ENDOCRINE AND REPRODUCTEVE CHANGES
DURING THE ESTROUS CYCLE OF THE BOVINE.

Thesis for the Degree‘of Ph. D.
MICHIGAN STATE UNIVERSITY
ARTHUR JAMES HACKETT
1968

 

"w’ ‘ k
g LIBR A R Y .
Michigan State

1,. University (‘3

THE-5:3

This is to certify that the
thesis entitled

Endocrine and Reproductive Changes During

The Estrous Cycle of the Bovine

presented by

Arthur James Hackett

has been accepted towards fulfillment
of the requirements for

Ph.D.

Dairy

degree in

   

1

Major professo;l

M
Date ay 8, 1968

 

0-169

ABSTRACT

ENDOCRINE AND REPRODUCTIVE CHANGES DURING
THE ESTROUS CYCLE OF THE BOVINE

by Arthur James Hackett

Thirty-five Holstein heifers were slaughtered during
the seventeenth month of age in groups of five at estrus
(day 0), metestrus (days 2 and A), diestrus (days 7, 11
and 18), and proestrus (day 20). Their body weight aver-
aged 377 kg and previous estrous cycle length was 20.6 t
0.2 days. The pituitary contents of prolactin, follicle
stimulating hormone (FSH) and luteinizing hormone (LH)
were estimated by the pigeon crop assay, ovarian weight
augmentation assay and the ovarian ascorbic acid deple-
tion assay, respectively. And the hypothalamic luteini-
zing hormone releasing factor (LRF) content was also
measured by the ovarian ascorbic acid depletion assay.

Pituitary contents of prolactin, FSH and LH were
highest on days 0, 18, and 20, respectively. Pituitary
prolactin decreased from 67 pg at day 0 to 19 ug at day
2 and then increased almost linearly to day 18, showing
a slight decrease at mid-cycle. Between days 18 and 20,
pituitary FSH content decreased 49 percent and further

decreased U6 percent by day 0; i.e., 73 percent decrease

Arthur James Hackett

from day 18 to 0. Then FSH increased to day 2 and declined
insignificantly at day 4, suggesting a biphasic pattern.
Similarly, pituitary LH decreased 71 and 61 per cent between
days 20 and O and between days 0 and 2, respectively; i.e.,
89 per cent from day 20 to day 2° Again there was evidence
of bimodality since, after an increase from day 2 to day
11, pituitary LH decreased between days 11 and 18.
Generally, changes in pituitary contents of prolac-
tin, FSH and LH mimicked each other except that FSH release
preceded prolactin release by 2 days and LH release preceded
prolactin.release by one day. Responses of all three showed
evidence of bimodality during the estrous cycle.
The hypothalamic LRF content was highest on days
20, O, 2, A, and 7, a period which included the lowest
pituitary LH. It was lowest on days 11 and 18, possibly
because of high levels of plasma progesterone. In any
event, high levels of hypothalamic LRF were associated
with decreased levels (apparent release) of pituitary LH.
Size of the largest ovarian follicle was greatest
at days ll and 0. But the 11-day follicle was not as
large as the ovulatory follicle, possibly because of
insufficient FSH and LH. The ovulatory follicle probably
ovulated because greater quantities of pituitary FSH and
LH were released and plasma progesterone content was
diminished at the time of estrus.
Progestin synthetic activity of the corpus luteum

increased from day 2 to day ll, remained relatively

Arthur James Hackett

constant to day 18 and then decreased dramatically as
the next estrus approached. However, in zit£g_synthetic
activity of corpora lutea homogenates remained high at
day 20.

The length of the vagina, cervix, body and both
horns of the uterus, and both oviducts were measured in
addition to the epithelial cell heights of these organs.
There were no differences in the lengths of these organs
which could be attributed to physiological changes during
the estrous cycle. However, heights of epithelial cells
of the vagina, cervix, and uterus varied during the
cycle. Whereas height of vaginal epithelial cell-layer
was highest at estrus and lowest at mid-cycle, heights
of cervical and uterine epithelial cells were biphasic—-
high at estrus and mid—cycle, suggesting stimulation by
estrogens from the ovulatory follicle and from the mid-
cycle follicle. Epithelial cell heights of oviducts
followed a pattern similar to corpus luteum progesterone.

DNA, RNA, protein and lipid measurements were made
for the vagina, cervix and uterus. Vaginal DNA, RNA and
protein contents were high at day 11 and estrus, showing
biphasic changes during the estrus cycle similar to those
for the largest ovarian follicle. Cervical RNA also
revealed a biphasic pattern, with peak values at estrus
and day 11. In contrast to uterine epithelial cell

height, which apparently was related to estrogen

Arthur James Hackett

secretion, uterine RNA appeared more related to secretion
of luteal progestogen.

In both the thyroid and thymus, there were no mean-
ingful trends during the estrous cycle with respect to
content of DNA, RNA, protein or lipid. Height of the
thyroidal acinar cells did not change during the estrous
cycle. Although these two glands are known to be asso-
ciated with reproduction, there was no evidence of physio-
logical changes in them during the estrous cycle in these
heifers.

One must conclude that high content of hypothalamic
LRF content is associated with release of pituitary LH
at estrus, which in turn is responsible for ovulation
and at least early growth (and function?) of the corpus
luteum. FSH is necessary for maximal follicular growth--
it is_released during the 2 or 3 days before estrus.
Prolactin may be part of a luteolytic complex in the
bovine because pituitary content is greatest during
luteal regression. That the mid-cycle follicle fails
to ovulate may be due to an inhibitory amount of circula-
ting progesterone which blocks release of FSH and LH.
Several of the measured criteria suggested that the mid-
cycle follicle may secrete estrogen, but in much smaller

quantity than is secreted by the ovulatory follicle.

ENDOCRINE AND REPRODUCTIVE CHANGES DURING THE

ESTROUS CYCLE OF THE BOVINE

By

Arthur James Hackett

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Dairy
1968

5 :‘f/ (7/ 7.3

"Meminisse iuvabit"

Vergil's Aeneid

ii

Dedicated to my parents

Walter and Elvira Hackett

iii

BIOGRAPHICAL SKETCH
of

Arthur James Hackett

I was born in Guelph, Ontario, Canada on August 26,
1938 and received my public and high school education in
that city. In September 1956, I entered the Ontario
Veterinary College, University of Toronto and studied
for my D.V.M., which I received in May 1961.

During my youth and collegiate days, I participated
in most sports, notably track and field, cross country
and hockey. I lettered in track and field and cross
country for the five years I was an undergraduate. And
while pursuing my Masters degree, I was again a member of
the varsity cross country team. I was fortunate to receive
the award for the outstanding member of the track and
field team for two consecutive years.

Following graduation in May 1961, I worked for the
Canadian Federal Government, testing cattle for tubercu-
losis. Beginning in August of that year, I worked for~
the Central Ontario Cattle Breeding Association at Maple,
Ontario, Canada. While diagnosing and treating cases
of bovine infertility I became more aware of the complexi-

ties of reproduction. So with the financial aid of the

iv

Ontario Association of Animal Breeders Fellowship, I
entered Graduate School at the University of Guelph. I
received my M.Sc. from that institute in May 1965. My
thesis was "Studies Related to the Freezability of
Bovine Spermatozoa."

In September 1965, I received a graduate research
assistantship in the Dairy Department, Michigan State

University where I completed my Ph.D. in June, 1968.

ACKNOWLEDGMENTS

I should like to thank the Chairman and the staff
of Dairy Department of Michigan State University for
providing facilities for my training. But without the
ever-guiding hand and encouragement of Dr. Harold Hafs,
many of my personal endeavours would have been futile.

I feel greatly indebted to him for his guidance and hOpe
that in the future I shall be a source of some satisfac—
tion to him.

I also owe sincere thanks to Dr. Allen Tucker who
capably guided me through my first year at Michigan State
University while Dr. Hafs was on sabatical at Harvard,
and for his continued interest, advice and assistance
throughout my sojourn in East Lansing. And I also am
grateful for the cooperation given to me by members of
my committee, Drs. E. P. Reineke, J. L. Gill, R. L.
Anderson and S. D. Aust.

Because a large portion of the work required to
obtain the data in this thesis was the result of team
cooperation, I should also like to thank those involved.
They include Mrs. Helga Hulkonen, Dr. L. J. Boyd, and
my student colleagues, Claude DesJardins, Ken Kirton,

Jock Macmillan, Max Paape, Yogi Sinha and Bill Thatcher.

vi

The gifts of hormones from the Ayerst, Squibb and
Upjohn Laboratories and the Endocrinology Study Section
of the National Institutes of Health, as well as the
financial support provided by the National Institutes
of Health (grant number HD 0137A) are recognized and
appreciated.

I should like to thank my wife, Ellen, and children
Lisa, Ruth, and A. J. for their tolerance and sacrifices
while being without husband and father for much of the
time I spent at the "lab."

For those who cannot read Latin, "Meminisse iuvabit"

means "A pleasure to recall."

vii

TABLE OF CONTENTS

Page
DEDICATION . . . . . . . . . . . . . . . iii
BIOGRAPHICAL SKETCTH . . . . . . . . . . . . iv

ACKNOWLEDGMENTS . . . . . . . . . . . . . V

LIST OF TABLES . . . . . . . . . . . . . . ix
LIST OF FIGURES o o o o o o o o o o o o 0 X
LIST OF APPENDICES . . . . . . . . . . . . Xi
INTRODUCTION . . . . . . ,. . . . . . . . 1
REVIEW OF LITERATURE . . . . . . . . . . . . A
A. Nature of the estrous cycle . . . . . . . A
1. Length of estrous cycle . . 4

2. Changes in the bovine reproductive tract
during the estrous cycle . . . . . 4
3. Regulation of the estrous cycle . . . 5

B. Changes in the hypothalamus and pituitary

gland during the estrous cycle . . . . . 7
l. Releasing factors . . . . . . . . 7
2. FSH and LH . . . . . . . . . . 8
3. Prolactin . . . . . . . . . . . 8
C. Ovarian changes during the estrous cycle . . 9
1. Growth of follicle . . . . . . . 9
2. Ovulation and corpus luteum formation . 10
3. Luteotrophic process . . . . . . . 12
A. Luteolytic process . . . . . . . . 15
D. Morphology of the reproductive tract . . . . 17
l. Vagina . . . . . . . . . . . . 17
2. Cervix . . . . . . . . . . . . 18
3. Uterus . . . . . . . . . . . . 19
A. Oviduct . . . . . . . . . . . 19

viii

E. Changes in other endocrine glands . . .
l O Therj-d o O o O O 0 0 0 O
2 O Thymus O 0 O O O O 0 O O O

F. DNA, RNA, proteins and lipid content of the

reproductive tract, thyroid and thymus
MATERIALS AND METHODS . . . . . . .
A. Experimental animals and management .

B. Slaughtering and retrieval of samples

1. Method of slaughter . . . . .
2. Hypothalamus and pituitary

3. Thyroid and thymus . . . . .

A Reproductive tract . . . . .

C. Bioassays of pituitary hormones and LRF
l. Follicle stimulating hormone . .
Luteinizing hormone . . . . .

20
3. Luteinizing hormone releasing factor
A

. Biochemical determinations . . .
5. Histological determinations . . .

RESULTS AND DISCUSSION . . . . . . . . .

Statistical analyses . . . . . . .
Length of estrous cycle . . . . . .
Body weights at slaughter . . . .
Hypothalamic and pituitary gland changes
during the estrous cycle . . . . .
l. Luteinizing hormone releasing
factor--LRF . . . . .
2. Anterior pituitary . . . .
a.) Weight . . . . .
b.) Prolactin . . .

c.) Follicle stimulating
hormone--FSH . . .
d.) Luteinizing hormone--LH .

UOCDS>

O 0 O O
9 O O 0

E. Ovarian changes during the estrous cycle
1. Follicle . . . . . . . . .
2. Corpus luteum . . . . . . .
a.) Growth . . . . . . .
b.) Progesterone . .
c.) 208- hydroxy- -pregn-A— en-3- one
(208-01) . .
3. Ovary weight with CL and follicle .

A. Ovary weight without CL and follicle

ix

Page
20

2O
2O

21

F. Vaginal changes

1. Weight .
2. DNA, RNA,
3. Height of
A. Length .
5. Summary

G. Cervical changes

1. Weight .
2. DNA, RNA,
3.
A. Length .
5. Summary
H. Uterine changes
. Weight .
DNA, RNA,

Length .
Summary

U'l-I-TUUNI-J

Epithelial cell height . . .

. Epithelial cell height .

during the estrous cycle
protein and lipid . .
epithelial cell—layer

O O O O O O O 0

during the estrous cycle

protein and lipid

O O O O O O 0

during the estrous cycle

protein and lipid

I. Changes in the oviduct during the

estrous cycle
1. Length .

2. Epithelial cell height

3. Summary

0 0 O

0 6 O O O

J. Changes in the thyroid during the

estrous cycle
. Weight .

.5me

Summary

. DNA, RNA, protein and lipid
. Epithelial cell height .

O 0 O O O O

0 O O O

K. Changes in the thymus during the

estrous cycle

GENERAL DISCUSSION .

O 0 O O O O O O O

O O O 0 O O O 0

Suggestions for future research . . . . .

SUMMARY AND CONCLUSIONS
BIBLIOGRAPHY . . .

APPENDICES . . . .

71
7I
71

73

73
75
100
102
106
119

Table

10.

ll.

l2.

13.

LIST OF TABLES

Hypothalamic LRF units for the two dose
levels . . . . . . . . . .

Pituitary weight and prolactin, FSH and LH.
Ovarian weight characteristics.

Corpus luteum weight and progesterone,
208-01, and progestin (progesterone
plus 208-01) content

Net synthesis of progesterone (prog) and
2OB—hydroxy—pregn—A-en-3-one (208-01)
mass during incubation of corpus luteum
homogenates . . .

Vaginal weight, DNA, RNA, protein and lipid
content and epithelial call-layer height.

Length of vagina, cervix, uterine body and
horns and oviducts . . . . . . .

Cervical weight DNA, RNA protein and lipid
content and epithelial cell height.

Uterine weight, DNA, RNA, protein and
lipid content and epithelial cell
height . . . . . . . . .

Oviducal epithelial cell height

Thyroid weight, DNA, RNA, protein and
lipid content and acinar epithelial
cell height . . . . . . . .

Thymus weight, DNA, RNA, protein and
lipid content. . . . . .

Correlations between gonadotrOphins and
luteal progestins or follicle size.

xi

Page

37

39
A6

A9

52

57

6O

62

67

71

72

7A

90

LIST OF FIGURES

Figure Page
1. Hypothalamic LRF during the estrous cycle . . 38
2. Pituitary prolactin during estrous cycle . . . A1
3. Pituitary FSH during estrous cycle . . . . . A3

A. Pituitary LH during estrous cycle . . . . . A5
5. Follicular size during estrous cycle . . . . 48
6. Luteal progestin during estrous cycle . . . . 51

7. Vaginal nucleic acids and epithelial
cell-layer height . . . . . . . . . . 58

8. Cervical RNA and epithelial cell height . . . 63
9. Uterine RNA and epithelial cell height . . . 66
10. Pituitary LH, FSH, and prolactin . . . . . 95
ll. Hypothalamo-pituitary-luteal relationships . . 96
12. Pituitary-follicular-cervical relationships . . 97

13. Pituitary prolactin and luteal progestin . . . 98

xii

Appendix

1.

2.

LIST OF APPENDICES

Age, weight and estrous cycles . . . .

Pituitary weight and prolactin, FSH and
LH concentration . . . . .

Ovarian weight characteristics, corpus
luteum weight and progesterone, 208-01
and progestin (progesterone plus
208-01) content . . . .

Net synthesis of progesterone (prog) and
2OB-hydroxy-pregn-A-en-3—one mass during
incubation of corpus luteum homogenates .

Vaginal characteristics . . . . .

Cervical characteristics . . . . . .

Uterine characteristics

Oviducal lengths and epithelial cell height

Thyroid characteristics . . . . . . .

Thymus characteristics . . .

xiii

Page
120

I21

I22

12A
126
128
130
132
133
135

INTRODUCTION

In 1967, infertility (85) ranked second as the
Michigan dairyman's greatest financial loss. For maximum
reproductive efficiency and economic production, a calving
interval of one year to 13 months is required. Since the;
advent of artificial insemination techniques and calfhood
vaccination against brucellosis, many transmissible and
infectious diseases (109, 117) have been eliminated. In
my opinion, and excluding poor management, hormonal
imbalances are now the major causes of bovine infertility.
Although we have been able to treat some cases success-
fully, we have been unsuccessful in determining the
specific causes of these conditions.

To better understand endocrine imbalances, we must
first quantify pituitary or gonadal and plasma levels
of the pertinent hormones, luteinizing hormone (LH),
follicle stimulating hormone (FSH), estrogen and proges-
terone during the normal estrous cycle. Most of our
present day concepts of physiological control of repro-
duction and reproductive cycles are based on evidence.
(117) established 30 or A0 years ago. Within the last
few years more specific and sensitive assays have

allowed us to measure quantitatively both LH and FSH

in an individual cow pituitary. For example, Rakha and
Robertson (102) determined both the FSH and LH content
in pooled pituitary glands of heifers at the 15th and
18th day of the estrus cycle, and during estrous and
after ovulation. Prior to 1965, most investigators
measured "total gonadotrophic" activity.

Although we base our working hypotheses of repor-
duction control on the pituitary-gonadal axis, we should
also be cognizant of the regulation by the central nerv-
ous system and production and release of gonadotrophins.
Using median-eminence extracts from rats, rabbits, and
cattle, Nikitovitch—Winer (92) induced ovulation in
"pentobarbital-blocked" proestrous rats and Campbell
g£_al. (17) induced ovulation in female rabbits. At
about the same time, McCann (79) induced release of LH
in the rat by injection of median-eminence-stalk extract.
Since that time, separate hypothalamic factors (A5) have
been demonstrated to control synthesis and release of
each of the anterior pituitary hormones.

That FSH and LH act directly and indirectly on
the reproductive tract is well established. FSH pro-
motes follicular development and LH stimulates ovula-
tion and formation of corpora lutea. Through the
ovarian hormones (estrogen and progesterone), FSH and
LH cause morphological changes in the reproductive;

tract. Now with the development of methods for

measuring biochemical components such as deoxyribonucleic
acid (DNA), ribonucleic acid (RNA), and protein, we can
quantify cell numbers and activity. These biochemical
measurements can supplement studies of anatomical changes.
The—chief objective of the present study was to
measure the individual pituitary gonadotrophins and some
meaningful biochemical components of the reproductive
tract of dairy heifers during the estrous cycle. A sec-
ond objective was to correlate these quantitative measure-
ments of pituitary gonadotrophins with follicular and
luteal growth. Finally, we wished to study the relation—
ship of the luteinizing hormone releasing factor (LRF) V

with the synthesis and release of pituitary LH.

REVIEW OF LITERATURE

A. Nature of the Estrous Cycle

 

1. Length of the Estrous Cycle

Duration of the estrous cycle in cattle averages
21 days and somewhat less than 21 days in heifers (7, 11,
A8, 73, 101, 117). Physiologically, infertile service,
effects of feed level, season of year and age may alter
estrual cycle length (32, 93, 109). Pathologically,
.diseases such as tuberculosis and Johne's disease may
alter the estrous cycle and embryonic mortality may
lengthen the estrous cycle (109). And experimentally,
drugs also alter the length of the cycle. For example,
oxytocin (7) administered during the first week of the
estrous cycle shortens the length of the cycle. Daily
injections of estradiol (200-300 ug) decrease the length
of the cycle (100) and 5 to 10 mg of progesterone (5A)
shorten the length of estrus and reduce the time from
the end of estrus to ovulation. Pregnancy (109) abruptly
terminates cyclicity of the pituitary and reproductive
tract.

2. Changes in the Bovine Reproductive Tract
During the Estrous Cycle ‘

 

The bovine estrous cycle is usually divided into

proestrus, estrus, metestrus and diestrus (9, 30, 109, 117).

A

Under the influence of estrogen, produced by the growing
Graafian follicle, proestrus (109) is a buildup phase for
cells and cilia lining the oviduct as well as for vaginal
vascularity and thickness of the vaginal mucosa. And the
increasing amounts of estrogen cause an increase in uter-
ine vascularity and stimulate muscular activity of the
uterus causing it to be turgid.

The highlight of the cycle is estrus (117), a
period lasting 12 to 18 hours and a time at which the
female is receptive to the male. During metestrus, the.
cow ovulates, usually about 12 hours following "standing
heat." Capillary haemorrhage, resulting from hyperemia
over the caruncles, may occur during early metestrus, and
the profuse secretion of cervical mucous ceases.

Throughout diestrus, progesterone exhibits its most
marked effects. For example, hypertrophy of the endo-
metrial glands and stricture of the cervix occur. Begin—
ning at about day 18 the corpus luteum (31) regresses
and progestin concentration decreases near the time of

the next estrus.

3. Regulation of the Estrous Cycle

Throughout the estrous cycle, the anterior pitui-
tary hormones (117), secreted into and carried by the
‘blood to the ovaries, modify the action of these target
<Drgans. The follicle, growing rapidly during the 3

days prior to estrus (50, 117), is under the influence

of FSH (102) which decreases by 27% in the pituitary about
18 hours before estrus. And the fluid of the follicle
follicular phase (82) has ten times as much estrogenic
activity than the luteal phase fluid. Although the
levels of endogenous estrogen in the peripheral blood
plasma (13) have not been unequivocably identified, the
results indicate highest levels (82) during the follic-
ular phase of the estrous cycle, which is in agreement
with bioassayed levels in follicular fluid. In the rat
(83), the increasing level of estrogen in the blood at
the time of proestrus feeds back negatively on the
pituitary, the hypothalamus, or both, blocking the
release of FSH from the pituitary. At the same time,
increasing amounts of circulating progesterone, produced
by the-maturing follicle, stimulate the release of LH,
and this surge of LH induces ovulation. But in the cow
there is no evidence for estrogenic feedback blocking
the release of FSH; however, there is evidence of lutein—
ization (27) of most follicles before estrus and that
follicles obtained near ovulation in the cow contained

3 ug of progesterone (117) per ml of fluid. Small
amounts of progesterone (5A) administered at the begin-
ning of estrus hasten ovulation. This suggests that a
rise in progesterone levels prior to ovulation may con-
tribute to the control of the estrous cycle. In the

cow, pituitary LH decreases 61% just prior to ovulation

(102) and preliminary results indicate blood plasma
levels (A, 61) of LH increase 6—7 hours before ovulation.
So it appears likely that LH is the hormone responsible
for ovulation in the cow. Moreover, LH stimulates
growth of the corpus luteum.
B. Changes in the Hypothalamus and Pituitary
Gland During the Estrous Cycle

1. Releasing Factors

That releasing factors (A5, 120) exist in hypo-
thalamic extracts of several (if not all) species has
been firmly established by several groups of investi-
gators. In fact, partial or complete purification (120)
of bovine, ovine and porcine luteinizing hormone releasing
factor (LRF) has been described. Column chromatography
has been used to separate FSH-RF from LH-RF and to par-
tially purify it (AA, 62). Besides FSH-RF and LRF, other
releasing factors (A5) control synthesis and release of
growth hormone, thyrotrophic hormone and ACTH. But pro-
lactin systhesis and release (8A) is chronically inhib-
ited by prolactin inhibiting factor (PIF) in the hypothalamus.

Although releasing factors have been obtained from
large animals (cow, sheep and pig), the LRF content (18)
has been measured only in the rat during the estrous
cycle. In this species, LRF content in the hypothalamus
increased during late diestrus and decreased markedly
during proestrus, synchronizing in an inverse relationship

'with the pattern of LH secretion during the estrous cycle.

2. FSH and LH

 

The total gonadotrophic content (39, 60, 113) of
pituitaries decreased at the time of estrus in all species
which have been studied. It was elevated during the
remainder of the cycle, suggesting release of gonadotro-
phin from the pituitary at the time of estrus. Following
development of more sensitive assay techniques to study
pituitary FSH (131) and LH (96) contents separately,
later studies (38, 59, 9A, 97, 102, 110, 111, 112, 119,
122) disclosed that FSH was released shortly before LH at
the time of estrus in the rat, pig, monkey, sheep, hamster
and cow. And preliminary data revealed that blood plasma
content of LH (A) increased rapidly about 12 hours prior
to ovulation in the gilt, 6-17 hours in_the cow, and
6-12 hours in the rat. Specifically, FSH and LH levels
in the bovine pituitary decreased 27 and 61% respectively
between the onset and end of estrus indicating that FSH
as well as LH (102) may have a role in reproductive

events near the time of ovulation in cattle.

3. Prolactin

 

Although an assay for prolactin (107) was developed
in 1937, there are very few reports concerning levels of
this hormone during estrous cycles. In the rat and guinea
pig (106, 107) pituitary prolactin concentration is higher
during estrus and proestrus than during metestrus and

diestrus, and in the gilt (23), pituitary prolactin

potency increases linearly with advancing stages of the
estrous cycle. Prolactin (6A) was measured by radio-
immunoassay in blood plasma of the mouse and found to
increase during proestrus and estrus. And prolactin
reaches detectable levels in the blood of women (125)
during the first five and last ten days of the menstrual
cycle.
C. Ovarian Changes During the
Estrous cycle

1. Growth of the Follicle

Formation of oocytes (101) is completed at birth,
and the nuclei of all oocytes have entered the pachytene
stage of the first meiotic division. In sexually mature
heifers, oocyte nuclei in the primordial follicles are
also in the pachytene stage. Primordial follicles, con-
sisting of an ovum and a single layer of epithelium,
proliferate to form growing follicles. During this stage
the theca forms from the multiplication of this single
layer of cells surrounding the ovum and by a special
development of the surrounding connective tissue cells
(53). The theca develops into two layers, externa and
interna and cells of the latter layer are believed to
secrete the estrogenic hormones. Granulosa cells, derived
from the proliferations of the single layer of cells sur-
.rounding the ovum, secrete the follicular fluid which

IPesults in the formation of an antrum in the follicle.

10

Large vesicular follicles are called Graafian follicles
and the cells surrounding the ovum, known as the cumulus
oophorus, eventually become separated from the granulosa
cells lining the cavity of the follicle.

Preovulatory growth (50, 101) of the bovine Graafian
follicle is most rapid during the 3 days prior to estrus.
During this follicular phase (95) there is ten times as
much estrogenic activity in the follicular fluid than in
the luteal phase, and estrogens in the peripheral blood
plasma (l3) follow a similar pattern. But consistent
results of estrogenic (82) activity in the urine of
cycling cows have not been obtained. In the sow (66,

99) however, urinary estrogen excretion was greatest
just before or at the onset of estrus. Apparently the
large follicle at mid-cycle (101) in the bovine does not
produce discernible changes in sexual behavior of the

animal.

2. Ovulation and Corpus Luteum Formation

Although the exact mechanism is unknown, ovulation
(50) involves the rupture of the largest follicle at the
surface of the ovary and release of the ovum into the
infundibulum of the oviduct. After ovulation there is
some haemorrhage (9, 50) at the point of rupture, but
any slight haemorrhage (9) in the follicle is confined
between theca interna and the granulosa. Occasionally

a clot (50) may form in the empty follicle. Also,

11

following ovulation, the walls of the follicle (27)
collapse and the granulosa and theca interna layers
become deeply folded. Approximately two days after ovu-
lation, the folds of the wall of the corpus luteum meet, and
by the third day after ovulation, the cavity produced by these
folds is obliterated. Cysts (11, A9, 101) of various
size have been noted in bovine corpora lutea, but are
usually part of normal development, or if they are of an
abnormal condition, they are of little consequence.
However, luteal cysts (80) over one cm in diameter may
be an important cause of infertility.
Contrary to a previous report (A9) suggesting
corpora lutea are derived from granulosa cells only,
more recent work (27, 36, 39, 81) supports the hypothesis
that they are also derived from theca interna cells.
Early luteinization of both layers occurs approximately
6 hours after the onset of estrus (and before ovulation),
and mitotic activity is seen in both layers but more
frequently in the granulosa layer. In addition to
mitosis, the granulosa and theca interna cells also
enlarge and have increasing amounts of lipid material.
The size of the corpus luteum increases between
days A and 7 and reaches a maximum (72) by about day
11. At days 9 and 11, the only mitotic activity (36)
appears in the connective tissue. Between days 11 and

17 (72) the weight of the corpus luteum is constant

l2

and it decreases (50) in size shortly before the cow

returns to estrus.

3. Luteotrophic Process

The luteotrophic process (115) has been defined as
the process "which promotes the growth of the corpus
luteum and a rate of progesterone secretion at least
sufficient to prevent ovulation and/or to permit implan-
tation to occur." In the cow (51, 52, 127), LH promotes
the growth of the corpus luteum and secretion of proges-
terone. When added to bovine corpus luteum slices incu-
bated in vitrg_(78), LH enhanced synthesis of progester-
one. In a similar experiment, Armstrong and Black (5)
demonstrated this phenomenon occurred only in functional
corpora lutea, i.e., corpora lutea capable of secreting
progesterone £2.1112- Bartosik gt_al. (1A) demonstrated
increased progesterone secretion from perfused luteal
ovaries and contralateral "follicular" ovaries, following
administration of LH in vitgg. They also observed
increased progesterone secretion from luteal ovaries,
but not from contralateral "follicular" ovaries, fol—
lowing prolactin administration. These researchers
suggested that prolactin as well as LH is involved in
the luteotrophic process of the bovine. When bovine
ovaries (A3) were perfused ih_vit£2_with a combination
of LH and prolactin, the corpus luteum synthesized

greater amounts of progesterone than during treatment

13

with either hormone alone. However, in viva prolactin
(7, 127) alone did not maintain the corpus luteum beyond
its normal life span. Nor did daily injections of pro-
lactin delay the onset of estrus. Some of these data
are contradictory but it is at least conceivable that
under certain circumstances, prolactin may indeed be
luteotrophic or part of a luteotrophic complex in cows.
Prolactin (12, 29, 33, 3A) has been conceded to
be the luteotrophic hormone in rats and mice. But pro-
lactin (77) administered to hypophysectomized rats,
requires synergistic action of LH to maintain corpora
lutea. Reminiscent of the effect of LH on progestin
secretion in the rabbit ovary (57), intravenous injec-
tions (77) of LH increased secretion of progesterone
2.5 fold. And prolactin increased the secretion of
20a— hydroxy-pregn-Aen-3-one, but to a lesser extent
than the LH effect on progesterone secretion. This
study also agrees with other investigators (8) who
suggest LH aids in regulation of ovarian functions in
the rat. Possible other synergisms exist in the luteo-
trophic process in rats. For example, progesterone
or estrogen alone (116) maintain growth of rat corpora
lutea for only 2 to 3 weeks, whereas the two hormones
together maintain luteal growth and function for at
least A weeks. Although corpora lutea respond to pro—

lactin, even in the absence of other pituitary hormones,

1A

this does not mean that other pituitary hormones are not
involved in the luteotrophic process in the rat. In the
mouse (16), there is also evidence for LH-prolactin
synergism.

In other species, the luteotrophic process varies.
A complex (A3) of prolactin, FSH, and LH is required for
luteal maintenance in hamsters hypophysectomized during
the first half of pregnancy. Exogenous prolactin alone
does not maintain pregnancy but it does preserve the
histologic integrity of the corpus luteum. But concur-
rent administration of FSH maintains functional corpora
lutea which sustain pregnancy. If a small amount of LH
is added to this minimal luteotrophic complex, the LH
enhances corpora luteal function as shown by increased
size and hyperemia of the corpora lutea.

Progesterone (2) injected into pigs from the time
of ovulation until day 10 to 13 of pregnancy does not
prevent formation of corpora lutea, whereas progesterone
injections from days 12 to 16 of pregnancy result in
regression of corpora lutea. Based on this evidence,
Nalbandov (88) suggested that discharge of a pituitary.
luteotrophin over a relatively short time (2-3 days)
maintains the life span of the corpora lutea during the,
estrous cycle. Furthermore, no additional release of
luteotrophin occurs unless the female becomes pregnant,

and, if such is the case, intrauterine events stimulate

15

a secondary release of the pituitary luteotrophin which
continues throughout gestation. Because exogenous pro-
lactin (2) is not luteotrophic in pigs, perhaps LH is
the luteotrophin. Neither FSH nor prolactin (19)
increased progesterone synthesis in porcine corpora
lutea in gitrg, whereas porcine, ovine or bovine LH
increased progesterone synthesis by 15%.

Except that the initial outpouring of luteotrophin
(possibly LH) lasts longer (1), the guinea pig parallels
the pig in formation and maintenance of corpora lutea.
That prolactin (86, 132, 136) is luteotrophic in sheep
has been questioned. Now most evidence indicates FSH

or LH, or both, is luteotrophic (12A).

A. Luteolytic Process

If one accepts the arguments (51) that LH is the
major luteotrophic hormone, the question arises whether
luteal regression is due to a decrease in plasma LH
level. Accurate measurements of blood levels of LH
during development and regression of the corpus luteum
are required to determine whether lowered LH in the
plasma is responsible for luteal regression. Regression
of corpora lutea (129) and interruption of pregnancy
occur in rabbits following treatment with rabbit anti-
serum to ovine LH. These observations suggest anti—serum
to ovine LH suppresses the secretion of endogenous

estrogen, thereby prompting regression of corpora lutea,

16

with a consequent shortage of progestin and interruption
of pregnancy.

Bovine corpora lutea (5) lose their ability to pro-
duce increased amounts of progesterone in response to
high levels of LH added in yitgg at the 18th day of the
cycle. But homogenates (A6) of corpora lutea obtained
from cows at day 18 or 20 of a 21 day estrous cycle
retain their ability to synthesize progesterone and the
altered appearance of their blood vessels suggests that
deficiencies of precursors account for decreased steroido-
genesis lE.YlY29

In many species (3, 67, 71), including cattle, the
uterus produces a luteolysin which appears to act locally
on the corpus luteum. Corpora lutea formed prior to
hysterectomy fail to regress following hysterectomy and
contain as much progesterone 20 days after hysterectomy
as those removed 9-11 days postestrus. In addition,
they also contain significant levels of 208-01. How;
ever, hysterectomy does not affect the ability of the
pituitary to secrete hormones required for follicular
growth, ovulation, and corpus luteum formation. Estra-
diol (51) does not cause luteal regression in hysterecto-
mized heifers to the same degree that it occurs in nor-
mally cycling animals. This_evidence provides further
support for the hypothesis that the uterus may elaborate

a-luteolytic factor at certain stages.

17

Recently, a luteolytic effect of the uterus has
been demonstrated more directly. When acetone dried
powder preparations of either late luteal or early estrual
bovine uteri were injected intraperitoneally into pseudo-
pregnant rabbits (13A, 135), they induced luteal regres-
sion and development of follicles. During in vitrg
incubations of ovaries, these powders depressed incor-
poration of acetate into progesterone. For controls,
the.same dosage of a similarly prepared abdominal muscle
powder was without effect. Although they hypothesized
a uterine luteolytic hormone, affecting ovarian function
without involving LH, these authors provide no evidence
that their preparation was indeed a hormone. And their
preparation was made up of an entire uterus which may
contain other factors which could adversely affect the
corpora lutea.

Because single-layer cultures of granulosa cells
provide an ideal test system for luteolytic substances,
Short (123) has tested various fractions of uterine
washings. His preliminary data indicated uterine washings
possessed no luteolytic substances. He attributed any
death of the culture cells to toxic factors in the test

substances.

D. Morphology of the Reproductive Tract
l. Vagina
As estrogen levels increase during the follicular

phase of the estrous cycle, the superficial layer (9, 76)

18

in the mucoid portion of the vagina becomes secretory.
At estrus the cell membranes rupture, extruding the cell
contents, leaving the underlying layer to continue
secretion. During the luteal phase, the superficial
layer of epithelium forms a layer of secretory low
cuboidal cells. In the vestibular region, the number

of leucocytes in the mucosa, increase and congestion

and edema of the epithelial layers occur during proestrus.
These changes subside by two days postestrus, but the
cells of the middle layer increase markedly in size and
number by day 8 to 11. At 10 days postestrus the super—
ficial layers tend to become squamous, but true cornifi-

cation does not occur (50).

2. Cervix

While the predominate cell type is the mucous-
secreting cell (7A), approximately 10% of the cells of
the bovine cervical epithelium are ciliated. These
mucous-secreting cells distend with mucous at estrus
and, following its discharge into the folds of the
cervix and into the cervical canal itself, become
cuboidal cells. Thus these cells, unlike those in the
vagina, are not lost. At the fifth day after estrus,
the mucous cells become larger, and mucous accumulates
in the cells. The peak of this stage occurs at the
eighth day after estrus when the first wave of follicles

developing in the ovaries is secreting estrogen. With

l9

secretions of this estrogen, the cells become smaller
again and remain small until the 16th day of the cycle.
The author suggests the discharge of mucin is controlled
by estrogen and the accumulation of mucous is controlled

by progesterone.

3. Uterus

Less than 1% of the uterine surface epithelium
cells (75), obtained from heifers at estrus are ciliated
and, of the remainder, cell height is lowest (15 u) at
estrus. By day 6 of the estrous cycle, the cell height
increases to 30 N. This high level remains until day
16, after which time it decreases. During proestrus
and estrus the uterine glands are fairly quiescent and
the lumina are straight. Two days postestrus, the
glands are more coiled, and the lumina are filled with
secretion. As-the corpus luteum grows, the glands
hypertrophy and reach maximum growth at about 12 days

postestrus; then they regress after the fifteenth day.

A. Oviduct

Increased epithelial cell height in the oviduct
accompanies an increase in the secretion from the fim-
briated region during estrus (68, 108). Two days
postestrus, more mucous and leucocytes are found than
at any other time (9). By day 8, the epithelium becomes

lower, and numerous globules of cytoplasm are found.

20

The cilia of the epithelial cells are more prominent

during proestrus than at any other time.

E. Changes in Other Endocrine Glands
l. Thyroid

That the thyroid gland is necessary for reproduction
has been established (21), because thyroidectomized
animals show no behavioral signs of estrus. However,
there are no reports (50) of changes of a cyclic nature
in the thyroid associated with the estrous cycle in the
cow. In sheep (69), diethylstilbestrol decreases total
pituitary gonadotrophin content while increasing thyro—
trophin content. This finding, coupled with the finding
that increased thyrotrophin (6) content is correlated
with increased thyroid activity, suggests changes may

occur in the ruminant thyroid during estrus.

2. Thymus
Although the thymus (109) has been assumed to be

an endocrine gland, its endocrine function is not well
documented and little understood. In the heifer (25),
thymus weight increases steadily from 1 to 12 months of
(age. Desjardins concluded that reproductive development
in heifers was not related to changes in the thymus
‘weight. These results are in contrast to those in
:female laboratory animals (2A), in which thymus weight

cieclines with advancing age. In the bull (70), the

21

weight of the thymus gland increases dramatically from
1 to A months of age and then shows a decline to 7
months of age. Between 7 and 12 months there is.a
slight increase with age. Macmillan concluded that
changes in the weight of the thymus gland in the.Hol-
stein bull do not appear to be associated with repro-
ductive develOpment. And these two studies show dif-
ferences in the development of the thymus between the
male and female.

F. DNA, RNA, Protein and Lipid Content of
the Reproductive Tract, Thyroid and Thymus

 

 

To the best of my knowledge, no one has measured
DNA, RNA, protein and lipid content of the bovine repro-
ductive tract during the estrous cycle. However, the
DNA, RNA, and lipid contents (126) have been measured
in the rat uterus; DNA being maximal at proestrus or
estrus, decreasing at metestrus and remaining at the
same level during diestrus. The RNA content paralleled
DNA. The ratio of RNA to DNA is often calculated to_
illustrate metabolic activity, and in the above study
with rats, this ratio, in the uterus decreased from a-
maximum at estrus to a minimum at metestrus. Between
metestrus and estrus, the ratio increased 27%. These
results suggested increased metabolic activity at the
time of estrus. Variations in lipid content also

paralleled that of RNA:DNA ratio, suggesting that

22

lipid synthesis was one of the earliest responses of the
uterus to estrogen.

There have been no reports about DNA, RNA, protein
and lipid content of the bovine thyroid and thymus during
the estrus cycle. After 7 months of age (25), thyroid
weight, total DNA and RNA paralleled each other. Between
6 and 12 months of age the thymus weight, total DNA and

RNA increased five fold.

MATERIALS AND METHODS

 

A. Experimental Animals and Management

This experiment was designed to study endocrine
and reproductive tract changes of dairy heifers during
the estrous cycle and to ascertain whether or not cyclic
changes occurred in the thyroid and thymus glands during
the estrous cycle. or a total of 35 Holstein heifers,
five each were slaughtered on day 0 of the estrous cycle
(estrus) days 2 and A (metestrus), days 7, 11, and 18
(diestrus) and day 20 (proestrus). These heifers were
born from registered Holstein sires and production
tested dams and came from Wisconsin or Michigan farms.
To have as uniform a group as possible, we selected
only animals born within the period between June 30 and
July 19, 196A. They were transported, within one week
of birth, to the Michigan State University Dairy Barn.
Each animal received a physical examination, the results
of which was-recorded on its health record.

Initially they were housed in individual pens
and fed an average of 8 lbs of milk, about 5 lbs of hay,
and grain increasing to about 2 1/2 lbs per day. Grad-
ually the milk was deleted and both hay and grain.

increased so that by four months of age they were fed

23

2A

12 lbs of hay and 5 lbs of grain daily. From this age
they were housed communally in box stalls. When they
reached their sixth month of age, they occupied a loose
housing arrangement and were fed corn silage and hay
free choice. They remained there until slaughtered in
their 16th or 17th month of age, the time at which most
Holstein heifers are customarily mated for dairy purposes.
Beginning in August 1965, each heifer was observed
for-signs of estrus twice daily—-between 7:30 a.m. and
8:30 a.m. and between A:30 p.m. and 5:30 p.m. Dates of
estrus were recorded for each animal. For determining
estrus, the following criteria were used: (1) an animal
standing when another animal mounted, (2) attempts to
mount another animal, (3) swollen external genitalia,
and (A) general uneasiness. Any post-estrus haemorrhage

further substantiated the observed estrus.

B. Slaughtering and Retrieval of Samples
1. Method of Slaughter

0n the day of slaughter, the heifers were trans-
ported to the University Meats Laboratory at approxi-
mately 5:30 a.m. and on arrival, each heifer was weighed.
A captive-bolt pistol was used to stun the animal in
the head, then the animal was exsanguinated. Usually,
slaughtering began at about 7:00 a.m. and was completed

by 11:00 a.m.

25

2. Hypothalamus and Pituitary

Within 5 minutes after stunning, the hypothalamus
(including the surrounding median eminence and pitui-
tary stalk) was removed, finely cubed, placed in a sample
bottle, covered with a minimal amount of 0.1N hydro-
chloric acid (HCl) and rapidly frozen on Dry Ice. And
at the same time a sample of cerebral cortex was similarly
treated for a control. Immediately afterwards the pitui—
tary gland was removed, trimmed, and weighed. Following
the separation of the two lobes, the anterior lobe was
weighed and placed in a polyethylene bag and frozen on

Dry Ice.

3. Thyroid and Thymus

Concomitantly with the removal of the hypothalamus
and pituitary, the thyroid gland was removed, trimmed
free of fat and connective tissue and weighed. A small
section (1—2 mm) was placed in Bouin's solution for
subsequent histological examination and the remainder
was covered in 0.25 M sucrose in a polyethylene bag and
frozen on Dry Ice for subsequent analysis of DNA, RNA,
protein and lipid content. Between 20 min and A5 min
after stunning, the thymus gland was removed, trimmed
and weighed and a portion was retained in 0.25 M sucrose

for analysis of DNA, RNA, protein and lipid.

26

A. Reproductive Tract

Both ovaries* were removed within 10 min after
stunning and placed in ice cold 0.9% NaCl. The corpus
luteum was dissected from each ovary, weighed, a portion
fixed in Bouin's solution for histological study and
about 1 qm homogenized in about A m1 Krebs-Ringer bicar-
bonate buffer in an all glass homogenizer. About 200 gm
of this homogenate was frozen on Dry Ice and stored at
-200C for subsequent analysis of nucleic acids, sterols
and steroids. Other portions of about 100 mg tissue
(0.5 ml homogenate), after gassing with 95% 02 and 5%
C02, were incubated for 1 hr at 37°C in a final volume
of 2.5 ml Krebs-Ringer bicarbonate buffer containing
1 mg/ml glucose and 5 no cholesterol-73H with or without
LH (A ug/ml) and with NADPH (0.2 umole/ml) or with
NADPH generating system (NADP + glucose-6-phosphate,
each 0.2 umole/ml). When sterol, LH or cofactors were
to be included, they were added to the incubation flask,
lyophilized and stored desiccated at -79°C until buffer
was added to the flask immediately before incubation
was begun.

When present, the largest follicle on the ovary
was isolated and its diameter measured. The follicle
‘was then weighed with and without follicular fluid and

by subtracting the weight of the follicular wall from

*A study of the growth of the corpora lutea and
progesterone synthesis has been reported by H. D. Hafs
and D. T. Armstrong (A6).

27

that of the total, the weight of the follicular fluid
wastobtained.

The vulva, vagina, cervix, uterus, and oviducts
were removed intact approximately 30 min after stunning.
After removing the connective tissue and fat, the vagina
was opened on the dorsal surface from the dorsal commis—
sure of the vulva to the dorsal fornix of the anterior
vagina and the length of the vagina was measured from
the ventral commissure of the vulva to the ventral for-
nix of the anterior vagina. After the vulva was removed
from the vagina posteriorly and the cervix was detached
anteriorly, the vagina was weighed, a small portion of
tissue (l-2mm), taken just anterior to the urethral
orifice, was fixed in Bouin's solution for histological
examination, and the remainder was covered with 0.25 M
sucrose in a polyethylene bag and frozen on Dry Ice for
analysis of DNA, RNA, protein and lipid content. The
(cervical canal was exposed and its length measured.
.After severing the cervix from the uterus, the freed
<3ervix was weighed, a small portion of tissue (1-2 mm)
ffiixed in Bouin's solution for histological examination
arni the remainder frozen in 0.25 M sucrose for DNA,
PETA, protein and lipid analysis. The body and both
hfixrns of the uterus were opened on the dorsal surface
anti, after taking their length, the entire uterus was

WEighed. A portion of tissue was taken from the middle

28

of one uterine horn for a histological section, and the
remainder was frozen in 0.25 M sucrose. After recording
the length of each oviduct, a sample was fixed in Bouin's
solution and the remainder discarded.

When slaughtering and dissecting had been com-
pleted, all samples frozen on Dry Ice were stored at
-2OOC until required for analysis. The histological.
specimens were processed immediately and kept imbedded

in paraffin.

C. Bioassays of Pituitary Hormones and LRF

The anterior pituitaries were removed from storage
(-2OOC), thawed, weighed to the nearest-0.1 mg and homo-
genized in a Potter-Elvehjem homogenizer in small por-
tions (about 100 mg) in pyrogen—free 0.85% saline. After
adjusting the volume of homogenate to 50 mg pituitary
equivalent per ml, the homogenate was centrifuged to
remove cellular debris and the supernatant fluid was

used in the assays for FSH and LH.

l. Follicle Stimulating Hormone

Because bovine pituitaries contain little FSH
relative to LH or relative to pituitary FSH potencies
in other species, doses of 50 and 100 mg equivalents
of pituitary tissue were used for the FSH bioassay (25,
70, 73). In addition, each rat received 20 IU of human
chorionic gonadotrophin (HCG-Upjohn Chorionic Gonado-

‘trophin) as previously outlined in the ovarian weight

29

augmentation bioassay (131). The unknowns were compared
to 30 and 60 pg levels of NIH-FSH-S2 and to a dose of
HCG alone. For each assay, five rats were used at each
dose level of the unknowns and standard and the total
dose was injected in 9 doses, given approximately 8 hrs
apart over a three day period.. Twenty-four hours after
the last injection, the rats were killed, both ovaries
removed, trimmed and weighed and the potencies were

estimated by the slope ratio procedure (15).

2. Luteinizing Hormone

 

To measure the pituitary levels of LH, the ovarian
ascorbic acid depletion method (OAAD) was used (96). At
25 days of age, the test rats (Sprague-Dawley strain
from Spartan Research Animals, Haslett, Michigan) were
injected with 50 IU of pregnant mare's serum (PMS-
Ayerst Laboratories, "Equinex") and with 25 IU of HCG
60 hrs later. Six days after the HCG injection, each
rat received 0.5 ml of either an unknown or standard
preparation via the femoral vein. Two dose levels,

0.1 mg and 0.A mg pituitary equivalent per rat, were
used for each pituitary and five rats were used at each
dose level. Four hours later, the left ovary was surgi-
cally removed, trimmed, weighed to the nearest 0.1 mg,
homogenized in 2.5% meta phosphoric acid and brought

to a concentration of 10 mg ovarian tissue/ml. This
homogenate was filtered and the ascorbic acid concen-

tration of the ovary was determined.

 

30

Each rat received approximately 30,000 IU of peni-
cillin at the time of surgery and again 2A hrs later.
Two days after surgery, the rats, which had been housed
together in a large cage, were reallotted at random
into groups of five and each rat again received 0.5 ml
of either unknown or standard preparations via the
femoral vein and the concentration of ascorbic acid was-
determined in the right ovary. The ovarian ascorbic
acid depletion of these rats was compared with the.
ovarian ascorbic acid depletion of five rats at eachv
level produced by 0.A ug and 1.6 ug of NIH—LH-B2 per

rat by the procedure for parallel-line assays (15).

3. Luteinizing Hormone Releasing Factor

Each hypothalamus was thawed and boiled in its
own sample bottle to destroy any LH-like activity.
After cooling, the samples were pooled according to
day of cycle and each sample bottle was rinsed three
times with 0.1 N HCI to remove any adhering material.
All pools were brought up to 75 ml with 0.1 N HCl,
placed in three dialysis bags (25 ml per bag), and
dialyzed against 225 ml distilled water. The dialysate.
‘was neutralized with 2 N NaOH, frozen, lyophilized and
stored at -20°C.

LH releasing activity of the hypothalamic extract
l~as determined in rats prepared similarly to those for

‘the LH bioassay. Each of the five rats in the low dose,

31

group received 0.2 equivalents of hypothalamus and those
in the high dose group 0.8 equivalents; all in 0.5 ml
solution. Four hours after injection of the test solu-
tions, the ascorbic acid concentration response to the
LRF was measured, and compared with the response obtained
from rats injected with 0.5 ml 0.85% saline. Other con—
trols were cerebral cortical extracts, treated similarly
to those of the hypothalamus, and a dose level equivalent
to 15 ug per rat of NIH-LH-S9 which had been boiled in
0.1 N HCl, dialyzed against distilled water, frozen and
lyophilized. Since the lyophilized hypothalamic extract
contained a large quantity of NaCl from the neutraliza-
tion of HCl and NaOH, it was dissolved in distilled
water and injected very slowly to prevent shock due to

the hypertonicity of the test solution.

A. Biochemical Determinations

DNA, RNA, protein and lipid, were measured for
the vagina, cervix, uterus, thyroid and thymus from
homogenates containing 50 mg tissue per ml saline. For
ITucleic acid analyses, a modification (133) of the
Schmidt and Thannhauser (121) method was employed.
lLipid content was estimated from the weight of the
:residue after evaporation of the combined alcohol,
Inethanol—chloroform and ether extractions obtained
:from the above nucleic acids analysis. And protein

<3ontent was based on the Biuret reaction (A1).

32

5. Histological Determinations

Sections from the vagina, cervix, uterus, oviducts,
and thyroid were stained with Harris' hematoxylin and
eosin and the heights of the luminal epithelial cells

were measured with a calibrated occular micrometer.

RESULTS AND DISCUSSION

A. Statistical Analyses
The analysis of variance appropriate to most criteria
of response in the heifers in this study included two
sources of variation——stages of estrous cycle and heifers
within stage of estrous cycle. To teSt for significant

differences due to endocrine changes at various stages of

 

the estrous cycle, I orthogonally partitioned the 6 degrees
of freedom for stage of cycle as follows.
a. days 20 + 0 versus the rest--proestrus and estrus
versus metestrus and diestrus
b. day 20 versus day 0--proestrus versus estrus
0. days 2 + A versus days 7, 11 and l8--metestrus
versus diestrus
d. day 2 versus day A——early metestrus versus late
metestrus
e. day 7 versus day 11 and 18—-early diestrus versus
later diestrus
f. day 11 versus day 18—-middle diestrus versus late
diestrus.
Conclusions in the following sections are based

lar“Sely upon these orthogonal contrasts.

33

3A

B. Length of the Estrous Cycle

Since observation of the heifers for behavioral mani-
festations of estrus began when the heifers were about 13
months old, the exact age at which first estrus occurred
is unknown. Desjardins (25) reported that first estrus
occurred between 5 and 11 months, the mean being 7.A i 0.3
mouths. And the animals in this study were raised under the
same management conditions, so we may assume first estrus
occurred in the majority of these heifers between 7 and 8
months.

The mean length of the estrous cycle was 20.6 i 0.2
days and this agrees very well with the values of 20.23 i

0.05 and 20.5 i 0.6 obtained by others (10).

C. Body_Weights at Slaughter
The mean body weight at slaughter of 377 kg is com-
parable to that of 373 kg (87) for growing dairy heifers in
their 16th month of age. Orthogonal contrasts revealed
some significant body weight differences between days of
the estrous cycle (P < 0.01), but as will be shown below,
these differences were not associated with endocrine changes

and therefore, appeared to be caused by other factors.

35

D. Hypothalamic and Pituitary Gland
Changes During the Estrous Cycle

 

 

1. Luteinizinngormone Releasing
Factor--LRF

 

 

Recent work (18) has shown LRF content to be high in
hypothalami of cycling rats in the evening of late diestrus.
Early in proestrus there is a decline in the.hypothalamic
content of LRF and shortly thereafter there is a decline in
the pituitary LH content, suggesting LRF stimulates release
of LH. The data obtained in this study are the first known
report of hypothalamic changes of LRF during the estrous
cycle of the bovine.

Ovarian ascorbic acid depletion activity exhibited by
the hypothalami was not depleted by injections of similarly
treated cerebral cortical preparations. These results sug-
gest that the responses produced by the hypothalami are not
due to substances produced elsewhere in the brain.

Another group of rats were injected with NIH-LH-S9
which had been inactivated by boiling (105) for 10 min in
0.1 N hydrochloric acid and then removed by dialysis. If
none of this LH had been inactivated and if all had been
found in the dialysate, each rat would have received 15 ug
LH. The rats receiving "inactivated LH" did not exhibit a
depression in ovarian ascorbic acid concentration. In fact
the average value of ascorbic acid was identical to that of
the saline controls. This result suggests that inactivation

of any LH which may have existed in the hypothalami was

36

complete. Injections of 0.2 pg and 0.A ug of NIH-LH-S9
depressed ascorbic acid concentration by 2A and 28 per cent
respectively. Any LH contamination of the hypothalami
before boiling and dialyzing would probably be minimal, so
the possibility of LH contamination altering the data for
the hypothalamic extracts is negligible (70).

The units of LRF activity were arbitrarily set as '1‘
being the per cent depression of ovarian ascorbic acid by I

the hypothalamic extracts compared with ovarian ascorbic .J

 

acid content of rats receiving saline injections. l
Since injections were made at two dose levels (0.20
hypothalamus and 0.80 hypothalamus), we can study the
effects of 2 estimates of LRF activity independently and
then combine the two. LRF activity for the low dose
(Table l and Fig. l) was high from days 2 to 7 of the
estrous cycle and decreased to days. 11 and 18. Orthogonal
contrasts revealed that the depression of hypothalamic
LRF at days 11 and 18 was significant (P < 0.01).
The response of the high dose generally followed that
of the low dose. Again LRF activity was high from day 20
to day 7 and low on days 11 and 18. Orthogonal contrasts
revealed that LRF was greater during metestrus than during
diestrus and declined significantly during diestrus (from

day 7 to days 11 and 18, P < 0.01).

TABLE l.--Hypotha1amic LRF units

37

8.

for the two dose levels.

 

Day of Estrous

Low Doseb

 

Cycle High Doseb Average
0 6.2 18.5 12.A
2 13.1 16.5 1A.8
A 10.8 20.5 15.7
7 15.0 l8.A 16.7
11 8.3 8.6 8.A
18 6.A 6.8 6.6
20 10.1 16.9 13.5

 

aPer cent ovarian ascorbic acid depletion, relative
to that caused by saline.

bLow and high doses were 0.2 and 0.8 hypothalamic
equivalents per rat.

2. Anterior Pituitary

 

a. Weight.--Anterior pituitary weight did not vary
greatly during the estrous cycle (Table 2). There was,
however, a relatively small decline during mid—cycle and
again at proestrus, but only the increase from day 11 to 18
was significant (P < 0.05). At all other stages of the
cycle, average anterior pituitary weight ranged only from
1.500 to 1.558 gm.~ As will be shown below, pituitaries,
prolactin, FSH and LH reach maximum levels late in the
cycle but in view of the quantity of hormones found, it

seems unlikely that these levels alone would be responsible

38

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for a significant change in total pituitary weight during
the cycle. In the rat (126), however, anterior pituitary
glands gained weight during proestrus and estrus and lost
weight during metestrus and diestrus.

b. ~Prolactin.—-Pituitary prolactin content (126)
paralleled prolactin concentrations (Table 2 and Fig. 2)
throughout the estrous cycle. Because of large variations
among animals, Sinha evaluated the data by regression
analysis and obtained a significant slope (P 5 0.08).
Although the statistical validity is questionable, he used
a t-test to compare the various stages of the estrous cycle
such as day 2 vs day 20. Although the changes in prolactin
concentration were of borderline statistical significance,
he concluded that there were some valid trends. For example,
from a minimum of 0.012 IU/mg at day 2 of the estrous cycle,
prolactin concentration increased to a maximal value of
0.0A5 IU/mg on the day of estrus-—an increase of 275 per
cent. Day (2A) also observed a significant linear increase
in pituitary prolactin potency from day 2 to day 18—19 of
the estrous cycle of sheep.

That prolactin is possibly luteotrophic in the bovine
was discussed above in "Review of Literature." The additional
evidence from the present heifers suggests that luteal func—
tion in the bovine may be related to prolactin as well as to
LH in that changes in pituitary prolactin content follow

by about 1 day changes in pituitary LH. Also, a recent

 

i-

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report revealed that injections of antibovine LH serum (128)
reduced corpus luteum weight and total progesterone but_did
not decrease progesterone content. Furthermore, in cows
with severed pituitary stalks (56), progesterone secretion
followed a curve identical to that for intact controls but
at a reduced level. In pituitary stalk-sectioned sheep
(132) progesterone secretion was comparable to intact
animals during the estrous cycle. Thus, our knowledge is
presently insufficient to determine the relationships of LH
and prolactin to luteal function.

0. Follicle Stimulating Hormone--FSH.—-Total content
of pituitary FSH paralleled pituitary FSH concentration
(Table 2 and Fig. 3). Pituitary FSH was highest on day 18
(late diestrus). Forty—nine per cent of pituitary FSH was
lost from day 18 to day 20 and A6% of the FSH present on
day 20 was lost by day 0. In other words, from day 18 to
day 0 there was a decrease of 73 per cent in pituitary FSH.
This stage of the cycle corresponds to the period of rapid
growth of the follicle (9, 30, 5h)_

From day A to day 18 there was a linear increase in
pituitary FSH and the maximum content was attained on day
18. There was little evidence of biphasic FSH release,
although as Desjardins and Hafs (26) also reported, there

was an insignificant depression in pituitary FSH on days A
and 7,

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d. Luteinizing Hormone-—LH.-—As with prolactin and
FSH, LH concentration and total LH content measured in
individual pituitary glands paralleled each other (Table 2
and Fig. A). Pituitary LH increased significantly from
metestrus to diestrus (P < 0.01). It then decreased from
day 11 to day 18 (P < 0.05) and increased to a maximum at
day 20. But the most dramatic change in pituitary LH
occurred from day 20 to day 0, when there was a significant
decrease (P < 0.01) of 71 per cent in pituitary LH. It
further declined 61 per cent from day 0 to day 2. Thus,
pituitary LH decreased 89 per cent from day 20 to day 2.

Unfortunately at the time of this study, meaningful
techniques for measuring LH in peripheral blood were
unavailable. Therefore, no data on plasma LH levels were
taken on these heifers. However, if LRF may be equated with
LH release and pituitary LH reflects LH storage, then the
ratio LRF/LH should be a rough measure of release/storage
ratio. These ratios (x 1000) for days 0, 2, A, 7, ll, 18
and 20 were 6.9, 21.5, 9.2, A.2, 1.6, 1.8 and 2.1, respec-
tively. Similar calculations could be done by equating LRF
with LH synthesis, but the important conclusion is that the
hypothalamic/pituitary relationship is relatively constant
during the last half of the estrous cycle. It changes to a
Peak on day 2. Whether an increment in these ratios repre-
sents release or synthesis, it suggests more plasma LH
between days 0 and 7. The low values at days ll, 18 and 20

may be due to high progesterone negative feedback on LRF.

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E. Ovarian Changes During_the
Estrous Cycle

 

 

1. Follicle

 

Of the four criteria of the follicle measured (Table 3
and Fig. 5), the weight of the follicle wall probably gives
the best estimate of follicular steroidogenic activity.

The weight of the follicle wall increased from day 2 to day
7, levelled betWeen day 7 and 11 and decreased significantly
(P < 0.05) from 388 mg on day 11 to 252 mg on day 20.
Follicles measured on day 20 may have been atrophying or
they may have been the new preovulatory follicles. The most
marked follicular growth occurred between day 20 and day 0.
The periods of follicular wall growth (days 2 to 7 and 18

or 20 to 0, Table 3) coincide with loss of FSH from the
pituitary (Fig. 3). When there was little follicular
growth (days 0 to 2 and 7 to 18 or 20), FSH accumulated in
pituitaries. Thus, follicular growth probably reflects
stimulation by FSH which has been released from the pituitary
gland between days 2 and A and 18 and 0.

Trends in follicular diameter, weight of follicle and
weight of follicular fluid are somewhat similar to that of
the weight of the follicle wall. Overall, the results
suggest a buildup in the size of the follicle from day 2
to day 11 and degeneration to day 20. But the greatest

change is the marked increase in size from day 20 to day 0.

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2. Corpus Luteum

corpora lutea obtained in this study and only highlights of

A9

those data will be given here.

the estrous cycle, weight changes followed a typical growth
curve (Table A) and estimated cell numbers (DNA) paralleled

luteal weight during luteal growth.

But the DNA concentra-

Growth.--A report (A6) has been published on the

During the first half of

tion increased from 2.A8 mg/gm at day 11 to A.30 mg/gm at

day 0.

sumably caused by aging of the corpora lutea.

This suggests more cells in a smaller volume, pre-

Ratios of

RNA/DNA which estimate synthetic activity were greatest

during the most rapid growth phase.

TABLE A.--Corpus luteum weight and progesterone, 20 B-ol,

progestin (progesterone plus 20 8-01) content.

 

 

Day of Weight Progesterone. 20 B-ol Progestin
Estrous of Content Content Content
Cycle CL Per CL Per CL Per CL
(ms) —— (us) -—
0 1,917 5.3 1.7 10.6
2 360 2.5 0.8 3.3
A 932 23.6 7.0 30.6
7 A,59A 186.7 16.6 203.3
11 6,328 266.7 62.8 329.5
18 5,0A2 239.1 23.1 262.1
20 3,A96 10A.A 30.1 13A.5

 

50

Histologically, large lutein cells which have been
associated with progesterone synthesis (130) were sparse in
corpora lutea on day 2 but increased through day 11 and
remained as the most striking micromorphological feature
through days 18 and 20. However, by day 18, these cells
were rounded as compared with their previous irregular
polyhedral shape and the small lutein cells had lost most
of their identity by day 18. Arterioles (A6) of corpora
lutea on days 18 and 20 possessed thickened walls, and the
endothelia appeared serrated.

b. Progesterone.-—Progesterone concentration paral-

 

leled total progesterone (Table A and Fig. 6), and both of
these parameters paralleled corpus luteum weight. These
data generally agree with previous research (72) but reveal,
for the first time, low progesterone concentration (7 Ug/Sm)
in corpora lutea on day 2 and a significant increase from
day A (25 ug/gm) to day 7 (Al ug/gm). At mid-cycle it is
maximal, decreasing gradually toward the time of next
ovulation.

However, net synthesis of corpus luteum progesterone
(Table 5) increased progressively from 17 ug/gm at day 2 to
136 ug/gm at day 11. Synthesis in vitrg'was relatively
constant from day 11 to day 20 but at day 0, it dropped to
A6 ug/gm. There was greater synthesis of progesterone in
incubations with NADPH (P < 0.001) than in incubations with

the NADPH generating system.

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53

c. 208—hydroxy:pregn-A-en-3-one (208-o1).--Total
208-01 content (Table A and Fig. 6) increased progressively
from day 2 (0.80) ug/CL to day 11 (63 ug/CL). At day 18,
208-01 content was 23 ug/CL, but unlike progesterone, the
content increased to 33 ug/CL at day 20, declining to 5 ug
at day 0. Hilliard et_al. (58) suggested that 20aeol, the
a isomer of 208-o1, acts as a positive feedback agent to
prolong and heighten LH discharge in mated rabbits. Their
results indicate that "coitus triggers a transient release
of LH sufficient to activate the synthesis and release of
200-01 and that this progestin acts as a positive feedback
agent to prolong and heighten gonadotrophin discharge."

In the rabbit, 20a-ol is a discreet hormonal entity derived
from sterol precursors stored by the ovarian interstitial
cells. However, in the present study 2OB-ol was measured
in the corpus luteum, and both the concentration and total
content increased at day 20 when pituitary LH content was
highest. Perhaps at this time sufficient LH was released
to stimulate synthesis and release of further LH. 208-01
measured at more frequent intervals and in ovarian venous
effluent, rather than in the corpora lutea of the present
heifers, would have been a better test of Hilliard's
hypothesis.

From an average of 3 ug/gm at day 2, net synthesis of
208-01 during incubation of corpus luteum homogenates

increased to 26 ug/gm at day 11, remained relatively constant

5A

to day 20 (22 ug/gm), and then declined to l ug/gm (Table 5)
at day 0. Average synthesis of 208-ol on days 11, 18 and

20 was significantly (P < 0.001) greater in incubations with
the NADPH generating system (19 ug/gm) than with NADPH

(8 ug/gm). In other words, glucose-6-phosphate dehydrogenase
was not rate limiting to synthesis of 208—01. But, this is
in contrast to the results with progesterone synthesis.

Total progestin (progesterone plus 208-01) synthesized

with the NADPH generating system was quite similar to the
amount synthesized with NADPH, and this was true at each

stage of the estrous cycle.

3. Ovary Weight with CL and Follicle

 

The weight of the whole ovary which contained the
corpus luteum and largest follicle (Table 3) increased
progressively from day 2 (8.18 gm) to day 18 (12.05gm) and
decreased to day 0 (7.22 gm). These changes are mainly due
to the corpus luteum weight, the largest follicle weight
and the weight of any other growing follicles. At day 18,
both corpus luteum weight and the weight of the largest
follicle are lower than at day 11, suggesting that the
larger ovarian weight on day 18 may be due to increased
interstitial tissue.

But this conclusion is untenable because one cannot
be certain which "large follicle" was measured on day 18.
And, besides the atretic mid-cycle follicle and new pre-

ovulatory follicle on day 18, there are other follicles

. Ta

 

55

of varying size and weight which increase the overall
ovarian weight. However, by day 20, the corpus luteum is
rapidly declining in weight, and possibly other follicles
are becoming atretic because the preovulatory follicle is
growing at an extremely rapid rate. At day 0, there is a
further decline by both the corpus luteum and smaller
follicles which have given way completely to the ovulatory
follicle.

A. Ovary Weight Without Corpus

Luteum and Follicle

When weights of both the corpus luteum and largest
follicle are removed from the weight of the ovary (Table 3),
the remainder is largest on day 18, but as outlined above,
this could be caused by structures other than interstitial
tissue. The corpus luteum from the previous cycle further
degenerates between day 2 and A, reducing the weight of the
ovary. The increase from day A to day 18 is probably due
to the growth of smaller follicles which will eventually
atrophy by day 20, possibly because of the increasing
demands for FSH by the new follicle. And, as before, they
will atrophy further by day 0.

From the preceding data, and the data on corpus
luteum, progesterone and 208-ol, it appears unlikely that
changes in the ovarian interstitium cause significant
changfis on the estrous cycle; i.e., unlike the rabbit, the

bovine ovarian interstitium probably does not produce any

56

hormones. However, the progesterone and 2OB—ol content
should be measured in ovarian venous blood and interstitium
to ascertain whether or not the interstitium of bovine
ovaries does actively secrete gonadal hormones.

F. Vaginal Changes During the
Estrous Cycle

1. Weight

 

The changes in weight of the vagina (Table 6) at
various stages suggest a biphasic pattern. For example,
the maximal weight of 207 gm occurred at day 2. Then it
declined to 169 gm at day 7, but increased to 189 gm at
day 11 in mid-cycle. There was a decline in weight by
day 18 (178 gm) and then it increased to 201 gm at day 0
(estrus). Orthogonal contrasts revealed a significant
(P < 0.05) decrease in vaginal weight from day 2 to A and
from metestrus to diestrus (days 7, 11 and 18). The
increase from days 7 to 11 approached significance (P < 0.09).
Thus, the data suggest that weight increases occur at the
time of estrus and around day 11, times when there are

large follicles on the ovary.

2. DNA, RNA,_Protein and Lipid

Vaginal DNA concentration was lowest on day 0 (1.55
mS/gm) and highest on day 11 (1.96 mg/gm) suggesting fewer
number of cells per given volume during estrus, perhaps as

a result of edema, possibly in the epithelial layer. But

57

TABLE 6.--Vagina1 weight, DNA, RNA, protein and lipid con-
tent and epithelial cell-layer height.

 

 

Day of
Estrous Weight DNA RNA Protein Lipid Epithelial
Cycle Height
(gm) — (ms) — (cm) (11)
0 201 310 622 31 58 57
2 207 287 655 33 57 39
A 182 326 675 30 A9 31
7 169 282 551 23 Al 22
11 189 371 692 3A A5 21
l8 178 331 598 31 37 27
20 18A 296 588 33 A0 29

 

total DNA (Table 6 and Fig. 7) was lowest on day 7 (282 gm)
and highest on day 11 (371 gm). Thus, DNA flucturates
during the estrous cycle. It is somewhat higher at estrus
and mid-cycle. And this is further reflected by vaginal
DNA/100 kg body weight which is highest at estrus and day
11 (92 and 9A mg/100 kg body weight respectively).

RNA concentration followed a pattern similar to DNA
concentration but there were fewer significant changes
between various stages of the estrous cycle. Changes in
total RNA mimicked those for total DNA. Consequently,

RNA also shows a biphasic pattern--high at estrus and

around day 11 (Table 6 and Fig. 7).

58

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59

As-with DNA and RNA concentration, vaginal protein
concentration does not follow the same trends as total
proteins; being lowest on day 7 (0.139 mg/gm) and highest
on day 19 (0.186 mg/gm). Total protein follows total DNA
and RNA; being highest at day 11 (3A mg) and lowest on day
7 (23 ms).

Lipid concentration and total lipid content paral-
leled each other fairly well, being highest at estrus (283.2
mg/gm and 57.8 gm, respectively) and lowest at day 20
(222.A mg/gm and A0.0 gm respectively). The total lipid
content did not show the biphasic pattern as did DNA, RNA
and protein. Rather, it was significantly greater (P < 0.01)
from metestrus to diestrus.

3. Height of Epithelial
Cell—Layer

 

 

At estrus, the height of the vaginal epithelial cell-
layer reached a maximum of 57 u (Table 6 and Fig. 7).
Thereafter it decreased steadily to day 1A (21 u), remained
relatively constant to day 20, and doubled from day 20 to
day 0. Thus, unlike some of the other vaginal criteria,
there was no evidence from the height of the epithelial cell-
layer to suggest appreciable estrogen secretion in mid-
cycle. On the other hand, this response would be expected
to require considerable time rather than being instantaneous.
If this was the case in these heifers, the vaginal ep1_
thelium may have responded to mid—cycle estrogen secretion

with increased thickness on days 12 to 15.

60

A. Length

 

The average length of the vagina was 29.A cm (Table 7).
Changes in length during the estrous cycle appeared to be
random. At least no known physiological substance could
be established for the minor changes in vaginal length

apparent in Table 7.

TABLE 7.--Length of vagina, cervix, uterine body and horns
and oviducts.

 

 

 

 

 

 

Uterus Oviduct
Day of
Estrous Vagina Cervix Body Right Left Right Left
Cycle Horn Horn
.“(Cm)_.l
0 28.6 5.6 2.0 28.8 31.6 21.2 19.8
2 29.8 5.A 2.6 29.6 29.2 21.0 21.2
A 28.8 5 2 2.A 31.0 31.6 20 A 19.8
7 28.6 5 O 2.6 33.8 3A.8 22.A 21.A
11 33.0 6.0 2.6 32.A 32.0 21.A 22.2
18 29.8 6 0 2.6 35.0 33.6 2A.0 21.6
20 27.2 5.2 2.8 30.0 30.A 21.A 23.2

 

5. Summary

Total DNA, RNA and protein content showed biphasic
patterns; high at estrus and day 11. But the RNA/DNA
ratio revealed no significant changes (P > 0.05),

suggesting RNA increases paralleled those of DNA. Both

61

the total lipid content and height of the epithelial layer
were highest at estrus and lower for the remainder of the
cycle.

G. Cervical Changes During the
Estrous Cycle

 

 

1. Weight

 

Whether the changes in cervical weight reflected
physiological changes or random variation were difficult
to ascertain (Table 8). The highest value occurred during
metestrus (A2 gm) and the lowest on day A (30 gm). The
only apparent physiological reason for this sudden increase
was the growing corpus luteum with its increasing levels of
progesterone. In fact, weight of the cervix follow a trend
almost the inverse of progesterone content of the corpus

luteum (Table 6).

2. DNA, RNA, Protein and Lipid

 

Total DNA content reached a maximal level at day 11
(Table 8) of 126 mg. It was lower at estrus (89 mg). At
day 20, total DNA reached a second peak, suggesting a
biphasic curve. The DNA/100 kg body weight did not follow
this pattern; it was high at day 11 (31.7 mg/100 kg body
weight) and decreased steadily to day A (20.1 mg/100 kg
body weight). The significant decrease between day 2 and A
(97 to 70 mg) for total DNA seemed to be due to the large
difference in cervical weight between these two days

(P < 0.01).

62

TABLE 8.—-Cervical weight, DNA, RNA, protein and lipid
content and epithelial cell height.

 

Day of

 

Eggrgus Weight DNA RNA Protein Lipid Hgight
(gm) ————(mE)——- (gm) (u)

0 39 89 191 5.3 7.5 28.6

2 A2 97 179 6.7 8.5 25.2

A 30 70 119 A.9 7.6 16.6

7 35 108 163 5.7 7.A 16.6

11 38 126 162 6.7 8.1 18.3
18 A1 111 169 5.A 10.9 15.8
20 A0 112 182 6.8 10.8 15.7

 

Basically, RNA concentration and total content of
the cervix paralleled each other (Table 8 and Fig. 8).
Total RNA was highest at estrus (191 gm) and declined to
minimal levels at day A (119 gm). The significant
decrease of the total RNA content reflected changes in
the weight of the cervix (P < 0.01). The increase in RNA
began at day 18 and continued through day 0. Although
there was a significant decrease in RNA concentration
throughout diestrus (P < 0.01), this was not reflected in
the total RNA, as the weight of the organ icnreased
steadily throughout this period. There was no evidence

suggesting bimodality of total RNA during the estrous cycle.

63

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Except for the low value of total protein at day A
(A.9 mg), protein concentration and total content paralleled
each other. Total protein showed a triphasic pattern with
high levels at days 2 (6.7 gm), 11 (6.7 gm) and 20 (6.8 gm).
The lipid concentrations and total content also paralleled
each other. The highest values occurred at day 18 and 20
(11.0 and 10.7 gm) followed by significant decreases at
estrus (P < 0.01). During metestrus there was an increase,
followed by a decrease to day 7 and then a steady increase

through day 20.

3. Epithelial Cell Height

Between days 20 and 0, cervical epithelial cell
heights almost doubled (15.7 N to 28.6 0 respectively,
Table 8 and Fig. 7). Following estrus there was a steady
decrease to day A (16.6) and from day A there was a steady
increase to day 11 when a second peak of 18.3 0 occurred;
this was followed by a significant decrease to day 18 of
15.8 p (P < 0.05). These data again showed a biphasic

pattern.

A. Length
The average length of the cervix was 5.A9 cm (Table
7). There do not seem to be any physiological differences

in the length of the cervix throughout the estrous cycle.

65

5. Summary

Based upon epithelial cell height, greatest synthesis
of mucus in the cervix took place at estrus. From estrus
to mid-cycle (day 11), synthetic activity diminished and
gradually increased from day 18 on. It followed the
pattern of total RNA. Cervical epithelial cell heights and
total DNA content revealed parallel biphasic curves, whereas
protein showed a triphasic curve. Lipid content was maxi-
mal on day 20 and minimal at day 7.

H. Uterine Changes During the
Estrous,§ycle

 

1. Weight
The uterine weights suggested no meaningful cyclic

pattern (Table 9).

2. DNA, RNA, Protein and Lipid

DNA concentration paralleled total DNA, reaching peak
levels at day 2 and day 11 and minimal levels at days 7,
l8 and 20. Here again there was a suggestion of a biphasic
trend (Fig. 9), with peaks occurring around estrus and
again at mid—cycle. There seemed to be a lag period from
the time of expected maximal estrogen blood levels to the
time of maximal DNA around estrus. Unfortunately, there
were no other estimates between day 11 and 18 to see if

such was the case at mid-cycle.

66

 

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67

TABLE 9.--Uterine weight, DNA, RNA, protein and lipid content
and epithelial cell height.

 

Day of
Estrous Weight DNA RNA Protein Lipid Cell Height
Cycle

 

(sm) (mg) (mg) (me) (Em) (A)

0 168 905 A86 18.8 36.0 25.1

2 173 113A A96 20.6 59.0 17.8

A 152 938 625 18.5 A5.2 23.3

7 177 883 731 25.3 AA.0 25.8

11 166 1106 672 19.9 A2.2 22.1
18 190 870 A8A 22.8 A6.o 2A.A
20 169 872 A60 23.A A3.A 2A.0

 

Uterine RNA concentration and total were lower at
proestrus and estrus than during the rest of the cycle and
this was also true for DNA (Table 9 and Fig. 9). Highest
RNA concentrations occurred at days A, 7, and 11 (A.l8,
A.01 and A.06 mg/gm reSpectively), and this trend was also
evident for total RNA. During early metestrus, RNA con-
centration was relatively low but increased markedly by
day A, and again this was evident for total RNA. From
days 7 to 18, both RNA concentrations and total RNA
decreased. By day 20, RNA concentrations began to increase

rapidly to estrus (2.90 mg/gm). Partly due to the variation

.2."

h

68

of the uterine weights, total RNA did not follow this
trend, but it increased at estrus.

Protein concentrations and total protein generally
paralleled each other, with peaks at days 7 and 20.
Initially the curve followed that of RNA, but when RNA
declined after day 11, total protein increased to day 20
(23.A mg) and fell by the day of estrus.

Although there was some variation between lipid con-
centrations and total lipid content, the overall trends
were similar. Lipid content was highest on day 2 (59.0 gm),
declined to day 11 (“2.2 gm), and remained relatively con-
stant to day 20 (A3.A gm). Between days 20 and 0, there
was a significant drop to 36.0 gm (P < 0.05), the lowest

value during the estrous cycle.

3. Epithelial Cell Height

Uterine epithelial cell heights were maximum at
estrus and day 7 (25.1 and 25.8 p respectively). There was
a significant increase in cell height during metestrus
(from 17.8 p on day 2 to 23.3 p on day A) which continued
until day 7 (P < 0.01). This was followed by a drop at
day 11 (22.1 u) and a gradual increase to day 0 (25.1 u).
These data also showed some evidence for a biphasic change

during the estrouscycle (Table 9 and Fig. 9).

B;_ Length
The average length for the uterine body was 2.51 cm

(Table 7). The shortest length occurred at estrus, possibly

69

because high estrogen titers increased the tone of the
uterus. There was no important difference between lengths
of right and left uterine horns; the right averaged 31.5

cm and the left 31.9 cm.

5. Summary

 

There seems to be no uniform pattern for all of the
biochemical criteria measured in the uterus. This, in part,
may be a result of the heterogeniety of this tissue which
included endometrium, myometrium and connective tissue.

But the data indicated there were cyclical changes in the
individual parameters measured during the estrous cycle.
With regards to the epithelial cell heights, the changes
inversely followed the progesterone content of the corpus
luteum. Actually, this criterion may have more accurately
and directly reflected estrogen titers, but estrogens were
not measured in this experiment. Uterine RNA and RNA/DNA,
on the other hand, appeared directly related to progesterone
content of the corpus luteum.

I. Changes in the Oviduct During the
Estrous Cycle

 

 

1. Length

The average lengths of the right and left oviducts
were 21.69 and 21.31 cm respectively; and the overall
average was 21.50 cm (Table 7). There were no significant

differences between lengths of left and right oviducts

 

70

within any of the days of the estrous cycle (P < 0.05).
And although there were significant differences in both
groups when orthogonal comparisons were made for various
stages of the estrous cycle, (P < 0.05) they have no known

physiological or anatomical meaning.

 

2. Epithelial Cell Height f
At proestrus, the epithelial cell heights (Table 10) F‘
were minimal (20.7 A). From day 0 t0 7, they increased to E;

reach a maximum of 25.7 u, and declined precipitously to
22.8 and 22.A u on day 11 and 18 respectively (P < 0.01).
These results disagreed with another report (10) which
cited that epithelial cells were highest (“5.3 U) at

estrus and are minimal (26.6 p) 6 days after estrus.

3. Summary

 

The lengths of the right and left oviducts were
similar and varied from 19.8 to 24 u. This agreed with the
range of about 20 to 35 cm previously reported (10). There
was disagreement between the present data and that of a
previous report (10) concerning the epithelial cell heights
and variations in heights during the estrous cycle. But
the heights reported here agreed more closely to those of
Desjardins (25), who measured these cell heights in
heifers from birth to puberty. For example at 12 months,

the epithelial cell heights averaged 28.4 i 0.2 u.

71

TABLE lO.--Oviducal epithelial cell height.

 

 

Day of Estrous Cycle Cell Height

(u)

0 22.6

2 22.3

A 23.5

7 25.6

11 22.8

18 22.A

20 20.7

 

 

J. Changes in the Thyroid During the
Estrous Cycle

1. Weight

 

Thyroid weight (Table 11) fluctuated throughout the
estrous cycle, but in no orderly fashion, so it was con—
cluded that there were no changes caused by the hormones

of the estrous cycle.

2;, DNA, RNA, Protein and Lipid

Unlike the reproductive organs, there were no pat-
terns to indicate a cyclical pattern for the.thyroid bio-
chemical parameters. The overall tendency was for a
Slight increase in DNA, RNA, protein and lipid content

around days 18 and 20, but this may have reflected the

72

TABLE ll.-—Thyroid weight, DNA, RNA, protein and lipid con-
tent and acinar epithelial cell height.

 

 

 

Day of Cell
Estrous Weight DNA RNA Protein Lipid Height
Cycle
(gm) (mg) (mg) (mg) (gm) (u)
0 16.3 70 103 3.2 3.3 11.6
2 17.8 68 112 3.7 3.7 10.7 ..
A 13.6 61 99 2.7 2.1 11.3 i;
7 16.3 76 90 3.6 2.8 10.9
11 1A.6 65 93 3.0 3.0 12.A
18 18.1 85 12A 3.7 3.8 11.6
20 17.A 80 111 3.3 A.0 10.9

 

slight increase in weight at this time which was sig-
nificantly higher than at day 18 (P < 0.01). Otherwise
there was no physiological pattern, nor for that matter
any important physiological reason for increasing thyroid
activity. But we must remember that a "normal" thyroid is

required for reproduction (89).

3. Epithelial Cell Height

 

Essentially, thyroid acinar epithelial cell heights

did not change during the estrous cycle.

73

A. Summary
Throughout the cycle, DNA, RNA, protein and lipid

content practically paralleled each other and in no

instance was there any reason to believe there was a

cyclical thythm corresponding to follicular or luteal

changes. The cell height data suggested there was no

increased activity during the estrous cycle and the RNA/ fl
1

DNA ratio, although showing some differences, further

3’: \-L 4

indicated no increased activity.
K. Changes in the Thymus During the
Estrous Cycle

As with the thyroid, the thymus did not seem to under-
go any cyclic changes associated with the estrous cycle
(Table 12). Although there was some variation in the
weight of the thymus, this may have been due to difficulty
in removing all of it. Because of anatomical variations,
in some cases it was more difficult to remove it quanti-
tatively than in others. And in some cases it was more
involuted than in others. So it was concluded that,
although gonadal hormones have an effect on thymus activity,
the thymus is not involved to any great extent in the

estrous cycle.

7A

TABLE l2.——Thymus weight, DNA, RNA, protein and lipid

 

 

content.
Day of
Estrous Weight DNA RNA Protein Lipid
Cycle
(gm) (gm) (gm) (mg) (gm)
0 A7A 2A.5 3.7 82.7 9A.2
2 A6A 22.3 3.2 78.6 77.A
A 580 27.1 A.2 lOA.O lO2.A
7 A36 22.9 3.2 82.A 72.8
11 553 26.9 A.0 9A.8 9A.2
18 516 27.9 A.0 88.0 83.8
20 A8A 22.0 3.3 93.1 90.8

 

GENERAL DISCUSSION

The relationship between the hypothalamus, pitui-
tary and gonads is one of negative feedback of gonadal
steroids on the pituitary, hypothalamus, or both (18)
and possibly a positive feedback of 20a-ol (or in cattle
208-01) on pituitary LH release (58) through both the~
"long loop" and "short loop." By definition the long
loop (35) is coupling of the target gland with that of
the control unit, for example, gonadal steroids acting
back on the hypothalamus, pituitary gland, or both.

And the short loop refers the action of a pituitary
gonadotrophin on its hypothalamic controller (20).

Many of the details of these relationships have been
described for rats, but not for other animals. For
example, releasing factors (120) have been isolated
from pig, sheep, and cattle hypothalami in relatively
pure form but there are no known reported studies of
changes involving hypothalamic levels during the_
estrous cycle in species other than rats. Hypothalamic
LRF content (70) measured in young Holstein bulls from
birth to puberty increased between 6 and 8 or 9 months
of age. This increased hypothalamic LRF was associated

with a marked increase in plasma LH, suggesting high

75

76

levels of hypothalamic LRF were associated with LH
release from the pituitary. In the present study, hypo-
thalamic LRF content was high on days 20, O, 2, A, and

7 (proestrus, estrus and metestrus) and low on days 11
and 18 (diestrus). These data suggest that LH was
released from the pituitary during proestrus, estrus

and metestrus and that LH was responsible for ovulation>
and for formation and early growth (and function?) of
the new corpus luteum.

Although the pituitary-ovarian relationship in
rats is not necessarily similar to that in cows, results
obtained from these animals may help to elucidate the
possible controlling mechanism for the hypothalamo—
pituitary-gonadal axis in other species. The peak LH-
releasing activity (or LRF) of rat stalk median eminence
occurred in the evening of late diestrus.(18). By noon
of the following day (proestrus) hypothalamic LRF had
significantly (P < 0.01) declined. This lower level
was maintained without any significant change through-
out the remainder of the estrous cycle. Except for
the duration of the high or low levels, these results
are somewhat similar to those of the present study,
in that high levels of hypothalamic LRF content are
present Just prior to estrus and ovulation. They dif-
fer, however, in the duration of the high levels; in

the rat, high levels lasted for a very short time,

77

whereas in the bovine they lasted somewhat longer. It
is well known, in contrast to cows, that rats develop
no functionsl corpora lutea during the estrous cycle,
and consequently have a very short diestrous.

The acute decline of rat hypothalamic-LRF (18)
was interpreted as being a release of stored releasing
factor which may initiate an increase in LH release
from the pituitary as indicated by plasma LH assays
(103). And the authors suggested that a further decline
in hypothalamic-LRF content on the afternoon of proes-
trus, when pituitary LH releasing is maximal, did not
occur because synthesis was equivalent to release of
LRF during this period. Since prolactin is luteotrophic
in rats, is it possible that no further LH is required
at this time so there is neither synthesis nor release
of hypothalamic LRF? This may also explain why the
level of hypothalamic LRF remained constant and low
for the remainder of the rat estrous cycle.

The exact mechanism for control of hypothalamic
LRF synthesis and release has not been completely
mapped. Many researchers hypothesize that LH can act
directly on the hypothalamus by negative feedback (18,
22, 103, 10A), thereby blocking synthesis and release
of LH. And estrogen from the blood plasma also can
feedback negatively on both the hypothalamus and anterior

pituitary. Furthermore, LRF activity (90) is detectable

78

in plasma of long-term hypophysectomized rats but van-
ishes following destruction of the median eminence
region--the site for storage of LRF. This result adds
further support to the theory of negative feedback,
since hypophysectomized rats also had atrophic uteri
and ovaries incapable of secreting estrogen.
Hypothalamic LRF content, measured 21 days fol-
lowing castration in female rats (98), decreased to 30
percent of control values and daily injections of 0.8
ug of estradiol benzoate further depressed hypothalamic
LRF content. Pituitary LH content increased about
four-fold following castration and was significantly
depressed by estrogen which was caused, the authors
suggested, by depressed hypothalamic LRF content. And,
since the reduction in hypothalamic LRF content after
ovariectomy did not parallel the observed increase in
pituitary LH content, they suggested that ovariectomy
elicited a greater release of hypothalamic LRF than
synthesis with consequent reduced levels of hypothalamic
LRF. In similar experiments, Chowers and McCann (18)
suggested that lowered steroid levels in blood plasma
elevate hypothalamic LRF and high levels of LH in the
blood counterbalance this effect, resulting in little
if any change in hypothalamic LRF. Similarly, in
steroid—treated rats, there would be an interaction

between the two negative feedbacks; high steroid levels

 

 

79

tending to decrease hypothalamic LRF, and low levels of
LH in the blood tending to increase it. Overall, the
net result of these two antagonistic forces would be
little or no change in hypothalamic LRF.

Estrogen in very small doses (7, 37, 11A) inhibits
synthesis and release of folliculotrophin (FTH-—a term
used to specify LH and FSH but not prolactin), and a

single injection of estrogen (37) may elicit LH release.

 

This is known as the "Hohlweg effect." Furthermore,
there is evidence suggesting the primary effect of pro-
gesterone is to suppress secretion of FTH, especially
that of LH. And this inhibition may act at the basal
tuberal region of the brain, at the anterior pituitary,
or both. But, on the other hand, small doses of proges-
terone elicit or at least hasten ovulation in rats (91)
and cows (A0, 55). Maximal pituitary LH release occurs
at proestrus, Just prior to ovulation, and even though
plasma LH is elevated, it can be further elevated by
low levels of progesterone (91). Thus it appears, in
the rat, that hypothalamic LRF may be regulated by
circulating steroids (progestogens and estrogens), at
least in certain ratios, and by LH by negative feedback.
When all nervous pathways to the hypophysiotrophic
area (a term used to designate connection between the
hypothalamus and anterior pituitary) of the median

eminence in rats are severed (A7), ovulation is inhibited,

80

whereas testicular function is maintained. In reviewing
this observation and those of his own research involving
androgen sterilization in female rats, Gorski (A2) sug-
gested testosterone may eliminate the cyclic LH releasing
properties of the preoptic area during the first post-
natal week. Thus, he concluded that the ovarian hor-
mones regulate the preoptic area as well as the ventro-
medial nucleus. And, in another review, Flerké (35)
concluded "that it is the preoptic-suprachiasmatic area
in the normal cycling female which responds under proper
environmental and hormonal circumstances by an activation
of the more terminal hypophysiotrophic area to elicit
an ovulatory discharge of FTH from the anterior lobe.
In the absence of this mechanism, the hypophysiotrophic
structures still function and FTH is secreted at a basal
rate which evokes the constant vaginal estrus syndrome."
The exact influence which LRF exerts over the
anterior pituitary gland is unknown, although an early
hypothesis (118) suggested it involved both synthesis
and release of pituitary LH. Recently, a conjecture
has been made that "LRF somehow affects the enzymes
involved in either the attachment or the synthesis of
the carbohydrate moiety of the LH polypeptide chain
and thereby controls the production of the glycoprotein,
active LH." Using lE.X$EEE incubations of pituitaries

from normal or castrated rats, recent work (118) has

 

81

shown hypothalamic LRF failed to incorporate either
luC-leucine or lL‘C-glucosamine into LH. The authors
concluded the main effect of the hypothalamic LRF was
on LH release.

That follicle-stimulating hormone (FSH) releasing
factor (63) is present in the hypothalami of various

species has been reported by many investigators. In

vivo results have shown FSH-RF acts directly on the

I p can“:
A

anterior pituitary, causing a reduction of pituitary

FSH and an increase in serum FSH. And release of FSH
from rat pituitaries cultured in yit§2_followed addition
of an extract of rat hypothalami to the incubation media.
In ovariectomized rats injected with estrogen, extracts

of beef stalk-median eminence depleted the pituitary

of FSH by 26 percent. Measurements of serum FSH indi-
cated that it increased as pituitary FSH fell. These
results indicated that FSH-RF obtained from cattle
stimulated release of FSH from rat pituitaries. There

is some evidence for a negative feedback between estro-
gen in the blood and FSH secretion from the pituitary,

and this is possible mediated through hypothalamic FSH-RF.
Although these results suggest a role for FSH-RF in con—
trolling FSH release from the pituitary gland, similar

to LRF in controlling LH secretion, the effects of FSH-RF,
extracted from the hypothalami of domestic animals on

reproductive function remain to be investigated. There

82

are no reports of changes in hypothalamic FSH-RF content
during the estrous cycle in any species.

In general, the experiments described above have
only outlined some of the cyclic changes occurring in
the hypothalamus and pituitary gland of rats during
the estrous cycle. And they only suggest a possible
approach to elucidate the changes occurring in the
bovine hypothalamus and pituitary during the estrous
cycle. For example, hypothalamic LRF content remained
high for a longer period at the time of estrus in the
bovine than for the corresponding period of estrus in
the rat. If we assume from a previous experiment (118)
that LRF mainly causes release of LH, and that LH is
luteotrophic or at least necessary for the initial
growth of the corpus luteum in the bovine, this may be
one function related to the persistently high levels
of hypothalamic LRF after estrus. On the other hand,
in rats, after the surge of LH which is required for
ovulation, no further LH may be required, and therefore,
there may be no further need for a high level of hypo-
thalamic LRF. This hypothesis assumes that high levels
of hypothalamic LRF indicate synthesis of LRF is at
least equivalent to release of LRF. This reasoning
differs from that of Chowers and McCann (18) who sug-
gested the reason for no further decline in hypothalamic

LRF on the afternoon of proestrus was the result of

83

synthesis of LRF keeping up with the release of LRF
and both were proceeding at a rapid pace although the
hypothalamic level was low.

In the present heifers, beginning at proestrus,
pituitary LH fell about 89 percent from day 20 to day
2, and at day 20 the hypothalamic LRF content was high,
having increased from day 18 (Fig. 11). The suggestion
that high levels of hypothalamic LRF are conducive to
pituitary LH release in the bovine seems to be supported
by the fact that from day 20 to day 2 most of the pitui-
tary LH was released. Now it has also been shown that
antibovine luteinizing hormone (128) administered daily
on day 2 through day 6 following estrus significantly
(P < 0.01) reduced corpus luteum weight and progesterone
(P < 0.01) in corpora lutea removed on day 11 of the
cycle. So a continuing release of LH must be required
to stimulate the growth of the new corpus luteum, which
increases rapidly both in weight and in progesterone
concentration and content between days A and 7. However,
as the corpus luteum is growing, the synthesis of LH
exceeds release from day 2 to day 11, and pituitary LH
content increases. The data in this thesis suggest
that, during metestrus and early diestrus, circulating
LH and progesterone may each feedback negatively on
the hypothalamus reducing release of hypothalamic LRF

and thereby reducing release of pituitary LH. The,

8A

hypothalamic LRF, measured at this time, may very well
be that which is synthesized in the hypophysiotrophic
center responsible for tonic release of LRF, if indeed
in the bovine a site exists such as has been advanced
in rats (A2).

Progesterone (100 mg) administered daily (65)
for 35 days starting at day 7 of the estrus cycle (estrus
being day l) to dairy heifers curtailed release of both
FSH and LH. These results substantiate the view that
LH release is inhibited by large amounts of progesterone,
but there is no indication in that report whether the
action occurs at the hypothalamic or pituitary level,
or both. And although these doses of progesterone may
not be physiological, the results reveal that proges-
terone can feedback negatively on the pituitary and
block synthesis release, or both, of LH and FSH.

By day 11 of the cycle of the present heifers,
the hypothalamic LRF content was low, pituitary LH and
progesterone concentration and content of the corpus
luteum were high, and there was a relatively large mid-
cycle follicle. The important question is, why does the
follicle not undergo final maturation and ovulate?
There may be several valid reasons. But first let us
review the effects of various hormonal treatments on
the estrus cycle. Firstly, a single injection of pro-

gesterone (10 mg), administered during estrus in the

85

cow (A0, A3, 55, 58), reduced the time to next ovulation
by about 10 hours, probably acting through the hypothalamo-
pituitary axis. Secondly, daily injections (A0) of pro—
gesterone (50 mg) inhibited ovulation. Thirdly, as men—
tioned in the preceding paragraph, high levels of
progesterone (100 mg) blocked synthesis, release, or
both of FSH and LH. These results suggest that minimal
amounts of progesterone are required to enhance release
of gonadotrophin, but larger amounts are inhibitory.
In fact, ratios of progesterone to estradiol of 12.5:1
to 75:1 appeared to be optimum for the induction of
estrus in ovariectomized cows (A0). Fourthly, oxytocin
(7), given early in the estrous cycle, inhibited forma-
tion and function of the corpus luteum resulting in
precocious estrus and ovulation at about day 12. This
result suggested that reduction of progesterone altered
the ratio of progesterone to estradiol in such a manner
as to promote ovulation. Finally, antibovine lutein-
izing hormone (128) reduced corpus luteum weight and
progesterone content. These treatments indicate to some
degree what possible relationships may exist during the
estrous cycle under normal physiological circumstances.
The mid—cycle follicle does not normally attain
size as large as that of the ovulatory follicle. In
part, this may be the result of lower plasma FSH levels

in the early stages of the cycle. Pituitary FSH content

86

decreased insignificantly early in the cycle (Fig. 3)

and a similar trend was evident in a previous study (25),
but by mid-cycle pituitary FSH was relatively high and
the increasing levels of circulating progesterone at

this time may block further synthesis, release, or both
of FSH, expecially release. Consequently, the mid-

cycle follicle does not grow as rapidly as the pre-

:h‘

ovulatory follicle which more than doubles in size

raw-:7

between day 20 and estrus under the influence of greater
quantities of plasma FSH (as evidenced by the precipi—
tous decrease in pituitary FSH between day 18 and 0).
And the failure of final follicular growth at day 11
may limit the amount of available estrogen which is
needed to form the correct ratio of progesterone to
estradiol, thereby failing to trigger pituitary LH
release. In addition, the high levels of circulating
plasma progesterone possibly block release of pituitary
LH at the hypothalamic or pituitary level, or both.

The synthesis and secretion of estrogen from the mid-
cycle follicle will be discussed later.

There was a significant decrease in pituitary LH
between days 11 and 18 and an increase from day 18 to
day 20 (P < 0.01), but we do not know from the present
data how great the depression in pituitary LH was, nor
exactly when the depression occurred because of the long

period between days 11 and 18 when no heifers were

87

studied. A similar depression around mid—cycle (Fig. A)
has been reported previously. Although not designed to
measure cyclic changes in pituitary FSH and LH, the
study by Desjardins (25) revealéd decreased pituitary
LH levels around the middle of the estrous cycle. On

a limited number of observations (61), blood levels of
LH increased around day 8, suggesting that increased
amounts of LH may indeed be released during the mid-
cycle; but in most cases, blood levels of LH were too
low to measure precisely.

The controlling mechanism of pituitary LH release
at day 11 is complicated. Many factors may be involved.
From the previous discussion, the release of pitui-
tary LH in mid-cycle does not appear to be associated
with any dramatic change in hypothalamic LRF, which is
low at this time. But our assay for LRF is insensitive
relative to the biological potency of LRF. In the rat
(18), estradiol benzoate (2-50 ug/day) did not alter
hypothalamic LRF, but decreased pituitary LH content.
So perhaps small amounts of estrogen may act directly
on the pituitary causing release of pituitary LH. And
as the mid-cycle follicle atrophies, the depressing
effect of estrogen is removed, and LH synthesis becomes
greater than release, resulting in further increase in
pituitary LH between days 18 and 20.

Recently, a functional role (58) for 20a-hydroxy-

pregn-A—en-3-one in the rabbit has been proposed. The

88

results suggest "that this progestin acts as a positive
feedback agent to prolong and heighten LH discharge in
the mated rabbit." Unfortunately, 208-01 was not
measured at various intervals between day 20 and day 0--
the period comparable to that after mating in rabbits,
so there was no indication as to whether 208-01 may be
related to LH release in cattle. Perhaps 208-01 has

a function in the dairy heifer similar to that proposed
for 20a-ol in rabbits and, if so, perhaps the lower
levels of 208-01 observed at day 11 (compared to day
20) may be another factor responsible for failure of
sufficient pituitary LH release for ovulation.

Dramatic endocrine changes occur after day 18.
Pituitary FSH and corpus luteum progesterone content
decrease precipitously between days 18 and 0. Following
presumed increased plasma FSH, the follicle more than
doubles in size and activity between days 20 and 0.
Between days 18 and 0, both hypothalamic LRF and pitui-
tary LH increase, but pituitary LH decreases by 89 per—
cent during the next 3 days, presumably due to stimula-
tion by LRF. Heifers exhibit psychic manifestations of
estrus on day 0. About 18 hours later, under the
influence of the "LH surge," she ovulates and the whole
chain of events recycles.

As an alternate method to illuminate the relation-

ship between pituitary LH and corpus luteum activity,

89

correlation coefficients were calculated between pitui—
tary LH and total progestogen (progesterone and 208—01)
content of the corpus luteum within each of the 7
selected days of the estrous cycle (Table 13). On day
2 there was a significant correlation of -0.90 (P < 0.10).
At this time pituitary LH was low, havingbeen released
from day 20 through day 2, while the corpus luteum was
increasing in size and synthetic activity presumably
under the influence of higher levels of blood plasma LH.
Although the correlation coefficient of 0.83 at day A
was not significantly different from O (P > 0.10), it
revealed a relationship between pituitary LH and corpus
luteum progestogen exactly opposite that which existed
on day 2. Comparison of pituitary LH levels revealed
net release of LH between days 20 and 2, but net storage
of LH between days 2 and 11. And indeed, it appeared
that pituitary LH synthesis exceeded LH release around
day 2 as evidenced by an increase in pituitary LH after
this time. Because corpus luteum progestogen content

is an indicator of plasma progestogens, it seems quite
likely that the increasing level of plasma progestogens
at day A negatively affects the hypothalamus, the pitui-
tary, or both, and thereby blocks or diminishes release
of LH from the pituitary, thereby causing an increase

in (or storage of) pituitary LH. However, it is likely

that some LH is still released from the pituitary, as

90

TABLE l3--Correlations between gonadotrophins and luteal
progestins or follicle size.

 

 

Day of LH FSH
estrous vs vs follicle

 

cyble progestin wall weight
0b 0.1A(3)a -0.90(3)
2 —0.90(A) 0.80(A)
A 0.83(A) -0.13(5)
7 -0.A0(A) -0.0A(5)
11 0 1A(5) -0.83(5)
18 0.19(5) -0.62(5)
20 -0.89(5) -0.13(5)

 

aNumbers in parentheses indicate number

bThe corpus leutem was 21 days old.

of samples.

 

91

evidenced by the increasing size of the corpus luteum.
The correlation coefficients were not significantly
different from O for days 7, 11, and 18 (P > 0.10).
This lack of statistical significance may be due to
the small number of heifers involved in each correla-
tion (3 to 5) or it may reflect LRF/LH ratios which
are relatively low at this time.

At day 20, for the negative correlation of -O.89,
there may be two different explanations. Firstly, by
this time some animals may have been close to estrus
and the corpus luteum was degenerating and in view of
its degenerate vascular system, probably incapable of
responding to LH, at a time when pituitary LH content
reached a maximum. Secondly, in synergism with FSH,
the LH probably stimulates the preovulatory follicle
causing it to produce increasing levels of estrogen
which in turn probably hasten degeneration of the old
corpus luteum.

Actually, it is surprising that such large cor-
relation coefficients were obtained because there is
undoubtedly a lag between pituitary LH release and
formation and steroidogenic activity of the corpus
luteum. And when a correlation coefficient was cal-
culated for all the data, i.e., across all days and
heifers, it was nonsignificant, further supporting
the hypothesized lag from LH release to progesterone

synthesis.

92

Luteal regression began about day 18. It was
precipitous after day 20. This period coincided with
the period of greatest pituitary prolactin content.

If one assumes that high pituitary prolactin reflects
high blood levels, as has been suggested (6A) in rodents
(and exactly the reverse of the relationships between
pituitary and blood levels of LH or FSH), then prolactin
may be associated with leuteolysis in heifers. Whether
such an effect of prolactin may be directly on the

aging corpus luteum or mediated through estrogen from
the growing preovulatory follicle is entirely specula-
tory. It could even be mediated through a possible
effect of estrogen upon the uterine luteolytic factor
(134, 135).

Although the large follicle present at mid-cycle
(101) does not produce discernible changes in sexual
behavior, there are several signs that several parts
of the sexual system display mid-cycle changes typically
caused by estrogen. For example, there was some evi-
dence for a biphasic increase in the weight of the
vagina--at estrus and at mid-cycle. Also, total
vaginal RNA/DNA and protein showed similar biphasic
patterns. However, the epithelial layer while thickened
at estrus was not affected at mid-cycle, probably
because the lower estrogen levels at day 11 than at day

0 did not stimulate the entire cell layer to any great

93

extent. In contrast, the single epithelial cell layer
in the cervix was significantly stimulated at estrus
and at day 11, revealing a biphasic pattern in the
cervix which also typified cervical DNA and paralleled
follicular size.

In the uterus and oviduct, neither the biochemical
parameters nor epithelial cell heights reflected pat-
terns indicative of bimodality corresponding to estrogen
secretion by the mid-cycle follicle. This does not come,
as any great surprise, since these two organs seem to be
more under the control of the luteal secretions while
the cervix and vagina seem more under the control of
follicular secretions.

Correlation coefficients between pituitary FSH
and follicle wall weight within each day of the cycle
(Table 13) were computed and none was significant
(P > 0.05), probably because of the limited number of
heifers. However, 6 out of 7 were negative and when
the correlation was computed over the entire group,
the correlation of -0.A0 was significant (P < 0.01).
These data suggested low pituitary FSH resulted in
high follicle wall weight. In other words, release
of pituitary FSH stimulated follicle wall growth.

A significant correlation of 0.A2 between fol-
licle wall weight and vaginal epithelial cell-layer

suggested stimulation of the epithelial layer by

9A

estrogen (P < 0.01) although the vaginal epithelial
cell-layer did not increase between day 2 and day 18
(Fig. 7). However, this may have been due to the
inability of small amounts of estrogens to signifi-
cantly stimulate the entire layer.

Correlations between total pituitary gonado-
trophins and total gonadal hormones and the various
biochemical parameters and cell heights measured in
the reproductive tract were computed within the days
of the estrous cycle. Most of these were not signifi-
cant (P > 0.05), and those that were significant could
not be interpreted from a sound physiological point
of view mainly because of two factors: 1) most of the
parameters measured are the result of stimulation and
interaction of two or more hormones and 2) a variable
lag period between endocrine stimulation of an organ
and response is normally expected. So it seems most
likely that any relationship between the pituitary
gonadotrophins and gonadal hormones and the biochemical
parameters and cell heights can most readily be inter-
preted by studying the various graphs and taking into
account both the interactions and lag periods involved
(Fig. 10, ll, l2, 13).

For the first time, prolactin, FSH, and LH were
assayed in individual bovine anterior pituitary glands

(Fig. 10) and LRF was assayed in pooled hypothalami

95

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during the estrous cycle. And at the same time, ovarian
follicular and luteal activity was measured. From these
results a mechanism for the regulation of the bovine
estrous cycle is proposed.

Sometime after day 18, blood plasma levels of
progesterone decrease thus freeing the hypothalamus,
pituitary, or both, from the depressing action of pro-
gesterone. FSH-RF stimulates release of pituitary FSH
which in turn synergizes with low plasma levels of LH
to stimulate growth of the preovulatory follicle. This
follicle more than doubles in size between day 20 and
estrus and secretes ever increasing amounts of estradiol
and smaller amounts of progesterone. The increasing
level of estradiol blocks further pituitary FSH release.
As follicular progesterone secretion increases, the-
ratio of progesterone to estradiol is such that around
day 20 it stimulates increased hypothalamic LRF, which
stimulates release of pituitary LH. Also around day
20, 208-01 content is high and perhaps also stimulates
hypothalamic LRF production. This surge of LH causes
ovulation, and stimulates mitosis of the granulosa and
theca interna cells for 2 to A days to form the corpus
luteum. As the newly formed corpus luteum continues
to grow, it secretes ever increasing amounts of proges-
terone, possibly under influence of prolactin (as well

as LH) which is also released from the pituitary after

 

100

day 0. These increasing levels of progesterone nega-
tively feedback on the hypothalamus, pituitary, or

both, decreasing LH release to a constant level, which
is controlled by the tonic releasing center of the hypo-
thalamus. Between days 7 and 18, the high level of
progesterone inhibits release of gonadotrophins thereby
causing the quiescent stage known as diestrus. Of

course, after day 18, the events recycle.

Suggestions for Future Research

 

Unfortunately there were no sensitive assay pro—
cedures available at the time this study was performed
to measure blood levels of the gonadotrophins. So it
is impossible to relate the exact blood levels of these
hormones to those in the pituitary throughout the estrous
cycle and blood levels are more important than pituitary
levels for controlling the estrous cycle. But, by
observing the effects on target organs, especially the
ovary, the pituitary hormone levels reflected fairly
accurately the proposed role of these hormones in regu—
lating the estrous cycle. In future experiments, meas—
uring blood levels of the pituitary gonadotrophins by
radio-immunoassay will further elucidate pituitary-
gonadal relationships and provide valuable clinical
methods upon which means of treatment of some endocrine

disorders can be based.

101

When more sensitive assays for the blood levels
of pituitary gonadotrophins become available, it will
be possible to obtain more precise measurements of the
releasing factors. For example, one possible method of
measuring hypothalamic LRF would be to use the "Parlow
rat" and measure blood levels of the rat's circulating
pituitary hormone before and after injection of the
test substance. Both the rat's pituitary LH and ovarian
ascorbic acid content could also be measured to add
precision to the assay. And possibly when better extrac—
tion and purification procedures become available,
releasing factors for individual hypothalami can be
measured. These improvements in techniques would add
considerable impetus to endocrine research.

Further possible additions to the present study
would be measurements of estrogen, progesterone and
203-01 from ovarian venous blood, or possibly peripheral
blood, especially around day 11 and around day 20.

These measurements would, among other things, enable
researchers to study the failure of the mid-cycle fol-
licle to ovulate. Also by labeling gonadal steroids

and gonadotrophins, the site of action of these hormones
at the ovarian, pituitary and hypothalamic levels could

be elucidated.

SUMMARY AND CONCLUSIONS

Thirty—five Holstein heifers, 16 months of age,
were killed in groups of 5 at days 2, A, 7, ll, 18, and
20 of the estrous cycle and at estrus (day 0).

The mean length of the estrous cycles (avg. 20.6
days) body weight at slaughter (avg. 377 kg) were similar
to those previously reported for dairy heifers. There-
fore these animals were representative of "normal" dairy
heifers at the age most are first mated commercially.

Hypothalamic LRF was high during proestrus (day 20),
estrus (day 0) and metestrus (days 2 and A) and low
during diestrus days 11 and 18. Although there was no
significant correlation between hypothalamic LRF and
pituitary LH (P > 0.01), the high level of hypothalamic
LRF was associated with pituitary LH release as evi-
denced by declining levels of pituitary LH from day 20
to day 2 and formation and growth of the corpus luteum
from day 2 through day 11. Thus it was concluded that
high levels of hypothalamic LRF were associated with
pituitary LH release.

Pituitary prolactin content was highest on day 0
(67 IU per mg fresh pituitary) and lowest on day A (18
IU per mg fresh pituitary). These results suggest pro-

lactin may be part of a luteotrophic complex.

102

103

Changes in pituitary FSH during the estrous cycle
appeared to be biphasic with peaks on days 18 and 2 and
depressions on days 0 and A. Pituitary FSH was signifi-
cantly correlated with follicle wall weight, when taken
over all days and hiefers (r = -0.A0, P < 0.01). These
results suggested that release of pituitary FSH on days
18 and A stimulated growth of the follicles, but the
follicle at mid-cycle failed to ovulate. Thus it
appeared that there was insufficient release of FSH
around day 11 to maximally stimulate the mid-cycle
follicle to maturity and in turn this follicle failed
to secrete sufficient estrogen to cause the dramatic
changes associated with estrus.

Pituitary LH declined 89% from 6,178 pg at day
20 to 689 ug at day 2, increased to 5,390 g at day 11
and decreased to 3,731 mg at day 18. Thus, like FSH,
changes during the estrous cycle in pituitary LH appeared
to be biphasic and pituitary LH was released during
proestrus, estrus, and metestrus and possibly to a les-
ser extent in mid-cycle. The pituitary gonadotrophin,
data indicated that FSH release precedes LH release by
about 2 days and LH release precedes prolactin release
by about 1 day (Fig. 10). Other than these delays,
changes in pituitary levels of these three gonadotrophins
during the cycle were quite similar.

Corpus luteum total progestogen content increased

from 3.3 pg at day 2 to 329.5 pg at day 11. But it

 

10A

declined from 268.1 pg at day 18 to 10.6 pg at day 0.
Luteal progestogen was negatively correlated with pitui-
tary LH on day 2 (r = -0.90) and positively correlated
on day A (r = 0.83). The secretory pattern of 208-01
differed from that for progesterone in that it was
increased on day 20 as well as on day 11.

Follicle wall weight more than doubled between
proestrus and estrus, during which period LH and FSH
disappeared from the pituitary. It also increased to a
lesser extent between days A and 7, just after the sec-
ond depression in pituitary FSH. Weight of the wall of
the largest follicle declined between days 7 and 18,
suggesting that the large mid-cycle follicle degenerated
and another, which was destined to ovulate, replaced it
as the largest after day 18.

Vaginal DNA, RNA and protein content showed
biphasic changes during the estrous cycle; high at
estrus and day 11. These results suggested that
increases in vaginal DNA, RNA and protein reflected
estrogen secreted by the mid-cycle follicle as well
as by the ovulatory follicle. Both cervical RNA and
cervical epithelial cell heights revealed a similar
biphasic pattern; with peak values at estrus and day 11.

Uterine epithelial cell heights were maximal on
days 0 and 7, when follicle wall weights were greatest.

In contrast, uterine RNA appeared more related to luteal

105

progestogen secretion than to follicular estrogen secre—
tion. Thus, the activity of the uterus appeared related
to both follicular and luteal influences.

Oviducal epithelial cell heights followed a pat-
tern during the cycle similar to corpus luteum proges-

terone content. No changes in thyroid or thymus were

“In

indicative of follicular or luteal changes during the
estrous cycle.

The data on vagina, cervix and uterus all suggest

'— Fo"~halh.‘flg '
i

that the largest mid-cycle follicle secretes some estro-
gen, but not at large levels typical of the ovulatory
follicle because heifers do not normally show signs of
estrus at mid-cycle. Release of pituitary FSH on day A
may cause growth of the mid-cycle follicle. Although
pituitary LH declined slightly after day 11, failure

of the mid—cycle follicle to mature is undoubtedly
associated with high levels of progesterone at mid-
cycle. In contrast, final growth of the ovulatory
follicle is associated with reduced levels of proges-
terone elevated hypothalamic LRF, and marked loss of

LH and FSH from the pituitary.

 

 

BIBLIOGRAPHY

106

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1".

APPENDICES

119

 

APPENDIX TABLE l.-—Age, weight and estrous cycles.

12C)

 

 

 

Day of Estrous Cycles
Estrous Animal Age Weight
Cycle NO' Length Avg.
(mo) (kg) (days) (days)
0 16 16.3 36 .3 17, 19, 19 18.33
AA 15.8 3A8.2 21, 20, 19 20.00
77 16.1 3A7 6 22, 20 20.33
88 16.0 307.3 20, 19 19.50
90 16.5 320.5 - - _ _
Mean 1 SE 337.A : 10.
2 91 16.7 351.8 20 — — 20.00
61 16.2 396.A 22, 22, 20 21.33
87 17.A A68.2 21, 22 21.50
A 16.6 A10 0 19, 21, 16, 19 18.75
11 16.2 A16 A 19, 23, 19 20.33
Mean 1 SE A08.6 : 18.
A 83 15.9 360.0 22, 21 21 50
71 16 2 352.7 21, 20, 19 20.00
7A3 — 270.9 — _ _
10 16.0 375.5 22, 2A 23.00
72 l6.A 358.2 25, 19, 22 22.00
Jean 1 SE 351.5 + 21.
7 58 15.8 3A2.7 26, 18, 19 21.00.
7A 16.A 390.9 39, 23 31.00
78 l6.A 383.6 20 20 20.00
60 15. 393.6 21, 1», 2 20 00
81 16.3 322.7 20, 18, 27 20.00
Mean : SE 366.7 + 1A.
11 27 15.9 386.A 22, 22 22.00
86 l6.A 378.2 13, 20 16.50
12 15.9 A09.1 19, 18, 21 19.33
69 16.0 A00.0 36, 2A 30.00
76 l6.A All.8 18, 21, 20 19.66
Mean i SE 397.1 1 6.
18 28 16.3 A16.A 5, 19 12.00
85 16.5 399.1 22, 23 22.50,
80 16.2 360.0 22, 23 22.50
8A 16.5 A15 5 21, 21 21.00
A1 15.9 363.6 19, 20, 21 20.00
Mean 1 SE 390.9 : 12.
20 73 16.6 352.7 22, 21 21.50
75 16.5 A2A.5 19 '19.00
29 16.3 390.1 21, 19 20.00
5 16.0 37A.5 22, 21 21.50
79 l6.A 3A9.1 3A, 23 28.50
Mean : SE 386.2 : 12.

 

 

 

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2122

APPENDIX TABLE 3.—-Ovarian weight characteristics, corpus luteum weight and
progesterone, 20 8—01 and progestin (progesterone plus 20 B-ol) content.

 

Ovarian Weight

 

 

 

 

Day of Weight Weight
Estrous Animal With CL Without CL of of Follicle
Cycle and Follicle and Follicle Follicle Wall
(gm) (gm) (mg) (mg)
0 16 6.2A 3.10* 2,510 A22
AA 6.92 3.02* 3,650 980
77 8.95 A 5 3,280 76A
89 8.39 l 51* 5,920 878
A0 5.58 A 16 1,890 388
Mean : SE 7.22 i 0.90 3 21 i 0.AA 3,A50 i 689 686 i 120
2 91 — - - -
61 10.01 3.68 261 56
87 — 7.A0* — —
A 3 lost 5.22* - 118
11 6.36 2.52* 166 A5
Mean 1 SE 8.18 i 1.82 A 70 + 1.06 21A 1 15 73 i 23
A 83 6. 2 3.7A* t50 1A6
71 9.38 5.05* 1,120 331
7A3 A.9A 2.87* 370 175
10 8.2’ 3.90* 371 107
72 7.37 3.97* A80 11A
Mean 1 SE 7 38 i 0.71 3.91 i 0 23 981 2 i 137 175 1 A1
7 58 6.92 3.17* 1,8 0 336
7A 7.76 3.39 1,781 315
78 9.26 3.35* 2,83 A96
60 6.12 5.17 2,030 620
81 12.36 5.59 1,120 283
Mean : SE 8.A8 : l 10 A 13 + 0.51 1,933 : 73‘ A10 : 6A
11 27 7.7A 8! 2,2A0 A96
86 9.73 A A5* 1,610 290
12 9.81 13* 2,A90 AA8
69 10.52 A 30* 1,520 355
76 9.16 3 15* 2,130 353
Mean : SE 9.37 : 0.A6 3.78 i 0.32 1,998 i 137 333 i 37
18 28 lost — - 280
85 10.70 A.75* 1,110 300
80 10.92 5.08 3,1A0 5A0
8A 8.60 A.70 1,5A0 193
Al 17.97 9.10 985 166
Mean 1 SE 12.05 i 2.01 5.91 + 0.95 1,69A i 272 296 + 66
2O 73 9.69 5.13 1,390 193
75 8.27 A.22 2,680 A07
29 11.91 5.Al 1,900 212
5 lost 5.A2* — 1A0
79 7.13 A.06 960 236
Mean 1 SE 9.25 i 1.03 A.85 :‘0.29 1,733 i 286 252 : A5
*
Ovary had no corpus luteum or follicle. All others had corpus luteum,

follicle, or both, but weights of these structures were subtracted from total
ovarian weight to give the weights listed in this column.

1123

 

 

Weight of Diameter Weight Progesterone Progestin
Follicular of of Content Content Content
Fluid Follicle CL per CL per CL per CL
(mg) (mm) (mg) (pg) (pg) (pg)
2,088 18 1,500 18.0 0 18.0
2,670 19 2,610 5.2 5.2 10.A
2,516 19 1,6A0 3 3 0 3.3
5,0A2 2A cystic - - -"
1,502 17 1,A10 - - -
2,76A : 60A 19.A 1 1.2 1,917 : 3A9 5.3 1 3 8 1.7 i 1.7 6.0 i 3.1
— 10 35 0.7 0 0.7
205 3 310 3.7 1.6 5.3
— 3 330 2.0 O 2.0
lost 8 A00 2.8 2.0 A.8
121 A A10 3.3 0.A 3.7
163 i A2 6.6 i 1.3 301 i 19 2.5 i 0.5 O.Q _ 0.A 3 3 + 0.9
50A 10 590 12.A 3.5 15.9
789 1A 1,590 35.0 22.3 57.2
195 9 710 1A 2 5.7 19.9
270 9 1,150 39 1 2.3 A1.A
366 10 67) 7 A 1.2 18.6
A25 1 105 10.A i 6 9 932 i 193 .3.6 i 5.6 7 0 i 3 9 30.6 + 8.0
1,5AA 17 A,170 216 8 12.5 229.A
1,A65 15 6,720 228.5 13.A 2A1.5
2,38A 19 3,930 133.6 7.9 1A1.5
1,A10 18 A,100 18A.5 A1 0 225.5
837 11 A, 50 170.1 8.1 178.2
1,528 i 2A7 16.0 i 1 A A,51A i 533 1.6 7 i 16.0 16.6 _ 6.2 203.3 1 18.8
1,7AA 18 6,700 301.5 120.6 A22.l
1,320 15 10,080 201.6 80.6 282.2
1,9A2 15 A,700 .67,0 A2.3 310.2
1,165 1A 5,300 333.9 31.8 365.7
1,777 17 A,dtO 228.A 38.9 276.3
1,590 i 1A7 16 A i 0.8 6,328 1 1,002 310.7 i 23 9 62 8 i 16.7 329.5 i 28.5
lost 17 A,980 AA.8 29.9 7A.7
810 16 A,860 306.2 A.9 311.0
2,600 19 1,700 20.A 0 20.A
1,3A7 16 6,A50 A77.3 51.6 528.9
719 13 7,220 3A6.6 28.9 375.A
-l.369 i A38 16.2 +1.0 5,0A2 i 9A7 239.1 1 88.8 23 1 i 9.A 262.1 1 9A.?
J.,197 1A 5,A00 19A.A 75.6 270.0
2?,273 18 3,720 37.2 37.2 7A.A
1,618 16 1,610 0 0 0
lost 13 2,960 32.6 1A.8 A7.A
72A 15 3,790 257.7 22.7 280.5
1-.A53 1 308 15.2 + 0.9 3.A96 i 617 10A.A : 51.0 30.1 i 12.9 13A.5 i 58.6

x

en-3—one (20 8-01) mass during incubation of corpus luteum homogenates.b

12A

APPENDIX TABLE A.--Net synthesis of progesterone (prog) and 20 B—hydroxy-pregn-A

 

NADP + G — 6 - P

 

 

 

 

Day of
Estrous Animal No LH LH
Cycle No.
Frog 20 8—01 Total Frog 20 6-01 Total
0 16 20 0 20 22 0 22
AA 78 0 78 80 0 80
77 10 0 10 A 0 1A
88 - - - - -
9o - - - — - -
Mean : SE 36 i 21 0 i 0 30 i 21 39 i 21 0 i 0 39 i 21
2 91 _ - - _ _ _
61 — - — - - -
87 — — — — - -
a _ _ _ _ - -
11 - — — — — -
Mean 1 SE
A 83 35 1 6 79 5 A“
71 52 3 55 ’7 0 A7
783 39 6 A5 13 A A7
10 73 0 73 75 3 78
72 76 0 76 6A 0 6A
Mean 1 SE 55 + 27 2 i 1 57 i 8 5A 1 7 2 i l 56 i 7
7 58 1A9 5 15A 128 6 13A
7A 76 A 80 HA 6 89
78 7A 9 ”3 75 7 C25
60 86 A3 129 71 28 99
81 72 10 52 62 23 85
Mean i SE 91 i 15 1A + 7 105 i 15 85 i 11 12 i 5 59 i 9
11 27 92 10 102 163 11 151
86 A9 17 66 A9 3A 83
12 1A5 23 268 156 9A 250
69 156 16 172 17A 19 193
76 132 32 16A 1A2 39 181
Mean 1 SE 115 i 19 A0 i 21 155 i 35 136 i 22 A0 1 15 176 + 27
18 28 180 18 198 1,0 19 189
85 106 8 11A 101 8 109
80 62 0 62 6A 0 6A
8A 158 27 18? 158 28 186
A1 13A 79 213 138 7A 212
Mean 1 SE 128 i 21 26 : 1A 15A : 29 126 i 20 26 i 13 152 i 28
20 73 180 71 251 176 7A 250
75 132 18 150 156 10 166
29 0 0 0 0 0 0
5 223 11 23A 21A 11 225
79 158 69 227 13A 81 215
Mean i SE 139 i 38 3A i 15 173 i 31 136 i 36 35 i 17 171 1 A5

 

aIncubation of about 100 mg corpus luteum for

b

Data from Hafs and Armstrong (A6).

1 hr at 370 in 2.5 m1 Krebs-
Ringer bicarbonate buffer with 1 mg/ml glucose and 5 uc cholesterol - 73H.

125

 

NADPH

 

No LH

 

L H

 

 

Frog 20 8-01 Total Frog 20 8—61’ Total
38 0 3? A2 A

106

5A + 28

18 + 5

1A6
1AA i 25
178
150
98
187
192
161 i 17

230
1AA

237
178

158 i A3

 

p.
I +
C

’1, A‘

3‘1 U1 1: H w

P
O
u;
I m l +
C

1...
5‘ O“. ".2

1A + 6

156 + 3‘

165 i 19

106

2D

*4]

f_J
(Q I“) C)

K-U LU LA) ‘N‘ _: I +

I +

11.4

LU

}_J

Mb—J
OCDFU

O\
O
+

1H1.»
TQKQDJ

H
KO

t—JTU
|+ \ON

7‘.—
.4.—

,‘l

'=‘\] 2.."
LU LLLU UNO

3'“
V

‘r—4
1‘.)

kA)
O

\)L';

 

APPENDIX TABLE 5.——Vaginal Characteristic

C!
u

126

 

Day of
Estrous
Cycle

Weight

DNA

 

Cons

Content per

 

 

ll

18

16
AA
77
88
90

Mean 1

91
61
87

u
11

Mean +

83
71
7A3
10

'7_)

l 1.

f-Ie an :

Mean +

Mean +

1:- (x10- tut-R)
,_. 1: <3 \fl CD

Mean :

7\)\}\1
\OUWMQLDLAJ

\1

SE

(" ‘
(J.

..\ 1“
5.1 I;

L")
[71

(gm)

200.

109.:

P—‘J F—‘ (.41 Q

TL:-

L1H

UT UT (,1;

C‘ [‘4

O

”I"

O—k.

(mg/gm

1C\NC\

b— F—J rm;- t—-‘ r—J
.
-" '1)-

3 1
L. o v
- o I

J

. 71

1 7 .

.L I L.‘
k a ..

HH.H
o
w

0.-
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u
I
1.1

m

F—P—F—‘F‘F’ -'!J"_)+-'
O O I
C.“ Lwr- C .TTC

ta
0

/\
I +
('3
o

)

LA;
'.-.‘

7 «* (v~’w
\I 1'); ,1 1 1: .. - 1
1‘ . ‘ -
3.; . . O J .
JV.) V.’ o :I o
l. 7 a 1
3L1- . ,, 1 1 ,
1-‘:‘.'J 1‘1.
“15' . 1' 1 1 J .
- 1 . ‘1 1
it) a 8+ :1 L: n k,‘ I L a +
a v 1
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11". '
1 .. u ‘ 0
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1’ \
f .1 l o 4‘ _, a I
I 1 I", ,.:‘ ,
V .2 a u , u
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‘L' o L +1.") I ‘1 o & +

7'7 .
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'fi .

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\_n

 

127

 

1
L

’thelia

v
.

1‘:

 

 

1"" '\
IL/Al‘-

k

wk

\.

O.’

 

(cm)

.11_ a). ma

(JJM¢J1AW

 

11. \_1 114/;
O O I
_/_
1.;
r; i
.3 V 1x
1 , 1 7..
.. I. 1
L. .
1 121
1 1
J \11 p
r . ,1!
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1 1O .1;
1, V ..2
L

. 1 7. .,

‘.1L+J.U1

2.1+0.2

/‘
\J

U.-51+

¢

‘ 1

v-‘J

+0.1

1
U

')
(.-

—.-

1.9+O.l

c 1 .4I.11. 11.
. . . 1. 1 1
1 1 .1.
n o o o o
v k . r
1 1 \L
.
. J
1 1 1.1. 1
V
1 1 A. z
1 .

4 1 11 J
O D 3 I I
u I (1 . A

<
1 4:. 1. , .4
i l. , l 1. a)
.V» ’5 « A _
A ll. ) 1

I“

1
\J

(.1. Lt.)+lv) o s)

0.1

I
1.4 .1
l
U I
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, 1
,', V1
.11 .1 J
I, .\IV
I. . .1
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J. 1
, a .
t, 11
1 1.
' I
.1} 1:,
7.

0.19

J

11'

2.11:0.1

 

APPENDIX TABLE 6.—-Cervical characteristic“

128

 

Day of
Estrous
Cycle

Heifer
No.

Weight

 

Cone

per

1C0

;‘\ .-.
. .in

 

 

\]

ll

18

20

Mean

C);C\\O |+
L‘Nb—‘F—J

{/3

F:

H
.-J

Jean

mean

Jean 1 SE

Mean : SE

79

Mean 1 8

(gm)

0

Louie“! to.)
b—Ji—chnlw
I I

OLTIQWO

.

C)

+
\J

39

I."
.
( ‘N

_I
a

LTLA,‘ \J‘ UV kg"
:: 1'}: k“

C I
Q \i') 0 KC?-

P
— I
L
[4--
UK

4 I.
o

H Y \
LA; CA: OZ ’ '.
Q

j .0
'J‘. 1
g. A. I K
.\ {- ( f ..
5:, ,3) 0 7+ 1 o J

4 .ng
O
-\_,‘ KIT

1 -,
\A’
O

l
x "
\.‘

T "4). '.
O

K.» (J: Lo R,“ W
C J V

O

m U‘

UL.
J:
L L

| +
PM
L)

37.5
42.5
38.0
38.7
35.3

“3.3+1.2

u9.0
3;.0
M3.1
no.0
33.5

CD
\1
| +
rt.)
0‘

EG‘NJTU‘I

:qung
rum3mmqon

u!
H

uo.5:3.u

(tug/gm)

7“ F1) \f1 O R)

P4 [‘J H LA} LA)
I

O
r
\

.\ ’

r‘ ¢
.
f—J {:1

Lu

" v

'71 7‘

L o J

a - \

J ;
p c J + } o ;_

(I 3

L o s.

. J I |

t... C I ‘

‘3- p2.

-. o x.'

. ‘ «

_,. 0 fl‘

.7] ‘ .

L. I :7

LA.) F- ;2.‘ KAJ KJ.‘
L

kA.’
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+
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5.4
3.1
2.0
3.5

2.810.“

|.4
‘ ‘v
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x

x”.
15C.H
?9.:
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‘7 ‘

I O

l u &
A‘M.Q

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4" . + ‘. ‘
LU .U
h V
Ag" .

i I i u H
7 1". ..' .4
1 Z {.I‘ n ‘V I
,7", J ‘1 1
L1}.

:34.9
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. +‘
1
4 +.
.'
..+
J
|
71‘1"
._ 1.4+ "
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M,
. {+1
90
‘VI
,)_ ,
L‘L Cy

+

\J
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‘ ,7
43")
LZQ.O
.; 1r,r..<
’ ‘ v ‘ ’—7 '*
-4;.1‘+.] '
Lil-.4
~ . I“
k I H
i ‘1" . q
i n In,
|

1~: 5
1"]; '4‘
i .
’1:
X ¥ 7+7-
1 4
.)
. .96;

. I ,3
I L r. JI 4+ 1‘ q A
. 1‘ 'u ,
l J x I ‘
7 j l 1 J
‘ . (a
. 4 ., W . ‘
w 1 V ‘71.
e _ . . 4/
’r J , ' . Jr

r « I, . :1
, , H ‘
J A J ' . >
r r— 4, . l
.— I.> . «1,4 ' p,
"1' “
. . ' l ' . 1
,7 . , 4‘
.. ‘
) . _ ~ ‘ L —
i ~ 7 V .1
,‘ o 4 j .l I J

 

1239

 

 

 

P'I‘O'L e in LE {T i Er fuel ‘E 1.5
RNA/DNA "4i4—i«j.z31' E~r=»;‘n
Cor": Crux-ten- Cone (33m em, Eire“: HY

 

. O)

a
,_
\

MHr—JMH
UOH‘-

H

HP—‘V—Jl—JH
O\CDO\O‘-*\J

H
x)
I +
C)
H

1:)“

J? O h.) \l

war—w—J
Ntccwo

H
LA)

[4'
C
i—J

V—‘P—‘f—‘P—‘H
WmmPQCX;

1.6102

G
b—d
+
C‘
,L

O.l&+fl.uu(

2’1”.)
0.;0
Q. ‘1')

0.17:0.01

0.15
0.20
0.17

0.17:0.01

a.
I /'
40!)
T. '.+.'"
4;._; J.
0.4
, .7
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L.
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r 7+»
O ‘4.
f.

l.

t‘.

2
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>1.:‘
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f

v
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4 I

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fi . >r
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L
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Tkfi
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6.8
\.'-l$+‘.).2

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re

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A I;
A . I ‘
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4 ..
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k.
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TA.

 

 

L

-r ‘ic

 

.Ti

1Q30

APPENDIX TABLE 7.--Uterine characteristics.

 

Day of DNA 131
Estrous Heifer Weight
Cycle N0. Cunc *Content per 100 kg Cone Content

 

 

 

(gm) (mg/gm) (mg) (mg/100 kg) (mgr-

O 16 195.0 U.“ 830.0 235.2 3.6 710.5
uu 156.0 5.1 795.1 226.3 1.6 255,;
77 190.0 5.2 1011.5 sgu,u .2 ”3?.8
88 118.0 6.3 7H5.3 2u3.5 ".' 111.9
90 1?6.0 6.3 1105.3 BHU.9 .3 ”05.5
Hean:SE 167.8:1u.u 5.5:o.u 900.5:67.0 263.3+32.9 3.7+1.3 u95,7+(9,
2 91 18u.O . 98U.Q 962.9 9.0 479.0

5 O k 4

61 166-0 9 3 1530.0 335,0 é'? L.q.1

07 210.0 6.9 lugg_3 ;1¢_{ 7.1 W?1.0
1* 57

150.0 . 053.1 200.1 1.9 ;*6.6
11 153.0 5.7 870.7 209.1 5.6 511.3

_Mean:3€‘ 173.u:11.8 6.5:0.8 1133.9:15u.u 377.0:3U.4 :.%:1.u di&.0+ww

U 83 178.0 5.2 931.0 256.6 _ 1.1.1
71 128.0 6.7 851.3 CH1.h x 1 i '.‘
7M3 169.0 6.3 107.7 145.6 . Tu .;
10 135.0 6.u 807.5 -11.1 ..w ».¢»,1
72 153.0 6.3 907.0 P~3.5 a, an ,{
Mean:SE 152.u:9.5 6.2:J.3 937.7:39.5 .,3.1:10. ..:*.. a G.v: I
7 58 199.0 6.3 1553.3 :1 .7 ... -rw.
7“ 179.0 6.3 1135.0 2 .6 » . . 1.
78 188.0 H.6 051.” g 1. 1. . ,
60 165.0 0.6 4&1.» 1. . ., .1 ,1
81 153.0 1.3 1+~.« *~,.1 . ; -.1

Mean:3€

H
\X
_\l
L-
|+
O
\C
+
L»
C
if
+
L
1
+
4.
1

11 27 65.0 0.; r:u.; n..7 1 1'.-
db {filfiotj 503 ..r .401) 1 ‘1' I L I J " t’°l
l ‘3 i (J Y, O 1‘ 4 J :1 ‘J o ‘ u . J n l

6‘
76

1— ,_. b—‘ 1‘-» 1—
r.) 91 H1 :

\o ‘ H U
I .
C C)
C" «2
U Q
1.: o
. I
1 7“
_ ‘
‘ V
‘1
O O
r
V
9‘- ‘4
-
' I
" a l;
A A
D C
1
O Q

Meanis

F—l
C
\_'1
O
K“
| +
H
,_.1
O
\C.
(s
I
«a
l +
k3
O
\A
| J
1...
J
i
F A
+
“v
C.
0
n
f
O
| +
,.
O
\w
0
,1
4.
t
1
F
I
+
)

18 2‘ 235.0 3.u 776.3 141.' .u 1* .u
85 l'{,£).0 7.0 L.:7.9 .JJ .5 J J ‘ ‘10‘
80 800.0 L.d -113.U ‘17.. u *
811 162.0 3.5 ”111.? 13.0.4 1. 4 1 .1
”1 135.0 %.L 013.t 1"5 K . 2’.‘

Mean18E . 190.2+20.H 0.6+0.7 869.<+1<5.3 3:3.3+57.1 C.5+7.: 8*s.y+-yq

20 73 15u.0 ;. 76”.0 .u".: 3.1 311.0
75 200.0 3. 6-3.5 1un.9‘ 1.7 111.0
29 l96.0 ’V . L’an‘} gt n?) J '1 OLJ

D 3 ‘., ‘i‘
5 159.0 0. 10H?.U 373.9 “.9 311.0

Mean+SE 169.5+13.5 5.3+O.7 871.0+77.9 33%.3+23.5 2.7+“.5 “50.0+95

' E

z —-\1 .
“\J F.) 1,1.) FL) I")

1...; I..—

4-

 

Protein

 

Conc

Content

 

1.321

r-c" 1: ‘
1.21.11
.1: .
301ght

 

 

(ms/gm)

0.12
0.11
0.11
0.11
0.11

0.11:0.003

0.12
0.11
0.11
0.11
0.11

0.11:0.009

0.12:0.000

0.15
0.13
0.10
0.1?
0.12

0.1U10.011

0. 1:1
0.11
0.12
0.12

0.12:0.00h

0.16
0.18
0.10
0.11
0.13

o.1u:o.o1u

x

o
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2132

 

 

 

 

APPENDIX TABLE 8.--Oviduca1 lengths an1 epitnu1111 uu?1 Ind,-
Day of
Estrous Wpifha‘11l
Cycle Hefiger (7:511 1v ‘ 1
' Right Left
(cm) (CM) (0)
0 16 22 16 3’ 9
UN 19 19 1% Y
77 21 21 .1.1
88 21 2 '5 5
90 23 22 1%».2
Mean : SE 21.2 i 0.7 19 8 i 1.1 2 r f <
2 91 21 2% 111m
61 21 21 2a.
87 18 11.1 fil.14
u 21“ 2” 111.1.)
11 21 20 92.7
Mean 1 SE 21.0 i 1.0 212 _+_ n '0‘ 13.5:
1 83 2M 23 ~u,5
71 15 15 2h.2
7&3 18 15 30,0
10 20 21 28.9
'72 25 25 2.5
Mean i SE 20.u i 1.9 19.8 i 2.1 1% 5 i 1 W
7 58 23 22 25.8
7“ 23 22 21,»:
78 19 .19 «/.r')
60 21 21 29.5
81 26 23 1‘1.”
Mean 1 SE 22 u + 1.2 21.1 i n Y ’ n i 1 i
ll 27 21 .33 $1.0
86 19 :3 .Ppn
12 21 :2: 2331 ‘i
69 25 23 1n 2
76 21 21 2'.0
Mean i SE 21.h : 1.0 23 2 i 0.“ ‘ 8 i 1
18 28 23 91 ‘h0
85 21 2%) Fj.H
80 26 28 2‘.)
8H 20 20 19.€
U1 25 22 32.1
Mean 1 SE 2U.0 : 1.5 21.0 i H 0 ’1 1 f 1
20 73 21 £3 [J.K
75 17 1] 1.1).“)
29 22 27 2/.0
5 20 £2 31.“
79 27 27 17.5
Mean : SE 21.“ i 1.6 23 1 i l H CH r i I H

133

APPENDIX TABLE 9.—-Thyroid characteristics.

 

Day of 03L

 

Estrous Heifer Weight
Cycle NO. Can: Junteuu

 

 

o 16 13.1 11.7 111.:
an 17,7 . 5”.1
1') o L) r,"

.71!
EB 10.

90 1A.9 8.0 71.9

Mean+3€ 16.1+1.1 ~.?+0.’ n1.'+%.

I“;

91 1 ' u a.) '3 . 1 1'9'7, . ‘1'
1' ' '

, 4 I
r11. .1 :1. ‘ .
1.1" i1 '7 V ”J :
.1 1 A . r ‘1. " .
I ' b I
A: L, L7 U “ '. I * l I

7“
h
\;
i: ‘J CC
KJ 9) LA) H LA:
1‘“
(fl‘.
0
. h»
,
i
\
o

xcp

1 .2 . . .1
(5 1 .5 3.0 ._
on 11.? .51 ' .
fil 1%.: .L .

1 1 27f

F4

.. 1 ‘ I
L U 11* . .1 ' . j .
"v ‘ ‘) l
1 ’ 4'. i o 1) )c 1 ‘1 A .
1 -
t") l 0 o a JD- ) " '

\‘
"Yx
\—

p.

”N x
.

r—1
-H
o

a

\v
w

n
C

1'18 (in:"h .1.” I ('4. L. I.) 11." +) 0 1 '~"1""+'I'c '

18 28 17.7 ”.5 :u.e
5:; 111.9,) ‘ ." 7 'i .

..
'\
I

.1
~-
,4

1‘; fi'y'i .1 I 0
H3 13,7 u,7 .,,‘
M1 17.3 n.n I u

Mean+JE 18.1+0.0 N.7+J.J “3.3+U.

Fu “.1

LA x.) \f‘ \O ‘31
L_.J

'J‘ ..

0‘ R. w P—‘ O
J.

O

.. \Q

\'\]

Mean:8E 17.u:1.3 h,6:u,3 79,7:5,;

+

4-

 

1314

 

RNA/DNA

Protein

 

 

Acinar

Epithelial

 

Cone Content thC CorLenL Cell Height
(mg/gm) (mg) (mg/gm) (gm) (0)
1.8 0.17 2.2 195 2.6 1M.5
1.5 0.2’ 3.0 — — 11.3
1.5 0.20 3.6 — - 12.3
1.2 0.2!: 24.0 :13 34.1.; 171.9
1.5 0.17 2.0 — — 9.1
1.5 + 0.1 0.20 1 0.01 3.2 + 0.“ ;20.5 + 12.9 3 3 i ).T 11.6 i 0.8
1.14 0.15 2.23 _ — 10.1‘
1.6 0.20 3.6 no 3.? 13m
1.“ 0.22 Q.» 237 3.7 l0.1
1.9 0.22 u.9 - — 3.?
1.9 0.29 0.0 — - 10.9
1.6 + 0.1 0.21 + 0.0: 3.7 + 0.14 .-;.):15.) 1 mi) 10.1 i (1.?
1.5 0.19 («.0 2116.5 AI: 11.-
1.2 0.20 2.1 197 2.C 10.0
1.2 0.20 2.1 20- 7 1 13.9
2.0 0.25 u.3 — - 1;.3
1.8 0.15 30L) - - 7'?
1.6 + 0.2 0.20 + 0.01 [.7 + 0.0 ’15.? + 5.5 ‘.1 + 0.0 11.3 + 1.1
1.U 0.80 ..9 191 I 9 H
1.0 (‘0'.‘9'1 1.0!.) _ _ \:‘ .I‘
1.“ 0.23 “.3 - - ‘ 1
l D 2 (W O :1) K“ O J 1’) i § I L.3
1.0 0.;0 33.3" - - .‘_' 4
1.2 i 0.1 0.33 i 1.0-. 3n. + 0.3 113'.0 + '3 - .92 + . 1:: ‘i 1
1.U 0.2 5 0 f-n .- ‘1 4
1.“ 0.23 3.” PC: I 3
1.5 0.30 3 -1" 9.. I“ ’
1.6 0.44 33 ‘31 3 1* »
1.9 0.20 3.0 17” E 13 0
13:. i 0.1 0.21: 0.00 3.0 i 1. w. .7 i: -; :1 .'. i
1.” 0.21 3.7 :05 ~.6 10.1
1.1 0.1: 3.0 ” N .h -3 i
1.14 0.2.1? 5:..3 - - -'~.-'s
1.7 0.21 “.1 £0? 5.1 . 0
1.7 0.2( 3.0 210 3.1 “.0
1.5 i 0.1 0.20 i 0.01 3.7 i 0.. ’10.5 i 3.7 2 Yr 1 1 01 1 E i 1.-
1.3 0.19 3.3 2:3 3.1 3
1.5 0.19 2.7 f - - a
107 001'] 'L).8 18's) figl ‘
1.“ 0.20 3.2 - - .3
1.2 0.21 u.3 975 ).H
1.“ 1 0.1 0.19 + 0.01 3.3 + 0.3 270.0 1 10.6 3 97 i 0.7 10 U i 0.8

 

135

APPENDIX TABLE 10.--Thymus characteristics.

 

Day of
Estrous Heifer We

'V"'r'\ u -“
irl’al‘. .uu“.

 

 

f-Ao
0’4

:3“

( f

\ x , ‘ ‘ v» r"
cycle A0. ffiF" “antent VB“ ;uw 2‘ k0"? puntent

 

O 15 “07.3 “9.5 EH 9 V i 7 : 2 n

y . p ,_ v .\ . .‘r. 7. ‘, _ ,
‘ 14 Ejbzhu 5.3.3 ‘ .‘J .. .' r‘ . a -'~.U
" “ Lfi r .1 ) ; '3 - ; _r ‘ ' ,, a ‘ ,
{7 jja.¢ 44., .7.) 4. .. _.n
d d h l!) a .2 i,‘ 2) . 1.’ :‘l f, a . I 1‘.) o ; 4‘“ o i. ,' I

90 350.5 ‘3.5 g2.2 . ",3 ?n

Mean + SE N73.S+Jd.0 51.2+1.9 3N.E+3.J T.w+..- .‘+ .1 :.5+5.’
2 {'11 k.) 3 b a C) ‘1' L) U f] ,‘ (x a 4) I" o r“ ,0, u {‘7‘ l; I l
61 175.5 9;.1 Y.r l. ‘ * l.’
5'! ’365.(; 1.8.9 3.9 a;
u 57( ‘3 M '4‘] ,I to I'.H ‘aj
ll “73.5 4?.w 32.: . r.1 .4
Mean + SE U6M.5+7U.2 u7.u+1.3 2;.3+;.7 a.i+;.” r. +9.: €.;+3.
u 83 715.0 UH.9 % .0 I. .“
71 MU6.6 HR.M 1,.0 ‘.W 3.: «.\
7}‘lj 'I’EOCD £‘|J.:»l J ‘0{ 110‘) 7 u‘ ‘-
10 593.6 yd.) A.~ .‘ j.4 ..
'2 399.5 if.. ‘1.“ . .‘ :.
:I‘lean : 81:; c— ‘j A) q :j:'/ (J I 6 2‘ r{’ I l:;\ I ‘1‘ L17]. 0 . :3 a J V” o ‘7.) :1 a .7 C" I ‘ + “I C “l h" 0 I: ‘I ' '3
7 53 380.u J,.: -V.‘ L. . 9
7M u“w.n N .» :5. '. .
78 u59.5 yfi.w :1.; .' . '
>J 575.3 “. :w.w ”.' . .
51 3JW.O pW.w 17.. . . .

Mean + :‘IE “5600+1I‘HI‘2) 1'5.';.‘V.r‘+l.l} :"J‘f .1+I.g y oi‘+[; . “ ' ’I . ;:.‘

¥ +

11

FF"

“v
o
I
~
0
a
V‘
a
.:

\OXHCYTr/
JMQPJqu
RAJ
V

L,‘
O
N}—
I
O
.

‘
L

¥
.
\ ‘
\1
D
C? \Ci
.
\
C
._4
.
.
‘
O

18 25 7flfl.l 5?.1 <y. 4. M.§
7 ’ ‘ ’ r "’ v!
85 doj.b 39.: n.fi '. I 3.“
3 ” ‘I‘ :Q' q 0 ‘3 r I o v" L- I u 1 o J :r' o [l I U
D! " * x -. ,
94 jiij’ {_.-’1 J . . Ha .'
1‘1 E) 11‘ D 3 :1 ‘3 o r u . i ' 1‘ ‘ O V l ‘
Mean + SE 515.&+L3.5 ;+.u+i.o S .;+-.‘ ,. +‘. 7. + . . +‘.
>0 1') 5(‘1 ( L: ~. '>.-, ‘ r ..
L I J .J I . J / ”‘1 ) _ '.. o A a L L o
75 gui.3 ;6.2 L5.0 a.) ,. -”
2r 6:2.7 “0.9 95.; Y.W . t.)
7 ~, i .. : A a. ~
5 3&4 O f ‘1‘. C.‘ 1‘,“ 0‘ /I 4‘ . 1 s u '
79 510.1 4".U m
., ‘ ,. 1"! Y‘ -) ‘ v 'v '1 * U, ‘, , :4» ~ 1". a‘ .‘ 4 ‘ _ ‘; . '_,
[‘Ie dn + " {‘4 u h "I O S + D '3 1 9 1“) O J + i a {'4 C) L.) I (I! + '.> a ',/ i) o I +1] 0 l (.1 o J + 1 . '1 f, I ‘j: J l L.

136

 

 

 

' 1'1). 5‘21!
(it -, ."~\ 9.
FL.IH,’L.i.lfi
.V'V ‘7‘ —. -, .
V2110 (021111.141:
. \ .~
(1.15;.1‘; . ,.)

..
\..’ J
o c
r<..
. r
.V

\0

u

r'v

J
V.

23‘

D 0

,. 4.

\ .4
._

My

0 I

r 1

L)
t—J
\"r‘
|+
C
|+
._.
L.
[-4-

0.111 + 0.000 1J.L'(’ + 0.11.1. (“LR + 13.1"

0.1 .19 If .1
0.1 'J. it.) 1" .
l C! . 1.11 - ‘lt 1.

.1 U.il,' 1"1.”
0.1 U 1".» '1...

0.16 i 0.007 0.15 i 0.0n, 114.0 + “J“

0.115 "}.1‘ 1o.’
O.L§ U. H '.
Holt; H.‘ ‘5 1
O.Lj 1.. ‘1'.'
0.13 0. ‘ . ,

0.3. 0.1“- 1.1.
0.15 0.1: ".

0.13 i 0.300 0.10 i 0.41. 10.1 i W.f

0.17 0.1? 131.0
0.13 1.x. Tt.%
0.16 :L1
0 u 114“ L.‘ . L 1!" f h
0.12 0.1? ‘
0.1“ i 0.007 0.1? + L 0nd *3 a + 10.1
0.13 0.1
0.16 0.15 7?.1
0.15 0.31 1D9.7
0.13 0.1% u7.3
0 . 1:5 0 . 1 1 .W

0.15 i 0.009 0.19 + 0.019 03.1 i 1H.U

 

 

! +

I 4—

‘ 4

| +-