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3 1293 00869 3909 LIBRARY
Michigan State
University
This is to certify that the
thesis entitled
The Squirrel Monkey (Saimiri sciureus) As A Model For
313 Vivo Immunocontraceptive Testing
presented by
Donna Lynn Pierce
has been accepted towards fulfillment
of the requirements for
M. S 0 degree in Animal SCience
mQ/WXW%J
Major professor
Date June 5, 1986
0-7639 MS U is an Affirmative Action/Equal Opportunity Institution
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' “ 'I ~ ,5:
THE SQUIRREL MONKEY (SAIMIRI SCIUREUS) AS A MODEL
FOR IN VIVO IMMUNOCONTRACEPTIVE TESTING
BY
Donna Lynn Pierce
A THESIS
Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Animal Science
1986
ABSTRACT
THE SQUIRREL MONKEY (SAIMIRI SCIUREUS) AS A MODEL
FOR IN VIVO IMMUNOCONTRACEPTIVE TESTING
bY
Donna Lynn Pierce
Fifty female squirrel monkeys (§aimiri sciureus) were
each immunized with 200 ug of the 55 K macromolecule (ZP—3)
from porcine zona pellucida. The effect of the ZP-3
antibodies on ovarian function and fertility of the
immunized monkeys was monitored over a 19 month period.
High antibody titers were found ( 75% binding levels as
determined by radioimmunoassay ) at approximately four
months post-immunization and remained high ( 68% binding
level) for the duration of the study. Initial disturbances
in normal ovarian steroid secretion and function were found
through hormone analyses and laparoscopic observations of
ovaries. An interference in follicular growth was found
through histological studies at 6-7 months post-
immunization. No pregnancies occurred in the immunized
monkeys during the first breeding season. Hormonal and
laparoscopic data indicated a recovery in ovarian function
at 10—15 months after the initial injection despite the
presence of high titer levels to ZP-3. These findings
demonstrate that purified porcine zona macromolecules have
potential as an immunocontraceptive vaccine and should
continue to be investigated.
ACKNOWLEDGEMENTS
I would like to express my gratitude to the following
individuals for their personal and professional support
over the last several years. My advisor, Dr. W.R. Dukelow,
who made this all possible by providing me with a great
opportunity and giving guidance and support when it was
needed the most. I would like to thank Mrs. Lavonda Cleeves
for her friendship (and the wild parties at her cottage!)
since she has made my stay at the Endocrine Research Center
even more enjoyable. My graduate committee, Dr. A.G. Sacco,
Dr. D.E. Ullrey and Dr. J.A. King who provided their
interest, help and encouragement which enabled this task to
be completed. I would also like to thank all of my friends
who helped me along the way especially Lori McCallum who is
one of the nicest friends a person could ever have. I am
eternally grateful to my gptirg family for their support,
encouragement and LOVE in helping me fulfill a dream.
Without them, this would not have been possible.
111
If we want to become all that is in us to become, we
have to use everything we've got-our feelings, our
intuition, our intelligence, and our will power—our
whole self. If we do, the payoff is enormous.
s
We can all help ourselves to change, to grow, to be-
come the person it is in us to be. We can learn to be
our own best friend. If we do, we have a friend for life.
We can buoy ourselves up, give ourselves comfort and
sustenance the times when there is no one else. We are
our best source of encouragement and good advice.
Mildred Newman and Bernard Berkowitz
The two greatest stimulants in the world are youth and debt.
Disraeli
iv
TABLE OF CONTENTS
Page
LIST OF TABLES .......................................... vii
LIST OF FIGURES ........................................ viii
INTRODUCTION .............................................. 1
LITERATURE REVIEW .............................. . .......... 4
Basic Reproductive Characteristics of the Squirrel Monkey
a. Cycle length ..................................... 6
b. Captive breeding and pregnancy diagnosis ......... 8
c. Seasonality ....................... . ............. 10
Ovulation Induction .................................. 12
Immunological Aspects of the Zona Pellucida-
Consideration as a Target Antigen ................... 14
a. Detection of Antibodies to the Zona Pellucida...17
b. Primate Studies on Immunocontraception
relative to the Zona Pellucida ................. 21
1. Human primates ............................ 21
2. Nonhuman primates ......................... 23
MATERIALS AND METHODS .................................... 25
Animals ................................................ 25
Ovulation Induction Regimen ............................ 25
Laparoscopic Recovery of Oocytes ....................... 26
Preparation and Administration of Vaccine .............. 27
Blood Sampling Procedure ................ . ........... ...28
Antiserum Titration .................................... 29
Hormone Analyses ........................... ..... ..... ..29
Statistical Analysis of Data ........................... 29
RESULTS .................................................. 30
Laparoscopic Observation and Oocyte Yields ............. 30
Ovarian Histology ...................................... 32
Antibody Titer Levels .................................. 32
Hormonal Profiles.. ....................... ..... ..... ...35
Fertility Study ........................................ 46
DISCUSSION ............................ . .................. 48
SUMMARY AND CONCLUSIONS .................................. 53
LITERATURE CITED ......................................... 54
V
TABLE OF CONTENTS (continued)
APPENDICES .............................................. 64
Publications by the Author ............................. 64
Abstracts by the Author ................................ 64
VITA ..................................................... 66
vi
Table
LIST OF TABLES
Page
Pregnancy detection in the squirrel monkey ........ 9
Laparoscopic observation of follicles and
oocytes retrieved from ZP-a-injected, adjuvant—
injected and untreated control monkeys at 4
and 15 months following initial immunization ..... 31
Maximum estradiol and progesterone levels in
blood samples in ZP—3, adjuvant-injected and
untreated controls at 114, 297 and 392 days
after initial immunization .......... .... ......... 45
The effect of ZP-3 immunization on fertility
during the first breeding season...... ........... 47
vii
LIST OF FIGURES
Figure.
10.
11.
Tissue sections from adjuvant-injected
and ZP—3 injected monkeys showing ovarian
histology ..................... . ...............
Cumulative antibody titration profile of the
ZP-3 injected monkeys .............. ...... .....
Hormone profile of untreated control monkeys
at 114 days post-immunization........ .........
Hormone profile of adjuvant-injected monkeys
at 114 days post—immunization ..................
Hormone profile of ZP—3 injected monkeys at
at 114 days post—immunization............. .....
Hormone profile of untreated control monkeys
at 297 days post-immunization.. ....... .. .......
Hormone profile of adjuvant—injected monkeys
at 297 days post—immunization ..................
Hormone profile of ZP-3 injected monkeys at
at 297 days post—immunization ..................
Hormone profile of untreated control monkeys
at 392 days post—immunization..................
Hormone profile of adjuvant-injected monkeys
at 392 days post-immunization ..................
Hormone profile of ZP—3 injected monkeys at
392 days post-immunization.. ...................
viii
Page
....33
....34
....36
....37
....38
....39
....4O
....41
....42
....43
....44
Introduction
The zona pellucida has been investigated for many
years by a number of laboratories as a possible target
antigen for immunocontraceptive purposes. The earliest
experiments involved passive immunization of hamsters
(Ownby and Shivers, 1972), mice (Jilek and Pavlok, 1975)
and rats (Tsunoda and Chang, 1976) using antisera developed
against ovarian antigens. Such antisera contained
antibodies directed against the zona and were demonstrated
to be effective in preventing fertilization.
Experiments describing the immunological similarity of
antigens in porcine and human zonae (Sacco, 1977) indicated
the potential of the porcine zona pellucida as a possible
target antigen in the development of a contraceptive
vaccine with human application. The cross-reactivity of
antigens in the porcine zona pellucida with that of many
other species, coupled with the availability of large
numbers of porcine ovaries from slaughterhouses , has made
this species extremely useful for immuncontraceptive
studies.
Recently, studies utilizing porcine zona antigens in
the active immunization of female rabbits (Wood et al.,
1981; Dietl et al., 1982; Skinner et al., 1985) and dogs
(Shivers et al., 1981; Mahi-Brown et al., 1982, 1985) have
been performed.
Preliminary work has been done in nonhuman primates
to assess the anti-fertility effects of active
immunization against zonae extracts (Shivers et al., 1978;
Gwatkin et al.,1977).
The squirrel monkey (Saimiri sciureus) is widely used
in research today due to its small size, ease of
handling and simple housing requirements. In addition, a
strong background of basic reproductive procedures exists
for this species. Zonae from rodent species cross-react
poorly with antisera produced against porcine zonae
pellucidae (Sacco et al., 1981a,b). Rabbits would
appear to be the next best alternative due to the
cross—reactivity of rabbit and porcine zonae but
antibodies against pig zonae do not inhibit
homologous sperm—zona interaction in the rabbit system
(Sacco et al., 1981a). Preliminary data from the squirrel
monkey demonstrated adequate species cross-reactivity as
well as in 21:59 contraceptive potential of antibodies to a
purified porcine zona antigen (PPZA) (Sacco et al., 1983).
The data from these studies has emphasized the
contraceptive effectiveness of antibodies formed against
the zona pellucida. However, some of these investigations
have noted disruptions in the menstrual cycle and
reproductive hormonal profiles. Histological examination of
ovaries from actively immunized rabbits with porcine zona
have indicated that the presence of zona antibodies were
interfering with normal ovarian folliculogenesis (Skinner
et al., 1985). Therefore, these recent studies have
suggested that infertility observed following active
immunization of females with zona antigens may be a
consequence of zona antibodies inhibiting normal ovarian
function as well as by preventing sperm-egg interaction. It
must be noted that the studies described above were of
short term duration and utilized crude or total zona
prepartions as immunogens.
This investigation is unique in that is the first on-
going, long term primate study using a large number of
immunized monkeys (50) that were administered a highly
purified 55K zona pellucida macromolecule as an immunogen.
The objectives of the present immunocontraceptive
studies were:
1) To determine the longevity of the immune response.
2) To examine the anti-fertility effects of active
heteroimmunization with ZP—3 in squirrel monkeys.
3) To assess the potential detrimental effects of such
immunization on normal reproductive characteristics.
LITERATURE REVIEW
It is known that antiserum raised against zonae
pellucidae from one species will cross react differently
with zona of other species. This suggests that there are
common antigens shared by the zonae of various animals and
for immunocontraceptive purposes involving active
immunization, the degree of cross reactivity and similarity
of zona antigens is important. Antisera produced against
porcine zonae antigens react poorly with rodent zonae
(Sacco et al., 1981) so mice, hamsters and rats cannot be
used to evaluate active immunization using porcine zona
antigens (Sacco et al., 1981a,b). The level of cross
reactivity of antibodies to rabbit zonae and pig zonae
seems sufficient (Sacco et al., 1981b) but antibodies to
porcine zonae do not inhibit homologous sperm—zona
interaction. Results from these types of data suggest that
a nonhuman primate might be the next likely test system to
evaluate the contraceptive efficacy of active immunization
with porcine zona antigens. The squirrel monkey was the
species chosen based on preliminary results obtained by
Sacco et a1. (1983).
Studies have shown the porcine zona pellucida is
biochemically and antigenically complex. When resolved by
2-dimensional polyacrylamide gel electrophoresis (2—D
PAGE), it is comprised of four major families of charge
heterogeneous glycoproteins with presumptive molecular
weights of 82K(ZP1), 61K(ZP2), 55K(ZP3) and 21K(ZP4)
(Subramanian et al., 1981; Dunbar et al., 1981). In
consideration of a contraceptive vaccine development using
the porcine zona, the approach was to employ the smallest
component of the porcine zona which was capable of
producing antibodies exhibiting contraceptive properties.
The 55K(ZP3) glycoprotein was selected for the following
reasons: a) preliminary work expressed it was the best
candidate for successful purification using chromatographic
procedures, b) it is the most predominant glycoprotein
accounting for 60% of the porcine zona, c) it appears to be
the most immunogenic glycoprotein (Sacco et al., 1983) and,
d) it probably is biologically active and exhibits sperm
receptor activity (Sacco et al., 1984).
The first attempts to purify the 55K(ZP3) constituent
resulted in a preparation that was free of the the other
major zona glycoproteins but was not totally homogeneous
(Yurewicz et al., 1983). This preparation called purified
porcine zona antigen (PPZA) containing primarily the 55K
component was further purified chromatographically to
electrophoretic homogeneity and was referred to as ZP—S
(Yurewicz et al., 1984).
BASIC REPRODUCTIVE CHARACTERISTICS
OE THE SQUIRREL MONKEY
Cycle Length
The cycle length of the squirrel monkey varies from 7-
12 days with an average of 9 days. Earlier studies
(Denniston, 1964; Srivastava et al., 1970; Richter, 1976)
described cycle lengths as high as 25 days using
cytological techniques to examine the vaginal cell
cornification cycle. Hutchinson (1978) reported that cycle
lengths might vary depending on the type of housing and
whether the monkeys were housed individually or in groups.
Rosenblum et a1. (1967) cited a cycle length of 7—8
days using vaginal cell cornification cycles and presence
of sperm in vaginal smears of females housed with males.
This study was confirmed by Gould et a1. (1973) and Travis
and Holmes (1974) who reported a peak in the karypyknotic
index at intervals of 10.9 days.
There have been many studies utilizing activity cycles
and behavioral characteristics to confirm the 8-10 day
period of the cycle (Richter, 1976; Latta et al., 1967;
Jarosz et al., 1977; Wilson, 1977).
Early work examined urinary steroids as a measure
of cycle length. Using animals showing a 10.9 day cycle
length, Travis and Holmes (1974) divided the cycle
into follicular and luteal phases based on a
karyopyknotic index that peaked on day 5. They found
that pregnanediol excretion increased sharply on the
sixth day of the cycle and the total amount excreted
throughout the luteal phase was greater than that found
for the follicular phase. With the development of
radioimmunoasssay (RIA) techniques, small blood samples
could be examined for circulatory steroid levels in the
squirrel monkey throughout the cycle. It was discovered
that these levels are strikingly higher than are found in
old world monkeys and humans. Furthermore, this
characteristic has been observed in other new world
species. Estradiol levels peak at 503:57.5, and 8118.2
pg/ml was found to be the minimum value. Serum progestins
reached maximum concentrations of 399 :27.7 ng/ml, 3-4 days
following the estradiol peak (Wolf et al., 1977; Wilson,
1977). In confirmation, Ghosh et al. (1982) determined the
cycle length to be 8-9 days using hormonal measurements of
serum estradiol 17-B, progesterone and luteinizing
hormone(LH). Estradiol and LH increased markedly on day 4
of the cycle while progesterone peaked on day 6 indicating
ovulation on day 5.
Recently, work was carried out to detect any seasonal
changes in estradiol and progesterone in the Bolivian
squirrel monkey (Diamond at al., 1984). The results
indicated a 6-12 day cycle based on the time interval
between consecutive estradiol peaks. It was noted that
serum estradiol and progesterone concentrations were lower
during the nonbreeding season (April to November) with an
absence of a preovulatory surge of estradiol.
Captive Breeding and Pregnancy Diagnosis
When squirrel monkeys have become acccustomed to
their captive habitat, a conception rate of 50-60% can be
expected (Kaplan, 1977; Dukelow, 1982). However, a high
rate of stillbirths or abortions (16.6%) and neonatal death
(34.3%) is observed which can possibly be attributed to a
high maternal body weight to infant birth weight ratio
(7:1). Many breeding systems house males and females in
groups with a maximum female to male ratio of 10:1. When
pregnancy occurs, the gestation period lasts from 135 to
175 days, with a mean length of 150 days (Jarosz et al.,
1977; Kerber et al., 1977).
Various techniques have been used to detect pregnancy
in the squirrel monkey (Table 1). Increased uterine size
and chorionic gonadotropin occur in the first trimester and
would be useful in detecting pregnancy as soon as possible.
A pregnancy test kit developed by the National Institutes
of Health can detect pregnancy in squirrel monkeys between
40 and 60 days of pregnancy with a 10% inherent risk of
false negatives (Hodgen et al., 1978).
TABLE 1
Pregnancy detection in the squirrel monkey
Characteristic Days After Reference
Conception
1. Uterine size 20—25 Goss et al., 1968
increase(laparotomy)
Chorionic gonadotropin
a. plasma
b. urine
Uterine size
increase(palpation)
Diabetes
Maternal weight
gain
Visual abdominal
enlargement
Fetal skeleton
by x-ray
insipidus
20-105
40-60
42-56
60
60-135
60-95
67-147
74~102
81
Nathan et al., 1966
Hodgen et al., 1978.
Rosenblum, 1968
Clewe, 1969
Travis and Holmes,
1974
Hopf, 1967
Travis and Holmes,
1974
Hopf, 1967
Nathan et al., 1966
10
Seasonality
Reproductive seasonality has been well documented in
the squirrel monkey and is expressed by behavioral changes,
dimorphic appearance of the adults ("fatted male syndrome")
and hormonal variations. In their natural environment,
squirrel monkeys mate from July to September with births
occurring from January to March. The mating season shifts
from January to March when squirrel monkeys are moved to
the northern hemisphere with births occurring from June to
September (Dumond, 1968). This marked change in the mating
and birth seasons was correlated to the amount of rainfall,
with the dry season triggering the onset of mating season.
The humidity effect was corroborated by Baldwin and Baldwin
(1971) in field studies.
In the laboratory, the seasonal effect of lowered
ovulation is apparent in the female response to ovulation
induction regimens (Harrison and Dukelow, 1973). Ovulation
can be induced in the anovulatory months by increasing the
dose of follicle stimulating hormone (FSH) before human
chorionic gonadotropin (HCG) administration (Kuehl and
Dukelow, 1975).
Seasonality has also been observed in the male and is
termed the "fatted male syndrome" (Dumond and Hutchinson,
1967). It is associated with an increase in body size,
fluffiness of pelage, maximum spermatogenesis and increased
mating activity.
11
In establishing a breeding colony,
captivity must be taken into consideration.
will adapt after nine months of captivity
Dukelow, 1973) while others require as
breeding seasons to adjust (Lorenz et
adaptation to
Some
animals
(Harrison and
long
al.
I
as
three
1973).
12
Ovulation Induction
A wide variety of ovulation induction protocols
have been effective in the squirrel monkey. Bennett (1967)
used a regimen of five days pregnant mares serum
gonadotropin (PMSG) followed by four days of a combination
of PMSG and human chorionic gonadotropin (HCG) to mimic the
luteal phase of the cycle. An average of 5.3 ovulations per
ovary was observed with this regimen. Later, studies were
performed to induce single or double ovulations in the
squirrel monkey in an attempt to accurately time ovulation
(Dukelow, 1970). The optimal treatment consisted of four
days of 1 mg FSH followed by a single injection of 250-500
IU HCG on the fourth day.
Seasonality is observed in ovulation induction
regimens as well. A seasonal responsiveness was reported
with the FSH-HCG regimen (Harrison and Dukelow, 1973) but
could be overcome by either increasing the dose of FSH or
by extending the time of FSH administration during the
anovulatory months of July, August and September (Kuehl and
Dukelow, 1975a). The minimum effective dose of HCG was
found to be between 100—250 IU (Dukelow, 1979), and
increasing the dose had no effect during the anovulatory
months (Kuehl and Dukelow, 1975a).
The question of whether oocytes produced from
ovulation induction regimens are capable of fertilization
13
was also examined. It was shown that these oocytes were
able to be fertilized both 13 vivo (Jarosz et al., 1977)
and in vitro (Kuehl and Dukelow, 1975b; 1979; Chan et al.,
1982).
14
Immunological Aspects g: the zona Pellucida- Consideration
as a 19:99: 5211929
The zona pellucida is a non-cellular, gelatinous
layer surrounding all mammalian oocytes and
preimplantation embryos. During oogenesis and unilaminar
follicular growth , the zona pellucida is formed in the
area between the developing oocyte and the‘ surrounding
granulosa cells. A controversy remains over the precise
site of origin of zona pellucida material due to the
biochemical complexity of the layer. Some researchers
consider the granulosa cells surrounding the oocyte to be
the sole site of zona synthesis (Oakberg and Tyrell, 1975;
Haddad and Nagai, 1977) while others ascribe its origin to
the oocyte (Chiquione, 1960; Flechon et al., 1984). A third
vieWpoint states that the granulosa cells may contribute
protein and polysaccharide components while the oocyte
contributes only polysaccharides (Hadek, 1965).
The zona pellucida has several roles in fertilization
and early embryonic development. Its functions in
fertilization include sperm recognition of the oocyte
(Yanagimachi, 1972; Hanada and Chang, 1972) and prevention
of polyspermy (Braden et al., 1954; Barros and Yanagimachi,
1972). The zona pellucida provides mechanical protection of
the embryo during its journey through the oviduct
(Gwatkin, 1963), osmotically regulates the environment
(Piko, 1969) and maintains normal cleavage patterns
(Mintz, 1962).
15
The structure of the zona pellucida has been examined
by light microscopy and transmission and scanning electron
microscopy. The zona appears as a translucent layer with
a granular structure when viewed by light microscopy with
thickness varying from 3 to 22 microns (Piko, 1969) and
a diameter of approximately 60 to 170 microns (Wright et
al., 1977). When viewed by transmission electron
microscopy, a substructure of filaments permeated by
numerous granulosa cell processes and egg microvilli are
visible (Piko,1969). Scanning electron microscopy of zonae
indicates an extensive fibrous network interspersed with
numerous pores of various sizes with the largest pores at
the outer surface (Dudkiewicz and Williams, 1977).
Biochemical studies on ~the zona pellucida have
demonstrated neutral or weakly acidic glycoproteins to be
the major constituent with amounts of sialic aid residues
(Soupart and Noyes, 1964) and phosphate and sulfate esters
(Dunbar et al., 1980). The structural integrity of the zona
pellucida is maintained by both covalent and noncovalent
bonds (Inoue and Wolf, 1974; Dunbar et al., 1980) and
disruption of these bonds by heat or altering pH or ionic
strength will result in a solubilized zona preparation.
This zona preparation is maintained by covalent bonds which
can be disrupted by reducing agents to prepare solubilized
preparations of individual zona macromolecules.
The zona pellucida was considered as a target antigen
16
for use as a contraceptive based on the above information.
It is also contains antigens specific to reproductive
tissue and has an optimum location within the reproductive
system to interrupt fertiliity since it represents a
structure through which sperm must pass in order to reach
and fertilize the egg. The zona pellucida is complex
structurally and biochemically and is both antigenic and
highly immunogenic. These characteristics would most likely
lead to prevention of fertilization rather than affecting a
developmental stage after fertilization. Finally, large
quantities of isolated zonae material can be collected
using new screening procedures (Dunbar et al., 1980;
Oikawa, 1978).
l7
Detection 9; Antibodies 39 Zona Pellucida
There are various methods used to measure and monitor
antibody activity against ZP antigens. These methods
include:
1) Zona precipitation reaction (ZPR)
2) Indirect immunofluorescence (II)
3) Prevention of zona digestion by proteolytic enzymes
4) Passive hemagglutination assay
5) Immunodiffusion and Immunoelectrophoresis
6) Radioimmunoassay (RIA)
7) Prevention of sperm attachment and penetration of the
zona pellucida
Zonae pellucidae treated with antibodies to zonae
pellucidae possess a precipitation layer on the outer
surface of the zona which alters its light—scattering
properties. When viewed through a bright-field microscope.
a dark layer is observed on the surface of the zona.
Antibody-treated zonae appear to be brighter than controls
when viewed using dark-field microscopy (Sacco, 1981). It
is believed that the binding of antibodies to the outer
portion of the zona account for this change in light—
scattering properties (Ownby and Shivers, 1972; Garvagno
et al., 1974). Using light microscopy, Garavagno (1974)
demonstrated that the precipitate was located only on the
outside of the hamster zona. Flechon and Gwatkin (1980)
used transmission electron microscopy to demonstrate that
antibodies to bovine zonae pellucidae are present on both
the internal and external surfaces of the zona pellucida.
When the precipitation layer of antibody-treated zonae was
18
observed with an electron microscope, aggregations of fine
to medium-coarse granules were seen to adhere to the
fibrous zona network everywhere but around the pores
(Dudkiewicz et al., 1976). The titer of the zona antisera
is measured by the formation of the precipitation
layer and is expressed as the reciprocal of the highest
dilution of antiserum which produces the precipitation
layer as compared to preimmune serum treated control zona
(Sacco and Shivers, 1978).
The indirect immunofluorescence technique is also used
to detect zona antibodies at the surface of the zona
pellucida. Antibody-treated zonae fluoresce after treatment
with a second fluorescein-labelled antibody that is
directed against the zona antibody. The reciprocal of the
highest antiserum dilution which causes a fluorescence on
the zona as compared to those treated with a preimmune
serum is termed the immunofluorescent titer (Sacco et
al., 1983). This procedure is more sensitive than the ZPR
and is able to detect antibodies to zona at titers
insufficient to produce a precipitate.
There are reports in the literature (Ownby and
Shivers, 1972; Sacco and Shivers, 1973) that the presence
of zona antibody on the zona surface makes the zona
resistant to digestion by proteases. Zona—bound antibodies
can be detected by exposing antibody and control-treated
zona-coated oocytes to proteolytic solutions and comparing
19
zona lysis times. Zona dissolution times are longer for
antibody-treated zona as compared to untreated or control-
treated zona (Sacco, 1981).
A passive hemagglutination assay has been used to
detect ovarian antigens (Tsunoda and Chang, 1976)
utilizing the method described by Herbert (1977). They
reported antibodies to ovary homogenate were detectable in
12
serum dilutions up to 2
Early studies on immunoprecipitation (Ownby and
Shivers, 1972; Sacco and Shivers, 1973 ) utilized
antibodies to whole ovarian tissue and due to the
heterogenity of the ovary, the exact ovarian antigens
that were studied is difficult to determine. Recently, more
precise methods have been developed that use antisera
containing antibodies from isolated porcine and rabbit
zonae (Dunbar et al., 1980; Dunbar and Raynor, 1980) and
bovine zonae (Gwatkin et al., 1980). ImmunoelectrOphoresis
techniques have been used to show a specific antigen in
ovarian homogenates recognized by goat antiserum to
isolated bovine zonae (Tsunoda et al., 1980). Using one—
dimensional (Dunbar and Raynor, 1980) and two-dimensional
(Woodard and Dunbar, 1981) gel electrophoresis, antibodies
have been isolated against purified zona proteins.
Immunoelectrophoretic techniques have demonstrated that
there are multiple zona-specific antigens associated with
porcine and rabbit zonae pellucidae and have allowed
20
characterization of these specific proteins.
The most sensitive technique for detecting antibodies
to zona antigens is radioimmunoassay (RIA) (Palm et al.,
1979; Gerrity et al., 1981; Subramanian et al., 1981). The
presence of zona antigen can be examined using RIA either
as a competitive inhibition assay or titration of zona
125
antiserum of iodinated ( I) solubilized zonae.
Sperm can be inhibited from attaching to the zona
pellucida by exposing zona-coated eggs i_ yitro to anti-
zona serum (Shivers et al., 1972). The degree of inhibition
is directly related to the concentration of antibody added.
21
PRIMATE STUDIES ON IMMUNOCONTRACEPTION
RELATIVE TO THE ZONA PELLUCIDA
Human Zona Pellucida Studies
Studies have been done to examine the antigenicity of
the human zona pellucida for its potential use in
regulating fertility (Sacco, 1977a). He reported that the
human ovary contains at least one antigen not found in 21
other human tissues and fluids. Antiserum reacting with
this ovarian antigen possessed antibody activity against
the human zona pellucida as demonstrated by the ZPR. Cross-
reactivity of human and porcine zonae pellucidae has been
demonstrated using agar gel diffusion and immunofluorescent
staining (Sacco, 1977,b; Shivers and Dunbar, 1977). Sacco
et a1. (1981) found antiserum to a purified pig zona
antigen (PPZA) formed a precipitation layer on the surface
of porcine and human oocytes and inhibited human sperm
adherence ig 31539. These results indicate that the porcine-
zona pellucida is a prime candidate as a target antigen for
the development of a human contraceptive vaccine.
Investigations on the role of autoantibodies to zona
pellucida was examined as a possible cause of idiopathic
infertility. Using immunofluorescence techniques, anti—zona
pellucida antibodies are found in the sera of infertile
women (Shivers and Dunbar, 1977; Mori et al., 1978).
Shivers and Dunbar (1977) speculated that zonae are
continually exposed to autoantibodies through egg atresia
22
in the ovary and absorption of ovulated oocytes in the
reproductive tract. Several investigators (Tsunoda and
Chang, 1979; Nishimoto et al., 1980) have found
autoantibodies to zona antigens to be present in the sera
of aging women and animals. Nishimoto (1980) observed
decreased zona binding activity in the sera of aging women
after pre—absorption with red blood cells. However, when
immunoflourescence techniques are used, zona binding
activity has been observed in the sera of infertile women
(Shivers and Dunbar, 1977) as well as in the sera of
fertile males and females (Sacco and Moghissi, 1979; Dakhno
et al., 1980). Sacco and Moghissi (1979) reported that
several of the infertile females studied, whose serum
possessed anti-zona activity, became pregnant. These
successful pregnancies could be due to an insufficient
titer level in the women to prevent fertilization. This was
found to be the case in animal studies (Tsunoda et al.,
1979; Sacco, 1979) where oocytes with bound zona antibodies
were fertilized, indicating a minimal amount of zona
antibody needs to be associated with the zona before
fertilization is inhibited.
23
Nonhuman Primate Studies
Recently, work has been done with marmosets (Shivers
et al., 1978), squirrel monkeys (Sacco et al., 1983) and
cynomologus monkeys (Gulyas et al., 1983a,b) involving
immunization with zona antigens. Initial studies (Shivers
et al., 1978) demonstrated the cross—reactivity between
human, chimpanzee, marmoset and porcine zona antigens as
determined by the zona precipitation reaction and
immunofluoresence methods. Following passive immunization
in marmosets, sperm attachment to eggs was prevented jg
213:9 and antibodies were located on the zonae of ovarian
oocytes.
Fox et a1. (1981) examined the antibody response using
an enzyme-linked immunosorbent assay (ELISA) in marmosets
innoculated with porcine zonae. Over a nine-week
immunization period, the antibody response profile obtained
by ELISA was similar to that found with indirect
immunoflouresence on intact zona. The ELISA method was
found to be useful when large numbers of samples were
screened and was advantageous over previously used methods.
Cross-reactivity of human and squirrel monkey oocytes
to a purified porcine antigen (PPZA) was shown by Sacco et
a1. (1983). Immunization of squirrel monkeys with PPZA
resulted in production and maintenance of high antibody
titers for at least one year. Antibody binding to monkey
zonae was detected by the presence of a precipitation layer
24
on the surface of the zona. Pretreatment of human and
squirrel monkey oocytes with anti—PPZA sera resulted in
total inhibition of homologous sperm attachment ip 31:39.
The jg giyg effects of PPZA antibodies observed on squirrel
monkey zonae were in sitg binding of antibodies on
the zonae forming a precipitate, and significantly fewer
oocytes were collected via laparoscopy from immunized
monkeys as compared to the controls.
Gulyas et al. (1983a) immunized cynomologus monkeys
(Macaca fascicularis) with heat-solubilized pig zonae and
examined the anti-fertility effects of the zona antibodies.
A rapid solid-phase radioimmunoassay (Gulyas et al., 1983b)
was used to monitor serum antibody titers which reached
maximum levels 6 to 10 weeks after the initial
immunization. Six of the twelve monkeys became pregnant at
the time of maximum antiserum titers, although the
remaining six that did not conceive had lower antibody
titers. The hormonal status was altered in five of the six
monkeys with the menses interrupted periodically and
midcycle estradiol peak was absent for several cycles. The
menses and midcycle estradiol peak returned to normal in
five of the monkeys 3 to 5 months after the last booster
injection. Histological studies showed atresia of small
follicles and accumulation of luteal tissue at the end of
the 18 month study.
MATERIALS AND METHODS
Animals
Adult squirrel monkeys (Saimiri sciureus) of Bolivian
and Guyanese origin (South American Primates, Miami,
Florida) were housed indoors on a 12:12 hour 1ight:dark
cycle. The temperature was maintained at 211 3°C and
relative humidity was not controlled. During the summer
months (June to October), the animals were maintained in
large colony cages outdoors (Jarosz and Dukelow, 1976). The
animals were fed a commercial, high protein monkey feed #
5047 (Ralston-Purina Co., St. Louis, Missouri) supplemented
with apple slices and fresh water ag libitum.
A total of 100 sexually mature female squirrel monkeys
were randomly divided into three treatment groups. Fifty
monkeys received the ZP-3 antigen plus Freund's adjuvant,
25 females received the adjuvant only, and the remaining 25
were used as untreated controls.
Ovulation Induction Regimen
Mature female squirrel monkeys received an ovulation
induction regimen consisting of four daily i.m. injections
of follicle stimulating hormone (1 mg, F.S.H.-P, Burns-
Biotec Laboratories Inc., Omaha, Nebraska) and a single
R
i.m. injection of HCG (250 IU, A.P.L. Ayerst Laboratories,
Inc. New York, New York) on the fourth day (Dukelow, 1970;
1979). During the anovulatory months (July to September),
five daily FSH injections, rather than four, were given
25
26
(Kuehl and Dukelow, 1975) followed by HCG.
Laparoscopic Recovery 9: Oocytes
The use of laparoscopy in reproductive studies
has been extensively reviewed by Harrison and
Wildt (1980). The laparoscopic technique for oocyte
recovery in squirrel monkeys has been described (Dukelow et
al., 1971; Dukelow and Ariga, 1976). The squirrel monkey
was anesthetized with sodium pentobarbital (27 mg/kg body
weight per adult female, i.m.) 15 to 16 hours after the HCG
injection. A small midline incision was made with a scalpel
and the trocar-cannula inserted. The trocar was then
removed and the laparoscope (4 mm diameter, Karl Storz Co.,
Tuttlingen, West Germany) inserted. To improve viewing, the
abdominal cavity was insufflated with carbon dioxide passed
through the cannula. A 25 gauge needle and 1 ml tuberculin
syringe were used to move the fimbria aside to expose the
ovaries. The ovarian follicles were counted according to
size (large >3 mm; medium 3mm to 1 mm; small <1 mm ) and
aspirations made using 25 gauge 5/8 inch needle. The
oocytes were aspirated into 0.05 ml of TC-199 culture
medium (with 25 mM Hepes buffer, Earle's salts and L-
glutamine, Gibco Laboratories, Grand Island, New York)
supplemented with 20% heat inactivated GG-free bovine serum
albumin (Gibco Laboratories), 1 mM pyruvate (Sigma Chemical
Co., St. Louis, Missouri), 100 mg Gentamicin sulfate
(M.A. Bioproducts, Walkersville,Maryland) per m1 and 1 unit
27
heparin per ml. The oocytes were placed into sterile 8—
chambered tissue culture slides (Lab-Tek Products,
Napierville, Illinois) and incubated at 37°C in a moist
atmosphere of 5% CO in air. The cultures were observed
through an invertedzmicroscope and the numbers of oocytes
as well as the stage of maturation recorded for each
monkey. The laparoscopy procedure was performed on all
female monkeys before and after receiving either adjuvant
or ZP-3 treatment.
Preparation and Administration 9: Vaccine
The first purified porcine zona antigen macromolecule
(PPZA) consisting primarily of the 55 K component used to
immunize squirrel monkeys (Sacco et al., 1983) was further
purified chromatographically (Yurewicz et al., 1984) and
referred to as ZP-3. Test tubes were prepared containing
350 ug of ZP-3 antigen in 5 ml of 0.1 M phosphate—buffered
saline (PBS) and were frozen until needed. Each test tube
contained enough antigen to inoculate 10 animals. The
antigen was prepared for injection by thawing the test tube
contents, pouring into a 20 ml Pyrex glass beaker and
removing any remaining antigen from the test tube
with a glass pipet. An equal volume (5 ml) of Freund's
adjuvant (Sigma Chemical Co., St. Louis, Missouri) was
added to the antigen in the beaker and emulsified using a
10 ml syringe and 18 gauge needle with a small piece of
tygon tubing attached to the tip. The female squirrel
28
monkey was prepared for innoculation with a sedating dose
of VetalarR (10 mg/600 gm monkey, Parke—Davis, Morris
Plains, New Jersey) and the hair on their backs was clipped
from the shoulders to the mid—thoracic region. Each animal
was injected intradermally with 1 ml of emulsion containing
35 ug ZP-3 antigen in multiple injection sites on the back
(20-25 injection sites per one ml of emulsion). The
injection regimen followed was three inoculations one week
apart followed by a booster injection approximately 100
days later which contained 100 ug of ZP—3 antigen with
Freund's adjuvant. The initial inoculations contained the
antigen emulsified with Freund's complete adjuvant while
the remaining inoculations were emulsified with Freund's
incomplete adjuvant. The total amount of antigen injected
per animal was 200 ug.
Blood Sampling Procedure
Blood samples were taken periodically to monitor
antibody titer levels and hormonal status. The monkeys
were sedated with VetalarR (10 mg/600 gm monkey) and the
femoral area cleaned with an alcohol swab. A 3 ml syringe
with a 25 gauge needle was inserted into the femoral vein
and 2-3 ml of blood withdrawn. The blood was stored
o
overnight at 4 C and allowed to clot. The clot was
0
centrifuged for 30 minutes at 4 C, the serum drawn
0 .
off and stored at —20 C in 12x35 mm, 1/2 dram screw
cap vials (Kimble, Toledo,0hio).
29
Antiserum Titration
A control blood sample was obtained from each animal
prior to inoculation. Beginning one week after the third
inoculation, blood was collected on a weekly basis for 14
weeks and processed as described previously. Subsequent
bleedings were taken at 1 month intervals to continue
monitoring antibody response. The serum samples were sent
to Wayne State University for analysis of the antibody
response by radioimmunoassay (RIA) titration methods (Sacco
et al., 1983).
Hormone Analyses
Daily blood samples were collected from monkeys in the
three treatment groups during the months of May, August and
November. Samples were taken at 9 AM each day for a period
of 8—12 consecutive days to monitor hormonal status. The
blood samples were processed as previously described and
were later shipped to Wayne State University for analysis
of serum estradiol and progesterone levels.
Statistical Analysis 9: Data
Analysis of variance (ANOVA) was used to evaluate
data and the student-Newman-Keuls procedure was used to
determine differences between groups. There was great
variability in P values so log transformation of the data
was performed before ANOVA. A chi-square test was used to
examine differences between groups in the fertility study.
RESULTS
Laparoscopic Observations and Oocyte Yields
Hormonally-primed squirrel monkeys were subjected to
laparoscopy in order to evaluate the possible adverse
effects of immunization. At laparoscopy, the ovaries were
observed, the number and size of the follicles counted and
aspirated for oocyte recovery. Laparoscopic examinations
were performed approximately four months after initial
immunization (Experiment 1) and 15 months (Experiment 2).
The results of laparoscopic observation and oocyte
retrieval at the two time intervals are shown in Table 2.
The number of follicles present and oocytes collected in
the injected group were significantly less than in the
control groups at four months post-immunization (p<0.01).
At 15 months following initial immunization (Experiment 2),
similar observations were noted in number of eggs retrieved
and follicles present with the exception of large
follicles where no significant difference was found
(p>0.05) between ZP-3-injected and control groups. Visual
inspection of the ovaries in the ZP-S-injected group at
four months revealed the ovaries were small, whitish in
color and non-responsive in appearance with respect to the
ovulation induction regimen. During the time of experiment
30
31
TABLE 2
Laparoscopic observation of follicles and oocytes
retrieved from ZP—S-injected, adjuvant-injected and
untreated control monkeys at 4 and 15 months following
initial immunization
a
Mean Mean Number
Exp Treatment n Wgt. of Follicles Eggs/
No. Group gms L M S Monkey
1 Control 8 636 1.1 6.3 7.5 2.1
Adjuvant 7 621 2.8 6.0 12.3 1.7
ZP-3 20 591 0.3* 0.6* 3.5* 0.4*
2 Control 14 644 0.9 2.0 6.4 1.1
Adjuvant 8 611 0.8 4.8 9.4 2.3
ZP-3 34 642 0.2# 1.0* 3.8* 0.3*
> 2mm
1-2mm
< 1mm
* sig. different from control (p<0.01)
# not sig. different from control (p>0.05)
(02!”
"ll"
32
2, most ovaries in the ZP—3—injected monkeys appeared
morphologically similar to the animals in the control
group.
Ovarian Histology
Ovaries have been obtained from each of two monkeys in
the ZP-3-injected and adjuvant—injected groups (total of
4 monkeys) and prepared for histological examination. Death
occurred approximately 180-210 days following
immunization with either ZP—3 or adjuvant alone. The
ovaries were removed immediately after death, fixed in
Bouin's solution and sections stained with hemotoxylin and
eosin. Tissue sections from the adjuvant-injected animals
exhibited normal ovarian histology (Figure 1). Numerous
oocyte-containing primordial follicles of various size were
observed in sections from one of the ZP—3-injected
monkeys (Figure 1). The second ZP—3—injected animal had
sections with oocyte-containing primordial follicles with a
few primary follicles and corpora lutea.
Antibody Titer Levels
A cumulative titration profile for all immunized
monkeys is shown in Figure 2. Antibody titer levels
increased significantly to approximately the 54% binding
level during the first 56 days after initial immunization.
During the next two months, titer levels continued to
increase to the 65% binding level at which time a booster
33
FIGURE 1
Tissue sections from adjuvant-injected and ZP-3
injected monkeys showing ovarian histology
'A. Section from Adjuvant-injected monkey
8.0. Section from ZP-3 immunized monkeys
Arrows indicate oocytes within follicles
34
wwmq mmHHH MnomHHz<
35
injection was administered. This resulted in further rapid
increase in titers which peaked about 3 weeks following the
booster at approximately the 77% binding level. In the
absence of additional boosters, titer levels decreased
slowly but remained at high levels (68% binding level) 493
days following the first immunization. The immune response
was consistent among the 50 monkeys as evidenced by the
small standard error (Figure 2). 'There was no anti-ZP—3
activity detected in sera from monkeys in the untreated
control and Freund's adjuvant injected control groups.
Hormonal Profiles
Series of daily blood samples were obtained from
randomly selected monkeys in the three treatment groups,
and estradiol (E ) and progesterone (P) levels were
measured by RIA.2 Figures 3 through 11 show the hormone
profiles from untreated control, adjuvant-injected and ZP-3
injected monkeys during November, August and May. These
samples represent hormonal status at approximately 114, 297
and 392 days following initial immunization, respectively.
Cyclical trends were detected in estradiol and progesterone
levels, although none of the monkeys had ovulatory cycles
as evidenced by the peak E and P levels (Table 3) obtained
(Diamond et al., 1984).2 The maximum levels of E were
significantly greater in the control group fp<0.05)
compared to adjuvant and ZP-3 injected groups during the
bleeding period prior to the onset of breeding season.
36
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37
PROGESTERONE (ng/ml) ----
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PROGESTERONE (ng/ml) ----
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(rm/3d) 'IOICIVHISH
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«'0‘
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(rm/3d) 'Iomvaisa
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41
if
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PROGESTERONE (ng/ml)
HH
MAUWU ho mw<2
mumN
(1111/ 1id) "1019991193
42
mAqu mo 9an
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45
TABLE 3
Maximum estradiol and progesterone levels in blood samples
from ZP-3, adjuvant-injected and untreated controls
at 114, 297 and 392 days after initial injection
Month of Bleeding
NOV MAY AUG
Treatment (# Days after 114 297 392
Group 1st injection)
Untreated E2 77:13*(5) 5710.5(6) 209:112(10)
(pg/ml)
P 2.8:1.0 (5) 5.710.5(6) 45.9:20(10)
(ng/ml)
Adjuvant E2 35:5.5(5) 71:19.2(5) 256:81(10)
(Pg/ml)
P 2.5:0.7(5) 22.417.6(5) 92.7:34(10)
(ng/ml)
ZP-3 E2 20:10(7) 95123.2(?) 2811132(10)
(pg/ml)
P 20:54#(7) 16:3.9(7)102.3:54 (10)
(ng/ml)
( )= number animals
* = signif. greater than adjuvant and ZP—3 injected groups
(p<0.05)
# = signif. greater than untreated and adjuvant injected
(p<0.05)
46
Animals in the ZP—3 injected group exhibited significantly
higher P levels than the untreated and adjuvant-injected
animals (p<0.05).
Fertility Study
Matings began in December 1984 by placing a male of
proven fertility with 10 females in a breeding cage. Male
monkeys were rotated to different cages every two weeks
throughout the breeding season. Through May 1985, 7
pregnancies were detected in the control group (Table 4) by
palpation and confirmed by elevated progesterone levels.
None of the pregnancies went to term due to either
stillbirths or abortions. No pregnancies occurred in either
the adjuvant-injected or the ZP—3-injected groups.
47
TABLE 4
The effect of ZP-3 immunization on fertility during
the first breeding season
Treatment Group n No. Pregnant % Pregnant
Control 24 7 29
Adjuvant 16 0* 0
ZP-3 34 0* 0
* (p< 0.05)
DISCUSSION
In this study, active immunization with porcine zona
antigen demonstrated an inhibitory effect upon the
fertility of the female squirrel monkey. Similar findings
were reported in female dogs (Mahi-Brown et al., 1982,
1985) and cynomolgus monkeys (Gulyas et al., 1983b) after
active immunization with porcine zona material. The
squirrel monkey responded immunologically to the ZP-3
immunization regimen and produced antibodies to the porcine
antigen possessing high antigen binding activity as
determined by RIA. This study has shown that the
circulating antibodies affected normal ovarian function by
reducing the number of developing follicles and oocytes
produced. In addition, reduced levels of estradiol and the
variable patterns of progesterone secretion indicate
disturbances in ovarian function. Previous work has shown
that squirrel monkey antiserum to ZP-3 to inhibit sperm-
zona interaction 13 giggg (Sacco et al., 1983a, 1984).
However, these i vivo studies suggest that antibodies to
the zona may be exerting their main effect at the ovarian
level as revealed by cycle disturbances rather than
preventing sperm attachment to the zona. Immunized monkeys
recover from the ovarian disruptions by 10 months after the
48
49
initial injection as demonstrated by the elevated estradiol
and progesterone levels between control groups and ZP—3
immunized group. Normal oocyte production in response to
the ovulation induction regimen had not recovered in the
ZP-3 immunized group at 15 months after initial injection
since significantly fewer oocytes were retrieved despite
the E2 and P recovery. The number of large follicles was
not significantly different between the ZP-3 injected and
control monkeys indicating that folliculogenesis may be
slowly recovering in the immunized monkeys. Histological
studies of ovaries from two of the ZP-3 injected monkeys
showed only minor alterations on ovarian histology at 6
months after immunization. The data presented in this
investigation indicate that the effects on ovarian function
in the squirrel monkey in response to anti-zona antibodies
are milder and probably reversible as compared to the
rabbit (Skinner et al., 1984).
The differences in observations between the rabbit
test system and the squirrel monkey could be due to
species or the use of a purified zona macromolecule (2P3)
as immunogen. In rabbits, complete inhibition of follicular
development resulted when heat-solubilized, total porcine
zona proteins were used as the immunogen (Skinner et al.,
1984). A follow-up study reported no effect on normal
ovarian follicular development with the use of a purified
zona protein (Skinner et al., 1984). In contrast to the
50
rabbit study, Gulyas et al. (1983) described a reversible
infertility with intermittant distubances in menstrual
cycles in response to immunization of cynomolgus monkeys
with heat—solubilized total zona protein. The milder effect
observed in the primate study following the use of total
porcine zona as immunogen suggests a species difference in
response to immunization.
Among individual monkeys,there was great variability in
steroid levels, cyclicity and, follicle and oocyte
production throughout the post-immunization period. It
was apparent that the ZP—3 immunization did not result
in complete inhibition of ovarian function since
immunized monkeys did exhibit follicle and oocyte
development despite the high antibody titer level.
Many of the reports previously discussed involving
active immunization of monkeys (Gulyas et al.,1983),
rabbits (Skinner et al., 1984) and dogs (Mahi-Brown et al.,
1985) with porcine zona have cited disturbances in estrous
cycles as well as disruptions in normal ovarian function
and steroid secretion. The tissue specificity of zona
antigens has been clearly demonstrated (Palm et al., 1979;
Gerrity et al., 1981) so the effects caused by circulating
antibodies are observed at the level of the ovary on zona
components. Due to the controversy over the site of zona
synthesis (Bleil and Wasserman, 1980; Wolgemuth et al.,
1984), the exact cellular targets of these antibodies and
51
their mechanism of action on steroid secretion and normal
ovarian function can only be hypothesized. Antibodies to
the zona might interfere with the communication of the
oocyte with its surrounding corona radiata cells. Another
possibility is that the antibodies react with the zona or
zona precursor material at the site of zona synthesis and
ultimately destroy the oocyte. This would account for the
lower number of follicles collected via laparoscopy and the
alteration in steroid secretion.
The maximum steroid levels (E2 and P) for all three
groups of monkeys was considerably lower than levels
reported in the literature (Ghosh et al., 1983; Diamond et
al., 1984; Aksel et al., 1985). The radioimmunoassay
procedures used in all of these studies was identical. The
short time of acclimatization to the laboratory, young age
of the colony and stress of handling might account for the
observed differences. An increase in the E2 and P levels
was noted with the most recent bleedings indicating the
monkeys in the colony are beginning to stabilize. Although
the steroid levels reported here are lower than those
previously cited in the literature, they are well within
the range described. Also, none of the serial bleedings
were performed during the breeding season when levels would
normally be elevated in order to allow matings to occur
undisturbed.
Early immunocontraceptive studies as well as this
52
investigation have demonstrated the contraceptive
effectiveness of antibodies directed against zona pellucida
macromolecules. Further work could examine the other zona
glycoproteins for antigenic activity and modified forms of
ZP-3 glycoporotein (deglycosylated, peptide fragments and
enzyme digests). The use of other adjuvants might also be
tested since Freund's adjuvant may have independently
affected normal ovarian function in this study.
Nevertheless, the findings of this study encourage further
investigation of purified zona macromolecules for
contraceptive vaccine development.
SUMMARY AND CONCLUSIONS
The squirrel monkey provides a model for the
examination of the purified 55K porcine macromolecule (ZP-
3)
are
1)
2)
3)
4)
as a contraceptive vaccine. The following conclusions
indicated:
Immunization of female squirrel monkeys with the
purified porcine zona macromolecule (ZP-3) results
in the disruption of ovarian function. This
disturbance is reversible as shown by the recovery
in follicular development and hormone levels at 10-15
months post-immunization.
Histological data indicate an interference in
folliculogenesis in the ZP-3 injected animals.
Production of high antibody titers (75% binding level)
can be achieved in immunized monkeys at 120 days
following initial injection. Anti-ZP—3 titers remained
high (68% binding level) throughout the study.
Administration of the 55K macromolecule produced an
anti-fertility effect on the immunized monkeys since
no pregnancies occurred in this group. A 29% pregnancy
rate was observed in the untreated control group.
53
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APPENDIX
299119911999 by the Aether
Full Papers
1)
Antigenic profile and impact of immunization with
zona pellucida antigens in primates. A.G. Sacco,
M.G. Subramanian, E.C. Yurewicz, D.L. Pierce and
W.R. Dukelow. In: Immunological Approaches to
Contraception and Promotion of Fertility. (C.P.
Talwar, ed.) Plenum Press. New York. In press,
1986.
Abstracts
1)
2)
3)
4)
5)
Ovulation control, sperm capacitation and
fertilization. W.R. Dukelow, J.A. Kontio, R.D.
Bates and D.L. Pierce. Proc. American Society of
Primatologists. 1984.
Contraceptive potential of a ZP-3 antigen vaccine
as tested in squirrel monkeys. D.L. Pierce,
A.G. Sacco and W.R. Dukelow. Proc. Michigan
Academy of Science. 1985.
Laparoscopic observation of the mouse uterus. W.R.
Dukelow, D.L. Pierce, J.A. Kontio and L.
Martin. Proc. Michigan Academy of Science.1985.
The effect of a ZP-3 antigen vaccine on the
fertility of squirrel monkeys (Saimiri sciureus).
D.L. Pierce, A.G Sacco, M.G. Subramanian and W.R.
Dukelow. Proc. American Society of Primatology.
1985.
In vitro fertilization normality of squirrel monkey
(Saimiri sciureus) embryos. W.R. Dukelow, D.L.
Pierce, W.E. Roudebush, J.K. Graham and T. Asakawa.
Proc. Fourth World Congress on In Vitro
Fertilization. 1985.
64
6)
7)
8)
65
Ovaries remain functional in squirrel monkeys
immunized with porcine zona pellucida (ZP).
A.G. Sacco, M.G. Subramanian, E.C. Yurewicz, D.L.
Pierce and W.R. Dukelow. Society for Gynecological
Investigation. 1986.
In vitro fertilization in nonhuman primates. W.
R. Dukelow, W.E. Roudebush, D.L. Pierce, J.K.
Graham and K. Sengoku. Proc. International
Primatological Society. 1986.
Immunization of squirrel monkeys with porcine zona
pellucida (ZP); effect on ovarian function. A.G.
Sacco. M.G. Subramanian, E.C.Yurewicz, D.L. Pierce
and W.R. Dukelow. Proc. 3rd International Congress
of Reproductive Immunology. 1986.
Name:
Born:
Birthplace:
Education:
Degrees Received:
VITA
Donna Lynn Pierce
August 9, 1961
Washington, D.C.
Dulaney Senior High School
Timonium, Maryland
Michigan State University
East Lansing, Michigan
Bachelor of Science (1984)
Michigan State University
66