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PCB EFFECTS ON MOUSE OOCYTES : THE POTENTIAL
ROLE OF CUMULUS CELLS IN THE MEDIATION
OF TOXICITY

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presented by
Charles Ryan Greenfeld

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1/” WM“

 

PCB EFFECTS ON MOUSE OOCYTES : THE POTENTIAL ROLE OF CUMULUS
CELLS IN THE MEDIATION OF TOXICITY

By

Charles Ryan Greenfeld

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

MASTER OF SCIENCE

Department of Zoology

1997

ABSTRACT

COMMERCIAL PCB EFFECTS ON MOUSE OOCYTES : THE POTENTIAL ROLE
OF CUMULUS CELLS IN THE MEDIATION OF TOXICITY

By

Charles Ryan Greenfeld

Aroclor 1254, a commercial PCB mixture, has previously been shown to decrease
mouse in vitro fertilization (IV F), by exerting toxicity upon the cumulus-enclosed oocyte
through an unknown mechanism. This study examined the effects of A-1254 on zona
pellucida (ZP) hardening in cumulus-free mouse oocytes. Oocytes were aged in the
presence or absence of 1.0 or 10.0ug/ml A-1254, for 1, 3 or 6 hours. A-1254 neither
induced nor inhibited ZP hardening. It did, however, reverse ZP hardening following 6 I
hours of aging. This indicates that A-1254 acted directly upon the ZP, altering its
structural integrity, by means of a novel mechanism. IVF was also examined using
cumulus-free mouse oocytes, aged for either 3 or 6 hours in the presence or absence of the
same concentrations of A-1254. No reduction in fertilization was seen. These results

suggest that cumulus cells may be involved in the mediation of PCB-induced toxicity.

DEDICATION

To my wife Lisa, for all your love and support, and for putting up with me through this
sometimes arduous process. To my parents, thanks for the same, and for not giving up on
me when it would have been easy to. And to Phish, whose music brought me solace

often.

iii

ACKNOWLEDGMENTS

I wish to thank Dr. Dukelow for giving me a chance at the Endocrine Research
Center. What I have learned there is invaluable. Also, thanks to my committee members,
Dr. Roy Fogwell and Dr. John Kaneene, for their assistance. Thanks to Bonnie Cleeves
for friendship, help, and for trusting me with account numbers, and to Dr. Bursian, Dr.
Aulerich, Debbie Powell, and all the other grad students at the Endocrine Research
Center. Thank you also to Dr. Gregory Fink for advice on statistical analysis. This work

was supported by NIEHS grant no. ESO4911.

iv

TABLE OF CONTENTS

INTRODUCTION .................................................................................................. 1
LITERATURE REVIEW ........................................................................................ 5
Mouse Development ................................................................................................................................... 5
PCBs : History, Production and Use ........................................................................................................... 7
PCBs : Chemical Properties ........................................................................................................................ 9
PCBs : Environmental Contamination ...................................................................................................... 11
PCBs : Accumulation, Metabolism, Elimination and Environmental Breakdown .................................... l3
Aroclor 1254 ............................................................................................................................................. l6
PCBs : Experimental Toxicity .................................................................................................................. 16
PCBs : Carcinogenic Potential .................................................................................................................. 21
Fertilization Events ................................................................................................................................... 24
MATERIALS AND METHODS ............................................................................ 28
Animals ..................................................................................................................................................... 28
Aroclor 1254 ............................................................................................................................................. 28
Superovulation .......................................................................................................................................... 28
In Vitro Fertilization (IVF) ....................................................................................................................... 29

V

Zona Pellucida Hardening Assay .............................................................................................................. 30

Statistical Methods .................................................................................................................................... 32
RESULTS ........................................................................................................... 33
DISCUSSION ..................................................................................................... 37
LIST OF REFERENCES ..................................................................................... 47
APPENDIX A ...................................................................................................... 62
APPENDIX B ...................................................................................................... 64
APPENDIX C ...................................................................................................... 67

vi

LIST OF TABLES

Table 1 - Effects of A-1254 on zona pellucida hardening in cumulus-free mouse

oocytes ................................................................................................. 62

Table 2 - The effects of A-1254 on in vitro fertilization in cumulus-free mouse

oocytes ................................................................................................. 63

vii

LIST OF FIGURES

Figure 1 - Rate of zona pellucida dissolution following a three hour incubation ......... 64
Figure 2 - Rate of zona pellucida dissolution following a six hour incubation ............ 65

Figure 3 - Zona pellucida hardening following various incubation times .................... 66

viii

Introduction

Polychlorinated biphenyls (PCBs) are a class of aromatic hydrocarbons that were
used in many industrial processes from 1929 until their ban in 1977. Several inherent
properties of these chemicals, including low reactivity, and resistance to degradation,
made them attractive for industrial use. These same properties however, also make them
highly persistent in the environment, and because they are lipophilic, they can be
bioaccumulated in food chains. Because of this, PCBs will continue to be present in the
environment, and in foods, and will continue to pose a health risk for many years to come
(Battershill 1994).

Experimental evidence of the toxicity of PCBs in animals has been mounting
since their detection in the environment in 1966. They have been found to cause
problems ranging from hyperpigmentation of the skin to carcinogenesis (N orback and
Weltrnan 1985; Guo et al. 1995). Due to extensive experimentally derived data, as well
as correlative evidence of carcinogenicity of PCBs in animals, the United States
Environmental Protection Agency (U SEPA) has listed the chemicals as probable
carcinogens (Kimbrough and Linder 1974; Bertazzi et al. 1987; Vater et al. 1995).

PCBs have also been shown to produce many reproductive problems. In mink,
PCBs produce complete reproductive failure (Aulerich et al. 1973; Bleavins et al. 1980).
They decrease pregnancies and litter size in the beagle, pig and rat (Earl et al. 1974;
Brezner et al. 1974). They also decrease oocyte fertility in vitro. Huang and Chou (1994)

found that in F1 female mice derived from PCB-exposed mothers, oocyte fertility was

2
reduced. Kholkute et al. (1994b) found that the commercial PCB mixture, Aroclor 1254,
when present in the in vitro fertilization (IV F) culture media, reduced the percentage of
mouse oocytes that were fertilized. The same response was produced following oocyte
incubation in the presence of A-1254, while incubation of spermatozoa with A-1254 had
no effect on fertility.

The mechanism responsible for decreased oocyte fertility following PCB exposure
in vitro, has yet to be determined. “Hardening” of the zona pellucida (ZP) is one
possibility. ZP hardening is a natural event in the fertilization process, following gamete
fusion, and the subsequent cortical reaction (Kalab et al. 1991). It is responsible for the
essential slow block to polyspermy. However, ZP hardening can occur prematurely
during in vitro maturation in serum-free medium, or during in vitro aging, and can be
induced by artiﬁcial activation of the oocyte (DeFilici and Siracusa 1982; Downs et a1.
1986; Vincent et al. 1990). When it occurs prematurely, ZP hardening can cause
infertility, as has been witnessed in human IVF programs (Ducibella et al. 1995).
Assessment of ZP hardening can be accomplished in vitro by exposing the oocyte to
proteolytic enzymes, or by performing IVF. It has been shown that following hardening,
the ZP develops a resistance to dissolution by such enzymes, and also becomes
impenetrable to sperm (DeFilici and Siracusa 1982; Gianfortoni and Gulyas 1985).

IVF, which is only one of many assisted reproductive techniques, has become well
established as a research tool for screening reproductive toxicants. In vitro toxicity assays
have many beneﬁts over in vivo tests (Frazier 1992). These beneﬁts include the speed at

which the assay can be performed. With thousands of chemicals being introduced every

3
year, rapidity of toxicological assessment is very important and is a hallmark of in vitro
tests. Cost is another beneﬁt, with in vitro tests being considerably cheaper than their in
vivo counterparts. Other beneﬁts are that no pain is caused to live animals during in vitro
tests, environmental conditions are controllable, variability between experiments is
greatly reduced, and the interaction between organ systems is eliminated.

Toxicology is not the only ﬁeld in which IVF has found application. It has been a
staple in clinical human fertility programs ever since the birth of the ﬁrst IV F human in
1978 (Fishel 1986). IVF is also receiving a great deal of attention from the ﬁeld of
conservation biology, where along with many other assisted reproductive techniques, it is
being used in the conservation of endangered wildlife. Some of these other techniques
include, but are not limited to, in vitro maturation of both primordial follicles and
immature oocytes, intracytoplasmic sperm injection, embryo transfer, and gamete/embryo
cryopreservation and banking (Schroeder et a1. 1988; Wildt 1989; Keefer 1990; Wildt
1991; Eppig and O’Brien 1996). It has also been recognized in the last few years that
immature oocytes recovered from aged, sick, dying, or dead animals can be matured and
fertilized in vitro, and can be used to obtain live offspring (Johnston et al. 1991;
Schroeder et al. 1991; Eppig and O’Brien 1995). This latter technique has tremendous
importance, as it can be used to salvage, or rescue genetic resources that would otherwise
be wasted.

The objective of the present research was to examine the functional effects of

PCBs on mouse oocytes, using both a ZP dissolution assay, and IVF. The hypotheses

4
tested were that A-1254 both induces ZP hardening, and causes decreased fertilization in

cumulus-free mouse oocytes.

Literature Review

Mouse Development

The gestational period for the mouse fetus is in the range of 19-20 days (Hogan et
a1. 1986). During a natural ovulatory cycle, eight to twelve eggs are ovulated over a 2-3
hour period (Hogan et al. 1986). Ten hours after fertilization has occurred, the ovum has
emitted its second polar body, and, by 24 hours, has reached the two-cell stage (Hogan et
al. 1986; Theiler 1989). The morula stage is reached by two days post coitus, and
development continues in the oviduct until day three when the embryo enters the uterus
(Hogan et al. 1986; Theiler 1989). Implantation begins at 4.5 days. By 7.5 days, the
amniotic cavity is sealed off, the neural plate is clearly delineated, both anteriorly and
laterally, and the head process is developing (Theiler 1989). The heart is developing
rapidly at 8 days, and the ﬁrst seven somites are formed. The oral plate is formed at this
point, and the brain is developing. Beginning in the region of the fourth and ﬁfth
somites, the neural folds close, and continue to do so both anteriorly and posteriorly. By
nine days the heart is capable of maintaining minimal circulation, and placental
circulation is being established. Both the forelimb and the hindlimb buds condense at 9.5
days. At the same time, the circulatory system further develops, as does the pancreas.
Primordial germ cells can already be seen lined up at the genital ridge by ten days. The
extremities and tail bud develop rapidly at 10.5 days, and the most cranial somites

become more indistinct.

6

The genital ridge contains numerous gonocytes by 11.5 days. The lens vesicle has
completed closure, and the cranial nerves and spinal ganglia are well developed. The
lung buds have developed secondary and tertiary bronchi, and the stomach becomes
greatly distended at 12 days. Sexual differentiation occurs at 13 days. Also at this time,
the lungs are completely subdivided into lobes. The liver is well developed, and the
kidney is developing. Hair follicles are present everywhere except the head by 14 days.
By 15 days, the atrioventricular and semilunar valves are developed in the heart, and the
arteries and veins have their ﬁnal fetal conﬁguration. The oral and nasal cavities are
separated by palatal processes which fuse with the nasal septum. The ovary contains
dividing gonocytes, and in the testes the seminiferous tubules are developed and
differentiated. Vertebral ossiﬁcation begins at 16 days, and blood cell production
increases in the liver. At 17 days, many dividing oocytes have entered prophase I, and at
18 days the ovaries become attached to the lower poles of the kidneys, and are almost
completely surrounded by the bursa ovarica.

The mouse is born at day 19-20. It is hairless, both its eyes and ears are closed.
They remain that way for about two weeks. The vagina does not open until 6 weeks after
birth, when the estrous cycle begins. Spermatogenesis is active at 24 days, which is also
the time of weaning. The ﬁrst successful mating can occur at 2-3 months. The mouse is

full grown by 3-4 months (Theiler 1989).

PCBs : History, Production and Use

Polychlorinated biphenyls (PCBs) were ﬁrst synthesized in 1881 by Schmidt and
Schultz, but did not have industrial application until 1929 (Tanabe 1988). The Monsanto
Corporation was the sole domestic producer of PCBs from this time until 1977 when the
manufacture of PCBs was discontinued (Hutzinger et al. 1974; Brown et al. 1991). Their
use had already been restricted once by this time, when Monsanto withdrew PCBs from
the market in 1970, for all but closed systems use (Trout 1972). This voluntary
withdrawal came after the identiﬁcation of PCB compounds in environmental samples by
Jensen and Widmark in 1966, and subsequent research showing widespread
environmental contamination and possible health hazards (Norton 1972). Today in the
United States, PCBs are no longer used commercially, but they are still present in many
active electrical systems, and in many ecological systems throughout the world. PCB
presence in the environment will continue for many years to come.

Production of industrially important PCB products was done by chlorination of
biphenyl with anhydrous chlorine, using iron ﬁllings or ferric chloride as a catalyst, until
a desired endpoint of chlorine content was reached (Cook 1972; Hutzinger et al. 1974).
This endpoint was determined by making speciﬁc gravity or softening point
measurements on samples that were withdrawn during the chlorination process. Crude
products were puriﬁed by treatment with alkali followed by distillation, to remove color
and traces of both hydrogen chloride and ferric chloride. The end product would be a

mixture of chlorobiphenyls containing different chlorine contents.

8

The Monsanto Corporation produced PCBs under the trade name of Aroclor®
(Hutzinger et al. 1974). PCB mixtures were designated using a two number system. The
ﬁrst number represented the type of compound, for example PCBs were 12, and the
second number represented the weight percentage of chlorine. Therefore, the name
Aroclor 1254 signiﬁes a PCB mixture with an average of 54% chlorine by weight. The
commercially important Aroclor mixtures were A-1221, 1232, 1242, 1248, 1254, 1260,
1262 (Kalmaz and Kalmaz 1979), and Aroclor 1016, which does not follow the
standardized nomenclature. A—1016 contains 42% chlorine by weight, and was
introduced by Monsanto in 1971, as a biodegradable alternative to A-1242 (Bowers et al.
1975; Brinkman and DeKok 1980). In the United States, total production and use of
PCBs from 193 0-1975 exceeded 1.4 billion pounds (Brinkman and DeKok 1980).

Polychlorinated biphenyls contained many chemical properties, which will be
discussed later, that made them useful for many industrial applications. These
applications included : use as dielectric ﬂuids in capacitors and transformers, industrial
ﬂuids for use in hydraulic systems, gas turbines and vacuum pumps, ﬁre retardents, heat
transfer systems, plasticizers, sealants, a component of the dye carrier in carbonless c0py
paper, and kill-life extenders in a variety of pesticides including Aldrin® and DDT (Trout
1972; Hutzinger et al. 1974; Gellert 1978). Prior to 1971, open-ended use, which
included such uses as pesticide extenders, represented 26% of the total use in the United
States, while nominally-closed, and closed system use represented 13% and 61%
respectively (Brinkman and DeKok 1980). Many of these uses, especially open-ended,

led to the introduction of PCBs into the environment.

PCBs : Chemical Properties

There are 209 theoretical PCBs. Any individual PCB is called a congener
regardless of the degree or position of chlorination. A subclass of PCBs of a speciﬁed
degree of chlorination (e. g. tetra- or pentachloro-) is a homolog, and a member of a
homolog is an isomer (N orstrom 1988). There have been approximately 100 congeners
identiﬁed in commercial PCB mixtures (Battershill 1994). Most of the individual
congeners are solids at room temperature, whereas the commercial mixtures are either
mobile oils (A-1221, A-1232, A-1242, A-1248), viscous liquids (A-1254), or sticky
resins (A-1260, A-1262) (Cook 1972; Hutzinger et al. 1974). The solubility of PCB in
water is very low and is inversely related to the degree of chlorination (Hutzinger et a1.
1974). Volatility is also inversely related to the degree of chlorination, and is overall very
low (Norton 1972; Kalmaz and Kalmaz 1979; Swain 1983). In general, PCBs have high
thermal stability, and high resistance to both oxidation and hydrolysis in industrial
settings (Hutzinger et al. 1974). These factors, combined with high general stability,
inertness, and excellent dielectric properties led to their widespread industrial usefulness
(Hutzinger et al. 1974). Ironically, these same factors contribute to their high
environmental stability and persistence.

Of the 209 possible congeners, 20 can attain a coplanar conﬁguration of the
biphenyl ring system due to a lack of ortho- substitution (Tanabe 1988). Structure-

function relationships show that only congeners substituted in both the para- and meta-

10
positions, and that lack ortho- substitution, can exhibit a maximum coplanar
conﬁguration and approximate the ﬂat structure of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(Safe et al. 1985; Koopman-Esseboom et a1. 1994). Congeners with high numbers of
ortho-chlorines have reduced coplanarity and thus reduced toxicity (Koopman-Esseboom
et al. 1994). The most active PCB congeners are : 3,3’,4,4’-T4CB; 3,4,4’,5-T4CB;
3,3’,4,4’,5-P5CB; and 3,3’4,4’,5,5’-H5CB (Safe et al. 1985; Tanabe 1988). They all lack
ortho- substitution, but are substituted at both para- positions and at 2 or more meta-
positions, and are therefore approximate stereoisomers of both 2,3,4,7,8-P5CDF, and
2,3,7,8-T4CDD (Safe et al. 1985; Tanabe 1988). These coplanar congeners are present in
low amounts in commercial mixtures and may be responsible for much of the overall
toxicity of the mixture (Golub et al. 1991).

PCDFs have been identiﬁed as contaminants in Japanese PCB preparations, in all
Aroclor preparations except A-1016, and have been found to be as high as 2 ppm in A—
1248 (Bowes 1975; Goldstein et al. 1978; Wakimoto et al. 1988). Along with the
PCDDs, the PCDFs are oxidation products and can be present in PCB preparations as
contaminants. They can be created through industrial use (Barlow and Sullivan 1982).
PCBs can be converted to PCDFs in temperatures as low as 300°C, and, in electrical or
PCB ﬁres, very complex mixtures of PCDFs along with a variety of other chlorinated
aromatic compounds are produced (Erickson 1989). DeFelip et al. (1990) found both
PCDFs and PCDDs to be present in residual ﬂuid in a transformer following accidental

leakage. Brown et al. (1988) also found PCDFs to be present in the dielectric ﬂuids of

11
capacitors and transformers, but did not ﬁnd evidence that they were produced during

use.

PCBs : Environmental Contamination

PCBs were present in environmental samples as early as the 19503. They were
found in many specimens that were analyzed for the presence of chlorinated hydrocarbon
pesticides, however they were ignored as unknown quantities until they were identiﬁed in
1966. Since then, PCBs have been found in the environment, and in organisms from pole
to pole (Holmes et al. 1966; Koeman et al. 1969; Musial et al. 1974; Subrarnanian et al.
1983; Tanabe et al. 1983; Tanabe et a1. 1987; Cummins 1988; Kipcic and Vukusic 1991;
Bordet et al. 1993; Quemerais et al. 1994).

Cummins (1988) estimated that of the 1.2 million tons of PCBs in the world, 65%
is either still in use in electrical systems, deposited in landﬁlls or stored on land, and 31%
percent has been released into the environment, the rest being degraded or incinerated.
Environmental contamination by PCBs has occurred by : discharge in urban sewage
efﬂuent (Schmidt et al. 1971), leakage from electrical equipment, spills during their
manufacturing and transport, vaporization or leaching from PCB containing formulations,
improper handling and disposal (N isbet and Saroﬁm 1972), stack emissions during
incineration (Kocan et al. 1991), agricultural and municipal runoff, and by direct
discharge into waterways by power plants (Kalmaz and Kalmaz 1979). Once in the

environment, movement of PCBs predominantly occurs by atmospheric transport and

12
deposition which serves to distribute the chemicals worldwide (Bidleman et al. 1976;
Kalmaz and Kalmaz 1979; Swain 1983; Tanabe 1988).

The most common route for PCBs to enter the food chain is by aquatic systems.
After deposition into waterways following atmospheric fallout, PCBs tend to reside in the
surface microlayer due to their hydrophobic nature (Kalmaz and Kalmaz 1979).
Microorganisms and plankton are common food chain entry points, as they reside and
feed in this region. This is where the greatest effects of PCB contamination are felt in the
aquatic system, as they inhibit the growth and photosynthesis of phytoplankton at levels
as low as 10-100 ppb (Norton 1972; Kalmaz and Kalmaz 1979). These organisms are
essential to the functioning of aquatic systems as they provide oxygen and nutrients to the
system.

Subsequent biomagniﬁcation occurs as consumers feed upon plankton, and the
chemicals move up the food chain until they reach a top consumer. Top consumers
generally have long lifespans, and can therefore be exposed to large doses of toxins over
time. Among the most sensitive organisms to the toxic effects of PCBs are marine
mammals, which can biomagnify the chemicals by factors as great as ten million times
(Tanabe et al. 1984; Cummins 1988). These organisms are highly sensitive to PCBs
because many have genetically inﬂuenced reductions in the ability to induce drug
metabolizing enzymes (Cummins 1988). Animals with high-fat diets, such as marine
mammals like polar bears, as well as humans, are at especially high risk. Polar bears rely
on seal blubber as a major dietary component. Blubber can contain very high levels of

PCBs and other organic toxins, as this is the tissue into which these chemicals tend to

13
partition. Levels of PCBs in the adipose tissue of polar bears increased four-fold from
1969 to 1984, and if this trend continues they will soon surpass the 50 ppm level
designating them as toxic waste (Cummins 1988). High levels of toxins in marine
mammals are also dangerous because they have fat rich milk, which facilitates their

transfer to nursing young (Tanabe 1988).

PCBs : Accumulation, Metabolism, Elimination and Breakdown

As stated earlier, PCBs are persistent in the environment, highly lipophilic and
can therefore be bioaccumulated (Bordet et al. 1993). This means that the toxin enters an
organism, either by ingestion, or by diffusion across the body surface and becomes
concentrated in tissues and organs that are high in lipids (Hutzinger et al. 1974). They do
not necessarily remain in adipose tissue permanently. Rather, they can be mobilized into
the plasma and travel to other parts of the body, eliciting toxicity, or they can be
metabolized and excreted. Mobilization tends to occur during pregnancy, lactation and
during periods of stress or starvation (Fuller and Hobson 1986).

Metabolism of PCBs is accomplished by hepatic drug metabolizing enzymes.
Induction of these enzymes by PCB tends to follow two different patterns. One pattern is
similar to that initiated by the drug phenobarbital (PB). PB induces the production of
cytochrome P-450, the terminal oxidase of hepatic drug metabolizing enzymes, and
enhances the metabolism of a wide variety of substrates (Goldstein et a1. 1977; Goldstein

et al. 1978). Congeners that follow this pattern are generally chlorinated in both the para-

14
and ortho- positions. The other pattern, of PCB metabolism, mimics that initiated by the
carcinogenic polycyclic aromatic hydrocarbons. A prototype of this pattern is 3-
methylcholanthrene, which induces both cytochrome P-448 and the associated aryl
hydrocarbon hydroxylase (AHH) (Goldstein et al. 1977; Goldstein et al. 1978).
Congeners of this type tend to be those that are chlorinated in both the para- and meta-
positions. These congeners can assume a coplanar conformation, and can elicit a
response similar to the highly toxic 2,3,4,7,8-pentachlorodibenzofuran, and 2,3,7,8-
tetrachlorodibenzo-p-dioxin, but to a lesser extent (Safe et al. 1985).

Most PCB congeners follow the PB pattern of induction and are metabolized by
cytochrome P-450 (Brown et al. 1989). The phenyl ring is hydroxylated at the 3- or 4-
position by 3,4-epoxidation. This hydroxylation facilitates elimination of the molecule.
Hydroxylation of the PCB congener can also follow an arene oxide intermediate (Barlow
and Sullivan 1982). In general, the ease of metabolism is inversely related to the number
of chlorines present (Hutzinger et al. 1974; Sugiura et al. 1976). It is also effected by the
position of chlorine substitution. A high hydroxylation rate is attainable for congeners
with no substitution at para- or meta- positions (Sugiura et al. 1976).

Biological half-lives and elimination of PCB are determined by the rate of
metabolism. PCBs with low chlorine content have short half-lives and are eliminated
fairly rapidly (Schmid et al. 1992). Niimi and Oliver (1983) found that following
ingestion of one dose of PCB, containing 31 different di- to decachlorobiphenyls, by
rainbow trout, half-lives were in the range of 5 days for the lower chlorinated congeners,

whereas there was no apparent elimination of the more highly chlorinated congeners. In

15
human milk samples, levels of mono- and diortho congeners were signiﬁcantly decreased
over a four week postpartum interval, while the mean level of coplanar congeners
containing para- and meta-, but no ortho-chlorines did not change (Koopman-Esseboom
et al. 1994). Brown et al. (1991), in monitoring the levels and distributions of individual
congeners in the sera of capacitor workers with chronic PCB exposure, found that most
lower congeners were rapidly metabolized and eliminated. However, congeners with
high numbers of chlorine persisted and could be used as near permanent indicators of
exposure. In this same population, the observed half-lives ranged from 1.4 years for
trichloro-, monoorthobiphenyls, to 12.4 years for hexachloro-, diorthobiphenyls (Brown
et al. 1989).

Environmental dechlorination of PCB is slow at best. This is due in part to the
fact that there are no known environmental chemical agents that have the negative
reduction potentials needed to reductively dechlorinate the toxin (Brown et al. 1987).
Bacteria can to some extent degrade PCB, but the ease of this process is inversely related
to the degree of chlorination, as with higher organisms (Kalmaz and Kalmaz 1979). In
recent years however, anaerobic microorganisms have been identiﬁed that can reductively
dechlorinate highly chlorinated congeners, and aerobic bacteria have been identiﬁed that

can oxidatively degrade more lightly chlorinated congeners (Abramowicz 1994).

16

Aroclor 1254

Aroclor 1254 is composed of a mixture of biphenyls containing an average of 4.96
chlorines per molecule (Hutzinger et al. 1974). It is a viscous liquid that contains 54%
(w/w) chlorine and is composed of 11% tetra-, 49% penta-, 34% hexa-, and 6%
heptachlorobiphenyls (Hutzinger et al. 1974; Linder et al. 1974). A-l254 was used in
electrical capacitors and transformers, vacuum pumps, hydraulic ﬂuids, adhesives, wax
extenders, dedusting agents, pesticide extenders, inks, lubricants, cutting oils, and as a
plasticizer in resins and in rubbers (Hutzinger et al. 1974). As with other commercial
PCB mixtures (James at al. 1993), polychlorinated dibenzofurans have been detected as a
contaminant in A-1254. Bowes (1975) detected the highly toxic contaminant in a
preparation of A-1254 at a level of 1.7 ppm. A-1254 has been shown to produce toxic
symptoms in non-human animal models (Earl et al. 1974; Merson and Kirkpatrick 1975;

Brezner et al. 1984; Kholkute et al. 1994b; Arnold et al. 1995).

PCBs : Experimental Evidence of Toxicity

Since the discovery of PCBs in the environment, the toxicity of the chemicals in
animal systems has been examined extensively. Virtually every animal studied is both
susceptible to contamination and shows a toxic response following exposure. These
responses are highly variable from species to species, and among tissues and organs

within an individual animal. Responses of young and old individuals within a species are

17
also variable, with the young generally experiencing greater toxicity, as PCBs impair
developmental processes.

This latter fact has serious implications, as young animals, both in utero and while
nursing, can face high levels of exposure to PCB. Many studies have shown that PCBs
are transferred from mother to young, either transplacentally to the fetus, or through milk
to the nursing infant. Maternal transfer of PCBs to neonates occurs in many species,
including, but not limited to, bats (Clark 1978), mice (Masuda et al. 1979), rhesus
monkeys (Allen et a1. 1980), rabbits (Seiler et al. 1994), and humans (Bailey et al. 1980;
Jacobson et al. 1984). Effects related to this passage of PCBs from mother to young,
include decreased egg hatchability in lake trout (Mac and Schwartz 1992), and in ring
doves (Peakall et al. 1972), and they have been correlated with premature births in
California sea lions (DeLong et al. 1973), and miscarriages in humans (Leoni et al. 1989).
Earl et al. (1974) found that feeding beagle bitches 5 ppm/day A-1254, from the day of
breeding, through parturition, caused decreased pregnancies and litter size at parturition
and at two weeks postpartum. There was also increased resorptions and teratogenic
abnormalities in these females. They also found the same response in pigs following oral
administration of A-1254, beginning 21 days prior to breeding. In rats, the number of live
pups per litter decreased following maternal exposure to A-l254 (Brezner et al. 1984).
Arnold et al. (1995) found that oral doses of A-l254 in the female rhesus monkey,
beginning 37 months prior to breeding, led to decreased conception rates, increased fetal
mortality, and decreased infant head size for a given back length. The researchers also

saw an increased incidence of nail lesions and gum recession in infants at 22 weeks

18

postpartum that had nursed with exposed females. In the human, infants born to women
in the lake Michigan region who had chronic PCB exposure due to ﬁsh consumption,
tended to be smaller in physical size, had reduced head circumferences and increased
startle reﬂexes (Swain 1988). Infants born to women with high occupational exposure to
A-1254, A-1242, and A-1016 have been shown to have decreased birth weights and
gestational lengths (Taylor et al. 1984). However, the decreased birth weights were likely
a result of the shortened gestation times. In another study, there was no relationship
between serum PCB levels and preterm births (Berkowitz et al. 1996). The cause of the
conﬂicting results in the above studies is not entirely clear, but may be a result of low
maternal exposure in the latter study (Berkowitz et al. 1996).

PCBs elicit their toxicity by inducing numerous enzymes, including the hepatic
and extrahepatic drug-metabolizing enzymes (Sanders et al. 1974; Safe et al. 1985).
Their action in the liver causes increased weight of the organ in mice (Merson and
Kirkpatrick 1975; Sanders and Kirkpatrick 1975), and rats (Linder et al. 1974). The
induction of these enzymes, as well as the hepatic microsomal enzymes, has been shown
to enhance the breakdown of steroid hormones. Sanders and Kirkpatrick (1975) found
that testosterone levels in A-1254 exposed males was reduced, as was the number of
sperm/mg testis. J onsson et al. (1976) found a reduction in plasma progesterone in
female rats following prolonged exposure to A-1242.

PCBs have been implicated to have mild estrogenic activity. Chronic exposure to
A-1254 or Clophen® A60 leads to increased estrous cycle lengths (Orberg et al. 1972;

Orberg and Kihlstrom 1973; Brezner et al. 1984). Gellert (1978) found that A-1221

19
signiﬁcantly advanced the age of vaginal opening in rats and induced persistent vaginal
estrus at a level of 10 ppm. In this study however, only a level of 1000 ppm A-1221
induced signiﬁcant uterine growth, and the weight was only half that of a uterus from an
animal given 1 ppb estradiol 175. Other studies have found no estrogenic activity
(J onsson et al. 1976; Gellert and Wilson 1979).

Aside from hormone-like activities, PCBs have the capability to induce more
acute effects in organ systems. These typically include thymic atrophy, immunotoxic
responses, reproductive problems, porphyria and related liver damage (Safe et a1. 1985).
They can also produce skin problems including hyperpigmentation, and chloracne
(Kuratsune et al. 1972). In vitro, PCBs induced aggregation of human platelets, and
increased the secretion of serotonin from these cells (Raulf and Konig 1991). They have
also been found to stimulate the production of inositol phosphates in polymorphonuclear
neutrophils, and they are capable of activating protein kinase C and altering calcium
homeostasis in rat cerebellar granule cells (Kodavanti et al. 1993; Kodavanti et al. 1994;
Tithof et al. 1995). It was shown that ortho-substituted congeners are most potent in
terms of activity in neuronal cells (Kodavanti et a1. 1995). Aroclor 1242 produced
complete reproductive failure in such sensitive species like the mink (Aulerich et al.
1973; Bleavins et al. 1980). Recently it was found that A-1242, A-1248 and A-1254 can
all induce uterine muscle contraction, which could potentially play a role in premature
births (Bae and Loch-Caruso 1996). Reproductive problems have also been shown to
occur in vitro. Huang and Chou (1994) found that in vitro fertilization of mouse oocytes,

taken from F1 females born to 3,3’,4,4’-TCB exposed females, was reduced. Kholkute et

20
al. (1994b) found that at levels of 0.1ug/ml and higher, A-1254 had adverse effects on
mouse IVF. These effects were produced only when the chemical was present in the IVF
media, or following a six hour incubation of oocytes in PCB-containing media. No
adverse effects occurred when spermatozoa were treated. This is cause for concern
because PCBs, along with many other industrial and pesticide pollutants, have been found
in human follicular ﬂuid, in which the oocyte develops and matures. In a Canadian study
of women undergoing in vitro fertilization, one group of women was found to have a
mean PCB level of 4.08 ppb in follicular ﬂuid (Jarrell et al. 1993). In two European
studies, PCBs have been found at levels of up to 142 ppb in Austrian women, and up to
27 ppb in German women (Trapp et al. 1984; Baukloh et al. 1985). Seiler et al. (1994)
found no reduction in fertilization rate in vivo in rabbits, following chronic oral exposure
to A-1260, though they found that the chemicals accumulate to a high degree in both pre-
and postimplantation embryos.

Human exposure to PCBs has been widespread, since the chemicals are both
present and persistent in the food chain, and the fact that humans are top consumers, but
there have been no deﬁnitive studies showing that adverse health effects are related to
non-occupational exposures (Swanson et al. 1995). Nor has a consensus been reached
about occupational exposures, but there have been adverse responses produced following
exposure in these settings (Swanson et al. 1995). With many of these studies, there are
confounding factors involved, such as multiple chemical exposures, especially in the
occupational setting (James et al. 1993). Historically, there have been few instances of

acute exposure involving large cohorts of people, unlike the large groups chronically

21

exposed by the situations discussed above. There have been two major poisoning
episodes however : the Yusho epidemic in Japan, and the Yu-Cheng epidemic in Taiwan,
both terms meaning oil disease.

The Yusho epidemic began in 1968, and affected 1,057 people (Kuratsune et al.
1972). The Yu-Cheng epidemic began in 1978 and affected over 2000 people (Lan et al.
1990; Guo et a1. 1995). Both poisonings occurred due to the consumption of PCB-
contaminated rice oil. There was also large consumption of PCDFs, which were created
as a result of heating the oil. Common symptoms included increased eye discharge,
swelling of the upper eyelids, chloracne, hyperpigmentation of the skin, and peripheral
neuropathy (Kuratsune et a1. 1972; Guo et al. 1995). A small sample of twelve babies
born to Yusho women included 2 stillborn children, as well as 9 who had unusually
gray/dark brown stained skin, ﬁve who had similar pigmentation of the gingiva and nails.
Most had eye discharge (Kuratsune et a1. 1972). Male school children affected with
Yusho had decreased gains in both weight and height, but this was not seen in girls.
Delayed development was also seen in children born 7-12 years after the Yu-Cheng

epidemic, who were born to exposed mothers (Guo et al. 1994).

PCBs : Carcinogenic Potential
Many PCB mixtures, especially those with low chlorination (<50%), along with
many individual congeners, have been seen to be minimally carcinogenic in many assay

systems, while higher chlorinated mixtures (>50%) have been shown to be

22

hepatocarcinogens in rodents (Safe 1989). A-1254 produced adenoﬁbrosis of the liver, as
well as hepatocarcinoma in mice, following chronic oral exposure (Kimbrough and
Linder 1974). Neoplastic lesions were produced in 170 of 184 livers, in rats chronically
exposed to A-126O (Kimbrough et al. 1975). Norback and Weltrnan (1985) found
hepatomegaly, neoplastic nodules on the liver, and hepatocarcinoma to be produced in the
rat following a long term feeding study of A-1260. The tumors produced did not
metastasize to distant organs, nor did they invade blood vessels. They also did not
increase mortality in the animals. A—1254 has also been shown to be a weak initiator in a
two-stage system of mouse skin tumorogenesis (DiGiovanni et al. 1977), and a promoter
of lung and liver tumors in the mouse (Anderson et al. 1994). Kerkvliet and Kimeldorf
(1977) found that A-1254 can also function as an inhibitor of tumor growth. They found
that A-1254, in a dose-dependent manner, inhibited the growth of tumors in rats
inoculated with the Walker 256 carcinoma.

Data involving PCB-induced carcinogenesis in the human is inconclusive, and is 1
for the most part correlative. Despite this, the Environmental Protection Agency
(U SEPA) has classiﬁed all PCB mixtures as probable carcinogens (V ater et al. 1995).
Moore et a1. (1994) claim that classifying all mixtures as having equivalent carcinogenic
potentials has no scientiﬁc foundation and should be reconsidered. After evaluating
seven studies in rats, involving PCB-induced liver tumorogenesis, they found that
mixtures containing 60% chlorine consistently produced a signiﬁcant increase of tumors,

while mixtures containing 42 or 54% chlorine did not increase tumorogenesis.

23

As stated above, most evidence of PCB-induced carcinogenesis in humans is
correlative, and has come primarily from studies of cohorts of electrical plant workers.
Occupational cohorts, however, represent small populations with unusually high
exposures (Ahlborg et al. 1995), and they often face exposure to many industrial solvents
concomitantly to PCBs, which can add confounding factors to these retrospective studies
(James et al. 1993). Nonetheless, many such studies have been undertaken in recent
years. In a cohort of workers from an Italian capacitor manufacturing plant, where PCB
mixtures containing 54 and 42% chlorine were used, the number of deaths due to cancer
were higher than expected, with the lymphatic tissue and GI tract being the most common
sites of tumorogenesis (Bertazzi et al. 1987). However, factors such as smoking history
and socioeconomic indicators were not taken into account, so the role of PCBs in cancer
initiation can neither be proved nor disproved. In a Canadian plant, where PCB-
containing ﬂuids were used in a small percentage of transformers that were produced, the
incidence of pancreatic cancer was higher than expected (Yassi et al. 1994). In this
cohort the exposure to mineral oil, which was reﬁned from predominantly naphthenic
base crudes, was much greater than their exposure to PCBs, so its role in carcinogenesis
can not be discounted. There have also been other studies suggesting a correlation
between PCB exposure and increased incidence of malignant melanoma, and cancer of
the brain (Bahn et a1. 1976; Sinks et al. 1992). Still more studies show no correlation
between PCB exposure and increased cancer incidence (Gustavsson et al. 1986).

There have also been studies in humans undertaken to ﬁnd a link between PCB

exposure and breast cancer. In one study, adipose tissue samples were taken from women

24
with mammary carcinoma as well as women with benign disease (F alck et a1. 1992).
Mean levels of both PCB and p,p’-DDE were 50-60% higher in the tissues of women
with breast cancer. Using logistic regression modeling, it was shown that PCB level was
signiﬁcantly correlated with breast cancer in all models, while p,p’-DDE was signiﬁcant
only when smoking was not included in the model. In another study, adipose tissue was
sampled from deceased women with or without breast cancer, at autopsy, and also from
living women with other breast disorders (U nger et al. 1984). The results showed no

correlation between PCB levels and breast cancer.

Fertilization Events

Every day a species speciﬁc number of primordial follicles begin growth to the
primary follicle stage. Follicle stimulating hormone (FSH), released ﬁ'om the pituitary
gland, plays a supportive role in the continued growth and development of these follicles.
Under continued FSH stimulation, folliculogenesis begins, enclosing the oocyte in layer
upon layer of granulosa cells. At the level of the tertiary follicle the ﬂuid ﬁlled antrum
has formed, and follicular cells have completed differentiation into an inner layer of
granulosa, and an outer layer of thecal cells, separated by the membrana granulosa.
Through the process of development, less dominant oocytes in the cohort, which are not
destined for ovulation, undergo atresia and are lost. The dominant follicle, or follicles in
the case of multiple ovulators such as the mouse, continue to grow, and produce estrogen,

until the luteinizing hormone (LH) surge occurs and stimulates ovulation. This surge also

25

stimulates the maturation of oocytes. Oocyte maturation is evidenced by germinal vesicle
breakdown, and involves the progression of meiosis from prophase I to metaphase II, the
extrusion of the ﬁrst polar body, and accompanying cytoplasmic changes (Eppig 1993).
Cytoplasmic maturation prepares the oocyte for activation, formation of pronuclei, and
preimplantation development (Eppig and O’Brien 1995).

Prior to ovulation, during the growth of the immature oocyte, the zona pellucida
(ZP) is produced (Dean 1992). The ZP is a thick extracellular coating which surrounds
the plasma membrane in mammalian eggs (Wassarman and Mortillo 1991). It is
responsible for the species-speciﬁcity of sperm binding, as well as the establishment of
the slow block to polyspenny following gamete fusion. It also protects the
preimplantation embryo as it travels through the oviduct (Wassarman and Mortillo 1991;
Dean 1992). The ZP is composed primarily of three proteins, ZPl, ZP2 and ZP3, which
represent 36%, 47%, and 17% respectively, of the total protein content (Bleil and
Wassarman 1980; Wassarman and Mortillo 1991). At the peak of production, ZP protein
synthesis represents 7-8% of the total protein produced in the oocyte, and this drops to
zero by the time of ovulation (Dean 1992). The principle function of ZPl is to maintain
the structural integrity of the ZP, whereas ZP2, and ZP3 are involved in gamete
interaction (Saling 1991). During oocyte maturation, the ZP also undergoes maturational
changes, which include disaggregation in the texture of ZP proteins, which are necessary
for sperm to penetrate the ZP (Tesarik et al. 1988; Rufas and Shalgi 1990; Henkel et al.

1995).

26

Fertilization begins when capacitated sperm contact the ZP. The ZP3 protein is
the primary sperm receptor, and both binds the sperm and induces the acrosome reaction
(Florrnan and Storey 1982; Bleil and Wassarman. 1983; Shalgi et al. 1989; Wassarman
and Mortillo 1991). For fertilization to be successﬁrl, the acrosome reaction must occur
at the surface of the ZP (Saling 1991). Following induction of the acrosome reaction,
ZP3 releases the spermatozoa, which then binds to ZP2, the secondary sperm receptor
(Wassarman and Mortillo 1991). After binding to ZP2, the acrosome-reacted
spermatozoa can penetrate through the ZP and enter the perivitelline space, where it then
fuses with the plasma membrane. Here, the fusion of the gametes stimulates transient
calcium increases, which trigger, among other things, the cortical reaction (Cuthbertson
and Cobbold 1985). The cortical reaction entails the migration of the cortical granules
(CG) to, and their ﬁrsion with, the plasma membrane of the oocyte (Wassarman and
Mortillo 1991; Dean 1992). CG exudate, which includes proteinases and glycosidases, is
released into the perivitelline space, and causes the alteration of ZP2 and ZP3, to ZP2f
and ZP3f respectively (Barros and Yanagimachi 1971; Bleil et al. 1981; Kalab et al. 1991;
Dean 1992). It is believed that this is due to protein cross linking via disulﬁde bonds
(Zhang et al. 1991). After alteration, the spermatazoa receptors are inactive, and the
essential block to polyspermy has been created, in a process termed the zona reaction
(Schroeder et a1. 1990). ZP “hardening”, the result of the zona reaction, can also occur
spontaneously during in vitro maturation in serum-free medium (Downs et al. 1986; Choi
et al. 1987; Fujii et al. 1990; Kalab et al. 1991), as CG exocytosis occurs to a small extent

during oocyte maturation (Ducibella et al. 1990), or due to oocyte aging (DeFelici and

27

Siracusa 1982; Dodson et al. 1989; Schiewe et al. 1995). ZP “hardening” manifests itself
by creating both a loss of oocyte fertility (Gianfortoni and Gulyas 1985), and resistance to
proteases (DeFelici and Siracusa 1982). It has recently been implicated as a cause of
reduced success in human in vitro fertilization (IV F) programs (Ducibella et al. 1995).

Fusion of the gametes not only stimulates the cortical reaction. It also stimulates
activation of the oocyte, which entails the resumption and completion of meiosis, as well
as the extrusion of the second polar body (Kurasawa et al. 1989). At this point, the
spermatozoa enters the cytoplasm of the now haploid egg, and releases its’ genetic
complement. The two haploid sets of chromosomes then join to form the ﬁrst

pronucleus, and mitosis, along with embryonic cell division, begins.

Materials and Methods

Animals

Female C57BL/6J, and male DBA/ZJ mice were obtained from the Jackson
Laboratory (Bar Harbor, ME). All animals were housed in a 12 hour light : dark
photoperiod at 23 j; 2 °C. Food and water were available ad lib. All females were at least

8 weeks of age and males were at least 12 weeks of age at the time of usage.

Aroclor 1254

A-1254 was purchased from Accustandard, Inc. (Lot #085-021, New Haven, CT)
in neat form, dissolved in methyl alcohol. It was diluted in culture media to attain ﬁnal
concentrations of 1.0 and 10.0 jig/ml. These concentrations were chosen as they were

previously reported to produce adverse effects in an in vitro fertilization system

(Kholkute et al. 1994b).

Superovulation

Female mice were injected i.p. with 10 I.U. pregnant mares serum gonadotropin
(PMS, Cat. #G-4877, Sigma Chemical Co., St. Louis, MO) between 4 and 5 PM. Forty

eight hours later they were injected i.p. with 10 I.U. human chorionic gonadotropin (hCG,

28

29
Cat. #C-5297, Sigma Chemical Co.). Cumulus-enclosed oocytes were collected 17-19

hours following the hCG injection, as described below.

In Vitro Fertilization (I VF)

Brinster’s medium for oocyte culture with 0.5% BSA, (BMOC-3, Cat. #11126-
034, Gibco BRL, Grand Island, NY) was used for all sperm and oocyte collection and for
IV F (Kholkute et al. 1994b). One milliliter of BMOC-3 was used in the inner wells of
Falcon organ tissue culture dishes (Cat. #3037, Becton-Dickinson and Co., Lincoln Park,
NY), and 3 ml were used in the outer wells. Media was prepared on the night before each
trial and was equilibrated overnight in a humidiﬁed incubator under an atmosphere of 5%
C02 in air at 37°C.

Females were superovulated as above. On the morning of the trial, one male was
sacriﬁced by cervical dislocation. Two more males were sacriﬁced over the course of the
experiment, 1-1 .5 hours prior to use. The cauda epididymides were excised and placed 1
into BMOC-3, where they were repeatedly punctured with a 25 gauge needle to release
spermatozoa. The dish was returned to the incubator for O.75-1.5 hours, to allow
capacitation to occur. Approximately 30 minutes following sperm collection, 8-10
superovulated females were sacriﬁced by cervical dislocation. Their ovaries, oviducts
and uteri were removed and placed into BMOC-3. Under a dissecting microscope, the
cumulus masses were collected by teasing them from the swollen oviducts, and they were

dispersed in 2mg/ml hyaluronidase (Cat. #H-23 76, Sigma Chemical Co.). The cumulus-

30

free oocytes were rinsed and then randomly distributed to the inner well of one of the
seven experimental group dishes. The ﬁrst group was the experimental control, in which
oocytes were neither aged, nor exposed to PCB prior to their insemination with ﬁfty to
seventy ﬁve microliters of sperm suspension (~2-3 million sperm). There was a set of
three groups that were incubated for three hours prior to insemination. These oocytes
were exposed to either 0, 1.0 or 10.0 ug/ml A-1254 in BMOC-3. The other set of three
was identical except that the oocytes were incubated for six hours prior to insemination.
Each group had between 8 and 37 oocytes.

Twenty four hours after insemination, the oocytes were analyzed for fertilization.
This was done visually under a dissecting microscope, and an oocyte was presumed to be
fertilized if either two polar bodies were present, or if it had developed to the 2-cell stage

(Kholkute et al. 1994b).

Zona Pellucida Hardening Assay

Following fertilization, and the subsequent cortical reaction, enzymes released
into the perivitelline space alter the zona pellucida (ZP) proteins, ZP3 and ZP2, during the
zona reaction. These proteins are responsible for primary sperm binding and the
induction of the acrosome reaction, and secondary sperm binding respectively, and they
are converted to ZP3f and ZPZf during this reaction (Schroeder et a1. 1990; Wassarman
and Mortillo 1991). This phenomenon inactivates these proteins, creating a block to

polyspermy. The zona reaction can also occur prematurely, during in vitro maturation in

31

serum-free medium (Schroeder et al. 1988), or as a result of oocyte aging (Gianfortoni
and Gulyas 1985). ZP “hardening”, a term used to describe the result of the zona
reaction, decreases fertilizability of unfertilized oocytes (Schroeder et al. 1990), and
creates resistance to dissolution of the ZP by proteolytic enzymes such as a-chymotrypsin
(DeFelici and Siracusa 1982). The present set of experiments were designed to detennine
whether a three or six hour exposure to A-1254 could induce ZP hardening, revealed by
resistance of the ZP to proteolytic enzyme-induced dissolution, and if that was
responsible for the reduced fertility of the oocytes in vitro.

The following protocol was identical for both the 3 and 6 hour exposure trials.
Eight to ten female mice per trial were superovulated as above. On the morning
following the hCG injection, females were sacriﬁced by cervical dislocation, and their
reproductive tracts were removed. Cumulus masses were collected directly into
Dulbecco’s phosphate buffered saline (PBS, Cat. #14280-036, Gibco) at pH 7.8, which,
along with all media used in these experiments, had been equilibrated ovemight in a
humidiﬁed incubator at 37°C under an atmosphere of 5% C02 in air. Following
collection, cumulus masses were dispersed in 2mg/ml hyaluronidase in PBS. The
cumulus-free oocytes were rinsed in PBS and transferred to one of the three 3 or 6 hour
groups (the 1.0ug/ml A-1254 group, the 10.0ug/ml A-1254 group, or the control group)
all of which contained 3mg/ml bovine serum albumin (BSA, Cat. #A- 2153, Sigma
Chemical Co.) in PBS, or directly to a 200111 droplet of 2mg/ml a-chymotrypsin (Cat. #C-
7762, Sigma Chemical Co.) in PBS. This ﬁnal group, which was unaged, was the

experimental control and was used to determine if effects seen were due solely to aging.

32

Following the 3 or 6 hour incubation period, oocytes were rinsed twice in PBS and then
transferred to 200 pl droplets (5-15 oocytes/droplet) of 2mg/ml or-chymotrypsin in PBS.

Once transferred to the a-chymotrypsin dr0plets, oocytes were assayed for the loss
of their ZP. They were checked every ﬁve minutes under a dissecting microscope for the
duration of the trial. ZP were considered solubilized when they had completely
disappeared and the oocyte adhered to the bottom of the dish (Downs et al. 1986). Dishes
were kept on a stage warmer at 37°C between examinations for ZP solubilization. The
time required for 50% of the ZP to become solubilized, the Lysisso, was used for
comparison among groups (Gianfortoni and Gulyas 1985; Downs et al. 1986; Shroeder et

al. 1990; Zhang et al. 1991).

Statistical Methods

The mean percentages of fertilization in the IVF trials were compared using
AN OVA, and pairwise comparisons among groups were made using Tukey’s test. The
Lysisso of each group in the ZP hardening assay, was compared using the Kruskal-Wallis
t sample test, which is a nonparametric rank-based test for use with data that are not

normally distributed (Freund and Wilson 1993).

Results

It was previously shown that following a 6 hour incubation of cumulus-enclosed
oocytes, in the presence of 0.1, 1.0 or 10.0ug/ml A-1254, IV F was signiﬁcantly reduced
(Kholkute et al. 1994b). It was also shown that lowered fertilization rates were due solely
to effects incurred by the oocyte, but that the viability of the oocytes was not
compromised. The present study was undertaken to examine the functional effects of
PCBs on mouse oocytes.

I chose to look at ZP hardening in cumulus-free oocytes cultured in serum-free
medium, and hypothesized that PCBs induce the cortical reaction and subsequent ZP
hardening. If this was true then the decreased fertilization rates seen previously could
have been due to a failure of sperm to penetrate the ZP. Studies from other cell systems
would seem to support this hypothesis : PCBs induce the formation of inositol phosphates
in polymorphonuclear neutrophils, and both elevate intracellular calcium levels and
activate protein kinase C in rat cerebellar granule cells (Kodavanti et al. 1993; Kodavanti
et al. 1994; Tithof et al. 1995). All three of these processes occur naturally in the oocyte
following gamete fusion, and are involved in the calcium-dependent signal transduction
pathway that leads to oocyte activation and CG exocytosis (Kurasawa et al. 1989; Bement
1992). If PCBs act similarly in oocytes, as they do in these other cell types, then one
would expect to see a high degree of ZP hardening in PCB-exposed oocytes. As shown
in Table 1 this was not the case. Oocytes were aged for either 3 or 6 hours, following

collection, in the presence of various concentrations (0.0, 1.0 or 10.0ug/ml) of A-1254.

33

34
These dose levels were chosen because they were shown to produce maximal reduction of
IV F (Kholkute et al. 1994b). The values given are the mean Lysissos for eight trials. This
value represents the time required to dissolve 50% of the ZPs in a group by or-
chymotrypsin. Larger values indicate greater resistance to dissolution, which is an artifact
of ZP hardening. Following three hours of incubation, A-1254 had no effect on ZP
hardening. Although slightly lower, hardening in the PCB groups did not differ from the
aged control (p>0.05). Both the 10.0ug/ml A-1254, and the aged control groups were
signiﬁcantly different from the unaged control (p<0.05), while the 1.0ug/ml A-1254
group approached signiﬁcance. Increased resistance to ZP dissolution was gained in the
aged control group due to limited CG exocytosis, which occurs spontaneously during in
vitro culture in denuded oocytes (De F elici and Siracusa 1982). These results suggest that
A-1254 has no effect on the cortical reaction.

The results from the 6 hour groups are also shown in Table 1. The Lysisso for the
aged control was the same as for the 3 hour group, indicating that spontaneous CG
exocytosis was completed by three hours of incubation. The Lysissos for the two A-1254
groups however, were reduced below the level of the unaged control, and were both
signiﬁcantly lower than that from the aged control (p<0.05). This result is unexpected
because the cortical reaction had already triggered ZP hardening by three hours of
exposure to A-1254, as evidenced by the increase in the Lysisso for the 3 hour groups
(Table 1), in a nonreversible reaction.

The kinetics of ZP dissolution by or-chymotrypsin are shown in Figures 1 and 2.

Figure 1 shows the curves for the 3 hour groups and illustrates that dissolution occurred

35
linearly over time. The rate of dissolution was fastest for the unaged control group. For
the aged control, the rate of dissolution was rapid in the beginning, and slowed over time.
Figure 2 shows that following 6 hours of incubation, dissolution also occurred linearly. It
also shows that the rate of ZP dissolution was increased in the A-l254 groups, over both
the unaged control, and the 3 hour A-1254 groups. It also shows that the rate of ZP
dissolution for the aged control slowed greatly following 6 hours of aging.

To further clarify what was occurring, I chose to do a 1 hour aging trial. This
would allow discrimination between the situation where A-1254 greatly increased ZP
hardening in the short term, or had no effect at all on this phenomenon. The results of
this trial are shown if Figure 3. The data in the graph is the ZP hardening relative to the
unaged control, and was determined using the following equation, which was adapted

from Gianfortoni and Gulyas (1985) :

ZP Hardening (min) = Lysisso at time x - Lysisso at time 0.

A-1254 had no effect on ZP hardening following 1 hour of exposure time. The Lysisso
for the 1.0ug/m1 A-1254 group did not differ from the unaged control, and that for the
10.0ug/ml A-1254 was only slightly elevated. For the 1 hour aged control however, ZP
hardening was evident. This shows that spontaneous cortical granule exocytosis begins
early during in vitro culture. The data also suggest that A-1254 slows the rate of
spontaneous cortical granule exocytosis, as it does not begin to occur until some time

after one hour of culture.

36

The results for the IV F trials are shown in Table 2. IV F trials were carried out to
evaluate the relationship between ZP hardening and oocyte fertilizability. Kholkute et al.
(1994b) previously reported that A-1254 had adverse effects on IVF, using cumulus-
enclosed oocytes aged for 6 hours prior to insemination. I felt it necessary to use
cumulus-free oocytes, in order to determine what effect cumulus cells had on PCB
toxicity in this system. Cumulus-free oocytes were aged for 3 or 6 hours, either in the
presence or absence of 1.0 or 10.0ug/ml A-1254, as above, and were then inseminated.
In contrast to Kholkute et al. (1994b), there were no differences in fertilization rates
between groups (p>0.8). This indicates that PCBs may elicit their toxicity through a
process involving the cumulus cells. Table 2 also shows that the percentage of
degenerated oocytes was elevated in the unaged control group. Degeneration in the 6
hour A-1254 groups was signiﬁcantly lower than in the unaged group (p<0.05). This also
differs from Kholkute et al. (1994a) who found that the percentage of degenerate oocytes

increased with increasing A-1254.

Discussion

ZP hardening trials. In this study I examined the effects of a commercial PCB
mixture, Aroclor 1254, on ZP hardening in cumulus-ﬂee mouse oocytes. The results
suggest that A-1254 does not directly inﬂuence CG exocytosis-induced ZP hardening. At
1 hour, the Lysisso for the PCB groups was not elevated, while for the aged control it was.
If A-1254 had a stimulatory effect on CG exocytosis than one would expect an increase in
ZP hardening at one hour. These results do suggest, however, that A-1254 was inhibitory
on spontaneous CG exocytosis at 1 hour, compared to the aged control where it began
immediately. Spontaneous CG exocytosis did occur in the PCB groups by three hours, as
shown by the increase in ZP hardening. This increase occurred to a lesser extent than in
the aged control. If A-1254 had a complete inhibitory effect on CG exocytosis, one
would not expect this increase at three hours.

The decrease in ZP hardening in the A-1254 groups at 6 hours, to below that for
the unaged control, gives further evidence that the toxin does not directly affect CG
exocytosis. CG exocytosis and the subsequent zona reaction are not reversible, so rather
than inﬂuencing either of these reactions, the results suggest that A-1254 was acting
directly upon the ZP to drop the Lysissos in these groups. Zhang et al. (1991) found that
the addition of B-mercaptoethanol, a reducing agent, to serum-free medium during in
vitro oocyte maturation, prevented ZP hardening. They suggested that ZP hardening
results from protein cross-linking by disulﬁde bonds. Cross-linking of the glycoproteins,

which undergo limited proteolytic cleavage during the zona reaction (Wassarman and

37

38
Mortillo 1991), results in conformational changes of the ZP such that ZPl becomes
shielded from the extracellular environment. ZPI acts as a ﬁlament crosslinker,
maintaining the integrity of the ZP by interconnecting ZP2 and ZP3 (Greve and
Wassarman 1985). It is also preferentially cleaved by a-chymotrypsin, resulting in the
loss of interconnections between ﬁlaments and dissolution of the ZP (Greve and
Wassarman 1985). It would appear that over time in culture, A-1254, by some unknown
mechanism, alters the conformation of the modiﬁed ZP, making ZPl more available to a-
chymotrypsin, and thus decreases the resistance of the ZP to dissolution. If ZP hardening
does in fact result from protein cross-linking by disulﬁde bonds, then A-1254 could act
by disrupting these bonds. Bond disruption and the alteration of membrane structural
integrity represents a novel mechanism of action of PCBs.

The fact that A-1254 does not stimulate CG exocytosis is surprising, based on the
activity of PCBs and similarly acting chemicals in other cellular systems. In rat cerebellar
granule cells, it was shown that PCBs alter intracellular calcium homeostasis (Kodavanti
et al. 1993). Following ten minutes of exposure to PCBs, intracellular calcium levels
were increased, and it was found that calcium uptake by mitochondria and other
organelles was inhibited, as was synaptosomal Ca2+-ATPase which is involved in calcium
extrusion from the cytoplasm. This resulted in a sustained rise, rather than a natural
transient rise, in intracellular calcium concentrations due to the inhibition of the cellular
buffering systems. A rise in intracellular calcium initiates a second messenger cascade,
culminating with the activation of protein kinase C (PKC). Activation of PKC has been

seen following PCB exposure in rat cerebellar granule cells (Kodavanti et al. 1994;

39

Kodavanti et a1. 1995), as well as in brain extracts from both rats and mice (Shukla and
Albro 1987). In the oocyte, PKC acts as a mediator of calcium-induced CG exocytosis,
by phosphorylating cellular substrates that are involved in the regulation of exocytosis
(Bement 1992). Compounds such as 4B-phorbol 12,13-didecanoate (4B-PDD), and 12-0-
tetradecanoyl phorbol 13-acetate (TPA), which are biologically active phorbol esters; and
sn-1,2-dioctanoy1 glycerol ((1ng), a diacylglycerol, are PKC activators in the mouse
oocyte and induce both CG exocytosis and ZP modiﬁcations (Endo et al. 1987; Ducibella
et al. 1993).

Others have reported activities of PCBs that would further suggest their role in
CG exocytosis in the oocyte. Tithof et al. (1995) found that in polymorphonuclear
neutrophils, A-1242 stimulated the production of inositol phosphates, among them
inositol 1,4,5-trisphosphate (1P3). 1P3 both stimulates calcium release from intracellular
stores, and activates PKC. It has been shown to initiate CG exocytosis and oocyte
activation (Cran et al. 1988; Ducibella et al. 1993). Intermediate weight Aroclor mixtures
(1248, 1254, and 1260) were shown to enhance high affinity binding of ryanodine to
ryanodine-sensitive calcium release channels, in the membrane of the sarcoplasmic
reticulum in both skeletal, and cardiac muscle cells (Wong and Pessah 1996). The PCB
mixtures were also shown to decrease the potency of inhibition of calcium release, by
calcium, through negative feedback. Ryanodine receptors have been located in both
mouse and bovine oocytes, and microinjection of ryanodine into mouse oocytes
stimulates CG exocytosis and ZP modiﬁcation, though to a lesser extent than 1P3 (Ayabe

et al. 1995; White and Yue 1996). The fact that PCBs can induce 1P3 production, and can

4O
enhance ryanodine binding to calcium-sensitive release channels, further argues for their
role in stimulating CG exocytosis.

There are several potential reasons to explain why PCBs do not induce CG
exocytosis, in spite of the evidence presented above which suggests otherwise. The ﬁrst
is simply that PCBs may act differently among different cell types. Differences in the
way that cells respond to toxic insult can lead to alterations in the way that PCBs behave.
In rat cerebellar granule cells, only ortho-substituted congeners, which are unable to
assume a coplanar conﬁguration, are capable of inducing PKC activity (Kodavanti et al.
1995). The same is true in polymorphonuclear neutrophils, with regards to inositol
phosphate production (Tithof et al. 1995), and in skeletal and cardiac muscle, with
regards to stimulating high afﬁnity ryanodine binding (Wong and Pessah 1996).
However, 2,3,7,8-tetrachlorodibenzo-p—dioxin (TCDD), a coplanar compound and
structural analogue to PCB, was found to stimulate PKC activity in plasma membrane
preparations from rat livers (Bombick et al. 1985).

Species-speciﬁc differences with regards to PCB behavior, have also been
demonstrated. Identical congeners were found to produce different ﬁrnctional responses
of PKC from rat and mouse brain extracts (Shukla and Albro 1987). In mice, PCBs
caused increased binding of phorbol esters, an indicator of PKC activity, but decreased
histone phosphorylation. In rats, PCBs caused decreased phorbol ester binding, but
increased histone phosphorylation. Species-speciﬁc differences in the behavior of other
PKC activators have been demonstrated. In murine oocytes, TPA was able to induce

transient calcium oscillations, which occur following gamete fusion, and sustain them for

41
up to three hours (Cuthbertson and Cobbold 1985). However, in the hamster oocyte,
calcium oscillations induced by guanosine-S'-0-(3-thiotriphosphate) (GTP[S]), were
inhibited by TPA (Swann et al. 1989). This led the researchers to suggest that there may
be species-speciﬁc differences in the mechanisms of control of the calcium-dependent
signal transduction pathway. All of this discussion serves to illustrate that behaviors seen
in one cell type are not necessarily produced in another cell type. The mouse oocyte
could be an example of this with regards to the induction of CG exocytosis.

Another reason why PCBs did not induce CG exocytosis may be a result of their
effects on calcium homeostasis. As discussed above, PCBs have been shown to induce
large and sustained increases in intracellular calcium levels (Kodavanti et al. 1993).
Following gamete fusion in mouse oocytes, transient oscillations in intracellular calcium
levels begin, and following the ﬁrst two transients, continue to occur at twenty minute
intervals for up to 4 hours (Cuthbertson and Cobbold 1985). These transient calcium
oscillations are essential for activation events, including meiotic resumption, and the
extrusion of the second polar body (Kline and Kline 1992). It has been shown that TPA
can induce similar calcium oscillations in mouse oocytes and can parthenogenetically
activate them (Cuthbertson and Cobbold 1985). Ethanol, dirnethylsulphoxide, and
calcium ionophore (A23 187) can induce CG exocytosis and ZP hardening (Gulyas and
Yuan 1985; Ducibella et al. 1988; Vincent et al. 1990). While these agents do not induce
calcium oscillations, they do induce a single transient rise in intracellular calcium levels
(Cuthbertson et al. 1981; Eusebi and Siracusa 1985; Miyazaki 1991). If PCBs do act in

the oocyte, as they do in rat cerebellar granule cells, and cause an increase in intracellular

42

calcium levels in a nontransient manner, then CG exocytosis would potentially be
inhibited. This could occur due to a desensitization, or inhibition of the enzymes
involved in the calcium dependent signal transduction pathway, or by uncoupling signals
between members in the pathway. Whatever the mechanism may be, a nontransient rise
in intracellular calcium could lead to a failure in the initiation of activation.

The lack of cumulus cells in this study could also explain why A-1254 did not
induce CG exocytosis. PCB-induced toxicity could potentially be mediated by a
mechanism requiring the oocyte to be cumulus-enclosed. This possibility will be

discussed below.

I VF trials. The IVF results that I obtained were different from those previously
reported (Kholkute et al. 1994b). In that study it was shown that A-1254 signiﬁcantly
decreased IVF in cumulus-enclosed oocytes. A-1254 had an inhibitory effect when
present in the IVF media, and also following a six hour incubation period of cumulus
masses in its presence. It was also shown that A-1254 increased the percentage of
degenerate oocytes (Kholkute et al. 1994a). Neither of these effects were replicated in
this study using cumulus-free oocytes. Compared to the control group, fertilization was
not affected at either 1.0 or 10.0ug/ml A-l254 dose level, nor was it affected by duration
of incubation prior to insemination. While the percentage of fertilized oocytes in this
study was lower than that reported by Kholkute et al. (1994b) (49.6% vs. 79.8%
respectively), it was not different from values reported elsewhere for cumulus-free

oocytes (Saeki et al. 1994). The percentage of degenerate oocytes was also unaffected by

43
A-1254 in this study. In fact, the percentage of degenerate oocytes in the unaged control
group was the highest, and was signiﬁcantly higher than both of the 6 hour A-1254
groups. The reason for this increase in degenerate oocytes in the unaged control is
unknown.

The fact that A-1254 did not affect IVF in cumulus-free oocytes gives further
support for the notion that it did not induce CG exocytosis and subsequent ZP hardening.
If it had, than fertilization should have been decreased in the treatment groups, due to a
failure of sperm to penetrate the ZP. The results also suggest that A-1254 did not induce
a rise in intracellular calcium. If it had induced this rise, than the gamete fusion-induced
calcium oscillations would not have been able to trigger activation events, and
fertilization would not have occurred. The same fertilization percentages could have been
obtained if the oocyte had become desensitized to PCBs with regards to inhibition of the
calcium buffer systems, and became able to lower intracellular calcium levels. Fusion-
induced calcium oscillations would have then been able to trigger activation. The same I
percentages could have also been obtained if it is assumed that what is important in
triggering activation is the attainment of some threshold calcium level, rather than a
relative change in intracellular calcium concentration. If the PCB-induced calcium rise
was below the threshold value, than gamete fusion could potentially elevate calcium

beyond the threshold level, and activation would occur.

The potential role of cumulus cells in PCB toxicity. Based on the results from

both IVF and the ZP hardening assay I suggest a role for the cumulus cells in PCB-

44
induced toxicity in the oocyte. This suggestion is made due to the fact that A-1254
neither stimulated nor inhibited CG exocytosis, nor did it lower IV F in cumulus-free
oocytes. I did not examine the effects of A-1254 on ZP hardening in cumulus-enclosed
oocytes, though it seems warranted, and should be examined in the future. The effects of
A-1254 on IVF in cumulus-enclosed oocytes have been examined, and the results
demonstrated adverse effects on fertilization.

There are several mechanisms by which cumulus cells could be involved in the
mediation of PCB-induced toxicity. The ﬁrst is that they could aid in PCB accumulation
within the oocyte. PCBs would enter the cumulus cells with ease, then pass into the
oocyte through gap junctions. This is unlikely, as the ZP is a highly porous membrane,
allowing passage of even small viruses (Gwatkin 1967), and due its lipophilicity, PCB
should ﬁnd ready access into the oocyte. Another possibility is that the effects of PCBs
are elicited by a second messenger, the production of which is stimulated in the cumulus
cell, which is passed to the oocyte through gap junctions. This messenger could then
produce toxicity. Conversely, PCB could inhibit the production and transfer of a factor
required by the oocyte, and the lack of this factor would be the cause of toxicity.

The regulation of PKC represents another potential site for cumulus cell mediated
effects. Colonna et al. (1989) found that a 74-kDa phosphorylated protein, the function of
which is unknown, was present only in granulosa cell-enclosed oocytes. They proposed
that a signal was sent to the oocyte from the granulosa cells, and that this signal regulated
protein phosphorylation by PKC. It is possible that PCBs could block this signal, thereby

inhibiting PKC activity, and therefore CG exocytosis, and could potentially cause an

45
excess of aneuploidy, as the slow block to polyspermy would not be created. This could
explain the results in Kholkute et al. (1994a), in which it was found that A-1254 caused
an increase in the percentage of degenerate oocytes. The potential for PCBs to inhibit
intercellular communication by gap junctions has been demonstrated in rat liver cells
(Hemming et al. 1991; Krutovskikh et al. 1995). The loss of intercellular communication
between the oocyte and granulosa cells, would then be responsible for the adverse effects
on IV F. While the oocyte does not require signaling ﬁom cumulus cells, it does play a
large role in oocyte functionality, as shown by a reduction in fertilization by 30% in
comparison to Kholkute et al. (1994b). Similar reductions in fertilization, between
cumulus-enclosed and cumulus-free oocytes, have also been reported elsewhere (Saeki et
al. 1994). PKC activity in the oocyte is still functional without cumulus-signaling, since
fertilization can occur, but the lack of signaling may cause the oocyte to be less
responsive to fertilization stimuli, as well as to toxins such as PCB.

The loss of communication between the cumulus cells and the oocyte, could also
be deleterious in and of itself. It is unknown whether it is more harmful to the oocyte to
inhibit communication between itself and attached cumulus cells, than it is to remove
them. That is, in both cases cumulus signaling is absent, but it is unknown if the
maintenance of contact with the cumulus cell creates more harm. If it is, than PCBs
could be toxic to the oocyte simply by inhibiting communication.

There is one ﬁnal potential mechanism that needs to be addressed as it helps to
explain both the role of cumulus cells in mediating PCB toxicity, as well as some of the

results obtained in Kholkute et al. (1994b). In that study, it was shown that IVF was

46

decreased to a greater extent (20-25%) when A-1254 was present in the IVF medium,
than when cumulus-enclosed oocytes were incubated for 6 hours in its presence, and then
inseminated in PCB-free media. A possible explaination is that upon initial exposure of
cumulus-enclosed oocytes to A-1254, calcium levels are increased in the oocyte, due to
signals induced in the cumulus cells by A-1254. Inhibition of the oocytes’ buffering
system would also occur, and so the rise in calcium would be nontransient. Thus far, the
mechanism is in agreement with the activity of PCBs in other cell types (Kodovanti et al.
1993). When inseminated in the presence of A-1254, gamete fusion-induced calcium
oscillations would not be able to induce activation events, due to elevated intracellular
calcium levels. This would yield low fertilization rates, and a high percentage of
aneuploidy in these oocytes. With regard to the 6 hour incubation of cumulus masses, the
initial rise in calcium also occurs. However, over time through the inhibition of
intercellular communication, the inhibitory effect of A-1254 on the oocytes’ buffer
system would slowly cease. This would allow the intracellular calcium levels to slowly
return to normal, and this could be what is responsible for the 20-25% improvement in
fertilization rates following a 6 hour exposure prior to insemination.

While all of these mechanisms are speculative, they deserve attention. Examining
the effects of PCBs on the mammalian oocyte is important, as PCB exposure continues to
pose a threat to the health of all animals, and has the potential to create severe

reproductive and developmental problems.

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APPENDICES

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APPENDIX C

APPENDIX C

VITA
Name : Charles R. Greenfeld
Born : October 6, 1972
Birthplace : Baltimore, Maryland

Degrees Received : Bachelor of Science, Ecology and Evolution

University of Pittsburgh, 1994

Masters of Science, Zoology
Michigan State University, 1997

Publications by the Author

. C Greenfeld, O Hernandez, WR Dukelow. 1996. The reproductive toxicity of a
commercial PCB (Aroclor 1254) in the mouse. Annual Research Day, MSU
Department of Zoology. (Abstract).

. Dukelow WR, Greenfeld CR, Hernandez O, Clemens LG, Chung Yu-Wen, Kaneene
J B. 1996. Toxic chemical inﬂuences on in vivo and in vitro reproduction. Proc
Forum on Environmental Remediation and Environmental Toxicology. Lansing
MI. (Abstract).

. Dukelow WR, Hernandez O, Greenfeld CR, Kholkute SD. 1996. In vitro fertilization
assessment of PCB effects in B6D2-Fl mice. Proc Amer Assoc of Laboratory
Animal Science. p5. (Abstract).

. Yu-Wen Chung, LG Clemens, WR Dukelow, O Hernandez, CR Greenfeld, JB
Kaneene. 1997. Reproductive physiology and behavior effects of PCBs. Proc
10th Ann of Superfund, North Carolina, February, 1997. (Abstract).

. Greenfeld C, Hernandez O, Dukelow WR. 1997. Inhibition of zona pellucida
hardening in the mouse oocyte following prolonged exposure to a commercial
PCB (Aroclor 1254). Annual Research Day, MSU Department of Zoology.
(Abstract).

67

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