Title: In vivo and in vitro responses of cattle to prostaglandin F2a
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Title: In vivo and in vitro responses of cattle to prostaglandin F2a
Physical Description: xiv, 168 leaves : ill. ; 28 cm.
Language: English
Creator: Chenault, John Robert, 1949-
Copyright Date: 1977
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Subject: Cattle -- Reproduction   ( lcsh )
Cattle -- Physiology   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- UF -- Animal Science
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non-fiction   ( marcgt )
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Statement of Responsibility: by John Robert Chenault.
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 154-167.
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General Note: Typescript.
General Note: Vita.
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 000010039
oclc - 02897140
notis - AAB2151

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IN VIVO AND IN VITRO RESPONSES OF
CATTLE TO PROSTAGLANDIN F2












By

JOHN ROBERT CHENAULT


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




UNIVERSITY OF FLORIDA


1977














ACKNOWLEDGMENTS


The author is deeply grateful to Dr. W. W. Thatcher,

chairman of the supervisory committee, for his concern,

support, patience, advice and encouragement during these

studies.

Drs. R. M. Abrams, F. W. Bazer, M. J. Fields, M. J.

Fregly, R. M. Roberts and C. J. Wilcox are acknowledged for

their help as members of the supervisory committee with spe-

cial thanks extended to Dr. C. J. Wilcox for his invaluable

assistance with statistical analyses and preparation of this

manuscript and to Drs. R. M. Abrams and M. J. Fields for

additional assistance with various aspects of these studies.

The author would like to acknowledge L. Owens, D. Starr,

D. Clarke and B. Patterson for their excellent help in the

laboratory and Drs. W. S. Cripe and H. N. Becker for their

assistance with ovariectomies.

All fellow graduate students and, in particular, R. W.

Adkinson, F. C. Gwazdauskas, H. Roman, L. C. Fernandes, R. M.

Eley and F. Bartol are thanked for technical and academic

assistance and friendship.







The author would like to express his deep felt appre-

ciation for the understanding, support and love received

from family and personal friends over the course of these

studies and to acknowledge that without such support these

studies may not have been completed.


iii













TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS . . . . . . . . . ii

LIST OF TABLES. . ... . . . . . vii

LIST OF FIGURES . . . . . . . . .. ix

ABSTRACT. . . . . . . . . . . xi

INTRODUCTION. .. .. . . . . . . . .1

SECTION

I LITERATURE REVIEW. . . . . . . . 3
Prostaglandins. . . . . . . . 3
Luteolytic Effect of Prostaglandin F 3
Pharmacological Effects of PGF2 . 13
Follicular Steroidogenesis . . . 18

II IN VIVO RESPONSES TO PFG2. THAM SALT ..... 40
Materials and Methods . . . . . 40
Experiment 1. Response of Dairy Heifers
to Two Injections of PGF2 12 Days
Apart . . . . . . 40
Experiment 2. Physiological Response of
Dairy Cattle to a Luteolytic Dose of
PGF2a Tham Salt Administered Intramus-
cularly . . . . . . . . 41
Thermocouple preparation and
calibration . . . . . 42
Surgical preparation and experimental
protocol. . . . ... .43
Experiment 3. Physiological Response to
a Luteolytic Dose of PGF2a Tham Salt
Administered Intravenously . . .. 45
Experiment 4. Effects of PGF2a Tham
Salt on Uterine Blood Flow in Sheep. 46
Results and Discussion. ... .. . . .. 49
Experiment 1. Response of Dairy Heifers
to Two Injections of PGF2( 12 Days
Apart. . . . . . . . .. 49








TABLE OF CONTENTS (continued)


SECTION Page

II Experiment 2. Physiological Response of
(cont.) Dairy Cattle to a Luteolytic Dose of
PGF2a Tham Salt Administered Intramus-
cularly. . .. . . . . . . 57
Experiment 3. Physiological Response to
a Luteolytic Dose of PGF a Tham Salt
Administered Intravenous y . . . 61
Experiment 4. Effect of PGF2a Tham Salt
on Uterine Blood Flow in Sheep . . 68

III IN VITRO BOVINE FOLLICULAR STEROIDOGENESIS . 73
Materials and Methods ... . . . 73
Experimental Design . . . . .. 79
Experiment 1. Estradiol Secretion by
Bovine Follicles In Vitro: Test of
Incubation System. . . . . ... 79
Experiment 2. Effects of PGF2( and LH
on In Vitro Estradiol Secretion by
Bovine Follicles . . . . .. 80
Experiment 3. Effects of PGF2a on In
Vitro Estradiol Secretion by Bovine
Follicles. . . . . . . .. 82
Experiment 4. Effects of FSH, Testoster-
one and FSH plus Testosterone on In Vitro
Estradiol Secretion by Bovine Follicles. 82
Experiment 5. Histology of Bovine Folli-
cles Induced with FSH-p: Before or After
In Vitro Incubation. . . . . ... 83
Results and Discussion. .. ..... . 84
Experiment 1. Estradiol Secretion by
Bovine Follicles In Vitro: Test of
Incubation System. . . . . ... 84
Experiment 2. Effects of PGF2a and LH
or In Vitro Estradiol Secretion by
Bovine Follicles . . . . . . 91
Experiment 3. Effect of PGF2a on In
Vitro Estradiol Secretion by Bovine
Follicles . . . . . . . 103
Experiment 4. Effects of FSH, Testoster-
one and FSH plus Testosterone on In Vitro
Estradiol Secretion by Bovine Follicles. 106
Experiment 5. Histology of Bovine
Follicles Induced with FSH-p: Before
or After In Vitro Incubation . . . 110








TABLE OF CONTENTS (continued)


SECTION

IV

APPENDICE

I

II

III

REFERENCE

BIOGRAPHI


SUMMARY AND CONCLUSIONS. . . . . .

S

MEDIUM 199 . . . . . . . .

HISTOLOGICAL SOLUTIONS AND PROCEDURES. .

HORMONAL DATA AND STATISTICAL ANALYSES

S . . . . . . . . . .

CAL SKETCH . . . . . . . .


Page

. 125


133

135

139

154

168













LIST OF TABLES


Table Page

1 Distribution of estrus in dairy heifers follow-
ing injection of PGF2a tham salt. . . . 53

2 Mean arterial blood pressure response of sheep to
8 mg PGF2a tham salt administered intravenously 69

3 FSH-p injection schedule . . . . . . 75

4 Estradiol biosynthesis in vitro by whole folli-
cles from FSH-p treated cows. . . . . ... 86

5 Total estradiol in half follicles . . ... 89

6 Medium 199 ingredients per liter. . . . ... 134

7 Bouins fixation solution. . . . . . ... 136

8 Dehydration, dealcoholization and infiltration
procedures. . . . . . . . . . 137

9 Hematoxylin and eosin staining procedures . . 138

10 Estradiol secretion by incubated bovine follicles in
Experiment 1, Section III . . . . . .. 140

11 Extradiol secretion by bovine follicles in
Experiment 2, Section III . . . .. ... . 141

12 Progestin secretion by bovine follicles in
Experiment 2, Section III . . . . . 142

13 Testosterone secretion by bovine follicles in
Experiment 2, Section III . . . .. . . 143

14 Estradiol secretion by bovine follicles in
Experiment 3, Section III . . . . . . 144

15 Estradiol secretion by bovine follicles in
Experiment 4, Section III . . . . . . 146


vii








LIST OF TABLES (continued)


Table Page

16 Analysis of variance for estradiol secretion in
Experiment 1, Section III . . . . . 148

17 Analysis of variance for estradiol secretion in
Experiment 2, Section III . . . . . 149

18 Analysis of variance for progestin secretion in
Experiment 2, Section III. . . . . ... 150

19 Analysis of variance for testosterone secretion
in Experiment 2, Section III . . . .. 151

20 Analysis of variance for estradiol secretion in
Experiment 3, Section III. . . . . ... 152

21 Analysis of variance for estradiol secretion in
Experiment 4, Section III. . . . . . 153


viii














LIST OF FIGURES


Figure Page

1 Estrogen biosynthetic pathways. . . . ... 24

2 Heifer distribution during the estrous cycle on
each injection day when two PGF tham salt
injections were given 12 days a art .. .. 51

3 Mean arterial blood pressure responses to
intramuscular injections of PGF2t tham salt
or saline . . . . . . . .. . 59
4 Mean arterial blood pressure and mean heart
rate responses to two intravenous infusions
of PGF2c tham salt given within the same cow. 62

5 Mean uterine temperature and mean arterial
blood temperature responses to two intravenous
infusions of PGF2a tham salt given within the
same cow. . . . . . . . . .... 65

6 Uterine blood flow response to intravenous
injection of PGF2a tham salt in estradiol
primed ovariectomized sheep . .. . .... 71

7 Estradiol secretion by FSH-p induced bovine
follicles in vitro. . . . . . . ... 87

8 Effects of PGF2a and LH on in vitro estradiol
secretion by bovine follicles . . . . 92

9 Effects of PGF2a and LH on in vitro progestin
secretion by bovine follicles . . . . 95

10 Effects of PGF2a and LH on in vitro testosterone
secretion by bovine follicles . . . . 99

11 Effects of PGF2a on in vitro estradiol secre-
tion by bovine follicles. . . .. . . 104

12 Effects of FSH, testosterone and FSH plus
testosterone on in vitro estradiol secretion
by bovine follicles . . .. . . 107








LIST OF FIGURES (continued)


Figure Page

13 Portion of a nonincubated, FSH-p induced
follicle . . . . . . . . . 112

14 High power of a punctured, unincubated follicle. 112

15 High power of a nonincubated follicle demon-
strating vacuoles. . . . . . .. 114

16 Portion of a punctured, unincubated follicle 116

17 Portion of an incubated follicle . . . . 119

18 High power of an incubated follicle. . . .. 119

19 Comparison of granulosa cells in a punctured,
nonincubated follicle with pyknotic granulosa
cells free floating in the antrum of an
incubated follicle ... . . . . . 120

20 High power of an incubated follicle. . . 122








Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IN VIVO AND IN VITRO RESPONSES OF
CATTLE TO PROSTAGLANDIN F2

By

John Robert Chenault

March, 1977

Chairman: W. W. Thatcher
Major Department: Animal Science

Dairy heifers (n=37) were treated twice with 33.5 mg

prostaglandin F2x tham salt (PGF2a) 12 days apart to deter-

mine if this management scheme increased percentage of

animals expressing estrus after the second injection.

Average plasma progestin concentrations were 3.33 and 6.51

ng per ml at the first and second injection. A greater per-

centage of heifers expressed estrus after the second injec-

tion (89 vs 60%). Consequently, treatment of cattle two

times, 12 days apart, is a management scheme that increases

percentage of animals undergoing corpus luteum regression

for subsequent insemination.

To determine if a luteolytic dose of PGF2. exerted any

physiological effects that may detract from its practical

usefulness, an experiment was conducted to determine if

heart rate, blood pressure, arterial blood temperature and

uterine temperature change after PGF2a injection. To








monitor these responses a polyvinyl catheter and a thermo-

couple were surgically placed into the external iliac artery

and an additional thermocouple placed into the uterine serosa

of two cows.

In response to intramuscular injections (IM) of either

33.5 mg PGF2 (n=3) or saline (n=3), there was a slight rise

in mean blood pressure and heart rate. No major alterations

in blood or uterine temperatures occurred over 3 hr follow-

ing PGF2a injection.

Following 2 min intravenous infusion of PGF2( (33.5 mg;

n=2), blood pressure increased from 140 mm Hg to 230 mm Hg,

whereas heart rate decreased from 34 to 16 beats per 30 sec.

Associated with these changes were an increase in uterine

temperature and a concurrent widening in the temperature dif-

ference between the uterus and arterial blood. These re-

sults are suggestive of a marked decrease in uterine blood

flow. A study utilizing electromagnetic blood flow trans-

ducer probes demonstrated that PGF2a (8 mg; intramuscularly)

decreased uterine blood flow (72 to 41 ml per min) in

estradiol primed (20 ig), ovariectomized sheep (n=3).

Experiments indicated that a luteolytic dose of PGF2.

administered IM caused no major alterations in the physio-

logical parameters measured, whereas intravenous infusion

caused major alterations in circulatory homeostasis.


xii








An in vitro incubation system was developed to study

bovine ovarian follicular steroidogenesis. Follicles were

obtained from cows treated twice daily with FSH-p (Armour

Baldwin Labs) on days 16 to 19 of the estrous cycle. After

follicle dissection from the ovary, follicular fluid was

aspirated and follicles incubated individually for 14 hr dur-

ing which the medium was changed every 2 hr. Hormonal treat-

ments were added to the medium, and treatment effects

analyzed by least squares analyses. Incubation medium

(100 ml) consisted of 80% Medium 199 with Hanks salts, 20%

fetal calf serum, 5.6 mg insulin, 5.6 mg ascorbic acid and

1.8 mg gentamicin.

Follicles (n=5) incubated for 14 hr secreted four

times more estradiol into the medium than extracted from

unincubated frozen follicles (n=5; P<.01). In Experiment 2,

PGF2a (5 ng per ml medium) and LH (50 ng per ml medium) had

no effect on in vitro estradiol secretion. Progestin secre-

tion increased during incubation in control follicles and

this increase was stimulated oy LH (P<.01). In control and

PGF2a treated follicles testosterone secretion increased

in the pretreatment periods and decreased gradually during

treatment periods. However, LH treatment stimulated testo-

sterone secretion (P<.01).


xiii








PGF2a added to medium at doses of 5, 100 and 1000 ng

per ml had no effect on estradiol secretion in Experiment 3.

Similarly, FSH (100 ng per ml), testosterone (5 X 10-7M)

or combination of both had no effect on estradiol secretion

in Experiment 4.

Histological examination of incubated follicles sug-

gested that this nonresponsiveness of estradiol secretion

may be due to disassociation of granulosa cells from the

basal lamina into the antrum.


xiv













INTRODUCTION


Major limitations to artificial insemination in cattle

are failure to detect estrus and improper timing of insem-

ination. In recent years there has been considerable inter-

est in establishing an ovulation control system which

includes a predetermined time for insemination. Optimally,

such a system would eliminate the need for estrous detection

and provide a precise time for insemination which maximizes

conception rates. Such reproductive management programs

would be of paramount importance in the Florida dairy

industry where large herd sizes hamper reproductive manage-

ment efficiency.

Recent studies indicated that prostaglandin F2(

(PGF2,) may be used for ovulation control in cattle. PGF2x

induces luteal regression which is followed by ovulation;

however, it is not effective during the first 5 days of the

estrous cycle. Therefore, its general usefulness as a

practical tool for ovulation control is limited unless

alternative management systems are developed. Very few

studies have been conducted in cattle to determine if PGF2(

has any detrimental physiological effects which may limit

its general usefulness.








Objectives of the first series of experiments were

twofold:

1. to test a dual injection management system for

PGF2a administration. This system was designed to

eliminate animals from being in the nonresponsive

first 5 days of the estrous cycle at the second

PGF2. administration.

2. to determine if PGF2. affects heart rate, mean

arterial blood pressure, arterial blood temperature

and uterine temperature when administered in a

luteolytic dose to dairy cattle.

PGF2o also has been implicated as a natural luteolytic

agent for luteal regression in several species and to affect

estradiol secretion in cattle. In a second series of experi-

ments an incubation system for bovine ovarian follicles was

developed to study the effects of PGF2. and gonadotropins on

follicular estradiol secretion in vitro. Results from these

studies may contribute to our understanding of follicular

steroidogenesis and corpus luteum regression. These

phenomena must be understood before luteal regression, fol-

licular maturation and ovulation can be controlled optimally.













SECTION I

LITERATURE REVIEW



Prostaglandins


Prostaglandins (PG) are unsaturated 20 carbon fatty

acids containing a cyclopentane ring and two aliphatic side

chains. This class of compounds is widely distributed in

animal tissues and has powerful pharmacological effects in

many biological systems. Recent findings indicate that PGs

are produced physiologically and may act as local hormones

or autocoids.


Luteolytic Effect of Prostaglandin F2


Prostaglandin F2a (PGF2a) has various physiological

effects, and one of these is its lytic action on the corpus

luteum. PGF2a induces luteal regression in many species,

including bovine, ovine and equine, and this response has

stimulated the interest of animal scientists. Inskeep (1973)

reviewed the luteolytic properties of PGF2a in domestic

animals, and they have been well documented in the bovine

(Chenault et al., 1976; Hafs et al., 1974, Thatcher and

Chenault, 1976).








In cattle, injection of PGF2a is followed by a sequence

of hormonal patterns very similar to those observed during

spontaneous corpus luteum regression, follicular maturation

and ovulation. Following a single administration of PGF2a,

plasma progestins decline rapidly reaching estrus concentra-

tions by 24 hr posttreatment, whereas estradiol concentra-

tions slowly increase and stimulate an ovulatory surge of

luteinizing hormone (LH) at 79+ 21 hr (Y+S.D.) posttreatment.

Ovulation occurs at 99.5+ 19 hr after PGF2. administration

(Chenault et al., 1976). Various workers have demonstrated

that fertility after the post-PGF2a ovulation is comparable

to fertility after spontaneous ovulations in untreated

herdmates (Ellicott et al., 1974; Lambert et al., 1975;

Lauderdale et al., 1974; Tobey and Hansel, 1975). Lauderdale

et al. (1974) also demonstrated that fertility in animals

bred twice at 72 and 90 hr post-PGF2x, regardless of estrus,

was comparable to fertility in both untreated controls and

PGF2a treated animals bred at the posttreatment estrus.

PGF2a induction of luteal regression followed by ovu-

lation and normal fertility provides the minimal require-

ments for ovulation control. However, there are several

factors associated with use of a single injection of PGF2(

which limits its practical usefulness for ovulation control.

It is well documented that PGF2a is effective only after

day 5 of the estrous cycle (Cooper, 1974; Lauderdale, 1972;








Henricks et al., 1974) when a functional corpus luteum is

present. Therefore on any random day of injection, assuming

a 21 day cycle and all animals are cycling, only a 76% maxi-

mum animal response would be expected. The other 24% would

be in the nonresponsive first 5 days of the estrous cycle.

To minimize the number of animals in this nonresponsive

stage of the estrous cycle (first 5 days), Lauderdale et al.

(1974) treated only animals with a palpable corpus luteum

and treated all others 1 week later. Despite this precau-

tion only 65% of the treated animals (n=239) displayed visual

signs of estrus in the 7 days following PGF2. administration.

This can be compared to an 80% level of estrous detection in

untreated herdmates during a concurrent 18 to 25 day interval.

Of the animals expressing estrus in the PGF2a treated group,

90% of these heats were distributed over a 3 day period.

Similar variation in onset of estrus following PGF2. treat-

ment has been reported by Chenault et al. (1976), Louis,

Hafs and Sequin (1973) and Louis, Hafs and Morrow (1974a).

These results indicate that management schemes must be de-

veloped which manipulate the estrous cycle so that all ani-

mals treated respond to PGF2a. Furthermore, the variability

in time of estrus and/or ovulation after PGF2a treatment

must be reduced if such ovulation control systems are to

include a timed insemination component after PGF2a injection.







Several management schemes have been utilized to over-

come the problem of nonresponse during the first 5 days of

the estrous cycle. Lambert et al. (1975) bred all animals

which came into estrus over a 4 day period and then treated

all remaining animals with PGF2a. This treatment scheme

would eliminate animals from being in the nonresponsive

stage of the cycle on the day of PGF2a treatment. Another

method would be to treat all animals with a progestogen for

5 to 7 days and then administer PGF2a The short-term pro-

gestogen treatment would suppress estrus and ovulation in

animals whose corporalutea regressed during the progestogen

treatment. These animals would return to estrus upon with-

drawal of the progestogen block. Cows that had corpora lutea

at the end of progestogen treatment would return to estrus

in response to PGF2. whereas those animals having developing

corpora lutea (days 1 to 5) at the beginning of progestogen

treatment also would have responsive corpora lutea to PGF2,.

This technique has been used for ovulation control, and the

induce ovulations are followed by normal fertility (Heersche

et al., 1974; Roche, 1976a).

An alternate to these management schemes is to treat

all animals twice with PGF2a, 10 to 12 days apart, as sug-

gested by Inskeep (1973). Theoretically, this would increase

the potential percentage of animals in the responsive stage

of the cycle at the second injection. All animals in the







responsive stage of the cycle, days 6 through 21, at the

first day of PGF2a injection will express an estrus and be in

the responsive luteal stage of the cycle at the second in-

jection, 10 to 12 days later. All animals in the nonrespon-

sive stage of the cycle, days 1 to 5, at the first injection

will not respond to PGF2a and will be in a responsive luteal

stage at the second injection. Using this dual injection

technique with an ICI analogue to PGF2a (ICI 80,996), Cooper

(1974) reported that only 2 of 175 animals failed to respond

to the second PGF2a treatment. Furthermore, 90% of the ani-

mals were in estrus between 48 and 72 hr after the second

treatment and fertility of this second estrus was normal.

In a large field study using this technique, Hafs, Mans

and Lamming (1975) reported sufficient synchronization of

ovulation after the second PGF2a injection to obtain fertil-

ity from one insemination at 80 hr after PGF2a comparable

to two timed inseminations at 70 and 80 hr. Furthermore,

fertility in both timed insemination groups was equivalent

to that of untreated herdmates. This large field study in-

dicated that treatment with PGF2a two times, 10 to 12 days

apart, improved potential responsiveness and precision of

synchronization to allow for a single timed insemination

after the second injection of PGF2a. A large field study

by Cooper and Jackson (1975) failed to support these results

concerning a single fixed time of insemination. Fertility








to a single insemination at 72 or 80 hr after the second

PGF2a treatment was 7% lower than fertility in untreated

controls or cattle bred twice at 72 and 96 hr after the

second injection. Both groups of workers agreed, however,

that there was equal fertility between controls and animals

bred twice.

Increased fertility to a single timed insemination

might be accomplished by controlling time of ovulation. An

LH surge is initiated in cattle by synthetic or natural

gonadotropin releasing hormones (Kalra et al., 1974; Zolman

et al., 1973) or estradiol (Hobson and Hansel, 1972; Short

et al., 1973). Injection of gonadotropin releasing hormone

(GnRH, a synthetic releasing hormone) at 48 or 60 hr after

PGF2a resulted in significantly higher plasma LH concentra-

tions than GnRH given at 0, 12 or 24 hr. However, plasma

progestins also were elevated following GnRH at 0, 12, 24

and 48 but not 60 hr after PGF2~(Kalra et al., 1974).

Rodriguez et al. (1975) reported that GnRH given at 48 hr

after PGF2, injection reduced significantly the frequency of

estrous behavior. It is possible that elevated plasma pro-

gestins or premature ovulation induced by GnRH suppressed

estrus in these animals. These two studies suggested that

GnRH should be administered no sooner than 60 hr after PGF2.

Graves et al. (1975) reported that GnRH given at 60 hr

after PGF2. was effective in reducing variation in ovulatory

time.








Welch et al. (1975) reported that injection of estradiol

benzoate (400 pg) at 48 hr after PGF2a reduced variability in

onset of estrus during the period of 56 to 88 hr post-PGF2 .

However, no advantage in fertility was seen in the estradiol

treated animals. No workers have reported an advantage in

fertility rate when PGF2. was used in combination with an LH

release as compared to PGF2a treatment alone (Graves et al.,

1975; Tcbey and Hansel, 1975; Welch et al., 1975).

Differences in reports (Cooper and Jackson, 1975; Hafs

et al., 1975) as to whether fertility at a single timed in-

semination is comparable to that obtained with two timed

inseminations indicates that optimal management systems for

the use of PGF2. have not been fully developed and additional

studies are warranted.

The mechanism by which PGF2a initiates or induces

corpus luteum regression is unknown. Pharriss and Wyngarden

(1969) suggested that PGF2a exerted a venoconstricting effect

on the ovarian vein which resulted in regression by anoxia.

Several authors since have disproved this theory by demon-

strating that the decline in plasma progesterone after PGF2(

could not be correlated with any change in total ovarian

blood flow (Behrman, Yoshingaga and Greep, 1971; McCracken,

Baird and Goding, 1971). However, Novy and Cook (1973) and

Thorburn and Hales (1972) demonstrated that PGF2. may redis-

tribute intra-ovarian blood flow. Using microsphere tech-

niques these workers reported that blood flow to the corpus

luteum was reduced, whereas blood flow to the stroma and








follicular component of the ovary was increased,following

PGF2a administration.

Morphological electron microscopic studies have indi-

cated that PGF2a induced regression of the corpus luteum is

comprised of functional followed by structural regression.

Functional regression is the termination of progesterone

secretion, whereas structural regression is the physical de-

struction of the luteal cell. Furthermore, PGF2a induced

regression of sheep corpora lutea mimics structural and func-

tional sequences characteristic of normal luteal regression

(Stacy, Gremmell and Thorburn, 1976). Lowered plasma pro-

gestin concentrations were observed at 3 hr postinfusion of

a luteolytic dose of PGF2a and occurred prior to any

noticeable disorganization of cell structure. However, by 6

hr postinfusion cellular signs of early regression were

present, and by 24 hr gross signs were observed. Early signs

of regression included lack of secretary granules and accumu-

lation of lipid droplets within the cells. Following in-

fusion of low doses of PGF2a plasma progestin levels decreased

for a short period of time. These decreases were associated

with early signs of functional regression; however struc-

tural regression did not occur.

Because structural regression always was preceded by

accumulation of lipid droplets, Stacy et al. (1976)









postulated that "During regression PGF2a may inhibit one or

more stages in steroidogenesis so that lipid, being diverted

from its normal synthetic pathway, accumulates as droplets

of cholesterol ester in the luteal cell" (p.290).Umo (1975) also

using electron microscopy observed functional regression

prior to any evidence of structural regression following

intramuscular injection of PGF2. in sheep. These results

clearly demonstrated that PGF2. does not cause termination

of progesterone secretion (functional regression) by mass

destruction of the luteal cells, but most likely acts.on

steroidogenic and other biochemical pathways. Structural

regression follows and may be a consequence of these bio-

chemical changes. It has been demonstrated that PGF2ain-

creases lysosomal fragility and this may be the mechanism

by which PGF2. induces structural regression (Weiner and

Kaley, 1972).
Henderson and McNatty (1975) presented a biochemical

hypothesis by which PGF2. may initiate corpus luteum regres-

sion. LH binds to cell membranes of luteal cells (Channing

and Kammerman, 1974) and has a stimulatory effect on pro-

gesterone synthesis. This response is mediated through

activation of the adenylate cyclase system in the cell mem-

brane to produce adenosine 3', 5'-monophosphate (CAMP;

Savard, Marsh and Rice, 1965) which acts as an intracellular

second messenger to stimulate progesterone synthesis.








Henderson and McNatty hypothesized that PGF2a binds to the

membrane of the luteal cell and directly or indirectly pre-

vents transmission of the activating signal from the LH

coupling component to the adenylate cyclase catalytic site.

This would prevent synthesis of CAMP and thereby inhibit pro-

gesterone synthesis. They also suggested that PGF2. may not

be effective during the first 5 days of the estrous cycle be-

cause LH from the ovulatory surge saturates the regulatory

units of the luteal cells, and this bound hormone protects

the young cells. Therefore, PGF2a would only be luteolytic

when sufficient LH molecules have been dissociated from re-

ceptors and thus have exposed the receptors to the action of

PGF2a.

An alternate explanation for the ineffectiveness of

PGF2. during the first 5 days of the estrous cycle can be

postulated. Specific receptors for PGF2. have been demon-

strated in the bovine corpus luteum (Kimball and Lauderdale,

1975; Rao, 1975) and preliminary data indicated that there

were fewer PGF2. receptors on day 4 than on days 8, 12, 14 or

16 (Kimball et al., 1976). These data suggested that PGF2a

may not be effective until specific PGF2a receptors have

been synthesized in the newly formed corpus luteum.

To date, the mechanisms by which PGF2. induces luteal

regression and the reasons for PGF2a being ineffective during

the first 5 days of the cycle are unknown. Additional








studies in this area are essential to further our understand-

ing of these phenomena. Answers to these questions may be

useful in designing ovulation control systems and may in-

crease our understanding of natural luteal regression.

Pharmacological Effects of PGF2.

PGF2a exerts many biological effects other than induc-

tion of luteal regression. These effects range from its

endocrine (Louis et al., 1974b) and previously mentioned

effects on the reproductive system to its potent smooth

muscle stimulatory action (Main, 1973; Nakano and Cole, 1969).

Plasma prolactin and growth hormone were increased

within 10 min and glucocorticoids within 30 min in dairy

heifers, following intramuscular (IM) injection of various

PGF2a doses, within the luteolytic range (15 to 60 mg). No

changes were observed in plasma insulin or free fatty acids

following intravenous (IV) injection (5 mg) or infusion

(15 mg at .5 mg/minute) of PGF2L, whereas plasma glucose

was elevated following IV administration (Louis et al.,

1974b). Plasma LH also was increased following IM injec-

tion, but the interval to the increase (1.5 to 6 hr) and the

amount of increase (2.7 to 17 ng per ml) was highly variable

among animals. More recently this increase has been at-

tributed to the removal of negative inhibition by proges-

terone on the hypothalamo-pituitary axis rather than a








direct stimulatory effect of PGF2a on LH release (Louis,

Stellflug and Hafs, 1975). Lauderdale et al. (1975) ob-

served an increase in plasma catecholamines following Il

administration of PGF2 in mares.

PGF2a also may affect estradiol concentrations in the

bovine. Chenault et al. (1976) observed spikes of estradiol

in peripheral plasma 2 to 9 hr following Ii injection of a

luteolytic dose (33.5 mg) of PGF2L tham salt. Hixon et al.

(1973) observed elevated peripheral plasma estrone and estra-

diol following intraluteal injection of PGF20. Louis et al.

(1974a) failed to detect any increase in estradiol secretion

immediately following administration of PGF2 PGF2a may

directly stimulate follicular estrogen synthesis, as Shemesh

and Hansel (1975) reported that PGF2a stimulated testosterone

secretion by bovine follicular slices.

Effects of PGF2a on peripheral blood pressure depend

on the species of interest. Intravenous injection of PGF2.

causes a fall in arterial blood pressure in rabbits and cats

(Horton and Main, 1964: Ang ard and BergstrBm, 1963), whereas

it has a pressor action in dogs (Nakano and McCurdy, 1968)

and rats (DuCharme, Weeks and Montgomery, 1968; Viguera and

Sunahara, 1969). Intravenous infusion causes constriction

of metarterioles of the mesocecum and cremaster muscles in

rats (Viguera and Sunahara, 1969). However, DuCharme et al.

(1968) suggested the pressor action of PGF2, in rats is








mediated by venoconstriction which is supported by the obser-

vation of Mark et al. (1971) that PGF2a induced constriction

of the saphenous vein in vitro. On the other hand, Nakano

and Cole (1969) concluded that PGF2. induced a vasoconstric-

tion in the regional arteries which resulted in increased

arterial pressure in dogs.

PGF2a does not appear to have a direct action on the

heart, as PGF2a had no effect on force or rate of contrac-

tion in isolated chicken (Horton and Main, 1967) or hamster

hearts (Lee et al., 1965). Neither did PGF2a affect heart

rate when injected into the canine sinus node artery (Nakano,

Chiba and Nakajima, 1971). Therefore, changes in heart rate

observed in several species following PGF2a treatment must

be an indirect effect, possibly through baroreceptor action,

as PGF2a does affect arterial blood pressure.

The gastroinestinal tract is stimulated by PGF2a'

PGF2a stimulates constriction of circular and longitudinal

muscles of the ileum and colon in guinea pig, rat and man

(Bennett and Fleshler, 1970) as well as the dog colon (Vana-

sin et al., 1970).

Uterine contractions are stimulated by PGF2a in the

ewe (Rexroad and Barb, 1975), mare (Capraro et al., 1976),

rat, guinea pig (Horton and Main, 1964, 1967) and human

(Karim et al., 1971).








The mechanism by which PGF22 affects smooth muscles

appears to be a direct action. Its effect on gastrointes-

tinal smooth muscle is not through -neural stimulation as

these effects are not affected by parasympatholytic (anti-

muscarinic) drugs or alpha or beta blocking agents (Main,

1973). Several workers have demonstrated that PGF2a may

affect smooth muscle contractibility through control of intra-

cellular calcium. PGF2. stimulates release of calcium from

the scarcoplasmic reticulum into the cell and inhibits ATP

dependent calcium binding for the reuptake of calcium by the

scarcoplasmic reticulum (Carsten, 1972; Coceani et al., 1969).

These two actions of PGF2a would increase the availability

of intracellular calcium which is essential for muscular

contraction.

Several workers also have indicated a role for PGF2

in thermoregulatory mechanisms. Hales et al. (1973) demon-

strated this action in a series of experiments in cool,

thermoneutral and warm environments. PGF2a injected into

the lateral cerebral ventricle in sheep stimulated brain

pathways involved in the stimulation of heat production. and

vasomotor tone, and inhibited heat loss pathways. Observa-

tions following PGF2. injection included increased rectal

temperature, decreased skin temperature (vasoconstriction)

and decreased respiratory rate.








Intramuscular injection of PGF2a in mares was followed

by increased plasma catecholamines, sweating and reduced

rectal temperature, whereas injection of epinephrine was

followed by sweating, shivering and no decrease in rectal

temperature (Lauderdale et al., 1975; Miller, Lauderdale

and Geng, 1976). These results indicate that PGF2. caused

release of epinephrine which resulted in sweating. However,

PGF2 also appeared to inhibit heat production mechanisms

(such as shivering) as evidenced by the drop in rectal

temperature following PGF2 This effect is opposite the

heat producing and conserving effects of PGF2a when adminis-

tered into the lateral ventricles of sheep (Hales et al.,

1973).

Reports on the pharmacological effects of PGF2a on

cattle are very limited and generally restricted to work

with calves except for the endocrine studies mentioned previously.

Lewis and Eyre (1972) reported that PGF2a increased systemic

blood pressure, pulmonary arterial pressure, abdominal venous

pressure, respiratory volume and heart rate in calves. Ait-

ken and Sanford (1975) also reported increased systemic blood

pressure and heart rate, but observed a decrease in respira-

tory minute volume in response to PGF2a in calves.

With the increased interest in the use of PGF2a as a

basis for an ovulation control system in cattle, additional

research on the pharmacological effects of PGF2a is warranted








to determine if PGF2 has any detrimental biological effects

which may limit its usefulness in cattle.


Follicular Steroidogenesis


Follicles generally are regarded as the primary source

of preovulatory estrogens (Hisaw, 1947). However, beyond

this point there is considerable debate in regard to tissues

and pathways involved in estrogen biosynthesis. There ap-

pears to be considerable species variability and contradic-

tory evidence within species to stimulate this debate.

Ovaries of cattle contain primary, secondary, tertiary

and mature Graafian follicles. Larger follicles consist of

ovum, granulosa cells and theca cells whereas primary folli-

cles consist of the oogonia surrounded by a single layer of

granulosa cells. Follicles are classified as secondary folli-

cles when the zona pellucida has been formed around the cell

membrane of the ovum and granulosa cells have multiplied

into several layers. The tertiary follicle is formed from

a secondary follicle when separation of granulosa cells

occur to form a cavity or antrum. Concurrent with the forma-

tion of the antrum the outer border of granulosa cells is

surrounded by a cell layer of stromal origin designated as

the thecal layer. With enlargement and filling of the antrum

with follicular fluid, the follicle grows and is designated

as a mature Graafian follicle. At the onset of estrus the








follicle which will ovulate is approximately 10 mm in

diameter and increases to 16 to 18 mm by ovulation. During

this time, the mature follicle consists primarily of granu-

losa and theca cells. Generally 6 to 10 layers of granulosa

cells line the antrum. This layer is avascular and is sep-

arated from the theca cells by a lamina propria. There are

four to five layers of theca cells which are highly vascular-

ized and are in opposition with the ovarian stromal tissue.

The theca cells classically are divided into two layers,

the theca internal and theca externa. The theca internal is

composed primarily of spindle shaped connective tissue cells

(fibrocytes); however, near ovulation some hypertrophy occurs

and cells become epithelioid or glandular in appearance. The

theca externa consists of spindle shaped connective tissue

cells (Marion, Gier and Choudary, 1968; Priedkalns and Weber,

1968; Rajakoski, 1960).

Falch (1959) first introduced the concept that granu-

losa and theca cells may interact for biosynthesis of estro-

gens. Since this suggestion, much attention has focused on

these two cell types to identify location and chemical bio-

synthetic pathways of estrogen production. Earlier work

utilized in vivo and in vitro incorporation of isotopically

labeled precursors to study pathways and cell types involved

in the synthesis of steroids. Since the advent of the

radioimmunoassay, many workers have turned their attention








to mechanisms controlling these pathways, primarily the role

of gonadotropins.

The primary purpose of this section is to review three

areas involved in steroid biosynthesis: pathways, cell types

and controlling mechanisms.

Much of the research has been done utilizing in vitro

systems and therefore is subject to criticism applicable to

all in vitro systems. Pathways shown to predominate in vitro

may not dominate and indeed may not be functional in the

intact animal. Precursors in the medium may not be freely

available in vivo. In vitro work with follicular tissue

can be criticized additionally due to the nature of the tissue

cells involved. Granulosa cells are avascular in vivo and

are separated from the blood supply by a lamina propria.

This may act as a barrier or otherwise limit availability

of certain substrates to the granulosa cells. The use of

follicular minces or slices would expose granulosa cells

directly to all substrates in the medium. Furthermore, granu-

losa cells from large preovulatory follicles have been shown

to undergo spontaneous luteinization in some in vitro sys-

tems, and granulosa cells from small follicles undergo

luteinization following addition of LH to culture medium

(Channing, 1970a, 1970b, 1974). Luteinization is the

morphological and functional transformation of granulosa

cells into luteal cells. Channing (1970a) stated that








granulosa cells have lutenized in culture when they accumulate

eosinophillic granules and lipid droplets in the cytoplasm,

increase in size and cytoplasmic-nuclear ratio, and secrete

large amounts of progesterone. Histological observations on

granulosa cells which luteinized in vivo revealed similar

changes as well as transformation of mitochondria cristae

from plate-like to tubular and villous forms, with the endo-

plasmic reticulum becoming agranular. These changes in

organelles are associated with steroid synthesis (Priedkalns

and Weber, 1968).

Several workers suggested that removal of the ovum

also results in luteinization of the follicle. Surgical

ovectomy in vivo resulted in luteinization and increased

progesterone secretion by follicles in rabbits and pigs,

whereas simple puncture of the follicle and loss of follicular

fluid had no effect on follicular steroidogenesis or

morphology (El-Fouly et al., 1970). In support of these

findings, rat granulosa cells cultured in monolayers

luteinized when cultured with limited oocytes, whereas

granulosa cells cultured with many ova retained their granu-

losa cell morphology (Nekola and Nalbandov, 1971). These

authors suggested that the ovum may produce a luteostatic

substance that inhibits luteinization of the granulosa cells.

This concept is not supported by the following observations.








Indomethacin blocks ovulation in the rabbit; however,

luteinization occurs in response to the ovulatory surge of

gonadotropins. The result is a fully functional corpus

luteum with an ovum trapped within it. This does not dis-

prove the presence of a luteostatic substance as the

ovulatory surge of LH may inhibit the synthesis of such a

substance by the ovum (Caldwell, Auletta and Speroff, 1973).

Moor, Hay and Seamark (1975) observed that cultured sheep

follicles did not luteinize following degeneration of the

ova. Linder et al. (1974) reported that progesterone

secretion was not increased when ova were removed from

incubated rat follicles. Pig, monkey and human granulosa

cells from small or medium follicles will not luteinize in

culture unless stimulated by LH (Channing 1970a, 1970b,

1974).

In attempts to overcome these inherent problems of

follicular tissue many workers have limited their studies to

short incubations or have worked with whole or intact

follicles. In this review tissue preparation and type of

system utilized will be identified. Any in vitro experi-

ment lasting less than 24 hr will be referred to as an

incubation, whereas any experiment longer than 24 hr will be

designated a culture.

There are two primary pathways for estrogen bio-

synthesis, the so called "A5 and A4" pathways (delta five








and delta four; Figure 1; Ryan and Smith, 1965). In the A5

pathway, pregnenolone is converted to androstenedione

through the A5-3B-hydroxy-steroids, 17-hydroxy-pregnenolone

and dehydroepiandrosterone, whereas in the A4 pathway,

pregnenolone is converted to androstenedione through the

44-3-keto steroids, progesterone and 17-hydroxy-progesterone.
Androstendione then can be converted to estradiol through

either estrone or testosterone.

Workers utilizing metabolites isolated from follicular

fluid (Short, 1962a) or in vivo incorporation of isotopically

labelled precursors (YoungLai and Short, 1970) postulated

that the A4 pathway predominates in the ovaries of mares.

Aakvaag (1969a) also using isotopically labelled precursors

reported that androstenedione is synthesised solely via the

A4 pathway in porcine ovarian tissue incubated in vitro.

The A4 pathway also has been shown to predominate in homoge-

nized mouse ovaries using similar incorporation techniques

(Kraiem and Samuels, 1974). However, various techniques

indicate the 65 pathway predominates in the human ovary

(Aakvaag, 1969b; Ryan and Smith, 1965; Patwordhan and

Lanthier, 1971). Lacroix, Eechaute and Leusen (1974) have

shown clearly that estradiol synthesis is through the 65

pathway in incubated bovine follicular tissue. Isotopically

labelled pregnenolone and 17-hydroxy-pregnenolone were con-

verted by bovine follicular tissue to androgens, whereas














A4 Pathway






Aromatase


Cholesterol

Side chain cleavage

Pregnenolone

/ \
Progesterone 17-OH-Pregnenolone

4i A5 Pathway

17-OH-Progesterone Dehydroepiandrosterone

Androstenedione Androstenediol

Testosterone


Estrone


Estrad
Estradiol


Aromatase


Figure 1. Estrogen biosynthetic pathways.








negligible progesterone or 17-hydroxy-progesterone were

transformed.

Considerable work has been conducted to determine cell

types which are responsible for the various steps within the

steroidogenic pathways. Short (1962a) suggested a "two cell

type" theory for ovarian steroid synthesis. This theory

postulated that all steps of steroidogenesis for estrogen

synthesis occur within the theca cells, and at the time of

ovulation the burden of steroidogenesis is shifted to the

granulosa cells which are only capable of progestin secretion.

Portions of this theory have since been challenged by con-

siderable in vitro investigations.

Human theca cells incubated alone or recombined with

granulosa cells, but not granulosa cells alone, produce

estradiol from 14C acetate in vitro (Ryan, Petro and Kaiser,

1968). In contrast, theca but not granulosa cells from

hamster follicles incubated in vitro produce high concen-

trations of androgens but very little estradiol. Recom-

bination of theca and granulosa cells resulted in five times

more estradiol accumulation in medium than either cell type

alone (Makris and Ryan, 1975). Similar findings were re-

ported in bovine follicular tissue incubations (Lacroix et

al., 1974). Both cell types were able to convert pre-

gnenolone to androstenedione; however, this capacity was

small in the granulosa cells compared to the theca cells.







Both cell types also were able to transform androstenedione

to estrogens, but granulosa cells were much more active in

this regard. These studies suggest that theca cells have

only limited aromatase activity in vitro. It also has been

demonstrated that incubated granulosa cells from equine

(Ryan and Short, 1965; YoungLai, 1972) porcine (Bjersing

and Carstensen, 1967) and rabbit (YoungLai, 1973) follicles

are capable of aromatization of exogenous androstenedione

or testosterone to estradiol. However, equine theca cells

have a higher conversion rate than do granulosa cells (Ryan

and Short, 1965). From these incubation studies it appears

that there is a positive interaction between these two cell

types for estradiol synthesis. It has been postulated that

theca cells, possibly in combination with granulosa cells,

synthesize steroids up to the androgens which are then

transported or simply diffuse to the granulosa cells where

they are aromatized to estrogens.

A recent study by Channing and Coudert (1976) suggested

that this is not the mechanism of in vivo estradiol syn-

thesis in the rhesus monkey. In this species plasma estradiol

concentrations remained unchanged for up to 120 min follow-

ing surgical aspiration of granulosa cells and follicular

fluid.

It is generally accepted that LH plays a major role

in the regulation of gonadal steroid secretion. LH is








stimulatory to steroid secretion, and this action has been

shown to be at a site common to all gonadal synthesis, the

conversion of cholesterol to pregnenolone (Armstrong, 1968;

Hall and Young, 1968). However this site of action has not

been shown directly in follicular tissue. The role of

follicle stimulating hormone (FSH) on estradiol secretion

generally has been attributed to its ability to stimulate

growth and development of follicles to a point where they

are competent of responding to the steroidogenic action of

LH (Lostroh and Johnson, 1966; Kraiem and Samuels,

1974). This concept was challenged recently by Moon,

Dorrington and Armstrong (1975) and Dorrington, Moon and

Armstrong (1975) who demonstrated a direct action of FSH on

estradiol synthesis.

Using the immature rat and mouse as a model, it has

been demonstrated that FSH and LH are both necessary for

complete growth and development of the follicle. Mechanisms

controlling early steps of follicle development have only

begun to be elucidated. FSH stimulated growth of follicles

in neonatal mice. This growth was characterized by prolif-

eration of granulosa cells and formation of a lamina propria.

However, absence of FSH diminished but did not completely

prevent granulosa cell proliferation. LH was necessary for

more extended growth such as secretary activity of the

granulosa cells, antrum formation and maintenance of the








thecal layer (Eshkol and Lunenfeld, 1972). These data

suggest that there are receptors for FSH in granulosa cells

of very small follicles.

Specific LH and FSH receptors have been demonstrated

in follicular tissue. Autoradrigraphic studies utilizing

mature follicles from mice and rats have shown that both FSH

and human chorionic gonadotropin (HCG) were specifically

bound to granulosa cells whereas only HCG was bound to theca

cells (Fraioli et al., 1972; Midgley, 1973; Rajaniemi and

Vanha-Pertulla, 1972). HCG and LH bind to the same receptor;

however,HCG is more stable under the conditions necessary

for this technique and therefore has been used routinely to

identify LH receptors. Apparently LH receptors are found

primarily in the theca cells of sheep as well (Moor, un-

published observations cited by Weiss et al., 1976).

Zeleznik et al. (1974) showed that there is a different

population of follicular gonadotropin receptors in immature

rats compared to the mature rat. In the immature rat FSH

bound only to the granulosa cells in the same manner as the

adult rat. However,HCG only bound to the theca cells in

immature rats and not to the granulosa cells as in the adult

rat.

The point in time or state of maturity when follicles

gain the ability to synthesize steroids is unknown. However,

there is ample evidence that LH and FSH both are necessary

for initiation and maintenance of steroidogenesis.








Moor et al. (1973) using an organ culture system re-

ported that the amount of estrogen secreted in vitro by

follicles removed from pregnant mare serum gonadotropin

(PMSg) treated sheep was higher than secretion by follicles

from untreated sheep. In untreated sheep only 5% of

follicles secreted high concentrations of estrogens in vitro.

This was increased to 25% of follicles after a 5 min exposure

to PMSg in vivo and 80% with a 24 hr exposure. This demon-

strates that PMSg,which has both FSH and LH activity, stimu-

lates estrogen synthetic pathways in sheep follicles.

Addition of LH to incubation medium caused variable

increases in estradiol synthesis by follicles from estrus

rabbits. However, 17-hydroxyandrogen synthesis was enhanced

many times that of estradiol (Mills and Savard, 1972;

YoungLai, 1974a, 1974b, 1975a). Androgen secretion also was

stimulated by LH in rat follicles incubated in vitro

(Lieberman et al., 1975). Estradiol secretion by quartered

ovaries of immature hypophysectomized rats in organ culture

was stimulated by FSH, FSH plus exogenous testosterone (an

aromatizable substrate) or LH plus testosterone. LH alone

had no effect on estradiol secretion and was not as effec-

tive as FSH when given in combination with testosterone

(Moon et al., 1975). Estradiol secretion by cultured

granulosa cells from hypophysectomized immature rats was

stimulated only by addition of FSH in combination with







testosterone. FSH, testosterone, and LH alone, or LH plus

testosterone, had no effect on estradiol secretion (Dorrington

et al., 1975). These data provide evidence for the first

time that FSH has a direct role in estradiol steroidogenesis.

FSH may initiate synthesis or activation of granulosa

aromatizing enzymes. These observations suggest that theca

cells under the influence of LH secrete androgens which then

are transported to the granulosa cells for aromatization

under the influence of FSH. This may explain the action of

low concentrations of LH and FSH on immature or developing

follicles; however, it does not explain the effects of high

concentrations of gonadotropins on large preovulatory

follicles. In mature follicles high concentrations of

gonadotropins have a biphasic effect on steroidogenesis. In-

itially all steroidogenesis, including estradiol synthesis,

is stimulated by gonadotropins. This stimulation is followed

by an inhibitory effect on estradiol secretion and a stimu-

lation of progesterone secretion. This biphasic effect has

been shown in several species and can be mimicked by L-I

alone. Mills and Savard (1973) reported that follicles re-

moved from rabbits 2 hr postcoitum incorporated signifi-

cantly more 14C acetate into steroids during incubation than

did follicles from unmated rabbits (estrus follicles). In-

corporation by estrus follicles was stimulated by addition

of LH to the incubation medium;however, LH had' no significant








stimulatory effect on incorporation by follicles from mated

rabbits. This clearly demonstrates the stimulatory effect

of LH on follicular steroidogenesis. These data also sug-

gested that follicles from mated rabbits may be maximally

stimulated by endogenous LH and FSH released at mating and,

therefore, exogenous LH had no further stimulatory effect.

Follicles removed 12 hr postmating incorporated very little

acetate into steroids and LH had no effect on incorporation.

Therefore, in rabbit follicles, LH initially stimulates

steroid synthesis, and this stimulation is followed by a rapid

decline in steroidogenesis by the follicle at 12 hr after

exposure to LH.

In incubated rat follicles estradiol secretion was

stimulated above controls the first 6 hr of incubation after

LH was added to the medium. However, estradiol secretion

ceased during the next 6 hr and progesterone secretion in-

creased (Linder et al., 1974). The inhibitory effect of LH on

estradiol secretion and stimulatory effect on progesterone se-

cretiun have been shown in several experiments with sheep

follicles. Estrogen secretion by whole sheep follicles can be

maintained for up to 7 days in organ culture (Moor, 1973; Sea-

mark, Moor and McIntosh, 1974). However, following the addition

of LH to the culture medium, estrogen secretion declined

rapidly and progesterone secretion increased over several days.

Similarly follicles removed from sheep after the preovulatory







surge of gonadotropins (Seamark et al., 1974) or following

infusion of LH (floor, 1974) produced insignificant amounts

of estrogens when cultured in vitro. In vivo infusion of LH,

into sheep in which the corpus luteum had been removed 18 hr

earlier, prevented ovaries from secreting the large amounts

of estrogens which otherwise would have occurred (Moor et

al., 1973).

In vivo observations in cattle also are suggestive of

a biphasic effect of LH on estradiol secretion. Chenault

et al. (1975) observed that peripheral plasma estradiol was

elevated during the initial phase of the ovulatory surge of

LH, declined 50% by 5 hr after the peak of LH, and reached

very low levels several hr prior to ovulation. It is dif-

ficult to determine if this is a response to LH or FSH as

Akbar et al. (1974) reported a concurrent peak of FSH at

this time. However, following injection of HCG peripheral

estradiol concentrations declined in cattle (Dobson, 1973).

This biphasic effect of LH may be explained very

simply High concentrations of LH endogenouss or exogenous)

may provide a burst of steroidogenic stimulation (presumably

through the side chain cleavage enzymes) thereby increasing

the general availability of precursors for estrogen synthesis.

The subsequent inhibition of estradiol secretion occurs

several hours after exposure to LH and most likely reflects

luteinization of granulosa cells and inhibition of theca








cells. This time delay may be necessary for initiation of

intracellular events associated with luteinization with one

end result being termination of estradiol secretion.

Several lines of evidence support this hypothesis.

Following addition of LH to cultured sheep follicles CAMP

synthesis increased rapidly (Weiss et al., 1976). When

mature sheep follicles were exposed to low levels of dibutyryl

CAMP estrogen production declined rapidly and all steroid

secretion ceased (Mclntosh and Moor, 1973). When higher

levels of CAMP were added, estradiol secretion was inhibited

and progesterone secretion was greatly stimulated. Channing

(1970a, 1970b, 1974) demonstrated that LH initiated

luteinization in rhesus monkey, human, swine and equine granu-

losa cells cultured over several days and that this effect

was mimicked by CAMP. In Channing's studies both morphologi-

cal and functional luteinization occurred. These studies

indicate that CAMP acts as a second messenger to cause

luteinization of granulosa cells.

The progesterone increase in incubated rat follicles

following the addition of LH is blocked by inhibitors of

protein and RNA synthesis, whereas estradiol synthesis is

stimulated. This suggests that the luteinizing effect of LH,

that is the stimulation of progesterone secretion and termin-

ation of estradiol secretion, requires protein and RNA syn-

thesis. However, the general steroidogenic effect of LH as







evidenced by the earlier stimulation of estradiol secretion

is not mediated through RNA or protein synthesis (Linder et

al., 1974). In immature rabbit follicles the general LH

stimulatory mechanisms appear to be different. Inhibitors

of protein synthesis inhibited LH induced androgen produc-

tion, whereas RNA synthesis inhibitors had no effect. Thus

the LH induced steroidogenesis is mediated through protein

synthesis possibly by activating translation of a stable

messenger RNA (YoungLai, 1975a, 1975b).

The preovulatory surge of LH does not luteinize small

or immature follicles. Luteinization in response to LH may

depend on presence of LH receptors in the granulosa cells.

Immature rat granulosa cells had no LH receptors (Zeleznik

et al., 1974), whereas mature follicle granulosa cells con-

tained LH receptors (Midgley, 1973). Channing and Kammerman

(1973, 1974) reported that granulosa cells from large,pig

follicles (6 to 12 mm) bound 10 to 1,000 times more HCG than

granulosa cells from small (1 to 2 mm) or medium (3 to 5 mm)

follicles, demonstrating that mature follicle granulosa cells

contain significantly more LH receptors than granulosa cells

from small or medium follicles.

Several lines of research demonstrated that FSH stimu-

lates LH receptors in granulosa cells. Following 2 days of

pretreatment with FSH in vivo, HCG bound to granulosa cells

of immature rat follicles whereas no HCG was bound to








granulosa cells of untreated immature rats (Zeleznik et al.,

1974). Granulosa cells from small,porcine follicles grown

in culture for 2 days with FSH bound HCG, whereas cells

grown without FSH bound no HCG (Channing, 1975). In

hypophysectomized immature rats treated for 1 to 4 days with

estradiol, LH receptors in granulosa cells declined while

rats receiving FSH instead of estradiol showed no change in

LH receptors (Richards and Midgley, 1976). However, granu-

losa LH receptors increased markedly in rats receiving both

FSH and estradiol. Furthermore, follicles in rats receiving

FSH or estradiol alone became atretic following exposure to

LH, whereas follicles from rats treated with FSH and estradiol

responded to LH by undergoing luteinization. These data

suggest that FSH and estradiol act synergistically during

follicle development to induce LH receptors and thereby pro-

vide mechanisms necessary for follicles to respond to the pre-

ovulatory surge of LH.

The mechanisms controlling estradiol secretion in the

bovine follicle are unknown. Only two studies, demonstrating

the effects of gonadotropins on bovine steroidogenesis in

vitro, have been found for this review. Results from one

study were too variable for the authors to draw any conclu-

sive statements: "These results would suggest that, in some

follicles, follicle stimulating hormone in vitro may modify

steroid biosynthesis" (Oakey and Stitch, 1968, p. 407). Shemesh and






Hansel (1975a reported that LH stimulated testosterone and

estradiol synthesis by bovine follicular slices.

The only other documented studies on estradiol secre-

tion by the bovine are studies quantifying peripheral blood

levels. However, most workers (Chenault et al., 1975;

Dobson and Dean, 1974; Glencross et al., 1973; Henricks,

Dickey and Hill, 1971; Shemesh, Ayalon and Linder, 1972;

Wettemann et al., 1972) concentrated their observations

during the follicular phase of the estrous cycle, with only

occasional samples, if any, taken throughout the cycle.

Estradiol increases for several days prior to estrus and

reaches a peak on the day of estrus with the highest concen-

trations occurring during the preovulatory LH surge.

Following the surge of LH, estradiol declines rapidly reach-

ing very low levels several hours before ovulation.

Estradiol concentrations are not as well documented during

the luteal phase of the cycle. Several authors (Dobson and

Dean, 1974; Glencross et al., 1973; Shemesh et al., 1972)

have described high concentrations of estradiol on different

days of the cycle. However, these peaks are variable from

study to study, which suggests that there are no consistent

increases in estradiol secretion during the luteal phase of

the cycle in cattle.

Changes in blood estrogens may be correlated with

ovarian follicular development. Large follicles (10 to 12 mm)








are found on the ovary at all stages of the estrous cycle,

but preovulatory follicles (16 to 18 mm) are found only on

days 20, 21, and 0. Follicular growth appears to be con-

tinuous and independent of stage of the cycle (Choudary et

al., 1968; Rajakoski, 1960). If these large transitory

follicles secrete estrogens this may explain the variable

increases in blood estrogen during the luteal phase of the

cycle which have been detected. With random growth, folli-

cles would reach a certain level of maturity and begin to

secrete estrogen. If the follicle has reached maturity dur-

ing the proestrus period, the estrogens, in the absence of

luteal progesterone concentrations, may stimulate a preovula-

tory surge of LH which causes the follicle to grow into a pre-

ovulatory follicle and ovulate. If the follicle reaches

maturity during a period of high progesterone concentration

(the luteal phase) it would secrete estrogen and then under-

go atresia without inducing either a preovulatory surge of

LH or ovulating.

Several authors have suggested that PGF2c also may

influence estrogen secretion in the bovine. Chenault et

al. (1975) observed spikes of estradiol following IM in-

jection of PGF2,. Hixon et al. (1973) observed elevated

peripheral blood estrone and estradiol following intraluteal

injection of PGF2,. These effects may be a direct effect of







PGF2, as Shemesh and Hansel (1975a) reported that PGF2a

stimulated testosterone but not estradiol secretion by

bovine follicular slices. This concept is not supported by

Louis et al. (1974a) who did not observe increases in

estradiol immediately following administration of PGF2.

These observations are of interest as it has been sug-

gested that there might be interplay between PGF2a and estro-

gen for spontaneous luteolysis. It long has been known that

estradiol shortens the length of the estrous cycle in intact

but not hysterectomized cattle (Brunner, Donaldson and Hansel,

1969). This suggests that estradiol works through the

uterus for luteolysis. It also is known that estrogens cause

release of PGF2a from the sheep uterus (Caldwell et al.,

*1972; Ford et al., 1975). Therefore, PGF2c released from

the uterus may stimulate estradiol synthesis in the follicle

which then stimulates additional release of PGF2. from the

uterus in a reinforcing manner. However, it should be

pointed out that the uterus is not necessary for luteolysis

in response to exogenous PGF2L (LaVoie et al., 1975;

Stellflug et al., 1975).

There is little known about the dynamics of follicular

steroidogenesis or luteolysis in cattle. These two functions

may indeed be tied together and mutually stimulatory. The

corpus luteum must be regressed to induce a controlled ovula-

tion, and steroid synthesis (estrogen) is essential for




39



proper conditioning of the reproductive tract for reception

of the egg and sperm. Considerable research is necessary

before these systems will be understood fully and this

knowledge is essential before these processes can be opti-

mally controlled.














SECTION II

IN VIVO RESPONSES TO PGF2. THAM SALT


Materials and Methods


Experiment 1. Response of Dairy Heifers
to Two Injections of PGF2
Tham Salt 12 Days Apart


This experiment was designed to determine if two in-

jections of PGF2, tham salt administered 12 days apart would

increase both the percentage of dairy heifers in the poten-

tially responsive phase of the estrous cycle at the second

injection and the number of animals having a synchronized

estrus after the second injection.

Thirty-seven cycling dairy heifers, 11 to 14 months of

age, were utilized. Before being assigned to this study, all

animals were examined per rectum to verify that reproductive

tracts were normal and ovaries were active. All animals

were treated twice, 12 days apart, with 33.5 mg PGF2, tham

salt* (IM) dissolved in 5 ml of .9% saline. Animals were

observed twice daily for signs of estrous behavior from 50



*PGF2a tham salt used in all experiments presented in
this dissertation was provided by Dr. J. W. Lauderdale,
Upjohn Company, Kalamazoo, MI.







days prior to the first injection until 30 days after the

second injection. Estrous detection was conducted prior to

the trial to determine the exact day of the estrous cycle

each animal was in at the time of the first injection.

A jugular blood sample (30 ml) was collected from each

animal 1 to 3 hr prior to each PGF2a injection. All blood

samples were collected via jugular puncture into heparinized

syringes, placed immediately into an ice bath, centrifuged

at 12,000 g for 15 min at 4 C, and plasma stored at -20 C

until analyzed for plasma progestin concentration.

Plasma progestins were measured by radioimmunoassay

procedures described by Abraham et al. (1971). The antipro-

gesterone was a gift of Dr. J. L. Fleeger of Texas A&M

University. Extraction and quantification procedures have

been validated in our laboratory by Chenault et al. (1973,

1975, 1976).

Statistical analyses for differences in response to

PGF2, and progesterone concentrations were done by Chi-square

and analysis of variance, respectively.


Experiment 2. Physiological Response
of Dairy Cattle to a
Luteolytic Dose of PGF2a
Tham Salt Administered
Intramuscularly


Objectives of this study were to determine if heart

rate, arterial blood pressure, uterine temperature and ar-

terial blood temperature were affected by a luteolytic dose







of PGF2a administered intramuscularly. Intramuscular injec-

tion is the recommended route for administration.


Thermocouple preparation and calibration


Thermocouple preparation and calibration have been

described extensively by Gwazdauskas (1974) and Gwazdauskas

et al. (1974). Lengths of 36 gauge, nylon coated, copper

constantan wire were pulled through polyvinyl tubing (V-7;

Bolab Inc., Derry, N.H.). The terminal thermojunctions were

heat sealed in polyvinyl and then coated with liquid tygon

(U.S. Stoneware Co.). Stranded, untinned copper extension

wires were soldered to divided copper wires leading to the

thermojunctions. All extension wires lead either to a milli-

volt potentiometer (#8686, Leads and Northrup, Philadelphia,

Pa.: limits of error of recording system + .075 C), or to a

dual channel strip chart recorder (Hewlett-Packard M 7100B;

limits of error of recording system + .03 C). Most, but not

all, of the potentials from the arterial ice water thermo-

couple' were suppressed by known amounts before being ampli-

fied and recorded.

Calibration of the thermocouples was made routinely by

use of a Bureau of Standards Certified Thermometer in a well

stirred, insulated water bath held at intervals between 36 to

40 C.








Surgical preparation and experimental protocol


Two dairy cows with histories of normally occurring

estrous cycles were used in these experiments. Prior to

surgery animals were placed on a 48 hr feed and water fast.

Animals were anesthetized with 2 to 4 g sodium thiopental

(Abbott Laboratories, North Chicago, Ill.) dissolved in

saline while standing and restrained. They then were placed

on a portable surgical table, tracheotomized, and maintained

under surgical anesthesia with methoxyfluorane (Pitman-Moore,

Washington Cross, N.J.). After removal of hair, the abdomin-

al and inguinal regions were scrubbed thoroughly with

Betadine (Purdue Fredrick Co., Norwalk, Conn.) and rinsed

with 70% alcohol.

An incision was made in the inguinal region and the

external pudic artery located. A 1.5 mm diameter polyvinyl

catheter (V-7; Bolab Inc., Derry, N.H.) and one thermocouple

then were inserted into this artery and passed upward into

the external iliac artery. The catheter was flushed and

maintained patent with a heparin solution (15U per ml of .9%

saline). The uterus then was exposed via a retroperitoneal

approach. Using small scissors, a 3 to 4 cm tunnel was made

under the serosa in the medial side of one uterine horn about

1 cm from the bifurcation. A thermocouple was inserted into

this tunnel and tied in place with 000 silk thread. The








thermocouple was secured by tying the extension wires two

or three times to the serosa along the uterine horn.

Thermocouple wires and catheters were tunneled under

the skin within a stainless steel cannula, exteriorized

through the flank, and maintained within a canvas pack which

was attached to the flank with two stainless steel pins

passed through a flap of skin. Thermocouple placements were

confirmed prior to their surgical removal 7 to 10 days after

completion of the experiments.

Experiments began 3 days after surgery to allow

animals to recover from the surgical preparation. Treatments

consisted of an intramuscular injection of either 5 ml of .9%

saline or 33.5 mg PGF2 tham salt dissolved in 5 ml saline.

To obtain within animal comparisons of treatment effects,

animals received on each experimental day a saline injection

followed several hours later by a PGF2a injection. One

animal was utilized on 2 experimental days whereas the other

was utilized on only 1 day to give a total of three saline

and three PGF2. injections.

Thermopotentials generated by the thermocouples were

amplified and recorded continuously during each experiment.

These were used to calculate external iliac blood temperature

and uterine temperature. Arterial catheters were connected

via miniature pressure transducers (RP-1500, Narco Bio-

systems, Inc., Houston, Tx.) to a physiograph recorder








(Physiograph Desk Model DMP-4B, Narco Bio-systems, Inc.,

Houston, Tx.). This allowed continuous recording of

arterial blood pressure and heart rate. Mean blood pressure

was taken arbitrarily as diastolic pressure plus one third

of the pulse pressure. For analysis and presentation of

mean arterial blood pressure, uterine temperature and

arterial blood temperature, recordings were averaged over 30

sec intervals for each experiment and means of all observa-

tions (n=3) for each 30 sec interval were plotted. Heart

rates were counted from the continuous strip chart record of

blood pressure.


Experiment 3. Physiological Response to a
Luteolytic Dose of PGF2 Tham
Salt Administered Intravenously


This experiment was conducted to determine effects of

a luteolytic dose of PGF2a tham salt, infused directly into

the jugular vein on heart rate, arterial blood pressure,

uterine temperature and arterial blood temperature. This

series of experiments was conducted with one cow which had

been used in Experiment 2 (Section II). One day prior to

initiation of this study, the animal was fitted with a poly-

vinyl catheter (V-7; Bolab) by jugular venipuncture. The

catheter was filled with a heparin solution (15 U per ml of

.9% saline), capped with a brass brad, and the external

catheter stored in an adhesive tape pocket glued to the neck

of the animal with branding cement.








Experiments consisted of infusion over a 2 min period

either with saline (n=2) or 33.5 mg PGF2. tham salt dissolved

in saline (n=2) via the jugular catheter. On each experi-

mental day (n=2) saline infusion was followed several hours

later by PGF2a infusion.

As in the previous experiment (Experiment 2, Section

II), thermopotentials generated by the thermocouples were

amplified and recorded continuously during each experiment.

These were used to calculate external iliac blood and

uterine temperatures. Arterial blood pressure and heart rate

were recorded continuously via the arterial catheters. Organ-

ization of data for analysis and presentation was the same as

described in Experiment 2 (Section III).


Experiment 4. Effects of PGF2 Tham Salt
on Uterine Bloob Flow in
Sheep


This experiment was conducted to determine the effect

of an intravenous injection of a luteolytic dose of PGF2a

tham aalt on uterine blood flow in sheep. Three sheep were

anethetized with methoxyfluorane, the abdominal region

cleaned with Betadine and rinsed with 70% alcohol, and a

midventral incision made to expose the reproductive tract.

Ovaries were removed and a small area of one miduterine

artery isolated in the broad ligament. The adventitia was

dissected from the vessel and the head of an electromagnetic









blood flow transducer (C and C Instrument, Los Angeles,

Calif.) was positioned around the vessel and secured in

place by suturing the transducer lead cables several times

to the broad ligament. Lead cables were exteriorized through

the flank area and maintained in a canvas pack sutured to

the skin. A polyvinyl catheter was placed into the femoral

vein, exteriorized through the flank and also maintained in

the canvas pack.

The principle of operation of the electromagnetic

flowmeter is based on Faraday's law of magnetic induction.

The principle states that if an electrical conductor tran-

verses a magnetic field, a voltage will be induced across

the field which is proportional to the strength of the mag-

netic field, length of the conductor, and the rate of trans-

versal. Within the head of the blood flow transducer is an

electrode and an electromagnet which are in contact with the

outer surface of the vessel wall. In this case, a magnetic

field is generated by the electromagnet and blood is the

electrical conductor. When blood flows through the magnetic

field a voltage is generated which is picked up by the

electrode in the head of the flow transducer. The voltage

from the pick-up electrode is a measurement of flow velocity,

but since the cross-sectional area of the blood column is

defined precisely, the device can be calibrated in terms of

volumetric flow.







During experiments transducer lead cables were con-

nected to a Narcomatic Electromagnetic Flowmeter (Model RT-

500, Narco Bio-systems, Houston, Tx.). A continuous re-

cording of blood flow (ml per min) was obtained by connecting

the flowmeter to a Narco Physiograph Recorder (DMP-4B)

equipped with Narco transducer couplers (model 7173) and

channel amplifiers (model 7070). Blood flow was contin-

uously recorded for a 2 hr period prior to treatment and 2

hr posttreatment. Flow transducers were calibrated in vitro

as described by Roman-Ponce (1977).

Experiments were initiated several weeks after sur-

gery. Animals had been ovariectomized to remove effects of

endogenous gonadal hormones on uterine blood flow. Therefore

without endogenous gonadal hormones blood flow to the

uterus was very low. To increase uterine blood flow, sheep

were injected intravenously with 20 ug estradiol 2 hr prior

to administration of experimental treatments. Roman-Ponce

et al. (1976) have shown that this dose of estradiol in-

creases blood flow to the uterus in ovariectomized ewes. In

that study, blood flow reached a peak approximately 2 hr

after estradiol administration and remained elevated for a

minimum of 6 hr.

Experimental treatment consisted of an intravenous

injection of 8 mg PGF2a tham salt dissolved in 5 ml saline

via the femoral catheter. No control was used in this








experiment as saline infusion in previous studies utilizing

these same animals had been shown to have no effect on

uterine blood flow (Roman-Ponce et al., 1977).

To determine if PGF2a has the same hypertensive effect

in ewes as observed in cattle, periodic measurements of

systemic blood pressure were made in two animals using a

manometer.


Results and Discussion


Experiment 1. Response of Dairy Heifers
to Two Injections of PGF2~
Tham Salt 12 Days Apart


It is well established that PGF2a is not effective

during the first 5 days of the estrous cycle. This lack of

responsiveness limits the usefulness of PGF2a as a practical

agent for ovulation control. This experiment was conducted

to determine if two injections of PGF2. tham salt (33.5 mg;

IM), 12 days apart, would increase both the percentage of

animals (n=37) in the potentially responsive phase of the

estrous cycle at the second injection and the number of

animals having a synchronized estrus after the second injec-

tion.

Distribution of heifers among the various phases of

the estrous cycle (day 0 to 5, 6 to 16, 17 to 21 and un-

known) on the 2 days of injection, 12 days apart, are shown








in Figure 2. The majority of animals (6 of 8) in the

unknown phase of the estrous cycle on the day of first in-

jection were not observed in estrus during the 50 days prior

to treatment. The remaining two animals were observed in

estrus twice within a short period of time just prior to the

first injection and therefore could not be assigned with

certainty to any group. Animals in the unknown phase at the

second injection had not been observed in estrus after the

first injection but had triggered heat mount patches indi-

cating they did respond to the first injection. Therefore,

these heifers could not be assigned conclusively to any

group.

A responsive stage of the estrous cycle and a response

are when a heifer is at a stage of the estrous cycle in

which PGF2, injection will induce corpus luteum regression

(responsive stage; days 6 to 21) allowing the animal to have

a detected estrus (response). A nonresponsive stage would be

between days 0 to 5 when a developing corpus luteum is

present which will not regress following PGF2,.

Distribution of heifers differed (P<.01) among phases

of the estrous cycle on the 2 days of injection. In a large

normal population of cycling cattle 76% of animals (based on

a 21 day cycle) would be expected to be in a potentially re-

sponsive stage of the cycle (not days 0 to 5) on any one

day selected at random. In this study 25 of 37 heifers (68%,















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which is very close to the expected 76%) were in a poten-

tially responsive phase on the day of first injection,

whereas 36 of 37 heifers or 97% were in this phase at the

second injection day. This clearly demonstrated that a

single injection of PGF2. alters the cycle so that animals

are in a potentially more responsive stage of the cycle 12

days later. This observation is substantiated by the

progestin data. Average (n=37) plasma progestin concentra-

tion was higher (P<.01) on injection day 12 (6.51 ng per ml

plasma) than on injection day 0 (3.33 ng per ml plasma).

The corpus luteum is mainly responsible for plasma progestin

concentrations. Thus the higher average concentration on

injection day 12 indicated that more animals had a functional

corpus luteum at the second injection day.

Any animal expressing estrous behavior within 8 days

after PGF2a was considered to have responded to PGF2 The

distribution of observed estrus, i.e., response to PGF2c,

following both injections is shown in Table 1. Since more

animals were in a potentially responsive stage of the cycle

at the second day of PGF2a injection, a greater number of

animals expressed estrus within 8 days after PGF2. compared

to the estrous response of the first injection (89 versus

60% respectively; Table 1). /In this study 89% of the animals

responded or had a detected estrus within 8 days after the

second PGF2. injection. This demonstrated that treatment of








Table 1.


Distribution of estrus in dairy heifers
following injection of PGF2a tham salt.


Day after
injection


1
2
3
4
5
6
7


First injection
Number of % of
animals animals


Second injection
Number of % of
animals animals


Total


Respondinga
Synchronizedb
Nonresponding


89**
84**
11


dResponding heifers were detected in estrus within 8 days
after PGF2a.
bSynchronized heifers were detected in estrus within 7
days after PGF2.a

*P<.01







cattle two times, 12 days apart, was a practical management

scheme to increase the percentage of animals responsive to

the second injection. These results agree with Cooper

(1974) who reported that 98% of heifers treated twice with

500 pg ICI 80,966 (a PGF2a analogue) 11 days apart responded

to the second injection.

If a synchronized estrus is defined as a detected

estrus within 7 days after PGF2a administration, then the

percentage of heifers expressing a synchronized estrus also

was greater after the second injection than after the first

(84 vs 60%, respectively; Table 1). The wide distribution

of estrus (8 days) following the second injection indicated

that a single timed insemination may result in lowered

fertility and therefore may not be practical. This indica-

tion substantiates results of Cooper et al. (1975), who

reported that fertility was lower (= 7%) following a single

insemination at 72 or 78 hr after the second of two PGF2n

treatments, 10 to 12 days apart, when compared to fertility

of untreated control animals or animals bred twice at 72 and

96 hr after PGF2a. Another study reported that 90% of

heifers were in estrus between 48 and 72 hr after the second

of two PGF2t injections (Cooper, 1974). This degree of

precision may allow for a single timed insemination with

normal fertility. However, the precision of onset of estrus

was not as good for our study (Table 1). Hafs et al. (1975)








reported fertility to a single insemination at 80 hr after

PGF2a was about equal to fertility of untreated controls or

PGF2a treated animals bred twice. Consequently use of a

single timed insemination after the second injection still

is questionable since results are not consistent among

studies in the literature. Data from the present study in-

dicated that there was considerable variability in the time

of onset of estrus after the second injection and thus a

single timed insemination may not be feasible using this

two injection regime. A single timed insemination at 72 or

78 hr would be considerably after the time at which 43%

(Table 1) of heifers were detected in estrus. These animals

probably would not conceive to a single breeding at these

times, however, measurement of fertility to a time insemin-

ation was not an objective of the present experiment. An

alternate insemination procedure is to inseminate all

animals 6 to 12 hr after onset of estrus. This would con-

centrate labor over a 7 to 8 day period which although is

not the optimal management scheme it probably would be

acceptable in commercial dairy operations.

Several systems utilizing LH releasers, either

estradiol (Welch et al., 1975) or GnRH (Graves et al., 1975)

have been used to synchronize time of ovulation further

after PGF2a injection. However, estradiol given at the same

time as PGF2a failed to increase fertility to a single








insemination at 72 hr after PGF2a over fertility in animals

which received PGF2a alone and were bred at the timed insem-

ination (Tobey and Hansel, 1975). GnRH administered at 60 hr

after PGF2a similarly had no effect on fertility rates to a

timed insemination (Tobey and Hansel, 1975) whereas, when

administered at 48 hr after PGF2a, fertility to a single

timed insemination was suppressed (Roche, 1976b). A neces-

sary assumption of these methods is that all animals have a

mature follicle present which can ovulate in response to

released LH. This may not be the case in all animals. The

proestrus increase in plasma estradiol, indicative of

follicular growth, is highly variable among animals follow-

ing PGF2a (Chenault et al., 1976; Louis et al., 1974a). This

may explain why studies using LH releasers have failed to

improve fertility to a timed insemination. The literature

to date suggests that a majority of animals have a follicle

which ovulates within a short period of time (99.5+ 19.2 hr)

following PGF2c injection. Use of an LH release would only

affect these animals and have no effect on animals which do

not have a mature follicle. More research utilizing LH

releasers is needed to determine if these drugs have any

practical value when used in combination with PGF2a. Addi-

tional methods which induce concise follicular development

in all animals may be needed before fertility to a single

timed insemination can be improved to that of or above








fertility in control animals. Such methods would eliminate

the human error associated with estrous detection and timing

of artificial insemination. Therefore fertility rates above

those presently obtained in control animals should be

feasible.


Experiment 2. Physiological Response of
Dairy Cattle to a Luteolytic
Dose of PGF2. Tham Salt
Administered Intramuscularly


Results from Experiment 1 (Section II) indicated that

PGF2t was effective in increasing the potential percentage

of animals responsive to the second of two PGF2a injections.

This experiment was designed to determine if a luteolytic

dose of PGF2a administered IM would have any detrimental

effect on heart rate, arterial blood pressure, uterine tem-

perature and arterial blood temperature in dairy cows (n=2)

which may limit the practical use of PGF2. for ovulation

control.

The mean external iliac blood pressure diastolicc

pressure plus one third pulse pressure) responses following

IM injection of either saline or 33.5 mg PGFa tham salt,

are illustrated in Figure 3. In response to both treatments

there was a slight increase in blood pressure. Following

saline injection blood pressure returned to pretreatment

values by 2 min posttreatment, whereas it remained slightly








elevated (P>.10; <10 mm Hg) above pretreatment values fol-

lowing PGF2 Associated with these initial increases were

transient increases in heart rate (from 62 to 74 beats per

min) following both treatments. Because saline and PGF2

injections both caused transient increases in heart rate it

was felt that the responses were due to injection and not

to the drug. Apparently in response to physical stress of

IM injection, catecholamines were released by the adrenal

medulla which stimulated heart rate and increased arterial

blood pressure. The slight elevation in blood pressure

after PGF2a injection may be due to a vasoconstricter effect

of PGF2.

Uterine and arterial temperatures were monitored as

changes in these temperatures and their differences reflect

changes in blood flow to certain body compartments and

specifically the uterus. Dilation of peripheral vessels

result in cooling of arterial blood whereas vasoconstriction

of peripheral vessels results in a quick shunt of cool blood

to the body core which may be followed by an elevation in

blood temperature. Either decreased or increased arterial

blood temperatures would indicate possible PGF2 effects on

peripheral vascular smooth muscle tone and/or alterations of

thermal regulation. Alterations in uterine temperature,

independent of changes in arterial blood temperatures, can be




























































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used as an indirect measurement of uterine blood flow.

Blood is the primary means of removing metabolic heat from

the uterus. Therefore if arterial blood temperature remains

constant, an increase in uterine blood flow results in a

decreased uterine temperature, whereas a decrease in flow

results in an increased uterine temperature. In this study

no changes in arterial blood or uterine temperatures could

be detected for the 3 hr period following IM injection of

either saline or PGF2a. Therefore, PGF2. appeared to have

no major effect on uterine blood flow. It also is likely

that PGF2a had no effect on the peripheral vascular smooth

muscle or thermoregulatory mechanisms of the animals as any

such effects would have resulted in some change in arterial

blood temperature. Consequently a luteolytic dose of PGF2.

tham salt (33.5 mg) administered by IM injection caused no

major alterations in circulatory homeostasis.

Two routes of PGF2a administration have been used

routinely in studies to date, either IM injection or intra-

uterine placement. When placed into the uterus lower doses,

1 to 5 mg of PGF2a (compared to 33.5 mg for intramuscular

injectionY are used (Inskeep, 1973; Louis et al., 1974a;

Welch et al., 1975). It is doubtful that this lower

uterine dose of PGF2a has any systemic effects, but it

appears to induce a local effect since corpus luteum regres-

sion occurs. From this experiment it appears that IM








injection is a safe, practical method for administration of

a luteolytic dose of PGF2( tham salt./


Experiment 3. Physiological Response
to a Luteolytic Dose of
PGF2 Tham Salt Adminis-
tereo Intravenously


Experiment 2 (Section II) indicated that a luteolytic

dose of PGF2a tham salt administered IM had no major effects

on circulatory homeostasis or arterial blood and uterine

temperatures. However, the classical smooth muscle effects

of PGF2a may have been masked by route of administration.

Given intravenously, the acute availability of PGF2a would

be increased. Furthermore, giving PGF2a as an intravenous

infusion over a 2 min period also would reduce the physical

stress of IM injection experienced in the previous experi-

ment. Therefore, the intravenous approach was conducted to

clarify further the effects of PGF2a in the bovine.

Saline infusion caused no detectable effects on

arterial blood temperature, heart rate, arterial blood tem-

perature or uterine temperature., Therefore, the following

discussion concerns only changes following a 2 min infusion

of 33.5 mg PGF2a tham salt.

Circulatory homeostasis (Figure 4) was affected within

70 sec from initiation of PGF2a infusion. Mean iliac blood

pressure rose rapidly from approximately 140 mm Hg to 230 mm





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Hg, and heart rate declined from 34 to 16 beats per 30 sec

by end of the first min postinfusion. Heart rate returned

to nearly pretreatment levels by 4 min posttreatment.

However, blood pressure did not return to pretreatment levels

until 30 min posttreatment. This increase in blood pressure

agrees with that observed by Lewis and Eyre (1972) and Aitken

and Sanford (1975) who reported increased blood pressure in

calves following intravenous injection of the same dose (on

a per kilogram basis) of PGF2 However, both of these

studies showed an increase in heart rate following PGF2 .

In the present study heart rate declined and this decrease

trailed the elevation in blood pressure. The decline in

heart rate may be due to a physiological response to in-

creased blood pressure and not due to a direct pharmacological

effect of PGF2 on the heart. PGF2a has been shown to have

no direct effect on the heart in several other species

(Horton and Main, 1967; Lee et al., 1965; Nakano et al.,

1971). No alterations in circulatory homeostasis were ob-

served following infusion of saline.

Following IM injection of saline or PGF2 heart rate

increased (Experiment 2, Section II) which is opposite from

the pharmacological response observed in the infusion experi-

ment. This provides additional support for the conclusion

that increase in heart rate following injection was a physio-

logical response to route of administration and not to PGF2?








treatment. Apparently IM administration reduced the acute

effective systemic concentration of PGF2 .

During PGF2a infusion and for 8 to 9 min postinfusion,

the cow exhibited frequent periods of defecation, urination,

abdominal straining and bulging of the eyes. These obser-

vations indicated that PGF2. probably stimulated smooth

muscle activity and were consistent with the reported effects

of PGF2 on gastrointestinal smooth muscle in other species

(Bennett and Fleshler, 1970; Main, 1973).

Effects of PGF2a on uterine temperature and external

iliac arterial blood temperature are shown in Figure 5.

Uterine temperature increased abruptly from 39.5 to 39.8 C

immediately following PGF2 infusion. Arterial blood tem-

perature began a slow decline 5 min after infusion and

reached a stable temperature 50 min postinfusion. By 15 min

postinfusion uterine temperature was also falling in parallel

with the decline in arterial blood temperature.

The initial increase in uterine temperature most

likely reflects an early reduced blood flow to the uterus.

With a lower blood flow, less metabolic heat is removed and

uterine temperature increases. A reduction in blood flow

could be due either to uterine vasoconstriction or to in-

creased transmural pressures resulting from uterine contrac-

tions. PGF2a does have vasoconstricting effects on certain

vascular beds (Viguera and Sunahara, 1969; Mark et al., 1971)






65





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and causes uterine contractions (Capraro et al., 1976;

Rexroad and Barb, 1975).

Infusion of PGF2a appeared to affect thermoregulation

as evidenced by the decline in arterial blood temperature.

Splanchnic blood temperature (external iliac) decreased .4 C

by 50 min postinfusion and remained stable at this tempera-

ture for the 3 hr of postinfusion recording. Saline in-

fusion caused no change in arterial blood temperature. The

mechanism by which PGF2a may affect body temperature is

unknown. However, peripheral vasodialation is a distinct

possibility. Miller et al. (1976) detected a sweating re-

sponse and lowered rectal temperature in mares following

PGF.- administration. In contrast, Hales et al. (1973) re-
2 a
ported that heat generating and conserving mechanisms were

stimulated by PGF2. administered into the lateral ventricle

in sheep.

Uterine and arterial temperature responses can be used

to provide additional information about the physiological

response of the animal to PGF2a. If arterial blood tempera-

ture changes (+ or -) and there is no change in rate of

blood flow to the uterus then the temperature difference

between the uterus and arterial blood (AT -A ) would be

expected not to change. However, if flood flow to the

uterus is reduced, less metabolic heat would be removed and

the ATuA would increase. Following infusion of PGF2a a








marked widening of the ATu-A was observed (Figure 5). This

suggests that uterine blood flow was reduced markedly. The

increase in ATuA reached a peak at 20 min postinfusion and

returned to near pretreatment levels by 60 min postinfusion.

This return to pretreatment levels indicated that PGF2( had

only a transient effect on uterine blood flow.

By all indications, PGF2a had no prolonged effects on

the animal, which is consistent with reports that PG's are

rapidly metabolized in the body (Ferreira and Vane, 1967;

Raz, 1972). The only data which contradict this are

arterial blood temperatures which did not return to pretreat-

ment levels. The rapid drop in blood temperature reflects a

treatment effect (peripheral vasodilitation?). However, the

stabilization at this lower temperature most likely is due

simply to a loss of deep core heat. Perhaps there is also an

alteration in thermoregulatory setpoint.

/This study demonstrates that PGF2a has strong smooth

muscle stimulatory actions in the bovine. The animal dis-

played signs of discomfort during the first 10 min after in-

fusion and the smooth muscle responses, in particular the

pressor effect, may prove hazardous to animals in poor

health. Therefore, the intravenous route of administration

should be avoided. This route is not recommended and care

should be taken when administering PGF2a intramuscularly to

avoid accidental injection into the vascular bed.








Experiment 4. Effect of PGF2 Tham Salt
on Uterine Blood Flow in
Sheep


Results from Experiment 3 (Section II), using an

indirect method to measure blood flow (ATuA indicated

that uterine blood flow in cattle was rapidly decreased by

intravenous infusion of PGF2a tham salt. This experiment

was conducted to measure, by use of blood flow transducers,

the direct effects of a luteolytic dose of PGF2a (8 mg,

intravenously), on blood flow through one miduterine artery

in sheep (n=3).

Exogenous estradiol (20 pg) had been administered to

increase uterine blood flow. At 2 hr after injection, the

uterine blood flow had increased from approximately 5 ml per

min to 70 ml per min. A similar response of uterine blood

flow to estradiol was described by Roman-Ponce et al. (1976).

Following infusion of PGF2a mean blood pressure (Table

2) was elevated. This response appears to be of short

duration as blood pressure was returning towards pretreatment

levels by 8 min postinfusion in one animal. A similar tran-

sient increase in mean blood pressure was observed in cattle

in Experiment 3 (Section II). This observation indicates

that in sheep and cattle responses of the circulatory system

to PGF2a are similar. This elevation in blood pressure most

likely was due to a vasoconstrictor effect of PGF2a on









Table 2. Mean arterial blood pressure (mm Hg) response of
sheep to 8 mg PGF2a tham salt administered
intravenously.


Time (min) post Animal
infusion 1 2


Pre 80 70
6 134 -
8 108 96
23 83








vascular smooth muscle and not to an increase in heart rate,

since heart rate was slowed following infusion of PGF2a in

cattle.

Uterine blood flow decreased dramatically following

infusion of PGF2, (Figure 6). Within the first min postin-

fusion, blood flow fell rapidly from 72 to 43 ml per min and

then rebounded to 63 ml per min. Flow then declined slowly

reaching its lowest flow (41 ml per min) at 10 min postinfu-

sion. During the remainder of the experiment flow increased

gradually to near pretreatment levels by 60 min postinfusion.

Immediate increases in uterine temperature and ATu-A

were observed following infusion of a luteolytic dose of

PGF2( in cattle (Experiment 3, Section II). Both of these

responses are indicative of decreased blood flow to the

uterus. It was concluded that PGF2. decreased blood flow to

the uterus in cattle. Results of this experiment in sheep

clearly substantiate these conclusions since PGF2a infusion

caused an immediate decrease in uterine blood flow.

The initial rapid decline in uterine blood flow doubt-

less occurred much too rapidly for PGF2. to be transported

from the femoral vein through the heart and lungs (site of

metabolism) and then to the uterus. Therefore, this response

may be a neural response elicited by PGF2a. PGF2a may stim-

ulate the sympathetic nervous system as epinephrine has been
























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shown to decrease uterine blood flow in sheep (Roman-Ponce,

1977) and Lauderdale et al. (1975) reported that plasma

epinephrine was elevated following administration of PGF2 .

This initial effect was short-lived, lasting less than 1 min,

which also is suggestive of a neural effect. In contrast to

this short term effect, the prolonged decline in blood flow,

initiated 1 min after infusion and lasting for 60 min, most

likely reflects a direct effect of PGF2c on vascular or

uterine smooth muscles. The mechanism by which PGF2X induces

this prolonged decrease in uterine blood flow is not clear

from this study. In other tissues, PGF2a has been shown to

stimulate constriction of both arteries (Viguera and Sunahara,

1969) and veins (Mark et al., 1971). Similarly, PGF2x induces

uterine contractions which may result in increased transmural

pressure. Any of these mechanisms could reduce blood flow

to the uterus.

In ewe and cow, effects of PGF2a were short-lived.

Uterine artery blood flow returned to near pretreatment

level; by 60 min posttreatment in ewes which is similar to

the 50 min period required for ATu-A to return to pretreat-

ment levels in the cow.













SECTION III

IN VITRO BOVINE FOLLICULAR
STEROIDOGENESIS


Materials and Methods


Elevated plasma estrogens have been observed in cattle

following injection of PGF2x (Chenault et al., 1976; Hixon

et al., 1973) which suggest PGF2a stimulated estrogen

synthesis. Hixon et al. (1973) proposed the ovarian fol-

licle as the site of PGF2a stimulated estrogen synthesis.

This theory is of interest as it also has been suggested

that PGF2a and estrogens interact in a reinforcing manner

during spontaneous luteolysis. Furthermore, among animal

plasma estrogen concentrations are highly variable following

PGF2a injection and approaching ovulation. High plasma

estrogen concentrations in association with low concentra-

tions of plasma progestins are essential for triggering the

ovulatory surge of LH. Therefore PGF2( stimulated estrogen

synthesis may influence the degree of synchronization of

ovulation following PGF2a injection. In addition, there is

very little known about the dynamics of follicular steriodo-

genesis in cattle. Objectives of these studies were to







determine the acute effects of PGF2 and gonadotropins on

bovine ovarian follicular estradiol secretion in vitro.

The in vitro approach was utilized to obtain treatment

responses of isolated follicular tissue, which is not obtain-

able utilizing in vivo approaches. Results from these experi-

ments should further our understanding of follicular steroido-

genesis which may contribute also to our understanding of

spontaneous luteolysis.

In order to obtain sufficient numbers of large fol-

licles and faced with a limited number of cattle, animals

were treated with FSH-p to induce follicular development.

FSH-p (Armour-Baldwin Laboratories, Omaha, Ne.) is purified

FSH obtained from pituitaries of swine and cattle. One mg

FSH-p is equivalent to 1 mg of Armour Standard FSH which is

a porcine FSH prepared as described by Steelman and Pohley

(1953).

Lyophilized FSH-p (50 mg) was dissolved in 10 ml of

.9% saline and injected intramuscularly two times daily on

days 16 to 19 of the estrous cycle, with doses shown in

Table 3. This treatment schedule was adopted from Bellows,

Anderson and Short (1969) who used FSH-p to induce follicular

development for superovulation studies.

By treating all animals in this manner it was thought

that all follicles, though taken from different animals,








Table 3.


FSH-pa injection schedule


Day
of Cycle


5.0 mg
5.0 mg
2.5 mg
2.5 mg
ovariectomy


2X daily
2X daily
2X daily
2X daily


a FSH-p Armour Baldwin Labs.


Amount








would have developed in, and been exposed to, a similar

hormonal milieu prior to harvesting. Twenty to 25 follicles

of fairly uniform size (7 to 15 mm in diameter) and weight

(100 to 200 mg) could be harvested per animal. Therefore

a sufficient number of follicles would be obtained from one

cow to run a complete experiment.

Animals were checked for estrous behavior twice daily

before and during FSH-p treatment. None of the treated

animals displayed estrous behavior during the injection

schedule. On day 20, animals were given a local anesthetic

while standing and restrained. A 10 to 15 cm incision was

made in the lumbar region and ovaries removed using a cutting

ecrasseur. Ovaries were placed immediately into an ice cold

solution of 50% saline (.9%) and 50% incubation medium.

Incubation medium consisted of 8 parts Medium 199 with

Hanks salts and glutamine (see Appendix 1; Difco Laboratories,

Detroit, MI.) and 2 parts fetal calf serum (GIBCO, Grand

Island, N.Y.). To each liter of incubation medium 56 mg

insulin (Iletin, Eli Lilly and Co., Indianapolis, Ind.), 56

mg ascorbic acid and 18 mg gentamicin sulfate (Schering Cor-

poration, Bloomfield, N.J.) were added. This medium has been

used successfully for culture of whole sheep follicles (Moor,

1973; Moor et al., 1973) and a similar medium was used by

YoungLai (1973, 1974a, 1974b) for incubation of whole rabbit

follicles.







Following ovariectomy, follicles were isolated and

trimmed of connective tissue, and follicular fluid was

aspirated with a 22 gauge needle; follicles then were

weighed and assigned randomly to treatment.

Follicles were incubated individually and free float-

ing in 5 ml of incubation medium in 25 ml Erlenmeyer flasks;

incubations were under a gaseous atmosphere of 50% N2, 45%

02 and 5% CO2 in a Dubnoff shaker at 37 C for 12.5 or 14 hr.
During incubation, follicles were agitated at one stroke per

sec. Incubation medium was changed every 2 hr and after

removal, the medium from each incubation period was stored

separately at -20 C until analyzed for steroid hormones.

This frequent changing of medium was conducted to allow

characterization of time trends in steroid secretion during

the incubation.

These studies were designed to determine acute effects

of different hormonal treatments on follicular estradiol

production. Several authors have utilized culture techniques

to den;onstrate acute treatment effects. However, in vivo,

follicles grow and develop over a short period of time and

then either ovulate or undergo atresia. Therefore culture

systems in which follicles are maintained in a static state

over long periods of time do not mimic physiological condi-

tions. The method of preparing follicles for incubation and

the incubation system used in these studies were developed








with a desire to keep the system as physiological as possible.

Due to this rationale, short term incubations were used

rather than culture techniques, and whole follicles were in-

cubated rather than tissue slices or minces. Use of whole

follicles maintained the integrity and interrelationship of

cell layers as found in vivo which includes the physical

isolation of granulosa cells from the nutrient supply.

However, follicular fluid was aspirated to remove the high

concentrations of steroids known to be present in follicular

fluid (Short, 1962b). Otherwise, these steroids may leach

out of the antrum during incubation and mask treatment

effects. Moor (1973) reported that steroid secretion by

whole sheep follicles in tissue culture was not affected if

the follicular fluid was removed by puncture of the follicle.

Similar in vivo observations have been reported in swine and

rabbit follicles (EI-Fouly et al., 1970).

Concentrations of estradiol, progestins and testoster-

one in the medium following incubations were determined by

radioimmunoassays. The progestin assay was described in

Materials and Methods, Experiment 1, Section II. Estradiol

was measured by the technique of Hotchkiss, Atkinson and

Knobil (1971). Extraction, purification and quantification

have been validated in our laboratory by Chenault et al.

(1973, 1975, 1976). The estrogen antibody was a gift of

Dr. V. L. Estergreen of Washington State University.









Testosterone was extracted with diethyl ether, isolated by

Sephadex LH-20 column chromatography, and assayed by the

method of Coyotupa, Parlow and Abraham (1972). The testos-

terone antibody (S741 #2) was acquired from Dr. G. E. Abraham

of Bal Harbor Hospital, Torrance, Cal. and has been described

by Coyotupa et al. (1972).

Experimental Design


Experiment 1. Estradiol Secretion by
Bovine Follicles in Vitro:
Test of Incubation System


This experiment was conducted to determine if bovine

follicles synthesize estradiol in this incubation system.

Ten follicles from one FSH-p treated cow were prepared

for incubation as described previously and then assigned

randomly to treatments. Treatment one consisted of immedi-

ate freezing of the follicles after aspiration of follicular

fluid (n=5), whereas treatment two follicles (n=5) were in-

cubated for 14 hr during which the medium was changed every

2 hr.

Follicles from treatment one were homogenized in a

Polytron Homogenizer (Kinematica, distributed by Brinkmann

Company, Westbury, NY.). The homogenized follicular tissue

of treatment one and medium from treatment two were assayed

for estradiol. Test of treatment effects on total estradiol








was conducted using analysis of variance. Least squares

regression was used to determine time trends of estradiol

secretion (pg estradiol per mg tissue per period of incuba-

tion, and ng estradiol per follicle per incubation period)

during incubation in treatment two. The statistical model

included follicle and incubation period (time) as a contin-

uous independent variable to the highest order of signi-

ficance up to the fifth order.


Experiment 2. Effects of PGF2( and LH on
In Vitro Estradiol Secretion
by Bovine Follicles


This experiment was conducted to determine the effect

of PGF2, and LH on estradiol secretion by incubated follicles.

Fifteen follicles from one FSH-p treated cow were assigned

randomly to one of three treatments. Treatments consisted

of control incubation medium (n=5), PGF2 tham salt at a con-

centration of 5 ng per ml incubation medium (n=5), and LH

(NIH-LH-B7) at a concentration of 50 ng per ml medium (n=5).

These concentrations were chosen because they are equivalent

to physiological blood levels present when dramatic changes

in estrogen biosynthesis occur in vivo. PGF2Q in the uterine

vein averaged under 5 ng per ml plasma on days 15 to 17 of

the estrous cycle (Shemesh and Hansel, 1975b), and plasma LH

often exceeds 50 ng per ml during the preovulatory surge of

LH (Henricks, Dickey and Niswender, 1970; Snook, Saatman and

Hansel, 1971; Thatcher and Chenault, 1976).







The incubation was for 14 hr during which the medium

was changed every 2 hr. During the first 4 hr, all fol-

licles were incubated in control medium. The first 2 hr of

incubation, designated preincubation, was included to allow

leaching of preexisting steroids. The second 2 hr, designated

control incubation period, was included to obtain a within

pretreatment measurement of steroid secretary capability.

The preincubation and control periods were followed by five

2 hr treatment incubations. Hormonal treatments were added

to the medium only during the treatment incubations. With

each bi-hourly change of medium, fresh medium with hormonal

treatments was added.

The initial design called for estradiol to be assayed

in the medium from each 2 hr period. However, due to dif-

ficulty in interpreting the estradiol secretary profile,

progestins and testosterone also were measured.

An extensive series of least squares analyses was con-

ducted to determine time changes in hormonal secretion. The

statistical model included treatment, follicle within treat-

ment and time, as a continuous independent variable, to the

highest order of significance. Test for significance of

treatment effects was a test of heterogeneity of regression

(Snedecor and Cochran, 1967). Using this test, treatment

effects are significant when there is significant gain in

fitting one curve for each treatment over fitting an overall

pooled curve.








Experiment 3. Effects of PGF2, on
In Vivo Estradiol Secre-
tion by Bovine Follicles


This experiment was designed to test PGF2a dose effects

on follicular estradiol secretion. Twenty follicles from one

FSH-p treated cow were assigned at random to treatments con-

taining 0, 5, 100 or 1000 ng PGF2a tham salt per ml incuba-

tion medium. The incubation was for 14 hr including a 2 hr

preincubation and six 2 hr treatment incubations. PGF2a was

added to the medium only during the treatment periods.

Medium was changed every 2 hr. A control incubation period

was not included in this experiment in order to begin treat-

ments early in the incubation when steroidogenesis is maximal.

Only estradiol was assayed in the medium and statistical

analyses were the same as described in Experiment 2,

(Section III).


Experiment 4. Effects of FSH, Testosterone
and FSH plus Testosterone on
In Vitro Estradiol Secretion
by Bovine Follicles


This experiment was conducted to determine if the

presence of testosterone, an aromatizable substrate, FSH or

testosterone plus FSH could stimulate estradiol secretion in

vitro.

Twenty follicles from one FSH-p treated cow were

assigned randomly to treatments in a 2 X 2 factorial design.







Treatments consisted of incubation medium alone (control),

5 X 10-' molar testosterone (Steraloids, Inc., Pauling, NY.;

144.2 ng/ml) added to incubation medium, 100 ng FSH (NIH-FSH

81) per ml incubation medium, or both FSH and testosterone

added to medium.

The incubation was for 12.5 hr which consisted of a

.5 hr preincubation, a 2 hr control incubation, and five 2

hr treatment periods. Hormonal treatments were added to the

medium during the five treatment periods (2.5 to 12.5 hr).

Estradiol was assayed in the medium from all periods

and statistical analysis conducted as described in Experi-

ment 2 (Section III).


Experiment 5. Histology of Bovine Fillicles
Induced with FSH-p; Before or
After In Vitro Incubation


In all preceding incubation experiments estradiol

secretion declined during incubation and failed to respond

to treatments. Purpose of this experiment was to examine

follicles for histological evidence to account for these

results.

Fifteen follicles from one FSH-p treated cow were

harvested. Five follicles were incubated following the

procedure described in Experiment 1 (Section III). Immed-

iately after the 14 hr incubation these follicles were fixed

in Bouins solution (Appendix 2). The additional 10 follicles








were fixed immediately in Bouins solution following their

isolation from the ovary. This allowed for histological

examination of follicles induced by the FSH-p injection

schedule as well as follicles postincubation.

Dehydration, dealcoholization and infiltration pro-

cedures used as well as minimum time requirements of each

step are given in Appendix 2. Follicles were embedded in

paraffin, sectioned on a Spencer 820 Microtome (American

Optical Corp., Buffalo, NY.), at 6 to 8 microns and stained

with eosin and hematoxylin (see Appendix 2). Microscopic

examination was conducted using a Nikon LUR-Ke microscope

(Nikon Inc., Garden City, NY.).


Results and Discussion


Experiment 1. Estradiol Secretion by
Bovine Follicles in Vitro:
Test of Incubation System


Purpose of this experiment was to test the ability of

follicles to synthesize estradiol in the incubation system.

If bovine follicles secreted significantly more estradiol

into the medium during a 14 hr incubation (n=5; treatment

two) than was extracted from homogenized tissue of unincu-

bated frozen follicles (n=5; treatment one) then estradiol

synthesis could be assumed to have occurred during incu-

bation.








The mean total estradiol in the medium after 14 hr of

incubation, for the five incubated follicles, was 64.3 pg

per mg tissue (Table 4). This was greater (P<.01) than the

mean of 16.1 pg estradiol per mg of unincubated follicular

tissue. Thus, estradiol was synthesized during incubation.

Time trends of estradiol secretion during the incuba-

tion periods were characterized by fourth order equations

shown in Figure 7. These data are expressed as both pg

estradiol per mg follicular tissue per period of incubation

(Y = -5.70 + 25.135X -10.875X2 + 1.7913X3 .10300X4; X

hr incubation 2) and ng estradiol per follicle per period

(Y = -1.03 + 4.241X-1.846X2 + .30711X3 .01783X4). As

shown in this example, hormonal data expressed either ad-

justed for follicular weight or on a whole follicle basis

resulted in nearly identical secretion curves. Furthermore,

data expressed either way provided identical interpretations

of treatment effects (Table 4). This was a consistent obser-

vation for all experiments and therefore hormonal results in

the incubation medium will be presented only as pg hormone

per mg follicular tissue.

Estradiol secretion increased initially during the

first 4 hr and then declined gradually during the remainder

of the incubation. Although estradiol secretion declined,

there was secretion of estradiol in each period of incuba-

tion. The reason for this decline was unknown; however,








Table 4.


Estradiol biosynthesis in vitro by whole follicles
from FSH-p treated cows


Follicle No.

Frozen follicles

1

2

3

4

5

Mean


Pq per mq tissue


9.5a

12.6

16.6

27.3

14.5

16.1


Ng per follicle


2.4a

1.7

3.3

7.2

2.7

3.5


Incubated follicles

6

7

8

9

10

Mean


b
63.8

61.8

53.6

68.0

74.5

64.3**


13.5b

9.5

6.3

12.8

10.0

10.4**


aEstradiol extracted from homogenized tissue of nonincubated
frozen follcles.
Total estradiol secreted during 14 hr incubation with medium
changed every 2 hr.

**P < .01 Significantly more estradiol in medium after 14 hr
incubation than extracted from nonincubated, frozen
follicles.


_




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