The Neuroendocrinology of ovine parturition

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Title:
The Neuroendocrinology of ovine parturition effect of estrogens and androgens on fetal ACTH secretion in late gestation
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Effect of estrogens and androgens on fetal ACTH secretion in late gestation
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Estradiol -- pharmacology   ( mesh )
Estradiol -- physiology   ( mesh )
Androstenedione -- pharmacology   ( mesh )
Androstenedione -- physiology   ( mesh )
Corticotropin -- metabolism   ( mesh )
Corticotropin -- drug effects   ( mesh )
Hydrocortisone -- metabolism   ( mesh )
Fetus -- physiology   ( mesh )
Labor -- physiology   ( mesh )
Sheep -- physiology   ( mesh )
Department of Physiology thesis Ph.D   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1995.
Bibliography:
Bibliography: leaves 118-135.
Statement of Responsibility:
by Christine Jean Saoud.
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Typescript.
General Note:
Vita.

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THE NEUROENDOCRINOLOGY OF OVINE PARTURITION:
EFFECT OF ESTROGENS AND ANDROGENS ON FETAL ACTH SECRETION
IN LATE GESTATION











By

CHRISTINE JEAN SAOUD


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

UNIVERSITY OF FLORIDA
















To my friends: thank you for standing beside me.
To my family: thank you for believing in me.
To my husband: thank you for loving me.

In memory of my Grandfather. I wish you could have been here.













ACKNOWLEDGMENTS


I would first like to thank my supervisory committee for their hard work, unending

patience, and confidence in this project. Becoming a member of a doctoral committee is

always an undertaking which requires a unique level of trust and I hope I have fulfilled

their expectations. Throughout my graduate program, a number of people were

instrumental in giving me support, guidance, and a helping hand or a patient ear. Their

efforts have not gone unnoticed. A heartfelt thank you goes out to Tim Cudd, Melanie

Pecins-Thompson, Sonny Singh, Tammy Gault, and Ellen Wolfe. Maureen Keller-Wood,

although not officially on any committee, was indispensable as a resource, mentor and

friend. Euni Lee and Scott Purinton have been dear friends whom I could always count

on in tight spots. I must also thank husband, Michael, who encouraged me to enter

graduate school in the first place.

Finally, I would like to express my extreme gratitude to Charles E. Wood. I first

began working in the lab as an undergraduate running assays and later became a graduate

student in reproductive physiology. I had never pictured myself in that role, but Dr. Wood

always exuded confidence in my abilities. After over six years under his guidance, I feel I

have gained not only a mentor and teacher, but a colleague and friend, whom I will miss

dearly.













TABLE OF CONTENTS



ACKNOW LED GM ENTS........................ ................................ ...................... iii

KEY TO ABBREVIATIONS.................................................................................vii

A B ST R A C T ................................................. .. ..........................................................x

CHAPTERS

1 IN TR O D U C TIO N ............................................................ ..........................1

2 REVIEW OF THE LITERATURE............................... ........................ 3

2.1 Control of Parturition.........................................................................3
2.1.1 Ontogeny and Maturation Within the HPA Axis..................3
2.1.2 Induction of Parturition by Stimulation of the HPA Axis.......6
2.1.3 Disruption of the HPA Axis Delays Parturition.................7
2.1.4 Alterations in Fetal Gonadal Steroids in Late Gestation.........8
2.2 Regulation of ACTH Secretion...................... .........................10
2.2.1 Corticotropin Releasing Factor..................... .................... 11
2.2.2 Arginine Vasopressin.................................. ................ 12
2.2.3 CRF and AVP as Regulators of ACTH Secretion...............16
2.2.4 Other ACTH Secretagogues.......................................17
2.3 Negative Feedback Regulation of ACTH Secretion by Cortisol.........18
2.4 Glucocorticoid Receptor Structure and Function.............................21
2.5 Gonadal Steroid Modulation of ACTH Secretion............................24
2.6 Sum m ary....................... .................................................... ..26

3 GENERAL METHODOLOGY........................................28

3.1 Animals and Surgery.................... .... .............................28
3.2 Blood Collection and Handling...................... ............................31
3.3 Infusion Experimental Procedures.................................................32
3.4 Hormone Assay Techniques............................ ......................34
3.5 Tissue Collection and Handling.................... .........................37









4 MODULATION OF OVINE FETAL ADRENOCORTICOTROPIN
SECRETION BY ANDROSTENEDIONE AND 17P-ESTRADIOL.............. 39

4.1 Introduction.................................................... .................. ......39
4.2 Materials and Methods................................. ........... ...........41
4.3 Results............................. ...................................45
4.4 D iscussion....................................................... ............................54

5 ONTOGENY AND MOLECULAR WEIGHT OF IMMUNO-
REACTIVE ARGININE VASOPRESSIN AND CORTICOTROPIN-
RELEASING FACTOR IN THE OVINE FETAL HYPOTHALAMUS...........59

5.1 Introduction................... ..... ................ ..... 59
5.2 Materials and Methods............................ .....................60
5.3 Results........................... .................................62
5.4 D iscussion......................................................... ...........................68

6 ONTOGENY OF PROOPIOMELANOCORTIN
POSTRANSLATIONAL PROCESSING IN THE OVINE FETAL
PITU ITA R Y ................................................................. ..................... .......72

6.1 Introduction............... ........... ............ ..................72
6.2 M materials and M ethods.......................................... ......................74
6.3 Results................................. .................................- ........... 76
6.4 D iscussion............. .. ............................. ...........................80

7 DEVELOPMENTAL CHANGES AND MOLECULAR WEIGHT OF
IMMUNOREACTIVE GLUCOCORTICOID RECEPTOR PROTEIN IN
THE OVINE FETAL HYPOTHALAMUS AND PITUITARY......................85

7.1 Introduction.................................................... ...........................85
7.2 Materials and Methods................................... ......................86
7.3 R esults.................................... ......... ............89
7.4 D iscussion.............. .. ................................................... 92

8 EXOGENOUS 17P-ESTRADIOL AND ANDROSTENEDIONE
ADMINISTRATION SHORTENS GESTATION LENGTH IN THE
OVINE FETU S.......... ................................................................................. 96

8.1 Introduction.................................................. .............................96
8.2 M materials and M ethods........................................ ........................97
8.3 Results..................... ..................................................... 99
8.4 Discussion.............. ... ........................ .....................104








9 SUMM ARY............................................................... ....................... 07

REFERENCES............................................................ ......................... 18

BIOGRAPHICAL SKETCH......................................... .............................136















































vi













KEY TO ABBREVIATIONS



ACTH..................... ....................... adrenocorticotropic hormone

ADH.......................a......................anti-diuretic hormone

ANOVA......................................... analysis of variance

ANP ..................................................atrial natriuretic peptide

Arg..................... .........................arginine

Asn...................... ...................... .. asparagine

AVP............................................arginine vasopressin

B/I...............................................bioactive to immunoreactive ratio

BID ........................................... twice a day

BSA ......................................... bovine serum albumin

cAMP............................................cyclic adenosine monophosphate

cDNA...........................................complementary deoxyribonucleic acid

CRF............................................corticotropin releasing factor

Cys.................... ..........................cysteine

dex......................................dexamethasone

DNA............................................deoxyribonucleic acid

F ............................................. ........cortisol

Gin............ .......... .......................glutamine


vii








Gly................................... ... ........glycine

GRE.................................................glucocorticoid response element

HC ............................................. hydrochloric acid

HPA............................................... hypothalamus-pituitary-adrenal

HPD................. ..............................hypothalamus-pituitary disconnection

HSP......................................... heat shock protein

HSS......................... .....................hydrocortisone sodium succinate

e .......................... .....................isoleucine

iACTH..... ................. .......................immunoreactive ACTH

iAVP ........................................ immunoreactive arginine vasopressin

iCRF .................. ......................immunoreactive corticotropin releasing factor

ID...............................................inner diameter

Ig........................ ......................... immunoglobulin

IM........................................... intramuscular

IV ................ ......................intravenous

kD........................................kilodalton

Leu.............................................leucine

M .......................... .........................m olar

MAP...........................................mean arterial pressure

mmHg............................................millimeters of mercury

N .. .............................. .......... ........norm al

NiPr...........................................nitroprusside

NO..............................................nitric oxide

viii








NPY............................................ neuropeptide Y

NTS.................... ...................... nucleus of the tractus solitarius

oCRF...................... ......................ovine corticotropin releasing factor

OD.................... ....................... outer diameter

O .D .................... .......................optical density

ovx ............................................ ovariectomized

P.CO ...........................................arterial partial pressure of carbon dioxide

P.2..........................................arterial partial pressure of oxygen

Phe.............................................phenylalanine

POMC...............................................proopiomelanocortin

Pro ......................... .....................proline

PVN.... ..................................paraventricular nucleus

RNA........................................ribonucleic acid

RIA................................................ radioimmunoassay

SON..................... ........... ............. supraoptic nucleus

Tyr.......................... ...................... tyrosine
















ix













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

THE NEUROENDOCRINOLOGY OF OVINE PARTURITION:
EFFECT OF ESTROGENS AND ANDROGENS ON FETAL ACTH SECRETION
IN LATE-GESTATION

By

Christine Jean Saoud

December 1995


Chairman: Charles E. Wood, PhD.
Major Department: Physiology



Parturition in the sheep is controlled by fetus. Near the end of gestation, plasma

adrenocorticotropic hormone (ACTH) and cortisol concentrations are increased,

stimulating estrogen production from the placenta which acts on the uterus to stimulate

contractions leading to labor and delivery. It has previously reported that cortisol negative

feedback efficacy is decreased at the end of gestation. We hypothesized that increases in

fetal plasma androgen and estrogen concentrations in the fetus might increase plasma

ACTH concentrations, either by stimulating ACTH secretion or by altering the negative

feedback effect of cortisol on ACTH. Fetal sheep were chronically catheterized and

implanted with 17I-estradiol and/or androstenedione pellets (approximately 250 pg per

day) between 110 and 125 days gestation. Fetal plasma ACTH and cortisol








concentrations were measured throughout the duration of the pregnancy and ACTH

responses to hypotension and the effectiveness of cortisol inhibition of ACTH secretion

were tested. The effect of estradiol and androstenedione were independent of each other

and significant. Estradiol treatment increased basal plasma ACTH concentrations as well

as stress- induced ACTH secretion (approximately 2-fold). Estradiol, however, did not

have a significant effect on cortisol negative feedback sensitivity. Androstenedione

significantly decreased the ability ofcortisol to inhibit plasma ACTH secretion without

influencing basal or stimulated plasma ACTH concentrations. When estradiol and

androstenedione were administered together, the combined effect was to increase plasma

ACTH and cortisol concentrations prematurely and significantly decrease the length of

gestation when compared to control fetuses (141.7 and 145.8, respectively). In summary,

increases in exogenous 17 P-estradiol concentrations augmented fetal plasma ACTH

secretion. Increases in exogenous androstenedione concentrations decreased cortisol

negative feedback efficacy of plasma ACTH secretion. When these two gonadal steroids

were given in combination, the mean gestation length was significantly decreased. These

results suggest that androgens and estrogens may play a role in stimulating fetal plasma

ACTH secretion and therefore contribute to the initiation of parturition in the sheep.













CHAPTER 1
INTRODUCTION


Parturition in the sheep is stimulated by activation of the fetal hypothalamus-

pituitary-adrenal (HPA) axis (Challis and Brooks, 1989; Liggins et al., 1973). At

approximately 85% gestation, drive to the fetal HPA is stimulated and fetal plasma

adrenocorticotropic hormone (ACTH) and cortisol concentrations begin to increase. At

approximately 95% gestation, there is a further increase in plasma ACTH and cortisol

secretion due to a decrease in cortisol negative feedback efficacy (Wood, 1988). Cortisol

acts at the placenta to increase cytochrome P45007 (17a-hydroxylase and 17,20 lyase

activities) which increases production of nineteen and eighteen carbon steroids from

progesterone. These steroids are subsequently converted to estrogens and androgens,

increasing fetal plasma concentrations of both. With this conversion, there is a shift in the

plasma ratio of progesterone to estrogen. Estrogen promotes uterine contractions by the

formation of gap junctions (see Chap. 2)(Garfield 1977, 1984; Garfield et al., 1978), as

well as direct and indirect actions on the uterus itself along with prostaglandins and

oxytocin (Lye et al., 1983a; Fuchs et al., 1982), promoting the initiation of labor.

In the sheep, the HPA axis must be intact for parturition to occur normally.

Destruction or inhibition of any part of the axis leads to an abnormal length of gestation.

In a review by Nathanielsz et al., it was stated that it is "necessary to look for a stimulus to

the fetal hypothalamus which is not present at an earlier stage of gestation and is removed






2

by parturition" (1972, p. 40). This is the key to determining what drives parturition in all

species, including the sheep. The hypothesis of these studies is that the maturation of the

fetal HPA axis involves gonadal steroids produced by the placenta. Increasing fetal

plasma cortisol concentrations then act on the placenta to induce cytochrome P450

enzyme action, shifting placental steroid production from progesterone to androgens and

estrogens. These gonadal steroids then can act on the fetal brain to augment plasma

ACTH secretion and possibly facilitate parturition.

To test this hypothesis, the effects of estradiol and androstenedione on the fetal

hypothalamus-pituitary-adrenal axis function were examined. The normal developmental

pattern of plasma ACTH, cortisol, estrogen and androgen secretion was examined as well

as the tissue content of releasing factors for ACTH and the processing of ACTH in the

pituitary. Glucocorticoid receptor protein in the fetal hypothalamus and pituitary was

examined as well. Finally, the combined effects of estrogen and androgen on the timing of

parturition were examined. With these studies, we believe that the role of estrogens and

androgens in the control of parturition in this species will be determined.













CHAPTER 2
REVIEW OF THE LITERATURE



2.1 Control of Parturition

After numerous observations and experiments, in 1973, Liggins et al. concluded

that parturition in the sheep is controlled by the fetus. This is accomplished by the

coordination of many maturational processes and endocrine factors, working in concert,

which culminate in the expulsion of a healthy, fully developed fetus. This process is

intricately linked to the hypothalamus-pituitary-adrenal axis in the fetus. Prior to the

pioneering studies by Liggins, a natural phenomenon in sheep was reported (Binns et al.,

1964). If ewes ate a particular plant (Veratrum califonicum) on day 14 of gestation, the

birth of the fetus was delayed indefinitely until caesarian section or death of the fetus. The

fetuses had a number of birth defects, particularly cyclopia and a dysfunctional

hypothalamus-pituitary (HP) axis due to dislocation of the pituitary from its normal

position. The connection between the malformed, dysfunctional HP axis and the delayed

parturition would soon be realized as a significant event.

2.1.1 Ontogeny and Maturation within the HvDothalamus-Pituitary-Adrenal Axis

Development of the ovine fetal hypothalamus-pituitary-adrenal axis begins during

the first third of gestation (term being 145-148 days) with the formation of the pituitary

and adrenal glands. The fetal pituitary gland can be detected as early as 31 days gestation

and differentiation of the anterior pituitary seen at about 40 days (Perry et al., 1982).

3








Staining of cells in the anterior pituitary indicate the presence of ACTH immunoreactivity

by as early as 50 days gestation and by 60 days gestation, processing of ACTH from

proopiomelanocortin (POMC) can be detected in the intermediate lobe of the pituitary in

the fetal sheep (Mulvogue et al., 1986).

The fetal adrenal can be identified by approximately 28 days gestation in the sheep

(Wintour et al., 1977). By 40-50 days gestation, in vivo experiments demonstrate that the

fetal adrenal readily secretes cortisol in response to ACTH (Wintour et al., 1975;

Glickman and Challis, 1980). Between 90 and 120 days gestation, the cells of the zona

fasciculata (which are responsible for the synthesis of cortisol) are relatively immature

(Robinson et al., 1979) and approximately 90%/ of fetal plasma cortisol is derived from the

maternal circulation (Hennessy et al., 1982). After 120 days gestation, fetal adrenal

sensitivity to plasma ACTH increases (Liggins et al., 1973; Rose et al., 1982), the

proportion of cortisol that is of fetal origin increases (Hennessy et al., 1982a), and the

correlation between fetal ACTH secretion and fetal cortisol secretion becomes significant

(Hennessy et al., 1982b). Adrenal weight also increases as a function of gestational age

(Comline and Silver, 1961).

Early studies involving fetal hypophysectomy (Liggins et al., 1967) and

adrenalectomy (Drost and Holm, 1968) with subsequent prolongation of pregnancy

suggested a link between adrenal steroid production and parturition. If this is the case,

fetal plasma cortisol secretion should be altered as gestation nears an end. Before about

130 days gestation, fetal corticosterone levels are low, but several days before parturition,

plasma levels increase and peak at birth (in the chronically instrumented animal; Bassett

and Thorbur, 1969). Nathanielsz et al. (1972) found that fetal plasma cortisol






5

concentrations began to increase about 3-4 days before parturition and then steadily

declined in the newborn lamb. A more elaborate study ofcortisol secretion was

performed by Magyar et al. (1980) in which exponential curves were fit to the data to

more accurately describe the increase in fetal plasma cortisol concentrations. This analysis

revealed fetal plasma cortisol concentrations increasing exponentially about 10-15 days

prior to parturition. Cortisol is secreted in response to ACTH binding to receptors at the

adrenal gland. In vivo experiments by Brown et al. (1978) demonstrated that not only did

glucocorticoids increase with development, but the ability of the fetal adrenal to secrete

cortisol in response to ACTH was observed around 120-129 days gestation. The increase

in cortisol secretion that occurs near the end of gestation is in part due to an increase in

adrenal sensitivity to stimulation by ACTH (Madill and Bassett, 1973) but also possibly

due to increased plasma ACTH secretion from the fetus.

Jones et al. (1977) presented some of the first data examining the changes in fetal

plasma ACTH concentrations during development. They demonstrated that fetal plasma

ACTH concentrations increase prior to parturition. However, they concluded that the

increase occurred after the increase in fetal plasma cortisol and therefore is probably not

the reason for the changes in fetal plasma cortisol concentrations. Experiments performed

later by other investigators (accounting for episodic secretion of ACTH, stress-induced

ACTH secretion, and cortisol negative-feedback inhibition of ACTH secretion) revealed

that fetal plasma ACTH concentrations increase much sooner than previously described by

Jones et al. (MacIsaac et al., 1985; Wintour, 1984; Norman et al., 1985). In fact, fetal

plasma ACTH concentrations increase during the last 30 days of gestation, well before the

increase in plasma cortisol concentrations which occur at approximately 120 days

gestation.








The results of increasing plasma ACTH concentrations in vivo when taken

together with the in vitro pituitary ACTH secretion studies support the presence of an

additional factor involved in the process of parturition. Basal output of ACTH from fetal

sheep pituitaries in culture does not increase as a function of gestational age (Durand et al,

1986). Pituitary ACTH secretion in culture does not increase during the last week of

gestation at a time when in vivo circulating plasma ACTH concentrations are increasing

exponentially (McMillen and Merei, 1993). These data suggest that the preparturient

increase in ACTH secretion is dependent on some other factor (hypothalamic or other) to

stimulate secretion and not a function of basal pituitary output. In addition, McMillen and

Merei (1993) also found no change in responsiveness of the fetal corticotroph to CRF as a

function of gestational age. However, prior exposure of the fetal pituitary to cortisol

increased its responsiveness to CRF. There appears to be a signal, possibly from the

hypothalamus, that increases the activity of the fetal pituitary and therefore the fetal

adrenal and is the trigger to parturition in the sheep.

2.1.2 Induction of Parturition by Stimulation of the HPA axis

The involvement of the fetal adrenal cortex in the initiation of parturition in the

sheep was suggested after in utero plasma concentrations of corticosteroids revealed

dramatic increases in these hormones prior to birth (Bassett and Thorburn, 1969). Before

this conclusion was made, a number of studies aimed at investigating the role of the

pituitary-adrenal axis in the birth process were performed in fetal sheep by Liggins (1968).

ACTH infused (0.1 mg/24hrs) into fetal sheep induced parturition within 4-7 days, along

with producing adrenal hypertrophy. Cortisol infusions into the fetus induced parturition

within 5 days. The same doses of ACTH or cortisol infused into the ewes did not induce

parturition. Although 17p-estradiol at 2mg/24hr had no effect, an infusion of cortisol at








25mg/24 hr plus the estradiol resulted in spontaneous delivery after about 4 days.

(Liggins, 1968).

Further studies by Liggins (1969) showed that it was glucocorticoid activity (and

not mineralocorticoid activity) that was important for initiation of parturition. Evidence

for this effect was shown by the inability of deoxycorticosterone or corticosterone to

induce parturition. Dexamethasone infused at rates of 0.06-4 mg/24hr in the fetus and

4mg/24hr in the ewe was ineffective in producing premature delivery.

Since the earlier experiments of Liggins, many other studies have been performed

which confirm the previous results. Parturition was induced by continuous IV cortisol

infusion at 130 days gestation (increasing the infusion rate from 2.8mg/24hours up to

44.8mg/24hr over 3 days) (Thomas et al., 1978). Activation of fetal adrenal function by

pulsatile ACTH administration in 125-127 day fetal sheep induced labor and delivery in 4-

5 days, resulting in 4-6 fold elevation in fetal plasma cortisol concentrations (Lye et al.,

1983). Hypophysectomized fetuses administered Dexamethasone or ACTH infusions

exogenously undergo parturition (Kendall et al., 1977).

2.1.3 Disruption of the HPA Axis Delays Parturition

Parturition can be induced artificially by increasing the activity of the fetal HPA (as

described above) or artificially delayed by blocking the normal process at one of the three

levels; at the hypothalamus, pituitary, or the adrenal gland. Adrenalectomy in the late

gestation fetal sheep delays parturition (Drost and Holm, 1968). Pituitary ablation also

inhibits the initiation of parturition (Liggins et al., 1967). Disconnection of the

hypothalamus from the pituitary (HPD) in fetal sheep between 108-112 days gestation

delayed birth by at least 8 days (Antolovich et al., 1990). Following hypophysectomy,






8

infusions of ACTH or glucocorticoid still induce parturition (Kendall et al., 1977).

Further studies from this group also demonstrated the necessity of the HP axis in

maturation of pituitary corticotrophs (Kendall et al., 1977). After HPD, fetuses were

infused with saline or cortisol. In the cortisol-infused fetuses, the proportion of fetal-type

corticotrophs was significantly lower than in the saline-infused fetuses however the

number of adult-type corticotrophs did not change. A direct effect of cortisol on pituitary

corticotroph maturation was seen but complete maturation required the presence of a

complete hypothalamus-pituitary axis (Antolovich, et al., 1992). More specifically,

following destruction of the fetal paraventricular nuclei of the hypothalamus, parturition

was delayed (McDonald and Nathanielsz, 1991). Therefore, the signal for parturition may

either be sent to the PVN (receives input from the NTS, amygdala and/or hippocampus)

or possibly be derived in the CRF and AVP producing neurons of the PVN.

2.1.4 Alterations in Fetal Gonadal Steroids in Late Gestation

In addition to the increase in fetal plasma ACTH and cortisol concentrations that

occur at the end of gestation, fetal plasma estrogen and androgen concentrations also

increase. Plasma estrone concentrations appear to increase over the last four days of

gestation while estradiol may be increasing over the last 8 days in the fetus and amniotic

fluid (Challis and Patrick, 1981) (Findlay and Cox, 1970). In a paper by Challis and

Patrick, fetal plasma estrone concentrations increased from 40 pg/ml at 14 days prior to

birth to near 400 pg/ml the day before birth. Fetal plasma estradiol concentrations

increased from 20 pg/ml at -14 days to 80 pg/ml on the day before parturition. Plasma

estradiol concentrations in the ewe increased over the last 2 days of pregnancy in the ewe

(Robertson and Smeaton, 1973), with a 10-fold increase on the day before parturition,






9

from 20-40 pg/ml up to 411 pg/ml (Challis, 1970). At the time plasma estrogens increase,

there was a decrease in plasma progesterone (Bedford et al., 1972). This is possibly due

to the conversion of estrogen from progesterone, with plasma progesterone acting as sink

for plasma estrogen production. Since progesterone is also a precursor for androgen

production, one would expect to see increases in plasma concentrations of these steroids

as well. Plasma androstenedione and testosterone concentrations do indeed increase in the

late gestation fetus in a manner similar to estrogen concentrations (Pomerantz and

Nalbandov, 1975; Yu et al.., 1983).

This increase in plasma androgen and estrogen concentrations is a result of the

actions ofcortisol on the placenta (Anderson et al., 1975; Steele et al., 1976). Although

the placenta has aromatase activity, the plasma concentrations of androgens and estrogens

are very low throughout gestation (Mann et al., 1975). Cortisol acts at the placenta to

induce an enzyme, cytochrome P450c,, which has 17a-hydroxylase and 17,20 lyase

activities. Upon induction, P450c,7 facilitates the production of estrogens and androgens

from progesterone, thus increasing plasma concentrations of these hormones.

Gap junctions in the myometrium, which are very important for synchronized

uterine contractions are believed to be formed at the time of progesterone withdrawal

(Garfield et al., 1977). It has been proposed that as a result of decreased progesterone

and increased estrogen, there is an augmentation in synthesis of proteins associated with

gap junctions (Garfield et al., 1978; Garfield, 1984, 1977). These gap junction proteins

are inserted into the plasma membrane of the myometrium and aggregate to form gap

junctions. Gap junctions are necessary for the uterus to contract in a unified, coordinated

fashion in order to expel the fetus. Prostaglandins and oxytocin, as well as estradiol (Lye

et al., 1983) act in concert to stimulate uterine contractions in the process of labor.








Tissue estrogen concentrations in sheep also increase towards term or after ACTH

induced labor, especially in the myometrium (Power and Challis, 1987). The ovine

placenta, through sulfatase and aromatase activities, converts estrone and estrone sulfate

to 17p-estradiol, the more potent estrogen for the target organ, the myometrium (Rossier

and Pierrepoint, 1974).



2.2 Regulation of ACTH Secretion

Secretion of ACTH is primarily controlled by corticotropin releasing factors of the

hypothalamus, namely CRF and AVP. CRF and AVP are synthesized in the parvocellular

neurons of the paraventricular nucleus of the hypothalamus and project to the median

eminence (Reeves and Andreoli, 1992). These CRFs are secreted into the external zone

of the median eminence. The peptides then diffuse from the pericapillary space into the

primary capillaries of the hypothalamo-pituitary portal circulation (Leranth et al, 1983).

CRFs are carried down the infundibular stalk via long portal vessels to the anterior

pituitary, where they are released into the extracellular space surrounding the cells of the

anterior pituitary and act on the corticotrophs via their specific receptors to increase

ACTH secretion. Corticotrophs produce ACTH through the step-wise processing of pro-

opiomelanocorticotropin (POMC) to produce ACTH,., which is secreted into the general

systemic circulation. Upon reaching the adrenal gland, ACTH stimulates adrenal cortical

secretion ofglucocorticoids. These glucocorticoids then feed back at the hypothalamus

and pituitary to inhibit ACTH secretion (Keller-Wood and Dallman, 1984). This is

demonstrated pictorially in Figure 2.1.




















Anterior
Ptuitary







Adrenal
- Gland


Negative
Feedback
Effects


Figure 2.1. Hypothalamic-Pituitary-Adrenal (HPA) Axis. (-) indicates action of cortisol
to inhibit ACTH secretion at the pituitary and inhibit AVP and CRF secretion at the
hypothalamus.

2.2.1 Corticotropin-Releasing Factor

Since 1955 it has been known that the hypothalamus contained substances that

acted at the pituitary gland to increase ACTH secretion in vitro (Guillemin and Rosenberg,

1955; Saffran and Schally, 1955). In 1981, Vale et al. characterized a 41-amino acid

peptide from sheep hypothalamus that stimulated ACTH secretion from corticotrophs

(Vale et al., 1981) and published the primary structure of ovine CRF (oCRF) (Spiess et

al., 1981). oCRF was further characterized when the cDNA was cloned and sequenced








(Furutani, 1983). At this time a similarity was discovered between the precursor proteins

for CRF, AVP and ACTH which implies a common evolutionary beginning. A 20

kilodalton immunoreactive form of CRF (from which CRF,.4 can be generated by

proteolytic cleavage) was identified from rat hypothalamus and is close to the value for

ovine and human pre-pro-CRF based on their cDNA sequences (Lauber et al., 1984).

CRF increases ACTH secretion from the anterior pituitary gland by binding to high

affinity receptors (Wynn et al., 1983) located on the corticotrophs (Leroux and Pelletier,

1984). Activation of the receptor complex increases adenylate cyclase activity (Perrin et

al., 1986) and cAMP, which results in an increase in ACTH secretion. In cultured rat

pituitary cells, CRF can enhance the rates of ACTH synthesis as well as release (Vale et

al., 1983). ACTH release can be modulated by down-regulation of CRF receptors in the

anterior pituitary. There is evidence that CRF (Wynn et al., 1988) AVP (Hauger and

Aguilera, 1993) and glucocorticoids (Hauger et al., 1987; Schwartz et al., 1986; Wynn et

al., 1985) can all act to regulate CRF receptor number. Therefore, an alteration in CRF

receptor number, receptor activity, receptor coupling or even corticotroph number can

affect the ability of CRF to stimulate ACTH secretion.

2.2.2 Arginine Vasopressin

The other major regulator of ACTH secretion of hypothalamic origin is arginine

vasopressin (AVP). Classically, AVP is known as anti-diuretic hormone (ADH) for its

role in renal regulation of fluid balance. An increase in plasma osmolality is the most

"potent" stimulus to AVP secretion in that very small increases in osmolality cause an

almost immediate secretary response from the posterior pituitary. AVP binds to receptors

on the basolateral membrane of the cortical and medullary collecting ducts of the nephron.








Binding to these receptors (V) results in activation of adenylate cyclase, which

subsequently increases cyclic AMP. This second messenger is then thought to facilitate an

increase in protein channels found in the luminal membrane, thereby increasing the

diffusion of water out of the nephron and concentrating urine while retaining fluid

(Vander, 1985). In addition to AVP's role in modulating fluid reabsorption in the renal

system, AVP is also a potent vasoconstrictor of the cardiovascular system (binding to V,

receptors). Significant decreases in blood volume (15-20%) produce large increases in

plasma vasopressin (as much as 20-fold). Decreases in blood volume are sensed as

decreased stretch of the arterial baroreceptors located in the carotid sinus as well as

receptors in the left atrium and AVP secretion is reflexively stimulated (Berne and Levy,

1986).

AVP is one of the two hormones secreted from the posterior pituitary gland

(oxytocin, being the other). The posterior pituitary, also called the neurohypophysis, is

comprised of axons and axon terminals, which account for 42% of its total volume

(Nordmann, 1977). Theses axons project from magnocellular neurons of the supraoptic

nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. AVP and

oxytocin are structurally similar hormones (differing by only 2 amino acids) with very

different functions. AVP has actions on the renal and circulatory systems while oxytocin

causes milk ejection and uterine contractions. AVP and oxytocin are produced in different

neurons of the same nuclei and are stored in secretary vesicles or granules with their

appropriate neurophysin (Silverman and Zimmerman, 1975).












Structure of 2 Tyr -
Arginine Vasopressin: NH C- ly- A -Pro Cy Aa- Gn

Structure of oxytocin: same as for AVP except that Leu replaces Arg
and Be replaces Phe.





AVP is produced from a large precursor protein containing not only AVP, but also

neurophysin and a glycopeptide, "signal" sequence (Sachs et al., 1969). The proposed

structure of this molecule is shown in Fig. 2.2, reproduced from Gainer et al. (1980).





Hy Olycopptide AVP Neurophya COOH



Figure 2.2. Proposed structure of the precursor protein for vasopressin. A sugar moiety
is found attached to the glycopeptide, or signal sequence. The (*) represent basic amino
acid residues, which serve as cleavage sites in the precursor molecule.


The precursor protein molecule is packaged in granules with the enzymes needed for

processing AVP to its final form. As the granules move down the axons, posttranslational

processing of the precursor molecule occurs within the granules. When the granules reach

the axon terminals, the nerve is depolarized and the granules are exocytosed, the contents

of the granules (AVP hormone, neurophysin, and fragments of precursor) are released

(Brownstein et al., 1980). Magnocellular neurons containing AVP project fibers to the








median eminence (external and internal zones) and therefore may be important in the

regulation of ACTH secretion (Holmes et al., 1986).

AVP is a potent modulator of pituitary ACTH secretion. In fact in sheep, AVP is

a more potent stimulator of ACTH secretion than CRF (Familari et al., 1989; Liu et al.,

1990). AVP binds to receptors on the anterior pituitary corticotroph to increase plasma

ACTH secretion. These receptors are different from the pressor receptors (subtype V,) or

antidiuretic (subtype V) that are found in the periphery. Data from two different groups

(Baertschi and Friedli, 1985; Jard et al., 1986) suggest that a subtype (classified as V,, or

V3), distinct from the peripheral receptors, exists in the brain with protein kinase C as its

second messenger.

AVP can effect anterior pituitary secretion in two ways; either by AVP secretion

from axons which terminate in the median eminence or by AVP secretion from the

posterior pituitary. Evidence for both possibilities exists. AVP containing neurons of the

SON and PVN are known to project to the external zone of the median eminence

(Hoffmann et al., 1989). A further distinction has been made in that AVP-containing

parvocellular neurons of the PVN were found to innervate the external zone of the median

eminence. The magnocellular neurons of the PVN pass through the internal zone of the

median eminence, to the neurohypophysis, but also contribute to the AVP found in the

median eminence (Holmes et al., 1986). AVP secretion from the posterior pituitary gland

has also been implicated in plasma ACTH secretion. In studies performed in dogs,

neurohypophysectomy attenuated the plasma ACTH response to hypotension. After

restoration of plasma AVP levels to those observed in the intact animal, plasma ACTH

levels were almost completely returned to normal (Raffet al., 1988). The action of AVP

appears to be through a direct effect at the pituitary gland.






16

2.2.3 CRF and AVP as Regulators of ACTH Secretion

The synergistic activity of CRF and AVP on ACTH secretion has been well

documented. In support of this is an overwhelming amount of anatomical data for the

interaction between these two secretagogues. CRF and AVP are found in the same

neurosecretory vesicles in the median eminence (Whitnall et al., 1985). In normal rats,

staining of the median eminence by immunocytochemistry revealed co-localization of AVP

in 50% of CRF axons (Whitnall et al., 1987). Repeated stress (immobilization in rats)

increases the co-localization of AVP in CRF nerve terminals in the median eminence (de

Goeij et al., 1991). Following adrenalectomy in rats, CRF immunostaining increases in

parvocellular neurons of the PVN and the amount of co-localization with AVP increases

(Sawchenko et al., 1984). The increase in CRF and AVP immunoreactivity following

adrenalectomy is prevented by intracerebral injection of dexamethasone (Sawchenko,

1987).

From in vitro studies performed in cultured pituitary cells, CRF is considered to be

the more potent secretagogue for ACTH secretion in rats while AVP appears to be a more

potent stimulus to ACTH release in sheep (Familari et al., 1989). However, the action of

each is potentiated when administered together in cultured pituitary cells (Gillies et al.,

1982), adult freely moving rats ( Rivier and Vale, 1983b) and in fetal sheep (Brooks and

White, 1990). In a study by Engler et al. (1989) performed in conscious sheep, stress-

induced ACTH secretion (audiovisual and insulin-induced hypoglycemia) was

accompanied by increases in hypothalamic CRF and AVP secretion. What is most

interesting is that the CRF:AVP molar ratio was altered with the stress. Portal plasma

AVP was increased above that of CRF, increasing the ratio of AVP to CRF. Since CRF








and AVP can be found in the same neurosecretory vesicles, this suggests that differential

regulation of each individual hormone also occurs. A synergistic effect of CRF and AVP

on ACTH secretion was also observed as seen by a lack of 1:1 concordance between

hypothalamic AVP/CRF secretion and pituitary ACTH secretion. Engler et al. suggested

that this effect may be due to secretion of other hypothalamic factors which increased

ACTH secretion.

2.2.4 Other ACTH Secretagogues

The control of adrenocorticotropin secretion is a complex process involving

numerous factors (neurotransmitters and neuropeptides) that augment ACTH secretion.

Rat pituitary corticotrophs in culture release ACTH in response to epinephrine and

norepinephrine acting on a,-adrenergic receptors (Giguere et al., 1981). Epinephrine has

also been identified in portal plasma suggesting a physiological role in the control of

anterior pituitary function (Johnston et al., 1983). Neuropeptide-Y injected centrally in

fetal and adult sheep increase plasma ACTH concentrations but does not stimulate the

pituitary directly (Brooks et al., 1994). This suggests that NPY acts centrally to increase

the activity of the HPA axis. Evidence for NPY stimulation of CRF secretion supports

this conclusion (Haas and George, 1987). Endogenous opioids are capable of stimulating

the HPA axis in late gestation fetal sheep but do not tonically stimulate the axis in a

regulatory manner (Brooks and Challis, 1988). Serotonin stimulates ACTH secretion in

humans, demonstrated by pharmacologically increasing serotonin with fenfluramine (Lewis

and Sherman, 1984). However, there are conflicting data as to the site of action or

seratonin (hypothalamus or pituitary). In rats, CRF secretion is decreased after

elimination of endogenous hypothalamic catecholamines suggesting a role for central






18

catecholinergic neurons in control of ACTH release (Guillaume et al., 1987). Angiotensin

I also increases plasma ACTH secretion by induction of CRF (Rivier and Vale, 1983).

Prostaglandin E2 alone does not increase ACTH secretion but enhances the ability of AVP

to stimulate ACTH secretion with no effect on CRF (Brooks and Gibson, 1992). In

addition to stimulating activity of the HPA axis, factors from the brain also inhibit the axis.

The dopaminergic system in the amygdaloid central nucleus has been found to inhibit

ACTH secretion by action on the anterior and lateral hypothalamus (Beaulieu, et al.,

1987). Atrial-natriuretic peptide (ANP) has also been shown to alter ACTH secretion.

Brain ANP is secreted into the hypophysial portal vessels from the hypothalamus and

physiological concentrations inhibit ACTH release from pituitary cells in vitro (Dayanithi

and Antoni, 1989; Lim et at., 1990; Sheward et al., 1991). In vivo immunoneutralization

of ANP significantly increased ACTH release but had no effect on release during ether

stress (Fink et al., 1991). These results suggest a role for ANP as a mediator in the

regulation of ACTH secretion.



2.3 Negative Feedback Regulation of ACTH Secretion by Cortisol

Glucocorticoids are very versatile steroids and necessary for survival in a number

of species, including sheep and primates. Binding of the glucocorticoid to its receptor

promotes binding to and transcription of DNA, production of mRNAs for synthesis of

enzymes, and eventually alteration of cell function. Glucocorticoids act in the body to

increase plasma glucose concentrations by increasing hepatic glycogenesis and

gluconeogenesis. They also increase protein catabolism and in the periphery,

glucocorticoids exert actions that counter the effects of insulin. Glucocorticoids are also






19

necessary for vascular reactivity. Without them, the vascular smooth muscle becomes

unresponsive to epinephrine and norepinephrine, the capillaries expand and their walls

become permeable to proteins in the plasma. Finally, glucocorticoids are released in

response to stress or noxious stimuli. In animals that lack normal secretion of

glucocorticoids, exposure to a stress can be life-threatening (Ganong, 1985).

Secretion of glucocorticoids is regulated by adrenocorticotropin hormone from the

pituitary. When a stimulus to ACTH secretion from the hypothalamus reaches the

pituitary gland, ACTH is secreted into the general circulation, binds to its receptor at the

adrenal gland and stimulates cortisol secretion. Cortisol then acts at target organs to

increase plasma glucose levels, etc. However, cortisol also acts at the levels of the brain

to reduce ACTH secretion. Cortisol acts at the hypothalamus and pituitary gland

negatively to inhibit further ACTH secretion. This is called cortisol negative feedback

inhibition of ACTH secretion.

The mechanism of action of cortisol to reduce ACTH secretion has been studied at

length in many species and by many investigators. A study by Canny et al. (1989),

performed in sheep, examined both hypothalamic and pituitary sites of action for

glucocorticoids. Measurements of hypophysial portal concentrations of AVP and CRF

and systemic measurements of ACTH and cortisol concentrations were made before and

after dexamethasone infusion with different stimuli to ACTH secretion. The data suggest

that glucocorticoids act in a site-specific manner to inhibit ACTH secretion (Canny et al.,

1989). During audiovisual stress, hypothalamic CRF and AVP secretion were unaltered

but ACTH secretion was inhibited suggesting a pituitary site of action of dexamethasone.

In response to hypoglycemia, dexamethasone inhibited both the hypothalamic and pituitary






20

responses to the stress. Studies in rats have shown that glucocorticoids inhibit CRF

secretion as well as CRF synthesis (Sato et al., 1975) suggesting a mechanism for a

hypothalamic site of action. On the other hand, glucocorticoids may interfere with CRF

activation of second messenger systems (cAMP) at the pituitary and therefore prevent

stimulation of ACTH secretion (Bilezikjian and Vale, 1983).

In the fetus cortisol has the ability to inhibit plasma ACTH secretion in response to

a hypotensive stimuli by approximately 90 days gestation (Hargrave and Rose, 1985).

High concentrations of glucocorticoids near the fetal PVN prevent increased ACTH

secretion in response to hypotension and hypoxemia (some effects at the level of CRH-

loss of staining in the median eminence, and loss of AVP staining in the external zone of

the median eminence; McDonald et al., 1990). Between 117 and 131 days gestation, fetal

sheep are extremely sensitive to the negative feedback effects of cortisol. This was

demonstrated by infusions of cortisol that caused less than 2ng/ml increases in plasma

cortisol concentrations but which completely inhibited the normal ACTH response to

hypotension (Wood, 1986). This knowledge predicts the existence of a normal feedback

response in the fetus, which is the case in late gestation fetal sheep. However, in near

term fetal sheep, cortisol negative feedback regulation of ACTH secretion becomes

ineffective. In experiments in which infusions of cortisol increased plasma cortisol

concentrations to approximately 60 ng/ml, fetal plasma ACTH secretion was still not

suppressed (Wood, 1987, 1988). The mechanism of this reduction in glucocorticoid

negative feedback efficacy is not fully understood at present.






21

2.4 Glucocorticoid Receptor Structure and Function

Biological effects ofglucocorticoids are dependent on the presence of functional

receptors (Miesfeld et al., 1986). The glucocorticoid receptor is a member of a

superfamily ofligand-dependent nuclear transcription factors (Evans, 1988). The

glucocorticoid receptor is a transcriptional activator which binds to cis-acting

glucocorticoid response element sequences and alters the rate of transcription of specific

genes (Yamamoto, 1985; Burnstein and Cidlowski, 1989). Binding studies indicate that

the glucocorticoid receptor (as well as other steroid and thyroid receptors) dimerize and

bind to their response elements with high affinity (Tsai et al., 1988). Upon activation by

hormone binding, the glucocorticoid receptor is thought to interact with one or more

components of the transcriptional machinery to increase or decrease the rate of

transcription of the genes with which it is associated.

The glucocorticoid receptor has separate and distinct binding domains (Wrange

and Gustafsson, 1978; Giguere et al., 1986). DNA binding occurs between amino acids

421-481 on the human receptor and this binding site is highly conserved among steroid

receptors (Giguere et al., 1986; Weinberger et al., 1985). This region consists of a

clustering of basic amino acid residues which can interact with DNA and a cysteine rich

motif which participates in the formation of zinc fingers, another aid in DNA binding

(Evans, 1988). Steroid binding occurs approximately between amino acids 500-777 at a

single binding site (Giguere et al., 1986; Weinberger et al., 1985). An amino acid region

located within the steroid binding domain (approximately a.a. 574-596) of the human

glucocorticoid receptor is thought to be involved in direct interaction with a 90 kD heat

shock protein (HSP 90) dimer to form the nonactivated 9S complex (Pratt et al., 1988).








Upon binding of glucocorticoid to its receptor and receptor activation, the HSP 90 dimer

dissociates from the receptor. However, loss of the HSP 90 dimer before receptor

activation results in lower affinity of the glucocorticoid receptor for its ligand (Denis and

Gustafsson, 1989). If bases are deleted from the steroid binding domain, the receptor

becomes constitutively active (Godowski et al., 1987).

Nonactivated glucocorticoid receptor with a sedimentation coefficient of

approximately 9S and a molecular weight of approximately 300 kD under non-denaturing

conditions has been found in the cytosol of target cells. Activated complexes have a

molecular weight of approximately 100 kD (Sherman and Stevens, 1984). These data

support the concept of a HSP 90 dimer binding the nonactivated receptor and dissociating

upon activation purely on the molecular weight data alone. It has been postulated that the

HSP binding to the receptor maintains proper conformation so after activation by ligand

binding, the receptor is able to associate with the DNA. Some studies have suggested that

receptor phosphorylation (Orti et al., 1989) and disulfide bond (Silva and Cidlowski,

1989) formation may also be involved in receptor action. Evidence suggests that

glucocorticoids are necessary for nuclear translocation (Picard and Yamamoto, 1987) and

transcriptional modulation by the glucocorticoid receptor (Picard et al., 1990), but their

role in regulating receptor DNA binding is uncertain.

One factor determining the cellular responsiveness to glucocorticoids is the

concentration of glucocorticoid receptors (Bougeios and Newby, 1979; Gehring et al.,

1986). Glucocorticoids can negatively regulate gene expression, possibly through

negative GREs which have been identified in a few genes. Binding of the GR to these

GREs results in inhibition of transcription of the gene with which the negative GRE is

associated (Beato et al., 1989).













A B C
94kD


90 kD 90kD




A BC



90 994kD
90 Ik D 90


No-activated GR
pprox. 300 kD



Glcoortoid


- Dissociated HSP 90 dimer


I ""A/B C




A BC



GRE


Activated Receptor


Figure 3: Proposed model for in vitro activation of the glucocorticoid receptor (GR)
(modified from Denis and Gustafsson, 1989). The non-activated receptor is bound to a
dimer of heat shock protein 90 (HSP 90), which masks the DNA binding domain (B).
Binding of ligand to the steroid binding domain (A) results in modification of the GR and
dissociation of the HSP 90 dimer. The activated receptor complex (as a dimer) is then
able to bind DNA at specific sites (GREs glucocorticoid response elements). Area 0 on
the receptor is the immunogenic region.






24

2.5 Gonadal Steroid Modulation of ACTH Secretion

It has long been known that female rats have greater activity of the HPA axis than

male rats. Studies in adult animals demonstrate that female rats have increased

corticosterone secretion in response to stress as compared with male rats. They also had

greater corticosterone secretion following ACTH administration and greater adrenal

responsiveness to tropic stimulation (Kitay, 1961). As the major difference between the

sexes is gonadal steroid production, experiments involving gonadectomy and replacement

of gonadal steroids were performed. After gonadectomy, testosterone depressed ACTH

content and steroid clearance in male rats but increased adrenal responsiveness to ACTH.

In female rats, estradiol had a consistent stimulatory effect on ACTH secretion (Kitay,

1963). In female rats, plasma ACTH and corticosterone responses to restraint stress were

enhanced during proestrus, when estradiol concentrations are highest (Kitay, 1963). In

ovariectomized (ovx) rats replaced with estradiol, this effect can be restored (Viau and

Meaney, 1991). Following ovariectomy, there was a decreased capacity of the pituitary to

synthesize ACTH and a decreased responsiveness to stimulation by hypothalamic extracts

(Coyne and Kitay, 1969). In ovx rats, estradiol implants into the area of the anterior

pituitary, arcuate nucleus and lateral mammillary bodies in rats facilitated pituitary-adrenal

activity, suggesting a central nervous system effect (Richard, 1965). However, estrogen

stimulation of corticosterone secretion in ovx rats may be due in part to a direct effect on

the adrenal cortex (Kitay et al., 1965). In ovx rats, plasma ACTH and corticosterone

responses to foot shock and ether vapor stress were lower than in estrogen-replaced ovx

rats (Burgess and Handa, 1992). From these results, it was concluded that the increased

activity of the pituitary-adrenal axis was due to an impairment of the glucocorticoid

negative feedback mechanism.








The mechanism of action of estrogen stimulation of HPA axis activity is not fully

understood. Based on the present data, estrogen may interact with a number of systems

impinging on the HPA axis. Estrogen uptake has been demonstrated in corticotrophs

isolated from anterior pituitary cells from orchidectomized, adrenalectomized rats (Keefer,

1981), suggesting a possible direct effect of estrogen on the pituitary. Estradiol has also

been shown to concentrate in tyrosine-hydroxylase containing neurons in the arcuate and

periventricular nuclei of the rat (Sar, 1984). In another study, estradiol-concentrating cells

have been found in the amygdaloid central nucleus (Beaulieu et al., 1987). Since this

dopaminergic system can inhibit ACTH secretion, this may be important in modulating

HPA axis activity. In rats given 100 pg estradiol exogenously for two weeks, there was

an increase in AVP in SON and PVN of the hypothalamus without any changes in

pituitary ACTH and AVP content or basal plasma ACTH or AVP concentrations

(Hashimoto et al., 1981). In another study, plasma AVP concentrations were found to be

greatest when estrogen concentrations were highest. If the rats were ovariectomized,

plasma AVP concentrations decreased but were restored when estrogen was replaced

(Skowsky et al., 1979). These data suggest a possible role ofhypothalamic releasing

factors in the mediation of estrogen stimulation of HPA axis activity.

Androgens have a different effect on the HPA axis. Gonadectomy in male rats

increases basal and stimulated plasma ACTH concentrations (Coyne and Kitay, 1971). If

androgens are replaced (testosterone or dihydrotestosterone), this increase in plasma

ACTH can be reversed (Handa et al., 1994). However, testosterone implants were

ineffective in altering corticosterone secretion (Telegdy et al., 1963). Sexual maturation in

male rats is accompanied by an increase in POMC gene transcript levels, but testosterone






26

given to immature male rats had no effect on POMC gene transcript levels (Kerrigan et al.,

1992). So it appears that androgens may play an inhibitory role in HPA axis activity. The

mechanism of this action is unknown. It has been demonstrated that orchidectomy had no

effect on hippocampal, hypothalamic, or pituitary glucocorticoid receptors, suggesting

that androgens have no effect on glucocorticoid negative feedback in adult rats (Handa et

al., 1994).



2.6 Summary

As has been described, the process of parturition in the fetal sheep is dependent

upon the HPA axis of the fetus. Near the end of gestation, a poorly understood signal

increases activity of the HPA axis. This has been described as an increase in drive to the

HPA axis and is reflected in increased plasma ACTH and cortisol concentrations of the

fetus beginning at approximately 120-125 days gestation. Plasma ACTH and cortisol

concentrations are further increased in late gestation by a decrease in glucocorticoid

negative feedback efficacy at approximately 140 days gestation. This alteration in

negative feedback sensitivity appears to be the final step in increasing plasma ACTH

secretion and initiating parturition. Understanding this process is crucial in understanding

the mechanism of parturition.

In this system, all aspects of the HPA axis seem to have been examined. However,

cortisol also increases the production of plasma androgens and estrogens in late gestation.

As stated previously, the gonadal steroids have a profound effect on plasma ACTH

secretion in the adult animal. We hypothesized that androgens and estrogens may also

play a role in pituitary-adrenal axis activity in the fetal sheep. If these gonadal steroids do






27

have the ability to influence fetal pituitary ACTH secretion, then they may also influence

the process of initiation of parturition. The following studies were designed to examine

the possibility of a role for gonadal steroids in the control of parturition in the ovine fetus.













CHAPTER 3
GENERAL METHODOLOGY



3.1 Animals and Surgery

The sheep used in these experiments were pregnant ewes (74 days gestation or

later) of various breeds (mixed western, Florida native, etc.). Animals were purchased

from three suppliers (Institute of Food and Agricultural Sciences, University of Florida,

Gainesville, FL; Arborlow Farms, Newberry, FL; Tom Morris, MD). Animals were

housed in approved pens in the Health Center Animal Resources Department or at the

University of Florida Veterinary School, Metabolic Unit and were maintained under

controlled lighting and temperature. Pens were cleaned daily by the husbandry staff and

ewes were offered food and water ad libitum.

Surgeries were performed using aseptic technique in the Animal Resources

Department surgery suites. There were no deaths in these experiments due to the surgical

procedure or lack of sterility during the surgery. Ewes were fasted 24 hours prior to

surgery and water was withheld for 12 hours. Anesthesia was induced and maintained

with 0.5% 3.0% v/v halothane or isoflurane in oxygen. The abdomen and flank were

shorn and aseptically prepared for surgery with povidone iodine. Following intubation

ewes were connected to a ventilator. Heart rate, mean arterial pressure, rectal

temperature, ventilatory CO2 and O2, and respirations were all monitored during the

surgery. When ewes were fully recovered, free access to food and water was permitted.

28






29

Surgery was performed between days 108 and 125 of gestation, depending on

experimental design. The ewes were monitored at all times during the surgery and

recovery until the animal could stand under its own effort.

An incision was made along the midline of the abdomen beginning at the umbilicus

and extending caudally approximately 10 cm. The hindlimbs of the fetus were located by

palpation and the uterine horn containing the hindlimbs was exteriorized. The uterus was

incised and a hindlimb was exteriorized from the uterus. An arterial polyvinyl chloride

catheter (.030" ID, .050" OD) was placed into the tibial artery and the tip advanced to the

descending aorta. A venous polyvinyl chloride catheter (.040" ID, .070" OD) was placed

into the saphenous vein and the tip advanced to the inferior vena cava. The limb was

returned to the amniotic cavity and the contralateral hindlimb was catheterized as was the

first. In each case, the distal end of the vessel was closed with suture ligatures and the

catheter was sutured in place with 2.0 silk. A steroid implant or placebo was inserted

subcutaneously into the area of the gluteus medius before suturing the second hindlimb. A

polyvinyl chloride catheter (.050" ID, .090" OD) with side holes cut into the tip was

attached to the second hindlimb with suture for delivery of antibiotics to the amniotic

space and for amniotic fluid pressure measurements. The second hindlimb was returned to

the amniotic cavity and the uterus was closed with 2.0 silk. 500 mg ampicillin (Polyflex@,

Fort Dodge Laboratories, Fort Dodge, IA) was administered into the amniotic cavity and

the catheters were exteriorized through a flank incision via trochar. The catheters were

filled with heparin (1000 units/ml, Elkins-Sinn, Inc, Cherry Hill, NJ) and closed with a

sterile brad inserted into the end. The abdomen was then closed with #3 polyamid suture

(Pitman-Moore, France) and 500 mg ampicillin was administered IM to the ewe. A cloth






30

pouch to house and protect the catheters was fixed to the ewe's flank with #1 polyamid

suture. A piece of spandage was then put around the ewes abdomen to prevent the

catheters from falling out of the pouch.

Some ewes also received maternal catheters at the time of surgery. An incision

was made at the femoral triangle and the femoral artery and vein were exposed and the

distal end of the vessel was ligated closed. Polyvinyl chloride catheters (.050" ID, .090"

OD) were inserted into both vessels. The arterial catheter was advanced to the descending

aorta and the venous catheter was advanced to the inferior vena cava. The catheters were

filled with heparin and closed with a sterile nail and exited subcutaneously via a separate

flank incision next to the incision for the fetal catheters.

In ewes with twin pregnancies, both fetuses were catheterized if they appeared

strong and healthy. If one of the fetuses displayed apparent growth retardation (i.e. was a

"runt"), it was not used in the experiments. This was done, in part, because the blood

vessels were too small to catheterize. In the case of twins, one twin received the steroid

implant and the other twin was a vehicle control. Following surgery, the ewes were

treated with 500 mg ampicillin IM BID for 5 days and their temperatures were monitored

as an indication of infection.

Steroid pellets were obtained from Innovative Research of America (Toledo, OH).

Pellets were 5 mg, 21 day releasing pellets or 15 mg, 60 day releasing pellets

(approximately 10 pg per hour). This dose was chosen in order to raise plasma estrogen

and androstenedione concentrations to levels that were seen prior to parturition.

The ewes were treated post-operatively with 500 mg ampicillin IM BID for 5 days.

Temperatures were also monitored BID for fever as an indication of infection. Post-






31

operative pain was determined on an individual basis and intervention was needed only in

a few cases (veterinary care provided by Animal resources).



3.2 Blood Collection and Handling

Catheters were removed from the cloth pouch and the distal ends were scrubbed

with povidone iodine (Betadine@, Purdue Frederick Co., Norwalk, CT) and alcohol. The

brad was removed and a sterile blunt adapter was inserted. Blood was withdrawn until it

replaced the catheter dead space (about 3 ml in the fetus and about 5 ml in the ewe). The

resultant fluid was discarded. Blood (3-5 ml) was then drawn and collected into chilled

tubes containing Na4EDTA (57 pg or .3M EDTA/ml bloodXSigma Chemical Co., St.

Louis, MO). Blood gas samples (1.5 ml) were drawn anaerobically into syringes coated

with heparin for measurement of fetal blood gases and pH (BMS3MK2 blood

microsystem and PHM73 pH and blood gas analyzer, 37C, Radiometer, Copenhagen,

Denmark). After sampling, the blood volume removed was replaced with 0.9%/ normal

saline (Baxter Healthcare Co., Deerfield, IL) and the catheters refilled with heparin.

Catheters were then plugged with sterile brads and returned to the cloth pouch. Each day

the catheters were opened, the fetus was administered 750 mg ampicillin via the amniotic

catheter.

Blood samples were kept on ice until transported to the lab (approximately 1-4

hours). Samples were centrifuged at 3000 x g for 20 minutes at 40C in a refrigerated

centrifuge (Sorvall RT6000B, Dupont, Newtown, CT). After centrifugation, the plasma

was transferred to a clean tube and stored at -20C until hormones were assayed.






32

3.3 Infusion Experimental Procedures

Experiments were performed between days 126 and 135 days gestation. All

experiments were started between 0900 and 1100 hours to minimize possible variation in

hormone concentrations between experiments. On the morning of the experiment, ewes

were loaded into a study cart and transported to a study room (either in the animal

resources facility or in the laboratory). Ewes remained in the cart for the duration of

sampling. Catheters were then flushed with heparinized saline (2.0% v/v) and connected

to transducers (Statham P231d, Statham Instruments, Oxnard, CA) for measurement of

fetal arterial and amniotic pressure. At least one hour was then allowed for the ewe to

adjust to the new environment. Arterial and amniotic fluid pressures were measured

during the final 20 minutes of the experiment using a Grass Model 7 recorder. The data

were digitized and stored using an IBM AT microcomputer and a Keithley analog-to-

digital converter on-line.

The experiments used to examine fetal ACTH responses were designed as

"feedback experiment." The fetal plasma ACTH response to a stress (in this case,

hypotension) was elicited by infusion of nitroprusside. This response is reproducible and

controllable by infusing at a known dose and rate. The dose and rate selected decreased

fetal mean arterial blood pressure significantly (approximately 50%). In order to assess

feedback efficacy of the hypothalamus-pituitary-adrenal axis, fetal plasma ACTH response

was examined under control conditions with an infusion of normal saline prior to the

hypotension stress. This response was then compared to the fetal plasma ACTH response

following infusion of cortisol (hydrocortisone sodium succinate, Upjohn, Kalamazoo, MI)

(0.20 ng/ml in 0.9% saline) infused into to fetal inferior vena cava at a rate of 5.1 ml/hr for






33

2 hr. This dose of cortisol inhibited the fetal HPA axis and decreased fetal plasma ACTH

secretion by approximately 50% in control fetuses. By comparing the control plasma

ACTH response to the inhibited response following the cortisol infusion, an estimate of

feedback efficacy was obtained.

The stimulus for fetal plasma ACTH secretion was sodium nitroprusside (NiPr,

Elkins-Sinn, Inc., Cherry Hill, NJ). NiPr is a nitric oxide (NO) donor. The NO acts on

the smooth muscle vascular epithelium causing relaxation and a subsequent decrease in

MAP. This decrease in MAP is sensed by the carotid sinus baroreceptors as a decrease in

stretch. This decrease in MAP results in decreased firing of impulses from the carotid

sinus through the nerve of Hering to the glossopharyngeal nerve to the nucleus of the

tracts solitarius in the medulla (Berne, Levy, 1986). In addition to increasing

sympathetic tone to restore blood pressure, the NTS also has projections to the

hypothalamus. With a decrease in MAP, the NTS signals the hypothalamus and plasma

ACTH secretion (as well as AVP) is increased. NO acts through its second messenger,

guanylate cyclase, to increase cGMP, which may also be an independent stimulus for

ACTH secretion.

In each experiment, vehicle (0.9% w/v normal saline) or cortisol was infused into

the fetal inferior vena cava at a rate of 5.1 ml/hr for 2 hours. One hour after the end of the

vehicle or cortisol infusion, NiPr was infused into the fetal inferior vena for 10 minutes at

a rate of 50 pg/min (0.5 ml/min) using a constant infusion syringe pump (Sage

Instruments, Orion Research, Inc., Model 341A, Cambridge, MA). Blood sample (5 ml)

was drawn from the fetal arterial catheter at the beginning of the vehicle or cortisol

infusion. Blood samples (3 ml) were drawn from the fetus after I and 2 hours of vehicle






34

or cortisol infusion and 0, 10, 20 minutes after the start of the NiPr infusion. Blood

samples were handled as described above.



3.4 Hormone Assay Techniques

Plasma ACTH concentrations were measured by radioimmunoassay (RIA) as

previously described (Bell et al., 1991) using an antibody raised in rabbits developed in

this laboratory to human-ACTH(1-24). Iodinated ACTH (I'M-ACTH) was prepared using

the chloramine-T method (Berson and Yalow, 1968) with human-ACTH (1-39) (Sigma,

St. Louis, MO) and radioactive sodium iodide (Amersham, Arlington Heights, IL). I'~-

ACTH was made fresh approximately every 6 weeks. ACTH was first extracted from

plasma before assaying. ACTH (500 pl) was extracted on glass (35 mg per tube)(100-

200 mesh glass, Coming Glass Works, Coming, NY) in 500 pl assay buffer (.05M

phosphate buffer, pH 7.4, with 0.2% w/v silicic acid-extracted bovine serum albumin

(BSA, Sigma, #9647). The supernatant was aspirated and the glass washed with assay

buffer. The ACTH was eluted from the glass with 1 ml acid:acetone (1 volume 0.25N

HCL: 1 volume acetone). The extracts were dried under vacuum (Savant Instruments,

Farmingdale, NY) and frozen at -200C until assayed. Extracts were reconstituted

overnight in 500 pl assay buffer (containing 0.5% v/v mercaptoethanol). Extraction

recovery was corrected by comparing samples to a standard curve prepared from standard

extracted with each set of samples.

Plasma AVP concentrations were measured as previously described (Raffet al,

1991) using an AVP antibody raised in rabbits. Iodinated AVP was purchased from

Amersham and synthetic AVP from Sigma. AVP was extracted from 500 ul plasma with






35

1 ml bentonite slurry (0.3% w/v in distilled water) and acidified with 500 pl IN HCI.

Extracts were eluted with 1 ml acid:acetone (20% IN HCI: 80% acetone) with sonication.

Samples were then evaporated to dryness and stored at -200C until assayed. Extracts

were reconstituted with 250 pl assay buffer (0.05 M phosphate buffer, pH 7.4 with 0.01

M EDTA (Sigma, #ED4SS) and 0.2% BSA w/v (Sigma, #A-7638)). Extraction recovery

was corrected by comparing samples to a standard curve prepared from standard extracted

with each set of samples.

Plasma cortisol concentrations were measured as previously described (Wood et

al., 1993) using an antibody raised in rabbits and tritiated cortisol purchased from

Amersham (#TRK-407) and cortisol standard from Sigma. Cortisol was extracted from

20 pl plasma (in duplicate) with 1 ml ethanol. Standard was prepared in ethanol, and

standards and samples were dried under vacuum with heat and immediately reconstituted

with 500 pl assay buffer [0.05 M phosphate buffer (using 0.06 M sodium phosphate

dibasic and 0.04 M sodium phosphate monobasic) pH 7.0, with 0.15 M NaCI, 0.1% w/v

gelatin, and 0.1% w/v sodium azide].

Plasma estrogen concentrations were measured as previously described (Kalra and

Kalra, 1974). 500 pl plasma was extracted twice in duplicate with 2 ml ethyl ether.

Extracts were dried under air and reconstituted to 200 pl with assay buffer (0.1M

phosphate buffer, pH 7.0, with 0.1% w/v gelatin, 0.9% w/v sodium chloride, and 0.1%

w/v sodium azide). Rabbit antiserum #244 was used at a dilution of 1:50,000 for a final

assay dilution of 1:200,000. Tritiated estradiol was obtained from Dupont NEN (# NET-

317). This antibody cross-reacted with estradiol (approximately 10%) and estrone,

therefore results were expressed as plasma estrogen concentrations. This antibody also






36

cross-reacted with a substance in sheep plasma which resulted in a background reading of

6-10 pg/ml in plasma from ovariectomized (ovx) ewes. When the plasma was stripped

with charcoal, this interference was eliminated. To correct for this blank problem, a pool

of ovx plasma was evaluated in each assay. The value obtained from this pool was then

subtracted from values obtained for the unknowns and results were calculated. A pool of

maternal plasma was also included as an internal standard.

Due to the normal physiological variations in plasma estrogen values and the high

background interference in the assay, plasma estradiol concentrations were further

analyzed after separation of estradiol from estrone. 1 ml plasma from each fetus

(estradiol-treated and sham-control) was extracted as previously described. After

evaporation, each sample was reconstituted in the solvent system used in the Quik-Sep@

LH-20 steroid separation system (Isolab inc., Akron, OH). Each sample was fractionated

by passing through the columns, the appropriate fractions collected, evaporated to

dryness, and reconstituted with assay buffer. Samples were then run in the estrogen assay

as before. This measurement was used to estimate the increase in plasma estradiol levels

by the administration of the steroid pellets. The plasma estrogen levels were used in the

ontogeny analysis, since the plasma estradiol was measured for only a single time point in

each fetus.

Tissue CRF concentrations were measured as previously described (Keller-Wood

and Wood, 1991) using an antibody to ovine CRF (oCRF). Iodinated oCRF was

purchased from Amersham and oCRF standard from Bachem (Torrance, CA). Tissue

samples were diluted in assay buffer (0.05 M phosphate buffer, pH 7.4 with 0.01 M

EDTA, 0.2% BSA w/v (fraction V, #A-7638), 0.1% w/v sodium azide, and 0.05% v/v








Triton X-100) and run unextracted. Samples from G-75 column were evaporated to

dryness and reconstituted in assay buffer to be run in the assay.

Plasma androstenedione concentrations were measured using a kit from Diagnostic

Products Corporation, Los Angeles, CA. 500 pl plasma was extracted with 5 ml ethyl

acetate and hexane (3:2 ratio, respectively). The liquid phase was frozen and the organic

phase was transferred to a clean tube and evaporated to dryness in a warm water bath

under nitrogen. Samples were then reconstituted in 600 pl assay buffer (provided in kit)

and 200 pl aliquots were assayed.



3.5 Tissue Collection and Handling

After experimentation, the ewes, fetuses and lambs were euthanized with sodium

pentobarbital (minimum of 1 grain/kg). Tissues were dissected as quickly as possible,

placed in a clean tube and quick frozen in a dry ice and acetone bath. Samples were

stored at -20C until processed. Tissues were later homogenized (Tekmar, Cincinnati,

OH) in Laemmli buffer (Laemmli, 1970)) (0.0625 M Tris with 2% w/v SDS, 10% v/v

glycerol, 0.72 M mercaptoethanol, and 0.3% w/v PMSF) in a ratio of 5 ml buffer per 1 g

tissue. Homogenates were centrifuged at 15,000 x g for 20 minutes at Oc.. to remove

particulate matter and supernatant was stored at -400C until analyzed by RIA or Western

blotting. Protein concentration of each sample was determined by the method of Bradford

(Bradford, 1976) using a kit purchased from BioRad (# 500-0002, Hercules, CA). Results

were expressed or corrected for the protein concentration in each sample.

Tissue samples were run on pre-cast polyacrylamide gels (BioRad) with 20-40 pg

protein loaded per lane for each sample. Molecular weights were estimated by comparing








bands to known molecular weight proteins (Amersham, Rainbow coloured markers,

#RPN-755 and #RPN-756) which were run on each gel. Separation of the proteins were

performed using a Mini-Protean II cell gel electrophoresis system (BioRad) at

approximately 100 V. Proteins were transferred to membranes overnight at 22 V.

Membranes were blocked with a 7.5% w/v non-fat powdered milk solution and stained

using anti-rabbit Ig, horseradish peroxidase, linked whole antibody (raised in donkey, #Na-

934, Amersham) diluted 1:700 and RenaissanceTM western blot chemiluminescence

reagent (Dupont). Individual protocols based on the protein being identified are as

follows:



Glucocorticoid receptor Samples were run on 10% (#161-0907) stacking gels.

Primary antibody was obtained from Affinity BioReagents (PAl-511, Neshanic

Station, NJ) and diluted to 10pg per blot for incubation.

Corticotropin-releasing factor Samples were run on 15% (161-0908) stacking

gels. Primary antibody was the same as was used in the RIA. A dilution of

1:80,000 was used for incubation.

Arginine vasopressin Samples were run on 15% stacking gels. Primary antibody

was the same as was used in the RIA. A dilution of 1:34,000 was used for

incubation.

Adrenocorticotropic hormone Samples were run on 16.5% (#161-0922) tris-tricine gels.

Primary antibody was obtained from Zymed Laboratories (# 18-0087, S. San

Francisco, CA) and diluted 1:250 for incubation.













CHAPTER 4
MODULATION OF OVINE FETAL ADRENOCORTICOTROPIN SECRETION
BY ANDROSTENEDIONE AND 17P-ESTRADIOL



4.1 introduction

In fetal sheep, parturition is stimulated by increased fetal hypothalamus-pituitary-

adrenal (HPA) axis activity (Challis and Brooks, 1989; Liggins et al., 1973). Parturition

can be prevented or delayed by destruction of the pituitary (Liggins et al., 1966, 1967;

Liggins and Kennedy, 1968) or stimulated by infusions ofACTH (Liggins, 1968, 1969) or

glucocorticoids (Jack et al., 1975; Wood and Keller-Wood, 1991). Between 100 and 125

days of gestation, fetal plasma ACTH and cortisol concentrations are low; during this time

a majority of the cortisol in fetal plasma originates from the maternal adrenal. Fetal

adrenal cortisol secretion slowly increases between 125 to 135 days of gestation. After

135 days of gestation, ACTH and cortisol continue to increase in a semilogarithmic

pattern (Bassett and Thorburn, 1969; Rose et al., 1978; Wintour et al., 1975; Wood,

1988) until parturition occurs at approximately 145 days (term in the chronically

instrumented animal). Increasing concentrations of cortisol in fetal plasma act at the

placenta to induce the enzyme, cytochrome P450c7, which has 17a-hydroxylase and 17,

20 lyase activities. Induction of this enzyme increases the production of androgens and

estrogens from progesterone (Anderson et al., 1975; Pomerantz and Nalbandov, 1975;

Steele et al., 1976; Yu et al., 1983). The resultant increase in estrogen-to-progesterone








ratio increases uterine contractility and eventually culminates in initiation of labor and

parturition (Liggins, 1974).

Understanding the mechanism of the increased fetal HPA axis activity at the end of

gestation is key to understanding the mechanism of spontaneous parturition in this species.

It has been previously demonstrated that fetal sheep are very sensitive to cortisol negative

feedback of ACTH secretion between 120 and 130 days (Wood, 1986) but then become

relatively insensitive to the effects of cortisol near term (Wood, 1988). It has been

proposed that this decrease in cortisol negative feedback efficacy after 135 days of

gestation may be important for the generation of the preparturient increase in plasma

ACTH and cortisol (Wood, 1988). The mechanism of this change in negative feedback

has not been identified. It is possible that increased fetal plasma estrogen and/or androgen

concentrations might be involved.

Studies in adult animals have shown that estrogen increases the activity of the

HPA axis. Female rats have higher basal and stimulated plasma corticosterone levels than

males rats (Kitay, 1961) and female rats have enhanced plasma ACTH and corticosterone

responses to stress during proestrus (Viau and Meaney, 1991). Several groups have

shown in rats that ovariectomy results in decreased plasma ACTH bioactivity and

corticosterone responses to stress that can be reversed by exogenous estrogen

replacement (Burgess and Handa, 1992; Coyne and Kitay, 1969; Kitay, 1963). Because

estrogens and androgens increase prior to parturition and estrogens are known to interact

with the HPA axis, we propose that androgens or estrogens might influence fetal ACTH

secretion at the end of gestation.

These experiments were designed to investigate the effects of exogenous

androstenedione and 17p-estradiol on ACTH secretion in late gestation fetal sheep.








These steroids were chosen because they are naturally-occurring steroids with known

increased concentrations at the end of gestation. Specifically, we tested the hypothesis

that elevated fetal androstenedione or estradiol would result in increased plasma ACTH

secretion through altered cortisol negative feedback.



4.2 Materials and Methods

Animals Twenty five pregnant ewes of known gestational ages were studied.

Seventeen of these ewes were included in all aspects of this protocol. An additional eight

ewes were included as supplementary animals used to estimate the effect of hormone

pellet implantation on fetal plasma steroid concentrations. Of the animals subjected to the

complete protocol, 11 ewes carried singleton fetuses and 6 ewes carried twins. In the

supplementary group, all 8 ewes carried singleton fetuses.

Fetal surgery. Aseptic surgery was performed for chronic implantation of vascular

and amniotic fluid catheters and steroid pellets on day 1220.9 (estradiol-treated group),

120.0t1.1 (androstenedione treated group) or day 122.4*1.0 (control group) days of

gestation (term is approximately 148 days in uncatheterized fetal sheep). Surgery was

performed as previously described (Wood, 1986, 1988). At the time of catheter

placement, a pellet containing 5 mg androstenedione or 173-estradiol (Innovative

Research of America, Toledo, OH) was implanted subcutaneously in the area of the

gluteus medius. Sham control fetuses were catheterized but did not receive a pellet

implant. Each fetus received 750 mg ampicillin via the amniotic fluid at the time of

surgery and again each day the vascular catheters were flushed. The ewes received 750

mg ampicillin IM post-operatively BID for 5 days. Catheters were flushed and






42

reheparinized at the time of blood collection or experimentation (at least once every 3

days).





Twin PRcsgmcy SigletonPgmawy





'rA' T B,

e4ated(n-3) 4h1m (n-3) ShMa conol (n-3)
j atrMd(n-5)
A4tled (n-3) A-4hm(n-3) A-trated(n-3)





Figure 4.1. Distribution of experimental animals.

The distribution of singleton and twin pregnancies and the distribution of estradiol,

androstenedione, or sham treatments are illustrated in Figure 4.1. In the 6 ewes carrying

twins, one twin received a pellet and the other twin was a sham control (3 estradiol and 3

androstenedione, each with a twin control). Eight singleton fetuses received pellets (5

estradiol and 3 androstenedione) and three were sham controls.

Experimental protocol Twenty-three fetuses were subjected to an experimental

protocol designed to test fetal ACTH responsiveness to stimulation and to test for

alterations in cortisol negative feedback sensitivity (previously described in Chapter 2).

The experimental design is illustrated in Figure 4.2. Briefly, vehicle (0.9% w/v saline) or

cortisol (HSS: hydrocortisone sodium succinate; 6.0 pg/ml in 0.9% w/v saline)






43

was infused into the fetal inferior vena cava at a rate of 5.1 ml/hr for 2 h. One hour after

the end of the vehicle or HSS infusion, NiPr was infused into the fetal inferior vena cava

for 10 min at a rate of 50 pg/min (0.5 ml/min) using a constant infusion syringe pump.

Blood samples (5 ml) were drawn from the fetal arterial catheters at the beginning of the

vehicle or HSS infusion. Blood samples (3 ml) were drawn from the fetus after I and 2 h

of vehicle or HSS infusion and 0, 10, and 20 min after the start of the NiPr infusion.

Blood samples were placed into chilled tubes containing EDTA. An additional fetal

arterial blood sample (1 ml) was drawn anaerobically at the beginning of each experiment

for measurement of fetal blood gases and pH. Arterial and amniotic fluid pressures were

measured during the final 20 min of the experiment. The data were digitized and stored










t t t ttt
Saline or HSS Infusion NiPr Infusion




-180 -120 -60 01020
Time (minutes)




Figure 4.2. Experimental design.

using an IBM AT microcomputer and a Keithley analog-to-digital converter on-line.

Not all of the 23 fetuses included in this protocol were subjected to both saline and

HSS infusion experiments. Seven fetuses were subjected to only one experiment because

of spontaneous delivery (n=3), complications not related to the experiment (n=3), and








pump failure (n=1). Although not all fetuses were studied in all parts of the experimental

design, all fetuses were healthy as defined by pH, P02, and PCO2 when they were studied

(Table 4.1).

A supplemental group of 8 fetuses were subjected to surgical implantation (n=2) or

sham-implantation (n=6) of androstenedione pellets and were allowed to recover from

surgery, as described above. These fetuses were not subjected to saline, HSS, or

nitroprusside infusion. They were used only to test the effect of pellet implantation or

sham pellet implantation on fetal plasma steroid concentrations. The addition of these

fetuses was necessary because we needed larger group sizes in order to increase the

statistical power in the assessment of statistical significance when analyzing fetal plasma

concentrations of estradiol and androstenedione.

Analysis of blood samples. Plasma ACTH, cortisol, and estradiol concentrations

were measured by RIA as previously described (Bell, et al., 1991; Kalra and Kalra, 1974;

Wood, et al., 1993). Plasma androstenedione concentrations were measured using a

commercial RIA kit (Diagnostic Products Corporation, Los Angeles, CA). Plasma ACTH

and cortisol concentrations were measured in all plasma samples; androstenedione and

estradiol were measured in the first plasma sample drawn in each experiment. In the

fetuses subjected to the complete protocol, the volume of plasma available for analysis

was limited, therefore we measured plasma androstenedione concentrations in all fetuses

treated with androstenedione, but only in 3 sham fetuses. To augment the number of

fetuses available for measurement of plasma steroid concentrations after pellet

implantation, we added a supplemental group of 2 androstenedione-treated fetuses and 6

sham fetuses. All estradiol samples were run in a single assay, as were the

androstenedione samples.








Calculations and statistical analyses Fetal mean arterial pressure was corrected by

subtraction ofamniotic fluid pressure. Data were analyzed using one- and two-way

analysis of variance (ANOVA) for equal or nearly equal "n" corrected for repeated

measures in one dimension (time) (Winer, 1971). The two-way ANOVA was used to

compare values of experimental variables in saline- and HSS-infusion experiments within

each treatment group (i.e., control, androstenedione, and estradiol treatment groups). A

posteriori pairwise testing of differences between group means was performed using

Duncan's multiple range test (Duncan, 1957). A significance level of0.05 was used to

reject the null hypothesis in all tests. Values are reported as the mean SEM.



4.3 Results

Blood eases. Fetal arterial blood gases and pH measured prior to the start of

experiments are reported in Table 4.1. The values in each group are similar to blood gases

that we have reported in healthy fetuses in previous experiments (Wood, 1986; Wood,

1988). There was no statistically significant differences in any of the measurements

between any of the treatment groups (data are mean 4 SEM).

Table 4.1. Fetal blood gases and pH at the time of experimentation.
P.O2 (mmHg) P.CO2(mmHg) pH.
Androstenedione + saline (n=6) 21.31.5 49.83.1 7.348-0.008
Androstenedione + HSS (n=6) 22.111.5 48.8*2.9 7.334-0.009
Estradiol+saline (n=6) 24.22.1 43.2-1.0 7.331+0.023
Estradiol + HSS (n=7) 21.31.6 43.10.9 7.3460.014
Sham + saline (n=8) 25.11.2 45.712.2 7.337+0.008
Sham + HSS (n=8) 21.41.2 45.0*2.6 7.350a0.012








Fetal plasma estradiol and androstenedione concentrations. Plasma estradiol

concentrations were significantly increased in estradiol-treated fetuses (39.111.1 pg/ml,

n=7) compared with sham-treated fetuses (11.43.9 pg/ml, n=12). Similarly, fetal plasma

androstenedione concentrations in androstenedione-treated fetuses (273.120 pg/ml, n=9)

were significantly elevated when compared to sham-treated fetuses (203.510 pg/ml,

n=8). The breakdown of twin and single fetuses along with their corresponding estradiol

or androstenedione concentrations are shown in Table 4.2. There was no significant

difference in steroid plasma concentrations between the twin control fetuses and the single

sham-control fetuses, indicating no significant steroid transfer in utero between the treated

twin and the control twin.

Table 4.2. Plasma steroid concentrations in the twin and sin leton fetuses.
Plasma Estradiol (pg/ml) Plasma A.Dione (pg/ml)
Steroid-Treated Twin 58.526.1 (n=3) 238.6a 25.3 (n=4)
Control Twin 21.110.0 (n=3) 209.4 21.8 (n=4)
Steroid Singleton 27.46.9 (n=5) 307.6 27.3 (n=4)
Control Singleton 8.13.7 (n=9) 198.8 8.1 (n=5)


Basal fetal plasma ACTH and cortisol concentrations. Basal or initial plasma

concentrations of ACTH and cortisol (prior to the start of saline infusion) are illustrated in

Figure 4.3. Estradiol treatment significantly increased fetal plasma ACTH (top panel) and

cortisol (bottom panel) concentrations (p<0.05). Androstenedione treatment did not

significantly alter fetal plasma concentrations of either ACTH or cortisol (data are

represented as mean SEM).











Fetal Plasma ACTH (pgl)


ieo0


aIRi
Estadkl 'dlone


Fetal Plasma Cortisol (ng/ml)


0 L--


Esiadil 'dan


Figure 4.3. Basal fetal plasma ACTH (top panel) and cortisol (bottom panel)
concentrations in sham-, estradiol-, and androstenedione-treated fetuses at time -180 in
the saline-infused animals. Data are represented as mean values and error bars represent 1
SEM.


ACTH and cortisol responses to saline or HSS infusion. Fetal plasma cortisol

concentrations before and during a two-hour infusion of HSS or saline are reported in

Table 4.3. In both the sham control and the androstenedione-treated fetuses, HSS


1400 /
1000 /
aw


200
400 -
0 m-EM
Shm






48

infusion produced statistically significant increases and saline infusion did not produce any

changes in fetal plasma cortisol concentration. In the estradiol-treated fetuses, however,

there were spontaneous, statistically significant (p<0.05), increases in fetal plasma

concentrations of cortisol during the infusion of saline. Because of this apparently

spontaneous activity of the HPA axis, we measured no apparent increase in fetal plasma

cortisol concentration during cortisol infusion, but a paradoxical increase during saline

infusion (Table 4.3a). The apparent increase in fetal plasma cortisol during saline infusion

was accounted for by spontaneously increased fetal HPA axis activity in 3 of the 5

estrogen-treated fetuses in this experimental group.



Table 4.3. Plasma cortisol (table 4.3a) and ACTH (table 4.3b) concentrations
during HSS or saline infusions. Data are expressed as mean SEM.

Table 4.3a. Fetal plasma cortisol concentrations (ng/ml).
TIME A'Dione A'Dione Estradiol Estradiol Control Control
(MIN) + Saline +HSS +Saline +HSS +Saline +HSS
-180 2.320.5 3.611.0 15.54.9 17.4+2.5 6.101.2 8.002.0
-120 1.910.4 7.07+3.6 25.49.8 17.93.0 7.041.9 14.13.6

-60 1.740.2 7.35+3.2 29.313.4 15.6a2.3 5.62*1.6 13.13.4
0 2.940.8 3.371.1 27.213.6 16.72.3 7.12-1.7 9.712.1

Table 4.3b. Fetal plasma ACTH concentrations (pg/ml).
TIME A'Dione A'Dione Estradiol Estradiol Control Control
(MIN) + Saline +HSS +Saline +HSS +Saline +HSS
-180 19913 18215 846618 12327 12739 8821
-120 19217 16714 1508*786 9419 10728 142+22
-60 15615 18711 1444569 11737 10933 16317

0 19534 18213 707423 12917 17241 11120






49

Fetal plasma ACTH was not significantly altered in any of the groups between -180 and 0

min, except in the estradiol-treated fetuses (Table 4.3b). During saline infusion, the

estradiol-treated fetuses exhibited an apparent increase (but not statistically significant) in

plasma ACTH concentrations compared to the HSS infused fetuses at the -120 and -60

min time points. The increases in both ACTH and cortisol concentrations in the plasma of

these fetuses was accounted for by apparently spontaneous increases in three of the five

fetuses.

Stimulus to fetal ACTH secretion: blood pressure responses to nitroprusside.

Nitroprusside infusion produced hypotension to a similar degree in both infusion groups

(saline vs. HSS) in each treatment group (Figure 4.4: sham, estradiol, and androstenedione

groups are shown in top, middle, and bottom panels, respectively). HSS infusion tended

to increase fetal mean arterial blood pressure in all treatment groups, although this effect

was statistically significant only in the androstenedione-treated fetuses.

ACTH and cortisol responses to nitroprusside infusion. The plasma ACTH and

cortisol responses to the nitroprusside infusion are shown in Figures 4.5 and 4.6.

Nitroprusside infusion significantly increased plasma ACTH concentrations following

saline infusion (peak values of 1237+311 pg/ml, 2239874 pg/ml, and 1045429 pg/ml in

androstenedione-, estradiol- and sham-fetuses at 10 min, respectively). Comparison of the

peak fetal plasma ACTH values in the sham- vs. estradiol-treated fetuses revealed that

estradiol treatment significantly augmented the ACTH response to nitroprusside.

Comparison of peak values in sham- and androstenedione-treated fetuses did not reveal

any significant alteration of the responsiveness to nitroprusside by androstenedione.








Control Fetuses


Estradiol fetuses


Androstenedlone fetuses




I I I I I
0 5 10 15 20
Time (minutes)


Figure 4.4. Fetal mean arterial pressure responses to nitroprusside infusion in sham-
control, estradiol, and androstenedione fetuses following 2 hour saline infusion (0) or
HSS (*) infusion. Nitroprusside was infused between 0 and 10 min. Data are
represented as mean SEM.


~5~rs~~(









Androstenedione


1000 -
-^


ff'-
2 10 -

I


1000
I-







1000



100


Estradiol


0 10
Time (min)


Figure 4.5. Fetal plasma ACTH responses (log scale) to nitropmrsside infusion in
androstenedione, estradiol, and sham control fetuses following 2 hour saline (0) or HSS
(0) infusion. Nitroprusside was infused between 0 and 10 minutes. Data are represented
as mean SEM.






52

The ACTH response to nitroprusside was significantly reduced by HSS infusion in

the sham control group when analyzed by two-way ANOVA corrected for repeated

measures. However, this analysis was not able to demonstrate statistical significance of

the effect of HSS on fetal ACTH responses to nitroprusside in either the estradiol-treated

fetuses or in the androstenedione-treated fetuses. When the sham control and estradiol-

treated fetuses were analyzed together in a three-way ANOVA corrected for repeated

measures, there was a significant interaction effect of HSS infusion and experimental

treatment (sham control vs. estradiol treatment). A similar comparison of the

androstenedione- and sham-treated fetuses by three-way ANOVA also demonstrated a

significant interaction between HSS infusion and experimental group. When aposteriori

analysis was performed after three-way ANOVA to compare peak ACTH responses to

nitroprusside, the peak values measured after HSS infusion in the estradiol- and sham-

treated fetuses (888450 and 40492 pg/ml, respectively) were significantly lower than

peak values in each group after saline infusion (2239874 and 1045429 pg/ml,

respectively). There was no significant difference between peak values after HSS- and

saline- infusion in the androstenedione-treated fetuses (95799 vs. 1237311 pg/ml,

respectively).

Nitroprusside infusion significantly increased fetal plasma cortisol concentrations

in all groups except the estradiol-treated, saline-infused fetuses (Figure 4.6). Analysis of

these data by two-way ANOVA revealed that the fetal plasma cortisol response to

hypotension was not suppressed in any group. In both sham and androstenedione-treated

fetuses, fetal plasma cortisol concentration was significantly higher in the HSS group than

in the saline group. In the saline-infused estradiol-treated fetuses, fetal plasma cortisol








was already elevated as the result of the spontaneous elevation in fetal HPA axis activity

prior to nitroprusside infusion. For this reason, the fetal plasma cortisol concentrations

were not increased further in response to the nitroprusside infusion, and there was no

apparent suppression of the fetal plasma cortisol response to nitroprusside after HSS

infusion.


40 Androtmnedion
30 -
20





10


20


40



30

20
10
0 ------------





0 10 20
Time (mln)


Figure 4.6. Fetal plasma cortisol responses to nitroprusside infusion in androstenedione,
estradiol, and sham control fetuses following 2 hour saline (0) or HSS (0) infusion.
Nitroprusside was infused between 0 and 10 minutes. Data are represented as mean
SEM.






54

4.4 Discussion

The results of this study demonstrate that 1) estradiol augments fetal ACTH

responses to hypotension; and 2) androstenedione decreases the efficacy ofcortisol

negative feedback inhibition of fetal plasma ACTH secretion. We propose that the

increase in hypothalamic-pituitary-adrenal axis activity resulting from physiological

increases in fetal plasma androgen and estrogen concentrations is an important component

in the preparturient increase in fetal ACTH and cortisol secretion at the end of gestation in

this species.

In the present experiments, the effect of exogenous androstenedione appeared to

be different from the effect of the exogenous estradiol. Estradiol augmented the plasma

ACTH response to hypotension, while androstenedione reduced cortisol negative

feedback efficacy. It is remarkable that, even though estradiol doubled the fetal ACTH

response to hypotension, HSS suppressed the fetal ACTH responses approximately 60%

in both the estradiol- and sham-treated groups. This was consistent with the observation

in adult animals that cortisol negative feedback suppression of ACTH responses to low-

and moderate intensity-stimuli were essentially independent of stimulus intensity (Keller-

Wood and Dallman, 1984). On the other hand, exogenous androstenedione completely

eliminated cortisol negative feedback. We believe that the effect of androstenedione is

similar to the spontaneous reduction in cortisol negative feedback efficacy observed at the

end of gestation (Wood, 1988).

These results are consistent with the known actions of estrogens in adult animals.

In adult rats, ovariectomy has been shown to decrease both basal and ether-stimulated

plasma ACTH concentrations and to decrease pituitary responsiveness to stimulation by






55

hypothalamic extracts in vitro (Coyne and Kitay, 1969). Female rats have increased

corticosterone responses to stress of exposure to ether vapors and have a greater adrenal

responsiveness to exogenously administered ACTH when compared to male rats (Kitay,

1961). In ovariectomized rats, exogenously administered estradiol increased plasma

ACTH bioactivity and adrenal responsiveness to ACTH (Kitay, 1963), and increased

plasma ACTH and corticosterone responses to stress (Burgess and Handa, 1992; Viau and

Meaney, 1991). However, our results differ from those of Burgess and Handa (1992)

who reported that estradiol administration to ovariectomized rats impaired glucocorticoid

negative feedback inhibition of ACTH secretion. We found that androgen, not estrogen,

impaired glucocorticoid negative feedback efficacy in sheep fetus.

The effect of androgen on ovine fetal ACTH secretion also differed from that in

the rat. Castration of male rats chronically increased both basal and stimulated plasma

ACTH concentrations (Coyne and Kitay, 1971; Handa et al., 1994). Treatment of

castrated rats with testosterone (Coyne and Kitay, 1971; Handa et al., 1994) or

dihydrotestosterone (Handa et al., 1994) reversed this effect. It is therefore apparent that

androgen is inhibitory to ACTH secretion in the male rat. The lack of effect of castration

on hippocampal, hypothalamic, or pituitary corticosteroid receptors suggests gonadal

steroids do not act through corticosteroid negative feedback in rats (Handa et al., 1994).

We found that administration of androstenedione does not by itself alter basal or

stimulated ovine fetal plasma ACTH, but did block corticosteroid negative feedback

effects on ACTH. Whether this represents a species difference or, perhaps, a difference in

experimental paradigm (intact fetal sheep exposed to both gonadal and placental steroids

versus gonadectomized adult rats) cannot be addressed without further experiments.








HSS infusion had a tendency to increase mean arterial pressure compared with

saline infusion. Excesses ofglucocorticoids were shown to be associated with

hypertension in man and in animals (Grunfeld, 1990). The HSS infusion used in these

experiments utilized minimal concentrations, increasing plasma cortisol concentrations

approximately 5 pg/ml in the androstenedione fetuses. In these fetuses, MAP was

increased approximately 5 mmHg, which was statistically significant. However, in all

experimental groups, there was no significant difference in the MAP drop, indicating a

similar degree of hypotension created by the nitroprusside infusion.

The doses of estradiol and androstenedione used in the present experiments are

physiological. The present results demonstrate a high sensitivity of the fetal

hypothalamus-pituitary-adrenal axis to estradiol and androstenedione. The release of

estradiol or androstenedione from implanted pellets (approximately 238 pg/day or 9.9

pg/hr, as stated by Innovative Research of America, the supplier of the pellets) elevated

plasma estradiol and androstenedione concentrations approximately 28 and 70 pg/ml,

respectively. These concentrations were well within the range of endogenous

concentrations reported at the end of gestation (Yu et al., 1983).

The apparent stimulation of the fetal HPA axis by estradiol increased fetal plasma

ACTH and cortisol concentrations unrelated to the infusions of saline, HSS, or

nitroprusside. Specifically, 3 of the 5 estradiol-treated fetuses displayed spontaneously

increased fetal plasma ACTH during saline infusion (at -60 min: up to 1670, 2560, and

2742 pg/ml). The result of this spontaneous HPA activity was elevated mean plasma

ACTH concentrations at time 0 in the saline-infused estradiol-treated fetuses compared to

HSS-infused fetuses (707473 vs. 12949 pg/ml, respectively). We believe that








spontaneous HPA activity also complicated measurement of changes in fetal plasma

cortisol in the HSS-infused group. We administered HSS at a rate of 0.5 pg/min for 2

hours, a dose which completely suppressed fetal ACTH responses to the same dose of

nitroprusside when infused for 5 hours (Wood, 1986). The expected increase in fetal

plasma cortisol concentration during this infusion was small (Wood, 1986), but was

completely obscured in the present experiments by the variable baseline in fetal cortisol

and the relatively infrequent blood sampling.

We were unable to demonstrate statistically significant differences in the fetal

plasma cortisol response to nitroprusside infusion. We believe that the fetal cortisol

response to nitroprusside infusion in the present studies was limited by the maximal

adrenal secretion rate of cortisol. The peak fetal plasma concentrations of cortisol

measured in each group were consistent with known maximal fetal plasma cortisol

concentrations in late-gestation fetuses (Rose et al., 1982).

A preparturient increase in maternal and fetal plasma estrogen concentration has

been documented by a number of investigators, although the time course of this increase

varies within studies. Maternal unconjugated estrogens have been reported to increase

over the last 40 hours (Robertson and Smeaton, 1973) to 4 days (Bedford et al., 1972)

with a very sharp increase immediately before parturition (Challis, 1971). In the fetus, a

similar pattern has been reported for conjugated and unconjugated estrone (Nathanielsz et

al., 1982); however, data in one study demonstrated a more gradual increase in fetal and

amniotic estradiol over approximately 8 days with a rapid increase in fetal, maternal and

amniotic concentrations in the final two days of fetal life (Challis and Patrick, 1981).

Increased estrogens are also seen during dexamethasone-induced parturition (Kendall et






58

al., 1977). The fetuses in this study were subjected to 17p-estradiol chronically over the

course of approximately two weeks, a pattern which does not faithfully mimic the pattern

of plasma estradiol concentrations at term in intact fetal sheep. While the present data

cannot be interpreted as evidence that fetal plasma estradiol represents a "trigger" to

parturition, the results suggest that estrogens play a facilitatory role in HPA axis activation

at the end of gestation by increasing the magnitude of the ACTH responses to stimuli.

We conclude that exogenous androstenedione and 17p-estradiol in physiological

concentrations modulate plasma ACTH secretion in late-gestation fetal sheep. Estrogen

increased plasma ACTH both during nitroprusside-induced hypotension and during

apparently "basal" conditions. Androstenedione decreased the negative feedback action of

cortisol on ACTH secretion (demonstrated by the lack of suppression of the ACTH

response to nitroprusside-induced hypotension following the infusion of HSS). We

therefore speculate that physiological increases in fetal plasma androgen and estrogen

concentrations at the end of gestation are an important component of the mechanism

which results in parturition in this species.













CHAPTER 5
ONTOGENY AND MOLECULAR WEIGHT OF IMMUNOREACTIVE ARGININE
VASOPRESSIN AND CORTICOTROPIN-RELEASING FACTOR IN THE OVINE
FETAL HYPOTHALAMUS


5.1 Introduction

In sheep, parturition is stimulated by an increase in the activity of the fetal

hypothalamus-pituitary-adrenal (HPA) axis (Liggins et al., 1973; Challis and Brooks,

1989). This is observed as an increase in plasma concentrations of adrenocorticotropin

hormone (ACTH) and cortisol in the fetus. Increased fetal plasma concentrations of

cortisol induce the activity of cytochrome P450,17 (Anderson et al., 1975; Steele et al.,

1976), an enzyme that catalyzes the synthesis of estrogens from progesterone. Near the

end of gestation, fetal plasma ACTH secretion is further increased as cortisol negative

feedback sensitivity decreases (Wood, 1988), thereby increasing fetal plasma cortisol

concentrations and placental estrogen production. An increase in the rate of production of

estrogens relative to progesterone increases uterine contractility and ultimately induces

labor and delivery (Liggins, 1974).

Both corticotropin releasing factor (CRF) and arginine vasopressin (AVP)

stimulate the release of ACTH from corticotrophs. In rats, CRF is more potent than AVP

in stimulating ACTH secretion. In sheep, AVP is more potent (Familari et al., 1989; Liu

et al., 1990). In both species, CRF and AVP are synergistic when corticotrophs are

exposed to both (Brooks and White, 1989). ACTH secretion in vivo is most likely the






60

result of combined CRF and AVP secretion. CRF- and AVP-containing neurons within

the hypothalamus are thought to be the primary controllers of ACTH secretion. In rats,

500% of the AVP-positive parvocellular neurons of the SON and PVN also stain for CRF

(Whitnal et al., 1987). AVP and CRF have been found in the same secretary vesicles in

these neurons (Whitnal et al., 1985). AVP from magnocellular neurons of the PVN is

thought to play a role in the stimulation of ACTH secretion, although this role is

secondary to that of the parvocellular AVP (Holmes et al., 1986; Raff et al, 1985).

We hypothesized that hypothalamic content of immunoreactive CRF and/or AVP

(iCRF and iAVP) increase at the end of gestation. We further hypothesized that the

hypothalamic content of iCRF and iAVP is comprised of both processed and unprocessed

forms of these hormones. The present experiments were designed to test these

hypotheses.



5.2 Materials and Methods

Tissues. Experiments were performed using tissues collected from pregnant ewes

of known gestational ages. Fetal hypothalami and pituitaries were harvested from healthy

fetuses at various gestational ages (74-101 days, 125-129 days, 135-139 days, 141 days-

term) and from 3- 4 week old healthy lambs as previously described. The tissues were

stored at -20C or -40"C until processed.

Tissues were later homogenized and processed as previously described. Protein

concentrations of the homogenates were determined and homogenates were stored at -

40*C until assayed or run on gels.

Hormone assays. Tissue content ofiCRF and iAVP was determined by

radioimmunoassay (RIA) ofpeptides in Laemmli buffer homogenates. Hypothalamic








tissues were diluted 1:100 with assay buffer and pituitary tissues were diluted 1:500-

20,000. Tissue content is expressed as pg or ng hormone/mg protein. Descriptions of

these radioimmunoassays were published previously (Keller-Wood and Wood, 1991, and

Raffet al., 1991, respectively). After dilution, we found no interference of the Laemmli

buffer with the binding ofpeptides to the antisera.

Sephadex chromatography. Three fetal hypothalami (ages 138, 138 and 143 days

of gestation) were homogenized in CRF assay buffer (.05M phosphate, .01M EDTA,

0.1% v/v Triton, 0.1% w/v sodium azide) with 5% v/v glycerol, then centrifuged to

remove the cellular debris. Samples were applied to a 49 x 1.5 cm Sephadex G75 column

(Pharmacia, Uppsala, Sweden). The column was primed with 2 ml charcoal stripped

sheep plasma, calibrated with molecular weight standards (Sigma), and equilibrated with

CRF assay buffer. 100gl of the tissue homogenate or synthetic oCRF standard was added

directly to the column in a mixture containing blue dextran and phenol red in CRF buffer

with 5% v/v glycerol. Fractions were collected every 2 min at a flow rate of 600 dl/min

and frozen at -20 until assayed. Both iCRF and iAVP concentrations were measured

from each fraction. To correct for interference by the CRF buffer system with the AVP

antibody, the immunoreactive AVP was calculated from a standard curve prepared in CRF

buffer and the assay was performed as described previously (Raff et al., 1991).

Western blotting. Western blots were performed as described previously. The

protein was then transferred to nitrocellulose membranes and examined for the presence of

iAVP and iCRF using the antibodies that were used in RIAs of these hormones. Blots

were visualized as previously described and analyzed by densitometry. Antibody

specificity was determined by preabsorption of the primary antibody with synthetic peptide

and identification of non-specific banding patterns.








Statistical analysis. Initial analysis of the data for heteroscedasticity (distribution

of error) revealed that they were not normally distributed. Overall comparison ofiCRF

and iAVP concentrations among the tested ages was performed using the Kruskal Wallis

H test (one way analysis of variance on ranks). Multiple comparisons were tested using

Dunn's test. Data are expressed as mean SEM. Significance was established at p<.05.

Statistical procedures were accomplished using SigmaStat (Jandel Scientific, San Rafael,

CA).



5.3 Results

iAVP. The ontogeny of pituitary immunoreactive AVP (iAVP) content is shown

in Figure 5.1. There was no statistically significant effect ofgestational age on pituitary

AVP content within the range of ages tested (H=7.62, df=4, p=. 10). The fetal ontogeny

of hypothalamic iAVP is shown in Figure 5.2. iAVP concentration in hypothalamic tissue

increased significantly as a function of age (H=13.6, df= 4, p < 0.01). The lowest iAVP

concentration was measured in the 125-129 day fetal age group, and the highest in the 3-4

week old lambs (Fig. 5.2). The iAVP concentrations at these two ages were significantly

different from each other when tested by Dunn's test (p< 0.05).

iCRF. The ontogeny of fetal hypothalamic immunoreactive CRF (iCRF)

concentration is shown in Figure 5.3. Hypothalamic iCRF concentration increased

significantly as a function of age (H=16.9, df=3, p<0.O01). The increase in hypothalamic

iCRF concentration appeared more gradual than the increase in hypothalamic iAVP

concentration. Thus, the concentrations in the term fetuses and the 135-139 day fetuses

were significantly greater than in the 74-101 day fetuses.































Figure 5.1. Pituitary content ofimmunoreactive arginine vasopressin. Gestational age
did not significantly effect pituitary content ofiAVP within the range of ages tested
(H=7.62, df-4, p=0.10).


Figure 5.2. Hypothalamic content ofimmunoreactive AVP. Tissue content ofiAVP
in the fetal hypothalamus increased significantly as a function of age (H=13.6, df=4,
p<0.01).


80
sao -






40
20



74-M.d 129-12 64 laU1 14AL4mtM IMe*
ANe


aso
o70

4o00





10o
400



74-1M& 13-1 d. 1t IM 41M Hn .

















2000



1500



S1000 -
LL
i T
.1
E 500 -


= r n=6 7 r7 nS

74-101 d. 125-129d. 135-139d. 141d.-trm lambs
Age





Figure 5.3. Hypothalamic content ofimmunoreactive CRF (iCRF). Tissue content of
iCRF in the fetal hypothalamus increased significantly as a function of age (H=16.9, df=4,
P<0.001).

Molecular weight analysis. The results of size exclusion chromatography

(Sephadex G75) for iCRF are shown in Figure 5.4. iCRF isolated from fetal hypothalamus

(fig. 5.4, top) eluted earlier than the synthetic ovine CRF peptide (fig. 5.4, bottom),












Hypothalamic ICRF

. A


oCRF
..


20 40 1
Fraction number


Figure 5.4. Hypothalamic iCRF eluted faster than synthetic oCRF. This indicates a larger
molecular weight form than the synthetic CRF.


demonstrating that the peptide found in this tissue had a higher molecular weight than fully

processed CRF. Hypothalamic tissue analyzed by Western blot revealed a band which

migrated with the 21.5 kD molecular weight marker (fig. 5.5), a weight which is

consistent with the molecular weight of pro-CRF, and another band with a molecular








weight which was approximately 45 kD. These bands were specific for iCRF since

preabsorption of the antiserum with synthetic oCRF reduced the intensity of the staining.



46kD-- "i
30kD-*--
21.5kD -- --- Tissue iCRF

14.3kD

6.5kD *

Synthetic oCRF




Figure 5.5. Western blot analysis ofhypothalamus homogenate. Bands specific for iCRF
are denoted by arrows.

iAVP was eluted from the Sephadex column in two molecular weight forms (fig.

5.6). The major peak of iAVP is consistent with fully processed AVP (~1 kD). A second

major peak was found in the void volume, suggesting the presence of a high molecular

weight moiety. The high molecular weight forms of iAVP were re-examined using

Western Blot. A band was clearly visible with a molecular weight of approximately 20 kD

and another band was visible with a molecular weight of approximately 40 kD (fig. 5.7).

These bands represent specific staining for iAVP, since the intensity of staining is reduced

after preabsorption of the antiserum with synthetic peptide.























Figure 5.6. Elution profile ofiAVP in hypothalamic homogenate.


46kD -*-

30kD -

21.5kD "-- --Tissue iAVP

14.3kD --


Figure 5.7. Western blot analysis ofhypothalamus homogenate. Bands specific for iAVP
are denoted by arrows.






68

5. 4 Discussion

The results of this study indicate that concentrations of ACTH releasing hormones

in hypothalamic tissue changed with time throughout development. Hypothalamic iAVP

did not increase significantly until postnatal life; however, the highest concentrations of

hypothalamic iCRF were measured at the end of gestation. The measured increases in the

concentrations of these hormones demonstrate that maturation of the hypothalamus-

pituitary-adrenal axis includes a maturation of the secretary capacity of hypothalamic

neurons. The pattern ofiCRF relative to that for iAVP suggests that changes in iCRF

secretary capacity at the end of gestation might be an important factor in the stimulation

of the preparturient fetal ACTH surge.

The ontogeny ofiCRF and iAVP measured in the present study was remarkably

similar to that reported by Brieu et al. (1989), although we were unable to demonstrate

any decrease in iCRF or iAVP in animals near term gestation (141 days to term, Figures

5.2 and 5.3). Our demonstration of increased iCRF as a function of fetal gestational age

was also similar to data reported by Watabe et al. (1991) and Currie and Brooks (1992),

as stated previously. We speculate that this developmental increase is important to

increased plasma ACTH secretion in fetal sheep observed near the end of gestation.

Watabe, et al. (1991) examined hypothalamic iCRF in fetal sheep ranging in age

from 48-140 days of gestation. Currie and Brooks (1992) examined hypothalamic iCRF in

fetal sheep ranging in age from 70-130 days of gestation. The results of our present study

agree with the results of the previous studies in that we demonstrated an increase in fetal

hypothalamic iCRF concentration at the end of gestation. The results of our study

differed, however, in that we found a high molecular weight form ofiCRF in fetal








hypothalamus. In the Watabe study, only one molecular weight species ofiCRF was

identified in hypothalamic extracts (Watabe et al., 1991). We detected iCRF with a higher

molecular weight than fully processed CRF using Sephadex gel chromatography. The

results of Western blot analysis demonstrated one molecular form of iCRF consistent with

the known molecular weight of pro-CRF (Lauber et al., 1984) and another with molecular

weight consistent with a dimer ofpro-CRF. We believe that there are two likely

explanations for the difference between our results and those reported by other

investigators. First, differences might be due to the binding characteristics of the

antibodies used in the studies. Our anti-CRF antibody recognized the N-terminal region of

ovine CRF (Keller-Wood and Wood, 1991) while Watabe, et al. used an antibody that

recognized the C-terminal end of rat CRF. Second, we quantified immunoreactive

peptides in tissue which was homogenized directly in a reducing buffer. The previous

studies quantified the peptides after acid extraction, a protocol which could have been

confounded by poor recovery of or proteolysis of the unprocessed forms of the peptides.

Experiments with bovine hypothalamic CRF, for example, have shown that lyophilization,

boiling for 15 min, or boiling at a low pH shifted CRF immunoreactivity and/or bioactivity

to a higher fraction, suggesting proteolytic cleavage of the peptide (Yasuda and Yasuda,

1985a; Yasuda and Greer, 1978). The evidence for large molecular weight forms of

iCRF in the present study is consistent with data from human fetal (Ackland et al., 1986),

human adult (Suda et al., 1984), and rat (Yasuda and Yasuda, 1985b) hypothalami.

Currie and Brooks (1992) found one molecular weight form of iAVP which

corresponded to synthetic AVP in their hypothalamic extracts. This contrasts with the

results of the present study, and with the known processing of AVP in other species.








Sephadex chromatography demonstrated a form ofiAVP with a molecular weight

consistent with fully processed AVP (fig. 5.5a), and a larger molecular weight form.

Western Blot analysis demonstrated one form with a molecular weight (-20 kD)

consistent with pro-AVP (Russell, et al., 1979), and another form with a molecular weight

of approximately 40 kD. It is likely that the 40 kD form is a dimer of pro-AVP. The

molecular weight of pro-AVP has been estimated to be 17,310 kD in cows (Land et al.,

1982) and 20,000 kD in rats (Russell et al., 1979). The presence of pro-AVP in

hypothalamic tissue is consistent with the known processing of AVP in rats (Russell et al.,

1979). We believe that Currie and Brooks measured only the fully processed AVP

because of the binding characteristics of the anti-AVP antiserum used in their study (ie., it

is possible that their antiserum did not cross-react with pro-AVP). It is also possible that

processed and unprocessed forms of AVP were differentially recovered in the hydrochloric

and acetic acid extraction procedure used in that study.

The observation that CRH mRNA in the hypothalamus is present and increases as

a function ofgestational age supports our results of CRF protein content (Matthews and

Challis, 1995). In examining the hypothalamic AVP tissue content, we did not

differentiate between magnocellular or parvocellular AVP. Matthews and Challis (1995)

found that magnocellular AVP mRNA increased in late gestation. Parvocellular AVP

mRNA did not change at this time, but the two increased in the newborn. These results

are similar to our study in which we did not detect a significant increase in hypothalamic

iAVP until after birth. In work performed by Keiger, et al., CRF message and peptide

were measured in the fetal ovine hypothalamus over 4 gestational age ranges (95-106,

120-123, 128-138, and 140-142 days). CRF mRNA levels in the 140-142 day group were








significantly different than the other groups analyzed. However, the CRF peptide

concentrations in the 140-142 day group were significantly greater than only the 95-106

and 128-138 day groups, showing a possible dissociation between message and protein

levels.

In summary, we have demonstrated that 1) fetal hypothalamus contains precursor

proteins for AVP and CRF; and 2) that hypothalamic iAVP and iCRF increases as a

function of fetal and neonatal developmental age when measured by radioimmunoassay.

An increase in tissue stores of ACTH releasing factors may reflect increased synthesis and

secretion of these hormones and therefore an increased stimulus to ACTH secretion. The

increased tissue stores of releasing hormones might complement the decrease in cortisol

negative feedback inhibition of ACTH secretion that occurs late in gestation during the

preparturient rise in fetal ACTH secretion. Therefore, this increase in iAVP and iCRF

might be an important component of the increase in hypothalamus-pituitary-adrenal

activity that initiates parturition in this species.













CHAPTER 6
ONTOGENY OF PROOPIOMELANOCORTIN POSTTRANSLATIONAL
PROCESSING IN THE OVINE FETAL PITUITARY



6.1 Introduction

In the sheep, parturition is stimulated by an increase in the activity of the fetal

hypothalamus-pituitary-adrenal (HPA) axis (Liggins et al., 1973; Challis and Brooks,

1989). In late gestation there is an increase in fetal HPA axis activity which increases

plasma adrenocorticotropin (ACTH) and cortisol concentrations. Cortisol acts at the

placenta to increase production of estrogens, which increase uterine contractility and

ultimately leads to labor and delivery (Liggins, 1974).

ACTH is initially synthesized as pro-opiomelanocortin (POMC), a precursor for

ACTH as well as P-endorphin, P-lipotropin, and a-melanocyte-stimulating hormone

(Eipper and Mains, 1982). After synthesis, POMC, a 32 kilodalton (kD) peptide, is

cleaved twice in order to release ACTH (Figure 6.1; Schnabel et al., 1989). The first

processing step yields a 22 kD fragment (so-called "pro-ACTH") which contains the

sequence for ACTH. The second step yields fully processed ACTH. Because POMC,

22kD fragment, and fully processed ACTH (ACTHI.3,) all contain the ACTH epitope,

anti-ACTH antisera generally recognize all three forms of ACTH.

Evidence from several laboratories has demonstrated that fully processed ACTHI.3

stimulates fetal adrenal secretion of cortisol; in addition, however, the larger molecular






73

weight forms of immunoreactive ACTH (iACTH) appear to antagonize the effect of

ACTHI, (Jones and Roebuck, 1980; Roebuck and Jones, 1980). This differential effect

of fully processed ACTH, and unprocessed and partially processed POMC produces

changes in the ratio of biologically active ACTH (bACTH) to iACTH in various

conditions.


Pro-opiomelanocortin (POMC)


hepdds






22 kD pro-ACTH



1 9



1 94


ACTH


beta-lipotropin



136 139 209


1-39


136
13 gamma -beta



139 176 179-209


Figure 6.1. Diagram of the stepwise processing of ACTH from POMC, including other
major products of POMC processing. Figure adapted from Schnabel et al. (1989).


COOH






74

In ovine fetal plasma, the concentration of fully processed ACTH increases relative to that

of the larger molecular weight forms (Thorbum et al., 1991). The present study was

designed to test the hypothesis whether the concentration of fully processed ACTH

increases in fetal pituitary relative to that of either POMC or pro-ACTH with

development. We also designed these experiments to test the hypothesis that

administration of exogenous estrogens and androgens increase the pituitary concentration

of fully processed ACTH observed in mature fetuses.



6.2 Materials and Methods

Tissue Experiments were performed using tissues collected as previously

described from healthy fetuses at various gestational ages (74-101 days, 125-129 days,

135-139 days, 141 days-term) and from 3-4 week old healthy lambs. Fetal tissues were

rapidly collected, quick-frozen, and stored at -200C. Tissues were processed as previously

described. Protein concentrations of the homogenates were determined and homogenates

were stored at -40C until assayed or run on gels.

Steroid treated fetuses. Fetuses were treated with 21 day, 5 mg 17p-estradiol or

androstenedione pellets implanted in the area of the gluteus medius on about day 120 of

gestation (approximately 238 pg per day). We had previously reported that implantation

of estradiol pellets increased fetal plasma estradiol concentration to 39.111.1 pg/ml

compared to control fetuses, in which fetal plasma estradiol concentration was 11.4+3.9

pg/ml (Chapter 4). Implantation of androstenedione pellets increased fetal plasma

androstenedione concentrations to 27320 pg/ml compared to control fetuses in which

fetal plasma androstenedione concentration was 20310 pg/ml (refer to Chapter 4). Fetal

pituitaries were harvested one to two weeks after pellet implantation.






75

Hormone assays. Tissue content ofiACTH was determined by radioimmunoassay

(RIA) ofpeptides in Laemmli buffer homogenates. Pituitary homogenates were diluted

1:10,000 with assay buffer and tissue content was expressed as ng hormone/mg protein.

The ACTH RIA was performed as previously described (Bell et al., 1991). After dilution

with assay buffer, we found no interference of the Laemmli buffer with the binding of

ACTH to the antisera. However, the concentration of ACTH as determined by the

immunoreactivity did not dilute parallel to the standard curve, so the assay was performed

with all the samples at the same dilution.

Western blotting. Western blots were performed as previously described on

16.5% Tris-tricine precast polyacrylamide gels. An equal amount of protein (20pg) was

loaded in each lane. The protein was then transferred to PVDF membranes and probed

for iACTH using the same antibody that was used in the RIA (dilution of 1:13,000). Blots

were visualized by a Western blot chemiluminescence reagent and analyzed by

densitometry. Antibody specificity for iACTH staining was determined by preabsorption

of the primary antibody with 100pg synthetic ACTH per blot and identification of non-

specific banding patterns. Synthetic ACTH was also run on each gel as a marker for

ACTH1.39

Statistical analysis. Statistical procedures were accomplished using SigmaStat

(Jandel Scientific, San Rafael, CA). Differences between pairs of means were assessed

using Student's t-test. Ontogenetic changes in measured or calculated variables were

assessed using simple linear regression or multiple linear regression. For the purpose of

performing regression analysis, we estimated the ages of the lambs as 175 days. In each

statistical test, the null hypothesis was rejected when p<0.05.






76

6.3 Results

There was a variable, yet statistically significant (p<0.05), relationship between

pituitary iACTH concentration and developmental age (Figure 6.2). The age-related

changes in pituitary iACTH concentration were further analyzed by Western Blot

technology to identify possible changes in the relative concentrations of the different

molecular species which comprise iACTH.


Figure 6.2. Pituitary immunoreactive ACTH content (ng/mg protein) as a function of
developmental age. There was a significant relationship between pituitary iACTH and
developmental age (p<0.05). Data are represented as mean (solid line) with a 95%
confidence interval (dashed lines).


2.0

1.8
**
1.6 -

*I 1.4 //*

. 1.2 -









0.2

M-
0-0 --i ii

60 80 100 120 140 160 180 200

Developmental Age (days)





77
As shown in Figure 6.3, we were able to separate the three major molecular forms

of iACTH: POMC, 22 kD fragment, and fully processed ACTH. The staining for these

peptides was immunologically specific, since preabsorption of the antiserum with synthetic

human ACTH1., decreased the intensity of the staining of all three bands (Figure 6.3).



Pituitary iACTH ACTH,39
+ Preabsorption +

I-l -POMC-
22 kD


age -A CM IACTH.




Figure 6.3. Western blot analysis of pituitary homogenate iACTH. Bands specific for
iACTH are denoted by arrows.


Although each gel contained tissue samples representing a wide range of

developmental ages, statistical analysis of the data was complicated by the variation in the
intensity of staining and variation in the exposure of the film during the chemiluminescence

reaction. We corrected for this variation by analyzing the optical density data for each
molecular form ofiACTH (POMC, pro-ACTH, and ACTHi., ) by multiple regression
analysis, using developmental age and gel number as independent variables. This analysis

revealed a statistically significant (p<0.05) influence of age on the intensity of staining in

the band representing fully processed ACTH (Figure 6.4), but no statistically significant








changes were shown in the other two bands (data not shown). In each analysis, the

variation attributable to the variations in gel density was statistically significant (p<0.001).


Figure 6.4. Multiple regression analysis of 5kD ACTH optical density (O.D.). Staining
of ACTHi.3 increased as a function of developmental age with respect to gel number
(p<0.05). There was no relationship between POMC and pro-ACTH staining with age
(data not shown).

To obtain an index of the changes in fully processed ACTH relative to the

abundance of 22 kD fragment and POMC, we calculated an index of the percentage of

iACTH which is fully processed to ACTH.39. This index is the ratio of the optical density

in the band containing fully processed ACTH.,, and the sum of the optical density in the

other bands. Analysis of this ratio by linear regression analysis revealed a statistically

significant increase as a function of developmental age (Figure 6.5). As can be seen by








comparison of Figures 6.4 and 6.5, calculation of this ratio eliminated the need to analyze

by multiple linear regression analysis, since variations in image density were normalized.





Ratio of [ACTH/(POMC + 22 kD)]
1.0
0.8




0.0 -


-0.4

60 80 100 120 140 160 180 200

Developmental Age


Figure 6.5. Linear regression analysis of the ratio of ACTHI.3 and the sum of POMC
and pro-ACTH.

As shown in Table 6.1, treatment of fetuses with estradiol or androstenedione

chronically did not alter the concentration ofiACTH in the fetal pituitary (measured by

RIA). The processing ofiACTH was studied in a subset of these steroid-treated animals

using Western blot analysis. The index of processing, calculated as shown in Figure 6.5,

was reduced during estradiol treatment but not by androstenedione treatment (calculations

not shown). In 4 estradiol-treated fetuses compared to 7 age-matched control fetuses, the

calculated index was 5118% of age-matched control fetuses. In 5 androstenedione-

treated fetuses compared to 8 age-matched control fetuses, the calculated index was

15985% of the age-matched control fetuses.






80

Table 6.1. Fetal pituitary iACTH concentration in estradiol-treated, androstenedione-
treated, and age-matched control fetuses.
Experimental Group # of Age of iACTH Ratio of
Fetuses Fetuses (ng/mg [ACTH /
protein) (POMC+22
kD)l

estradiol-treated 4 1380.9 1.14 0.17 0.12 0.05
control fetuses 7 139 0.9 1.13 0.14 0.27 0.08
androstenedione-treated 5 128 0.5 1.31 0.11 0.22 0.10
control fetuses 8 1271.1 1.10 0.15 0.130.05


6.4 Discussion

The results of this study demonstrate that the steady state concentration of iACTH

in ovine fetal pituitary tissue was relatively constant throughout the last half of gestation,

but that fully processed ACTH represented a significantly greater proportion of the total

pituitary iACTH in late-gestation fetuses and lambs. Despite obvious variability in the

data, there was a significant difference in the concentration ofiACTH in the pituitary as a

function of developmental age. However, chronic treatment of the fetuses with estradiol

reduced the proportion of iACTH which is processed.

In sheep, the fetal hypothalamus-pituitary-adrenal axis acts as a "trigger" which

initiates parturition. Late in gestation (approximately 120 days or approximately 80% of

full-term gestation), plasma ACTH concentrations begin to increase. Fetal plasma ACTH

and cortisol concentrations slowly increase until approximately 140 days (approximately

95%) gestation. At this time, cortisol negative-feedback efficacy decreases and cortisol is

no longer able to "turn off' ACTH secretion from the pituitary. We believe that

simultaneous with the decrease in cortisol negative feedback sensitivity is the onset of a






81

stimulation, or "drive", to ACTH secretion. Together, these changes in drive and

feedback sensitivity increase fetal plasma concentrations of both ACTH and cortisol.

Both fetal and adult ovine corticotrophs secrete a mixture of fully processed

ACTH.,, and larger forms ofiACTH. It has been proposed by other investigators that the

relative proportions of processed and unprocessed forms of iACTH are important

variables in determining adrenal responsiveness to circulating ACTH. It has been shown

that high molecular weight precursors of ACTH inhibit the response to ACTHI.3, by fetal

sheep adrenal cells (Jones and Roebuck, 1980; Roebuck and Jones, 1980). POMC is

somewhat less effective than 22 kD iACTH, but both are effective inhibitors of ACTH

action. Schwartz and colleagues have demonstrated that the 22 kD fragment inhibited the

fetal adrenal response to ACTH,.,3 in a 2.3 molar excess, while POMC inhibited the

response in a 26 molar excess (1995). This interaction was not demonstrable in

experiments using adrenal cortical cells from adult sheep (Schwartz et al., 1995). In the

perfused fetal rat adrenal cells, POMC did not block the action of 4.5 kD ACTH. In fact,

both ACTHi.3, and 22 kD fragment were steroidogenic, and the adrenal response to these

peptides increased with fetal gestational age (Chatelain and Cheong, 1987).

The changing proportions of processed and unprocessed forms ofiACTH in the

ovine fetal pituitary probably explains the developmental changes in the so-called

bioactive-to-immunoreactive ACTH ratio, or b/I ratio, during stimulation in fetal sheep.

In the plasma of unstimulated fetal sheep, the b/I ratio for ACTH is low (Castro et al.,

1993). In other words, the plasma concentration of ACTH measured by bioassay is low

when compared to the plasma concentration of ACTH measured by RIA. When

subjected to hemorrhage, the b/I ratio increases (Castro et al., 1993), probably reflecting






82

the secretion of ACTHi., from the fetal pituitary. However, the b/I ratio during

stimulation changes as a function of fetal gestational age (Castro et al., 1992). That is, the

b/I ratio in plasma samples from stimulated fetuses is higher late in gestation compared

with stimulated fetuses which are younger and less well developed (Castro et al., 1992).

Changes in POMC processing are reflected in the molecular forms circulating in plasma

from unstimulated fetuses. Hollingworth and Thorburn have demonstrated that the

proportion ofiACTH in fetal plasma which is fully processed increases late in gestation

(Thorbur et al., 1991).

We (refer to Chapter 5) and others (Brieu et al., 1989; Watabe et al., 1991; Currie

and Brooks, 1992) have reported developmental increases in ovine fetal hypothalamic

concentrations of ovine fetal hypothalamic concentrations of ovine corticotropin releasing

factor (oCRF) and arginine vasopressin (AVP), the two hypophysiotropic releasing factors

for ACTH in the sheep. The ontogenetic changes in hypothalamic releasing factor

concentrations complement the ontogenetic changes in pituitary ACTH concentration and

processing reported in this study. Together these changes would support an increase in

the activity of the hypothalamus-pituitary-adrenal axis as the fetus matures. In other

words, these changes in tissue peptide concentrations most likely create a greater reserve

for activity of the HPA axis. This reserve might be important during the preparturient

surge in activity which precipitates parturition. The reserve could also be important

during the neonatal period in which HPA axis responses to stress are important for

survival.

We tested the effect ofestradiol or androstenedione treatment on fetal pituitary

iACTH concentration and on POMC processing to test the hypothesis that either steroid






83

would produce changes in the measured or calculated variables similar to the spontaneous

developmental changes. We found that estradiol produced changes in POMC processing

which were opposite those observed endogenously. Therefore, it is unlikely that increases

in fetal plasma estrogen concentrations constitute the stimulus to increased POMC

synthesis and processing. We have reported that chronic estradiol treatment increases

both basal- and stimulated- fetal plasma ACTH concentrations (refer to Chapter 4). We

have also reported that chronic androstenedione administration dramatically reduces the

sensitivity of cortisol negative feedback inhibition of fetal ACTH secretion. The decrease

in pituitary POMC processing produced by estradiol might simply reflect stimulation of

pituitary secretion (increased secretion at the expense of intracellular stores). If so, the

developmental increase in iACTH and POMC processing which occurs in the absence of

exogenous steroid must be dependent upon another stimulus (ie., increased hypothalamic

oCRF or AVP secretion).

Understanding changes in fetal pituitary posttranslational processing of POMC into

the different molecular forms of iACTH are likely to be important in completely

understanding the mechanism of parturition. However, it is important to recognize that

the pituitary is not the only potential source of iACTH in fetal plasma. It has been

demonstrated that the fetal lung contains iACTH (Cudd et al., 1993). When tested by

Western blot analysis, pulmonary tissue contained only large molecular weight forms of

iACTH, but not any measurable amounts of ACTHI.3, (Cudd et al., 1993). In vivo, the

lung secreted iACTH under both basal (Cudd and Wood, 1995) and highly stimulated

(Cudd et al., 1993) conditions. In addition to secreting iACTH, the lung clears

approximately 50% of the ACTH in the pulmonary arterial plasma (Cudd and Wood,






84

1995). While we do not know the molecular form ofiACTH secreted by the lung, it is

estimated that a significant proportion of the iACTH measured in the plasma of

unstimulated fetal sheep could potentially originate in the fetal lung (Cudd and Wood,

1995). This suggests that, in addition to developmental changes in pituitary processing,

developmental changes in pulmonary POMC biosynthesis and processing might be

important in the timing of parturition.

In summary, we have demonstrated that the concentration ofiACTH increases as a

function of fetal gestational age in ovine fetal pituitary, and that the proportion of the total

immunoreactivity which is fully processed to ACTH.39 also increases. Estradiol

pretreatment of the fetuses significantly decreases the proportion which is fully processed,

an action which might be the result of estrogen-induced stimulation of fetal ACTH

secretion.













CHAPTER 7
DEVELOPMENTAL CHANGES AND MOLECULAR WEIGHT OF
IMMUNOREACTIVE GLUCOCORTICOID RECEPTOR PROTEIN IN THE OVINE
FETAL HYPOTHALAMUS AND PITUITARY




7.1 Introduction

The glucocorticoid receptor (GR) belongs to a superfamily of steroid and thyroid

hormone receptors that are ligand-dependent, nuclear transcription factors (Evans, 1988).

The DNA sequence for the human GR has been identified (Hollenberg et al., 1985) as has a

partial sequence for ovine GR (Yang et al., 1991), which has 92% identity to bases 143-453

of the human GR. Based on the deduced amino acid sequence, a number of new antibodies

have been produced to peptide sequences in the amino-terminal end of the receptor. Based

on the deduced amino-acid sequence, a number of new antibodies have been produced to

peptide sequences in the amino-terminal end of the receptor. These antibodies recognize

multiple forms of the receptor: unactivated receptor, activated DNA-binding receptor, and

unliganded receptor. Previously available antibodies only recognized activated DNA-binding

forms of the receptor protein, possibly due to an interaction of the antibody with an epitope

of the receptor protein exposed after ligand binding. Cidlowski et al. (1990) produced two

rabbit anti-human polyclonal antibodies (#57 and #59) using peptide sequences that are

independent of the steroid and DNA binding domains as well as receptor oligomeric state.

The binding characteristics of the antibody was determined using Hela cell cultures and






86

immunocytochemistry (McGimsey et al., 1991; Burnstein et al., 1991; Burstein et al., 1990).

In these systems, antibody #57 recognized a 97 kilodalton protein when analyzed by Western

blot gel electrophoresis. The purpose of the present study was to use this GR antibody in a

tissue homogenate preparation for identification of ovine immunoreactive GR protein in the

fetal hypothalamus and pituitary.

In the sheep, parturition is stimulated by an increase in the activity of the fetal

hypothalamus-pituitary-adrenal (HPA) axis (Liggins et al., 1973; Challis and Brooks, 1989).

Prior to spontaneous parturition, plasma concentrations of adrenocorticotropin hormone

(ACTH) and cortisol increase in the fetus. Near the end of gestation, fetal plasma ACTH

secretion is further increased as cortisol negative feedback sensitivity decreases (Wood,

1988), thereby further increasing fetal plasma. cortisol concentrations. Cortisol increases

placental conversion of estrogens from progesterone, increasing uterine contractility, and

initiating labor and parturition (Liggins, 1974). We hypothesized that the decrease in cortisol

negative feedback efficacy that is seen late in gestation may be due to a reduction in

glucocorticoid receptors in the fetal hypothalamus or pituitary.



7.2 Materials and Methods

Tissues Experiments were performed using tissues collected from pregnant ewes of

known gestational ages. Fetal hypothalami and pituitaries were harvested from healthy

fetuses at various gestational ages (74-101 days, 125-129 days, 135-139 days, 141 days-term)

and from 3- 4 week old healthy lambs. Fetal tissues were rapidly collected and snap-frozen

using a dry ice-acetone bath. The tissues were stored at -20*C or -40C until processed.

Tissues were later homogenized in reducing buffer (Laemmli, 1970) and prepared as








previously described. Protein concentrations of the homogenates were determined and

samples were stored at -40*C until analyzed by electrophoresis.

Steroid-treated fetuses. Fetuses were treated with 5 mg 17p-estradiol,

androstenedione, or tamoxifen (estrogen antagonist) pellets designed to release over 21 days,

implanted in the area of the gluteus medius on about day 120 of gestation (release rate of

approximately 238 pg per day). Estradiol-, androstenedione- and tamoxifen-treated fetuses

were sacrificed at 1381 (n=4), 1281 (n=8), and 1281 (n=5) days gestation, respectively.

The hypothalamus from one androstenedione-treated fetus and the pituitary from one

tamoxifen-treated fetus were not used because of errors in tissue preparation or collection.

Age-matched controls for these groups consisted of fetuses which were 128+1 (n=14) days

gestation for the androstenedione- and tamoxifen-treated fetuses and 140.1 (n=13) days

gestation for the estradiol-treated fetuses. In other experiments, we found that implantation

of estradiol pellets increased fetal plasma estradiol concentration from 11.43.9 (n=12) to

39.111.1 (n=7) pg/ml (Chapter 4). Implantation of androstenedione pellets increased fetal

plasma androstenedione concentrations to 27320 (n=8) to control fetuses 20310 (n=9)

pg/ml (Chapter 4). Fetal hypothalami and pituitaries were harvested one to two weeks after

pellet implantation.

Western blotting. Western blot analyses were performed as described previously on

10% precast polyacrylamide gels. An equal amount of protein (40pg) was loaded in each

lane. The protein was then transferred to nitrocellulose membranes and probed for iGR.

Binding of the antibody to the protein on the membrane was visualized by a Western blot

chemiluminescence reagent and analyzed by densitometry (Bio-Rad). Antibody specificity

for iGR staining was confirmed by preabsorption of the primary antibody with 10 pg of the






88

protein sequence used to create the antibody (Cat# PEP-001) and identification of specific

and non-specific banding patterns. A diagram of this peptide sequence and proposed

structure of the human glucocorticoid receptors shown in Figure 7.1 (Giguere et al., 1986;

Weinberger et al., 1985).

Statistical analysis. Statistical procedures were accomplished using SigmaStat (Jandel

Scientific, San Rafael, CA). Developmental changes in measured or calculated variables were

assessed using simple linear regression or multiple linear regression. Student's t-test was used

to test for differences between the treatment groups and their controls. For the purpose of

performing the regression analysis, we assigned a value of (147 + postnatal age) in days to

each lamb. Data were expressed as.% of control in the steroid-treated fetuses. The null

hypothesis was rejected and statistical significance determined when p<0.05.




l 346-367 a 421-481 5O0-T7

Domsaa Domain

ptipde seoquents used aus dpiB
D .Q-K-I-f-N-V-Ir--I-P-V-G-S-L-N-fW-N--C





Figure 7.1. Proposed structure of the human glucocorticoid receptor. The peptide
sequence from amino acids 346-367 was used as antigen to produce antibody #57
(Cidlowski, et al., 1990), which was used in this study. This peptide sequence was also
used in the preabsorption experiments to determine specific staining ofiGR. Drawing was
adapted from published data (Affinity BioReagents, Neshanic Station, N.J.; Giguere, et al.,
1986; Weinberger, et al., 1985).






89

7.3 Results

The Western blot shown in Figure 7. 2 is representative of the staining observed in

these studies. Panel A illustrates specific staining for immunoreactive glucocorticoid receptor

protein at approximately 90-94kD and staining of a smaller protein at 46-50 kD. Panel B is

a Western blot of the same tissue with the antibody preabsorbed with peptide sequence to

which the antibody was raised. Decreased staining intensity in both bands in response to

preabsorption demonstrates specificity of staining for the epitope of GR to which the antibody

was raised. Protein-antibody binding was determined to be specific by preabsorption of the

antibody and a subsequent decrease in staining intensity of protein bands.

In performing the Western blots, the fetal tissue samples were distributed among the

gels so that each gel contained a subset of the developmental ages studied. However,

statistical analysis of the data was complicated by variation in staining intensity and because

of variation in exposure of the film during the chemiluminescence reaction. We corrected for

these variations by analyzing the optical density data by multiple regressional analysis, using

developmental age and gel number as independent variables. This analysis did not detect a

significant influence of developmental age on the full-length receptor in either the pituitary

or the hypothalamic tissue homogenates (data not shown). The variation attributable to the

variations in gel density was statistically significant (p<0.05).

Multiple regression analysis of the 46-50 kD protein in both the pituitary and

hypothalamic tissue homogenates revealed a significant influence of developmental age on

optical density ofthe immunoreactive band (data not shown). We further analyzed the results

by calculating the ratio of the optical density of the 46-50 kD protein band to the optical

density of the 90-94 kD GR band. Analysis of this ratio by linear regression analysis revealed




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