TESTICULAR AND BLOOD PLASMA ANDROGEN LEVELS ' V
IN. THE MALE BOVINE FROM BIRTH THROUGH PUBERTY

Thesis for the Degreeof M. S‘
MICHIGAN STATE UNIVERSITY
NORMAN C. RAWLINGS
1970

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ABSTRACT

TESTICULAR AND BLOOD PLASMA ANDROGEN
LEVELS IN THE MALE BOVINE FROM
BIRTH THROUGH PUBERTY

BY

Norman C. Rawlings

A technique was develOped to quantify the low levels
of testosterone and androstenedione found in the blood and
testes of bulls from birth through puberty. The technique
involved the conversion of the steroids to heptafluorobuty-
rate derivatives and quantification by gas liquid chroma-
tography with electron capture detection. A total of
sixty-five Holstein bulls were killed in groups of five at
monthly intervals from birth to 12 months of age. Twenty-
nine Hereford bulls were bled at the eleventh, twelfth,
and thirteenth month and slaughtered two weeks after the
last bleeding.

In the Holstein bulls plasma concentration of
testosterone increased from birth to 5 months of age, fell
to 6 months of age, rose until ll months and finally
declined at 12 months of age. Testicular testosterone
concentration showed a similar trend from 4 months of age.
Androstenedione concentration in the testicle fell from

4 months of age to a very low level at 6 and 7 months, then

Norman C. Rawlings

rose slowly until 11 months and finally declined to a low
level at 12 months of age. In the plasma, androstenedione
was very variable for the first 5 months and then almost
zero until a spurious rise in concentration at 12 months
of age. The plasma concentration of androstenedione did
not reflect or parallel testicular concentration. The
ratio of testosterone to androstenedione in the testis
varied from 0.65:1 at 4 months of age to 48:1 at 8 months,
and 8:1 at 12 months of age.

Testosterone and androstenedione concentrations were
higher in the Hereford bull serum than in the Holstein bull
plasma, but tended to decrease with age. Values for the
testes and serum at slaughter were also higher, but this
elevation may have been due to stress of slaughter. In
the serum the difference could have been a breed difference
or perhaps that these beef bulls were less mature physio-
logically.

The changes in concentrations of testosterone and
androstenedione in both tissues with age were compared to
changes for various other endocrinological and reproductive
criteria by correlation analysis. These analyses sug-
gested the following conclusions. Testosterone synthesis
and secretion increased with age, stimulated by increased
circulating levels of LH. Testosterone in turn stimulated
the seminal vesicular content of fructose and citric acid.
The biphasic pattern of increased circulating LH and

increased seminal vesicular secretions (rising from 2 to

Norman C. Rawlings

4 months of age, declining to 6 months, and increasing to
12 months of age) was paralleled somewhat by testosterone
synthesis and secretion. Androstenedione did not appear
to be involved in these relationships and probably lacked
androgenic properties. It may have been more involved
with the growth of the testis, and its synthesis and
secretion by the testis did appear to be stimulated by LH
in bulls older than 6 months of age. The role of both
androgens in the initiation of spermatogenesis was not
clear, but testosterone did appear to have some relation-
ship to gonadal sperm numbers, eSpecially at initiation
of spermatogenesis.

FSH was probably not related to the stimulation of
steriodogenesis during puberty; its role may have been
more involved with growth of the testis and reproductive
tract and the initiation of spermatogenesis. Testosterone
and androstenedione levels declined to stable adult levels
no sooner than 11 months of age, but growth and activity
of other reproductive criteria of these same animals

indicated that puberty ended at about 9 months of age.

i'h't‘.5l

 

TESTICULAR AND BLOOD PLASMA ANDROGEN
LEVELS IN THE MALE BOVINE FROM

BIRTH THROUGH PUBERTY
By

Norman C.1Rawlings

A THESIS

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

MASTER OF SCIENCE
DEPARTMENT OF DAIRY

1970

\Ex'

B IOGRAPH I CAL SKETCH

Norman C. Rawlings was born in Bristol, England, on
July 10, 1946. Lived in the small village of Dundry,
Somerset, where he attended a nursery and junior school
run by the Church of England. At age 11, he moved to a
new comprehensive school at Chew-Stoke, Somerset, but
due to a promotion his father received he moved to
Leicester where he initially attended Lancaster boys
school. He later passed the 13 plus examination and was
again moved to Moat Road Intermediate school, Leicester,
where he obtained passes in eight subjects at the General
Certificate of Education ordinary level. This allowed
him to transfer to Gateway boys school, Leicester (a
grammar school) to study for the advanced level GCE in
Chemistry, Botany and Zoology obtaining passes in Botany
and Zoology and also a special subject, Use of English.

He was accepted by the University of London, Wye
College, in 1964 to study for a degree in Agriculture.
He graduated in June 1968 with a BSc pass degree (second
division) after three years at College and one year

mandatory practical work on a farm in South Wales.

ii

In September 1968 he was awarded a graduate research
assistantship by the Department of Dairy, Michigan State
University. This position allowed him to study for a
Masters degree, with a major in Reproductive Physiology

and Endocrinology, which he completed in the fall of 1970.

iii

ACKNOWLEDGEMENTS

My initial decision to undertake post graduate
studies in the United States of America was partially
stimulated and always encouraged by my Director of Studies
at Wye College (London University, England), Dr. G. F.
Pegg. His encouragement and continued interest is
appreciated.

I am very grateful to the Dairy Department at
Michigan State University for the opportunity to study
for a Masters degree, and also for financial support
(NIH Research Grant HD-03039) without which it would
not have been possible for me to attend graduate school
in America.

My most sincere thanks are extended to my Major
professor Dr. Harold Hafs. If I have moved closer to
being the kind of person that can conduct useful and
imaginative research it is because of the constant
encouragement and advice received from him.

I should also like to thank my advisory committee
men for their advice and assistance; they are Dr. Spike
and Dr. Sweeley. I am grateful to Dr.'s Boyd, Convey,
Edgerton and Tucker for their interest and advice through-

out my stay at Michigan State University.

iv

My graduate colleagues are to be thanked for their
interest and moral support, special thanks are due to
Mr. L. V. Swanson, and Mr. R. Wetterman for their assist-
ance with the statistical analysis and interpretation of
the data.

Special thanks are also due to Miss Pat Kaneshiro for
skilled and enthusiastic technical assistance throughout
the development of the assay and the analysis of the
samples.

Last but not least, I should like to thank my wife,
Jeannie for her never failing understanding and encourage-
ment, and for the many hours spent helping to prepare the

data and the manuscript.

TABLE OF CONTENTS

BIOGRAPHICAL SKETCH . . . . . . . . . . .

ACKNOWLEDGEMENTS . . . . . . . . . . . .

LIST OF TABLES . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . . .

INTRODUCTION 0 O C C O O C O O . C O O 0

REVIEW OF LITERATURE . . . . . . . . . . .

1. GonadotrOpins . . . . . . . . . . .
2. Development of the Testis . . . . . . .
(a) Growth and Spermatogenesis . . . . .
(b) Steroidal Secretory Activity . . . .
3. The Hypothalamic-Pituitary-Testis Axis . .
(a) Maturation of the Axis . . . . . .
(b) Correlations and Interactions between
the Pituitary, Testis, and Hypothalamus
4. Attempts to Accelerate the Process of
Sexual Maturation . . . . . . . . .
5. Development of the Accessory Sex Glands . .
6. Testosterone and Androstenedione in the
Testes and Plasma as Measures of
Androgenic Status . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . .
1. Development of Techniques . . . . . . .
(a) Objective . . . . . . . . . . .
(b) Introduction . . . . . . . . . .
(c) Use of the Hydrogen Flame Detector . .
(d) Electron Capture Detector . . . . .

vi

Page

ii
iv
ix

xii

00qu

15
15

18

21

26

29
35
35
35
35

39
42

mesa

 

(e)
(f)

(9)
(h)
(i)

Trimethylsilyl Ether of Testosterone . .
O-Methoxime-Trimethylsilyl Derivatives
of Testosterone and Androstenedione .
Halogen Derivatives of Androgens . . .
Monoheptafluorobutyration of Testosterone
Development of an Extraction and
Purification Scheme for Plasma
and Testicular Testosterone and
Androstenedione . . . . . . . .

2. Final Materials and Methods for the
Routine Quantification of Testicular
and Plasma Testosterone and Andro-
stenedione using Gas Liquid Chroma-
tography with Electron Capture
Detection . . . . . . . . . . .

(a)
(b)

RESULTS .

Preparation, Storage and Treatment
of Materials and Samples . . . . .
Methodology . . . . . . . . . .

l. Assay for Testicular and Plasma Levels
of Testosterone and Androstenedione . . .

(a)

Introduction . . .

(b) Linearity of the Detector Response . .
(c) Sensitivity . . . . . . . . . .

(d) Stability of Heptafluorobutyrate
Derivative . . . . . . . . . .
(e) Recoveries . . . . . . . . . .
(f) SpGCifiCity o o o o o o o o o o
(g) Accuracy . . . . . . . . . . .
(h) Precision . . . . . . . . . . .
2. Holstein Bulls . . . . . . . . . .

(a) Testicular Testosterone and

Androstenedione . . . . . . . .
(b) Plasma Testosterone and Androstenedione .
3. Hereford Bulls . . . . . . . . . .
GENERAL DISCUSSION 0 O O O O O O O O O O 0
SUMMARY AND CONCLUSIONS . . . . . . . . . .

vii

Page
44
45

48
54

59

66
66
70
79

79
79
79
80
83
83
83
85
85
89
89
93
94
96

118

Page
BIBLIOGRAPHY O 0 0 O O O 0 0 O O O O 0 O O 124

APPENDICES . . . . . . . . . . . . . . . 137

viii

LIST OF TABLES

Table
l. Gonadotropin Levels in Plasma of Human at
Various Ages . . . . . . . . . .
2. Gonadotropin Levels in Anterior Pituitary
of Male Rats at Various Ages. . . . .
3. Variation of Plasma and Testicular Testos-

terone and Androstenedione with Age in
the Rat O O O O O O O O O O O O

4. Variation of Plasma Testosterone with Age
‘ in the Human . . . . . . . . . .

5. Variation of Plasma and Testicular Testos-
terone and Androstenedione with Age in
the Guinea Pig . . . . . . . . .

6. Normal Adult Levels of Testosterone and
Androstenedione from Plasma and
Testicles of Different Species as
Estimated by Gas Liquid Chromatography .

7. Normal Adult Levels of Testosterone and
Androstenedione in Free and Conjugated
Form from Plasma of Man as Estimated
by Various Techniques . . . . . . .

8. Retention Data for Testosterone (Test.)
and Androstenedione (Androst.) on
Various Gas Liquid Chromatographic
Columns and at Various Conditions . . .

9. Some Retention Time Data for Trimethylsilyl
and O—methoxime-Trimethylsilyl Derivatives
of Testosterone and Androstenedione . .

10. Reaction Conditions and Products for the

Preparation of Testosterone and Andro-
stenedione heptafluorobutyrates (HFB) .

ix

Page

11

12

13

14

32

41

47

51

Table Page

11. Solvent Fractions Containing Various
Steroidal Hormones and their Hepta-
fluorobutyrate Derivatives as
Eluted from Silica Gel Microcolumns . . . . 56

12. Fractions Containing Various Steroids and
Their Heptafluorobutyrate Derivatives
as Eluted from Florosil Microcolumns . . . . 58

13. Average Distribution of Radioactivity in
Various Discarded Fractions and Con-
tainers for a Total of Twenty Samples . . . 65

14. Replicate Analysis of Standard Testosterone
and Androstenedione added to Saline . . . . 86

15. Duplicate Analysis of Bull Testes for
Testosterone and Androstenedione . . -. . . 87

16. Analysis of Variance of the Duplicate
Androgen Analysis in Table 15 . . . . . . 87

17. Average Testicular and Plasma Concentration
of Testosterone and Androstenedione in
Holstein Bulls from Birth to 1 Year of Age . . 90

18. Testosterone Androstenedione Ratio from
Birth to 1 Year of Age in the Testes
and Plasma of Holstein Bulls . . . . . . 92

19. Average Plasma Concentrations of Testosterone
and Androstenedione in Hereford Bulls of
11, 12 and 13 Months of Age, with Serum and
Testicular Levels of Testosterone and Andro-
stenedione at Slaughter . . . . . . . . 95

20. Various Overall Correlation Coefficients (r)
for Holstein Reproductive and Endocrinologi-
cal Data from Birth to 12 Months of Age . . . 99

21. Various Within Month Correlation Coefficients
(r) for Holstein Reproductive and Endocrinol-
ogical Data from Birth to 12 Months of Age . . 100

Table
22. Various Correlation Coefficients (r) for
Material Collected at Slaughter from
Hereford Bulls . . . . . .' . .
23. Correlation Coefficients (r) for Serum

Androgens and Serum Gonadotropins from
Herefords at 11, 12 and 13 Months of
Age . . . . . . . . . . . .

xi

Page

. 101

. 102

LIST OF FIGURES

Figure Page

1. Identification of Steroidal Constituents
of a Bovine Testicular Extract on Thin
Layer Chromatography Plates . . . . . . . 63

2. Positions of Steroids and Various Contami—
nants on Silica Gel Thin Layer Plates
after Development in Ether:dimethyl
formamide:acetic acid (99:1:1 V/v) (l)
and Chloroform:methanol (99:1 v/v) (2),
as Revealed by Ultra Violet Light and
Concentrated Sulphuric Acid . . . . . . . 73

3. Gas Liquid Chromatographic Tracing of
Testosterone from 10 ml of Plasma . . . . . 81

4. Gas Liquid Chromatographic Tracing of
Testosterone. From 4 g of Testis with
10 ng of a Progesterone Internal
Standard . . . . . . . . . . . . . 82

xii

LIST OF APPENDICES

Appendix Page
1. Plasma and Testicular Testosterone and
Androstenedione Values for Individual

Holstein Bulls by Month . . . . . . 138
2. Plasma Testosterone and Androstenedione

Values for Individual Hereford Bulls by
Month and at Slaughter, and Testis
Testosterone and Androstenedione Values. 141

3. Average Plasma Concentrations and Total
Plasma Content of Luteinizing Hormone
and the Average Ratio Between Total
Plasma Content and Total Pituitary
Content of Luteinizing Hormone in
Holstein Bulls from Birth to 12 Months

Of Age I O O O O O O O O O O O 143
4. Gonadal Sperm Concentration and Sperm

Numbers in Holstein Bulls from 5 to

12 Months of Age . . . . . . . . 145
5. Changes in Testicular Nucleic Acid Concen-

tration and Content and the RNA:DNA
Ratio for Holstein Bulls from Birth to
12 Months of Age . . . . . . . . 147

6. Changes in the Weight of the Paired
Seminal vesicles and Their DNA. RNA:
Citric Acid and Fructose Contents for
Holstein Bulls from Birth to 12 Months
of Age. . . . . . . . . . . . 149

7. Plasma Concentration and Content of
Luteinizing Hormone in Holstein Bulls
from Birth to 12 Months of Age as
Measured by Radioimmunoassay . . . . 151

xiii

IN TRODUCT ION

The demands on modern commercial animal production
necessitate the utmost efficiency of the producer if he
is to profit or even survive. Even small improvements
in any aspect of animal production may bring large
rewards. Genetic improvement of stock whether for beef
or dairy purposes is permanent, but slow. Sons of
superior sires must be kept unproductive for several
years while they mature and are progeny tested, and even
then they may be discarded as inferior. Studies have
been performed in our laboratory to describe pituitary
hormonal and reproductive changes during sexual matura-
tion in bulls. Knowledge likely to come from studies
such as these may allow us to hasten the process of
sexual maturation, allowing commercial AI centers to
obtain "proofs" on bulls at an earlier age and signifi-
cantly reduce the unproductive portion of the life of
the bull. By the same token, sperm production might be
increased and fertility enhanced by treatment with
exogenous hormones.

An important approach to these ends would be to

identify causal relationships between levels of various

hormones and sperm production or fertility. A more com-
plete description of the reproductive and endocrine
changes from birth through puberty in the male bovine is
prerequisite to these goals. The present study on andro-
gen levels complements reproductive and endocrine data
obtained earlier on the same bulls. To the author's
knowledge no one has attempted previously to measure
androgen levels from birth through puberty in the bull.
As recently as ten years ago, analysis of steroidal
compounds by gas liquid chromatography was realized. In
the last three or four years, methods for the quantifi-
cation of steroid hormones involving gas liquid chromato-
graphy have appeared. These techniques were insensitive
or not amenable to the routine analysis of large numbers
of biological samples. The initial part of this study
was development of a sensitive and accurate assay for
androgens that could be performed routinely and with some

Speed.

REVIEW OF LITERATURE

Between birth and one year of age, the bull goes
through many endocrine and physiological changes associated
with sexual development. At birth the testis consists of
solid sex chords but by one year of age the seminiferous
tubules have attained mature form, spermatogenesis occurs
and the young bull is capable of paternity (Abdel-Raouf,
1960: Baker‘gt_al., 1955).

Among many definitions, puberty has often been
defined as the age at which sexual maturity is attained.
But Donovan and van der Werrf ten Bosch (1965) defined it
as including the entire period when the gonads secrete
hormones in amounts sufficient to cause accelerated growth
of the genital organs and the appearance of secondary
sexual characters.

Many endocrine, physiological and behavioural cri-
teria may be used to define the termination of puberty.
In Holstein bulls ejaculation can occur at 38 weeks of
age (Bratton §E_§1., 1959), but many bulls lack libido
at this age. Bratton §t_al. also showed that at 43 weeks
semen production had started as determined by ejaculation
into an artificial vagina. Sperm in the epididimis and

proximal ductus differentia were obtainable by

electroejaculation at this age. (Wolf §t_al., 1965.)
Some consider puberty complete when sperm are found in
these regions. All of these criteria vary with genetic
and environmental factors. This subject is reviewed by
Macmillan (1967) and will not be developed further as

the purpose of this review is to consider the role of
androgens and their interactions with other endocrine

and physiological factors during puberty, principally in
the male bovine. Since data on the bovine are limited,
other species will be included where necessary to develop

hypotheses.

l. Gonadotropins

 

Clark (1935); McQueen--Williams (1935) and Lauson
gt_al. (1939), were among the first to measure total
gonadotropins in the pituitary. Pituitary total gona—
dotropin potency in male rats increased gradually during
the first two weeks of life, faster in the third and
fourth weeks and then declined.

Burr et_al. (1970) reported that luteinizing hormone
(LH) levels in the plasma of boys rose at ten years of age
and reached a plateau at 13 years of age (see Table 1).
Similarly, Yen (1969) detected LH in the plasma of boys at
eight years of age and LH levels rose until 14 years of
age with a very rapid increase between 10 and 12 years.

He estimated a threefold increase in plasma LH between 8
and 14 years of age and concluded that sexual maturity

was attained at 14 years of age. Skinner et a1. (1968),

TABLE 1.--Gonadotropin Levels in Plasma of Humans at
Various Ages.

 

 

Age Plasma LH Plasma FSH
(years) ------- (miu/ml) -------- (ug FSH/ml)
5 4.03a -- 1.18a
6 4.02 -— 1.40
7 3.97 -- 1.27
8 4.27 1.1 220.29b 1.24
9 4.08 0.63:0.04 1.30
10 4.42 0.86:0.31 1.67
11 4.44 1.2 £0.11 1.77
12 4.80 2.1 £0.27 2.09
13 4.55 4.2 :0.83 3.11
14 4.99 4.1 £0.30 2.86

15 5.46 4.0 £0.45 2.25

 

aBurr et a1. (1970)

bJohannan et a1. (1969)

in an extensive survey of sexual maturation in the ram,
noted that LH levels in the blood fell for the first 42
days of life and then rose markedly. However, levels
of plasma LH were only weakly correlated with other
parameters.

Burr gt_al. (1970) reported rising levels of plasma
follicle stimulating hormone (FSH) in boys to 12 years of
age, after which FSH levels declined (see Table 1). This
fits well with data of Yen and Vicia (1970) who found that
plasma FSH rose to 14 years of age and then declined.
Kragt gt_al.(l968) determined that levels of FSH in the

pituitary of rats increased fifteenfold from 10 days to

35 days of age and then stabilized at levels typical of
adults. Gonadotropins remain fairly constant in man
from 20 years through 90 years (Kent and Acone, 1965).
The variation of plasma FSH levels with age for the rat

are shown in Table 2.

TABLE 2.--Gonadotr0pin Levels in Anterior Pituitary of
Male Rats at Various Ages.

 

 

Age Pituitary FSH Age Pituitary FSH
(days) (ugFSH/gland) (days) (ugFSH/gland)
11 18a 45 221

16 43 50 206

21 30 60 374

22-40 79-166 70 744

40 195

 

aPearce and Brown (1970)

Reece and Turner (1937) reported that pituitary pro-
lactin potency appeared to increase slowly from birth to
one year of age in bulls and then reached a plateau. In
male guinea pigs, pituitary prolactin potency increased
parallel to body weight from 170 to 880 9, but prolactin
potency in male rats did not change between 80 and 340 g
body weight. Reece and Turner (1937) also made observa-
tions on the male rabbit, here pituitary prolactin potency

in immature animals was three times greater than in adults.

2. Development of the Testis

 

a) Growth and Spermatogenesis

 

A log log plot of testis weight as a function of body
weight in rats and man revealed two distinct slopes with a
change in slope at puberty (Spencer, 1968). In rats, the
second part of the slope was less steep than the first.
That is, testis growth showed positive allometric growth
in the first phase and negative allometric growth in the
second phase, but the picture was reversed in man. This
result is substantiated by Burr §E_al. (1970) who observed
that testicular growth in boys was slow up to 12 years of
age and then proceeded more rapidly. Skinner (1968)
showed a sharp rise in the testicular weight of ram testes
at 42 days of age. Thus testicular development in rams
resembles that in man.

The changing allometric growth for rat testes agrees
well with nucleic acid data. The DNA concentration of
rat testes declined rather rapidly from 2 to 4 weeks of
age and then more gradually to 10 weeks (Fujii and Koyama,
1962). Desjardins gt_al. (1968) noted a similar trend,
but a marked increase from birth to 15 days. RNA varied
similarly. Fujii and Koyama (1962) concluded that sexual
function of the testis commenced at 3 weeks of age
based on the fact that the RNA:DNA ratio increased after

3 weeks of age.

Abdel-Raouf (1960) delineated five stages of repro-
ductive develOpment of bulls based on the morphology of
the seminiferous tubules. The infantile stage consisted
of the first two postnatal months and was marked by
solid sex chords and foetal type cells. The proliferative
stage lasted from 2 to 4 or 5 months. Spermatogonia
appeared during this period. Lumen formation occurred
and primary spermatocytes appeared in the third or pre-
pubertal stage. Spermatids and later sperm appeared dur-
ing the pubertal stage which lasted from 32 to 44 weeks.
During the final post-pubertal or adult stage, mainly
quantitative development took place and the testis
increased in size.

Martig (1969) observed that the fertility of beef
bulls and percentage of normal sperm increased from 1 to
2 years. Most bulls were relatively fertile at one year
near completion of puberty. In man spermatogenesis is

initiated between 12 and 14 years of age.

b) Steroidal Secretroy Activity

Experiments have been conducted to determine the
type and site of steroid hormone secretion. Knapstein
(1968) injected radioactive acetate or cholesterol into
the spermatic artery of an adult male human. Blood
collected from the spermatic vein revealed incorporation
of radioactivity into testosterone and androstenedione.
Incubation experiments by Bell (1968) and Hall (1969)

revealed conversion of progesterone or pregnenalone to

androgens by the seminiferous tubules. As cholesterol is
the major substrate for steroidogenesis it was concluded
that the Leydig cells produced most of the androgens
secreted by the testis.

In bulls, Leydig cells differentiate from intertubu-
1ar mesenchymal cells in some cases by four months of age
(Hooker, 1944), increase in number until two years of age,
and increase in size in the mature bull mainly due to
vacuolation.

The androgen secretory activity of the Leydig cells
also increases during the period of sexual maturation.
Lindner (1959) and Lindner and Mann (1960) observed that
the ratio of androstenedione to testosterone changed from
1:1 in testes of calves at four months of age to 1:10 at
nine months of age. Lindner (1961b) could detect only
testosterone in the spermatic vein blood taken from a
ram and two boars all between 3 and 4 months of age.
Skinner (1968) detected androgens in the ram testis at
birth; the ratio of androstenedione to testosterone at
this time was 1:1. The testicular content of testosterone
increased with testicular size but was highly variable.
Androstenedione content fell until 56 days of age and then
rose slightly to mature levels.

In vitro incubation studies provided evidence to
support the findings of Lindner and Skinner. Becker and
Snipes (1968) incubated radioactive androstenedione and

testosterone with guinea pig testicular tissue. Testis

10

from a lO-week old animal showed a steady state equili-
brium of androgens favoring androstenedione. However
testosterone predominated in testis from 7-month old
animals. Studies on the adult Rhesus monkey (Resko,
1967) showed a 10.1 ratio of testosterone to androstene-
dione. Pre-pubertal monkeys had small amounts of both
steroids in their testicles, but testosterone was unde-
tectable in the blood. By three years of age, andro-
stenedione was detected in the blood and testosterone
approached adult levels. The immature rat testis con-
tained much androstenedione and little testosterone
(Strickland, 1970). Androstenedione did not accumulate
in the immature testis. Thus there was no block to its
conversion to testosterone. It was concluded that
testosterone reductase may play a role in puberty. In
the mature testis, testosterone accumulated and andro-
stenedione formation decreased.

Tables 3, 4 and 5 summarize testicular and plasma
androstenedione and testosterone levels at different ages
for three species. Little comment is needed, but it is
interesting to note the large variability of androgen
levels during sexual maturation. The increase in plasma
testosterone in guinea pigs (Resko, 1970) between days
15 to 30 coincides with the first behavioural signs of
sexual maturity. Plasma testosterone levels in man from
20 through 90 years are fairly constant but the utilization

and metabolic clearance rate may drop with age (Kent and

11

Acone, 1965). Some normal adult levels of testosterone
and androstenedione from the plasma and testis of several
species are given in Table 6.

Probably the most interesting hypothesis of this
section is that the testis gains the ability to convert
androstenedione to testosterone in large amounts begin-
ning at puberty. This agrees with the hypothesis of
Strickland (1970) that testosterone reductase may be an

important enzyme of puberty.

'TABLE 3.--Variation of Plasma and Testicular Testosterone
and Androstenedione with Age in the Rat.

 

 

 

 

 

Age Testosterone Androstenedione
Plasma Testicle Plasma Testicle
(day8) (ng/ml) (pg/g) (ng/ml) (ug/g)
1 0.27a 0.194 NDd 0.096
5 0.21 0.134 ND 0.043
10 0.09 0.122 ND ND
15 0.10 Traceb ND ND
30 0.15 Trace 0.13 0.008
40 0.64 0.006 0.18 0.004
60 1.10 0.043 0.15 0.003
90 2.04 0.111 0.12 0.015
120 1.14 -- 0.57 -—d
a

Resko et a1. (1968)
bResponse not great enough to quantify
CNone detected

dSample lost

12

TABLE 4.-—Variation of Plasma Testosterone with Age in
the Human.

 

 

 

 

Age Plasma Age Plasma
Testosterone Testosterone

(years) (ng/ml) (years) (ng/ml)
<10 <o.2-o.5a 30-39 5.6

10-16 <0.2-5.4 40-49 4.7

22-59 3.4-14.9 50-59 5.9

20-40 5.0b 60-69 5.9

31 0.03:0.10C 70—79 5.8

20—29 6.6d 80-93 2.8

 

aVan der Molen (1965)
bPanicucci (1965)
CSaez and Bertrand (1968)

dKent and Acone (1965)

13

TABLE 5.--Variation of Plasma and Testicular Testosterone
and Androstenedione with Age in the Guinea Pig.

 

 

 

 

Age Testosterone Androstenedione
Plasma Testicle Plasma Testicle

(days) (mg/ml) (pg/g) (ng/ml) (09/9)
1 0.09a 0.092 0.49 0.043

5 NDb --d 0.42 -—
15 Tracec 0.082 0.18 0.118
30 0.42 0.049 0.45 0.030
40 0.25 0.014 -- 0.016
60 0.70 0.066 0.25 0.016
90 1.69 0.121 -- 0.022
120 1.13 0.194 0.34 0.018

250 1.98 -- -- --

 

aResko et a1.

b

CResponse not great enough to quantify

d

Sample lost

(1970)

None detectable

l4

 

 

 

 

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15

3. The Hypothalamic-Pituitary-Testis Axis

a) Maturation of the Axis

 

In immature animals, the pituitary gland is a rela-
tively labile structure and its future pattern of gonado-
tropin secretion is determined by the effect of steroidal
hormones working through and with the modifying influence
of the hypothalamus. Harriss and Jacobson (1952) trans-
planted pituitaries from immature to mature animals and
vice versa. They found that the activity of the trans-
planted glands was dependent on the age of the host, not
the gland. This suggested a modifying influence of the
hypothalamus.

Androgen sterilization of female rats within the
first few days of life is a fairly well known phenomenon
(Gorski, 1966) and leads to constant estrus. This treat-
ment is not effective after ten days of post natal life.
These observations suggest the possibility of modification
of the hypothalamic-pituitary axis during early life. From
work undertaken by Harris and Levine (1965) with the male
rat, it appeared that the normal mechanism underlying the
future patterns of sexual behaviour and gonadotropin secre-
tion are organized in the first few days of life by testi-
cular secretion in male rats. If no testicular secretion
is present then this pattern was fixed in a female form.

The gonads exert a continuous effect on the pituitary
in the rat, probably principally via the hypothalamus.

Libman et a1. (1953) castrated male rats at birth and

16

found pituitary baSOphils enlarged by seven days and even
more by 15 days. However pituitary castration cells,
similar to those seen in the castrated adult, did not
appear until 45 days of age. This suggested a build-up
of gonadotropins in the pituitary in the absence of
modifying influence of the gonads. Immature males had
lighter pituitaries than immature females (Van Rees and
Paesi, 1955). Castration caused male pituitary weight

to be increased by 32% but that of the female by only 7%.
This suggested that androgens may retard pituitary growth.
Some more recent evidence agrees with the hypothesis that
the hypothalamus is involved with steroidal modification
of pituitary activity. Domer and Staudt (1969) histolo-
gically investigated the effect of castration in various
areas of the hypothalamus and found that only the ventro
medial nucleus was affected. Immediately after birth,
this nucleus was large in females and smaller in males,
and castration of the male allowed it to enlarge towards
a female status.

Recent evidence utilizing new techniques to measure
plasma LH substantiates pituitary and histological evi-
dence of a build-up of pituitary gonadotrOpins after
castration. Ramirez and McCann (1963) observed that
gonadectomy of male rats increased the levels of plasma
LH. This effect was twice as large in the immature as
compared to the mature male rat. They also showed that

the increase in LH was suppressed by administration of

17

testosterone propionate, and the suppression was greater
in the immature rat. The differences between mature and
immature rats suggest a change in the sensitivity of the
hypothalamus to androgens during puberty. Kurcz (1969)
produced some important evidence that supports this
hypothesis. Male rats injected with large doses of
testosterone immediately after birth were incapable of
an increase in gonadotropin output. If the treatment was
delayed until five days after birth, however, gonadotro-
pin secretion developed normally. Kurcz (1969) concluded
that the early injection desensitized the hypothalamus
and damaged gonadotropin secretory cells. The very
drastic long term effects of steroids on the sensitive
early postnatal hypothalamus were demonstrated in work
by Ladosky and Kerikowski (1969). One-day old rats
injected with either estradiol benzoate or testosterone
propionate showed delayed spermiogenesis at 35 to 60
days. This had recovered by 90 days but the accessory
reproductive glands were still very small. Ulrich (1968)
suggested that the rate of utilization of sex steroids
did not alter at puberty, probably because the hypotha-
lamus became less sensitive to the minute amounts of
these steroids circulating during infancy and higher
levels were needed to inhibit gonadotrOpin output.

Hence higher levels of steroids were found in the blood.
The increased levels of protein and steroidal hormones

together stimulated sexual maturation.

18

The ability of the liver to metabolize steroids
increases at puberty. Thus the high sensitivity of the
immature hypothalamus to steroids may be due in part to
the slower rate of inactivation of steroids by the liver.
Denef (1968) showed that testosterone given immediately
after birth in female and castrated male rats prevented
the development of a female type steroid metabolism.
Treatment later in life had only a temporary effect. This
suggests that the liver has the potential to develop a
female pattern of steroid metabolism unless modified by
androgens, and also that the liver may play some role in
the maturation of the hypothalamus.

Changes in levels of steroid secretion and parallel
changes in the hypothalamus do not seem to be associated
with increases of secretion of luteinizing hormone
releasing factor (LHRF) by the hypothalamus. Ramirex and
Mcann (1963) showed no difference in LHRF activity in the
median eminence in mature or immature rats. However Mac-
millan (1967) could not detect LHRF in bulls up to four
months of age but found a sudden increase at five months,
a decline at six months, an increase at eight months and
little change to welve months of age. When viewed with the
changing levels of plasma LH a possible role of LHRF in
sexual maturation is suggested.

b) Correlations and Interactions between the
Pituitagy: Testis, and Hypothalamus

 

Tables 1, 2, 3, 4 and 5 provide a comparison of

gonadotropin levels and androgen levels in various species

19

during sexual maturation. Burr (1970) found that LH
levels in the plasma of boys rose at ten years and reached
a plateau at 13 years, this was associated with a small
increase in testicular growth. FSH rose in the plasma at
12 months of age. Testicular size rose slowly at first,
but much more rapidly during the period of rapidly in-
creasing FSH levels. Later FSH levels fell while LH
levels continued to rise. This suggests that maintenance
of the testis requires less FSH than the active growth
phase. At the peak of LH secretion, testosterone levels
rose in the blood, plasma levels of FSH increased and
spermatogenesis was initiated.

Testicular weight was associated with plasma levels
of FSH in the rat (Kragt and Ganong, 1968) but Skinner
(1968) reported that FSH in the ram was not highly corre-
lated to testosterone plasma levels after 40 months of
age.

The relationships described have more meaning in
terms of the roles of hormones in sexual maturation.
Lostroh (1969) investigated some of these roles in an
experiment where male rats were hypophysectomized and
treated with FSH and LH two months later. These treat—
ments initiated spermatogenesis and androgen secretion
as noted by accessory organ growth. Separately, neither
hormone exerted this effect. High sperm output was cor-
related with high androgen titers, but testosterone did

not initiate spermatogenesis.

20

FSH has been shown to stimulate testicular RNA and
protein synthesis and this prOperty seemed fairly speci—
fic for FSH and gonadotropins with FSH activity (Means,
1969). FSH also has been shown to stimulate phospholipid
synthesis in the testis even if the LH activity was des—
troyed with urea (Yokoe and Hall, 1970).

The mechanism of stimulation of the testis by FSH
and LH at the molecular level, albeit in different struc-
tures, are probably similar. Connell and Eiknes (1968)
and Murod gt_§l. (1969) performed in vitro incubation
studies with rat and canine testis, respectively, and
demonstrated that FSH and LH stimulated the production
of cyclic AMP from ATP, and also that FSH, LH and cyclic
AMP stimulated testosterone synthesis. It appears as if
the major role of FSH is in stimulation of testicular
growth via protein synthesis.

Kar (1966) injected ovine FSH and LH into Rhesus
monkeys. There was a small increase in the size of the
seminiferous tubules and Leydig cells with concomitant
stimulated testosterone secretion. No greater effect was
noted with combined FSH and LH, and he concluded that LH
had its effect on the interstitial cells and FSH on the
seminiferous tubules. Similar experiments in the rat
yielded less conclusive results, and he concluded that
the timing of gonadotropin injections with the phase of

development of the testis was very important. These

21

experiments associated LH with the Leydig cells, the major
site of androgen production.

Several investigators have reported that gonadotro-
pins stimulate testicular steroidogenesis by enhancing the
ZOQ-hydroxylation of cholesterol to pregnenolone (e.g.
Dorfman, 1969; Hall and Young, 1968). Oshima (1967)
injected radioactive progesterone into male rats followed
by an injection of testosterone propionate. The androgen
markedly reduced the utilization of the progesterone sub-
strate by the testis. Upon withdrawal of testosterone
propionate treatment progesterone utilization increased,
but the testis only converted it as far as androstenedione
and l7a-hydroxy-progesterone. After a longer period of

testosterone withdrawal, the l7a-hydroxylase and C C

17’ 20
lyase enzyme system recovered by l7B-hydroxy steroid dehy-
drogenase was much slower to recover. Oshima suggested
that LH stimulates these enzyme systems.

Literature reviewed in this section suggests that
FSH stimulates protein synthesis and general growth of the
testis, laying the foundation for steroid secretion while
LH acts directly on the enzyme systems involved in andro-
gen secretion. Both gonadotropins seem necessary to
initiate Spermatogenesis.

4. Attempts to Accelerate the Process
of Sexual Maturation

 

 

Most approaches to this problem have involved admin-

istration of hormones implicated in initiation and

22

maturation of spermatogenesis, or maturation of the repro-
ductive tract, or in stimulation of endoqenous levels of
these hormones. It has been known for a long time that
mating increases the circulatory and testicular levels of
testosterone. This was shown recently by Herz gt_al. (1969).
This reSponse probably reflects a neuroendocrine reflex
mediated by the hypothalamus causing release of LH which
stimulates synthesis of testosterone.

Lindner (1961a) increased spermatic vein levels of
androstenedione eight fold in a 90-day old calf, but could
not stimulate testosterone output with HCG administered
for 50 days prior to androgen determination. Saez and
Bertrand (1968) attained a twentyfold increase in plasma
testosterone levels in prepubertal boys with injections
of HCG, but in adults only a two or threefold increase
was noticed. He hypothesized that the lower levels of
LH in the prepubertal boys allowed much more capacity for
stimulation. Fariss (1969) reported that injections of
5 or 10 in HCG gave the maximum testosterone response
in hypophysectomized male rats, testosterone levels in
the plasma peaked 45 minutes after injection and then
declined rapidly. However, when male rats were given HCG
immediately after birth during the period of hypothalamic
differentiation (Dorner, 1969) testicular size and andro-
gen secretory activity increased only to result in hypo-
gonadism and small accessory sex organs in the adult.

Data from adult male rabbits (Haltmeyer, 1969; Saginar and

23

Horton, 1968) showed that either HCG or copulation increased
plasma levels of androstenedione and testosterone very
rapidly. Morkow g£_§l. (1968) examined the effect of HCG

on the testis in more detail. He injected 100 in HCG for

15 days into adult male guinea pigs and noted an increase

in size and number of Leydig cells. Within these cells,

the smooth and rough endoplasmic reticulum and the Golgi
apparatus were increased thus indicating a potential for

a vast increase in metabolic activity.

Wakeline (1959) injected pregnant mare serum gonado-
tropin (PMS), HCG, testosterone propionate and human post
menopausal gonadotropin (HMG) for seven days into intact
male rats from 25 or 28 days of age. All of the hormones
increased accessory organ weight, presumably by stimulating
testosterone synthesis. Only PMS accelerated appearance
of Spermatids in the testis but none of the treatments
accelerated initiation of spermatocytogenesis. Woods and
Simpson (1961) reported that ovine FSH and LH did not
hasten sexual maturity in intact young male rats, but a
combination of the two gonadotropins hastened maturity in
hypophysectomized animals. They maintained that the
immature pituitary produces an "anti-gonadotropin." The
rat testis was responsive to gonadotropins between birth
and six days of age (Price and Ortiz, 1944), but the maxi-
mum response to exogenous gonadotropins as measured by
seminal vesiculat weight was between 20 and 26 days. More

recently Sandler and Hall (1968) administered LH to male

24

rats at 20, 70, 240 and 360 days of age. There was no
difference in response between the latter three ages,

but injection of LH at day 20 resulted in greater conver-
sion of sholesterol to testosterone and androstenedione
with testosterone predominating. Sandler (1968) hypothe-
sized that the greater response in the young rats was due
to lower levels of endogenous LH. Mann §t_al. (1960)
could detect no change in testis weight, seminal vesicular
weight, or fructose and citric acid in calves given HCG
before 12 weeks of age. However these responses were
increased by testosterone propionate in calves of the
same age. The lack of response to HCG was probably due
to the lack of ability of the young testis to secrete
testosterone before 12 weeks of age.

Clomiphene acetate stimulates testosterone secretion
from the testis by blocking the feedback control mechanism
at the hypothalamus. Bardin gt_§l. (1967) injected 100-
200 mg of clomiphene acetate into adult male humans for
6 to 9 days and caused a small increase in plasma LH and
testosterone levels within 2 to 6 days. But after six
days there was a large increase in both criteria. In
adult human males, clomiphene citrate stimulates plasma
levels of FSH and testosterone (Kulin gt_§l. 1969). But
in the prepubertal boy, FSH and testosterone levels are
suppressed. The young hypothalamus is very sensitive to
low levels of steroids which reduce gonadotropin output.

Treatment with clomiphene citrate probably does not compete

25

completely for binding sites in the hypothalamus thus
allowing some steroid feedback to occur.

Kalra and Prasad (1967) demonstrated that clomi-
phene also inhibits spermatogenesis at the primary sperm-
atogenesis at the primary spermatocyte stage in the adult
male rat. Injections of 75 ug of FSH per day could not
counter this. However 200 ug per day of TP advanced
Spermatogenesis to the spermatid stage and when the two
treatments were combined spermatogenesis was complete.
The level of testosterone propionate administered was
sufficient to inhibit gonadotropin secretion, but for
testosterone propionate alone to maintain spermatogenesis
in the face of clomiphene citrate it required injections
of 1 mg per day. Both FSH and testosterone propionate
were required to restore testicular weight, but only 15
pg per day was necessary to maintain the accessory organs.
Work by Heller (1970) seems to qualify the previous com-
ments. He found that in normal men clomiphene citrate
had two primary actions in sperm production. At low dose
levels there was a stimulation of cell numbers but at
high dose levels he observed many abnormal Spermatids and
a fall in the number of ejaculated spermatozoa.

Methalibure has prOperties similar to clomiphene.
Skinner (1969) found that it increased testicular and
accessory organ growth rate in pubescent male rabbits.
Petry gt_al. (1968) experimented with mesteralone (l-

methyl-androstane-l7B-ol-3-one) a strong androgen which

26

exhibits no inhibition of gonadotropin secretion nor any
deleterious effect on sperm numbers. This unusual com-
bination of properties may be because the A ring of this
steroid is saturated and thus cannot be converted to
estrogens which have strong inhibitory effects at the
hypothalamus.

This survey is by no means complete as there are a
whole spectrum of drugs that have been investigated for~
properties that would hasten sexual maturity or at least
early production of fertile sperm. An attempt has been
made to outline some of the techniques, and physiological
and endocrinological interactions that offer some promise.
It is clear that the dose levels of drugs and hormones
and the period of sexual maturity at which they are admin-

istered are of prime importance.

5. Development of the Accessroy Sex Glands

The accessory reproductive glands develop under the
influence of testosterone. Before assays for blood and
testicular levels of testosterone were available, seminal
vesicular fructose, citric acid and nucleic acid were
taken as indicators of the androgenic status of animals.

Androstenedione administered to a castrated male
calf from 2.5 to 6 months of age delayed seminal vesicular
growth and secretion (Skinner gt_§l. 1968). He concluded
that androstenedione was devoid of any androgenic proper-
ties, being mainly an anabolic steroid. This view is

corroborated by Baranas (1969) who castrated 6-month old

27

calves and injected testosterone or androstenedione. Both
promoted growth and nitrogen retention, but only testos-
terone showed any androgenic properties.

Desjardin §t_§l, (1968) observed that rat seminal
vesicular RNA and DNA increased beginning around day 20
after birth. This was before the animals were sexually
mature and before the onset of fructose secretion. Rabin-
ovitch and Lutwak-Mann (1951) injected testosterone into
castrated rats, the earliest response of the seminal
vesicles was an increase in RNA levels. Ritter (1969)
performed a similar experiment, he noticed a rise in RNA
one or two days after the start of testosterone treatment
with a return to control levels by two to six days. Semi-
nal vesicular DNA started to rise one or two days after
the start of testosterone treatment and was complete by
six days. Protein concentration remained constant in the
glands. Ritter noted that similar responses occurred in
the prostrate gland, but they were somewhat more delayed.

Measurements of the growth of the seminal vesicles
(Pearce and Brown, 1970) and prostate gland (Kragt and
Ganong, 1968) of the rat revealed a rapid postnatal
growth phase for the seminal vesicle for 21 days. From
then on the growth curve was fairly flat until 40 days.
The prostrate gland had two periods of grwoth, 10 to 20
days, and 30 to 40 days; it is interesting to note that

pituitary LH declined rapidly during the last period.

28

Secretory activity of the seminal vesicles does not
seem to be correlated highly with any changes in DNA or
RNA. Porter and Melampy (1952) and Levey and Szego (1955)
could not detect a significant amount of fructose until
30 to 37 days in the rat. Seminal vesicles in guinea pigs
commenced secretory activity at 20 days, long before
spermatozoa appeared in the testis. In the bull, Abdel-
Raouf (1960) observed a marked rise in citric acid and
fructose secretion of the seminal residue at 24 weeks of
age. Fructose levels rose rapidly to 42 weeks, whereas
the rate of increase in citric acid was negligible after
36 weeks. Lindner and Mann (1960) showed an increase of
seminal vesicular secretory activity due to increasing
blood testosterone levels. Asdell (1955) noticed that
bull seminal vesicles grew slower than the body until
such a time when higher testosterone levels were expected,
but Abdel-Raouf (1960) believes the seminal vesicles
maintain a steady rate of growth until 68 months. In the
rams, Skinner (1968) detected fructose in the seminal
vesicles at birth, however citric acid was not detected
until 14 days of age and even then there existed a 10:1
ratio between fructose and citric acid. During sexual
maturation seminal vesicular fructose and citric acid
content and concentration were both correlated to plasma
testosterone levels.

Recently more evidence has been gathered to support

the hypothesis of androgenic stimulation of the growth

29

and secretion of accessory reproductive glands. Treter
(1969) demonstrated binding of testosterone by the seminal
vesicle of the rat. Unhjem and Treter (1968) injected
radioactively labelled testosterone into male rats and
found it became associated with macromolecules of the
prostrate gland, but not of the kidney or muscle. Belham
(1969) demonstrated that testosterone was taken up by the
prostate gland, but not bound specifically before it was
converted to dihydrotestosterone. In an experiment Unhjem
and Treter (1968) and Kouarski gt_al. (1969) found that
the highest concentration of testosterone was in the
prostrate gland and that dihydrotestosterone was found
nowhere else except in reproductive accessory glands.

The literature reviewed in this section indicates
that the growth and secretory activity of accessory re-
productive organs are very much dependent on circulating
androgen levels.

6. Testosterone and Androstenedione in
the Testes and Plasma as Measures
of Androgenic Status

Until recently, the androgenic status of an animal
was estimated by the growth and secretory activity of the
accessory reproductive organs because they were found to
respond uniformly to exogenous testosterone and to treat-
ments thought to stimulate endogenous testosterone secre-
tion. In intact animals, the response of the accessory

glands to androgenic stimulation is to the total androgenic

30

stimulation and may not represent one or two steroid
hormones.

In the present study testosterone and androstenedione
were measured in the blood and testes as a reflection of
the androgenic status of the bull. Several factors could
confuse the inferences which may be made from this study,
and these will be briefly discussed in light of the current
literature. Of all the androgenic compounds produced in
the body testosterone is the most potent. As discussed
earlier, androstenedione has little or no androgenic pro-
perties and most other metabolites are less potent than
testosterone. Van der Molen g£_al. (1965) considered free
testosterone in plasma reflected the androgenic status of
the male. Horton and Tait (1965) estimated that the bulk
of testosterone was secreted by the testis and not pro-
duced in the blood. Only 7% of the blood androstenedione
was converted to testosterone, however 36% of blood andro-
stenedione was formed in the blood from testosterone. The
rest was secreted by the testis. The pulmonary site is an
important site for the conversion of testosterone to andro-
stenedione. Kirschner gt_al. (1965) estimated that 20% of
the blood testosterone was produced by the adrenals, either
by direct synthesis and secretion or by conversion of
peripheral metabolites. Van de Wiele and Mcdonald (1963)
agreed with this. This value seems high and disagrees

with Horton's estimates. Rosenfeld et a1. (1969) found

31

no relationship between secretory function of the adrenal
gland and growth and development of the testis.
All of the testosterone in the blood may not be in
an active form; some may be bound to proteins. The tes—
tosterone binding affinity in the blood varies with age
in man (August, 1969; Pohlman gt_al., 1969). It is low
in newborn infants, increases in prepubertal boys, but
falls in adults. There does not seem to be any relation-
ship between testosterone levels in the blood and the
binding affinity of blood proteins for testosterone.
Several conjugated forms of the androgens are present
in the blood. These are generally regarded as excretory or
inactive forms of the steroids, but recently the steroid
conjugates and their hepatic mesenteric circulation have
been suggested as an important mechanism for control of
blood levels of these steroids. Among the conjugates,
only the sulfates of androstenedione and testosterone are
secreted by the testis (Laatikainen et_gl., 1969). The
glucuronides probably form the main excretory form (Ismail
to Loraine, 1969; Robel §E_al., 1965). Little testosterone
glucuronide is excreted. Most testosterone is reduced at
the A ring to a 58 androstane 3a-17B-doil before conjuga-
tion. Table 7 shows the levels of androgen conjugates in
the blood. It is interesting to note that the quantities

are almost as large as the free androgens.

32

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33

There are several techniques available to quantificate
androgen levels in biological fluids and probably two major
fluids from which meaningful estimates could be made.
Table 7 lists normal adult levels of testosterone and
androstenedione from the plasma of man as estimated by
various techniques. The techniques used are gas liquid
chromatography with hydrogen flame or electron capture
detection, enzymatic techniques, double isotope dilution
and derivative methods and the protein binding assay. All
of these techniques yield surprisingly similar results
considering the large variation that exists between human
subjects. Blood and urine are the two major biological
fluids in which androgen levels have been studied. Mea-
suring levels of androgens in the urine are not reflective
of levels available to the tissues.

Sampling of the blood pool may affect the androgen
picture. Ismail and Loraine (1969) demonstrated a cir-
cadian rhythm of testosterone secretion in the blood of
man. Seiki et_gl. (1968) showed that testosterone levels
are higher in the inferior vena cava as compared to the
carotid artery in rabbits. These factors and stress
factors could all add some variability to the levels of
androgens estimated from the blood sample.

Taking everything into consideration, levels of
testosterone in the plasma and testes are perhaps the
most reliable estimates of the androgenic status of an

animal. Androstenedione is probably eliminated in this

34

respect and blood levels of this steroid are confusing
because of its adrenal secretion and the conversion of

testosterone to androstenedione for excretory purposes.

MATERIALS AND METHODS

1. Development of Techniques

 

a) Objective

 

Information on methodology for the assay of plasma
and testicular levels of testosterone and androstenedione
was scarce and incomplete. No routine methods were
available for fast accurate assay of these two androgens
in tissues for any species. The reported methods are
tedious, time consuming, and untested with sufficient
numbers to render them generally acceptable. Most of
the analytical problems were related to the extreme
purification of the androgens required by the high sensi-
tivity required to measure the minute quantities of andro-
gens in bulls. My aim was to develop an accurate techni-
que that would be relatively simple in operation and
sufficiently rapid that it could be operated routinely

for the assay of large numbers of biological samples.

b) Introduction

 

The quantification of testicular and plasma andro-
gens demands a very sensitive technique as the levels of

these hormones found in bull tissues are in the range of

35

36

nanograms per milliliter of blood. Previous to 1960, no
such technique existed. ug and mg levels of androgens
had been assayed in bioassays such as the Cockerel comb
assay and secretory responses of various accessory repro-
ductive organs, these bioassays are not sufficiently
sensitive for measurement of androgens in bulls. Spec-
trophotometric techniques seemed feasible, but they also
lacked the sensitivity required by the low levels in
most biological samples.

Chromatography in general works on the basic prin-
ciple that the material to be purified or the mixture to
be resolved is repeatably distributed between two phases,
one is stationary, while the other flows past it. In gas
chromatography, the mobile phase is gaseous.

In 1952 James and Martin showed that it was possible
to separate compounds using gas liquid partition chroma-
tography. Various compounds were investigated following
this, such as fatty acids and organic amines. Several
gas phase detectors were developed including the hydrogen
flame ionization detector between 1957 and 1958.

During 1959-1960 the possibility of achieving a
separation of steroids by gas liquid chromatography was
under study in several laboratories. The main problem
was that the solid phases then available would bind
steroids tenaciously except at very high temperatures

when the steroids would break down.

37

Vandenheuvel, Sweeley and Horning, 1960, demonstrated
that thin films (1 to 3% of liquid phase) could be used
for separation of many steroids without loss of functional
groups at temperatures only a little higher than 200C and
with retention times of as little as 15 minutes. This
paved the way for the use of thin film columns, prepared
with deactivated supports such as silanized acid washed
diatamaceous earth.

The use of gas liquid chromatography for separation
and quantification of steroids quickly evolved. A simpli—
fied scheme showing the adaptation of gas liquid chroma-
tography for isolation and purification of steroids
follows. A gas column 3 to 6 feet long, 0.4 mm i.d. is
filled with finely divided inert solid, such as diatama-
ceous earth or ground fire brick impregnated with a non-
volatile liquid phase. The mixture of compounds to be
analyzed is introduced to the column, and flash evaporated
at one end of the column which is held at constant tempera-
ture throughout its length. The volatilized compounds are
swept through the column by a constantly flowing stream
of an inert gas such as argon, helium or nitrogen. Each
component of a mixture of compounds moves on the column
at a rate determined by its ratio of partition between
the gas phase and the nonvolatile liquid (stationary)
phase. Individual compounds in the gas emerging from the
column are usually detected by physical or chemical means.

Data are automatically recorded on a chart as a series of

38

peaks. The area under each peak is proportional to the
quantity of that component in the mixture, which is
identified by standards.

The hydrogen flame detector was used in our early
studies and a brief introduction to the theory of its
operation is warranted at this point. The sample is
swept by the carrier gas through the column and into the
hydrogen flame. As it enters the flame, molecules are
ionized, forming positive and negative ions. The extend
of this ionization depends on the nature of the compound
(molecular structure, degree of unsaturation, etc.), and
the temperature of the flame. An electric potential is
applied between the hydrogen jet and a collector ring
above it so that the jet is negatively charged and the
collector positively charged. When a compound is ionized
the electrons are attracted to the collector and flow via
a biasing circuit and create a voltage drop across the
input load resistor. This is amplified by the electro-
meter and the output is presented on a potentiometric
recorder in the form of a chromatographic peak. The
response of the ionization detector depends on the number
of molecules per unit time entering the detector. Thus,
increasing the flow rate or increasing the flame tempera-
ture would increase sensitivity. The degree of ioniza-
tion is roughly prOportional to the number of carbon atoms
per molecule in any given organic compound. Inorganic

compounds such as hydrogen, nitrogen, carbon dioxide and

39

and water are not ionized and hence not detected. A general
rule is that a compound must have either a carbon-carbon
linkage or a carbon-hydrogen linkage to be detectable with
hydrogen flame detectors.

According to Guerra-Garcia et_al. (1963) sensitivity
of the hydroqen flame detector for adequate testosterone
detection is 0.05 mg. This was obviously not sensitive
enough to accurately quantify plasma levels of androgens,
but seemed sufficiently sensitive to quantify testicular
levels. Based on estimates by Lindner and Mann (1960) I
expected to find approximately 6 pg of testosterone in 4 to
5 g of testicular paranchyma. I thought this may be enough,
with careful calculation to produce a quantifiable response

using the hydrogen flame detector.

c) Use of the Hydrogen Flame Detector

 

We homogenized 4 to 5 g of testicular tissue and
extracted it with diethyl ether following the purification
scheme of Armstrong §t_al. (1964). The extract was dried
and transferred to a silica gel thin layer plate which
was developed in two dimensions; first in hexane:ethyl
acetate (5:2 vv) and second in methylene dichloride:
diethyl ether (5:2 vv). The areas corresponding to stan-
dard androstenedione and testosterone on the thin layer
plates were scraped from the plate. Steroids in the
scraped silica gel were eluted and the androgens were

taken up in methanol for gas liquid chromatographic analysis.

40

Standard curves for testosterone and androstenedione gave
a linear response from 50 to 1,000 ng. I did not test
less than 50 ng. My extracts of testes contained severe
organic contamination. The most sensitive setting on

the gas chromatograph (without completely masking the
androgen peaks with contaminant peaks) revealed approxi-
mately 4 ng of testosterone. At a 1 ml dilution, this
was equivalent to 4 ug of testosterone in the 4 to 5 g

of testis extracted. Under these conditions of purifi-
cation, 16 ng injected into the gas liquid chromatograph
(peak of 2 cm2) was the lower limit for accurate quanti-
fication. Further work revealed that the bulk of contami-
nants were from the testis extract, and the extraction
efficiency was less than 10%. Thus, it appeared that a
better method of purification and a more sensitive detec-
tion system were required to quantify testicular and
especially plasma levels of androgens.

This preliminary work did however reveal retention
data. The data for testosterone and androstenedione on
various columns and at various conditions are summarized
in Table 8. Column packing, carrier gas flow rate and
oven temperature all influence retention time. Liquid
phases can be divided into nonpolar or nonselective phases,
and polar or selective phases. OV-l and SE-30 are non-
selective phases separating steroids mainly on a basis of
molecular size and shape. Thus retention times for any

percentage liquid phase are shorter with less difference

41

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42

between steroids than for selective phases such as QF-l
or XE-60. The latter phases separate steroids more on
the basis of charge properties of the molecules. Both
increasing the carrier gas flow rate and increasing oven

temperature decrease the retention time.

d) Electron Capture Detector

 

Browrie §t_§l. (1964) suggested that the electron
capture detector was 1,000 times more sensitive than the
hydrogen flame detector when haloacetates of steroids
were quantified.

The functional mechanism of electron capture detec-
tion is based on reduction of an electric current flow due
to the removal of free electrons from the system by sam-
ple components eluted from a gas chromatographic column.
The current is produced by a radioactive tritium foil
emitting electrons (beta radiation) which flow between
an anode and a cathode. The drop in current due to capture
of electrons by materials eluted from the column is
recorded, amplified and presented as a peak on the
recorder. The potentialacross the cell is applied as a
pulse. This increases the dynamic range of linear
response as it prevents large ion migration and plating
out of large ions on the cell foil which could reduce
sensitivity. An inert nonelectron capturing carrier gas
must be used such as 90% argon, 10% methane. No purge
gas is necessary unless quantities of sample greater than

1 09 are introduced onto the column. The response of

43

substances in this detection system depends on their
ability to capture electrons. Cargon and hydrogen do not
capture electrons readily. Thus many organic solvents

can be used because they give no response. Oxygen captures
electrons readily, and alcohols to a lesser extent; thus
most steroids should produce a reasonable response. Water
and many inorganic and organic contaminants will also
produce a response; thus the carrier gas must be very dry
and the samples extremely pure. Halogens capture elec-
trons readily and consequently linking of halogens to the
steroid molecule increases their electrol capture response,
as will be developed later. One disadvantage of the
tritium foil detector is its breakdown and release of
radioactivity at high temperatures. The temperature of
the detector should be kept below 225C. Unfortunately

this causes long retention times and necessitates columns
with a low percentage of nonpolar liquid phases.

Free androstenedione and testosterone standards
quantified using the electron capture detector showed that
the detector was approximately 25 times more sensitive to
androstenedione and 15 times more sensitive to testosterone
than hydrogen flame detection. The difference between the
two androgens is probably due to the extra oxygen group on
androstenedione.

With the increased sensitivity of the electron cap-
ture detector I decided to try to quantify free testo-

sterone and androstenedione. The main problem was

44

the large solvent and contaminant front produced by the
biological samples. I planned to separate the two andro-
gens from the contaminants and from each other at rela-
tively low temperatures on nonselective columns by selec-
tive derivitization of steroids only. I decided to
investigate the properties of trimethylsilyl and O-methyl-
oxime trimethyl silyl derivatives. The derivatives were

prepared by the method of Luukainen et al. (1961).

e) Trimethylsilyl Ether of Testosterone

 

Pyridine (0.2 ml) and 0.15 ml of hexamethyl disila-
zine were added to microgram amounts of testosterone,
0.05 ml of trimethyl chlorosilane also was added as a
catalyst. The reaction continued over night in a
dessicator.

The mixture was extracted with 0.5 to 0.3 m1 of
hexane and centrifuged, and the hexane was transferred
to another tube. The mechanism of reaction with the
hydroxyl group is unknown. According to Chambaz and
Horning (1969), the hydroxyl on the seventeenth carbon of
testosterone should be completely derivatized with the
20% trimethyl chlorosilane catalyst mixture that I
employed. Gas liquid chromatographic analysis on a 1%
SE-30 column, however, did not show one clear peak.
Whether this was due to incomplete reaction or separation
of the isomers etc. was not determined as I was not so
much interested in the mechanics as using the derivatives

as a tool in quantitative analysis.

45

f) O-Methoxime-Trimethylsilyl Derivatives
of Testosterone and Androstenedione

 

0.2 m1 of a solution of 14 mg of methoxyamine hydro—
chloride in 1 ml of pyridine were added to the sample.
The reaction mixture was left at room temperature in a
dessicator overnight. Hexamethyl disilazine (0.5 ml) was
added the next morning. After a further six hours, the
reaction mixture was dried under nitrogen and extracted
with hexane. In the second part of the reaction the
methoxyamine and hydrochloride acts as a catalyst.
Ketones at the three and seventeen positions should be
readily converted to the O-methyloxime-trimethylsilyl
derivatives.

A second technique of adding the hexamethyl disila-
zane with the pyridine and methoxyamine hydrochloride
overnight gave a better conversion of the steroids to
the O-methoxime-trimethylsilyl derivative.

Both androstenedione and testosterone-O-methoxime-
trimethyl silyl derivatives gave a single peak with elec-
tron capture on a 1% SE-30 column. The reaction mecha-
nisms are not clear. Testosterone forms an O-methoxime-
trimethylsilyl derivative at carbon 3. I did not investi-
gate the derivative to ascertain whether a trimethylsilyl
derivative also had formed at carbon 17. The position of
derivative groups on the androstenedione molecule also
were unknown; both carbon 3 and 17 are easily derivatized

in this reaction.

46

All derivatives gave a lower response with the
electron capture detector than the free steroids. The
lowered response seemed to be proportional to the removal
of the oxygen and hydroxylelectron capturing groups.
Greatest response was seen at a pulse interval of 50
and a carrier gas flow rate of 40 ml per minute. Reten-
tion data for these derivatives are listed in Table 9.

It will be noted that the trimethylsilyl group increases
the retention time markedly but the combined O-methoxime-
trimethylsilyl group does not increase the retention time
much more.

On a nonselective column, retention times depended
on molecular shape and size, and the increased molecular
size of the derivatives would enhance retention times and
aid separation. This property would be more applicable
to group separations and qualitative analysis. As the
derivatives did not enhance sensitivity nor the problem
of contaminants, I used them no more. An important use
of trimethylsilyl derivatives is to prevent thermal deom-
position. An example of this is the loss of the side
chain for steroids of the andrenocorticoid group containing
the cortisone or cortisol side chain. Direct gas liquid
chromatographic analysis of these results in loss of
the side chain, but the O-methoxime-trimethylsi1y1 deri-
vatives of these steroids are stable under most gas liquic

chromatographic conditions.

47

TABLE 9.--Some Retention Time Dataa for Trimethylsilyl and
O-methoxime-Trimethylsi1yl Derivatives of
Testosterone and Androstenedione.

 

 

Carrier gas Compound Analyzed Retention
flow rate times
(ml/min) (mins)
35 Androstenedione 12.5b
40 Androstenedione 12.0
60 Androstenedione 10.1
40 Testosterone 14.0
30 Testosterone trimethylsilyl ether 22.0
40 Testosterone trimethylsilyl ether 16.0
60 Testosterone trimethylsilyl ether 14.0
40 Testosterone methoxamine 15.0
40 Testosterone methoxamine
trimethylsilyl ether 17.0
40 Androstenedione methoxamine
trimethylsilyl ether 18.0
30 Androstenedione methoxamine 15.0
40 Androstenedione methoxamine 14.8

 

aColumn temperature, 180C; electron capture detector, 212C;
flash heater, 207C; and 1% SE-30 columns.

b . . .
Retention times are averages of several observations.

48

At this point it seemed necessary to try to reduce
the levels of organic and inorganic contamination of the
extracts analyzed by electron capture gas liquid chroma-
tography, and to try to find some means of increasing
the response of testosterone and androstenedione in the
electron capture detector. The latter problem was tackled

first.

9) Halogen Derivatives of Androgens

 

As stated before halogens are strongly electron
capturing. If a large molecule containing many halogen
units could be attached to the steroid nucleus extremely
high sensitivity could be attained.

Brownie gt_al. (1964) described a technique for the
determination of plasma testosterone utilizing gas liquid
chromatography with electron capture detection. This
technique involved forming a chloracetate derivative of
testosterone. The lowest level that could be accurately
quantified was stated to be 1 ng.

For nanogram quantities of testosterone the techni-
que for monochloracetylation was as follows. One-half
m1 of monochloroacetic anhydride in tetrahydrofuran
(100 mg/10 ml) and 0.1 ml pyridine were added to the
purified androgen extract. The reaction continued over
night at room temperature in a dessicator. One ml of
water was added to stOp the reaction. The reaction solu-
tion was extracted with 1 ml ethyl acetate three times.

The pooled extracts were washed in 1 m1 of 6 N

49

hydrochloric acid twice, and twice in 1 ml of distilled
water. Finally it was dried and taken up in hexane.

This was a reaction with the hydroxyl group at carbon 17.
The yield of derivative was high but to remove any underi-
vatized steroid Brownie purified the derivatives on thin
layer chromatography. However my experience showed the
chloracetate derivative to be unstable on thin layer
chromatography. The monochloracetate of testosterone
had an extremely long retention time on most gas liquid
chromatographic column phases. For these reasons and
the fact that new halogen derivatives existed with
shorter retention times, greater molar response, greater
ease of preparation and greater stability, I decided to
experiment with heptafluorobutyric anhydrides. This
steroid derivative depended for its sensitivity on
attaching a 7 fluorine chain at one or more positions on
the steroid nucleus. It was claimed that derivatives of
this type could lower the limit for accurate quantifica-
tion to 0.1 ng, a level that should make the assay of
blood androgen levels feasible.

Clark and Wotiz (1963) first used heptafluorobutyrate
esters. Several methods of preparation have been suggested.
Clark and Wotiz (1963) dried the extract of 1 m1 of plasma
under nitrogen and reacted this with 2 pl of heptafluoro-
butyric anhydride and 1 ml of tetrahydrofuran in 1 ml of
hexane for 30 minutes at 60C. The reagents were then

driven off under nitrogen at 60C and the derivatized

50

samples were dissolved in 50 pl of hexane. This method

gave the 3, 17-diheptafluorobutyrate of testosterone and
3-monoheptafluorobutyrate of androstenedione. Table 10

summarizes several techniques and their sources.

When preparing the monoheptafluorobutyrates the
level of pyridine was critical. If it was too low, the
diheptafluorobutyrate formed. If it was too high,
recoveries were very low. In my initial attempt to pre-
pare heptafluorobutyrate derivatives of testosterone and
androstenedione, varying amounts of steroid were used,
from 0.5 to 500 pg. A 1:1 ratio of benzene to hepta-
fluorobutyric anhydride was added to the steroids which
were then heated to 70C for 40 minutes. The yield of
this reaction was believed to be greater than 75% but
tritiated androgens were included with each reaction to
check yields. The reagents were evaporated under nitro-
gen at 60C and the residue was transferred with 0.5 m1
of acetone three times to a cellulose thin layer plate
(Whatman CC .41). The thin layer plates were developed
in acetone:water (3:1 v/v) and required 2.25 hours to
develop. Under ultra violet light, many ultra violet
absorbing areas were apparent, and the radioactivity was
spread from the origin continuously to the solvent front.
These observations suggested that either the derivatization
had failed or that the derivatives were unstable on cellu-

lose thin layer plates.

l
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52

Next, I attempted to use a derivatization procedure
using 1 ml of benzene, 100 pl of pyridine and 20 pl of
heptafluorobutyric anhydride. Again the reactants were
heated at 70C for 1 hour. The reaction was stopped by
addition of N hydrochloric acid, and the mixture was
washed three times with distilled water. The derivatives
were finally extracted with chloroform and transferred to
a silica gel thin layer plate which was developed in
benzene:ethyl acetate (4:1 v/v).

Inspection of the plates under ultra violet light
revealed no ultra violet light absorbing areas with an
RF similar to that of underivatized testosterone, but
two areas were found with RF greater than underivatized
testosterone. These were probably the mono- and
diheptafluorobutyrate esters, as the replacement of
hydroxyl or ketone groups by less polar groups should
decrease the polarity of the steroid, hence increasing
its RF on silica gel. Androstenedione gave an ultra
violet light absorbing area with an RF greater than
underivatized androstenedione. This was probably the
monoheptafluorobutyrate ester. However the yield was very
low and most of the ultra violet light absorbing material
migrated with the RF of underivatized androstenedione.
Derivatives prepared by the former method (involving only
benzene and heptafluorobutyric anhydride) also gave the
same ultra violet absorbing areas on silica gel thin layer

plates developed as above. The plates still showed some

53

evidence of breakdown products, various Spots of ultra
violet light absorbing areas appearing between the major
areas mentioned above for the derivatives prepared by
the technique involving pyridine.

Gas liquid chromatographic analysis of these samples
showed major peaks with retention times similar to those
later proven to be the mono- and diheptafluorbutyrate
esters of testosterone. Many contaminant peaks were
present, their nature was unknown but they were probably
breakdown products and various contaminants from glassware,
solvents, etc. The best results were obtained for
testosterone by increasing the amount of pyridine from
100 pl to 300 p1 in the second technique outlined at the
beginning of page 52. A very dense monoheptafluorobutyrate
area was noted on the thin layer plate, while no free
steroid and very little diheptafluorobutyrate were found.
Much of the interfering ultra violet material was elimi-
nated by prerinsing all glassware with acetone. The
efficiency of the reaction was improved by using only
freshly glass distilled benzene, (used for 1 week and then
redistilled). Pyridine was also glass distilled and
stored over potassium hydroxide. Only fresh heptafluoro-
butyric anhydride was used (used with 2 to 3 months of
preparation). Thus the following technique was evolved

for testerone.

54

h) Monoheptafluorobutyration of Testosterone

 

To approximately 10 pg of testosterone, 2 or 3 drops
absolute ethanol was added and dried to remove water and
1 m1 of a solution of heptafluorobutyric anhydride in
benzene (0.2 m1 of heptafluorobutyric anhydride in 10 ml
of benzene) was added to the evaporated eluate. Pyridine
(0.1 ml) was originally added but 0.2 or 0.3 ml or more
seemed necessary to prevent the formation of the dihepta-
fluorobutyrate. The reaction was performed in glass
stoppered tubes for 1 hour at 70-80C. The mixture was
then washed with 1 ml of l N hydrochloric acid and then
twice with 1 m1 of distilled water, vortexing each time
for at least 15 seconds. The lower acid and water layers
were removed with a Pasteur pipette and the tubes were
centrifuged in a milk testers centrifuge for five minutes
after removal of the bulk of the last wash to get the
tubes as dry as possible. The remaining sample was then
dried under nitrogen at 60C and taken_up in acetone for
thin layer chromatography on silica gel benzene:ethyl
acetate (4:2 v/v).

The monoheptafluorobutyrate of testosterone appeared
as a single peak with a retention time of 13 minutes on a
1% SE-30 column, an oven temperature of 203C, and a carrier
gas flow rate of 40 ml/minutes. The introduction of the
heptafluorobutyrate group to the testosterone molecule
increased its response with the electron capture detector

1,000 fold. Having improved my techniques it was then

55

possible to prepare the androstenedione monoheptafluoro-
butyrate by the method of Exley gt_§l. (1967) using equal
amounts of benzene and heptafluorobutyric anhydride. The
derivative was purified on cellulose thin layer plates or
silica gel plates in the systems previously outlined

(see page 52) with only minor breakdown. It gave a single
peak on a 1% SE-30 column with a retention time of 6.5
minutes, under the same conditions outlined (see page

54) for testosterone. Blanks run through the derivatization
techniques gave no response on analysis by gas liquid
chromatography. Greatest reSponse to the androgen deri-
vatives was obtained with a pulse interval of 50 micro-
seconds and a gas flow rate of 40 ml/minute or less.

Both techniques of thin layer chromatography still caused
some breakdown of the derivatives with a resultant loss

of steroid.

Challis and Heap (1969) used gel filtration on
sephadex LH-20 to purify the heptafluorobutyrate of
estrone, l7-B-estradiol and 20-8 hydroprogesterone. They
showed that the heptafluorobutyrates were stable under
purification, probably because they were never exposed to
the atmosphere. Sephadex LH-20 was not practical for our
use as the androgens were similar in molecular size,
shape, and polarity and I could not separate them from
their heptafluorobutyrates on a molecular sieve basis in
a range of solvent systems. Assuming that exposure on a

thin surface was responsible for the breakdown of the

56

derivatives, various solid phases were experimented to
develop a useful separational procedure. Columns (5 x

50 mm) were packed with silica gel GF-254—E (Merk A.G.
Darmstadt). A mixture of benzene:ethyl acetate (95:5 v/v)
was chosen after many attempts with other solvents, as it
seemed to separate the heptafluorobutyrates from the unde-
rivatized steroids. The column had a void volume of

0.5 m1 and ran at a rate of 0.1 ml per minute. Table 11
summarizes the fractions in which various compounds were

eluted from the column.

TABLE 11.--Solvent Fractions Containing Various Steroidal
Hormones and their Heptafluorobutyrate Deriva-
tives as Eluted from Silica Gel Microcolumns.a

!

 

!

 

Steroid Fraction Steroid Fraction
Testosterone 2.5 to 3 ml b
testosterone MHFB l to 2 ml
Androstenedione 3.5 to 4.5 ml
androstenedione MHFB 1.5 to 2.5 ml
Progesterone 2.7 to 3.2 m1
progesterone MHFB 1 to 2 ml

 

aSilica gel column (5 x 50 mm) eluted with benzene:ethyl
acetate (95:5 v/v).

bMonoheptafluorobutyrate.

57

Fractions (0.5 ml) were collected and the elution
patterns were ascertained by incorporating tritium labelled
steroids and by gas liquid chromatography of the frac-
tions. Although there was no breakdown of the derivatives
during purification with silica gel and no contaminants
were found, the chromatographic separation of the deriva-
tives from free steroids was not good and not always
repeatable.

Consequently columns were packed with 60 to 80 mesh
florosil (Fisher Scientific Company, New Jersey, USA) to
the same dimensions as the silica gel columns. Steroids
on these columns were eluted with various solvent systems
and Table 12 shows the solvent systems and separations
achieved.

The underivatized steroids came off very slowly and
had to be flushed off with a more polar solvent such as
methanol. Gas liquid chromatographic analysis (electron
capture detection) showed that there had been no breakdown
of the derivatives during purification on florosil micro—
columns. The samples were however heavily contaminated
and this was remedied by washing the florosil in methanol
and distilling all solvents in glass before use. It was
also noted that even tygon tubing was susceptible to
extraction by organic solvents giving intolerable contami-
nation.

This system of purification was used only in the

proof of our assay because so little free steroid was

.mumnmu590H05Hmmummnocon

.AEE cm x mv QESHOOOHUHE HHmouonm

 

 

8 as m ou H mmmz macapmsmpmonosm HE NHA macetmsmumontcd A>\> m.o"muHmv
5 Hmumsumumumom Hanumnmcmncmm
HE n cu H mmmz mcoumumoumms HE mHA msoumumoumme H>\> m.oumuHmv
Hmumzumumumom Hanumumcmusmm
HE m on H mmmz msoumummmoum HE mHA msoumummmoum A>\> mummy
Q mumumom Hanumumcmucmm
GOHuomum owoumum soauomum owouwum Emummm ucm>Hom

 

m.mcezHooouon HHmouon Eoum omusHm mm mm>Hum>Humo
mumuxusnouosHmmummm HHmca can mpwoumum msoHHm> mcHCHmucoo mcoHuomumll.mH mHm<B

59

ever detected when the heptafluorobutyrate was purified.
The yield of this reaction was probably better than 95%.
Mass spectral analysis and the repeatability of estimates
as shown in the Results and Discussion section made it
unnecessary to purify the heptafluorobutyrate derivatives
from unreacted free steroids as long as extreme care was
taken in their preparation (see page 74).

The preparation procedures were modified before the
assay was used routinely. All extracted and isolated
steroids were dried under nitrogen at 60C and vacuum
dessicated for a minimum of 12 hours before derivatization
to remove all moisture. Androstenedione monoheptafluoro-
butyrate was prepared as previously outlined and proges-
terone monoheptafluorobutyrate was prepared in the same
way. The original method for preparing testosterone mono-
heptafluorobutyrate was discarded as pyridine was very
difficult and noxious to work with, also traces of dihepta-
fluorobutyrate nearly always formed. I decided to use the
same technique as for androstenedione to prepare the
diheptafluorobutyrate of testosterone. The final scheme
of preparation is shown in the section below on the final

methods used.

1) Development of an Extraction and
Purification Scheme for Plasma
and Testicular Testosterone and
Androstenedione

 

 

 

 

From my experiences with preparing and analyzing

standard androgen heptafluorobutyrate ethers by gas liquid

60

chromatography and electron capture detection, reagents
and glassware used for routine laboratory work required
scrupulous purification and cleaning to keep contamination
at an acceptable level. Reagent requirements and techni-
ques for preparation of glassware are outlined in the
final materials and methods for routine assay section
below.

My original scheme for the purification of andro-
stenedione and testosterone (see page 39) was obviously
unacceptable for gas liquid chromatography with hydrogen
flame or electron capture detection. The main problems
were poor extraction efficiency and poor removal of
organic contaminants.

Surace et_al. (1965) presented a scheme for purifi-
cation of plasma testosterone. Briefly it was as
follows. Blood plasma was extracted three times with
three volumes of diethyl ether. The pooled extracts were
then washed with 10 ml normal sodium hydroxide three times,
once with 10 ml of 3% sodium bicarbonate and three times
with 10 ml of water. The extract was then dried and
redissolved in 10 m1 of 70% methanol. This was stored
for 12 hours at -18C and centrifuged at the same tempera-
ture, and the supernatant fluid was removed. The precipi-
tate of neutral lipids was washed with 5 m1 ice cold 70%
methanol. The supernatant fluid was washed twice with
5 ml of petroleum ether. The petroleum ether was re-

extracted with 10 m1 of 70% methanol. The pooled methanolic

61

extracts were diluted with an equal volume of water and
back extracted three times with 20 ml of benzene. The
benzene extract was dried and chromatographed by ascending
thin layer chromatography using the solvent system
benzene:ethyl acetate (60:40 v/v).

The main advantage of this scheme seemed to be the
early removal of neutral lipids. It was modified initially
as follows. Four to five g of testicular parenchyma were
homogenized in saline and extracted with a minimum of two
volumes of ether three times. A ratio of ether to homo-
genate less than 2:1 resulted in emulsions. The ether
extracts were dried and taken up in 25 ml of 70% methanol
and stored at -15C overnight. The next day they were
centrifuged at 1,200 g and -20C for 20 minutes. At first,
an attempt was made to dry the 70% methanol supernatant,
but as this took so long it was diluted with an equal
volume of water and back extracted with 30 m1 of benzene
three times. This partition often resulted in emulsion,
but 30 ml of benzene minimized this problem while volumes
less than this nearly always resulted in an emulsion. The
benzene was evaporated and the extract applied, relatively
free of neutral lipids, to silica gel thin layer plates.
The plates were developed using ascending thin layer
chromatography. The first solvent system was hexane:
ethyl acetate (5:2 v/v) to remove any remaining traces of
neutral lipids from the steroids, and the second at an

angle of 90° to the first was benzene:ethyl acetate

62

(60:40 v/v), to separate the androgens from each other and
other steroid hormones. Testosterone and androstenedione
were eluted, derivatized and analyzed by gas liquid
chromatography with electron capture detection. These
samples still gave a large solvent front on the gas
chromatograph when analyzed. Inorganic contaminants from
the silica gel were suspected of contributing to this.
Before further use all silica gel was washed three times
with methanol. Analysis of extracts of silica gel before
and after washing showed that a large contaimination-was
contributed by silica gel and that this could be removed
by methanolic washing.

Several thin layer plates were sprayed with concen-
trated sulphuric acid to visualize steroids and possible
contaminants (see Figure l). The neutral lipids had
migrated further than androstenedione or testosterone
but the latter two were not separated well and were con-
taminated by lipoproteins.

Nienstadt (1967) proposed a technique for defatting
steroids. This technique was tested in an attempt to
remove every nonsteroidal lipid component of the ether
extracts. The dried ether extract was dissolved in 5 ml
of 5% octanol in hexane and applied to a 5 x 50 mm florosil
microcolumn. Fatty impurities were eluted from the column
with 20 m1 of 5% octanol, and the octanol was displaced
from the active sites of the florosil with 5 ml of 1%

ethanol in hexane. The steroids were then eluted with

 

Z:solvent L—l_°'_,—

front

III

solvent
“from
solvent
system:

origin

 

 

 

Solvent system:

Figure l.—-Identification of Steroidal Constituentsa of a
Bovine Testicular Extract on Thin Layerb
Chromatography Plates.

 

aAreas showing colour reaction with concentrated sulphuric
acid are (l) testosterone, (2) androstenedione, (3) pro-
bably conjugated lipo proteins, (4) dehydroepiandrosterone,
(5) cholesterol and (6) esterified cholesterol.

bSilica gel GF-254.

CSolvent systems are (1) hexane:ethyl acetate (5:2 v/v) and
(2) benzene:ethyl acetate (6:4 v/v).

64

10 m1 of ethyl acetate. Based upon addition of isotopic
testosterone and androstenedione to the ether extracts

and liquid scintillation counting of the various extracts,
I estimated that 90% of the androgens were eluted in the
ethyl acetate fraction. This fraction was develOped on
thin layer chromatography (as outlined above), and visuali-
zation of the various components was achieved with concen-
trated sulphuric acid. The quantity of cholesterol was
much reduced as was the component that previously over-
lapped androstenedione and testosterone. However gas
liquid chromatographic analysis of the two androgens still
revealed some interferring peaks with retneion times
similar to the two androgens and a smaller but still
unacceptable front.

As this technique was tedious and the resultant
androgenic fractions required further purification, I
decided to try to improve the methanolic precipitation of
lipids and design a new thin layer chromatographic system
to purify and separate steroidal hormones. The results
of this work comprise the present method used for routine
assay of testosterone and androstenedione outlined on
page 66 to page 78.

Recovery of androstenedione and testosterone as
measured by the recovery of labelled steroids ranged from
35% to 65% (with a few extreme values), with a mean of
50% over twenty samples. Table 13 shows the counts per

minute collected from the various fractions and containers

65

TABLE l3.--Average Distribution of Radioactivity in Various
Discarded Fractions and Containers for a Total
of Twenty Samples.

 

Fraction/Container Counts per minute

 

Cylinders in which testicular extraction

performed 29a
Heptane (pooled) 880
Diluted methyl alcohol 960
Fatty Precipitates 2,000
Tubes from which residue transferred to a
TLC plates 36
Amount of radioactive steroid added to
testicular tissue,
3H-l,2-testosterone 8,700a
3H-7-androstenedione 6,050
Total 14,750
Total losses 3,840 (26%)

 

aConsidered to be background.

66

after they had been used in the extraction and purification
system. The fatty precipitate accounted for most of the
androgen lost and the heptane and methanol partitions
accounted for additional losses. The remaining loss was

in the thin layer chromatography step. Not all of the
androgens were scraped from the plates, but the loss was
not estimated.

Several water blanks were extracted and run through
the assay. None produced any response upon analysis by
gas liquid chromatography with electron capture detection
at a retention time similar to that of either androgen
assayed. Steps taken to check the authenticity, accuracy
and precision of the assay are discussed in the Results
section.

2. Final Materials and Methods for the Routine

anntification of Testicular and Plasma
Testosterone and Androstenedione

using Gas Liquid Chromatography
with Electron Capture Detection

 

 

 

 

a) Preparation, Storage and Treatment
of Materials and Samples

 

 

Reagents.--All solvents were nanograde (Mallinckrodt),
with the exception of heptane which was spectrophotometric
grade (Mallinckrodt), and all were used without further
distillation. Acetic acid was analytical grade (Mallin-
ckrodt), and water used was double glass distilled.

Heptafluorobutyric anhydride was obtained from K and

K Laboratories (Plainview, New York) and Peninsula

67

Biochemicals (Florida) in 10-g lots, and was stored

in brown glass bottles in a dessicator. A portion of the
heptafluorobutyric anhydride was prepared in our laboratory
by the method of Clark and Wotiz (1963). Freshly distilled
heptafluorobutyric acid (5 ml) was refluxed with a large
excess of phosphorus pentoxide (60 g) for 1 to 2 hours.
Heptafluorobutyric anhydride was distilled over phosphorus
pentoxide and the fraction which boiled between 108 to

110C was collected.

When stored unopened the heptafluorobutyric anhy-
dride was used up to nine months after purchase with no
detectable deterioriation, however when the bottle was
opened, signs of breakdown of the reagent appeared over
a period of five months. Benzene to be used in the
reaction to derivitize standards also was stored in the
dessicator.

Glassware.--All glassware was washed four times with

 

Alconox detergent, (Alconos Incorporated, New York) and
rinsed in tap water five times, in distilled water five
times, and in double distilled water five times, and then
air dried. Finally, before use, all glassware was rinsed
three times in nanograde acetone and air dried.
Steroids.--Standard testosterone (A4-androsten-l7 B
ol-3one), androstenedione (A4-androsten-3, l7-dione) and
progesterone (A4-pregnen-3, 20-dione) were purchased from
Nutritional Biochemicals Corporation (Cleveland, Ohio) and

. . . . H
used without further purification. 3 -7-progesterone and

68

3H--7-A4 androstenedione were purchased from Nuclear
Chicago (Des Plaines, Illinois), 3H-l,2-progesterone from
New England Nuclear (Boston, Massachusetts), and 3H-1,2-
testosterone from Amersham Searle (Des Plaines, Illinois).
All isotopes were repurified by thin layer chromatography
in the same systems as used in the assay system.
Measurement of Radioactivity.--Radioactivity was
measured in a Mark 1 liquid scintillation spectrometer,
manufactured by Nuclear Chicago (model number 6860). This
was equipped with a teletypewriter print out system (Tele-
type Corporation, Stokie, Illinois) with facilities to
punch counts per minute onto a computer tape. This sytem
was used throughout the study and two scintillation
fluids were employed. Brays solution, consisting of 4 g
PPO, 0.2 g dimethyl POPOP, 60 g napthalene, 20 m1 ethylene
glycol, 100 m1 methanol and 816 ml of paradioxane, in
approximately 1 litre of solution, was used when the
sample to be counted contained water or could not be
dried. It was found to be excellent in minimizing quench-
ing and gave most efficient radioactivity counting under
these conditions. The second scintillation fluid was
designed eSpecially for efficient counting of steroid
isdtopes and consisted of 7.5 g PPO, 75 g POPOP, 120 g
napthalene, 500 ml xylene and 500 ml paradioxane. All
samples were counted in glass vials (various sources),
generally 10 ml of Brays solution or 5 ml of steroid

scintillation fluid were used.

69

Gas Ligpid Chromatography.--A Hewlett Packard Model

 

402 gas chromatograph was used throughout this study. The
instrument was a dual column type, equipped with two
hydrogen flame detectors. A tritium foil electron capture
detector was mounted on one column. This column had two
exit ports allowing the use of either the hydrogen flame
or the electron capture detector.

Experimental Animals, Collection and Storage of

 

Samples.--a)Holstein Bulls. Sixty-five Holstein Friesian
bulls were used in this study which was part of a larger
study conducted by K. L. Hacmillan to investigate endo-
crine and reproductive development of the Holstein bull
from birth through puberty. Five bulls were slaughtered
at birth followed by five per month until 12 months of
age. Blood was collected at slaughter in heparinized
tubes and centrifuged at 1800 g. The plasma was frozen
and stored at -15C until assayed. Testes were removed
at slaughter, frozen whole and stored at -15C until
assayed.

b) Hereford Bulls. Thirty-four Hereford bulls, part
of a long-term genetic study, were available to be bled
at 11, 12 and 13 months of age. Twenty-nine of the.
animals were slaughtered at 13 months of age, one week
after the last bleeding. All Hereford blood samples were
allowed to form a clot and then were centrifuged. The
serum was frozen and stored at -15C until assayed. Testes

were removed at slaughter, frozen whole and stored at -15C

70

until assayed. Several other endocrine and reproductive

criteria were measured.

b) Methodology.

 

Sergm.--To a 100 ml glass stoppered cylinder, approx-
imately 4,000 cpms each of tritium labelled testosterone
and androstenedione were added (to account for extraction
losses) with a Lambda pipette. Serum was then added
followed by ether at a ratio of two volumes of ether to
one of serum. The contents of the cylinder then were
shaken for two minutes and the ether extract removed with
a 10 ml pipette into a 40 ml polypropylene centrifuge
tube. The extraction was repeated twice more and the
extract then dried thoroughly under nitrogen. The tube
walls were washed down three times with ether (to con-
centrate the extract to the base of the tube), and again
thoroughly dried.

Five ml of 70% methanol was added to the extract
and gently vortexed, a further 20 ml of 70% methanol were
then added by running the methanol down the walls while
rotating the tube. The tubes were capped with parafilm
and stored overnight at -15C. This step served to preci-
pitate most of the neutral lipids in the extract. The
tubes had to remain in the freezer for at least 12 hours
and no longer than 24 hours to achieve an effective lipid
precipitation. After this period the tubes were centri-
fuged at approximately 6,000 g for 0.5 hour at -15C. Care

was taken when transferring the tubes from the freezer to

71

the centrifuge to ensure that they did not warm. Follow-
ing centrifugation, the 70% methanol was immediately
decanted into a 100 ml glass stoppered cylinder.

Ten ml of heptane was added to the 70% methanol
and the cylinders were gently everted 5 to 10 times. The
heptane (upper layer) was removed with a 10 ml pipette and
discarded. The operation was repeated. This partition
removed most of the remaining neutral lipid material.

Following the heptane partition the 70% methanol was
further diluted with 40 ml of water, to render any steroids
present fairly insoluble. This solution was then back
extracted with methylene dichloride to remove steroidal
material as follows. Twenty ml of methylene dichloride
were added and the cylinders were everted five to ten
times as before. The methylene dichloride (lower layer)
was removed with a 10 ml pipette and transferred to a 40
ml conical tube. This extract was then dried under nitro-
gen. Care was taken not to remove any of the diluted
methanol as this would greatly hinder drying at this
stage. The tube walls were washed down three times with
methylene dichloride:benzene (1:; v/v) to concentrate the
residue to the tip of the tube, and the dried extract was
taken up in 0.5 ml of the same solvent mixture.

The extract was transferred to a silica gel thin
layer plate with a Pasteur pipette, and the tube was
rinsed twice in the same solvent. Washings also were

transferred to the thin layer plate. The sample was

72

spotted in the lower left hand corner of the plate, and
standard androstenedione and testosterone were spotted

in the lower right hand corner. The plate was developed
by ascending chromatography in the solvent system ether:
dimethyl formamide:acetic acid (99:1:1 v/v). This system
was designed to drive all remaining neutral lipids above
and away from the androgens I was concerned with.

The plates were dried and turned 90° so that the
androgen sample spots were again on its lower edge. Once
again standard steroids were applied away from the sample
androgen areas and the plate was developed in chloroform:
methanol (99:1 v/v). This system served to separate the
two androgens concerned, and to separate them from other
major neutral steroids (see Figure 2).

The acetic acid in the first solvent system tended
to alter the developmental pattern of the lower two edges
of the plate. Thus a 2-inch corridor to the right of the
plate in the second system was isolated by scoring a line
down the plate with a Pasteur pipette. This gave a flat
solvent front in the second system as compared to one
sloping up to the right on plates not scored as above. The
leping solvent front was due to faster migration of the
solvents on the right hand side of the plate.

The standard androgens were visualized on the plates
under ultra violet light. A line was scored from the front
edge of each standard spot across the plate, parallel with

the appropriate edge. An area about 3 cm2 was scraped below

73

I?

°_.——-————71

 

 

 

 

solvent

sYstem.: 0 66) ”#:33-4

GO

 

O
Lorigin

solvent systema

 

\V

 

Figure 2.--Positions of Steroids and Various Contaminants
on Silica Gel Thin Layer Plates after DevelOp-
ment on Etherzdimethyl formamide:acetic acid
(99:1:1 v/v) (l) and Chloroformzmethanol
(99:1 v/v) (2), as Revealed by Ultra Violet
Light and Concentrated Sulphuric Acid.

 

Areas showing colour reaction with concentrated sulphuric
acid are (l) 20 hydroxyl progesterone, (2) testosterone,
(3) androstenedione, (4) progesterone, (5) lipOprotein or
phospholipid, (6) dehydroepiandrosterone, (7) cholesterol,
(8) free fatty acids, (9) cholesterol palmitate.

74

each line at the point of intersection. It was necessary
to scrape well behind the standard areas because the
sample androgens nearly always run slower than the stan-
dard androgens. Care was taken to leave at least 0.25
inch of silica gel between the two areas scraped to ensure
no overlap.

The silica gel bearing the sample androgens was
placed in a 12 ml conical centrifuge tube and eluted with
2 ml of ethyl acetate three times. Each tube was vor-
texed for 15 seconds and centrifuged at 298 g for 1 to 2
minutes. The extract was poured off into either a 12 or
15 ml conical centrifuge tube with a ground glass stopper.

The pooled extracts were dried and the androgens
were carefully concentrated to the tip of the tube with
two to four washes of acetone. The extracts at this stage
had to be thoroughly dried under nitrogen. If storage of
the samples was necessary it was done by simply storing
the silica gel scrapings in ethyl acetate in a sealed
tube.

The dried extracts were vacuum dessicated overnight.
When the vacuum was broken, nitrogen rather than damp air
was allowed to fill the dessicator. To each tube of either
androstenedione, testosterone or progesterone, 50 p1 of
benzene was added, followed by 50 pl of heptafluorobutyric
anhydride. The tubes were then stOppered and heated in a
water bath at 75C for 30 minutes. The reaction coupled a

7-fluorine chain onto the steroid nucleus.

75

The steroids were concentrated to the tip of the tube
and taken up in hexane. The quantity of hexane depended on
the suspected concentration of steroid; 500 p1 was usually
used for most serum and testicular extracts. Between 1
and 4 pl of this solution was injected into the gas liquid
chromatograph column, again the amount depended on the
estimated concentration of steroid.

A 3-foot, 1 to 5% OV-l column was used with an oven
temperature between 190 and 198C. The flash heater and
the electron capture detector were set between 200 and
210C. A pulsed DC current was applied to the detector
with a pulse interval of 50 microseconds. The electro—
meter was operated at range 10, and attenuations between
4 and 128; again this depended on the concentration of
steroid suspected in the sample. Argon-methane carrier
gas was used with no purge gas. The carrier gas was
supplied at 40 p/si and between 55 and 65 ml per minute
flow rate. Care was taken not to apply to the Column
amounts of steroid exceeding 1 pg. If this amount was
to be exceeded, a purge gas or hydrogen flame detector
had to be used. With such large quantities of steroid,
if no purge gas was used with the electron capture detec-
tor, severe contamination of the cell foil could result.
Chart recorder speed was set at 0.25 inches per minute.

With each batch of samples derivatized duplicate
10 pg amounts of standard testosterone, androstenedione

and progesterone were derivatized. Before each set of

76

of samples were chromatographed, 10 ng of each duplicate

of testosterone and androstenedione were chromatographed
with 10 ng of progesterone as an internal standard. This
established the ratios between the internal standard and

the androgens which were used in the quantification formula
described later. It also established retention times for
the steroids in question. The latter was important as
retention times vary from day to day. It was also necessary
to have the data to separate the androgen and internal
standard peaks from interfering peaks of contaminants.

The carrier gas flow rate and oven temperature con-
trolled the retention times on a particular column, the
amount of steroid injected however may have given minor
fluctuations. At a carrier gas flow rate of 60 ml/min
(rotameter reading of 2.85 at 38.75 p/si), an oven tempera-
ture of 193C and flash heater 199C, the retention time of
androstenedione and testosterone was 6.1 minutes; and of
progesterone was 10.2 minutes. These settings gave a
reasonable compromise between sensitivity and an accept-
able retention time. Maximum response at pulse 50 was
gained at a rotameter reading of 2.3 and an oven tempera-
ture of 194C, but the reduced carrier gas flow rate gave
an intolerably long retention time for routine use.

Each sample was injected with a known quantity of
internal standard. The area under each peak was measured
by the method of height times width at half height; the

technique although not perfect gave the most accurate

77

assessment of the area as compared to such methods as
triangulation or planimeter measurements.

An aliquot was removed for liquid scintillation
counting to calculate extraction losses, usually before
gas liquid chromatographic analysis. But if only a small
quantity was removed for the latter analysis, the remain-
ing sample was eluted into a liquid scintillation vial with
three washings of acetone.

All peak areas were standardized to a 1 pl injection
at a sensitivity of 10 x 64 and quantification calculated
from the formula on page 78.

Testicular Homogenates.--A section of testicular

 

parenchyma was weighed, diced, and placed in a 7 m1 glass
homogenizer. Approximately 4,000 cpms each of tritium
labelled testosterone and androstenedione were added to
this with a Lambda pipette to account for extraction
losses. Five m1 of saline also was added, and the material
was homogenized. Up to 3 g of tissue could be homogenized
in this way. The homogenate was poured into a 100 ml

glass stoppered cylinder and the homogenizer was thoroughly
rinsed three times with ether. The rinsings were poured
into the cylinder. Ether was added to the homogenate in
the cylinder at a rate of three volumes of ether to one

of tissue, and sometimes at a ratio of 4:1 for very fresh
tissue samples. The contents of the cylinder were shaken
and extractions performed as for the serum samples. The
testicular homogenate extract was treated exactly as the

serum extract from this point on, however the techniques

78

outlined to remove neutral lipids had to be rigidly

followed as testicular parenchyma, especially fresh sam-

ples, apparently contained more lipid.

Quantification Formula.--Quantity (in pg) of testos-

 

terone injected into the gas liquid chromatograph column

was calculated from the following formula:

Testosterone =<§S><CS X>ET:; 832:4] (0.01) , where

Xs

XP

Cs
Cx

TS

Tx

288.4

484

 

 

Cpm of testosterone ( 3H) initially added
to plasma,

cpm of 3H) in the aliquot removed prior
to GLC,

area (cm2) of 20 ng of internal standard,
area (cm2) of internal standard from sample,

area (cm2) of 0.01 pg testosterone mono-
heptafluorobutyrate,

area (cm2) of testosterone monoheptafluoro-
butyrate from the sample,

MW of testosterone, and

MW of testosterone monoheptafluorobutyrate.

The value obtained from the formula above has to be

corrected for dilution and for the amount of tissue used

to express the result on a per g or per m1 basis.

RESULTS

1. Assay for Testicular and Plasma Levels
of Testosterone and Androstenedione

 

 

a) Introduction

 

Success of the quantitative determination of steroids
in biological samples by gas liquid chromatography with
electron capture detection depends on scrupulous atten-
tion to detail. Although the technician or researcher
with average skill can successfully use this technique
in routine analysis, if he is not willing to adhere
rigidly to the necessary standards of cleanliness and
accuracy then it is futile to even commence work. Puri-
fication of the desired steroids from other steroids as
well as organic, and inorganic contaminants is important.
The success of the assay also depends on the precision
at each stage of the procedure, the stability of the

derivative, and the linear response of the detector.

b) Linearity of the Detector Response

 

The response of the electron capture detector to
testosterone diheptafluorobutyrate and androstenedione

monoheptafluorobutyrate was found to be linear in the

79

80

range of quantities that were measured. For testosterone
it was 0.001 to 10 ng, and for androstenedione it was

0.003 to 10 ng.

c) Sensitivity

 

The quantities of steroid that gave a peak of suf-
ficient area to accurately quantify were 0.001 ng for
testosterone diheptafluorobutyrate and 0.003 ng for andro-
stenedione monoheptafluorobutyrate. Levels less than this
could be detected but not quantified with acceptable
accuracy. Biological samples giving a response in this
category were recorded as having "trace" levels of the
particular hormone.

Most biological samples were diluted in 500 pl of
solvent. If 1 pl of this solution was introduced onto
the gas liquid chromatographic columns, then the quanti-
fication limits were 0.1 ng testosterone and 0.3 ng
androstenedione per ml of blood plasma. Larger quantities
could be injected and the samples could feasibly be con-
centrated to a smaller volume but the solvent front and
background noise became severe problems.

Figures 3 and 4 show representative traces of tes-
tosterone diheptafluorobutyrate and androstenedione mono-
heptafluorobutyrate from plasma and testicular extracts
with progesterone monoheptafluorobutyrate as an internal

standard.

81

 

G-O

 

 

‘

iiSZ'iiiémfififififiw

 

Injection. Time (minutes)

Figure 3.--Gas Liquid Chromatographic Tracing of
Testosteronea from 10 ml of Plasma.

 

Testosterone peak equivalent to approximately 16 pg.
Conditions were oven temperature 195C, flash evaporator
and electron capture detector 200C, 3-foot, 1% OV-l
column, argon/methane 95:5 carrier gas, supplied at
40 p/si and 60 ml/minute, sensitivity attenuation 10 x 4
and a pulse interval of 50 microseconds.

82

23$fi

 

 

 

 

 

 

Wifvfivvv

— m
f 1234 56 78 910111213141516

Iniection. Ti me (mi n u tes)

Figure 4.--Gas Liquid Chromatographic Tracing of Testoster-
one.b From 4 g of Testis with 10 ng of a
Progesterone Internal Standard.C

 

Testosterone peak equivalent to approximately 470 pg.
Conditions were as for Figure 3 except oven temperature,
flash evaporator, and electron capture detector tempera-
ture were elevated 5C, also attenuation was 10 x 64.

83

d) Stability of Heptafluorobutyrate Derivative
Purification of testosterone diheptafluorobutyrate,
androstenedione monoheptafluorobutyrate and progesterone
monoheptafluorobutyrate on florosil microcolumns, as
discussed in the Materials and Methods section (page 35)
revealed no significant breakdown of the derivative and
better than 95% derivative formation. These observations
were based on the fact that isotopes added to the three
steroids yielded virtually no activity in the fractions
from the microcolumns that should contain the free
steroids if they were present. The derivatives were often
stored in hexane at 5C for several weeks without any
evidence of degeneration as shown by the size and

appearance of peak traces.

e) Recoveries

 

These are dealt with in the Materials and Methods

section (page 35 and Table 13).

f) Specificity

 

Specificity of determination of androgens by gas
liquid chromatography depends on purifying the steroids
of interest and thus removing all organic and inorganic
contaminants plus all other steroids that could interfere
with the response of the desired steroids.

During the development of the procedure, all glass-
ware and reagents used were rinsed or extracted with

organic solvents and the residues were analyzed by gas

84

liquid chromatography with electron capture detection
after reaction with derivatized reagents. These
extraction procedures were repeated after attempts had
been made to clean and purify glassware and reagents

by the techniques described in the methodology section,
and none of the rinsings or extractions gave a response
when analyzed except silica gel, which gave a large
front but no peaks and with retention times similar to
either androgens or the progesterone derivative.

During the routine analysis of biological samples
no interfering peaks were observed and it was assumed
that all other steroids that could interfere had been
eliminated by the purification procedure. The peaks
from the biological samples believed to be androstene-
dione monoheptafluorobutyrate or testosterone dihepta-
fluorobutyrate gave identical retention times as standard
preparations of the steroids on several columns with
different liquid phases (see Materials and Methods
section). Analysis of these standard steroids by gas
liquid chromatography mass spectrometry revealed typical
scans for androstenedione monoheptafluorobutyrate, tes-
tosterone diheptafluorobutyrate and progesterone mono-
heptafluorobutyrate. Finally water blanks were analyzed
by the routine procedure, none of these gave any response

to electron capture detection.

85

g) Accuracy

The precision of the complete extraction and analy-
sis method was estimated by replicate analysis of known
amounts of testosterone or androstenedione (10 ng) added
to 5 ml of saline. The range of values obtained is
shown in Table 14. The average estimate and standard
error of this mean for testosterone was 10.6610.34 ng

and for androstenedione was 11.48:0.76 ng.

h) Precision

 

Replicate analyses were performed on testis samples,
these values are shown in Tables 15 and 16. The standard
deviation for duplicates was 0.17. Thus, the precision
of measurement of testosterone and androstenedione from
biological samples was reasonable. One source of error
in this estimate may have been the varying mass of hormone
present as the radioactive tracer. Radioactively labelled
steroids of high specific activity were used; 1.050
Curies/mM for 3H-7-androstenedione and 44.1 curies/mM
for 3H-l,2-testosterone. After purification few biologi-
cal samples exceeded a combined amount of 6,000 cpms of
3H-l,3-testosterone and 3H-7-androstenedione. Isotopes
(5,000 cpm) of each hormone (10,000 Cpm combined) deriva-
tized, diluted in 500 p1 of hexane, and 1 pl injected
onto the gas liquid chromatographic column produced no
response with electron capture detection. Most samples
contained less than 2,000 cpm 3H-7-androstenedione and

2,000 cpm 3H-l,2-testosterone. This was equivalent to

86

TABLE l4.--Replicate Analysis of Standard Testosterone and
Androstenedione added to Saline.

 

 

Replicate Testosterone Androstenedione
Determinations
----ngs ----------- ngs ------
Amount added 10 10
Replicate
Determinations, 1 12.67 14.68
2 10.10 13.32
3 9.19 10.97
4 10.84 14.14
5 11.52 10.94
6 8.01 8.71
7 9.49 12.39
8 11.43 9.43
9 13.24 8.74
10 10.83 ---
11 10.57 ---
12 10.74 ---
13 9.62 ---
14 10.80 ---
15 10.91 ---

Mean plus
standard i
error x ex

 

TABLE lS.-—Duplicate Analysis of Bull Testes for Testerone

and Androstenedione.

 

 

 

Testosterone Androstenedione

Bull l 2 1 2
-------------------- (pg/gm)-----------------------
127 2.072 1.462 0.108 0.133
132 0.205 1.231 0.010 0.009
134 0.255 0.197 0.015 0.028
121 1.343 1.316 0.035 0.058
124 1.854 1.829 5.172 4.539
109 0.212 0.176 0.030 0.025

 

TABLE l6.--Analysis of Variance of the Duplicate Androgen
Analysis in Table 15.

 

 

Source df 53 ms
Testosterone
Among bulls 5 6.44 --
Within bulls 5 0.19 0.03a
Total‘ 11 6.63 --
Androstenedione
Among bulls 5 38.56 --
Within bulls 6 0.20 0.03a
Total 11 38.76 --

 

aStandard deviation between duplicates 0.17 for both
steroids.

88

0.0008 ng of androstenedione introduced to the gas liquid
chromatograph system and 0.000023 ng of testosterone,
assuming dilution in 500 pl and a 1 ul injection. The
limit for reasonably accurate quantification was 0.003

ng for androstenedione and 0.001 ng for testosterone. It
was obvious that the mass of testosterone would give no
interference in the quantification of 3H-l,2-testosterone.
Some question existed for 3H-7-androstenedione. Calcula-
tions of mass made from the specific activity given by
the supplier suggested it should give a GLC response at
the levels that were used. However repeated analysis of
up to 5,300 cpm of the tracer gave no response. Levels
much higher (20,000 cpm) gave a small response equivalent
to those classified as "trace" among biological samples.
Consequently, no correction was made on biological sam-
ples for the mass of radioactive tracers. From the Speci—
ficity and accuracy of the assay for androstenedione, and
the fact that 3H-7-androstenedione gave no response upon
analysis at the levels used, it was assumed that the
specific activity of the Tracer was greater than indi-
cated by the supplier.‘ The latter could be explained by
the fact that the radioactively labelled steroids were
constantly repurified by thin layer chromatography,

before and during routine use.

89

2. Holstein Bulls

 

a) Testicular Testosterone and Androstenedione

 

Testosterone and androstenedione concentrations in
the testes of the Holstein bulls are presented as monthly
averages with standard errors in Table 17. These data
are presented only from 4 to 12 months of age because
the testes of the younger bulls were lost previously.

The values among bulls of similar age were very
variable (Table 17, Appendix Table 1). Analysis of
variance for testicular testosterone over the period of
4 to 12 months of age revealed significant differences
at the 10% level of probability. Analysis of the testi-
cular testosterone data by orthogonal contrasts showed
(1) an increase from 4 to 5 months (P<0.05), (2) a
depression from 4 and 5 months to 6 and 7 months (P<0.01),
(3) a general increase from 6 to 11 months and (4) a
decrease at 12 months (P<0.010). In other words, testi-
cular concentration of testosterone was high at 4 months
of age, plunged to a low level at 6 months and increased
to 11 months of age. After 11 months, it fell to the
lowest level at 12 months of age.

An analysis of variance for testicular androstene-
dione concentrations over the period 4 to 12 months of
age gave an F value only approaching significance (P<0.10).
Orthogonal contrasts revealed a significant reduction in

testicular concentrations of androstenedione from months

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91

4 to 7 to the last 5 months (P<0.05). Concentrations at
months 6 and 7 were lower than at months 4 and 5 (P<0.025),
and the difference in testicular androstenedione between
months 4 and 5 approached significance (P<0.10). The
concentration of testicular androstenedione fell sharply
and continuously to 7 months of age, remained stable at

a low level until 9 months of age and then appeared to
rise until 11 months, only to fall back to a low level

at 12 months of age.

It should be pointed out that the orthogonal con-
trasts for analyses of testicular androgens were set up
after the data were collected because the original con-
trasts were designed to include observations at birth,

1, 2 and 3 months of age. This restriction does not apply
to any of the other data in this thesis.

Table 18 shows the change of the ratio of testicular
testosterone to androstenedione with age from 4 to 12
months of age. At 4 months of age there appeared to be
more androstenedione than testosterone, but the ratio
increased until 8 months of age when the ratio of testos-
terone to androstenedione was 48:1. The data suggested
a decrease in the ratio of 12 months of age (9:1), due to
a fall in testosterone concentration between 8 and 9 and
between 11 and 12 months, and the gradual increase of

androstenedione concentration to 11 months of age.

92

TABLE l8,--Testosterone Androstenedione Ratio from Birth

to 1 Year of Age in the Testes and Plasma of
Holstein Bulls.

 

 

 

Age Testosterone/Androstenedione
Testis Plasma
Birth Lost NDa
1 Lost 0.072
2 Lost 1.26
3 Lost 0.32
4 0.65 NDb
5 4.31 0.41
6 1.83 NDb
7 14.95 NDb
8 48.29 482.00
9 15.87 240.00
10 13.34 NDb
11 8.34 NDb
12 8.76 0.27

 

aTestosterone and androstenedione were not detected.

b

Androstenedione was not detected.

93

b) Plasma Testosterone and Androstenedione

Table 17 lists the monthly means and standard errors
for testosterone and androstenedione concentrations in
the plasma. The plasma concentrations of testosterone and
androstenedione were highly variable among bulls within
age.

Analysis of variance of testosterone concentrations
in the plasma revealed that variations from birth to 12
months of age were significant at the 20% level of pro-
bability. Orthogonal contrasts revealed one significant
contrast (P<0.01); plasma concentrations of testosterone
were significantly higher in the last 6 months than during
the first 7 months. Despite the large fluctuations, there
seemed to be a gradual increase of testosterone concentra-
tion in the plasma from birth to 11 months of age followed
by a precipitous drop at 12 months of age.

Plasma concentrations of androstenedione were
extremely variable and there were no significant differ-
ences between months as revealed by analysis of variance
or orthogonal contrasts. DeSpite enormous fluctuations
during the first 6 months, no androstenedione was detected
in the blood again until 12 months of age. The testoster-
one to androstenedione plasma concentration was extremely

variable (Table 18).

94

3. Hereford Bulls

 

Table 19 shows the serum concentrations of testos-
terone and androstenedione at 11, 12, and 13 months of
age, with values from slaughter blood (13 months of age
plus 2 weeks). Also presented are the testicular con-
centrations of testosterone and androstenedione from
testes removed at slaughter. Serum concentrations of
testosterone appeared to decline from 11 months to 12
and 13 months, but this difference was not significant.
Concentrations of plasma androstenedione appeared to dr0p
over the 2-month period, but again an analysis of variance
revealed no significant difference among these values.

The slaughter values for both parameters were higher than
the value for the 13 month bleeding, but analysis of
variance showed that only the difference between testos-
terone concentration at slaughter and the value obtained
from the 13 month bleeding approached significance (P<0.10).
This suggested a general release of steroids from the

testis caused by the stresses associated with confinement

and slaughter.

95

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GENERAL DISCUSS ION

The changing ratio of testosterone to androstene-
dione in the testes with age of the Holstein bulls was in
good agreement with other investigators and in several
Species. Lindner (1959) and Lindner and Mann (1960)
observed that the ratio of testosterone to androstenedione
in the testis changed from 1:1 in a male calf at 4 months
of age to 10:1 at 9 months of age. Skinner (1968) showed
that this ratio was 1:1 in the ram testis at birth. He
observed an increase of testicular testosterone with age,
but found that levels were highly variable as was apparent
in the present study. He found that androstenedione con-
tent fell until 56 days of age and then rose slightly to
mature levels. This seemed to generally parallel the
findings of the present study in the testes of Holstein
bulls.

Studies in the guinea pig (Becker and Snipes, 1968),
in the Rhesus monkey (Resko, 1967), and in the rat
(Strickland, 1970) also substantiated the findings of
this study. The Hereford bulls slaughtered at 13 months

of age had a ratio of testicular testosterone:androstenedione

96

97

of 12:1 but slightly higher levels than found in the
Holstein bulls at 12 months of age.

This is the first report of androgen levels in bull
blood, but results were not clear. The Holstein bulls
generally showed an increasing plasma concentration of
testosterone to 11 months, and then a decline to 12
months, but the rise to 11 months was by no means smooth.
There was no smooth decline of plasma androstenedione
concentration from the early months to the later ones.
Rather, two or three large peaks of androstenedione
occurred until 5 months of age with values much higher
than any testosterone values, and in most cases due to a
large androstenedione value calculated for one bull.
After 5 months however no androstenedione was detected
in the blood until 12 months of age when a large but non-
significant rise occurred.

Resko gt_al, (1968) demonstrated a general rise in
androstenedione plasma concentrations with age until a
mature level was reached in the male rat. However,

Resko (1970) in the guinea pig seemed to show a picture
somewhat similar to the present study with rather eratic
androstenedione plasma concentrations declining to 60
days and then increasing slightly to a mature level at
120 days.

The plasma androstenedione and testosterone concen—
trations for the 3-month1y bleedings of the Hereford bulls

(11, 12 and 13 months) seemed to be higher than equivalent

98

Holstein values, as were the testicular values. This was
probably a breed difference. The Hereford bull plasma
androstenedione did not show a rise between 11 and 12
months, but rather a steady decline to 13 months estab-
lishing approximately a 2:1 ratio of testosterone to
androstenedione, in agreement with other investigators.
The Hereford bull androgen levels could be higher because
they were less mature than the Holstein bulls; it is known
that beef bulls mature slower than Holstein bulls. Aside
from the elevation at slaughter, the plasma testosterone
concentrations suggested a leveling off at what could be
a mature adult concentration.

Correlations were analyzed between various androgen
values and between androgen levels and other endocrinolo-
gical and physiological criteria concerned with reproduc-
tive development in the bull (see Tables 20, 21, 22 and
23). The Holstein bull androgen data collected in this
study complemented the larger study conducted by Macmillan
(1967). This study was entitled "Endocrine and Reproduc-
tive Development of the Holstein Bull from Birth through
Puberty." Criteria measured by Macmillan (1967) that are
pertinent to the present study are as follows: (1) gona-
dal and extra gonadal sperm numbers (Macmillan and Hafs,
l968a),(2) testicular DNA, seminal vesicular fructose and
citric acid (Macmillan and Hafs, 1969); and (3) pituitary
FSH and plasma LH (Macmillan and Hafs, 1968b). Plasma LH

was estimated by the method of acetone precipitation and

99

 

 

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101

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102

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103

ovarian ascorbic acid depletion, but the recovery of LH
from the plasma of the bulls was only 20%. More recently
(Swanson, 1969) re-quantified plasma LH by radioimmuno-
assay. There is some variance between the two estimates
and these will be discussed (see Table 20).

Data presented in this study for androgen levels in
Hereford bulls were also analyzed parallel to other repro-
ductive and endocrimological criteria (see Tables 22 and
23). These criteria were estimated by Swanson §E_gl. (1970)
and included: (1) gonadal and extra gonadal sperm numbers;
(2) seminal vesicular fructose and citric acid; and (3)
pituitary FSH and plasma LH.

The correlation between plasma testosterone concentra-
tion and testicular testosterone concentration for Holstein
bulls was significant. This suggested that as testosterone
rose, plasma testosterone also did and not vice versa as
is often the case for the secretions of other endocrine
glands. The within month correlations between plasma and
testicular testosterone revealed a non significant nega-
tive correlation at months 4, 5 and 6, however the correla-
tions were positive from months 7 to 12, thus months 7, 8
and 9 were significant (see Table 21). This qualifies the
overall correlation and suggests that during months 4, 5
and 6 testosterone secretion has not reached a maximum and
that release into the blood depletes the testicular stores
of testosterone. The overall correlation between testi-

cular androstenedione and plasma androstenedione

104

concentration was not significant (see Table 20). This
indicated that increased levels of androstenedione in the
testis after 4 months of age were only partially released
into the blood, the larger portion being rapidly converted
to other steroids such as testosterone. Also, that high
levels of androstenedione in the blood after 4 months of
age did not reflect secretion by the testis, but probably
metabolism of plasma testosterone or perhaps secretion
from the adrenal gland. Horton (1965) estimated that 36%
of blood androstenedione was produced by the metabolism
of blood testosterone. Evidence by Kirschner (1965) also
suggested the adrenals as being an important source of
androstenedione.

The relationship between testicular and plasma con-
centrations of androgens for Hereford bulls at slaughter
(13.5 months of age) showed similar relationships.

Plasma testosterone concentration was significantly corre-
lated with testicular testosterone concentration (see

Table 22), and plasma concentration of androstenedione
showed a small non-significant correlation with testicular
androstenedione concentration (see Table 22). For the
Holstein bulls the correlation between plasma testosterone
concentration and plasma androstenedione concentration
approached significance (see Table 20), and the correlation
of testicular testosterone concentration and androstene-
dione concentration, although not significant was positive

(see Table 20). After 4 months of age, increased synthesis

105

of testosterone in the Holstein bull testis seemed to be
accompanied by small increases in androstenedione synthe-
sis. This seemed feasible as androstenedione is synthe-
sized as a precursor for testosterone and some of the
intermediate metabolite was likely to be detected.

As for androgens in the blood, as a major portion of
the testosterone was apparently converted to androstene-
dione possibly in preparation for excretion, increases in
androstenedione concentration could reflect increases in
testosterone concentration. The correlation of plasma
testosterone and androstenedione (see Table 20), although
not highly significant, was larger than the similar
testicular correlation (see Table 20), probably because
it included the earlier months when androstenedione was
the predominant steroid. Increased concentrations of
blood androstenedione at this early date were probably
accompanied by smaller increases in testosterone, as
little testosterone was produced from the androstenedione
pool in the testis at this time.

Testicular concentration of androstenedione and
plasma concentration of testosterone were negatively
correlated, but the correlation was small and non-signi-
ficant (see Table 20). A similar non-significant corre-
lation was noted for testicular concentration of testos-
terone and plasma concentration of androstenedione (see
Table 20). This seemed to confuse the issue, but may be

explained by the fact that the correlations were only

106

made on values from 4 to 12 months of age and during this
period the two hormones were only vaguely correlated in
the testis or within the blood. However, in the blood,
the testosterone and androstenedione concentrations seemed
to follow each other more closely and this is acceptable
if the bulk of blood androstenedione is regarded as a
metabolite of blood testosterone, when the steroidal
levels were cross correlated the correlation was negative.
The lack of significant correlations suggest no important
relationship between testicular production and plasma con-
centration of androstenedione.

Plasma LH (Macmillan and Hafs, 1968c, Appendix Table
3) in the Holstein bulls, as measured by the ovarian
ascorbic acid depletion method, rose from 2 months of age
to 4 months, declined to 6 months and then increased to
12 months of age. This biphasic pattern of secretion was
not clearly reflected in plasma testosterone concentration
but appeared to be reflected in the testicular values.
When measured by radioimmunnoassay plasma LH appeared to
rise from 1 month of age to 2 months, declined to 4 months,
rose again to 5 months, fell to 6 months and then rose
slowly to 9 months of age. A sharp rise was noted between
10 and 11 months but the levels of LH in the plasma fell
again at 12 months of age (Swanson, 1970; Appendix Table
3). It appears that the levels of LH in the plasma for
the first 6 months were higher on average than for months

6 to 10. This is interesting as the most rapid reproductive

107

development was made in the latter period. The values as
measured by radioimmunoassay are probably more realistic
as the assay procedure is more sensitive and accurate
than the ovarian ascorbic acid depletion bioassay. Also,
the LH precipitated from the blood of the bulls to be
used in the latter assay was accomplished at a very low
recovery rate.

A correlation over all months showed that testicular
and plasma content of testosterone were both significantly
correlated to plasma content of luteinizing hormone in
the Holstein bulls, no matter which set of LH figures were
used (see Table 20). Within month correlations between
plasma LH (as measured by radioimmunoassay) and testicu-
lar or plasma testosterone levels show a more or less
continuous stimulation of testosterone synthesis in the
testis of Holstein bulls within each age group from 5 to
12 months of age (see Table 21). Release of testosterone
into the blood appears to be related, within monthly age
groups, to plasma LH levels during two periods, 1 to 3
months and 9 to 12 months (see Table 21). Testosterone
levels in the plasma generally rose between these two
periods, thus it seems feasible to conclude that release
of testosterone from the testis from 4 to 8 months occurs
without LH control.

A correlation over all months revealed that andro-
stenedione content of the testis and plasma were negatively

correlated to plasma content of LH, as measured by ovarian

108

ascorbic acid depletion, but the correlations were far
from significant (see Table 20). Using LH figures from
radioimmunoassay a similar correlation resulted with
plasma androstenedione, but with testis androstenedione
a positive correlation approaching significance was
revealed (see Table 20).

Within month correlations between plasma LH and
testicular or plasma androstenedione levels were also
analyzed (see Table 21). Plasma levels of androstene-
dione in the early monthly age groups (1 to 6 months)
seemed to show little correlation with LH levels in the
plasma. In the testis it appeared that within the early
months (4 to 7 months) androstenedione levels decreased
with increasing levels of LH in the plasma, but in older
bulls they increased parallel with plasma LH.

These correlations between androgen levels in the
testis and plasma of Holstein bulls suggest that LH
stimulates testosterone synthesis mainly by stimulating
the testicular enzymes concerned with the conversion of
androstenedione to testosterone. Also, that considerable
stimulation is seen, especially in older animals, at an
earlier stage in steroidogenesis.

Oshima (1967) and Strickland (1970) both suggested a
stimulatory role for LH on the enzyme system responsible
for hydroxylation at the 17 carbon of androstenedione.
High levels of androstenedione in the testes of young

animals suggested that ample availability of cholesterol

109

and acetate substrate, but it was not until later stages
of sexual maturity that the chain of enzyme reactions
were carried as far as testosterone in large quantities,
probably due to the stimulation of increasing LH titers
in the blood.
uata from the Hereford bulls bled at 11, 12 and 13
months of age revealed no significant correlations between :00
plasma concentrations of LH (see Table 23). If anything
these correlations suggested a decreasing dependence of
testosterone secretion on circulating LH with age. As

shown by the Holstein data, the dependency was probably 2;

greater in developing animals when increasing circulating
levels of LH were stimulating androgen synthesis and
secretion. However, within month correlations between
testis or plasma androgens and circulating LH levels sug-
gested a lower correlation between the criteria in the
more mature bull, especially at month 12 (see Table 21).
Androstenedione concentrations in the blood of the
Hereford bulls at 11, 12 and 13 months of age showed no
significant correlations with LH concentrations in the
blood (see Table 23). At these ages LH probably only
maintained testicular androgen secretion, unlike pubertal
animals where large increases of both criteria were seen.
if this is true, then in mature animals, a more critical
assessment of androgen and LH are necessary to detect
relationships between the two, above the large between

animal variations that exist. It is interesting to note

110

the apparent lack of stimulation of plasma testosterone
levels by plasma LH levels between 4 and 8 months of age
in the Holstein bulls (see Table 21). It may be that
testis release of androgens at this period and in mature
bulls is not under any LH control. In older animals,
androstenedione seemed to be more prominent in the blood,
and the ratio of testosterone to androstenedione was only
2:1. Less androstenedione may have been converted to
testosterone in the testis and/or more was released into
the blood. This may reveal, as was suggested for the
Holstein bulls, a stimulatory effect of LH earlier in the
steroidogenic pathway than conversion of androstenedione
to testosterone. Alternately, that enzyme systems con-
verting androstenedione to testosterone may reach a
steady state in the mature animal. Production of andro-
stenedione would not be rate limiting to testosterone
synthesis and a steady state secretion of androstenedione
could be established.

Data from Hereford bulls at slaughter revealed a
significant correlation between testicular concentration
of testosterone and serum LH concentration (see Table 22),
as well as a significant correlation between testicular
androstenedione concentration and serum LH (see Table 22).
Similar correlations for serum concentrations of testos-
terone and androstenedione with serum LH concentration
approached significance (see Table 22). These data

probably reflected a general stimulation of hormone

111

synthesis due to stress. Several investigators suggested

that gonadotropins stimulated steroidogenesis by enhancing
the 200hydroxylation of cholesterol to pregnenalone (e.g.

Dorfman, 1969; Hall and Young, 1968).

Pituitary FSH potency showed no relationship to
androgen synthesis or secretion in bulls. Correlations
of pituitary FSH potency in the Holstein (Macmillan and
Hafs, 1968) and Hereford (Swanson gE_al., 1970) bulls
with testicular and plasma concentrations of testosterone
and androstenedione were all non-significant except for
the correlation with plasma androstenedione concentration
at slaughter for the Hereford bulls (see Tables 20 and
22).

Means (1969) demonstrated the ability of FSH to
stimulate testicular protein biosynthesis and concluded
that the activity was specific for FSH and gonadotropins
with FSH activity. After experiments involving adminis-
tration of exogenous FSH and LH into Rhesus monkeys, Kar
(1966) concluded that the major role of FSH in the testis
was to stimulate growth via protein synthesis. Thus,
although FSH may be important in preparing the machinery
for androgen secretion the role of supervising secretion
is probably more in the hands of LH.

Plasma prolactin concentration was determined for
the Hereford bulls (Swanson §E_al., 1970) but it was not
significantly correlated to any of the androgen criteria

(see Table 23). Correlations of plasma prolactin with

112

androgen data at slaughter were highest and androstene-
dione criteria gave consistently negative correlations
with plasma prolactin concentration. The role of pro-
lactin in the bull is not clear.

Sperm were not detected in the testis or epididymis
until 6 or 7 months of age for the Holstein bulls studied,
all bulls showed sperm in these areas by 8 months of age
(Macmillan and Hafs, 1968; Appendix Table 4). For these
bulls, no significant overall correlations were found
between testicular or epididymal sperm content and testi-
cular testosterone and androstenedione or plasma testos-
terone concentration (see Table 20). However, plasma
androstenedione concentration was significantly correlated
to gonadal sperm concentration and total spididymal sperm
(see Table 20).

The correlations between androgen levels and testi-
cular sperm numbers were examined on a within month basis
(see Table 21). The correlation between testis or plasma
testosterone and testicular sperm numbers was greatest
at 8 and 9 months of age, decreasing from then on. This
suggests that testosterone may play a role in the initia-j
tion of spermatogenesis and to a lesser extent with the I
following increase in sperm numbers. Androstenedione did
not appear to be consistently related in any way to the
initiation and maturation of spermatogenesis in Holstein

bulls.

113

The concentration of testosterone in the slaughter

serum of the Hereford bulls was not significantly corre-

lated with gonadal sperm concentration or total epididymidal'

sperm (see Table 22). However, androstenedione was signi-
ficantly correlated to gonadal sperm concentration and the
correlation with total epididymidal sperm numbers approached
significance.

A combination of FSH and LH was necessary to stimu-
late and maintain Spermatogenesis in hypophysectomized
rats (Lostrah, 1969). It is possible that a combination
of gonadotropins and androgens were necessary for the
initiation of Spermatogenesis in bulls. The DNA concen-
tration of Holstein bull testes declined to 10 months of
age (Macmillan and Hafs, 1968; Appendix Table 5) due to
increases in the size of the seminiferous tubules. This
trend was reversed at 10 months of age when large numbers
of sperm appeared in the testis. Changes in testosterone
levels did not appear to parallel these changes, but testi-
cular androstenedione concentration was significantly
correlated to the concentration of testicular DNA over
the whole period (see Table 20), and the correlation of
plasma androstenedione and testicular DNA concentration
approached significance (see Table 20). Analysis of
within month correlations between plasma or testis andro-
stenedione concentrations and testis DNA concentration
was made (see Table 22). It appears from this that

testicular androstenedione may be involved with DNA

‘50.;

114

synthesis in the testis and that this could be a local
effect not mediated via the systemic circulation. Andro-
stenedione may be involved with protein synthesis and
testicular growth. No clear relationship between testos-
terone and androstenedione and spermatogenesis was
revealed by this study, but testosterone appeared to be
involved in at least the initiation of spermatogenesis,
if not maturation and maintenance. 1‘

Accessory gland growth and secretory criteria are

-_—- III-2435;. - V’wr

acknowledged classically to reflect the androgenic status

of an animal. In this study, seminal vesicular secretory

 

activity was shown to reflect the changing levels of
plasma testosterone but androstenedione did not seem to

be related to accessory sex organ activity. The fructose
and citric acid contents of the seminal vesicles of the
Holstein bulls increased from 1 month of age to 1 year
with a more rapid increase during the last 6 months
(Macmillan and Hafs, 1969; Appendix Table 6). Over the
whole period testicular testosterone concentration was

not significantly correlated with either seminal vesicular
citric aCid concentration (see Table 20), or fructose
concentration. But plasma testosterone was significantly‘
correlated to seminal vesicular fructose concentration

and citric acid concentration. Plasma testosterone content<
was also significantly correlated to seminal vesicular
fructose content. Plasma androstenedione concentration was

not significantly correlated to seminal vesicular fructose

.4

115

content. Plasma androstenedione concentration was not
significantly correlated to seminal vesicular fructose
content or citric acid concentration (see Table 20).
Testicular androstenedione concentration however was
significantly negatively correlated to seminal vesi-
cular fructose concentration, and to citric acid concen-

tration (see Table 20). Plasma testosterone concentration

was positively correlated to seminal vesicular fructose {5%
concentration within each month, with large and even some

significant correlations (see Table 21) occurring

biphasically at 2, 3 and 4 months and 8 and 9 months of (“J

 

age. Within month correlations between testis testos—
terone concentration and seminal vesicular fructose con-
centration were basically positive with significant
correlations at 9 and 10 months of age (see Table 21).
The correlation was negative at 4 and 5 months of age,
this may reflect the negative correlation between testis
and plasma testosterone concentrations that existed at
this age.

The correlations between testicular androstenedione
and seminal vesicular secretions were not surprising as
testicular androstenedione decreased with age in Holstein
bulls and was very low after 6 months of age, or the time
when the most rapid increases in seminal vesicular acti-
vity occurred. The picture for blood levels of andro-
stenedione was less dramatic, probably because levels of

androstenedione to 6 months of age were highly variable

116

and then almost zero until 12 months of age. Neither were
correlations between testosterone and seminal vesicular
secretions surprising; plasma testosterone concentration
did not seem to increase as biphasically as testicular
testosterone concentration, but there was a low point at

6 months which coincided with the end of the first phase
of increasing seminal vesicular secretory activity and

the beginning of the second phase. The overall relation- I
ship of testicular testosterone to seminal vesicular

secretion is difficult to explain, however the within

 

month correlations show the expected relationships. It LJj
would be expected that blood levels would be more corre-

lated to target organ activity but the blood levels of

testosterone were strongly correlated to testicular values.

One can only surmise that had all of the testicular values

from birth to 1 year been available then the correlation

would have been greater.

However testosterone in the blood apparently stimu-
lated seminal vesicular secretory activity, and androstene-
dione played no such role. This view is corroborated by
Baranas (1969) who castrated 6-month old bovine males and
injected testosterone or androstenedione. Both promoted
growth and nitrogen retention but only testosterone showed
any androgenic properties. Skinner (1968) found that
androstenedione depressed seminal vesicular growth and

secretion.

117

The relationships between androstenedione or testos-
terone and seminal vesicular secretory activity for the
Hereford bulls was not clear at all (see Table 22). All
of the correlations were made on slaughter material which
appeared to be confused by an elevationof hormonal levels

.. ,-._._

due to stress. None of the correlations made were signi-
00—WWWHH H-
ficant, testicular testosterone concentration showed, as
for Holstein bulls, a small positive correlation with
seminal vesicular fructose concentration, and a small
negative correlation with citric acid concentration.
Testicular androstenedione gave small positive correlations
with seminal vesicular concentration of fructose and citric
acid. Serum testosterone concentration was poorly corre-
lated with either secretory product (see Table 22).
Androstenedione concentration in the plasma gave a nega-
tive correlation with citric acid concentration in the
seminal vesicle, and a small positive correlation with

fructose concentration in the same accessory gland (see

Table 22).

ELM—A I a

 

SUMMARY AND CONCLUS IONS

A technique for routine assay of androstenedione
and testosterone from biological samples has been evolved.
The salient features of this assay were extraction of
tissue with diethyl ether, purification by methanolic
precipitation of lipids and thin layer chromatography to
isolate the androgens. Quantification was performed on

the diheptafluorogutyrate derivative of testosterone and

M

 

31-h": 5%.. can’t. {'0- ‘\ ‘ "
.
‘3‘“;
..

the monoheptafluorobutyrate derivative of androstenedione
utilizing gas liquid chromatography with electron capture
detection. The sensitivity of the assay was good enough
to allow quantification of plasma levels of testosterone

and androstenedione to as little as 0.001 ng. Specifi-

rfi—mnn ,

city, accuracy and precision of the technique also were
acceptable. This assay was employed in a study of testos-
terone and androstenedione in the blood and testis of bulls
from birth through puberty.

A total of sixty-five Holstein bulls were killed in
groups of five at monthly intervals from birth to 12 months
of age. The testicular parenchyma and plasma from these
animals were assayed for testosterone and androstenedione.

A group of twenty-nine Hereford bulls were bled at 11, 12

118

119

and 13 months of age and slaughtered two weeks after the
last bleeding. Androgens in tissues from these animals
were assayed as for the Holstein bulls.

Testicular concentration of testosterone from Hol-
stein bulls increased during months 4 (1.08 ug/g) and 5
(3.15 ug/g), and during months 7 (0.17 ug.g) and 11

(1.86 ug/g). A decline was noted at 6 (0.32 ug/g) and

12 (0.29 ug/g) months. The concentration of testicular f) '

androstenedione fell sharply from months 4 (1.66 09/9)

to 7 (0.03 ug/g), remained stable at a low level until 5

9 months Of age and then appeared to rise until 11 months 3’;
.

 

(0.22 ug/g), only to fall to a low level at 12 months of
age. These fluctuations in concentration produced a

ratio of testosterone to androstenedione of 0.65:1 at 4
months of age, 48:1 at 8 months and 8:1 at 12 months of
age. Despite large fluctuations there seemed to be a
gradual increase of testosterone concentration in the
plasma of Holstein bulls from 0.00ng/m1 at birth to
3.73ng/m1 at 11 months of age followed by a precipitous
drOp to 0.50 ng/ml at 12 months of age. Despite great
fluctuations during the first 6 months, no androstenedione
was detected in the blood again until 12 months of age.
Testerone and androstenedione levels in the Hereford bulls
were generally higher than in Holsteins even though the
concentrations in the blood of Herefords appeared to fall
from 11 (testosterone 5.5 ng/ml, androstenedione 4.25 ng/ml)

to 13 (testosterone 4.32 ng/ml, androstenedione 1.83 ng/ml)

120

months. It was not clear if this was a difference due to
breed or physiological maturity. The plasma androgen
concentrations recorded at slaughter may have been ele-
vated due to stress. However a 2:1 ratio of testosterone
to androstenedione was established in the blood.

The relationships between the plasma and testicular
steroids quantified were examined over the first year of
life in bulls. Correlation analysis was also performed
between testicular and plasma androstenedione or testos—
terone and several endocrinological and physiological 3

criteria associated with sexual maturation. Among these

 

 

criteria were plasma LH concentration, pituitary FSH
potency, gonadal and extra gonadal sperm numbers and
seminal vesicular secretory activity.

In the Holstein bulls, plasma testosterone concen-
tration reflected testicular testosterone concentration
from 4 to 12 months of age. No such relationship existed
for androstenedione. After the first 4 months of age,
little androstenedione was released from the testis.

Most was probably converted to testosterone. In the
blood, the major part of androstenedione probably origi-
nated from metabolism of testosterone or from adrenal
secretion.

Testosterone synthesis and secretion increased with
the rising titers of plasma LH during the first year of
life in Holstein bulls, but there was an indication that

testosterone secretion from the testis may be independent

121

of LH between months 4 and 8 and in mature bulls. Andro-
stenedione and LH appeared to be negatively correlated

in young bulls but after 6 or 7 months of age this rela-
tionship was positive. Few of the within month correla-
tions were significant. It was suggested that_LH stimulated
steroidogenesis mainly by stimulating the conversion of

androstenedione to testosterone, but some stimulation at

”—0.

MW’

an earlier stage of steroidogenesis was also apparent
eSpecially in the older bulls.

After one year of age, data from the Hereford bulls y

 

suggested little correlation of LH to testosterone or ;J
androstenedione. Thus LH may only maintain androgen
secretion in the mature animal and not directly stimulate

Ma

it. ”The biphasic pattern of increasing LH secretion (ris

M.

.....

ing from 2 to 4 months of age, declining to 6 months, and
then increasing to 12 months of age) was reflected more
noticeably in plasma than in testicular testosterone concen-
trations. Large between animal and between month variations
tended to obscure this result.

Pituitary FSH potency was not significantly corre-
lated with either androstenedione or testosterone in
either breed. FSH may be involved with general stimula-
tion of growth and protein synthesis in the testis of
young bulls during sexual maturation.

No significant relationships were found between serum

prolactin and plasma or testicular androstenedione or

122

testosterone in the Herefords. The role of prolactin in
the male was not clear.

Gonadal and extra gonadal sperm numbers during
maturation of the Holstein bulls were shown to be signi-
ficantly correlated only to plasma androstenedione concen-
tration on the basis of overall correlations. The Here-

ford slaughter data suggested a negative relationship
between blood levels of androstenedione and gonadal and

extra gonadal sperm numbers. Testosterone concentration

in the plasm seemed to have little relationship with

sperm numbers in this breed. Correlations made within

 

each age group between testosterone or androstenedione
and gonadal Sperm numbers revealed a positive relation-
ship between testis and plasma testosterone and gonadal

sperm numbers. Although few of the correlations were

0
”—0

significant, a role was suggested for testosterone in

the initiation of spermatogenesis. Androstenedione did
no: exhibit any clear relationship at all with spermato-
genesis. It is probable that a combination of gonadotro-
pins and androgens is necessary for the initiation of
spermatogenesis in bulls.

The fructose and citric acid content of the seminal
vesicles of the Holstein bulls increased from 1 month of
age to 1 year, with a more rapid increase during the last
6 months. The changing levels of plasma testosterone

were found to reflect this pattern. Androstenedione

levels in the maturing bull did not appear to be related

123

to increased secretory activity of the accessory glands.
The correlations made between Hereford bull seminal
vesicular activity and serum or testicular testosterone
and androstenedione levels showed no significant rela-
tionships. This was confusing, it could be that the

stress of slaughter had altered hormonal levels or that

m1" “"1. A ,

when maintenance rather than active growth in the mature .
' r4515:
11”“ 1, ,1- . i

animalfwas the case then no strong relationship between

*"*%--..
~ ._,_.1

gonadal and accessory glands could be seen.
‘W-Pubgrty based on testicular and circulating levels

of testosterone and androstenedione would appear to con-

r" _".|.'.. r , -
‘
%

tinue until at least 11 months of age in bulls. After
this age, the levels of testosterone and androstenedione
decline to adult levels. Testosterone appears to be the
major androgenic steroid in bulls responding to increas-
ing circulating levels of LH and stimulating accessory
gland secretion in a biphasic pattern over the first year
of life. Androstenedione lacks androgenic properties,
and any specific function during puberty is unclear

although it may promote testicular growth.

BIBLIOGRAPHY

124

'uifa‘h .

_—. —_- —_T..‘.v‘
-

 

i?

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n
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40"-

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APPENDICES

137

APPENDIX TABLE 1

PLASMA AND TESTICULAR TESTOSTERONE AND

ANDROSTENEDIONE VALUES FOR INDIVIDUAL

HOLSTEIN BULLS BY MONTH

138

139

 

 

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APPENDIX TABLE 2

PLASMA TESTOSTERONE AND ANDROSTENEDIONE VALUES
FOR INDIVIDUAL HEREFORD BULLS BY MONTH AND
AT SLAUGHTER, AND TESTIS TESTOSTERONE

AND ANDROSTENEDIONE VALUES

141

142

 

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APPENDIX TABLE 3

AVERAGE PLASMA CONCENTRATIONS AND TOTAL PLASMA
CONTENT OF LUTEINIZING HORMONE AND THE AVERAGE
RATIO BETWEEN TOTAL PLASMA CONTENT AND TOTAL
PITUITARY CONTENT OF LUTEINIZING HORMONE IN
HOLSTEIN BULLS FROM BIRTH TO 12

MONTHS OF AGE

143

 

144

APPENDIX TABLE 3.--Average Plasma Concentrations and Total

Plasma Content of Luteinizing Hormone and the Aver-
age Ratio Between Total Plasma Content and Total
Pituitary Content of Luteinizing Hormone in Holstein
Bulls from Birth to 12 Months of Age.

 

 

 

LH
Age Plasma Plasma Plasmac
Concentration Content Pit. Ratio
(months) (ug/l)a (ug/animal)b
Birth 0.48d 0.59d 1.97
1 0.41d 0.63d 0.33
2 0.17 i 0.03 0.43 i 0.08 0.34
3 0.34 t 0.09 1.17 i 0.36 0.49
4 0.35 i 0.09 1.72 i 0.45 0.88
5 0.29 i 0.08 1.51 i 0.41 0.53
6 0.24 i 0.06 1.50 i 0.43 0.61
7 0.30 i 0.09 2.14 i 0.59 1.50
8 0.42 i 0.10 3.32 i 0.77 2.38
9 0.42 i 0.12 3.90 i 1.22 1.51
10 0.50 i 0.14 5.08 t 1.48 2.33
11 0.38 i 0.10 4.31 i 1.07 1.87
12 0.47 i 0.11 5.47 i 1.18 2.22

 

aug NIH-LH-B3 equivalent per liter.

bug LH per liter x body wt.

(kg) x 0.035.

cug plasma LH per animal % mg pituitary LH per animal.

d

Estimates derived from pooled samples.

APPENDIX TABLE 4

GONADAL SPERM CONCENTRATION AND SPERM NUMBERS

IN HOLSTEIN BULLS FROM 5 to 12 MONTHS OF AGE

145

146

APPENDIX TABLE 4.--Gonadal Sperm Concentration and Sperm
Numbers in Holstein Bulls from 5 to 12 Months of

 

 

Age.
Age Number Sperm Total
of Bulls Concentration Sperm
(months) (106 sperm/g (109 sperm/
parenchyma)a testis)
5 l 4.24 0.15
6 2 6.80 0.34
7 3 4.26 0.27
8 5 27.87 i 4.52b 2.62 i 0 62
9 5 50.37 t 3.80 7.80 i 1.39
10 5 38.99 i 1.77 5.41 + 0 62
ll 5 52.83 i 4.19 8.59 t 1.38
12 5 57.33 i 5.97 10.69 t 1.97

 

aInclude spermatids (stages VI to VIII) and Spermatozoa.

bi SE: only computed when average included 5 bulls.

APPENDIX TABLE 5

CHANGES IN TESTICULAR NUCLEIC ACID CONCENTRATION

AND CONTENT AND THE RNA:DNA RATIO FOR HOLSTEIN

BULLS FROM BIRTH TO 12 MONTHS OF AGE

147

148

APPENDIX TABLE 5.--Changes in Testicular Nucleic Acid
Concentration and Content and the RNA:DNA Ratio
for Holstein Bulls from Birth to 12 Months of

 

 

Age.
Age DNA RNA Total Total RNA/DNA
Conc.a Conc.a DNA RNA
(months) (mg/g) (mg/g) (mg/testis) (mg/testis)
Birth 7.18 5.05 13.64 9.71 0.70
l 6.26 5.32 17.28 15.39 0.90
2 5.43 5.94 34.93 37.80 1.11
3 4.64 5.19 43.40 49.26 1.13
4 4.09 6.13 79.72 120.35 1.54
5 3.95 5.56 111.23 157.19 '1.43
6 4.01 5.55 168.12 235.30 1.39
7 3.35 4.94 194.59 290.15 1.47
8 3.34 4.56 302.30 414.56 1.38
9 3.28 4.59 510.86 722.27 1.40
10 3.27 4.34 448.40 599.16 1.33
11 3.95 4.67 607.15 732.70 1.27
12 3.60 4.72 666.83 873.30 1.31

 

a .
Concentration

APPENDIX TABLE 6

CHANGES IN THE WEIGHT OF THE PAIRED SEMINAL
VESICLES AND THEIR DNA, RNA, CITRIC ACID AND
FRUCTOSE CONTENTS FOR HOLSTEIN BULLS FROM

BIRTH TO 12 MONTHS OF AGE

149

 

150

APPENDIX TABLE 6.--Changes in the Weight of the Paired
Seminal Vesicles and Their DNA, RNA, Citric Acid
and Fructose Contents for Holstein Bulls from
Birth to 12 Months of Age.

 

 

Weight Total Total Total Total
DNA RNA Citric Fructose
Acid
(months) (9) ----------------- (mg) -----------------
Birth 3.29 11.93 12.37 2.17 0.26
1 1.95 5.45 4.38 1.43 0.12
2 3.08 10.77 11.43 2.86 0.51
3 5.95 21.29 27.85 5.09 1.61
4 12.56 42.75 60.09 11.11 4.82
5 16.24 45.79 62.74 16.56 8.89
6 15.61 40.58 58.68 17.07 12.80
7 21.12 55.68 69.91 46.26 20.74
8 22.71 50.79 67.25 54.79 31.82
9 40.18 87.49 132.37 76.11 101.90
10 36.20 86.65 114.19 81.81 59.52
11 44.15 86.08 135.27 108.83 98.52
12 44.61 103.90 148.90 130.88 81.50

 

APPENDIX TABLE 7

PLASMA CONCENTRATION AND CONTENT OF LUTEINIZING
HORMONE IN HOLSTEIN BULLS FROM BIRTH TO 12
MONTHS OF AGE AS MEASURED BY

RADIOIMMUNOASSAY

151

 

152

APPENDIX TABLE 7.--P1asma Concentration and Content of
Luteinizing Hormone in Holstein Bulls from Birth
to 12 Months of Age as Measured by Radioimmuno-

 

 

 

assay.
Plasma LH
Age Bull No. Conc. Content
(months) (ug/l) (pg/bull)
l 0.38 0.39
2 1.00 1.22
0 3 0.40 0.63
4 1.74 2.05
5 0.00 0.00
MeaniSE 0.71:0.21 0.86:0.36
180 0.66 0.98
183 0.52 0.80
l 179 0.13 0.19
182 0.65 0.92
184 0.55 0.81
MeaniSE 0.5010.06 0.74:0.14
175 1.51 3.83
176 2.66 7.10
2 177 1.85 4.42
178 1.40 3.38
185 0.40 1.05
MeaniSE 1.6310.23 3.96:0.97
164 -- --
165 0.73 2.49
3 166 1.73 6.19
167 0.89 2.83
168 1.04 3.81
MeaniSE 1.10:0.15 3.83:0.83
163 0.21 1.01
170 0.19 0.94
4 171 0.97 4.97
172 0.00 0.00
174 1.89 8.60
MeaniSE 0.65:0.24 3.10:1.62
156 0.58 3.04
158 0.88 4.61
5 160 0.82 4.18
161 1.54 8.07
162 0.84 4.49
MeaniSE 0.93:0.11 4.88:0.84

153

APPENDIX TABLE 7.--con't.

 

 

 

Plasma LH
Age Bull No. Conc. Content
(months) (Hg/l) (Hg/bull)
150 0.43 2.66
153 0.41 2.37
6 154 0.81 5.36
155 0.12 0.84
157 0.36 2.07
Mean:SE 0.42:0.08 2.66:0.74
143 -- --
144 0.06 0.43
7 145 0.55 3.70
146 0.30 2.04
147 0.65 4.00
Mean:SE 0.39i0.09 2.54:0.82
137 0.57 4.70
138 0.08 0.63
8 136 0.68 5.74
142 0.60 4.75
148 0.03 0.23
Mean SE 0.40:0.10 3.21:1.15
120 0.00 0.00
121 1.07 9.61
9 122 0.00 0.00
125 0.02 0.18
126 0.76 7.46
Mean SE 0.3710.15 3.45:2.10
124 0.70 6.63
127 0.61 6.39
10 123 0.00 0.00
132 0.30 3.15
134 0.00 0.00
Mean SE 0.32:0.10 3.23:1.46
108 1.66 19.32
113 2.13 25.61
11 118 1.68 18.64
117 1.48 15.86
119 1.13 12.42
Mean:SE 1.61:0.12 18.37:2.18

__._ Emmi—3

APPENDIX TABLE 7.--con't.

154

 

 

 

Plasma LH
Age Bull No. Conc. Content
(months) (pg/1) (ug/bull)
101 1.15 13.91
102 0.92 10.59
12 104 1.82 22.80
107 0.46 6.17
109 1.54 15.63
MeaniSE 6.17:0.17 13.82:2.76

 

 

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