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ABSTRACT

IMMUNOBIOLOGICAL INHIBITION OF

LUTEINIZING HORMONE ACTIVITY
by Edward M. Convey

The object of this investigation was to determine
whether antisera to equine luteinizing hormone (Armour
PLH) could be prepared in goats, and whether this antisera
would neutralize the hormonal activity of luteinizing hor-
mone (LH) ig_yizg, A preliminary study employing rabbit
antisera to equine luteinizing hormone (RAELH), which had
been previously characterized, was designed to test the
feasibility of such an investigation.

Twenty-five adult rats, exhibiting normal estrous
cycles, were divided into five equal groups: (1) five
rats were injected with 0.85% saline; (2) five with con—
trol serum; (3) five with RAELH 12 hr before ovulation
(4) five with RAELH 24 hr before ovulation; and (5) five
with RAELH 36 hr before ovulation. Injections were admin-
istered daily for 10 days. Vaginal smears of rats injected
with RAELH fluctuated between those typical of proestrus
and/or estrus, while the control groups cycled normally.
Ovary, oviduct, uterus, adrenal and thyroid weights were

not effected significantly by the treatments (P)’0.05). In

by Edward M. Convey
contrast, the average weight (4.67 mg/100 g body weight) of
the pituitaries from the three groups that received RAELH
was significantly less than the comparable average (5.58
mg/100 g of body weight) for the two control groups (P( 0.05).
Pituitary LH activity, as measured by the ovarian ascorbic
acid depletion assay, was significantly higher in the
antisera treated rats (.521 ug of ascorbic acid/mg of
ovary), than in the control rats (.714 ug of ascorbic acid/
mg of ovary), (P( 0.05). These results probably reflect a
partial neutralization of endogenous LH.

In view of these encouraging results, the main body
of this research was undertaken. Two female goats were
immunized with PLH. Five precipitin lines were observed,
in agar, when the goat anti-equine luteinizing hormone
(GAELH) was titrated with PLH. A single precipitin line
developed after absorbtion of the antisera with normal goat
sera. This line was presumed to be due to an antibody
specific for LH. Absorbed GAELH did not cross react, in
agar, with crude extracts of rat or rabbit pituitary gland,
or NIH preparations of ovine LH, follicle stimulating hor—
mone, growth hormone, thyroid stimulating hormone, prolactin
or bovine LH.

As little as 0.1 m1 of GAELH was capable of neu—
tralizing i2_vitro the ascorbic acid depleting activity of
50 ug of PLH or 2.0 mg of crude saline extracts of rat
pituitary gland, In contrast, GAELH did not neutralize the

LH activity of ovine or bovine NIH—LH.

by Edward M. Convey

Sixty adult female rats, exhibiting normal estrous
cycles, were used to determine whether GAELH would inhibit
the estrous cycle. The rats were assigned to one of three
treatment groups: (1) 20 rats received 1.0 m1 of 0.85%
saline; (2) 20 rats received 1.0 m1 of control sera; (3) 20
rats received 1.0 m1 of GAELH. One half of the rats in
each group were killed after 6 days of treatment. The re-
maining rats were continued on treatment for an additional
10 days. All rats including those treated with GAELH ex-
hibited normal estrous cycles throughout the treatment
period. Ovary, uterus, adrenal and pituitary weights were
not effected by the treatments (P>v0.05). Similarly,
pituitary LH and FSH of the GAELH treated rats did not
differ significantly (P)>0.05) from those of the controls.
Failure of GAELH to effect the estrous cycle was probably
the result of a low titer of specific antibodies to LH.

Thirty adult female rats were used to determine
whether GAELH would inhibit ovulation. Rats were selected
on the basis of a normal 4-5 day estrous cycle pattern,
divided into five equal groups, and treated as follows:
(l) five received 0.85% saline 36 hr before ovulation;
(2) five received control sera 36 hr before ovulation;
(3) five received GAELH 12 hr before ovulation; (4) five
received GAELH 24 hr before ovulation; and (5) five re-
ceived GAELH 36 hr before ovulation. GAELH injected 24 or
36 hr before the next expected ovulation effectively in-

hibited ovulation. In contrast, GAELH did not inhibit

by Edward M. Convey
ovulation when injected 12 hr before expected ovulation.
Since 50%, instead of the expected 100% of the control rats
ovulated, the effects of GAELH on ovulation remain in

doubt.

IMMUNOBIOLOGICAL INHIBITION OF

LUTEINIZING HORMONE ACTIVITY

BY

Edward Michael Convey

A THESIS

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

MASTER OF SCIENCE

Department of Dairy

1965

BIOGRAPHICAL SKETCH

The author was born in Hicksville, New York on
October 12, 1939. He received his elementary education
at Jericho Grade School and Glen Cove High School. In
September of 1957 he entered Long Island Agricultural and
Technical Institute at Farmingdale, New York and was
graduated in 1959 with an Associate of Applied Science
degree. In September of 1960 he entered Michigan State
University and received his Bachelor of Science degree
in the field of animal husbandry in 1963. He began his
graduate work in the Department of Dairy in September of
1963. In July of 1964 he was awarded a National Insti-
tue of Health predoctoral fellowship. He received his
Master of Science degree in September 1965 in the De—

partment of Dairy with a major in Reproductive Physiology.

ii

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to
Dr. H.D. Hafs and Dr. H.A. Tucker for their aid and
guidance throughout the research and preparation of this
thesis.

Sincere appreciation is extended to Dr. Lon
McGilliard and Dr. E.M. Smith for their generous help
in preparing this manuscript.

Thanks are due to Claude Desjardins for his ad-
vice concerning the bioassay and immunological aspects
of this work, M.J. Paape, Y.N. Sinha, K.T. Kirton, J.K.
MacMillan and H. Hulkonen for their generous assistance
with the many laboratory tasks associated with this
research.

Appreciation is expressed to the National Insti-
tutes of Health for a predoctoral fellowship and for
the hormone preparations used in this investigation.

The help, motivation, and encouragement of Linda

Convey is greatly appreciated.

iii

TABLE OF CONTENTS

PAGE

 

BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . ii
ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . iii
LIST OF TABLES. . . . . . . . . . . . . . . . . . . . Vi
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . Vii

LIST OF APPEIJDICES. O O O O O O O O O O O O O O O O O Viii

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE. . . . . . . . . . . . . . . . . 3
History of Research on Antihormones . . . . . . . 3
Antigenicity of Gonadotrophins. . . . . . . . . . 5
Luteinizing hormone . . . . . . . . . . . . . 5
Follicle stimulating hormone. . . . . . . . . 7

Methods Used for the I2_Vitro Study of Hormone
Preparations. O O O O O O O O O O O O O O O O 9

In‘Vivo Neturalization of Hormones by Specific

Antisera. . . . . . . . . . . . . . . . . . . 10
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 13
Preliminary Experiment. . . . . . . . . . . . . . 13
Collection of Control Sera. . . . . . . . . . . . 14
Preparation of Antisera . . . . . . . . . . . . . 14
Agar Gel Double Diffusion . . . . . . . . . . . . 15
£3_Vitro Neutralization of Hormones . . . . . . . 18

iv

Antisera
Antisera
Bioassay
RESULTS. . .

Agar Gel

Inhibition of

Inhibition of

of LH and FSH

Estrous Cycles.

Ovulation . . .

Double Diffusion Characterization

GAELH. . . . . . .

IEIVitro

Antisera
Antisera
DISCUSSION .
SUMMARY. . .
APPENDIX . .

BIBLIOGRAPHY

Neutralization of Hormones. .

Inhibition of

Inhibition of

Estrous Cycles.

Ovulation . . .

19

20

21
27
32
39
45
52
55

58

TABLE

10.

11.

12.

13.

LIST OF TABLES

Pituitary luteinizing hormone activity of
rats treated with control sera or RAELH. .

Organ weights after administration of
rabbit anti PLH. . . . . . . . . . . . . .

Effect of goat anti—equine luteinizing

hormone on the biological activity of puri-

fied LH preperations or pituitary extracts

Effect of goat anti-equine LH on the bio-
logical activity of purified ESH and the
FSH present in PLH (equine LH) . . . . . .

Organ weights of rats treated with saline,
control sera, or GAELH for 6 days. . . . .

Organ weights of rats treated with saline,
control sera, or GAELH for 16 days . . . .

Pituitary luteinizing hormone activity of
rats treated with saline, control sera, or
GAELH fo r 6 day S O O O O O O O O O O O O O

Pituitary luteinizing hormone activity of
rats treated with saline, control sera, or
GAELH for 16 days. . . . . . . . . . . . .

Relative FSH potency of pituitary tissue
from control and GAELH— treated rats . . .

Inhibition of ovulation with GAELH . . . .

Ovarian and pituitary weights of rats treated

with saline, control sera or GAELH . . . .

Pooled pituitary LH activity of rats treated

with saline, control sera, or GAELH. . . .

Pooled pituitary FSH activity of rats

treated with saline, control sera, or GAELH.

vi

 

24

25

30

31

34

35

36

37

38
40

41

42

44

 

 

 

LIST OF FIGURES

FIGURE

1.

Number of rats exhibiting estrus or proestrus
vaginal smears before and after treatment
with rabbit anti-equine LH. . . . . . . . . .

Agar gel diffusion plate: Titration of
GAELH with equine LH. . . . . . . . . . . . .

Agar gel diffusion plate: Titration of
GAELH (absorbed) with equine LH . . . . . . .

Agar gel diffusion plate: Titration of
GAELH with crude saline extracts of rat
pituitary gland . . . . . . . . . . . . . . .

Agar gel diffusion plate: Titration of
GAELH (absorbed) with crude saline extracts
of rat pituitary gland. . . . . . . . . . . .

Agar gel diffusion plate: Titration of
GAELH with crude saline extracts of rabbit
pituitary gland . . . . . . . . . . . . . . .

Agar gel diffusion plate: Titration of

GAELH (absorbed) with crude saline extracts
of rabbit pituitary gland . . . . . . . . . .

vii

PAGE

 

23

29

29

29

29

29

29

LIST OF APPENDICES

APPENDIX PAGE

 

 

A. Assay of luteinizing hormone. . . . . . . . . 55

B. Assay of follicle stimulating hormone . . . . 57

viii

INTRODUCTION

Early reports concerning the physiological func-
tion of anterior pituitary hormones were based primarily
upon clinical observations of endocrine qkmd ablation as
a result of disease. The use of surgical and chemical
techniques for endocrine gland ablation, chemical extrac-
tion of hormones, replacement therapy, radioisotopic
tracer methods, and bioassays has advanced our knowledge
of pituitary hormone function to its present level.

Many previous investigations of hormone function
were undertaken using crude glandular extracts or semi—
purified preparations. Interpretation of hormone function
under these conditions is complicated by the interactions
of the several hormones which may be augmentative or
supressive. Therefore it would seem necessary, if indi—
vidual pituitary hormone functions are to be elucidated,
to study each hormone as a separate entity. This may be
accomplished by replacement of a single purified hormone
under conditions of hypophysectomy, or selective ablation
by specific inhibitory substances. Since selective in-
hibition allows the use of animals that more closely
approach the physiological norm, it would seem best suited

for investigations in this area.

2

Recent advances in immunology, as applied to hor—
mone research, have made such a technique available.
Since the anterior pituitary hormones are proteins, they
may cause suitable animals, to produce antibodies. These
antibodies are usually specific, in that they do not
react with other pituitary hormones and, as such, may be
used to selectively inhibit the biological activity of
the antigen (hormone) when injected into a test animal.
The physiological activity of this hormone may then be
studied by virtue of its specific elimination. Thus, the
present study was undertaken to investigate the estrous
cycle and ovulation inhibiting properties of antisera to

equine luteinizing hormone.

REVIEW OF LITERATURE

History of Research on Antihormones:

 

To the author's knowledge, the first report of a
hormone antagonist was published by Mobius in 1903. He
described a substance in the blood of thyroidectomized
sheep that neutralized the biological activity of thyroid
stimulating hormone (TSH). The sera of those sheep were
used clinically as a treatment of Grave's disease.

The discovery of insulin in 1922 by Banting and
Best was followed by numerous attempts to purify this
factor. De Jongh (1924) demonstrated a double—peaked dose
response curve with certain insulin preparations in vari-
ous stages of purification. In an effort to explain the
nature of this response he postulated the presence of an
"anti-insulin" that masked the biological response at an
intermediate level but was overcome when the insulin pre-
paration was administered in much higher doses. He removed
this "anti—insulin" by further purification of the hor-
mone, and demonstrated a normal dose response curve.

A report of altered gonadal physiology due to
chronic administration of hormone preparations was pub—
lished by Evans and Long (1921, 1922). While studying the
effects of intraperitoneally administered bovine anterior
pituitary extracts on growth rates in rats, they discovered

3

4
significant changes in the reproductive organs. Estrus,
as determined from vaginal smears, occurred infrequently
or not at all. Uterine weights were reduced and ovarian
weights augmented in the treated animals. Histological
examination of the ovaries revealed the formation of luteal
tissue about the ova in unruptured, and atretic follicles.
Collip (1932), reported that human chorionic gonadotrophin,
(HCG) produced greatly enlarged ovaries in rats. However,
after the initial period of stimulation, the ovarian
weights regressed to control levels or below. McPhail
(1933), reported a similar gonadal weight response with
pituitary implants in male and female rats.

There are many other reports in the early litera-
ture that refer to the development of a refactory condition
upon chronic administration of a hormone preparation. In
an effort to explain the nature of this phenomenon, Collip
(1934, 1935) formulated the theory of antihormones;

For each hormone there may be an opposite or antagon—
istic principle. This antagonist is present in the
normal subject but may not be demonstrated until it
exceeds in amount the hormone substance with which it
is balanced.
These antihormones were considered by Collip to be true
hormones and not the results of an antigen—antibody reac-
tion. Collip defended his theory of antihormones, as
opposed to an immunological response, on the following
basis: 1. Selye et a1. (1934) reported the presence of
an inhibiting substance in the serum of rats after pre-

treatment with homologous hormones. This was not consis—

tent with the nature of an immunological response.

5
2. Collip (1935) observed the presence of a substance
antagonistic to the anterior pituitary hormones in the
serum of certain individuals, never exposed to previous
hormone treatment. 3. Bachman (1935) studied the im—
munological nature of the refactory response. He con—
cluded that the antihormone effect did not parallel the
immunological effect and was probably due to a factor
other than an antigen—antibody response. 4. Animals made
refactory to hormone extracts of one species were also
refactory to hormone preparations of other species.

Although Collip's theory of antihormones was given
little support, and was finally replaced by the immunolog-
ical concept of antihormones, the work of these early in—
vestigators provided a working hypothesis to pursue the
nature of this phenomenon. As such, the theory of anti—
hormones has played an important role in the study of
endocrine physiology, and should be considered the genesis
of immunological techniques as a method in hormone research.
Antigenicity of Gonadotrophins
Luteinizing Hormone (LH):

Early investigations designed to determine the
antigenicity of hormone preparations were hindered by the
impurity of the hormones available. Attempts to obtain
antibodies to hormones EE£.§E usually resulted in the
development of heterogenous or multiple systems of anti-
bodies that confused attempts to determine antihormone
specificity. As more highly purified hormone preparations

were made available, immunological studies became more

6

meaningful. These purified hormones allowed for the pro—
duction of specific antibodies, of high titer, with little
or no extraneous antibody contamination.

Chow (1942) investigated the immunological pro-
perties of a preparation of luteinizing hormone "metaken—
trin" that satisfied several of the criteria of protein
homogenity. This hormone preparation stimulated formation
of specific antibodies in rabbits, as determined by pre—
cipitin and complement fixation reactions. Failure of
this antiserum to react with ovine and bovine luteinizing
hormone (LH), extracted by identical procedures, indicated
that porcine LH was immunologically species specific. This
lack of immunological similarity between porcine LH and
heterologous LH preparations was substantiated by Henry
and Van Dyke (1958). They reported that antisera, pre—
pared in rabbits, to ovine LH did not cross react im—
munobiochemically or immunobiologically with porcine LH.
This antisera did, however, neutralize the biological
activity of, and form precipitin bands with bovine and
ovine LH. More recently, Moudgal and Li (1961) isolated
ovine LH in a highly purified state. Zone electrophoresis,
ultracentrifugation, chromatography, and biological inves—
tigation were used to determine the homogenity of this
preparation. Immunological analysis substantiated the
purity of this hormone as evidenced by a single precipitin
band in agar gel double—diffusion and immunoelectrophoretic
systems. Homologous anterior pituitary hormones and serum

proteins did not cross react in agar. Analysis of cross

7

reactivity demonstrated a lack of immunological similarity
with chicken and salmon pituitary extracts, pregnant mares
serum gonadotrophin (PMSG), HCG and purified human LH.
However, this antisera did neutralize the LH activity of
PMSG, and human LH which indicated an antigen-antibody
reaction at other than the active site. Contrary to the
investigations of Segal et a1. (1962) and Henry and van
Dyke (1958), immunological and immunobiological cross
reactions with porcine and rat pituitary LH were demon—
strated. This report, however, did substantiate that HCG
and ovine LH do not manifest identical immunological be—
havior.

Chemical alterations in one or more of these hor-
mone preparations, variations in antibody titer, or dif—
ferences in investigating techniques may account for the
lack of agreement among these reports. Desjardins and
Hafs (1965) investigated the immunological properties of
rabbit antisera to equine LH (PLH). This antisera, after
absorption with equine blood sera, resulted in a single
precipitation in agar when titrated against its homologous
antigen (PLH). Neutralization of the biological activity
of equine, bovine and ovine LH, as well as saline extracts
of bovine and ovine pituitaries was demonstrated after in-

cubation with the antisera to PLH.

Follicle Stimulating Hormone (FSH)

The antigenicity of purified FSH preparations were
J

demonstrated by Van Dyke et a1. (1950). Purified FSH, of

8

ovine and porcine origin, stimulated (in rabbits) the for—
mation of antibodies that precipitated their respective
antigens. Failure to cross react with the heterologous
antigen indicated porcine and ovine FSH to be immunolog—
ically dissimilar. Further evidence for the antigenicity
of FSH was reported by Maddock et a1. (1953) in a_c1inical
investigation. Realizing the antigenicity of hormone pre-
parations, they caused the development of antibodies to
porcine FSH in women suffering ovarian hyperfunction.
Plasma from these patients inhibited the FSH activity of
human pituitary gonadotrophin, HCG, and porcine FSH in
immature female rats. They concluded that temporary
ovarian quiesence may be achieved by the production of
antigonadotrophins.

More recently Segal et a1. (1962)and Ely and
Tallberg (1964), have investigated the immunological pro-
perties of purified preparations of ovine FSH. Both lab—
oratories reported the formation of doublet precipitin
bands, when the absorbed antisera were reacted with their
antigens, indicating two antigenic components. No cross
reaction occurred with other pituitary trophic hormones
of ovine origin or with HCG and PMSG. In addition, Segal
et a1. (1962) reported that neither crude extracts of rat
hypophysis (with known FSH activity) nor human menopausal
urine gonadotrophin, reacted with the antisera in agar gel
diffusion plates. Since both of these crude preparations
demonstrated FSH activity, it appeared the hormonal and

antigenic sites were different.

9

The antigenicity of human FSH was reported by
McCarry and Beck (1963). Hemagglutination tests failed
to demonstrate any immunological similarity between human
FSH and homologous pituitary trophic hormones. Human
chorionic gonadotrophin, when used in high quantities,
did cross react with the antisera in hemagglutination
tests. With the sole exception of monkey FSH, all
heterologous hormone preparations tested failed to cross
react. As reported with ovine FSH, human FSH appeared to
have two antigenic components. In a subsequent report,
Saxena and Henneman (1964), verified the antigenicity of
purified human FSH preparations as well as its immunolog-
ical similarity to HCG. They did not, however, report two
antigenic components.
Methods Used for the In Vitro Study of Hormone
Preparations

Recent developments in immunological methodology
have stimulated its use in several areas of research. Once
limited to a branch of bacteriology, immunology is now em-
braced as a useful analytical tool in many diverse areas of
research. Some immunological techniques are directly
applicable to hormone research and as such have gained pop-
ularity with workers in this field. These techniques in-
clude complement fixation (Trenkle et al., 1960); (Brody
and Carlstrom, 1961); hemagglutination (Boyden, 1951; Wide
and Gemzell, 1960); anaphylaxis and quantitative precipitin
reactions (Hayashida and Li, 1959) and diffusion—in-gel

rnethods, Ouchterlony, (1958). These methods of hormone

10
analysis are simple to perform, require a minimum of equip—
ment, and yet are specific, sensitive, and usually repeat-

able.

In Vivo Neutralization of Hormones by Specific Antisera

A primary advantage of the antigen—antibody reaction
is its specificity. However, physiological investigation
based on this specificity must be preceded by experiments
designed to establish the specificity of the antibody for
its homologous antigen in each series of experiments. Once
this is established, the application of these antisera to
.the investigation of physiological phenomenon is justified.
Specific antisera have been applied to the detection of
minute quantities of hormones in body fluids (immunoassay),
elimination of contaminating hormones in hormone purifica—
tion procedures, and of particular interest here, to effect
altered endocrine physiology by partial immunological
hypophysectomy.

Ely (1957) utilized antigonadotrophic serum to
determine whether the changes that occur in female mice
after whole body irradiation were due to a copious release
of pituitary gonadotrophins. Rabbit antisera to sheep
gonadotrophins prevented the expected increases of ovarian
and uterine weight. Estrous cycles, as determined by vag-
inal smears, were irregular in the antisera-treated rats
Inhereas the control rats cycled normally. He concluded
“that the hypergonadotrophic condition which followed irradi-

Eation was prevented by the antigonadotrophin administration.

11

Antisera, prepared in rabbits, to purified ovine
LH, (Henry and Van Dyke, 1958; and Moudgal and Li, 1961)
neutralized the activity of their homologous antigen (ovine
LH) when both the hormone and the antisera were injected
into immature hypophysectomized rats. In addition, Henry
and Van Dyke (1958) investigated the ability of the
antisera to neutralize the biological effects of endogenous
rat LH. They could not, however, demonstrate effective LH
neutralization.

Bourdel (1961) reported neutralization of endo-
genous rat LH activity in immature female rats. Antisera,
administered intraperitoneally daily for 4 days, resulted
in decreased ovarian and uterine weights which were cor—
related with the amount of antisera injected. Histolog—
ically these organs bore a striking resemblance to those
of hypophysectomized rats of the same age. This report
prompted a subsequent investigation to determine the effect
of rabbit antisera to ovine LH in the adult female rat
(Bourdel and Li, 1963). Antisera injections, initiated l,
2 and 3 days before the next expected estrus and continued
for 12-day period, resulted in a 25% decrease in ovarian
weight, and a 60% decrease in uterine weight. Ovarian
histology revealed normal healthy follicles in all the
antisera-treated animals. However, none of these follicles
were as large as those normally seen in a proestrous
female. When antisera injections were administered 12 hr
before the next expected estrus, the estrus occurred

normally but failed to reoccur for the remainder of the

“xi

12
treatment period. When antisera treatment was initiated
24 or 36 hr before the next expected estrus, cornified
cells were never detected in the vaginal smears. Kelly gt
“al.,(1963L investigated the ovulation inhibiting prOper—
ties of rabbit anti-ovine LH. Antisera was administered
intraperitoneally to female rats at approximately the
time of release of LH from the pituitary gland, i.e.
(approximately 12 hr before the expected ovulation).
Ovulation was effectively inhibited in 10 of 11 animals
while all control rats treated with control rabbit sera
ovulated as expected. Although ovulation was blocked,
estrus occurred normally. This was in agreement with the
results of Bourdel and Li (1963).

That rabbit anti—ovine LH will effectively neutralize
the biological effects of endogenous LH was demonstrated by
Hayashida (1963) in male rats. Daily injections of anti-
sera resulted in a 79% depression of testis weight and
complete suppression of spermatogenesis. Similarly, anti-
sera treatment of mature males resulted in a 45% suppres—
sion of testicular weight, an 86% suppression of prostate
weight, and a parallel decrease in seminal vesicle weight.
Spermatozoa obtained from the vas deferens of control—sera—
treated rats showed normal mobility while spermatozoa from
antisera-treated rats were completely immobile. Perhaps
the most interesting effect demonstrated in this investi-
<gation, was the complete withdrawal of the testis into the

.inguinal canal in all antisera—treated immature males.

MATERIALS AND METHODS

Preliminary Experiment

 

To determine the feasibility of using anti—LH pre—
parations to inhibit the physiological activity of endo—
genous LH, a preliminary experiment was conducted. Rabbit
antisera to equine LH (Armour PLH),1 prepared and immunolog-
ically characterized by Desjardins and Hafs (1965), was
used in this trial. Twenty-five adult female rats were
selected on the basis of regular 4 or 5 day estrous cycles
as determined from the vaginal smears. Thereafter five
control rats were injected with saline; five controls with
normal rabbit serum; five with rabbit anti-equine lutein-
izing hormone (RAELH) beginning 12 hr before expected
ovulation; five with RAELH beginning 24 hr before expected
ovulation; and five with RAELH beginning 36 hr before ex—
pected ovulation. Injections were administered daily in
0.5 m1 quantities. The injection period was 10 days and
the animals were killed 24 hr after the final injection.

At autOpsy the ovaries, uteri, oviducts, adrenals and)
pituitaries were isolated and weighed. The pituitaries
were stored at —20°C and subsequently assayed for LH activ-
ity by the rat ovarian ascorbic acid depletion assay

(Parlow, 1961) described in Appendix A.

—____

lArmour Pharmaceutical Company: Omaha, Nebraska.

13

14
On the basis of the encouraging results of this
preliminary experiment, the main body of this research
was undertaken. Since large volumes of antisera would be
needed to extend these preliminary studies to the bovine,
the possibility of using an animal which would permit the
collection of large volumes of antisera was investigated.

Thus, goats were immunized with PLH.

Collection of Control Sera

 

Prior to immunization, five female goats were bled
by venipuncture to obtain control sera. The blood was
collected in 50 m1 beakers and incubated at room termpera—
ture for 30 min. At the end of this period the clots were
separated from the beaker wall with a hardwood stick, and
stored overnight at 5°C to allow the clot to contract. The
sera were decanted, centrifuged at 15,000 x g, and 1% of a
1:1,000 solution of Merthiolate2 added to retard bacterial
growth during subsequent analysis.

The sera were pooled and stored at -20°C in 10 m1

aliquots.

Preparation of Antisera

 

Two of the five goats were immunized with a com—
mercial preparation of equine LH (Armour PLH). This hor—
mone was diluted with physiological saline and emulsified

with an equal quantity of Freund's adjuvant3 in a Servall

 

2Bios. Laboratories Inc., N.Y., New York.

3Difco Laboratories, Detroit 1, Michigan.

15
omnimixer. The adjuvant was utilized to enhance antibody
production. The preparation was considered satisfactory
for injection when a drop of the emulsion would not disperse
on water. The first injections employed Fruend's complete
adjuvant while subsequent injections employed Freund's in-
complete adjuvant. The complete adjuvant contained
Arlacel A (mannide monooleate) 1.5 ml; Bayol F (paraffin

oil), 8.5 m1; and Mycobacterium butyicium, 5 mg. Incom-

 

plete adjuvant contained the same quantity of mannide
monooleate and paraffin oil but no bacteria.

Initially, each goat received a total of 300 mg of
PLH, contained in 15 m1 of a saline—adjuvant emulsion,
administered in three equal biweekly injections. An addi—
tional 100 mg of PLH in 5 ml of emulsion was administered
5 months following the third injection. All injections
were administered subcutaneously in the supra scapular re—
gion. Two weeks after the third and fourth injections,
blood (approximately 400 m1 from each goat) was obtained
by venipuncture. Immune sera was obtained by the procedure
previously described for control sera. The antisera were

pooled and stored at -20°C for subsequent use.

.Agar Gel Double Diffusion

The technique of double diffusion in gels, as des-
cribed by Ouchterlony (1958), is contingent upon the
characteristic diffusion rates of both antigen and antibody
in a gel matrix. The object of this technique is to bring

together, by diffusion, optimal concentrations of antigen

l6
and antibody which precipitate and appear as lines. If
the concentrations are optimal, the number of lines re-
solved is indicative of the minimal number of antigen—
antibody systems present. Because the PLH preparation
used to immunize the goats was not homogeneous, the
double diffusion plate technique was used to investigate
the heterogeniety of the antibody spectrum.

The plates used in the agar gel diffusion analyis,
were prepared by folding pieces of Whatman no. 1 filter
paper (2.5 x 0.5 cm) over the lip of the male half of a
90 mm petri dish. The pieces were held against the wall
of the dish with a ring of stainless, 26 guage wire approx-
imately 24" long.

The agar gel was prepared by mixing 8.5 gm of
Oxoid Ionagar4 no. 2 in one liter of 0.85% NaCl buffered
with 0.005 M phosphate buffer at pH 7.4. The mixture was
autoclaved at 15 psi for 30 min. The hot agar was filtered
through Whatman no. 1 filter paper and 30 ml aliquots de-
livered into each of the previously prepared plates. The
agar was allowed to solidify, covered, and stored at 5°C
in a water saturated container until used.

Just prior to use, wells were cut in the agar
with a Feinberg (no. 1801) template4 and the agar plugs
removed. One drop of molten agar was used to seal the
base of each of the peripheral wells and two drops to seal

the base of the center well. In the event an air bubble

 

4Consolidated Laboratories, Inc., Chicago
Heights, Illinois.

17
developed while sealing the wells, the well was recut and
resealed with molten agar. Each plate was assigned an
identification number, placed above and to the right of
the number one peripheral well, thus serving to identify
the position of the wells as well as the plate. A 1.5%
solution of Alcian blue in 3.0% acetic acid was used for
marking purposes. Antigen and antibodies were introduced
into the wells and allowed to diffuse at 5°C. With the
first development of precipitin lines the plates were
photographed with Kodak high contrast film. Subsequent
changes in precipitin lines were also photographed, thus
establishing a permanent record of development of each
plate.

To determine the relative amounts of precipitating
antibody, it was desirable to determine that ratio of
antigen to antibody that would achieve the maximal number
of precipitations. Thus, various dilutions of antigen
were placed in the peripheral wells and reacted against a
constant concentration of antisera in the center well.

The Bjorkland (1952) modification of Ouchterlony's
double diffusion method was also utilized. This involved
the addition of normal horse sera to the center well 24
hr prior to the addition of immune sera against equine LH.
Antibodies against normal horse sera components were thus
absorbed (precipitated in the well) while other antibodies,
including those specific for equine LH, diffused into the

surrounding agar.

18
Ouchterlony plates were also designed to deter—

mine the presence or absence of contaminating antibodies
in GAELH, that would form precipitins with ovine or
bovine LH, TSH, growth hormone (GH), FSH, prolactin, or
HCG.5 Serial dilutions of the hormone to be analyzed
were placed in the peripheral wells and reacted against
GAELH which was placed in the center well. Plates were

photographed at varying intervals as previously described.

In Vitro Neutralization of Hormones
The capacity of GAELH to neutralize the ovarian
ascorbic acid depleting properties of ovine and bovine
LH, PLH, and rat pituitary extracts, as well as the
ovarian weight augmenting properties of PLH and FSH,5
was investigated according to the following proceedure.
Each hormone to be assayed was mixed in a glass
centrifuge tube with 0.1 ml of either saline, control goat
sera, or GAELH. The resulting dilution was incubated in
a.water bath at 37°C for 30 min and then at 5°C for 48 hr.
.Amy resulting floccules were sedimented by centrifugation

eat 15,000 x g for 30 min, and the supernatant fluids were

eassayed for LH and FSH hormone activity (Appendix A, B).

—_¥

5The Endocrinology study section of the National
IInstitutes of Health supplied the following highly puri-
Ified hormone preparations for use in these studies:

Ovine TSH-SZ

Ovine FSH—S3

Ovine LH -S8

Ovine GH -S6

Ovine prolactin-S6

Bovine LH—B3

19

Antisera Inhibition of Estrous Cycles

Sixty normal adult female rats, weighing between
150 and 200 gm were used to determine whether or not
GAELH would inhibit estrous cycles. Rats were selected
for treatment on the basis of cornified cells in the
vaginal smears which were taken to be an indication of a
normal estrous cycle (Long and Evans, 1922). The rats
were assigned to one of three treatment groups: (1) 20
rats received physiological saline; (2) 20 rats received
normal goat serum; and (3) 20 rats received GAELH. All
injections (1.0 ml) were administered daily, intra-
peritoneally. Vaginal smears were observed daily between
9AM — 11AM. At the end of 6 days of treatment, 10 rats in
each group were killed. The remaining 10 rats in each
group were continued for an additional 10 days. From the
9th to 15th day, an antisera, obtained from the original
two immunized goats after the final booster dose, and be—
lieved to contain a higher titer of antibodies, was sub—
stituted for the original antiserum. On the 16th day of
'treatment the remaining rats were killed and autopsied 24
fir after the last injection. All rats were weighed at
‘the beginning and end of the treatment periods.

At autopsy, the ovaries, uterus, vagina, and
aadrenals were isolated and weighed on a torsion balance.
VVith the exception of the adrenals all tissues were sub-
ESequently fixed in Bouin's fluid, embedded in paraffin,

Shectioned at 8 u and stained with hematoxylin and eosin.

20
Pituitary glands were dissected from the sella
cica and weighed to the nearest 0.2 mg. One-half of the
gland was frozen, stored at —20°C, and subsequently used
for LH and FSH bioassay (Appendix A, B). The remaining
portion of the pituitaries were fixed, embedded and sec—

tioned as given above, then stained with acid fuchsin and

aniline blue (Russel, 1939).

Antisera Inhibition of Ovulation

Thirty normal adult female rats, weighing between
200 and 250 gm were used to determine whether or not
GAELH would inhibit ovulation when injected at varying
intervals before the time of expected ovulation.

Prior to the initiation of this treatment, estrous
cycles of 150 adult female rats were checked for regularity
using the vaginal smear technique (Long and Evans, 1922).
Thirty animals were selected on the basis of a normal 4—5
day cycling pattern for at least five cycles and divided
into the following groups: (1) five rats were injected
(with physiological saline 36 hr before ovulation; (2) five
vvith normal goat sera 36 hr before ovulation; (3) five
Vvith GAELH 12 hr before ovulation; (4) five with GAELH 24
lir before ovulation; and (5) five with GAELH 36 hr before
(ovulation. In all cases 1.0 m1 of the treatment material
Vvas injected via the intraperitoneal route, in a single
iJijection. Estimation of the time of ovulation was based
CH1 the investigations of Everett (1948, 1956). He reported

tfliat ovulation occurred between 2 and 4 AM on the morning

21

following the day of proestrus. Thirty two hr after the
time of expected ovulation the rats were killed.

At autopsy, the oviducts were isolated and flattened
between glass slides. The flattened oviducts were then
examined microscopically and the number of ova within the
oviducts was recorded.

The pituitary glands were dissected from the sella
turcica, weighed, frozen at -20°C, and subsequently
assayed for LH and FSH as described in Appendix A and B,

respectively.

Bioassay of LH and FSH

 

LH activity was determined by the ovarian ascorbic
acid depletion assay (Parlow, 1961) as described in
Appendix A. FSH activity was assayed by the method of

Steelman and Pohley (1953) as described in Appendix B.

RESULTS

Rats injected with RAELH 12 hr before the next
expected ovulation exhibited one complete cycle after
treatment was initiated but failed to cycle thereafter.
When RAELH was administered 24 or 36 hr before the next
expected ovulation, the rats exhibited normal estrous
patterns until the next proestrus and/or estrus. There—
after, these rats continued in either proestrus or estrus,
throughout the remainder of the treatment period. Fig. 1
shows the number of rats (RAELH treated) exhibiting
estorus or proestrous smears prior to and during the
treatment period. The saline—injected control rats, and
rats injected with normal rabbit serum continued to ex—
hibit regular estrous cycles throughout the treatment
,period.

The results of the pituitary LH bioassay, of the
{preliminary experiment, are presented in Table l. The
Lbooled pituitaries from the control rats contained signif—
:icantly less LH activity (0.714 ug ascorbic acid/mg ovary)
'than the pooled pituitaries from rats treated with RAELH
(p.521 ug ascorbic acid/mg ovary).

Ovary, oviduct, uterus, adrenal, thyroid and body
Weights were not significantly affected (P)0.5) by the
FGAELH treatments (Table 2). In contrast, the average

22

 

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TABLE 1. Pituitary luteinizing hormone
activity of rats treated with
control or RAELH.a

 

Treatment No. of rats Ovarian ascorbic
' acid (ug/mg)

 

x':_S.E.

5 ug NIH LH 51 5 .841 i .05
20 ug NIH LH 51 5 .207 1 .03
320 ug RAELHb 5 .521 1 .07
320 ug ControlC 5 .714 i.°07

 

aRabbit anti-equine luteinizing hormone.
bPooled pituitaries from rats treated with antisera.

CPooled pituitaries from control rats.

25

.Hwflumpme HmncmEHquxm HE m.o nuflz pmpuwncfi pmu comm

Q

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.N mnm<e

26

weight of the pituitaries (4.67 mg/100 g body weight)

from the three groups that received RAELH was signif—
icantly less (P<:0.05) than the comparable average (5.58
mg/100 g body weight) of the two control groups. The
uterine weights from the saline controls were signif—
icantly heavier than the uterine weights from the control-
sera—, or the RAELH-treated rats. However, the uteri from
rats treated with control sera were not significantly dif—

ferent from the RAELH—treated rats (P)'0.05).

Agar Gel Double Diffusion Characterization
of GAELH

Fig. 2 illustrates the precipitation reaction
that developed when GAELH was titrated with its homologous
antigen (equine LH). Although all the precipitin lines
cannot be seen in the photograph, a minimum of five pre—
cipitins developed over a period of 2 weeks. Because the
antigen was known to be heterogeneous, the Bjorklund (1952)
modification was used to determine if any of the pre-
cipitations ovserved were specific for equine LH. When
(SAELH was absorbed with normal equine blood sera a single
Ebrecipitin developed. This plate is illustrated in Fig. 3.

Fig. 4 and 6 illustrate the precipitins that de-
\reloped when GAELH was titrated against crude saline
(0.85%) extracts of rat and rabbit pituitary glands respec-
‘tively. In each case a single diffuse precipitin line
developed. These lines did not develop, however, when the

GAELH was preabsorbed with normal equine blood sera, Fig.

5 and 7.

27

No precipitin lines developed when GAELH was
titrated with purified ovine FSH, LH, TSH, GH, prolactin,

or bovine LH.5

In Vitro Neutralization of Hormones

 

The LH activity of equine LH (50 ug), and rat
pituitary extracts (2.0 mg), were significantly reduced
when these preparations were incubated with 0.1 ml of
GAELH (P<(0.05). Analysis of variance indicated that the
LH activity of equine LH, after pretreatment with GAELH,
did not differ significantly (P) 0.05) from that of saline.
This indicates that as little as 0.1 m1 of GAELH completely
neutralized the biological activity of 50 ug of equine LH.
In contrast, normal goat sera or saline failed to signif-
icantly influence the ovarian ascorbic acid depletion
effects of these hormones.

Purified preparations of ovine and bovine LH were
not significantly affected by the GAELH treatment (P>»0.05).
These results are listed in Table 3.

FSH bioassay of the equine LH used in this study
revealed that FSH was present as a contaminent 6.11 ug
equivalents5 (ug of equine LH). Incubation of equine LH
with 0.1 m1 of GAELH did not, however, significantly reduce
the FSH present in this preparation. Similarly, purified
preparations of ovine FSH5 was not significantly affected

when incubated with GAELH (Table 4).

Fig. 2 and 3

 

Titration of GAELH and absorbed GAELH with equine
LH. The peripheral wells contained a serial dilution of
equine LH (0.1 ml). The center wells (CW) contained 0.25

ml of GAELH (Fig. 2) or 0.25 ml of absorbed GAELH (Fig. 3).

(A) 1000 ug/ml (D) 125 ug/ml
(B) 500 ug/ml (E) 62.5 ug/ml
(C) 250 ug/ml (F) 31.25 ug/ml

Fig. 4 and 5

 

Titration of GAELH and absorbed GAELH with crude
saline extracts of rat pituitary gland. The peripheral
wells contained a serial dilution of rat pituitary gland
(0.1 ml). The center well (CW) contained 0.25 ml of

GAELH (Fig. 4)or 0.25 ml of absorbed GAELH (Fig. 5).

(A) 10 mg/ml (D) 1.25 mg/ml
(B) 5 mg/ml (E) 0.625 mg/ml
(C) 2.5 mg/ml (F) 0.3125 mg/ml

Fig. 6 and 7

 

Titration of GAELH and absorbed GAELH with crude
saline extracts of rabbit pituitary gland. The peripheral
wells contained a serial dilution of rabbit pituitary
gland (0.1 ml). The center well (CW) contained 0.25 ml of
GAELH (Fig. 6) or 0.25 ml of absorbed GAELH (Fig. 7).

(A) 10 mg/ml (D) 1.25 mg/ml

(B) 5 mg/ml (E) 0.625 mg/ml

(C) 2.5 mg/ml (F) 0.3125 mg/ml

29

 

30

TABLE 3. Effect of GAELHa on the biological activity of
purified LH preparations or
pituitary extracts.

 

 

 

Hormone or No. Ascorbic acid content
Pituitary extract of of the ovary
rat Mgan S.E.
ug/mg
Saline 5 .958d 2,-05
Ovine LH (1.6 ug) + GAELHa 5 .536d :_.04
Ovine LH (1.6 ug) + NGSb 5 .516d I.°O3
Ovine LH (1.6 ug) 5 .483d :_.03
Equine LH (50 ug) 5 .622d 1 .01
Equine LH (50 ug) + GAELHa 5 .850d 1 .01
Equine LH (50 ug) + NGSb 5 .658d 1 .04
Bovine LH (1.6 ug) 5 .283eli .02
Bovine LH (1.6 ug) + GAELHa 5 .2838 i .04
Bovine LH (1.6 ug) + NGSb 5 .311e :_.03
rat PEC (2.0 mg) ‘ 5 .573e : .06
rat PEC (2.0 mg) + GAELHa 5 .618e :_.06
rat PEC (2.0 mg) + NGSb 5 .550e : .03

 

aGoat anti—equine luteinizing hormone.

bNormal goat sera.

CRat pituitary extract 2.0 mg wet weight.
dFirst ovary used for analysis (see Appendix A).

eSecond ovary used for analysis (see Appendix A).

31

TABLE 4. Effect of GAELHa on the biological activity
of purified FSH or the FSH present
in PLH (equine LH).

 

 

Hormone No. ug FSH standardb/ug of
Preparation of hormone preparation
rats
NIH—FSHa 5 1.000
NIH—FSH + 0.1 ml GAELH 5 .974
PLH 5 .11367
PLH + 0.1 ml GAELH 5 .17259

 

aGoat anti—equine luteinizing hormone.

bNIH-FSH—S3.

32
Antisera Inhibition of Estrous Cycles

The vaginal cycle patterns of rats treated with
GAELH for 6 days, demonstrated no significant differences
from those of either saline — or normal-goat—sera injected
controls. All rats exhibited regular 4 or 5 day cycles
throughout the treatment period. Similarly, other female
rats treated with the above antisera for 6 days, and with
an antisera believed to contain a higher titer of anti-LH
for an additional 10 days, did not exhibit any significant
differences in estrous cycle patterns.

Histological examination of the vaginal epithelium
supported the vaginal smear data. Vaginal epithelial
histology suggested that rats from both the antisera—
treated, and control—group were in all stages of the
estrous cycle. Similarly, the uteri from all groups ex—
hibited a normal appearance with all stages of the estrous
cycle represented.

The ovaries of all groups revealed many developing
follicles, several of which had developed to the antrum
stage. Mitotic activity in the granulosa suggested that
normal follicular development was occurring. Corpora
lutea were also present in all the ovaries examined. No
significant changes in the interstitial tissue could be
discerned.

In view of the preceding data, no detailed cell
counts were made on the pituitary glands. However, both
acidophils and basophils appeared to be of normal size and

shape with no vacuolization or other gross histological

33

alterations. No differences in cell staining intensity
could be discerned in the pituitaries from any of the
treatment groups.

It is evident from Tables 5 and 6, which sum-
marizes body weights and organ weights obtained from the
different treatment groups during the 6 and 16 day injec—
tion periods, respectively, that GAELH did not signif—
icantly effect body weight gains (PT>0.05). Similarly,
the weights of the adrenals, ovaries, uteri and pituitaries
were unaffected by the treatments (P:>0.05).

Tables 7 and 8 summarize the LH activity of the
pituitary glands obtained from those rats treated with
GAELH for 6 and 16 days, respectively. No significant
difference (P)>0.05) was found in the LH activity of the
pituitary glands from control- and antisera—treated rats
when injections were continued for 6 days. Similarly,
substitution of a more highly potent antisera and contin-
uation of treatment for an additional 10 days, did not
significantly effect pituitary LH activity with respect
to controls (P > 0.05) .

Due to the small quantity of pituitary tissue
available, FSH determinations were made on the basis of
three assay rats per point. Table 9 lists the relative
potency estimates based on NIH—FSH—S3 as a standard. It
is apparent from this table that the quantity of FSH pre—
sent in the pituitary glands obtained from the different
treatment groups, did not differ significantly, whether

treatment was continued for 6 or 16 days.

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35

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36

TABLE 7. Pituitary luteinizing hormone activity of rats
treated with saline, NGSa, or GAELHb for 6 days.

 

Treatment No. of rats Ovarian ascorbic
acid (ug/mg)

 

Ԥ.i S.E.

0.4 ug NIH—LH—S8 5 .750 i.°04
1.6 ug NIH-LH—S8 5 .524 i .02
200 ug saline 5 .750 I.‘01
200 ug NGSa 5 .744 3:. .03
200 ug GAELHb 5 .744 + .02

 

aNormal goat sera.

bGoat anti-equine luteinizing hormone.

37

TABLE 8. Pituitary luteinizing hormone activity of rats
treated with saline, NGSa, or GAELHb for 16 days.

 

Treatment No. of rats Ovarian ascorbic
acid (ug/mg)

 

£.i S.E.

0.4 ug NIH—LH—S8 5 .661 i .02
1.6 ug NIH-LH—S8 5 .570 :_.04
100 ug saline 5 .648 i .02
100 ug NGS 5 .666 i .05
100 ug GAELH 5 .718 i .02

aNormal goat sera.

bGoat anti-equine luteinizing hormone.

38

TABLE 9. Relative FSH potency of pituitary tissue of
rats treated with saline, NGSa, or
GAELHb for 6 or 16 days.

 

Treatment No. of rats ug FSH standardC/mg
pituitary tissue

 

Saline — 6 days 3 14.086
NGSa - 6 days 3 11.664
GAELHb — 6 days 3 8.248
Saline — 16 days 3 15.110
NGSa — 16 days 3 11.632
GAELHb — 16 days ' 3 14.3106

 

aNormal goat sera.

bGoat anti—equine luteinizing hormone.

CNIH—FSH s3

39

Antisera Inhibition of Ovulation

 

The effect of injecting saline, control sera,
GAELH 12 hr before ovulation, GAELH 24 hr before ovulation,

GAELH 36 hr before ovulation, on the number of ova released

from the ovary is summarized in Table 10.

No ova were found in the oviducts when GAELH was
injected 36 hr before the next expected ovulation. Sim—
ilar results were observed when GAELH was injected 24 hr
before ovulation in that four of the five rats failed to
ovulate. When GAELH was injected 12 hr before expected
ovulation (at the time of release of LH from the pitui—
tary) four of the five rats ovulated. Contrary to expec—
tations, however, only three of the saline control, and
two of the control sera group ovulated when injected 36 hr
before the next expected ovulation.

Whether ovulation had occurred was, in all cases,
reflected in the condition of the uterus. When the uterus
was distended with luminal fluid, no ova were found in the
oviducts.

The weights of the ovaries and pituitaries obtained
from rats in the different treatment groups are summarized
in Table 11. Ovary and pituitary weights were not affected
significantly by the GAELH treatments (P:>0.05).

The LH activity of the pooled pituitary glands,
from each of the different GAELH-treatment groups, are
listed in Table 12. Analysis of variance indicated no sig—
nificant differences due to treatments (PI>0.05). Similarly,

GAELH injected 12, 24 or 36 hr before the next expected

4O

 

 

TABLE 10. Inhibition of ovulation with GAELHa
Time injected No. of No. of
Treatment before expected rats rats that
ovulation (hr) Ovulated

b

Saline 36 5 3
b

Control sera 36 5 2
GAELHa’b 12 5 4
GAELHa’b 24 5 l
GAELHa’b 36 5 0

 

a . . . . .
Goat anti—equine luteinizing hormone.

b

All materials injected in 1 m1 quantities.

TABLE 11.

Ovarian and pituitary weights of rats
treated with saline, NGSa or GAELHb.

 

 

 

Time injected No. Weight (mg)
Treatment before ovula— of
tion (hr) rats Ovary Pituitary
E :. S.E. $6 _+_ S.E.
SalineC 36 5 74.7 _+_ 4.90 13.4 i .64
a,c
NGS 36 5 73.1 .t 6.23 12.3 i .89
GAELHb’C 12 5 72.6 i. 2.92 12.7 i .24
GAELH‘b’C 24 5 77.5 1 3.56 13.3 2‘. 1.14
GAELHb’C 36 5 73.2 1. 3.22 12.2 2:. .59

 

aNormal goat sera.

b

CEach rat injected with 1.0 m1 of experimental

Goat anti—equine luteinizing hormone.

material.

42

.AHSGHEB umBV uomquo >HMHHSMHQ mcHHmmU

.mcoeuor mEHNHCHmpSH mafisqmlflbcm umown

.mumm umom Hmeuozm

 

 

No. + mam. m oom mm nmqm<o
mo. H_oom. m oom 6N piqued
mo. H.aoe. m oom NH nmqm<o
no. H.nme. m com om emoz
so. H emm. m oom om ocaaom
.m.m H.m
Aurv
AmE\msv UHum mama omSmEHu COHumHs>o muomwn uEmEpmmuB
UHQuoumm cmflum>o hmmmm mo .02 mumpHSpHm Umaoom m: cmpumnCH mEHB

 

o.mqm<o no mmoz .ocaamn spa: cannon» nnnu Ho
soa>anoo mcoenoc moanacamnaa snnpaanaa oofiooo .NH mqm<e

43
ovulation did not significantly alter the quantity of FSH

present in the pituitary gland (Table 13).

.mmlmmmlmHZU

.Abcmez pmzv nomnnxm humpflduflm mafiammo
.mEoEnoz mcHNHcHGHSH ECHSUEIHHCH pmown
.mnmm umom HmEnozm

 

mbNH.HH m m mm qum<w

4.

A. mmno.m m m an nmqmao
mmHH.m m m NH qum<o
momm.OH m m mm mmoz
mbe.wH m m mm mcHHmm

Logo Anna
hocmuom mnmn Uddmmap COHHHHS>O whom #cmEpmmnH
Ummm 0>Hpmamm wommm mo .02 NMMpHSpHQ pmaoom Ion UmnuanH mEHB

 

n.mqm<o no cmoz .mcnnnn annz oonnmnn nnmn no
mnflbflpum ECOEMOQ UEHumHSEHHm mHUHHHom humpflsuHm pmaoom .mH mum<e

DISCUSSION

Desjardins and Hafs (1965) reported that anti—
bodies, produced in rabbits against equine LH (Armour
PLH), when titrated with PLH resulted in a heterogenous
antibody spectrum of six precipitin lines in agar. Simi—
lar titrations after absorption with normal equine blood
sera resulted in a single precipitin line which was pre-
sumed to be due to an antibody specific for equine LH.
This antisera was capable of neutralizing the biological
activity of equine, bovine and ovine LH as well as crude
saline extracts of rat pituitary glands. This is in
general agreement with the results presented in this study
for GAELH.

The five precipitins that developed when GAELH
was titrated against its homologous antigen, represented
at least five antigenic components present in equine LH.
Similar titrations with equine LH after absorbtion with
normal equine blood sera resulted in the development of a
single precipitin line. This indicated that four of the
five antibodies present in GAELH were due to normal equine
blood sera components. Rapidity of development, sharp
definition, and intensity of these lines indicated that
these blood sera components comprised the major antigenic
factors present in equine LH. The single diffuse line

45

46

that develOped after specific absorbtion, with normal
equine sera probably represented an antibody specific for
LH. However, this precipitin line lacked intensity and
clarity even when antigen concentrations as high as 1000
ug/ml were employed. This indicated that GAELH contained
a low titer of antibodies specific for LH.

The single precipitin line that developed when
GAELH was titrated with crude saline extracts of either
rat or rabbit pituitary was evidence that at least one
antigenic component in each of these extracts was
immunologically similar to an antigen found in equine LH.
Failure of these lines to develop after specific absorp—
tion of the antisera with equine blood sera, indicated
that these antigenic components were not LH. It is sug-
gested that these lines may represent some component pre—
sent in both rat and rabbit blood sera that was immunolog—
ically similar to antigenic factors present in equine blood
sera. These results, which are contrary to those of
Desjardins and Hafs (1965), are probably caused by a low
concentration of antibodies specific for LH in GAELH.

Incubation of 0.1 m1 of GAELH in vitro with 50 ug
of its homologous antigen (equine LH) effectively, and
completely neutralized the LH activity of this preparation.
Similarly, this antisera was effective in neutralizing the
LH activity of crude saline extracts of rat pituitary
gland. This is an interesting development, since GAELH
did not counteract the physiological effects of rat LH, and

gave no precipitin reaction when titrated with dilutions of

47
rat pituitary extracts. These results introduce the
question of whether the observed in vitro neutralization
of equine and rat pituitary LH was indeed active neutral—
ization or merely passive sedimentation. There is general
agreement that the anterior pituitary hormones are trans—
ported through the vascular system conjugated to blood
sera proteins. As previously mentioned, GAELH contained
at least four antigenic components that were immunolog—
ically similar to factors found in equine blood sera.
Furthermore, the precipitin line that developed when
absorbed GAELH was titrated against rat and rabbit pitu—
itary extracts were attributable to blood sera components.
Thus, it is quite possible that the observed neutraliza-
tion was due to a non—specific sedimentation of blood
serum proteins with LH molecules attached to their surface,
rather than a specific reaction involving LH per s3.
Furthermore, failure of GAELH to neutralize the biological
activity of NIH ovine and bovine LH, hormones purified as
to be free of blood sera components, lends further credence
to the idea of passive sedimentation. This question might
be resolved by determining whether absorbed GAELH retains
the ability to neutralize these hormones.

Daily injections, for a period of 10 days, of a
rabbit antisera prepared against PLH, was capable of in-
hibiting the estrous cycle of normal, adult, female rats,
when injected 24 hr before the time of expected ovulation.
These results are in agreement with those presented by

Bourdel and Li (1963) and Young, et a1. (1963), with

48
respect to the time of inhibition. In contrast, these
investigators did not report estrous cycle arrest in the
estrous and/or proestrous stages but rather in the
diestrous phase of the estrous cycle.

Fevold (1939) and Greep, et a1. (1942) reported
that injection of LH into immature hypOphysectomized rats,
treated with FSH, resulted in growth and maturation of the
reproductive tract. This effect is due to the stimulation
of estrogen secretion. In the hypOphysectomized rat the
cells of theca interna of the ovary, which secrete estrogen
remained atrophic underFSH stimulation and acquired the
cytological characteristics of actively secreting cells
only if LH is also administered. Since LH is required for
estrogen production which in turn produces vaginal cornifi-
cation, the presence of cornified cells in the vaginal
smear is indicative of the presence of LH. Thus, it must
be concluded that the antisera used in this preliminary
study was not sufficiently potent, or was to rapidly
metabolized, to completely neutralize all the endogenous
rat LH. Estrous cycle inhibition, however, indicated a
hormonal imbalance which was probably due to partial neu—
tralization of the endogenous rat LH.

The observed increase in pituitary LH, in rats
treated with RAELH, is consistant with this idea of partial
neutralization of endogenous LH. It is generally agreed
that the physiological relationship between the ovary and
the pituitary is reflected in the condition of the vaginal

epithelium. Since the estrous cycle was arrested in the

49
estrus and/or proestrus phases it is not unreasonable to
assume that the pituitaries of those rats treated with
RAELH, resembled those of rats just prior to ovulation.
If this was indeed the case, a high pituitary LH content
would be expected.

The amount of unneutralized LH was apparently
sufficient to maintain the integrity of the reproductive
organs, as evidenced by the ovarian and uterine weights.
Failure of the antisera to significantly effect the
adrenal, thyroid, and body weights is evidence that the
antisera preparation used did not contain appreciable
quantities of antibodies to adrenal corticotrOpic hor—
mone, TSH or GH. FSH bioassay revealed the presence of
trace quantities of FSH in equine LH. However, no
appreciable antibody titer developed against this FSH
fraction as evidenced by the ineffectual neutralization
of either purified FSH, or the FSH present in equine LH.
The decrease in pituitary weight when rats were treated
with RAELH was apparent after the pituitary weights were
corrected for body weight. Since a decrease in pituitary
weight was not consistent with the observed increase in
pituitary LH content, it is suggested that this decrease
was a function of body weight variation and not a true
pituitary weight decrease.

In contrast to the results reported in the pre—
liminary experiment with RAELH no significant effect on

the estrous cycle could be attributed to GAELH. Estrous

cycles, organ weights, and histology, as well as LH and

50

FSH content of the pituitaries, were unchanged after GAELH
treatment for 6 or 16 days. It would therefore appear
that GAELH did not affect the endogenous LH in adult fe-
male rats as measured by these criteria of response.

Kelly, et a1. (1963), investigated the ovulation
inhibiting properties of rabbit anti—ovine internizing
hormone (RAOLH). These investigators reported that RAOLH,
injected intraperitoneally into female rats, at approx—
imately the time of release of LH from the pituitary gland,
effectively inhibited ovulation. Although ovulation was
inhibited, estrus occurred normally. The results of
these workers, with respect to estrous cycle inhibition,
agree with the results of the present investigation. Treat-
ment of rats with GAELH 24 or 36 hr before the next expected
ovulation, inhibited the expected ovulation. Also, as
reported above, the GAELH did not effect the estrous cycle.
In contrast, to the reports of Kelly, et a1. (1963), GAELH
injected at the time of release of LH from the pituitary
gland, did not inhibit the next expected ovulation. Bourdel
and Li (1963) reported that RAOLH did not prevent the
occurence of the next expected estrus when injected 12-16
'hr in advance. This would indicate that this antisera did
not prevent the release of LH prior to ovulation and sup-
ports the results of this study using GAELH. Although
rats treated with GAELH, 24 or 36 hr before the next ex-
pected ovulation, failed to ovulate, this result should be
treated with caution because only one-half of the rats

treated with control sera or saline ovulated. It was

51
assumed that all control rats would have ovulated. Know-
ledge of the percent of females ovulating over a typical

estrous cycle would be useful in evaluating these results.

S UMMA RY

Intraperitoneal injections of 0.5 ml of rabbit anti-
equine luteinizing hormone (RAELH) successfully in-
hibited the estrous cycle of adult female rats in the
estrous and/or proestrous stages. These results sug—
gest that RAELH partially neutralized endogenous rat
LH.

Ovary, oviduct, uterus, adrenal and thyroid weights
were not significantly effected by RAELH (P)’0.05).
The average weight (4.67 mg/100 g body weight) of the
pituitaries from rats treated with RAELH, at varying
intervals before expected ovulation, was significantly
less than the comparable average (5.58 mg/100 g body
weight) for the two control groups (P<£0.05).

Pooled pituitaries from control rats contained sig—
nificantly less LH activity (0.714 ug of ascorbic
acid/mg of ovary) than the pooled pituitaries from
rats treated with RAELH.

Injection of 400 mg of equine LH (Armour PLH) at
irregular intervals over a 7 month period resulted in
antibody production in goats.

Antibody analysis by agar gel diffusion revealed five
precipitin lines when goat anti-equine luteinizing hor-
mone (GAELH) was titrated with its homologous antigen.

52

10.

53

This was evidence that PLH contained at least five
antigenic components.

When GAELH, was absorbed with normal equine blood
sera and subsequently titrated with PLH, a single
precipitin line developed. This line was probably
due to an antibody specific for LH. Failure of the
other four precipitin lines to develop indicated that
these precipitin lines represented antigens in PLH
which were immunologically similar to normal equine
blood serum components.

A single precipitin line developed when GAELH was
titrated with either rat or rabbit pituitary saline
extract. Failure of these lines to develop when
GAELH was preabsorbed with normal equine blood sera
suggested that these lines may not be due to anti-
bodies specific for LH.

No precipitin lines developed when GAELH was titrated
with purified ovine FSH, LH, GH, TSH, prolactin or
bovine LHS.

The LH activity of equine LH (50 ug) and rat pitui—
tary extracts (2.0 mg) were significantly reduced
after incubation with 0.1 m1 of GAELH (P<;0.05). The
LH activity of equine LH, after pretreatment with
GAELH did not differ significantly from that of saline
(P2>0.05), indicating complete neutralization of LH
activity. In contrast, saline and normal goat blood
sera did not effect the LH activity of these prepar-

ations.

11.

12.

13.

14.

54
NIH preparations of ovine and bovine LH were not
significantly effected by incubation with GAELH
(P>0.05).
GAELH did not significantly effect the FSH activity
of PLH or ovine FSH when incubated with GAELH.
Estrous cycles of rats treated with GAELH (1.0 ml)
for either 6 or 16 days did not differ significantly
from those of the controls. Similarly, organ weights
and histology as well as pituitary FSH and LH content
of the GAELH-treated rats did not differ significantly
from the corresponding data of the controls (P>>0.05).
GAELH injected 24 or 36 hr before the next expected
ovulation inhibited ovulation in 9 out of 10 cases.
In contrast GAELH injected at the time of release of
LH from the pituitary (12 hr before expected ovula-
tion) did not inhibit ovulation. Ovary and pituitary
weights were not significantly effected by GAELH
(P)'0.05). Similarly, FSH and LH activity of the
pooled pituitary glands from the GAELH-treated rats
did not differ significantly from those of the con—

trols (P >o.05) .

APPENDIX A

Assay of luteinizing hormone (Parlow, 1961).

A. Preparation of the rats for bioassay.

1.

Immature female rats, 24—26 days of age, were pre—
pared for bioassay with a single subcutaneous in-
jection of pregnant mares serum (PMSG, 50 IU)
followed, 55—56 hr later, with a single sub—
cutaneous injection of human chorionic gonado-
trophin (HCG 25 IU). The rats were used for
bioassay 6—8 days following the administration

of HCG.

B. Assay procedure.

1.

At the time of assay the animals were anesthe—
tized with ether. The material to be assayed,
dissolved in 0.5 ml of saline, was injected into
the femoral vein over a period of 10—15 sec.

Four hr': 10 min after the intravenous injection,
the right ovary was removed surgically. The
ovary was dissected free of surrounding fat and
the periovarian capsule was removed. Blotting on
a paper towel removed adherent blood and moisture.

The ovary was then weighed to the nearest 0.2 mg.

55

56

After weighing, the ovaries were homogenized in
2.5% metaphosphoric acid with a glass and teflon
tissue homogenizer. The homogenate was filtered
through Munktell's No. 00 filter paper and the
homogenizer and filter paper washed with 2.5%
metaphosphoric acid until the final dilution of
tissue was 10 mg of tissue extract/l ml of 2.5%
metaphosphoric acid.

Two levels of NIH—LH—S8 standards (0.4 — 1.6 ug)

and two levels of unknowns were determined.

Analysis for Ascorbic Acid.

1.

Samples were analyzed for ascorbic acid by the
method of Mindlin and Butler (1938). The
ascorbic acid concentration was expressed as mg/

100 gm of ovarian tissue (mg %).

Statistical Analysis.

1.

The results of the assay were analyzed statis-

tically by the method of Bliss (1952).

APPENDIX B

Assay of Follicle Stimulating Hormone.
(Steelman and Pohley, 1953).

Assay Animals.

1.

In all experiments 21 day old Sprague—Dawley

female rats were used.

Assay Procedure.

1.

The FSH to be tested was mixed with HCG in
physiological saline.

The resulting mixture was injected three times
daily for 3 days in a volume of 0.5 ml per in-
jection (total of nine injections, total volume

4.5 ml per rat).

Injection dose of HCG: 20 IU total (20 IU/4.5 ml).
Animals were killed 8 hr after the final injection;
the ovaries were removed and weighed to the nearest
0.1 mg.

Two levels of NIH-S3 standard (60 — 120 ug) and

two levels of unknowns were run for determina-

tion of relative potency.

Statistical Analysis.

1.

The results of the assay were analyzed statis—

tically by the method of Bliss (1952).

57

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normal rats.