Estrogen-central nervous system interactions in cardiovascular control and parturition

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Estrogen-central nervous system interactions in cardiovascular control and parturition
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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
        Page v
    Abstract
        Page vi
        Page vii
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
        Page 4
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    Chapter 2. Literature review
        Page 10
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    Chapter 3. General materials and methods
        Page 39
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    Chapter 4. Hormonal responsiveness in an estradiol, hypotensive, carotid sinus denervated ovine model
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    Chapter 5. Neuronal activation in an estradiol, hypotensive, carotid sinus denervated ovine model
        Page 69
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    Chapter 6. Ontogeny of estrogen sulfatase and estrogen sulfotransferase in brain regions important for hypothalamus-pituitary-adrenal axis control
        Page 95
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    Chapter 7. Summary and conclusions
        Page 121
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    References
        Page 138
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    Biographical sketch
        Page 158
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Full Text












ESTROGEN-CENTRAL NERVOUS SYSTEM INTERACTIONS IN
CARDIOVASCULAR CONTROL AND PARTURITION














By

SCOTT CHRISTOPHER PURINTON


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


1999























To the following, I dedicate this dissertation.
Without them, none of this would have been possible.


To my family: Thank you for your support and encouragement
To LeighAnn: Thank you for believing in and standing by me

To David M. Bush: Thank you for your friendship
Wish you could have been here













ACKNOWLEDGMENTS


First and foremost I would like to thank Dr. Charles E. Wood, chair of my

supervisory committee, for his guidance, wisdom, and friendship. Dr. Wood has

certainly been a true mentor to me as a scientist, as well as a role model to anyone

wishing to balance a fulfilling life with a successful career in science. For this and

everything else he has done for me, I am very grateful.

I would like to thank the other members of my supervisory committee, Drs.

Maureen Keller-Wood, Pushpa Kalra, and James Simpkins for their guidance and advice.

A special thanks goes to Dr. Maureen Keller-Wood for her extensive knowledge and for

being an indispensable resource. Also, a special thanks goes to Sherry McDaniel for her

assistance with the surgery and care of the animals.

Last but certainly not least, I would like to extend my gratitude to my family for

their never-ending love and support. They have always stood by me in whatever tasks I

set forth to accomplish. I especially would like to thank Dr. LeighAnn Stubley, my best

friend and fiancee. Without LeighAnn's love and support, I can truly say that none of

this would have been possible. She has taught me more about myself than I could ever

have learned from graduate school. For this I will be forever indebted to her.














TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS..................................................................................................... iii

ABSTRACT ............................................................................................................................. vi

CHAPTERS

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

General Background and Significance.............................................................................. 1
Specific Aims and Hypotheses .......................................................................................... 4
Experimental Protocol........................................................................................................ 5
Experimental M ethods ....................................................................................................... 7

2 LITERATURE REVIEW ......................................................................................... 10

Control of Parturition....................................................................................................... 10
The Hypothalamus-Pituitary-Adrenal Axis............................................................. 13
The Regulation of ACTH Secretion.........................................................................6......
The Development of the Fetal HPA Axis ....................................................................... 24
The Importance of Gonadal Steroids in Parturition and Cardiovascular Control........27
Cardiovascular Reflex Responsiveness.......................................................................... 33

3 GENERAL MATERIALS AND METHODS ........................................................39

Surgical Preparation of Fetal Sheep................................................................................. 39
In Vivo Experimental Procedures ...................................................................................42
Peptide Assays.................................................................................................................. 44
Steroid Assays.................................................................................................................. 45
Estrone Sulfatase Activity................................................................................................ 45
W western Blotting.........................................................................................4.. ..........47
Immunohistochemical Techniques............................................................................. 48

4 HORMONAL RESPONSIVENESS IN AN ESTRADIOL, HYPOTENSIVE,
CAROTID SINUS DENERVATED OVINE MODEL.......................................... 50

Introduction................................................................................................................ 50
M ethods and M aterials.................................................................. ...................52








Results.............................................................................................. .................58
D iscussion.................................................................... 60

5 NEURONAL ACTIVATION IN AN ESTRADIOL, HYPOTENSIVE,
CAROTID SINUS DENERVATED OVINE MODEL.......................................... 69

Introduction.......................... 69
M ethods and M aterials.............................................................................................. 70
Results ............................................................................ ........................................... 72
D discussion ......................................................................................................................... 76

6 ONTOGENY OF ESTROGEN SULFATASE AND ESTROGEN
SULFOTRANSFERASE IN BRAIN REGIONS IMPORTANT FOR
HYPOTHALAMUS-PITUITARY-ADRENAL AXIS CONTROL ....................95

Introduction...............................................................95
M ethods and M aterials.............................................................................................. 97
Results.......................................................................................................... ... 100
Discussion............................................................................................... 102

7 SUMMARY AND CONCLUSIONS..................................................................... 121

REFEREN CES .................................................................................................................... 138

BIOGRAPHICAL SKETCH........................................................................................ 158














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

ESTROGEN-CENTRAL NERVOUS SYSTEM INTERACTIONS IN
CARDIOVASCULAR CONTROL AND PARTURITION

By

Scott Christopher Purinton

May 1999

Chairman: Charles E. Wood
Major Department: Physiology

In the fetal sheep, parturition is triggered by an increase in the activity of

the fetal hypothalamus-pituitary-adrenal (HPA) axis. Parturition can be delayed by

destruction of the pituitary or stimulated by infusions ofadrenocorticotropin (ACTH) or

glucocorticoids. The last days of gestation are marked by an increase in the activity of

the fetal hypothalamus as seen by elevated levels of fetal plasma ACTH. Estrogen has

been shown to trigger this preparturient increase in ACTH. I hypothesized that

estrogen's actions on fetal cardiovascular reflex responsiveness to hypotension will be

measurable in intact fetuses but not in baroreceptor/chemoreceptor denervated fetuses.

Research has previously demonstrated that denervation attenuates the reflex hormonal

and hemodynamic responses to moderate reduction in arterial blood pressure. I found

that interruption of this afferent pathway eliminated the effect of estrogen on reflex

cardiovascular responsiveness. This was shown by measuring hormone responsiveness








(ACTH, vasopressin, and cortisol) to hypotension by brachiocephalic occlusion. I further

hypothesized that estrogen's actions within the fetal central nervous system are centered

within the nucleus of the tractus solitarius (NTS), the paraventricular nucleus (PVN), or

within the components of cardiovascular regulatory centers receiving input from

baroreceptors or chemoreceptors. I used immunohistological techniques to identify the

neuroanatomical regions which are activated by hypotension and, subsequently, those

areas modified by estrogen's action and baroreceptor/chemoreceptor denervation. The

use of these techniques allows for the measurement of c-fos expression, an early response

gene which can be used as a marker of neuronal activity. I found that estradiol implanted

animals had more c-fos abundance, and hence more c-fos staining in relevant brain areas

(NTS, PVN, etc.) as compared to control animals. Furthermore, it was revealed that c-fos

staining was negligible in denervated animals. Finally, c-fos staining was significantly

elevated in all hypotensive animals as compared to normotensive animals. These results

suggest that estrogen works within the ovine fetal baroreceptor/chemoreceptor afferent

pathway in brain regions relevant for HPA axis control in order to augment ACTH

secretion in response to hypotension as well as play a role in the mechanism of

parturition.














CHAPTER 1
INTRODUCTION


General Background and Significance

There is a wealth of information concerning the influence of estrogen on the

cardiovascular system of the adult. The use of high dose estrogen contraceptives has

stimulated a great deal of interest in the role that exogenous estrogens play in the

pathogenesis of hypertension and stroke. However, it should be noted that little work has

been conducted regarding the influence of either endogenous or exogenous estrogen on

the control of cardiovascular function by the central nervous system. Furthermore, with

the exception of the studies conducted in Dr. Charles E. Wood's laboratory at the

University of Florida, Gainesville, Florida, there are no data concerning the influence of

estrogen on cardiovascular function in the fetus.

Increases in plasma estrogen concentration are thought to be an important

component of the maternal adaptation to pregnancy. During gestation, the growing fetus

and enlarging uterus exhibit an increasing demand for oxygen. For this reason, the utero-

placental blood flow constitutes an ever-increasing proportion of maternal cardiac output

(Rudolph, 1974). The increased flow demands are supported by an increase in maternal

blood volume and an increase in vascular compliance (Longo, 1983). This typically

produces a physiological condition in which maternal cardiac output and blood volume are

higher than in the nonpregnant state; however, maternal arterial pressure and central









venous pressure are lower (Keller-Wood, 1994; Longo, 1983). Though this response is

not entirely consistent among mammalian species, it has been proposed that estrogen plays

an important role in this phenomenon (Ueda, 1986). No studies have yet been conducted

that investigate the effects of estrogen on cardiovascular reflex responsiveness. By doing

such studies in fetal sheep, it is possible to investigate the mechanism behind parturition,

because an increase in the activity of the hypothalamus-pituitary-adrenal (HPA) axis has

been shown to trigger parturition (Liggins et al., 1973).

Both androgen and estrogen receptors can be found in the anterior pituitary as well

as in various regions of the brain, including the hypothalamus and the brainstem.

Receptors, and the mRNA encoding for these receptors, can be found in the various

structures, including the arcuate nucleus and the preoptic nucleus (Simerly et al., 1990). In

addition to the structures known for control of the hypothalamus-pituitary-gonadal axis

and for reproductive behavior, androgen and estrogen receptors are found in regions

better known for control of the hypothalamus-pituitary-adrenal axis, including the
/
paraventricular nucleus (PVN) and hippocampus (Lehman et al., 1993; Pomerantz and

Sholl, 1987). Estrogen receptors have been localized in the magnocellular portion of the

PVN, and may therefore influence either oxytocin, vasopressin (AVP), or both (Lehman et

al., 1993; Simerly et al., 1990). Both androgen and estrogen receptors are also found at

sites which are known to be relay centers on afferent pathways [the nucleus of the tractus

solitarius or (NTS)] mediating adrenocorticotropic hormone (ACTH) responses to

stresses such as hypoxia, hypercapnia, and hypotension (Simerly et al., 1990). Finally,

estrogen receptors have been localized in GABA-ergic cells, suggesting the possible








interaction of estrogen and ACTH secretion via this neuronal system within the brain

(Herbison et al., 1993).

The focus of the experiments is the neuroendocrine (ACTH, AVP) responsiveness

to hypotension. Of particular interest to me at the present time is the relationship between

neuroendocrine mechanisms controlling AVP and ACTH and how they regulate the

cardiovascular system of the fetus as well as trigger parturition. These two hormonal

systems are inextricably linked. In the fetus (as in the adult), afferent baroreceptor

pathways are shared by both endocrine systems. It is likely that the central pathways

carrying afferent information from the NTS to the hypothalamus are also mostly shared by

the two systems. AVP is synthesized in the PVN of the hypothalamus and can be found in

both magnocellular and parvocellular neurons (projecting to the posterior pituitary and

median eminence, respectively). Magnocellular AVP is secreted into the bloodstream by

the posterior pituitary. Parvocellular AVP is secreted into the hypothalamo-hypophyseal

portal system and acts at the anterior pituitary to induce the release of ACTH. ACTH, via

stimulation of cortisol, act to maintain vascular reactivity, promote plasma protein

biosynthesis, and indirectly alter fetal blood pressure and distribution of combined

ventricular output. Parvocellular neurons, in addition to AVP, synthesize and release

corticotropin releasing hormone (CRH) which also stimulates the release of ACTH from

the anterior pituitary. Parvocellular CRH neurons project axons to various parts of the

brain in addition to the median eminence. Most notably, these neurons project to the

cardiovascular control centers in the hypothalamus and medulla. When released from

these neurons, CRH stimulates increases in sympathetic efferent tone: measured in the

cardiovascular system as an increase in blood pressure and heart rate and a redistribution








of cardiac output (Lenz et al., 1987). Accordingly, it has been suggested that CRH is the

transmitter which coordinates all of the endocrine and cardiovascular responses to stress,

including hypotension (Lenz et al., 1987).

Preliminary experiments suggest that estrogen augments ACTH response to

hypotension, but that it does not alter AVP responses. Furthermore, estrogen augments

the ACTH response to cardiovascular stimuli, but not to purely endocrine stimuli.

Because of this difference between ACTH and AVP and between cardiovascular and non-

cardiovascular stimuli, I hypothesized that estrogen acts within the central nervous system

to affect the parvocellular CRH biosynthesis and secretion. Thus, it is proposed that

estrogen affects either CRH neurons or the ascending pathways which stimulate CRH

neurons, as well as affecting the activity of the descending (toward cardiovascular

regulatory centers) CRH pathways.

Specific Aims and Hypotheses

In the following experiments, a combination of in vivo (whole animal) and in vitro

(immunohistochemistry) techniques was employed.

Aim 1 The actions of estrogen on fetal cardiovascular reflex responsiveness to

hypotension will be measurable in intact fetuses but not in baro- and chemo-denervated

fetuses. Research has previously demonstrated that the combined baro- and chemo-

denervation attenuates (approximately 50%) the reflex hormonal and hemodynamic

responses to moderate (50 %) reduction in arterial blood pressure. I proposed that the

interruption of the afferent pathways would eliminate the effect of estrogen on the reflex

cardiovascular responsiveness. If so, it could concluded that estrogen acts on, within, or

requires input from, the afferent baroreceptor and chemoreceptor pathways.






5

Aim 2 The actions of estrogen within the fetal central nervous system are centered within

the nucleus of the tractus solitarius, the parvocellular neurons of the paraventricular

nucleus, or within the components of the cardiovascular regulatory centers receiving input

from baroreceptors or chemoreceptors. I used immunohistological techniques to identify

the neuroanatomical regions which were activated by hypotension and, subsequently,

those areas modified by estrogen's action and baro- and chemo-denervation. The use of

these techniques allows for the measurement of c-fos expression, the protein product of

the early response gene which can be used as a marker of neuronal activity. I proposed

that estradiol implanted animals will have more Fos activity, and hence more c-fos staining

in relevant brain areas (NTS, PVN, etc.) as compare to control animals. Furthermore, it is

expected that c-fos staining will be negligible in denervated animals. Finally, c-fos staining

should be significantly elevated in all hypotensive animals as compared to normotensive

animals.

In addition to the c-fos immunohistochenmical studies, the action of estrogen

sulfatase and estrogen sulfotransferase was investigated. Since the concentration of

estrogen sulfate precedes the increase in HPA axis activity (Nathanielsz et al., 1982), and

since conjugated estrogens circulate in much higher concentrations than unconjugated

estrogens (Carnegie and Robertson, 1978; Tsang, 1974), I hypothesized that these

enzymes would be present in brain areas important for HPA axis control. If true, a local

mechanism would be in place for conversion of biologically inactive to active estrogen.

Experimental Protocol

Design A total of 40 pregnant ewes were studied (5 per experimental group). Animals

were randomly assigned to the following groups:









1. placebo implant, intact (sham-denervated),normotensive fetuses

2. placebo implant, carotid sinus denervated, normotensive fetuses

3. placebo implant, intact (sham-denervated) fetuses subjected to hypotension

4. placebo implant, carotid sinus denervated fetuses subjected to hypotension

5. estradiol implant (5 mg/21 day release), intact (sham-denervated), normotensive

fetuses

6. estradiol implant (5 mg/21 day release), carotid sinus denervated, normotensive

fetuses

7. estradiol implant (5 mg/21 day release), intact (sham-denervated) fetuses

subjected to hypotension

8. estradiol implant (5 mg/21 day release), carotid sinus denervated fetuses

subjected to hypotension

Fetal sheep were chronically prepared with vascular catheters and implants on

around day 115 and allowed post-surgical recovery for 5 days. Fetuses were subjected to

experiments on day 1203, during which time each fetus was subjected to a 10 min period

of hypotension produced by brachiocephalic occlusion (no occlusion for control animals).

Fetal arterial blood samples (5 ml) were drawn at 0 min, +10 min, +20 min, and +60 min

relative to the initiation of hypotension. An additional 1 ml blood sample was drawn

anaerobically for the measurement of fetal blood gases and hematocrit at 0 min, +10 min,

and +20 min time points. Immediately after drawing the +60 min blood sample, the ewes

and fetuses were euthanized using an overdose of sodium pentobarbital. In each

experimental group, five fetuses will be studied, then prepared for immunohistochemistry

as detailed below. Immunohistochemistry was performed for the detection of c-fos. A










subset of sections was double-stained for ACTH and c-fos (pituitary), AVP and c-fos

(hypothalamus), and CRH and c-fos (hypothalamus).

Analysis The neuroanatomical areas which express c-fos in response to hypotension

(NTS, anterior hypothalamus, PVN, and pituitary) were identified. Immunohistochemistry

of c-fos was analyzed in a semi-quantitative manner (counting c-fos positive cells in the

relative regions). The number of cells expressing immunoreactive c-fos in each

experimental group at each site was be compared using two- and three-way ANOVA. I

further classified neuronal activation by counting cells which express/co-express c-fos and

ACTH (anterior pituitary), c-fos and CRH and c-fos and AVP (PVN).

Experimental Methods

Aim 1 Prior to surgery, food was withheld from the pregnant ewe (2-6 years old of mixed

breed, mostly Columbia-Ramboillet and Suffolk) for 24 hours. Before and during surgery,

the ewe was anesthetized with halothane (0.5-2.0%/) in oxygen. Fetal catheters were

routed to one flank of the ewe and held in place with a synthetic pocket and an expandable

bandage.

After exposing the uterus and the fetal hindlimbs via a midline incision, fetal

catheters were placed in the femoral arteries where their tips are advanced to the

subdiaphragmatic aorta. An estradiol or control implant was placed subcutaneously at this

time. An amniotic fluid catheter was sewn to the hindlimb before returning the fetus to the

amniotic cavity. Catheterization of fetal vessels is routine in Dr. Wood's laboratory

(Keller-Wood and Wood, 1991; Raff and Wood, 1991; Raff and Wood, 1992; Wood et

al., 1990).










For carotid sinus denervated fetuses, the common carotid artery was exposed and

stripped of nerves and connective tissue between the carotid-occipital arterial junction and

the carotid-lingual arterial junction. The occipital artery was ligated and divided and the

lingual artery was stripped of all nerves and connective tissue. Before closure of the fetus,

catheters were passed into the lingual arteries and advanced approximately 1 cm into the

carotid artery toward the heart. This entire procedure is conducted bilaterally. Dr.

Wood's laboratory has had extensive experience with this procedure and has published

descriptions of this technique (Wood, 1989, 1995).

Hypotension was achieved via brachiocephalic occlusion. A 10 minute period of

hypotension is necessary for activation of the HPA axis. Access to the artery was gained

through the second intercostal space on the left side of the chest. The occluder was tested

and deflated before the ribs, skin, and uterus were sutured closed.

Throughout the experiments, intravascular and amniotic pressures as well as heart

rate were measured utilizing a polygraph. Monitoring blood pressure assured that I

achieved a consistent and appropriate (50%) state of hypotension via brachiocephalic

occlusion. Blood pressure measurements were analyzed utilizing a computer program in

order to help quantify the level of estrogen augmentation of reflex responsiveness. Blood

gases and hematocrits were measured immediately after completion of each experiment.

Blood samples were processed (centrifuged, etc., to obtain plasma) and frozen until

hormone levels were measured via radioimmunoassay or enzyme immunoassay techniques

used routinely in Dr. Wood's laboratory. Plasma hormone concentrations (ACTH, AVP,

cortisol, and estradiol) were analyzed by 2- and 3-way ANOVA.










Aim 2 In preparation for immunohistochemistry, the head of the fetus was perfused with

heparinized saline followed by 4% paraformaldehyde. The fetal hypothalamus and

brainstem were dissected and processed accordingly (alcohol dehydrated and embedded in

paraffin). Tissues were sectioned (6-10 micron) using a Zeiss rotary microtome and

mounted on subbed slides. Staining for ACTH, AVP, and CRH was performed using

antisera produced in Dr. Wood's laboratory. Staining for c-fos was done using

commercially available antibodies from Oncogene Sciences. Staining for estrogen

sulfatase and estrogen sulfotransferase were done using custom made antibodies from

Alpha Diagnostic. Staining was visualized using a Histostain-SP kit (Zymed streptavidin-

biotin system).














CHAPTER 2
LITERATURE REVIEW


Control of Parturition

Though it has been known for some time that the fetus controls parturition in the

sheep, the precise mechanism has yet to be fully understood. However, much is known

about the major endocrine axis that initiates this process as well as controls blood pressure

in the fetus. These processes are controlled by the hypothalamus-pituitary-adrenal (HPA)

axis. One of the first pieces of evidence that linked the fetal HPA axis to the control of

parturition was reported by Binns et al. (1991). This was a natural phenomenon that

occurred when sheep ate a particular plant on day 14 of gestation. This plant, Veratrwnum

californicum, delayed the birth of the fetus indefinitely until caesarian section or death of

the fetusor ewe. The fetus had a number of birth defects, including cyclopia and a

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

normal position. It was realized later that this malformed, dysfunctional axis was

responsible for delayed parturition. This observation and others led Liggins (1973) to

conclude that the fetus controls parturition in the sheep. Liggins has credited Hippocrates

with first suggesting this idea when he wrote that the fetus pushes its way out of the

womb when the nutrition supplied by the mother is no longer sufficient for further growth.

Malpus (1933) put forth a more modem observation suggesting a fetal role in the timing

of birth when he reported the association of fetal ancephally and prolonged gestation in








women. These ideas laid the groundwork for much of the ongoing research to determine

the precise mechanism of parturition.

Though many have contributed to this idea of a fetal role in the initiation of

parturition, no one has contributed to this field of research more than Liggins. Liggins

was the first to hypothesize and directly test that the fetal pituitary is intricately involved in

this process (Liggins et al., 1967). This was done by first hypophysectomizing fetal sheep

by surgical electrocoagulation. In fetuses where 70% or more of the pituitary was ablated,

gestation was significantly prolonged and delivery was achieved only after caesarian

section. Fetal adrenalectomy was also found to prolong gestation (Drost and Holm,

1968). Disconnection of the hypothalamus from the pituitary in the fetal sheep between

108-112 days gestation delays birth by at least eight days (Antolovich et al., 1990).

Following hypophysectomy infusion of adrenocorticotropin (ACTH) or glucocorticoid still

induces parturition (Kendall et al., 1977). Further studies from this group also

demonstrate the necessity of the HP axis in maturation of pituitary corticotropes (Kendall
s
et al., 1977). After disconnection of the hypothalamus from the pituitary, fetuses were

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

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

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

corticotropin maturation requires the presence of complete HP axis (Antolovich et al.,

1992). More specifically, following destruction of the fetal paraventricular nucleus (PVN)

of the hypothalamus, parturition was delayed (McDonald and Nathanielsz, 1991).

Therefore, the signal for parturition may either be sent to the PVN which receives input

from the nucleus of the tractus solitarius (NTS), amygdala, or hippocampus, or possibly be









derived in corticotrophin releasing hormone (CRH) and arginine vasopressin (AVP)

producing neurons in the PVN.

Whereby disruption of the fetal HPA axis causes a delay in the timing of birth,

stimulation of this endocrine axis can result in premature parturition. The involvement of

the fetal adrenal cortex in the initiation of parturition in sheep was suggested after in utero

plasma concentration of corticosteroid revealed dramatic increases in these hormones

prior to birth (Bassett and Thorbumrn, 1969). Before this conclusion was made a number

of studies aimed at investigating the role of the HPA axis in the birth process were

performed in the fetal sheep by Liggins (1968). ACTH infused into fetal sheep induced

parturition within four to seven days along with producing adrenal hypertrophy. Cortisol

infusion into the fetus induced parturition within five days. The same doses of ACTH or

cortisol infused into ewes did not induce parturition. Although estradiol at 2 mg/24 hr had

no effect, infusion of cortisol at 25 mg/24 hr plus estradiol resulted in delivery after four

days (Liggins, 1968). Further studies by Liggins (1969) showed that it was glucocorticoid

activity (not mineralocorticoid) that was important for the initiation of parturition,

evidence of this effect was shown by the inability of deoxycorticosterone or corticosterone

to induce parturition. Dexamethasone infused at rates of 0.06 4.0 mg/24 hr in the fetus

and 4 mg/24 hr 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 intravenous

cortisol infusion at 130 days gestation (Thomas et al., 1978). Activation of fetal adrenal

function by pulsatile ACTH administration in 125-127 day fetal sheep induced labor and

delivery in four to five days, resulting in four to six fold elevation in fetal plasma cortisol








concentrations (Lye et al., 1983). Furthermore, it was shown that hypophysectomized

fetuses that were administered dexamethasone or ACTH infusions exogenously would

undergo parturition (Kendall et al., 1977).

The Hypothalamus-Pituitary-Adrenal Axis

The HPA axis integrates a variety of neuroendocrine inputs to regulate the

synthesis and secretion of the adrenocorticosteroids which are required for the

maintenance of life. These steroid hormones exert effects to minimize any disturbance in

homeostasis. The critical role of adrenocorticosteroids can clearly be observed after

adrenalectomy or during hypoadrenocorticism either induced by drug or due to a disease

state. Without adrenal corticosteroids disrupted electrolyte balance or carbohydrate

metabolism leads to circulatory collapse of hypoglycemic coma and death. Physical,

emotional, and chemical stresses such as pain, trauma, hypoxia, acute hypoglycemia, cold

exposure, and vasopressin administration have all been shown to stimulate ACTH and

cortisol secretion (Gannet al., 1981).

The Hypothalamus The hypothalamus contains several nuclei of neuronal cells. The PVN

of the hypothalamus, located bilaterally on the ventricle, contains specialized

neurosecretory cells which synthesize CRH and AVP. The relatively large-celled (hence,

termed magnocellular) neurons in the PVN contain AVP and project to the posterior

pituitary (Lechan, 1987; Reichlin, 1992). In addition to the large projection to the

posterior pituitary, it has been established that small-celled (parvocellular) neurons in the

PVN contain CRH and AVP and project to the median eminence and are involved in the

regulation of ACTH release (Sawchenko and Swanson, 1980). The increase in plasma

ACTH concentration during stress is mediated by CRH as well as AVP from the










hypothalamus. These two factors are released into the hypothalamo-pituitary portal

circulation and diffused into the anterior pituitary to act on corticotropes to stimulate the

release of ACTH.

The Pituitary The pituitary is divided into the anterior lobe, the intermediate lobe, and the

neural or posterior lobe. Vessels of the hypophyseal-pituitary portal system deliver blood

from the median eminence of the hypothalamus to the anterior pituitary. This system

delivers hormones released from hypothalamic neuronal axons in the median eminence to

the anterior pituitary. CRH and AVP diffuse into the anterior pituitary and bind to their

receptors on specialized cells called corticotropes, which represent 15-20% of

adenohypophyseal cells. Once the releasing hormone binds to its receptor in the

corticotrope, a single mRNA the directs the synthesis of the large precursor molecule

called proopiomelanocorticotropin (POMC). POMC is then processed to produce the

smaller biologically active fragment ACTH. ACTH is then released into the systemic
/
circulation where it acts on adrenocortical cells to stimulate synthesis and secretion of

glucocorticoids (Reichlin, 1985).

The Adrenal Cortex The adrenal glands are endocrinologically complex organs that are

composed of two distinct endocrine tissues derived from different embryologic sources.

The outer zone is called the adrenal cortex and constitutes 80-90% of the gland. The

cortex is the source of the steroid hormones. The smaller inner zone is the adrenal

medulla which is the major source of circulating catecholamines. The adrenal cortex is

highly vascularized and receives its main arterial supply from branches of the inferior

phrenic artery, the renal arteries, and the aorta. There are three major groups of hormones

produced by the adrenal cortex: the mineralocorticoids, the glucocorticoids, and the sex










steroids. Histologically the adult cortex is composed of three zones: an outer zona

glomerulosa, a zona fasciculata, and an inner zona reticularis. The primary product of the

zona glomerulosa is the mineralocorticoid aldosterone. The zona fasciculata and

reticularis produce cortisol and androgens as their primary products (Pescovitz et al.,

1990). ACTH stimulates the secretion of glucocorticoids, mineralocorticoids, and

androgenic steroids from the adrenal cortex. ACTH binds to the receptors on the adrenal

cortex and provokes steroidogenesis through stimulation of cAMP production. cAMP

activates protein kinase A, which catalyzes the phosphorylation of a variety of proteins

thereby producing cholesterol. ACTH also stimulates synthesis of new adrenal proteins

and this increases adrenal weight. Glucocorticoids exert negative feedback at the

pituitary, hypothalamus, and other neural sites (Keller-Wood and Dallman, 1984).

Cortisol is carried in blood bound to transcortin (59%) and albumin (19%), while

about 22% is free in ovine plasma (Patterson and Hills, 1976). The basal production rate

of cortisol is 600 ,g/hr and the metabolic clearance rate of cortisol is about 51 L/hr in

sheep (Panaretto, 1974). The liver and the kidney are the principle organs involved in

clearing the steroid hormones from the circulation. Although most tissues can metabolize

steroids, the liver is the primary site of steroid hormone metabolism and the kidney is the

primary site of steroid hormone excretion. The plasma half-life of cortisol is 60-100

minutes in the human (Pescovitz et al., 1990).

Cortisol produces a number of diverse physiological actions to maintain

homeostasis. As the term homeostasis implies, an excess or deficiency ofglucocorticoids

affects every tissue of the body. Glucocorticoids are essential for survival (Baxter, 1972;

Gann et al., 1981; Pescovitz et al., 1990). The term glucocorticoid refers to the glucose










regulating properties of these hormones. However, glucocorticoids have multiple effects

that include important roles in carbohydrate, lipid, and protein metabolism (Baxter, 1972).

These increase blood glucose by increasing gluconeogensis in the liver and kidneys,

increasing hepatic glycogenesis and decreasing glucose uptake in tissues. The

glucocorticoids increase lipolysis and proteolysis. The glucocorticoids also have

stimulatory effects on cardiovascular function by increasing cardiac output and increasing

vascular response to catecholamines. At high concentrations, the glucocorticoids inhibit

most immunologic and inflammatory responses. Although these effects may have

beneficial aspects, they may also be detrimental to the host by inducing a state of

immunosuppression that will predispose the host to infection (Parrillo and Fauci, 1979).

The glucocorticoids also influence growth, development, bone metabolism, and central

nervous system activity.

The Regulation of ACTH Secretion
i
Corticotropin Releasing Hormone (CRH) Since 1955 it has been known that the

hypothalamus contains substances that acted at the pituitary gland to increase ACTH

secretion in vitro (Guillemin and Rosenberg, 1955; Saffi-an and Schally, 1955). In 1981,

Vale et al. (1981) characterized a 41 amino acid peptide from sheep hypothalamus that

stimulated ACTH secretion from corticotrophins and published the primary structure of

ovine CRH. Ovine CRH was further characterized when the cDNA was cloned and

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

precursor proteins for CRH, AVP, and ACTH which implies a common evolutionary

beginning. A 20 kD immunoreactive form of CRH was identified from rat hypothalamus










and is close to the value for ovine and human pre-pro-CRH based on their cDNA

sequences (Lauber et al., 1984).

CRH 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 CRH can enhance the rates of ACTH synthesis as well as

release (Vale et al., 1983). ACTH release can be modulated by down-regulation of CRH

receptors in the anterior pituitary. There is evidence that CRH (Wynn et al., 1988), AVP

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

1986; Wynn et al., 1985) can all act to regulate CRH receptor number. Therefore, an

alteration in CRH receptor number, receptor activity, receptor coupling, or even

corticotrophin number can affect the ability of CRH to stimulate ACTH secretion.
/
Arginine Vasopressin (AVP) The other major regulator of ACTH secretion of

hypothalamic origin is AVP. Classically, AVP is known as antidiuretic 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 basal-lateral 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 cAMP. 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. Decreases in blood volume are sensed as decreased stretch of the

arterial baroreceptors located in the carotid sinus as well as the receptors in the left atrium

and AVP secretion is reflexively stimulated (Berne and Levy, 1986).

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

is 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).

These axons project from magnocellular neurons of the supraoptic nucleus (SON) and

PVN from the hypothalamus. AVP and oxytocin are structurally similar hormones 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).

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 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, post-translational 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 and the contents of the

granules are released (Brownstein et al., 1980). Magnocellular neurons containing AVP










project fibers to the median eminence and therefore maybe 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 than CRH (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 V1) or anti-

diuretic receptors (subtype V2) that are found in the periphery. Data from two different

groups (Baertschei and Friedli, 1985; Jard et al., 1986) suggest that a subtype (classified

as Vib 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 exist. AVP-containing neurons of the

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

(Hoffminann et al., 1991). 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.

Synergism of CRH and AVP The synergistic activity of CRF and AVP secretion has been

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

the interaction between these two hormones. CRH 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 immunohistochemistry revealed co-localization of

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

rats) increases the co-localization of AVP in CRH nerve terminals in the median eminence

(de Goeij et al., 1991). Following adrenalectomy in rats, CRH immunostaining increases

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

increases (Sawchenko et al., 1984). The increase in CRH and AVP immunoreactivity

following adrenalectomy is prevented by intracerebroventricular injection of

dexamethasone (Sawchenko, 1987).

From in vitro studies performed in cultured pituitary cells, CRH 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, 1983a) and in fetal sheep (Brooks and

White, 1990). In a study performed in conscious sheep, stress-induced ACTH secretion

(audiovisual and insulin-induced hypoglycemia) was accompanied by increases in

hypothalamic CRH and AVP secretion (Familari et al., 1989). What is most interesting is

that CRH:AVP molar ratio was altered with the stress. Portal plasma AVP was increased










above that of CRH, increasing the ratio of AVP to CRH. Since CRH 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 CRH and AVP on ACTH

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

AVP/CRH secretion and pituitary ACTH secretion. This effect may be due to secretion of

other hypothalamic factors that increase ACTH secretion.

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

epminephrine and norepinephrine acting on a l-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 (Johnson et al., 1983). Neuropeptide Y (NPY)

injected centrally in fetal and adult sheep increases 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 CRH

secretion supports this conclusion (Haas and George, 1987). Endogenous opiates are

capable of stimulating the HPA axis in late gestational fetal sheep but do not tonically

stimulate the axis in a regulatory manner (Brooks and Challis, 1988). Serotonin stimulates

ACTH secretion in humans, demonstrates by pharmacologically increasing serotonin with

fenfluramine (Lewis and Sherman, 1984). However, there are conflicting data as to the

site of action of serotonin (hypothalamus or pituitary). In rats, CRH secretion is

decreased after elimination of endogenous hypothalamic catecholamines suggesting a role

for central catecholinergic neurons in the control of ACTH release (Guillaume et al.,










1987). Angiotensin II also increases plasma ACTH secretion by induction ofCRH (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 CRH (Brooks and

Gibson, 1992). In addition to stimulating the 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 hypophyseal portal vessels from the

hypothalamus and physiological concentrations inhibit ACTH release from pituitary cells

in vitro (Dayanithi and Antoni, 1989; Lim et al., 1990; Sheward et al., 1991). In vivo

immunoneutralization of ANP significantly increases ACTH release but has 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.
/
Negative Feedback Control of ACTH Secretion 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 mRNA 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 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 and 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 of 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)
t
performed in sheep, examined both hypothalamic and pituitary sites. of action for

glucocorticoids. Measurements of hypophyseal portal concentrations of AVP and CRH

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

1989). During audio-visual stress, hypothalamic CRH 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

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

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










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

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 stimulus 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 (McDonald et al., 1990). Between

117 and 131 days gestation, fetal sheep are extremely sensitive to negative feedback

effects of cortisol. This was demonstrated by infusions of cortisol that caused less than 2

ng/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 gestational

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). This mechanism of this reduction

of glucocorticoid negative feedback efficacy is not fully understood at present.

The Development of the Fetal HPA Axis

Development of the ovine fetal HPA axis begins during the first third of gestation

(term being 145-148 days gestation) 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 can be seen at about 40 days (Perry et al., 1982).

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










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

POMC can be detected in the intermediate lobe of the pituitary of 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 90-120 days gestation, the cells of the zona

fasciculata (which are responsible for cortisol synthesis) 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., 1982), and the correlation between fetal

ACTH secretion and fetal cortisol secretion becomes significant (Hennessy et al., 1982).

Adrenal weight also increases as a function ofgestational age (Comline and Silver, 1961).

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

adrenalectomy (Droust and Holm, 1968) with prolongation of pregnancy suggested a link

between the adrenal steroid production and parturition. If this is the case, fetal plasma

cortisol secretion should be altered as gestation nears and ends. Before about 130 days

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

plasma levels increase and peak at birth (Bassett and Thorburn, 1969). Nathanielsz et al.

(1972) found that fetal plasma cortisol concentrations began to increase about three to

four days before parturition and then steadily declined in the newborn lamb. A more

elaborate study of cortisol 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 concentration 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 concentrations. Experiments performed later by

other investigators revealed that the fetal plasma ACTH concentrations increase much

sooner than previously described by Jones et al. (Maclssac 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

occurred 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 ofgestational 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 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 CRH as a function of

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

responsiveness to CRH. 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.

The Importance of Gonadal Steroids in Parturition and Cardiovascular Control

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

occur at the end of gestation, the 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 eight 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 increase 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 prior to birth to 80 pg/ml on the day before

parturition. Plasma estradiol concentrations in the ewe increase over the last two days of

pregnancy in the ewe (Robertson and Smeaton, 1973), with a ten-fold increase on the day

before parturition, from 20 to 40 pg/ml up to 411 pg/ml (Challis, 1971). At the time










plasma estrogens increase, there is a decrease in plasma progesterone (Bedford et al.,

1972). This is possibly due to a conversion of estrogen to progesterone, with plasma

progesterone acting as a reservoir 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 gestational fetus in a manner similar to

estrogen concentrations (Pomerantz and Nalbandov, 1975; Yu et al., 1983).

This increase in plasma estrogen and androgen 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 estrogens and androgens

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

induce an enzyme, cytochrome P450c7, which has 17a-hydroxylase and 17,20 lyase

activities. Upon induction, P450c17 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, act in

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










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 estradiol and estrone sulfate

to 173-Estradiol and estrone, the more potent estrogen for the target organ, the

myometrium (Rossier and Pierrepoint, 1974).

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 following ACTH administration and greater adrenal

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

sexes is gonad and 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 were highest (Kitay, 1963). In ovariectomized (ovx) rats replaced with

estradiol, this effect can be restored (Viau and Meaney, 1991). Following ovx, 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 mammilary

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 in the pituitary-adrenal axis was due to

an impairment of the glucocorticoid negative feedback system.

The mechanism of action of estrogen stimulation on 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 corticotrophins

isolated from anterior pituitary cells from 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 100lg estradiol exogenously for two weeks, there was an increase

in AVP in the 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 ovx, plasma AVP concentrations decreased

but were restored when estrogen was replaced (Skowsky et al., 1979). These data

suggest a possible role of hypothalamic releasing factors in the mediation of estrogen

stimulation of HPA axis activity.

The results of this dissertation demonstrate that there is significant estrogen

sulfatase activity in ovine fetal hypothalamus, hippocampus, and brainstem, and that there










are statistically significant ontogenetic changes in activity of this enzyme in the

hippocampus. Also shown is the presence of estrogen sulfotransferase in the fetal

hypothalamus and brainstem. It has previously been demonstrated that estrogens in fetal

plasma increase both basal- and stimulated- fetal plasma ACTH secretion. The present

results suggest a mechanism by which the most abundant form of estrogen in ovine fetal

plasma, estrone sulfate, might be made available to areas within the fetal brain known to

be involved in the control of the fetal HPA axis.

Mathew and Balasubramanian (1982) and Lakshmi and Balasubramanian (1979)

have previously demonstrated estrogen sulfatase and sulfotransferase activity in adult

sheep brain tissue. Other investigators have demonstrated these enzymatic activities in

adult brain tissue from rats (Connolly and Resko, 1989; Kawano and Aikawa, 1987), mice

(Hobkirk, 1987), non-human primates (Lakshmi and Balasubramanian, 1981), and human

beings (Platia, 1984). Hobkirk and coworkers demonstrated that enzyme activities are
/
transiently increased postnatally in the brain of the mouse (1987). While the development

of brain estrogen sulfatase and sulfotransferase activity have not been studied in sheep, the

development of activities in mice suggests the possibility that this might be an important

developmental process in the perinatal period.

Using a histochemical technique, Kawano and Aikawa found that sulfatase activity

is highest in pineal gland, choroid plexus, and pars distalis of the pituitary in adult rats

(1987). I investigated the activity in hypothalamus, brainstem, and hippocampus because

these areas are known to contain nuclei involved in integration, afferent signal relay, or

negative feedback inhibition within the HPA axis (Grizzle et al., 1974; Keller-Wood and

Dallman, 1984; Maran, 1978; Ward, 1978). The presence of activity in any of these areas










could be important for the deconjugation of sulfated estrogens in the blood perfusing the

brain. Rosenfeld et al in 1980 reported that the majority of estrogen produced by the

ovine placenta is sulfoconjugated and thus protected since sulfatase in not present. My

data suggest otherwise given that sulfoconjugates in the fetal compartment may have

specific regional roles. The effect of estrogen on both basal- and hypotension stimulated-

concentrations of ACTH could be the result of an action of estrogen on the PVN in the

hypothalamus, an action on the hippocampus (which mediates some of the negative

feedback actions of corticosteroids on ACTH secretion), an action on the NTS (which

relays neural traffic from visceral afferents), or an action on any part of the pathways

leading from the NTS to the PVN (e.g., the RVLM). Estrogen receptors have been

demonstrated in the NTS and hippocampus (Lehman, 1993). While estrogen receptors

within the hypothalamus are most concentrated in the arcuate nucleus, estrogen receptors

have been demonstrated in the PVN (Lehman, 1993; Simerly, 1990). The results of the

present experiments identify the cellular location of the sulfatase activity which is

consistent with these centers for HPA axis control. I found widespread staining

throughout nuclei and fiber tracts of the hypothalamus and brainstem. Neuronal staining

was much more concentrated than fiber tract staining, however both were observable.

While estrogen sulfatase may be responsible for decongugating estrone sulfate

locally within the fetal to increase HPA axis activity directly, the role of estrogen

sulfotransferase is probably more indiscrete. Naturally one such role of the enzyme is to

maintain high levels of circulating conjugated estrogens that cannot be readily degraded.

A less obvious role of estrogen sulfotransferase might be to conjugate cortisol so that










inhibition of HPA axis negative feedback is achieved. This, concomitant with local

activation of estrogens via estrogen sulfatase, would increase ACTH release.

Cardiovascular Reflex Responsiveness

Arterial baroreceptors are mechano-receptors located in the walls of large systemic

arteries including the aortic arch, brachiocephalic artery, and carotid sinuses (Boss and

Green, 1956; Green, 1954). Afferent signals from the arch of the aorta are transmitted

through the left and right aortic depressor nerves to the vagus nerve and ultimately to the

nucleus of the tractus solitarius in the medullary area of the brain stem (Boss and Green,

1956; Nakayma, 1965; Nonindez, 1935). Sensory input from the carotid sinus region

travels to the nucleus of the tractus solitarius as well, but via the sinus (Hering's) and

glossopharyngeal nerves.

Barosensitive nerve endings are found in areas of the arteries with large quantities

of elastic tissue (Muratori, 1967). Approximately 40% of the tissue comprising the walls

of the aortic arch is an elastin-collagen mixture (Bader, 1963) which is almost free of

smooth muscle (Gregoreva, 1962). Similarly, the carotid sinus is thinner (Adams, 1958;

Addison, 1944; Rees, 1968; Rees and Jepson, 1970), contains less smooth muscle

(Addison, 1944; Bagshaw and Fischer, 1971; Muratori, 1967; Rees and Jepson, 1970),

and shows a higher elastin content than other areas of the carotid artery (Addison, 1944;

Rees and Jepson, 1970).

Although their name implies a pressure-sensitive quality, baroreceptors are stretch

receptors which respond to deformation of the vessel wall in which they are located

(Hauss et al., 1949; Angell-James, 1971). There is evidence which shows that the degree

of wall deformation determines the electrical activity of the carotid sinus and aortic arch










baroreceptors. Hauss et al. (1949) demonstrated that the reflex fall of blood pressure

produced by an increase in carotid sinus pressure is abolished if the stretching of the

carotid artery is prevented by a plaster cast applied to the outside of the sinus region.

Additionally, Angell-James (1971) reported that increased baroreceptor activity produced

by elevation of intrathorasic pressure could be prevented by simultaneously increasing the

extramural pressure by the same amount. Experiments in man have shown that changing

pressure in a chamber surrounding the neck results in reflex changes in heart rate and

blood pressure and provide further evidence that altered transmural pressure is a stimulus

for baroreceptor activation (Bevegard and Shepard, 1966; Emrnestine and Parry, 1957).

Koch (1931) was the first to demonstrate that when carotid sinus pressure was

changed in a stepwise manner, mean arterial pressure exhibited an inverse sigmoidal

response to the change in intrasinus pressure. Bronk and Stella (1932) who observed that

the impulse frequency in Herring's nerve exhibited a positive sigmoidal, relationship to

changes in sinus pressure later substantiated these findings. These early findings identified

that baroreceptors can be characterized as having a threshold pressure range for which the

discharge rate increases with a rise in mean arterial pressure, as an asymptotic saturation

pressure beyond which there is little increase in baroreceptor activity (Koushanpour,

1991).

The aortic and carotid baroreceptors exhibit different threshold and saturation

characteristics. Carotid sinus baroreceptors are silent at arterial pressures between 0 and

60 mmHg, but above 60 mmHg, they respond progressively and reach maximum discharge

capacity at approximately 180 mmHg (Koushanpour, 1991). Aortic baroreceptors

respond in a manner similar to that of the carotids except that they exhibit a threshold










pressure approximately 30 mmHg higher (Koushanpour, 1991). Therefore, in the normal

operating range of approximately 100 mmHg (80 to 180 mmHg), slight changes in

pressure elicit strong baroreceptor-mediated autonomic reflexes to return arterial pressure

to within homeostatic limits.

Blood pressure control in the fetus is similar with some differences. Decreases in

blood pressure increase the secretion of ACTH, cortisol, AVP, and rein (Robillard et al.,

1979; Rose et al., 1981; Wood 1989). As in the adult, the magnitude of the responses are

proportional to the magnitude of the decrease in arterial pressure (Wood et al., 1982), and

responses are attenuated by sinoaortic denervation (Wood, 1989). Chemoreceptors have

a critical role in maintaining blood pressure as well by monitoring the levels of oxygen,

carbon dioxide, and hydrogen ions in the blood.

Effective baroreceptor function is necessary to respond to transient alterations in

arterial pressure (Brown, 1980). In the face of increasing pressure, baroreceptor-

generated signals ascend afferent pathways and enter the nucleus tractus solitarius where

secondary signals inhibit the medullary vasoconstrictor center and excite the vagal center

stimulating vasodilation and decreased myocardial ionotropic and chronotropic response

(Brown, 1980). These actions lead to lowered peripheral resistance, cardiac output, and

ultimately, lower blood pressure. Conversely, a sudden fall in arterial pressure leads to

reflex actions which increase cardiac output and systemic resistance to raise blood

pressure.

The bradycardic response to baroreceptor stimulation in humans is mediated

through vagal cholinergic mechanisms. Several investigators (Eckberg et al., 1971;

Pickering et al., 1972; Simon et al., 1977; Takeshita et al., 1979) have demonstrated that










elongation of the R-R interval which accompanied a rise in arterial pressure following

administration ofphenylephrine was not reduced by propranolol (Jose and Taylor, 1969),

but was abolished by atropine. Others have reported similar observations in response to

stimulation of the carotid baroreceptors by neck suction (Eckberg, 1977; Eckberg et al.,

1976).

In contrast, there is a lack of consensus regarding the autonomic mechanisms

mediating the tachycardic response to arterial baroreceptor unloading. It has been

observed that the early tachycardia observed after administration of vasodilators was

unaffected by propranolol, but abolished by atropine, suggesting a predominant vagal

mediation of this response (Leon et al., 1970; Mancia et al., 1979; Mroczek et al., 1976;

Pickering et al., 1972). Contrary to these reports, others have demonstrated that the

increase in heart rate produced during infusions of nitroglycerin was reduced by atropine,

but could only be abolished by combined administration of atropine and a beta-adrenergic
/
blocker (Goldstein et al., 1975; Robinson et al., 1966). In addition, it has been

demonstrated that during lower body negative pressure, tachycardia was diminished 52%

by propranolol with the remaining response abolished by atropine (Bjurstedt et al., 1977).

Therefore, it appears there is a significant vagal component to the cardioacceleration

which accompanies baroreceptor unloading. However, an increase of sympathetic cardiac

influence may contribute to the more sustained component ofbaroreflex-mediated

tachycardia (Mancia and Mark, 1983). Taken together, these results suggest redundancy

in mechanisms by which the autonomic nervous system mediates baroreflex-induced

tachycardia.










The baroreceptor system markedly reduces daily variation in arterial pressure.

This phenomenon is readily demonstrated in sinoaortic denervated animals who exhibit

elevated blood pressure (Cowley et al., 1973). Controversy exists regarding the

persistence of this hypertension with some authors arguing it eventually subsides (Guyton

et al., 1974) while others suggest elevated blood pressure persists (Scher and Ito, 1978;

Alexander, 1979; Touw et al., 1979; Werber and Fink, 1979).

Studies involving the measurement of substances released from the hypothalamus,

the pituitary or the adrenal require the evaluation of blood pressure as blood pressure is

reflexively defended at least in part by hypothalamic, pituitary and adrenal responses. In

both the adult and the fetus, responses to hypotensive stimuli involve neural and hormonal

changes that are responsible for restoring blood pressure. In the adult, decreases in blood

pressure are detected by stretch receptors in the atria (Cryer and Gann, 1974) and by

baroreceptors in the high pressure circulatory system (Roseetal, 1981) and result in the
/*
increased release of ACTH, cortisol (Gann, 1979), renin and AVP (Claybaugh and Share,

1973). In the fetus the ACTH, cortisol, AVP and renin responses to hypotension are not

vagally mediated but instead are thought to involve changes in blood pH and central

chemoreceptors (Wood et al., 1989). ACTH is important in both adult and fetal animals

for inducing the appropriate adrenocortical responses to noxious stimuli such as

hypotension (Wood and Rudolph, 1983). Cortisol is essential for the restitution of blood

volume following hemorrhage and as a permissive substance for appropriate

vasoconstriction following hypotension in both the adult (Grimes et al., 1987; Pirkle et al.,

1976) and fetus (Brace, 1983). Vasopressin is responsible for vasoconstriction,






38



redistribution of blood flow and antidiuresis in the adult (Cowley et al., 1974) and fetus

(Iwamoto et al., 1979).













CHAPTER 3
GENERAL METHODS


Surgical Preparation of Fetal Sheep

The sheep used in this study were all pregnant ewes of 115 days gestation or later.

Animals were purchased from various suppliers (Institute of Food and Agricultural

Sciences, University of Florida, Gainesville, Fl; Tom Morris, MD) and were of various

breeds (Florida Native, Mixed Western, etc.). Prior to surgery, all animals were housed in

approved pens in the Health Science Center or the 34'h Street facility at the University of

Florida. Prior to experimentation, all animals were housed in Animal Resources at the

Health Science Center and were maintained under controlled lighting and temperature.

Pens were cleaned daily and ewes were given food and water ad libitum.

Aseptic fetal surgery was performed in Animal Resources or at the 34th Street

facility under general anesthesia with 0.5% 2.0% halothane. All ewes were between 115

and 125 days gestation at the time of surgery. Food and water were withheld from ewes

24 hours prior to surgery. Ewes were sheared close to the skin around the abdomen and

prepared for surgery with povidone iodine (Betadine, Purdue Fredrick Co., Norwalk,

CT). Animals were intubated and connected to a respirator to allow for constant

anesthesia. Heart rate, blood pressure, ventilatory 02 and CO2, respirations per minute,

and rectal temperature were all monitored at the time of surgery. Animals were closely

monitored from the time of intubation until recovery when the animal could stand on its








own effort. Ewes were allowed free access to food and water throughout the post-

operative period.

The uterus was exposed using a midline incision beginning at the umbilicus and

extending caudally approximately 10 cm. Once the hindlimbs were located, a small

incision was made in the uterus. Hindlimbs were delivered through the uterine incision

one at a time for the purpose of placing a polyvinyl chloride catheter (0.03" ID, 0.05" OD)

into each femoral artery. Later, these catheters would be used for blood sampling and

blood pressure recording. The tips of each femoral catheter were advanced to the

subdiaphragmatic aorta. At this time an estradiol implant (5 mg/21 days or 250 gig/day;

Innovative Research of America, Toledo OH) or placebo was inserted subcutaneously into

the area of the gluteus medius before suturing the incised hindlimbs. An amniotic catheter

made of polyvinyl chloride (0.05" ID, 0.09" OD) was sutured to the exterior of a hindlimb

for the purpose of antibiotic delivery as well as amniotic fluid pressure measurements.

Hindlimb and uterine incisions were closed using 2.0 silk suture. Hindlimb incisions were

closed using a simple continuous suture pattern. All uterine incisions were closed first

with a locking simple continuous pattern followed by umbrication of the uterus.

Using a technique similar to the one described above, catheters were placed in the

lingual arteries and advanced 1 cm into the carotid artery toward the heart. Upon closure

of the neck incision, lingual catheters were anchored to the chin of the fetus with 2.0 silk

suture. Lingual catheters were of the same material and size and femoral catheters.

Depending upon the experimental setup, carotid sinus denervation was employed

at this time. After exposing the common carotid artery, denervation was accomplished by

stripping all nerves and connective tissue between the carotid-occipital arterial junction








and the carotid-lingual arterial junction. The occipital artery was ligated, for this is the

only method by which all of the carotid sinus baroreceptor and chemoreceptor afferent

fibers are cut. The lingual artery was stripped rostrally of all nerves and connective tissue

for approximately 1 cm from the carotid-lingual junction. This denervation was conducted

bilaterally.

Before returning the head of the fetus to the uterus, an occluder was placed around

the brachiocephalic artery. These silastic occluders resemble miniature blood pressure

cuffs and were purchased from In Vivo Metric (Cat. # OC8, Healdsburg, CA). The left

forelimb of the fetus was delivered through a uterine incision. An incision was made into

the second intercostal space and the brachiocephalic artery was located. Once the

occluder was sewn in place, the incision under the left forearm was closed and the fetus

returned to the uterus. The uterine incision was closed once again using a locking simple

continuous suture pattern followed by an umbrication technique. 750 mg ampicillin

(Polyflex, Ft. Dodge Laboratories, Ft. Dodge, IA) was administered into the amniotic

cavity before closure of the maternal linea alba and skin.

All catheters were filled with heparin (1000 units/ml, Elkins-Sinn, Inc., Cherry Hill,

NJ) and closed with a sterile brad inserted into the end. Catheters and occluders were

flanked and exteriorized via a trochar. Catheters were held in place with an elastic

bandage. The linea alba was closed with #3 polyamide suture (Pitmann-Moore, France)

while the skin was closed with #1. 750 mg ampicillin was administered intramuscularly to

the ewe.

It should be noted that in ewes with twin pregnancies, both fetuses were surgically

manipulated the same way. At the time of experimentation however, only one of the








animals was made hypotensive while the other animal served as a control. All ewes were

treated with 750 mg ampicillin twice per day for five days post-operatively. In addition,

rectal temperatures were taken to monitor for infection. All ewes were monitored closely

for any indication of poor health.

In Vivo Experimental Procedures

All ewes were given five days to recover from surgery. On the day of

experimentation, catheters were removed from the elastic bandage and the distal ends

were cleaned with povidone iodine and alcohol. Each brad was removed and a sterile

blunt adapter with a three-way stopcock was inserted. This procedure was always done

for both femoral catheters and the amniotic catheter. Lingual catheters were only utilized

if the fetus was to be made hypotensive. All catheters to be used were flushed with

heparinized saline (2.0%/ v/v). One femoral catheter and the amniotic catheter were

attached to transducers (Statham P23Id, Statham Instruments, Oxnard, CA) for

measurement of fetal arterial and amniotic fluid pressure. As stated previously, if the fetus

was made hypotensive lingual pressure was also monitored to assure proper occlusion.

Arterial and amniotic fluid pressures were measured for the first 35 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.

All experiments were performed between 120 and 135 days gestation to minimize

variation in hormone concentrations between experiments and animals. All animals were

studied in their pens utilizing six sections of PVC tubing to limit movement of the ewe.

Once catheters were removed from the elastic bandage ewes were not touched in order to

limit the amount of external stress placed upon the animals. Experiments lasted one hour










with blood samples taken at 0, 10, 20, and 60 minute time points. If the fetus was to be

made hypotensive to activate the HPA axis, the brachiocephalic occluder was inflated after

collection of the 0 time point sample for 10 minutes. The brachiocephalic occluder was

inflated via an infusion of saline through the silastic tubing. This, in turn, causes a

hypoperfusion of blood to the fetal brain, which activates the HPA axis. Five ml of blood

were taken at each time point and collected in chilled tubes containing Na4 EDTA (50 tg

EDTA/ml blood, Sigma Chemical Co., St. Louis, MO). An additional 1.5 ml of blood was

drawn anaerobically into syringes coated with heparin for measurement of fetal blood

gases using a Ciba-Corning 288 Blood Gas System. A small portion of this blood was

used to measure hematocrit using an IEC microhematocrit centrifuge. After sampling,

volume of the catheter was restored with 0.9% normal saline with 2.0% v/v heparin.

Blood samples (5 ml) were kept on ice until further analysis for hormone levels.

Samples were centrifuged at 3000 x g for 30 minutes at 4 C in a refrigerated centrifuge
i
(Sorvall RT 600B, DuPont, Newtown, CA). After centrifugation, the plasma was

transferred and aliquotted to polystyrene tubes and stored at -20 C until hormones were

assayed.

Upon conclusion of each experiment, ewes were sacrificed with an overdose of

sodium pentobarbital via the jugular vein. Fetuses were immediately removed for

perfusion of the brain. The chest cavity of each fetus was opened and the brachiocephalic

artery was located and cannulated. Either by means of a pump or by syringe, brains were

perfused first with one liter of phosphate buffered saline (pH 7.4, 2.0% v/v heparin)

followed by two liters of 4% paraformaldehyde. Brains and pituitary glands were

removed and stored in 4% paraformaldehyde until processing for immunohistochemistry.










Peptide Assays

Arginine Vasopressin (AVP) Plasma AVP concentrations were measured using an

antibody raised in rabbits (Raffet al., 1991). lodinated AVP was purchased from

Amersham and synthetic AVP from Sigma Chemical Co. AVP was first extracted from

0.5 ml plasma with 1.0 ml bentonite slurry (0.3% w/v in distilled water) and acidified with

0.05 ml 1 N 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 -20 C

until assayed. Extracts were reconstituted with 0.25 ml 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.

Adrenocorticotropin (ACTH) Plasma ACTH concentrations were measured by

radioimmunoassay (RIA) as previously described (Bell et al., 1991) using an antibody
t
raised in rabbits developed in Dr. Wood's laboratory to human-ACTH (1-24). lodinated

ACTH (125 ACTH) was prepared using the chloramine-T method (Berson and Yalow,

1968) with human-ACTH (1-39)(Sigma Chemical Co., St. Louis, MO) and radioactive

sodium iodide (Amersham, Arlington Heights, IL). ). I125 ACTH was made fresh

approximately every six weeks. ACTH was first extracted from plasma before assaying.

ACTH (0.5 ml) was extracted on glass (35 mg/tube) (100-200 mesh glass. Corning

Glassworks, Comrning, NY) in 0.50 ml assay buffer (0.05 M phosphate buffer, pH 7.4 with

0.2% w/v silicic acid-extracted bovine serum albumin (BSA, Sigma Chemical Co.,

#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 0.25 N HCI: 1 volume









acetone). The extracts were dried under vacuum (Savant Instruments, Farmingdale, NY)

and frozen at -20 C until assayed. Extracts were reconstituted overnight in 0.5 ml 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.

Steroid Asays

Cortisol Plasma cortisol concentrations were measured as previously described (Wood et

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

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

extracted from 20 Wi 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 0.5 ml 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 NaCl,

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

Estradiol Plasma estradiol concentrations were measured utilizing an enzyme

immunoassay (EIA) kit from Oxford Biochemical Inc. (#EA70). 2 ml of plasma was

extracted with 16 ml ethyl ether. Extracts were dried under air and reconstituted to 200

4l with assay buffer (provided with kit). 50 u.l of extract was assayed for estradiol in

duplicate. The values obtained were divided by 10 to give ng/ml concentrations. The

antiserum in this particular EIA kit had a very low cross-reactivity with estrone (0.10%).

Estrone Sulfatase Activity

I studied fetuses (86-147 days gestation, term= 147 days), 4 lambs (3-4 weeks old),

and 4 adult nonpregnant ewes to determine estrone sulfatase activity. The sheep were










sacrificed using an intravenous overdose of sodium pentobarbital. Gestational ages of the

fetal sheep were calculated from known breeding dates. Whole brains were rapidly

removed, dissected into discrete regions, and quickly frozen on dry ice or in a slurry of dry

ice and acetone. All tissues were stored at -20C or -40C until studied.

Hypothalami, brainstems, and hippocampi were then processed to determine

estrone sulfatase activity. Each tissue sample was homogenized in medium 199 (Sigma,

St. Louis, MO) containing 25 mM HEPES. Homogenization was performed using a

Polytron homogenizer (Tekmar, Cincinnati, OH). The concentration of each tissue in the

homogenate was 0.5 g tissue in 5 mL medium.

Tissues were centrifuged at 1200 rpm for 5 min; supernatant was then collected

and assayed immediately. A sample of each homogenate was assayed for protein

concentration using the method of Bradford (1976) using a commercially-available assay

kit (Bio-Rad Laboratories, Hercules, CA). Homogenate (0.1 mL) was' aliquotted in

duplicate into borosilicate tubes (16 x 150 mm) containing 0.8 mL of a mixture of 3H-

[6,7]-estrone sulfate (DuPont-NEN, Wilmington, DE) and unlabeled estrone sulfate

(EiS04, Sigma, St. Louis, MO). All reactions were run at 37C. Reactions were

terminated by immediate cooling on ice, addition of 5 volumes of ethyl acetate:hexane

(3:2), and vigorous mixing for 30 seconds. The aqueous phase was frozen by submersion

of the reaction tube into a dry ice and acetone slurry. Subsequently, the organic phase

containing the 3H-estrone was decanted into 13x75 mm borosilicate glass tubes and dried

under a gentle stream of room air. Dried extracts were reconstituted in scintillant

(Cytoscint, ICN Corp., Costa Mesa, CA) and counted in a scintillation counter (LKB

Corp., Gaithersburg, MD).










Enzyme activities at different developmental ages and in different tissues were

measured using a substrate concentration of 3 pM and 3H-estrone sulfate specific activity

of approximately 0.67 gCi/nmol. For this experiment, reactions were allowed to run for 5

min. Using these conditions, less than 20% of the substrate was converted to 3H-estrone.

Western Blotting

Hypothalami and brainstems were harvested from fetuses, lambs, and adults of

known gestational and postnatal ages. The number and ages of animals varied slightly

between hypothalami and brainstem but 11-12 fetuses, 3-4 lambs and 2 adults were used

per tissue type. These tissues were originally obtained and homogenized for other studies

(Saoud and Wood, 1996). Unfortunately, hippocampi from these animals were not

available. All tissue was homogenized in reducing buffer and boiled for 5 minutes. The

samples were centrifuged to remove particulate matter and supernatant was recovered.

Protein.concentrations were obtained utilizing the Bradford technique (1976). Western

blots were performed using a mini-Protean electrophoresis system (Bio-Rad, Hercules,

CA) on 10% pre-cast polyacrylamide gels purchased from Bio-Rad laboratories. Samples

were diluted so that an equal amount of protein was loaded per lane (20 jg for brainstem

and 40 jg for hypothalami). The protein was then transferred to a nitrocellulose

membrane and probed for either estrogen sulfatase or estrogen sulfotransferase using

custom-made rabbit polyclonal antibodies (Alpha Diagnostic, San Antonio, TX). The

peptide sequence used from the human sulfatase gene, amino acids 294-309, was NH2-

FSSKDFAGKSQHGVYGC-COOH (Simerly et al., 1990). The peptide sequence used

from the bovine sulfotransferase gene, amino acids 273-295, was NH2-

RERFEEHYQQQMKDC-COOH (Nash et al., 1988). Primary antibodies were diluted to










a concentration of 1:1000 in antibody diluent (1 % BSA in phosphate buffered saline with

0.05 % Tween 20). Visualization of the protein-antibody complex was accomplished

utilizing a chemiluminescence detection system (Renaissance, DuPont NEN, Boston, MA)

and analyzed by densitometry (Bio-Rad). Antibody specificity was confirmed by

preabsorption of the primary antibodies with peptides (1 .g/ml) also supplied by Alpha

Diagnostic. Developmental changes were calculated using multiple linear regression in

order to control for differences between gel running conditions (SigmaStat, Jandel

Scientific, San Rafael, CA).

Immunohistochemical Techniques

Fetal brains were perfusion fixed with 4% paraformaldehyde, dissected and cut

into gross tissue regions (hypothalamus, midbrain, pons, medulla, spinal cord, etc.).

Tissue was processed for embedding by dehydration with progressively increasing

concentrations of ethanol, followed by xylene. All tissue was embedded in paraffin and

cut into 10 pm sections using a Zeiss microtome. Sections were mounted on poly-L-

lysine slides, deparaffinized with xylene and rehydrated in decreasing concentrations of

ethanol. Immunohistochemistry and visualization were made possible utilizing a

Histostain-SP kit from Zymed and metal-enhanced DAB (Pierce, Rockford, IL). Sections

were stained for estrogen sulfatase, estrogen sulfotransferase, c-fos, ACTH, AVP, and

CRH (see Table 3.1). Primary antibodies were diluted in antibody diluent (1% BSA in

phosphate buffered saline with 0.01% Triton X-100). Specific staining was confirmed by

dilution tests, as staining decreased as primary antibodies were further diluted. Specific

staining was absent upon replacing primary antibodies with 10% normal goat serum. All








slides were dehydrated prior to mounting of coverslips with Permount (Fisher Scientific,

Pittsburgh, PA).


Primary antibody Vendor (Source) Dilution
Estrogen Sulfatase Alpha Diagnostics (same as 1:500
for Western Blots)__________
Estrogen Sulfotransferase Alpha Diagnostics (same as 1:500
for Western Blots)
c-fos Oncogene Biomedical Inc. 1:5000
~~~____~_______ (cat. PC38)
ACTH Wood Laboratory (same as 1:10,000
for RIA)
AVP Wood Laboratory (same as 1:20,000
~~____~________for RIA) ______I___
CRH Keller-Wood Laboratory 1:100,000


Table 3.1: Primary antibodies used in immunohistochemical experiments. All antibodies

were diluted in 1% BSA in phosphate buffered saline with 0.01% Triton X-100.













CHAPTER 4
HORMONAL RESPONSIVENESS IN AN ESTRADIOL, HYPOTENSIVE, CAROTID
SINUS DENERVATED OVINE MODEL


Introduction

In the fetal sheep, parturition is triggered by an increase in the activity of the fetal

HPA axis (Challis and Brooks, 1989; Liggins et al., 1973). Parturition can be 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). The last few days of gestation are marked by an

increase in the activity of the fetal hypothalamus as seen by elevated levels of fetal plasma

ACTH. This increase in plasma ACTH causes a corresponding increase in plasma

cortisol.. Along with this increase in HPA axis activity is a decreased sensitivity of the axis

to cortisol negative feedback (Wood, 1988). It is well known that in the sheep, cortisol

acts at the placenta to increase the activity of an enzyme, cytochrome p450 (17-

hydroxylase and 17,20 lyase activities), which in turn, increases the ratio of estrogen to

progesterone (Anderson et al., 1975; Pomeranz and Nalbandov, 1975; Steele et al., 1976;

Yu et al., 1983). This cascade of events essentially increases the total amount of estrogen.

Estrogen is known to be an important factor in the initiation of parturition by

causing the uterus to contract (Liggins, 1974). 173-estradiol has been shown to increase

the activity of the HPA axis in sheep and rats. In a study by Saoud and Wood (1995),






51

estrogen was found to augment fetal plasma ACTH secretion in response to stress. Other

studies have shown similar results in adult animals (Viau and Meaney, 1991).

Understanding the mechanism of the increased fetal HPA axis at the end of

gestation is key to understanding the mechanism of spontaneous parturition in sheep.

These experiments were conducted to see if estradiol has it's stimulatory effect on HPA

axis activity through the afferent baroreceptor and chemoreceptor pathway. More

specifically, I hypothesized that estradiol's actions on fetal cardiovascular reflex

responsiveness to hypotension will be measurable in intact fetuses but not in baro- and

chemo- denervated fetuses. This augmentation of HPA axis activity will be assessed by

using a surgically manipulated ovine model. ACTH, AVP, estradiol, and cortisol levels

will be measured and compared across eight different treatment groups. These groups

include fetuses which are: (1) estradiol treated; (2) estradiol treated, hypotensive; (3)

estradiol treated, carotid sinus denervated; (4) estradiol treated, carotid sinus denervated,

hypotensive; (5) placebo treated; (6) placebo treated, hypotensive; (7) placebo treated,

carotid sinus denervated; (8) placebo treated, carotid sinus denervated, hypotensive.

Research has previously demonstrated that the combined baro- and chemo- denervation

attenuates (approximately 50%) the reflex hormonal and hemodynamic responses to

moderate (50 %) reduction in arterial blood pressure. I proposed that the interruption of

the afferent pathways would eliminate the effect of estrogen on the reflex cardiovascular

responsiveness. If so, it could concluded that estrogen acts on, within, or requires input

from, the afferent baroreceptor and chemoreceptor pathways.










Methods and Materials

Surgical Procedures Aseptic fetal surgery was performed in Animal Resources or at the

34th Street facility under general anesthesia with 0.5% 2.0% halothane. All ewes were

between 115 and 125 days gestation at the time of surgery. A total of 40 ewes were set

up and studied (n=5 per group). Food and water were withheld from ewes 24 hours prior

to surgery. Ewes were sheared close to the skin around the abdomen and prepared for

surgery with povidone iodine (Betadine, Purdue Fredrick Co., Norwalk, CT). Animals

were intubated and connected to a respirator to allow for constant anesthesia. Heart rate,

blood pressure, ventilatory 02 and CO2, respirations per minute, and rectal temperature

were all monitored at the time of surgery. Animals were closely monitored from the time

of intubation until recovery when the animal could stand on its own effort. Ewes were

allowed free access to food and water throughout the post-operative period.

The uterus was exposed using a midline incision beginning at the umbilicus and
f
extending caudally approximately 10 cm. Once the hindlimbs were.located, a small

incision was made in the uterus. Hindlimbs were delivered through the uterine incision

one at a time for the purpose of placing a polyvinyl chloride catheter (0.03" ID, 0.05" OD)

into each femoral artery. Later, these catheters would be used for blood sampling and

blood pressure recording. The tips of each femoral catheter were advanced to the

subdiaphragmatic aorta. At this time an estradiol implant (5 mg/21 days or 250 tg/day;

Innovative Research of America, Toledo OH) or placebo was inserted subcutaneously into

the area of the gluteous medius before suturing the incised hindlimbs. An amniotic

catheter made of polyvinyl chloride (0.05" ID, 0.09" OD) was sutured to the exterior of a

hindlimb for the purpose of antibiotic delivery as well as amniotic fluid pressure










measurements. Hindlimb and uterine incisions were closed using 2.0 silk suture. Hindlimb

incisions were closed using a simple continuous suture pattern. All uterine incisions were

closed first with a locking simple continuous pattern followed by umbrication of the

uterus.

Using a technique similar to the one described above, catheters were placed in the

lingual arteries and advanced approximately 1 cm into the carotid artery toward the heart.

Upon closure of the neck incision, lingual catheters were anchored to the chin of the fetus

with 2.0 silk suture. Lingual catheters were of the same material and size and femoral

catheters.

Depending upon the experimental setup, carotid sinus denervation was employed

at this time. After exposing the common carotid artery, denervation was accomplished by

stripping all nerves and connective tissue between the carotid-occipital arterial junction

and the carotid-lingual arterial junction. The occipital artery was ligated, for this is the

only method by which all of the carotid sinus baroreceptor and chemoreceptor afferent

fibers are cut. The lingual artery was stripped rostrally of all nerves and connective tissue

for approximately 1 cm from the carotid-lingual junction. This denervation was conducted

bilaterally.

Before returning the head of the fetus to the uterus, an occluder was placed around

the brachiocephalic artery. These silastic occluders resemble miniature blood pressure

cuffs and were purchased from In Vivo Metric (Cat. # OC8, Healdsburg, CA). The left

forelimb of the fetus was delivered through a uterine incision. An incision was made into

the second intercostal space and the brachiocephalic artery was located. Once the

occluder was sewn in place, the incision under the left forearm was closed and the fetus










returned to the uterus. The uterine incision was closed once again using a locking simple

continuous suture pattern followed by an umbrication technique. 750 mg ampicillin

(Polyflex, Ft. Dodge Laboratories, Ft. Dodge, IA) was administered into the amniotic

cavity before closure of the maternal linea alba and skin.

All catheters were filled with heparin (1000 units/ml, Elkins-Sinn, Inc., Cherry Hill,

NJ) and closed with a sterile brad inserted into the end. Catheters and occluders were

flanked and exteriorized via a trochar. Catheters were held in place with an elastic

bandage. The linea alba was closed with #3 polyamide suture (Pitmann-Moore, France)

while the skin was closed with #1. 750 mg ampicillin was administered intramuscularly to

the ewe.

It should be noted that in ewes with twin pregnancies, both fetuses were surgically

manipulated the same way. At the time of experimentation however, only one of the

animals was made hypotensive while the other animal served as a control. All ewes were

treated with 750 mg ampicillin twice per day for five days post-operatively. In addition,

rectal temperatures were taken to monitor for infection. All ewes were monitored closely

for any indication of poor health.

In Vivo Experimental Procedures All ewes were given five days to recovery from

surgery. On the day of experimentation, catheters were removed from the elastic bandage

and the distal ends were cleaned with povidone iodine and alcohol. Each brad was

removed and a sterile blunt adapter with a three-way stopcock was inserted. This

procedure was always done for both femoral catheters and the amniotic catheter. Lingual

catheters were only utilized if the fetus was to be made hypotensive. All catheters to be

used were flushed with heparinized saline (2.0% v/v). One femoral catheter and the










amniotic catheter were attached to transducers (Statham P23Id, Statham Instruments,

Oxnard, CA) for measurement of fetal arterial and amniotic fluid pressure. There were

eight different experimental groups in this study: (1) estradiol treated; (2) estradiol

treated, hypotensive; (3) estradiol treated, carotid sinus denervated; (4) estradiol treated,

carotid sinus denervated, hypotensive; (5) placebo treated; (6) placebo treated,

hypotensive; (7) placebo treated, carotid sinus denervated; (8) placebo treated, carotid

sinus denervated, hypotensive. As stated previously, if the fetus was made hypotensive

lingual pressure was also monitored to assure proper occlusion. Arterial and amniotic

fluid pressures were measured for the first 35 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.

All experiments were performed between 120 and 135 days gestation to minimize

variation in hormone concentrations between experiments and animals.- All animals were

studied in their pens utilizing six sections of PVC tubing to limit movement of the ewe.

Once catheters were removed from the elastic bandage ewes were not touched in order to

limit the amount of external stress placed upon the animals. Experiments lasted one hour

with blood samples taken at 0, 10, 20, and 60 minute time points. If the fetus was to be

made hypotensive to activate the HPA axis, the brachiocephalic occluder was inflated after

collection of the 0 time point sample for 10 minutes. The brachiocephalic occluder was

inflated via an infusion of saline through the silastic tubing. This, in turn, causes a

hypoperfuision of blood to the fetal brain, which activates the HPA axis. Five ml of blood

were taken at each time point and collected in chilled tubes containing Na4 EDTA (50 jig

EDTA/ml blood, Sigma Chemical Co., St. Louis, MO). An additional 1.5 ml of blood was










drawn anaerobically into syringes coated with heparin for measurement of fetal blood

gases using a Ciba-Comrning 288 Blood Gas System. A small portion of this blood was

used to measure hematocrit using an IEC microhematocrit centrifuge. After sampling,

blood volume was restored with 0.9% normal saline with 2.0% v/v heparin.

Blood samples (5 ml) were kept on ice until further processing. Samples were

centrifuged at 3000 x g for 30 minutes at 4 C in a refrigerated centrifuge (Sorvall RT

600B, DuPont, Newtown, CA). After centrifugation, the plasma was transferred and

aliquotted to polystyrene tubes and stored at -20 C until hormones were assayed.

Upon conclusion of each experiment, ewes were sacrificed with an overdose of

sodium pentobarbital via the jugular vein. Fetuses were immediately removed for

perfusion of the brain. The chest cavity of each fetus was opened up and the

brachiocephalic artery was located and cannulated. Either by means of a pump or by

syringe, brains were perfused first with one liter of phosphate buffered' saline (pH 7.4,

2.0% v/v heparin) followed by two liters of 4% paraformaldehyde. Brains and pituitary

glands were removed and stored in 4% paraformaldehyde until processing for

immunohistochemistry (Chapter 5). The experimental design can be viewed pictorially in

Figure 4.1.

Estradiol Assay Plasma estradiol concentrations were measured utilizing an enzyme

immunoassay (EIA) kit from Oxford Biochemical Inc. (#EA70). 2 ml of plasma was

extracted with 16 ml ethyl ether. Extracts were dried under air and reconstituted to 200

ll with assay buffer (provided with kit). 50 ul of extract was assayed for estradiol in

duplicate. The values obtained were divided by 10 to give ng/ml concentrations. This

particular EIA kit had a very low cross-reactivity with estrone (0.10%).










Adrenocorticotropin (ACTH) Assay Plasma ACTH concentrations were measured by

radioimmunoassay (RIA) as previously described (Bell et al., 1991) using an antibody

raised in rabbits developed in Dr. Wood's laboratory to human-ACTH (1-24). lodinated

ACTH (I125 ACTH) was prepared using the chloramine-T method (Berson and Yalow,

1968) with human-ACTH (1-39)(Sigma Chemical Co., St. Louis, MO) and radioactive

sodium iodide (Amersham, Arlington Heights, IL).). I125 ACTH was made fresh

approximately every six weeks. ACTH was first extracted from plasma before assaying.

ACTH (0.5 ml) was extracted on glass (35 mg/tube) (100-200 mesh glass, Coming

Glassworks, Coming, NY) in 0.50 ml assay buffer (0.05 M phosphate buffer, pH 7.4 with

0.2% w/v silicic acid-extracted bovine serum albumin (BSA, Sigma Chemical Co.,

#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 0.25 N HCI: 1 volume

acetone). The extracts were dried under vacuum (Savant Instruments,,Farmingdale, NY)
/
and frozen at -20 C until assayed. Extracts were reconstituted overnight in 0.5 ml 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.

Arginine Vasopressin (AVP) Assay Plasma AVP concentrations were measured using an

antibody raised in rabbits (Raffet al., 1991). lodinated AVP was purchased from

Amersham and synthetic AVP from Sigma Chemical Co. AVP was first extracted from

0.5 ml plasma with 1.0 ml bentonite slurry (0.3% w/v in distilled water) and acidified with

0.05 ml 1 N HCI. Extracts were eluted with 1 ml acid:acetone (20% IN HC1:80%

acetone) with sonication. Samples were then evaporated to dryness and stored at -20 C










until assayed. Extracts were reconstituted with 0.25 ml 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.

Cortisol Assay Plasma cortisol concentrations were measured as previously described

(Wood et al., 1993) using an antibody raised in rabbits and titrated cortisol purchased

from Amersham (#TRK-407) and cortisol standard from Sigma Chemical Co. 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 0.5 ml 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 NaCl,

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

Statistical Analyses ACTH, AVP, and cortisol levels were analyzed via three way

ANOVA. Estradiol treated fetuses were analyzed separately from placebo treated fetuses

by this means using hypotension/normotension, CSD/intact, and time as factors. Student

Newman-Keuls Test was employed as a multiple comparison procedure for statistically

significant groups within each grouping. Estradiol treated fetuses were compared to

placebo treated fetuses relative to time and treatment group by a t-test. Estradiol level

comparison between estradiol treated fetuses and placebo treated fetuses was made using

a t-test.

Results

Estradiol Assay Since my hypotheses depend upon the a difference in estradiol levels

between treatment groups, it seems only logical to discuss the results of the estradiol assay










first. There were a total of 40 animals studied in this experiment. Half(20) were

pretreated with an estradiol implant during surgery. These implants release estradiol at a

constant rate of 5 mg/21 day period or around 250 utg/day. Estradiol levels were

measured by EIA and statistical analyses showed that the estradiol treated fetuses were

significantly different than the placebo treated fetuses by a t-test (n=20 per group,

p<0.001). Result can be seen in Figure 4.2 which shows group means measured in units

of pg/ml +SEM.

Adrenocorticotropin (ACTH) Assay Plasma ACTH levels are shown in Figure 4.3.

Results are plotted as group means +SEM. Placebo treated fetuses and estradiol fetuses

were analyzed separately by three way ANOVA with hypotension / normotension, carotid

sinus denervation / intact, and time (0min., 10min., 20min., and 60min.) as experimental

factors. In both cases (placebo and estradiol treatment), groups were found to be

statistically different (n=5 per group, p<0.001). Specifically, ACTH levels in the
t
hypotensive animals at 10min. and 20min. as well as hypotensive, carotid sinus denervated

animals at 10min. and 20min. were found to differ significantly from the rest of the

treatment groups when further analyzed by Student Newman Keuls Comparison (n=5 per

group, p<0.001). This was true for both placebo treated fetuses and estradiol treated

fetuses. Also, all placebo treated fetuses differed significantly from their corresponding

estradiol treated fetuses by t-test (n=5 per group, p<0.01).

Arginine Vasopressin (AVP) Assay Plasma AVP levels are shown in Figure 4.4. Results

are plotted as group means +SEM. Placebo treated fetuses and estradiol fetuses were

analyzed separately by three way ANOVA with hypotension / normotension, carotid sinus

denervation / intact, and time (Omin., 10min., 20min., and 60min.) as experimental factors.










In both cases (placebo and estradiol treatment), groups were found to be statistically

different (n=5 per group, p<0.001). Specifically, AVP levels in the hypotensive animals at

10min. and 20min. as well as hypotensive, carotid sinus denervated animals at 10min. and

20min. were found to differ significantly from the rest of the treatment groups when

further analyzed by Student Newman Keuls Comparison (n=5 per group, p<0.001). This

was true for both placebo treated fetuses and estradiol treated fetuses. Also, all placebo

treated fetuses differed significantly from their corresponding estradiol treated fetuses by

t-test (n=5 per group, p<0.01).

Cortisol Assay Plasma cortisol levels are shown in Figure 4.5. Results are plotted as

group means SEM. Placebo treated fetuses and estradiol fetuses were analyzed

separately by three way ANOVA with hypotension / normotension, carotid sinus

denervation / intact, and time (Omin., 10min., 20min., and 60min.) as experimental factors.

In both cases (placebo and estradiol treatment), groups were found to be statistically
/
different (n=5 per group, p<0.001). Specifically, cortisol levels in the hypotensive animals

at 10min. and 20min. as well as hypotensive, carotid sinus denervated animals at 10min.

and 20min. were found to differ significantly from the rest of the treatment groups when

further analyzed by Student Newman Keuls Comparison (n=5 per group, p<0.001). This

was true for both placebo treated fetuses and estradiol treated fetuses. Also, all placebo

treated fetuses differed significantly from their corresponding estradiol treated fetuses by

t-test (n=5 per group, p<0.01).

Discussion

Understanding the mechanism of the increased fetal HPA axis at the end of

gestation is key to understanding the mechanism of spontaneous parturition in sheep.










These experiments were conducted to see if estradiol has it's stimulatory effect on HPA

axis activity through the afferent baroreceptor and chemoreceptor pathway. More

specifically, I hypothesized that estradiol's actions on fetal cardiovascular reflex

responsiveness to hypotension will be measurable in intact fetuses but not in baro- and

chemo- denervated fetuses. This augmentation of HPA axis activity was assessed by using

a surgically manipulated ovine model. ACTH, AVP, estradiol, and cortisol levels were

measured and compared across eight different treatment groups. These groups include

fetuses which are: (1) estradiol treated; (2) estradiol treated, hypotensive; (3) estradiol

treated, carotid sinus denervated; (4) estradiol treated, carotid sinus denervated,

hypotensive; (5) placebo treated; (6) placebo treated, hypotensive; (7) placebo treated,

carotid sinus denervated; (8) placebo treated, carotid sinus denervated, hypotensive.

This study has shown that that estradiol does have an effect on cardiovascular

responsiveness. Figures 4.3-4.5 show that pretreatment with estradiol increases even
I
basal ACTH, AVP, and cortisol levels. This effect of estradiol is seen best perhaps after a

ten minute period of hypotension. At this point, hormonal responsiveness has been greatly

augmented with regard to ACTH, AVP, and cortisol levels. Carotid sinus denervation,

essentially eliminates this augmented HPA axis activity in response to brachiocephalic

occlusion. Any hormone response leftover after denervation may be due in part to

incomplete denervation at the time of surgery. Of course another explanation lies with the

fact that other pathways or systems might be responsible for hormone secretion. Carotid

sinus denervation by itself, however, caused no HPA axis activity or augmentation,

deeming the surgical elimination of baroreceptor / chemoreceptor responsiveness an

appropriate and effective control. Research has previously demonstrated that the










combined baro- and chemo- denervation attenuates (approximately 50%) the reflex

hormonal and hemodynamic responses to moderate (50 %) reduction in arterial blood

pressure. This study supports this finding demonstrated by the fact that ACTH, AVP, and

cortisol levels at 10min. and 20min. time points in hypotensive, carotid sinus denervated

animals are approximately half their corresponding levels with hypotension alone. At the

very least, it can be concluded that estrogen acts on, within, or requires input from, the

afferent baroreceptor and chemoreceptor pathways.

This study utilized an artificial stimulus for HPA axis activation. This taken with

the fact that the animals in this study are at about 85% gestation makes direct comparison

to the time of parturition impossible. However, this study is useful in that it sheds light on

how an ovine fetus responds to bought of hypotension. Furthermore, the effect of

estradiol on cardiovascular responsiveness has been shown to greatly augment HPA axis

activity in response to such hypotension as controlled through the baroreceptor /

chemoreceptor pathway. These are important findings because although this ovine model

done not exactly mimic the process of parturition, important conclusions can be made. All

of the animals in this study are at least at 125 days gestation. Before around 120 days

gestation, the HPA axis is fairly unresponsive to stimuli. The estradiol implants used in

this study raised plasma estradiol levels to within a physiological range. Also, plasma

estradiol levels were comparable to time just prior to parturition, when estradiol levels are

thought to activate the HPA axis. For these reasons, it is evident that the model system

used in this study is appropriate for deciphering not only fetal responsiveness to

cardiovascular stimuli, but also the mechanism of parturition itself










Though the results of this study elude to an estradiol responsive baroreceptor /

chemoreceptor afferent pathway participation in cardiovascular reflex control and trigger

for parturition, they do not by themselves answer account for total control over these

processes. As stated before, research has previously demonstrated that the combined

baro- and chemo- denervation attenuates (approximately 50%) the reflex hormonal and

hemodynamic responses to moderate (50 %) reduction in arterial blood pressure. This

means that 50% of the mechanism / pathway responsible for cardiovascular reflex

responsiveness and parturition is unaccounted for. It is plausible that the remaining

compensatory mechanisms exist within the central nervous system itself Since the

cerebral circulation of the fetus exhibits autoregulation, one could hypothesize that

hypotension would cause reduced cerebral blood flow. This reduced flow may be

defended against by a local release ofvasoactive substances. Prostanoids represent one

such group of compounds that may if fact be responsible for this response. Although it

has not been quantified at this time, our lab has shown that estradiol treatment does in fact

increase prostaglandin E2 (PGE2) within areas of the brain that are responsible for HPA

axis control. Other studies have eluded to a role of thromboxane A2 (TxA2) in this

process. TxA2 acts within the area perfused by the cerebral vasculature to stimulate

ACTH secretion in the fetus (Wood et al., 1993), which suggests that local generation of

TxA2 would effectively stimulate the HPA axis. Whatever the mechanism, it is obvious

that further research needs to be done in order to fully understand the inner-workings of

this complex system.













Blood sampling time points






Omin. 1 Omin. 20min.


Hypotension
(Brachiocephalic Occlusion)


Euthanize and
perfuse brain
(Prepare for
Immunohistochemistry)


Figure 4.1: Experimental design for in vivo studies. 5ml blood samples are taken at
0min., lOmin., 20min., and 60min.(additional blood is taken at 0min., 10min., and
20min. for blood gas and hematocrit measurement). Hypotension via brachiocephalic
occlusion takes place between 0min. and 10min. Blood pressure and heart rate are
recorded for the first 35min. of the experiment. Animals are sacrificed at 60min. and
brains are extracted for immunohistochemistry.


60min.






































Estradiol treated
fetuses


Placebo treated
fetuses


Figure 4.2: Estradiol levels +SEM for fetuses treated with a 5 mg/21day estradiol
implant and fetuses treated with a placebo implant. The group means are
significantly different (n=20 per group, p<0.001).







1600 -

1400 -

1200 -

1000 -
-SD
S800 -

G 600-

400 -

200 -

0 -


1600 -

1400 -

1200 -

S1000 -

S800-

S600-
.<

400 -

200 -

0-


0 10 20 30 40 50 60


Estradiol treated fetuses


-Control
I IHypotensive
- CSD
-- Hypo/CSD


fI I


II


I~I


Time (minutes)



Figure 4.3: ACTH plasma levels plotted as group means +SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p<0.001). All estradiol groups
were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).


111 i1l i I I r, i


III I







Placebo treated fetuses


- Control
' IHypotensive
- CSD
aHypo/CSD


in i I


+ *

jA ,HEr


.non


I I I I I
0 10 20 30 40 50 60
60
Estradiol treated fetuses Control
50 I Hypotensive
SCSD
----I Hypo/CSD
40 -

30 + *

20 -



': oii ____________
0 10 20 30 40 50 60
Time (minutes)


Figure 4.4: AVP plasma levels plotted as group means +SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).








Placebo treated fetuses


M Control
Zi Hypotensive
- CSD
Hypo/CSD


*
15
+
10 +

5

0
0 10 20


InI
60


0 10 20 30 40 50 60


Time (minutes)


Figure 4.5: Cortisol plasma levels plotted as group means +SEM. Top graph
shows placebo treated fetuses and bottom graph shows estradiol treated fetuses.
*,+ denotes statistical significance (n=5 per group, p<0.001). All estradiol groups
were significantly different from placebo groups relative to treatment and time
(n=5 per group, p<0.01).


30 -

25 -

20 -


I














CHAPTER 5
NEURONAL ACTIVATION IN AN ESTRADIOL, HYPOTENSIVE, CAROTID
SINUS DENERVATED OVINE MODEL


Introduction

Parturition in the sheep has been shown to be controlled by the fetal HPA axis

(Challis and Brooks, 1989; Liggins et al., 1973). The last few days of gestation are

marked by an increase in the activity of the fetal hypothalamus as seen by elevated levels

of fetal plasma ACTH. This increase in plasma ACTH causes a corresponding increase in

plasma cortisol. Along with this increase in HPA axis activity is a decreased sensitivity of

the axis to cortisol negative feedback (Wood, 1988). It is well known that in the sheep,

cortisol acts at the placenta to increase the activity of an enzyme, cytochrome p450 (17-

hydroxylase and 17,20 lyase activities), which in turn, increases the ratio of estrogen to

progesterone (Anderson et al., 1975; Pomeranz and Nalbandov, 1975; Steele et al., 1976;

Yu et al., 1983). This cascade of events essentially increases the total amount of estrogen.

Estrogen is known to be an important factor in the initiation of parturition by

causing the uterus to contract (Liggins, 1974). 1703-estradiol has been shown to increase

the activity of the HPA axis in sheep and rats. In a study by Saoud and Wood (1995),

estrogen was found to augment fetal plasma ACTH secretion in response to stress. Other

studies have shown similar results in adult animals (Viau and Meaney, 1991).

Understanding the mechanism of the increased fetal HPA axis at the end of

gestation is key to understanding the mechanism of spontaneous parturition in sheep.








These experiments were conducted to see at what point in the HPA axis that estradiol has

in augmenting ACTH secretion. More specifically, I hypothesized that neuronal activity

will be highest in areas important for HPA axis control in estradiol treated, hypotensive

animals (see Chapter 4). I hypothesized that the baroreceptor / chemoreceptor afferent

pathway is involved, thus, carotid sinus denervation will eliminate the augmented HPA

axis activity. Neuronal activity was assessed by measuring the level of c-fos, any early

response gene, in brain areas important for HPA axis control. This method has been used

in numerous studies to assess neuronal activation due to physiological stress (Hoffinan et

al., 1991; Shen et al., 1992; Chan et al., 1993).

Methods and Materials

Immunohistochemical Techniques Fetal ovine brains were perfusion fixed with 4%

paraformaldehyde, dissected and cut into gross tissue regions (hypothalamus, midbrain,

pons, medulla, spinal cord, etc.). These brains were the obtained from the experiments

discussed in Chapter 4. All animal were euthanized via an overdose of'sodium

pentobarbital one hour after the beginning of the experiment (50 minutes after a ten

minute hypotensive or corresponding normotensive period). There were a total of 24 fetal

brains used for the histological experiments (n=3 per group). The fetal ovine groups were

as followed: (1) estradiol treated; (2) estradiol treated, hypotensive; (3) estradiol treated,

carotid sinus denervated; (4) estradiol treated, carotid sinus denervated, hypotensive; (5)

placebo treated; (6) placebo treated, hypotensive; (7) placebo treated, carotid sinus

denervated; (8) placebo treated, carotid sinus denervated, hypotensive. Tissue was

processed for embedding by dehydration with progressively increasing concentrations of

ethanol, followed by xylene. All tissue was embedded in paraffin and cut into 10 pm










sections using a Zeiss microtome. Sections were mounted on poly-L-lysine slides,

deparaffinized with xylene and rehydrated in decreasing concentrations of ethanol.

Immunohistochemistry and visualization were made possible utilizing a Histostain-SP kit

from Zymed and metal-enhanced DAB (Pierce, Rockford, IL). Sections were stained for

c-fos, ACTH, AVP, and CRH (see Table 3.1). Primary antibodies were diluted in

antibody diluent (1% BSA in phosphate buffered saline with 0.01% Triton X-100).

Specific staining was confirmed by dilution tests, as staining decreased as primary

antibodies were further diluted. Specific staining was absent upon replacing primary

antibodies with 10%/ normal goat serum. All slides were dehydrated prior to mounting

coverslips with Permount (Fisher Scientific, Pittsburgh, PA).

Fetal brains regions important in HPA axis control were measured for c-fos

generation by means of Microcomputer Imaging Device (MCID) from Imagining Research

Inc. Cerebellum and cortex were also measured to verify if c-fos activity was specific or

just a general activation of the central nervous system. The following brain regions were

analyzed: (1) paraventricular nucleus (PVN), (2) nucleus of the tractus solitarius (NTS),

(3) rostral ventral lateral medulla (RVLM), (4) hippocampus, (5) cerebellum, (6) cortex,

and (7) pituitary gland. For each of the eight treatment groups (listed in the Materials and

Methods section), n=3. As paraffin blocks were cut on the microtome, the fifth section

was used once the region of interest was identified. This assured homogeneity among

fetal brains.

All densitometry was performed in a similar mannor. The region of interest was

outlined designating the scanned area. The computer would then count the number of

cells stained positive depending upon the assigned criteria. This value or target number










was multiplied by the mean target area (calculated by the computer) to establish the total

target area. Finally, the total target area was divided by the scanned area to establish the

proportion of positive stained cells in each brain section. Values were then analyzed via

three way Analysis of Variance (ANOVA) followed by a multiple comparison procedure.

The multiple comparison procedure employed was Student-Newman-Keuls Method. All

statistics were run using Sigma Stat.

Results

PVN C-fos staining was found to be significantly different among the eight treatment

groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values

+SEM are plotted in Figure 5.1. A further analysis of the data revealed statistically

significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.

normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method

revealed-the following significant differences among treatment groups: '(1) mean values of

c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated

fetuses was significantly different (n=3 per group, p<0.001), and (2) mean values of c-fos

staining among the different levels of hypotensive fetuses vs. normotensive fetuses was

significantly different (n=3 per group, p<0.001). Representative photomicrographs of the

PVN are shown in Figure 5.2. It can be seen visually that estradiol treated fetuses have

more c-fos generation in the PVN when compared to control fetuses (panels A and B).

Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses

compared to placebo treated, hypotensive fetuses (panels C and D). As stated before

hypotensive animals also have more c-fos generation compared to normotensive animals

(panels A and C).










Carotid sinus denervation by itself did not cause significant c-fos generation. This

is show in Figure 5.3 which shows a representative estradiol treated fetus along with a

representative estradiol, carotid sinus denervated fetus. The region shown is the PVN and

visually, as well as statistically, there is no difference in the amount of staining. Figure 5.4

shows the effect of carotid sinus denervation on hypotension. The PVN of a

representative placebo, hypotensive fetus along with a representative placebo,

hypotensive, carotid sinus denervated fetus. Photomicrographs show that denervation

eliminates the effect of hypotension. These effects of carotid sinus denervation were true

of all brain regions studied.

Along with c-fos staining in the PVN, serial sections were also stained for AVP,

and CRF. Though statistics were not performed for the relative levels of these hormones,

the pattern of staining seemed to be consistent with that of c-fos. Not only was the

staining of AVP and CRF co-localized with c-fos, but also the levels of these hormones
t
seemed to increase with estradiol treatment as well as in hypotensive animals.

NTS C-fos staining was found to be significantly different among the eight treatment

groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values

+SEM are plotted in Figure 5.5. A further analysis of the data revealed statistically

significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.

normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method

revealed the following significant differences among treatment groups: (1) mean values of

c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated

fetuses was significantly different (n=3 per group, p<0.001), and (2) mean values of c-fos

staining among the different levels of hypotensive fetuses vs. normotensive fetuses was










significantly different (n=3 per group, p<0.001. Representative photomicrographs of the

NTS are shown in Figure 5.6. It can be seen visually that estradiol treated fetuses have

more c-fos generation in the NTS when compared to control fetuses (panels A and B).

Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses

compared to placebo treated, hypotensive fetuses (panels C and D). As stated before

hypotensive animals also have more c-fos generation compared to normotensive animals

(panels A and C). These results coincide with those of the PVN.

RVLM C-fos staining was found to be significantly different among the eight treatment

groups by three way ANOVA (n-3 per group, p<0.001). The mean densitometry values

+SEM are plotted in Figure 5.7. A further analysis of the data revealed statistically

significant interactions between all three factors (placebo vs. estradiol, hypotensive vs.

normotensive, and carotid sinus denervated vs. intact). Student Newman Keuls Method

revealed, the following significant differences among treatment groups: (1) mean values of

c-fos staining among the different levels of placebo treated fetuses vs. estradiol treated

fetuses was significantly different (n=3 per group, p<0.001), (2) mean values of c-fos

staining among the different levels of hypotensive fetuses vs. normotensive fetuses was

significantly different (n=3 per group, p<0.001. Representative photomicrographs of the

RVLM are shown in Figure 5.8. It can be seen visually that estradiol treated fetuses have

more c-fos generation in the RVLM when compared to control fetuses (panels A and B).

Also shown is significantly more positive staining in estradiol treated, hypotensive fetuses

compared to placebo treated, hypotensive fetuses (panels C and D). As stated before

hypotensive animals also have more c-fos generation compared to normotensive animals

(panels A and C). These results coincide with those of the PVN and the NTS.











Hippocampus C-fos staining was found to be significantly different among the eight

treatment groups by three way ANOVA (n-3 per group, p<0.001). The mean

densitometry values +SEM are plotted in Figure 5.9. Note that the y axis is smaller than

that of the PVN, etc. because the level ofc-fos staining is lower in the hippocampus.

Student-Newman-Keuls Method revealed the that the mean values of c-fos staining among

the different levels ofhypotensive fetuses vs. normotensive fetuses was significantly

different (n=3 per group, p<0.001). However, mean values of c-fos staining among the

different levels of placebo treated fetuses vs. estradiol treated fetuses was not significantly

different (n=3 per group, p=0.407). Representative photomicrographs of the

hippocampus are shown in Figure 5.10. It can be seen visually that hypotensive fetuses

have more c-fos generation in the hippocampus when compared to normotensive fetuses

(panels A and B). These results resemble those of the PVN, NTS, and RVLM with regard

to the effect of hypotension, however, the absence of an estradiol effect is novel.
n
Cerebellum Cerbellum was analyzed as a peripheral tissue that does not integration in the

system being studied. As suspected, analysis by three way ANOVA did not yield any

differences among treatment groups (n=3 per group, p=0.721). Mean group values

+SEM of the densitometry analysis are shown in Figure 5.11. Note that the y axis is much

smaller than that of the PVN, etc. because of the absence of any significant staining.

Representative photomicrographs of fetal ovine cerebellum are shown in Figure 5.12.

Panel A shows a control animal while panel B shows a hypotensive animal. Visually, as

well as statistically, there is no difference. Though not shown pictorially, the same is true

of estradiol treated fetuses vs. placebo treated fetuses.










Cortex As the case with cerebellum, cortex was analyzed as a peripheral tissue that does

not integration in the system being studied. As suspected, analysis by three way ANOVA

did not yield any differences among treatment groups (n=3 per group, p=0.399). Mean

group values +SEM of the densitometry analysis are shown in Figure 5.13. Note that the

y axis is much smaller than that of the PVN, etc. because of the absence of any significant

staining. Representative photomicrographs of fetal ovine cortex are shown in Figure 5.14.

Panel A shows a control animal while panel B shows a hypotensive animal. Visually, as

well as statistically, there is no difference. Though not shown pictorially, the same is true

ofestradiol treated fetuses vs. placebo treated fetuses.

Pituitary gland Pituitary glands were stained for c-fos as well as ACTH and AVP.

Though statistics were not performed for the relative levels of these hormones, the pattern

of staining seemed to be consistent with that of the PVN, NTS, and RVLM. Not only was

the c-fos increased with estradiol treatment and hypotension, but the levels of ACTH and

AVP seemed to mimic the pattern and level of c-fos staining. ACTH was seen in the

anterior pituitary in and increased with both estradiol treatment and hypotension. AVP

was seen mostly in the posterior pituitary in areas of increased c-fos generation.

Discussion

Understanding the mechanism of the increased fetal HPA axis at the end of

gestation is key to understanding the mechanism of spontaneous parturition in sheep.

These experiments were conducted to see at what point in the HPA axis that estradiol has

in augmenting ACTH secretion. More specifically, I hypothesized that neuronal activity

will be highest in areas important for HPA axis control in estradiol treated, hypotensive

animals (see Chapter 4). I hypothesized that the baroreceptor / chemoreceptor afferent










pathway is involved, thus, carotid sinus denervation will eliminate the augmented HPA

axis activity. Neuronal activity was assessed by measuring the level of c-fos, any early

response gene, in brain areas important for HPA axis control. This method has been used

in numerous studies to assess neuronal activation due to physiological stress (Hoffmnan et

al., 1991; Shen et al., 1992; Chan et al., 1993).

The results of this study support to my original hypothesis. Estradiol augments

HPA axis activity through baroreceptor / chemoreceptor pathways and this augmentation

seems to be within brain regions important for HPA axis activity. Furthermore, the

elimination of increased neuronal activity in carotid sinus denervated animals shows that

the pathway involved is probably this central cardiovascular reflex pathway.

The immunohistochemistry results show that the PVN, NTS, and RVLM respond

similarly with regard to specific treatments. This finding comes as no surprise as all of

these areas are involved in HPA axis control. The PVN is the main collection of neuronal
f
cell bodies in the hypothalamus where CRH and AVP are synthesized and released to act

at the anterior pituitary to cause ACTH release (Lehman et al., 1993; Pomerantz and

Sholl, 1987). The NTS is the first synapse point in the pathway connecting the afferent

baroreceptors with the PVN. The RVLM is a cardiovascular regulatory center which

coordinates information from the periphery. It has been shown that all of these brain

regions have estrogen receptors, enabling them to respond to estradiol treatment prior to

brachiocephalic occlusion (Lehman et al., 1993; Simerly et al., 1990). Estradiol caused a

significant increase in neuronal activity in the PVN, NTS, and RVLM. In fetuses made

hypotensive via brachiocephalic occlusion, this effect was further augmented as shown by










statistical analyses. Carotid sinus denervation eliminated increases in c-fos activity in these

HPA axis regulatory centers.

An interesting finding of this study was the absence of an estradiol effect in the

hippocampus. Like the PVN, NTS, and RVLM, an increase in c-fos activity was seen in

hypotensive animals and this effect was eliminated with carotid sinus denervation.

However, basal levels as well as stressed levels of neuronal activity in estradiol treated

animals compared to placebo treated animals did not differ statistically. This reveals that

estradiol has no effect at the level of the hippocampus in augmenting HPA axis activity in

response to hypotension. This is interesting because there have been estrogen receptors

reported within the hippocampus (Lehman et al., 1993; Simerly et al., 1990). Perhaps the

answer to this discrepancy lies in fact that the hippocampus is a classic site of cortisol

negative feedback. Wood has further shown that the HPA axis is insensitive to inhibition

via cortisol negative feedback towards the end of gestation (1987). Possibly, the

hippocampus is unresponsive to an estradiol augmentation of ACTH secretion at this time.

Of course, this may be in the best interest of the fetus since an increase in neuronal activity

at the level of the hippocampus may lead to inhibition rather than stimulation of the axis.

It was certainly encouraging that in peripheral brain regions, an increase in

neuronal activity was observed in neither estradiol treated nor estradiol treated,

hypotensive fetuses. This lack of c-fos staining in the cortex and cerebellum indicate that

the response to treatment within the CNS was specific. All things being equal, only brain

regions important in HPA axis control seemed to respond to estradiol treatment and

brachiocephalic occlusion.










Pituitary glands were stained for c-fos as well as ACTH and AVP. Though

statistics were not performed for the relative levels of these hormones, the pattern of

staining seemed to be consistent with that of the PVN, NTS, and RVLM. Not only was

the c-fos increased with estradiol treatment and hypotension, but the levels of ACTH and

AVP seemed to mimic the pattern and level of c-fos staining. ACTH was seen in the

anterior pituitary in and increased with both estradiol treatment and hypotension. AVP

was seen mostly in the posterior pituitary in areas of increased c-fos generation. All of

this information fits with my hypothesis. One would expect to see an increase in staining

in these areas within the pituitary with both estradiol treatment and hypotension. There

are estrogen receptors within the pituitary and an increase in activity of the pituitary would

be logical during hypotension. It is possible that ACTH and AVP synthesis is increased

during estradiol treatment. This would also be true in the normal fetus towards the end of

gestation when estradiol is increasing. If these hormone levels are in fact increasing, The

fetus would be able to better respond to episodes of hypotension as well as trigger

parturition with an augmented ACTH and AVP response.

I have reported that within the PVN, NTS, RVLM, hippocampus, and pituitary of

fetal sheep, an in crease in c-fos activity is observed with estradiol treatment and

brachiocephalic occlusion. It may seem logical that rather than an increase in neuronal

activity, a decrease in c-fos staining at the level of the NTS would accompany

hypotension. Hypotension causes a decrease in the rate of firing of the baroreceptors, and

thus a decrease in signal at the NTS. Perhaps what is being observed here is a

chemoreceptor mediated effect. As the brachiocephalic artery is occluded, the blood

perfusing the head becomes acidotic and hypoxic. This is revealed through elevated levels











of hydrogen ions, elevated partial pressure of carbon dioxide (PCO2), and a decrease in the

partial pressure pressure of oxygen (PO,) in the blood. All of these factors were in fact

monitored throughout the duration of the experiment. I believe that the fetal PO2 level is

strictly monitored in the carotid artery and in a state of hypotension, the compensatory

response is an increase in HPA axis activity in order to re-establish adequate blood flow to

the brain. This will have the inevitable response of bringing blood gas levels back to a

homeostatic level.

It is important to realize that the HPA axis is not only integral in controlling blood

pressure, but it is also the endocrine axis of parturition. This is a fact which has been

proven many times without actual confirmation of a precise mechanism. This study sheds

light on the internal working of this system. I have shown that an intact afferent

baroreceptor / chemoreceptor pathway is necessary for an ovine fetus to respond to a state

of hypotension. I have further shown that estradiol augments this response through this
t
cardiovascular reflex pathway. More precisely, I have shown through the utilization of

immunohistochemistry, the brain areas involved in this process. This study used an

artificial stimulus to increase HPA axis activity, however, it is not disputed that the

pathway described is in fact involved in triggering parturition.












0.08







+,+









20.04



0.03



0.02



0.01









Figure 5.1: C-fos immunohistochemistry staining in the fetal ovine paraventricular
nucleus (Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated). There is a significant difference between Plac vs. E2
(*, p<0.001) and Control vs. Hypo (+, p<0.001).






82









A\ I ~; <"" *-*




,
























nucleus (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol treated,
hypotensive). All photomicrographs are at a magnification of 40X.






83





A





I









B


4J
b i

It





















Figure 5.3: Photomicrographs of the fetal ovine PVN comparing c-fos staining in an
intact estradiol treated fetus (A) and an estradiol treated, carotid sinus denervated
fetus (B). A and B were not statistically different showing that denervation alone did
not cause c-fos generation.
















































Figure 5.4: Photomicrographs of the fetal ovine PVN comparing c-fos staining in a
hypotensive fetus (A) and a hypotensive, carotid sinus denervated fetus (B). A and B
were statistically different (p<001) showing that denervation diminished c-fos
generation in hypotensive animals.










0.08 -



0.07


0.06*+
0.06 -


0.05 -



0.04 -



0.03 -


0.02 -



0.01 -



0.00 -


0


V\CW


I ~ -T
~f~VO


Figure 5.5: C-fos immunohistochemistry staining in the fetal ovine nucleus
of the tractus solitarius (Plac= placebo implant, E2= estradiol implant, Hypo=
hypotensive, CSD=-- carotid sinus denervated). There is a significant difference
between Plac vs. E2 (*, p<0.001) and Control vs. Hypo (+, p<0.001).


V0\


















*0






q.


n




*b


B












em,





& 4
5'
-a


*1k


S.

4 .*


Figure 5.6: C-fos immunohistochemistry staining in the fetal ovine nucleus of the
tractus solitarius (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol
treated, hypotensive). All photomicrographs are at a magnification of 100X.


I.
St
a-
S


A


Am










0.08



0.07



0.06


0.05 -



0.04 -



0.03 -


0.02 -



0.01 -



0.00-


CH ',


Figure 5.7: C-fos immunohistochemistry staining in the fetal ovine rostral
ventral lateral medulla (Plac= placebo implant, E2= estradiol implant, Hypo=
hypotensive, CSD= carotid sinus denervated). There is a significant difference
between Plac vs. E2 (*, p<0.001) and Control vs. Hypo (+, p<0.001).








































Figure 5.8: C-fos immunohistochemistry staining in the fetal ovine rostral ventral
lateral medulla (A- Control; B- Estradiol treated; C- Hypotensive; D- Estradiol treated,
hypotensive). All photomicrographs are at a magnification of 100X.


A B







C D.v ^-^
..*.., A'
"- *..
: i,-
A ,, I, D d "
aI I










0.040



0.035 -



0.030 -

CC

-. 0.025 -
S+
U +

o 0.020



0.015



0.010



0.005



0.000 -





Figure 5.9: C-fos immunohistochemistry staining in the fetal ovine hippocampus
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD= carotid
sinus denervated). There is a significant difference between Control vs. Hypo
(+, p







90














i' .'. ;'. ?' >*', '*


*,. > .,. , ,.
.... . . .. . .








.,N .. ,





































Figure 5.10: C-fos immunohistochemistry staining in the fetal ovine hippocampus
(A- Control; B- Hypotensive). All photomicrographs are at a magnification of 40X.













0.0020


0.0018 -


0.0016 -


0.0014 -


0.0012
0













"\c *^*
C 0.0010


0.0008
0

0.0006


0.0004


0.0002


0.0000





Figure 5.11: C-fos immunohistochemistry staining in the fetal ovine cerebellum
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated).




















































Figure 5.12: C-fos immunohistochemistry staining in the fetal ovine cerebellum
(A- Control; B- Hypotensive). All photomicrographs are at a magnification of 40X.
A and B are not statistically different.














0.0014 -

0.0012 -

0.0010 -

0.0008 -

0.0006 -

0.0004 -

0.0002 -

0.0000 -


,s~# ~c~0\C5~'9


Figure 5.13: C-fos immunohistochemistry staining in the fetal ovine cortex
(Plac= placebo implant, E2= estradiol implant, Hypo= hypotensive, CSD=
carotid sinus denervated).


0.0020 -

0.0018 -

0.0016 -


IL
p 0


IL.1
I > --r