Download Immunocontrol in dogs

Animal Reproduction Science 60–61 Ž2000. 365–373
www.elsevier.comrlocateranireprosci
Immunocontrol in dogs
R.A. Fayrer-Hosken a,) , H.D. Dookwah b, C.I. Brandon a
a
Department of Large Animal Medicine and Physiology and Pharmacology, College of Veterinary Medicine,
UniÕersity of Georgia, Athens, GA 30602-7385, USA
b
Department of Anatomy and Radiology, College of Veterinary Medicine, UniÕersity of Georgia, Athens,
GA 30602-7385, USA
Abstract
Population control in dogs and cats is an important goal for many groups. Control measures
over the years has included surgery, hormonal therapy and more recently immunological control.
The current presentation discusses dog population control with an emphasis on immunologic
control. Specifically, vaccination with purified zona pellucida ŽZP. glycoproteins leads initially to
immunocontraception and then to the profound and irreversible changes of immunosterilization.
The preliminary studies are extremely encouraging on developing a vaccine for lasting canine
population control. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Immunocontraception; Immunosterilization, Zona pellucida
1. Introduction
Mammalian fertilization involves a series of intricate events. One of the most crucial
steps that must take place prior to fertilization is the ovulation of an ovum with an intact
and mature zona pellucida ŽZP.. The ZP glycoproteins are highly glycosylated and these
carbohydrates moieties are necessary for the process of fertilization. Generally, the
sperm cell must penetrate the cumulus matrix, recognize and bind to the specific surface
zona carbohydrates then penetrate the ZP.
As the target of sperm binding the ZP plays a pivotal role in fertilization. It has
therefore become a desirable target for immunocontraception. Mechanistically, disruption or blocking of the carbohydrate andror protein receptor sites or chemical modification of the ZP generally will result in immunocontraception. Specifically, the production
)
Corresponding author. Tel.: q1-706-542-6451; fax: q1-706-542-8833.
E-mail address: [email protected] ŽR.A. Fayrer-Hosken..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 1 3 9 - 1
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of anti-ZP antibodies and their binding to the ZP results in immunocontraception.
However, some species need irreversible population control and in these instances
immunosterilization is the primary goal. Immunosterilization results after vaccination
with a specific target protein that leads to a massive T-cell reaction. If the reaction is
substantial enough the reproductive integrity of the ovary will be destroyed and the
female is sterilized by an immune reaction. This immunosterilization is the best
technology for dog and cat population control. In order to understand the pathophysiology of immunocontraception and immunosterilization, the biology of the canine ovary,
the oocyte and zona pellucida is reviewed. This provides the background for the
discussion of the mechanism of canine immunocontraception and immunosterilization.
2. Structure of the carnivore ovary
Morphologically, the canine and feline ovaries appear similar to those of many other
domestic species in that it is composed of an outer cortical region and an inner medulla.
The internal region or medulla is characterized by the presence of nerves, large coiled
blood vessels and lymph vessels embedded in loose connective tissue that contains
strands of smooth muscle. Retie ovarii, which are networks of irregular channels lined
by cuboidal epithelium or solid cellular cords, are prominent in the medulla of
carnivores. These, when appositioned to an oocyte, are thought to differentiate into
follicular cells.
The peripheral or cortical region of the ovary is occupied by follicles in various
stages of development or corpora lutea embedded in a loose connective tissue stroma.
The corpora lutea of carnivores and rodents, contain cords of polyhedral interstitial
endocrine cells. Additionally, the ovary of the bitch contains narrow, cuboidal epithelium-lined channels called cortical tubules, which are continuous with the layer of
cuboidal epithelium that overlies the thick connective tissue layer, the tunica albuginea
of the ovary ŽPreidkalns and Leiser, 1998..
2.1. Classification, structure and deÕelopment of follicles and oocytes
Oogenesis, the production of oocytes from primordial germ cells, begins prenatally
by mitotic proliferation of the internal epithelial cell masses in the ovarian cortex. The
internal epithelial cell masses are considered to arise from interaction of several cell
types. These include the cortical stroma, ovarian surface epithelium or the rete ovarii
and primordial germ cells which embryologically arrive in the gonadal ridge from the
entoderm of the yolk sac ŽNoden and DeLahunta, 1985.. Unlike other species, oogenesis
in the dog may occur for about 2 months after birth since proliferating cells, resembling
germ cells, have been observed in small clusters throughout the ovarian cortex ŽMcDougall et al., 1997..
Follicles are classified variously, based on size, numbers of layers of follicle cells
surrounding the oocyte, the appearance of the zona pellucida and the appearance of the
follicular antrum ŽTesoriero, 1981.. In its most basic form, an ovarian follicle is
composed of an oocyte enclosed by specialized epithelial cells. As the follicle undergoes
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further differentiation, an additional layer of specialized stromal cells envelops the
oocyte with subsequent differentiation of a fluid-filled cavity or follicular antrum within
the stroma.
2.2. Primordial follicles
The earliest recognizable form of the female gamete, the primordial follicle, is seen
from about 3 weeks after birth and occurs in clusters in the cortex of the ovary
ŽMcDougall et al., 1997.. The follicles are small and contain a primary oocyte
surrounded by a single layer of squamous epithelial cells Žfollicular cells. that rest on a
basement membrane. The oocyte contains a large dictyate nucleus Žarrested in diplotene
stage of first meiotic division. with a distinct nucleolus. The ooplasm contains large
rounded mitochondria, smooth endoplasmic reticulum and small Golgi bodies ŽTesoriero,
1981.. Gap junctional contacts are also present between the microvilli of follicle cells
and the oocyte membrane. Desmosomes anchor the follicle cells to each other ŽAnderson
and Albertini, 1976..
2.3. Primary follicles
The next stage in the development of the solid growing follicles is the primary or
preantral or unilaminar follicle. The follicle is characterized by an increase in size and
enclosure of the oocyte by a layer of simple cuboidal epithelial follicular cells
ŽPreidkalns and Leiser, 1998.. The zona pellucida is immunologically detectable,
although it may not be completely circumferential ŽFayrer-Hosken, 1999 personal
communication..
2.4. Secondary follicle
The progression of development to the secondary follicle is characterized by significant changes in morphology. Specifically the enclosure of the oocyte by a stratified
epithelium of polyhedral follicular cells termed granulosa cells. These morphological
changes include dilatation and budding of the outer lamellae of the nuclear membrane as
well as increases in mitochondria, endoplasmic reticulum, Golgi bodies and granularity
of the matrix of the ooplasm. All of these organelles are consistent with increased
synthetic activity within the cell. A unique feature of the dog, cat and porcine ova at this
stage is the appearance of large amounts of lipid yolk material. The yolk material first
appears in association with a single centriole and is directly associated with lamellae of
the smooth endoplasmic reticulum. At this stage, the zona pellucida continues to form as
aggregates of material that coalesce to form a continuous layer between the oocyte and
the adjacent granulosa cells. Focal areas of contact are maintained through the zona
pellucida by cytoplasmic extensions of the granulosa cells to the oocyte ŽTesoriero,
1981; Preidkalns and Leiser, 1998..
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2.5. Tertiary follicles
As oocyte development progresses to the tertiary follicle, the nature of the follicle
changes to an antral or vesicular body which characterizes the Graafian follicle. At this
stage, the oocyte is enclosed by a layer of stratified epithelium of granulosa cells, which
are delimited by a basement membrane. On the exterior of this membrane a multilaminar
layer of stromal cells called thecal cells develops. The thecal cells differentiate into an
external layer which is essentially supportive, and an inner layer which is vascular. As
the follicle develops, small fluid filled clefts develop among the granulosa cells and
these later coalesce to form the large antrum which fills with fluid, the liquor folliculi.
As the antrum develops, the oocyte becomes enclosed in a collection of granulosa cells
termed the cumulus oophorus that later become columnar and radially disposed, forming
the corona radiata. The corona radiata is believed to supply nutrients to the oocyte
ŽPreidkalns and Leiser, 1998.. Within the oocyte cytoplasm, adjacent to the granulosa
cell-contact areas, are large Golgi, numerous mitochondria and dense granular vesicles
containing cortical granules. Dense cortical granule-like vesicles are also noted within
lamellar spaces and are thought to be associated with a mechanism whereby material is
added to the yolk bodies. As the oocyte continues to grow, the amount and size of the
lipid accumulation in vesicular bodies increase but do not coalesce ŽTesoriero, 1981..
3. Ovulation
While monovular follicles are the most prominent feature of the ovary until just
before first estrus, several polyovular follicles, containing two or more ova, are present
in the ovary at all ages ŽMcDougall et al., 1997.. Additionally, the increases in the total
number of monovular follicles and in oocytes found in monovular follicles at first estrus
may be indicative of the establishment of a population of follicles and oocytes for
subsequent ovulations at mature ages ŽMcDougall et al., 1997.. Ovulation is characterized by rupture of several mature follicles in the dog and results in the development of
several corpora lutea in the cortex of the ovary. Subsequent to increased follicular blood
capillary pressure and permeability associated with estrus there is an increased accumulation of liquor folliculi, which leads to follicular rupture. As the follicular wall swells it
becomes thinner and more transparent at the stigma, which is the site of future rupture of
the follicle ŽPreidkalns and Leiser, 1998.. Rupture is thought to be related to the release
of collagenase as a result of prostaglandin activity mediated by the release of luteinizing
hormone ŽPreidkalns and Leiser, 1998.. At ovulation, the follicle collapses and blood
fills the antrum resulting in the formation of a corpus hemorrhagicum. After ovulation,
the stratum granulosum becomes highly vascularized via capillaries from the theca
interna. The granulosa cells undergo hypertrophy and hyperplasia with accumulation of
lipid Žyellow pigment. which is characteristic of luteinization and formation of the
corpus luteum. Luteal regression is characterized by condensation of lutein pigment,
fibrosis and resorption of most of the corpus luteum resulting in a connective-tissue scar
remnant termed a corpus albicans ŽPreidkalns and Leiser, 1998..
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The unique feature of ovulation in the dog, in contrast to other mammalian species, is
the release of an immature oocyte containing a germinal vesicle, which has to mature
within the oviduct ŽTsutsui, 1989.. The immature germinal vesicle is characterized by a
vesicular nucleus with a distinctive nucleolus surrounded by fine filaments ŽHewitt and
England, 1998.. This immature oocyte requires at least 48 h to complete its meiotic
maturation ŽTsutsui, 1989; Hewitt and England, 1998. and the ovulated canine oocyte
can remain fertile, in vivo, for up to 108 h ŽTsutsui, 1989.. Accordingly, to ensure
fertilization of mature oocytes, spermatozoa may remain viable for as long as 268 h in
the estrous female genital tract after mating ŽDoak et al., 1967.. Further, in vitro studies
have demonstrated that canine sperm can penetrate homologous immature oocytes ŽMahi
and Yanagimachi, 1976., which suggests that in vivo sperm penetration may occur in
the oviduct prior to completion of oocyte maturation. In contrast to previous reports,
histological examination of ovarian oocytes in situ has demonstrated the presence of
germinal vesicle breakdown nuclear material in some oocytes. This suggests follicular
maturation may occur to a limited extent within the ovary; however, these may simply
represent a transitory state or material obtained from atretic follicles ŽHewitt and
England, 1998..
The ZP is an extracellular glycoprotein matrix which surrounds the canine oocyte and
serves to protect the underlying ooplasm and contains specific receptors for spermatozoal binding. The canine zona pellucida, in common with other mammals, consists of
three glycoproteins, ZP1, ZP2 and ZP3. The sequences of these proteins have been
reported ŽHarris et al., 1994. and they have significant homology with the ZP glycoproteins of other species. Specifically, there is homology with the ZP proteins of the pig.
But of greatest interest to immunocontrol of the dog, there are significant distinctive
differences in the glycosylation of the dog ZP glycoproteins ŽBarber et al., 1999.
compared to the pig glycoproteins.
4. Immunocontraception
Interest in the ZP as a potential target for mammalian immunocontraceptive and
immunosterilant vaccines has arisen because of its importance in fertilization, its unique
expression in oocytes, and its strong immunogenicity. If the ZP is masked or structurally
altered, fertilization will not occur and one would have an immunocontraceptive
vaccine. Thus, much research has focused on the generation of anti-ZP antibodies to
specific epitopes that inhibit fertilization without altering ovarian function. Specifically,
there has been the administration of porcine zona pellucida ŽpZP. glycoproteins, which
has resulted in immunocontraception in many species of mammals ŽKirkpatrick et al.,
1996, 1997; Fayrer-Hosken et al., 1997a,b.. Immunocontraception can be defined as the
ability to use a reproductive protein to produce a humoral immune response that leads to
the animals immunocontraception for a defined time period. At the end of this period the
amount of circulating antibodies, IgG, decreases and the animal becomes fertile.
In theory, the general mechanism of immunocontraception is simple and we have
hypothesized that antigens, which in this case are ZP glycoproteins, are presented in a
manner that results in the production of anti-ZP IgG antibodies. These antibodies then
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block fertilization primarily at the site of sperm–zona interaction. In reality, the
underlying mechanism of immunocontraception is actually quite complex. Immunocontraception probably interferes with one or several mechanisms that cause a cascade of
biochemical events leading to infertility.
It has been shown ŽHenderson et al., 1988. that anti-pZP antibodies bind to sperm
receptor sites of the host zona and ultimately inhibit spermatozoal binding. These
findings are further supported by earlier studies ŽSacco et al., 1989. in which pretreatment of porcine oocytes with antibodies to pZP3a Žprimary sperm receptor. blocked
sperm binding, while pretreatment of oocytes with antibodies specific to pZP3b
Žsecondary sperm receptor. did not inhibit sperm binding. It has also been suggested
ŽEast et al., 1985; Mahi-Brown et al., 1985. that anti-pZP antibodies do not simply
inhibit sperm binding to the oocyte, but in fact result in an inhibition of sperm
penetration. In the study by East ŽEast et al., 1985. using a mouse model, monoclonal
antibodies to both the primary and secondary sperm receptors in the oocyte ŽZP3 and
ZP2, respectively. did not interfere with sperm binding, but did reduce sperm penetration. The study by Mahi-Brown ŽMahi-Brown et al., 1985. in that same year reported
similar findings in the bitch in that inhibition of sperm penetration was the primary
finding that explained the immunocontraception in these animals. In fact, these authors
suggested that perhaps sperm penetration was inhibited via an inhibition of the acrosome
reaction of the spermatozoa. From this it could be argued that the immunocontraception
may be a consequence of altered sperm–zona attachment or a modification of the
sperm’s ability to penetrate the zona pellucida, or across species a combination of both
precepts.
Another potential mechanism of immunocontraception may involve a change in
structure of the zona pellucida itself. In most species, after sperm bind to and penetrate
the oocyte, a cascade of biochemical events results in the release of peripheral cortical
granules, the cortical reaction. The release of the cortical contents into the perivitelline
space induces zona hardening and ultimately prevents penetration by additional spermatozoa Žzona block.. It has been proposed ŽDucibella, 1996. that binding of anti-ZP
antibodies to the oocyte cause a premature activation of the oocyte. Thus the potential
oocyte activation leads to the cortical reaction which leads to zona hardening and the
spermatozoa are incapable of binding or fertilizing the egg.
The vaccination of dogs with pZP initially causes a rise in serum IgG levels. These
levels are enough to block fertilization and immunocontracept the bitch. Bitches that
have been vaccinated with our vaccine have been immunocontracepted for several
months. However, the vaccinated dogs with significantly elevated serum IgG levels also
show marked ovarian pathology. This pathology is the preliminary evidence of impending permanent ovarian damage and eventual immunosterilization.
5. Immunosterilization
Immunosterilization involves the ability to use pZP glycoproteins to produce an
immune response that leads to the animals sterilization by destroying oocytergranulosa
cell complexes or causing ovarian follicular atrophy. However, the pathophysiology of
immunosterilization is not completely elucidated for all affected species. The pathophys-
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iology of mammalian ovarian disease is multifactorial. There are clearly several distinct
clinical presentations, ranging from premature ovarian atrophy to polycystic ovarian
disease to autoimmune oophoritis. The pathogenesis of the naturally occurring disease
involves the humoral and cell mediated immune responses. However, the pathophysiology of immutable, induced ovarian disease is not as clear. Also the immunological and
pathological differences among affected species is paradoxical.
The genetic predisposition of an animal to the disease mediates the severity of the
immune response ŽTung et al., 1997.. Thus, some species or certain population groups
within a species might be the most susceptible to ovarian pathology. In all affected
animals, the final disease is a result of antigen presentation and the sequence and
severity of the immune response.
Antigen presentation is the most important facet of current research. Most researchers
are trying to define a synthetic, immunocontraceptive molecule. This would be a small
epitope that would not cause any ovarian pathology. In our research, we are investigating a safe, yet permanent immunosterilization and therefore aggressive antigen presentation is essential. The molecule itself must be antigenic, so we use a heterologous
molecule. Also, the presented antigen is enhanced by an adjuvant. For our studies the
pZP molecules provide the necessary prerequisite features for a immunosterilant vaccine.
For the vaccinated animals, the individual immune response is also pivotal to ensure
an immunosterilant effect. In several species, female subjects immunized with heterologous ZP developed ovarian pathology, but the mechanism of the ovarian diseases
remained undefined ŽSkinner et al., 1984; Mahi-Brown et al., 1985; Henderson et al.,
1988.. However, over time excellent studies by Tung et al. Ž1997. showed that ovarian
pathology was a result of Ža. immunoglobulin priming and T-cell response, and Žb.
B-cell mediated responses. In a study on murine ZP peptide, outbred CD1 mice
immunized with a 15-mer murine ZP peptide developed reversible infertility ŽMillar et
al., 1989.. However, this ZP peptide was subsequently shown to possess T-cell epitopes
and to induce oophoritis, an autoimmune disease induced by CD4 q T cells ŽRhim et
al., 1992; Luo et al., 1993..
An additional problem regarding ZP vaccine development is the genetic polymorphism of autoimmune responses to the ZP glycoproteins. Previous reports had shown the
use of a chimeric peptide with a promiscuous T-cell epitope as a effective vaccine
formulation ŽGreenstein et al., 1992; Partidos et al., 1992; Su and Caldwell, 1992; Lopez
et al., 1994.. Tung et al. Ž1997. modified this strategy by altering the ZP epitope so that
the T-cell epitope of ZP was completely eliminated. This produced an antibody to ZP for
immunocontraception induction, yet insufficient for induction of autoimmune oophoritis.
However, for an immunosterilant vaccine retention of the T-cell epitopes has been
important to produce adequate ovarian pathology. Pathologically, the cytotoxic T-cells
lead to autoimmune disease of the ovary and secretion of cytokines by the immune cells
probably have an adverse effect on ovarian steroidogenesis. Therefore, the immunosterilant vaccine has an effect on the female reproductive system by affecting the endocrine
and the immune systems.
For our studies enzyme-linked immunosorbent assays ŽELISAs. revealed that the
serum antibody levels of vaccinated, immunosterilized dogs were significantly Ž p - 0.05.
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elevated when compared to controls vaccinated with a placebo. Antibody levels rose
slightly after the initial vaccination and then significantly Ž p - 0.05. after the first and
second boosters. These data clearly show that there is a significant immune response to
the pZP vaccine and adjuvant. Furthermore, normal dog ovaries have distinct oocytes
with a zona pellucida, especially in primary, secondary and tertiary follicles. Immunohistochemical studies of these normal ovaries indicated that there was marked staining of
the zona pellucida in these follicles with anti-pZP antibodies. This can be compared and
contrasted to ovaries of vaccinated immunosterilized dogs. The ovaries of immunosterilized dogs revealed histologically that the integrity of all follicles has been breached. All
follicles have been significantly invaded by leucocytes and specifically neutrophils,
lymphocytes and macrophages. With immunohistochemical studies it was clear that
there was an immunologic response at the site of the zona pellucida of the oocytes, and
in fact no immunodetectable zona pellucida glycoprotein material remained. It is evident
from these studies that the oocyte–granulosa cell complexes in the canine ovary have
been disrupted.
6. Conclusion
In conclusion, recent reports have indicated that immunocontraception via zona
pellucida immunization is a highly promising avenue for providing alternative means of
contraception. We present preliminary data that permanent immunosterilization can also
be achieved in dogs. Immunosterilization would be an effective and practical technology
for controlling dog populations throughout the world.
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