treatment of progesterone and estrogen resulted in the highest response to oxytocin challenge. Ovariectomized ewes, which have low basal concentrations of both progesterone and estradiol, have also been used to study the factors which control oxytocininduced PGF 2a release (Beard and Lamming, 1994; Beard et al . , 1994). In these ewes, expression of the endometrial OTr was constitutively present in high levels in luminal and superficial glandular epithelium, and treatment with progesterone initially caused a complete loss of OTr expression (Wathes and Lamming, 1995; Stevenson et al . , 1994; Sheldrick and Flint, 1985). However, chronic progesterone treatment for 12 days resulted in an increase in endometrial phospholipid stores, OTr expression, prostaglandin synthase activity, and oxytocin-induced pulsatile release of PGF (Salamonsen, 1992; Spencer et al . , 1995b; Vallet et al., 1990). The OTr that develop as

PAGE 53

38 progesterone down-regulates Pr are localized exclusively to luminal and superficial glandular epithelium, which is essentially identical to the spatial expression of OTr during luteolysis (Wathes and Lamming, 1995) . Estradiol, like progesterone, affects the enzymes involved in the synthesis of PGF 2a . Estrogen treatment on Days 9 and 10 of the cycle results in increased inositol phosphate metabolism (an indication of phospholipase-C activity) within 12 h, over that of controls. However, in ovariectomized ewes which received physiological doses of steroids for 16 days, there was a decrease in phospholipaseC activity in ewes treated with estrogen alone, or estrogen plus progesterone (Raw and Silvia, 1991) . Prostaglandin synthetase mRNA was decreased in ovariectomized ewes treated with estrogen alone, however, prostaglandin synthetase mRNA was not different from controls when ewes were treated with progesterone and estrogen (Salamonsen et al . , 1991). In this study, prostaglandin synthetase mRNA was decreased, but there was no difference in the levels of immuno re active prostaglandin synthetase in luminal and glandular epithelial cells of ewes that received progesterone or progesterone plus estrogen. Summary The results of these studies clearly indicate that there is an effect of the ovarian steroids on PGF 2a synthesis and secretion. While this effect is apparent

PAGE 54

39 after progesterone treatment alone, estrogen appears to facilitate the complete response in the normal, cyclic ewe. Endometrial Oxytocin Receptor The release of PGF 2a is initiated by oxytocin after release from large luteal cells (Rodgers et al . , 1983), and binding to its endometrial receptor (Flint and Sheldrick, 1983; Roberts et al., 1976). The appearance of OTr on endometrial epithelium (surface primarily) about Day 14 of the cycle is believed to set up the oxytocin/PGF 2a feedback loop which initiates and insures luteolysis. Pulses of PGF 2a have been shown to coincide with pulses of plasma oxytocin (Flint and Sheldrick, 1983; Hooper et al . , 1987). The binding of oxytocin initiates the phosphoinositidephospholipase-C transduction signal, one action of which is to mobilize arachidonic acid, which results in the formation of PGF 2a . Endometrial OTr concentrations rise rapidly 48 h prior to estrus (Sheldrick and Flint, 1985). Endometrial OTr begin to increase on Day 14, reach maximum values at estrus and decline by Day 5 of the subsequent cycle. This is coincidental with the decrease in plasma progesterone, Pr, the rise in estrogen and Er (Sheldrick and Flint, 1985 ; Wallace et al . , 1991) during proestrus. Through the use of receptor binding studies it was shown that endometrial OTr are more concentrated in the intercaruncular than in the

PAGE 55

40 caruncular tissue of ewes in estrus, while Days 13 through 15 there were more receptors located in caruncular tissues (Shledrick and Flint, 1985) . Autoradiographic studies have shown that at the time of luteolysis, endometrial OTr are found only on the luminal epithelial cells (Ayad et al., 1991a, 1991b; Wallace et al . , 1991). The appearance of receptors in stromal tissues and glandular epithelial cells occurs after luteolysis. It is proposed that the apparent differential regulation of these receptor populations may be important in the process of luteolysis (or lack of luteolysis in the pregnant ewe) . The effect of oxytocin on its own receptor was examined by Flint and Sheldrick (primarily for therapeutic purposes; 1985) . In this study, systemic infusion of oxytocin resulted in a lengthening of the interestrous interval. Upon examination there was no apparent lysis of luteal tissues. It was proposed by others that the action of oxytocin was to down-regulate its own receptor and that the increase in endometrial OTr at the time of luteolysis was due to removal of this down-regulation by decreasing concentrations of oxytocin in plasma. However, since plasma oxytocin concentrations decrease prior to this time, this seems unlikely. Down-regulation was also proposed as a possible explanation for the lack of OTr in pregnant ewes in response to oxytocin produced by the conceptus (Lacroix et al., 1988). This does not appear likely since ovine

PAGE 56

41 trophoblast cells do not express oxytocin (Parkinson et al . , 1991) and intrauterine infusions of oxytocin have no effect on cycle length or endometrial OTr expression (Parkinson et al., 1991; Ayad et al . , 1993). It might be argued that uterine proteases denatured the infused oxytocin and this was the reason for the difference in cycle extension noted between these studies. However, OTr expression has been shown to be absent on the luminal epithelium if oxytocin was infused into the uterine lumen (Ayad et al . , 1993). Thus, OTr may be localized to the basolateral domain of the epithelial cells so that intraluminal oxytocin may not have access to those receptors. Since the conceptus does not express oxytocin (Parkinson et al., 1991), down regulation does not appear to be a controlling factor in OTr concentration at the time of maternal recognition of pregnancy. The fact that endometrial OTr concentrations are highest when plasma oxytocin concentrations are low (Flint and Sheldrick, 1985) indicates that oxytocin effects are exerted at low receptor occupancy and suggests that control of uterine responsiveness to oxytocin is through control of OTr gene expression, rather than control of oxytocin secretion. Oxytocin receptors are clearly under steroid hormone control. Uterine OTr increase (in rodents) in response to estrogen (Soloff, 1975). As indicated by PGF 2a secretion, prolonged progesterone treatment followed by estrogen

PAGE 57

42 stimulation resulted in maximum OTr formation (McCracken et al., 1981). It was proposed (using a hamster model) that estrogen stimulates endometrial OTr formation and that progesterone exerts inhibitory actions through progesteroneinduced inhibitors which block estrogen binding to its receptor (Okulicz et al., 1981). These findings led McCracken et al. (1984) to propose that there was a progesterone block to uterine OTr formation during Days 5 to 14 of the cycle in ewes. At the end of this period the endometrium became refractory to the progesterone block, probably because of down-regulation of Pr by progesterone. Removal of the progesterone block, along with the stimulatory influence of rising estrogen, caused upregulation of endometrial OTr. Vallet et al. (1990) found that endometrial OTr were high in ovariectomized ewes, and treatment with progesterone initially caused a complete loss of OTr expression (Wathes and Lamming, 1995; Vallet et al . , 1990; Lau et al . , 1992) which indicates that the role of ovarian steroids, in control of endometrial OTr, is inhibitory. Interestingly, these receptors 'were apparently uncoupled from PGF 2a synthesis as noted by the lack of PGFM response after challenge with exogenous oxytocin. Since progesterone increases phospholipid stores (Boshier et al . , 1987) and prostaglandin synthase activity is necessary for conversion of arachidonic acid to PGF 2a (Eggleston et al . , 1990) this "uncoupling" phenomenon was apparently related

PAGE 58

43 to a reduction in the machinery necessary for the increased PGF synthesis. Progesterone treatment alone, for a period of 12 days, was sufficient to increase endometrial phospholipid stores, OTr expression, prostaglandin synthase activity, and oxytocin-induced pulsatile release of PGF 2a (Salmonsen, 1992; Spencer et al . , 1995b; Vallet et al . , 1990). Treatment with progesterone for 5 days was not sufficient to stimulate endometrial OTr formation. A regime meant to closely mimic a normal estrous cycle (progesterone pretreatment , estradiol, progesterone for 12 days, and estradiol on Days 11 and 12 of progesterone treatment) , resulted in only a slight increase in OTr number over that of progesterone alone. Estradiol alone was inhibitory to receptor formation. It was proposed by these authors that formation of endometrial OTr was under the inhibitory control of progesterone alone. Also, estrogen's action may be biphasic, in that estrogen may be stimulatory on Days 1 through 2 then inhibitory on Days 5 through 7. Similar findings were reported in studies utilizing ovariectomized ewes (Lamming et al . , 1991; Zang et al., 1992; Lau et al., 1992; Lau et al., 1993) and uterine explant tissues (Sheldrick and Flick-Smith, 1993) . The overall effects of estrogen on the timing, magnitude and pattern of PGF 2a response to oxytocin may be mediated through increases in OTr gene expression (Beard and Lamming, 1994; McCrackin et al., 1984), enhanced coupling of OTr to its second messenger

PAGE 59

44 signal transduction system (Bouvier et al . , 1991) and increased activity of the machinery which drives prostaglandin synthase activity (Eggleston et al., 1990; Huslig et al . , 1997) . Maternal Recognition Of Pregnancy The term "maternal recognition of pregnancy" was first used in 19 69 in a review published by R.V. Short. However, it had been known for some time that the conceptus in some way affected CL life-span. This was first alluded to in 1945 by Casida and Warwick who found that CL were maintained in pregnant ewes and that the conceptus was dependent on the CL for continued development until Day 55 of gestation. It was not until embryo transfer studies showed that synchronous transfers (Day 12 embryo into a Day 12 uterus) were successful, but embryo transfers on Day 13 were not, that the conceptus was directly implicated as having antiluteolytic effects. To determine that the Day 13 conceptus was not affected by the transfer procedure and that this was the reason there was not cycle extension in these ewes, Day 13 conceptuses were transferred to Day 12 cyclic sheep and the result was extension of the cycle. These results showed there was some affect of the conceptus that had to occur on Day 12 to prevent luteal regression in the pregnant ewe (Moor and Rowson, 1964; 1966a). A similar

PAGE 60

45 study also detected the antiluteolytic effect of the ovine conceptus (Niswender and Dziuk, 1966) . To further examine the antiluteolytic effect of the conceptus, Moor and Rowson (1966b) removed conceptuses on Days 5 through 15 of pregnancy. They found that removal of the conceptus on Day 12 or before resulted in a cycle length that was normal (-17 days) . However, if the conceptus was removed on Day 13 or after there was an extension in the life-span of the CL on average to Day 25. This study clearly indicated that the conceptus was affecting CL lifespan, and that this effect occurred on Day 12 of pregnancy. Since it was known that the uterus-initiated CL regression (see Luteolysis section) and that this was a local affect, Moor and Rowson (1966c) questioned whether the effect of the conceptus was also local. The authors found when conceptuses were transferred to an isolated ipsilateral horn the CL was maintained, but when the transfer was to an isolated contralateral horn the CL regressed. However, if embryo transfers to the contralateral horn coincided with removal of the ipsilateral horn the CL was maintained. These studies indicated that the conceptus exerts a local unilateral antiluteolytic effect. This was further indicated by the fact that embryo transfers to one isolated horn of a uterus in ewes with a CL on each ovary resulted in maintenance of the ipsilateral CL, but not the contralateral CL. Taken together, these studies clearly indicate that the

PAGE 61

conceptus overcomes the local luteolytic effect of the uterus . The next question was how does the conceptus exert its antiluteolytic effect. To examine this, Day 14 and Day 15 conceptus were collected, homogenized and frozen/thawed or heat treated. These homogenates were infused into the uterine lumen on Day 12 or daily for the treatment period. Infusions of conceptus homogenates on Day 12 alone or of heat-treated conceptus homogenates were not effective in extending the estrous cycle. Repeated daily infusions of frozen/thawed conceptus homogenates extended the cycle, on average to 22 days. Infusion of Day 25 sheep conceptuses homogenates or Day 14 pig homogenates had no effect on cycle length (Rowson and Moor; 19 67) . These studies indicated that the antiluteolytic effect was apparently species specific and time dependent. Furthermore, the fact that the antiluteolytic effect was present in frozen/thawed, but not heat-treated conceptus homogenates led the authors to suggest that the responsible factor was chemical in nature and heat labile. Similar studies indicated that there was an antiluteolytic effect of intrauterine infusion of bovine conceptus homogenates in the cow (Northly and French, 1980) . Mccracken's report (1971) that PGF 2a is released into the uterine vein by endometrial tissues and that the timing of this release coincides with CL regression, initiated a great deal of interest in the effect of the conceptus on

PAGE 62

47 PGF 2a release. Moore and Watkins (19 82) found that on Days 12 and 13, cyclic ewes responded with a pulse of PGFM for each pulse of oxytocin neurophysin and that this effect was absent in pregnant ewes. Pratt et al . (1977) reported that when PGF 2a was injected into the largest follicle on the ovary bearing the CL of cyclic and pregnant ewes the cyclic ewes had a 1.5 day shorter interestrous interval, while none of the pregnant ewes returned to estrus. In this same report, intrauterine infusions of prostaglandin-E into pregnant ewes resulted in longer cycle lengths than for control ewes. Silvia and Niswender (19 84) found that less PGF 2a (4 mg/58 kg body weight as compared to 10 mg/58 kg) was required to cause luteolysis in cyclic compared to pregnant ewes. In further studies by these same authors, the protective properties the conceptus bestowed on the CL, in the presence of PGF 2a challenge, did not occur until Day 13 and was lost by Day 26. Similar results were found when ewes were challenged with oxytocin (Fairclough et al . , 1984; Silvia et al . , 1992). Estradiol treatment also induces luteolysis in cyclic, but not pregnant ewes (Kittok and Britt, 1977) , indicating an affect of estradiol in PGF^ production which the conceptus is able to block. Mccracken (1984) first proposed that estradiol-induced formation of endometrial OTr, which bound oxytocin, of pituitary or luteal origin, to induce pulsatile release of PGF 2a . it was this process that began the feedback loop

PAGE 63

48 which resulted in luteolysis (see Luteolysis section) . Sheldrick and Flint (1985) reported that in pregnant ewes, the increase in OTr numbers detected in proestrus of cyclic ewes was attenuated. Oxytocin receptor expression is high from Day 14 of one cycle to Day 2 of the next cycle, but is almost completely absent in caruncular and intracaruncular endometrium of pregnant ewes (Flint and Sheldrick, 1986) . Similar differences were found between cyclic and pregnant cows on Day 17 (Jenner et al . , 1991). Autoradiological studies of endometrial receptors determined that labeled oxytocin was concentrated in luminal epithelium, glandular epithelia and caruncular stroma of Day 15 cyclic ewes. However, in pregnant ewes there was no labeling of endometrial tissues (Ayad et al., 1993). Intrauterine injections of conceptus homogenates increase the interestrous interval in ewes (Rowson and Moor, 1967) . Since the conceptus was directly affecting the maternal environment it was believed that this effect was through a secretory product. To determine if conceptusconditioned culture media contained a pregnancy recognition factor, culture medium from incubations of Day 15 and 16 conceptuses were injected into the uterine lumen of cyclic ewes from Days 12 to 18 of the estrus cycle (Godkin et al., 1984b) . The CL life-span was prolonged in all ewes treated with oCSP based on maintenance of progesterone secretion and CL which had previously been marked with India ink. One

PAGE 64

49 treated ewe maintained a functional CL until Day 52 when the project was terminated. In comparison, all the ewes treated with SP, as controls, ovulated by Day 25 when CL were checked at hysterectomy, and progesterone secretion had fallen by Day 19. This was the first study to conclusively show that oCSP could prolong CL life-span. Similar findings were reported by Vallet et al. (1988). To determine the effect of oCSPs on uterine PGF 2a responsiveness after estradiol or oxytocin challenge, oCSP or plasma proteins were infused into the uterine lumen of ewes from Day 12 to Day 14 of the cycle (Fincher et al., 1986) . On Day 14 all ewes were challenged with estradiol. Jugular blood samples were drawn hourly over a 10 hour period and assayed for PGFM . On Day 15 one-half of the ewes from each treatment group were challenged with oxytocin or saline. Blood was drawn for PGFM analysis. The PGFM response was lower in ewes which received oCSP, compared to SP-treated controls, after estradiol and oxytocin challenge. These results indicated that the factor in oCSP which affects the endometrium is a secretory protein produced by the conceptus and that it can induce changes in the uterine environment which prevent normal cyclic responsiveness to estradiol and oxytocin. Vallet et al . (1988) and Mirando et al. (1990a) also reported that PGF 2a secretion after challenges with estradiol or oxytocin, was reduced significantly in oCSP-treated ewes.

PAGE 65

50 The lack of PGF 2a responsiveness has been shown in the pregnant animal to be due to low oxytocin (Flint and Sheldrick, 1986) and estrogen (Findlay et al . , 1982) receptors on the luminal epithelium. An indirect measure of the OTr can be obtained by measurement of IP which is a factor in the signal transduction pathway stimulated by activation of the OTr (Flint et al., 1986; Hixon et al . , 1987). Mirando et al . (1990a, 1990b) reported that in Day 16 pregnant ewes there was an attenuation of IP hydrolysis within endometrial tissues after in vitro stimulation with oxytocin, while in Day 16 cyclic ewes there was an increase in IP hydrolysis. The pregnant ewes also had high plasma progesterone concentrations (Mirando et al . , 1990a). Intrauterine injections of oCSP on Days 11 through 15 also resulted in lower IP hydrolysis after in vitro stimulation of endometrial tissues with oxytocin as compared to SPtreated controls (Mirando et al., 1990a, 1990b). Maintenance of plasma progesterone concentrations noted in pregnant ewes may be important in sustaining the proposed progesterone block to OTr formation (McCracken et al., 1984). Ott et al. (1992) utilized an ovariectomized ewe model to indirectly (i.e. PGFM response to oxytocin challenge and IP hydrolysis) examine the interaction of oCSP and progesterone on OTr formation. Ovariectomized ewes received intrauterine injections of SP or oCSP from Day 11 through 14 (ewes were ovariectomized on Day 4 of the cycle)

PAGE 66

51 and daily progesterone injections (im) were administered from Day 4 through Day 10 or Day 4 through Day 15. The oxytocininduced PGFM response was completely blocked in oCSP-treated ewes which received progesterone until Day 15. Endometrial tissue of these ewes was not responsive to oxytocin-induced IP hydrolysis in vitro, while there was a doubling of the rate of IP hydrolysis in ewes treated with SP and progesterone. However, oCSP did not block endometrial responsiveness (either PGFM or IP hydrolysis) if progesterone treatment was stopped on Day 10. Interestingly, oxytocin-induced PGFM increases and IP hydrolysis were also blocked by treatment with progesterone alone until Day 15 (i.e., without intrauterine injection of oCSP) . This supports Mccracken's proposal that progesterone acts to block the increase in OTr formation until the time of luteolysis, but this does not support Mccracken's proposed requirement for estrogen. There is some evidence that ovine conceptuses secrete a factor (not oIFNi) that directly affects the luteal cells to attenuate PGF 2a -induced luteolytic effects. Wiltbank et al . (1992) found that in vitro cultures of separated large and small luteal cells were affected by oCSP. When large cells were cultured with oCSP and PGF 2a the normal anti-steroidal effect of PGF 2a alone was blocked. There was no effect of oCSP on progesterone secretion by large luteal cells. However, oCSP increased progesterone secretion from small

PAGE 67

52 luteal cells regardless of whether the small luteal cells were stimulated by LH or not. The anti-PGF 2a factor in oCSP, while not fully characterized is apparently a protein as the protective action was lost when oCSP was heated. The anti-PGF 2a effect was shown not to be oIFNt when progesterone secretion was not decreased in the presence of PGF 2a and oCSP with oIFNt removed. While prostaglandin-E has been reported to possess luteoprotective properties (Henderson et al . , 1977), prostaglandin-E was not considered the anti-PGF 2a factor since their dialysis procedure should have removed all prostaglandin-E. Also, there was no increase in progesterone secretion by the large luteal cells as noted when prostaglandin-E is added to large luteal cell cultures (Fitz et al., 1984). The anti-PGF 2a protein in oCSP apparently does not competitively inhibit binding of PGF 2a to its receptor as there was not a decrease in binding of 3 H-PGF 2a in the presence of oCSP. Therefore, it was proposed that the inhibitory action of this putative protein factor was at the post-receptor level, possibly at the level of the second messenger system. This study indicated that a factor in oCSP is capable of protecting progesterone production by luteal cells in the presence of PGF 2a , and may explain why progesterone secretion is not decreased in pregnant ewes with higher basal levels of PGF 2a (Ellinwood et al., 1979; Fincher et al . , 1986; Zarco et al . , 1988a; Vallet et al., 1989a; Burgess et al . , 1990) and why

PAGE 68

53 luteolysis in pregnant ewes requires higher doses of exogenous PGF 2ci (Inskeep et al . , 1975; Silvia et al., 1984b; Silvia et al., 1986). However, this factor is not oIFNt, shown to be the only protein in oCSP to act on endometrial tissues to prevent pulsatile secretion of PGF 2a , thus maintaining pregnancy (Vallet et al . , 1988). A supportive role of this, to-date unknown protein, to oIFNx in the maintenance of pregnancy should not be ruled out. Since intrauterine injection of oCSP attenuates oxytocin-induced PGF 2a secretion (Fincher et al . , 1986; Vallet et al . , 1988) and IP hydrolysis (Mirnado et al . , 1990b) by endometrium, it has been proposed that OTr formation is blocked in pregnant ewes by conceptus -mediated events (Flint et al . , 1994, 1995; Flint, 1995). Vallet and Lamming (1991) reported that intrauterine injection of oCSP blocked oxytocin-induced PGF 2a secretion and endometrial OTr formation. This was the first direct measure which confirmed that conceptus secretory products had an effect on OTr formation. The proposed progesterone block to OTr formation was suggested to be maintained by the conceptus to prevent normal down regulation of the Pr on Days 12 to 14 (McCracken et al., 1984). In the pregnant ewe, Pr concentrations in endometrial tissues does not change from Day 10 to Day 16 (Ott et al . , 1993b). However, during this time Pr mRNA actually decreased by 50 percent. Estrogen receptor protein

PAGE 69

54 and mRNA decreased in pregnant ewes from Day 10 to Day 16. Mirando et al . , (1993) reported that intrauterine injection of oCSP decreased Er protein and mRNA, as well as OTr in endometrial tissues. These results support an earlier hypothesis proposed by Ott et al. (1993) that Pr are stabilized in pregnant ewes and that Er formation is blocked. This in turn prevents the formation of endometrial OTr and, therefore, release of luteolytic pulses of PGF 2a in response to oxytocin by endometrium. Progesterone dependence has also been reported for uterine responsiveness to oxytocin (as measured by IP metabolism; Vallet et al . , 1989b; Ott et al . , 1992). However, as indicated previously, others have shown that continuous exposure of the endometrium to progesterone down-regulates endometrial Pr mRNA and protein abundance in the luminal epithelium, shallow glandular epithelium, and stroma (Wathes and Hamon, 1993; Spencer and Bazer, 1995). The mechanism responsible for this is currently poorly understood, but may involve Prmediated decreases in Pr gene transcription (Alexander et al., 1989; Read et al . , 1988). Results from studies performed by Spencer et al . (1995) have shown that negative regulation of the Pr gene in the endometrial epithelium occurs in both cyclic and pregnant ewes, because Pr mRNA abundance and immunoreactive Pr protein declined in the endometrial luminal epithelium and shallow glandular epithelium after Day 6. Thus, results of this study were

PAGE 70

55 used to modify the hypothesis to indicate that pregnancy does not stabilize or up-regulate Pr gene expression in the endometrium. Ovine Trophoblast Protein1 To determine whether ovine conceptuses produce a pregnancy recognition factor, ovine conceptuses were collected on Days 13 through 21 and cultured in the presence of 3 H-leucine for 24 h (Godkin et al . , 1982). Twodimensional polyacrylamide gel electrophoresis of the dialyzed medium reveled one major protein product with three isoelectric species. The isoelectric species had pi's of 5.5 to 5.7 and an estimated molecular weight of 17,000 to 20,000. This conceptuses product was initially referred to as protein X (Wilson et al . , 1979; Godkin et al . , 1982; 1984a) Protein X production could be detected by gel filtration chromatography 2D PAGE between Days 13 and 21 of pregnancy (Wilson et al . , 1979; Godkin et al . , 1982). In situ hybridization studies later showed that oTP-1 mRNA could be detected on Day 12, but full scale production of oTP-1 was not up-regulated until Day 13 (Hansen et al . , 1985; Farin et al . , 1990). Because this protein was produced transiently by the conceptus during the period of maternal recognition it was proposed to be the pregnancy recognition factor (Godkin et al., 1982). This same protein

PAGE 71

56 had been partially characterized by Martal and coworkers in 1979 and named Trophoblastin . Godkin et al . (1984a) further characterized the actions of oTP-1 in a series of experiments which showed that oTP-1 in Day 16 pregnant sheep was localized within the uterus. Ovine trophoblast protein1 was associated with the trophectoderm cells of the blastocyst and with the surface and upper glandular epithelium of the uterus. Uterine infusion of I25 I-oTP-l into Day 12 cyclic ewes indicated that oTP-1 was retained in the uterine tissues with little reaching the vasculature draining the uterus. When tissues from the CL and other ovarian structures were examined no oTP-1 was found. This indicated that the action of oTP-1 is local, at the level of the endometrium. Ovine trophoblast protein1 did not stimulate progesterone production by dispersed luteal cells from Day 12 cycling ewes. However, there was an oTP1-induced increase in protein production in vitro from uterine tissue acquired from ewes on Day 12 of their cycle. In competition assays, oTP-1 did not compete with oPRL in rabbit mammary cell cultures. Ovine trophoblast protein1 also did not compete with hCG or bLH in sheep luteal cell cultures. As a result of these studies, the authors suggested three possible functions for oTP-1: first; (1) induce the uterus to produce proteins to meet the nutritional requirements of the conceptus until attachment occurs; (2) induce endocrinological changes

PAGE 72

57 within uterine tissues which control the synthesis, release, or sequestration of PGF 2a ; or (3) induce secretion of particular proteins from the endometrium which would act in a luteotropic fashion at the level of the ovary. The second and third functions have been shown to be incorrect (for oTP-1) . For this reason oTP-1 is considered to be an antiluteolytic hormone and not a luteotropic hormone. A protein similar to oTP-1 is produced by the bovine and caprine conceptuses, and termed bTP-1 and cTP-1, respectively. The bTP-1 is the major protein produced by the bovine conceptus during the period of maternal recognition of pregnancy (Day 16 to 24) . Bovine trophoblast protein1, like oTP-1, posses several molecular weight and isoelectric variants ranging from 20 to 26 kDa and pi of 4.5 to 6.5, respectively (Bartol et al., 1985). However, unlike oTP-1, bTP-1 is glycosylated. Caprine trophoblast protein-1 has at least two isoforms with molecular weights of about 17,000 and pis of 5.2-5.7. The cTP-1 is the major protein produced by the goat conceptus during the time of maternal recognition of pregnancy (Day 17) (Gnatek et al., 1989). Ovine Trophoblast Protein-1 is an Interferon The identification of oTP-1 as an I FN came about as result of molecular cloning of cDNA and protein sequencing techniques (Imakawa et al . , 1987; Stewart et al . , 1987; Imakawa et al . , 1989; Charpigny et al . , 1988). These

PAGE 73

58 reports identified oTP-1 (which has also been called Trophoblastin, Protein-X, oTP-1, oIFNa n l, oTIFN-omega) , as a Type I IFN. Ovine trophoblast protein1 is reportedly closest in homology to the omega IFNs (originally refereed to as alpha n l IFNs) . Trophoblast interferons of different species (bTP-1, cTP-1 and oTP-1) are more closely related to one another than they are to the omega IFNs of their own species (i.e. oTP-1 and other ovine omega IFNs). The trophoblast interferons are apparently functionally related as well, as indicated by extension of the cycle in goats when sheep conceptuses were transferred to the goats uterus, prior to the period of maternal recognition or when roIFNi was injected into the uterine lumen (see Bazer et al., 1993) . In cattle, the transfer of trophoblastic vesicles (Heyman et al . , 1984) or intrauterine injection of roIFNx also results in extension of the cows estrous cycle (see Bazer et al . , 1993). It has been proposed, for these reasons, that the nomenclature IFNt be used to refer to all the trophoblast interferons (ovine, oIFNt; bovine, blFNt ; and caprine, cIFNx) Genes for IFNs are believed to exist in all ruminants of the order Artiodactyla and are believed to have diverged from IFN-omega genes 30 to 65 million years ago (Roberts et al . , 1992; Leaman et al . , 1992). Several cDNA sequences have been published for oIFNt since the first reports; however, they all have common characteristics

PAGE 74

59 (Stewart et al . , 1989b; Klemann et al . , 1990; Charlier et al . , 1991) . All IFNts are 172 amino acids in length. Trophoblast interferons, like other Type I IFNs, are intron-less. They are coded for by a 595 base pair open reading frame which codes for a 195 amino acid preprotein with a 23 amino acid signal sequence that is cleaved to produce the 172 amino acid mature protein. There are two disulfide bridges at highly conserved Cys residues. The first is between the Cys 1 residue and Cys". The second is between Cys 29 and Cys 139 . This last pair of Cys residues have been identified in all alpha, beta and omega IFNs and appears to be important for biological activity of the molecule (see Roberts et al., 1992) . There are several other conserved residues found in all Type I IFNs, including the IFNts. They are the four Cys just discussed, as well as Leu 3 , Leu 30 , Arg 33 , Phe 38 , Pro 39 , Glu 50 , Glu 52 , Ser 73 , Gin 92 , Leu 96 , Tyr 123 , Tyr 130 , Leu 131 , Ala 140 , Trp 141 , and Val 144 (Klemann et al . , 1990). Hydrophilicity-hydrophobicity plots of the different isoforms of Type I IFN are very similar in spite of the differences noted in the sequences as a whole. Secondary structures are also very similar. It is believed that there are five a-helices arranged in an anti-parallel manner with connected loop regions (see Roberts et al . , 1992). This arrangement is similar to that reported for IFNS, interlukin-1, interlukin-4 , growth hormone and granulocyte

PAGE 75

60 macrophage -colony stimulating factor (see Bazer et al . , 1993) . Interestingly, a recent report indicates that granulocyte macrophage-colony stimulating factor may play a role in modulation of production of oIFNx (Imakawa et al., 1993) The oIFNt molecule is not glycosylated; however, there is a site of potential glycosylation in some of the isoforms at Asn 78 (Godkin et al . , 1982; Anthony et al . , 1988) This is in contrast to bIFNT and some isoforms of cIFNt which are glycosylated (Helmer et al., 1987; Helmer et al., 1988; Baumbach et al . , 1990). Why oIFNt is not glycosylated and other IFNts are is not currently known. Trophoblast IFNs are biologically similar to other Type I IFNs. Ovine IFNt affords antiviral protection to cells cultured in the presence of virus just as IFNa (Pontzer et al., 1988). Ovine IFNt decreases proliferation of several cell lines as does IFNa (Pontzer et al., 1991). Incorporation of tritiated thymidine into lymphocytes following mitogen exposure is blocked by both oIFNt and IFNa (Newton et al., 1989). Finally, oIFNx up-regulates 2,5oligoadenylate synthetase in endometrial tissues (Mirando et al., 1991). The major difference between the IFNts and other Type I IFNs is their apparent lack of cytotoxicity. Even in large concentrations, the IFNts exert little or no cytotoxic effects. This lack of cytotoxicity has generated

PAGE 76

61 considerable interest in IFNt for use as a therapeutic drug (see Bazer, 1991) . For future experiments in the area of maternal recognition of pregnancy, and as a therapeutic drug, IFNt must be produced in larger quantities than those obtained from 30 hour cultures of a Day 16 conceptus (Godkin et al . , 1982) . For this reason, recombinant forms of oIFNx have been developed using synthetic oligonucleotides and cDNAs (Ott et al., 1991 and Martal et al., 1990, respectively). The roIFNt produced by Ott et al . (1991) was derived from a synthetic gene which was edited to include 17 unique restriction sites not found in the natural oIFNx sequence (Imakawa et al., 1989). While roIFNt was designed for expression in E. coli, yeast were used to overproduce the product. The use of yeast has allowed large amounts of roIFNt to be produced (Ott et al . , 1991; Van Heeke et al., 1996) . The restriction sites will allow for the easier production of mutants to investigate structure/function of oIFNt domains in the future. Biological activities for roIFNx and oIFNt have been shown to be identical (Ott et al . 1991) . Materna l Recognition Effects of Ovine Interferon Tau and Type I Interferons While it had been shown that the maternal pregnancy recognition factor produced by the ovine conceptus was contained within the milieu of oCSPs, and that oTP-1 (now

PAGE 77

62 referred to as oIFNi) was the major protein produced by the conceptus during the pregnancy recognition period (Godkin et al . , 1984b), studies with purified oIFNt were required to demonstrate that oIFNt is the maternal pregnancy recognition factor. The ability of partially purified oIFNt to extend the interestrous interval was initially demonstrated by Godkin et al. (1984b). The purified oIFNt was obtained by pooling culture media from a large number of conceptus cultures and passing the media over DEAE cellulose ion-exchange and S-200 Sephacryl columns. The resultant purified product was injected into the uterine lumen via catheters surgically placed into the tip of each uterine horn on Days 12 through 21. Plasma progesterone was used to determine CL life-span. A decrease in plasma progesterone concentrations to below 1 ng/ml was used to indicate luteal regression. Progesterone levels in ewes treated with partially purified oIFNt remained elevated 4 days longer than controls. While this was significant it was not nearly as long as the cycle extension noted for ewes (from this same report) which received intrauterine injections of oCSP. The differences in the extension of cycle between ewes which received oCSP and those that received oIFNt could have been due to several factors. The purified oIFNt may have been degraded by uterine protease or the estimate of the amount used per infusion, calculated from conceptus production in culture

PAGE 78

63 during a 24 hour period, may have been too low to elicit the same response as oCSP. The other major question was whether the difference between the two treatments could be due to some other factor which acted in concert with the 0IFN1 in the total oCSP to cause the longer cycle extension. To answer these questions, Vallet et al . (1988) prepared highly purified oIFNi . In addition, the oCSP remaining after oIFNt had been removed using an anti-oIFNt column several times to remove all traces of oIFNt, was used to treat ewes. Ewes were fitted with uterine catheters and injected (intrauterine) with either SP, oCSP, oIFNt, or oCSP minus oIFNt. There was no difference in the cycle length (19 days each; determined by fall in plasma progesterone) between ewes treated with SP and oCSP with oIFNt removed. From these findings, it is apparent that proteins other than oIFNt in oCSP are not involved in maternal recognition of pregnancy. Further, there was no difference in the intraestrous interval between the ewes which received oCSP or highly purified oIFNt (27 and 25 days, respectively). These results indicate that there is not a synergistic effect between other conceptus products and oIFNt in maternal recognition of pregnancy. Intrauterine injections of recombinant forms of oIFNt also increase the interestrous interval in ewes. Martal et al. (1990) reported an extension of up to 64 days (confirmed by marked CL at slaughter) when large amounts of roIFNi were

PAGE 79

64 administered (intrauterine; 340 /xg/day) . There was also an extension of the cycle in ewes which received levels of roIFNt comparable to that produced by Day 16 conceptus in culture (170 jug/day) , as shown in more recent studies in which roIFNt was administered from Day 11.5 through Day 16 and resulted in an extension of the interestrous interval to that of about 31 days (OTT et al . , 1993a). Recombinant forms of other Type I IFNs (rblFNct) also extend the interestrous interval; however, larger doses were required (2 mg/day) (Stewart et al . , 1989a; Parkinson et al., 1992). It is interesting to note that intramuscular injections of rblFNa increased the lambing rate in treated ewes (Schalue et al., 1989, 1991; Nephew et al . , 1990; Martinod et al . , 1991) . Although, it is not known how rblFNa aided in pregnancy recognition it was proposed that it supplemented the activity of endogenous oIFNt to ensure recognition of pregnancy in ewes in which conceptuses may have been retarded in growth, or for some reason producing suboptimal levels of oIFNt. Effects on prostaglandin Synthesis Intrauterine injections of oIFNt affect endometrial prostaglandin responsiveness to oxytocin and estrogen in a manner similar to that following intrauterine injections of oCSP in studies by Fincher et al. (1986). Vallet et al. (1988) reported that a challenge with estradiol on Day 14

PAGE 80

65 resulted in a subsequent rise of plasma PGFM concentrations in ewes treated with intrauterine injections of SP as controls while there was no such rise in ewes which received oIFNt . These same ewes, when challenged with oxytocin on Day 15, responded with a lower PGFM response when treated with oIFNt . There was no effect of treatment on prostaglandin-E production. This indicates that the prostaglandin production is not shunted from production of PGF 2a in cyclic ewes, to production of prostaglandin-E in pregnant ewes, to prevent luteolysis as proposed by McCracken et al . (1984). Attenuation of the PGF 2a response to exogenous oxytocin in pregnant ewes is acquired over a period of several days. Endometrium collected from ewes on Day 15, after treatment with oIFNt or SP by intrauterine injection on Days 12 through 14 (Vallet et al., 1989a) was perifused with buffer plus oxytocin and the medium assayed for PGF 2a content. There was a higher PGF 2a response to oxytocin for SP-treated than for oIFNttreated ewes. This supported results from a previous in vivo study (Fincher et al., 1986). However, when Day 15 endometrium was obtained from ewes not treated with oIFNt in vivo, the in vitro PGF 2a response was higher for endometrium treated in vivo with oIFNt. This is exactly opposite from the effects reported with long term treatment with oIFNt. These results imply that oIFNt does not act directly to competitively interfere with binding of oxytocin

PAGE 81

66 to its receptor or by other means to block the PGF 2c( response. Rather, oIFNt , prevents development of endometrial sensitivity to oxytocin-induced PGF 2o , secretory response. It has been shown that Type I interferons increase arachidonic acid metabolism. If oIFNt has the same affect on uterine tissues the increase in PGF 2a secretion reported in the short term perifusion experiments may be attributed to an increase in arachidonic acid metabolism within the tissues. However, several reports in the literature indicate that, in uterine tissues, this may not occur (Salamonsen et al . , 1988) and that arachidonic acid mobilized by IFNa is shunted away from the cyclooxygenase pathway (Hannigan and Williams, 1991). As mentioned previously, I FN produced by the bovine conceptus is very similar to that of oIFNt . Bovine IFNa and other Type I IFNs , like oIFNt , extend CL life-span when administered at the time of maternal recognition in the cow (Thatcher et al., 1989; Helmer et al . , 1989a: Plante et al . , 1991) . However, unlike the ewe, basal PGF 2a is lower in both pregnant cows, and cows which receive IFN treatment, than in cyclic cows. It is not known why basal levels of PGF 2a are lower in blFNx treated cows compared to controls, while there is no difference in basal levels of PGF 2a noted in oIFNttreated and control ewes. It may be that the cow blocks luteolysis by inhibition of PGF 2a synthesis early in the PGF 2a /oxytocin feedback loop to prevent a rise in OTr.

PAGE 82

67 There is evidence for this mode of action of blFNx in cows (Shemesh et al . , 1981; Basu and Kindahl, 1987; Gross et al . , 1988a; Helmer et al . , 1989b) The action of a prostaglandin inhibitor in sheep has not been well studied, but endogenous prostaglandin inhibitors have been detected in endometrium (Basu, 1989) and in allantoic fluid (Harper and Thornburn, 1984; Rice et al . , 1987) of sheep. It is not known, however, if an inhibitor of PGF 2a synthesis plays a role in maternal recognition pregnancy in ewes. In the cow, blFNt may induce synthesis of a prostaglandin inhibitor (DanetDesnoyers et al., 1993). Interferons affect arachidonic acid metabolism. Also, inhibitors of the cyclooxygenase or lipoxygenase pathways of arachidonic metabolism increase activities of the transduction signal for IFNa (Hannigan and Williams, 1991). Type I IFNs have been reported to attenuate prostaglandin secretion. Interferon-a was reported to attenuate prostaglandin production in human cells (Dore-Duffy et al . , 1983; Browning and Ribolini, 1987) The prostaglandin affected in this study was prostaglandin-E which is produced via the same pathway (cyclooxygenase) as PGF 2a . Human IFNa incubated with endometrial cells from ovariectomized ewes, maintained on a steroid regime that mimics that of a normal estrous cycle, caused a decrease in PGF 2a secretion (Salamonsen et al . , 1988; Salamonsen et al., 1989). Vallet et al . (1991) reported that intrauterine injection of rblFNa on Days 12 through 14 was as effective

PAGE 83

68 in blocking oxytocininduced PGF 2a secretion from the uterus as was oCSP; however, this was believed to be through progesterone attenuation of the OTr. The Ovine Interferon Tau Receptor and Signal Transduction Ovine IFNt , like other Type I IFNs (alpha, beta, and omega) bind to a high affinity (Godkin et al . , 1984a;) Type I IFN receptor (Stewert et al . , 1987). Type I I FN receptors are distributed throughout endometrial tissues of the ewe and their expression may be influenced by ovarian steroids (Knickerbocker and Niswender, 19 89) . Type I receptors are also present in other tissues of the body (Knickerbocker and Niswender, 1989). Pontzer et al. (1990) reported that high concentrations of the NT of oIFNt attenuated antiviral effects of oIFNt, but not IFNa, in cell co-cultures. Conversely, a synthetic peptide corresponding to amino acids 139-172 (CT) blocked antiviral effects of both oIFNt and IFNa in cell co-cultures. The authors concluded that NT of oIFNt binds to a unique domain in the Type I IFN receptor while the CT binds to a domain common to Type I IFNs. This may explain the unique actions of oIFNt . Ovine IFNt -induced hormone action is initiated by the transduction of signal via activation of the JAK/STAT system (see Willians, 1991a) and is believed to act in the same manner as other Type I interferons (see Interferon Receptor/Signal Transduction section) .

PAGE 84

69 Ovine IFNt and other IFNx ' s increase endometrial protein production dramatically (Gross et al . , 1988b; Sharif et al., 1989; Ashworth and Bazer, 1989). Included in these is the enzyme 2 ' , 5 ' -oligoadenylate synthetase (Mirando et al., 1991; Short et al . , 1991). Estrogen receptors are increased in endometrial adenocarcinoma cells by IFNa 2b and in human breast cancer tissue and human endometrium. In rabbit endometrium Er expression is increased by IFNa. Progesterone receptors may be increased by IFNa 2b in endometrial adenocarcinoma and by IFNa in AE-7 endometrial cancer cells (see Bazer et al., 1993). Full length Er and Pr genes for ruminants have not been cloned to determine the presence of an interferon stimulated response element in the 3 'or 5' flanking region; however, analysis of partial clones of genomic DNA from human and rabbit Er and Pr indicate their presence (see Bazer et al . , 1993). If an interferon stimulated response element (s) is present in the Er and Pr genes they may allow oIFNx to negatively regulate expression of Er and OTr within the endometrium during early pregnancy (Mirando et al., 1993; Ott et al., 1993b) to allow for establishment of pregnancy.

PAGE 85

CHAPTER 3 THE EFFECTS OF RECOMBINANT OVINE INTERFERON TAU AND SYNTHETIC PEPTIDES, CORRESPONDING TO PORTIONS OF RECOMBINANT OVINE INTERFERON TAU, ON OXYTOCINSTIMULATED ENDOMETRIAL INOSITOL PHOSPHATE METABOLISM AND ENDOMETRIAL OXYTOCIN RECEPTOR CONCENTRATION. Introduction The establishment of pregnancy in ewes requires that the conceptus, by Day 12 of pregnancy (Moor and Rowson, 1966a), activate a mechanism (s) (oIFNt; Godkin et al . , 1982; Vallet et al., 1988) to prevent luteolysis. Luteolysis in ruminants is initiated by pulsatile secretion of PGF 2a by endometrial tissues (McCracken et al . , 1981; Hooper et al., 1986) . Pulsatile secretion of uterine PGF 2a is thought to be responsible for luteolysis due to the fact that continuous infusion of PGF 2a , or immunization against PGF 2a , results in extension of the cycle (Scaramuzzi and Baird, 1976; Fairclough et al . , 1981). During luteal regression, PGF 2a is secreted from the endometrium in a series of five to eight, high amplitude, short duration episodes (Thornburn et al., 1973; Barcikowski et al . , 1974; Flint and Sheldrick, 1983; Zarco et al . , 1988b) with 6 to 8 h between each episode. McCracken et al. (1984) have shown that the CL must be exposed to approximately 5 pulses of PGF 2a over a 25 70

PAGE 86

71 h period to undergo complete luteolysis. The pulsatile secretion of PGF 2a may be initiated by the secretion of oxytocin from the posterior pituitary and is escalated by oxytocin secreted by the CL (Flint et al . , 1990). Oxytocin from the CL and PGF 2a from the uterus act together in a positive feedback loop to generate the luteolytic pulses required for luteolysis (Flint and Sheldrick. , 1986; Hooper et al., 1986; see Silvia et al., 1991 ;McCracken et al . , 1991) . However, the ability of the endometrium to secrete PGF 2o in response to oxytocin does not develop until Day 13 to 14 of the cycle (Roberts et al., 1976; Roberts and Mccracken, 1976; Fairclough et al . , 1984; Silvia et al . , 1991) when OTr concentrations increase (Sheldrick and Flint, 1985) . It is the coupling of oxytocin to specific endometrial OTr sites that stimulates PGF 2a synthesis, through activation of the inositol phosphate/diacylglycerol signal transduction pathway (Flint et al . , 1986; Silvia and Homanics , 19 88) . During early pregnancy, pulsatile secretion of PGF 2a secretion by the uterus is attenuated or absent (Thornburn et al., 1973; Barcikowski et al . , 1974; Moore and Watkins, 1982; Hooper et al . , 1987; Zarco et al . , 1988a) with a disruption of the oxytocin/PGF 2a positive feedback loop. The attenuation of the loop in the pregnant ewe is primarily due to the absence of expression of endometrial OTr and Er

PAGE 87

72 (McCracken et al . , 1984; Sheldrick and Flint, 1985, Spencer et al. , 1995b) . Ovine IFNt is the antiluteolytic protein secreted by the conceptus (Godkin et al., 1992; Vallet et al . , 1988; see Bazer et al., 1991; Godkin et al . , 1984a; 1984b; Bazer et al., 1995). Endometrial IP metabolism (Mirando et al., 1990 a,b; Ott et al . , 1992), PGF 2a secretion (Vallet et al . , 1988; Mirando et al . , 1990a; Ott et al . , 1992) as well as endometrial OTr and Er expression (Vallet and Lamming, 1991; Mirando et al., 1993; Spencer et al., 1996) are inhibited by intrauterine injection of oIFNx. Intrauterine injection of roIFNT is as effective as oIFNt purified from conceptus culture medium in blocking luteolysis (Ott, 1992; Ott et al., 1993a). It is believed that oIFNt acts through its Type I IFN receptor, and activation of its transduction signal to prevent endometrial expression of Er and OTr. The Pr and progesterone are considered permissive to antiluteolytic effects of oIFNt , but the mechanism is not known (Spencer 1995) . Several questions have been raised as to whether or not the oxytocininduced IP metabolism, previously reported from our laboratory, was due to stimulation through the OTr, or could it be through the AVP receptor. Experiment 1 was designed to answer this question, by determining if oxytocininduced IP metabolism within endometrial tissues is mediated through the OTr or the AVP receptor. This was

PAGE 88

accomplished by means of an IP metabolism assay that examined all possible combinations of OT, and AVP stimulation with all possible combinations of the receptor antagonist for the OTr and AVP receptor. Other primary objectives of these experiments (as they relate to IP metabolism and endometrial OTr concentration) , were to determine what effect treatment of cyclic ewes, with various synthetically produced peptides corresponding to overlapping segments of oIFNt, had on oxytocin-induced endometrial IP metabolism, and on endometrial OTr concentration. The NT and CT peptides were first examined (Experiment 2) due to the fact that they were the most extensively examined of the peptides (Pontzer et al . , 1990, 1994) . It has been proposed that the NT possesses the properties which makes oIFNt different from other IFNas, and that CT is the portion common to the IFNas. Experiment 3 examined the effect of the remaining peptides (2-5) on oxytocin-induced IP metabolism and endometrial OTr concentration. Experiments 4, also examined the effect of NT treatment on endometrial OTr concentration, but this experiment was specifically designed to determine what effect oxytocin challenge (in vivo) on Days 13 and 15 would have with regards to the main treatment of NT or roIFNi. The final experiment, Experiment 5, was designed to determine what effect NT has on endometrial OTr concentration through Day 18, which is two days longer than

PAGE 89

oIFNt had been examined. This was to determine if NT has the ability to hold OTr concentration at a low level for an extended period of time. Materials and Methods Animals Ewes of primarily Rambouillet breeding were checked daily at 07:30 am for 20 min with vasectomized males of St. Croix or mixed Rambouillet breeding. Ewes which had previously exhibited at least two normal estrous cycles (16 to 17 days in length) were assigned to experimental groups. Ewes for experiments 1, 2, and 3 were housed at the Sheep Research Facility, University of Florida, Gainesville. Ewes for experiment 4 and 5 were housed at the Sheep Research Center, Texas A&M University, College Station. Protein And Peptide Preparation Ovine conceptus secretory protein preparation Ovine conceptus secretory proteins were prepared as previously reported by our laboratory (Vallet et al . , 1988 and Mirando et al., 1990b). Briefly, ovine conceptuses were collected at laparotomy on Day 16 of pregnancy by flushing the uterus with 20 ml minimum essential medium (Earl's salts; Gibco/Life Technologies, Grand Island, New York). The conceptuses were cultured for 30 h in minimum essential medium as reported by Godkin et al . (1982). The resultant

PAGE 90

75 oCSP-conditioned medium was collected, pooled and stored at -20°C until used. Medium containing oCSP was thawed, pooled and dialyzed (3500 Mr cutoff) at 4°C against 4 liters of 0.9% NaCl (w/v) , changed three times (4L each change) . Ovine conceptus secretory proteins were concentrated to one-tenth the original volume using an Amicon ultrafilter (500 Mr cutoff; Amicon Co., Danvers , MA). The concentration of oIFNt in oCSP was determined by RIA (Vallet et al., 1988). Ovine conceptus secretory proteins were diluted with in 0.9% NaCl (w/v) to an oIFNt concentration of 25/ng/ml oIFNt . The concentration of total protein in oCSP was determined by the method of Lowry et al . (1951). The oCSP was diluted with SP in 0.9% NaCl (w/v) to a total protein concentration of 0.75 mg/ml, and stored at -20°C in 2 ml aliquots until just prior to use when they were thawed under running water. Recombinant interferon tau preparation Recombinant oIFNt , provided by Dr. Troy Ott, was produced as described by Ott et al . (1991). Antiviral units of roIFNx were determined by antiviral assay using Madin Darby bovine kidney cells challenged with vesicular stomatitis virus (Pontzer et al . , 1988). Protein concentration of roIFNx was determined by protein assay (Lowry et al., 1951). Recombinant oIFNt was diluted to 25 ptg/ml for Experiment 3 and 5 0 jiig/ml for Experiments 4 and 5. The total protein concentration was brought up to 0.75 mg/ml

PAGE 91

76 by the addition of SP in 0.9% NaCl (w/v) . Aliquots of 2 ml each were stored in glass scintillation vials at -20°C until just prior to use at which time they were thawed under running water. Serum protein preparation Blood was collected from the jugular vein of a pregnant ewe on Day 16 (07:00 am) and allowed to clot 1 h at room temperature and then overnight at 20°C. Serum was collected and dialyzed (3500 Mr cutoff) at 4°C against 4 L 0.9% NaCl (w/v) , with three changes (4 L each change) . Protein concentration was determined (Lowry et al . , 1951) and diluted to a protein concentration of 0.7 5 mg/ml with 0.9% NaCl (w/v) and stored in glass scintillation vials at -20°C in 2 ml aliquots until just prior to use when they were thawed under running water. Synthetic peptide production Synthetic peptides corresponding to the amino and carboxylterminus of oIFNt were produce by Dr. Carol Pontzer in Dr. Howard M. Johnson's laboratory as described by Pontzer et al. (1990). Briefly, peptides were synthesized on a Biosearch 9500AT automated peptide synthesizer using f luorenylemthyloxycarbonyl chemistry. Peptides were cleaved from resins using trif luroacetic acid/ethanedithiol/thioanisole/anisole . Cleaved peptides were extracted in diethyl ether and ethyl acetate, dissolved in water and lyophilized. Reverse phase HPLC was used to

PAGE 92

77 determine purity of the peptides. Peptides produced were: to the NT (aa 1-37) ; CT (aa 139-172) : as well as four overlapping internal peptides: Pep 2 (aa 34-64); Pep 3 (aa 62-92); Pep 4 (aa 90-122); and Pep 5 (aa 119-150). All peptides were reconstituted in 0.9% NaCl (w/v) to 0.5 mg/ml. This concentration had previously been shown to block oIFNtinduced antiviral activity (Pontzer et al . , 1990). Serum proteins were added to bring total protein concentration to 0.7 5 mg/ml in a 2 ml volume. Aliquots of 2 ml were stored in glass scintillation vials at -20°C until just prior to use when they were thawed under water. Experimental Design Experiment 1 To determine if oxytocin-induced IP metabolism within endometrial tissues is mediated through the OTr or the AVP receptor inositol phosphate metabolism was examined in endometrium from three ewes after in vitro stimulation of oxytocin or AVP. Ewes received no in vivo treatment. On Day 16 of their estrous cycle ewes, were anesthetized with halothane and ovariectomized-hysterectomized by mid-ventral laparotomy. Caruncular tissue was collected from the entire uterus into 100 mm petri dishes, maintained on ice and minced into fine pieces. Tissue (0.999 gm) was transferred to 16 separate 20 ml glass scintillation vials and placed on ice until the oxytocin/AVP IP assay was begun (~ 10 min) .

PAGE 93

78 Experiment 2 Twentyfour ewes were randomly assigned in a 2 X 3 factorial arrangement to receive SP, SP+NT, SP+CT, oCSP, oCSP+NT or oCSP+CT (n=4/treatment ; Fig. 3.1). On Day 6, ewes were anesthetized with halothane and the uterus exteriorized by laparotomy. The number and location of CL were recorded, and a catheter (8' in length; V6 tubing, Bolab, Lake Havasu City, Az) placed into each uterine horn (~ 2 cm) via the oviduct at the utero-tubal junction (Vallet et al . , 1988). Catheters were secured to the oviduct at the utero-tubal junction, and the uterine body at the external bifurcation, with suture on either side of a set of cuffs (~2 cm and 3 0 cm from one end; VI 0 Tubing, Bolab, Lake Havasu City, AZ) fused to the catheter with a drop of cyclohexanone (Fisher) . Catheters were exteriorized through an incision in the flank, and attached to the skin by suturing cloth tape, which was wrapped around the tubing, to the skin. The catheters were flushed with sterile 0.9% saline to determine that there were no blockages and the ends sealed to prevent air from entering the tubing. The external portion of the tubing was stored, wrapped in betadine soaked 4X4 gauze, and placed in a pouch attached by suture to the skin at the point of exit (Vallet et al . , 1988) . Ewes were allowed to recover for 24 h. They were then returned to their respective group pens until where they were placed into individual pens located within group

PAGE 94

'-d tQ (= H fD W H3 fD a fD U) HIQ Hi O i-l M X t) fD Prt to

PAGE 95

>» E o •*-> o 2> CD 4-> (0 80 c o c o E Q. CO CD CO CM CM CO > c a> E +» CO CD CO •a X CM C k. o CD c mwum a 3 O) E Q. CO (0 CO >» >% "Jo CO X X CM CM c c o o CD CD C C wmm 9mm 3 3 Z O E E in m * o o + + Q_ 0. CO CO CO T3 X CM c k. o c MM k. s 3 <0 (0 o "D X X CM CM C c v_ k. o o CD a> C c mmm k. MB CD CD 3 3 H H Z O See + + 0.0.0. CO CO CO o o o o o o _ E 0. TCO
PAGE 96

81 pens until Day 11. This arrangement limited movement of the ewes during the treatment period while allowing them constant contact with the other ewes in their respective pens. Hay and water were available ad libitum and about 120 gm concentrate feed was provided each morning. Hay, water and food were withheld 12 h prior to surgery. On Day 12, treatments began and continued through the morning of Day 16. Each ewe received, per uterine horn, twice daily injections (06:00 and 18:00 h, respectively) of one of the following, according to group: 1.5 tag SP; 0 . 5 mg NT plus 1.0 iag SP; 0.5 mg CT plus 1.0 mg SP; 0.7 5 mg oCSP (containing 2 5 /j.g oIFNt by RIA) plus 0.7 5 mg SP; 0.7 5 mg oCSP plus 0 . 5 mg NT; or 0.7 5 mg oCSP plus 0.5 mg CT. All injections were balanced to a total protein concentration of 1.5 mg with SP and were adjusted to 2 ml in volume with 0.9% NaCl (w/v) . Each injection also contained 50 mg ampicillin (Polyflex; Aveco Co. Inc., Fort Dodge, IA) in 0.1 ml 0.9% NaCl (w/v) which was mixed with the thawed treatment sample just prior to injection. Each catheter was flushed with 1 ml 0.9% NaCl (w/v) after injection, resulting in a total infused volume of 3.1 ml. The ends of the each catheter was resealed without introducing air and the catheters returned to the pouch in fresh 4X4 gauze sponge soaked in betadine. On Day 15, all food and water was removed from the ewes and on the morning of Day 16, after the last intrauterine injection, the ewes were ovariectomized-hysterectomized.

PAGE 97

82 Endometrial tissue (-1.2 gm; primarily of caruncular origin) from the uterine horn ipsilateral to the CL was collected into ice-cold KRB for determination of IP metabolism. The remainder of the endometrium was collected into bags, snap frozen in liquid nitrogen, and stored at -80°C for use in oxytocin receptor assays (filter method) and Er mRNA analysis (Chapter 5). Experiment 3 Twenty-eight ewes were randomly assigned to one of seven treatment groups to receive SP, roIFNt , NT, Pep 2, Pep 3, Pep 4, or Pep 5 (n=4/treatment ; refer to the section on synthetic peptide preparation for amino acid determination of each peptide; Fig. 3.2). On Day 8 or Day 9 of the estrous cycle ewes were anesthetized and the uterus exteriorized by laparotomy, the number and location of CL noted and catheters placed as described in Experiment 2. Ewes were housed as described in Experiment 2 with the exception that individual crates were located in an adjoining pen in sight of ewes in their respective group pens. Intrauterine injections were administered as in Experiment 2 except that they began on Day 11. Treatments, per horn, consisted of 1.5 mg SP, 0.25 ,ug roIFNT , 0.5 mg NT, 0 . 5 mg Pep 2, 0 . 5 mg Pep 3, 0 . 5 mg Pep 4, or 0 . 5mg Pep 5. The total protein concentration per treatment was brought to 1.5 mg with SP and the total volume adjusted to 2 ml with 0.9% NaCl (w/v) . Each injection, also contained 50 mg

PAGE 98

cd M •0 CD H3 0) 0 rt 0) CD W HiQ Hi o M X TJ CD H3 CD rt

PAGE 99

CO O CO 3 im (0 LU £ Q. CD co IO CO (N ^ O ^ CM CM tS (O O) t' fO i i i o> • Tf CM O rtCO tO O tco (0 (0 CO (0 (0 CO CO CO (0 CM CO *o Q. Q. Q. Q. 0) 0) 0) 0) Q_ Q_ Q_ Q_ i2 C CD E (0 "D X CM c o CO » g a & 3 O CO .C "O 0) Q).E ^ 3 E§ O IO (0 "D X CM C c o CD c CO +-» "O 3 O) O) E E CO IO Q. CD QCO

PAGE 100

85 ampicillin in 0.1 ml 0.9% NaCl (w/v) which was added with the sample just prior to injection. Each catheter was flushed with 1 ml of 0.9% NaCl (w/v) after injection, to clear the catheter, so the volume injected was 3.1 ml. On Day 15 all food and water was removed from the ewes and on the morning of Day 16, after the last injection, the ewes were ovariectomized-hysterectomized. Endometrial tissue (-1.2 gm; primarily of caruncular origin) from the uterine horn ipsilateral to the CL was collected into icecold KRB for determination of IP metabolism. The remainder of the endometrium was placed in plastic bags, snap frozen in liquid nitrogen, and stored at -80°C for use in oxytocin receptor assays (filter method) and Er mRNA analysis (Chapter 5) . Experiment 4 Twelve ewes were randomly assigned to receive intrauterine injection of either SP, roIFNx or NT (n=4/treatment ; Fig. 3.3 and 3.4). On Day 8 or 9 of the estrous cycle ewes were anesthetized and the uterus exteriorized by laparotomy and catheters placed as described in Experiment 2. Ewes were allowed to recover for 24 h and then returned to a pen, separate from, but in sight of, ewes in their respective group pen until Day 10, when they were placed into individual pens located within sight of ewes in their group pens. Hay and water were available ad libitum. On Day 11 treatments began, and continued twice daily (06:00

PAGE 101

HiQ CD OJ O < rt> PI to X -d fD g rt I — 1 rt) CO HU3 3 Ml o w t) fD H H3 rt> 3

PAGE 103

piq c CD 4*. d fD rt CD HH fD CL Cb fD W P£l O hh rt P fD fD rt •0 fD P* 0 Hi O M X TJ fD H P§ rt

PAGE 104

89 CO C o CO CM CO 0) "D C hi * I a a. iS c CD E +> (0
PAGE 105

90 and 18:00 h) through the morning of Day 16. Treatments, per horn, consisted of 1.5 mg SP, 0.50 /ig roIFNt or 0.5 mg NT. Total protein concentration per treatment was balanced to 1 . 5 mg with SP and the total volume adjusted to 2 ml with 0.9% NaCl (w/v) . Each injection also contained 50 mg ampicillin in 0.1 ml 0.9% NaCl (w/v) which was mixed with the thawed sample just prior to injection. Each catheter was cleared by flushing with 1 ml of 0.9% NaCl after each injection, resulting in a total injected volume of 3.1 ml. All ewes were challenged with an injection of oxytocin (10 iu) into the jugular vein on Day 14. Jugular blood samples were collected into heparinized tubes, 10 min prior to oxytocin injection at time 0 and at 10, 20, 30, 45, and 60 min post-oxytocin injection. Samples were maintained on ice until they were centrifuged (15 min at 1800 x g and 4°C) , plasma transferred to plastic scintillation vials for storage at -20°C until used in PGFM assays (Chapter 4) . On Day 15 all food and water was removed from the ewes and on the morning of Day 16, after the last injection, the ewes were ovariectomized-hysterectomized. Endometrium was collected into plastic bags, snap frozen in liquid nitrogen, and stored at -80°C for use in oxytocin receptor assays (PEG method) , as well as analyzed to determine Er mRNA , Er and Pr protein concentrations (Chapter 5) .

PAGE 106

Experiment 5 Twentyfour ewes were randomly assigned to a 2 x 3 factorial design to receive intrauterine injections of either SP or NT and be hysterectomized on either Day 16, 17 or 18 (n=4/treatment*day ; Fig. 3.5). On Day 8 or 9 of the estrous cycle ewes were anesthetized and the uterus exteriorized by laparotomy and catheters placed in the uterine lumen as described in Experiment 2. Ewes were housed as described in Experiment 4. Treatments began the morning of Day 11, and continued through the morning of Day 16. Treatments, per horn, consisted of 1.5 mg SP or 0.5 mg NT. The total protein concentration per treatment was balanced to 1.5 mg with SP and the total volume adjusted to 2 ml with 0.9% NaCl (w/v) . Each injection, also contained 50 mg of ampicillin in 0.1 ml 0.9% NaCl (w/v) which was mixed with the thawed sample just prior to injection. Each catheter was cleared by flushing with 1 ml of 0.9% NaCl (w/v) after injection, resulting in a total infused volume of 3.1 ml. On the morning prior to surgery all food and water was removed from the ewes. Four ewes from each treatment group were ovariectomized-hysterectomized on each of the days examined (Day 16, Day 17 and Day 18). Endometrium was collected into plastic bags, snap frozen in liquid nitrogen, and stored at -80°C for use in oxytocin receptor assays (PEG

PAGE 107

92 method) , and analyzed for Er mRNA, Er and Pr protein concentration (Chapter 5). Inositol Phosphate Metabolism Arginine vasopressin/OT-induced IP metabolism To determine if changes in IP metabolism were induced within endometrial tissues through AVP receptor-mediated processes, the following assay was modified from that reported by Flint et al . (1986) and by Vallet and Bazer (1989) . Caruncular tissue from the ipsilateral uterine horn was collected (Experiment 1) into ice-cold KRB containing 10 mM glucose and 10 myo-inositol , minced into approximately 5 mg pieces (Fig. 3.6) Tissue pieces were weighed and 0.1 p transferred into each of 16 separate glass scintillation vials (see Table 1 for treatments) and washed with 1 ml icecold KRB. Tissue incubations were carried out under an atmosphere of 95% 0 2 , and 5% C0 2 ; at 37°C in a shaking water bath. Vials were re-gassed and returned to the water bath after each change of buffer until the assay was terminated and vials were placed on ice. Initially, tissues were incubated for 2 h in 1 ml KRB plus 10 fiCx myo[ 2 3 H] inositol (specific activity 18.9 Ci/mmol; Amersham, Arlington Heights, IL) . After incubation, KRB was removed and fresh KRB without labeled inositol was added, samples were

PAGE 108

Hc fD M t3 fD O rt fD W HIQ Hi o Hi w X fD H3 fD

PAGE 110

Figure 3.6: Schematic of the AVP/OT-stimulated IP assay. Individual treatments listed by vial number are shown in Table 1.

PAGE 111

96 Control (n=1 vial) 100 mg Tissue/Vial 1 ml KRB + 100 nM 3 H-lnositol Treatments (n=15 vials) 120 min, 95% 0 2 , 5% C0 2 , 37°C in a shaking, metabolic incubator 1 ml fresh KRB, 30 min LiCI (10mM), 10 min 0.1 M Na 2 C0 2 or 0.1 M Na 2 C0 2 + 500 nM OTA &/Or 500 nM AVPA, 20 min 0.1 M Na 2 CO z or 0.1 M Na 2 C0 2 + 100 nM OT &/Or 100 nM AVP, 20 min Cells were lysed and IP's separated on anion exchange columns

PAGE 112

97 incubated for an additional 3 0 min. Buffer was replaced again and 20 /Ltl LiCl (0.51 M) was added (to inhibit inositol 1-monophosphatase activity) and tissues incubated 10 min. Following this 10 min incubation, appropriate vials were incubated 15 min with 20 /Ltl KRB + 20 fxl OTr antagonist (26 jitM in NaC0 3 ; L-365,209; generously supplied by Dr. D.J. Pettibone; Merck, Sharp and Dohme Research Laboratories, West Point, PA) , 20 (J.1 KRB + 20 Ail AVPr antagonist (26 jitM in NaC0 3 ; Peninsula Laboratories, Inc.), or 20 /Ltl OTr antagonist + 20 /Ltl AVPr antagonist . All other vials were balanced with KRB buffer. Following the 15 min incubation, appropriate vials were incubated 20 min with 20 /Ltl oxytocin (5.2 /uM in NaC0 3 ; Sigma Chemical Co., St. Louis, MO), or 2 0 /Ltl AVP (5.2 /LtM in NaC0 3 ; Sigma Chemical Co., St. Louis, MO). All other vials were balanced with KRB buffer. Following the 20 min incubation, buffer was replaced with ice-cold 15% TCA (w/v) and vials placed on ice for 20-30 min to stop the reaction. The TCA was transferred to boracilicate glass tubes and extracted five times with 5 ml water saturated with diethyl ether. Extracts were discarded each time and the residual diethyl ether was removed from the aqueous phase under a stream of N 2 . The samples were neutralized with 25 (Ltl Tris (hydroxymethyl) aminome thane (0.5 M; pH 7.5) and stored at -20°C. Inositol phosphates (IP 1( IP 2 , IP 3 ) were separated on Dowex columns. Dowex anion-exchange resin (1x8 -200; Sigma

PAGE 113

03 03 Ph CQ Pi Pi > p> ^ tn l-H L 1 .p Q_i L 1 v; o i ~± -i. ?s CD CD p) no no IN IN 1 ) 4—' r* M i i T "T i— d \ vL> rn rn rn m W |1) M-l M-l 1 CS cS cr* rv* K K K ffi r , r m 4—' v v v v HH r-H r~*i LJ LJ U) rrt (1) m 1 1 1 1 1 1 1 1 , i . i L_| Li P_t L CD CD O CD CD CD ^st 1 ^ ^ r\i no CO rn r-n fd rH 1 1 1 1 g CD a r-\ H •-{ r-\ H 0 0 0 0 /-< r1 H H M M .,_| i 1 i 1 i 1 i \ 4~ J 4— ' 4— ' 4-J f-n (Jl i— 1 t~* r-* t— ' C G C G M r\ r*\ rs r\ U U CJ U *l LJ LJ LJ rH CD — H £3 rc cd •H E-i > h (N n ^ ID LO < < < rH m ca a, 03 CQ P, oi &> > pel Pi > i*; ^ < ^ < >— | 1— | H H H H H H 3. 3. 3. 3. 3. 3. 3 3 o o o o o o o o CN CN CN CN CN CN CN CN + + + + + + < * < u < < rH < < rH < Oh >H DP rH rH PQ rH Ol rH ^ f_| ^> f_| PH ^> fH v; (~) n n^n «-H fVl fVI fXI ^J 1 In l. n In IN ' N 1 N | j 1 1 1 1 rH, lilt u rH a. a. < < < X 1 T H ^ l-M rH r< Li Qj Li rH MH rH Li n, Li L-t rH t-M rH r^ r, r . U r 1 ^ r 1 n> r, -«-«^ r . CJ LJ »7 n ^ n * ^ r-^ < < < < HrHHH i 1 i 1 S >4 <4 <4 -i. -i -i 1 1 1 1 1 1 1 1 1 1 1 1 —A. —A. o o o o ~i *i ~i *i —A. — L. ^i, — i. CD CD no no no no IN IN vN IN CD CD CD CD CN CN CN CN CN CN + + + + _i_ j_ r i J_ _1_ _L _L 1 T T I ft dl Oh Oi > > > > Eh FH Fh Eh tS" ~s >-> ^ 1-^ P-, P-, P-, P-, CH tH tH tH o o rrf 1 rf* 1^ O O C\ c\ w ^ ^ w O rH CN lo CTl H H H rH rH rH rH

PAGE 114

99 Chemical Co.; St. Louis, MO.) was swelled in distilled water and poured into 6 x 0.6 cm columns (n=16) . Resin was converted to the formate form by sequential washing with five volumes of 1 M HC1, 1 M NH 4 OH , 1 M formic acid, and 0.1 M formic acid in 2 M NH 4 formate. Columns were washed with 15 volumes distilled water and samples were applied to the columns. Sample tubes were rinsed with 0 . 2 ml distilled water and rinse added to the column. Columns were eluted with 5 x 5 ml distilled water, 3 x 5 ml 25 mM Na 2 B 4 0 7 in 60 mM Na formate, 3 x 5 ml 0 . 1 M formic acid in 0.2 M NH 4 formate, 3 x 5 ml of 0 . 1 M formic acid in 0.4 M NH 4 formate and 3 x 5 ml 0 . 1 M formic acid in 1.0 M NH 4 formate. Each of the elution series were pooled following final 5 ml elution. Inositol, glycerophosphoinositol , IP 1( IP 2 , and IP 3 were collect in the five eluents, respectively. Incorporation of [ 3 H] inositol into IPs was determined by liquid scintillation spectrometry. Oxvtocin-induced IP metabolism Oxytocin-induced endometrial IP metabolism was determined as described by Flint et al . (1986) and as modified in our laboratory by Vallet and Bazer (1989) . Caruncular tissue from the ipsilateral uterine horn was collected (Experiment 2 and 3) into ice-cold KRB containing 10 mM glucose and 10 ^M myo-inositol , minced into approximately 5 mg pieces (Fig. 3.7). Tissue pieces were weighed and 0 . 1 gm transferred into duplicate glass

PAGE 115

100 scintillation vials and washed with 1 ml ice-cold KRB. One vial served as a control and did not receive oxytocin in the assay and the other had oxytocin added to determine oxytocin-induced IP metabolism. Tissues were incubated under an atmosphere of 95% 0 2 ; and 5% C0 2 ; at 37°C in a shaking water bath. Vials were re-gassed and returned to the water bath after each change of buffer until the assay was terminated and vials placed on ice. The tissues were incubated for 2 h in 1 ml KRB plus 10 /iCi myo[23 H] inositol (specific activity 18.9 Ci/mmol.; Amersham, Arlington Heights, IL.). After incubation KRB was removed and fresh KRB without labeled inositol was added and incubated 3 0 min. Buffer was replaced again with 20 ^1 LiCl (0.51 M; final concentration = 10 mM) added to inhibit inositol 1monophosphatase activity and tissues were incubated 10 min. After 10 min, vials were incubated for 20 min with no oxytocin (control vial) or with 5 . 2 /u.M oxytocin (oxytocininduced vial) in 20 jil NaC0 3 (0.1 M; final oxytocin concentration = 0 or 100 nM, respectively) . After 20 min, buffer was replaced with ice-cold 15% TCA (w/v) and vials placed on ice for 20-30 min to stop the reaction. The TCA was transferred to boracilicate glass tubes and extracted 5 times with 5 ml water saturated diethyl ether. As much diethyl ether as possible was removed with a pipet and discarded each time. Residual diethyl ether remaining after the final wash was removed from the aqueous phase

PAGE 116

Figure 3.7: Schematic of the 0Tstimulated IP metabolism assay.

PAGE 117

Control 102 OT-Stimulated 100 mg Tissue/Vial 1 ml KRB + 100 nM 3 H-lnositol 120 min, 95% 0 2 , 5% C0 2 , 37°C in a shaking, metabolic incubator 1 ml fresh KRB, 30 min LiCI (10mM), 10 min 0.1 M Na 2 C0 2 or 0.1 M Na,CO, + 100 nM OT 2 NSNS 2 Cells were lysed and IP's separated on anion exchange columns

PAGE 118

103 under a stream of N 2 . The samples were neutralized with 25 111 Tris (hydroxymethyl) aminome thane (0.5 M; pH 7.5) and stored at -20°C. Inositol phosphates were separated on Dowex columns as described previously in the AVP/OT-induced IP metabolism section . Endometrial Oxytocin Receptor Assay Filter procedure Concentrations of endometrial OTr were determined in Experiment 2 and 3 by the filter method (Sheldrick and Flint, 1985) . Tissues previously frozen at -80°C were removed from the freezer and maintained on ice while approximately 1.5 gm of tissue was removed. The remainder of the tissue was returned to the freezer. While frozen, approximately 1 gm of tissue was weighed and minced with a razor blade into fine pieces. The minced tissue was transferred to a 50 ml conical tube maintained on ice and rinsed with 5 ml homogenization buffer (25 mM Tris-HCl, 250 mM Sucrose, 1 mM EDTA ; pH 7.4 at 4°C) . Buffer was replaced with 10 ml homogenization buffer and endometrium homogenized for 5 sec with a polytron homogenizer (Brinkman Instruments, Westbury, NY) at a speed setting of 8. The homogenate was filtered through two layers of gauze into a chilled ground glass homogenizer, and further homogenized by 10 strokes with a ground glass homogenizer. The homogenate was

PAGE 119

104 decanted into a clean 50 ml conical tube, and the homogenizer rinsed with 2 ml of homogenization buffer which was added to the conical tube. Homogenates were centrifuged at 3,000 x g for 10 min at 4°C. The supernatant was transferred to ultracentrif uge tubes and centrifuged at 196,000 x g for 90 min. After centrif ugation the supernatant was discarded, the pellet rinsed twice with 25 mM Tris-HCl (pH 7.4 at 4°C) and 1 ml of this buffer then used to resuspend the membrane preparation. Membrane preparations were transferred to polypropylene tubes, a 200 fj.1 aliquot was removed for protein determination by bicinchoninic acid assay (Smith et al . , 1985), and the remainder was divided equally into two separate tubes for storage (-80°C) until assayed. The assay was validated by measuring the number of receptors in a pool of endometrium collected from six ewes in estrus. Receptor binding was maximal at 60 min at 25°C. At membrane protein concentrations of 25, 50 and 75 mg, there was a linear increase in receptor binding (Fig. 3.8). There was no detectable binding of FSH, LH, or roIFNi by the membrane preparations when added to the assay (data not shown) . The final assay was with membrane preparations (50 /ig/100 fil) incubated for 60 min at 25°C with 5 fmol to 16 pmol [tyrosyl-2 , 6-3H] -0T (New England Nuclear, Boston, MA; specific activity 37.1 Ci/mmol) in 100 jul 25mM TrisHC1 , 20

PAGE 120

Figure 3.8. Validation of the OT binding assay (filter method) . Percent specific binding of OT was measured in endometrial pools obtained from estrous ewes. A) temperature validation. Binding was determined at various times (0 to 240 min) following incubation at 20°C (diamonds) , 25°C (triangles) and 39°C (heptagon) . Binding was maximal at 6 0 min and 2 5°C. B) dissociation kinetics. Membrane protein (5 0/zg) was incubated with 3H-OT for 60 min at 25°C. At equilibrium, 1 ixg of radioinert OT was added and the samples assayed for radioactivity bound at the indicated times. C) protein validation. Specific binding was determined using increasing concentrations of membrane protein. There was a linear increase in OTr binding when membrane concentrations of 25, 50, and 7 5 mg/ml were examined.

PAGE 121

1400 r O 1200 o 1000 I>X o X CO Q z o CO 800 600 400 200 0 106 120 -i 100 80 CD m 60 SO 40 20 0 £ 1600 -i % 1400 D) 1200 c 1000 H T3 C CO o o 0 Q. 800 600 400 200 50 100 150 TIME (MIN) 200 250 1 2 3 5 10 15 Incubation Time (hours) 25 50 75 Protein/tube (mg) 100

PAGE 122

mM MnCl 2 , 0.2% BSA, and 0.02% NaN 3 . Non-specific binding was estimated in the presence of 800 pM radioinert oxytocin. Following incubation, tubes were placed on ice and 2 ml 2 5 mM Tris-HCl, 10 mM MnCl 2 , 0.1% BSA, and 0.01% NaN 3 was added. Receptor fractions were collected onto 0.2 ^m GVWP filters (Millipore Corp, Bedford, MA.). Filters were rinsed twice, placed into 5 ml plastic scintillation vials with 4 . 5 ml Scintiverse II (Fisher) , allowed to equilibrate 4 h and receptor-bound [ 3 H] -oxytocin quantified by liquid scintillation spectrometry. Number and K d of receptors were determined by Scatchard analysis using LIGAND (Munson and Rodbard, 19 80) . PEG assay Concentrations of endometrial OTr were determined in Experiment 4 and 5 by a PEG assay method modified from procedures reported by Sernia et al. (1991) and Lau et al. (1992) . Tissues frozen at -80°C were removed from the freezer and maintained on ice while approximately 1.5 gm tissue was removed. The remainder of the tissue was returned to the freezer. While frozen, 1 gm of tissue was weighed and minced with a razor blade into very fine pieces. The minced tissue was transferred to a 50 ml conical tube maintained on ice and rinsed with 5 ml homogenization buffer (50 mM TrisHCl, 250 mM sucrose, 4 mM EDTA; pH 7.4 at 4°C) . Buffer was

PAGE 123

108 replaced with 10 ml hornogenization buffer and endometrium was homogenized for 5 sec with a polytron homogenizer (Brinkman Instruments, Westbury, NY) at a speed setting of 8. The homogenate was filter through two layers of gauze into a chilled ground glass homogenizer, and further homogenized 10 strokes with the ground glass homogenizer and poured into a clean 5 0 ml conical tube. The ground glass homogenizer was rinsed with 2 ml hornogenization buffer and the rinse added to the conical tube. Homogenates were centrifuged at 3,000 x g for 10 min and 4°C. Supernates were transferred to ultracentrifuge tubes and centrifuged at 196,000 x g for 90 min. After centrif ugation the supernate was discarded, the pellet rinsed twice with 50 mM Tris-HCl (pH 7.4 at 25°C) and 1 ml of this buffer was replaced to resuspend the membrane preparation. Membrane preparations were transferred to polypropylene tubes, a 200 fil aliquot was removed for protein determination by bicinchoninic acid assay, and the remainder was divided equally into two tubes for storage (-80°C) until assayed. The assay was validated by measuring the number of receptors in a pool of endometrium collected from four ewes at estrus. Receptor binding was maximal at 2 h and 25°C in 20% PEG (Fig. 3.9). There was a linear increase in receptor binding when membrane protein concentrations of 25, 50, 100, and 200 jig/ml were examined (Fig. 3.10). There was no

PAGE 124

109 detectable binding of FSH, LH, or roIFNt to membrane preparations (data not shown) . The endometrial OTr within the membrane preparations were measured by radioreceptor assay. This assay consisted of 100 /il (200 fig) membrane preparation from a frozen aliquot, 100 /il (0.321 pM) [tyrosyl-2 , 6-3H] -oxytocin (New England Nuclear, Boston, MA; specific activity 39.0 Ci/mmol) and 100 jul assay buffer (50 mM Tris-HCl, 20 mM MnCl 2 , 0.3% BSA (w/v) and 0.2% NaN 3 (w/v); pH 7.6 at 25°C) containing 0, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, 20, and 80 pM radioinert oxytocin. The membrane preparations were incubated at 25 °C for 2 h. Bound oxytocin was precipitated by the addition of 100 /il 7-globulin (8 mg/ml; pig derived; Sigma Chemical Co., St Louis, MO) and 1 ml 20% (w/v) PEG followed by centrif ugation at 3,000 x g for 10 min. The precipitate was dissolved in 400 jul 50 mM Tris-HCl and precipitated a second time with 1 ml 20% PEG followed by centrifugation at 3,000 x g for 10 min. The precipitate was dissolved in 400 /il 50 mM Tris-HCl, to which 4 ml scintillation fluid (Scintiverse II; Fisher Scientific, Inc.) was added. After equilibrating for 1 h, samples were quantified by liquid scintillation spectrometry. Statistical Analysis Data were subjected to least squares ANOVA using the GLM procedure of SAS (SAS Institute, 1985). Data for IP

PAGE 125

Figure 3.9. Temperature validation of the polyethylene glycol (PEG) binding assay for the measurement of endometrial OTr. Percent specific binding of OT was measured in endometrial pools obtained from estrous ewes. Binding was determined every 3 0 min though 4.5 h and again at 24 h following incubation at 25°C (squares) and at 39°C (triangles) . Binding was maximal at 2 h and 25 °C.

PAGE 126

Ill TIME (hours)

PAGE 127

Figure 3.10. Protein validation of the polyethylene glycol (PEG) binding assay for the measurement of endometrial OTr. Percent specific binding (squares) , total binding (circles) and nonspecific binding (NSB; triangles) of OT was measured in endometrial pools obtained from estrous ewes. Binding was determined using increasing concentrations of membrane protein. There was a linear increase in OTr binding at membrane concentrations of 50, 100, and 200 jug/ml .

PAGE 128

113

PAGE 129

114 metabolism analysis were analyzed untransf ormed and logtransformed to alleviate statistical problems associated with heterogeneity of variance. Least squares means and standard errors were obtained using the least squares means statement of the GLM procedure. Ewes were nested within treatment for Experiments 2 through 5. All tests of hypothesis were performed using the appropriate error terms according to the expectation of the mean squares (Snedecor and Cochran, 1980). Data presented are least squares means ± SEM. Results Inositol Phosphate Metabolism In Experiment 1, there was no detectable IP metabolism induced by AVP stimulation of Day 16 ovine endometrium. As indicated in Fig. 3.11, tissue exposed to AVP (vials 9-12; see Table 1) did not respond with an elevation in IP metabolism over basal levels (vial 1) . Both the OTr antagonist and the AVP receptor antagonist cross-reacted with the OTr to prevent an increase in IP metabolism induced by oxytocin. Also, after treatment with either OTr antagonist or AVP receptor antagonist alone there was no detectable stimulation of IP metabolism over that of controls. There was no synergistic effect between oxytocin and AVP (vial 13) on IP metabolism when compared to the

PAGE 130

gure 3.11. IP metabolism by endometrium from estrous ewes in response to OT, AVP , and OTrA and AVPrA (Experiment 1) . Tissues from Day 0 ewes were pretreated with no antagonist (control; no A) , OTrA, AVPrA or both the OTrA and the AVPrA. Tissues were subsequently exposed to control medium alone, OT, AVP, or OT+AVP. Bars with the same letters are not different (P>0 . 05) .

PAGE 131

116 70 _ 65 111 Z> 60 CO CO 55 w 50 j| 45 Q_ Q 40 T O 35 & 30 _l < 25 IO 20 hQ_ 15 T 10 00 5 0 b b b T b 5 no A OTr A 222 AVPr A OTr + AVPr A b b b b b b ,. m 5 ° O H > < "0 3 o O H + > < TJ 5 O O H " 7 > > > < T) > < TJ 5 o O H > < TJ + + > < "0 CONTROL OT AVP OT+AVP

PAGE 132

117 level of IP metabolism induced by oxytocin alone (vial 5). These results indicate that Class I AVP receptors are either not present in Day 16 ovine endometrium or that they are present in inadequate numbers to permit AVP stimulation of IP metabolism. Results also indicate that there is no cross-reactivity between AVP and oxytocin to stimulate OTr to increase IP metabolism. Therefore, it appears that the increase in IP metabolism in these experiments was due to oxytocin stimulation of OTr. Oxytocin induced IP metabolism Oxytocin-induced IP metabolism in endometrial tissues of ewes treated with SP, the synthetic NT peptide of oIFNt, or oCSP is shown in Fig. 3.12. Intrauterine injection of NT alone blocked oxytocin-induced IP metabolism after oxytocin stimulation of endometrial tissues. Treatment of ewes with NT + oCSP also attenuated oxytocin-induced metabolism of IPwithin endometrial tissues, but the effect was not additive. Interestingly, the CT had no effect on oxytocininduced IP metabolism when given alone, but when given in conjunction with oCSP it negated the inhibitory effect of oCSP on oxytocin-induced IP metabolism. The results of oxytocin-induced IP metabolism for Experiment 3 are shown in Fig. 3.13. Inositol phosphate metabolism in endometrial tissues from ewes treated with SP was higher (P<0.05) than in tissues from ewes treated with

PAGE 133

Figure 3.12. In vitro oxytocin induced endometrial IP metabolism in ewes receiving intrauterine injections of SP, oCSP, synthetically produced peptides of oIFN r , NT and CT, or combinations of the proteins and peptides (Experiment 2) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.5 mg NT + 1.0 mg SP, 0.5 mg CT + 1.0 mg SP, 0.7 5 mg oCSP + 0.7 5 mg SP, 0.7 5 mg oCSP + 0.5 mg NT or 0.7 5 mg oCSP + 0.5 mg CT. Treatments began on Day 12 and continued through the morning of Day 16. Zero (0) Pep indicates ewes were treated with SP or oCSP only. Bars that share letters are not different (P>0.05).

PAGE 134

119 0T CT NT 0T CT NT SP oCSP

PAGE 135

120 NT (aa, 1-37), Pep 2 (aa, 34-64), Pep 3 (aa, 62-92), Pep 4 (aa 90-122) or roIFNi . Inositol phosphate metabolism within endometrial tissues collected from ewes treated with Pep 5 (aa, 119-150) was not different from that for SP-treated ewes. Furthermore, IP metabolism within endometrium was not different for treatment with Pep 5 vs. NT, Pep 2-4 or roIFNx . These results indicate that peptides, corresponding to amino acids 1-122, blocked oxytocin-induced IP metabolism in Day 16 ovine endometrium. There was, however, no effect of Pep 5, corresponding to amino acids 119-150, on IP metabolism. Endometrial Oxytocin Receptor Assay The results from Experiment 2 showing endometrial OTr concentrations expressed in fmol/mg protein are presented in Fig. 3.14. The main treatment effects of oCSP and SP are shown in Fig. 3.14A, while the peptide main effects and the treatment by peptide interactions are shown in Fig. 3.14B, and Fig. 3.14C, respectively. Regardless of peptide treatment, intrauterine injection of oCSP blocked the formation of endometrial OTr (Fig. 3 . 14A) . Intrauterine injection of NT alone or with oCSP almost completely attenuated development of endometrial OTr (Fig. 3.14C). Intrauterine injection of CT also attenuated the formation of endometrial OTr, but was not as effective as NT (Fig.

PAGE 136

121 3.14C). The CT treatment, when given with SP, was only half as effective in blocking OTr formation in endometrial tissues as NT. Results from Experiment 3 are presented in Fig. 3.15. Intrauterine injections of NT, Pep 2, Pep 3, or Pep 4 almost completely attenuated the formation of endometrial OTr, as did treatment with roIFNi . In several instances receptor numbers were undetectable in the assay. Treatment of ewes with Pep 5 blocked OTr formation compared to controls with about a 2fold difference in endometrial receptor number between that for SP-treated ewes and ewes treated with Pep 5. However, Pep 5 was not as effective in blocking receptor formation as were the other peptides or roIFNi . The endometrial OTr concentration results expressed as percent specific binding for Experiment 4 are shown in Fig. 3.16. Results are expressed as specific binding because receptor numbers were so low in the NT and roIFNi treated groups that receptor number determination by Scatchard analysis was not possible. However, specific binding was determined to be an acceptable response in the determination of differences between treatments. The OTr concentrations within endometrial tissues of ewes treated with NT and roIFNi were not different from each other, but they were almost 10 -times less than for ewes treated with SP.

PAGE 137

gure 3.13. Endometrial IP metabolism in ewes receiving intrauterine injections of SP, synthetically produced peptides of oIFN,, or roIFN, (Experiment 3) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.25 /ng roIFN,, or 0.5 mg each of synthetic peptide NT, 2, 3, 4, or 5. The total protein concentrations per treatment were balanced to 1.5 mg with SP. Treatments began on Day 12 and continued through the morning of Day 16. Bars with the same letters are not different (P>0 . 05) .

PAGE 138

123 5p \777A Pep 3 (aa 62-92) NT (aa 1 -37) Pep 4 (aa 90-1 22) \ 1 Pep 2 (aa 34-64) Egg Pep 5 (aa 11 9-1 50) 1=1 rolFNi a b

PAGE 139

Figure 3.14. Endometrial OTr concentrations in ewes receiving intrauterine injections of SP, oCSP, synthetically produced peptides to domains of oIFN,, or combinations of the proteins and peptides (Experiment 2) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0 . 5 mg NT + 1 . 0 mg SP , 0. mg CT + 1.0 mg SP, 0.7 5 mg oCSP + 0.7 5 mg SP, 0.7 5 mg oCSP + 0.5 mg NT or 0.75 mg oCSP + 0.5 mg CT per uterine horn. Treatments began on Day 11 and continued through the morning of Day 16. A, main effects of treatment. B, main effects of peptide treatment. C, effects of treatment by peptide interaction. Bars with the same letters are not different (P>0 . 05) .

PAGE 140

c o 0 A) o o O | 75 o £ £ E o "O c Hi 1000 900 800 700 600 500 400 300 200 100 0125 SP oCSP B) C) £ O mmm O c o O (0 500 =§ 400 " E 300 w 200 100 0 a -Eb OPep NT CT O 1000 -i a 2 900 0 C 800 c £ 700 ^ o o O ^_ 600 t 5? 500 O E ^ 400 H 5 E 300 E 200 § LU 0 NT CT SP c b b 0 NT CT OCSP

PAGE 141

Figure 3.15. Endometrial OTr concentrations in ewes receiving intrauterine injections of SP, synthetic peptides to specific domains of oIFN,, or roIFN, (Experiment 3) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 25 jug roIFN,, or 0.5 mg each of synthetic peptide NT, 2, 3, 4, or 5 per uterine horn. The total protein concentrations per treatment were balanced to 1.5 mg with SP. Treatments began on Day 12 and continued through the morning of Day 16. Bars with the same letters are not different (P>0 . 05) .

PAGE 142

127 1200 r C o H o c o o OJ (0 o 5 E E o "O C LU 1100 1000 900 800 700 600 500 400 300 200 100 b b — SP NT 2 3 4 5 rolFNx PEPTIDE FRAGMENTS

PAGE 143

Figure 3.16. Endometrial OTr concentrations in ewes receiving intrauterine injections of SP, NT, or roIFN r (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 5 0 fig roIFN, or 0.5 mg NT per uterine horn. The total protein concentrations per treatment were balanced to 1.5 mg with SP. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Days 13 and 15. Bars with the same letters are not different (P>0.05).

PAGE 144

129 o M 2 C O) o c si o .E O -O »o 12 h 10 o 15 o o a (0 o T3 C LU 8 « 6 4 2 0 a SP NT rolFNx

PAGE 145

130 Endometrial OTr concentrations in tissues from ewes collected on Days 16, 17 or 18 after intrauterine injections with either SP or NT (Experiment 5) are shown in Fig. 3.17A, B and C. Treatment with NT attenuated formation of OTr in ewes on all days examined, compared to ewes treated with SP. Receptor concentrations were lowest in ewes treated with NT and collected on Day 16. There was a 4-fold increase in OTr numbers from Day 16 to Day 17 in ewes treated with NT; however, this number went down slightly from Day 17 to Day 18. In ewes treated with SP there was a steady increase in OTr concentrations. While values were not different between Day 16 and Day 17, the increase between Day 16 and Day 18 was different. The OTr concentrations in tissues collected from ewes treated with SP and collected on Day 17 were intermediate to values on the other two days. Discussion Ovine IFNt , the conceptus secreted maternal pregnancy recognition factor (Godkin et al . , 1984b; Valletet al . , 1988), is assumed to bind to high affinity (Godkin et al., 1984a) Type I I FN receptors (Stewert et al., 1987) which are distributed throughout endometrial tissues of the ewe (Knickerbocker and Nisewinder, 1989) . Trophoblast IFNs are biologically similar to other Type I IFNs and like the other IFNxs, oIFNx displays both antiviral and antiproliferative properties (Pontzer et al., 1988).

PAGE 146

Figure 3.17. Endometrial OTr measurements, expressed as percent specific binding, in ewes receiving intrauterine injections of SP or NT (Experiment 5) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP or 0.5 mg NT + 1.0 mg SP per uterine horn. Treatments began on Day 11 and continued through the morning of Days 16, 17 or 18 when tissues were collected. A, main effects of treatment. B, main effects of day. C, effects of treatment by day. Bars with the same letters are not different (P>0.05).

PAGE 147

c o C D) 0 C o .E O -Q Oo — o .5 o. i(/) o C LU A) 14 r 12 10 8 6 4 2 0132 SP NT c o MM 2 +-* C 0 O c o o O MM +-» 0 E o T3 C UJ 14 n — 12 U) c =5 10 c O 8 o 6H & 4 — 2 0 B) C) a b 16 17 18 Day of Estrus Cycle ion 14 5 12 c 0 c one indi 10 O J2 8 o immm MO o 6 0 75 mmm a i**-» (0 4 0 s« E o 2 TJ C LU 0 16 17 18 16 17 18 SP NT

PAGE 148

133 Synthetic NT peptides and CT peptides of oIFNt (Pontzer et al . , 1991) as well as two internal peptides (aa 62-92 and aa 119-150) inhibit oIFNt receptor binding and antiviral activity in a dose dependent manner (Pontzer et al., 1994) indicating specific competition between those specific peptides and oIFNt. The NT has no effect on antiviral activity of IFNct. Collectively, these findings indicate that the NT acts through a novel domain of the oIFNt receptor while the other peptides may act through another more common domain of the Type I receptor. This may explain the unique actions of oIFNt . Ovine IFNt and other Type I IFNs increase endometrial protein production dramatically (Gross et al., 1988b; Sharif et al . , 1989; Ashworth and Bazer, 1989) . Our current working hypothesis for pregnancy recognition, supported by recent work by Spencer et al . (1995a, 1995b, 1996) , is that in the pregnant ewe, oIFNt acts to prevent luteolysis, by preventing transcription of the Er gene which in turn prevents the formation of endometrial OTr on the luminal and superficial glandular epithelium. The mechanism by which oIFNt alters OTr upregulation and reduces endometrial responsiveness to oxytocin is not known, but is most likely due to negativeacting transcriptional factors such as interferon regulatory factor-2. In the cyclic ewe, long term exposure to progesterone down-regulates Pr allowing expression of Er and

PAGE 149

134 OTr (McCracken et al . , 1984;) especially in the luminal endometrium and superficial glandular epithelium (Spencer and Bazer, 1995). In the pregnant ewe, progesterone downregulates endometrial Pr in luminal endometrial and superficial glandular epithelium, just as it does in the cyclic ewe, but unlike in the cyclic ewe, Er and OTr are undetectable in these tissues (Spencer et al . , 1995). Pr and Er are detectable only in low levels in the stroma and deep glandular epithelium in pregnant ewes. Therefore, it is believed that oIFNt actions are inhibitory to Er gene expression. By this action, OTr formation is attenuated and the positive feedback loop of ovarian oxytocin and pulsatile uterine PGF 2a is blocked in the pregnant ewe. Without the effects of pulsatile PGF 2a to regress the CL, progesterone production is maintained, and it can act on Pr-positive stroma and deep glandular epithelium to continue the suppression Er and OTr genes. Oxytocin stimulates endometrial production of PGF 2a (Fairclough et al . , 1984; Vallet et al . , 1988, Chapter 4) and IP metabolism in oxytocinstimulated endometrium of cyclic and SP-treated ewes (Mirando et al., 1990). These results show that oxytocin stimulates uterine secretion of PGF 2a during luteolysis via activation of the IP/diacylglycerol signal transduction system. Oxytocin's specific action through the IP/diacylglycerol signal transduction system is further indicated by results of the

PAGE 150

135 present study with oxytocin, AVP and their antagonists which indicate that IP metabolism is due to effects of oxytocin acting via OTr and not through the AVP receptor in the ewe. IP metabolism, however, is an indirect measure of OTr in endometrial tissues. While it is not currently known how oIFNt blocks expression of Er and OTr genes, Er upregulation is apparently required for OTr gene expression in endometrial luminal epithelium (McCracken et al . , 1980; Vallet et al . , 1990; Silvia et al., 1991; Beard et al . , 1994; Spencer et al . , 1995a, 1995b, 1996). Estrogen is necessary for development of the luteolytic mechanism and, in the absence of estrogen, oxytocin is unable to initiate luteolytic PGF2a release (Zhang et al., 1992). Furthermore, treatment of cyclic ewes with estrogen up-regulates OTr expression and initiates premature luteolysis (Hixon et al., 1987). Intrauterine injections of oCSP (Mirando et al . , 1993; Chapters 3 and 5) and roIFNi (Spencer et al., 1995; 1996; Chapters 3 and 5) blocks estradiol-induced Er and OTr expression. Results of the present study indicate that NT and oIFNt are equally affective in attenuating formation of endometrial OTr. The ability of NT to effectively block upregulation of endometrial OTr continued for up to 48 h after the last intrauterine injection was administered. However, treatment with CT of oIFNt did not attenuate the increase in endometrial OTr. These findings support those of Pontzer

PAGE 151

136 et al. (1994) which indicated that the NT of oIFNt has a unique interaction with the Type I oIFNt IFN receptor in endometrium.

PAGE 152

CHAPTER 4 THE EFFECTS OF RECOMBINANT OVINE INTERFERON TAU AND SYNTHETIC PEPTIDE DOMAINS OF INTERFERON TAU ON OXYTOCINSTIMULATED PROSTAGLANDINF METABOLITE CONCENTRATIONS IN PLASMA OF EWES. Introduction Prostaglandin F 2a produced by endometrial luminal epithelium causes luteal regression in ewes (McCracken et al . , 1972). Luteal regression is achieved by the high amplitude, short duration pulsatile secretion of PGF 2a (Thornburn et al., 1983; Barcikowski et al . , 1974; Flint and Sheldrick, 1983; Zarco et al . , 1984, 1988) initiated by the pulsatile release of oxytocin (Flint et al . , 1986). Together uterine PGF 2a and ovarian oxytocin form a positive loop, with 5 0% (Walker et al., 1997) to 97% of all episodes of uterine PGF 2a secretion being coincidental with pulses of oxytocin in the blood (Hooper et al., 1987). Estrogen enhances endometrial secretion of PGF 2a in response to oxytocin (McCracken et al., 1984). Endometrial OTr are increased by estrogen (Hixon and Flint, 1987) and although, chronic progesterone treatment will allow OTr receptor formation after Pr are downregulated, endometrial OTr formation is stimulated 137

PAGE 153

138 optimally by a regime of progesterone followed by estrogen (Vallet et al., 1990, see Spencer et al . , 1996). During early pregnancy the pulsatile pattern of PGF 2a secretion is attenuated (Thornburn et al., 1973; Barcikowski et al., 1974; Moore et al., 1982; Hooper et al., Zarco et al . , 1988a) by disruption of the oxytocin/PGF^ positive feedback loop. The normal response of increased PGF 2a secretion in response to exogenous estrogen (Fincher et al . , 1986) and oxytocin (Fairclough et al . , 1984) also is blocked during early pregnancy. The attenuation of the loop in the pregnant ewe is due primarily to a blockage of endometrial OTr formation seen in the cyclic ewe just prior to luteolysis (McCracken et al., 1984; Sheldrick and Flint, 1985) . It is this reduction in endometrial Er and OTr which is responsible for the decreased endometrial responsiveness to oxytocin during early pregnancy in ewes (McCracken et al., 1984; Fairclough et al . , 1984; Mirando et al . , 1990a, 1990b) Ovine IFNt is the primary protein component of oCSP and the antiluteolytic protein secreted by the conceptus (Godkin et al., 1992; Vallet et al., 1988; see Bazer et al . , 1991; Ott et al . , 1993a). Intrauterine injection of oCSP, highly purified oIFNt or roIFNi are effective in blocking the luteolytic mechanism (Vallet et al . , 1989b). Therefore, these studies were undertaken to determine; (1) if roIFNx is as effective as native olFNi in blocking oxytocin-stimulated

PAGE 154

PGF 2a secretion (as determined by measuring the metabolite of PGF 2a , PGFM) and (2) if peptides representing major domains of oIFNi elicited the same response as roIFNi . Materials and Methods Animals Ewes of primarily Rambouillet breeding were checked fo estrous behavior daily at 07:30 AM for 20 min with vasectomized males. Ewes which had exhibited at least two normal estrous cycles (16 to 17 days in length) were assigned to experimental groups. Ewes in this study were housed at the Sheep Research Center, Texas A&M University. Protein And Peptide Preparation Recombinant ovine interferon tau preparation Recombinant ovine interferon tau was produced and prepared as described in Chapter 3. Serum protein preparation Serum proteins were prepared as described in Chapter 3 Synthetic peptide production A synthetic peptide corresponding to the NT portion of roINFi was produced as described in Chapter 3.

PAGE 155

140 Experimental Design Experimental design for Experiment 4 is discussed in Chapter 3 . Prostaqlandin-F metabolite assay Plasma, processed from jugular vein blood samples obtained from ewes after they had been challenged with oxytocin in Experiment 4, was assayed for PGFM. The PGFM assay had been validated previously for sheep by Fincher et al . (1986) and cross-reactivities of the antibody with other prostaglandins had been determined (Guilbault et al . , 1984; Knickerbocker et al., 1986). Plasma used for preparation of all standards was obtained from banaminetreated sheep (100 mg/injection, two injections 12 h apart) to inhibit prostaglandin synthesis. Each standard was measured in triplicate and each unknown in duplicate. The standard curve measured PGFM at 50, 25, 10, 0.5, 0.25, 0.10, 0.05, 0.025, 0.01, and 0.005 ng/ml . The PGFM was measured by incubating (15 min at 25°C) 200 (Ltl plasma in 100 ,Lil Tris-HCl (0.05 M; pH 7.5 at 4°C) with 100 jil 0.5% human 7-globulin. After incubation, 100 jul PGFM anti-serum (goat anti-PGFM 23; 1/6,000; Supplied by Dr. Ken Kirton, Upjohn Co., Kalamazoo MI) was added and allowed to incubate 30 min at 25°C. Radiolabled PGFM (100 ^1 3 HPGFM ; -18,000 dpm) was added, incubated 1 h at 25°C, and then overnight at 4°C. Bound

PAGE 156

141 radiolabled PGFM was precipitated with 750 jul PEG 8000 (40 % w/v; 4°C) and centrifuged 30 min at 3,000 x g at 4°C. The supernate was discarded, the pellet redissolved in 750 /nl Tris-HCl (0.05 M pH 7.5 at 4°C) , bound PGFM reprecipitated with 750 /il PEG, and the sample centrifuged for 30 min at 3,000 x g at 4°C. The supernate was again discarded and the pellet resuspended in 1 ml Tris-HCl. The solution was transferred to plastic scintillation vials and 4 ml scintillation cocktail (Bio-HP) added. Scintillation vials were allowed to equilibrate 1 h and counted. The results were expressed in pg/ml PGFM in plasma. Statistical Analysis Data were subjected to least squares ANOVA using the GLM procedure of SAS (SAS Institute, 1985) . Tests of homogeneity of regression were performed to detect differences in patterns in PGFM secretion over the days examined for the treatment groups. Least squares means and standard errors were obtained using the least squares means statement of the GLM procedure. Ewes were nested within treatment and all tests of hypothesis were performed using the appropriate error terms according to the expectation of the mean squares (Snedecor and Cochran, 1980) .

PAGE 157

Results Results of individual ewe PGFM responses are shown in Fig. 4.1 through 4.3. Fig. 4.1 shows the PGFM response after oxytocin stimulation in individual ewes treated with OT on Days 13 and 15. Two SP-treated ewes responded to oxytocin challenge on both Day 13 and Day 15. Figures 4.2 and 4.3 indicate responses of individual ewes treated with NT and roIFNi , respectively. Ewes that received intrauterine injections of NT or roIFNi had small or undetectable increases in PGFM on Day 13 in response to the oxytocin challenge. On Day 15 two of the roIFNi -treated ewes also were unresponsive to oxytocin challenge. Mean plasma PGFM concentrations after oxytocin challenge for all treatments are shown in Fig. 4.4. Plasma PGFM concentrations were lower (P<0.02) in ewes treated with roIFNi and NT compared to SP on both Day 13 and Day 15 (Fig. 4.4) . The mean PGFM response to oxytocin was lower on Day 13 than Day 15 for all treatments (Fig. 4.5). The mean PGFM responses ( treatment *day) for all treatments are shown in Fig. 4.6. Analysis of regression curves ( treatment*day* time ; Fig. 4.7) indicated that attenuation of PGFM responsiveness on Day 13 or Day 15 by NT-treated ewes was not different from that of roIFNi -treated ewes. However, ewes which received either NT or roIFNi had lower PGFM responses than ewes treated with SP.

PAGE 158

Figure 4.1. Plasma PGFM of individual ewes which received intrauterine injections of SP (Experiment 4). Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP per uterine horn. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Days 13 and 15 at time zero. Plasma samples were obtained 10 min prior to OT challenge, at time zero, and at 10, 20, 30, 45 and 60 min post-challenge. PGFM profiles for ewe 15 (E15) ; ewe 35 (E35) ; and ewe 39 (E39) on Days 13 (circles) and Day 15 (squares) are shown in panels A, B and C, respectively.

PAGE 159

A) 144 U) CL (D Q_ (0 £ en Ql 1600 -| 1400 1200 1000 800 600 400 200 0 • E15 D13 E15 D15 \ / \ i — i — i — i — i — i 100 102030405060 TIME (min) O) q. a. £ Q. 1600 1400 1200 1000 800 600 400 200 0 E39 D13 E39 D15 1 — I — I — I — l — l 10 0 102030405060 TIME (min) C) E35D13 E35D15 10 0 1020 3040 50 60 TIME (min)

PAGE 160

gure 4.2. Plasma PGFM in individual ewes receiving intrauterine injections of NT (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 0.5 mg NT. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Days 13 and 15 at time zero. Plasma samples were obtained 10 min prior to OT challenge, at time zero, and at 10, 20, 30, 45 and 60 min post-challenge. PGFM profiles ewe 88 (E88) ; ewe 111 (Elll) ; ewe 114 (E114) on Days 13 (circles) and Day 15 (squares) are presented in panels A, B, and C, respectively.

PAGE 161

a Q. CO E Q_ 1600 1400 1200 1000 800 600 400 200 0 A) 146 E88D13 E88D15 » ••HI i — i — i — i 10 0 10 20 30 40 50 60 TIME (min) a LL O CL E V) Q. 1600 I 1400 1200 1000 800 600 400 200 0 B) E111 D13 E111 D15 I I I I I 10 0 10 20 30 40 50 60 TIME (min) Q. Ll_ O Q_ CD E TO 1600 -i 1400 1200 1000 800 600 400 200 0 C) E114 D13 E114D15 -10 0 i — i — r 10 20 30 40 50 60 TIME (min)

PAGE 162

Figure 4.3. Plasma PGFM in individual ewes receiving intrauterine injections of roIFN, (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 50 fj,g roIFN, per uterine horn. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Day 13 and Day 15 at time zero. Plasma samples were obtained 10 min prior to OT challenge, at time zero, and at 10, 20, 30, 45 and 60 min postchallenge. PGFM profiles ewe 11 (Ell) ; ewe 37 (E37) ; ewe 85 (E85) ; ewe 96 (E96) on Day 13 (circles) and Day 15 (squares) are presented in panels A, B, C, and D, respectively .

PAGE 163

148 a LL o Q_ «j E J2 Q. 1600 -i A ) 1400 1200 1000 800 600 400 200 E11 D13 E11 D15 \ \ 1 1 1 1 1 1 10 0 10 20 30 40 50 60 TIME (min) O Cl E V) Q_ 1600 1400 1200 1000 800 600 400 200 0 E37D13 E37D15 1 I I I 1 1 10 0 10 20 30 40 50 60 TIME (min) D) Q. O Q_ CD E c/) Q. 1600 1400 1200 1000 800 600 400 200 0 C) E85D13 IE85D15 10 0 10 20 30 40 50 60 1*1600 -. a)1400 ^1200 O CL 03 E (/) 03 Q_ 1000 800 600 400 200 0 D) E96D13 E96D15 1 1 1 T 1 1 1 -10 0 10 20 30 40 50 60 TIME (min) TIME (min)

PAGE 164

Figure 4.4. Mean plasma PGFM in ewes receiving intrauterine injections of SP, NT, or roIFN, (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.5 mg NT, or 50 \iq roIFN r per uterine horn. The total protein concentrations per treatment were balanced to 1.5 mg with SP. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Day 13 and Day 15 at time zero. Plasma samples were obtained 10 min prior to OT challenge, at time zero, and at 10, 20, 30, 45 and 60 min post-challenge. These values represent the mean main effects of treatment. Bars with the same letter are not different (P>0.05).

PAGE 165

149 Discussion In cyclic ewes, pulsatile release of PGF 2a from the endometrium between Days 14 and 15 of the estrous cycle causes luteolysis and ewes return to estrus. Establishment of pregnancy in the ewe requires abrogation of uterine release of luteolytic pulses PGF 2a to prevent luteolysis, maintain CL function and luteal cell production of progesterone (Roberts et al . , 1990; Bazer, 1991). During the period of pregnancy recognition in the ewe (Days 9-15; Moor and Rowson, 1966a, 1966b, 1966c; Farin et al . , 1990), the conceptus secretes oIFNt (the pregnancy recognition factor in ewes) . Ovine IFNt inhibits development of the endometrial luteolytic mechanism and prevents pulsatile release of endometrial PGF 2a (Fintcher et al., 1986; Vallet et al., 1988; Ott et al . , 1992; Beard et al . , 1994; Beard and Lamming, 1994; Stevenson et al., 1994; Spencer et al . , 1996; Spencer and Bazer, 1996) . The cyclic increase of endometrial OTr on Day 14 in the ewe is blocked by intrauterine injection of oIFNt (Mirando et al . , 1993; Spencer et al . , 1995, 1996; Spencer and Bazer, 1996). Progesterone, estrogen, and oxytocin, acting through their individual receptors mediate production of the pulsatile pattern of PGF 2a release by the endometrium (McCracken et al., 1980, 1984; Silvia et al . , 1991; Wathes and Lamming,

PAGE 166

500 -i a SP (6.0 mg/day) 1=1 NT (2.0mg/day) dD rolFNx (200^g/day) 400 E 300 O) Q. (i) 200 100 SP NT rolFNx

PAGE 167

gure 4.5. Mean plasma PGFM in ewes receiving intrauterine injections of SP, NT, or roIFN, (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.5 mg NT, or 5 0 fig roIFN f per uterine horn. The total protein concentrations per treatment were balanced to 1.5 mg with SP. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Day 13 and Day 15 at time zero. Plasma samples were obtained 10 min prior to OT challenge, at time zero, and at 10, 20, 30, 45 and 60 min post-challenge. These values represent the mean main effects of day. Bars with the same letter are not different (P>0.05).

PAGE 169

Figure 4.6. Mean plasma PGFM in ewes receiving intrauterine injections of SP, NT, or roIFN r (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.5 mg NT, or 50 /ig roIFN, per uterine horn. The total protein concentration per treatment were balanced to 1.5 mg with SP. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Day 13 and Day 15 at time zero. Plasma samples were obtained 10 min prior to OT challenge, at time zero, and at 10, 20, 30, 45 and 60 min post-challenge. These values represent the mean effects of the interaction of treatment by day. Bars with the same letter are not different (P>0.05).

PAGE 170

EgggS SP (6.0 mg/day) l=l NT (2.0 mg/day) nTTTI rolFNi (200ng/day)

PAGE 171

Figure 4.7. Mean plasma PGFM in ewes receiving intrauterine injections of SP, NT, or roIFN, (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP (circles), 0.5 mg NT (squares) , or 50 jig roIFN, (triangles) per uterine horn. The total protein concentration per treatment were balanced to 1.5 mg with SP. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Day 13 and Day 15 at time zero. Plasma samples were obtained 10 min prior to OT challenge, at time zero, and at 10, 20, 30, 45 and 60 min postchallenge. These values represent the interaction of treatment by day by time interactions for Day 13 and Day 15 are represented in panels A and B, respectively .

PAGE 172

A) 157 D) Q. o CL 1200 n 1100 1000 900 800 700 600 500 400 300 200 100 -i 0 SP Day 13 NT Day 13 rolFNx Day 13 10 0 10 20 30 40 50 60 Time (min) a CL 1200 -. 1100 1000 900 800 700 600 500 400 300 200 100 0 B) i / / SP Day 15 NT Day 15 rolFNx Day 15 -10 0 10 20 30 40 50 60 Time (min)

PAGE 173

158 1995, Spencer et al . , 1996). In cyclic ewes, development of the endometrial luteolytic mechanism involves progesteroneinduced down-regulation of Pr, which in turn allows upregulation of Er and OTr on the endometrium. In pregnant ewes, Pr expression by endometrial epithelium is also downregulated; however, up-regulation of Er and OTr is blocked by an as yet unknown effect of oIFNx (Wathes and Hamon, 1993; Spencer et al . , 1995a; Spencer and Bazer, 1996). Through this mechanism the oxytocin-induced pulsatile release of PGF 2a by the endometrium is blocked. Results from the present study indicate that the NT peptide of oIFNt is as effective as roIFNt in attenuating the oxytocininduced PGFM response in ewes.

PAGE 174

CHAPTER 5 THE EFFECTS OF OVINE CONCEPTUS SECRETORY PROTEINS, RECOMBINANT OVINE INTERFERON TAU AND SYNTHETIC PEPTIDES CORRESPONDING TO PORTIONS OF INTERFERON TAU ON ENDOMETRIAL CONCENTRATIONS OF ESTROGEN AND PROGESTERONE RECEPTOR PROTEIN AND mRNA Introduction Establishment of pregnancy in ewes requires that the conceptus, by Day 12 of pregnancy (Moor and Rowson, 1966a) sends a message (oIFNt; Godkin et al . , 1982; Vallet et al . , 1988; see Spencer et al . , 1996) to prevent luteolysis. Luteolysis is induced in ruminants by pulsatile secretion of PGF 2a by endometrial tissues (Hooper et al . , 1986; McCracken et al., 1981, 1991). The pulsatile secretion of PGF 2a is initiated by secretion of oxytocin from the posterior pituitary and is escalated by oxytocin of ovarian origin (Flint et al . , 1990). Oxytocin from the ovary and PGF 2a from the uterus act together in a positive feedback loop to generate the luteolytic pulses required for luteolysis (Flint and Sheldrick. , 1986; Hooper et al . , 1986; see Silvia et al., 1991; McCracken et al . , 1991). However, the ability of endometrium to secrete PGF 2a in response to oxytocin does not develop until Day 13 to 14 of the cycle (Roberts et al . , 1975; Roberts and McCracken, 1976; Fairclough et al., 1984; 159

PAGE 175

160 Silvia et al . , 1991). Changes in endometrial responsiveness to oxytocin are due to differences in OTr concentrations within the uterus during the estrous cycle (Sheldrick and Flint, 1985; Beard and Lamming, 1994; Spencer et al . , 1995a; Wathes and Lamming, 1995) . During early pregnancy the pulsatile pattern of PGF 2a secretion is attenuated (Thornburn et al., 1973; Barcikowski et al., 191 A; Moore et al . , 1982; Hooper et al . , Zarco et al., 1988a) by the disruption of the oxytocin/PGF 2a positive feedback loop. Attenuation of the loop in the pregnant ewe is due to a block in endometrial expression of Er and the subsequent formation of OTr seen in the cyclic ewe just prior to luteolysis (Fairclough et al . , 1984; McCracken et al., 1984; Sheldrick and Flint, 1985; Mirando et al., 1990a, 19 9 0b; Beard and Lamming, 1994; Wathes and Lamming, 1995; see Spencer et al . , 1996) Ovine IFNt acts through its transduction signal to prevent the cyclic increase in endometrial Er and OTr. Within cyclic ewes Er is abundant in the endometrial luminal epithelium just before and after estrus. Receptor numbers fall dramatically from Day 3, are low from Days 10 to 14, increase dramatically beginning on Day 14, and peak at estrus (Koligian and Stormshak, 1976; Miller et al., 1977; Zelinski et al., 1980; Cherny et al . , 1991; Ott et al . , 1993b). Cherny et al . (1991) reported that endometrial Er expression under steroidogenic control is not homogenous

PAGE 176

161 throughout endometrial tissue. However, throughout these tissues, estrogen has been reported to be stimulatory to it's own receptor formation (Anderson et al . , 1975; Bhaloo and Katzenellenbogen, 1977; Zelinski et al . , 1980; Cherny et al., 1991). Progesterone, on the other hand, is inhibitory to Er formation in several species (Brenner et al . , 1974; Hsueh et al . , 1975, 1976; Tseng and Gurpide, 1975; West et al . , 1976; Bhakoo and Katzenellenbogen, 1977) including sheep (Koligian and Stormshak, 1977b; Zelinski et al., 1980; Cherny et al . , 1991). Control of expression of Er in the cyclic ewe may involve several factors (Cherny et al . , 1991) but in the pregnant ewe control of the Er is controlled by oIFNt (Spencer et al., 1995a, 1995b; Spencer and Bazer, 1996) . Progesterone in the cyclic animal is inhibitory to it's own endometrial receptor, through down-regulation mechanisms (Milgrom et al . , 1973; Leavitt et al . , 1974; Vu Hai et al . , 1977). In the cyclic, ewe progesterone ' s downregulation of its own receptor in luminal epithelial and superficial glandular endometrium initiates removal of the proposed progesterone block (McCracken et al., 1984), allowing Er upregulation, and formation of the OTr, which when bound by oxytocin, initiates pulsatile PGF 2a production and luteolysis (Wathes and Hamon, 1993; Beard and Lamming, 1994; Spencer and Bazer, 1995, 1996; Wathes and Lamming, 1995; Spencer et al . , 1995a, 1995b, 1996, 1996). In pregnant

PAGE 177

162 ewes, the Pr is also downregulated by the extended period of elevated progesterone during diestrus (Ogle et al . , 1989, 1990; Ott et al . , 1993b; Mirando et al . , 1993; Wathes and Lamming, 1993; Spencer and Bazer, 1995) while at the same time the Er gene is prevented from the cyclic up-regulation of the Er, by removal of the progesterone-block, through the actions of oIFNx (Wathes and Hamon, 1993; Spencer et al . , 1995a, 1995b; Spencer and Bazer, 1996) . This in turn prevents the formation of endometrial OTr and, therefore, release of luteolytic pulses of PGF 2a in response to oxytocin by endometrium (see Wathes and Lamming, 1995; Spencer et al . , 1996) . It is not known how oIFNt blocks Er up-regulation but it is believed that oIFNt signals the activation of transcription suppressors to block Er formation (see Spencer et al., 1996). Also unknown, is whether the entire oIFNt molecule is required to elicit this resopnce, or if a portion of oIFNt is able to induce the same response as the whole protein. This study was undertaken to determine: (1) if roIFNx is as effective as native oIFNt in blocking endometrial Er and Pr expression; and (2) if oIFNt, NT and CT are equally effective in blocking expression of Er and Pr. In particular these experiments were to determine what effect treatment of cyclic ewes, with various synthetically produced peptides corresponding to overlapping segments of oIFNt , had on endometrial Er mRNA , Er and Pr concentration.

PAGE 178

163 The NT and CT peptides were first examined (Experiment 2; Er mRNA concentration only was examined for this experiment) due to the fact that they were the most extensively examined of the peptides (Pontzer et al . , 1990, 1994). It has been proposed that the NT possesses those properties which makes oIFNt different from other IFNas, and that CT is the portion common to the IFNas. Experiment 3 examined the effect of the remaining peptides (2-5) on endometrial concentration of Er mRNA. Experiments 4, also examined the effect of NT treatment on endometrial concentration of Er mRNA, Er and Pr, but this experiment was specifically designed to determine what effect oxytocin challenge (in vivo) on Days 13 and 15 would have with regards to the main treatment of NT or roIFNi. The final experiment, Experiment 5, was designed to determine what effect NT has on endometrial concentration of Er mRNA, Er and Pr through Day 18, which is two days longer than oIFNt had been examined. This was to determine if NT has the ability to hold OTr concentration at a low level for an extended period of time. Materials and Methods Animals Ewes of primarily Rambouillet breeding were checked daily at 07:30 AM for 20 min with vasectomized males of St. Croix or mixed Rambouillet breeding. Ewes which had

PAGE 179

164 exhibited at least two normal estrous cycles (16 to 17 days in length) were assigned to experimental groups. Ewes for Experiments 1, 2, and 3 were housed at the Physiology Unit, Department of Animal Science, University of Florida. Ewes for Experiments 4 and 5 were housed at the Sheep Research Center, Texas A&M University. Protein And Peptide Preparation Ovine conceptus secretory protein preparation Ovine conceptus secretory proteins were produced and prepared as described in Chapter 3. Recombinant ovine interferon tau preparation Recombinant ovine interferon tau was produced and prepared as described in Chapter 3. Serum protein preparation Serum protein was produced and prepared as described in Chapter 3 . Synthetic peptide production Synthetic peptides corresponding to the NT and CT of oIFNx were produced and prepared as described in Chapter 3. Experimental Design See Chapter 3 for the experimental designs of Experiments 2, 3, 4, and 5.

PAGE 180

165 Estrogen Receptor mRNA Ribonucleic acid isolation Endometrial tissues for mRNA analysis were stored in plastic bags at -80°C until used. Tissues storage time was approximately 1.5 years for Experiment 2, 6 months for Experiment 3 and 2 months for Experiments 4 and 5 . During this period of time homologous Er probes were developed for use. Ribonucleic acid was isolated by means of a single step guanididium thiocyanate-phenol-chlorof orm extraction method modified from the method described by Puissant and Houdebire (1990). Briefly, 1 gm of tissue was homogenized in 10 ml 4 M guanidine thiocyanate, 2 5 mM sodium citrate, sarkosyl 0.5% (w/v) , pH 7 . 0 , 0.1 M 2 -mercaptoethanol at 4°C within a 50 ml conical tube. One ml of 2 M sodium acetate (pH 4.0), 10 ml of water-saturated phenol was added sequentially and gently mixed by inverting the tube after each addition. Two milliters of chloroform: isoamyl alcohol (49:1) was added and vortexed to mix. Tubes were centrifuged at 3,000 x g for 15 min. The upper phase was collected, and the RNA allowed to precipitate overnight at 20°C in 10 ml of isopropanol. Total RNA was pelletized by centrifugation at 3,000 g for 10 min, the supernate discarded, and the tubes inverted to drain. The pellet was resuspended in 2 ml LiCl (4 M) and total RNA repelletized by centrifugation at 3,000 x g for 15 min. The supernate was discarded and the tube inverted to drain. The pellet was

PAGE 181

166 resuspended in 2 ml 10 mM Tris-HCl (pH 7.5), ImM EDTA, 0.5% SDS (w/v) , and transferred to a 15 ml conical tube. Two milliters of chloroform was added, vortexed to mix and centrifuged at 3,000 x g for 10 min. The upper phase was collected, transferred to a sterile tube and 0.2 M sodium acetate added. Two milliters of isopropanol was added, and RNA allowed to precipitate overnight at -80°C. Total RNA was pelletized by centrif ugation at 3,000 x g for 30 min. The supernate was discarded and the tubes with pelletized RNA inverted to drain. The pellet was washed with 1 ml 7 0% ethanol and recentrif uged at 3,000 x g for 10 min after which the tubes were inverted and allowed to dry for 15 min. Once dried, the pellet was suspended in 200 /txl 10 mM TrisHCl (pH 7.5), 1 mM EDTA and transferred to a 1.5 microcentrifuge tube. A 5 /il aliquot was taken for quantification by spectrophotometric absorbance measurement at 260 nm. The remainder was stored in aliquots, at -80°C for northern blot and slot blot analyses. Northern blot procedure A 40 ill aliquot of each RNA sample was thawed and f reeze-dried. The RNA was denatured in 15 /xl 24 mM HEPES , 6 mM sodium acetate, 1.2 mM EDTA (pH 7.0), 50% formamide (v/v) , and 2.2 M formaldehyde at 65°C for 15 min then rapidly chilled. Loading buffer (5 pi; 20 mM phosphate buffer, 50% (v/v) glycerol, 0.05% (w/v) bromophenol blue) was added to samples just prior to gel loading. Samples

PAGE 182

167 were electrophoresed through a 1.5% agarose, 2.2 M formaldehyde gel (12 h at 70 volts). Location of 18s and 28s ribosomal RNA bands as well as RNA integrity were determined by staining the gel in running buffer with 5 jil ethidium bromide (10 mg/ml) . For northern bolt analysis, the RNA was transferred to a 0.4 5 jm. nylon membrane (Biotrans, Irvine, CA) by capillary blotting. RNA was cross-linked to the filters by exposure to short-wave ultraviolet light for 2 min and then baked at 80°C for 2 h Membranes were stored in sealed hybridization bags until use. A northern blot was hybridized with each slot-blot as a control. Slot blot procedure Nylon membranes (Biotrans, Irvine CA) were prepared for RNA by soaking in water 5 min then in 10X SSC prior to being mounted on slot blot apparatus. After mounting each slot was rinsed with 500 jil 10X SSC. Total RNA (each sample in aliquots of 5 , 10 and 20 /Lig RNA; yeast RNA was added to each blot as a control) was freeze dried, 125 fil denaturant (50% formamide, 6% formaldehyde, 2 0 mM Tris-HCl pH 7.5) was added and incubated at 65°C for 5 min. Twenty X SSC (125 ^1) was added and the sample loaded on the membrane. Each slot was then rinsed with 500 /nl 20X SSC. RNA was cross-linked to the filters by exposure to short-wave ultra-violet light for 2 min and then baked at 80°C for 2 h. Membranes were stored in sealed hybridization bags until use.

PAGE 183

168 Isolation of ovine Er The ovine Er cDNA probe (360 bp) was developed in our laboratory (Spencer et al., 1993) and cloned using polymerase chain reaction with primers to the human Er mRNA sequence and reverse transcribed template from Day 16 cyclic ovine endometrial RNA. A clean probe was obtained by use of Geneclean (Bio 101 Inc, La Jolla, CA) . Briefly, the linearized plasmid was run on a 1% TAE (Tris-HCl, acetate, EDTA) gel, the band excised, chopped into 2 mm 2 cubes and transferred to a 1.5 ml microcentrifuge tube. Impurities were removed from the DNA by the addition of sodium iodide (2.5 volumes of 6 M; supplied with Geneclean) to the gel and the addition of Glassmilk (silica matrix in water, supplied with Geneclean; 5 ^1) to the gel mixture after melting at 55°C for 5 min. The Glassmilk was allowed to bind to the DNA for 5 min on ice and then pelletized by microcentrifugation for 5 sec to separate it into a clean fraction. The pellet was resuspended in 200 ^1 ice-cold "NEW" (NaCl, ethanol, water, supplied with Geneclean) three times and pelletized by centrif ugation (5 sec) . The pellet was resuspended in TE buffer (10 mM Tris-HCl pH 7 . 5 , 1 mM EDTA) and Glassmilk pelletized by microcentrifugation (30 sec) to yield the pure DNA in the supernatant. The final concentration of DNA after the Geneclean procedure was 0.172 jj,g/n± as determined through quantification by spectrophotometric absorbance measurement at 280 nm.

PAGE 184

169 The RNA probe was produced from the clean cDNA through the use of the Riboprobe Genmini System II kit (Promega Corp., Madison WI . ) with T7 polymerase and labeled with 32 P (New England Nuclear, Boston, MA. ; 800 Ci/mMol) . Free radioactivity was separated from the specifically labeled probe by means of spin-quick chromatography columns (SelectD(RF); 5 Prime 3 Prime Inc., Bolder, CO.). A 1 jul aliquot of the labeled probe was counted to determine the radioactivity of the probe. Only probes with greater than 60 x 10 6 cpm were used for hybridization. Hybridization Northern blots and slot blots were pre-hybridized 2 h at 55°C in 20 ml hybridization buffer (50% f ormamide , 50 mM Na 2 P0 4 , 5X SSC, 0.1% SDS, 1 . 0 mM EDTA, 0 . 5X Denhardts , 200 /Lig/ml Herring sperm DNA) . Pre-hybridization buffer was replaced with hybridization buffer (pre-hybridization buffer with radiolabled ovine Er probe; 2 0 X 10 6 dpm) and the membranes hybridized 19 h at 55°C. After hybridization the northern blot and slot blot membranes were washed sequentially, three times (20 min each wash; 68°C) in 0 . IX SSC and 0.1% SDS, then three times (5 min each wash; room temperature) in 2X SSC. Hybridization signals were quantified using a Batascope 603 Blot Analyzer (Betagen) with the results given as CPM above background. To correct for loading differences, membranes were stripped by boiling in 500 ml 0.5% SDS and 0.01X SSC for 20

PAGE 185

170 min three times. The northern and slot blots were rinsed in 100 ml 0 . IX SSC two times. The membranes were rehybridized to a human 28s rRNA cDNA and the signals quantified on blot analyzer. For each Er mRNA sample data, the corresponding 28s data was used as a covariate in analyses of covariance by SAS. Estrogen Receptor Assay Estrogen receptor concentration within endometrial tissues was determined in the laboratory of Dr. Tom Ogal (Medical College of Georgia) . All receptor measurements were performed under conditions of endogenous steroid exchange which have been reported previously in sheep (Ott et al . , 1993b; Mirando et al . , 1993). Endometrial tissues stored at -80°C (Experiments 4 and 5) were rapidly thawed and homogenized (ice-cold conditions were maintained throughout all procedures unless otherwise noted) by three 10 sec bursts of a tissumizer (Tekmar Co., Cincinnati, OH) followed by 30 sec periods of cooling in TEDSL (0.05M Tris-HCl (pH 7.8 at 4°C) , 1.5 mM EDTA, 0.5 mM dithiothreitol, 0.25 M sucrose and 0 . 2 mM leupeptin) buffer (5mg/ml) . Centrif ugation at 800 x g for 20 min resulted in a pellet fraction and a supernatant fraction. The supernatant was recentrif uged at 105,000 x g. The 800 x g pellet was washed three times by resuspension in TMDSL (0.05 M Tris-HCl (pH 7.8 at 4°C) , 2 . 5 mM MgCl 2 , 0 . 5 mM

PAGE 186

171 dithiothreitol , 0.25 M sucrose and 0.2 mM leupeptin) buffer, followed by centrif ugation at 800 x g for 20 min (each wash) , and finally rehomogenization in TEDSL buffer using a Dounce homogenizer . A 0.25 ml aliquot of the supernatant fraction was incubated in a final volume of 0.3 ml in TEDSL. Estradiol exchange was performed by incubating receptor preparations at 22°C for 2 h then 4°C for 18 h with incubation buffer containing six concentrations of 3 Hestradiol (0.3-3.0 nM; Dupont NEN Research, Boston, MA) Non-specific binding was determined in the presence of a 100-fold molar excess of unlabeled estradiol. Steroid-bound to the receptor was separated from free steroid by dextrancoated charcoal. Following charcoal extraction, free steroid was removed from the pellet fraction by three rinses with Tris-sucrose buffer, and finally suspended in 1 ml ethanol warmed to 30°C for 1 h. The suspension was repelletized by centrif ugation at 1600 x g for 10 min and the supernate decanted into a scintillation vail for counting in a Beckman LS-1800 liquid scintillation counter (Beckman Instruments, Palo Alto, CA) . Protein concentrations in homogenates were assayed by the method of Lowry et al . (1951), and DNA levels were determined using a fluorometric procedure (Hill and Whatley, 1975) .

PAGE 187

172 Progesterone Receptor Binding Assay Progesterone receptor concentrations within endometrial tissues was determined in the laboratory of Dr. Tom Ogal (Medical College of Georgia) . All receptor measurements were performed under conditions of endogenous steroid exchange which have been previously reported in sheep (Ott et al., 1993b; Mirando et al . , 1993). Homogenous aliquots of frozen (-80°C) endometrial tissues were rapidly thawed and mechanically homogenized at 0°C in TGDL buffer (0.01 M Tris-HCl, pH 7.8, 30% glycerol, 1 mM dithiothreitol and 0.2 mM leupeptin) by three 10 second bursts with a Tissumizer (Tekmar Co., Cincinnati OH) each followed by 30 seconds of cooling. Unless otherwise stated all procedures were carried out under ice-cold conditions. Following the final homogenization step the homogenate was filtered through two layers of gauze and centrifuged at 800 X g for 15 min. The supernatant was recentrifuged for 40 min at 157,000 X g. The receptors were purified partially by differential precipitation with ammonium sulphate, and 100 ^1 of partially purified cytosolic receptor were incubated with 0 . 2 ml TDGL for 20-22 h. Bound steroid was separated from free steroid by use of dextran-coated charcoal. The nuclear pellet was rinsed twice with fresh TDGL, suspended in TDGL buffer. Aliquots of 0.1 ml containing 0.1-0.2 mg DNA were incubated for 22 h at 4°C. Free steroid was removed from the pellet fraction by

PAGE 188

I 173 repeated rinsing with TDGL and subsequently resuspended in 0.6 ml buffer. Protein concentrations in homogenates were assayed by method of Lowery et al . (1951), and DNA levels were determined using a fluorometric procedure (Hill and Whatley, 1975) . The receptor exchange assay was performed by incubating receptor preparations with incubation buffer containing six concentrations of 3 H-labeled ligand (1.0-17 nM progesterone; Dupont NEN Research, Boston, MA) . Nonspecific binding was determined in the presence of a 100 fold molar excess of unlabeled ligand. All receptor measurements were performed under conditions of endogenous steroid exchange (Ogle et al . 1989, 1990). Sample radioactivity was measured by liquid scintillation spectrometry (LS-1800, Beckman Instruments, Palo Alto, CA) . Statistical Analysis Values for Er mRNA were measured from radiographs of slot blots in CPM above background. Values were corrected for loading variation prior to analysis by dividing each Er mRNA CPM by the corresponding 28s CPM value from the same striped membrane (Ott et al . , 1993b). The resulting relative unit value was subjected to analysis. Data were subjected to least squares means analysis of variance using the general linear models procedure of the Statistical Analysis

PAGE 189

System (SAS Institute, 1995) . Ewes were nested within treatment and all tests of hypothesis were performed using the appropriate error terms according to the expectation of the mean squares (Snedecor and Cochran, 1980) . Data are expressed in least squares means ± S.E.M. Within status comparisons among days were made using orthogonal contrasts of the means. Steroid hormone receptor assays were analyzed by computerized curvefitting programs described previously by Ogle et al . (1989). Measurements of receptor binding were normalized for DNA and expressed as total receptor content. Data were subjected to least squares means analysis of variance using the general linear models procedure of the Statistical Analysis System (SAS Institute, 1995) . Ewes were nested within treatment and all tests of hypothesis were performed using the appropriate error terms according to the expectation of the mean squares (Snedecor and Cochran, 1980) . Data are expressed in least squares means ± S.E.M. Within status comparisons among days were made using orthogonal contrasts of the means.

PAGE 190

Results Estrogen Receptor mRNA Endometrial tissues were collected for determination of Er mRNA concentration in Experiments 2, 3, 4, and 5. The results are shown in Figures 5.1 through 5.4, respectively. Although only one level of RNA is shown in the figures (5 jug RNA) there was a linear increase in CPM over the three levels of RNA loaded onto each membrane. All CPM values were corrected for loading variation with 28s CPM values from the striped membranes prior to analysis of the data. In Experiment 2 there was no difference in endometrial Er mRNA concentration between SP and oCSP treatment (Fig. 5 . 1A) . Treatment with no peptide (0 Pep), the NT peptide of oIFNt or the C-terminus of oIFNx also were not different in their effect on endometrial Er concentration (Fig. 5. IB) and there was no effect of the interaction of treatment by peptide (Fig. 5 . 1C) . The results for Experiment 3 are shown in Figure 5.2. There were no differences in endometrial Er mRNA concentration in tissues collected from ewes treated with SP or Peptides 1 through 5 . Endometrial Er mRNA concentration was lower in tissues collected from ewes treated with roIFNi as compared with SP, Pep 2and Pep 3 -treated ewes.

PAGE 191

176 Furthermore, Er mRNA in ewes treated with roIFNi was not different from ewes treated with NT, Pep 4 and Pep 5. Oxytocin challenge as in Experiment 4 apparently did not alter the overall findings of Experiment 3 as they relate to endometrial Er mRNA concentration (Fig. 5.3). Inthis study endometrial Er mRNA concentration was lower in ewes treated with roIFNt compared to SP-treated control ewes and ewes treated with NT. There were no differences in mRNA concentrations in endometrial tissues collected from ewes treated with NT and ewes treated with SP. The results for Experiment 5 are shown in Fig. 5.4 A, B, and C. No difference was found in mRNA concentrations within endometrial tissues collected from ewes treated with SP compared with ewes treated with NT. There also was no change in Er mRNA concentration across the days examined (Day 16, 17 and 18) and there was no differences found after contrasting the means of the treatment*day interaction Estrogen Receptor Binding Assay Results from the analysis of endometrial Er binding evaluation indicating total endometrial Er concentration for Experiment 4 and Experiment 5 are shown in Fig. 5.5 and 5.6A, B, and C, respectively. Er binding was lower in ewes treated with roIFNx or NT as compared with SP-treated animals in Experiment 4. There was no difference in the Er concentrations between ewes treated with NT and roIFNi in

PAGE 192

Figure 5.1. Endometrial Er mRNA in cyclic ewes receiving intrauterine injections of SP, oCSP, the peptides CT and NT, or combinations of the proteins and peptides (Experiment 2) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0 . 5 mg NT + 1 . 0 mg SP , 0.5 mg CT + 1.0 mg SP, 0.7 5 mg oCSP + 0.7 5 mg SP, 0.7 5 mg oCSP + 0 . 5 mg NT or 0 . 75 mg oCSP + 0.5 mg CT. Treatments began on Day 12 and continued through the morning of Day 16. Main effects of treatment, peptide treatment, and the interaction of protein by peptide are represented in panels A, B and C, respectively. Zero (0) Pep indicates ewes were treated with the main treatment of SP or oCSP only. Bars with the same letters are not different (P>0.05).

PAGE 193

178 < z on o +» D) 3 i O a: E LU 200 r 180 |160 | 140 120 100 E 80 60 40 20 0A) a a 1 SP oCSP C) B) a l i OPep NT CT 0 NT CT 0 NTCT SP oCSP

PAGE 194

Figure 5.2. Endometrial Er mRNA in ewes receiving intrauterine injections of SP, synthetically produced peptides of oIFN f , or roIFN r (Experiment 3). Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.25 jug roIFN,, or 0.5 mg each of synthetic peptide NT, 2, 3, 4 , or 5 . The total protein concentration per treatment were balanced to 1.5 mg with SP. Treatments began on Day 12 and continued through the morning of Day 16. Bars with the same letters are not different (P>0 . 05) .

PAGE 195

180

PAGE 196

Figure 5.3. Endometrial Er mRNA in ewes receiving intrauterine injections of SP, NT, or roIFN, (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.5 jig roIFN f or 0.5 mg NT. The total protein concentration per treatment were balanced to 1.5 mg with SP. Treatments began Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of oxytocin (10 i.u.) on Day 14. Bars with the same letters are not different (P>0.05).

PAGE 197

1000 -i < ^ 800 H o G) 600 3 IO i O E UJ 400 200 a SP NT rolFNx

PAGE 198

183 this study (Fig. 5.5) In Experiment 5, endometrial Er concentration also was lower in ewes treated with NT as compared with SP-treated ewes (Fig. 5 . 6A) . Endometrial Er concentration increased over the days examined (Day 16, 17 and 18) with Er concentration being greater in tissues collected from ewes on Day 18 than in tissues collected from ewes on Day 16 or 17 (Fig. 5.6B). This increase in endometrial Er concentration was due to an increase in the Er numbers in tissues collected from ewes treated with SP from Day 16 to Day 18. There was no increase in Er numbers over the days examined in tissues collected from ewes treated with NT (Fig. 5.6C). The NT was effective in blocking the rise in endometrial Er concentration seen in ewes treated with SP. Progesterone Receptor Binding Assay Results for the analysis of Pr binding determination indicating total receptor concentration within endometrial tissues examined in Experiment 4 and Experiment 5 are shown in Figures 5.7 and 5.8, respectively. As expected, due to down-regulation of the Pr prior to Day 16, there was no difference in Pr concentration within endometrial tissues collected from ewes treated with SP, NT or roIFNx in Experiment 4 (Fig. 5.7). In Experiment 5 there was also no difference in receptor concentration between treatments (Fig. 5.8A) or between days on which tissues were collected

PAGE 199

Figure 5.4. Endometrial Er mRNA m ewes receiving intrauterine injections of SP or NT (Experiment 5). Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP or 0.5 mg NT + 1 . 0 mg SP . Treatments began on Day 11 and continued through the morning of Day 16, 17 or 18 when tissues were collected. Main effects of treatment, main effects of day and the effects of the interaction of treatment by day are presented in panels A, B, and C, respectively. Bars with the same letters are not different (P>0. 05) .

PAGE 200

185 < z o -*-» D) 3 lO i CL o a: E I— LU 2000 F 1800 1600 I 1400 1200 1000 800 600 400 200 0 A) a T a SP NT < a: 75 <+-> o O) D i Q. O E LU 2000 -i 1800 1600 1400 1200 1000 800 600 400 200 0 B) a 16 17 18 < +j o +» 1 CL o E LU Day of Estrous Cycle 161718 1617 18 SP NT

PAGE 201

Figure 5.5. Total endometrial Er in ewes receiving intrauterine injections of SP, NT, or roIFN, (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.5 /xg roIFN, or 0.5 mg NT. The total protein concentration per treatment were balanced to 1.5 mg with SP. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of oxytocin (10 i.u.) on Day 14. Bars with the same letter are not different (P>0.05).

PAGE 202

90 -i 80 70 C 3 o k. Q_ ^ i< 60 LU Z « Q 50 0 E 11 40 c a 30 LU ^ O H 20 10 a SP NT rolFNx

PAGE 203

Figure 5.6. Total endometrial Er in ewes receiving intrauterine injections of SP or NT (Experiment 5) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP or 0.5 mg NT + 1.0 mg SP. Treatments began on Day 11 and continued through the morning of Day 16, 17 or 18 when tissues were collected. Main effects of treatment, effects of day and effects of the interaction of treatment by day are represented in panels A, B and C, respectively. Bars with the same letters are not different (P>0 . 05) .

PAGE 204

c 2 o t< LU Z |o LU 3 o A) 189 90 c 80 r 70 j60 r 50 r 40 r 30 j20 f10 a SP NT c 0 +-> o CL LU 0 E o "O c LU 5 o 90 80 -70 J 2 60 H O O) 50 -| | 40 o £ 30 S 2 0H 10 0 B) 16 17 18 Day of Estrus Cycle C) c +» O k. Q_ iLU 140 n 120 < 100 O D) 3 o 80 .2 £ E E| 60H C Q40 LU w 20 0 be a abab a T b 161718 161718 SP NT

PAGE 205

190 regardless of treatment (Fig. 5.8B). Contrasts of the means of the treatment*day interaction revealed that the receptor concentration on Day 16 from ewes treated with NT was greater than from ewes treated with SP, regardless of the day examined, and also was greater than the receptor concentration found within tissues of ewes treated with NT that were collected on Day 18. While there was no change in the receptor concentration within endometrial tissues collected from ewes treated with SP across the days examined the Pr numbers fell dramatically within tissues from ewes treated with NT between Day 16 and Day 18. Discussion In ewes, pulsatile production of PGF 2a by the endometrium is primarily controlled by the steroid hormones progesterone and estrogen, which act in concert to regulate OTr formation (Soloff, 1975; McCracken, 1984; Sheldrick and Flint, 1985; Meyer et al . , 1988; Zang et al., 1992; Beard et al . , 1994; Spencer and Bazer, 1995). McKracken et al. (1984) was the first to suggest that progesterone acts to block OTr formation. Until recently our working hypothesis had been that oIFNt stabilized or up-regulated the endometrial Pr population to prolong the progesterone block (McCracken, 1984) to Er formation and the subsequent OTr formation (Ott

PAGE 206

gure 5.7. Total endometrial Pr in ewes receiving intrauterine injections of SP, NT, or roIFN, (Experiment 4) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP, 0.5 m roIFN r or 0.5 mg NT. The total protein concentration per treatment were balanced to 1.5 mg with SP. Treatments began on Day 11 and continued through the morning of Day 16. All ewes were challenged with a single injection of OT (10 i.u.) on Day 14. Bars with the same letters are not different (P>0.05).

PAGE 207

c o 75° o E 11 C Q. Ill w 3 O 9 -i 8 £ 76 5 4 3 2 1 a a SP NT i-oIFNt

PAGE 208

Figure 5.8. Total endometrial Pr in ewes receiving intrauterine injections of SP or NT (Experiment 5) . Each ewe received twice daily intrauterine injections (06:00 and 18:00 h, respectively) of 1.5 mg SP or 0.5 mg NT + 1 . 0 mg SP . Treatments began on Day 11 and continued through the morning of Day 16, 17 or 18 when tissues were collected. Main effects of treatment, effects of day, and effects of the interaction of treatment by day are represented in panels A, B and C, respectively. Bars with the same letters are not different (P>0 . 05) .

PAGE 210

et al . , 1993; Mirando et al . , 1993). However, the current hypothesis is oIFNx does not stabilize or up-regulate Pr, but that the actions of oIFNx are to block Er gene expression and subsequent increase in endometrial OTr concentration which allows for the luteolytic release of PGF 2a (Spencer and Bazer, 1995; Spencer et al., 1996). How pregnant ewes prevent upregulation of endometrial Er is not fully understood. Clearly the developing embryonic secretory product, oIFNx , prevents development of the luteolytic mechanism and pulsatile release of PGF 2a (Vallet et al . , 1988). In pregnant ewes the increased concentration of the Er (Findlay et al . , 1982; Mirando et al., 1993; Ott et al . , 1993) and OTr (Roberts et al., 1976; Ott et al., 1993) are not seen as observed in the cyclic ewe after 10 days of progesterone exposure. Intrauterine injection of oCSP inhibits oxytocininduced endometrial phosphoinositol hydrolysis (Mirando et al. 1990; Ott et al., 1992; Chapter 3) and PGF 2a secretion (Vallet et al . , 1990 ; Mirando et al., 1990; Chapter 4). While Mirando et al., (1993) reported that oCSP intrauterine injection prevented increases in Er mRNA and protein in cyclic ewes, no such block was found in Experiments 2 . In Experiment 2 the endometrial concentration of Er mRNA was the same for SPtreated ewes as it was for oCSPtreated ewes. However, in Experiments 4 and 5, intrauterine injection of roIFNx did block the increase in Er mRNA as noted by others for cyclic

PAGE 211

196 ewes. The difference between these two current studies could be attributed to the days in which the intrauterine injections were administered, in the case of the oCSP the injections were given from Day 12 to Day 16 while in the roIFNt study the injections were administered from Day 11 to Day 16. Since Day 12 has been identified as the "critical" day for maternal recognition to occur (Moor and Rowson, 1964; 1966a) it could be that the ewes receiving oCSP were on the boarder line of this critical period and were not as receptive to the effects of the oCSP. Alternatively, these results could be attributed to the fact that these samples were stored at -80°C for a prolonged period, while homologous probes were being developed, and this could have affected the stability of the mRNA. Interestingly, NT decreased Er protein without affecting levels of Er mRNA or gene expression. This could be attributed to either destabilization of the transcripts as well as other posttranscriptional effects on the mRNA, or prevention of translation. The RNA could be affected by the activation of 2' -5' A synthetase, which activates endonucleases that degrade RNA. Translation could be adversely effected by the actions of IFN to inactivate the translation initiation factor elF-2a (Singer and Berg, 1991) . All of these actions could be explained by partial agonist activity of NT. The question then becomes, how is this partial agonist signal by NT, at least in regards to Er

PAGE 212

197 mRNA, seen by the endometrium as different from that of roIFNt . There is no data to convincingly answer this question, but it would seem plausible that the NT, in binding to the Type-I IFN receptor initiates a conformational change in the receptor, or at least some change in receptor signal, that would account for these differences . Collectively, these results support our current working hypothesis that oIFNx prevents the cyclic increase in endometrial Er but this effect is not through an effect of roIFNi to stabilize or up-regulate Pr. Also, all instances examined in the present study, except for the case of the Er mRNA, the NT acted the same as did the complete roIFNx molecule indicating that the specific maternal recognition properties of oIFNx reside within the amino -terminal portion of this molecule.

PAGE 213

CHAPTER 6 GENERAL DISCUSSION Working Model of Maternal Recognition of Pregnancy in the Ewe A working model for the events involved in the maternal recognition of pregnancy in the ewe has been developed based on the findings of Spencer and others (Bazer, 1992; Spencer and Bazer, 1996; Spencer et al . , 1995a, 1995b; Bazer et al . , 1995) . According to this model, it has been proposed that for cycling ewes, Er are present in the occupied state on uterine epithelium during metestrus, suggesting that OTr are present in this tissue as well. Pr are also present, but insufficient numbers of Pr are occupied to suppress synthesis of OTr due to low circulating levels of progesterone. During diestrus, endometrial Er and estradiol in circulation are low and occupied Pr initiate and maintain the progesterone block to synthesis of Er and OTr for approximately 10-12 days. During late diestrus, progesterone down-regulates Pr which allows up-regulation of Er and OTr which is accompanied by increasing secretion of estradiol by the ovarian follicles. The pulsatile release 198

PAGE 214

199 of oxytocin from the CL and posterior pituitary initiates the release of luteolytic pulses of prostaglandin from the endometrium to destroy the CL. In the cyclic ewe during late diestrus (Fig. 6.1), Pr is undetectable in the luminal and superficial glandular epithelium. In the absence of suppression by progesterone, Er gene transcription increases in the epithelium which allows for Er-mediated increases in OTr formation. When pregnancy occurs (Fig. 6.2), the conceptus produces antiluteolytic signal (s) between Days 10 and 21 which acts on the endometrial steroid hormone receptors during pregnancy recognition to inhibit the formation of OTr and the subsequent pulsatile release of luteolytic prostaglandins. This luteolytic signal in the ewe has been demonstrated as being oIFNt , which acts to prevent OTr formation in the Pr-negative luminal and superficial glandular epithelium. Ovine IFNx attenuation of OTr prevents pulsatile production of luteolytic PGF 2a , this in turn maintains CL production of progesterone. Continued progesterone production in turn acts on the Pr-positive stroma and deep glandular epithelium to suppress estrogeninduced increases in Er and OTr gene expression in these tissues. These actions prevent the pulsatile production of PGF 2a by cells of the stroma and deep glandular epithelium. The mechanisms by which oIFNt acts (Fig. 6.3) are presumed to be mediated through the binding to Type -I INF receptors

PAGE 215

Hd HIQ c coCDQJrJ'dQjfDHjft) trn-m 0,0 (d h-iq cti CflrtfDfDiQCOrtCCHIPnrtCHrt fD Prt (D W HHW 3 c o d 4 con H 33 33 CD PHi W (D (t -O 3 3 PC eli-3 Hi QJ n h-bi tro rt (D 5; m a) —on QJ Hft) rt n H co33cnH , i-' k 3 trw^ 3 o n fD H. QJ fD fD 3 fD Qj 1 *0 H • QJ T5 ft) T3 3 M a) rt t) C fD O QJ CO QJ O Ht) H(D d H C & rt QJ QJ CO Q) H fD Hrt rt rt co Q HQj X 3 HCD fD 3T H33 fDTJua HDiHUOiQfDH fD O I — 1 I — ' fD QJ H 3 H rt co HiSfDontriQcos: fD n X O HH HO H H 3 cn Qj o H fDCDMiqn 33 33 ?r QJ QJ H H ft) H Q HCO CO Ql p pc p3 (DfDiQtlrtgHPiQ co (D a hj rD qj 0 p c gj • h rt 3 Hi PfD H (t tr O 3 QJ Hn fD fD Qj T) rt H O Pti fD OOH 3 4 p-PP TlrrOJfDConrtd £ H 33 C P 3 H CO HHH fD HO Hinrt30)P34 O H P n rt p p) Hi HfD H hC H rt 3 TJ H CD 3 3 QJ rt HQJ IQ QJ fD rt hd co ^3 H-03fDI— 'CDQjfD O 3 < • (DO < 33 < rt CO fD HQJ fD rt tr 33 3 3 tl HO pi rt qj n Qj M co 3 p to — H Qj fD HH0 0 HQJ 33 CO Qj s 33 rt Co C < tr fD rt Hi tQ 0 fD CO he HH H 33 QJ < HIQ 33 fD Q Q H C rr (U P P 3T rt QJ 33 0 fD P

PAGE 217

iQ C Hj CU TJ (D i-i rt) 3 Hj tJ fD rt CD piQ CTi p-iq rt e • MP tTHW C 0) fD 0) •• ft 3 M rt O O OJ O !< H P3 •< H rt rt) — O hn i-3 Hi n o o iQ t3 U 3 o HiQ rt OJ 3 3 H3 O O O Q.3 f— ' 3 fD to Hrt M 3 h; r& o n H rt 3 (t hj oj tJ 3 rt) rt OJ o w Po rt) Hn 3" 0 h< CL rt) rt) tl rt> Hj O t) rt) a d to n ^ iQ w rt HH H01 3 Pi O IQ 3 3 rt Ch O rt d Hto h01 C to H rt TJ 3" -rj rt rt) rt) p H-t) (D3H-H to rt) rt C to * 3* 3 rt) Hrt rt H 3 tr h-oj rt> d h s 0) — 3 rjrj Q, W n fD 3 CU rt Hn a HQJ IQ M OJ 3 a fD tJ H" n rt HiQ DJ O hj * P3 £ B to H OJ rt) IM 0) to fD rt rt 1 to rt g 0 to p w O 5 rt 3^0 OJ 3 Hj . 01 ; § " n 8 Hj 3 1 p. n o irt fD rt to rt TJ rt 3" Hrt O OJ O C 3 H 3 to — H O hh Ji ts O 4iQ rt & HH tr c 3 01 ft nq 3 fD Hj d t— Hi Hh O rt fr fD fD < fD 3 rt co P< O H < fD P3

PAGE 219

0) Hi H cn (D (D rtppj (D (D Q H CO PfD rt cn 3 3 rt 0) Cb 5? O Cn P53 o D n> h triQ pqj rt CT rt 5J PPCD rt < fD rt n 2 rt fD rt TJ Hi CD CD P < TJ ft) fD P fD CD n PiQ tJ rt rt PP rt PH 3 53 iQ I H to 53 U3 i fD fD rt t-3 TJ 5Tk; 5jfD 5V O CO CO TJ 53Z O 53P p.l< o h h rt 53 P l 53 i-» cn CD M co O O PCD rt po 53 rt Q 53C fD P PH 53 C iQ 3 CD p 53 O rt h3 fD P 53 CD Hi 53 O n p fD 3 CD O rt Hi po TS 53 P 0 LQ fD Co rt fD P 0 53 fD O 53 O CD 53 cn n O Hi P Hi Pt3 rt rt 53fD O 53 co TJ O CO Prt P< fD H *S I CD n rt co & CO O fD hi n c rt fD 5353 Prt cn H 0J ft H n p 53 rt cu iQ O 53 cn rt n p p p o cr p p53 CT cn W Qi fD n p cn o tj o a C H n c rt rt PfD O O 53 H >< rt 0) 53 & 3 cn o C ^ n M n » fD cn co rt O rt 5T fD Qi P PCD TJ 53 rt a rt p cu 53 P53 cn O ^ P CD rt fD a n p53 M P iQ fD 53 fD w o Hi rt 53fD rt O rt 53(D 53 C n H fD cn 2 fD P fD rt 5Jp-tj 53 P CD fD H iQ 53 CD CD 53 53 a o cn TJ fD P P fD n o Hi tQ 53 Pc p O fD H 2 • U) P " 2 H P CD g rt 3 HCD O rt a a O |-3 5^ P53 CD cn rt ca fD cn CD 53 CD c. Qi H H' W TJ 53 rt M pCD 53 rt 5T fD t-3 > P3 to CD 53 H PI P2 C 3 cr Hi Qi rt W 5? rt fD o 2 p Q n Q fD TJ rt o p cn TJ P fD co 3 fD P P PC 3 CD 53 CD fD 53 rt H 2 O P P53 iQ 3 O Dj fD H 5 Hi fD O Qj 5^ O fD 5 rt < P 2 p 5. CD rt p. cn fD H" TJ 2 rt O fD 2 fD p. a gp3 53

PAGE 221

206 present on the luminal and superficial glandular epithelium of the endometrium. Changes in confirmation of the IFN receptor activate two cytoplasmic tyrosine kinases, Jakl and Tyk2. These activate through phosphorylation, proteins within the cytoplasm which comprise the interferon stimulated gene factor3 complex. The interferon stimulated gene factor3 complex, subsequently translocates to the nucleus. It has been proposed that translocation of the signal into the nucleus requires a sequence which facilitates that translocation of the signal transduction complex, interferon stimulated gene factor3. There is a nuclear translocation sequence located on the CT of the full length oINFx molecule. There may also be other nuclear translocation sequences located on the cellular portion of the Type -I IFN receptor, on various components of the JAK/STAT signal transduction pathway, or on, as yet unknown, components of the signal cascade. Regardless of where the nuclear translocation sequence is located, once the signal has made its way into the nucleus, it binds to specific interferon stimulated response elements on DNA which direct IFN-induced transcriptional responses . One of these responses is the ultimate transcription of interferon regulatory factor1 and interferon regulatory factor-2, transcription factors that act either positively or negatively, respectively. It has been proposed that oIFNt causes an increase in the

PAGE 222

207 interferon regulatory factor-2 to interferon regulatory factor1 ratio, which may then suppress the expression of Er and Otr. Thus, the combined actions of oIFNx and progesterone result in suppression of OTr formation in the entire endometrium, reduction in pulsatile secretion of PGF, maintenance of the CL, and the successful establishment of pregnancy. This model has been used as to generate a working hypothesis that has been tested herein. Specifically, studies were designed to determine the function of oIFNx on factors associated with maternal recognition and to examine the functional properties of specific domains of oIFNx responsible for maternal recognition of pregnancy effects. Effects of oIFNt on IP metabolism and OTr Concentrations Our working hypothesis predicts that roIFNi or NT, working through the signal transduction pathway for oINFi, would decrease pulsatile PGF 2a secretion. We have shown that IP metabolism is directly associated with the mechanism of OTr action. IP metabolism is attenuated by the actions of oCSP, roIFNi and NT on endometrial tissues. Furthermore, endometrial OTr concentration is also reduced by these treatments. Collectively these results support the

PAGE 223

208 hypothesis that oIFNt blocks OTr formation leading to prevention of the luteolytic release of PGF 2a . Effects of oIFNt on OxytocinStimulated PGFM According to our working hypothesis pregnancy recognition ultimately requires control of pulsatile prostaglandin secretion by the uterine epithelium which is controlled by oxytocin through OTr. We have demonstrated, by measuring PGFM in plasma, that roIFNx reduces pulsatile release of PGF 2a . Similar results were obtained following treatment of ewes with NT. In addition, both roIFNx and NT had the same affect on plasma PGFM as did oCSP in previous studies from this laboratory. This further supports our hypothesis that there is a direct link between oIFNx and the control of PGF 2a . Effects of oIFNt on Er Protein and mRNA The current model for maternal pregnancy recognition signaling by oIFNx predicts that modulation Er expression plays a fundamental role in the mechanisms by which oIFNt prevents OTr expression and ultimately pulsatile PGF 2a secretion. Results from these studies have confirmed that in ewes roIFNx decreases both the message and the protein for Er, but does not affect Pr. This is the first report that NT decreased Er protein without affecting Er mRNA.

PAGE 224

209 The fact that NT decreased Er protein without affecting levels of Er mRNA or gene expression, brings about an interesting question. How does NT induce endometrial cells to act differently than roIFNt in regards to Er mRNA and identical to roIFNt as it relates to Er. This could be attributed to either destabilization of the transcripts as well as other posttranscriptional effects on the mRNA, or prevention of translation. The RNA could be affected by the activation of 2' -5' A synthetase, which activates endonucleases to degrade RNA. Translation could be adversely effected by the actions of IFN to inactivate the translation initiation factor elF-2a (Singer and Berg, 1991) . All of these actions could be explained by partial agonist activity of NT. The question then becomes, how is this partial agonist signal by NT, at least in regards to Er mRNA, seen by the endometrium as different from that of roIFNt . There is no data with which to answer this question, but it could be possible that the NT, in binding to the Type-I IFN receptor initiates a conformational change in the receptor, or at least some change in receptor signal, that would account for these differences. In light of the fact that NT has no nuclear translocation signal (it is found on the CT of the full length oINFt molecule) these results also indicate that there is an additional signal somewhere within the pathway. It could be located on the cytoplasmic side of the Type-1 IFN receptor, one of the

PAGE 225

components of the JAK/STAT signal transduction pathway, or a component of the pathway yet to be found. In any case, the differences between the action of NT and oIFNt warrants further study. Summary During the process of maternal pregnancy recognition in the ewe, control of PGF 2a release is critical to the maintenance of pregnancy. As discussed earlier, oIFNt accomplishes this by preventing Er expression in luminal epithelium and superficial glandular endometrial tissues. By controlling Er expression in these cells oIFNx's actions set into motion a series of events which not only prevents the luteolytic release of PGF 2a , but also maintains production of progesterone by the CL. Continued production of progesterone by the CL is also important in the stromal and deep glandular endometrium to bind Pr and prevent expression of Er and OTr in these tissues, completing the block to pulsatile PGF, a release. While the experiments discussed in this dissertation were not designed to address the temporal and spatial effects of oIFNt , they support the current hypothesis which delineates the mechanism of action of oIFNt as the maternal pregnancy recognition factor in the ewe. Specifically, in endometrial tissues roIFNi and NT decrease Er expression

PAGE 226

protein, thereby preventing up-regulation of OTr, which in turn prevents oxytocininduced pulsatile secretion of PGF 2a

PAGE 227

APPENDIX A PROTOCOL FOR PGFM ASSAY Standards (triplicates) 1. Pipette 200 /ill Banaminetreated plasma into standard tubes, NSB and BO (not TCT) . 2. Pipette 100 fxl standards into appropriate tubes. 3. Pipette 200 fxl Tris-HCl buffer into NSB tubes and 100 ill Tris buffer into B0 tubes. 4. Pipette 400 |Ul Tris buffer into TCT tubes. Samples and Reference Plasma (duplicates) 1. Pipette 200 /xl sample or reference plasma into appropriate tubes. 2. Pipette 100 \xl Tris buffer into sample and reference tubes . Samples and Standards; General Assay Procedure 1. Add 10 0 jxl 0.5% human globulin to all tubes. 2. Incubate for 15 min at room temperature. 3. Add 100 ixl rabbit anti-PGFM J53 (1/15,0000) or J57 (1/8,000) or goat anti-PGFM 23 (1/6,000) to all 212

PAGE 228

213 tubes except NSB and TCT. Anti-PGFM is stored at 1/100 in Tris-HCl. 4. Incubate for 30 min at room temperature. 5. Add 100 jul 3 H-PGFM (20 jih of stock (prepared 1-4-93) in 5 ml Tris) to all tubes (-18,000 dpm) . 6 . Incubate for lh at room temperature and then overnight at 4°C. 7. Add 750 fxl PEG to all tubes and vortex for 1 min 8. Centrifuge for 30 min at 3,000 rpm at 4°C. 9. Put tubes in foam racks, invert to drip dry (10 min) . 10. Redissolve pellets in 750 jul Tris and vortex for 1 min . 11. Add 750 ,ul PEG to all tubes and vortex for 1 min. 12. Centrifuge for 30 min at 3,000 rpm at 4°C. 13. Put tubes in foam racks, invert to drip dry (10 min . ) 14. Redissolve pellets in 1 ml Tris and vortex for 3 min . 15. Transfer entire solution to scintillation vials. 16. Add 4 ml Bio HP to all vials and count. Solutions Buffer: 0.05 M Tris-HCl (7.88 gm/L Tris-HCl; )

PAGE 229

0.1 % Na azide (1 gm/L; ) Adjust pH to 7.5 and store at 4°C. Banaminetreated plasma: Stored in 2 ml aliquots at -20°C. Reference plasma: High: 6.86 ml Banamine-treated plasma 140 nl 5,000 pg/100 fj,l Standard (50 ng/ml) final concentration = 1 ng/ml Low: 4 . 5 ml Bananimetreated plasma 0.5 ml High plasma (1 ng/ml) final concentration = 100 pg/ml Human Globulins : 0.5% w/v in 0.05 M Tris-HCl (fraction II, III from Sigma) Polyethyleneglycol 8000 (PEG; Fisher Scientific, Inc.): 40% (w/v) in distilled water. Standard Curve Stock Solution: 1 /ig/ml PGFM in Tris-HCl buffer Stored at -20°C

PAGE 230

215 Standard Curve Working Solutions: 100 jUl (1 ng/ml)+ 1.9 ml Tris buffer = 5,000 pg/0.1 ml 500 fil (5,000 pg/0.1 ml)+ 0.5 ml Tris = 2,500 pg/0.1 ml 400 /zl (5,000 pg/0.1 ml)+ 1.6 ml Tris = 1,000 pg/0.1 ml 500 Ml (1,000 pg/0.1 ml) + 0.5 ml Tris = 500 pg/0.1 ml 500 jul (500 pg/0.1 ml)+ 0.5 ml Tris = 250 pg/0.1 ml 150 fil (1,000 pg/0.1 ml) + 1.35 ml Tris = 100 pg/0.1 ml 500 jul (100 pg/0.1 ml)+ 0.5 ml Tris = 50 pg/0.1 ml 500 nl (50 pg/0.1 ml)+ 0.5 ml Tris = 25 pg/0.1 ml 100 Ml (100 pg/0.1 ml)+ 0.9 ml Tris = 10 pg/0.1 ml 500 ij,1 (10 pg/0.1 ml)+ 0 . 5 ml Tris = 5 pg/O.l ml

PAGE 231

APPENDIX B PROTOCOL FOR OXYTOCIN RECEPTOR ASSAY (PEG PROCEDURE) TISSUE PREPARATION wear gloves 1. Weigh 1 gm of tissue into weigh dish (keep frozen; return remaining tissue to -80°C immediately) mince with razor blade. 2. Transfer to 50 ml conical tube. 3. Rinse with 5 ml of homogenization buffer (HB; keep buffer on ice) and replace with 10 ml of HB. 4. Keep tube on ice until after the ultracentrifuge run, especially during homogenization. 5. Homogenize (5 sec on high; until smooth but not to the point that the mixture begins to turn grey or dark, grinding too long will denature the receptors) . Transfer to ground glass homogenizer. 6. Homogenize 10 strokes with ground glass homogenizer. Pour into clean 5 0 ml tube. Rinse with 2 ml HB buffer. 7. Centrifuge at 3,000 X g for 10 min. Keep pellet for DNA assay (freeze in tube at -20°C) . 8. Transfer supernate to ultracentrifuge tube. Balance exactly. Spin at 196,000 X g for 9 0 min. 9. Discard supernate.

PAGE 232

217 10. Wash pellet twice with Membrane Diluting Buffer (MD) , 1 ml . 11. Add 1 ml MD buffer, loosen pellet with transfer pipet, transfer to small ground glass homogenizer, homogenize until it is in solution. 12. Transfer to a polypropylene tube, rinse homogenizer with 1 ml MD buffer. Take out an aliquot for bicinchoninic acid assay. Equally divide the remainder into 2 separate tubes. 13. Freeze receptor tubes at -80°C and bicinchoninic acid assay tubes at -20°C. Procedure for Oxytocin Receptor Assay 1. Label tubes (in triplicate) in order of; Total Count Tubes (TCT) , Non-specific Binding Tubes (NSB) , Total Binding (Bo) (for Day 0 membrane preparation) , Total Displacement (Bo+80) (for Day 0 membrane preparation) , Total Binding (Bo) (for experimental tissue membrane preparation) , Total Displacement (Bo+80) (for experimental tissue membrane preparation) , Various levels of cold oxytocin for curve (experimental tissue) . * (start numbering tubes at #2. The 1st tube will be a blank tube) .

PAGE 233

218 2. Make up radio-labeled oxytocin just prior to use (0.321 pM/100 Ml) . 3. Dilute Day 0 and experimental membranes (200 ug/100 til) just prior to use. 4. Add 400 ill MD buffer to TCT , 200 jul MD buffer to NSB tubes, 100 /iil MD buffer to Bo tubes. 5. Add 100 Ml cold oxytocin at the appropriate level to the appropriate tubes (Bo+80 and curve) . 6. Add 100 jil radio-labeled oxytocin (0.321 pM/100 jul) to all tubes. 7. Add 100 /il Day 0 membrane preparation. (200 jug/100 jul) to Day 0, Bo and Bo+80. 8. Add 100 /il experimental tissue membrane preparation. (200 ^g/100 iil) to experimental tissue Bo, experimental tissue Bo+80, and curve tubes. 9. Incubate at room temperature (Make up gamma globulins (8 mg/ml) during incubation) . 10. Set aside TCT (they will not have gamma globulins and PEG added or be centrifuged with the other tubes) . 11. Add 100 iil gamma globulin (8 mg/ ml) to all tubes except TCT. 12. Mix by shaking rack by hand. 13. Add 1 ml 20% PEG (8000) to all tubes except TCT. 14. Vortex on speed 1.5 for 1 min. 15. Centrifuge at 3,000 X g for 10 min. (Label scintillation tubes while waiting) .

PAGE 234

219 16. Transfer spun tubes to foam racks. Drain supernatant into radioactive waste bucket. Drain tubes byinverting in foam racks over absorbent paper for 10 min. 17. Return tubes to tube racks. Add 400 /Ltl 50 mM Tris vortex (speed 2) for 2 min. 18. Add 1 ml 2 0% PEG. Vortex (speed 2) 1 min. 19. Centrifuge and pour off as before. 20. Add 1 ml 5 0 mM Tris and vortex (speed 1.5) for 5 min. 21. Add 600 ill 5 0 mM Tris to TCT and move them back with the rest of the tubes. 22. Add 1 ml 50 mM Tris to the first scintillation tube to be used as a blank. 23. Pour off assay tubes into scintillation tubes. Add 400 ul 5 0 mM Tris to all assay tubes, vortex, and pour off into scintillation tubes again. 24. Add 4 ml Scintiverse II to scintillation tubes, cap, shake, and equilibrate 2 h 25. Count for 2 min. Buffers For Oxytocin Receptor Assay Homogenization buffer (HB) 50 mM Tris-HCl 250 mM Sucrose 4 mM EDTA

PAGE 235

220 1. Adjust ph to 7.4 at 4°C (adjust pH close to 7.4, allow to equilibrate in cold room, and then adjust to final pH) . 2. Keep sample on ice. If the temp, goes above 4° the pH will change considerably. Membrane diluting buffer (MP) 50 mM Tris-HCl 1. Adjust pH to 7.4 at room temperature. 2. Use at room temperature. If used at cold room temperature the pH will harm the membrane preparations. Oxytocin diluting buffer (OP) 50 mM Tris-HCl 0.2% NaN 3 0.3 % BSA 2 0 mM MnCl 2 1. Add Tris, NaN 3 , and BSA to 980 ml H,0 (if making 1L) . Adjust pH to 7.5, equilibrate in cold room. 2. Add MnCl 2 to -15 ml of water, once in solution equilibrate in cold room. 3. When both solutions are cold, slowly add MnCl 2 with a transfer pipet (if it falls out of solution it must be started again) 4. Check pH which must be at 7 . 4 . If it is not then start over .

PAGE 236

APPENDIX C PROTOCOL FOR BICINCHONINIC ACID PROTEIN ASSAY Assay Procedure 1. Use microtiter plates and run duplicates of each sample across the plate. 2. Pipet 100 /il SPB into wells Al and A2 . 3. Pipet 100 ju.1 standard curves into wells A3-A12, B1-B4. 4. Pipet 100 ^1 known reference into wells B5 and B6. 5. Pipet 100 ^1 of unknown samples into wells B7-H12. (Dilute samples to between 5 and 10 ^g/100 /nl data points of the standard curve.) 6. Mix the working bicinchoninic acid (BCA) assay reagents just prior to use. 7. Pipet 100 BCA working solution into all wells. 8. Incubate 1 h at 60°C, cool to room temperature and read on microtiter plate reader Mode 89 at 540 nm wavelength or incubate 1 h at room temperature and read every 5 min for 1 h or until readings fall in center of curve. 9. Calculate mg protein/ml. Solutions Reagent A: 221

PAGE 237

222 Na 2 C0 3 6.84 w/v (Fisher Scientific, S-263-1) NaOH 1.60 w/v (Fisher Scientific, S318B) Sodium Tartrate Dihydrate 1.6 w/v (Sigma, S8640) NaHC0 3 w/v (Sigma, S5761) ddH 2 0 Adjust pH to 11.25 with NaHCo 3 . Store at room temperature. Reagent B: Bicinchoninic Acid Disodium Salt 4.0% w/v (C 20 H 10 N 2 O4Na 2 ; Sigma S-8284) ddH 2 0 Store at room temperature Reagent C: Cupric Sulphate Pentahydrate 4.0% w/v (Fisher Scientific, C-493) ddH 2 0 Store at room temperature Sodium Phosphate Buffer (SPB) 0.1 M: To prepare 1 liter of SPB (0.1 M) : 77.4 ml Stock A + 22.6 ml Stock B + 900 ml ddH,0 Stock A (1 M) : 14.196 gm Na 2 HP0 4 / 100 ml ddH 2 0 (Fisher Scientific, BP 332-500) Stock B (1 M) : 11.996 gm Na 2 H,Po 4 / 100 ml ddH 2 0

PAGE 238

(Fisher Scientific, BP 329-500) Store at room temperature or 4°C Standard Curve: BSA Stock Solution (50 jug/100 pi): 0.05 gm BSA /100 ml SPB (Fraction V RIA grade; United Biochemical Corp. , 10868) 1. 50 /il/100 ul 2. 25 jLig/10 0 jul: 25 ml #1 + 45 ml SPB 3. 10 jug/100 ul: 10 ml #1 + 40 ml SPB 4. 5 jug/100 Ml: 5 ml #1 + 45 ml SPB

PAGE 239

APPENDIX D PROTOCOL FOR OXYTOCIN RECEPTOR ASSAY (FILTER PROCEDURE) TISSUE PREPARATION wear gloves 1. Weigh 1 gm of tissue into weigh dish (keep frozen; return remaining tissue to -80°C immediately) mince with razor blade. 2. Transfer to 50 ml conical tube. 3. Rinse with 5 ml of homogenization buffer (HB; keep buffer on ice) and replace with 10 ml of HB. 4. Keep tube on ice until after the ultracentrifuge run, especially during homogenization. 5. Homogenize (5 sec on high; until smooth but not to the point that the mixture begins to turn grey or dark, grinding too long will burn the receptors) . Transfer to ground glass homogenizer. 6. Homogenize 10 strokes with ground glass homogenizer. Pour into clean 5 0 ml tube. Rinse with 2 ml HB buffer. 7. Centrifuge at 3,000 X g for 10 min. Keep pellet for DNA assay (freeze in tube at -20°C) . 8. Transfer supernate to ultracentrifuge tube. Balance exactly. Spin at 196,000 X g for 90 min. 9. Discard supernatant. 224

PAGE 240

10. Wash pellet twice with Membrane Diluting Buffer (MD) , 1 ml. 11. Add 1 ml MD buffer, loosen pellet with transfer pipet, transfer to small ground glass homogenizer, homogenize until it is in solution. 12. Transfer to a polypropylene tube, rinse homogenizer with 1 ml MD buffer. Take out an aliquot for BCA assay. Equally divide the remainder into two separate tubes. 13. Freeze receptor tubes at -80°C and BCA tubes at -20°C. Procedure for Oxytocin Receptor Assay 1. Label 18 12 x 75 mm borosilicate glass tubes (in duplicate) as Total Count Tubes (TCT) for each level of 3 H-oxytocin to be used (0.05, 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 4.0, 8.0 pM/ 5 0 jul) and 3 6 tubes (4 tubes at each concentration of 3 H-oxytocin) for each experimental tissue sample. 2. Make up 3 H-oxytocin at concentrations listed above just prior to use. 3. Make up cold oxytocin (80 0 pM/ 5 0 ^1) 4. Thaw experimental membranes preparations just prior to use . 5. Add 50 ixl of each concentration of 3 H-oxytocin to appropriate tubes (2 tubes per concentration for TCT

PAGE 241

and 4 tubes per concentration for experimental tissues) . 6. Add 150 Ml OD buffer to all TCT 6. Add 50 ixl OD buffer to the first 2 tubes of 4 at each concentration of 3 H-oxytocin for experimental tissues to be used as an estimation of total binding. 7. Add 50 /il cold oxytocin to the last 2 tubes of 4 at each concentration of 3 H-oxytocin to be used as to estimate non-specific binding. 8. Add 100 jul experimental tissue membrane preparation all 4 of the tubes at each concentration of 3 H-oxytocin. 9. Incubate at room temperature for 60 min. 10. After incubation put on ice. Add 2 ml ice cold AW buffer to all tubes (TCT and experimental) . 11. Transfer contents to Millipore GVWP 22/j, membrane filters in filter manifold and apply vacuum. 12. Place filter in scintillation vial and add 4.5 ml Scintiverse II to scintillation tubes, cap, shake, and equilibrate 3 h or overnight. 13. Count for 5 min. 14 Calculate receptor concentration of receptors using the program Ligand or Scatchard analysis by hand. DPM bound = DPM bound at each 3 H-oxytocin concentration NSB at each 3 H-oxytocin concentration. DPM total = total DPM at each concentration NSB at each 3 H-oxytocin concentration.

PAGE 242

227 DPM free = DPM total DPM bound. DPM are converted to mass and then to concentration using the specific activity of the radioligand and 2.22 x 10 12 DPM/Ci. The regression of Y on X, where Y= [bound] /[ free] and X= [bound] , gives the receptor concentration (X intercept) and 1/slope (Ka) gives the Kd. Buffers For Oxytocin Receptor Assay Homoqenization buffer (HB) 25 mM Tris-HCl 250 mM Sucrose 1 mM EDTA 1. Adjust pH to 7.4 at 4°C (adjust pH close to 7.4 then let equilibrate in cold room, before making final adjustment to pH 7.4). 2. Keep sample on ice. If the temperature goes above 4° the pH will change considerably. Membrane diluting buffer (MP) 25 mM Tris-HCl 1. pH to 7.4 at 4°C. Oxytocin diluting buffer (OP) 25 mM Tris-HCl 0.02 % NaN 3

PAGE 243

228 0.2 % BSA 20 mM MnCl, 1. Add Tris, NaN 3 , and BSA to 980 ml H 2 0 (if making 1 L) . Adjust pH to 7.5 at 4°C. 2. Add MnCl 2 to -15 ml water and once in solution, equilibrate in cold room. 3. When both are cold slowly add MnCl 2 with a transfer pipet (if the MnCl 2 falls out of solution the procedure must be started again) 4. Check pH. It must be -7.4, if not start over. Assay Wash Buffer (AW) 25 mM Tris-HCl 0.02 % NaN 3 0.1 % BSA 10 mM MnCl 2 1. Prepare as OD buffer.

PAGE 244

APPENDIX E PROTOCOL FOR INOSITOL PHOSPHATE ASSAY Preparation of Tissue 1. Each experimental tissue will be incubated in a pair of 20 ml glass vials. For each experimental tissue sample, thaw 2 . 5 ml KG I buffer and 2.5 ml BC buffer. Mix together and add 20 ml ddH 2 0 to make 25 ml KRB . Gas 5 min with 9 5% 0 2 and 5% C0 2 by bubbling gas through buffer. Cap tightly and chill on ice. 2. After dissection of caruncular endometrium, immediately place approximately 0.5 gm tissue into 5 ml ice cold KRB in a petri dish on ice. 3. Cut tissue into pieces approximately 5-10 mg in size and place into petri dish with 5 ml (fresh) KRB on ice. 4. Remove tissue from buffer with forceps and rapidly blot on kim-wipe. Weigh 100 mg tissue directly into 20 ml borosilicate glass scintillation vials and place on ice . Incubation 1. Add 10 jUCi 3 H-inositol (50 jul) to each vial, swirl contents to break-up tissue clumps, gas as before, cap and return to ice. 229

PAGE 245

230 2. Begin each incubation at 60 sec intervals bytransferring vials to a Dubnoff metabolic shaking incubator at 37°C. Incubate 120 min to incorporate 3 Hinositol into cell membrane phospholipids. 3. After 120 min, remove KRB and discard. Replace with 1 ml fresh KRB previously warmed to 37°C, gas, cap and incubate for an additional 30 min. 4. After 3 0 min, remove KRB and discard. Replace with 1 ml fresh KRB warmed to 37°C. Add 20 fil 0.51 M LiCl (final concentration = 10 mM) , gas, cap and incubate for 10 min. 5. Add 20 ^1 0.1 M Na 2 C0 3 to the first vial of each pair and 20 /Lil 5.2 oxytocin in 0.1 M Na 2 C0 3 to the second vial of each pair (final concentration of oxytocin 100 nM) , gas, cap and incubate 20 min. 6. Remove KRB and discard. Terminate incubation and lyse cells by adding 1 ml ice cold TCA. Place on ice for 30 min . 7. Transfer TCA to 15 x 85 mm borosilicate glass tubes. Rinse tissue with 200 jUl ddH 2 0. 8. Add 5 ml H 2 0 saturated diethyl ether to TCA tubes, cap with 15 mm polypropylene caps and extract with vigorous shaking for 5 sec. Carefully remove as much ether as possible with a pasteur pipette and discard. Repeat extraction procedure 4 times.

PAGE 246

9. Move tubes to a 37°C H,0 bath. Dry off residual ether from aqueous phase under a stream of N 2 for 5-10 min. Neutralize with 25 (J.1 0.5 M Tris-HCl and store at -20°C until chromatographic separation of inositol phosphates . Chromatographic Separation of Inositol Phosphates 1. Immediately prior to use, wash columns with three 5 ml volumes of ddH 2 0. 2. Thaw samples and transfer to individual columns (12 samples can be eluted simultaneously) . Wash each sample tube with 200 jil ddH 2 0 and transfer wash to its respective column. 3. Elute sample with three 3 ml volumes. Collect sample (contains inositol) and transfer 5 ml into 20 ml plastic scintillation vials. 4. Elute columns sequentially with 5 ml volumes of elution buffers #1, #2, #3, and #4, collecting each (containing glycerophospho-inositol , inositol monophosphate, inositol bisphosphate , and inositol trisphosphate , respectively) directly into 20 ml plastic scintillation vials . 5. Add 15 ml scintillation cocktail (Scintiverse II) to each vial, shake, let equilibrate 2 h or overnight and count 5 min. 6. Values are expressed as DPM/gm wet tissue.

PAGE 247

7. After use wash columns with two 5 ml volumes of elution buffer #5 and plug bottom. Add an additional 1 ml to top of column and cap. Columns can be stored as such and used for up to 10 elution procedures. Buffers and Solutions Kreb's Glucose Inositol (KGI) : 9.0 gm Sodium Chloride (NaCl 2 ) 0.375 gm Potassium Chloride (KC1 2 ) 0.3 gm Calcium Chloride (CaCl 2 ) 0.035 gm Potassium Phosphate (KH 2 P0 4 ) 0.03 gm Manganese Sulphate (MgS0 4 ) 1.80 gm Glucose 1.80 mg Myo-inositol Bring up to 10 0 ml with ddH 2 0 . Store in 10 ml aliquots at -20°C Bicarbonate (BC) : 2.18 g Bicarbonate Bring to 100 ml with ddH 2 0 . Store in 10 ml aliquots at -20°C 3 H-Inositol : Bring 1 . 0 mCi to 5.0 ml in ddH,0 Store at 4°C

PAGE 248

Lithium Chloride (LiCl) 0.51 M: 0.51 M LiCl in ddH 2 0 Store at 4°C Sodium Bicarbonate (0.1 M NaHC0 3 ) 0.1 M NaHC0 3 in ddH 2 0 , adjust pH to Aliquot and store at -20°C Oxytocin (5.2 /xM in 0.1 M NaHC0 3 ) 0.1 M NaHC0 3 (Prepared as above) 5.2 jU.M Oxytocin Aliquot and store at -20°C Trichloroacetic Acid 15% (v/v) in ddH 2 0 Store at 4°C Neutralization Buffer 0.5 M Tris-HCl, pH to 8.0 Store at 4°C Elution Buffers #1 25 mM Sodium Tetraborate 60 mM Formic Acid Sodium Salt #2 0.1 M Formic Acid 0.2 M Formic Acid Sodium Salt

PAGE 249

234 #3 0.1 M Formic Acid 0.4 M Formic Acid Sodium Salt #4 0.1 M Formic Acid 1.0 M Formic Acid Sodium Salt #5 0.1 M Formic Acid 2.0 M Formic Acid Sodium Salt Store at room temperature Column Preparation 1. Add Dowex-1 anion exchange resin (Sigma: 1x8 -200 Chloride Form) to an excess of ddH 2 0. 2. Add slurry to Bio-Rad Poly-Prep columns with glass pipet to a volume of 0.6 ml. 3. Convert resin to formate form by sequential washing with 6 ml 1 N HC1, 6 ml 1 M NH 4 OH and 6 ml elution buffer #5. 4. Store columns capped with enough elution buffer #5 to cover resin.

PAGE 250

REFERENCE LIST Alexander, I.E., Clarke, C.L., Shine, J., and Sutherland, R.L., 1989. Progestin inhibition of progesterone receptor gene expression in human breast cancer cells. Mol. Endocrinol. 3:1377-1386. Alila, H.W. , Dowd, J. P., Corradino, R.A. , and Harris, W.V. , 1988. Control of progesterone production in small and large bovine luteal cells separated by flow cytometry. J. Reprod. Fertil., 82:645-655. Anderson, J.N., Peck, E.J., and Clark, J.H., 1975. Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei. Endocrinology, 96:106-167. Anthony, R.V. , Helmer, S.D., Sharif, S.F., Roberts, R.M. , Hansen, P.J., Thatcher, W.W. , and Bazer, F.W., 1988. Synthesis and processing of ovine trophoblast protein1 and bovine trophoblast protein1, conceptus secretory proteins involved in the maternal recognition of pregnancy. Endocrinology, 123:1274-1279. Aronica, S.M., and Katzenellenbogen , B.S., 1991. Progesterone receptor regulation in the uterine cells: stimulation by estrogen, cyclic adenosine 3', 5'monophosphate and insulin-like growth factor1 and suppression by antiestrogens and protein kinase inhibitors. Endocrinology, 128:2045-2052. Ashworth, C.J., and Bazer, F.W., 1989. Interrelationships of proteins secreted by the ovine conceptus and endometrium during the periattachment period. Anim. Reprod. Sci., 20:117-130. Ayad, V.J., Guldenaar, S.E.F., and Wathes , D.C., 1991a. Characterization and localization of oxytocin receptors in the uterus and oviduct of the non-pregnant ewe using an iodinated receptor antagonist. J. Endocrinol., 128 : 187-195 . Ayad, V.J., Matthew, E.L., Whathes, D.C., Parkinson, T.J., and Wild, M.L., 1991b. Autoradiographic localization 235

PAGE 251

236 of oxytocin receptors in the endometrium during the oestrous cycle of the ewe. J. Endocrinol., 130:199206. Ayad, V.J., Parkinson, T.J., Matthews, E.L., and Wild, M.L., 1993. Effects of pregnancy and systemic or intrauterine oxytocin infusion on the distribution of endometrial oxytocin receptors in the ewe: an autoradiographic study. J. Endocrinol., 137:423-431. Baird, D.T., 1978. Pulsatile secretion of LH and ovarian estradiol during the follicular phase of the sheep estrous cycle. Biol. Reprod. , 18:359-364. Baird, D.T., Land, R.B., Scaramuzzi, R.J., and Wheeler, A. G . , 1976. Endocrine changes associated with luteal regression in the ewe; the secretion of ovarian oestradiol, progesterone and androstenedione and uterine prostaglandin F 2a throughout the oestrous cycle. J. Endocrinol., 69:275-286. Barcikowski, B., Carlson, AJ.C, Wilson, L . , and McCracken, J. A. , 1974. The effect of endogenous and exogenous estradiol1715. on the release of prostaglandin F 2a from the ovine uterus. Endocrinology, 95:1340-1349. Bartol, F.F., Roberts, R.M. , Bazer, F.W., Lewis, ACS., Godkin, J.D., and Thatcher, W.W. , 1985. Characterization of proteins produced in vitro by periattachment bovine conceptuses. Biol. Reprod., 32: 681-693 . Basu, S., 1989. Endogenous inhibition of arachidonic acid metabolism in the endometrium of the sheep. Prostaglandins Leukotrienes and Essential Fatty Acids, 35: 147-152. Basu, S. and Kindhal, H. , 1987. Prostaglandin biosynthesis and its regulation in the bovine endometrium: A comparison between nonpregnant and pregnant status. Theriogenology , 28:175-193. Baumbach, G.A. , Duby, R.T., and Godkin, J.D., 1990. NGlycosylated and unglycosylated forms of caprine trophoblast protein1 are secreted by preimplantation goat conceptuses. Biochem. Biophys. Res. Comm., 172 : 16-20 Bazer, F.W., 1991. Type I conceptus interferons: Maternal recognition of pregnancy signals and potential therapeutic agents. Am. J. Reprod. Immunol., 26:19-22.

PAGE 252

237 Bazer, F.W., 1992. Mediators of maternal recognition of pregnancy in mammals. Proc . Soc. Exp. Biol. Med., 199: 373-384. Bazer, F.W., Spencer, T.E., Ott, T.L., and Johnson, H.M. , 1995. Cytokines and pregnancy recognition. In: Immunobiology of Reproduction . Hunt, J.S. editor, Serono Symposia, USA, Norwell, Massachusetts, pp 37-56. Beato, M. , 1991. Transcriptional control by nuclear receptors. FASB 5:2044-2051. Beato, M. , Chavez, S., and Truss, M. , 1996. Transcriptional regulation by steroid hormones. Steroids, 61:240-251. Beard, A. P., Hunter, M.G., and Lamming, G.E., 1994. Quantitative control of oxytocininduced PGF2a release by progesterone and oestradiol in ewes. J. Reprod. Fertil. 100:143-150. Beard, A. P., and Lamming, G.E., 1994. Oestradiol concentration and the development of the uterine oxytocin reseptor and oxytocin-induced PGF2a release in ewes. J. Reprod. Fertil. 100:469-475. Berridge, M.J., 1984. Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J., 220 : 345-360 . Bhakoo, H.S., and Katzenellenbogen , B.S., 1977. Progesterone modulation of estrogen-stimulated uterine biosynthetic events and estrogen receptor levels. Mol. Cell. Endocrinol., 8:121-134. Bindon, B.M., Blanc, M.R., Pelletier, J., Terqui, M. , and Thimonier, J., 1979. Periovulatory gonadotropin and ovarian steroid patterns in sheep of breeds with differing fecundity. J. Reprod. Fertil., 55:15-25. Boshier, D.P., Fairclough, R.J., and Holloway, H., 1987. Assessment of sheep blastocyst effects on neutral lipids in the uterine carnucular epithelium. J. Reprod. Fertil. 79:569-573. Boshier, D.P., Holloway, H., and Millener, N.M. , 1981. Triacylclycerols in the rat uterine epithelium during the oestrous cycle and early pregnancy. J. Reprod. Fertil., 62:441-446.

PAGE 253

238 Bouvier, C, Lagace, G., and Collu, R.G., 1991. Protein modulation by estrogens. Mol. Cell. Endocrinol. 79 : 457-467 . Branden, T.D., Gamboni , F. , and Niswender, G.D., 1988. Effects of prostaglandin F-2 alpha-induced luteolysis on the populations of cells in the ovine corpus luteum. Biol. Reprod., 39:245-252. Brenner, R.M., Resko, J. A., and West, N.B., 1974. Cyclic changes in oviductal morphology and residual cytoplasmic estradiol binding capacity induced by sequential estradiol-progesterone treatment of spayed rhesus monkeys. Endocrinology, 95:1094-1104. Bresnick, E.H. , Dalan, F.C., Sanchez, E.R., and Pratt, W.B., 1989. Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor. J. Biol. Chem. , 264 : 4992-4998 . Brinsfield, T.H., and Hawk, H.W. , 1973. Control by progesterone of the concentration of lipid droplets in epithelial cells of the sheep endometrium. J. Anim. Sci. 36:919-922. Browning, J.L., and Ribolini , A., 1987. Interferon blocks interleukin 1-induced prostaglandin release from human peripheral monocytes. J. Immunology, 138:2857-2863. Burgess, K.M. , Ralph, M.M. , Jenkin, G. and Thornburn, G.D., 1990. Effect of oxytocin and estradiol on uterine prostaglandin release in nonpregnant and early-pregnant ewes. Biol. Reprod., 42:822-833. CarsonJurica, M.A. , Schrader, W.T., and B.W. O'Malley. Steroid receptor family: Structure and functions. Endocrine Reviews, 11 (2) : 201-220 . Casida, L.E., and Warwick., B.J., 1945. The necessity of the corpus luteum for the maintenance of pregnancy in the ewe. J. Anim. Sci., 4:34-36. Charlier, M. , Hue, D., Boisnard, M. , Martal, J., Gaye , and P., 1991. Cloning and structural analysis of two distinct families of ovine interferon a genes encoding functional class II and trophoblast (OTP-1) interferons. Mol. Cell. Endocrinol., 76:161-171. Charpigny, G., Reinaud, P., Huet, J.-C, Guillomot, M. , Charlier, M. , Pernollet, J.C., and Martal , J., 1988. High homology between a trophoblastic protein

PAGE 254

239 (trophoblastin) isolated from ovine embryo and alphainterferons. FEBS Lett., 228:12-16. Cherny, R . A . , Salmonsen, L.A., and Findly, J.K., 1991. Immunocytochemical localization of oestrogen receptors in the endometrium of the ewe. Reprod. Fertil. Dev., 3: 321-331. Cook, B., Karsh, F.J., Foster, D.L., and Nalbandov, A.V. , 1974. Estrogen-induced luteolysis in the ewe: possible sites of action. Endocrinology, 94:1197-1201. Danet-Desnoyers , G. , Johnson, H.W. , O'Keefe, S.F., and Thatcher, W.W. , 1993. Characterization of a bovine prostaglandin synthesis inhibitor (EPSI) . Biol. Reprod., 48:(Suppl. 1)115 (abstr.). Darnell, J.E., Kerr, I.M., and Stark, G.R., 1994. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415-1421. David, M. , and Larner, A.C., 1992. Activation of transcription factors by interf eron-alpha in a cellfree system. Science, 257:813-815. Dore-Duffy, P., Perry, W. , and Kuo, H.-H., 1983. Interf eron-mediated inhibition of prostaglandin synthesis in human monuculear leukocytes. Cellular Immunology, 79:232-239. Eggleston, D.L., Wilken, C, van Kirk, E.A. , Slaughter, R.G., Ji, T.H., and Murdoch, W.J., 1990. Progesterone induces expression of endometrial messenger RNA encoding for cyclooxygenase (sheep) . Prostaglandins 39:675-683. Ellinwood, W.E., Nett, T.M., and Niswender, G.D., 1979. Maintenance of the corpus luteum of early pregnancy in the ewe. II. Prostaglandin secretion by the endometrium in vitro and in vivo. Biol. Reprod., 21: 845-856. Fairclough, R.J., Moore, L.G., Peterson, A.J., and Watkins, W.B., 1984. Effect of oxytocin on plasma concentrations of 13, 14-dihydro-15-keto prostaglandin F and the oxytocin-associated neurophysin during the estrous cycle and early pregnancy in the ewe. Biol. Reprod. , 31:36-43. Fairclough, R.J., Smith, J.F., and McGowan, L.T., 1981. Prolongation of the oestrous cycle in cows and ewes

PAGE 255

240 after passive immunization with PGF antibodies. J. Reprod. Fert., 62:213-219. Farin, C.E., Imakawa, K. , Hansen, T.R., McDonnell, J.J., Murphy, C.N., Farin, P.W., and Roberts, R.M. , 1990. Expression of trophoblastic interferon genes in sheep and goats. Biol. Reprod., 43:210-218. Findlay, J.K., Ackland, N. , Burton, R.D., Davis, Walker, F.M.M., Walters, D.E., and Heap, R.B., 1981. Protein, prostaglandin and steroid synthesis in caruncular and intracaruncular endometrium of sheep before implantation. J. Reprod. Fertil., 62:361-377. Findlay, J.K. , Clarke, I.L., Colvin, N. , and Doughton, B., 1982. Oestrogen receptors and protein synthesis in caruncular and intercaruncular endometrium of sheep before implantation. J. Reprod. Fertil., 64:329-339. Fintcher, K.B., Bazer, F.W., Hansen, P.J., Thatcher, W.W. , and Roberts, R.M. , 1986. Proteins secreted by the sheep conceptus suppress induction of uterine prostaglandin F-2a release by oestradiol and oxytocin. J. Reprod. Fertil., 76:425-433. Fitz, T.A. , Mayan. M.H., Sawyer, H.R., and Niswender, G.D., 1982. Characterization of two steroidogenic cell types in the ovine corpus luteum. Biol. Reprod., 22: 703711 . Fitz, T.A. , Mock, E.J., Mayan, M.H., and Niswender, G.D., 1984. Interactions of prostaglandins with subpopulations of ovine luteal cells. II. Inhibitory effecto of PGF 2a and protection by PGE 2 . Prostaglandins, 28:127-138. Fitzpatrick, F.A., and Murphy, R.C., 1988. Cytochrome P-450 metabolism of arachidonic acid: formation and biological actions of "epoxygenase" -derived eicosanoids. Pharmacol. Rev., 40:229-241. Flint, A.P.F., 1995. Interferon, the oxyticin receptor and the maternal recognition of pregnancy in ruminants and Non-runimants : a comparative approach. Reprod. Fertil. Dev., 7:313-318. Flint, A.P.F., Lamming, G.E., Stewart, H.J., and Abayasedara, D.R.E., 1994. The role of the endometrial oxytocin receptor in determining the length of the sterile oestrous cycle and ensuring maintenance of

PAGE 256

241 luteal function in early pregnancy in ruminants. Phil. Trans. R. Soc . Lond. B, 344:291-304. Flint, A.P.F., Leat, W.M.F., Sheldrick, E.L. , and Stewart, H.J., 1986. Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium. Biochem. J., 237:797-805. Flint, A.P.F., Riley, P.R. , Kaluz , S., Stewart, H.J., and Abayasekara, D.R.E., 1995. The sheep endometrial oxytocin receptor. In: Oxytocin. I veil, R. and Russell, J., editors. Plenum Press, New York. pp. 281-294. Flint, A.P.F. and Sheldrick, E.L., 1982. Ovarian secretion of oxytocin is stimulated by prostaglandin. Nature, 297 : 586-588. Flint, A.P.F. , and Sheldrick, E.L., 1983. Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep. J. Reprod. Fertil. 67:215-225. Flint, A.P.F., and Sheldrick, E.L., 1985. Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes. J. Reprod. Fertil., 75:623-631. Flint, A.P.F, and Sheldrick, E.L., 1986. Ovarian oxytocin and the maternal recognition of pregnancy. J. Reprod. Fertil. 76:831-839. Flint, A.P.F., Sheldrick, E.L., McCann, T.J., and Jones, D.S.C., 1990. Luteal oxytocin: Characteristics and control of synchronous episodes of oxytocin and PGF 2a secretion at luteolysis in ruminants. Domestic Animal Endocrinology, 7:111-124. Ford, S.P., Weems, C.W., Pitts, R.E., Pexton, J.E., Butcher, R.L., and Inskeep, E.K., 1975. Effects of estradiol17S and progesterone on prostaglandins F in sheep uteri and uterine venous plasma. J. Anim. Sci. 41(5): 14071413 . Freedman, L. , 1992. Anatomy of the steroid receptor zinc finger region. Endocrine Reviews, 13 (2) : 129-145 . French, L.R., and Spennetta, B., 1981. Effects of antibodies to progesterone on reproduction of ewes. Theriogenology , 16:407-418.

PAGE 257

242 Fu, X.-Y., 1992. A transcription factor with SH2 and SH3 domains is directly activated by an interferon ainduced cytoplasmic protein tyrosine kinase (s) . Cell, 70:323-335. Fu, X.-Y., Schindler, C, Improta, T., Aebersold, R. , and Darnell, J., 1992. The proteins of ISGF-3, the interferon a-induced transcriptional activator, define a gene family involved in signal transduction. Proc. Natl. Acad. Sci. USA, 89:7840-7843. Furlow, J.D., Murdoch, F.E., and Gorski, J., 1993. High affinity binding of the estrogen receptor to a DNA response element does not require homodimer formation or estrogen. J. Biol. Chem. , 268 (17) : 12519-12525 . Ginther, O.J., 1969. Length of the estrous cycle and size of the corpus luteum in guinea pigs and sheep treated with progesterone at different days of the estrous cycle. Am. J. Vet. Res., 30:1975-1978. Gnatek, G.G. , Smith, L.D., Duby, R.T., Godkin, J.D., 19 89. Maternal recognition of pregnancy in the goat: Effects of conceptus removal on interestrous intervals and characterization of conceptus protein production during early pregnancy. Biol. Reprod. , 41:655-663. Goding, J.R. Cumming control Fertil . Godkin, J.D. Roberts Blockey, M.A.B., Brown, J.M. , Catt, K.J., and I. A., 1970. The role of oestrogen in the of the oestrus cycle in the ewe. J. Reprod. 21:368-369. Bazer, F.W., Moffatt, J., Sessions, F., and R.M. , 1982. Purification and properties of a major, low molecular weight protein released by the trophoblast of sheep blastocyst at Day 13-21. J. Reprod. Fertil., 65:141-150. Godkin, J.D., Bazer, F.W., and Roberts, R.M. , 1984a. Ovine trophoblast protein, an early secreted blastocyst protein, binds specifically to uterine endometrium and affects protein synthesis. Endocrinology, 114(1) :120130. Godkin, J.D., Bazer, F.W., Thatcher, W.W. , and Roberts, R.M. , 1984b. Proteins released by the cultured Day 1516 conceptuses prolonged luteal maintenance when introduced into the uterine lumen of cyclic ewes. J. Reprod. Fertil., 71:57-64. Goodman, R.L., 1994. Neurocrine control of the ovine conceptus. In: Knobil, E., and Neil, J.D. (eds.). The

PAGE 258

243 Physiology of Reproduction. New York: Raven Press, 659-710 . Gorski, J., Toft, D., Shyamala, G. , Smith, D., andNotides, A., 1968. Hormone receptors: Studies on the interaction of estrogen with the uterus. Rec . Prog. Hormone Res., 24:45-80. Gross, T.S., Thatcher, W.W. , Hansen, P.J., Johnson, J.W. , and Helmer, S.D., 1988a. Presence of an intracellular endometrial inhibitor of prostaglandin synthesis during early pregnancy in the cow. Prostaglandin, 35:359-378. Gross, T.S., Plante, C., Thatcher, W.W. , Hansen, P.J., Helmer, S.D., and Putney, D.J., 1988b. Secretory proteins of the bovine conceptus alter endometrial prostaglandin and protein secretion in vitro. Biol. Reprod., 39:977-987. Guilbault, L.A, Thatcher, W.W. , Drost, M. , and Hopkins, S.M., 1984. Source of F series prostaglandins during the early postpartum period in cattle. Biol. Reprod., 31: 879-887 . Hannigan, G.E., and Williams, B.R., 1991. Signal transduction by interferon-a through arachidonic acid metabolism. Science, 251:204-207. Hansen, P.J., Anthony, R.V. , Bazer, F.W., Baumbach, G.A. , and Roberts, R.M., 1985. In vitro synthesis and secretion of ovine trophoblast protein1 during the period of maternal recognition of pregnancy. Endocrinology, 117:1424-1430. Hansen, T.R., Kazemi , M. , Keisler, D.H., Malathy, P.-V., Imakawa, K. , and Roberts, R.M. , 1989. Complex binding of the embryonic interferon, ovine trophoblast protein1, to endometrial receptors. J. Interferon Res., 9:215-225. Harada, H. , Takahashi, E.-I., Itoh, S., Harada, K. , Hori, T.-A., and Taniguchi, T., 1994. Structure and regulation of the human interferon regulatory factor 1 (IFN-1) and IRF-2 genes: implications for a gene network in the interferon system. Molec . Cell. Biol. 14: 1500-1509. Hard, T. , Kellenbach, E. , Boelens, R. , Maler, B.A., Dahlman, K. , Freedman, L.P., Carsted-Duke , J . , Yamamoto, K.R., Gustafsson, J., and Kaptein, R. , 1990. Solution structure of the glucocorticoid receptor DNA-binding domain. Science, 249:157-160.

PAGE 259

244 Harper, C.M.L., and Thornburn, G.D., 1984. Inhibition of prostaglandin synthesis by ovine allantoic fluid: acute reduction in inhibitory activity during late gestation. Can. J. Physiol. Pharmacol., 62:1152-1157. Hawk, H.W. , and Bolt, D.J., 1970. Luteolytic effect of estradiol176 when administered after midcycle in the ewe. Biol. Reprod. , 2:275-278. Hawkins, D.E., Belfiore, C.J., and Niswender, G.D., 1993. Regulation of mRNA encoding 3b-hydroxysteroid dehydrogenase/delta5 delta4 isomerase (3b-HSD) in the ovine corpus luteum. Biol. Reprod. 48:1185-1190. Helmer, S.D., Hansen, P.J., Anthony, R.V. , Thatcher, W.W. , Bazer, F.W., and Roberts, R.M. , 1987. Identification of bovine trophoblast protein1, a secretory protein immunologically related to ovine trophoblast protein1. J. Reprod. Fertil., 79:83-91. Helmer, S.D., Hansen, P. J., and Thatcher, W.W. , 1988. Differential glycosylation of the components of the bovine trophoblast protein1 complex. Mol. Cell. Endocrinology, 58:103-107. Helmer, S.D., Hansen, P.J., Thatcher, W.W. , Johnson, J.W. , and Bazer, F.W., 1989a. Intrauterine infusion of highly enriched bovine trophoblast protein1 complex exerts an antiluteolytic effect to extend corpus luteum lifespan in cyclic cattle. J. Reprod. Fertil., 87:89101. Helmer, S.D., Gross, T.S., Newton, G.R., Hansen, P.J., and Thatcher, W.W. , 1989b. Bovine trophoblast protein-1 complex alters endometrial protein and prostaglandin secretion and induces an intracellular inhibitor of prostaglandin synthesis in vitro. J. Reprod. Fertil., 87:421-430. Henderson, K.M. , Scaramuzzi, R.J., and Baird, D.T., 1977. Simultaneous infusion of protaglandin E 2 antagonizes the luteolytic action of prostaglandin F 2a in vivo. Endocrinology, 72:379-383. Heyman, Y. , Camous, S., Fevre , J., Meziou, W. , and Martal, J., 1984. Maintenance of the corpus luteum after uterie transfer of trophoblastic vesicles to cyclic cows and ewes. J. Reprod. Fertil., 70:533-540. Hill, B.T., and Whatley, S., 1975. A simple, rapid microassay for DNA. FEBS Letters, 56:20-23.

PAGE 260

245 Hixon, J.E., and Flint, A.P.E., 1987. Effects of a luteolytic dose of oestradiol benzoate on uterine oxytocin receptor concentrations, phosphoinositide turnover and prostaglandin F-2a secretion in sheep. J. Reprod. Fertil., 79:457-467. Hollenberg, S.M., Giguere, V., Segui, P., and Evans, R.M. , 1987. Co-localization of DNA binding and transcriptional activation functions in the human glucocorticoid receptor. Cell, 49:39-46. Homanics, G.E., and Silvia, W.J., 1988. Effects of progesterone and estradiol17S on uterine secretion of prostaglandin F 2a in response to oxytocin in ovariectomized ewes. Biol. Reprod. 38:804-811. Hooper, S.B., Walker, D.W., and Thornburn, G.D., 1986. Cannulation of the utero-ovarian vein in intact ewes: Hormone concentrations and blood gas levels during the oestrous cycle and pregnancy. Acta Endocrinol., 112: 507-526. Hooper, S.B., Watkins, W.B., and Thornburn, G.D., 1987. Oxytocin-associated neurophysin, and prostaglandin F-2a concentrations in the utero-ovarian vein of pregnant and non-pregnant sheep. Endocrinol. 119:2590-2597. Hsueh, A.J.W., Peck, E.J., and Clark, J.H., 1975. Progesterone antagonism of the oestrogen receptor and oestrogen-induced uterine growth. Nature, 245:337-339. Hsueh, A.J.W., Peck, E.J., and Clark, J.H., 1976. Control of uterine estrogen receptor levels by progesterone. Endocrinology, 98:438-444. Huslig, R.L., Fogwell, R.L., and Smith, W.L., 1979. The prostaglandin forming cyclooxygenase of ovine uterus: Relationship to luteal function. Biol. Reprod. 21:589-600. Imakawa, K. , Anthony, R.V. , Kazemi, M. , Marotti, K.R., Polites, and Roberts, R.M. , 1987. Interf eron-like sequence of ovine trophoblast protein secreted by embryonic trophectoderm. Nature, 330:377-379. Imakawa, K. , Hansen, T.R., Malathy, P-V. , Anthony, R.V., Polites, H., Marotti, K.R., and Roberts, R.M. , 1989. Molecular cloning and characterization of complementary deoxyribonucleic acids corresponding t bovine trophoblast protein1: a comparison with ovine

PAGE 261

246 trophoblast protein1 and bovine interf eron-a n . Mol. Endocrinol., 3:127-139. Imakawa, K. , Helmer, S.D., Nephew, K.P., Meka, C.S.R., and Christenson, R.K., 1993. A novel role for GM-CSF: Enhancement of pregnancy specific interferon production, ovine trophoblast protein1. Endocrinology, 132:1869-1871 InsKeep, E.K., Smutny, W.J., Butcher, R.L., and Pexton, J.E., 1975. Effects on intraf ollicular injections of prostaglandins in nonpregnant and pregnant ewes. J. Anim. Sci., 41:1098-1102. Jenner, L.J., Parkinson, T.J., and Lamming, G.E., 1991. Uterine oxytocin receptors in cyclic and pregnant cows. J. Reprod. Fret., 91:49-58. Jensen, E.V., Suzuki, T . , Kawashima, T. , Stumpf, W.E., Jungblut, P.W., DeSombre, E.R., 1968. A two-step mechanism for the interaction of estradiol with rat uterus. Proc. Natl. Acad. Sci., 59:632-638. Johnson, H.M., Bazer, F.W., Szente, B.E., and Jarpe, M.A. , 1994. How interferons fight disease. Scientific American, May: 68-75. Jones, D.S.C., and Flint, A.P.F., 1986. Oxytocinneurophysin mRNA in the corpus luteum of the sheep during the oestrus cycle and in pregnancy. J. Endocrinol. Ill (suppl) : 140 , 1986. Kerr, I.M., and Stark, G.R., 1991. The control of interferon-inducible gene expression. FEBS, 285:194198. Kessler, D.S., and David, E.L., 1991. Protein kinase activity required for an early step in interferon-a signaling. J. Biol. Chem. , 266:23471-23476. King, W.J., and Greene, G.L., 1984. Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature, 307:745-747. Kittok, R.J., and Britt, J.H., 1977. Corpus luteum function in ewes given estradiol during the estrous cycle or early pregnancy. J. Anim. Sci., 45 (2) : 336-341 . Klemann, S.W., Imakawa, K. , and Roberts, R.M. , 199 0. Sequence variability among ovine trophoblast interferon mRNA. Nucleic Acids Res., 18:6724-

PAGE 262

247 Knickerbocker, J.J., and Niswender, G.D., 1989. Characterization of endometrial receptors for ovine trophoblast protein1 during the estrous cycle and early pregnancy in sheep. Biol. Reprod. , 40:361-369. Knickerbocker, J.J., Thatcher, W.W. , Bazer, F.W., Drost, M. , Barron, D.H., Fincher, K.B., and Roberts, R.M. , 1986. Proteins secreted by Day 16 to 18 bovine conceptuses extend corpus luteum function in cows. J. Reprod. Fertil., 77:381-391. Ko, Y. , Lee, Y, Ott, T.L., Davis, M.A. , Simmen, R.C.M., Bazer, F.W., and Simmen, F.A., 1991. Insulin like growth factors in sheep uterine fluids: Concentrations and relationship to ovine trophoblast protein1 production during early pregnancy. Biol. Reprod., 45: 135-142. Koligian, K.B., and Stormshak, F. , 1977a. Nuclear and cytoplasmic estrogen estrogen receptors in ovine endometrium during the estrous cycle. Endocrinology, 101:524-533. Koligian, K.B., and Stormshak, F., 1977b. Progesterone inhibition of estrogen receptor replenishment in ovine endometrium. Biol. Reprod., 17:412-416. Kramer, R.M., Hession, C, Johansen, B., Hayes, G. , Chow, CP., Tizard, R. , and Pepinsky, R.B., 1989. Structure and properties of a human non-pancreatic phospholipase A2. J. Biol. Chem. , 264:5768-5775. Lacroix, M.C., Charpigny, G., and Reinaud, P., 1988. Is oxytocin of conceptus origin involved in inhibition of luteal regression in early pregnant ewes? J. Endocrinol., 118:R17-R20. Lamming, G.E., Vallet, J.L. and Flint, A.P.F., 1991. Progestational control of endometrial oxytocin receptors determines cycle length in sheep. J. Reprod. Fertil. (Suppl.), 43:53-54. Larner, A.C., David, M. , Feldman, G.M., Igarashi, K. , Hacket, R.H., Webb, D.S.A., Sweitzer, S.M., Petricoin III, E.F., and Finbloom, D.S., 1993. Tyrosine Phosphorylation of DNA binding proteins by multiple cytokines. Science, 261:1730-1733. Lau, T.M., Kerton, D.J., Gow, C.B., and Fairclough, R.J., 1992. Increase in concentration of uterine oxytocin receptors and decrease in response to 13 , 14-dihydro-15-

PAGE 263

248 keto-prostaglandin F 2a in ewes after withdrawal of exogenous progesterone. J. Reprod. Fertil., 95:885893 . Lau, T.M. , Kerton, D.J., Gow, C.B., and Fairclough, R.J., 1993. Role of progesterone in the control of endometrial oxytocin receptors at luteolysis in sheep. J. Reprod. Fertil., 98:229-233. Leaman, D.W. , and Roberts, R.M. , 1992. Genes for the trophoblast interferons in sheep, goat, and musk ox and distribution of related genes among mammals. J. Interferon Res., 12:1-11. Leavitt, W.W. , Okulicz, W.C., McCracken, J. A. , Schramm, W. , and Rubidoux W.F., 1985. Rapid recovery of nuclear receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal. J. Steroid Biochem. , 22 : 687-691 . Leavitt, W.W. , Toft, D.O., Strott, C.A., and O'Mallay, B.W. , 1974. A specific progesterone receptor in the hamster uterus: physiologic properties and regulation during the estrous cycle. Endocrinol., 94:1041-1053. Lefevre, F., Martinat-Botte , F., Guillomot, M. , Zouari, K. , Charley, B. , and LaBonnardiere , C, 1990. Interferon-g gene and protein are spontaneously expressed by porcine trophectoderm early in gestation. Eur. J. Immunol. 20 : 2485-2493 . Legan, S.J., I'Anson, H., Fitzgerald, B.P., and Fitzovich, D. , 1985. Does the seasonal increase in estradiol negative feedback prevent luteinizing hormone surges in anestrous ewes by suppressing hypothalamic gonadotropin-releasing hormone pulse frequency? Biol. Reprod., 23:404-413. Levy, D.E., Lew, D.J., Decker, T., Kessler, D.S., and Darnell, J.E., 1990. Synergistic interaction between interferon-a and interferonx through induced synthesis of one subunit of the transcription factor ISGF3. EMBO J., 9:1105-1111. Loeb, L.A. , and Gross, R.W. , 1986. Identification and purification of sheep platelet phospholipase A2 isoforms. Activation by physiologic concentrations of calcium ion. J. Biol. Chem. , 261:10467-10470.

PAGE 264

249 Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J., 1951. Protein Measurement with the folin phenol reagent. Journal of Biological Chemistry, 193:265-275. MacDonald, J.I.S., and Sprecher, H. , 1991. Phospholipid fatty acid remodeling in mammalian cells. Biochem. Biophys. Acta., 1084:105-121. Mader, S., Kumar, V., Verneuil, H. , and Chambon, P., 19 89. Three amino acids of the estrogen receptor are essential to its ability to distinguish on oestrogen from a glucocorticoid-responsive element. Nature, 338:271-274. Mamimekalai, S., Umapathy, E., and Govindaraj ulu , P., 1979. Hormonal influence on uterine lipids. Hormone Res., 10:213-221. Martal, J., Lacroix, M-C, Loudes, C, Saunier, M. , and Wintenberger-Torres , S., 1979. Trophoblastin, an antiluteolytic protein present in early pregnancy in sheep. J. Reprod. Fertil., 56:63-73. Martal, J., Degryse, E., Charpigny, G. , Assal, N. , Reinaud, P., Charlier, M. , and Lecocq, J. P., 1990. Evidence for extended maintenance of the corpus luteum by intrauterine infusion of a recombinant trophoblast ainterferon (trophoblastin) in sheep. J. Endocrinol., 127 :R5-R8 . Martin, T.W. , and Wysolmerski, R.B., 1987. Constraints on prostaglandin biosynthesis in tissues. J. Biol. Chem. , 262:3510-3517. Martinod, S., Maurer, R.R., Siegenthaler , B., Gerber, C, and Hansen, P.J., 1991. The effects of recombinant bovine interferon-a on fertility in ewes. Theriogenology 36:231-236. Marx, J., 1992. Taking a direct path to the genes. Science, 257:744-745. McCracken, J. A., 1971. Prostaglandin F 2a and corpus luteum regression. Prostaglandins, 180:456-472. McCracken, J. A. , 1980. Hormone receptor control of prostaglandin F-2a secretion by the ovine uterus. Adv. Prostaglandin Thromboxane Res., 8:1329-1344. McCracken, J. A., Schramm, W. , Barcikowski, B., and Wilson, L., 1981. The identification of prostaglandin F 2a as a

PAGE 265

250 uterine luteolytic hormone and the hormonal control of its synthesis. Acta Vertinaria Scandinavia. 77:71-88. McCracken, J. A., Schramm, W. , and Okuliez, W.C., 1984. Hormone receptor control of pulsatile secretion of PGF2a from the ovine uterus during luteolysis and its abrogation in early pregnancy. Anim. Reprod. Sci., 7:31-55. McCracken, J. A. , Smith, T.T., Lamsa, J.C., and Robinson, A.G., 1991. The effect of estradiol176 and progesterone on the oxytocin pulse generator in the ovexed sheep. Biol. Reprod., 30(Suppl. 1):154 McGuire, W.J., Hawkins, D.E., and Niswender, G.D., 1991. Activation of protein kinase (PK) -C inhibits progesterone production in vivo. Biol. Reprod. 46 (Suppl) : 84. McKinizie, F.F., and Terrill, C.E., 1937. Estrus, ovulation, and related phenomena in the ewe. Missouri Agr. Exper. Sta. Res. Bull., 264:5-88. Meidan, R. , Girsh, E., Blum, 0., and Aberdam, E., 1990. In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: Morphological and functional characteristics. Biol. Reprod., 43:913-921. Milgrom, E., Thi , L . , Atger, M. , and Baulieu, E.E., 1973. Mechanisms regulating the concentration and conformation of progesterone receptor (s) in the uterus. J. Biol. Chem. , 248:6366-6374. Miller, B.G., Murphy, L . , and Stone, G.M. , 1977. Hormone receptor levels and hormone, RNA and protein metabolism in the genital tract during the oestrous cycle of the ewe. J. Endocrinol., 73:91-98. Mirando, M.A. , Ott, T.L., Harney, J. P., and Bazer, F.W., 1990a. Ovine trophoblast protein-one inhibits development of endometrial responsiveness to oxytocin in ewes. Biol. Reprod., 43:1070-1078. Mirando, M.A. , Ott, T.L., Vallet, J.L., Davis, M. , and Bazer, F.W., 1990b. Oxytocinstimulated phosphate turnover in endometrium of ewes is influenced by stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins. Biol. Reprod. , 42:98-105.

PAGE 266

251 Mirando, M.A. , Harney, J. P., Zhou, Y. , Ogle, T.F., Ott, T.L., Moffatt, R.J., and Bazer, F.W., 1993. Changes in progesterone and oestrogen receptor mRNA and protein and oxytocin receptors in endometrium of ewes after intrauterine injection of ovine trophoblast interferon. J. Mol. Endocrinol., 10:185-192. Mirando, M.A. , Short, E.C., Geisert, R.D., Vallet, J.L., and Bazer, F.W., 1991. Stimulation of 2 ' , 5 ' -oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of oTP-1, and by intramuscular administration of recombinant bovine interferon-ajl . J. Reprod. Fertil., 93:599-607. Miyamoto, T. , Ogino, N. , Yamamoto, S., and Hayaishi, 0., 1976. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. Biol. Chem. , 251:2629-2636. Moor, R.M. , and Rowson, L.E.A., 1964. Influence of the embryo and uterus on luteal function in the sheep. Nature, 201:522-523. Moor, R.M. , and Rowson, L.E.A., 1966a. The corpus luteum of the sheep: Functional relationship between the embryo and the corpus luteum. J. Endocrinology, 34:233-239. Moor, R.M. , and Rowson, L.E.A., 1966b. The corpus luteum of the sheep: Effect of removal of embryos on luteal function. J. Endocrinology, 34:497-502. Moor, R.M. , and Rowson, L.E.A., 1966c. Local maintenance of the corpus luteum in sheep with embryos transferred to various isolated portions of the uterus. J. Reprod. Fertil., 12:539-550. Moore, L.G., Choy, V.L., Elliot., R.L., and Watkins , W.B., 1986. Evidence for the pulsatile release of PGF, a inducing the release of ovarian oxytocin during luteolysis in the ewe. J. Reprod. Fertil., 76:159-166. Moore, L.G., and Watkins , W.B., 1982. Embryonic suppression of oxytocin-associated neurophysin release in early pregnant sheep. Prostaglandins, 24(l):79-88. Morgan, G.L., Geisert, R.D., McCann, J. P., Bazer, F.W., Ott, T.L., Mirando, M.A. , and Stewsrt, M. , 1993. Failure of luteolysis and extension of the interoestrous interval in sheep treated with progesterone antagonist mifepristone (RU486). J. Reprod. Fertil. 98:451-457.

PAGE 267

252 Mueller, G.C., Herranen, A.M. , and Jervell, K.F., 1958. Studies on the mechanism of action of estrogens. Recent Progress in Hormone Research, 14:94-129. Munson, P.J., and Rodbard, D., 1980. Ligand: a versatile computerized approach for characterization of ligand binding system. Anal. Biochem. , 108:193-208. Nephew, K.P., McClure, K.E., Day, M.L., Xie, S., Roberts, R.M., and Pope, W.F., 1990. Effects of intramuscular administration of recombinant bovine interf eron-alpha^ during the period of maternal recognition of pregnancy. J. Anim. Sci., 68 (9) : 2766-2770 . Newton, C.R., Vallet, J.L., Hansen, P.J., and Bazer, F.W., 1989. Inhibition of lymphocyte proliferation by ovine trophoblast protein1 and a high molecular weight glycoprotein produced by the peri -implantation sheep conceptus. Am. J. Reprod. Immunol., 19:99-103 Niswender, G.D., and Dziuk, P.J., 1966. A study of the unilateral relationship between the embryo and the corpus luteum by egg transfer in the ewe. Anat. Record, 154:394-395. Niswender, G.D., and Nett, T.M., 1994. Corpus luteum and its control in infraprimate species. In: Knobil, E. , Neil, J.D., eds. The Physiology of Reproduction. New York: Raven Press, 781-816. Northly, D.L., and French, L.R., 1980. Effect of embryo removal and intrauterine infusion of embryonic homogenates on the lifespan of the bovine corpus luteum. J. Anim. Sci., 50 (2) : 298-302 . Ogle, T.F., Mills, T.M., and Costoff, A., 1990. Progesterone maintenance of the placental progesterone receptor and placental growth in ovariectomized rats. Biol. Reprod., 43:276-284. Ogle, T.F., Mills, T.M., and Soarcs, M.J., 1989. Changes in cytosolic and nuclear progesterone receptor during pregnancy in the rat placenta. Biol. Reprod., 40:10121019 . Okulicz, W.C., MacDonald, R.G., and Leavitt, W.W. , 1981. Progesterone-induced estrogen receptor-regulatory factor in hamster uterine nuclei: Preliminary characterization in a cellfree system. Endocrinol, 109:2237-2275.

PAGE 268

253 Orit, E. , Bodwell, J.E., and Munck, A., 1992. Phosphorylation of steroid hormone receptors. Endocrine Reviews, 13 ( 1 ) : 105 128 . Ott, T.L., Mirando, M.A. , Davis, M.A. , and Bazer, F.W., 1992. Effects of ovine conceptus secretory proteins and progesterone on oxytocinstimulated endometrial production of prostaglandin and turnover of inositol phosphate in ovariectomized ewes. J. Reprod. Fertil., 95: 19-29. Ott, T.L., Van Heeke, G., Fliss, F.M.V., Johnson, H.M. , and Bazer, F.W., 1991a. Antiluteolytic and antiviral activities of recombinant ovine trophoblast protein1 (roTP-1) overproduced from a synthetic gene in S. cerevisiae. Biol. Reprod., 44:89 (abstr.). Ott, T.L., Van Heeke, G. , Hostetler, C.E., Schalue, T.K., Olmstead, J.J., Johnson, H.M. , and Bazer, F.W., 1993a. Intrauterine injection of recombinant ovine interferon tau extends the interestrous interval in sheep. Theriogenology , 40:757-769. Ott, T.L. , Van Heeke, G, Johnson, H.M. , and Bazer, F.W., 1991b, Cloning and expression in Saccharomyces cerevisiae of a synthetic gene for the type-1 trophoblast interferon ovine trophoblast protein1: Purification and antiviral activity. J. Interferon Res., 11:357-364. Ott, T.L., Zhou, Y. , Mirando, M.A. , Stevens, C., Harney, J. P., Ogle, T.F., and Bazer, F.W., 1993b. Changes in progesterone and oestrogen receptor mRNA and protein during maternal recognition of pregnancy and luteolysis in ewes. J. Mol. Endocrinol., 10:171-183. Ottobre, J.S., Lewis, G.S., Thayne , W.V., and Inskeep, E.K., 1980. Mechanism by which progesterone shortens the estrous cycle of the ewe. Biol. Reprod., 23:1046-1053. Pagels, W.R., Sachs, R.J., Marnett, L.J., Dewitt, D.L., Day, J.S., and Smith, W.L., 1983. Immunochemical evidence for the involvement of prostaglandin H synthase in hydroperoxide -dependent oxidations by ram seminal vesicle microsome. J. Biol. Chem. , 258:5617-5623. Parkinson, T.J., Lamming, G.E., Flint, A.P.F., and Jenner, L.J., 1992. Administration of recombinant bovine interf eron-a, at the time of maternal recognition of pregnancy inhibits prostaglandin F 2a secretion and

PAGE 269

254 causes luteal maintenance in cyclic ewes. J. Reprod. Fertil. , 94:489-500. Parkinson, T.J., Stewart, H.J., Hunter, M.G., Jones, D.S.C., Wathes, D.C., and Flint, A.P.E., 1991. Evidence against a role for blastocyst-secreted oxytocin in early pregnancy maintenance in sheep. J. Endocrinol., 130 : 443-449 . Parsons, S.D., and Hunter, G.L., 1967. Effect of the ram on duration of oestrus in the ewe. J. Reprod. Fertil., 14 : 61-70 . Pelham, H. , 1988. Coming in from the cold. Nature, 332:776-778. Pfeiffer, L.M. , and Tan, Y.M. , 1994. Do second messengers play a role in interferon signal transduction? Trends Biol. Sci. 16:321-323. Plante, C, Thatcher, W.w. , and Hansen, P.J., 1991. Alteration of oestrous cycle length, ovarian function and oxytocininduced release of prostaglandin F-2a by intrauterine and intramuscular administration of recombinant bovine interferon-a to cows. J. Reprod. Fertil., 93:375-384. Platanias, L.C., Uddin, S., and Colamonici, O.R., 1994. Tyrosine phosphorylation of the a and £ subunits of the type I interferon receptor. J. Biol. Chem. , 269:1776117764 . Pratt, B.R., Butcher, R.L., and Inskeep, E.K., 1977. Antiluteolytic effect of the conceptus and of PGE 2 in ewes. J. Anim. Sci., 46 (4) : 784-791 . Pontzer, C.H., Bazer, F.W., and Johnson, H.M. , 1991. Antiproliferative activity of a pregnancy recognition hormone, ovine trophoblast protein1. Cancer Res., 51:5304-5307. Pontzer, C.H., Ott, T.L., Bazer, F.W., and Johnson, H.M. , 1990. Localization of an antiviral site on the pregnancy recognition hormone, ovine trophoblast protein-1. Proc. Natl. Acad. Sci. USA, 87:5945-5949. Pontzer, C.H., Ott, T.L., Bazer, F.W., and Johnson, H.M. , 1994. Structure/function studies with interferon tau: Evidence for multiple active sites. J. Interferon Research, 14:133-141.

PAGE 270

255 Pontzer, C.H., Torres, B.A., Vallet, J.L., Bazer, F.W., and Johnson, H.M., 1988. Antiviral activity of the pregnancy recognition hormone ovine trophoblast protein-1. Biochem. Biophys. Res. Comm., 152:801-807. Puissant, C. and Houdebire, L.M., 1990. An improvement of the single step method of RNA isolation by acid guanidinum thiocyanate phenol chloroform extraction. Biotechniques , 8:147-149. Ramwell, P.W., Leovey, E.M.K., and Sintetos, A.L., 1977. Regulation of the arachidonic acid cascade. Biol. Reprod. , 16 : 70-87 . Raw, R.E., Curry, T.E., and Silvia, W.J., 1988. Effects of progesterone and estradiol on the concentration and activity of cyclooxygenase in the ovine uterus. Biol. Reprod., 38(Suppl. 1):104 (abst.). Raw, R.E., and Silvia, W.J., 1991. Activity of phospholipase C and release of prostaglandin F 2a by endometrial tissues from ovariectomized ewes receiving progesterone and estradiol. Biol. Reprod., 44:404-412. Read, L.D., Snider, C.E., Miller, J.S., Greene, G.L., and Katzenellenbogen, B.S., 1988. Ligand-modulated regulation of progesterone receptors messenger ribonucleic acid and protein in human breast cancer cell lines. Mol. Endocrinol. 2:263-271. Rhodes, L. , and Nathanielsz, P.W., 1990. Myometrial activity and plasma progesterone and oxytocin concentrations in cycling and early pregnant ewes. Biol. Reprod. 42:834-841. Rice, G.E., Wong, M.H., Ralph, M.M., and Thornburn, G.D., 1987. Ovine allantoic fluid inhibition of prostaglandin synthesis in cotyledonary microsomes. J. Endocrinol., 114:295-300. Roberts, J.S., and McCracken, J. A. , 1976. Does prostaglandin F 2a released from the uterus by oxytocin mediate the oxytocic action of oxytocin? Biol. Reprod., 15:457-463. Roberts, J.S., McCracken, J. A. , Gavagan, J.E., and Soloff, M.S., 1976. Oxytocinstimulated release of prostaglandin F 2a from ovine endometrium in vitro: correlation with oestrous cycle and oxytocin receptor binding. Endocrinol., 99:1107-1114.

PAGE 271

256 Roberts, R.M. , 1993. Interferontau . Nature, 362:593. Roberts, R.M. , Cross, C.C., and Leaman, D.W. , 1992. Interferons as hormones of pregnancy. Endocrine Rev. 13 : 432-452 . Roberts, R.M. , Farin, C.E., and Cross, J.C., 1990. Trophoblast proteins and maternal recognition of pregnancy. In: Oxford Reviews of Reproductive Biology. Milligan, S.R., editor. Oxford University Press, Oxford, U.K.. pp. 147-180. Robinson, T.J., 1959. The estrous cycle of the ewe and doe. In: Reproduction in Domestic Animals, Cole, C.C., and Cupps , P.T., editors. Academic Press, New York., pp. 291-333 . Rodgers, R.T., O'Shea, J.D., Findlay, J.K., Flint, A.P.E., and Sheldrick, E.L., 1983. Large luteal cells the source of luteal oxytocin in the sheep. Endocrinology, 113 : 2302-2304 . Rowson, L.E.A., and Moor, R.M. , 1967. The influence of embryonic tissue homogenate infused into the uterus, on the life-span of the corpus luteum in the sheep. J. Reprod. Fertil., 13:511-516. Salamonsen, L.A. , 1992. Local regulators and the establishment of pregnancy: a review. Reprod. Fertil. Dev. 4:125-134. Salamonsen, L.A. , Hampton, A.L., Clements, J. A. , and Findly, J.K., 1991. Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus. J. Reprod. Fertil., 92: 393-406. Salamonsen, L.A., Manikhot, J., Healy, D.J., and Findly, J.K., 1989. Ovine trophoblast protein-1 and human interferon alpha reduce prostaglandin synthesis by ovine endometrial cells. Prostaglandins, 38:289-306. Salamonsen, L.A. , Stuchbery, S.J., O'Grady, CM., Godkin, J.D., and Findly, J.K., 1988. Interferon-a mimics effects of ovine trophoblast protein 1 on prostaglandin and protein secretion by ovine endometrial cells in vitro. J. Endocrinol., 117:R1-R4. Samuelsson, B., 1987. An elucidation of the arachidonic acid cascade. Discovery of prostaglandins, thromboxane and leukotrienes . Drugs, 33 (Suppl. l):2-9.

PAGE 272

257 SAS. Users guide. Cary, NC: SAS Institute Inc.; 1985. Sawyer, H.R., Niswender, K.D., Braden, T.D., and Niswender, G.D., 1990. Nucleaqr changes in ovine luteal cells in response to PGF2a. Dom. Anim. Endocrinol. 7:229-238. Scaramuzzi , R.J., and Baird, D.T., 1976. The oestrus cycle after active immunization aganinst prostaglnadin F-2 alpha. J. Reprod. Fertil. , 46:39-47. Scaramuzzi, R.J., Baird, D.T., Boyle, H.P., Land, R.B., and Wheeler A.G., 1977. The secretion of prostaglandin F from the autotransplanted uterus of the ewe. J. Reprod. Fertil., 49:157-160. Schalue, T.K., Cross, J.C., Keisler, D., Sikes, J.D., and Roberts, R.M. , 1989. Bovine interferon alpha1 increases pregnancy rates in ewes. Biol. Reprod., 40:85 (abstr.). Schalue, T.K., Farin, P.W., Cross, J.C., Keisler, D. , and Roberts, R.M. , 1991. Effect of injected bovine interferon-c^l on oestrous cycle length and pregnancy success in sheep. J. Reprod. Fertil., 91:347-356. Schindler, C, Shuai, K. , Prezioso, V.R., and Darnell, J.E., 1992. Interf eron-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science, 257:809-812. Sernia, C, Thomas, W.G., and Gemmell, R.T., 1991. Oxytocin receptors in the mammary gland and reproductive tract to a marsupial the brushtail possum (Trichosurus vulpecula) . Biol. Reprod., 45:673-679. Sharif, S.F., Francis, H. , Keisler, D.H., and Roberts, R.M. , 1989. Correlation between the release of ovine trophoblast protein1 by the conceptus and the production of polypeptides by the maternal endometrium of ewes. J. Reprod. Fertil., 85:471-476. Sharma, S.C., and Fitzpatrick, R.J., 1974. Effectt of oestradiol1715 and oxytocin treatment on prostaglandin F alpha release in the anoestrous ewe. Prostaglandins, 6:97-105. Sheldrick, E.L., and Flick-Smith, H.C., 1993. Effect of ovarian hormones on oxytocin receptor concentrations in explants of uterus from ovariectomized ewes. J. Reprod. Fertil., 97:241-245.

PAGE 273

258 Sheldrick, E.L., and Flint, A.P.E., 1985. Endocrine control of uterine oxytocin receptors in the ewe. J. Endocrinology, 106:249-258. Shemesh, M. , Ailenberg, M. , Lavi , S., and Mileguir, F . , 1981. regulation of prostaglandin biosynthesis by an endogenous inhibitor from bovine placenta. In: Dynamics of Ovarian Function. Scwartz, N.B., and Hunzicker-Dunn , M. , editors. Raven Press, New York. pp. 161-166 . Short, E.C., Geisert, R.D., Helmer, S.D., Zavy, M.T., and Fulton, R.W. , 1991. Expression of antiviral activity and induction of 2 ' , 5 ' -oligoadenylate synthetase by conceptus secretory proteins enriched in bovine trophoblast protein-1. Biol. Reprod. , 44:261-268. Short, R.V. , 1969. Implantation and the maternal recognition of pregnancy. In: Foetal Autonomy. Wolestenholme , G.E.W., and O'Connor, M. , editors. Ciba Foundation, New York. pp:2-31. Short, R.V. , McDonald, M.F., and Rowson, L.E.A., 1963. Steroids in the ovarian venous blood of ewes before and after gonadotrophic stimulation. J. Endocrin. , 26:155169. Silvennoinen, 0., Ihle, J.N. , Schlessinger , J., and Levy, D.E., 1993. Interf eron-induced nuclear signaling by Jak protein tyrosine kinases. Nature 366:583-585. Silvia, W.J., Fitz, T.A., Mayan, M.N. , and Niswender, G.D., 1984a. Cellular and molecular mechanisms involved in luteolysis and maternal recognition of pregnancy in the ewe. Animal Reproduction Science, 7:57-74. Silvia, W.J., and Homanics, G.E., 1988. Role of phosphollipase C in mediating oxytocin-induced release of prostaglandin F 2a from ovine endometrial tissue. Prostaglandins, 35:435-548. Silvia, W.J., Lewis, G.S., McCracken, J. A. , Thatcher, W.W. , and Wilson, L., 1991. Hormonal regulation of uterine secretion of prostaglandin F 2a during luteolysis in Ruminants. Biol. Reprod., 45:655-663. Silvia, W.J., and Niswender, G.D., 1984. Maintenance of the corpus luteum of early pregnancy in the ewe. III. Differences between pregnant and nonpregnant ewes in luteal responsiveness to prostaglandin F 2a . J. Anim. Sci. , 59 (3) :746-753.

PAGE 274

259 Silvia, W.J, and Niswender, G.D., 1986. Maintenance of the corpus luteum of early pregnancy in the ewe. IV. Changes in luteal sensitivity to prostaglandin throughout early pregnancy. J. Anim. Sci., 63:12011207 . Silvia, W.J., Ottobre, J.S., and Inskeep, E.K., 1984b. Concentrations of prostaglandins E 2 , F 2a and 6-ketoprostaglandin FjO. in the uterine venous plasma of nonpregnant and early pregnant ewes. Biol. Reprod. , 30:936-944. Silvia, W.J., and Raw, R.E., 1993. Activity of phospholipase C and release of prostaglandin F 2o by endometrium tissue from ewes during the oestrous cycle and early pregnancy. J. Reprod. Fertil., 97:529-537. Silvia, W.J., Raw, R.E., Aldrich, S.L., and Hayes, S.H., 1992. Uterine secretion of prostaglandin F 2a in response to oxytocin in ewes: changes during the estrous cycle and early pregnancy. Biol. Reprod., 46: 1007-1015. Singer, M. , and Berg, P., 1991. The Logic and Machinery of Gene Expression . In : Genes and Genomes. Singer, M. , editor. Plenum Press, New York. pp. 183-186. Smith, P.K., Krohn, R.I., Hermanson, G.T., Millia, A.K., Gartner, F.H., Provenzano, M.D., Fu j imoto , E.K., and Goeke, N.M. , Olson, B.J., 1985. Measurement of protein using bicinchoninic acid [published erratum apperas in Anal. Biochem. 1987 May 15; 163:279]. Anal. Biochem. , 150:76-85. Smith, W.L., 1986. Prostaglandin biosynthesis and its compartmentation in vascular smooth muscle and endothelial cells. Annu. Rev. Physiol., 48:251-261. Smith, W.L., 1989. The eicosanoids and their biochemical mechanisms of action. J. Biochem., 259:315-324. Smith, W.L., Marnett, L.J., and DeWitt, D.L., 1991. Prostaglandin and thromboxane biosynthesis. Pharmac . Ther., 49:153-179. Snedecor, G.W. , and Cochran, W.G., 1980. In: Statistical Methods, 7th ed. Ames, IA: Iowa State University Press, pp. 298-333.

PAGE 275

260 Soloff, M.S., 1975. Uterine receptor for oxytocin: effects of estrogen. Biochem. Biophys . Res. Comm., 65:205212. Spencer, T.E., and Bazer, F.W., 1995. Temporal and spacial alterations in uterine estrogen receptor and progesterone receptor gene expression during the estrous cycle and early pregnancy in the ewe. Biol. Reprod. , 53:1527-1543. Spencer, T.E., Becker W.C., George, P., Mirando, M.A. , Ogle, T.F., and Bazer, F.W., 1995a. Ovine interferon-t inhibits extrogen receptor up-regulation and estrogeninduced luteolysis in cyclic ewes. Endocrinology, 136:4932-4944. Spencer, T.E., Becker, W.C., George, P., Mirando, M.A. , Ogle, T.F., and Bazer, F.W., 1995b. Ovine interferon-t regulates expression of endometrial receptors for estrogen and oxytocin but not progesterone. Biol. Reprod., 53:732-745. Spencer, T.E., Ott, T.L., and Bazer, F.W., 1996. tInterferon: pregnancy recognition signal in ruminants. Proc. Soc. Exp. Biol. Med., 37:215-229. Stabenfeldt, G.H., Holdt, J. A., and Ewing, L.L., 1969. Peripheral plasma progesterone levels during the ovine estrous cycle. Endocrinol., 85:11-14. Stevenson, K.R., Riley, P.R., Stewart, H.J., Flint, A.P.F., and Wathes, D.C., 1994. Localization of oxytocin receptor and mRNA in the ovine uterus during the oestrus cycle and early pregnancy. J. Molec. Endocr. 12:93-105. Stewert, H.J., Flint, A.P.F., Lamming, G.E., McCann, S.H.E., and Parkinson, T.J., 1989a. Antiluteolytic effects of blastocyst-secreted interferon investigated in vitro and in vivo in the sheep. J. Reprod. Fertil., 37:127138. Stewert, H.J., McCann, S.H.E., Baker, P.J., Lee, K.E., Lamming, G.E., and Flint, A.P.F., 1987. Interferon sequence homology and receptor binding activity of ovine trophoblast antiluteolytic protein. J. Endocrinol., 115:R13-R15. Stewert, H.J., McCann, S.H.E., Northrop, A.J., Lamming, G.E., and Flint, A.P.F., 1989b. Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels. J. Mol. Endocrinol., 2:65-70.

PAGE 276

261 Stormshack, F., Kelley, H.E., and Hawk, H.W. , 1969. Suppression of ovine luteal function by 17ft-estradiol . J. An. Sci. , 29:476-478. Thatcher, W.W. , Hansen, P.J., Gross, T.S., Helmer, S.D., Plante, C. , and Bazer, F.W., 1989. Antiluteolytic effects of bovine trophoblast protein-1. J. Reprod. Fertil., 37:91-99. Thornburn, G.D., Cox, R.I., Currie, W.B., Restall, R.J., and Schneider, W. , 1973. Prostaglandin F and progesterone concentrations in the utero-ovarian venous plasma of the ewe during the oestrous cycle and early pregnancy. J. Reprod. Fertil., 18:151-158. Tuo, W. , Ott, T.L., Bazer, and F.W., 1993. Natural killer cell activity of lymphocytes exposed to ovine, type I, trophoblast interferon. Am. J. Reprod. Immunol., 29 : 26-34. Tseng, L, and Gurpide, E., 1975. Effects of progestins on estradiol receptor levels in human endometrium. J. Clin. Endocrinol. Metab., 41:402-404. Turzillo, A.M., Junegal, J.L., and Nett, T.M., 1995. Pulsatile gonadotropin-releasing hormone (GnRH) increases concentrations of GnRH receptor messenger ribonucleis acid and numbers of GnRH receptors during luteolusis in the ewe. Biol. Reprod. 53:418-423. Umesono, K. , and Evans, R.M. , 1989. Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell, 57:1139-1146. Vallet, J.L, and Bazer, F.W., 1989. Effect of ovine trophoblast protein-1, oestrogen and progesterone on oxytocin-induced phosphatidylinositol turnover in endometrium of sheep. J. Reprod. Fertil., 87:755-761. Vallet, J.L., Bazer, F.W., and Roberts, R.M. , 1987. The effect of ovine trophoblast protein-one on endometrial protein secretion and cyclic nucleotides. Biol. Reprod. , 37 : 1307-1316 . Vallet, J.L., Bazer, F.W., Fliss, M.F.V., and Thatcher, W.W. , 1988. Effect of ovine conceptus secretory proteins and purified ovine trophoblast protein-1 on interoestrous interval and plasma concentrations of prostaglandins F-2a and E and of 13 , 14-dihydro-15-keto prostaglandin F-2ct in cyclic ewes. J. Reprod. Fertil., 84:493-504.

PAGE 277

262 Vallet, J.L., Gross, T.S., Fliss, M.F.V., and Bazer, F.W., 1989. Effects of pregnancy, oxytocin, ovine trophoblast protein1 and their interactions on endometrial production of prostaglandin F 2a in vitro in perifusion chambers. Prostaglandin, 38:113-124. Vallet, J.L. , and Lamming, G.E., 1991. Ovine conceptus secretory proteins and bovine recombinant interferon a,l decreased endometrial oxytocin receptor concentrations in cyclic and progesteronetreated ovariectomized ewes. J. Endocrinol., 131:475-482. Vallet, J.L., Lamming, G.E., and Batten, M. , 1990. Control of endometrial oxytocin receptor and uterine response to oxytocin by progesterone and oestradiol in the ewe. J. Reprod. Fertil., 90:625-634. Van Heeke, G. , Ott, T.L., Strauss, A., Ammaturo, D. , and Bazer, F.W., 1996. High yield expression and secretion of the ovine pregnancy recognition hormone interferontau by pichia pastor is . J. Interferon and Cytokine Research, 16:119-126. Vu Haj , M.T., Logear, F., Warembourg, M. , and Milgrom, E., 1977. Biochemical actions of progesterone and progestin. Ann. N.Y. Acad. Sci., 286:199-209. Wahli, W. , and Martinez, E. , 1991. Superfamily of steroid nuclear receptors: Positive and negative regulators of gene expression. FASEB J., 5:2243-2249. Walker, D.L., Weston, P.G., and Hixon, J.E., 1997. Influence of estradiol and progesterone withdrawal on the secretion of the temporal correlation between pulses of oxytocin and prostaglandin F 2a in ewes. Biol. Reprod., 56:1228-1238. Wallace, J.M. , Helliwell, R. , and Morgan, P.J., 1991. Autoradiographical localization of oxytocin binding sites on ovine oviduct and uterus throughout the oestrous cycle. Reprod. Fertil. Develop., 3:127-135. Wathes, D.C., and Denning-Kendal , P. A., 1992. Control of the systhesis and secretion of ovarian oxytocin in ruminants. J. Reprod. Fertil., 45 (Suppl .): 39-52 . Wathes, D.C., and Hamon, M. , 1993. Localization of oestradiol, progesterone and oxytocin receptors in the uterus diring the oestrus cycle and early pregnancy of the ewe. J. Encocrin. , 138:479-491.

PAGE 278

263 Wathes, D.C., and Lamming, G.E., 1995. The oxytocin receptor, luteolysis and the maintenance of pregnancy. J. Reprod. Fertil., (Suppl.) 49:53-67. Wathes, D.C. and Swann, R. , 1982. Is oxytocin an ovarian hormone? Nature, 297:225-227. Welshons, W.V. , Krummel, B . A . , and Gorski, J., 1985. Nuclear localization of unoccupied receptors for glucocorticoids, estrogens, and progesterone in GH 3 cells. Endocrinology, 117 (5) : 2140-2147 . Welshons, W.V. , Lieberman, M.E., and Gorski, J., 1984. Nuclear localization of unoccupied oestrogen receptors. Nature, 307:747-749. West, N.B., Verhage, H.G., and Brenner, R.M. , 1976. Suppression of the estradiol receptor system by progesterone in the oviduct and uterus of the cat. Endocrinol., 99:1010-1016. Williams, B.R.G., 1991a. Signal transduction and transcriptional regulation of interf eron-a-stimulated genes. J. Interferon Res., 11:207-213. Williams, B.R.G., 1991b. Transcriptional regulation of interf eron-stimulated genes. Eur. J. Biochem. , 200:111. Wilson, L. , Butcher, R.L., and Inskeep, E.K., 1972. Prostaglandin F 2a in the uterus of ewes during early pernancy. Prostaglandins, 1:479-484. Wilson, M.E., Lewis, G.S., and Bazer, F.W., 1979. Proteins of ovine blastocyst origin. Biol. Reprod. , 20 (Suppl. ) : 101. Wiltbank, M.C., Diskin, M.G., and Niswender, G.D., 1991. Differential actions of second messenger systems in the corpus luteum. J. Reprod. Fertil., 43:65-75. Wiltbank, M.C., Knickerbocker, J.J., and Niswender, G.D., 1989. Regulation of the corpus luteum by protein kinase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells. Biol. Reprod., 40:1194-1200. Wiltbank, M.C., Wiepz , G.J., Knickerbocker, J.J., Belfiore, C.J., and Niswender, G.D., 1992. Proteins secreted from the early ovine conceptuses block the action of

PAGE 279

264 prostaglandin F 2a on large luteal cells. Biol. Reprod. , 46:475-482. Zarco, L. , Stabenfeldt, G.H., Basu, S., Bradford, and G.E., Kindahl, H. , 1988a. Modification of prostaglandin F2 2a synthesis and release in the ewe during the initial establishment of pregnancy. J. Reprod. Fertil., 83: 527-536 . Zarco, L., Stabenfeldt, G.H. , Quirke, J.F., Kindahl, H. , Hughes, J. P., and Bradford, G.E., 1988b. Release of prostaglandin F 2a and the timing of events associated with luteolysis in ewes with oestrous cycles of different length. J. Reprod. Fertil., 83:517-526. Zelinski, M.B. , Hirota, N.A. , Keenan, E.J., and Stormshak, F., 1980. Influence of exogenous estradiol17S on endometrial progesterone and estrogen receptors during the luteal phase of the ovine estrous cycle. Biol. Reprod., 23:743-751. Zhang, J., Weston, P.G., and Hixon, J.E., 1992. Role of progesterone and oestradiol in the regulation of uterine oxytocin receptors in ewes. J. Reprod. Fertil., 94:395-404.

PAGE 280

BIOGRAPHICAL SKETCH Tammie K. Schalue received her B.S. degree from the University of Missouri in 1987. She received her M.S. degree from the University of Missouri in 1990 under the direction of Dr. J.D. Sikes and Dr. R.M. Roberts. In 1990 she began a Ph.D. program under the guidance of Dr. F.W. Bazer and Dr. W.W. Thatcher at the University of Florida. In 1994 she continued her studies in the field of assisted reproductive technologies and preimplantation embryo diagnosis under the direction of Dr. S. Williams and Dr. K. Drury in the Ob/Gyn Department at the University of Florida. While in the Department of Ob/Gyn at Florida she collaborated with Dr. S. Kippersztok, in the Department of Ob/Gyn, on several reproductive toxicology studies. She is currently Director of the Preimplantation Embryo Genetic Diagnosis Laboratory and Research Assistant Professor at the University of Kansas School of Medicine in Wichita, Kansas. 265

PAGE 281

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. , Fuller W. Bazer, Co-chairman Graduate Research Professor of Animal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. William W. Thatcher, Co-Chairman Graduate Research Professor of Dairy and Poultry Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Ph~±Apsophy . William C. Buhi Professor of Biochemistry and Molecular Biology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Howard M. Jdhnsoi Graduate Research Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor -pfr Philosophy. JD C Dan C. Sharp III Professor of Animal Science

PAGE 282

This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 1997 Pean, College of Agriculture Dean, Graduate School


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EQAO5UKRT_5Q1BW2 INGEST_TIME 2015-04-02T21:30:37Z PACKAGE AA00029955_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES