I

.
a\

n

c

o .
.

.

u \
A

 

 

 

a V

VII

 

    
      
          

.v'uw.‘ 1&2.
m ,, .
u‘

    

  
 
 
      
    

  

. '.V ‘
‘<. - h x“
L
5})?“ ”my-‘9' .
. g,

 

 

    

 
  

 

     

 

 

   
 

,‘.L= ' 4936 "
:égxr‘
‘ I

 

    
  
    

,
o ,-. .Ltw. .
'1". ;$_ '1‘.""<—‘~‘-' n:
C‘
. 7‘
g

    
  

v' -' v ‘ n L:

«sum
"1.9:; n'vI-w.-."v)_~_v ~ »
'I ' 1 3

  

  

. L
. (J
-- (E5313

   

  
 
 

v-

‘ I II
I .
0‘
‘ , ‘
‘ ' ' ~u . I. .
. , "n
‘ ‘
‘ " H
.. ", . I V
‘ - ‘ ,
‘. .

 

 

This is to certify that the

thesis entitled

SERUM HORMONE CONCENTRATIONS, IN VITRO RESPONSE
TO LUTEINIZING HORMONE AND TESTICULAR GONADOTROPIN
BINDING IN BULLS DURING SEXUAL DEVELOPMENT

presented by

Michael S. McCarthy

has been accepted towards fulfillment
of the requirements for

PhrD. Dairy Science

 

 

degree in

 

Major professor /

DateOmLsL-L [1/ (7,, /978

0-7 639

SERUM HORMONE CONCENTRATIONS, IN_VITRO RESPONSE
TO LUTEINIZING HORMONE AND TESTICULAR GONADOTROPIN

BINDING IN BULLS DURING SEXUAL DEVELOPMENT

By
Michael S. McCarthy

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Dairy Science

1978

6' / o a 74/5

ABSTRACT

SERUM HORMONE CONCENTRATIONS, IN_VITRO RESPONSE
TO LUTEINIZING HORMONE AND TESTICULAR GONADOTROPIN

BINDING IN BULLS DURING SEXUAL DEVELOPMENT

BY

Michael S. McCarthy

The objectives of these experiments were to describe serum hormone
changes in bulls from birth through puberty, to examine negative feed-
back relationships between testes and hypothalamus/pituitary and to
examine testicular changes during the peripubertal period.

In the first experiment serum growth hormone, testosterone, pro-
lactin and luteinizing hormone (LH) concentrations were measured over
a 9—hr period in bulls and steers before and after puberty. Steers
had higher serum LH concentrations (9.9 ng/ml) than bulls (2.1 ng/ml)
and a greater frequency of LH peaks (6.8/9 hr) than bulls (2.3/9 hr),
but LH did not change with age. Testosterone was greater in pubertal
than prepubertal bulls (5.4 vs 1.4 ng/ml). Prepubertal bulls had
higher growth hormone concentrations than other groups.

In experiment 2, two-week-old bulls were assigned to be bulls,
steers, or short scrotum bulls (SS bulls) and blood samples from a 24-
hr period were collected monthly to measure follicle stimulating hormone

(FSH), LH, testosterone and androstenedione. Treatment did not affect

Michael S. McCarthy

growth rates. By 10 months of age bulls had epididymidal sperm, but
spermatogenesis was abolished in SS bulls. Serum LH and FSH concentra-
tions were greater in steers than bulls or SS bulls from 3 to 10 months
and LH was greater in SS bulls than bulls at S and 6 months. Episodic
LH peaks occurred more frequently in bulls than in steers from 2 to 10
months and in SS bulls at 3 and 4 months. FSH was not released episodi-
cally, did not differ between bulls or SS bulls, and did not change
with age in bulls or $8 bulls. Serum testosterone concentrations were
< 1 ng/ml until 4 months in bulls and 5 months in SS bulls. Increased
frequency and amplitude of testosterone peaks and increased baseline
concentrations contributed to increased mean concentrations. Serum
androstenedione increased transiently at 4 months in bulls and SS bulls.
In Experiment 3 blood samples were collected frequently for 24 hr
from 1-,3-,4-,5-,7- and 9-month-old bulls on a single day to measure
the effect of age on serum hormones independent of season or photo-
period. Serum LH concentrations and frequency of LH peaks increased
from 1 to 4 months and then declined. Mean concentrations, peak fre-
quency and height of testosterone peaks increased from 5 to 9 months.
Androstenedione increased transiently in 4-month-old bulls. FSH did
not change with age and had no episodic peaks. Average serum prolactin
increased from 1 to 5 months while peak frequency increased from 1 to
4 months. Testicular tissue from bulls of different ages produced
equivalent amounts of testosterone in response to LH in_vitrg,
Androstenedione production was greater in l-,3- and 4- than 5-,7- and
9-month-old bulls. LH and FSH binding to testicular homogenates was
greater in 1- to 3-month-old bulls than in 5- to 9-month-old bulls,

but total gonadotropin binding/testis increased from 1 to 9 months.

Michael S. McCarthy

Limited in_vixg_responsiveness of testes to gonadotropins is not due
to absence of gonadotropin binding sites.

In conclusion, the major hormonal change during puberty is increased
testosterone production. Increased frequency of LH peaks at 4 months
may induce maturation of testicular steriodogenesis. Growth hormone,
prolactin and FSH may participate in puberty but the role of FSH is
difficult to assess because serum concentrations do not change during
puberty. Testicular products control gonadotr0pin secretion as early
as 2 months of age, because FSH and LH concentrations were greater after
castration and LH was slightly greater in SS bulls than the normal bulls.
Sertoli cells, the principal occupant of seminiferous tubules in SS
bulls, apparently produced inhibin since serum FSH did not differ in

SS bulls and bulls.

ACKNOWLEDGMENTS

I would like to express my appreciation to Dr. H. D. Hafs for
allowing me to attend Michigan State University, for serving as my
major professor and for directing my graduate program. Dr. E. M.
Convey deserves thanks for invaluable assistance and advice given to
me during my graduate studies. I also am grateful to Drs. J. L. Gill
and W. L. Smith for serving on my graduate committee.

I would like to acknowledge the technical assistance of Barb
Hogarth, Judi Martin and Kathy Wood whose c00peration, diligence and
perserverance were extremely valuable to me in completing over 17,000
steroid assays needed for this thesis. Dr. R. Neitzel and Larry Chapin
also deserve my gratitude for computer assistance which made the
mammoth project of data tabulation and analysis a manageable task.

Finally and most important, my wife Susan deserves special thanks
for continued encouragement and support given to me during my graduate

program and for assistance in typing and compiling this thesis.

ii

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES .
INWRODUCTION

REVIEW OF LITERATURE

Introduction .
Factors Influencing the Onset of Puberty in .Bulls

Testicular Development .

Sperm Production . . .
Hormonal Control of Spermatogenesis
Hormone Action in the Testis .

mmmcnw>

LH . . .

FSH . .
Prolactin
Growth hormone .
Androgens
Estrogens

GUI-huts)»-

H. Concentrations of Pituitary Hormones in Serum
During Puberty .

I. Concentrations of Steroid Hormones During Puberty .

J. Hormones in the Pituitary and Hypothalamus
During Puberty . . . . .

K. Hormonal Influences on Testicular Function in Bulls

L. Hypothalamic- Pituitary-Gonadal Interactions

1 Castration and Cryptorchidism
2. Effects of Gonadal Steroids
3. Inhibin
4

A Possible Explanation for the Onset of Puberty

MATERIALS AND METHODS .
A. General Methods

1. Animals . . . . . . .
2. Blood Collection Procedures . . . .

iii

Body Growth and Development of Reproductive Organs .

Page

vi

(N

OWCDO‘U‘IMM

11

15
15
16
17

18
20

22
23
25

25
26
27
28
30
30

30
30

Page

3. Radioimmunoassays . . . . . . . . . . . . . . . . . . 31
4. Histological Preparation . . . . . . . . . . . . . . . 35
5. Statistical Analyses . . . . . . . . . . . . . . . . . 35

B. Experiment 1 . . . . . . . . . . . . . . . . . . . . . . . 36
C. Experiment 2 . . . . . . . . . . . . . . . . . . . . . . . 37
D. Experiment 3 . . . . . . . . . . . . . . . . . . . . . . . 38

1. Blood Collection . . . . . . . . . . . . . . . . . . . 38
2. Testis Incubations . . . . . . . . . . . . . . . 39
3. Testicular Gonadotropin Binding . . . . . . . . . . . 39

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 50

FSH Radioimmunoassays . . . . . . . . . . . . . . . . . . 50
Experiment 1 . . . . . . . . . . . . . . . . . . . . . . . 52
Experiment 2 . . . . . . . . . . . . . . . . . . . . . . . 56
Experiment 3 . . . . . . . . . . . . . . . . . . . . . . . 72

an>

Body and Testicular Growth . . . . . . . . . . . . . . 72
Serum Hormone Concentrations . . . . . . . . . . . . . 72
Testis Incubations . . . . . . . . . . . . . . . . 81
Testicular Gonadotropin Binding . . . . . . . . . . . 86

AMNH

GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . 93

SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . 97

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

iv

Table

LIST OF TABLES

Age and weight for bulls and steers in Experiment 1 .

Comparison of two radioimmunoassays for FSH in bovine
sermn O I O O O O O O O D O O O C O O O O O O O 0

Serum hormone characteristics for prepubertal and pubertal
bulls and steers . . . .

Frequency and height of episodic peaks and basal concen-
trations of LH in bulls, steers and short scrotum bulls
(SS bulls) . . . . . . . . . . . . . . . . . . .

Frequency and height of episodic peaks and basal concen-
trations of testosterone in bulls and short scrotum
bulls (SS bulls) . . . . . .

Frequency and height of episodic peaks and basal concen-
trations of androstenedione in bulls and short scrotum
bulls (SS bulls) . . .

Serum FSH concentrations (ng/ml) in bulls, steers and bulls
with shortened scrotums (SS bulls) at 3, 6 and 9 months
of age . . . . . . . . . . . . . . . . . . . .

Body and testicular weight in l-, 3-, 4-, 5-, 7- and
9-month-old bulls . . . . . . . .

Affinity constants (K ) for 125I-HCG and 125I—FSH binding
to bovine testicular fiomogenates . . . . . . . . .

Page

37

51

55

60

64

66

71

73

91

LIST OF FIGURES

Figure Page

1. Elution profiles of HCG (top) and FSH (bottom) iodination
reaction mixtures on Bio-Gel P-60 columns. Each point
represents the radioactivity in ZO-ul aliquots of l-ml
column fractions. The first peak (left) represents hor-
mgge bound 1251 and the second (right) represents free

I . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2. Time-course of 125I-HCG (top) and 125I-FSH (bottom)
binding to testicular tissue. A weighed amount of
tissue pellet obtained from centrifugation of crude
testicular homogenates for 10 min at 2000 x g was
rehomogenized and used in these studies . . . . . . . . . . 46

3. Saturation curves for 125I-HCG and 125I-FSH binding to
bovine testicular tissue. A weighed amount of tissue
pellet obtained from centrifugation of crude testicular
homogenates for 10 min at 2000 x g was rehomogenized
and used in these studies . . . . . . . . . . . . . . . . . 48

4. Comparison of NIH-FSH-Bl standard curves with serum and
media curves for two FSH radioimmunoassays. In the
heterologous rat FSH assay (top), curves are shown for
NIH-FSH-Bl (o--o), media from pituitary cell cultures
(I--I), bull serum (a--D), pregnant cow serum (O--O),
ovariectomized heifer serum (A--A), pregnant cow serum
plus 1000 ng FSH/ml (A--A) and steer serum (X-—X). In
the homologous bovine FSH assay (bottom), curves are
shown for NIH-FSH-Bl (o--o), media from pituitary cell
cultures (l--I), bull serum (n-—o), postpartum cow serum
(O--O), ovariectomized heifer serum (A—-A) and bull
serum plus 1000 ng FSH/ml (A--A) . . . . . . . . . . . . . . 54

5. Mean LH, testosterone and androstenedione concentrations
in bulls (0--O), steers (A--A) and short scrotum bulls
(o--o). Values are means of 48 samples from a 24-hr
period for LH and testosterone and 16 samples from an
8-hr period for androstenedione for four animals in
each group . . . . . . . . . . . . . . . . . . . . . . . . . 59

vi

Figure Page

6. LH (O--O), testosterone (o--o) or androstenedione (A--A)
concentrations in a representative bull, steer and short
scrotum bull at I, 4, 5 and 10 months of age . . . . . . . . 62

7. Serum FSH concentrations in bulls (O--0), steers (A--A)
and short scrotum bulls (o--o) in two FSH radioimmuno-
assays; heterologous rat FSH assay (top) or homologous
bovine FSH assay (bottom). Values are means of three
samples collected at hourly intervals from four animals
in each group . . . . . . . . . . . . . . . . . . . . . . . 69

8. Serum LH averaged for 72 samples/bull (top), mean number
of LH peaks/24 hr (middle) and mean LH peak height in
five bulls at each age (bottom) . . . . . . . . . . 75

9. Androstenedione (O--O) and testosterone (o--o) concentra-
tions averaged for 25 samples/bull (top); mean number
of androstenedione and testosterone peaks/8 hr (middle);
and mean androstenedione and testosterone peak height of
five bulls at each age (bottom) . . . . . . . . . . . . . . 78

10. Serum FSH concentrations averaged for eight samples at
hourly intervals in each of five bulls at each age (top).
Prolactin concentration averaged for 25 samples/bull;
mean number of prolactin peaks/8 hr; and mean prolactin
peak height of five bulls at each age . . . . . . . . . . . 80

11. Testosterone from testicular tissue (tissue plus media)
unincubated or incubated for 3 hr with 0, 5 and 50 ng
LH/ml of media; five bulls at each age (top).
Androstenedione release into media from testicular
tissue incubated for 3 hr with 0, 5 or 50 ng LH/ml of
media; five bulls at each age (bottom) . . . . . . . . . . . 82

12. Specific binding of 125I-Hcc; (top) and 125Lisa (bottom)

to testicular tissue. Values are the means of triplicate
determinations of five animals at each age . . . . . . . . . 88

vii

INTRODUCTION

Economic and social factors encourage increased efficiency in
animal agriculture. Major advances in animal production can be made
using genetically superior sires available through artificial insemina-
tion (AI) and by manipulating hormonal and nutritional factors which
result in greater efficiency and rate of growth.

AI organizations require proven sires; sires with known superior
genetic potential for a particular trait. Proving dairy sires requires
5 to 6 years and costs AI organizations up to $100,000/bu11. Any
reduction in the interval required to prove bulls would result in
savings to the AI organization and to producers. It also would decrease
generation interval, allowing more rapid genetic progress. Induction
of early puberty in bulls could reduce the interval needed to prove
sires. At present, however, the mechanisms involved in the onset of
puberty are not clear and attempts to advance age of puberty have failed.
Increasing our understanding of the physiological events occurring
around the time of puberty in bulls, may lead to ways to induce early
puberty.

Nutritional, metabolic and hormonal factors influence the rate and
efficiency of growth. By increasing our basic understanding of hormonal
changes during development in bulls, we may be able to manage pituitary

hormones or anabolic testicular steroids to increase growth.

2
Studies described within this thesis were designed to increase
understanding of the endocrine regulation of sexual maturation in bulls.
The objectives were to describe serum hormonal changes in bulls from
birth until after puberty; to examine negative feedback relationships
between the testes and the hypothalamus/pituitary using bulls, steers
and bulls in which the scrotum had been shortened; and to examine

testicular changes which probably affect spermatogenesis.

REVIEW OF LITERATURE

A. Introduction

 

The aim of this literature review is to discuss reproductive
development in bulls, accompanying hormonal events and hormonal control
of testicular function. Information cited will be predominately from
studies in bulls, but data from other species will be used where informa-

tion on bulls is unavailable.

B. Factors Influencingthe Onset of Puberty in Bulls

 

During the first year of life bulls deve10p from reproductive
quiescence, incapable of spermatogenesis to near maturity with sperm
production per gram of testis equal to mature rats (Macmillan and Hafs,
1968a). This is puberty.

Puberty has been defined in many different ways in males. These
include the time of appearance of the first sperm in the seminiferous
tubules or in an ejaculum, first motile sperm in an ejaculum, first
sexual interest and first completed mating (Lunstra gt_al,, 1978).
Donovan and Van der Werff ten Bosch (1965) defined puberty as "the
phase of bodily deveIOpment during which the gonads secrete hormones
in amounts sufficient to cause accelerated growth of genital organs
and appearance of secondary sex character." Foote (1969) suggested that
puberty in the fullest sense occurs when spermatogenesis is complete,
when libido is present, and when penile development is adequate for

3

4
intromission. Probably the most widely used definition for puberty in
bulls is the presence of 50 x 106 total spermatozoa in an ejaculum
(Wolf gt al,, 1965; Killian and Amann, 1972; Barber and Almquist, 1975).
The bull's capacity to impregnate a female is most important in a func-
tional definition of puberty (Foote, 1969).

The age at which bulls reach puberty depends upon genetic (Foote,
1969, Lunstra §t_al,, 1978) and nutritional factors (Bratton gt_al,,
1959; Abdel-Raouf, 1960). Beef breeds generally mature more gradually
and reach puberty later than dairy breeds (Abdel-Raouf, 1960; Macmillan
and Hafs, 1968a; Swanson §t_al,, 1971; Lunstra g£_al,, 1978). The
species also influences the rate of sexual maturation. Bos indicus
mature later than Bos taurus cattle (Plasse §t_al,, 1968). In addition,
specific lines within a breed show wide variations in the age at
puberty.

The level of nutrition influenced the age at puberty in Holstein
bulls (Bratton §t_§l,, 1959); average age at onset of sperm production
(500 x 106 sperm/ml with 50% mobility) was 37, 43 and 51 weeks for bulls
receiving high, medium or low levels of nutrition, respectively.
According to Abdel-Raouf (1960), low nutrition retarded puberty by 8
weeks. Level of nutrition during puberty did not affect subsequent
fertility when bulls were fed normally after 1 year of age, but pro-
longed underfeeding can prevent bulls from ever achieving normal ferti-
lity (VanDemark §£_al,, 1964).

Other than nutrition, environmental influences on age at puberty
in bulls have been investigated little and information on environmental
effects on fertility in cattle is limited. Amann §t_al, (1966) reported

monthly variation in sperm output in bulls while Mercier and Salisbury

5
(1947) demonstrated highest fertility in bulls in summer and fall,
decreased fertility in winter and gradually increasing fertility in the
spring. High temperatures (85 F) for 5 weeks decreased spermatogenesis
(Casady, 1953) in young bulls. Jersey bulls raised at 35 to 36 C and
80 to 90% humidity for 8 hr daily beginning at 26 weeks of age, reached

puberty at 55 weeks, 7 weeks later than controls (DeAlba §£_al,, 1966).

C. Body Growth and Development of Reproductive Organs

 

Rate of body growth to 1 year of age is linear in Holstein bulls
(Bratton g£_al,, 1959; Macmillan and Hafs, 1968a). The relationship
Y = 22.5 + 26.5X, where Y is body weight (kg) and X is age (mo), described
by Macmillan and Hafs (1968a) agrees with the growth rate of bulls fed
a high plane of nutrition by Bratton g£_al, (1959). A reduced level of
nutrition decreased the slope of this relationship (Bratton gt_al., 1959).

In contrast to the linear relationship described above, Abdel—Raouf
(1960) found a concave quadratic growth curve in Swedish red and white
cattle. This difference was probably due to Swedish red and white
cattle attaining mature size earlier than Holsteins.

Seminal vesicular growth does not parallel body growth; after an
initial lag period (birth to 2 months), seminal vesicles grow more
rapidly than the rest of the body from 2 to 4 and from 7 to 9 months,
with an intervening plateau. Seminal vesicular RNA and DNA fellow
similar patterns, but RNA increased more rapidly until 4 months, after
which both increased at the same rate. The early increase in RNA demon-
strates a proportionately greater increase in protein synthesizing

potential as compared to hyperplasia (DNA content). Seminal vesicular

fructose and citric acid content, reflecting androgen production (Mann

6
§£_al,, 1949), increased slowly until 6 months and then increased
markedly to 9 months (fructose) and 12 months (citric acid; Macmillan,
1967).

The length of the penis increased linearly until 9 months when it
nears mature size (Abdel-Raouf, 1960; Macmillan, 1967). The separation
of the penis from the preputial sheath at 34 to 40 weeks of age
(Ashdown, 1962) is an important change occuring during puberty because
it permits intromission (Foote, 1969).

Epididymidal growth is curvilinear, with a slow initial growth
rate from birth to 4 to 5 months and a more rapid growth rate to 12
months (Macmillan, 1967). Testicular growth follows a similar pattern;
a quadratic growth curve to 9 months followed by decreased growth rate

until 12 months (Abdel-Raouf, 1960; Macmillan, 1967).

D. Testicular Development

 

At birth the seminiferous tubules are solid chords about 40 to 50
microns in diameter (Abdel-Raouf, 1960; Macmillan, 1967). The diameter
increases at a rate of approximately 5 microns per month and lumina
become apparent at 4 to 6 months.

The progressive development of germ cells in the seminiferous
tubules of young bulls has been studied (Hooker, 1944; Santamarina and
Reece, 1957; Abdel-Raouf, 1960, 1961; Fossland and Schultze, 1961; Attal
and Courot, 1963). At 1 week of age the primary components of the
tubules are precursor supporting cells (bis-cells), abundantly distributed
around the periphery, and gonocytes located more centrally (Abdel-Raouf,
1961). Work by Abdel-Raouf established that by 4 weeks some bis-cells

have given rise to more advanced supporting cells (cis-cells). The

7
gonocytes double in size and undergo nuclear changes suggestive of
cellular degeneration. Both types of supporting cells increase in
number by 8 weeks, the gonocytes disappear and spermatogonia, larger
than the gonocytes from which they arise, are present. Few changes
take place from 8 to 12 weeks, although the number of gonocytes is re-
duced. By 16 weeks, lumen formation usually begins, no gonocytes re-
main and the cell types present are cis-cells, bis-cells and sperma-
togonia. At 20 and 24 weeks, primary spermatocytes and more sperma-
togonia and cis—cells are present, and few bis-cells exist. Primary
spermatocytes are more numerous and small round spermatids and Sertoli
cells are present by 28 weeks. By 32 to 44 weeks all eight cell types
are present, including bis-cells, cis-cells, Sertoli cells, spermato-
gonia, primary spermatocytes, secondary spermatocytes, spermatids, and
sperm. However, during this period there is a progressive decrease in
the numbers of the two types of precursor support cells and an increase
in Sertoli cells.

An important change that takes place in seminiferous tubules during
the onset of spermatogenesis is the formation of tight junctions between
adjacent Sertoli cells (Vitale-Calpe g£_§l:, 1973). This phenomenon
has not been studied in bulls but has been extensively investigated in
rats. Tight junctions result in formation of the blood-testis barrier
and create basal and adluminal compartments within the tubule. These
compartments contain different germinal elements and provide two separ-
ate environments for germ cell development.

The deve10pmental changes in the intertubular tissue have not been
so carefully documented. Abdel—Raouf (1960) stated that it was too com—

plicated to estimate the degree of metamorphosis of Leydig cells or to

8
estimate their absolute or relative numbers. Hooker (1944) described
differentiation of mesenchymal cells into Leydig cells and fibroblasts.
Few Leydig cells are present from 1 to 3 months in bulls; subsequently
the number increases due to division of precursor cells. Leydig cells

appear fully functional and secretory by 4 months (Hooker, 1944).

E. Sperm Production

 

Sperm are absent in testicular homogenates from bulls between
birth and 4 months of age (Macmillan and Hafs, 1968a). Few sperm are
present between 5 to 7 months, while a pronounced increase occurs be-
tween 7 and 8 months (4 x 106/g of testis to 28 x 106/g). By 12 months
the concentration (57 x 106/g) is equivalent to that in mature bulls
(55 x 106/g) reported by Almquist and Amann (1961). Increases in testi-
cular weight between 12 months and full maturity account for a two-fold
increase in total sperm/bull. Total daily sperm production calculated
from testicular homogenates was 0, 168 x 106, 3230 x 106 and 5900 x 106
at 0 to 4, 5 to 7, 8 to 10 and 11 to 12 months, respectively, and con-

tinued to increase until 7 years (Amann, 1970).

F. Hormonal Control of Spermatogenesis
Hypophysectomy causes gonadal atrophy in rats while replacement
therapy with pituitary extracts restores testicular function (Smith,
1930). Zondek (1930) discovered "Follicle ripening factor" (FSH) and
"Luteinizing factor" (LH) and knowledge of these two factors led Greep
gt_§1, (1936) to postulate FSH controls spermatogenesis and LH controls
testicular steroidogenesis. The effects of androgens on spermatogenesis

confused this originally neat hypothesis. Androgens maintain

9
spermatogenesis in hypophysectomized rats (Walsh gt_al,, 1934; Nelson
and Merckel, 1938), but do not reinitiate the process after atrophy of
the germinal epithelium following hypophysectomy (Nelson, 1937; Simpson
g£_§l,, 1942).

The roles of gonadotropins were re-evaluated in hypophysectomized
rats (Woods and Simpson, 1961) using replacement therapy with more puri-
fied materials than were available previously. LH at "low doses" could
maintain spermatogenesis but testicular weight was reduced. At "low
doses," FSH with no significant LH contamination produced only slight
effects on testes weight and development. High FSH doses with signifi-
cant LH contamination produced heavier testes than either hormone alone,
indicating that the two hormones acted synergistically.

The hormonal requirements for spermatogenesis have been reviewed
by Steinberger (1971) and Lostroh (1976). FSH in male rats functions
primarily during sexual deve10pment. It is synergistic with LH in initi-
ating androgen production and in development of spermatids and immature
spermatozoa. FSH also stimulates Sertoli cell function. LH is required
for androgen production by Leydig cells, synergistic with FSH in initi-
ating andrOgen production, required for maturation of spermatozoa and
synergistic with growth hormone in stimulating testosterone production.
Apparently the only actions of LH on spermatogenesis are via testosterone
production. Testosterone is required to stimulate meiosis of primary
spermatocytes and for spermiogenesis.

Madhwa Raj and Dym (1976) and Dym and Madhwa Raj (1977) selectively
withdrew FSH or LH using highly specific antisera. FSH antiserum treat-
ment for 14 days reduced testes weight, tubular diameter and spermatid

and spermatocyte numbers without interfering with testosterone

10
production (Madhwa Raj and Dym, 1976). LH antiserum injections decreased
testosterone in rete testis fluid, Leydig cell area and Leydig cell
endoplasmic reticulum. In the seminiferous tubules, LH antiserum
caused late spermatids to be retained by Sertoli cells, pachytene sperma-
tocytes to degenerate and Sertoli cell nuclei to become sperical, a sign
of degeneration.

Woods and Simpson (1961) found that prolactin increased the rate
of spermatogenesis in LH treated rats. In agreement with these studies,
prolactin treatment of hypophysectomized rats potentiated LH action on
spermatogenesis, but did not potentiate spermatogenesis when given with
testosterone propionate (Bartke,'1971a). These results suggest that
prolactin may augment LH effects on steroidogenesis, but not testosterone
effects on spermatogenesis. This was confirmed when Hafiez §t_al, (1972)
showed that hypophysectomized rats had higher plasma testosterone after
prolactin plus LH than after LH alone.

Woods and Simpson (1961) also observed that growth hormone potenti-
ates the action of gonadotropins on spermatogenesis in hypophysectomized
rats. Later Lostroh (1976) indicated that the stimulatory effect of
growth hormone was caused by increased androgen production. Growth hor-
mone plus LH resulted in larger testes and greater accessory gland

weights (an indirect measure of androgen production) than LH alone.

G. Hormone Action in the Testis

 

Few studies have been conducted on the mechanism of hormone action
in the bovine testis. Most information in this section is from experi-

ments in rats.

11

1. LH

 

The mechanism of LH action on testicular steroidogenesis has been
reviewed recently by Catt and Dufau (1976). Dufau gt_§1, (1976) and
Cooke (1976). Summarizing these reviews, LH first binds specifically
to Leydig cells on a membrane bound receptor. This receptor has been
purified to 50% homegeneity and is composed of two subunits of 90,000
molecular weight (Dufau et_al:, 1975). Binding to the receptor results
in activation of adenylate cyclase at the cell membrane and in most
cases, production of cyclic adenosine monophophate (CAMP) in the Leydig
cells. The fate of the hormone-receptor complex has been determined.
in luteal cell preparations and Leydig tumor cells. After binding of
HCG to the plasma membrane of ovine luteal cells, significant quantities
of hormone are internalized, localized in lysosomes (Chen et_al,, 1977a)
and degraded by acid hydrolases (Chen 33 31., 1978). In Leydig tumor
cells the fate of the bound LH is similar to that of HCG in luteal cells.
Blockade of lysosomal activity does not interfere with HCG-induced
steroidogenesis, indicating that lysosomal degradation is not prerequi-
site for steroid production (Ascoli and Puett, 1978). The LH internali-
zation phenomenon may explain decreased responsiveness of testicular
tissue (testicular desensitization) that occurs transiently after L“ or
HCG (Hsueh g£_al:, 1977).

Cyclic AMP formed as a result of LH stimulation binds to two pro-
tein kinase holoenzymes (4S and 6.5S; Dufau 93 E13: 1976). Each of
these holoenzymes dissociates into two regulatory and one catalytic sub-
unit. The catalytic subunit of protein kinase phosphorylates a variety
of protein substrates, but the function of phosphorylation is unclear

at present. Active protein kinase translocated to the nucleus has been

12
demonstrated to phOSphorylate histones (Walsh §t_al., 1968) and ribosomal
RNA's in other tissues (Barden and Labrie, 1973). These events may alter
transcription or translation processes. Changes in total protein syn-
thesis in Leydig cells could not be detected after LH treatment, but
treatment with the protein synthesis inhibitor, cycloheximide, prevented
increased testosterone synthesis (Cooke gt_al:, 1974).

Since LH stimulates increased conversion of cholesterol to pregnen—
olone, protein phosphorylation probably influences the activity of
cholesterol-side-chain-cleavage-enzyme complex or increases cholesterol
transport into mitochondria. Subsequent conversion of pregnenolone to
testosterone requires at least five enzymatic reactions in the smooth
end0p1asmic reticulum. Prolonged LH treatment can stimulate these reac-
tions (Dufau SE.§E:» 1976).

Direct involvement of cAMP in the mechanism of LH action has been
questioned. Treatment of Leydig cells with low levels of LH can increase
steroidogenesis without detectable changes in cAMP (Catt and Dufau, 1973).
Catt and Dufau (1976) suggested that LH may enhance steroidogenesis
through mechanisms other than cAMP, but they also point out that small
(undetectable) increases in cAMP, or cAMP translocated within intra—
cellular compartments as a result of LH stimulation may stimulate
steroidogenesis. Treatment of Leydig cells with cAMP or dibutyryl cAMP
results in steroidogenesis, additional proof of the importance of cAMP

(Dufau g_t_ 31., 1976).

2. FSH
FSH action in the testes has been reviewed by Hansson 22.21: (1976),

Means gt El. (1976) and Bartke §t_al, (1978). Sertoli cells are the

13
primary target for FSH in the testis and specific binding sites for FSH
are found on Sertoli cell plasma membranes (Means and Vaitukaitis, 1972;
Castro g£_al,, 1972; Steinberger gt_al,, 1974; Orth and Christensen,
1977). Events following FSH binding resemble those after binding of LH
to Leydig cells. The interaction of FSH with its receptor results in
activation of adenylate cyclase, production of cAMP and activation of
cAMP-dependent protein kinase in Sertoli cells (Means, 1973; Means and
Huckins, 1974). After rats are 21 to 24 days old, testicular response
to FSH is diminished, but hypophysectomy results in renewed sensitivity
to FSH. Decreased sensitivity to FSH was thought to be due to specific
isoenzyme of phosphodiesterase, appearing during development and pre-
venting cAMP accumulation in response to FSH. This was proven incorrect,
but an inhibitor of protein kinase under the control of FSH increases
rapidly during the first 20 days of age in rats, and may be one factor
responsible for the loss of sensitivity of Sertoli cells to FSH (Tash
gt_al,, 1978). Another possibility is that decreased sensitivity to
FSH in mature rats is due to decreased adenylate cyclase activity (Van
Sickle §£_al,, 1977).

The specific function of protein kinase in Leydig and Sertoli cells
is unknown, but following activation by cAMP, protein phosphorylation
occurs in every subcellular fraction. Protein and RNA synthesis in-
crease as do RNA polymerase I and 11 (Means gt_al,, 1976).

A specific protein induced by FSH is androgen binding protein (ABP).
ABP is produced by Sertoli cells (Dorrington gt 31,, 1974), secreted in-
to the lumen of seminiferous tubules and transported through efferent
ducts to the epididymis. The function of ABP is not known but it is

suspected to concentrate androgens in the tubule and epididymis for

l4
spermatocytogenesis and spermiogenesis (Ritzen 33 al., 1971; French and
Ritzen, 1973). Hypophysectomy causes a loss of ABP, but replacement of
FSH restores its production (Sanborn 93 21,, 1975).

An age difference in ABP production in response to FSH has been
reported (Kotite g£_al,, 1978). Fourteen-day-old rats produced ABP
after FSH treatment whereas adult rats did not respond to FSH with ABP
production. Age-related refractoriness to FSH in terms of ABP produc-
tion resembles FSH effects on cAMP.

Testosterone alone maintains spermatogenesis for extended periods
and this led some to suggest that the primary action of FSH is on
steriodogenesis (Bartke £3 31,, 1978). FSH treatment of immature hypo-
physectomized rats increased testicular responsiveness to LH (Odell
et_al,, 1973; Catt, 1977) and increased LH binding per testis (Chen
g£_al,, 1976; Catt, 1977). These findings suggest that the role of
increasing serum FSH concentrations during puberty is to increase
steroidogenesis in response to LH. Payne 3; a1, (1977) postulated that
FSH might affect steroidogenesis by stimulating 17B-hydroxysteroid
dehydrogenase activity.

The inability of FSH to bind to Leydig cells complicates hypotheses
of its action on steroidogenesis. FSH possibly alters steroidogenesis
indirectly via Sertoli cell products such as estradiol (Fritz, 1977).
FSH stimulates aromatase synthesis in Sertoli cells and subsequent
conversion of testosterone to estradiol (Dorrington and Armstrong, 1975).
Part of the steroidogenic stimulation induced by FSH may be its action
on steroidogenesis within the seminiferous tubules (Bell and Vinson,

1975; Welsh and Wiebe, 1976).

15

3. Prolactin

 

Demonstration of prolactin binding to isolated Leydig cells
(Rajaniemi £5 31,, 1974) and autoradiographic localization of prolactin
around Leydig cells (Charreau et_al,, 1977) suggests that prolactin
acts Specifically at Leydig cells. Suggestions of several actions of
prolactin on Leydig cells include regulation of LH binding and mobiliza-
tion of cholesterol for steroidogenesis. Decreasing serum prolactin
concentrations in hamsters by shortening the photoperiod resulted in a
loss of LH receptors (Bex and Bartke, 1977a). When rats were given
ergocryptine to reduce prolactin secretion, LH binding to Leydig cells
was reduced (Aragona gt_al,, 1977). Prolactin administration restored
LH binding in dwarf mice (Bohnet and Friesen, 1976), hypophysectomized
rats (Zipf and Payne, 1977) and hamsters on reduced photoperiod (Bex
and Bartke, 1977a).

Prolactin increased Leydig cell cholesterol esters (Bartke, 1969,
1971) which serve as a precursor pool for steroidogenesis (Bartke, 1971,
Bartke et al., 1973a). Prolactin also increases conversion of acetate
to cholesterol in_vit£g_and increases 38- and l7B—dehydrogenase

activity (Hafiez gt al., 1971; Musto et al., 1972).

4. Growth Hormone

 

Growth hormone treatment of testes-regressed hamsters increased
testicular and seminal vesicular weights (Bex and Bartke, 1977b), and
treatment of hyp0physectomized rats with growth hormone plus LH stimu-
lated greater reproductive organ growth than LH alone (Lostroh, 1976).
LH binding, plasma testosterone and testicular responsiveness to LH

treatment increased after growth hormone treatment (Bex and Bartke,

16

1977b; Zipf and Payne, 1977; Swerdloff and Odell, 1977).

5. Androgens

 

The ability of androgens to maintain spermatogenesis is undisputed
in rodents (Steinberger, 1971). How androgens are involved in sperma-
togenesis is less clear. Leydig cells are the main source of testosterone
in the testes (Cooke gt_al,, 1976), but Sertoli cells have also been
suggested to be capable of d§_ngvg synthesis of testosterone (Lacy,

1973). Several investigators have demonstrated conversion of pregneno-
lone, progesterone and dehydroepiandrosterone to testosterone in semi-
niferous tubular preparations (Christensen and Mason, 1965; Bell gt_al,,
1968), and many have shown that the seminiferous tubules or isolated
Sertoli cells are effective in converting testosterone to Sa-androstanediol,
38-androstanediol, dihydrotestosterone and estradiol (Rivarola and Podesta,
1972, Folman gt_al,, 1973; Dorrington and Armstrong, 1975; Welsh and

Wiebe, 1976; Tence and Drosdowsky, 1976).

The androgen target tissue in the testis is primarily Sertoli cells
(Sanborn gt_§1,, 1976), but androgen binding to germ cells has also been
demonstrated (Sanborn, 1977). In a nuclear exchange assay, the androgen-
receptor complex in germ cells was not translocated to the nucleus, which
questions the biological significance of germ cell binding of androgens.
In Sertoli cells, androgens bind to a cytoplasmic receptor (Mainwaring
and Mangen, 1973; Galena gt_al,, 1974; Mulder gt_al,, 1974; Hansson et_
al., 1974; Bardin et_al,, 1973; Means g£_al,, 1976). The cytoplasmic
androgen receptor has been determined to be different than ABP by a
variety of physical and chemical properties (Means gt_al,, 1976); After

binding of androgens to the receptor, the androgen-receptor complex is

l7
translocated to the nucleus where it stimulates synthesis of numerous
proteins, a major one being ABP.
Both FSH and testosterone can independently increase ABP synthesis.
The relative importance of these two hormones in regulating ABP levels

in normal animals remains to be elucidated.

6. Estrogens

 

The testes were known to produce estrogens as early as 1934 (Zondek,
1934). Since then many studies have been conducted to determine the
testicular compartment producing estrogens, as well as a testicular role
for estrogens. Interstitial tissue has been shown to be a major site
of aromatization and estrogen production after HCG stimulation in human
and rat testes (Payne §t_al,, 1976; Valladares 93 31,, 1978). In addi-
tion, Dorrington and Armstrong (1975) demonstrated that isolated-
cultured Sertoli cells produce estradiol in response to FSH when testo-
sterone was added as a precursor. Estrogen production in response to
FSH was age-dependent and decreased from maximum at 5 days to almost un-
detectable concentrations by 30 days of age (Dorrington et al., 1978).

The role of estrogen in testicular fUnction is unknown. However,
estradiol decreased testosterone production in_vitrg_($amuels gt_al,,
1964; Sholiton, 1975; Bartke et_al,, 1977) and in_vivg_(Danutra et_§1:,
1973; Tcholakian et_a1,, 1974). Estrogen effects may occur by direct
action on cytoplasmic estrogen receptors found in isolated Leydig cells
(Mulder gt_al., 1976).

Some are skeptical of evidence that estrogens act directly on Leydig
cells. Van Beurden gt_al, (1977) showed that estrogens affected testi-

cular steroidogenesis indirectly by negative feedback on gonadotropins.

18
Also, Bartke et_al, (1977) stated that non-physiological estrogen doses

(1000 x physiological) were used to alter steroidogenesis in_vitro.

 

Estradiol affected testicular function in hypophysectomized immature
rats; a model in which estradiol can not influence gonadotropin secre-
tion. In these animals estradiol inhibited FSH-increased testicular
responsiveness to LH (Van Beurden §t_al:, 1976; Chen gt 31:, 1977b).
From the age-dependent decline in estrogen production in response to
FSH by Sertoli cells (Dorrington et_al:, 1978), lower concentrations of
testicular estrogens in 30-vs l6-day-old rats (Chen gt_al,, 1977b) and
the inhibitory action of estrogens on FSH-increased responsiveness to
LH; it is tempting to suggest that high intratesticular estrogen concen-

trations early in sexual maturation may inhibit testicular development.

H. Concentrations of Pituitary Hormones in Serum DuringPuberty
Changes in serum LH, FSH and prolactin in the peripubertal period
have been extensively investigated in rats (Swerdloff et_al,, 1971;
Dohler and Wuttke, 1975; Dussault gt_al:, 1977a; Dussault §t_al:, 1977b;
Payne g£_§l,, 1977). Various authors disagree about LH concentrations
during this period. Payne g£_al: (1977) and Dussault §t_al: (1977a)
feund no change in LH concentrations with advancing age, but character-
ized the LH pattern as highly variable during puberty in rats. Dohler
and Wuttke (1975) found increased LH concentrations between 40 and 50
days, while Swerdloff gt_al, (1971) found a progressive rise between 21
and 91 days of age. In contrast to highly varied results found in LH
concentrations, most workers have found an increase in FSH at 32 to 35
days of age and then a decline coincident with the onset of spermato-

genesis (Swerdloff gt_al,, 1971; Payne gt_al,, 1977; Dussault gt_§l:,

l9
1977a). Serum prolactin concentrations increase from the neonatal period
to about 35 days of age and then decline with maturity (Dohler and Wuttke,
1975; Dussault et_al:, 1977b).

In bulls, serum LH concentrations during sexual maturation vary
between investigations similar to rat studies. No changes in serum LH
were observed between birth and 1 year in Brown Swiss (Karg gt al., 1976)
and 3/4 Limousin cattle (Schanbacher, In press). However, Lacroix gt a1.
(1977) found Charolais bulls had low LH concentrations between 1 to 5
weeks, higher and more variable from S to 20 weeks, and lower and stable
after 20 weeks. Holstein bulls had a similar LH profile; 2.9 ng/ml at
1 month, 6.7 at 3 months and 4.2 at 15 months (Barnes gt_al,, 1976).
Macmillan and Hafs (1968b), also using Holstein bulls, reported slightly
lower and more variable LH concentrations between 2 to 7 months and
higher more stable concentrations from 8 to 12 months. They also con-
verted LH concentrations to total LH content per bull and saw a marked
increase from 2 to 4 months, a plateau to 6 months and a gradual in-
crease to 12 months. The increase in LH content with age is expected.

It reflects increased body weight and blood volume but not necessarily
increased LH available at the testes. Lunstra §t_al: (1978) in several
beef breeds, Gombe §t_al: (1973) in Angus, Holsteins and Guernseys, and
Mori gt_al, (1974) in Holsteins found that LH concentrations gradually
increased from 7 months to maturity. So, similar to data in rats, re-
ported LH data during the peripubertal period are inconsistent in bulls.

Technical problems with radioimmunoassay have prevented extensive
investigations of serum FSH concentrations in bulls. Only recently has
FSH been measured in bulls of different ages (Karg gt_al,, 1976;

Schanbacher, In press). Both these studies reported no significant

20
elevations in serum FSH between birth and 1 year of age.

Season influences prolactin concentrations in cattle (Koprowski
et_al,, 1972; Karg and Schams, 1974; Tucker gt_al,, 1974). More speci-
fically, increased ambient temperatures and photoperiods raise serum
prolactin concentrations while reduced temperatures and photoperiods
decrease prolactin (Wetteman and Tucker, 1974; Bourne and Tucker, 1975).
Therefore, studies conducted across seasons should not be used to deter-
mine age effects on prolactin. Two such studies determined prolactin
concentrations in bulls from birth to 20 and 12 months, respectively
(Schams and Reinhardt, 1974; Lacroix et_al:, 1977). Prolactin concen-
trations were high in summer and low in winter, but were not affected
by age. These studies did not definitively assess age effects on pro-
lactin in bulls because they confOunded age with season.

Plasma growth hormone in bulls from 1 to 12 months of age was high
at birth, decreased in the first month and was constant to 12 months.
Plasma growth hormone was not related to growth criteria (Purchas et_§1,,

1970).

1. Concentrations of Steroid Hormones During Puberty

 

The concentration of androgenic steroids in testicular tissue,
spermatic vein plasma and peripheral plasma is low in bull calves less
than 4 months of age (Lindner and Mann, 1960; Lindner, 1961; Rawlings
gt_al,, 1972; Karg §£_al:, 1976; Seechiari et_al:, 1976). As bulls in-
crease in weight and age, there is a shift in type and amount of steroids
produced. The androstenedione to testosterone ratio in testicular tissue
was greater than one before 4 months, less than one from 4 to 6 months

and less than one-tenth from 6 months onward (Linder and Mann, 1960).

21
The spermatic vein concentration of these steroids followed a similar
pattern (Lindner, 1961). In bulls 4 to 6 months old, androstenedione
secretion rate (ug/hr) increased from 62 for an 89-kg bull to 217 for
a 123-kg bull, and then decreased to 4 for a ZOO-kg bull. Testosterone
secretion rate (pg/hr) for these animals was 14 (89-kg), 30 (123-kg) and
55 (ZOO-kg). The same type of change in androstenedione:testosterone
ratio seen in testes tissue was observed in spermatic vein effluent.

Peripheral concentrations of testosterone and androstenedione have
been studied more completely during maturation in bulls (Rawlings et_al:,
1972; Karg §t_al., 1976; Seechiari gt_§1., 1976). Plasma testosterone
increased erratically to 11 months, and androstenedione was high and
variable until 6 months, then declined (Rawlings et_al:, 1972). In
other reports (Karg gt_al,, 1976; Seechiari g£_al,, 1976), testosterone
remained <1 ng/ml for 5 months and increased markedly to 3 to 5 ng/ml
by 12 months.

Changes in the activity of enzymes related to steroidogenesis
during maturation have not been studied in bulls. Experiments in rats
have shown that the activity of enzymes essential to steriodogenesis in-
crease with age, especially during puberty. The enzymes 3B-hydroxy-
steroid dehydrogenase, l7B-hydroxylase and C17-C20 lyase all increase
sharply in male rats from 20 to 30 days of age while l7B-hydroxysteroid
dehydrogenase increases more gradually from 20 to 60 days of age (Inano
§t_al,, 1967). Similarly, Payne EEIEl: (1977) found increased 38-hydroxy-
steroid dehydrogenase between 10 and 30 days of age and l7B-hydroxy-
steroid dehydrogenase from 20 to 60 days. In conclusion, maturation of
enzyme systems in the testes is required for mature levels of steroid

production. The mechanisms responsible for maturation of these pathways

22
are unknown, but gonadotropins (LH and FSH) have been implicated (Payne
§£_al,, 1977; Wiebe, 1978). Payne gt_al, (1977) suggested that FSH
induces l78-hydroxysteroid dehydrogenase production while Wiebe (1978)
demonstrated that HCG induced 3B-hydroxysteroid dehydrogenase.

In mature bulls the major steroid product of the testes is testos-
terone (Schanbacher, 1976; Amann and Ganjam, 1976). Schanbacher (1976)
reported that bull plasma had 3.9 ng/ml of testosterone and 0.1 ng/ml
of dihydrostestosterone (dihydrotestosterone equalled approximately 3%
of testosterone). Testosterone, androstenedione, progesterone, estrogen.‘
(estradiol plus estrone), 5-androstenediol, 3a-androstanediol, and 3B-
androstanediol were present in spermatic vein blood (Amann and Ganjam,
1976). The presence of androstenedione and androstenediol indicates
that both the A4 and A5 steroid pathways are functional and contribute
to testosterone production. Presence of 3a- and 38- androstanediol and
estrogen indicate that considerable metabolism of testosterone occurs
in bulls. Progesterone, androstenedione, S-androstenediol, 3a-andro-
stanediol and SB-androstanediol each represented from 3 to 7% of testo-

sterone (78 ng/ml) while estrogens were only 15 pg/ml.

J. Hormones in the Pituitary and Hypothalamus During Puberty

 

Pituitary LH concentration and content increase in bulls from birth
to 1 month, then fluctuate randomly, not associated with the onset of
puberty. Pituitary FSH concentration and content increase from 1 to 2
months, then decline at 8 to 9 months and remain low (Macmillan, 1967).
Pituitary growth hormone content and concentration peaked at 3 and 4
months, declined, and remained constant to 12 months (Purchase gt_al:,

1970).

23
Few have studied hypothalamic hormone content in bulls during
puberty. Macmillan (1967) found increased hypothalamic luteinizing hor-
mone-releasing factor (LRF) activity at 5 months. LRF decreased at 6

months, increased until 8 months and was stable thereafter.

K. Hormonal Influences on Testicular Function in Bulls

LH is released into peripheral circulation in bulls in short secre-
tory surges or episodes, each persisting l to 2 hr (Katongole gt_a1,,
1971; Mongkonpunya §t_al,, 1974a; Kiser §t_al:, 1976). Mongkonpunya
et_al: (1974a) concluded that there were 3.7 episodic LH surges per 24
hr in 9-month-old bulls, while Katongole gt_al, (1971) found from 4 to
9 per 24 hr in mature bulls. Each increase in LH was followed within 1
hr by increased serum testosterone. The existence of a temporal rela-
tionship does not causally relate LH to testosterone secretion. However,
studies using HCG (Lindner, 1961; Katongole gt_al,, 1971) and LH
injections (Smith gt_§1:, 1973; Kiser gt_al., 1978) demonstrated a
direct cause and effect relationship between LH and testosterone secre-
tion. Also, treatment of bovine testicular tissue in_vitrg_with LH
promotes synthesis of testosterone (Kiser gt_al,, 1974).

Gonadotr0pin releasing hormone (GnRH) injections also demonstrated
tha causative role of gonadotropins in testosterone production. Treat-
ment of bulls with GnRH increased serum FSH and LH, followed by increased
serum androstenedione and testosterone at 4 hr post-injection (Zolman
and Convey, 1973). Kiser gt a1, (1978) demonstrated that by elevating
serum LH concentrations with prostaglandin an (PGan), serum testo-

sterone increased approximately 1 hr following LH peaks.

24

The testicular response to gonadotropins is age-dependent (Lindner,
1961; Mongkonpunya gt_al,, 1975a). HCG or pregnant mare serum gonado-
tropin (PMSG) administered to immature calves resulted in more andro-
stenedione than testosterone production, whereas HCG resulted primarily
in testosterone production in mature bulls (Lindner, 1961). Kesler and
Garverick (1977) reported that testosterone increased more in 10- to 24-
day-old calves than in 2- to S-day-old calves after GnRH treatment. A
similar age difference in testicular response was seen by Mongkonpunya
§t_al. (1975a) after treatment of 2-, 4- and 6-month-old bulls with
GnRH. The increase in LH following GnRH did not differ between ages,
but serum testosterone concentrations were significantly greater in 6-
month-old bulls compared with 2- and 4-month-old bulls. The andro-
stenedione to testosterone ratio was greatest in Z-month-old bulls.
In_vit£g_results (Kiser gt_§l,, 1974) agree with in_vivg_experiments.
Testes explants from 1- and 3-month-old bulls produced predominately
androstenedione in response to LH whereas explants from S-month-old
bulls produced predominately testosterone.

The role of FSH in steroidogenesis in bulls has not been investi-
gated. In other species, FSH increases androgen production in response
to an LH stimulus (Bartke gt_al,, 1978).

Few studies have been conducted to investigate prolactin's role in
testicular function in bulls. Smith §t_al, (1973) determined that a
single prolactin injection did not increase serum testosterone in bulls,
and prolactin plus LH did not increase testosterone more than LH alone.
Serum prolactin is related with seminal vesicular fructose, an indirect
measure of androgen secretion, and to sperm numbers in yearling bulls

(Swanson et_al,, 1971). Extended prolactin treatment can stimulate

25
androgen production in rats by increasing the sensitivity of the testes
to LH (Bartke §£_§1,, 1978). Whether a similar mechanism exists in
bulls has not been studied.

The effect of growth hormone on testicular function in bulls also
has not been studied. In other species growth hormone increased the
sensitivity of the testes to LH (Woods and Simpson, 1961; Swerdloff and
Odell, 1977).

Attempts to stimulate early testicular function and cause early
puberty have been unsuccessful. Treatment with GnRH (Mongkonpunya 33_
31,, 1975b) and GnRH plus thyrotropin releasing hormone (Haynes §E_§l:,
1977a) caused pituitary hormone release but failed to increase testi-

cular size or sperm numbers in young bulls.

L. Hypothalamic-Pituitary-Gonadal Interactions

 

l. Castration and Cryptorchidism

 

Castration results in increased serum gonadotropin concentrations
in a number of species including rats (Gay and Midgley, 1969; Swerdloff
et al,, 1971), monkeys (Atkinson gt_al,, 1970), rams (Pelletier, 1968;
Crim and Geschwind, 1972) and bulls (Odell gt_al,, 1971; Mongkonpunya
gt_al., 1974a; McCarthy and Swanson, 1976; Tannen and Convey, 1977).

As early as 1 month of age, castration results in increased serum LH
concentrations (Odell gt_al,, 1971; Tannen and Convey, 1977). Five- to
6-month-old (McCarthy and Swanson, 1976) and 9-month-old bulls re-
sponded similarly (Mongkonpunya §t_al,, 1974a).

Induced cryptorchidism also results in increased LH and FSH in

rats (Swerdloff 35 31,, 1971; Rager gt_al,, 1975) and rams (Schanbacher

26
and Ford, 1977), but these LH and FSH increases are not as pronounced

as in castrates.

2. Effects of Gonadal Steroids

Testicular steroids are involved in negative feedback on gonado-
tropins in intact males (Gay and Dever, 1971). Testosterone injections
reduce LH concentrations in rams (Bolt, 1971) and castrate rams
(Pelletier, 1970, 1973; Crim and Geschwind, 1972). Bolt (1971) found
that estradiol and progesterone also reduced LH concentrations in intact
rams. Schanbacher and Ford (1977) demonstrated that dihydrotestosterone
was ineffective in castrates and cryptorchid rams, while testosterone
reduced LH and FSH in castrates only. Estradiol reduced gonadotropins
in castrate and cryptorchid rams. FSH was reduced much less than LH and
it was suggested that factors other than estradiol alone are required
for maintenance of normal serum FSH concentrations.

Likewise, steroids reduce LH concentrations in bulls. Estradiol
was very effective in reducing LH levels in steers, while testosterone
propionate influenced LH concentrations only when administered at high
doses for several days (McCarthy and Swanson, 1976). Testosterone
treatment also reduced LH concentrations in l- and 9—month-old steers
(Mongkonpunya gt al., 1974a; Tannen and Convey, 1977).

Not only do castrates have higher basal LH concentrations, but
they also have a heightened response to GnRH. One- and 9-month-old
steers released more LH in response to GnRH than bulls of similar ages
(Mongkonpunya et_al,, 1974a; Tannen and Convey, 1977).

Testosterone treatment initiated 21 days post—castration and con-

tinued fOr 6 days failed to return the heightened GnRH-incuded LH

27
response to normal intact levels (Mongkonpunya 35 al., 1974a). One-
and 9—month-old bulls castrated and immediately given testosterone
responded with less LH secretion to a series of three GnRH injections
than did bulls. However, initiation of testosterone treatment to steers
at 21 days post-castration failed to reduce the exaggerated LH response
to GnRH (Mongkonpunya gt_al:, 1974a; Tannen and Convey, 1977). Thus,
testosterone treatment started immediately post-castration prevents
castration-induced increase in LH biosynthesis, while testosterone
treatment started later after castration cannot reduce LH biosynthesis
to levels in intact bulls. In agreement with these conclusions testo-
sterone propionate treatment of chronically castrate steers failed to
decrease LH response to GnRH (McCarthy and Swanson, 1976).

Twelve-hour continuous infusion of testosterone reduced LH concen-
trations and inhibited episodic LH surges (Haynes gt_al:, 1977b). GnRH
or PGan injection during testosterone infusion increased serum LH con-
centrations. These results support the concept that acute testosterone
treatment inhibits LH release by action at the hypothalamus or higher
centers and that the block is not at the pituitary. In this same study,
aspirin, a known prostaglandin synthesis inhibitor, administered for
12 hr, blocked episodic LH and testosterone secretion. Administration
of PGan overcame the block and caused LH release. Haynes gt_al, (1977b)
suggested that normal circulating testosterone concentrations may inhi-

bit LH by inhibiting prostaglandin production.

3. Inhibin

 

McCullagh (1932) postulated that a testicular substance from the

seminiferous tubules suppresses FSH production. Since then, numerous

28
observations have supported the idea that seminiferous tubular elements
produce "inhibin." Serum concentrations of FSH but not LH are elevated
in conditions in which the germinal epithelium has been destroyed as
after irradiation, induCed cryptorchidism, viral infections, antiferti-
lity agents or idiopathic infertility with normal Leydig cells
(Franchimont §t_gl:, 1975).

Water soluble rather than lipid soluble testicular extracts prevent
castration-induced changes in FSH. Extracts of bull and rat testes
(Lee gt_al., 1974; Braunstein and Swerdloff, 1977), ram rete testis
fluid (Setchell and Sirinathsinghji, 1972), human and bull seminal
plasma (Franchimont gt_al,, 1975) and media from Sertoli cell cultures
(Steinberger and Steinberger, 1977) reduced FSH concentrations in
several bioassay systems including castrate male rats, rabbits and rams
and pituitary cell cultures.

Chemical properties of inhibin have not been fully characterized.
Steinberger and Steinberger (1977) have shown that the Sertoli cell
product capable of decreasing FSH production from pituitary cell cul-
tures is a heat-labile molecule with a molecular weight greater than
12,000. This compound has inhibin-like properties, suggesting that
Sertoli cells are a source of inhibin. This does not exclude other

tubular elements as possible inhibin sources.

4. A Possible Explanation for the Onset of Puberty

 

Reduction in the sensitivity of hypothalamus and pituitary to nega-
tive feedback by gonadal products has been suggested as a possible cause
for increased serum gonadotropins and the initiation of puberty in rats

(Ramirez, 1973). Ramirez and McCann (1965) showed that smaller dosages

29
of testosterone on a body weight basis were capable of inhibiting LH
release in prepubertal than in adult rats. Negro-Vilar §t_al: (1973)
also demonstrated that testosterone propionate (10 pg per 100 g body
weight) suppressed elevated post-castrational LH concentrations in 15-
and 28-day-old rats, but not in 58- to 88-day-old rats.

The previous experiments did not insure that circulating testo-
sterone levels achieved by treatment were similar between animals in
different age groups. A recent study was conducted to fulfill that
objective (Negri and Gay, 1976). Adult and immature rats were implanted
at orchidectomy with polydimethylsiloxane capsules of varying size con-
taining enough testosterone to bring circulating testosterone levels
to equivalent concentrations in all age groups. These circulating
levels of testosterone suppressed LH concentrations in immature but not
mature rats. These findings substantiate the theory that reduced
sensitivity to negative feedback occurs as animals mature. The impor-

tance of this mechanism in bulls has not been assessed.

MATERIALS AND METHODS

A. General Methods

 

1. Animals

 

All animals used in these experiments were Holstein bull calves
purchased locally at about 1 week of age or obtained from the Michigan
State University dairy herd. Except during experiments, the bulls
were housed in an enclosed calf barn until about 3 months of age and
in a dry lot thereafter. Bulls were tied with halters or stanchioned
in a cold, enclosed barn for 2 to 3 days to allow them to adjust to

the surroundings before each experiment or blood collection period.

2. Blood Collection Procedures

 

Cannulae were inserted in the jugular vein 2 to 24 hr before start-
ing blood sample collection. Cannulae 30 inches in length (SLV-105#-18,
PCV cannula, ICO Rally Co., Palo Alto, Calif.) were inserted into the
jugular vein through a 2 inch-12 ga thin-walled needle (Becton and
Dickinson and Co., Rutherford, N.J.). The outside free end was plugged
and placed in a 2 inch by 2 inch Elastoplast enve10pe (Beiersdorf, Inc.,
South Norwalk, Conn.), secured to the neck with branding cement at the
exit point of the cannula from the epidermis. To prevent blood coagula-
tion, cannulae were filled with sodium citrate (3.5% in sterile water)

between blood collections.

30

31
After blood samples were collected they were allowed to clot at
room temperature for 4 to 6 hr and at 4 C for 24 hours. Blood was cen-
trifuged at 2000 x g for 20 to 30 min; serum was decanted and frozen at

-20 C until radioimmunoassays were completed.

3. Radioimmunoassays

 

Serum prolactin, growth hormone, luteinizing hormone and testo-
sterone were estimated by previously described double antibody radio-
immunoassays (Koprowski and Tucker, 1971; Purchas gt_gl,, 1970; Convey
§t_al:, 1976; Haynes gt_al,, 1977c).

Androstenedione was determined as described earlier (Mongkonpunya
gt_al,, 1975b), except a second antibody was used to separate free from
bound androstenedione similar to the testosterone assay described by
Haynes et_al, (1977c). Briefly, 200 pl of rabbit anti-androstenedione1
serum diluted to 1:2000 in .lM phosphate buffered (pH 7.4) saline with
.l% gelatin (PBS-G) and 1:100 normal rabbit serum was added to the
benzene:hexane (1:3) extract from each serum sample. Then 200 pl of
l,2,6,7-3H-androstenedione (5000 cpm) in PBS-G was added, the samples
were vortexed briefly and incubated for 24 hr at 4 C. Sheep anti-rabbit
gamma globulin serum (400 pl) at a dilution of 1:20 in PBS-G was added
to each tube. After incubation for 24 to 48 hr at 4 C, tubes were

centrifuged for 30 min at 2000 x g and a .5-ml aliquant was taken for

assessment of radioactivity by liquid scintillation spectrometry. The

 

1Rabbit #866-8-17-70 anti-androstenedione serum prepared against
6B-succinyl androstenedione conjugated to bovine serum albumin gener-
ously supplied by Dr. G. D. Niswender; Dept. Physiology and Biophysics,
Colorado State University, Fort Collins, Colo.

32
useful range of the androstenedione standard curve was from 10 to 500
pg. Fifty picograms of standard androstenedione reduced 3H-androstene-
dione binding by 50%. The average intra- and inter-assay coefficients
of variation calculated from standard serum assayed six times in each
of nine assays were 21.3% and 10%, respectively.

Bull and steer serum with added androstenedione (10 ng/ml) were
assayed in quadruplicate with and without isolation by column chroma-
tography. Benzene:hexane (1:3) extracts were chromatographed on Lipidex
5000 columns, 11 cm in length. Petroleum etherzchloroform (95:5) sol-
vent eluted androstenedione at 5 to 8 ml. After correction for proce-
dural losses, assay values for direct extraction were in good agreement
with chromatographed extracts. The bull serum averaged .15 :_.01 ng/ml
without and .16 :_.06 ng/ml with chromatography. Comparable values
for steer serum with 10 ng/ml added androstenedione were 10.111342 and
9.05:,55 ng/ml. When .5, 1.0, 2.0 and 5.0 ng/ml androstenedione were
added to steer serum, assays (quadruplicate) detected 101%, 107%,107% and
87% of the added mass.

Two different radioimmunoassays for FSH were used in Experiment 2
for comparative purposes and to obtain more confidence in results. Re-
sults from both assays were similar so only one assay was used in
Experiment 3. The first was a heterologous assay using anti-rat FSH
serum, highly purified rat FSH for iodination and bovine FSH for

standard.2

 

2Rabbit anti-rat FSH serum (s-7), rat FSH (1-3) and NIH-FSH-Bl
(.49 x NIH-FSH-Sl) were obtained from NIAMD of NIH.

33

Rat FSH was iodinated by the chloramine-T method (Greenwood et al.,
1963). Ten micrograms FSH in 5 ul of double distilled water was mixed
with 1.0 mCi Nalzsl (10 ul NaOH, pH 7 to 11) in 25 ul .5M phosphate buf-
fer (pH 7.5). Chloramine-T (5 ug/S ul .05M phosphate buffer, pH 7.5)
was mixed vigorously with FSH and 1251 for 2 minutes. The reaction was
stapped with sodium metabisulfite (12.5 ug/S ul .05M phosphate buffer,
pH 7.5). After adding 25 ul PBS-G and 100 pl of 1% potassium iodide
the mixture was placed on a 15 cm x .8 cm Biogel P-60 column to separate
free 1251 from FSH-1251.

FSH assays were conducted as follows: 200 pl rabbit anti-rat FSH
serum (1:2000 in 1:400 normal rabbit serum-.05M EDTA-PBS, pH 7.0) was
added to 500 pl standard FSH (NIH-B1) or 500 pl sample (serum plus PBS-G)
and incubated for 24 hr at 4 c. FSH-1251 (20,000 cpm) in 100 pl pas-0
was added to each tube and incubated at 4 C, followed in 24 hr by 200 pl
sheep anti-rabbit gamma globulin serum diluted 1:20 in .05M EDTA-PBS
(pH 7.0). After 72 hr, 3 m1 PBS was added and tubes were centrifuged
at 2000 x g for 30 minutes.

The second FSH radioimmunoassay was a slight modification of the
homologous bovine FSH assay developed by Cheng (1978). This assay uti-
lized an antiserum prepared in rabbits against highly purified bovine
FSH, both prepared by Cheng (1976, 1978).3 Iodination of bovine FSH
was conducted similar to that for rat FSH (above), not by the lactoperoxi-

dase method as used by Cheng (1978).

 

3Highly purified bovine FSH (160 x NIH-FSH-Sl) and rabbit anti-
bovine FSH serum were kindly provided by Dr. K. W. Cheng; University of
Manitoba, Winnipeg, Manitoba, Canada.

34

Two hundred microliters of rabbit anti-bovine FSH serum (1:120,000
in 1:400 normal rabbit serum-1% bovine serum albumin (BSA) -PBS) was
added to 500 pl standard FSH (NIH-Bl) in BSA-PBS or 500 pl of sample
(serum plus BSA-PBS) and incubated fOr 24 hr at 4 C. FSH-1251 (20,000
cpm) in 100 pl BSA-PBS was added to each tube and incubated at 4 C,
followed in 24 hr by 100 pl sheep anti-rabbit gamma globulin serum
diluted 1:20 in BSA-PBS. After 48 to 72 hr, 3 ml of cold PBS was added
and tubes were centrifuged at 2000 x g for 30 minutes.

Cross-reaction with prolactin (NIH-B4), growth hormone (NIH-BIZ),
TSH (NIH-B6), LH (NIH-B8); recovery of FSH-Bl added to serum and
parallelism of serum curves with FSH-Bl standard curve were tested
for both FSH radioimmunoassays.

Also luteinizing hormone-releasing hormone (500 ug Beckman Lot #
D1018) was administered iv to four lO—month-old bulls (300 kg body
weight) and serum samples were collected (-20, 0, 10, 15, 20, 30, 45,
60, 90, 120, 180, 240 min) to measure FSH response as an additional
validation step for both assays.

Androstenedione and testosterone in media from in_vitrg_incubations
were assayed by assays as described above. Before assay of tissue
testosterone, samples were homogenized and testosterone was isolated
chromatographically (Bartke gt al., 1973). Testicular tissue was homo-
genized in PBS (10 to 20 mg tissue/m1 buffer) with a Brinkman Polytron
for_two 10-sec blasts. To account for procedural losses, 25,000 cpm
3H-testosterone was added to 200 pl testicular homogenate. Five milli—
liters benzenezhexane (1:3) was added to homogenates and mixed for 1
minute. After freezing the aqueous phase, the solvent phase was de-

canted and evaporated in a vacuum oven. The residues were dissolved in

35
200 pl of column solvent (iso-octane:benzenezmethanol, 90:5:5) and
transferred to 11 x .5 cm Sephadex LH-20 columns with two 200 ul-solvent
rinses. Ten milliliters column solvent was added to the column and dis-
carded. Then 10 ml was added, saved, evaporated, and the residue was
redissolved in PBS-G. Aliquants were taken for testosterone assay and
calculation of procedural losses. Testosterone was assayed as described
by Haynes §t_al, (1977c) except 1% dextran-.5% charcoal was used to

separate bound from free testosterone.

4. Histological Preparation

 

Immediately after castration, testes were cut into 1 mm3 pieces
with a razor blade and fixed for 2 hours in a modified Karnovsky's fixa-
tive (Karnovsky, 1965) containing .5% formaldehyde and 1.3% glutaralde-
hyde in .lM phosphate. Post-fixation was done in phosphate buffered
osmium tetroxide and samples were dehydrated in a series of alcohols,
washed in propylene oxide and embedded in Epon 812-Araldite 502 Resin
(50:50). Sections of .5 to 1.0 u were made and stained with Azure II

for examination under a light microscope.

5. Statistical Analyses

 

Data were analyzed by split plot analysis of variance (Gill and
Hafs, 1971). Logarithmic transformations were made where heterogeneous
variance existed. Specific comparisons of means were by orthogonal con-
trasts and Scheffe's interval. Regression procedures were used to test
for linear, quadratic or cubic trends in data with advancing age (Gill,
1978).

An objective method to select episodic peaks was developed and used

to evaluate these data. A standard serum sample containing concentrations

36
that we judged to represent basal concentrations of each hormone was
assayed eight times in each assay. Values obtained were used to esti-
mate a 95% confidence interval around basal concentrations. An increase
greater than two 95% confidence intervals above a preceding value in a
hormone pattern was considered to be a peak. Two 95% confidence inter-
vals were .64 ng, .28 ng, .12 ng and 2.54 ng for LH, testosterone,
androstenedione and prolactin, respectively. The frequency of hormone
peaks, mean peak height (peak height = highest value in each peak) and
mean baseline concentration (lowest value between peaks) were calculated
for each animal and each age group. The objective of this method was
to insure that peaks reflected actual hormonal changes in animals, not
assay variation. This procedure was used to evaluate hormone profiles
in Experiments 2 and 3, but not in Experiment 1. Subjective evaluation

of graphs of hormone data was used to determine peaks in Experiment 1.

B. Experiment 1

 

The objective was to determine patterns of LH, prolactin, growth
hormone and testosterone in serum before and after puberty in bulls and
steers. Eight prepubertal and eight pubertal bulls were balanced for
age and weight within groups, four to be left intact and four to be
castrated 2 weeks before the experiment (age and body weight in Table l).
Jugular blood was collected via cannulae at .S-hr intervals from 0800
to 1700 hr for determination of growth hormone, prolactin, testosterone

and luteinizing hormone concentrations.

37

Table 1. Age and weight for bulls and steers in Experiment 1.

 

Treatment Groups

 

 

 

 

Prepubertal Pubertal
Parameter Bulls Steers Bulls Steers
Age (days) 113 1 243 157 1 o 270 1 32 286 1 35
Weight (kg) 80 1_12 100 1_7 229 1_36 184 1_16

 

aMean 1_SE of four animals.

C. Experiment 2

 

The objective was to compare at montly intervals from 1 to 10
months of age, patterns of LH and FSH secretion in bulls, steers and
bulls in which the scrotum had been shortened (SS bulls).

Twelve bulls were 2 weeks of age on April 1 when assigned randomly
four each to be left intact, castrated, or made short scrotum. Bulls
were made short scrotum by retaining the testes at the top of the scro-
tum with an elastic band. The purpose of making bulls short scrotum
was to interfere with seminiferous tubular function without effecting
Leydig cell function.

Body weight for all animals and scrotal circumference for bulls
were recorded at montly intervals from 1 to 10 months. Each month blood
was taken at .S-hr intervals from 1000 hr until 0930 hr of the following
day. All samples were assayed for LH and testosterone. Androstenedione
was measured in samples from the last 8 hr of each monthly bleeding in

bulls and SS bulls. FSH was measured by both radioimmunoassays in three

38
samples taken at hourly intervals (0700, 0800, 0900) from each animal
at each month. In addition, FSH concentrations were determined using
the rat FSH assay in two animals from each group at 3, 6 and 9 months
in all serum samples from the last 8 hr of the collection period. These
samples were measured to estimate FSH variability between groups and
ages.

At 10 months of age, testes and epididymides were removed and
weighed; and testes were fixed for histological evaluation to compare
seminiferous tubular development in normal and SS bulls. Epididymides
were homogenized in .9% saline-Triton X-100 buffer and spermatozoa were
counted in a hemocytometer with phase contrast microscopy (Kirton eg_

111., 1967).

D. Experiment 3

 

The objectives were to describe serum hormone profiles, testicular
responsiveness to LH and binding of gonadotropins to testicular tissue
in bulls of different ages. Serum samples were collected from all bulls
on a single day to permit determination of age effects independent of

season .

1. Blood Collection

 

Bulls at l, 3, 4, 5, 7 and 9 months of age and weighing 55, 79,
132, 176, 237 and 269 kg, respectively, were used in this experiment.
Jugular cannulae were installed 24 hr before the experiment. On
September 29 at 0800 hr, blood samples were collected at ZO-min inter—

vals for 24 hours.

39
Testosterone, androstenedione, prolactin and FSH were assayed in
samples collected for an 8-hr period (1200 hr to 2000 hr) and LH was
quantified in samples collected over 24 hours. FSH was estimated with

the rat FSH radioimmunoassay.

2. Testis Incubations

 

Testicular tissue from bulls of different ages was incubated 12_
EEEIE.t° measure testosterone and androstenedione production after LH
treatment. Testicular tissue from each bull was cut into explants
(1 mm3) in a petri dish containing Media TC 199. Five testis explants
were placed in each of 15 flasks for each bull. .Tissue was also frozen
immediately to serve as unincubated control. Three milliliters Media
TC 199 was added to each flask and treatments resulting in 0, 5, 50 ng
LH/ml of media were added to five flasks each in 300 ul .1% gelatin-
PBS. Flasks were incubated for 3 hr at 34 C in a Dubnoff Metabolic
Shaker while being gassed with 95% 02-5% C02. After 3 hr, tissue and

media were separated and frozen until assayed.

3. Testicular Gonadotropin Binding_

 

The amount of specific 1ZSI-FSH and 125I-HCG binding to testicular
tissue homogenates from bulls of different ages was determined. Three
to five l-cm3 pieces of testicular tissue were placed in 15 m1 of 20%
glycerol-PBS buffer, frozen in dry ice-methanol and stored at -60 C.
Later, testes tissue was thawed, blotted dry, weighed, placed in 20%
glycerol-PBS at a concentration of 500 mg tissue/ml and homogenized in
a Sorvall Omnimixer fOr 5 to 10 sec at top speed at 4 C. The homogenate

was filtered through two layers of cheese cloth, dispensed into 2-ml

40

aliquots, frozen in a dry ice—methanol bath and stored at -60 C until
assayed.

Human chorionic gonadotropin (HCG) and rat FSH were iodinated by
the chloramine-T method of Greenwood 23-31, (1963).“ HCG or FSH (10
ug/S ul double distilled water) was added to 1.0 mCi Nalzsl in 10 ul
NaOH (pH 7 to 11) containing 20 ul of .5M phosphate buffer (pH 7.5).
Chloramine-T (5 ug/S ul double distilled water) was added and allowed
to react for 2 min with gentle mixing. Sodium metabisulfite (10 mg/5
pl double distilled water) was added, followed by 100 pl of 1%
potassium iodide and 25 ul of PBS-G. The contents were placed on a 16
cm x .8 cm Biogel P-60 column which had been equilibrated with .05M
phosphate buffer (pH 7.5, column buffer). One rinse of 100 pl potassium
iodide was added to the iodination vial, mixed, and added to the column.
Free 125I was isolated from hormone-bound 125I by elution with column
buffer. Fractions of 1 ml were collected into tubes containing 1 ml
PBS-G. Aliquots of 20 ul of the collected fractions were counted for
.1 min in a gamma counter to characterize the elution pattern (Figure 1).

Iodinated hormone preparations were evaluated as described by
Ireland and Richards (1978). Specific activity of labeled hormones was
estimated to be 150 cpm/pg for HCG and 55 cpm/pg for FSH. This was
determined using labeled and unlabeled HCG (CR 119) or rat FSH (I-3)
and bovine testes homogenates as a source of binding sites. A constant
amount of receptor (35 to 70 ug of protein/tube for FSH and 1 to 1.5

mg protein/tube for HCG) was used to generate two types of parallel

 

“HCG (CR 119; 11,600 IU/mg) and rat FSH (1-3; 150 x NIH-FSH-Sl)
were obtained from NIAMD of NIH.

41

Figure 1.--Elution profiles of HCG (top) and FSH (bottom) iodination
reaction mixtures on Bio-Gel P-60 columns. Each point
represents the radioactivity in ZO-ul aliquots of l-ml
column fractions. The first peak (left) represents hor-
mone bound 1251 and the second (right) represents free

1251

cm xIO4

CPM no“

250 P
200V
I50 "
I00 '-

50"

 

350 -

300 -

250-

200 -

150-

IOO "

 

42

HCG-I099

 

 

 

 

50*- i
O 1141 11

I 3 5 7 9 ll l3 IS
F raction Number

43

standard curves. In one type increasing concentrations of labeled hor-
mone were used. In the other, counts per min of labeled hormone were
kept constant and the concentration of unlabeled hormone was increased.
The 50% inhibition point on each curve was used to calculate total cpm/
ug of active hormone. The active hormone or "active fraction" was
defined as the portion of labeled preparation which was specifically E‘
bound in the presence of excess receptor. This was 30 to 40% for HCG 1
and 20 to 30% for FSH, thus the specific activity of active labeled HCG
was 52.2 cpm/pg and labeled FSH was 14 cpm/pg. '

Binding assays were conducted in polypropylene tubes (12 x 75 mm)

 

precoated with 5% BSA-PBS. Specific binding was determined by sub-
tracting non-specific binding (binding in the presence of 1000-fold ex-
cess of cold hormone) from total binding for each tissue sample.

Ninety microliters (total binding tubes) or 60 ul of PBS (non-specific
binding) were added to each tube. Thirty microliters of PBS containing
unlabeled hormone (LH-NIH-BS, 30 ug; FSH-NIH-Bl, 75 pg) was added to
non-specific binding tubes. Saturating amounts of iodinated hormones

in 40 ul PBS were added to each tube including "total count tubes"
(HCG-1251, 250,000 cpm/tube; FSH-1251, 500,000 cpm/tube). Homogenized
tissue samples in 100 pl PBS were added, mixed and incubated in a shaker
at room temperature for 24 hours (time determined from time-study
described below). At the end of the incubation, 1 ml cold PBS was added
to each tube and the contents were centrifuged at 2000 x g for 30 minutes.
The supernatant was discarded; the pellet was resuspended in 1 ml cold
PBS by vigorous mixing; and tubes were recentrifuged, supernatant dis-
carded and the 125I in the tubes containing the pellet was quantified

in a gamma counter.

44

A time-study was conducted to determine the period required for
binding to reach equilibrium. Testicular tissue, homogenized as pre-
viously described, was incubated with iodinated hormone at room tempera-
ture for .17, .5, l, 2, 6, 12, 24, 36 and 48 hours. Tubes were centri-
fuged and the specific binding was determined. From results in Figure
2, maximum binding occurred in 12 to 36 hours. "

Saturation studies were conducted to determine the amount of iodi-
nated hormone required to saturate a given amount of tissue. Increasing

amounts of iodinated hormone were added to a constant amount of tissue

 

and specific binding was determined (Figure 3). Fifty milligrams of
tissue required approximately 40,000 cpm HCG-1251 for saturation. One
and 5 mg of tissue required approximately 125,000 cpm and 600,000 cpm
of FSH-1251 for saturation, respectively.

Before determining specific binding of testes homogenates from
Experiment 3, samples were thawed in cold water (4 C) and centrifuged
at 2000 x g for 10 minutes. The supernatant was discarded, the pellet
was weighed and resuspended in PBS at a concentration of 20 mg of pellet/
m1 (1 to 1.5 mg protein) fOr HCG binding assays and 1 mg of pellet/ml
(35 to 70 ug protein) for FSH binding assays. Resuspended tissue was
rehomogenized with 20 strokes in a glass homogenizer and 100 pl was used
to assess binding. During resuspension and rehomogenization, tissues
and buffers were maintained at 4 C. Specific binding was determined as
described above and expressed as hormone bound per ug of protein, per
ug of DNA or per testis x 2. Protein was measured with the Bio-Rad
Protein Assay (Bio-Rad Laboratories, Richmond, Calif.) and DNA was

estimated by the method of Burton (1956).

45

Figure 2.-—Time-course of 125I-HCG (tOp) and 125l-FSH (bottom) binding
to testicular tissue. A weighed amount of tissue pellet
obtained from centrifugation of crude testicular homo-
genates for 10 min at 2000 x g was rehomogenized and used
in these studies.

46

 

 

HCG-4O mg tissue
IOP
to
9 e-
X
2 6-
3
8
41-
2
a.
o 2..
O 1 1 L 1 1+
FSH-
25m tissue
IO- V 9
n
9 e-
X
a _ lmq tissue
5 6 f
8
z 4'
a.
U 21-
0 1 4 1 1 1

 

 

0 l2 24 36 48
Tlmelht)

47

Figure 3.--Saturation curves for 125I-HCG and 125I-FSH binding to
bovine testicular tissue. A weighed amount of tissue
pellet obtained from centrifugation of crude testicular
homogenates for 10 min at 2000 x g was rehomogenized
and used in these studies.

48

 

 

 

 

 

 

IIZ‘iHCG-SO mg tissue
IO-
"o ’__...---o-e
: 8' I
I
'0 I
S 6- 7’
a ,t
2 4" I,
n. O
o 2_
O 1 1 1 I l ,
20 4O 60 80 IOO
IIZ‘ZFSH
100-
n
9 30..
x (51119 tissue
'0 A
g 60- +
m 40-
: 1mg tissue
u 20* f " ,,
xfl—F—r— r
I I I I I I I _I

 

200 400 600 800 1000120014001600
cm ADDED x103

49

In addition to determining specific binding, saturation curves
using tissue from one to three bulls in each age group were used to

determine the affinity of hormone binding. These assays required a

constant amount of tissue (same as used previously) and varying amounts
of iodinated hormone. Results were evaluated by the method of Scatchard

(1949) and the affinities were estimated from the slope of these plots.

RESULTS AND DISCUSSION

A. FSH Radioimmunoassays 1’

 

Both radioimmunoassays were suitable to determine concentrations

of FSH in bovine serum (Table 2). The homologous FSH assay was 3 to 4

 

times more sensitive than the heterlogous FSH assay, but exhibited high
apparent cross-reactivity with NIH-TSH-B6. TSH cross-reaction was not
unlike that reported previously for bovine gonadotrOpin assays (Oxender
g£_§1,, 1972; McCarthy and Swanson, 1976). It is suspected that the
TSH standard preparation was contaminated with gonadotrOpins rather
than FSH antiserum reacting with TSH. The contamination left in TSH
preparations after purification has been suggested to be immunologically
active, but not biologically active gonadotropins (Niswender g£_§1,,
1969). To prove that this phenomenon is not true TSH cross-reaction,
serum containing low and high concentrations of TSH (pre- and post-
injection of a 50 ug, iv dose of thyrotropin-releasing hormone) were
assayed and did not have different LH concentrations (McCarthy and
Swanson, 1976). More highly purified bovine TSH, as well as bovine
TSH-a and TSH-B preparations obtained from Dr. J. G. Pierce (University
of California, Los Angeles, Calif.) did not cross—react significantly
in the homologous FSH assay (Cheng, 1978). Consequently, the observed
TSH cross-reaction originates from immunological contamination of NIH-

TSH-B6 standard, not nonspecificity of FSH antiserum.

50

51

 

Table 2. Comparison of two radioimmunoassays for FSH in bovine serum.
Heterologous rat Homologous bovine
Criterion FSH assay FSH assay

 

Antisera (rabbit) Anti-Rat-FSH serum

(S-7; NIAMD)
Antisera dilution 1:2000

Hormone for iodination Rat FSH (I-3; NIAMD)

CPM 1251.an added 20,000
a of 1251-an bound 25-30%
Standard NIH-FSH-Bl
Cross-reaction(%)1
TSH-NIH-B6 3
LH-NIH-BS <1
Prolactin-NIH-B4 <1
Growth hormone-NIH-Blz <1
Sensitivity2 9.5 ng

Coefficient of variation
Interassay (%) 4,4
Intra-assay (%) 8 2

Recovery (%)3 100.3
GnRH response“
Preinjection FSH (ng/ml) 100.8
Maximum FSH (ng/ml) 132.9
Time to peak (min) 60

Anti-Bovine-FSH-serum
(Cheng)

1:120,000
Bovine FSH (Cheng)
20,000
20-25%
NIH-FSH-Bl
60.7
4.3
<1
<1

2.5 ng

44.0
180.3
45

 

1A ratio of amount of FSH required to reduce binding of 125I-FSH equiva-
lent to reduction achieved by 1000 ng of other hormone was multiplied

by 100 to get percent cross-reaction.

2Sensitivity (ng) is defined as the amount of hormone required to dis-
place 125I-FSH binding equivalent to two standard deviations from zero.

3Mean recovery (%) of l ug/ml of FSH (NIH-Bl) added per ml of bull serum

from 50, 100 and 200 pl serum.

I'Mean preinjection FSH concentration and FSH response of four bulls to a
500 ug iv dose of luteinizing hormone-releasing hormone (GnRH).

52
Several pools of bovine serum and media from pituitary cell cul-
tures displaced 125I-FSH parallel to that of NIH-FSH-Bl in both assays

(Figure 4).

B. Experiment 1

 

Serum LH concentrations and number of secretory peaks per 9 hr
(Table 3) were greater in steers than bulls at both ages (P<.05), but
average serum LH did not differ significantly between ages. These re-
sults demonstrate that, in 4-month-old as well as 9-month-old bulls,
testicular products normally maintain low serum LH concentrations, since
removal of the testes at both ages causes increased serum LH.

Number of episodic peaks and mean testosterone concentrations
(Table 3) were four-fold greater in pubertal than in prepubertal bulls,
but these changes only approached significance (P=.10). Wide variation
in the testosterone secretion profiles between bulls within an age-group
prevented these groups from differing significantly. One bull in the
prepubertal group had serum testosterone concentrations as high as the
mean of bulls in the pubertal group and one pubertal bull had low testo-
sterone concentrations. There is no question through, that testosterone
concentrations increase with advancing age (Rawlings et_31,, 1972; Karg
g£_31,, 1976).

Average prolactin concentrations and episodic release patterns
were not affected significantly by castration or by age, although
prolactin tended to be higher and more variable in older animals.

Growth hormone concentrations and number of peaks were greater in pre-
pubertal bulls than in pubertal bulls (P<.07), but this age effect was

not evident in steers.

 

53

Figure 4.--Comparison of NIH-FSH-Bl standard curves with serum and
media curves for two FSH radioimmunoassays. In the hetero-
logous rat FSH assay (top), curves are shown for NIH-FSH-Bl
(e--e), media from pituitary cell cultures (I--I), bull serum
(D--o), pregnant cow serum (O--O), ovariectomized heifer
serum (A-—A), pregnant cow serum plus 1000 ng FSH/ml (A--A)
and steer serum (x--x). In the homologous bovine FSH assay
(bottom), curves are shown for NIH—FSH-Bl (e--e), media from
pituitary cell cultures (l--I), bull serum (o--o), post-
partum cow serum (O--O), ovariectomized heifer serum (A--A)
and bull serum plus 1000 ng FSH/ml (I--A).

54

Heterologous Rot FSH ossoy

 

 

 

 

IOO '-
80 '-
60 _-
40 -
m
3 20 -
2
o
‘ o
:6 I I I J I I I I I I p
:2 2.5 5 IO 25 50 I00 200 400 IOCX)
E
2 Homologous Bovine FSH Assay
.1 IOO -
o
O
0‘ oo-
60 _-
40 - ‘g
20 -
‘A
O " 1 1

 

 

I J I I I I
2.5 5 IO 25 50 I00 250 400
N I H-FSH-81 (no/tube) or Somple(u|)

SS

 

 

 

Table 3. Serum hormone characteristics for prepubertal and pubertal
bulls and steers.
Prepubertal Pubertal
Hormone Bulls Steers Bulls Steers
LH Concentration1 2.911. 10.517.3 3.3:1.8 9.2:3.l
Episodic peaks.2 2.01 . 6.31 .3 2.51 .s 6.81 9
Testosterone Concentration 1.411. -- S.4:3.5 --
Episodic peaks .5:_. -- 2.3:_ 3 --
Prolactin Concentration 7.713. 8.412.8 16.617.9 l8.018.8
Episodic peaks 1.5:_. 2.0:_ 7 2.5:_ 8 3.0:_ 8
GH Concentration 14.914. 8.0:}.9 6.7:2.4 9.8:4.2
Episodic peaks 4.3+ . 3.3:1.0 2.0+ 0 3.0+ 7

 

1Mean + SE of hormone concentration (ng/ml) for all samples taken

9 hr for four animals.

over

2Mean number (1 SE) of episodic peaks observed per animal per 9 hours.

56

Bartke (1976) and Bartke and Dalterio (1976) demonstrated the im-
portance of prolactin in testicular function, especially steroidogenesis
in several rodent species. Prolactin was greater in postpubertal bulls
than prepubertal bulls. However, we cannot determine from this investi-
gation whether prolactin caused increased testosterone production in
older animals, or if testosterone stimulated prolactin synthesis (Nolin
g: 31., 1977).

Prepubertal bulls had greater growth hormone concentrations than
did postpubertal bulls. Whether high prepubertal growth hormone concen-
trations participate in testicular maturation in bulls cannot be deter-
mined from this investigation. However, growth hormone has recently
been reported to increase testosterone production by immature male rats

in response to LH (Swerdloff and Odell, 1977).

C. Experiment 2

 

Body weights for bulls, steers and SS bulls did not differ signifi-
cantly. Averaged over treatments they were 50 :_3 (X 1_SE) kg at 1
month and 273 :_12 kg at 10 months. Scrotal circumference increased
linearly (P<.05) with age in intact bulls from 106 mm at 1 month to 277
mm at 10 months. Testicular weight (including epididymides) of bulls
and SS bulls at 10 months did not differ significantly (186 :_29 vs
140 1_19 g).

At 10 months, bulls had spermatogonia, Sertoli cells, spermatocytes
and spermatids, whereas short scrotum bulls had Sertoli cells and a few
spermatogonia. Epididymidal sperm were present in bulls (7 x log/epi-

didymis) but not SS bulls.

57

LH increased from 1 (1.1 1_1.6 ng/ml) to 3 months (5.6 :_3.3 ng/ml)
in steers and from 1 (.8 1_l.l ng/ml) to 2 months (2.4 1_3.1 ng/ml) in
SS bulls (P<.05), and remained unchanged thereafter (Figure 5). In nor-
mal bulls, LH was greater (P<.05) at 4 months (2.7 :_2.6 ng/ml) than at
1 month (.6 :_.5 ng/ml), but did not differ significantly among other
months.

Serum LH concentrations were greater (P<.05) in steers than in
bulls or SS bulls from 3 to 10 months. Also, SS bulls had higher serum
LH than bulls at 5 months (2.9 :_l.l vs 1.5 :_1.7 ng/ml) and 6 months
(2.2 :_1.2 vs 1.2 1_2.6 ng/ml), but not thereafter. Interestingly,
only at months (Figure 5), was testosterone less in SS bulls (.3 :_.2
ng/ml) than bulls (1.2 :_.2 ng/ml).

Serum testosterone concentrations in steers were low (100 pg/ml)
and did not change with time of day or age, thus they were excluded from
statistical analysis of results. The first significant increase in
serum testosterone (P<.05) occurred between 4 (.5 :_.2 ng/ml) and 5
months (1.2 :_.9 ng/ml) in normal bulls but not until 5 (.3 :_.2 ng/ml)
to 6 months (1.4 1_1.4 ng/ml) in SS bulls. Average androstenedione
concentrations (Figure 5) did not differ significantly between bulls
and SS bulls; it increased (P<.05) transiently at 4 months in both
groups.

Frequency of LH peaks increased (P<.05) from 1 to 4 months in bulls,
steers and SS bulls (Table 4; Figure 6). Thereafter the frequency of
LH peaks declined (P<.05) in bulls and SS bulls, but not steers. This
decreased frequency occurred coincident with increased testosterone
S€>cretion (Figure 5). Frequency of LH peaks did not differ between treat-

Hfirnts at 1 month, but at 2 months steers had a higher (P<.05) frequency

58

Figure 5.--Mean LH, testosterone and androstenedione concentrations in
bulls (O--O), steers (A--A) and short scrotum bulls (o--e).
Values are means of 48 samples from a 24-hr period for LH
and testosterone and 16 samples from an 8-hr period for
androstenedione for four animals in each group.

 

 

59

 

   

0 A.-A

BULLS SHORT SCROTUM BULLS

     

 

 

:Exocv I;

 

 

 

S
u
U 0
B |
M
U 9
S T
a m
U C 8
B S
M T 7
U R
.. mm
m L8 6
C U e
S B/_ 5
T /
m x
H R! 4.
S I
I,
S 3
L
o. 2
B

 

- b P n h

e 4 2 ..o. 4. 2 o
25:95 Ergo:
392333 326823.34

 

 

Age (mo.)

60

.mfigwg Hfiom MO mflmwfi 0:9 GHM m®5dw>m

 

 

 

 

 

o.H o.v n. H.m H.m v.0 m.w m.m~ o.n oa
o.H m.v m. o.v m.m m.m m.n m.m~ m.n m
m. w.m v. H.m w.o o.v w.m m.oH o.m w
o.~ o.m o. m.m N.o v.v o.o w.oH w.c n
m.~ H.m m. v.m m.m c.v w.n o.o~ w.v o
o.~ o.m m. v.v ~.n m.v o.oH m.v~ w.w m
H.N m.m ~.~ w.v m.n ~.o m.m~ m.n~ o.H~ v
~.H H.m m. 0.5 o.w m.m o.o~ m.oH m.m m
n. m.~ o. H.@ v.m v.w w.o m.m m.v N
o. o. m. v.v H.m m.m m.m w.m m.~ H
- 2595 - .. 2535 - - $242281“ 65 - 32v
mfiaanmm whooum maasm maasnmm muooum mafism madsnmm whooum mfiasm .Ammfl
Hmmmm usmflo: xmom xocozconm

 

o.aadfizammv means asoouom
unogm use whooum .mHHSD ca :4 mo m:oaumnucoocoo Hmmmn can mxwom aflvomfimo mo “swam: wad Aocosc0Hm .v enamR

61

.owm mo mnueoa OH wee m .v .H on aasa azuoaom uuonm use nooum .HH3n
o>fipmucomoamou m :a macaumnucoocoo m<nu<V o:oauoaoumonw:w no aonuou ozonomeHmou .mOunov $411.0 enamam

2
.B

62

1111/ Du ( v-v )ouogpeuusoipuv

N

O V

 

[ Shonscrommeuu 571 Monml

e.‘

 

Month 4

 

 

Month 5

fi

1 V

 

 

 

l6 IO 20 22 24

l2

1

02468

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

fl
3
2
0
n
L
a
o
..
(n
A A I A A L I
1 V I V l'
. .
. .
o: - -
. .
0"- -~---.
0 4 -4
«:0
J 0LT? - ‘
- a Q ,0 n 9
1: o::::' z 051-- ' £ 5 ‘
- "0! .-
0 C 1:
8 S __..o’ o g
z 2 on::_-___-_.- 4 5 ‘
8 . 1
D -...-- "'
_ o-°-'::::'.-- "3
—= 1 -~- I
D
L A 1 1 1 1 ‘ P 1 1 e
N o v (_\_1 a: Q C 2 Q o C

tut/bu (0-0 “.11 JO ( H ) euonuouu

14

IO
Hours

IO 20 22 24

12 l4 l6
Hours

IO

02468

IO 20 22 24

12 I4 16
Hours

IO

02468

63
of LH peaks than bulls (Table 4). At 3 and 4 months steers and SS bulls
had more (P<.05) peaks than bulls and from 5 to 10 months steers had
more (P<.05) peaks than bulls.

LH peak height doubled (P<.05) from 1 to 2 months (Table 4) in each
treatment. When averaged over all months of the experiment, steers had
higher (P<.05) LH peaks than bulls or SS bulls (7.4 ng/ml vs 4.9 ng/ml
and 5.0 ng/ml). From 3 to 10 months, steers had higher (P<.01) basal
LH concentrations (lowest value between peaks) than bulls and from 8 to
10 months basal LH in steers exceeded (P<.05) that in SS bulls. Basal
LH concentrations were elevated in steers because peaks occurred so
frequently that new peaks were initiated before serum LH declined from
the previous peak.

Increased (P<.05) frequency and amplitude of testosterone peaks
and increased (P<.05) baseline concentrations (Table 5; Figure 6) con-
tributed to increasing average testosterone (Figure 5). Only a few
small testosterone peaks (.3 to .6 ng/ml) occurred in bulls from 1 to 3
months and in SS bulls from 1 to 5 months. After this age, the number
and size of testosterone peaks increased (P<.05). In bull 559 (Figure
6) at 5 months, testosterone peaks of 2 ng/ml followed each LH peak and
by 10 months peaks of 5 ng/ml occurred. Frequency of surges differed
(P<.05) between bulls and SS bulls only at 5 months.

Basal testosterone concentrations in both normal and SS bulls in-
creased (P<.05) linearly with age, approximately 10- and 30-fold, re-
spectively (Table 5). Basal testosterone did not differ significantly
between bulls and SS bulls.

The increase in mean concentrations of androstenedione in bulls at

4 months (Figure 5) resulted from an increase (P<.05) in frequency and

64

Table 5. Frequency and height of episodic peaks and basal concentra-
tions of testosterone in bulls and short scrotum bulls
(SSbulls).a

 

 

 

 

 

Frequency Peak Height Basal
JHEL. Bulls SSbulls Bulls SSbulls Bulls SSbulls
(M0) -(N0. peaks/24hr)- -(ng/ml)- -(ng/ml)-
l 3 O .4 -- 1 O
2 1 3 1.8 5 4 l 1
3 5 3 .6 4 l l
4 2 3 1.8 l 8 7 2 2
5 7 5 1 0 2.1 8 5 2
6 4 5 2 8 4.2 2 l 4 l 3
7 6 3 3 5 3.7 2 3 4 5
8 4 5 3 0 3.4 2 4 5 9
9 6 3 9 O 6.5 7 4 8 2 3
10 6.8 8.5 9.3 10.1 1.0 3.1

 

8Values are the means of four animals.

65
amplitude of peaks and increased (P<.05) baseline (Table 6; Figure 6).
In 88 bulls, an increase (P<.05) in peak height at 4 months contributed
to the increased mean. However, frequency of peaks or baseline did not
differ significantly at 4 months from other months.

These results support those of Rawlings g£_§13 (1972) and Karg 23.
a1. (1976) who also found that increased serum testosterone concentra-
tion was the most pronounced hormonal change during sexual maturation
in bulls. Concomitant changes in gonadotropins or other pituitary
hormones have not been previously observed in bulls (e.g., Karg g; 31,,
1976). In this study, mean serum LH concentrations increased at 4 months,
associated with an increase in frequency of LH episodic peaks. Andro-
stenedione also increased at 4 months, 1 month prior to the onset of
significant testosterone production. Exposure of the testes to more
LH around 4 months may have been the stimulus that resulted in greater
androstenedione production at 4 months and increased testosterone pro-
duction at 5 months.

Frequency of episodic LH peaks increased in bulls, steers and SS
bulls from 1 to 4 months, in agreement with results reported in rams
(Foster g£_§13, 1978). What caused the increased frequency of LH is
not known, but it may be related to maturation of the hypothalamus.
Since increased frequency of LH peaks occurred in all treatments, irre-
spective of the steroid milieu, hypothalamic maturation presumably
occurs independent of circulating steroid concentrations.

The frequency of LH peaks was greater in steers than in bulls
throughout the experiment. Factors involved in negative feedback on

LH before puberty may not be testosterone or androstenedione, because

66

Table 6. Frequency and height of episodic peaks and basal concentra-
tions of androstenedione in bulls and short scrotum bulls
(SSbulls).a

 

 

 

 

 

Frequency Peak Height Basal
1351 Bulls SSbulls Bulls SSbulls Bulls SSbulls
(M0) -(N0. peaks/8 hr)- -(ng/m1)- -(ng/m1)-
1 0 .3 -- 1 -- 1
2 0 .0 -- —- -- --
3 0 .8 -- 2 -- 1
4 l 8 8 .7 6 3 2
5 1 0 .3 4 2 2 1
6 1 0 .0 3 -- 1 --
7 8 8 .3 2 1 l
8 3 5 .2 4 l 1
9 5 8 .4 2 1 l
10 .5 .8 .4 .2 .1 .1

 

aValues are the means of four animals.

67
even at 2 months when these steroids were low, bulls had fewer LH peaks
than steers. ‘

Until 6 months, frequency of LH peaks in SS bulls resembled that
in steers more closely than that in bulls, but after testosterone secre-
tion commenced in SS bulls the LH pattern resembled that in bulls.
Testosterone and androstenedione did not differ between bulls and SS
bulls until 5 months, so some factor other than these steriods, lacking
in young SS bulls must be involved in negative feedback on LH. Reduc-
tion in seminiferous tubular function in SS bulls could be the cause of
greater frequency of LH peaks in this group. Inhibin or steroids pro-
duced by the tubules (estradiol or Sa-reduced androgens) might be in-
volved (Dorrington and Armstrong, 1975; Dorrington and Fritz, 1975;
Franchimont 35H31., 1977).

The increased frequency of LH peaks which occurred in all three
treatment groups around 4 months of age occurred during months with
the longest photoperiod. Changes in secretion of other pituitary hor-
mones due to photoperiod and temperature have been reported in cattle
(Peters and Tucker, 1978). Whether increased LH secretion at 4 months
was due to seasonal hormone variation or age was unknown. This question
was investigated in Experiment 3.

Both radioimmunoassays were used to estimate serum FSH concentra-
tions (Figure 7). Absolute hormone concentrations varied slightly
between assays, but this did not alter the physiological interpretation
of the data. Data from each assay were analyzed separately by analysis
of variance. Treatment and age effects were similar between assays.
FSH increased (P<.01) in serum of steers from 1 to 2 months then did

not change significantly through 10 months. Serum FSH did not change

68

Figure 7.--Serum FSH concentrations in bulls (O--O), steers (A--A) and
short scrotum bulls (e--e) in two FSH radioimmunoassays;
heterologous rat FSH assay (top) or homologous bovine FSH
assay (bottom). Values are means of three samples collected
at hourly intervals from four animals in each group.

69

A./$TEERS

 

 

300- '6’"! \. .A°\A
./ \ .A
7‘ or”
A ZOO” ,' swonr SCROTUM BULLS 2
ob I00 MW
:1: 41
_ I: BULLS
Z O 1 1 1 1 1 1 1 L 4 J
E 300- ,srecas A
a .A'*, IA" '\
c -’ ‘A‘Nam-d' A
; 200- f
m I! SHORT scaoruu aw?
"i WWW-W
BULLS} "o"
O 1 1 1 1 1 1 4 L

 

 

11
I23456789|

Age (mo)

0

70
with age in normal or 88 bulls. Steers had higher (P<.05) serum FSH
concentrations than bulls or SS bulls from 2 to 10 months.

Serum samples collected at .5 hr-intervals for 8 hr from bulls,
steers and SS bulls (two each at 3, 6 and 9 months) were assayed in the
heterologous FSH assay to determine if episodic FSH peaks occurred.
Episodic peaks were not observed, but FSH concentrations did fluctuate
within an animal at a particular month (Table 7).

These results agree with Karg g£_§1, (1976), who also observed no
change in serum FSH concentrations during puberty. This does not mean
that FSH plays no role during sexual maturation in bulls! Serum FSH
increases before the onset of spermatogenesis in lambs and rats (Lee
e:flg1., 1976; Payne §£_213, 1977). Also, FSH sensitizes the testes to
respond to LH with testosterone production in rats (Swerdloff and Odell,
1977). This effect may occur as a result of FSH stimulation of Sertoli
cell secretions which affect the interstitium (Bartke g£_§13, 1978).
Constant serum FSH concentrations might accomplish these functions in
bulls.

Serum FSH concentrations were greater in steers than in bulls or
SS bulls by 2 months, demonstrating the importance of negative feedback
on FSH during the first few months of life. Although the factors in-
volved are not known, inhibin or steroids other than testosterone or
androstenedione produced by the prepubertal testes could be important.

The short scrotum procedure stopped spermatogenesis, yet did not
alter serum FSH levels by comparison with bulls. Only Sertoli cells
and scattered spermatogonia remained in the tubules. Sertoli cells are
a source of inhibin and estradiol,lxnfliof which are capable of reducing

serum FSH concentrations (Dorrington and Fritz, 1975; Schanbacher and

71

.H: w How m~m>uoucfl

Harm. um :oxmu moamemm pecan no xmmmm :mm mnemofionouo: may now: woeflsnopov one: meowuauucoucoo :mm scheme

 

 

 

 

 

 

Nea-aoH wea-waa Acm-3m~ eom-emm Hea-oma NmH-am emcee
m.~.u mNH H.N.u one c.e.u new o.~.n mom w.a.u Hoe ~.m.u eaa mm.u.w a
me~-eHH mma-am mam-oam emm-mem Hma-mm mea-em omeom
w.a.n mma w.~.u nHH m.a.u eom e.m.u mow e.m.u AHH m.m.u Nae mm.umm o
ema-caa wma-ooa one-~oe oo~-Hm~ mw-me ema-ea owed“
mow mi Wm.“ H3 fiend”? m.m.+. EN EN” cc mduo: mmnw m
Hem mom mam mam oem mom noooeoooa nozv
.mwfl.
oaaoomm maooom oaaam
psoEuwouh

 

nmamanmmv masuouom vocouuogm spa: mfifinn use

mHOQHm

m.omw mo mspcos m one o .m we

.mHHBD :fi nas\w:v mcofluonusoocoo 2mm Edhom .5 ofinmh

72
Ford, 1977; Steinberger and Steinberger, 1977). The short scrotum pro-
cedure may not have altered Sertoli cell function sufficiently to reduce
production of these compounds, hence low serum FSH concentrations were

maintained.

D. Experiment 3

 

1. Body and Testicular Growth

 

Body and testicular weight increased as a third degree polynomial
with age (Table 8). The curves for body weight and testicular weight
differed (P<.001), primarily due to slower testicular growth from 1 to
4 months and more rapid testicular growth from 5 to 7 months. The
changing ratio of testicular weight to body weight reflected the greater
relative increase in testicular growth from 5 to 7 months (Table 8).

Changes in testicular weight and body weight for these Holstein
bulls agree with similar observations reported by Macmillan and Hafs
(1968a). The rapid increase in testicular weight occurring after 4
months probably reflects increased seminiferous tubular diameter and
proliferation of spermatogenic cell types (Macmillan and Hafs, 1968a)

as well as Leydig cells hyperplasia and hypertr0phy (Hooker, 1970).

2. Serum Hormone Concentrations

 

Mean LH concentration (Figure 8) increased (P<.05) from 1.4 ng/ml
at 1 month to <2 ng/ml at 3, 4 and 5 months and then decreased to 1.4
ng/ml at 7 and 9 months. This increase in mean LH resulted from an
increase (P<.05) in the number of LH peaks/24 hr (Figure 8), from 4.4
at 1 month to 11.8 at 4 months; then LH peak frequency decreased to 6.0

at 5 months. LH peak height (Figure 8) was relatively constant in l-,

73

Table 8. Body and testicular weight in l-, 3-, 4-, 5-, 7- and 9-month-
old bulls.a

 

Testicular weight:

 

1&51 Body weight Testicular weight body weight ratio
(M0) (kg) (8) (g/kg)

1 55.4 :_3.9 12.8 :_1.2 .23

3 79.0 1 3.8 25.4 1_2.1 .32

4 131.5 :_5.6 48.3 1 2.5 .37

5 176.3 :_6.5 96.5 :_2.1 .55

7 236.6 1_10.3 247.9 :_9.1 1.05

9 268.5 :_ll.4 304.8 + 16.0 1.14

 

aValues are the mean 1’SE of five bulls per group.

74

Figure 8.--Serum LH averaged for 72 samples/bull (top), mean number of
LH peaks/24 hr (middle) and mean LH peak height in five
bulls at each age (bottom).

75

 

 

 

fl I I
.
.
1..
~
4
M
~
a I
_
fl I I
I
I
I
y 1 1
r! _ _ _ r. _ _ _ r21
.3 9. .I no mm m” .o a. .4
:59: e8: zeuxofioa 3.3.52

T...

 

 

 

’
’
c...
x n
\
\
A 1
I
H. ..
fix
_
.
.
o ..
a a. I
2235222. no...

Ageimo)

76
3-, 4- and S-month-old bulls and then decreased (P<.05) from 5 months
(7.6 ng/ml) to 7 months (3.5 ng/ml). Baseline concentrations (lowest
point between peaks) varied from .8 ng/ml at 1 month to 1.3 ng/ml at 4
months, but did not differ significantly between bulls of different ages.

The increase in mean LH concentrations and frequency of peaks pre-
ceded increased testosterone production (Figure 9). Mean testosterone
was <1 ng/ml at l, 3 and 4 months and then increased markedly at 5, 7
and 9 months. Frequency and peak height (ng/ml) increased linearly
(P<.05) from .4 and .9 at 1 month to 3.0 and 13.2 at 9 months, respec-
tively (Figure 9). Baseline concentrations followed a similar pattern;
a linear increase (P<.05) from 1 month (.2 ng/ml) to 9 months (2.8 ng/ml).
Decreased LH concentrations, number of peaks and peak height from 4 to
7 months may have been due to increased testosterone secretion at this
time.

Mean serum concentrations of androstenedione, frequency of peaks,
height of peaks or baseline did not change significantly with age.
However, there was a trend for an increase (P=.10) in mean concentration
at 4 months (Figure 9).

In contrast to LH, mean serum FSH (Figure 10) did not change with
age, and remained between 99 and 120 ng/ml. In addition, FSH profiles
within a bull did not have distinct episodic releases similar to those
observed for LH. Only small changes in FSH were seen during an 8-hr
period.

Mean prolactin concentrations and peak height increased (P<.01)
from 1 to 5 months, while frequency increased (P<.01) from 1 to 4 months

(Figure 10). Baseline prolactin concentrations increased (P<.01) from

77

.Aeouuonv own some on

mfiasn o>Hm mo pnwfio: xmom ocouopmoumop paw ocoflvocoumOchm came one Maofippflev

A: w\mxmom ocououmoumou can ocofiwocoawOchm mo Hones: come ”among Hasn\mofimamm
mm How vommpo>m mcoflpmypcoocoo flourov ocopoumoumou paw Aonro occawocoumOch<ru.m opswwm

78

Testosterone (H)

 

 

 

Peek helghting/ml) Peeks per 8hr Moon (no/ml)
o A a 75 o o N «5 do
I I 1 l I I T 1
9 - <1
I \
. \
I ‘1
.‘1

      

 

 

 

 

 

 

 

I
F
I
I
1
1
" i
1
I
|
I
- 6
1 1 J 1 1 1 1 1 J
o 'N lb or O - N u o 2. is) a,
Pool: height (no/ml) Peaks per 8hr

Moon (no/ml)
And rostenedione (O----°)

79

Figure 10.--Serum FSH concentrations averaged for eight samples at hourly
intervals in each of five bulls at each age (top). Prolactin
concentration averaged for 25 samples/bull; mean number of
prolactin peaks/8 hr; and mean prolactin peak height of five
bulls at each age.

‘80

 

 

 

 

 

 

 

 

 

.J 0 I. a J w J
4 . ~
. ~
. s
r 1 c 1 a - .1 1
. ~
. ~
~ ~
.. a, .. 1 P ..
I II
—
. a
. a
_ I
.. w 1 1 r 1
_L p _ _ _ P _ b _ _ .1
Mu mu 0 :0 .14. 8 4 O 32l0 m m 8 111.1
m P. 8 2E3... £99.23 :63...
:5on cans. zoos. .o .3652 232. soon.

1mm c_._oo_o.n_

Age (mo)

 

81
4.8 ng/ml at 1 month to 8.9 ng/ml at 5 months and then remained rela-

tively constant.

3. Testis Incubations

 

Testosterone concentration in unincubated control tissue increased
linearly (P<.05) from 75 ng/mg at 1 month to 303 ng/mg testis protein
at 9 months (Figure 11). Incubation of testes for 3 hr in zitrg_re-
sulted in accumulation of testosterone (media plus tissue) greater
(P<.05) than in unincubated control tissue at all ages. When all treat-
ments were considered (0, 5, 50 ng/ml), tissue from 4-, 5- and 7-month-
old bulls produced more (P<.05) testosterone during incubation than
tissue from 1- and 3-month-old bulls. Both LH doses increased testo-
sterone production above incubated controls (P<.05) at all ages, and
testes from bulls of different ages responded similarly to LH.

LH treatment caused testicular tissue to release androstenedione
into media (P<.05) when data were pooled over all ages, but large varia-
tion among animals prevented detection of significant LH responses with-
in each age (Figure ll). However, androstenedione released into media
when all treatments were considered was greater from testicular tissue
from l-, 3- and 4-month-old bulls than from S-, 7- and 9-month-old
bulls (P<.05).

Increased frequency of peaks and mean concentrations of LH at 4
months agrees with results from Experiment 2 and with results of Foster
gt_§l, (1978) with rams. These findings also support the contention
that changes in LH peak frequency occur due to age, independent of

photoperiod or seasonal changes.

82

Figure ll.--Testosterone from testicular tissue (tissue plus media)
unincubated or incubated for 3 hr with 0, S and 50 ng LH/ml
of media; five bulls at each age (top). Androstenedione
release into media from testicular tissue incubated for 3
hr with 0, 5 or 50 ng LH/ml of media; five bulls at each
age (bottom).

83

     

     

500

        
       
 

I W//////.///4///./////4
.H.n.u.u.H.n.u.u.u.n.u.u.“.u.u.m

Ivoo.

 

  

vw////////////A

. ....... :0...
u%%%%%%%%%%%%%

H /.
BLEII
mm

f

n" '".I| II. II
Ofmmm
Chl/l
630.90.
Ndnnn
00050
b... 5
“a

0.0

.3

n

Uum

 

b

 

 

—
m 0
2
2:30... 8.0252 9505
00220208.

IOOO

Age (mo)

an.

mmm 1M9
mmm m
990 l.\
nnn g 7e
05% M.

 

 

   

  

712%

0.... ...... ¢oooooooooo
q%%%%%%%%%%%%%%%%%%%%%

 

      

1 1......»......................v..

1

2.203 3.3:»: oExoc.
000.02.200.02 0.002

 

Aoehno)

84

The mechanisms involved in increasing the frequency of LH peaks
prior to puberty in bulls are not known. However, it seems unlikely
that increased frequency is due to increased availability of LH.

Bulls respond to GnRH treatment with equivalent LH release at 2, 4 and
6 months of age (Mongkonpunya et_al:, 1975b). In addition, pituitary
LH content in bulls iSEN:mature levels by 1 month (Macmillan and Hafs,
1968b). These results suggest that changes in LH release with age may
be due to maturation of the hypothalamus, or involve alterations in
sensitivity of the brain to negative feedback by gonadal factors.

LH peak height is reduced between S to 7 months, indicating less
LH released per peak at 7 months than at earlier ages. This change
occurred during the period that serum testosterone levels increased.
Negative feedback of testosterone or other gonadal steroids may have
decreased the rate of LH synthesis or possibly influenced GnRH synthesis
or release.

Serum androstenedione was maximal at 4 months and decreased at 5
months, while serum testosterone increased significantly at 5 months
and continuously to 9 months. Testosterone peaks following LH peaks
increased in size especially between 4 and 5 months, and this increase
is attributed to the transient increase in frequency of LH peaks. This
outcome is identical to results from Experiment 2.

Decreased androstenedione production between 4 and 5 months in_vi!g.
and in_vitrg_is indicative of increasing l7B-hydroxysteroid dehydrogenase
activity. Payne 3; a}, (1977) showed increasing activity of this enzyme
in male rat testes between 22 and 50 days of age. They suggested that
this enzyme is the major factor limiting testicular responsiveness to

LH and that the activity of this enzyme may be increased by FSH.

85

The absence of markedly increased serum testosterone production in.
vitrg_in response to LH (Figure 11) at puberty does not agree with the
marked increased in serum testosterone at 4 to 5 months of age in_vivg_
to LH. Furthermore, equivalent testosterone production ig_vitrg in
response to LH at all ages is surprising. Evidently, a population of
mature Leydig cells was present as early as 1 month. In contrast,
Hooker £E.El: (1970) in histological studies fOund that competent secre-
tory cells were not present in the interstitium until 4 months of age.
While testicular tissue responds to LH with greatly increased testo-
sterone production in_vitrg_as early as 1 month, very little testosterone
is released in response to LH in_vivg, Possibly blood flow to the
interstitium is inadequate before 4 to 5 months. Alternatively, inhibi-
tory factors in 1119 may have been removed in_vitrg_or the increase in
testicular to body weight ratio with age would give older bulls a
greater relative amount of testicular tissue to respond to LH. 0n the
other hand, testicular tissue from older bulls may not have an increased
response to LH in_vitrg_per mg testicular protein, because the relative
volume of the testes occupied by seminiferous tubules increases during
puberty (Macmillan and Hafs, 1968a), presumably reflecting a reduced
proportion of interstitial tissue.

Serum FSH concentration did not change significantly with age and
no distinct episodic FSH peaks were observed. These observations are
consistent between Experiments 2 and 3 and with results of Karg §t_§l,
(1976). Failure to observe changes in FSH is difficult to explain,
particularly in view of the known role of FSH during puberty in other
species (reviewed by Bartke gt_§l,, 1978) and the increase in serum FSH

that occurs in male rats prior to initiation of spermatogenesis

86
(Swerdloff et_al,, 1971; Dohler and Wuttke, 1975; Payne et_al:, 1977).
Aside from the possibility that FSH is of little importance in bulls,
perhaps FSH secretion need not change to affect spermatogenesis or
possibly changes in FSH were not detected in this study. These data
support the view that constant FSH levels in the presence of changes
in other pituitary hormone concentrations may be sufficient to initiate
testicular function.

Schams and Reinhardt (1974) reported no change in serum prolactin
with advancing age in bulls and heifers. In contrast, Leining gt_al,
(1976) and Davis gt_al: (1977) reported greater prolactin in serum of
heifers at 3 than at 1 month of age. In this study mean prolactin,
peak frequency and peak height increased at 4 to 5 months of age.

These changes resembled those for LH, but the function of increased
prolactin at this age is unknown. It may participate in increased
steroidogenesis as described by Bartke et_al, (1978) for rodents. How-
ever, increased prolactin may be a result of increased testicular
androgen production (Nolin gt_§l,, 1977).

Increased serum testosterone concentrations and decreased andro-
stenedione at 5 months in bulls may result from more frequent LH peaks
at 4 months. In addition, constant FSH levels and increasing prolactin
may aid in increasing testicular sensitivity to LH as suggested in other

species (Bartke EE.§l:’ 1978; Swerdloff gt_a1:, 1977).

4. Testicular Gonadotropin Binding

 

125I-HCG binding to testicular homogenates per ug DNA was greater
(P<.05) in tissue from 3- and 4-month-old than from 7- and 9-month-old

bulls (Figure 12). HCG binding per ug protein was greater at 1, 3 and

87

Figure 12.--Specific binding of 125I-HCG (top) and 125I-FSH (bottom)
to testicular tissue. Values are the means of triplicate
determinations of five animals at each age.

88

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TIW 9 11.1 9
I 7 TI. 7
L 5 I 5
I 4 .111- 4
I 3 T1 3
I TFII1I
- p . L . . p p p
m w 0 m 0 0 .al m 8 6 4 2 m
I 6 2
N x 0.333.500 00.0.: c N x 0:00: 0.500 8.06:
9 .I 9
M. h? o M H“?
.m 5 m .m 17 5
m I {IA—3 O B I 3
G If . b p _ A w . P . L .
mm w M“ ”w M m” n. .r m a. .0 .4 9. 0
5203 030.500 00.080 530:. 030.500 3.0:...
an”? 9
.7 7
T? 5 I 5
I 4. I 4
L 3 1 3
I I l I
p b b F . u . b _F p p . .
2 O O O
m. 3 5 A . o m 6 w w w 2 I o

<20 35.0000 00.06 a 4 z 0 91:00:00 3.0:...

Ago I mo)

89

4 months than at S, 7 and 9 months. These data are difficult to inter-
pret because testes contain a heterogeneous cell population and LH binds
specifically to Leydig cells (DeKretser §t_al:, 1971; Castro §£_al,,
1972). Thus changes in number and size of other cell types could alter
LH binding per pg DNA or protein in the absence of a change in the num-
ber of LH binding sites per Leydig cell. The decreased binding capacity
of testicular tissue after puberty was probably due to increased semini-
ferous tubular diameter and proliferation of germ cells that occur after
5 months in bulls (Macmillan, 1967), causing Leydig cells to represent
a smaller percent of the total cell population. Total 125I-HCG bound
per testis x 2 increased with age and was greater (P<.05) in 9-month-
old bulls than in l-month-old bulls (Figure 12). The increase in total
LH (HCG) binding sites per bull could be due to more receptor sites per
cell or an increase in the number of Leydig cells. Hooker (1970) found
an increase in the number and size of Leydig cells in bulls during
puberty. Thus, it is possible that both a greater number of cells and
an increased number of sites per cell could contribute to increased
125I-HCG binding/testis in older bulls. In contrast to these results,
several studies using rat testicular tissue found that tissue from
rats at 50 days or older bound more 125I-HCG or LH (per mg testis) than
tissue from 10-, 20- and 30-day-old rats (Frowein and Engel, 1975);
Thanki and Steinberger, 1976). The reason for the discrepancy between
changes in bovine and rat 125I-HCG binding with age is not understood.

Although these results do not clearly define changes in receptor
numbers per Leydig cell with advancing age, they do suggest that absence
of LH receptors is probably not the cause of low testicular response to

endogenous LH episodes in_vivo in prepubertal bulls.

9O

125I-FSH binding to testicular homogenates was greater in 1- and 3-
month-old bulls than 4-, 5-, 7- and 9-month-old bulls (P<.05) when ex-
pressed on a per pg DNA or protein basis (Figure 12). FSH binds speci-
fically to Sertoli cells (Castro et al., 1972; Means and Vaitukaitis,
1972; Steinberger et al., 1974), but loss of binding per ug of DNA or
protein during puberty did not result from a loss of Sertoli cells or
binding sites per Sertoli cell, but an increase in the other testicular
cellular components during puberty. Total binding per testis x 2 was
constant for months 1, 3 and 4, and then increased to 9 months (P<.05).
Since Sertoli cell populations remain stable and do not undergo mitotic
divisions after 2 weeks of age in rats (Steinberger and Steinberger,
1977), I suspect that increased 125I-FSH binding per testis is due to
increased binding per Sertoli cell. The relatively high concentration
of FSH binding sites in testicular homogenates from bulls before puberty
suggests that the ability of testicular tissue to bind FSH is not a
limiting factor to the initiation of spermatogenesis.

Affinity constants for binding of 125I-HCG and 125I-FSH to bovine
testicular tissue are in agreement with those found previously using
rat tissue (Table 9). Catt et al. (1972) and Means and Vaitukaitis
(1972) found affinities of 2.4 x 1010M’1 and .14 x 10911'1 for HCG and
FSH, respectively. I found no instances where affinities have been
shown to change with age or physiological state. However, there is a
trend in these data fer 7-month-old bull testes to have a higher affinity
for HCG and 1-month-old bulls to have a lower affinity for FSH. A
sufficient number of animals was not examined to estimate variability

within an age group. This question merits further investigation

91

Table 9. Affinity constants (KA) fer 1251-Hcc and 125I-an binding to

bovine testicular homogenates.

 

 

 

KA x 10914‘1
JEEL Animal
(M0) (ID) HCG FSH
1 6091 9.9 1.1
6092 -- 0.7
6104 -- 0,4
3 6046 8.3 3.3
4 6048 10.3 2.4
5 783 11.0 2.7
7 766 36.6 3.2

762

21.1 --

 

92
because this could be a mechanism whereby equivalent serum hormone con-

centrations in bulls of different ages could elicit different responses.

GENERAL DISCUSSION

The major hormonal change observed in these studies in bulls
during maturation was the pronounced increase in basal serum testo-
sterone concentrations and in peak testosterone concentrations fellow-
ing endogenous LH peaks. Increased androgen production is unquestion-
ably the most important factor in sexual maturation, and it is involved
in spermatogenesis, sex accessory gland function and secondary sex
characteristics (Steinberger, 1971). Episodic LH peaks were present
in l-month-old bulls, but they did not elicit increases in testosterone
secretion. By 4 months episodic LH peaks were followed by androstene-
dione peaks, but by 5 months the number and size of androstenedione
peaks was reduced. Testosterone peaks followed LH episodes at 5 months
and these peaks increased in size through 10 months of age. The hormonal
requirements for increased androgen production are not known, but in-
creased frequency of LH peaks at 4 months could stimulate differentia-
tion of precursor cells into Leydig cells (Chemes et_al,, 1976) and
maturation of the steroidogenic capacity of Leydig cells (Dufau et_§l:,
1976). Increased frequency of LH peaks before puberty could be the
major trigger to the onset of puberty.

LH is released in distinct episodes in bulls but the physiological
importance of this release pattern is unknown. Episodic release may be
biologically the most efficient way to deliver serum hormone to a target
organ. Temporarily elevated hormone level would load testicular LH

93

94
receptors and prolonged half-life of LH on receptors (Mendelson §t_al,,
1975) would provide constant hormonal stimulation long after serum
hormone levels had declined. In addition, episodic release is possibly
the best method to overcome "testicular’desensitization"(Hsueh gt_§l,,
1977) in which testicular response to LH is reduced immediately follow-
ing LH exposure.

The mechanism responsible for increased frequency of LH peaks is
not known. It has been suggested that the increase in serum gonadotro-
pin concentrations that occurs in male rats before puberty is due to
maturation of the hypothalamus and decreased sensitivity of the hypo—
thalamus to circulating steroids (Ramirez and McCann, 1965). Increased
frequency of LH peaks before puberty in bulls could result from a
similar mechanism, but the fact that bulls, steers and SS bulls all
had increased LH peak frequency at 4 months irrespective of circulating
steroid concentrations suggests maturation of the hypothalamus and in-
creased LH production is independent of alterations in steroid negative
feedback.

The limited responsiveness of the testis to LH in_vivg_before
puberty seems not due to a lack of LH binding sites. Although older
bulls had a greater LH binding capacity/testis; l-, 3- and 4-month-old
bulls had a higher concentration of binding sites. Also, testes from
1-month-old bulls responded ig_vitrg to LH with elevated testosterone
production, indicating that adequate numbers of functional LH binding
sites and some mature Leydig cells were present.

By contrast to the in_vitro testicular response to LH, in vivo

 

endogenous LH peaks are followed by testosterone release in bulls only

after 4 months of age. In_vitro, testes responded to LH at all ages.

95
Reasons for this discrepancy are unknown. Testicular vasculature early
in life in bulls may limit access of gonadotropins and other substances
to the interstitium, or Leydig cell products may not have free access to
peripheral circulation. In addition, testicular substances (e.g.,
estrogens) may inhibit production of testosterone in response to LH
in_vi!9, but these substances may be diluted in_vitrg_to allow testo-
sterone secretion. A greater volume of testicular tissue is present
relative to body size in older bulls. More steriodogenic tissue is
available to respond to LH and could result in greater elevation of
peripheral testosterone concentrations. In_vi££g_results indicate that
so far as testosterone secretion is concerned, a mature population of
Leydig cells is present as early as 1 month of age in bulls.

Serum FSH concentrations did not differ between bulls of different
ages. If FSH is involved in the induction of spermatogenesis as in
other species, constant serum concentrations of FSH probably perfbrm
this function in bulls. Possibly a change in the production of other
compounds (i.e., steroids or pituitary hormones) with age causes con-
stant serum FSH concentrations to become stimulatory to Sertoli cells
and spermatogenesis. Unlike LH, FSH was not released episodically in
bulls, steers or SS bulls. Different negative feedback on FSH than LH
secretion may partially account for this difference. Also, a longer
half-life for FSH in serum (Van Hall §t_al,, 1971) might make FSH
episodes more difficult to detect.

Serum FSH concentrations were greater in steers than in bulls or
SS bulls. Lack of elevated FSH concentrations in SS bulls was sur-
prising, especially since histological preparations indicated that

spermatogenesis was halted. The only cell type abundant in the

96

seminiferous tubules was the Sertoli cell. This suggests that Sertoli
cells are a major source of inhibitory compounds that influence FSH
secretion. This agrees with work by Steinberger and Steinberger (1977)
who demonstrated that media from Sertoli cell cultures inhibited FSH
release when placed in pituitary cell cultures.

To establish the function of pituitary hormones other than LH and
FSH in the onset of puberty will require further study. Growth hormones
and prolactin concentrations change during puberty in bulls and roles
for these hormones in testicular function have been demonstrated in
other species. A complex of pituitary hormones acting concurrently or
sequentially may be required to initiate full steroidogenesis and

spermatogenesis.

SUMMARY AND CONCLUSIONS

The objective of these experiments were to describe serum hormone
changes in bulls from birth until after puberty; to examine negative
feedback relationships between the testis and hypothalamus/pituitary
using bulls, steers and bulls in which the scrotum had been shortened
(SS bulls); and to examine testicular changes during the peripubertal
period.

The first experiment was designed to determine serum hormone con-
centrations and the importance of negative feedback in prepubertal and
pubertal bulls. Eight prepubertal and eight pubertal bulls were assigned
four each to be left intact and four to be castrated 2 weeks before the
experiment. Serum growth hormone, prolactin, testosterone and luteiniz-
ing hormone concentrations were determined from a 9-hr sampling period.
Steers had higher mean serum LH concentrations and a greater frequency
of LH peaks per 9 hr (P<.05) than bulls, but serum LH did not differ
between ages. The frequency of testosterone peaks and mean testosterone
concentrations were greater in pubertal than prepubertal bulls (P=.lO).
Prolactin concentrations did not differ between ages or castrational
state, but prolactin in older animals tended to be higher and more
variable. Prepubertal bulls had greater mean serum growth hormone
concentrations and a greater frequency of growth hormone peaks (P<.O7)

than pubertal bulls, steers and prepubertal steers.

97

98

The second experiment was designed to describe hormone patterns
during development in normal bulls, bulls with altered negative feed-
back (SS bulls) and bulls with no negative feedback (steers). Two-week-
old bulls were assigned four each to be bulls, steers or SS bulls.
Serum samples were collected during a 24-hr sampling period, monthly
for 10 months, and were assayed for LH, FSH, testosterone and androstene-
dione. Bulls, steers and SS bulls grew at similar rates. Bull reached
puberty by 10 months; indicated by the presence of spermatozoa in
epididymidal homogenates and active spermatogenesis apparent histologi-
cally in the seminiferous tubules. SS bulls had no spermatozoa in the
epididymides, and only Sertoli cells and scattered spermatogonia were
seen in the seminiferous tubules.

Mean LH concentrations were greater in steers than bulls or SS
bulls from 3 to 10 months, while SS bulls had greater LH concentrations
than bulls at S and 6 months (P<.05). In steers and SS bulls, average
LH concentrations increased from 1 to 3 months (P<.05) and then re-
mained constant. In bulls, LH was greater at 4 months than at 1 month,
but not different among other months. Frequency of LH peaks increased
from 1 month to 4 months in bulls, steers and SS bulls and then de-
clined in bulls and SS bulls. Steers had a greater frequency of LH
peaks than bulls from 2 to 10 months, while SS bulls had a greater fre-
quency of LH episodic peaks than bulls at 3 and 4 months.

Serum testosterone concentrations were less than 1 ng/ml in bulls
from 1 to 4 months and increased (P<.05) abruptly at 5 months. In SS
bulls, testosterone increased from 5 to 6 months, later than in bulls.
Increased frequency and height of testosterone peaks and increased

baseline concentrations each contributed to the increase in mean

99
testosterone concentrations. Small testosterone peaks (<1 ng/ml) fol-
lowed episodic LH peaks in bulls from 1 to 3 months and SS bulls from
1 to 5 months, but after this age the amplitude of testosterone peaks
increased to 2 ng/ml and continued to increase to 5 ng/ml by 10 months.
Average androstenedione concentrations did not differ between bulls and
SS bulls; it increased transiently at 4 months in both treatments.

Serum FSH concentrations increased from 1 to 2 months in steers,
did not change through 10 months and were greater in steers than in
bulls or SS bulls from 2 to 10 months. Serum FSH did not change with
age in normal of SS bulls, and did not differ between these two treat-
ments at any month.

I conclude from Experiment 2 that increased mean LH concentrations
and frequency of LH episodic peaks at 4 months probably stimulated the
increase in testosterone secretion that occurred by 5 months. Slightly
greater LH concentrations in SS bulls during puberty indicates reduced
negative feedback from 55 bull testes. In addition, comparable FSH
concentrations in bulls and SS bulls suggests that Sertoli cells are
the major source of compounds that feedback on FSH secretion.

The major objective of Experiment 3 was to examine serum hormone
changes due to age, independent of season and photoperiod. A second
objective was to examine in_vitrg_changes in testicular response to LH
and in_vitrg_gonadotropin binding in bulls of different ages. Blood
samples were collected from five bulls each at l, 3, 4, 5, 7 and 9
months of age for a 24-hr period on a single day. On the following day
testes were removed for in 33239 studies.

Serum LH concentrations increased from 1.4 ng/ml in l-month-old

bulls to <2 ng/ml at 3, 4 and 5 months and then decreased to 1.4 ng/ml

100
at 7 and 9 months. The frequency of LH peaks increased from 4.4 at 1
month to 11.8 at 4 months and then decreased to 6 months. LH peak
height was constant through 5 months, and then decreased to 7 months.
Increased LH secretion preceded increased testosterone production by lg
month. Testosterone was <1 ng/ml at l, 3 and 4 months and then in-
creased markedly at 5, 7 and 9 months. Frequency and peak height
(ng/ml) increased linearly from .4 and .9 at 1 month to 3.0 and 13.2
at 9 months, respectively. I suggest that a major factor responsible
fer turning on testosterone production at 5 months is the increased
frequency of LH episodic peaks in bulls at 4 months of age.

Androstenedione tended to be higher in 4-month-old bulls. FSH did
not change with age and did not have distinct episodic releases similar
to those observed fer LH. The absence of elevated serum FSH concentra-
tions before puberty is surprising. Changing FSH concentrations appear
not to be a requirement for the onset of puberty in bulls. Mean pro-
lactin and prolactin peak height increased from 1 to 5 months while
peak frequency increased from 1 to 4 months. Increased prolactin
concentrations around puberty may stimulate steroidogenesis, or could
be stimulated by rising androgen concentrations.

Testosterone concentration in testicular tissue increased linearly
in bulls from 1 to 9 months. Incubation in_vi£rg_caused a significant
increase in testosterone production. Over all treatments, 4-, 5- and
7-month-old bulls produced more testosterone than 1- and 3-month-old
bulls. LH caused equivalent testosterone production from testicular
tissue of bulls of all ages. LH caused androstenedione release into
media, but tissue from l-,3- and 4-month-old bulls produced significantly

more androstenedione than tissue from 5-, 7- and 9-month-old bulls.

101

Similar in_vi££g response to LH in testicular tissue from bulls
at all ages was unexpected and differs with in_vivg_findings where only
minimal amounts of testosterone are produced in response to endogenous
LH episodic peaks before 5 months of age. In_vitrg_results suggest
however, that mature, responsive Leydig cells are present in bulls as
early as 1 month of age. Decreased androstenedione production in_vivg_
and in_vi£rg_between 4 and 5 months suggests maturation of l7B-hydroxy-
steroid dehydrogenase system.

125I-HCG binding to testes homogenates per ug DNA was greater in
tissue from 3- and 4-month-old bulls than from 7- and 9-month-old bulls.
Binding per ug protein was greater at l, 3 and 4 than at 5, 7 and 9
months. Total HCG bound per testis increased from 1 to 9 months. FSH
binding was greater in l- and 3-month-old bulls than in 4-, 5-, 7- and
9-month-old bulls when expressed on a per ug DNA or protein basis.
Total FSH binding per testis was constant at l, 3 and 4 months, and
then increased to 9 months.

Gonadotropin binding studies demonstrate increased binding per
testis in older bulls, but higher concentration of sites in younger
bulls. I conclude that limited testicular responsiveness to gonadotro-
pins in_vivg_is not due to absence of binding sites. Binding studies
are difficult to interpret without being able to equate changes in
binding with changes in a specific cell population (i.e., Leydig cells).

Results of these experiments prove that the major hormonal change
during puberty is increased testosterone production. Increased andro-
stenedione at 4 months and a decrease at 5 months indicates that
maturation of testicular steroidogenic enzymes takes place to favor

testosterone production. I conclude that increased frequency of LH

102

peaks at 4 months probably induces testicular maturation and increases
testosterone production by 5 months. Other pituitary hormones may
participate but the role of FSH is difficult to assess because the
concentration in serum does not change during puberty.

FSH and LH concentrations were greater in steers, demonstrating
the importance of negative feedback of testicular products on gonado-
tropin secretion as early as 2 months of age. LH in SS bulls was only
slightly greater than that in normal bulls showing a change in negative
feedback in SS bulls. FSH concentrations in SS bulls were not different

than bulls, suggesting that Sertoli cells may produce inhibin in bulls.

BIBLIOGRAPHY

BIBLIOGRAPHY

Abdel-Raouf, M. 1960. The postnatal development of the reproductive
organs in bulls with special reference to puberty. Acta
Endocrinol., Copenhagen, Suppl. 49.

Abdel-Raouf, M. 1961. The proliferation of germ cells in the testes
of bull calves and young bulls. Acta Vet. Scand. 2:22.

Almquist, J. 0. and R. P. Amann. 1961. Reproductive capacity of dairy
bulls. II Gonadal and extragonadal sperm reserves as determined
by direct counts and depletion trials; dimensions and weight of
genitalia. J. Dairy Sci. 44:1668.

Amann, R. P. 1970. Sperm production rates. IE_A.D. Johnson, W.R.
Comes and N.L. VanDemark (Ed.) The Testis. Vol. 1. Development,
anatomy, and physiology. Academic Press, New York and London.
p. 433-483.

Amann, R. P., J. O. Almquist and J. T. Lambiase. 1966. Seasonal
cycles in bull semen characteristics. J. Anim. Sci. 25:916.

Amann, R. P. and V. K. Ganjam. 1976. Steroid production by the bovine
testis and steroid transfer across the pampiniform plexus. Biol.
Reprod. 15:695.

Aragona, C., H. C. Bohnet and H. G. Friesen. 1977. Localization of
prolactin binding in prostate and testis: The role of serum
prolactin concentration on the testicular LH receptor. Acta
Endocrinol. 84:402.

Ascoli, M. and D. Puett. 1978. Fate of the receptor-bound human
choriogonadotropin in Leydig tumor cells. Sixtieth Annu.
Meeting Endocrine Soc. (Abstr. 338).

Ashdown, R. R. 1962. Development of penis and sheath in young dairy
bulls, and its relationship to age and body weight. J. Agr. Sci.
58:65.

Atkinson, L. E., A. N. Bhattacharya, S. E. Monroe, D. J. Dierschke and

E. Knobil. 1970. Effects of gonadectomy on plasma LH concentra-
tion in the rhesus monkey. Endocrinol. 87:847.

103

104

Attal, J. and M. Courot. 1963. Développement testiculaire et
etablissement de la spermatogenése chez le taureau. Ann. Biol.
Animale, Biochim, Biophys. 3:219.

Barber, K. A. and J. 0. Almquist. 1975. Growth and feed efficiency
and their relationship to puberal traits of Charolais bulls. J.
Anim. Sci. 40:288.

Barden, N. and F. Labrie. 1973. Cyclic adenosine 3',S'~monophosphate
dependent phosphorylation of ribosomal proteins from bovine
anterior pituitary gland. Biochem. 12:3096.

Bardin, C. W., L. P. Bullock, R. J. Sherins, I. Mowszowicz and W. R.
Blackburn. 1973. Part II. Androgen metabolism and mechanism of
action in male pseudohermaphroditism: a study of testicular
feminization. Rec. Prog. Horm. Res. 29:65.

Barnes, M. A., J. V. Longnecker, J. W. Riesen and C. 0. Woody. 1976.
Plasma LH and testosterone in Holstein bulls. J. Anim. Sci. 43.
(Abstr. 246).

Bartke, A. 1969. Prolactin changes cholesterol stores in the mouse
testis. Nature 224:700.

Bartke, A. 1971. Effects of prolactin on spermatogenesis in hypo-
physectomized mice. J. Endocrinol. 49:311.

Bartke, A. 1976. Pituitary-testis relationships. lfl.P-O- Hubinont
(Ed.) Sperm Action; Progress in Reproductive Biology. Vol. I.
Karger, Basel. p. 136-152.

Bartke, A. and S. Dalterio. 1976. Effects of prolactin on sensiti-
vity of the testis to LH. Biol. Reprod. 15:90.

Bartke, A., A. A. Hafiez, F. J. Bex and S. Dalterio. 1978. Hormonal
interactions in regulations of androgen secretion. Biol. Reprod.
18:44.

Bartke, A., N. Musto, B. V. Caldwell and H. R. Behrman. 1973a. Effects
of a cholesterol esterase inhibitor and of prostaglandin F20 on
testis cholesterol and on plasma testosterone in mice.
Prostaglandins 3:97.

Bartke, A., R. E. Steele, N. Musto and B. V. Caldwell. 1973b. Fluctua-
tions in plasma testosterone levels in adult male rats and mice.
Endocrinol. 92:1223.

Bartke, A., K. I. H. Williams and S. Dalterio. 1977. Effects of
estrogens on testicular testosterone production in_vitro. Biol.
Reprod. 17:645.

105

Bell, J. B. G. and G. P. Vinson. 1975. Secretion of testosterone by
components of the rat testis in_vitro, and the effects of
gonadotropin stimulation. J. Endocrinol. 67:11P (Abstr.).

Bell, J. B. G., G. P. Vinson, D. J. Hopkin and D. Lacy. 1968. Path-
ways for androgen biosynthesis from (70-3H) pregnenolone and

(4-1“C) progesterone by rat testis interstitium in vitro. Biochem.
Biophys. Acta 164:412.

Bex, F. J. and A. Bartke. 1977a. Testicular LH binding in the hamster:
modification by photoperiod and prolactin. Endocrinol. 100:1223.

Bex, F. J. and A. Bartke. 1977b. Effects of prolactin (PRL) and
growth hormone (GH) on testicular LH receptor levels in the
hamster. Fifty nineth Annu. Meeting Endocrine Soc. (Abstr. 224).

Bohnet, H. G. and H. G. Friesen. 1976. Effect of prolactin and growth
hormone of prolactin and LH receptors in the dwarf mouse. J.
Reprod. Fert. 48:307.

Bolt, D. J. 1971. Changes in the concentration of luteinizing hormone
in plasma of rams fellowing administration of oestradiol, proges-
terone or testosterone. J. Reprod. Fert. 24:435.

Bourne, R. A. and H. A. Tucker. 1975. Serum prolactin and LH responses
to photoperiod in bull calves. Endocrinol. 97:473.

Bratton, R. W., S. D. Musgrave, H. 0. Dunn and R. H. Foote. 1959.
Causes and prevention of reproductive failures in dairy cattle.
11. Influence of underfeeding and overfeeding from birth to 80
weeks of age on growth, sexual development, and semen production
of Holstein bulls. Cornell Univ. Agr. Sta. Bull. 940.

Bratton, R. W., S. D. Musgrave, H. O. Dunn and R. H. Foote. 1961.
Causes and prevention of reproductive failures in dairy cattle.
III. Influence of underfeeding and overfeeding from birth through
80 weeks of age on growth, sexual development semen production,
and fertility of Holstein bulls. Cornell Univ. Agr. Exp. Sta.
Bull. 964.

Braunstein, G. D. and R. S. Swerdloff. 1977. Effect of aqueous ex-
tracts of bull and rat testicles on serum FSH and LH in the
acutely castrate male rat. In_P. Troen and H.R. Nankin (Ed.)
The Testis in Normal and Infertile Men. Raven Press, New York.
p. 281-291.

Burton, K. 1956. A study of the conditions and mechanism of the
diphenylamine reaction for the colorimetric estimation of
deoxyribonucleic acid. Biochem. J. 62:315.

Casady, R. B., R. M. Myers and J. E. Legates. 1953. The effect of
exposure to high ambient temperature on spermatogenesis in the
dairy bull. J. Dairy Sci. 36:14.

106

Castro, A. E., A. Alonso and R. E. Mancini. 1972. Localization of
folicle-stimulating and luteinizing hormones in the rat testis
using immunohistological tests. J. Endocrinol. 52:129.

Catt, K. J. 1977. Data presented at the fourth Testis Workshop,
Toronto, Ontario, Can.

Catt, K. J. and M. L. Dufau. 1973. Spare gonadotrOpin receptors in
rat testis. Nature New Biol. 244:219.

Catt, K. J. and M. L. Dufau. 1976. Basic concepts of the mechanism of
action of peptide hormones. Biol. Reprod. 14:1.

Catt, K. J., T. Tsuruhara and M. L. Dufau. 1972. Gonadotropin binding
sites of the rat testes. Biochim. Biophys. Acta. 279:194.

Charreau, E. H., A. Attramadal, P. A. Torjesen, K. Purvis, R. Calandra
and V. Hansson. 1977. Prolactin binding in rat testis: specific
receptors in interstitial cells. Mol. Cell. Endocrinol. 6:303.

Chemes, H. E., M. A. Rivarola and C. Bergada. 1976. Effect of HCG on
the interstitial cells and androgen production in the immature
rat testis. J. Reprod. Fert. 46:279.

Chen, T. T., J. H. Abel, Jr., M. C. McClellan, H. R. Sawyer, M. A.
Diekman and G. D. Niswender. 1977a. Localization of gonadotrophic
hormones in lysosomes of ovine luteal cells. Cytobiologie 14:412.

Chen, T. T., D. B. Endres, J. H. Abel, Jr. and G. D. Niswender. 1978.
Binding and degradation of human chorionic gonadotropin by ovine
luteal cells. Sixtieth Annu. Meeting Endocrine Soc. (Abstr. 337).

Chen, Y.-D. I., A. H. Payne and R. P. Kelch. 1976. FSH stimulation of
Leydig cell fUnction in the hypophysectomized immature rat. Proc.
Soc. Exp. Biol. Med. 153:473.

Chen, Y.-D. 1., M. J. Shaw and A. H. Payne. 1977b. Steroid and FSH
action on LH-receptors and LH-sensitive testicular responsiveness
during sexual maturation in the rat. Mol. Cell. Endocrinol. 8:291.

Cheng, K. W. 1976. Purification and properties of bovine pituitary
follitropin. Biochem. J. 159:651.

Cheng, K. W. 1978. Development and characterization of a homologous
radioimmunoassay for bovine follicle-stimulating hormone. J.
Endocrinol. 77:185.

Christensen, A. K. and N. R. Mason. 1965. Comparative ability of
seminiferous tubules and interstitial tissue of rat testes to
synthesize androgens from progesterone-4-1“C in_vitro. Endocrinol.
76:646.

107

Convey, E. M., W. E. Beal, B. E. Seguin, K. J. Tannen and Y. C. Lin.
1976. Gonadotropin releasing hormone induced luteinizing hormone
release after prostaglandin an in heifers. Proc. Soc. Exp. Biol.
Med. 151:84.

Cooke, B. A. 1976. Mechanism of LH and FSH action on the testis. In_
J.E. Dumont gt_al, (Ed.) Eukaryotic Cell Function and Growth.
Plenum Pub. Corp. New York. p. 653-666.

Cooke, B. A., F. H. A. Janszen, F. H. de Jong, F. F. G. Rommerts and
H. J. van der Molen. 1976. Comparison of steroid production and
trophic hormone control mechanisms in testis Leydig cells and
seminiferous tubules. Excerpta Medica Internatl. Congress Series
No. 402. Endocrinol. 1:429.

Cooke, B. A., F. F. G. Rommerts, J. W. C. M. van der Kemp and H. J. van
der Molen. 1974. Effects of luteinizing hormone, fellicle
stimulating hormone, prostaglandin E1 and other hormones on
adenosine-3':S'-cyclic monophosphate and testosterone production
in rat testis tissues. Mol. Cell. Endocrinol. 1:99.

Crim, L. W. and I. I. Geschwind. 1972. Patterns of FSH and LH secre-
tion in the developing ram: the influence of castration and

replacement therapy with testosterone propionate. Biol. Reprod.
7:47.

Danutra, V., M. E. Harper, A. R. Boyns, E. N. Cole, 8. G. Brownsey and
K. J. Griffiths. 1973. The effects of certain stilboestrol
analogues on plasma prolactin and testosterone in the rat. J.
Endocrinol. 57:207.

Davis, S. L., R. G. Sasser, D. L. Thacker and R. H. Ross. 1977. Growth
rate and secretion of pituitary hormones in relation to age and
chronic treatment with thyrotrOpin-releasing hormone in prepubertal
dairy heifers. Endocrinol. 100:1394.

DeAlba, J. and S. Riera. 1966. Sexual maturity and spermatogenesis
under heat stress in the bovine. Anim. Prod. 8:137.

DeKretser, D. M., K. J. Catt and C. A. Paulsen. 1971. Studies on the
in_vitro testicular binding of iodinated luteinizing hormone in
rats. Endocrinol. 88:332.

Dohler, K. D. and W. Wuttke. 1975. Changes with age in levels of serum
gonadotropins, prolactin, and gonadal steroids in prepubertal male
and female rats. Endocrinol. 97:898.

Donovan, B. T. and J. J. Van der Werff ten Bosch. 1965. Physiology of
Puberty. Edward Arnold Ltd., London.

Dorrington, J. H. and D. T. Armstrong. 1975. Follicle-stimulating hor-
mone stimulates estradiol-17B synthesis in cultured Sertoli cells.
Proc. Natl. Acad. Sci. U.S.A. 72:2677.

108

Dorrington, J. H. and I. 8. Fritz. 1975. Androgen synthesis and metabo-
lism by preparations from the seminiferous tubule of the rat testis.
In_F.S. French, V. Hansson, E.M. Ritzen and S.N. Nayfeh (Ed.)
Hormonal Regulation of Spermatogenesis. Plenum Press, New York.

p. 37-52.

Dorrington, J. H., I. B. Fritz and D. 1. Armstrong. 1978. Control of
testicular estrogen synthesis. Biol. Reprod. 18:55.

Dorrington, J. H., N. F. Roller and I. B. Fritz. 1974. The effects of
FSH on cell preparations from the rat testis. 12_M. Dufau and A.R.
Means (Ed.) Hormone Binding and Target Cell Activation of the
Testis. Vol. I. Plenum Press, New York. p. 237-241.

Dufau, M. L., E. Podesta and K. J. Catt. 1975. Physical characteristics
of gonadotropin receptor—hormone complexes fermed in_vivo and in_
vitro. Proc. Natl. Acad. Sci. U.S.A. 72:1272.

 

Dufau, M., E. Podesta, T. Tsuruhara, A. Hsueh, J. Harwood and K. Catt.
1976. Mechanisms of luteinizing hormone action in the testis.
Exerpta Medica Internatl. Congress Series No. 402. Endocrinol.
1:441.

Dussault, J. H., P. Walker, J. D. Dubois and F. Labrie. 1977a. The
development of the hypothalamo-pituitary axis in the neonatal rat:
sexual maturation in male and female rats as assessed by hypothala-
mic LHRH and pituitary and serum LH and FSH concentrations. Can. J.
Physiol. Parmacol. 55:1091.

Dym, M. and H. G. Madhwa Raj. 1977. Response of adult rat Sertoli cells
and Leydig cells to depletion of luteinizing hormone and testosterone.
Biol. Reprod. 17:676.

Folman, Y., N. Ahmad, J. G. Sowell and K. B. Eik-Nes. 1973. Formation
in_vitro of Sa-dihydrotestosterone and other Sa-reduced metabolites
of 3H-testosterone by the seminiferous tubules and interstitial
tissue from immature and mature rat testis. Endocrinol. 92:41.

Foote, R. H. 1969. Physiological aspects of artificial insemination.
12_H.H. Cole and P.T. Cupps (Ed.) Reproduction in Domestic Animals
Second Ed. Academic Press, New York and London. p. 313-353.

Fossland, R. G. and A. B. Schutze. 1961. A histological study of the
postnatal development of the bovine testis. Neb. Agr. Exp. Sta.
Res. Bull. 199.

Foster, D. L., I. H. Mickelson, K. D. Ryan, G. A. Coon, R. A. Drongowski
and J. A. Holt. 1978. Ontogeny of pulsatile luteinizing hormone
and testosterone secretion in male lambs. Endocrinol. 102:1137.

Franchimont, P., S. Chari and A. Demoulin. 1975. Hypothalamus—pituitary-
testis interaction. J. Reprod. Fert. 44:335.

109

Franchimont, P., S. Chari, M. T. Hazee-Hagelstein, M. L. Debruche and
S. Duraiswami. 1977. Evidence for the existence of inhibin. In_
P. Troen and H.R. Nankin (Ed.) The Testis in Normal and Infertile
Men. Raven Press, New York. p. 253-270.

French, F. S. and E. M. Ritzen. 1973. Androgen-binding protein in
efferent duct fluid of rat testis. J. Reprod. Fert. 32:479.

Fritz, I. B. 1977. Data presented at fourth Testis Workshop, Toronto,
Ontario, Can.

Frowein, J. and W. Engel. 1975. Binding of human chorionic gonadotro-
phin by rat testis: effect of sexual maturation, cryptorchidism
and hypophysectomy. J. Endocrinol. 64:59.

Galena, H. J., A. K. Pilla and C. A. Terner. 1974. Progesterone and
androgen receptors in nonflagellate germ cells of the rat testis.
J. Endocrinol. 63:223.

Gay, V. L. and N. W. Dever. 1971. Effects of testosterone propionate
and estradiol-benzoate -- alone or in combination -- on serum LH
and FSH in orchidectomized rats. Endocrinol. 89:161.

Gay, V. L. and A. R. Midgley. 1969. Response of the adult rat to
orchidectomy and ovariectomy as determined by LH radioimmunoassay.
Endocrinol. 84:1359.

Gill, J. L. 1978. Design and analysis of experiments. Iowa State
University Press. Ames, Iowa.

Gill, J. L. and H. D. Hafs. 1971. Analysis of repeated measurements
of animals. J. Anim. Sci. 33:331.

Gombe, S., W. C. Hall, K. McEntee, W. Hansel and B. W. Pickett. 1973.
Regulation of blood levels of LH in bulls: influence of age,
breed, sexual stimulation and temporal fluctuation. J. Reprod.
Fert. 35:493.

Greenwood5 F. C., W. M. Hunter and J. S. Glover. 1963. The preparation
of 1 1I-labeled human growth hormone of high specific radioactivity.
Biochem. J. 89:114.

Greep, R. 0., H. L. Fevold and F. L. Hisaw. 1936. Effects of two hypo-
physeal gonadotropic hormones on the reproductive system of the
male rat. Anat. Rec. 65:261.

Hafiez, A. A., C. W. Lloyd and A. Bartke. 1972. The role of prolactin
in the regulation of testes function. The effects of prolactin
and luteinizing hormone on the plasma levels of testosterone and
androstenedione in hypophysectomized rats. J. Endocrinol. 52:327.

Hafiez, A. A., J. E. Philpott and A. Bartke. 1971. The role of prolactin
in the regulation of testicular function: The effect of prolactin

110

and luteinizing hormone on 38-hydroxysteroid dehydrogenase activity
in the testes of mice and rats. J. Endocrinol. 50:619.

Hansson, V., R. Calandra, K. Purvis, M. Ritzen and F. S. French. 1976.
Hormonal regulation of spermatogenesis. Vit. and Hormones. 34:187.

Hansson, V., W. S. McLean, A. A. Smith, D. J. Tindall, S. C. Weddington,
S. N. Nayfeh, F. S. French and E. M. Ritzen. 1974. Androgen
receptors in rat testis. Steroids 23:823.

Haynes, N. B., H. D. Hafs and J. G. Manns. 1977a. Effect of chronic
administration of gonadotrophin releasing hormone and thyrotrOphin
releasing hormone to pubertal bulls on plasma luteinizing hormone,
prolactin and testosterone concentrations, the number of epididy-
mal sperm and body weight. J. Endocrinol. 73:227.

Haynes, N. B., T. E. Kiser, H. D. Hafs and J. D. Marks. 1977b.
Prostaglandin an overcomes blockade of episodic LH secretion with

testosterone, melengestrol acetate or aspirin in bulls. Biol.
Reprod. 17:723.

Haynes, N. B., T. E. Kiser, H. D. Hafs, T. D. Carruthers, W. D. Oxender
and M. S. McCarthy. 1977c. Effect of intracarotid infusion of
prostaglandin ancniplasma luteinizing hormone, testosterone and
glucocorticoid concentrations in bulls. J. Anim. Sci. 45:1102.

Hooker, C. W. 1944. The postnatal history and function of the inter-
stitial cells of the testis of the bull. Amer. J. Anat. 74:1.

Hooker, C. W. 1970. In A.D. Johnson, W.R. Gomes and N.L. Van Demark
(Ed.) The Testis, Vol. 1. Development, Anatomy and Physiology.
Academic Press, New York. p. 483-550.

Hsueh, A. J. W., M. L. Dufau and K. J. Catt. 1977. Gonadotropin-induced
regulation of luteinizing hormone receptors and desensitization of
testicular 3':5'-cyc1ic AMP and testosterone responses. Proc.

Natl. Acad. Sci. U.S.A. 74:592.

Inano, H., Y. Hori and B. Tamaoki. 1967. Effect of age on testicular
enzymes related to steroid bioconversion. Ciba Found. Collo.
Endocrinol. 16:105.

Ireland, J. J. and J. S. Richards. 1978. Acute effects of estradiol
and follicle-stimulating hormone on specific binding of human
(1251) iodo-follicle-stimulating hormone to rat ovarian granulosa
cells in_vivo and in_vitro. Endocrinol. 102:876.

Karg, H., T. Gimenez, M. Hartl, B. Hoffmann, E. Schallenberger and D.
Schams. 1976. Testosterone, luteinizing hormone (LH) and follicle
stimulating hormone (FSH) in pheripheral plasma of bulls: levels
from birth through puberty and short term variations. Zbl. Vet.
Med. 23:793. -

111

Karg, H. and D. Schams. 1974. Prolactin release in cattle. J. Reprod.
Fert. 39:463.

Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high
osmolality for use in electron microscopy. J. Cell Biol. 27:137a.

Katongole, C. B., F. Naftolin and R. V. Short. 1971. Relationship
between blood levels of luteinizing hormone and testosterone in
bulls, and the effects of sexual stimulation. J. Endocrinol.
50:457.

Kesler, D. J. and H. A. Garverick. 1977. Luteinizing hormone and
testosterone concentrations in plasma of bull calves treated with
gonadotropin releasing hormone. J. Dairy Sci. 60:632.

Killian, G. J. and R. P. Amann. 1972. Reproductive capacity of dairy
bulls. IX. Changes in reproductive organ weights and semen charac-
teristics of Holstein bulls during the first thirty weeks after
puberty. J. Dairy Sci. 55:1631.

Kirton, K. T., C. Desjardins and H. D. Hafs. 1967. Distribution of
sperm in male rabbits after various ejaculation frequencies. Anat.
Rec. 158:287.

Kiser, T. E., H. D. Hafs and W. D. Oxender. 1976. Increased blood LH
and testosterone after subcutaneous injection or intravenous infu-
sion of prostaglandin F20 in bulls. Prostaglandins 11:545.

Kiser, T. E., R. A. Milvae, H. D. Hafs, W. D. Oxender and T. M. Louis.
1978. Comparison of testosterone and androstenedione secretion
in bulls given prostaglandin an or luteinizing hormone. J.
Anim. Sci. 39:214 (Abstr.).

Kiser, T. E., K. Mongkonpunya, Y. C. Lin and W. D. Oxender. 1974. In_
vitro androgen biosynthesis in bull testes after LH. J. Anim.

Sci. 39:214 (Abstr.).

Koprowski, J. A. and H. A. Tucker. 1971. Failure of oxytocin to
initiate prolactin or luteinizing hormone release in lactating
dairy cows. J. Dairy Sci. 54:1675.

Koprowski, J. A., H. A. Tucker and E. M. Convey. 1972. Prolactin and
growth hormone circadian periodicity in lactating cows. Proc. Soc.
Exp. Biol. Med. 140:1012.

Kotite, N. J., S. N. Nayfeh and F. 8. French. 1978. FSH and androgen
regulation of Sertoli cell function in the immature rat. Biol.
Reprod. 18:65.

LaCroix, A., D. H. Garnier and J. Pelletier. 1977. Temporal fluctua-
tions of plasma LH and testosterone in Charolais bull calves during
the first year of life. Ann. Biol. Anim. Biochim. Biophys. 17:1013.

112

Lacy, D. 1973. Androgen dependency of spermatogenesis and the physio-
logical significance of steroid metabolism in vitro by seminiferous
tubules. In_V. James, M. Serio and L. Martini (Ed.) The Endocrine
Function of the Human Testis. Vol. 1. Academic Press, New York.

p. 493-532.

Lee, V. W. K., I. A. Cumming, D. M. DeKretser, J. K. Findlay, 8. Hudson
and E. J. Keogh. 1976. Regulation of gonadotropin secretion in
rams from birth to sexual maturity. 1. Plasma LH, FSH and testo-
sterone levels. J. Reprod. Fert. 46:1.

Lee, V. W. K., E. J. Keogh, D. M. DeKretser and 8. Hudson. 1974.
Selective suppression of FSH by testis extracts. Int. Res. Commun.
Syst. 2:1406.

Leining, K. B., H. A. Tucker, J. H. Britt and D. T. Vines. 1976. Pro-
lactin and growth hormone in calves selected for milk yield. J.
Anim. Sci. 43:293 (Abstr. 316).

Lindner, H. R. 1961. Androgens and related compounds in the spermatic
vein blood of domestic animals. 1. Neutral steroids secreted by
the bull testis. J. Endocrinol. 23:139.

Lindner, H. R. and T. Mann. 1960. Relationship between the content of
androgenic steroids in the testes and the secretory activity of
the seminal vesicles in the bull. J. Endocrinol. 21:341.

Lostroh, A. J. 1976. Hormonal control of spermatogenesis. In_C.M.
Spilman et_al, (Ed.) Regulatory Mechanisms of Male Reproductive
Physiology. Excerpts Medica, Amsterdam, The Netherlands. p. 13-27.

Lunstra, D. D., J. J. Ford and S. E. Echternkamp. 1978. Puberty in
beef bulls: hormone concentrations, growth, testicular develop-
ment, sperm production and sexual aggressiveness in bulls of dif-
ferent breeds. J. Anim. Sci. 46:1054.

Macmillan, K. L. 1967. Endocrine and reproductive development of the
Holstein bull from birth through puberty. Ph.D. Thesis. Michigan
State University, East Lansing.

Macmillan, K. L. and H. D. Hafs. 1968a. Gonadal and extra gonadal
sperm numbers during reproductive development of Holstein bulls.
J. Anim. Sci. 27:697.

Macmillan, K. L. and H. D. Hafs. 1968a. Pituitary and hypothalamic
endocrine changes associated with reproductive deve10pment of
Holstein bulls. J. Anim. Sci. 27:1614.

Madhwa Raj, H. G. and M. Dym. 1976. The effects of selective with-
drawal of FSH or LH on spermatogenesis in the immature rat.
Biol. Reprod. 14:489.

113

Mainwaring, W. I. P. and F. R. Mangen. 1973. A study of androgen
receptors in a variety of androgen sensitive tissues. J.
Endocrinol. 59:121.

Mann, T., D. V. Davies and G. F. Humphrey. 1949. Fructose and citric
acid assay in the secretions of the accessory glands of reproduc-
tion as indicator tests of the male sex hormone activity. J.
Endocrinol. 6:75.

McCarthy, M. S. and L. V. Swanson. 1976. Serum LH concentration
following castration, steroid hormone and gonadotropin releasing
hormone treatment in the male bovine. J. Anim. Sci. 43:151.

McCullagh, D. R. 1932. Dual endocrine activity of the testis.
Science 76:19.

Means, A. R. 1973. Specific interaction of 3H-FSH with rat testis
binding sites. Adv. Exp. Med. Biol. 36:431.

Means, A. R., J. L. Fakunding, Claire Huckins, D. J. Tindall and R.
Vitale. 1976. Follicle-stimulating hormone, the Sertoli cell,
and spermatogenesis. Rec. Prog. in Hormone Res. 32:477.

Means, A. R. and Claire Huckins. 1974. Coupled events in the early
biochemical actions of FSH on the Sertoli cells of the testis.
ln_M.L. Dufau and A.R. Means (Ed.) Hormone Binding and Target
Cell Activation in Testis. Plenum Press, New York. p. 145-165.

Means, A. R. and J. Vaitukaitis. 1972. Peptide hormone "receptors":
specific binding of 3H-FSH to testis. Endocrinol. 90:39.

Mendelson, C., M. Dufau and K. Catt. 1975. Gonadotropin binding and
stimulation of cyclic adenosine 3':5'-monophosphate and testo-
sterone production in isolated Leydig cells. J. Biol. Chem.
250:8818.

Mercier, E. and G. W. Salisbury. 1947. Fertility level in artificial
breeding associated with season, hours of daylight, and the age
of cattle. J. Dairy Sci. 30:817.

Mongkonpunya, K., H. D. Hafs, E. M. Convey, W. D. Oxender and T. M.
Louis. 1974a. Luteinizing hormone release by gonadotropin re-
leasing hormone befbre and after castration in bulls. Proc. Soc.
Exp. Biol. Med. 147:873.

Mongkonpunya, K., Y. C. Lin, P. A. Noden, W. D. Oxender and H. D. Hafs.
1974b. Androgens in the bovine fetus and dam. Proc. Soc. Exp.
Biol. Med. 148:489.

Mongkonpunya, K., H. D. Hafs, E. M. Convey, H. A. Tucker and W. D.
Oxender. 1975a. Serum luteinizing hormone, testosterone and
androstenedione in pubertal and prepubertal bulls after gonadotro-
pin releasing hormone. J. Anim. Sci. 40:682.

114

Mongkonpunya, K., H. D. Hafs, E. M. Convey and H. A. Tucker. 1975b.
Serum LH and testosterone and sperm numbers in pubertal bulls
chronically treated with gonadotropin releasing hormone. J.
Anim. Sci. 41:160.

Mori, J., J. Masaki, K. Wakabayashi, T. Endo and T. Hosoda. 1974.
Serum luteinizing hormone levels in cattle under various reproduc-
tive states. Theriogenology 1:131.

Mulder, E., M. J. Peters, W. M. 0. van Beurden, M. Galdieri, F. F. G.
Rommerts, F. H. A. Janszen and H. J. van der Molen. 1976. Andro-
gen receptors in isolated cell preparations obtained from rat
testicular tissue. J. Endocrinol. 70:331.

Mulder, E., M. J. Peters, W. M. 0. van Beurden and H. J. van der Molen.
1974. A receptor for testosterone in mature rat testes. FEBS
Lett. 47:209.

Musto, N., A. A. Hafiez and A. Bartke. 1972. Prolactin increased 178-
hydroxysteroid dehydrogenase activity in the testis. Endocrinol.
91:1106.

Negri, Ann and V. L. Gay. 1976. Differing effects of comparable serum
testosterone concentration on gonadotropin secretion in pre- and
postpuberal orchidectomized rats. Biol. Reprod. 15:375.

Negro-Vilar, A., S. R. Ojeda and S. M. McCann. 1973. Evidence for
changes in sensitivity to testosterone negative feedback on
gonadotropin release during sexual development in the male rat.
Endocrinol. 93:729.

Nelson, W. O. 1937. Some factors involved in the control of gameto-
genic and endocrine functions of the testis. Cold Spring Harbor
Symp. Quant. Biol. 5:123.

Nelson, W. O. and C. E. Merckel. 1938. Maintenance of spermatogenesis
in hypophysectomized mice with androgenic substances. Proc. Soc.
Exp. Biol. Med. 38:737.

Niswender, G. D., L. E. Reichert, Jr., A. R. Midgley, Jr. and A. V.
Nalbandov. 1969. Radioimmunoassay for bovine or ovine luteini-
zing hormone. Endocrinol. 84:1166.

Nolin, J. M., G. T. Campbell, D. D. Nansel and E. M. Bogdanove. 1977.
Does androgen influence prolactin secretion? Endocrinol. Res.
Commun. 4:61.

Odell, W. D., M. A. Hescox and C. A. Kiddy. 1971. Studies of hypo-
thalamic-pituitary-gonadal interrelations in prepubertal cattle.
ln_W.R. Butt gt_al. (Ed.) Gonadotropins and Ovarian Development.
Livingstone, Edinburgh, Scotland. p. 371-385.

115

Odell, W. D., R. S. Swerdloff, H. S. Jacobs and M. A. Hescox. 1973.
FSH induction of sensitivity to LH: one cause of sexual matura-
tion in the male rat. Endocrinol. 92:160.

Orth, Joanne and A. K. Christensen. 1977. Localization of 1251-
labeled FSH in the testes of hypophysectomized rats by autoradio-
graphy at the light and electron microscope levels. Endocrinol.
101:262.

Oxender, W. D., H. D. Hafs and L. A. Edgerton. 1972. Serum growth
hormone, LH and prolactin in the pregnant cow. J. Anim. Sci.
35:51.

Payne, A. H., R. P. Kelch, E. P. Murono and J. T. Kerlan. 1977. Hypo-
thalamic, pituitary and testicular function during sexual matura-
tion of the male rat. Endocrinol. 72:17.

Payne, A. H., R. P. Kelch, S. S. Musich and M. E. Halpern. 1976. Intra-
testicular site of aromatization in the human. J. Clin.
Endocrinol. Metab. 42:1081.

Pelletier, J. 1968. Elevation due taux de LH dans 1e plasma sanguin
du belier apres castration. Ann. Biol. Anim. Biochim. BiOphys.
8:313.

Pelletier, J. 1970. Mode of action of testosterone propionate on the
secretion and release of luteinizing hormone (LH) in the castrated
ram. Acta Endocrinol. 63:290.

Pelletier, J. 1973. Decrease of pituitary responsiveness to LH-RH
in castrated rams pretreated with testosterone propionate. J.
Reprod. Fert. 35:601.

Peters, R. R. and H. A. Tucker. 1978. Prolactin and growth hormone
response to photoperiod in heifers. Endocrinol. 103:229.

Plasse, D., A. C. Warnick and M. Koger. 1968. Reproductive behavior
of B05 Indicus females in a subtropical environment. I. Puberty
and ovulation frequency in Brahman and Brahman x British heifers.
J. Anim. Sci. 27:94.

 

Purchas, R. W., K. L. Macmillan and H. D. Hafs. 1970. Pituitary and
plasma growth hormone levels in bulls from birth to one year of
age. J. Anim. Sci. 31:358.

Rager, K., J. Zarzycki, M. Eichner and D. Gupta. 1975. Effects of
experimental bilateral cryptorchidism and castration on the plasma
gonadotropins in male rats during sexual maturation. Res. Exp.
Med. 165:55.

H., A. Oksanen and T. Vanha-Perttula. 1974. Distribution
I-prolactin in mice and rats. Studies with whole body and
microautoradiography. Hormone Res. 5:6.

Ra‘aniemi
J Of 155

116

Ramirez, V. D. 1973. Endocrinology of puberty. In_R.O. Greep (Ed.)
Handbook of Physiology. Vol. 11. Female Reproductive System,
Part I. Waverly Press, Baltimore, Maryland.

Ramirez, V. D. and S. M. McCann. 1965. Inhibitory effect of testo-
sterone on LH secretion in immature and adult rats. Endocrinol.
76:412.

Rawlings, N. C., H. D. Hafs and L. V. Swanson. 1972. Testicular and
blood plasma androgens in Holstein bulls from birth through
puberty. J. Anim. Sci. 34:435.

Ritzen, E. M., S. N. Nayfeh, F. S. French and M. C. Dobbins. 1971.
Demonstration of androgen-binding components in rat epididymis
cytosol and comparison with binding components in prostate and
other tissues. Endocrinol. 89:143.

Rivarola, M. A. and E. J. Podesta. 1972. Metabolism of testosterone-
1"C by seminiferous tubules of mature rats: fermation of 5 -
androstan-3a, l7B-diol-1“C. Endocrinol. 90:618.

Samuels, L. T., J. G. Short and R. A. Huseby. 1964. The effect of
diethylstilbestrol on testicular l7a-hydroxy1ase and 17-desmolase
activities in BALB/c mice. Acta Endocrinol. 45:487.

Sanborn, B. M. 1977. Data presented at the fourth Testis Workshop,
Toronto, Ontario, Can.

Sanborn, B. M., J. S. H. Elkington, M. Chowdhury, R. K. Tcholakian
and E. Steinberger. 1975. Hormonal influences on the level of
testicular androgen binding activity: effect of FSH following
hypOphysectomy. Endocrinol. 96:304.

Sanborn, B. M., A. Steinberger and E. Steinberger. 1976. Nuclear-
bound androgen in cultured Sertoli cells and germ cells from
rat testes. Fifty eighth Annu. Meeting Endocrine Soc. (Abstr 77).

Santamarina, E. and R. P. Reece. 1957. Normal development of the
germinal epithelium and seminiferous tubules in the bull. Amer.
J. Vet. Res. 18:261.

Scatchard, G. 1949. The attractions of proteins for small molecules
and ions. Ann. of New York Acad. of Sci. 51:660.

Schanbacher, B. D. 1976. Rapid chromatography for quantitation of
radioimmunoassayable Sa-androstane-l7B-ol-3-one and testosterone
in ram, bull and boar serum. Endocrine Res. Commun. 3:71.

Schanbacher, B. D. 1979. Relationship of in_vitro gonadotropin binding
to bovine testes and the onset of spermatogenesis. J. Anim. Sci.
(In press).

117

Schanbacher, B. D. and J. J. Ford. 1977. Gonadotropin secretion in
cryptorchid and castrate rams and the acute effects of exogenous
steroid treatment. Endocrinol. 100:387.

Seechiari, P., F. Martorana, S. Pellegrini and M. Luisi. 1976.
Variation of plasma testosterone in developing Friesian bulls.
J. Anim. Sci. 42:405.

Setchell, B. P. and D. J. Sirinathsinghji. 1972. Antigonadotrophic
activity in rete testis fluid, a possible 'inhibin'. J.
Endocrinol. 53:9.

Shams, D. and V. Reinhardt. 1974. Influence of the season on plasma
prolactin level in cattle from birth to maturity. Hormone Res.
5:217.

Sholiton, L. J. 1975. The in vitro and in vivo effects of diethlystil-
bestrol on testicular synthesis of testosterone. Steroids 26: 797.

Simpson, M. E., C. H. Li and H. M. Evans. 1942. Biological properties
of pituitary interstitial-eell-stimulating hormone (ICSH).
Endocrinol. 30:969.

Smith, P. E. 1930. Hyp0physectomy and a replacement therapy in the
rat. Amer. J. Anat. 45:205.

Smith, 0. W., K. Mongkonpunya, H. D. Hafs, E. M. Convey and W. D.
Oxender. 1973. Blood serum testosterone after sexual preparation

or ejaculation, or after injections of LH or prolactin in bulls.
J. Anim. Sci. 37:979.

Steinberger, E. 1971. Hormonal control of mammalian spermatogenesis.
Physiol. Rev. 51:1.

Steinberger, A. and E. Steinberger. 1977. Inhibition of FSH by a
Sertoli cell factor in vitro. In P. Troen and H. R. Nankin (Ed. )

The Testis in Normal and Infertile Men. Raven Press, New York.
p. 271-279.

Steinberger, A., K. J. Thanki and B. Siegal. 1974. FSH binding in
rat testes during maturation and fellowing hypophysectomy. Cellu-
lar localization of FSH receptors. In M. L. Dufau and A. R. Means
(Ed. ) Hormone Binding and Target Cell Activation in the Testis.
Plenum Press, New York. p. 177-191.

Swanson, L. V., R. P. Wettemann, N. C. Rawlings, H. D. Hafs and W. T.
Magee. 1971. Pubertal relationships of some endocrine and
reproductive criteria in Hereford bulls. J. Anim. Sci. 33:823.

Swerdloff, R. S. and W. D. Odell. 1977. Modulating influences of FSH,
GH, and prolactin on LH- stimulated testosterone secretion. In
P. Troen and H. R. Nankin (Ed. ) The Testis in Normal and Infertile
Men. Raven Press, New York. p. 395- 401.

118

Swerdloff, R. S., P. C. Walsh, M. 5. Jacobs and W. D. Odell. 1971.
Serum LH and FSH during sexual maturation in the male rat: effect
of castration and cryptorchidism. Endocrinol. 88:120.

Tannen, K. J. and E. M. Convey. 1977. Gonadotropin releasing hormone-
induced change in serum luteinizing hormone, testosterone and
androstenedione in bulls, steers and steers given testosterone.

J. Anim. Sci. 44:1080.

Tash, J. S., J. R. Dedman and A. R. Means. 1978. Specific regulation
by FSH of protein kinase inhibitor in Sertoli cell-enriched testis
of the rat. Sixtieth Annu. Meeting Endocrine Soc. (Abstr. 341).

Tcholakian, R. K., M. Chowdhury and E. Steinberger. 1974. Time of
action of oestradiol-l7B on luteinizing hormone and testosterone.
J. Endocrinol. 63:411.

Tence, M. and M. Drosdowsky. 1976. Biosynthesis and metabolism of
testosterone by Sertoli cell-enriched seminiferous tubules.
Biochem. Biophys. Res. Commun. 73:47.

Thanki, K. H. and A. Steinberger. 1976. 125I-LH binding to rat testes
at various ages and posthypophysectomy. Endocr. Res. Commun. 3:49.

Tucker, H. A., J. A. Koprowski, J. H. Britt and W. D. Oxender. 1974.
Serum prolactin and growth hormone in Holstein bulls. J. Dairy
Sci. 57:1092.

Valladares, L. E., D. Sarkar and Anita H. Payne. 1978. Induction of
aromatase by HCG in testes of adult rats. Sixtieth Annu. Meeting
Endocrine Soc. (Abstr. 129).

Van Beurden, W. M. 0., E. Mulder, F. H. DeJong and H. J. van der Molen.
1977. The effect of estrogens on luteinizing hormone plasma
levels and on testosterone production in intact and hypophysecto-
mized rats. Endocrinol. 101:342.

Van Beurden, W. M. 0., B. Roodnot, F. H. DeJong, E. Mulder and H. J. van
der Molen. 1976. Hormonal regulation of LH stimulation of testo-
sterone production in isolated Leydig cells of immature rats: The
effect of hyp0physectomy, FSH and estradiol-17B. Steroids 28:847.

Van Demark, N. L., G. R. Fritz and R. E. Mauger. 1964. Effect of energy
intake on reproductive performance of dairy bulls. II. Semen pro-
duction and replenishment. J. Dairy Sci. 47:898.

Van Hall, E. V., J. L. Vaitukaitis and G. T. Ross. 1971. Immunological
and biological activity of HCG following progressive desialylation.
Endocrinol. 88:456.

119

Van Sickle, M., J. Oberwetter, L. Birnbaumer and A. Means. 1977.
Developmental changes and characterization of adenyl cyclase in
the testis Sertoli cell. Biol. of Reprod. Tenth Annu. Meeting
Soc. Study Reprod. (Abstr. 27).

Vitale-Calpe, R., D. W. Fawcett and M. Dym. 1973. The normal develop-
ment of the blood-testis barrier and the effects of clomiphene
and estrogen treatment. Anat. Rec. 176:333.

Walsh, E. L., W. K. Cuyler and D. R. McCullagh. 1934. Physiologic
maintenance of male sex glands; effect of androtin on hypophysecto-
mized rats. Amer. J. Physiol. 107:508.

Walsh, D. A., J. P. Perkins and E. G. Krebs. 1968. An adenosine 3',
5'-monophosphate dependent protein kinase from rabbit skeletal
muscle. J. Biol. Chem. 243:3763.

Welsh, M. J. and J. P. Wiebe. 1976. Sertoli cells from immature rats:
In_vitro stimulation of steroid metabolism by FSH. Biochem.
Biophys. Res. Commun. 69:936.

Wettemann, R. P. and H. A. Tucker. 1974. Relationship of ambient
temperature to serum prolactin in heifers. Proc. Soc. Exp. Biol.
Med. 146:908.

Wiebe, J. P. 1978. Steroidogenesis in rat Leydig cells: Effect of
gonadotropins on the activity of 5-ane and S—ene 3a- and 38-
hydroxysteroid dehydrogenases during sexual maturation.
Endocrinol. 102:775.

Wolf, F. R., J. 0. Almquist and E. B. Hale. 1965. Prepuberal
behaviour and puberal characteristics of beef bulls on high
nutrient allowance. J. Anim. Sci. 24:761.

Woods, M. C. and M. E. Simpson. 1961. Pituitary control of the testis
of the hypophysectomized rat. Endocrinol. 69:91.

Zipf, W. B. and A. H. Payne. 1977. Modulation of LH receptors by
luteinizing hormone (LH), prolactin (PRL) and growth hormone (GH).
In_vivo effects of these hormones on testicular responsiveness
to LH. Fifty nineth Annu. Meeting Endocrine Soc. (Abstr. 62).

 

Zolman, J. and E. M. Convey. 1973. Effect on serum FSH and androgens
in bulls. J. Anim. Sci. 37:334 (Abstr.).

Zondek, B. 1930. Uber die hormone des hypophysen vorderlappens.
Klin. Wochschr. 6:245.

Zondek, B. 1934. Mass excretion of oestrogenic hormone in the urine
of the stallion. Nature 133:209.

IHMIHIH

2 1