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thesis entitled

THE EFFECT OF AROCLOR-1254 ON THE
DEVELOPMENT OF 8 CELL MOUSE EMBRYOS

presented by

Oscar Hernendez Maldonado

has been accepted towards fulfillment
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Masters degree in Animal Science

 

 

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1/98 c/Clmw-pjd

THE EFFECT OF AROCLOR-1254 ON THE DEVELOPMENT OF
8 CELL MOUSE EMBRYOS

By

Oscar Hernandez Maldonado

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

MASTER OF SCIENCE

Department of Animal Science

1997

ABSTRACT

THE EFFECT OF AROCLOR-1254® ON THE DEVELOPMENT OF
8-CELL MOUSE EMBRYOS.

By

Oscar Hernandez Maldonado

Polychlorinated biphenyls (PCBs) accumulate in the environment from past human
activities, and continue to disperse globally even though their applications are restricted .
In the United States, Aroclor-1254, a commercial PCB mixture was among the most
widely used and more toxic than other higher chlorinated Aroclors. Exposure to PCBs
has caused adverse effects on reproduction in a wide range of laboratory animals. For this
investigation three different concentrations of Aroclor-1254 (0.1, 1.0, and 10 ul/mL) were
tested. Eight cell mouse embryos were recovered from superovulated C57BL/6J females
at approximately 57 hours. Observations were made every 24 hours after collection for up
to 96 hours. The results indicated a significant effect (P< 0.05) at the three concentrations
of Aroclor-1254 (A-l254), on the development of the 8-cell mouse embryo, at 48 hours
during the morula stage. Also, a significant effect was obtained at 72 hours (P< 0.05) at

the 1.0 ul/mL concentration of A-1254.

To my wife Brenda, my parents, and to my brothers and sisters in Pnerto Rico for

their unconditional, love, support and understanding now and always.

ACKNOWLEDGMENTS

My most sincere appreciation to my major professor, Dr. W. Richard Dukelow, who
took me as one of his graduate students. His guidance and advice will be useful at both
my professional and personal levels. I wish to thank Dr. Melvin Yokoyama, Dr. Lynwood
Clemens and Dr. Richard Aulerich for being part of my graduate committee.

Thanks to my good fiiends Manuel Jime'nez and Nayda Santiago for all their help and
advice during this time. A special thanks to Dr. Gregory Fink for his recommendations on
the statistical analysis of this investigation.

Last but not certainly least, thanks to the group at the Endocrine Research Center;
Bonnie Cleeves, Chuck Greenfeld, Dr. Steve Bursian and Debbie Powell, their help and
friendship always will be remember and appreciated.

This work was supported by NlEHS grant no. ES- 049] 1.

iv

TABLE OF CONTENTS

LIST OF TABLES ......................................................................................................... vi
INTRODUCTION .......................................................................................................... 1
LITERATURE REVIEW ................................................................................................ 3
Polychlorinated Biphenyls (PCBs) ................................................................................ 3
Mouse Development .................................................................................................... 6
Pre-Implantation Mouse Embryo Development ............................................................ 7
Development of the 8-Cell Mouse Embryo ................................................................. 10
MATERIALS AND METHODS ................................................................................... 12
Animals ...................................................................................................................... 12
Aroclor 1254 .............................................................................................................. 12
Culture Media ............................................................................................................ 12
Superovulation ........................................................................................................... l3
Embryo Collection and Culture .................................................................................. 13
Statistical Analysis ...................................................................................................... 14
RESULTS ..................................................................................................................... 15
DISCUSSION ............................................................................................................... 24
SUMMARY AND CONCLUSIONS ........................................... . ................................. 30
BIBLIOGRAPHY ......................................................................................................... 32
APPENDIX A: CURRICULUM VITA ......................................................................... 39

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

LIST OF TABLES

Estimated early cleavage time table. .................................................................. 8
Specific cell stage and time used for 8-ce11 embryo classification during
experiment ...................................................................................................... 17

Embryo development to compacted 8-cell during the first 24 hours of in vitro

culture ............................................................................................................ 18
Embryo development to morula stage at 48 hours of in vitro culture ................ 19
Embryo development to blastocyst stage at 72 hours of in vitro culture ........... 20
Embryo development to the hatching stage at 96 hours of in vitro culture ........ 21
Embryos degenerated embryos at 24 hours of in vitro culture .......................... 22
Embryos degenerated embryos at 48 hours of in vitro culture .......................... 23

vi

INTRODUCTION

Polychlorinated biphenyls (PCBs) were first synthesized in 1881 and commercial
production began in the United States in 1929. Since their first commercial production, an
estimated 1.2 million tons of PCBs have been produced worldwide (Tanabe, I988). The
properties of improved chemical stability, low solubility in water, low flammability, and
excellent insulating properties, provide many commercial benefits for the use of PCBs as
heat transfer fluids (e.g., in electrical transformers and capacitators), flame retardants,
lubricating and hydraulic oils, additives in plastic, and innumerable other applications
(Pomerantz et al., 1978). These extensive uses, together with their physical/chemical
properties, led to their extensive distribution in the environment in the late 19605.

Due to the widespread occurrence of PCBs in the ecosystem and their potential
adverse effects on animals, including humans, significant efforts have been invested by
government and industry throughout the world to understand how PCBs function and
what possible hazard could occur from their presence in the environment.

Reproduction is considered one of the most sensitive methods of testing compound
toxicity. Maternal consumption of PCB contaminated fish in humans has been reported to
cause reduced birth weight, diminished head circumference, and reduced gestational age
(Fein et al., 1984; Swain, 1991).

Exposure to PCBs has caused adverse efl‘ects on reproduction in a wide range of
laboratory animals. In rodents, these effects have included embryo and fetal toxicity,

decreased fertility in both males and females, and decreased survival of offspring (EPA,

2
al., 1987; Golub et al., 1991). Previous studies in this laboratory (Kholkute et al., 1994a)
demonstrated that Aroclor-1221, 1254 and 1268 adversely affected in vitro fertilization in mice,
Aroclor-1254 being the most toxic. The primary objective of this study was to investigate the

effects of Aroclor -1254 on the development of the 8-cell mouse embryo.

LITERATURE REVIEW

Polychlorinated Biphenyls (PCBs)

Polychlorinated biphenyls (PCBs) are among the most extensive and constant
environmental contaminants (Swain, 1983). These industrial chemicals were marketed
under trade names such as Aroclor, Clophen, and Phenclor. They were first described and
synthesized in 1881, with commercial production and application beginning in 1929. The
world’s major producer of PCBs was The Monsanto Company. They stopped production
of PCBs in 1971, but continued production for closed uses (e.g.; closed heat-transfer
systems, electrical transformers, condensers). In 1977, Monsanto stopped production
completely. Since then the sale and use of these chemicals in the United States have been
strictly controlled by federal law.

Commercial PCB products were mixtures of up to 209 congeners, with different
physical and chemical characteristics. In the United States, the major registered trade
name for commercial PCB mixtures was Aroclor. The name Aroclor was followed by a
four digit number. The first two digits referred to the carbon atoms of the biphenyl ring
and the last two digits referred to the percent chlorine by weight. Aroclor-1254 was
among the most widely used and more toxic than other higher chlorinated Aroclors, such
as A-1260, A-1262, and A-1268 (Kimbrough et al., 1978) .

PCBs are colorless, liquids or solids which are inflammable, electrically insulating, and
chemically stable, which accounts for their persistence in the environment. These

characteristics also made PCBs resistant to acid-base reactions, hydrolysis, chemical

oxidation, photodegradation and thermal changes, in addition to being extremely soluble in
oils and fat. Because of this, they tend to partition out of the aquatic ecosystem into
biological tissue.

Some of the uses for PCBs include; electrical transformers, capacitators, heat transfer
plasticizers, paints, printing inks, suspension vehicle for the pigment in carbonless copy
paper, sealants, adhesives, flame retardants, microscope immersion oil, and lubricants.

Human exposure to PCBs in the environment is widespread because these persistent
chemical mixtures have entered the food chain. Occupational and accidental exposures
have occurred due to the use of these chemicals in a variety of product applications and
manufacturing processes (Swanson et al., 1995). Food has been shown to be the most
important route of exposure for the general population, and milk products have been
shown to contribute significantly to the total exposure (Krokos et al., 1996).

PCBs were first detected in milk in 1970 (Willett & Hess, 1975). The principal
source of residues was farm silos in the eastern United States that had been coated with a
PCB-containing coating (Krokos et al., 1996; Willett, 1983). Similarly treated silos
have also resulted in milk contamination in Switzerland (DeAlencastro et al., 1984).
Since human milk is part of the food chain, breast-fed infants will consume the highest
concentration of persistent chemicals accumulating in the environment (Drijver, et al.,
1988). PCBs in human milk fat are correlated to the corresponding levels in depot fat.
Thus, supporting the view that comparisons of the levels of PCB contaminants in human
milk should be based on the milk fat content (Koidu, 1983).

In Michigan, predominantly an industrial state, environmental contamination by PCBs

as well as other toxic substances, has been a widely reported problem (Wickizer et al.,

1981). Reports of elevated levels of PCBs in fish in the Great Lakes (Willford et al.,
1976), lead to controls being placed on commercial fishing and to recommendations by
public health officials that persons reduce or eliminate their consumption of such fish.

Developmental effects attributed to PCB exposure include low birth weight and
retarded growth in mice (McCoy et al., 1995). Prenatal exposure to PCBs has been
linked to teratological effects such as cleft palate in the mouse and pig (Fuller & Hobson,
1986). Adverse human reproductive and developmental deficits have been linked to PCB
exposure from consumption of contaminated fish or by direct agricultural contact
(Hansen, 1994).

Subacute or chronic exposures to PCBs pre- or postnatally, pre- or postpubertally, as
adults, or during pregnancy, have been found to be detrimental to reproductive fiinctions
in laboratory and wild mammalian species (Fuller & Hobson, 1986). Measurable
concentrations have been detected in human ovarian tissue (Mes, 1990) and human
embryos and fetuses (Nishimura et al., 1977).

Chemically induced reproductive disorders have been documented in different
mammalian species including humans. Some of these effects have been attributed to PCBs
(Lindenau & Fischer, 1996). In mice and rats, PCBs have been reported to affect the
estrous cycle (Orberg & Kihlstrom, 1973) and uterotrophic activity (Gellert, I978).

Embryo transfer experiments showed that momlae exposed to Sng Aroclor-1260 had
clear signs of degeneration after 24 hours in vitro culture but were able to implant
(Lindenau & Fischer, 1996). One of the primary concerns, regarding PCB exposure in

mammals, is the deleterious effects on many phases of reproduction in both sexes (George

& George, 1990). Studies of in vitro fertilization in mice indicated detrimental effects of

PCBs on oocytes, fertilization, and early cleavage (Kholkute et al., 1994).

Mouse Development

The mouse is well established as an experimental mammal for developmental and
genetic studies (Hennig, 1992). Mouse embryonic development has been studied in detail
and has been well described (Theiler, I989; Rugh, 1990).

The estrous cycle is 4-5 days and the gestation period is 19-20 days, depending on the
strain. Mouse lifespan in the laboratory is 1.5-2.5 years. The average litter size is
approximately 6-8 pups. In order to maintain regular cycling, a day-night photoperiod
should be kept constant. Under this cycle females tend to ovulate once every four to five
days, 3-5 hours after the onset of the dark period (Hogan et al., 1996).

Males under the same lighting conditions will copulate with females in estrus.
Ovulation and fertilization are assumed to occur about 2:00 am, under our photoperiod
at about the mid-point of the dark period. The morning afler mating, the females can be
checked for the presence of a copulation plug in the vagina (vaginal plug) as an indication
of mating. This consists of coagulated proteins from the male seminal fluid and can easily
be seen.

Males reach maturity at 6-8 weeks of age depending on the strain. Once used for
mating, females they should be kept in individual cages, or they will fight. The sexual
maturity of females is a major factor affecting the number of oocytes that are ovulated.
The best age for superovulation usually falls between 3 and 5 weeks. Age, however, is
not always a reliable indicator of the sexual maturity of the female; the nutritional status

and health of a female mouse can also affect follicular maturation (Hogan et al. , 1986).

Pre-Implantation Mouse Embryo Development

Fertilization, the fusion of the female and male gametes, activates the oocyte to enter
the complex process of embryogenesis. Preimplantation development takes about 4-5
days in the mouse. Cleavage (cell division) occurs while the cells are still surrounded by
the zona pellucida, resulting in the production of many smaller cells, the blastomeres.

Cleavage takes place much more slowly in mammals than in most lower vertebrates or
invertebrates. Frog eggs cleave about once an hour, and goldfish eggs every 20 minutes ',
but a mouse egg takes 24 hours for its first cleavage division, and 10-12 hours for each
succeeding division. (Austin & Short, 1982). Table I (Rugh, 1990) indicates the time
and stage for the development of the pre-implantation mouse embryo.

During the first two days of development, blastomeres are spherical cells which are
loosely attached at their sites of contact. At the eight cell stage blastomeres alter their
adhesive behavior and the embryo undergoes a profound morphological change. The cells
flatten towards each other, maximize cell-cell contacts and the former grape-like structure
is transformed into a compact aggregate of cells. This phenomenon is called compaction
and is dependent on the presence of calcium (Whitten, 1971) One major component of
this process is uvomorulin (UM) , a trans-membrane glycoprotein which is also known as
E-cadherin (Hyafil et al., 1980; Vestweber & Kemler, 1984; Yoshida-Noro et al.,
1984). Uvomorulin is involved in Caz ° dependent cell-cell adhesion not only in
preimplantation embryos, but also in many epithelial cells later during development in

adult tissues.

Table 1: Estimated early cleavage time table. '

 

 

 

 

 

 

 

 

 

 

 

Stage of Development Time Required (Hours)

Coitus 0

First cleavage spindle 21-28

Two cell 21-43

Four cell 38-50

Eight cell 50-64

Sixteen cell morula 60-70
Blastocyst 66-82
Transport to uterus 66-72
Implantation 4-5 days

 

lFrom Rugh, 1990.

 

After compaction, fluid accumulates between intercellular spaces and around the 32-
cell stage the blastocoel (cavity of the blastula) becomes evident. Outer cells pump fluid
into the blastocoel which rapidly expands. Two distinct cell populations are present in the
blastocyts: an outer layer of trophectodermal cells which represents a true epithelium
surrounding the blastocoel, and the inner cell mass (ICM) cells, a group of cells which is
attached to one side of the inner surface of the trophectoderrn (Hogan et al., 1986) .

The trophectodenn and inner cell mass cells remain totally distinct lineages from the
onset of cavitation (blastocyst formation) (Dyce et al., 1987). The trophectodermal cells
give rise exclusively to extraembryonic tissue. Shortly before implantation some of the
inner cell mass cells differentiate into a second epithelial cell type, the primitive endoderrn,
which arises on the free surface of the ICM facing the blastocoel. The remaining ICM will
give rise to the embryo prOper and to the extraembryonic mesodenn (Austin & Short
1982).

Implantation occurs on day 4-5 of development. Before the blastocyst can be
implanted it has to escape from its zona pellucida. This process is called hatching and is
brought about by localized proteolysis of the zona pellucida and contraction and expansion
of the blastocyst. A small hole is lysed into the protein matrix of the zona pellucida by a
trypsin-like protease. The blastocyst hatches through this hole and once freed from its
zona pellucida, attaches to the epithelium of the lateral uterine wall opposite from the
ICM. A detailed analysis of the orientation of mouse embryos during implantation and a
discussion of how this might be related to embryonic axis formation is given by Smith

(1980,1985)

10

Development of the 8-cell mouse embryo

The 8-cell stage of mouse development is characterized by a number of morphological
changes that are described under the general heading of compaction (Johnson & Maro,
1984). Whereas in the early 8-cell stage individual blastomeres are rounded, during
compaction the cells flatten against one another to maximize cell contacts, the intercellular
boundaries becoming indistinct at the level of the optical microscope (Lewis & Wright,
1935; Lehtonen, 1980).

At the same time, the cells develop an apical “epithelial-like” localization of microvilli
(Callarco & Epstein, 1973; Ducibella et al., 1977; Reeve & Ziomek, 1981). They
develop specialized cellular junctions (Ducibella & Anderson, 1975; Magnuson et al.,
1978; Lo & Gilula, I979; Goodall & Johnson, 1984) and show a redistribution of
intracellular organelles (Ducibella, et al., 1977; Reeve, 1981; Reeve & Kelly, 1983).

During compaction of the 8-cell embryo, the microtubules play a constraining role,
being involved in the regulation of change in cell shape and organization and in their
timing, rather than being actively involved in the actual mechanism of cell flattening and
polarization (Maro & Pickering, 1984).

According to Hogan et al., (1986), one of the essential features of compaction is the
polarization of the blastomeres, so that they show distinct apical and basolateral membrane
domains. These domains are clearly seen by scanning electron microscopy of compacted
embryos. Polarity-inducing ability arises at the two cell stage and increases up to the 8 to
16 cell stage.

Bavister (1987) reported that the permanent cellular polarity established at the 8-cell

stage may be cmcial for successful embryonic deveIOpment since it provides the

ll

foundation for the formation of the two distinct cell lineages leading to the trophectoderrn

and inner cell mass of the blastocyst.

MATERIALS AND METHODS

Animals

Female C57BU6J and male DBA/ZJ mice both eight weeks old were used for the
production of 8-cell embryos. They were purchased from The Jackson Laboratory (Bar
Harbor, ME).

Mice were housed in Plexiglass boxes 7” x 11 '/2” x 5” (Allentown Caging, Allentown,
NJ) containing Bed-o-cobs® (Anderson Co., Maumee, OH). They were provided Teklad
Rodent diet® (Madison, WI) and water ad libitum. All animals were kept at 12 hours
light : dark photoperiod and maintained in an air conditioned room at 23 i 2° C. The
housing, maintenance and care conditions were in accord with state and federal

regulations.

Aroclor 1254

Aroclor-1254 was purchased from AccuStandard, Inc. (New Haven, CT), dissolved in
ethyl alcohol, and serially diluted in culture medium to obtain the desired concentrations.
Three different concentrations of A-1254 (01,10 and 10 ug/mL) were studied, control
culture dishes contained Minimal Essential Medium Eagle (MEM, Sigma Chemical Co.,

St. Louis, MO).

Culture Media

For embryo culture, MEM was supplemented with 0.75g/L penicillin-G sodium salt,

0.75g/L, streptomycin sulfate, 2.2g/L sodium bicarbonate, and 0.1091 g/L calcium lactate.

12

l3

Brinster’s medium for oocyte culture with 0.4% Bovine Serum Albumin (BMOC-3,
Gibco, Grand Island, NY) was used for embryo collection. The same medium was used in
the outer well of Falcon 3037 Organ Tissue Culture Dishes (Lincoln Park, NY). The
MEM medium was prepared with cell culture grade distilled water (Gibco, Grand Island,
NY) sterilized using a 0.22pm filter (Millipore, Bedford, NY) and aliquots were stored at
4°C in sterile 500ml plastic bottles. The pH of the medium was 7.30 to 7.45. Fresh media

was prepared every third week.

Superovulation

Females were superovulated by an intraperitoneal (ip) injection of 10 IU of pregnant
mare serum gonadotropin followed by 10 IU of human chorionic gonadotropin (hCG,
both fi'om Sigma Chemical Co., St. Louis, MO) 46-48 hours later.

Immediately following hCG injection, females were housed with males overnight at a
ratio of one to one. Females were separated from males the moming after, checked for

vaginal plugs, and placed back in individual boxes with food and water ad libitum.

Embryo Collection and Culture

Culture dishes were prepared using BMOC-3 (3ml) and MEM (lml) in the outer and
inner well respectively for each culture dish containing various concentrations of A-l254
and the control. The culture dishes when prepared were then equilibrated overnight in a
humidified incubator with 5% CO: in air at 37°C.

Eight-cell embryos were collected from mated females at approximately 57 hours after

hCG injections. Female mice were sacrificed by cervical dislocation, the abdominal cavity

14

exposed, and both oviducts and uterine horns removed and placed in a Costar cell culture
cluster dish (Costar Corporation, Cambridge MA) containing 2 ml of BMOC-3 medium.

Embryos were removed by simply tearing the oviduct at several points along its length
with a fine forceps, and flushing the isthmus of the oviducts through the uterine horn,
using BMOC-3 media in a lml syringe attached to a 25 gauge (5/8 inch) hypodermic
needle. Embryos were collected using a lOul disposable micro pipet with a mouthpiece
adaptor (Dade Diagnostic, Inc., Aguada PR). The embryos were then washed once in
fresh MEM .

For every trial, three concentrations of Aroclor-1254 (0.1, 1.0 and 10.0 pg/mL) were
prepared. Only embryos at the 8-cell stage not compacted and showing normal
morphology and characteristics, with no signs of degeneration and/or abnormalities were
used. From 6-10 embryos were used with every concentration in every trial for clear and
easy identification of any changes in their development.

After the collection and embryo distribution, the culture dishes were placed in the
incubator (5% CO: in air at 37°C) and observations were made every 24 hours up to 96
hours using a dissecting microscope to determine the number of embryos that developed
further, indicated by the presence of compaction, morula, or blastocyst stages at the

different concentrations of A-1254.

Statistical Analysis

Individual Chi square test (x2) using the Microsoft excel program, was performed on the
data in order to determine if there was any significant difference (P<0 .05) between the

treatment and the control groups, due to the effect ofAroclor- 1254.

RESULTS

Table 2 indicates the specific cell stage and time used for 8-cell embryo classification
during the experiment.

Table 3 indicates the development to compacted 8-cell embryos at 24 hours of
incubation. No statistical significant effects (P >0.05) were found between the treatments;
0.1, 1.0, 10 ug/ml of Aroclor-1254, and control.

Table 4 indicates the development to morula stage at 48 hours of incubation.
Statistically significant effects (P<0.05) were found between the treatments; 0.1, 1.0, 10
ug/mL of Aroclor-1254, and control group. At this particular stage, there was a clear
evidence of a dose response relationship.

Table 5 indicates the percent development to the blastocyst stage at 72 hours of
incubation. A statistically significant effect (P<0.05) was only found between the control
and 1.0 ug/mL concentration of Aroclor-1254.

Table 6 shows the development of hatched embryos at 96 hours of incubation. No
statistically significant effects were found (P>0.05) between the three treatments; 0.1, 1.0
10. ug/mL of Aroclor-1254, and control group.

Table 7 indicates the degenerated embryos at 24 hours of incubation. No statistically
significant effects (P>0.05) were found between the three treatments; 0.1, 1.0 ,10. ug/mL

of Aroclor-1254, and the control group.

15

16

Table 8 indicates the degenerated embryos at 48 hours, no statistically significant
effects were found (P>0.05) in all the different treatments (0.1, 1.0 and 10. ug/mL) of

Aroclor-1254);

17

Table 2: Specific cell stage and time used for 8-cell embryo

classification during experiment.

 

 

 

 

 

 

Observation Days after Stage of Development
Period Recovery
(Hours)
24 1 Compacted 8-cell
48 2 Morula
72 3 Blastocyts
96 4 Hatched

 

 

18

Table 3: Embryo development to compacted 8-cell
during the first 24 hours of in vitro culture.

 

 

 

 

 

 

 

Group Total Total Percent
Embryos Compacted Development
Control 69 44 64
0. 1nglmL/12541 66 51 77
1 .0ug/mL/1254‘ 62 42 68
10.0ug/mU1254‘ 56 27 48
Totals 253 164 -

 

1Chi-square revealed no significant difference from the control (P>0.05).

 

19

Table 4: Embryo development to morula stage at 48 hours of in vitro culture.

 

 

 

 

 

 

 

Group Total Total Percent
Embryos Morulae Development
Control 44 40 91
0.1pg/mL 12541 51 26 51
1.0ug/mL 1254‘ 42 18 43
10.0ug/mL 12541 27 15 56
Totals 164 99 --

 

1Chi-square revealed significant difference fi'om the control (P <0.05).

 

20

Table 5: Embryo development to blastocyst stage at 72 hours of in vitro culture.

 

 

 

 

 

 

 

Group Total Total Percent
Embryo Blastocyts Development

Control 40 23 58

0.1 ug/mL 1254 26 19 73

1.0ug/mL 1254‘ 18 18 100

10.0ug/mL 1254 15 10 67

Totals 99 7O --

 

1Chi-square test revealed significant difference from the control (P<0.05).

 

21

Table 6: Embryo development to the hatching stage at 96 hours of in vitro culture.

 

 

 

 

 

 

 

Group Total Total Percent
Embryos Hatched Development
Control 23 5 22
0.1ug/mL 12541 19 4 21
1.0ug/mL 1254‘ 18 1 6
10.0ug/mL1254‘ 10 0 0
Totals 70 10 --

 

 

‘Chi-square revealed no significant difference from the control (P>0.05).

22

Table 7: Embryos degenerated at 24 hours of in vitro culture.

 

 

 

 

 

 

 

Group Total Total Percent
Embryos Degenerated Degenerated
Control 69 6 9
0.1ug/mL 1254‘ 66 5 8
1.0pg/mL 1254‘ 62 5 8
10.0pg/mL 1254‘ 56 6 11
Totals 253 22 --

 

‘Chi-square revealed no significant difference from the control (P>0.05).

 

23

Table 8: Embryos degenerated at 48 hours of in vitro culture.

 

 

 

 

 

 

 

Group Total Total Percent
Embryos Degenerated Degenerated

Control 69 7 10

0.1 pg/mL1254‘ 66 8 12

1.0ug/mL1254‘ 62 6 10

10.0pg/mL1254‘ 56 9 16

Total 253 30 --

 

‘Chi-square test revealed no significant difference fiom the control (P>0.05).

 

DISCUSSION

The results in this study demonstrated a significant effect of Aroclor-1254 on the
development of the 8-cell mouse embryo, specifically at 48 hours for all the concentrations
(0.1, 1.0, and 10 ug/mL) of 1254. Only the concentration of 1.0ug/mL of 1254 at 72
hours (blastocyst stage) indicated a significant effect. Aroclor -1254 also was detrimental
for embryo development in a dose-dependent manner.

The criteria for the selection of the data to be analyzed statistically was based on time-
specific stages; compacted 8-cells at 24 hours, morula at 48 hours, blastocysts at 72 hours
and hatched at 96 hours. Establishing this selection criteria helped make a better judgment
at the time of observations in order to determine if further development occurred based on
morphological and structural changes in the 8-cell embryos.

The presence of other stages of deveIOpment, in addition to the specific stage expected
at that particular time, is due the fact that division of the blastomeres does not occur at the
same phase for all the embryos (Bavister, 1987). Some of them could be developing at a
faster or slower rate than other embryos that attained the appropriate cell stage at a
specific time.

In the present investigation those, out-of phase embryos represented a small
percentage of the total embryos. Another group of embryos observed were the
degenerated, abnormal or dying embryos. These embryos showed a variety of

morphological abnormalities such as fragmentation, irregular blastomeres, and cracked,

24

25

empty zonae pellucidae among many others. As expected, the number of degenerated
embryos gradually increased, as the period of incubation increased from 24 to 96 hours.

There was no statistically significant effect, (P>0.05) on the 8-cell embryo during the
first 24 hours of in vitro culture. At this stage the embryo is just starting the process of
compaction. The blastomeres are distinct, therefore no gap-junction communications are
established completely between the blastomeres.

These gap-junctions serve in the transformation of developmental information among
blastomeres (Ziomek & Johnson, 1980). The lack of communication between the
blastomeres of the 8-cell embryo could be a reason why Aroclor-1254 did not cause a
significant effect at the three different treatments (0.1, 1.0, and 10 ug/mL) of Aroclor-
1254.

Individual blastomeres could incorporate and metabolize a specific amount of the
different concentrations of Aroclor-1254, at a specific rate independent of each other.
The disadvantage of not having gap-junctional communications at this particular stage,
may turn into an advantage for the embryo, granting some degree of protection (at 24
hours) against the toxic effect of Aroclor-1254.

At 48 hours, the results indicated a significant effect of the A-1254 affecting the
morula stage at all concentrations. Previous to and after 48 hours there was no significant
effect except for the 1.0ug/ml of 1254 at 72 hours (blastocyst stage), this could be an
indication that the development of morula or the morula stage at 48 hours is more
susceptible to the toxic effect of Aroclor-1254 . Additionally an indication of a dose

response relationship was demonstrated with the number of 8-cell embryos that

26

developed into morula during the first 48 hours of invitro culture. As the concentration
of Aroclor-1254 increase, the number of embryos developing into morula decreased.

Prior to the process of compaction the blastomeres are spherical and lack specialized
intercellular junctions. The 8-cell embryos at 48 hours are firlly compacted. During
compaction cells flatten upon one another to maximize intercellular contact, establishing
gap-junctional communications for the first time.

During the stage of compaction, polarization of the blastomeres first becomes evident
(Ziomek & Johnson, 1980). This important process on the development of the 8- cell
embryo occurs at the morula stage. In contrast to the embryos at 24 hours, the 8-cell
embryos at the morula stage (48 hours) are more susceptible to the toxic effects from
Aroclor-1254.

The main reason for this significant effect could be the establishment of the gap-
junctional communications between blastomeres of the 8-cell embryo. As the individual
blastomeres are exposed to the concentrations of Aroclor-1254, the chances of the other
blastomeres receiving the same exposure of Aroclor-1254 are greater because all the
blastomeres are connected through the junctions. Therefore, the toxic effect of Aroclor-
1254 will end up affecting the entire embryo and its fiirther development.

In the case of the blastocyst at 72 hours, only the concentration of 1.0ug/ml of
Aroclor-1254 revealed a statistically significant difference (P<0.05). Considering that
100% development occurred only at this concentration (1.0 ug/mL of Aroclor-1254), and
also that an effect is usually expected at higher concentration, suggests that this significant

effect is basically coincidental.

27

Since there was a significant effect at 48 hours, most of the damage to the embryo that
could afi'ect its development due to toxic effects of Aroclor-1254, already occurred.
Therefore, this could be an explanation, why no additional significant effect was indicated
at the other two concentrations (0.1, and 10.0 ug/mL Aroclor-1254).

During the 96 hours observation period, there was no significant effect (P>0.05) on
the percent of embryos hatched. During this particular stage of development, the embryo
is ready to start the process of implantation. The results of non-significant effects were
almost the results expected at this particular time, not because of the lack of toxicity of the
Aroclor-1254, but mostly because, considerations such as variation in culture condition
and media requirements for the implantation period were not applied in this experiment.
The purpose of the observations at 96 hours, was to determine mostly if any significant
change could be observed at the end of the pre-implantation period. As indicated in Table
6, the percent embryos hatched at 96 hours was much lower than any other period of
observation.

Another mechanism by which Aroclor-1254 could affect the development of the
embryos could be by reducing the number of gap junctions. Studies conducted by
Krutovskikh et al., (1995), using four different tumor-promoting agents including Aroclor
-1260, caused inhibition of intercellular communication. Krutovskikh et al., (1995),
developed a dye-transfer technique to evaluate cell-coupling fiinction in fresh liver slices,
and used it to show that inhibition of intercellular communication is associated with rat
liver tumor progression. Hemming et al., (1991) used the dye-transfer technique with rat
liver white blood cells to measure the ability of polychlorinated biphenyl congeners to

inhibit intercellular communication. Bager et al., (1994) demonstrated that PCB 1260

28

dramatically reduce gap junction protein expression in rat liver after 20 weeks of
promotion treatment.

These studies were conducted using liver cells instead of embryos, however the
fimction of the gap junction in both types of cells is believed to be involved in regulation
of normal cell growth and differentiation. Further investigations need to be conducted in
order to determine the specific mechanism by which Aroclor-1254 affects the development
of the embryo.

There was no statistically significant difference (P>0.05) among the percentage of
embryos degenerated fiom the 24-96 hours observation period, even when the percent of
degenerated embryos gradually increased . Since the major effect on development of the
8-cell embryo occurred during the morula stage at 48 hours, the percentage of embryos
degenerated before and after this particular period, was considered more important than
the other observation periods. This information is indicated in Table 7 and Table 8.

The results in this study, contribute and support some of the most recent findings in
other studies related to the toxicity of PCBs on the pre-implantation embryos. Kholkute
et al. (1994b) examined the effect of A-1254 on the development of 2-cell embryos to the
4-cell stage at 48 hours, and they found that increasing the concentration of A-1254 in
the culture medium significantly reduced the progression of 2-cell embryos to 4-cell stage
or greater at 48 hours. The development of 4-cell embryos to expanded blastocyst was
also significantly suppressed in 0.1, 1.0 and 10.0ug/mL of Aroclor-1254 groups.

Lindenau & Fischer, 1996 reported that Aroclor-1260, is embryotoxic in a dose-
dependent manner . The study was conducted on one-day-old cleavage stages and three

day-old rabbit morulae. This investigation further demonstrated the toxic effect of A-1254

29

at later stages other than 2-4 cell embryos. At these stages (2-4 cells) the mouse embryo
is more susceptible to disruption on development by changes in the culture environment.
Some of the trials in this investigation were conducted for up to 120 hours and one up
to 144 hours (data not shown), but no changes occurred. For this reason and also to have
a more uniform set of data for the purposes of statistical analysis, data at 120 and 144
hours was not analyzed statistically. Also, these observations, were beyond the pre-

implantation period.

SUMMARY AND CONCLUSIONS

PCBs have been demonstrated to be detrimental to reproductive fisnctions in
laboratory animals and wild mammalian species. Studies with Aroclor-1254 indicated
adverse effects on early cleavage in the mouse embryo.

The 8-cell stage of the mouse embryo represents a very critical and important stage in
the further development of the embryo. During the first 48 hours at the morula stage, the
embryo undergoes major physical reorganization, including the process of compaction, the
formation of gap-junctions and the process of polarization by which the components of
the cell are re-organized.

This present in vitro study demonstrated the effect of Aroclor-1254 on the
development of the 8-cell mouse embryo. The following conclusion were drawn from
this investigation:

1. Aroclor-1254 did not affect the development of the 8-cell mouse embryo at 24
hours of culture in vitro, in all three concentrations tested.

2. Aroclor-1254 demonstrated a significant effect on development of the 8-cell
embryo at 48 hours during the morula stage at the three concentrations tested
(0.1, 1.0 and 10 ug/mL). In addition a dose response relationship was clearly
observed.

3. Aroclor-1254 did not affect the development of the 8-cell mouse embryo at 72

hours during blastocyst stage at the concentrations of 0.1, and 10.0 ug/mL.

30

31

Only the 1.0 ug/mL concentration indicated a significant effect which may have!
been coincidental.

. Aroclor-1254 did not affect the development of the 8-cell mouse embryo at 96
hours of culture in vitro, at all three concentrations tested.

. Aroclor-1254 caused no significant effect, at all three concentrations tested,
during the periods of observations (24-96 hours) in relation to the number of

8- cell embryos degenerated.

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Name:
Born:

Citizenship:

Education:

Professional Positions:

APPENDIX A

CURRICULUM VITA

Oscar Hernandez Maldonado
Rio Piedras, Puerto Rico

USA

University of Puerto Rico

Arecibo, Puerto Rico 00613
Major: Animal Health

Degree: Associate Degree (1985)

Kansas State University
Manhattan, Kansas 66506
Major: Animal Science Pre-Vet

Iowa State University

Ames, Iowa 50011

Major: Animal Science

Degree: Bachelor of Science (1994)

Michigan State University

East Lansing , Michigan 48824
Major: Animal Science

Degree: Masters in Science (1997)

Veterinary Technician
Caribe Veterinary Hospital
Isla Verde, Puerto Rico

Laboratory Technician
Kansas State University
Veterinary Medicine School
Diagnostic Laboratory
Virology Department

39

Papers Published

40

Research Aide II
Iowa State University
Animal Science Department

Laboratory Technician
Michigan State University
Animal Science Department
Dairy Nutrition

W. R. Dukelow, O. Hernandez, C. R. Greenfeld,
& S.D. Kholkute. In vitro Fertilization
Assessment of PCB Effects in B6D2-FI Mice.
Proc. Amer. Assoc. Lab. Anim. Sci.

p.5, 1996 (Abstract)

W. R. Dukelow, C.R. Greenfeld, O. Hernandez,
L.G. Clemens, Yu-wen Chung & J .B. Kaneene
Toxic Chemical Influences on In vivo and In vitro
Reproduction. Proc. Forum on Environm.
Remediation & Environm.Toxicol. Lansing

MI. September 19-20 1996 (Abstract)

C. R. Greenfeld, O. Hernandez, & W. R. Dukelow.
Reproductive Toxicity of a Commercial PCB
(Aroclor 1254) in the Mouse. Annual Research
Day Department of Zoology. April 20, 1996.
(Abstract).

Yu-wen Chung, L. G. Clemens, W.R. Dukelow
O. Hernandez, C.R. Greenfeld & J.B. Kaneene.
Reproductive Physiology and Behavior Effects of
PCBs. Proc. 10‘” Ann. Of Superfirnd, North
Carolina. Feb. 1997.(Abstract)

C. Greenfeld, O. Hernandez, & W.R. Dukelow.
Inhibition of Zona Pelucida Hardening in the
mouse oocyte following prolonged exposure

to a commercial PCB (Aroclor 1254).

Annual Research Day Department of Zoology
March 22, 1997. (Abstract)