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ABSTRACT
EFFECTS OF HYPO- AND HYPERTHYROIDISM ON THE REPRODUCTIVE SYSTEM
OF THE FEMALE RAT
By
Ingrid Zimmerman

The effects of hypothyroidism and hyperthyroidism on the female rat
reproductive system were studied in three separate experiments.

1. The purpose of Experiment 1 was to determine the effects of early
hypo- and hyperthyroidism induced by feeding propylthiouracil (PTU) or
injecting thyroxine (T4) on body growth, age of onset of puberty, regularity
of the estrous cycle, and pituitary prolactin content. PTU treatment,
begun prior to and continuing after birth, resulted in dwarfed, sexually
immature animals with relatively unstimulated ovaries and uteri. Puberty
was not reached in the majority of these rats. In comparison, control
untreated rats reached puberty at 36.7 t 0.6 days of age, and thyroxine-
treated animals at 44.4 f 1.2 days of age. Precocious puberty (31.7 t 1.0
days) resulted when goitrogen treatment was withdrawn at 21 days of age.
Anterior pituitary prolactin content was significantly decreased in the
severely hypothyroid rats.

2. The purpose of Experiment 2 was to determine the effects of early
hypo- and hyperthyroidism on anterior pituitary follicle stimulating
hormone (FSH) and lutenizing hormone (LH) content and on the hypothalamic
content of FSH-RF. Also, thyroxine treatment was withdrawn in a second
group of rats after 21 days to determine the effects of early treatment
on the onset of puberty. PTU treatment begun prior to and continuing
after birth resulted in decreased pituitary content of FSH and LH, while

the content of both hormones increased greatly in the hyperthyroid rats.

Ingrid Zimmerman

Hypothalamic FSH-RF content did not vary significantly between groups.
Puberty was found to be significantly delayed after discontinuing
thyroxine treatment.

3. The third experiment was designed to determine whether oronot a
critical period exists after which time thyroxine and goitrogen treat-
ments have no effect on the onset of puberty. Rats treated with thyroxine
from the let day of life until puberty showed no significant delay of

vaginal opening. Hypothyroidism also had no effect on vaginal opening.

EFFECTS OF HYPO- AND HYPERTHYROIDISM
ON THE REPRODUCTIVE SYSTEM or
THE FEMALE RAT.
3?

j? _ Ingrid Zimerman

A THESIS
Submitted to
‘Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Physiology

1971

TABLE OF CONTENTS

Page
INTRODUCTION 1.
LITERATURE REVIEW 3
General Effects of Thyroid Hormones 3
Thyroid Abnormalities 4
Mechanism of Action of Thyroid Hormones 5
Thyroid Influence on Brain Development 6
Control of the Reproductive System 7
EXPERIMENT I. Effects of Early Hypo- and Hyperthyroidism
on Reproductive Function 15
Prolactin Assay 17
Results 17
EXPERIMENT 2. Effects of Hypo- and Hyperthyroidism on
Pituitary FSH and LH and Hypothalamic FSH-RF 25
Preparation of Hypothalamic Extract 26
Incubation of Pituitaries with Hypothalamic Extract 26
FSH Assay , \ 28
LH Assay 28
Results 29
EXPERIMENT 3. Effects of Treatment with PTU or T4
Beginning at 21 Days of Age on Onset of
Puberty 35
Results 35

ii

GENERAL DISCUSSION

REFERENCES

iii

37

42

LIST OF TABLES

Table ' Page
1. AGE OF VAGINAL OPENING IN CONTROL, HYPERTHYROID AND

HYPOTHYROID RATS o o o o o o o o o o o o o o o o o o o o o o 20
2. ORGAN WEIGHTS OF CONTROL AND PTU'TREATED RATS. . . . . . . 23
3. PROLACTIN CONCENTRATION AND CONTENT IN PTU'TREATED AND

CONTROL RATS o o o o o o o o o o o o o o o o o o o o s o o o 24
4 o PROCEDURES FOR FSH-RF INCUBATION o o o o o o o o o o o o o o 2 7

5. THE EFFECT OF EARLY THYROXINE TREATMENT ON THE ONSET OF
PUBE RTY. O I I 0 I O O O O O I O O O O O O l O O O O O O O O 30

6. ORGAN WEIGHTS OF ANIMALS TREATED WITH THYROXINE AND
PROPYLTHIOURAC IL 0 C . C . . O O C O . . . . . . . C O . . . 31

7. EFFECTS OF PROPYLTHIOURACIL AND THYROXINE TREATMENT ON
PITUITARY FSH CONTENT AND CONCENTRATION AND HYPOTHALAMIC
FSH-RF CONTENT o o o o o o o o o o o o o o o o ‘0 o o o o o o 33

8. EFFECTS OF PROPYLTHIOURACIL AND THYROXINE TREATMENT ON
PITUITARY LH CONTENT AND CONCENTRATION. . . . . . . . . . . 34

9. AGE OF VAGINAL OPENING IN ANIMALS TREATED WITH THYROXINE
AND PROPYLTHIOURACIL FROM THE TWENTY-FIRST DAY OF LIFE. . . 36

iv

LIST OF FIGURES

Figure Page

1.

BODY WEIGHTS OF CONTROL, HYPERTHYROID AND HYPOTHYROID .
RATS O O O O O O O O O O O O C O O O O O O O O O O O O O O O 18
ESTROUS CYCLES OF CONTROL, HYPOTHYROID AND HYPERTHYROID

RATS. O O O O O O O O O O O O O O O O 0 O O O O O O O O O O 21

INTRODUCTION

The thyroid gland of all vertebrates secretes thyroid hormones into
I the circulation. In man and in higher animals, these substances have a
profound influence on the development of the body. Although much is
known about the biochemistry and biological effects of the principle
thyroid hormones, L-thyroxine (t4) and 3,5,3'-L-triiodothyronine (t3),

' little work has appeared on their effects on the gonadotropins of the
pituitary and on reproduction in general.

In humans, it is known that decreased thyroid activity or hypothy-
roidism from infancy leads to sexual immaturity and infertility. Hypo-
thyroidism in the adult woman is commonly associated with loss of libido,
failure to ovulate and irregular menstrual patterns.

Hyperthyroidism, or increased thyroid hormone in the circulation,
also causes abnormal sexual development. Female patients with this
condition, (thyrotoxicosis), often have disturbed menstrual cycles which
in severe cases cease altogether. When ovulation does occur, fertility
is low and abortion is common. It has also been reported that puberty
is delayed in some of these patients. It is a reasonable working
hypothesis to assume that abnormalities in sexual development of human
subjects with thyroid disorders are directly related to altered secretion
of pituitary gonadotropins, although direct effects of the thyroid on
the gonads are also possible. While some anomalies remain, the changes
in sexual deveIOpment observed in the rats in these studies generally

support this contention.

2

Although there have been many investigations on the effects of
abnormal thyroid secretion on the reproductive system, there is little
in the literature to explain the mechanisms by which thyroid hormones
influence the gonads. The studies described here were designed to
further elucidate the mechanism of thyroid action at the pituitary and
hypothalamic levels. The pituitary content of gonadotropins and of
prolactin in the female rat were measured under normal, hyperthyroid
and hypothyroid conditions. The hypothalamic content of follicle
stimulating hormone releasing factor (FSH-RF) was also measured, and
ovarian and uterine weights were recorded. The pattern of the estrous

cycles for each rat and the age of onset of puberty were recorded.

REVIEW OF THE LITERATURE

general Effects of Thyroid Hormones

In warm-blooded animals thyroid hormones accelerate energy production
and processes related to increased metabolism in most normal tissues
(Lardy, 1955). Without thyroid hormones, protein synthesis is decreased
and profound shifts in protein stores occur leaving such organs as the
liver and kidneys small and protein deficient (Pitt-Rivers and Tata, 1959).

The effects of thyroid hormones on carbohydrate metabolism are
modified by other hormones, such as insulin and epinephrine, and are
dependent upon the levels of thyroid hormones present. Small doses of
T4 stimulate glycogen synthesis while large doses cause hepatic glycogen
depletion (Wolff and Wolff, 1964). In general, thyroid hormones tend to
increase the blood concentration of glucose by increasing intestinal
absorption of glucose, by depleting liver glycogen and by increasing
insulin degradation (see Hoch, 1962). This is somewhat offset by the
increased energy and oxygen requirements that T4 is known to invoke.

Thyroid hormones also influence lipid metabolism. T4 stimulates
lipid synthesis, mobilization and degradation although the predominant
effect of high levels of T4 is catabolic. In hypothyroidism an increase
in blood cholesterol and phospholipids is generally found (Kritchevsky,
1964).

The metabolism and excretion rate of hormones are also influenced
by the thyroid hormones, although this has not been thoroughly investigated.
In hypothyroidism, androgen secretion has been found to decrease and

3

4
testosterone and dehydroepiandrosterone have been reported to be trans-
formed into etiocholanolone rather than androsterone (Gallagher et al.,
1960). Thyroid treatment also increases the excretion of androsterone in
the male, and patients with untreated myxedema excrete decreased amounts
of androsterone (Campbell et al., 1965). Fishman et al., (1965) found
that hypothyroidism also alters the metabolism of estradiol, converting
it into estriol more often than into 2-hydroxyestrone. The same authors
reported that hyperthyroidism causes a decrease in the conversion of
estradiol to estriol and an increase in the 2-methoxyestrone fraction
(Fishman et al., 1962).

Thyroid Abnormalities.

 

The absence of thyroid hormones during early post-natal life causes
a child to become a cretin. When left untreated, such a child developes
into a short, dwarf-like person with definite and irreversible mental
retardation. Thyroxine replacement therapy is effective in reversing
this condition only when administered at or near birth (Ingbar and Woeber,
1968).

The severe shortage of thyroid hormones affects the development of
nearly every system in the cretin: growth and maturation are retarded,
the rate of metabolism is decreased, there is reduced activity in the
alimentary and renal systems, blood flow and blood volume are decreased,
muscle contraction and relaxation time is slowed and the reproductive
system functions poorly (Ingbar and Woeber, 1968). Cretins rarely
exhibit normal sexual develOpment, and in the case of the female, rarely
have normal menstrual cycles or successful pregnancies (Steinbeck, 1963).

Some of the symptoms of hypothyroidism may be caused by changes in
other endocrine glands such as the ovaries or the pituitary. Lack of

thyroid hormones is thought to depress secretion of adrenocorticotropic

5
hormone (ACTH), growth hormone (GH), prolactin (LTH), and the gonado-
tropins (LH and FSH) of the pituitary, while thyroxine is thought to
stimulate secretion of these hormones (Eartly and Leblond, 1956; Nicoll
and Meites, 1963; Rall et al., 1964). However, a lack of thyroid
hormones does not decrease anterior pituitary metabolism (Reichlin, 1966).

When hypothyroidism occurs in humans after infancy, it is referred
to as myxedema. Physical lethargy and mental dullness mark this condition,
although in severe cases most of the symptoms of cretinism are present.
Because the disease does not occur during the critical period of early
development the symptoms are reversible with proper hormone replacement
therapy (Ingbar andWoeber, 1968).

Hyperthyroidism produces rapid blood flow, increased metabolism and
increased nervous responses. This condition does not lead to increased
strength and enhanced ability to function, however, and weakness and
fatigue are common symptoms (Ingbar and Woeber, 1968).

Hyperthyroidism is also known to affect the reproductive system. In
early life hyperthyroidism may cause delayed puberty and in later life
may cause abnormal menstrual cycles, decreased fertility and increased
risk of abortion in women (see Steinbeck, 1963).

Mechanism of Action of Thyroid Hormones

Cretinism and thyrotoxicosis have been mimicked in laboratory animals
and in tissue culture in an effort to elucidate the way in which thyroid
hormones act. Such mechanisms are studied on two levels: 1) the organ
or system level, and 2) the cellular or biochemical level.

Little is known about the true action of thyroid hormones at the
cellular level. Following administration of moderate doses of T4, changes
in mitochondrial structures have been seen (Wolf and Wolf, 1964), and

enhanced protein synthesis has been observed for T4 stimulated mitochondria

6

(see Tata, 1964). Recently it was discovered that nuclear RNA turnover
rate increases upon T4 stimulation as does DNA dependent RNA polymerase.
All classes of RNAs are apparently effected by this stimulation (Tata,
1970). During the last decade the hypothesis has been generated that the
ubiquitous constituent of plasma membrane, adenyl cyclase, is directly
involved in the action of hormones (Robison, 1968) and that cyclic 3' -5'
AMP mimics the effects of hormones on their target cells. It has been
shown that hormones can act on tissues to increase the levels of 3'5'-AMP.
These hormones include the catecholamines, glucagon, ACTH, vasOpression,
LH, and TSH (thyroid stimulating hormone) (Sutherland, 1965). It is
thought that hormones such as the steroids and T4 may relay their
messages by similar systems (Sutherland, 1965). The effects of thyroid
hormone on the brain, the pituitary and the gonads will be reviewed.
Thyroid Influence on Brain Deve10pment

In the rat, a species whose brain is poorly developed at birth,
there appears to be a critical period before 15 days postpartum when the
presence of thyroid hormones is necessary for normal cerebral maturation
(Campbell, 1965). If thyroid hormones are absent at this time, the
capacity to learn is retarded (anrs, 1961), and the age at which innate
responses can first be elicited is increased (anrs, 1964b). During the
past twenty years the etiology of thyroid-related brain disturbances has
been studied. It has been found that there is a decrease in cell
processes and impairments in the axonal networks of the cortex, reducing
the probability of axo-dendritic interaction in the hypothyroid rat
(Uttley, 1955). Hypothyroidism has also been found to alter the shape of
the endocranium and to cause abnormal brain vascular pattern, increasing
the probability of brain damage (anrs, 1954).

An excess of thyroid hormone has been found to cause hyperplasia of

7
the cerebellar folia and a hastened regression of the fetal cortex in
young rats (Tusques, 1956).

Thyroid hormones also act on various enzyme systems in brain tissue.
Hamburgh and Flexner (1957), demonstrated that the development of cere-
bral cortical succinic dehydrogenase was irreversibly impaired in animals
made hypothyroid before the 15th day of age. Others have reported de-
creased gamma-amino butyric acid dehydrogenase activity in the cerebrum
of thyroidectomized rats (Argiz et al., 1967). According to Rall et al.,
1964, thyroid hormones probably act mainly as chelating agents, binding
metals that normally inhibit enzymes such as the dehydrogenases.

Control of the Reproductive System
The reproductive system in mammals has long been represented by the

'negative feed-back' or servo' hypothesis first applied to the pituitary-
thyroid interrelationship (Hoskins, 1949). The gonads and the pituitary
were thought to secrete hormones that effected each other, achieving a
balance within which reproduction could occur. It is now understood

that the hypothalamus, located in the brain above the pituitary, secretes
small descrete polypeptides known as 'releasing' or 'inhibiting' factors
which directly influence the pituitary, and hence reproduction.

The hypothalamic factors related to reproduction are follicle
stimulating hormone releasing factor (FSH-RF) and luteinizing hormone
releasing factor (LH-RF). Prolactin inhibiting factor (PIF) may also
be concerned with reproduction.

The pituitary hormones related to reproduction are known as FSH
and LH in the female. Prolactin or lactogenic hormone (LTH) is concerned
with the maintenance of corpora lutea in animals such as rats and mice

and with milk production in all mammals.

It is known that hormones from the hypothalamus stimulate (in the

8

case of PIF, inhibit) the anterior pituitary which in turn produces
hormones that stimulate the gonads (and the mammary glands). Until
recently, the gonads were thought to produce steroids that selectively
inhibited or stimulated pituitary production of one or both gonado-
tropins (FSH or LH) (see Creep, 1961). It is now known that the control
of mammalian reproduction is much more complex.

The following hypothetical model depicts the control of reproduction
in the adult female rat as it is now understood (see Schwartz, 1967;
Schwartz and Hoffman, 1967; McCann et al., 1967; Motta et al., 1969;

Flerko, 1966; and Davidson, 1969).

 

 

 

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A MODEL OF THE CONTROL OF REPRODUCTION IN THE FEMALE RAT

E = estrogen
P = progestrone
LH = luteinizing hormone
FSH = follicle stimulating hormone
LTH = prolactin, lactogenic hormone
-RF = releasing factors
PIF = prolactin inhibiting factor

9

In the preceding model the hypothalamus is divided into two areas;
an anterior area, the preoptic area, and the hypophysiotropic area
including the median eminence (ME). The preoptic area controls the dynamic
flow of pituitary LH and FSH, allowing a large discharge of these hormones
to occur which precipitates ovulation in the mature female rat (see
Flerko, 1966). In the ME region the production and release of LHRF and
FSHRF ensure a static level of pituitary stimulation (Flerko, 1966).
Ablation of this area (Szentagothai and Halasz, 1964) or removal of the
pituitary from beneath the ME (Nikitovitch-Winer and Everett, 1957)
depresses the production of LH and FSH, causing the ovaries to atrophy.
Pituitary production of prolactin, however, is not inhibited when
pituitary-hypothalamic connections are severed (Everett, 1956).

Prior to ovulation, LH and especially FSH stimulate the ovaries to
produce estrogen and some progesterone (see Schwartz, 1967). Estrogen
appears to feed back directly on the pituitary to stimulate LTH secretion
(see Meites, 1959; Nicoll and Meites, 1962) and may directly affect the
pituitary content of the gonadotropins (Bogdanove, 1963, 1964). However,
the major site of action for estrogen and progesterone is thought to be
at the hypothalamic level. It is not known whether different dose
levels of estrogen and progesterone pg£_§g_control the hypothalamic centers,
or whether it is the effect of the changing ratios of the two steroids.
Both steriods have been reported to stimulate and to inhibit the same
centers (Sawyer & Everett, 1959; see Davidson, 1969). When the estrogen-
progesterone ratio increases to a certain level the hypothalamic centers
appear to release a surge of LHRF which triggers ovulation and causes
the development of corpora lutea from the ovulation site (see Schwartz,
1967). If mating has occurred and plasma LTH is relatively high

(Amenomori et al., 1970), the corpora lutea are maintained and systemic

10
progesterone levels increase (see Everett, 1961). It is thought that
LH (see Rothchild, 1965) or in some species a uterine substance
(Nalbandov, 1964) may limit the life of the corpora lutea.

Other central nervous system inputs influence the reproductive
cycle. Schwartz and Bartosik (1962) have shown that there is an internal
regulator governing the ovulatory surge of LH and it has been shown that
the length of daylight sets this internal clock (Everett and Sawyer,
1950). Lactation and suckling also influence the release of LH, FSH
and prolactin via the central nervous system (see Meites, 1966).

Recent experiments have further complicated the story. Corbin,
(1966) discovered that there was short feedback effect of FSH on its own
releasing factor. Later Fraschini et al., (1968) confirmed this. Corbin,
1966 showed that implantation of small amounts of LH into the ME region
(of the hypothalamus selectively decreased pituitary and plasma LH.
MacLeod, (1966) showed, via disk electrophoresis,that prolactin secreting
pituitary tumors suppressed endogenous pituitary prolactin and Chen et al.,
(1967) independently observed this using the pigeon crop method for LTH
determination. The latter also reported an increase in hypothalamic PIF.
These results were confirmed in the same laboratory by different tech-
niques (Clemens and Meites, 1968; Welsch et al., 1968).

Superimposed upon this basic model are the other endocrine glands
concerned with reproduction. The thyroid will be reviewed here.
Interactions of the Thyroid and the Reproductive System

The interactions between the thyroid and the reproductive system
have been studied in various experimental animals. Soliman and Reineke,
(1954), reported that the female rat thyroid gland fluctuated in its
iodine uptake in accordance with the estrous cycle, collecting the

maximum iodine during estrus and reaching a minimum at proestrus. The

11
same authors found that endogenous estrogen treatment had similar effects
on the thyroid while progestrone antagonized the stimulatory effects of
estrogen (Soliman and Reineke, 1955). Others have found similar effects
of estrogen on radioactive iodine uptake in the rat (Feldman, 1956;
Brown-Grant, 1962). Iodine release rates appear unaffected in the rat,
_ however, (Brown-Grant, 1962). A review of the literature (see Feldman,
1956; Florsheim, 1958) reveals extensive work on this subject with many
conflicting results, partly explained by the variety of dose levels of
estrogen used and the degree of inanition resulting.

The effects of estrogen on the thyroid may be direct (Feldman,

1956; Florsheim, 1958) although Soliman and Reineke (1955) found no
stimulatory effect of estrogen or progesterone on the thyroids of hypo-
physectomized rats. More recently, the pituitary and higher centers have
been implicated. Brown-Grant (1963) has shown that the administration

of nembutal on the day of proestrus blocked the ovulatory surge of LH and
suppressed the increase in thyroid gland activity usually seen at estrus.
It has been suggested by the same author that there may be a spread of
impulses from the LHRF center to the TRF (thyroid stimulating hormone
releasing factor) center and that this is blocked by nembutal. Yamada

et al., (1966) has also reported that the pituitary is involved in the
thyroidal response to estrogen. Thyroid weight gains usually seen with
estrogen stimulation are not seen in similarly treated hypophysectomized
rats and only slight iodine uptake is noted in these animals.

In humans, estrogen stimulation has been found to increase the
thyroxine-binding proteins (Ingbar and Freinkel, 1960) and the level of
P31 (Engbring and Engstrom, 1959) while leaving total thyroid function
unaltered (Dowling et al., 1959).

It has long been known that the thyroid also influences the

12
reproductive system. In humans, thyroid therapy is one of the most
effective means of correcting menstrual abnormalities (Foster and
Thornton, 1939). In 1921, Evans and Long reported that thyroidectomy
caused a pause in the estrus cycles of rats. Others have found that
either thyroidectomy or goitrogen administration causes irregularities
of the cycle in many experimental animals (see Reineke and Soliman, 1953).
Thyroid feeding has been reported to delay impregnation in rats
(Gudernatsch, 1915) and to suppress ovulation when fed in large quanti-
ties (Evans and Long, 1921).

In acute hypothyroidism, the ovarian picture in the rat is one of
follicular growth (see Reineke and Soliman, 1953), and in the human with
myxedema, ovarian cyst formation is often found (Leathem, 1959). In
patients with myxedema due to destruction of the thyroid, precocious
menstruation accompanied by breast development and galactorrhea have
been reported (Nalbandov, 1963).

In acute hyperthyroidism the luteal cells predominate. Weichert and
Boyd (1933) reported that rats fed thyroid powder appeared pseudo-pregnant.
Long term treatment with thyroid powder, however, has been reported to
inhibit the normal growth and maturation of the ovaries (Ershoff, 1948).

There has been some investigation on the mechanism of action of
thyroid hormones on reproduction. Fluhmann (1934) found that thyrotropic
hormone (TSH) contamination decreased the stimulatory effects of
pituitary gonadotropin on ovarian weight in the rat. In 1936, Leonard
reported that thyroidectomy augmented the ovarian and uterine response to
FSH. Johnson and Meites (1950) showed that hyperthyroidism in rats de-
creased the ovarian response to pregnant mares' serum, while short term
treatment with the goitrogen, thiouracil, augmented the response.

Chronic administration of the goitrogen, however, reduced the ovarian

13
response was stimulated by hyperthyroidism in mice, suggesting that this
species reacted differently from the rat and secreted less than an optimal
amount of thyroid hormone for maximal response of the ovaries to gonado-
tropin administration. '

Severinghaus (1937) reported that hypothyroidism caused a decrease
in pituitary acidophil granulation, although there has been disagreement
as to whether the growth hormone (GH) content of such pituitaries
decreased. Schooley et al., (1966) pointed out that this discrepancy may
be due to the synergistic stimulation of the assay animals by contaminat-
ing TSH. McQueen-Williams (1935) and Meites and Turner (1947) have shown
that pituitary prolactin (also produced by acidophils) is decreased in
hypothyroidism. Further, Nicoll and Meites (1962) showed that thyroid
hormones accelerate pituitary prolactin release i§_gi££g, Macleod (1965)
found that thyroid hormones stimulate the growth of the pituitary tumor
MtTWS’ a predominantly acidophilic tissue which secretes both prolactin
and growth hormone.

Contopoulos et a1. (1958) did a series of experiments on gonadecto-
mized and thyroidectomized male rats. It was found that thyroidectomy
alone increases plasma TSH and decreases plasma CH. The anterior
pituitaries from these animals were found to be deficient in FSH, ICSH
(interstitial cell stimulating hormone), GH and TSH. Gonadectomy super-
imposed upon thyroidectomy reinstated the low levels of plasma and
pituitary gonadotropins, showing that the pituitary of thyroidectomized
animals is capable of responding to a decrease in steroid feedback.
Measurements were not specific for ICSH and FSH and were accomplished by
the method of ovarian and uterine weight response in hypOphysectomized,
immature female rats.

There is general agreement that experimental hyperthyroidism causes

14
an increase in the size and number of pituitary basophils (Kojima, 1917;
Halmi, 1952 and Severinghaus, 1937). Several investigators have
attempted to correlate a pituitary change in gonadotropin content with
hyperthyroidism. In 1930, Evans and Simpson thyroidectomized female rats
and after five weeks assayed the pituitaries for total gonadotropin.
The pituitaries from the hypothyroid animals were compared with normal
pituitaries and with pituitaries from a third group of animals fed
thyroid powder for five weeks for their relative ability to advance
sexual maturity in immature host rats. It was found that hypothyroidism
decreased the potential of the pituitaries, and hyperthyroidism stimulated
it. Smith and Engle (1930) however, reported no change in anterior
pituitary gonadotropin content when female rats were thyroidectomized.
In 1931, Van Horn observed that pituitaries taken from female rats fed
large doses of thyroid powder and then implanted into immature female
rats stimulated the gonads of the host more than did pituitary grafts
taken from untreated animals. However, direct measurements of pituitary

LH and FSH after thyroid hormone manipulation have not been recorded.

15.
EXPERIMENT I
Effects of EarlypHypo- and Hyperthyroidism on Reproductive Functions

The purpose of this experiment was to determine the effects of early
hypothyroidism and hyperthyroidism on the reproductive system in the
female rat. The hypothyroid animals were compared with euthyroid and
hyperthyroid animals for age of puberty, regularity of the estrous cycle
and body weight. Organ weights and pituitary prolactin content were
measured in the hypothyroid and control rats.

Mature male and female rats of the Spraque-Dawley strain (Spartan
Animal Farms, Haslett, Michigan) were housed in a constant temperature
room (25 TC) with automatically controlled lighting (14 hours light,10
hours dark), and were given food and water 3g lib. One male and five
females were placed in each of six cages and allowed to breed. The
females were checked daily for vaginal plugs and for the appearance of
sperm in the vaginal smears at the time of estrus, as an indication of
insemination. Estrous cycles were followed until a definite diestrous
pattern was noted, indicating pregnancy, at which time each female was
placed in a separate cage with adequate nest-building material. These
cages were not kept in temperature-controlled animal rooms and the room
temperatures fluctuated greatly. On the fifteenth day of pregnancy the
females were separated into three groups and treated as follows:

Group 1. control rats. 8 animals, 4 injected with saline, 4 not

injected.

Group 2. hypothyroid rats. 15 animals, all fed 0.1% PTU

(propylthiouracil) in feed.
Group 3. hyperthyroid rats. 7 animals, all injected with 10 ug
L-T4 (thyroxine) per 100 gm body

weight.

16

At parturition all the male young were discarded. The young females
of mothers in the control group were counted, marked, weighed when
necessary and allowed to remain with their mothers until 21 days of age.
Half of the young were injected with 0.1 m1 saline daily.

All the young of the hypothyroid mothers received the goitrogen (PTU)
from the milk of their mothers who were fed 0.1% PTU for 21 days after
parturition. Fifteen young rats were injected with 5 mg PTU/day for 20
days. However, all of the hypothyroid young appeared similarly affected
regardless of the route of PTU administration, and were combined at 20
days of age. On the 21st day of life, 10 of the hypothyroid young were
placed in separate cages and fed a regular diet of Wayne Lab Blox. These
animals were not treated with PTU further. The remaining animals were
placed in new cages (approximately 10 rats per cage) and continued on a
diet of 0.1% PTU in ground Wayne Lab Blox. Those young that were severely
hypothyroid were allowed to remain with their mothers for several
additional weeks. All hypothyroid young that continued to be treated
were kept in cages with nesting materials to conserve body heat.

The nursing mothers in the hyperthyroid group were injected daily
with 10 ug T4 per 100 gm body weight until weaning. The pups of the
hyperthyroid mothers were allowed to suckle until their let day of life,
at which time they were placed in new cages. A dose of 10 ug T4/100 gm
body weight was injected subcutaneously in the pups from day 15 until
sacrifice.

The young from all groups were weighed and checked for vaginal
opening and the stage of the estrous cycle. At approximately 60 days of
age, the animals were sacrificed and the pituitaries, thyroids, ovaries,
and uteri of 17 hypothyroid and 11 euthyroid (control) rats were dissected

out and weighed. The pituitaries were quickly weighed, frozen and stored

17

for prolactin assay. Body weight and length were also measured.
Prolactin assay

Prolactin content in the control and hypothyroid pituitaries was
assayed in six to eight week old White King squabs by the intradermal
pigeon crop sac method of Lyons, (1937). A direct comparison was made
between samples by injecting the control sample over one side of the
pigeon crop sac and the experimental sample over the opposite side. The
prolactin responses in each bird were rated by the method of Reece and
Turner (1937) and were converted into IU from a standard dose-response
curve established with NIH-B1 prolactin in pigeons of similar age and
breed. The results of the assays were analyzed by the 't'-test for

paired experiments.

:Results

The female rats that were bred became pregnant within two weeks of
each other. In the control group, 6 females littered a total of 25
female pups. In the hypothyroid group, 12 mother rats gave birth to 57
females, 27 of which died during the course of the experiment. The
severity of the hypothyroid condition varied greatly from animal to
animal but was independent of the route of PTU administration. In the
hyperthyroid group, only 3 female rats gave birth to live young. A total
of 12 female pups was obtained from these animals.

Body weights were measured daily in all groups of young rats prior
to, during and after puberty. These data are shown in Figure 1. Both
T4 and PTU treatments decreased body weight. The T4-treated animals did
not increase in weight as linearly as did the controls, but they did not
differ from the control animals greatly in their overall weight.

Continuous PTU treatment significantly retarded growth, and so severely

dwarfed the animals that their body weights averaged only 55 grams at

18

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92¢ o_om>_._._.on_>z 024
o_om>z._.mma>z .Jomhzoo “.0 9:55? >oom

.mmaorm

one? an:

 

 

: hm
nag-.23

Ugo-002v
.- h .-

.08....»

a;

 

(I)

"on I when lug shun

 

l9
forty days of age. The young animals whose PTU treatment was terminated
after 21 days gained weight rapidly. However, they never became as
heavy as either the control rats or the Té-treated animals.

'The age at which puberty was attained was recorded as the day of
vaginal opening. A vaginal smear of each animal was taken at that time.
All of the animals showed either cornified cells or a mixture of
cornified cells and leukocytes, confirming the presence of estrogen. The
average ages of vaginal opening for all groups are shown in Table l. The
average age of vaginal opening in the control group was 36.7 t .6 days
while the range was from 34 to 41 days. Puberty was first observed in
the T4-treated animals at 39 days and seen as late as 51 days with a
mean of 44.4 T 1.2 days of age. These animals were slightly heavier than
the controls at puberty. Continuous PTU treatment retarded body growth
and caused almost complete cessation of puberty. Several such animals
showed no signs of vaginal opening after 100 days of age. The ten
animals from the hypothyroid group taken off PTU feedings after 21 days
of age showed precocious signs of puberty. The first animal to exhibit
vaginal opening in this group was 27 days old; the average age of puberty
for this group was 31.7 t 1.0 days. The size and body weights of these
animals were significantly below those of the control rats at puberty.

For one month beginning on the first day of vaginal Opening, vaginal
smears were taken and a record of the estrous cycle was kept. In
Figure 2, three representative cycles from each group are shown. The
peaks represent the time of estrus (E) when the smear showed only cornified
cells, and the lowest levels represent diestrus (D) when the majority of
the cells were leukocytes. In the control animals approximately 48% of
the cycles are represented by estrus-proestrus, and neither the T -

4

treated nor the animals with discontinued PTU-treatment differed from this

20

TABLE 1

AGE OF VAGINAL OPENING IN CONTROL,
HYPERTHYROID AND HYPOTHYROID RATS

 

 

No. of rats Body weight at Age at

 

Treatment per group vag. opening vag. opening
Controls 18 110.2 36.7 I 0.61
Thyroxine _ 12 121.0 44.4 I 1.20
treated *1
Continuous 20 - >50 *3
PTU *2
PTU 10 64.0 31.7 I 1.0
discontinued

(at 21 days

of age)

 

*1. 10.0 Mg T4 injected/day after 15 days of age. Mothers of these
rats treated with 10 ug/lOOgm body weight/day from 15th day of
gestation until weaning at 21 days.

*2. 0.1% PTU placed in feed

*3. In several animals vaginas were not open at 100 days of age.

stages of estrous cycle

W‘W "VFW?" “P"WP-WVWW I

'-

21

controls

PTU
dusconhnusd

W

W

W
ir—x.

\. / \ GOfl;l:;OUO
«A. /" '\ .. z .
W
W

W: M T4 treated

doys (one month)

FIGURE 2

ESTROUO CYCLES OF CONTROL,HYPOTHYROID
AND HYPERTHYROID RATS

22
significantly. However, in the group of rats treated continuously with
PTU, those animals that did cycle were very irregular and exhibited
estrus-proestrus only about 30% of the time.

The weights of the various organs of 11 animals from the control
group and 17 animals from the hypothyroid group are shown in Table 2.

The hypothyroid animals had dwarfed bodies with weights less than one-third
that of the controls. The ovaries of these Jcretins' were greatly reduced
in weight and the uteri were small and showed no sign of estrogen stimu-
1ation. The thyroids were approximately 5 times as large as the control
thyroids, when expressed as mg/lOOg body weight and the pituitaries,

when similarily expressed, weighed slightly more than did pituitaries

from control rats.

Anterior pituitary prolactin concentration and content for the 11
control and 17 hypothyroid rats can be seen in Table 3. Prolactin levels
were significantly higher in the cycling, control females than in the
sexually immature, PTU-treated rats of the same age.

From the data in Experiment 1, it may be concluded that chronic
hypothyroidism depresses body growth and retards sexual maturity.
Pituitary prolactin concentration and content are also significantly
decreased in such animals. In those animals that do reach puberty the
estrous cycles are irregular and show a lack of normal follicular develop-
ment. Puberty is also significantly delayed in female rats made
continuously hyperthyroid by T4 administration from an early age, although
body growth and sexual deve10pment apparently are not seriously impaired.
From this experiment it can also be seen that precocious puberty follows

the removal of PTU in female rats under certain experimental conditions.

23

ORGAN WEIGHTS OF CONTROL AND PTU-TREATED RATS

 

 

 

 

 

 

 

 

ORGANS CONTROL PTU
Ovary (single) 31.4 I 1.6 6.6 t 1.1
(m8)
Uterus 379.6 I 26.8 83.1 I 11.9
(mg) '
Thyroid 15.2 I 0.7 21.8 I 0.5
(m8)
Thyroid . 6.6 I 0.3 31.4 T 0.6
(mg/IOOgm body wt.)
. . + +
Pituitary 11.8 - 0.8 4.8 - 0.4
(m8) ‘
Body weight 230.7 I 2.8 71.8 I 1.1
(8)
Body length 21.7 0.2 13.5 I 0.3

.I+

(cm)

 

 

 

24

TABLE 3.

PROLACTIN CONCENTRATION AND CONTENT
IN PTU-TREATED AND CONTROL RATS

 

CONTROL PTU

 

 

 

No. of rats 11 ' 17
per group '

 

Pituitary 11.8 4.8
weight
(m8)

 

Reece-Turner Units

(RTU) 0.50 0.28
LTH per mg
pituitary

 

RTU
LTH per 5.92 1.33
pituitary '

 

Significance 1%
level

 

 

No. of birds 10 10
per group '

 

 

25
EXPERIMENT 2
Effects of Hypo-and Hyperthyroidism on Pituitary FSH and LH and Hypo-

thalamic FSH-RF

 

The purpose of Experiment 2 was to determine the effects of hypo-
thyroidism and hyperthyroidism on pituitary FSH and LH content and on the
hypothalamic content of FSH-RF. It is known that the continuous state of
hyperthyroidism may change the metabolism of steroids and increase their
breakdown in the body. The effect of such an event on estrogen would
mimic pituitary gonadotropin inhibition. A group of rats in Experiment 2
was treated with T4 from the 15th day of gestation until 21 days of age
and allowed to reach puberty without further treatment. The purpose of
this was to determine the effects of early treatment with T4 on sexual
maturation without the metabolic effects of excess T4 in the circulation
at the time of puberty.

Sixty adult Spraque-Dawley rats were housed, bred and fed as in
Experiment 1. On the 15th day of pregnancy the females were placed in
separate cages and treated as follows:

Group 1. control rats. 15 animals per group, no treatment.

Group 2. hypothyroid rats. 16 animals per group, all given 0.1%

PTU in feed. -
Group 3. hyperthyroid rats. 20 animals per group, all injected with
10 ug T4/100 gm body weight.

At parturition all the male young were discarded and the female
young were treated as in the first experiment, with one exception.
Several days after giving birth, 5 mothers, with a total of 24 female
pups, were set aside. These nursing females were injected subcutaneously
daily with 10 ug T4/100 gm body weight until the pups were weaned at 21

days. The pups were then allowed to mature without further treatment.

26
The time of puberty was measured in these young and in a corresponding
control group by observing the day of vaginal opening by observing an
estrous smear.

At 45 days of age 10 rats from the control group, 17 hypothyroid rats,
and 12 rats treated continuously with T4 were sacrificed. The pituitaries
were quickly removed, weighed and frozen for future FSH and LH assay.

The hypothalami were rapidly removed and stored in 0.1N HCL at ~20°C.
The ovaries, uteri and adrenals were removed and weighed and the ovaries
were fixed, sectioned and stained for histological examination.

Preparation of Hypothalamic Extract

 

Prior to incubation, the hypothalami were thawed, homogenized in
chilled 0.1N HCl and centrifuged at 12,000 xG for 40 minutes at 4°C.
The supernatants were added to protein free medium 199 (Difco Labs.,
Detroit, Michigan) containing NaHCO3, and the pH was adjusted to 7.4 by
the addition of 0.1N NaOH. (Refer to Table 4 for exact amounts of all
reagents used). After neutralization, the hypothalamic extract was
immediately added to the incubating pituitaries.

Incubation of Pituitaries with Hypothalamic Extract

 

Male rats were killed by decapitation and each anterior pituitary
(AP) was removed, separated from the posterior lobe and placed in a
petri dish on filter paper moistened with cold medium 199. The incubation

procedure for FSH-RF (Mittler and Meites, 1966) was as follows:

Amount No. AP's No. flaSks Pre-incub. Incub.
VHE/AP per flask per group time (min.) time (hrs.)
0.25 4 I 4 30 6

In order to minimize the differences between donor pituitaries, each
AP was bisected and the halves distributed so that each group had halves
from a different pituitary. The equivalent of 16 pituitaries or 32

halves was placed in groups 1 and 2; one-half of each anterior pituitary

27

TABLE 4.

PROCEDURE FOR FSH-RF INCUBATION

 

CONTROL

 

T -TREATED

 

PTU-TREATED

 

No. of hypothalmi
in 3.0 m1 of
0.1N HCL per group

10

 

Hypothalamic
equivalents after
centrifugation

7.3

 

Amount of
Medium 199 added

(ml)

0.81

 

Amount of 1.0N
NAOH added

(ml)

0.22

 

Amount of 10%
NaHCO added
(m?)

0.10

 

Amount of H20
added

(ml)

4.78

 

Total volume
after additions

(ml)

8.1

 

Amount added
to each
incubation flask

(ml)

1.0

 

 

12

8.8

0.98

0.22

0.12

6.28

9.8

1.0

17

13.0

1.40

0.22

0.17

10.39

14.5

1.0

 

28

was placed in a flask in group 1, the second half in a flask in group 2.
Sixteen more pituitaries were halved and similarly placed in flasks of
group 2 and group 3. A final 16 pituitaries were halved and placed in
flasks of group 1 and group 3. The incubation was carried out in a
Dubnoff metabolic shaker (60 cycles/minute) under constant gassing with
95% 02 - 5% C02 at 37°C. The pituitaries were preincubated in medium
199 for 30 minutes and the medium was discarded. Fresh medium and
hypothalamic extract were then added to each flask to initiate the 6 hour
incubation. At the termination of the incubation, the pituitary halves
were weighed and discarded, and the medium was stored at -20°C until
assayed several days later.
FSH Assay

FSH activity in control, hyperthyroid, and hypothyroid animals was
. measured by the HCG augmentation method of Steelman and Pohley (1953)
as modified by Parlow and Reichert (1963). The incubation media collected
from each of the three groups was diluted with saline and HCG so that
each assay animal received a total dose of 3 ml of medium containing 50
IU HCG. NIH-FSH-S4 reference standard was tested at 50 and 100 microgram
levels. The mean and 95% confidence limits were calculated for each
group and students' "t" test was used to determine the significance of
differences in FSH activity between groups.
LH Assay

LH activity was assayed by the ovarian ascorbic acid depletion
methOd of Parlow (1961). NIH-LH-SB reference standard was assayed at
doses of 0.4 and 1.78 mg, the equivalent of 0.0013 and 0.0003 NIH-LH-Sl
units. The pituitary material from control, hyperthyroid and hypothyroid
rats was homogenized in saline and injected at a dose of 1 mg/assay

animal into 5 animals per group. The mean and 95% confidence limits were

29
calculated for each group and the results were analyzed by students' "t"
test.
Results

In the control group, 4 of the 15 females did not become pregnant;
the remaining 11 rats bore a total of 47 live female pups. In the hypo—
thyroid groups, 13 of the 16 breeding females littered a total of 53
females. Twenty-one of these pups died before termination of the experi-
ment. In the hyperthyroid group, 15 out of 20 animals gave birth to
live young, bearing a total of 56 female pups. One of the young rats
was eaten and 5 died shortly after birth.

TWenty of the 24 young rats that discontinued receiving T at 21

4
days of age developed into healthy adult animals. Four rats succumbed
to respiratory infections. All of the surviving rats that received T4
until 21 days of age showed significant delays in sexual maturation.

The age of vaginal opening for these rats and their controls is shown in
Table 5.

At the time of sacrifice, at 45 days of age, all of the control rats
were cycling normally. None of the 12 rats kept continuously on T4 had
reached puberty and only 2 of the 17 hypothyroid animals were cycling.

The body and organ weights from the PTU-treated, continuously T4-
treated, and control animals, at the time of sacrifice, are shown in
Table 6. As in Experiment 1, the body and organ weights of the hypo-
thyroid animals were smaller than in the controls. The ovaries of the non-
cycling animals in this group were very small with few developed follicles
and no corpora lutea, and the uteri showed no significant Sign of estrogen
stimulation. Although the T4-treated animals did not appear dwarfed,

they weighed significantly less than the control animals and had immature

ovaries and uteri. The ratio of adrenal weight/body weight did not

30

TABLE 5.

THE EFFECT OF EARLY THYROXINE TREATMENT

ON THE ONSET OF PUBERTY

 

 

 

Treatment No. of rats Age at vaginal opening
+
Intact 24 38.4 - 0.61
controls
Hyperthyroid 20 46.0 "f 1.10
animals*

 

* The rats in this group suckled females that were injected with 10 ug
T per lOOgm body weight from the 15th day of gestation until weaning.
Wfien the pups were 21 days of age thyroxine treatment was discontinued.

31

TABLE 6.

ORGAN WEIGHTS OF ANIMALS TREATED WITH

THYROXINE AND PROPYLTHIOURACIL

 

 

 

 

 

 

 

CONTROL PTU-TREATED CONTINUOUS T4
. + + +

Body weight 185.3 _ 5.2 56.7 - 2.5 103.5 - 3.0
(gm)

Anterior +

pituitary 8.4 - 0.5 3.6 ”E 0.2 3.5 I 0.4
(m8)

Ovaries 46.2 I 2.7 8.6 I 0.8 14.9 I 0.7
(m8)

Uterus 331.4 r 42.6 53.1 I 11.5 30.3 I 1.0
(m8)

Adrenals 47.2 I 2.5 16.5 I 0.8 38.5 I 1.5
(m8)

State of sexual 10/10 15/17 12/12

maturity cycling immature not cycling

 

4—
_

32
change significantly between the treated and control animals. Both
hypothyroidism and hyperthyroidism decreased the gross weight of the
pituitaries. However, when expressed as mg wet AP tissue/100gm body
weight, hypothyroidism increased the average weight from 4.53 to 6.35
while hyperthyroidism decreased AP weight to 3.38.

The results of the assays for FSH and FSH-RF in the control, hypo-
thyroid and hyperthyroid groups are presented in Table 7. In the PTU-
treated group, pituitary FSH concentration was slightly decreased and,
due to the small size of the pituitaries, total pituitary FSH content
was significantly lower than in the control group. In the TA-treated
animals, pituitary FSH concentration was more than twice that of the
. controls. Pituitary FSH content was also significantly greater in the
T4-treated group. As can be seen from Table 7 there was no significant
difference between the hypothalamic FSH-RF contents of the three groups.

Anterior pituitary LH concentration and content were measured in
the same rats as above. The results are summarized in Table 8. Thyroxine
treatment greatly increased the LH concentration and content while PTU
depressed LH content significantly.

It may be concluded from Experiment 2 that early treatment with T4
delays sexual maturation in the female rat and that this delay is not
dependent upon the presence of excess T4 at the time of puberty. Rats
continuously treated with T4 from the 15th day of gestation until
sacrifice at 45 days of age have increased FSH and LH per mg AP tissue,
as compared with control pituitaries, but show no signs of normal
estrogen secretion. The high FSH and LH in the pituitaries may represent
storage rather than release. Continuous PTU treatment decreases the AP
content of LH and FSH. Because neither hyperthyroidism nor hypothyroidism

had an appreciable effect on the hypothalamic content of FSH-RF, it is

possible that T4 and PTU may act directly on the pituitary gland.

33

Table 7.

EFFECTS OF PROPYLTHIOURACIL AND THYROXINE TREATMENT 0N
PITUITARY FSH CONTENT AND CONCENTRATION AND HYPOTHALAMIC

FSH-RF CONTENT.

 

 

 

 

 

 

 

 

 

CONTROLS PTU-TREATED T4-TREATED

No. of rats
per group 10 17 12
Pituitary.
weights .

(mg) 8.35 3.56 3.52
Pituitary FSH*1
concentration + +

(ug/mg) 7.67I0.07 6.97-0.35 23.00-2.25
Pituitary FSH*1
content

(Mg/gland) 64.0 24.8 81.0

_ Hypothalamic FSH-RF

(ug*2/mg pit./m1) 10.21 9.43 11.12

 

*1. NIH-FSH-S standard.

4

Relative potency = 1.37 U/mg.

*2. FSH-RF is expressed as ug of FSH released into incubation medium.

34

TABLE 8.

EFFECTS OF PROPYLTHIOURACIL AND THYROXINE TREATMENT

ON PITUITARY LH CONTENT AND CONCENTRATION.

 

 

 

 

 

 

CONTROLS PTU-TREATED T4-TREATED
No. of rats . 10 17 12
per group
Pituitary 8.35 3.56 3.52
weights
(m8)
Pituitary LH* (.5-1.3) (.43-.57) (9.1-11.0)
concentration
0.80 0. 0 10.00
(us/m8) 5
Pituitary LH*
content 6.68 1.78 35.20

(Hg/gland)

 

 

*. NIH-LH-S8 standard. Relative potency a 0.73 U/mg.

35

EXPERIMENT 3

 

Effects of Treatment with PTU or T4 beginning at 21 Days of Age on Onset

of Puberty

 

The third experiment was designed to determine whether or not a
critical period exists after which time the administration of T4 and PTU
would have no effect on the onset of puberty.

Fifty-three 21-day-old female Spraque-Dawley rats were placed in cages
housing approximately 5 rats each, were separated into 4 groups, and were
treated as follows:

Group 1. control rats. 12 rats per group, no treatment.

Group 2. TA-treated rats. 20 rats per group, 10 ug T4/100 gm

 

body weight/day until puberty.
Group 3. continuous PTU- 11 rats per group, 0.1% PTU placed in

treated rats. feed until puberty.

 

Group 4. discontinuous 10 rats per group, 0.1% PTU placed in
PTU-treated rats. feed until 28 days of age.

The animals were given food and water 29.122: Daily checks for
vaginal opening were made and at puberty, vaginal smears were taken.
Results

None of the animals in the four groups differed significantly from
each other in age of sexual maturation. Table 9 shows the body weights
of the rats at the onset of puberty. The rats that were continuously
treated with PTU lost weight and showed a slight but insignificant delay
in the onset of puberty.

It is concluded from this experiment that the changes in the onset
of puberty seen in female rats where thyroid treatment was begun before
the let day of life, are not observed in similar animals whose treatment

began after the 20th day of life.

36

Table 9.

AGE OF VAGINAL OPENING IN ANIMALS TREATED
WITH THYROXINE AND PROPYLTHIOURACIL FROM
THE TWENTY-FIRST DAY OF LIFE.

 

 

 

 

TREATMENT NO. OF RATS BODY WEIGHT AT AGE AT
PER GROUP VAGINAL OPENING VAGINAL OPENING
(gm) (Days)
CONTROLS 12 ' 153.1 ' 34.7
T4-TREATED*1 20 156.1 1 35.5

 

CONTINUOUS PTU
TREATMENT*2 11 103.7 37.2

 

PTU-TREATMENT
DISCONTINUED*3 10 159.7 35.2

 

 

*l. 10.0ug T4/100gm body weight injected daily until puberty.
*2. 0.1% PTU placed in feed until puberty.
*3. 0.1% PTU treatment discontinued at 28 days of age.

37
GENERAL DISCUSSION

The effects of early abnormal thyroid function on the reproductive
system in the female rat were studied in Experiments 1 and 2.. The number
and viability of young born to pregnant rats treated with T4 was reduced
as compared with the control rats in experiment I. Continuous administra-
tion of T4 to the surviving female pups did not significantly reduce their
growth rate as compared to control rats but did decrease their total
body weight somewhat. Although hypothyroidism did not affect the number
of viable young, many of the young pups that suckled PTU-treated mothers
became weak and died. The continuous administration of PTU severely
retarded growth and reproductive development.

It can be concluded from these experiments that the age at which
treatment is begun is important. Treatment with T4 from the 15th day of
gestation until 21 days of age delayed puberty to the same extent as
similar treatment continued beyond 50 days. Thyroxine treatment given
after 21 days of age had no effect on the onset of puberty. Continuous
PTU given from the 15th day of gestation until 60 days of age caused
severe growth retardation and a delay or complete cessation of puberty.
The same doses of PTU given to weanling rats for a period of approximately
15 days had no effect on the onset of puberty. In Experiment I, rats
made hypothyroid from gestation until day 21 responded to the withdrawal
of PTU by exhibiting precocious puberty and this was followed by normal

estrous cycles.

It has been shown that climate, stress and changes in temperature
can advance puberty (Donovan and van der Werff ten Bosch, 1965), as can
the presence of males, increased handling and crowded housing conditions.
It is also known that nutrition influences puberty and it has been

stated that the attainment of sexual maturity in most species corresponds

38

closely to the attainment of adult size (Donovan and van der Werff ten
Bosch, 1965). More recently, puberty has been advanced experimentally
by the administration of various median eminence extracts (Gellert et al.,
1964; and Andjus and Kamberi, 1966), estrogen treatments (Rameriz and
Sawyer, 1965) and anterior hypothalamic implants of estrogen (Smith and
Davidson, 1967). In the rats in the experiments described herein
puberty normally occurred between 34-41 days of age. The advancement of
puberty to 31.7 f 1.0 days by the withdrawal of long term PTU treatment
cannot be explained by stress or nutrition, and puberty occurred long
before the rats reached adult size. There are, however, several
rational explanations. It is possible, although unlikely, that as the
blood level of PTU decreased the ensuing mild hypothyroid condition
stimulated pituitary gonadotropin release. Another explanation might be
that during the hypothyroid period, when the pituitary gonadotropins
were suppressed, an increase in gonadotropin-RF'S was produced (due to
the decrease in the 'short' negative feedback of LH and FSH on the
hypothalamus). When the pituitary was no longer suppressed, following the
removal of PTU, it over-responded to the increase of gonadotropin-RF's
and the ovaries were prematurely stimulated. A third possibility is
that a severe lack of T4 may influence both the pituitary and the ovaries,
making the latter more sensitive to gonadotropins while inhibiting the
growth and function of the pituitary. After the removal of PTU, increased
ovarian response to pituitary LH and FSH could result in increased
estrogen secretion and the advancement of puberty.

The literature presents what appears to be contradictory evidence
concerning the effective role of T4 in murine reproduction. Study of
ovarian cytology shows that short-term hypothyroidism stimulates

follicular growth (implying either increased plasma FSH or increased

39
ovarian sensitivity to FSH), while mild hyperthyroidism augments luteal
deve10pment (implying either increased plasma LH or increased ovarian
sensitivity to LH and increased prolactin secretion). Johnson and Meites
(1950) found that hyperthyroid rats did not respond to exogenous pregnant
mares' serum (FSH) as much as did untreated control rats and that thyroid-
ectomy evoked the Opposite response. Theirresults suggest that the
effect of thyroid hormone is primarily on the ovaries. Others have
reported, however, that pituitary LH (ICSH in males) and FSH is decreased
in hypothyroid male rats (Contopoulos et al., 1958). Attempts have been
made to show that hyperthyroidism has the opposite effect on pituitary
gonadotropins but changes in these hormones have not been measured
directly.

It appears from the present data that severe hypothyroidism in the
rat, as in'the human cretin, stunts body growth and all of the processes
related to growth and maturation. The onset of puberty is delayed or
totally absent and there is little sign of ovarian maturation and steroid
production. The pituitaries, on first examination, appear small with
decreased LH and FSH content (Experiment 2) and prolactin (Experiment 1)
content. However, when the hormone content of these small pituitaries is
expressed in terms of 100 gm of body weight, only prolactin content
remains decreased. The content of LH and FSH per average body weight per
group does not differ significantly between the controls and those
treated with PTU. Since the pituitary weights of these rats decreased in
direct prOportion to the decrease in body weights, the most valid measure-
ment appears to be hormone concentration rather than content. As seen
from Tables 3, 7 and 8, only prolactin concentration decreased significantly.
The delay of puberty and the sexual immaturity of the severely hypothyroid

rats may, in fact, reflect the general retardation of cellular processes

40
and not specific inhibition of pituitary gonadotropin synthesis or
release.

Hyperthyroidism appears to act differently. Although pituitaries
from TA-treated animals are as small as those from PTU treated rats,
body growth and general appearance are approximately normal. The concen-
tration of LH and FSH is greatly increased in the hyperthyroid rats
although there is no sign of gonadotropin release.

Kragt and Ganong (1967) and Corbin and Daniels (1967) have shown
that pituitary FSH concentration increases from about the 15th day of
life until the 25th day, peaking around day 20, and then falls precipi-
tously prior to puberty. The average pituitary content of FSH in the
cycling rat returns to the levels seen around day 15 but the concentra-
tion of FSH remains low. A drop in the hypothalamic content of FSH-RF at
the onset of puberty has also been reported (Rameriz and Sawyer, 1966).
The highest levels of FSH-RF found prior to puberty are also found in
the normal cycling adult in late proestrus (Rameriz and Sawyer, 1965).

In Experiment 2 it was shown that chronic T4-treatment delayed puberty
significantly. The pituitaries from these animals showed a 3-fold
increase in FSH concentration as compared to normal, cycling 45-day-old
rats. The increase corresponded to the high levels of FSH previously
reported for normal 15-25-day-Old rats. Since the drop in FSH-RF at
puberty (Rameriz and Sawyer, 1966) and during estrus (Rameriz and Sawyer,
1965) is thought to be due to the increased negative feedback by estrogen
on pituitary LH, causing a decrease in the negative 'short' feedback loop
between LH and the hypothalamus, one might expect to find an increase in
FSH-RF in those rats that remained immature with little sign of pituitary
LH release. However, the slight increase observed for FSH-RF in the T -

4

treated group was not statistically significant for the number of animals

used.

41
It has been reported by Rameriz and McCann (1963) that LH concentra-
tion is approximately 3-fold higher in immature female rats than in
cycling adults. The same investigators found that ovariectomy of adult
rats increased pituitary LH concentration approximately 2-fold. In the
present experiments it was found that T4 treatment increased the concen-
tration of LH more than 10 times that of normal cycling controls.

Because the rats treated with T from the 15th day of gestation to

4
the let day of life, or until sacrifice, exhibited marked delay in

puberty, indicating a lack of ovulation, and because pituitaries from
T4-treated rats contained abnormally large amounts of LH and FSH as in

prepubertal rats, it is concluded that T acts at least in part to prevent

4
the release of LH and FSH from the anterior pituitary.*1 The possibility

remains that T4 also acts directly on the ovaries to render them less

sensitive to the gonadotropins.

 

*1. This author (unpublished data) has found that low doses of T4
increase pituitary LH content in adult male rats. High
doses of T or treatment with PTU did not change the pituitary
LH concentration significantly.

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