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This is to certify that the

thesis entitled

STUDIES ON THE HYPOTHALAMIC REGULATION
OF FOLLICLE-STIMULATING HORMONE RELEASE

presented by

James C. Mittler

has been accepted towards fulfillment
of the requirements for

PhoDo degree in PhXSiOIOQX

AWK/7 2147/
or professor

Date July 6, I966

 

0-169

 

 

 

 

ABSTRACT.

STUDIES ON THE HYPOTHALAMIC REGULATION OF

FOLLICLE-STIMULATING HORMONE RELEASE

by James C. Mittler

The regulation of adenohypophysial function by the
central nervous system has been shown to be by neurohumoral
rather than by direct nervous pathwayso Neurohumoral cells
in the hypothalamus liberate chemical mediators which are
conveyed by the hypothalamo-hypophysial portal blood to the
anterior pituitary, where they influence secretion of anterior
pituitary hormones. In early 1963, when the work reported
in this thesis was begun, some evidence had already been
reported that hypothalamic extracts contained substances
which stimulate synthesis or release of pituitary ACTH, TSH,
LH, and STH, and also inhibited synthesis and release of
prolactino Direct evidence for a substance with stimulatory
action on secretion of follicle—stimulating hormone (FSH)
however, seemed scanty and unconvincing, although there was
abundant circumstantial evidence for the existence of such
a substance in the hypothalamuso The search for an FSH-
releasing factor (FSH-RF) seemed of interest and of practical

importance°

James C. Mittler

FSH secretion is known to be regulated by feedback
mechanisms involving gonadal steroid hormones, possibly
acting through the hypothalamus, or directly on the pituitary.
or at both sites. The site(s) of action of steroid hormones
is currently the subject of much controversy. Study of
pituitary tissue ig_yit£g seemed to be the ideal method for
demonstrating the existence of an FSH-RF in the hypothalamus
and for localizing the site(s) of action(s) of steroid
hormones.

Explants of rat anterior pituitary tissue were found
to release significant amounts of FSH only during the first
3 days of a 9-day culture period. Cultures were performed
at 37°C in tissue culture medium 199 containing 10% calf
serum and under an atmosphere of 95% O2 - 5% C02. Hypo-
thalamic stimulation of FSH release was demonstrated by
means of paired cultures from opposite halves of 72 adult
female anterior pituitaries. At the end of the 3rd and
6th days of culture, the medium was removed. Fresh medium
containing neutralized 0.1N. HCl acid extract of rat hypo-
thalamus (including pituitary stalk and median eminence) was
added to one-half of the culture dishes and medium containing
similarly prepared cerebral cortical extract was added to
the remainder. Each pituitary equivalent was cultured with
an equivalent of 4 hypothalami from ovariectomized female
rats. Brain extracts were not added during the first 3 days
of culture. After termination of culture, the medium was

assayed by the method of Steelman and Pohley using medium

James C. Mittler

equivalent to 2 pituitaries and 50 I.U. of HCG per assay
rat. Medium from the first 3 days of culture was assayed
separately. Pituitary tissue cultured with cerebral corti-
cal extract (control) did not release significant amounts

of FSH, whereas pituitary cultured with hypothalamic extract
(experimental) released highly significant amounts of FSH

(p < .001).

During a 6-hour incubation period, neutralized
acidic extracts of rat hypothalamic tissue stimulated re—
lease of FSH from adult male rat pituitary tissue. A
graded response was observed in release of FSH by pituitary
tissue in response to doses of extract ranging from l/32
to 1/4 hypothalamic equivalent per incubated pituitary.

Castration was found to increase and testosterone
proponate injections (2 mg/day) to reduce the FSH—releasing
activity of hypothalamic extracts.

Brain extracts had no effect upon the activity of
purified FSH Lg vitro in tissue culture or 6-hour incubation,
nor when injected together with FSH into assay rats.

Testosterone (l ugm/ml), estradiol (0.25 ugm/ml),
progesterone (l ugm/ml) and the last two in combination
failed to inhibit FSH release in tissue culture. Estradiol
failed to antagonize the stimulatory effects of hypothalamic
extract in both tissue culture and in incubation experiments.
Testosterone failed to antagonize hypothalamic extract
during 6-hour incubation experiments. Steroids were added

to media dissolved in a minute volume of absolute ethanol.

James C. Mittler

An appendix describes studies on the hypothalamic
Content of prolactin inhibiting factor (PIF). Epinephrine
in oil (0.25 mg) and acetylcholine bromide (25 mg per kilogram
body weight) were found to decrease the amount of PIF in
the hypothalamus. Both treatments were administered twice
daily and one hour before removing the hypothalami. Hypo-

physectomy had no effect on PIF content.

STUDIES ON THE HYPOTHALAMIC REGULATION OF

FOLLICLE-STIMULATING HORMONE RELEASE

BY

\" ‘\
._ '0
0x

James C? Mittler

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physiology

1966

ACKNOWLEDGEME NTS

The author wishes to express his gratitude to Dr.
Joseph Meites, Professor in the Department of Physiology,
for his guidance, advice, and assistance throughout the
course of this study. He also wishes to express his appre-
ciation to Miss Barbara Brace, Mrs. S. Polityka, Dr. Donald
Schmidt and Dr. Theodore Staley for invaluable technical
assistance; to the Michigan Agricultural Experiment Station
and the Michigan Cancer Foundation, for funds: to the
Endocrinology Study Section, Nationa1.Instituteswmf Health,
for funds and for generous supplies of FSH-Sl and FSH—$2:
and to Mr. Merlyn Swab and Mr. Richard Kennedy for attending

to the experimental animals.

ii

Chapter
I.
II.

III.

IV.

VI.

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . .
REVIEW OF LITERATURE . . . . . . . . . . . .
TISSUE CULTURE EXPERIMENTS ON FSH RELEASE .
A. Materials and Methods
B. Results on Release of FSH lg vitro

C. Stimulation of FSH Release by
Hypothalamic Extract jg vitro

D. Effects of Steroid Hormones on FSH
Release in vitro

INCUBATION EXPERIMENTS ON FSH RELEASE . . .
A. Materials and Methods

B. Assay of FSH-RF Activity by 6-
hour Incubation Technique

C. Effects of Castration and
Androgen Treatment on Hypo-
thalamic FSH-RF Content

D. Additional Control Experiments

GENERAL DISCUSSION . . . . . . . . . . . . .

REFERENCES . . . . . . . . . . . . . . . . .

iii

Page

21
21

24

26

37
44

44

48

53
58
64

69

LIST OF TABLES
Table Page

1. Effects of acid extracts of rat cerebral

cortical extract and hypothalamus on

FSH—release by rat pituitary ig.yiggg . . . 29
2. Effects of incubating FSH with and without

brain extracts . . . . . . . . . . . . . . 34
3. Effects of injections of hypothalamic and

cerebral cortical extracts on response

of assay animals to FSH . . . . . . . . . . 34
4. Effects of steroids added directly to the

medium on FSH release in vitro . . . . . . 39
5. Effects of acid extracts of rat cerebral

cortex and hypothalamus on FSH release

by adult male rat pituitary 13.21EEQ . . . 50
6. Effects of treating hypothalamus donors on

FSH-RF activity . . . . . . . . . . . . . . 54
7. Effects of adding steroids to media together

with hypothalamic extract on FSH release

in yitgg . . . . . . . . . . . . . . . . . 59
8. Effects of incubating NIH-FSH-SZ with and

without brain extracts . . . . . . . . . . 61
9. Effects of injections of steroids and brain

extracts on the response of assay

animals to FSH . . . . . . . . . . . . . . 63

iv

LIST OF FIGURES

Figure Page
1. Release of FSH by Pituitary Tissue In Vitro
In Relation to Time . . . . . . . . . . . . 25
2. Dose-Response Curves: Tissue Culture Medium

FSH and NIH-FSH-Sl . . . . . . . . . . . . 27

3. Photomicrograph of Section of Uncultured
Female Rat Pars Anterior Tissue . . . . . . 31
4. Photomicrograph of Section of Female Rat

Pars Anterior Tissue Cultured for 9 Days . 32
5. Effects of Graded Doses of Hypothalamic Extract
on FSH Release by Pituitary Tissue

In Vitro . . . . . . . . . . . . . . . . . 51

LIST OF APPENDICES

Appendix
I. STUDIES ON PROLACTIN INHIBITING FACTOR
(PIFﬁ . . . . . . . . . . . . .. . . .
TABLE 1A: EFFECTS OF TREATMENTS ON
HYPOTHALAMIC PIF CONTENT . . . . .
FIGURE 1A: EFFECTS OF GRADED DOSES OF
HYPOTHALAMIC EXTRACT ON PITUITARY

PROLACTIN RELEASE . . . . . . . .

II. LIST OF PUBLICATIONS BASED ON WORK

PRESENTED IN THIS THESIS . . . . . . .

vi

Page

99

102

103

108

I. INTRODUCTION

Much research in the past few decades has been
devoted to elucidating the function of the anterior pituitary
gland. It has recently become apparent that the functions
of the anterior pituitary are regulated to a major extent
by the central nervous system. The portion of the central
nervous system most closely associated with control of
anterior pituitary function is the hypothalamus.

The control of the adenohypophysis by the central
nervous system has been shown to be by neurohumoral rather
than by direct nervous means. Neurohumoral cells in the
hypothalamus liberate chemical mediators which are conveyed
by the hypothalamo-hypophysial portal blood to the anterior
pituitary, where they influence secretion of anterior pitui—
tary hormones. In early 1963, when the work reported in
this thesis was begun, some evidence had already been re-
ported that hypothalamic extracts contained substances which
stimulate synthesis and release of pituitary ACTH, TSH,

LH, and STH and also inhibit synthesis and release of pro—
lactin. Direct evidence for a substance with stimulatory
action on secretion of follicle—stimulating hormone (FSH)
however, seemed scanty and unconvincing, although there was
abundant circumstantial evidence for the existence of such

a substance in the hypothalmus. The search for an

p4-

FSH-releasing factor (FSH—RF) seemed of theoretical interest
and also of practical importance.

FSH secretion is known to be regulated by feedback
mechanisms involving gonadal steroid hormones, possibly
acting through the hypothalamus, or directly on the pitui-
tary, or at both sites. The site(s) of this action of
steroid hormones is currently the subject of much contro-
versy. Study of pituitary tissue 13 vitro seemed to be the
ideal method for demonstrating the existence of an FSH-RF

in the hypothalamus and for localizing the site(s) of action(s)

of steroid hormones.

(1)

‘01

.(P

II. REVIEW OF LITERATURE

Neural control of the secretions of the anterior
pituitary, first proposed by HOhlweg and Junkmann (1932),
is now well established. Harris (1948, 1955, 1960) reviewed
evidence that release of hormones is influenced by the hypo-
thalamus and that the mechanism of control is neurohumoral
by way of the hypothalamo-hypophysial portal system.
Szentagothai et al. (1962) and Nalbandov, ed. (1963) have
reviewed more recent literature.

Benoit and Assenmacher (1953, 1955) have published
extensive reviews of the enormous clinical and experimental
literature on neural regulation of reproductive functions
of the anterior pituitary. More recent, specialized reviews
are those of Everett (1964) and Bogdanove (1964). Meites,
et al. (1963, 1966) reviewed the work on neural regulation
of prolactin secretion. Greep (1963) reviewed anatomical
relationships of the brain and pituitary and the complex
vascular connections between them. Some of the more important

and more recent findings will now be discussed.

1. Evidence from Effects of Brain Lesions

 

Atrophy of the gonads and different parts of the
genital tract after electrolytic lesions involving the median

eminence and adjacent parts of the basal tuberal region of

the hypothalamus has been reported in both sexes in the rat
(Bogdanove and Halmi, 1953; Bogdanove et al., 1957;
Bogdanove, 1957; Slusher, 1958; D'Angelo, 1959, and Taleisnik
and McCann, 1961). The basal tuberal region of the hypo—
thalamus may be defined as including the pituitary stalk,
median eminence, arcuate nucleus, and ventromedial nucleus.
D'Angelo (1959), Bogdanove and Schoen (1959), and Lisk (1960)
found that lesions which completely destroy the arcuate
nucleus result in gonadal atrophy in male and female rats.
Corbin (1963) found that bilateral lesions of the ventro-
medial nucleus-mammilary body area result in atrophy of
testes, seminal vesicles,tand ventral prostates in male rats.
Similar findings have been reported for the female guinea
pig (Dey, 1941, 1943), female cat (Laqueur et al., 1955),
male dog (Daily and Ganong, 1958), Davidson and Ganong, 1960;
Davidson et al. 1960b); and ewe (Clegg and Ganong, 1960).
David et al. (1965) presented convincing evidence
that the effects of lesions and stalk section cannot be
explained as the result of impaired blood supply tothe
anterior pituitary. Studies on partially hypophysectomized
rats showed that 70% of the adeno hypophysis may be removed
without impairment of function. Stalk section causes much
less loss than this. Furthermore, Haun and Sawyer (1960);
McCann and Friedman (1960), Nikitovitch-Winer (1965) and
others found that prolactin secretion actually rises after

such lesions.

An impressive body of evidence, accumulated in
recent years, indicates that neural elements involved in the
control of LH as well as FSH secretion must be located out-
side the basal tuberal region of the hypothalamus. Constant
estrous rats with ovaries containing developing and some-
times cystic follicles, but no corpora lutea, have been
observed by several authors following anterior hypothalamic
lesions (for review, see Flerko, 1961). Similarly, Clegg
and Ganong (1960) produced constant estrus in the ewe with
anterior hypothalamic lesions.

Donovan and van der Werff ten Bosch (1959a) found
that anterior hypothalamic lesions could hasten onset of
puberty in the female rat, and Donovan and van der Werff
ten Bosch (1959b) obtained early onset of heat in the ferret
after anterior hypothalamic lesions were placed during the
winter anestrous period.

Bogdanove and Schoen (1959) obtained precocious
puberty in female rats with anterior hypothalamic lesions;
the animals became estrous. In those animals in which the
arcuate nucleus was damaged, luteinization occurred also:
but complete destruction of the arcuate nucleus resulted in
atrophy of the gonads. Curiously, male rats were not affect—
ed by these lesions. However, Bogdanove et al. (1964)
later observed hypertrophy of prostate and seminal vesicles
(but not testes) after large periventricular lesions which

had little apparent effect in females.

t2

(7'

Y‘; E

a]

(D

D'Angelo and Kravatz (1960) and Flerko and Bardos
(1961) observed that female rats made "constant estrus"
by anterior hypothalamic lesions failed to show compensa—
tory hypertrophy after unilateral ovariectomy. These writers
suggest that receptors sensitive to "estrogen deficiency"
are located in the anterior hypothalamus. However, these

findings conflict with those of Desclin et a1. (1962).

2. Evidence from Behavior of Pituitary
Tissue Implanted in the

Hypothalamus

 

Flerko (1963) described and diagramed the region of
the hypothalamus in which anterior pituitary implants main—
tain normal function and cytology. This region is designated
the "hypophysiotrophic" area. Implants into the "hypophysio-
trophic area" maintained target organs, especially the gonads,
in hypophysectomized rats. Pituitary tissue implanted out-
side this area rapidly lost its normal cytology and function.

The "hypophysiotrophic area" is of considerable
length orocaudally. It extends from the anterior and para—
ventricular nuclei ventrally to the region of the basal
portions of the ventromedial nuclei and extends posteriorly
to the mammilary bodies. It includes the whole of the
arcuate nuclei and is very thin in the dorso-ventral direction.

The most complete studies of pituitary implants into
the hypothalamic hypophysiotrophic area were done by Halasz

et a1. (1962, 1965). Also, Knigge (1962) obtained castration

cells and testicular maintenance with such implants in
hypophysectomized male rats.

Flament-Durand (1964, 1965) obtained particularly
impressive results with pituitary implants into the region
of the arcuate nucleus in female rats. Castration cells
resulted in these implants when the animals were ovariecto-
mized. She had poor results with male rats, and did not

confirm Knigge's (1962) results.

3. Evidence from Use of Hypothalamic Extracts

Campbell et al. (1961, 1964) demonstrated that
direct infusion of median eminence extracts into the pitui—
taries of rabbits was effective in inducing ovulation, and
Nikitovitch-Winer (1962) similarly induced ovulation by
infusion of median eminence extract into the pituitaries
of pentobarbital-blocked proestrus rats. Nikitovitch-Winer
and Everett (1958, 1959) found that rat pituitary tissue
which had lost its normal functions and characteristic
cytological appearance after being transplanted under the
kidney capsule, could be restored to normal appearance and
functions by retransplantation under the median eminence.
Similarly, Evans and Nikitovitch-Winer (1965) found that
pituitary grafts under the kidney capsule could be restored
to normal cytology and function by continuous infusion with
median eminence extracts. Some of their animals went into

constant estrus.

McCann et a1. (1960) and McCann (1962a) first demon-
strated the presence of a luteinizing hormone-releasing
factor (LH—RF) in acid extracts of stalk-median eminence
tissue of rats using the ovarian ascorbic acid depletion
method of Parlow (1958). These results have been confirmed
by Courrier et al. (1961) and Guillemin et al. (1963).
Courrier et al. (1961) and Johnson (1963) observed also
that hypothalamic extracts could induce ovulation in andro-
gen-sterilized female rats. Schiavi et al. (1963) used
acid extracts of sheep hypothalami to produce ovulation in
rats rendered anovulatory by hypothalamic lesions. Ramirez
and McCann (1963) devised a sensitive test for luteinizing
hormone—releasing activity. Rats which had been ovariecto—
mized for 6 weeks were treated with a single injection of
estrogen (50 ugm of estradiol benzoate) and progesterone
(25 mg). This treatment blocked LH release and plasma LH
levels fell markedly within 3 days. Hypothalamic extracts
were then injected and caused a marked rise in plasma LH.
Johnson (1964) found that hypothalamic extract caused re-
lease of hypophysial LH in immature male rats. Schally and
Bowers (1964) demonstrated stimulation of LH release lg
yitrg. LH-RF has now been purified (Guillemin et al., 1963:
Schally and Bowers, 1964; Ramirez et al., 1964, and
Nikitovitch—Winer, 1965).

Igarashi and McCann (1964b) and Igarashi et al.
(1964a) presented evidence for the presence of a follicle-

stimulating hormone-releasing factor (FSH-RF) in rat

hypothalamus using in yiyg methods, e.g. the estrogen—
progesterone blocked ovariectomized rat described above

and also used rats with hypothalamic lesions. These results
were confirmed by Kuroshima et al. (1965). These workers
employed an HCG augmented mouse uterine weight assay for

FSH devised by Igarashi and McCann (1964a) but not properly
validated. Later, de Rivieres and Mauléon (1965) demonstrated
that this method was sensitive to LH, growth hormone, and
prolactin, as well as FSH.

Gellert et al. (1964) have also presented evidence
for the existence of FSH—RF by producing precocious puberty
in female rats with injections of hypothalamic extracts.
Independently of the work presented in this thesis, Kuroshima
et al. (1965) demonstrated stimulation of release of FSH by
a hypothalamic extract in vitro.

Kobayashi et al. (1963a) reported that crude saline
extracts of hypothalamus induced an increase in "total
gonadotropin" release from monolayer cultures of rat anterior
pituitary tissue. Gonadotropin content of cells was in-
creased also. Cerebral cortical extract, vasopressin, and
oxytocin were without effect. These workers used the immature
mouse uterine weight assay method, which measures the combined
effect of FSH and LH. While FSH alone may cause some growth
of ovarian follicles, Fevold (1941) and Greep et al. (1942)
demonstrated that LH is necessary for estrogen stimulation
and consequent uterine growth. The results of Kobayashi

et al. (1963a) may be considered of doubtful significance

10

since they never injected hypothalamic extract alone into
mice as a control. Furthermore, Courrier et al. (1963c)
reported that saline extracts of sheep hypothalamus contain
substantial quantities of FSH activity, whereas FSH activity
is absent in acid extracts of hypothalamus.

Kobayashi et al. (1963) demonstrated that hypothalamic
extracts stimulated the uptake of tritiated leucine by
PAS positive cells of the anterior pituitary lﬂ.!l££2°
Hypothalamic extracts seem therefore to stimulate the synthe-
sis of FSH, LH, and/or thyrotropic hormone in giggg.

Moszkowska (1959) and Moszkowska and Kordon (1961)
reported that more gonadotropin was released when pituitaries
and hypothalami were grafted together on ovaries than when
pituitaries were grafted alone. Similarly, Moszkowska
(1959) and Moszkowska and Scemama (1964) reported that
incubation media contained more FSH and LH when pituitaries
and hypothalami were incubated together than when pituitary
tissue was incubated alone. These authors did not do control
experiments to test the possibility that pars tuberalis
tissue adherent to the hypothalami might have contributed
to gonadotropin release.

Suzuki et al. (1961) claimed that slices of male
rat hypothalamic tissue stimulated gonadotropin synthesis
by slices of male rat pituitary during a 3-hour incubation.
Castration of the donor increased the potency of the hypo-

thalamic slices.

11

In an excellent study, Talwalker et al. (1963)
demonstrated that the hypothalamus contains a substance
which inhibits prolactin secretion i3 vitro. This finding

was confirmed by Schally et a1. (1965).

4. Steroid Feedback

A classical review on the steroid control of anterior
pituitary gonadotropic functions by feedback mechanisms was
that of Greep and Chester Jones (1950). The gonadal steroids
are conceived as regulating pituitary FSH and LH secretion
by acting on the pituitary and/or hypothalamus to inhibit
gonadotropin secretion. Reciprocal relationships between
rates of sex steroid and pituitary gonadotropin secretions
are required for certain phases of normal gonadal function.

In the extensive review by Greep (1961) the con-
clusion is drawn that it is now firmly established that
gonadectomy results in a marked increase in gonadotropin
secretion and in increased storage of gonadotropic hormones
in the hypophysis. Among the more recent literature,

Couzens and Nelson (1961) found a 3 to 4-fold increase in
pituitary FSH content as well as an approximately 4-fold
increase in pituitary LH concentration within 7 days after
ovariectomy in female rats. Paesi et al. (1955) reported
that ovariectomy causes a 5-fold increase in pituitary

FSH content in the female rat within 3 months. Male rat
pituitaries contained more than 5 times as much FSH as

females and did not increase significantly in content after

 

()

30

an.

ova
hat.

int

no:

COR

12

castration. In contrast, Hellbaum et al. (1961) found that
gonadectomy caused a rise in pituitary FSH and in LH in
both sexes in the rat.

Gans (1959a, b) and Gans and van Rees (1962) were
able to detect FSH and LH in serum in gonadectomized male
and female rats but not in intact rats. Gans and de Jongh
(1963) found that serum FSH was much higher in gonadectomized
male and female rats than in intact animals, and that serum
of castrated male rats contained more FSH than that of
ovariectomized female rats. Intact male rats were found to
have much larger amounts of FSH in their pituitaries than
intact female rats.

Parlow (1964a) found a 5-fold increase in FSH
content in the pituitary of the mouse after ovariectomy.
Serum FSH rose from undetectable to very substantial levels,
9 to 12 ugm NIH-FSH—Sl equivalents per m1. LH content did
not rise. Davidson et al. (1960a) found that the FSH
content of the male dog pituitary rises as much as 16-fold
after castration, and LH content doubles.

Parlow (1964c) used much improved assay techniques,
e.g., the OAAD assay for LH and a greatly improved version
of the Steelman-Pohley assay for FSH. He found that both
FSH and LH levels rose in the pituitary and blood of the
female rat after ovariectomy; 0.1 to 0.4 ugm estradiol
injected each day inhibited the pituitary and serum response
to gonadectomy; 2.0 ugm estradiol per day were required to

prevent the FSH response to castration. The duration of

'0

.17

r
O

13

this eXperiment was 16 days. Beyler and Potts (1962) found
that estradiol treatment (4 to 400 ugm/kg body wt/day for

14 days) caused decreased pituitary FSH content in the male
rat. Testosterone treatment (0.2 to 20 mg/kg/day) antagonized
this effect of estradiol and by itself did not decrease
pituitary FSH potency. Gans (1959a, b) found that estradiol
benzoate (2 ugm/day) or testosterone propionate (100 ugm/day)
inhibited the post-castration rise in serum FSH and LH in
male and female rats. Paesi et a1. (1955) and Hellbaum et a1.
(1961) obtained similar results. The parabiotic technique
was used by Miyake (1961) to demonstrate inhibition of
gonadotropin secretion by estrogens or androgens.

Parlow (1964c) also found that treatment of male
rats with testosterone propionate (1 mg/day for 3 weeks)
starting immediately after castration prevented the post-
castration rise in serum FSH and serum and pituitary LH.
Pituitary FSH content rose with testosterone treatment.
Hellbaum et al. (1961) treated gonadectomized male and
female rats with testosterone propionate (3 mg/day).
Pituitary LH content (but not FSH content) decreased.
Hoogstra and Paesi (1957) found that testosterone propionate
treatment (2 mg/day) increased the pituitary FSH content in
intact and gonadectomized male and female rats. Estradiol
benzoate (2 umg/day) did not modify this response.

Ramirez and McCann (1965) found that only 1/3 or
1/4 as much testosterone propionate was required to inhibit

the post-castration rise in plasma LH in the immature male

rats

auth

per

for

14

rats as compared with the post-puberal male rats. These
authors claimed that 75 to 100 ugm testosterone propionate
per 100 gm body weight per day was a "physiological" dose
for an adult male rat.

Van Rees (1961) studied release of FSH by rat pitui-
taries ig vitro after treatment in yiyg. Ovariectomy caused
enormously increased FSH release during a 2-hour incubation.
Intact male rat pituitaries contained much more FSH than
those from intact females, but released much less in vitro.
Treatment of intact and gonadectomized rats with testosterone
propionate in yiyg caused decreased release 33 vitro.

McCann (1962b) concluded that, in the ovariectomized
female rat, progesterone alone has only a feeble inhibitory
effect on secretion of LH, but estradiol benzoate treatment
sensitizes the animal to this inhibitory action. Similarly,
Sager et a1. (1966) found that estrone and progesterone
synergize in inhibiting the rise in plasma FSH after ovariec—
tomy. Ramirez and McCann (1963a) found that treatment with
a single injection of estradiol benzoate (50 ugm) and pro-
gesterone (25 mg) caused a marked fall in plasma LH activity
in 3 days in female rats which had been ovariectomized for
6 weeks. The pituitary of such an animal seems, however,
to be sensitized to the effects of substances which stimulate
release of LH. Igarashi and McCann (1964b) similarly obtained
inhibition of FSH release and sensitization to FSH—RF.

Testosterone alone apparently cannot account for the

testicular feedback mechanism. Bogdanove (manuscript in

pre
co:
ass

nat

15

preparation) studied pituitary cytology and pituitary FSH
content (using an improved version of the Steelman-Pohley
assay) in male rats after castration and testosterone propio-
nate treatment. This excellent work demonstrated increased
FSH stores in the pituitary and increased FSH in the serum

2 weeks after castration. With testosterone propionate
treatment (3.0 mg each 2 days), the FSH stores in the
pituitary increased still further while FSH content of the
serum decreased markedly. Pituitary cytology changed with
castration and became still more abnormal with testosterone
propionate treatment, indicating that some testicular factor
other than androgen participates in regulation of FSH
secretion in the male rat. In addition, Johnsen (1964)
concluded from a study of human clinical material that a
testicular-hypophysial feedback mechanism occurs separately

from testosterone.

5. Evidence from Implantation of Steroid Depots

Another approach to localizing the control of
gonadotropic function in the brain has been to implant
crystalline steroids into various regions. Such steroids
are slowly absorbed; thus they provide a means of localizing
sites of negative feedback action.

The region most often identified with steroid sensi-
tivity is the basal-tuberal portion of the hypothalamus,
particularly the median eminence. Davidson and Sawyer

(1961a) implanted estradiol benzoate into various regions

creas
ovari
alSo

in th
Et al
emine

in ra

16

of the brain in the female rabbit. Failure of copulation-
induced ovulation, and eventual ovarian atrophy, followed
implants into the basal tuberal region, especially the
posterior median eminence. Negative results were reported
for implants in the mammillary bodies, anterior pituitary.
etc. These findings were extended by Kanematsu and Sawyer
(l963d). Kanematsu and Sawyer (l963b, c) found that such
brain implants caused cytological changes indicating de-
creased gonadotropic stimulation in the pituitaries of
ovariectomized rabbits. Kanematsu and Sawyer (1964) found
also that these implants caused decreased amounts of LH

in the plasma of ovariectomized rabbits. Similarly, Ramirez
et al. (1964) found that implants of estradiol in the median
eminence prevented the rise in plasma LH after ovariectomy
in rats.

Lisk (1960) implanted estradiol benzoate into the
hypothalamus in male and female rats. The sensitive area,
in which implants caused atrophy of male and female tracts
and gonads, included the arcuate nucleus, surrounding ventral
hypothalamus and mammillary bodies. Lisk (1963) found that
estradiol implants into the arcuate nuclei in spayed rats
prevented the characteristic cytological changes in the
pituitary following gonadectomy. Implants in the mammillary
bodies and anterior lobe of the pituitary did not alter
the castration reaction. Control substances were ineffective
also. Lisk and Newlon (1963) found that estradiol implants

in the arcuate nucleus caused decreased size of nucleoli

in t

t85t1

of me
semir
from

the l
impla
dogs.

media

hYPotl
Small
regior

Uterir

17

in the cell bodies accompanied by atrophy of ovaries, uteri,
testes, etc.

Lisk (1962) implanted testosterone into the brains
of male and female rats. Atrophy of ovaries, uteri, testes,
seminal, vesicles, and prostates 30 days later was obtained
from implants into the median eminence and other parts of
the basal-tuberal region. Davidson and Sawyer (1961b)
implanted testosterone propionate into the brains of male
dogs. Testis atrophy resulted from implants to the posterior
median eminence and posterior tuberal regions.

An estrogen-sensitive structure in the anterior
hypothalamus was reported by Flerko and Szentagothai (1957).
Small fragments of ovarian tissue were implanted into the
region of the paraventricular nuclei and caused decreased
uterine size. Liver tissue was used as a control.

The question of steroid effects directly upon anterior
pituitary gonadotropic function remains controversial.

Lisk (1962) implanted testosterone into the pituitaries and
brains of male and female rats and obtained gonadal atrophy
only from implants in certain regions of the hypothalamus.
Davidson and Sawyer (1961b), however, obtained some evidence
of inhibition of gonadotropin secretion from effects of
intrapituitary implants of testosterone propionate in male
dogs.

Flerko and Szentagothai (1957) found that implanta-
tion of small fragments of ovarian tissue into the pituitary

had no effect on gonadotropin secretion whereas similar

 

(1963
inpla
impla
in CV
(1964
tarie:

0% r if

OI“Pro
Sehted
(1964b:

(1964)

18

implants in certain regions of the hypothalamus were highly
effective. Davidson and Sawyer (1961a) and Kanematsu and
Sawyer (l963b) reported similar results using estradiol
benzoate implants.

These findings conflict sharply with those of Bogdanove
(l963b) who found that ovarian or estradiol dipropionate
implants were effective directly on the pituitary. Such
implants produced localized regression of castration cells
in ovariectomized female rats. In addition, Ramirez et a1.
(1964) found that estradiol implants in the anterior pitui-
taries of female rats prevented the rise in plasma LH after
ovariectomy, and Rose and Nelson (1957) obtained inhibition
of the castration response from estradiol injected into the
hypophysial fossa. Kanematsu and Sawyer (1964), in contrast
to some of their earlier work, reported an increase in
plasma LH following estradiol benzoate implants in the
anterior pituitaries of ovariectomized rabbits.

Evidence for a direct stimulatory action of estrogen
on prolactin secretion by the anterior pituitary was pre-
sented by Kanematsu and Sawyer (l963d), by Ramirez and McCann
(l964b),and by Flament-Durand (1965). Nicoll and Meites
(1964) obtained in yi§£9_evidence for a direct effect.

Attramadal (1964) reported localization of tritium
labeled estradiol injected systemically into gonadectomized
rats of both sexes. Estradiol was taken up most rapidly
by pituitary, uterus, and vagina. In the pituitary, estra-

diol was found in the cytoplasm of the basophils. In the

 

Sec:
Pia:
thes
(196
9X15
exCE
Cone
Cent

and

el€c1

l9

hypothalamus, estradiol was found in the nuclei of cells

in the supraoptic and paraventricular nuclei. Eisenfeld
and Axelrod (1966) reported that tritiated estradiol is
selectively accumulated by the hypothalamus as well as by
anterior pituitary, uterus, and vagina. These findings of
selective affinities by both pituitary and hypothalamus for
estradiol are considered evidence for direct action on both

organs.

6. Neural Control of Ovulation

In the female rat, the immediate cause of ovulation
appears to be a marked rise in plasma LH on the afternoon
of proestrus, and it clearly is closely controlled by the
central nervous system (Ramirez and McCann, 1964a), as are
other phases of the ovarian cycle. The hypothalamus-pitui-
tary system of the male rat, unlike the female, does not
secrete gonadotropins in a cyclic fashion. Ovaries trans-
planted to castrated male rats develop large follicles, but
these do not ovulate. Harris (1964) and Gorski and Wagner
(1965) reviewed literature demonstrating that this difference
exists in the hypothalamus, not in the pituitary. Several
excellent and thorough reviews on the extensive literature
concerned with the nervous control of ovulation have re-
cently appeared; those of Sawyer (1959), Villee (1961),
and Everett (1961a, 1964) are particularly distinguished.

Sawyer (1959) found that ovulation results from

electrical stimulation of the hypothalamus, but not of the

 

An a
with
vent

EStI’

that
tO Cc
for c

(194%

20

pituitary in the rabbit. In the rat, Barraclough and Gorski
(1961) localized two regions concerned with stimulation of
LH secretion by means of electrical stimulation experiments.
An anterior (suprachiasmatic) region appeared to be concerned
with the ovulatory burst of LH, and a posterior (arcuate-
ventromedial) region appeared to be concerned with basal,
estrogen-regulated secretion of LH.

Sawyer et al. (1947) and Markee et al. (1948) found
that the neural mechanism involved in the ovulation response
to copulation in the rabbit has adrenergic links. Evidence
for cholinergic links also was presented by Sawyer et al.

(1949, 1950, 1951).

 

of
had
USec

ind.

neut
tiSs

and

III. TISSUE CULTURE EXPERIMENTS ON FSH RELEASE
A. Materials and Methods

Preparation of Brain Extracts: Five month old rats
of the Wistar strain (Wilson & Son, Acton, Indiana) which
had been ovariectomized for approximately 6 weeks, were
used as donors for hypothalamic tissue. Preliminary trials
indicated that ovariectomy resulted in increased FSH-RF
activity by the hypothalamus. Each hypothalamus, including
pituitary stalk and median eminence, was removed rapidly
and placed on dry ice. The hypothalamic tissue was homo-
genized in 0.1 N HCl (0.2 ml/hypothalamus) and centrifuged
at 12,000 x g for 45 minutes at 40C. The supernatant was
neutralized with l N NaOH and incorporated directly into
tissue culture medium 199 (Difco Laboratories, Detroit,
Michigan) containing 10% calf serum, 25 U/ml penicillin,
andfﬂlugm/ml streptomycin. Medium containing similarly
prepared extracts of rat cerebral cortical tissue served as
a control in each experiment in which hypothalamic extracts
were used. Each 3 m1 of culture medium contained extract

equivalent to 2 hypothalami (about 20 mg of brain tissue).

Culture Procedure: The procedure outlined by Nicoll
and Meites (1963) was adapted for these experiments. Mature

virgin female rats of the Wistar strain were used as donors

21

22

for cultured pituitary. The rats were stunned and decapi-
tated, and the posterior pituitary was removed and discarded.
The anterior pituitary was removed and cut into 8 explants,
of approximately equal size, with a single-edge razor blade
which had been soaked in ether and absolute alcohol.

The cultures were performed in 3.5 cm x 1 cm sterile
disposable plastic Petri dishes (Falcon Plastics, Inc.),
each containing 3 ml of culture medium 199. Medium 199 is
the formulation of Morgan, Morton, and Parker (1950) and was
purchased from Difco Laboratories, Detroit, Michigan.

Stock solutions of antibiotics were sterilized by filtra-
tion through Millipore filters with pore size of 0.45 u
and were stored at —200C. In each dish, 8 explants were
placed upon a strip of washed (95% alcohol overnight, 100%
alcohol overnight, then anhydrous ether overnight) lens
paper, which was supported on a 1 cm x 2 cm x 0.4 cm plat-
form of #46 grid stainless steel mesh (United Surgical Supply
Co.). The cleaning procedure for the stainless steel
platforms included soaking in 1/2 conc. HCl for 15 min.,
then immersing in distilled water, absolute alcohol and
anhydrous ether, and then drying in air.

Explants from opposite halves of the same anterior
pituitary were placed in a control and an experimental dish,
so that approximately equal cell populations were obtained.
The cultures were maintained in an air-tight Plexiglass
chamber for 9 days at 36°C under continuous gassing with

95% 02 - 5% CO2 at a flow rate of about 200 ml/minute.

23

The gas was humidified by passing through a sintered glass
filter which was immersed in a cylinder of distilled Water.

At the end of the 3rd and 6th days of culture, the
medium was removed and fresh medium was added. The lst,
2nd, and 3rd 3-day medium samples were collected separately
and stored at -20°C until assayed. Upon termination of
culture the eXplants were weighed, fixed, and studied
histologically. Aldehyde-fuchsin (Gomori, 1950, as
modified by Elftman, 1959b) and periodic acid - Schiff
(Elftman, 1959a) staining methods were used. Standard
recommendations were followed in washing and sterilizing
all glassware and instruments. Merchant et al. (1960)

and Paul (1960) were used as references.

Assanyrocedure: FSH activity was measured by the
human chorionic gonadotropin (HCG) - augmentation method
of Steelman and Pohley (1953). Weanling female rats
(Holtzman Co., Madison, Wisconsin), 25 days old, were
injected subcutaneously with the test materials to which
had been added a total dose of 50 IU HCG (NUtritional Bio-
chemicals Corporation, Cleveland, Ohio). The animals re—
ceived 3 injections per day for 3 days. The material for
each group of animals was divided into 3rds and frozen
until day of use. Injection solutions were kept at 40C
between injections. Approximately 72 hours after the
lst injection, the ovaries were removed and weighed. All
ovaries (each pair were weighed together) were recorded as

mg/100 gm body weight.

 

In

tn

(3

LA)

24

The standard error of the mean was calculated for

each combined group using the following equation:

g

S :ﬁQZXZ) - (XX)2
52. 2

N (N—l)

 

where N is the number of observations (X).
Students "t" test was used to determine the signifi-
cance of the differences between groups as described by

Snedecor (1956).

B. Results on Release of FSH In Vitro

Twenty male rat pituitaries were cultured for 9
days. Medium was collected each 3 days. At the end of the
9-day period, half of the explants were fixed in Bouin's
solution and studied histologically; the remainder were
frozen at -20°C for assay. Survival was good.

The level of FSH activity released in each 3-days
of culture is indicated by the results presented in Figure 1.
Each assay rat received tissue culture medium from the equi-
valent of 0.4 cultured male rat pituitaries. There were 6
assay animals per group. Data are presented as bar graphs;
standard errors of the mean for each group are indicated
also. Animals receiving HCG only had ovaries weighing
94.: 6 mg/100 gm body weight.

Significant FSH activity was present only in the
culture medium from the first 3 days of culture. FSH
activity was not detected in medium from the 2nd or 3rd

3-day period or in the explants at the end of 9 days.

 

rh Fh F

lupc. FL

hLUE \

Mn .1. £~uv 1.5 ﬂea

: .5. H L F..\a Av

~ . 5.. VJ:

I

WU an... 7. 9.... a ..Jv~

H N WHEN

TC

FSH Release -— Mean Ovarian Weights (mg per 100 gm body weight)

200

190

180

170

160

150

140

130

120

110

100

90

80

Fig.

25

 

 

 

 

 

 

 

 

 

 

 

7' 'r
4
q
‘F
__L_
‘._._—..p— ——_--—____—-__Tr-_—-
H G O 1
11: C “Y
a .1-
“""EE'"""2‘nE-- “ -’- =— ‘-
3‘daY 3'day ' Sipéigtif
Medium Medium 3rd 3-day 9 Da 3
_ p < .025 Me ium Y
1. Release of FSH by Pituitary Tissue in vitro in Re-

lation to Time. Lines on bars refer to standard

,error of the means, as do dotted lines.

Tht

NI}

7.60

‘ A
W

equ

Pur

of

it

..3
QC

1

7».
.l

r

26

These results indicate that pituitary tissue releases FSH
in vitro, but in the absence of stimulation it does not
synthesize or release appreciable quantities of FSH for
longer than 3 days.

Dose-response curves of tissue culture medium and
NIH—FSH—Sl are shown in Figure 2. Pooled tissue culture
medium samples, representing tissue cultured for 9 days,
were administered at 2 dose levels (0.2 or 0.4 pituitary
equivalents per assay rat) to groups of 6 assay animals.
Purified sheep FSH was similarly administered at dose levels
of 75 ugm or 150 ugm. The curves are plotted with a common
intercept (threshold dose).

The virtually identical dose—response curves ob-
tained indicate that living rat pituitary tissue releases
ig_vitro a substance which is biologically identical with
the highly purified FSH extracted from sheep pituitaries
collected at slaughterhouses, and that, therefore, it is

proper to make quantitative comparisons between the two.

C. Stimulation of FSH Release by Hypothalamic

Extract In Vitro

1. Effects of Hypothalamic and Cerebral

Cortical Extracts on FSH Release

In this eXperiment, brain extracts were added to the
media only during the last 6 days of a 9-day culture. The
medium from each group of 4 culture dishes, representing the

hormone released by the equivalent of 4 anterior pituitaries,

 

A _...._..).) >7:;~ 21a A»... 4...... Izzy ut.....-r...._.\$ .-..~L .z.~).d .a...._.<

Mean Ovarian Weights (mg per 100 gm body weight)

27

260-

240-

 

220-

200-

180-

160d

140-

120-

100-
94

 

 

I I l _ ,1
0 M1 31 M2 S2
Doses of Medium (M) and Purified Sheep FSH (S).

Fig. 2. Dose-Response Curves: Tissue Culture Medium FSH and
NIH-FSH-Sl.

28

was injected into 2 assay animals after a portion (l/l6th)
was removed for assay of luteinizing hormone. A total of
9 paired groups of assay animals was used; hence there were
18 rats given medium from pituitary explants cultured with
hypothalamic extract and 18 rats given medium from pituitary
cultured with cerebral cortical extract. A total of 72
dishes were used.

The data for the 9 paired groups are presented in
Table 1. In every group the medium from pituitary exposed
to hypothalamic extract produced a greater ovarian response
than the medium from tissue exposed to cerebral cortical
extract. Twenty assay rats treated with the augmentation
dose of 50 IU HCG alone had ovarian weights of 100 i 5
mg/100 g body weight, while 9 animals treated with 50 IU
HCG and neutralized acid extract of hypothalamus (7.5 hypo-
thalami per assay rat) had ovarian weights of 93‘i 7 mg/lOO
body weight. Thus, the mean responses to the augmentation
dose alone, to hypothalamic extract, and to the medium from
eXplants cultured with cortical extract were virtually
identical. This indicates that no detectible amount of FSH
was present in the control medium during the last 6 days
of culture. Only the culture medium from explants cultured
with hypothalamic extract contained significant FSH activity
(p < .001).

The tissue culture medium from the first 3 days of
culture was assayed subsequently. The anterior pituitary

explants were not cultured with either hypothalamic or

29

Table 1. Effects of acid extracts of rat cerebral cortex
and hypothalamus on FSH release by rat pituitary
lg vitro.

 

 

Mean Ovarian Weights of Assay Rats (mg/100 g body wt.)

 

 

Medium from anterior Medium from anterior
pituitary culture with pituitary culture with
Group cortical extract* hypothalamic extract*
1 76 166
2 78 125
3 89 94
4 94 125
5 89 150
6 130 142
7 122 145
8 98 225
9 114 173
Average 99 i.7+ Average 149 i 10T

 

*Medium from 4 anterior pituitary equivalents
injected into 2 assay rats.

TStandard errors were calculated on the basis of 18
assay rats. Cortical vs. Hypothalamic Groups:

t=4.1 p< .001.

cerebral cortical extracts during this period. Medium
equivalent to 1.87 cultured anterior pituitaries was injected
into each assay rat. Medium from the control and experi-
mental culture dishes produced average ovarian weights of

130 i 11 and 149 i 8 mg/100 g body weight, respectively.

This indicates that significant amounts of FSH were released

by the anterior pituitary explants during the first 3 days

30

of culture, and these amounts did not differ significantly
between the control and experimental culture dishes. These
ovarian weights cannot be strictly compared with those of the
assay rats given medium from the last 6 days of culture,
since there is some variation in response among different
shipments of assay rats.

The pituitary explants at the end of 9 days of
culture consisted of a wide rim of viable tissue and a necrotic
core. Acidophils and a small number of periodic acid-

Schiff positive cells were clearly visible. Photomicro—
graphs are presented in Figures 3 and 4 of sections of
fresh tissue and 9-day explants, respectively, stained with
aldehyde-fuchsin. No obvious differences were seen in the
explants cultured with hypothalamic extract as compared to
those cultured with cerebral cortical extract.

According to Purves and Griesbach (1955), there are
two types of gonadotropic cells in the anterior pituitary.
These are termed central gonadotrophs which produce luteiniz-
ing hormone and peripheral gonadotrophs which are scattered
close to the surface of the pituitary and produce FSH.

If so, there was virtually complete survival and healthy
appearance of FSH-secreting cells in the tissue culture and

incubation systems described in this thesis.

2. Effects of Incubating a Purified FSH
Preparation With and Without Brain

Extracts on FSH Activity

 

This experiment was performed to determine whether or not

31

 

Fig. 3. Photomicrograph of Section of Uncultured Female
Rat Pars Anterior Tissue. Aldehyde-Fuchsin X630.

52

 

Fig. 4. Photomicrograph of Section of Female Rat Pars
Anterior Tissue Cultured for 9 Days. Aldehyde-
Fuchsin X650.

33

FSH activity could be increased or decreased as a result of
incubation alone or incubation with brain extracts. Ovine
FSH (NIH-Sl) was incubated alone or with neutralized acid
extracts of rat hypothalamus or cerebral cortex for 3 days
in culture medium and injected into assay animals. The
dose in each case was 150 ug, and all substances were incor—
porated in culture medium 199. The results are presented
in Table 2. Eight animals (group 4) received FSH which
had been incubated with cerebral cortical extract from
ovariectomized rats in doses equivalent to 75 mg of fresh
tissue per assay rat. The rats in group 5 each received
FSH which had been incubated with extract equivalent to 75
mg of fresh hypothalamus from ovariectomized rats. The
animals in group 3 received FSH which had been incubated
alone. Eight animals (group 2) received non-incubated FSH.
These results indicate that cerebral cortical and hypo-
thalamic extracts had no significant effect on FSH activity

in vitro.

3. Effects of Injections of Hypothalamic And

 

Cerebral Cortical Extracts on Response of

 

Assay Animals to FSH

 

Each of the 6 assay animals received 150 ug of ovine
FSH mixed with a neutralized acid extract of 75 mg of rat
cerebral cortical tissue. Six other rats received FSH
together with extract equivalent to 7.5 rat hypothalami
(about 75 mg of brain tissue). Six animals received FSH

alone. The results are presented in Table 30 Neither

34

Table 2. Effects of incubating FSH with and without brain
extracts.

 

 

 

Grou and Treatment No. of Assay Mean Ovarian Wt.
p Rats (mg/100 g body wt.)*
1. Untreated Assay
Rats 10 24 i.1
2. HCG only 10 87.: 4
3. Non-incubated FSH 8 186 i 14
4. Incubated FSH 6 171 i 18
5. FSH incubated with
cerebral cortical
extract 8 150 i 6
6. FSH incubated with
hypothalamic extract 8 156 i 6

 

*Mean and standard errors.

Table 3. Effects of injections of hypothalamic and cerebral
cortical extracts on response of assay animals to
FSH. (50 IU HCG given to all groups.)

 

 

 

No. of Assay Mean Ovarian Wt.

Group and Treatment Rats (mg/100 g body wt.)*
1. HCG only 5 91.: 9
2. FSH 6 215 i 28
3. FSH and Hypothalamic

extract 6 188_: 13
4. FSH and Cerebral

cortical extract 6 184 + 10

 

*Mean and standard errors.

35

hypothalamic nor cerebral cortical extracts influenced the

ovarian response of the assay rats to FSH.

4. Discussion

 

These results demonstrate the existence of a factor(s)
in crude acid extract of hypothalamus from ovariectomized
rats which acts directly on the anterior pituitary to stimu-
late release of FSH. Stimulation cannot be attributed to
any effects of hypothalamic extracts directly on FSH in
the medium, since such extracts did not significantly acti-
vate or inactivate FSH in vitro. The hypothalamic extract
also did not significantly influence the response of the
assay animals to FSH injections. The first 3 days of culture
medium from the control and experimental groups were assayed
and found not to differ significantly in the amounts of FSH
released, thus confirming the equivalence of the control
and experimental pituitary explants. The presence of FSH
activity in the control medium from the first 3 days of
pituitary culture and its absence, under the conditions of
our assay, in the medium from the last 6 days culture,
indicate that FSH is released in detectible amounts only
for a few days in the absence of hypothalamic stimulation.
Incorporation of a hypothalamic extract into the culture
medium at the end of the third and sixth days of culture
apparently can maintain FSH release by the pituitary ex-

plants.

36

Our 13 vitro results appear to be in agreement with
the report of Igarashi and McCann (1964b), in which rat
hypothalamic extracts were used to stimulate FSH release

ig_vivo. The specificity of their FSH assay method in the

 

mouse requires further study and evaluation, whereas the
Steelman-Pohley assay method has been shown to be highly
specific for FSH. Our results are also in apparent agree-
ment with an independent study by Kuroshima et a1. (1965)

in which stimulation of FSH release was obtained by incu-
bating sheep and beef hypothalamic extracts with rat
pituitaries. Igarashi et al. (1964b) have reported partial
purification of the FSH-releasing factor and obtained evi-
dence that it is a small polypeptide different from vaso-
pressin and corticotropin-releasing factor. Kobayashi et al.
(1963a) found that cerebral cortical extracts, purified
vasopressin and oxytocin had no effects on gonadotropin
release 13.21EEQ- Igarashi and McCann (1964b) and Kuroshima
et al. (1965) also reported that cerebral cortical extract
had no effect on FSH-release. Lack of FSH activity in

391d extracts of hypothalamus was reported also by Igarashi
and McCann (l964b), Kuroshima et al. (1965) and Courrier

et al. (1963).

The question has been asked whether these results
represent stimulation of release or release secondary to
stimulation of synthesis or both. Since it was decided to
do a thorough histological study onithe explants in expecta—

tion of finding cytological changes caused by hypothalamic

37

factors and since this work was handicapped by a relatively
insensitive assay, there was insufficient explant tissue
left over to bioassay and therefore to prove a net increase
in activity in the system. Such results would not be con-
clusive, however; no one has yet determined whether or not
intracellular FSH has the same specific activity as FSH
that has passed through the cell membrane. The possibility
remains, therefore, that an activation process could take
place without protein synthesis. Kobayashi et a1. (1965)
have demonstrated that hypothalamic extract stimulates
uptake of tritiated leucine by pituitary basophil cells

in vitro; therefore, it appears that protein synthesis is

being stimulated.

D. Effects of Steroid Hormones on FSH

Release IQ Vitro‘

In the first five of the following experiments, a
total of 174 pituitaries from ovariectomized rats were
cultured for 6 days in medium 199 without calf serum.

Each culture dish contained explants derived from 4
pituitary halves. Half of each pituitary was cultured in
plain medium; the opposite half was exposed to steroid(s).
The experimental medium in experiment 2 contained estradiol
(0.25 ugm/ml). The experimental medium in experiment 3
contained progesterone (1.0 ugm/ml). Experimental medium
number 4 contained the above doses of estradiol and pro-

gesterone in combination. Experimental medium number 5

(n

m

m

Pf"!

n;

38

contained testosterone propionate (1.0 ugm/ml). Each assay
rat received medium from the equivalent of 1/2 ovariectomized
rat pituitary cultured for 6 days.

In the last experiment, 24 intact female rat pitui-
taries were cultured for 6 days in 199 medium without calf
serum. Explants from 2 pituitary halves were in each dish.
During the first 3 days, the pituitaries were cultured in
plain medium. During the last 3 days, the control medium
contained hypothalamic extract representing 3 adult male
rats per pituitary equivalent. The experimental group re—
ceived hypothalamic extract and estradiol (0.25 ugm/ml).

Culture media and injection solutions in each experi-
ment were kept in independent groups and injections were
arranged so that data on independent groups of 3 control
assay rats, with a matching group of 3 experimental assay
rats were obtained. Combined data are presented in Table 4.

The steroids used came from stock solutions pre-
pared in absolute ethanol. The progesterone (Smith, Kline,
and French, Philadelphia, Pa.) and testosterone propionate
(NUtritional Biochemicals Corp., Cleveland, Ohio) were each
prepared in solutions to contain 400 ugm/ml. Those of
estradiol (American Steroids Co., Hato Rey, Puerto Rico)
were made up to contain 100 ugm/ml. Control amounts of

ethanol were added to control medium.

Results: Data obtained in cultures with steroids
are presented in Table 4. Estradiol (No. 2), progesterone

(No. 3), estradiol and progesterone in combination (No. 4)

39

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ADmH momma mom .uﬂm N\HV
oumcmmoaon 05mmﬂu amoum

 

.Humxm HOHDCOO mumm mmsouw

 

>mmm¢ mo ponﬂmm
somMOHUCH unmﬂoz .oz Hmuoa mo .02

CMHHM>Q ENG:

mumu wmmmm mo ucmEummHB

OOZ
.uaxm

 

 

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.¢ OHQmB

In

(“f

('f

40

and testosterone propionate (No. 5) failed to inhibit FSH
release from ovariectomized rat pituitary tissue in vitro.
Ovarian weights from a group (No. l) of assay rats receiving
injections of a fresh tissue homogenate are included also.
Explants were stained with aldehyde-fuchsin and
studied histologically. Survival was excellent. No
obvious differences between control and experimental pitui-
tary halves were obtained with any treatment. Signet cells
were maintained 6 days lg vitro.
In an experiment with somewhat different design
(No. 6), estradiol failed to antagonize the stimulatory
effect of hypothalamic extract on intact rat pituitary tissue.
In Section IVD of this thesis, experiments were done in which
estradiol and testosterone failed to antagonize hypothalamic

extract during a 6—hour incubation period.

Discussion: The doses of steroids added to the
media were rather high. Actual content of steroid in the
medium was not measured. Uptake of steroids by pituitary
tissue (see Attramadal, 1964, and Eisenfeld and Axelrod,
1966), binding to proteins in solution, binding to
plastic in the Petri dishes, etc., probably caused sub—
stantial reduction in the actual amounts present.

The maximum possible total doses of steroids which
the assay animals could have received were 1.5 ugm of
testosterone propionate, 1.5 ugm of progesterone, 0.375
ugm estradiol, or the combination of the last two. These

levels are generally considered negligible.

41

These results seem to corroborate the findings of
Flerko and Szentagothai (1957) Davidson and Sawyer (1961c)
Lisk (1962) and Kanematsu and Sawyer (1963b) whose results
indicate that steroid hormones do not exert a direct negative
feedback effect on the pituitary. However, these results
seem incompatible with the findings of Rose and Nelson (1957),
Davidson and Sawyer (1961b) and Bogdanove (1963b), who found
evidence for a direct negative feedback effect.

The possibility remains, however, that changes in
experimental design might produce different results. For
example, data indicating whether or not a long preculture
with steroids would inhibit the effect of later addition of
hypothalamic extract were not obtained, but it is note-
worthy that Igarashi and McCann (1964b) and Kuroshima et al.
(1965) found that a single injection of large doses of
estrogen and progesterone in_yiyg actually seemed to sensitize
the pituitary to FSH-RF three days later. In addition,
unpublished work from two laboratories indicates that testo—
sterone treatment sensitizes the pituitaries of castrated
male rats to FSH—RF.

A number of findings, which can be interpreted as
indicating a direct action of steroid(s) on FSH release,
require comment. Hoogstra and Paesi (1957) reported that
testosterone treatment causes increased pituitary FSH
content in intact and castrated male and female rats in
doses which are known to cause decreased release. Parlow

(1964c) reported that testosterone treatment (1 mg/day)

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and
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42

caused increased pituitary FSH and decreased serum FSH in
castrated male rats. Bogdanove (manuscript in preparation)
and Beyler and Potts (1962) obtained similar results. On
the other hand, Paesi et al. (1955) found that estradiol
treatment causes decreased pituitary FSH content in intact
and castrated male and ovariectomized female rats,aand
Parlow (1964c) found that estradiol treatment lowered FSH
activity in both pituitary and serum. Van Rees (1961)

found that treatment with testosterone in vivo caused in-

 

creased pituitary FSH content but decreased release in vitro
during a 2-hour incubation period whereas estrogen treat-
ment caused decreased pituitary FSH content but actually
seemed to increase the rate of release ig_vitro. It is
noteworthy that these workers all injected steroids for 2

or more weeks.

The above findings can be reconciled with the con-
cept that there is no steroid feedback on pituitary FSH
release if we assume that FSH—releasing factor(s) stimulates
both synthesis and release of FSH, and therefore can produce
two dose-response curves. Testosterone is known to be
less effective than estradiol as an inhibitor of FSH secre-
tion (see Miyake, 1961) and may not completely inhibit it.
In the work quoted above, testosterone may be presumed to
reduce FSH-RF secretion until FSH release is almost com-
pletely suppressed while FSH synthesis occurs to a small
degree; therefore, pituitary content rises. The findings

of Kobayashi et a1. (l963a, 1964, 1965) in which hypothalamic

43

extract caused increased gonadotropic activity in cultured
cells, and the tissue culture findings in this thesis support
the idea of stimulation of FSH synthesis. Evidence for a
distinct stimulation of release arises from the depletion
of pituitary FSH content observed after intracarotid injection
of hypothalamic extract by David et al. (1965) and Schally
et al. (manuscript in preparation).

The possibility of direct feedback on the pituitary
cannot be excluded on the basis of evidence presently avail-

able. More work is needed.

IV. INCUBATION EXPERIMENTS ON FSH RELEASE

A. Materials and Methods

Experimental Animals: Rats of the Sprague-Dawley

 

strain (Spartan Research, Haslett, Michigan) were used in
all experiments. Donors of hypothalami and incubated pitui-
taries were sexually mature male rats weighing 250-350
grams. Unless otherwise noted, female rats, 22 days old,
were used for the FSH bioassays. The diet consisted of
Wayne Lab-Blox (Allied Mills, Inc., Chicago, Illinois).
Assay rats received bread and milk as a supplement. The
animals were maintained in a temperature controlled

(75 i 10F) and artificially illuminated room.

Preparation of Acid Extracts: Animals were killed
by guillotine. The hypothalami (including pituitary stalk
and median eminence) were removed rapidly and placed in
ice-cold 0.1 N. HCl (0.2 ml per hypothalamus). Hypothalami
and acid were later homogenized and centrifuged at 12,000
x g for 40 minutes at 40C. Supernatents (acid extracts)

were stored at -200C.

Incubations: A short term incubation method derived
from the method of Saffran and Schally (1955) was used.
Rats were killed by decapitation and the heads were rapidly
opened. Pituitaries were placed on filter paper soaked in

44

45

incubation medium and dissected. Anterior pituitary tissue
was separated from posterior pituitary, sliced longitudinally
and placed into 25 ml flasks, each containing 2 m1 of medium
199 (Difco Laboratories, Detroit, Michigan). Opposite halves
from each pituitary were placed in flasks for different exper-
imental groups. Each flask contained 8 pituitary halves.
Incubations were carried out in a Dubnoff metabolic shaker

(60 cycles per minute) under constant gassing with 95%

0 - 5% CO at 37°C.

2 2

Thirty minutes after the last pituitary was dissected
out, the medium in the flasks was replaced with fresh
medium. Next, the acid extracts were neutralized to approxi-
mately pH 7.0 with 1. molar NaOH and added to the flasks
as rapidly as possible. Six hours later, the incubation was
terminated; the incubated pituitary tissue was weighed,

and the media were frozen for storage.

Agggy: FSH activity was measured by the method of
Steelman and Pohley (1953) as modified by Parlow and Reichert
(1963). The specificity and reliability of this method seemed
well established (Parlow, 1964b). Weanling female assay
animals received test materials in a total volume of 3.0
ml containing 50 IU of HDman Chorionic Gonadotropin
(Nutritional Biochemicals Corporation, Cleveland, Ohio).
Animals received injections of 0.5 m1 twice daily, 8 to
10 hours apart, for 3 days. All treatments were administered
in medium 199, and solutions were kept at 40C between

injections. The animals were ovariectomized or killed on

46

the fourth day and the ovaries were freed from adherent
fat and connective tissue and weighed on a torsion balance.

Most of the materials were tested at two dose levels
since it was realized that demonstration of a dose-response
curve with each preparation provides strong evidence for the
validity of the results. Each assay rat received medium
from the equivalent of g-or g-pituitaries. Purified
ovine FSH (NIH-FSH-SZ) at two dose levels (100 ugm or
200 ugm) was used as a standard preparation. These doses
represent the lower portion of the dose-response curve.
Assay animals were randomly distributed among experimental
groups. Neutralized acidic extracts of cerebral cortical
and hypothalamic tissue have been shown to be without signifi-
cant effect on the assay when administered subcutaneously
(Kuroshima et al., 1965; Courrier et al., 1963; also see
Sections IIIC and IVD of this thesis).

All data obtained were plotted on graph paper and
regression lines were plotted for preliminary analysis.
The standard errors of the mean were calculated for each
point and t—tests were carried out. Estimation of relative
potency by graphical means and of statistical significance
by t-tests gave good agreement with potencies and confidence
limits computed by the following method of statistical
analysis derived from Bliss (1956).

When, as in the Steelman-Pohley assay, the response
y can be plotted as a straight line against arithmetic

dosage units, and when the plotted lines for the standard

47

(subscript l) and for each of the m—l unknowns (subscripts

2 to m) meet at zero dose within the sampling error, the
relative potency is given by the ratio of their slopes.

The doses of each preparation are spaced at equal arithmetic

intervals and coded: x = 0, l, or 2. The slopes are com-

puted with a common intercept a' at x = 0, so that Yl

I _ I _ I
a + bl x1, Y2 - a + b2 x2 . . . Y' — a + b x .

m m m
In coded units the relative potency P'*, for unknown 2, for
example is the ratio:

b
P'*='b—2'
1

To recover the potency P* in original units requires only
simple arithmetic.

When the assay is fully balanced with the same
number of assay animals at each of 2 dose levels, and when

a zero dose is not included the common intercept is determined

as:

_ I
'_5>3y 3Tb

a — f m
Where the total number of doses is 2m, each treatment has f

replicate responses, y, which total Tt, and Tg = Z (x Tt)'

The slope for each preparation is then computed as

_.L
101-5

The error variance about the m lines is determined as
2 .

2 2 y — a

_ I
s = 2 y 2(bi Tb)

n

 

48

where the number of degrees of freedom n = 2fm - m - l.

The index of precision, A, is computed as

 

The 95% confidence limits of P'* are next computed

 

 

 

as
_ I _ _ n2 _ I
xp.* _ CP*i K :fm 1) “Pu + 1) + x (K 2 cpﬂ)
where K = (C - l) ( 9 l
9 + m
bi
C =
2 2 2
bi - C11 8 _t
= .L _9__
C11 5f + 5fm
t = tabular value of student's t at p = 0.05

for n degrees of freedom.

B. Assay of FSH-RF Activity by 6-Hour

Incubation Technique

In these experiments FSH—release was studied in a
6-hour incubation system. Attempts were made to quantitate
the amount of FSH released by tissue in contact with hypo-
thalamic extract or cerebral cortical extract. A study was
done also on response of anterior pituitary tissue to dif-

ferent doses of hypothalamic extract.

49

l. Stimulation of FSH Release by Male

Rat Hypothalamic Extracts

The results of three similar experiments are shown
in Table 5. Each pituitary equivalent was incubated in the
presence of hypothalamic extract equivalent to 2.0 adult
male rat hypothalami. Similarly prepared cerebral cortical
extracts from equivalent amounts of tissue were used as
osmotic controls since previous work had indicated that
these had no effect on FSH release.

In experiments 1, 2, and 3, 40, 24, and 32 pituitary
halves respectively released readily measurable amounts of
FSH during incubation. Their opposite halves, in the
presence of hypothalamic extract, released significantly

greater amounts of hormone.

2. Effect of Graded Doses of Hypothalamic
Extract on FSH Release py Male Rat

Pituitary In Vitro

The effect of varying doses of hypothalamic extract
were evaluated in two separate experiments whose combined
data are shown in Figure 5. In these experiments, each
assay animal received medium from the equivalent of 2 adult
male rat anterior pituitaries. Doses of hypothalamic
extract, drawn from pooled groups of 24 animals, were 0,
1/32, 1/16, 1/8, 1/4 and 1/2 of a hypothalamus per incubated
pituitary equivalent. The data are presented as the mean

ovarian weights. Hypothalamus donors, pituitary donors,

50

.muHEHA oucopﬂmcou Xmm pom Goose

 

 

Amm.mu~a.mv 6H.m uomnuxm
UHEmamsuom>m
oem. Ame.mnma.mv am.a
Aoo.Humm.ov ms.o pumuuxm Hmoﬂuuoo a m
Loo.sumo.mv sm.m pumuuxm
UHEmHmcuommm
mom. Aoa.m1¢m.mv ©@.m
Amm.mueo.av Hm.m uomuuxm Hmoﬂuuoo m m
Amm.numm.mv No.8 uumuuxm
DAEmamcuomxm
mam. Ama.~nom.ﬂv NH.N Amo.aunm.mv ma.m nomuuxm Hmoﬂunoo m H
K smsﬁmamzuommm endow mom osmmHB ucoEumoHB Ho>oa omom mom HmQESZ

.m> xounou
“ommoaom 0>Humaom

humuﬂsuﬂm mE mom
mmummmlmHZ\Em1
mm ommmaom mmm

msouw mom mamEHcd ucoﬁﬂuomxm
>mmm¢ MO HOQEDZ

 

 

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.ouuﬂ> mm humuHDUHQ umn mama adopm an

.m magma

51

 

 

 

90—
«r
o
80.
l
m
4.)
n
.S‘
a) 70-
3
a
m
-H
H
m
>
O 60 ..
c
8
2‘.
50.
40‘
i T T f v
1/32 1/16 1/8 1/4 1/2

Hypothalamic Equivalents per Incubated Pituitary Equivalent

Fig. 5. Effects of Graded Doses of Hypothalamic Extract on
FSH Release by Pituitary Tissue ip vitro.

52

and assay animals were carefully chosen for uniform body
weight. vertical bars refer to standard errors of the mean,
and the student's "t" test was used to compare groups.

The dose of 1/32 hypothalamus per incubated pitui-
tary produced no significant stimulation of FSH release.
With 6 assay animals per group, the dose of 1/8 hypothalamus
produced a significant (p < .001) increase in ovarian weight
when compared with the dose of 1/32. With 4 assay rats per
group, the dose of 1/16 hypothalamus produced a significant
(p < .005) stimulation when compared with 1/32; and the
dose of 1/4 produced a significantly (p < .001) greater
reSponse than the dose of 1/16. Doses of 1/2 and 2
hypothalami per incubated pituitary were used also and were

not associated with further increases in ovarian weights.

Discussion: Previous work on FSH-RF used the long-
term ovariectomized female rat pretreated with 50 ugm
estradiol benzoate and 25 mg progesterone 3 days before use
ip Kipp (Igarashi and McCann, 1964b; Igarashi et al. 1964a;
Kuroshima et al. 1965) or ip vitro (Kuroshima et al.,

1965) systems. Also used previously were ovariectomized
rats with median eminence lesions (Igarashi and McCann,
1964b) and long term tissue cultures of pituitary tissue
from normal female rats (Kobayashi et al., 1963a, 1965).
The present study indicates that male rat pituitaries
provide a satisfactory means of demonstrating FSH-RF
activity. The graded response observed in the second part
of this work indicates a potential usefulness of this

method in quantitatively assaying FSH—RF.

53

In the 3rd experiment in Table 5, pituitaries from
old rats (body weights, 400-450 grams) with atrophic testes
released significantly less FSH in incubation than did those
from younger rats (experiments 1 and 2). The relative
release, however, was significantly greater in the old rats.
The low absolute release perhaps reflects the influence of
low endogenous FSH-RF activity before the pituitaries were
removed. The high relative release indicates that the pitui-
taries were still highly responsive. These observations
are consistent with the idea that reproductive failure with
age in the male rat is the result of hypothalamic rather

than pituitary deterioration.

C. Effects of Castration and Androgen Treatment

On Hypothalamic FSH-RF Content

Data on the effects of testosterone injections and
castration on FSH-RF activity in rat hypothalamus are
tabulated in Table 6. Data are presented as the ratios of
FSH activity in the control and experimental media.

In the first two experiments, hypothalami from groups
of animals which had been injected with 2 mg of testosterone
propionate (Nutritional Biochemicals Corporation, Cleveland,
Ohio) in corn oil each day for two weeks were compared with
those receiving control oil injections. Each incubated
pituitary equivalent was incubated with the equivalent of
0.2 hypothalami from adult male rats weighing 275-300 grams.
Pituitary tissue incubated with extracts from the steroid
treated rats released significantly less FSH than did the

controls.

.muﬂeﬂq moaaeﬂmaoo amm new cams.

 

 

 

 

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mmm. Amm.muea.av mm.a m m o.m m Seouumﬂum>o
4
.o amm. Aom.almm.av b¢.H v ab mo.o a
AmxooB NV
wmm. Abm.alho.av mm.a a no mo.o m coﬂumuummo
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. mop Hem mE NV
oDMCOHmOHm
mmm. Amm.OIoo.ov mm.o m Nb N.o H ocououmoumoe
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mﬂpm2\.pxm UHEmamzuommm omon Hon QUOHO poumnsucH mom mucmam ucoeﬂuomxm
Hmucoﬁﬂuomxm sues capo: mom mameﬂcﬁ I>aovo UHEmHmnuommm

"mucouom 0>Hpmamm mmmm< mo “09852

 

.muﬂ>ﬂuom mmlmmm co mHOCOp mDEmamnuom>£ mcﬂpmonu mo muoommm .o magma

55

In the second two experiments, pituitary tissue
incubated with hypothalamic extracts from male rats which
had been castrated for two weeks released significantly more
FSH than tissue incubated with control extracts. Each pitui-
tary equivalent was incubated with the equivalent of 0.05
hypothalami.

One experiment (No. 5) is shown comparing hypothalami
from ovariectomized rats with those from intact female rats.
Gonadectomy appears to cause increased hypothalamic FSH-RF
content in the female also. 2.0 hypothalami per incubated
pituitary were used, a dose which causes maximal stimulation
when male hypothalami are used. This experiment suggests,
therefore, that intact female hypothalami contain much
less FSH-RF than intact male hypothalami. This result may
not be reproductible, however. FSH—RF content seemed to
vary widely among different shipments of female rats but
not of male rats. With female hypothalami the ovarian
weights frequently indicated that there was no significant
FSH-RF activity in control or experimental groups, or that

both caused maximal stimulation.

Discussion: The finding that a steroid (testosterone
propionate) can cause reduced FSH-RF content in the hypo-
thalamus of the male rat is consistent with the observation
of David et al. (1965) that estradiol treatment causes de-
creased hypothalamic FSH—RF content in the hypothalamus

of the ovariectomized female rat.

56

A disturbing point arises however, since it is
known that testosterone not only participates in negative
feedback relationships with the hypothalamus-pituitary axis
but also has gonadotropic properties. Greep (1961) reviews
the extensive literature on this topic. Although FSH
plays an essential role in the growth of spermatogenic
tissue (see Greep and Fevold, 1937; Simpson, Li, and Evans.
1951; and Woods and Simpson, 1961), Albert (1961) reviews
work in which it was demonstrated that testosterone alone
can maintain testicular weight. More recently, Nelson
and Merckel (1938) and Randolph et al. (1959) found that
testosterone and testosterone propionate maintained complete
spermatogenesis in the hypophysectomized mouse. Ludwig
(1950) concluded that doses of testosterone and testosterone
propionate of about 0.1 mg/day caused decreased weight and
histological evidence of degeneration in the testes of the
male rat through effects on pituitary function, whereas
implants into the testes and doses of 1.0 to 3.0 mg per
day maintained testicular weights and normal histology by
direct action.

Greep and Fevold (1937) and Randolph et al. (1959)
found that LH alone could maintain spermatogenesis, pre—
sumably by stimulating the Leydig cells to secrete testo-
sterone which in turn acted on the seminiferous tubules.

In the normal animal, growth and maintenance of spermato-
genic tissue appears to result from the combined and syner-

gistic effects of FSH and LH (Simpson, Li, and Evans, 1951).

57

As was discussed in the literature review, testoster-
one alone cannot account for the negative gonadotropic feed-
back system in the male. Therefore, it is possible that the
observed decrease in hypothalamic FSH-RF content resulted
both from direct action on the hypothalamus and from re-
lease of some unidentified substance from the testes.

‘ The finding of increased FSH-RF content in the
hypothalamus of male and female rats after castration cor-
roborates the work of David et al. (1965) who found in—
creased FSH-RF activity in the hypothalamus of the ovariecto-
mized female. The increases in total—gonadotropin stimulating
activity after gonadectomy reported by Kobayashi et al.
(l963a) in the female rat and by Suzuki et al. (1961) in the
male rat are also compatible with these results.

Variations in light intensity during the moving of
our laboratory in the fall of 1964 are a possible source
of the apparent sex difference in hypothalamic FSH-RF
content. Fiske (1941) reported that constant light hastened
the advent of puberty in female rats. She also used the
HCG augmentation phenomenon to demonstrate that this re-
sulted specifically from an increase in FSH secretion.

Male rats were less affected. Dempsey and Searles (1943)
reported that constant illumination produced constant estrus
without ovulation in rats. Dempsey and Searles (1943),
Desclin (1961a, 1961b), and Ifft (1962) reported that con-
stant illumination caused increases in size of nucleoli in

many of/the nuclei of the female rat.

58
Variations in diet are a conceivable source of
variations in hypothalamic function. It is well known
that nutritional deficiency causes regression of gonadal
function. Srebnik et al. (1961), Srebnik and Nelson
(1962) and Srebnik (1964) studied this phenomenon in rats

and found a marked sex difference. Gonadal function in

female rats was much more severely affected than in males.
D. Additional Control Experiments

1. Effects of Steroid Hormones on the Hypothalamic

Stimulation of FSH Release In Vitro

Adult male rat pituitary tissue was incubated with
hypothalamic extract. In all groups, the dose of hypothalamic
extract was 0.2 hypothalamic equivalent per pituitary equiva-
lent. In the experimental groups, steroids were dissolved
in a minute volume of absolute ethanol and were added to the
medium together with the hypothalamic extract. Steroids
used were testosterone (0.5 or 1 ugm/ml) or estradiol
(0.25 ugm/ml). These steroids were obtained from American
Steroid Co., Hato Rey, Puerto Rico. All media were assayed
at 2 dose levels for FSH.

The results are presented in Table 7. No evidence
for an effect of steroids directly on pituitary tissue was
noted. These results indicate that steroids carried over
in hypothalamic extract did not influence the results ob-
tained in the hypothalamic FSH-RF content studies in the

previous section. This evidence is particularly important

59

.mucoEHHmmxo N Eoum mump meHQEOOs

mm I mameﬂce wmmmm mo .02 Hmuoa

.mumuﬂsuﬂm poquSUCH mom mucoam>ﬂsvm OHEmamnuommm N.o

 

 

 

 

 

mmﬂ. «gm.alwm.o emb.o w AHE\Emi mm.ov Hoﬂpmupmm
mmm. om.alm¢.o ¢H.H m KHE\Em1 m.ov mcououmoumoe
was. .em.aumm.o sam.o a AHE\emi av muonmumoumme
K muHEHA Houucoo Ho>oq pcoEummHB
oocopﬂmcou ﬁmm Hmucoeﬂuomxm mmoa mom QSOHO mom
mameﬂcd mmmmd Hmuoe
.OHUH>.mﬂ ommoaou
mmm co pomupxo DAEmamzuom>£ nuﬂ3 nonummou meoE ou mpﬂououm mcﬂppm mo muoomwm .h oHQmB

60

since Eisenfeld and Axelrod (1966) reported that the hypo-

thalamus selectively accumulates tritiated estradiol.

2. Effects of Incubating a Purified FSH
Preparation With and Without Brain

Extract on FSH Activipy

Ovine FSH (NIH-82) was incubated for 6 hours. Each
incubation flask contained 300 ugm of FSH. Assay animals
received 100 ugm of FSH. Assay animals received 100 ugm
or 200 ugm of FSH in each treatment group. Unincubated
FSH served as the control. There were 3 experimental groups:
FSH incubated alone, FSH incubated with neutralized cere-
bral cortical extract equivalent to 8 mg of fresh tissue
per flask, and FSH incubated with extract equivalent to
0.8 hypothalami per flask.

The data are presented in Table 8. FSH activity was
not increased or decreased as a result of incubation alone
or incubation with brain extracts. These findings corro-

borate results presented above in this thesis.

3. Effects of Injections of Steroids or Brain

Extracts on Response of Assay Animals to FSH

Ovine FSH (NIH-$2) was mixed with estradiol, testo-
sterone, cerebral cortical extract, or hypothalamic extract
in doses designed to serve as controls for previous experi-
ments. The doses were as follows:

Testosterone: Assay animals received 100 ugm FSH and 2/3 ugm

 

61

 

 

 

can. u H oH . mHmeHc< mammamnu.oz Hmuoe
am.Humm.o mo.H m uomuuxm 0HEmHmeuoo>m
QUHZ poquDUCH mmm
hm.H|mh.o 0H.H m pomupxm Hmoﬂuuou
nqu poquSUCH mmm
mm.H|Nw.o no.H N ocoH¢ poumﬂsocH mmm
muHEHH Houucoo Ho>oq omoa mom ucoEumoHB
oocopﬂmcoo *mm HmucoEHHooxm msouo Mom mHmEHc¢ Smmmﬂ

 

 

. mu.Uw.Hu.vnO CHMHQ

usozuHa mam euHs mmnmmmumHz mcHumnsucH Ho muummmm .m oHnme

62

testosterone or 200 ugm FSH and 4/3 ugm testosterone.

Estradiol: Assay animals received 100 ugm FSH and 1/6

 

ugm estradiol or 200 ugm FSH and 1/3 ugm estradiol.
Cerebral cortical extract: Assay animals received 100 ugm
FSH and neutralized acid extract equivalent to 8/3 mg. of
cerebral cortical tissue or twice these doses.
Hypothalamic extract: Assay animals received 100 ugm FSH
and neutralized acid extract equivalent to 0.267 rat hypo-
thalami or twice these doses.

Controls: Assay animals received 100 or 200 ugm NIH-FSH—SZ.

 

All treatments were administered in tissue culture medium 199.

The data are presented in Table 9. No indication
was observed that steroids or brain extracts could influence
the assay animals. The data on brain extracts corroborate
results presented earlier in this thesis as well as those
of Courrier et al. (1963), Igarashi and McCann (1964b) and
Kuroshima et al. (1965), and indicate that results obtained
in this work cannot be attributed to effects of hypothalamic
extracts directly on assay animals.

The negative findings with steroids corroborate the
results of Schuetz et al. (l964a, b) who found that steroids
failed to inhibit the HCG augmentation of FSH effects.

Payne and Runser (1958) obtained some FSH-augmenting activity
with doses of estrogen far higher than those used here, but
no effect at all with testosterone propionate. Smith and
Bradbury (1963) obtained effects on the ovary, but only

with very high doses of diethylstilbestrol.

63

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V. GENERAL DISCUSSION

Work reported in this study clearly demonstrates that
rat hypothalamus contains FSH-RF activity. Pituitary tissue
exhibited a graded response to different doses of FSH-RF.
Gonadectomy raised hypothalamic FSH-RF content and testo-
sterone treatment lowered it. Steroid hormones did not
inhibit FSH release or antagonize the stimulatory action of

hypothalamic extract 13 vitro.

Comparison With Other Work

 

The results obtained in the 9-day tissue culture
part of this work are similar to the results of Kobayashi
et al. (1963a) in which hypothalamic extract caused increased
gonadotropin both in the tissue culture medium and in the
cells. Hewever, the use of a nonspecific assay for gonadotro-
pin makes it difficult to assess the significance of these
results. LH and/or FSH may have been stimulated. In addition,
their work lacked proper controls. Kobayashi et al. (1965)
reported that hypothalamic extract stimulated uptake of
radioactive leucine by pituitary basophils, but again this may
be related to one or a combination of three hormones.

The hypothalamic stimulation of FSH release obtained
in the 6-hour release obtained in the 6-hour incubation ex—

periments (Chapter VII) is similar to an independent study

64

65

by Kuroshima et al. (1965) in which pituitary tissue was
incubated 1 hour. These workers did not present evidence
that their method was suitable for quantitative measure-
ments of FSH-RF. The work presented in this thesis cor-
roborates the findings of Igarashi and McCann (l964b),
Igarashi et al. (l964a), Gellert et al. (1964) and Evans
and Nikitovitch-Winer (1965) who found evidence for
FSH-RF activity in hypothalamic acid extracts by means

of ip_yiyp systems of questionable specificity.

Content studies of hypothalamic releasing factors
remain few in number. Ratner and Meites (1964) demonstrated
that suckling and estrogen treatment could reduce the
hypothalamic content of prolactin inhibiting factor (PIF).
vernikos-Danellis (1964, 1965) demonstrated that adrenal-
ectomy and stress caused increases in hypothalamic content
of corticotropin-releasing factor (CRF), whereas treatment
with cortisol causes a reduction. Sinha and Meites (1965)
found that thyroidectomy causes an increase in hypothalamic
content of thyrotropin-releasing factor (TRF). Fiel and
Meites (1965) reported that nutritional deficiency can cause
a reduction in hypothalamic somatotropin—releasing activity
(SRF) and Pecile et al. (1965) found a reduction in SRF
with age. Ramirez and Sawyer (1965) and Chowers and McCann
(1965) reported fluctuations in hypothalamic luteinizing
hormone-releasing activity (LRF) during the estrus cycle
in the rat. Piacsek and Meites (in press) also reported that
gonadectomy and injections of gonadal steroids altered LRF

content.

66

Recently, David et a1. (1965) reported that FSH-RF
activity in hypothalamic extracts, as measured by a carotid
injection system, was approximately doubled when the hypo-
thalamus donors were ovariectomized. If the ovariectomized
donors were then treated with estradiol benzoate, FSH-RF
content declined to less than 1/4 of the control value.
Unfortunately, their results were presented simply as bar

graphs, without statistical analysis.

Significance of This Work

The work presented here provides strong evidence
for the existence of a substance(s) in hypothalamic
extracts which stimulate the release of FSH from the
pituitary. This work is the first, so far as I know,
to demonstrate stimulation of FSH release (specifically)
from male rat pituitary tissue. The dose-response curves
obtained here demonstrates a potentiality for quantitative
assay of FSH-RF. Since all or almost all established hor—
mones exhibit graded dose-response relationships, whereas
nonSpecific agents usually do not, demonstration of a dose-
response curve for FSH-RF is circumstantial evidence for
its physiological significance. Both Dr. G. Duncan (The
Upjohn Co., Kalamazoo, Mich.) and Dr. S. M. McCann (Dept.
of Physiology, Southwestern Medical School, Dallas, Texas)
have successfully used my 1p.y;p£p FSH-RF assay (private

communication to J. Meites).

67

The demonstrated ability of castration and testo-
sterone propionate treatment to influence the hypothalamus
is evidence that the site of negative feedback action by
steroids is the hypothalamus. Failure of steroids to
directly inhibit FSH release ip 23:39 or to antagonize the
stimulatory effect of hypothalamic extract is inconsistent
with the concept of negative feedback directly on the
pituitary. Ability to alter the hypothalamic content of
FSH-RF by treatment of the donor is also further evidence
that this substance has physiological significance and is
not merely an artifact of chemical and physical extraction

of the hypothalamus.

Suggestions For Further Work

 

There is a large practical need for the purification,
structural determination, and synthesis of FSH-RF for use
in medicine and agriculture. Once this is done, and pure
FSH-RF is available, it will be practical to demonstrate
unequivocally that FSH-RF stimulates synthesis, as well as
release.of FSH. It also will be possible to do satisfactory
studies on the effects of FSH-RF on pituitary cytology and
to study effects on cell metabolism, including oxygen con—
sumption. In addition, pure FSH-RF could be used to firmly
establish the shape of the dose-response curve.

Since my results do not completely rule out the
possibilities of steroid effects directly on the pituitary,

more experiments are needed. For example. a long pre-culture

68

with steroids might influence the response to hypothalamic
extract added later.

Much work remains to be done on hypothalamic FSH-
RF content in female rats. The effects of ovariectomy and
treatment with steroids should be studied more completely.
Effects of nutritional deficiency, melatonin, and changes
in light intensity are of interest also. Differences in
hypothalamic FSH-RF content between male and female rats
are suggested by my data and should be investigated
thoroughly.

Developmental and comparative aspects are of
interest. The hypothalamus should be studied from fetal

stages to senility, and from fish to reptiles and birds.

Albert,

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-‘ _-‘

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APPENDIX I
STUDIES ON PROLACTIN INHIBITING FACTOR (PIF)

Meites (1959) found that virgin female rats injected
subcutaneously for 10 days with 10 ugm estradiol daily
followed by twice daily injections of 25 mg acetylcholine
iodide/kg body weight or 0.25 mg epinephrine in oil for 5
days, showed extensive lobulo-alveclar mammary growth and
lactation. Meites, Talwalker, and Nicoll (1960) obtained
similar results with epinephrine, acetylcholine iodide, and
serotonin in estrogen-primed female rabbits. Desclin (1960)
confirmed the finding of Meites (1959) that epinephrine
could initiate mammary secretion in estrogen-primed rats.
Meites, Nicoll, and Talwalker (1963) reviewed work demon-
strating that epinephrine, acetylcholine, and serotonin
can initiate lactation, maintain mammary secretion and pro-
duce pseudopregnancy in rats under certain experimental
conditions. These results are interpreted as indicating
that these neurohumors stimulate, directly or indirectly,
the release of prolactin from the pituitary.

Talwalker, Ratner and Meites (1963) demonstrated
the presence of a prolactin-inhibiting factor (PIF) in
acid extracts of rat hypothalami. The method developed by
these authors was applied by Ratner and Meites (1964) as

an ig_vitro assay method for PIF and used to obtain evidence

99

100

indicating that certain stimuli, including the suckling
stimulus, act indirectly on the pituitary by depleting the
hypothalamus of its PIF content.

Since Talwalker, Ratner and Meites (1963) found that
epinephrine and acetylcholine had no effect on the pitui-
tary in yitgg, it seemed of interest to attempt to explain
the lactational effects of these neurohumors by demonstrating
effects on the PIF content of the hypothalamus. Effects
of graded doses of hypothalamic extract on pituitary pro-
lactin release and of hypophysectomy on hypothalamic PIF

content were considered worth studying also.

Materials and Methods

 

The work reported here follows in detail the methods
used in the paper by Ratner and Meites (1964) except where
otherwise noted. For each of 2 experiments designed to
investigate the effects of epinephrine, experimental hypo-
thalamus donors were 12 Carworth CFN female rats treated
with epinephrine in oil (0.25 mg per injection) twice
daily for five days and killed 1 hour after the last
injection. The Parke-Davis Co. supplied most of the epine-
phrine used. Twelve CFN female control animals received
injections of 0.1 m1 of corn oil twice daily.

For each of 2 experiments designed to study the
effects of acetylcholine, 12 CFN female rats comprising the
experimental group received 25 mg acetylcholine bromide
(Eastman Co., Rochester) per kilogram body weight twice

daily. Control animals received saline injections.

101

Twelve intact female Sprague-Dawley rats were used
as control hypothalamus donors incnueexperiment. Twelve
hypophysectomized rats of the same age and strain also
served as controls.

The incubation procedures involved use of neutraliz-
ed acid extract equivalent to 2 hypothalami for each
pituitary equivalent incubated. In each incubation, 12
adult Spartan rat pituitaries were divided among three
flask pairs, i.e., there were 2 pituitary equivalents per
flask. The medium from each incubation was injected into
12 pigeons. In an experiment in which graded doses of hypo-
thalamic extract were used, 6 incubated pituitaries and 6
pigeons Were used for each dose level. Dose levels of hypo-
thalamic extract ranged from 1/2 to 4 hypothalami per
incubated pituitary. Control pituitary halves were incu-

bated with equivalent amounts of cerebral cortical extract.

Results: The results from the content study experi-
ments are presented in Table 1A. Effects of graded doses
of hypothalamic extract are presented in Figure 1A as
differences between control and experimental ratings for
each bird: vertical bars represent standard errors of the
mean.

Epinephrine treatment produced a definite reduction
in hypothalamic PIF content. Both experiments are signifi-
cant at the 0.5 level. Acetylcholine bromide produced
results which achieved acceptable levels of statistical

significance only when the 2 experiments were combined.

102

Table 1A. Effects of treatments on hypothalamic PIF content.

 

 

Epinephrine:

 

 

Exp. No. of Pigeons Mean Responses (RTU) P.

1 11 c = 0.500 E = 0.955 < .05

2 12 c = 1.000 E = 1.708 < .05
Combined
Data 23 c = 0.761 E = 1.308 < .0025

Acetylcholine Bromide:

 

 

 

Exp. No. of Pigeons Mean Responses (RTU) P.
1 12 c = 0.480 E = 0.750 < .06
2 12 c = 0.833 E = 1.083 < .10
Combined
Data 24 c = 0.656 E = 0.917 < .006
Hypophysectomy:
No. of Pigeons Mean Responses (RTU) P.
10 C = 0.950 E = 0.925 Not
(intact) (hypophy- signifi-

sectomized) cant

 

103

. 90¢ "

.80-

70- "

.60-

 

.50. '7 ii

.40.

i

.20-

Negative RTU

 

1
.10- i

 

 

.00

 

 

I l I

1/2 1 2 4

Hypothalamic Equivalents per Incubated Pituitary
(log log Scale)

Fig. 1A. Effects of Graded Doses of Hypothalamic Extract
' "on Pituitary Prolactin Release.

104

Hypophysectomy had no effect on hypothalamic PIF content.
Treatment of data from the PIF dose-response curve
poses certain problems. It is known that the Reece-Turner
unit (RTU) is linearly related to the logarithm of the dose
of prolactin in ugm or International Units (I.U.) within
a certain dose range, as are all but a few hormonal dose-
response relationships. It is postulated that the log dose
of PIF is inversely related to the amount of prolactin re-
leased in ugm or I.U. within a certain range of dose.
Therefore, the RTU should be related inversely to the log

log PIF within a certain dose range, i.e.,

RTU = -K1 log 16g PIF + K2

where K1 and K2 are constants related to threshold effects.
For the purposes of this study, it is convenient to analyze
these negative RTU's by first subtracting each experimental
crop rating from the matched control, then graphing results
as means and standard errors of the mean, as is done in
Fig. 1A.

Because of small numbers and birds differing con-
siderably in age and physical condition, the attempt to
demonstrate a graded response (dose-reSponse curve) was
not conclusive. However, the results do suggest a relation—
ship between amount of hypothalamic extract and degree

of inhibition.

105

Discussion

 

Results obtained in this work indicate that epine-
phrine and acetylcholine can reduce the hypothalamic PIF
content and presumably, its rate of release. It appears,
therefore, that the lactogenic effects of these neurohormones
are indirect and mediated through the hypothalamus. The
failure of hypophysectomy to modify PIF content may be con-
sidered as preliminary evidence that there is no feedback
of prolactin on the hypothalamus. However, this work is by
no means conclusive.

Because of the small numbers and ladk of uniformity
in the birds, the graded response experiment could not be
conclusive. It is, however, encouraging, Since almost
all hormones exhibit a log-dose response relationship,
whereas non-specific agents rarely do, demonstration of a
logarithmic relationship for PIF on prolactin release would
be further evidence that PIF is of physiological signifi-
cance and is not merely an artifact of extraction of the
hypothalamus. More recent work by Kragt (Ph.D. thesis,
Michigan State University, Dept. of Physiology, 1966)
definitely establishes that PIF shows a definite dose-

response relationship to pituitary prolactin release in vitro.

Desclin,

Meites,

Meites,

Meites,

Ratner,

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Urbana, 1963.

J., P. K. Talwalker, and C. S. Nicoll, Induction of
mammary growth and lactation in rabbits with epine-

phrine, acetylcholine and serotonin, Proc. Soc°

 

Exptl. Biol. Med., 104: 192-194, 1960.

A. and J. Meites, Depletion of prolactin-inhibiting
activity of rat hypothalamus by estradiol or suckling

stimulus, Endocrinology, 25: 377-382, 1964.

106

107

Talwalker, P. K., A. Ratner, and J. Meites, In vitro
inhibition of pituitary prolactin synthesis and

release by hypothalamic extract, Amer. J. Physiol.,

 

205: 213-218, 1963.

 

APPENDIX II

LIST OF PUBLICATIONS BASED ON WORK PRESENTED IN THIS THESIS

l.

Mittler, J. C. and J. Meites, Stimulation of Pituitary
Release by Hypothalamic Extract ig vitro, in Program,
46th Annual Meeting, The Endocrine Society, 1964.

(Abstract No. 8).

Mittler, J. C. and J. Meites, In Vitro Stimulation of
Pituitary Follicle-Stimulating-Hormone Release by
Hypothalamic Extract, Proc. Soc. EXptl. Biol. Med.,

117: 309-313, 1964.

Mittler, J. C. and J. Meites, Effects of Castration and
Androgen on FSH-releasing Activity of Hypothalamic

Extracts in Rats, Endocrinology, 18: 500-504, 1966.

108

   

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