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Reproductive Biology, Toxicology and
Epidemiology
Reproductive Biology
Sperm Cell Biology and Contraceptive Development (James
Overstreet). The research effort to identify the functions of PH-20, the sperm
surface hyaluronidase, and to develop immunocontraceptive vaccines
that target this protein have been described in previous progress
reports. The culmination of this research effort was a fertility
trial in female macaques immunized with PH-20. Although high titers
of anti-PH-20 antibodies were attained in all females, no contraceptive
effect could be demonstrated. As a result, this line of investigation
was ended.
A major focus of current investigation is the contraceptive
potential of lignin-derived macromolecules (LDMs). LDMs are biologically
active compounds that affect a variety of cell to cell interactions
including the inhibition of fertilization and embryo development
in a number of non-mammalian species. Lignosulfonic acid (LSA)
is a water-soluble LDM that is derived from lignin and is highly
sulfonated. The effect of LSA on cynomolgus macaque sperm-oocyte
interaction was evaluated with a zona pellucida binding assay
and
by in vitro fertilization (IVF). Prior to gamete interaction,
sperm were centrifuged through a 6 cm column of 80% Percoll,
followed
by 2 consecutive washes with capacitation medium, overnight incubation,
and activation with dibutyryl cyclic AMP and caffeine. Sperm
were treated with LSA (1.5mg/ml) either prior to washing (pre-capacitation)
or after activation (post capacitation). The zona binding assay
was performed using immature zona pellucida-intact oocytes adhered
to the center of a glass “binding chamber.” Sperm were introduced
to the chamber following activation and the number of sperm that
attached to the zona over a 3 minute period was recorded. Sperm
attachment to the zona was significantly inhibited by LSA as compared
to controls, whether treatment was post capacitation (92.5%; p < 0.001)
or pre-capacitation (82.5%; p < 0.001). When sperm were treated
similarly with fucoidin, a sulfated polysaccharide known to inhibit
sperm-oocyte interaction, sperm-zona binding was significantly
inhibited by post-capacitation treatment but not by pre-capacitation
treatment. Both pre- and post-capacitation treatment of sperm with
LSA completely blocked fertilization. In four IVF cycles, no oocytes
were observed to form pronuclei or cleave after co-incubation with
LSA-treated sperm. No LSA-treated sperm were observed on the surface
of lightly rinsed oocytes after 4 hours of co-incubation. The fertilization
rate following insemination with control sperm was 65 ± 17%.
LSA treatment appeared to have no effect on percentage of motile
sperm,
sperm movement characteristics, or the development of hyperactivated
motility. LSA is a compound with great potential as a contraceptive
agent for vaginal application.
To evaluate the mechanism of the anti-fertility effect of LSA,
changes in sperm-zona pellucida binding affinity, acrosomal status,
and the corresponding changes in sperm motility on the zona pellucida
were observed directly and analyzed with videomicroscopy. A single
zona was air-dried and rehydrated on a microscope slide, and
a cover slip supported by glass beads was added. Capacitated
sperm
were added together with SBTI-Alexa, a probe for acrosin, which
can detect the acrosome reaction. In control preparations, the
heads of loosely attached sperm oscillated on the zona and the
flagella beat symmetrically with a sigmoid-shaped waveform. After
16 seconds tight binding was observed as the sperm head became
fixed in place on the zona. The shape of the flagellar beat simultaneously
shifted to a more rigid, “c”-shaped waveform. Within 11 seconds
of tight binding the first signs of the acrosome reaction were
detected. Rapid flushing removed approximately 65 % of sperm that
were loosely attached but only 2 % of tightly bound sperm. In the
2 minutes following the onset of tight binding, the lateral displacement
of the flagellum increased by approximately 30% and the beat frequency
decreased by 25%. LSA inhibited loose sperm attachment and the
development of tight binding. LSA had no effect on the time of
the acrosome reaction following tight binding or on changes in
motility that followed tight binding. These data suggest that LSA
affects initial attachment or “docking” of sperm to the zona,
a step that may align or recruit specific zona receptor(s) responsible
for mediating the acrosome reaction.
In other work, we are continuing to search for sperm proteins
that may be vulnerable targets for immunocontraception. To identify
a sperm-surface component that is highly antigenic, we immunized
female cynomolgus macaques with glycosylphosphatidylinositol (GPI)-anchored
sperm surface proteins that were released following treatment with
phosphatidylinositol-specific phospholipase C (PI-PLC). Five different
adjuvants were used in combination with the PI-PLC-released proteins,
and three of these proteins (24, 48 and 53 kDa) were shown to be
potent antigens for immunization of female monkeys. The 53 kDa
protein was found to be a surface coating protein and not a GPI-anchored
protein. Polyclonal antibodies to the 24 kDa protein and the 48
kDa protein were produced in rabbits. The two antibodies recognized
both proteins on Western blots. The same rabbit antibodies recognized
28 kDa, 18 kDa and 10 kDa bands on a Western blot of chemically
reduced PI-PLC-released proteins, suggesting that the 48 kDa protein
is a dimer of the 24 kDa protein, which we refer to as MAK248.
Rabbit polyclonal antibodies developed to reduced fragments of
the 24 kDa protein showed that the 18 kDa and 10 kDa bands are
proteolytic peptide fragments of the 24 kDa protein. Screening
of tissues from male macaques showed that MAK248 is expressed only
in the epididymis. Microsequencing of two proteolytic fragments
of the 18 kDa component showed 100% amino acid homology to a 233
deduced amino acid sequence previously identified in human testes
genome. Antibodies to MAK248 recognized a 24 kDa protein released
from human sperm exposed to PI-PLC. Antibodies to MAK248 recognized
the equatorial segment and posterior head regions of capacitated
cynomolgus macaque sperm. Structural analysis suggests that MAK248
is a novel CRISP protein and a member of the CAP family of proteins.
Based on amino acid sequence homology, it is possible that MAK248
functions as a protease inhibitor.
Reproductive Toxicology
Mechanism of Action of Environmental Hazards on Early
Pregnancy Loss (Bill Lasley, James Overstreet, Allen Conley,
Francisco Moran). Studies conducted over the past six years in this laboratory have
shown that dioxin induced early fetal loss in the monkey model,
and that this induction of fetal loss is associated with abnormal
trophoblastic function. These data suggest that early pregnancy
losses caused by environmental hazards in animal experiments have
biological similarities to spontaneous pregnancy loss in humans.
This year’s extension of these studies have shown that the effect
of dioxin is similar when human trophoblast cells are exposed in
vitro and the effects of these exposures are compared to the in
vivo results using the nonhuman primate model. These in vitro studies
show that dioxin does not affect the secretion of immunoreactive
CG but decreases the bioactivity of the apparent intact molecule.
Additional studies using monkeys show that this effect in vivo
occurs following the ability of the early placenta to produce CG
with full biological activity. Taken together our results show
the trophoblast to be a primary target of dioxin’s toxicity with
one of the adverse effects to change the ability of secreted
CG to have reduced capacity to transducer its biological signal.
In order to understand the dioxins effects at the molecular level
we have cloned monkey chorionic gonadotropin (mCG) and expressed
the beta subunit in vitro. We have also synthesized a small amino
acid chain from mCG and made antibodies to it.
In a parallel study, the effect of dioxin on ovarian function
was studied using human luteinized granulose cells in vitro. The
results of these studies demonstrate that dioxin has a direct effect
on ovarian estradiol production by targeting the P450c17 enzyme.
This finding is particularly important as it identifies for the
first time the specific target of toxicity for dioxin in the human
ovary and provides string evidence that P450c17 is the rate limiting
step for estradiol production in this organ.
The Effect of Environmental Hazards on Ovarian Function
(Bill Lasley, James Overstreet, Susan Shideler). Our
studies this year provide evidence that biomarker assays
can be used to more precisely
identify the day of ovulation as well as predict fecundity.
These assessments are important in epidemiologic studies
that are designed
to identify environmental hazards to women’s reproductive health.
Previous studies indicated that abnormal ovarian function can
be induced in women by modest stressors acting at specific
time intervals
during the normal menstrual cycle. Stressors that perturb the
secretion of follicle stimulating hormone (FSH) during the
luteal-follicular
transition have a much higher likelihood of perturbing subsequent
ovarian function than would the same stressor occurring at
different times of the menstrual cycle. Perturbation of ovarian
function
is associated with reduced estrogen production, delay of ovulation
and evidence of a transient, increased bone loss.
The monkey model was used during the past year to better understand
the relationship between physical stress and ovarian function.
A urinary assay for cortisol was developed and used in conjunction
with assays for ovarian function. The patterns of urinary hormone
profiles were used to determine if a cycle-stage specific interval
does exist and if the perturbation of FSH causes subsequent aberrations
in ovarian function. The results of these studies demonstrate a
concomitant release of cortisol and follicle stimulating hormone
in cycling but not non-cycling females but subsequent perturbations
were not observed.
Parallel studies of young Chinese women who work rotating shifts
are now being completed and provide evidence that subtle stressors,
such as abnormal shift work schedules, lead to prolonged subsequent
follicular phase lengths and a significant increase in bone loss.
Endocrine Changes Associated with the Menopausal Transition
(Bill Lasley and Susan Shideler). A great deal of controversy surrounds
the hormone changes associated with menopause. We have conducted
experiments in the nonhuman model and made comparisons of hormone
changes in longitudinal studies of mid-aged women. Our results
indicate that adrenal function changes markedly in most monkeys
and some women during the latter stages of the menopausal transition.
This change in adrenal function results in increased circulating
androgens and possibly increased estrogens. These changes, which
are not uniform between women, may explain observed differences
in symptoms that accompany the menopausal transition and provide
insights to develop more individual therapies.
Effect of Time of Surgery and Recurrence of Breast Cancer
(Bill Lasley). Recent clinical evidence has suggested that the timing
of breast cancer surgery in respect to the phase of the menstrual
cycle can influence the recurrence of breast cancer. Unfortunately,
the current data is based on the patient's recall of her menstrual
cycle during the month of her surgery. In order to more precisely
stage breast cancer surgery, we are evaluating daily urine samples
from breast cancer patients immediately following their surgery.
These data, while difficult to obtain, will permit a precise staging
of the time of surgery which can then be correlated to final health
outcome.
Development of Biomarkers for Environmental Estrogens
(Bill Lasley). Recognition of a group of compounds
that can disrupt endocrine function has stimulated interest
in specific environmental compounds
that have estrogenic properties. To date, however, attempts to
develop biomarkers of exposures have failed because the endogenous
estrogens are more potent and swamp all functional assay systems.
To obviate this concern, we have developed methods for removing
endogenous estrogens from biological samples in order to be able
to detect and quantify the exogenous or "xeno-" estrogens in
a sample. We have shown that the immuno-precipitation of steroidal
estrogens may be effective in removing most if not all endogenous
estrogens from a sample so that functional assays can be employed
to evaluate that sample for xenoestrogen content. This biomarker
assay is now being applied to monkey samples in controlled experiments
and in archived human samples to detect natural exposures.
Effects of TCDD on Nonhuman Primate Embryos Following
Exposure in Early Pregnancy (Andrew Hendrickx). This study examines the
embryotoxic effects and endocrine alterations associated with early
pregnancy failure induced by an environmental toxin, TCDD, in the
cynomolgus macaque, a well-documented reproductive/developmental
model for humans. Pregnant cynomolgus macaques were treated once
with TCDD during the post-implantation period (gestation days [GD]
15 and 20). The embryos were surgically removed on GD 23-28 and
processed for immunohistology. Step serial sections were examined
for cellular morphology (apoptosis, necrosis, and vascular congestion)
and immunohistochemistry was employed to identify endothelial cells,
proliferating cells, and apoptotic cells. Mild to moderate apoptosis
was evident in embryos exposed to TCDD on GD 20. The neural and
surface epithelium were the most common areas of increased apoptosis,
particularly in the dorsal region of the neural tube containing
premigratory neural crest cells. The foregut endoderm and migratory
crest cells in the head mesenchyme were occasional sites of abnormal
levels of apoptosis in treated embryos. Increased intercellular
spaces in the neural tube as well as vascular congestion were also
observed in several embryos. These results indicate that TCDD has
deleterious effects on early embryonic development.
Genetic Toxicology
Heritable Effects from Parental Exposures To Environmental
Agents (Janet Baulch, James Overstreet and Otto Raabe). Mice conceived
6 weeks after paternal irradiation with attenuated 137Cs gamma
rays were fathered by sperm that were Type B spermatogonia at the
time of irradiation. Previous studies showed this paternal F0 germ
cell irradiation led to biological effects at several stages in
the lifecycle of mice including decreased preimplantation embryo
cell proliferation rates in F1 and F2 embryos using chimera assays,
altered enzyme activity levels and protein levels in juvenile F3
offspring, and reduced whole-body weights in adult F1 animals.
In this study we examined four generations of CD1 mice following
paternal F0 irradiation of the Type B spermatogonia (1.0 Gy attenuated
137Cs gamma rays) to obtain information regarding the stability
of the previously observed heritable biological effects. Livers
from juvenile offspring with paternal F0 radiation history were
found to have significantly altered a) liver-weight to body-weight
ratios in F2 and F3 generations, and b) cytosolic protein kinase
C activity in F1 and F3 generations. Adult male F2, F3, and F4
offspring also had significantly altered whole-body weights. These
results support the hypothesis that irradiated Type B spermatogonia
develop a capacity to transmit heritable effects to three or more
generations of offspring.
In other experiments, nineteen-day-old F3 CD1 mice with and without
a 1.0 Gy paternal F0 radiation history received doses of 1.0 Gy
from acute irradiation. Kidney PKC and MAPK activities, and p53
protein levels were evaluated immediately following F3 irradiation.
The same endpoints and DNA damage were evaluated in kidney-derived
fibroblast primary cell cultures three weeks after irradiation.
Kidneys had significantly decreased PKC and MAPK activities and
p53 protein levels related to F0 irradiation and increased PKC
and MAPK activities following F3 irradiation irrespective of F0
radiation history. Kidney-derived fibroblasts had significant changes
or strong trends for all selected endpoints based upon cross-interaction
of F0 radiation history with F3 irradiation. Comet assays demonstrated
significantly increased DNA damage in fibroblasts related to F0
irradiation and following F3 irradiation. Significantly decreased
damage was demonstrated based upon cross-interaction of F0 radiation
history with F3 irradiation. These data suggest that irradiation
of paternal F0 Type B spermatogonia resulted in cellular reprogramming
causing offspring with this radiation history to have altered responses
to acute somatic gamma irradiation.
Wildtype and p53 null C57Bl/6J male mice have also been irradiated
using 0.1 Gy of 137Cs gamma radiation and bred to obtain three
generations of offspring. The same animal and biochemical endpoints
used in previous studies are being evaluated to test the hypothesis
that the heritable effects of paternal F0 germline irradiation
observed in CD1 mice are also observed in the C57Bl/6J strain of
mice. Additionally, these data will provide information to suggest
whether the absence of p53 at the time of paternal irradiation
affects the correlative animal and biochemical endpoints. These
data, together with an analysis of microsatellite DNA stability
will provide us with important information regarding the mechanism
underlying the heritable phenotype. This project is supported by
the National Institutes of Health and will draw to a close at the
end of 2002. Impact of Genetic Factors on the Heritable Effects
of Paternal Exposure to Low-Dose Radiation (Janet Baulch and Otto
Raabe). There has long been concern about potential heritable effects
of low dose radiation exposure. This study uses the ATM mutation
in 129/SvEv mice to evaluate the impact of genetic factors on heritable
effects of paternal germ line exposure at 0.1 Gy where the target
is Type B spermatogonia. Research in our laboratory has demonstrated
in two strains of mice that paternal F0 irradiation of the radiation
sensitive Type B spermatogonia, 6 weeks prior to conception leads
to a high incidence of heritable effects in the F1 offspring. We
have demonstrated changes in multiple endpoints for up to three
generations following paternal F0 137Cs gamma irradiation with
doses of 1.0 Gy in CD1 mice. Endpoints evaluated in these studies
include alterations in signal transduction biomarkers (PKC and
MAPK activities, p53 and p21waf1 protein levels), adult body weights,
and 19 day-old liver-weight to body-weight ratios. These data support
the hypothesis that paternal germline irradiation induces genomic
instability in offspring.
To test the hypothesis that a radiation-sensitive genetic factor
increases the magnitude of the transgenerational effects transmitted
to the offspring by the paternal F0 irradiated Type B spermatogonia,
ATM heterozygous male mice and 129/SvEv wild-type mice will be
irradiated with 0.1 Gy of 137Cs gamma rays and controls will be
sham-irradiated. Three generations of offspring will be evaluated
for changes in PKC and MAPK activities and p53 and p21waf1 protein
levels. These biomarkers are essential components in signal transduction
pathways modulating cellular proliferation and DNA repair. DNA
damage will be evaluated using comet and micronuclei assays of
nucleated cells in peripheral blood, and minisatellite repeat DNA
loci will be evaluated for polymorphism as an indicator of genomic
instability. Liver-weight to body-weight ratios will be evaluated
as a general indicator of altered enzyme or protein levels and
adult whole-body weights will be evaluated as an indicator of overall
health and vigor.
We will measure differences between offspring of paternal F0
irradiated ATM heterozygotes and of paternal F0 irradiated wild-type
mice in the magnitude of effect on signal transduction endpoints,
and genomic instability. This result will test our hypothesis that
ATM heterozygosity is a genetic factor increasing individual susceptibility
to the transmission of heritable effects following paternal irradiation.
This study will further characterize the heritable biological phenotype
resulting from paternal F0 germline irradiation and, using the
ATM mutation, extend our understanding of the mechanism that underlies
this phenomenon. This 3-year project is supported by the U.S. Department
of Energy.
Reproductive Epidemiology
Biomarkers of Male Reproductive Function (James Overstreet). In response to concerns about a possible worldwide decline in sperm
counts, a coordinated set of international studies is being undertaken
to estimate geographic variability of semen parameters in the partners
of pregnant women recruited in European, Asian and U.S. cities.
The secondary aims of these studies are to evaluate the geographic
variability of serum hormones in this population of fertile men,
as well as testicular and other physical abnormalities, and the
relationships between these outcomes and semen quality. To evaluate
the health consequences of the variability, the studies will assess
the strength of the relationships between these male reproductive
parameters and time to conception, after controlling for confounding
factors in both partners. These studies should provide unbiased
estimates of variability among cities that have been reported to
differ widely in semen quality, provide baseline levels of male
biomarkers for future studies, and generate hypotheses of environmental
causes of variation in these parameters. This laboratory serves
as the Andrology Coordinating Center for a NIEHS-sponsored study
of 1,200 fertile men in four different US cities, and participates
in the coordination of quality control procedures for the international
program, insuring that similar laboratory methodologies are used
in all of the international studies.
Data from the first 493 male study subjects demonstrate significant
between-center differences in semen quality. Men recruited from
the most rural and agrarian of these centers (Columbia MO) have
significantly lower sperm counts and motility than men recruited
from each of the urban centers (CA, MN and NY); these differences
are particularly apparent when MO is compared with MN and NY. When
subjects in CA are stratified by ethnicity, semen quality of Hispanics
is similar to that of subjects from MO, while semen quality of
other ethnic groups more closely resembles that of MN and NY. Because
strict quality control measures were employed throughout the study,
these findings are unlikely to be the result of between-center
differences in study methods and initial data on potential confounders
indicate that these differences are unlikely to be the result of
confounding.
Lifestyle and Ovarian Function in Midlife Women (Ellen
Gold and Bill Lasley). This project is part of the multi-center Study of
Women Across the Nation (SWAN), which is funded by the National
Institute on Aging and consists of seven clinical sites, one central
laboratory and a coordinating center. One objective of the project
is to investigate ethnicity and the perimenopause. Over 3,000 women
have been recruited nationally representing Caucasian, Chinese,
Japanese and African-American ethnic groups. Baseline and annual
follow-up serum samples have been collected and analyzed and daily
urine samples have been collected on selected subjects. The relationship
between reproductive status (ovarian function) and lifestyle (smoking,
exercise, occupation, diet, etc.) and cultural aspects of life
(ethnic origin and religion) are being examined in regard to other
health outcomes (weight, bone mass, cardiovascular function and
cancer). These data will provide new information regarding the
cause and severity of symptoms associated with the menopausal transition.
Cross-sectional analyses of the relation of lifestyle factors to
age at menopause and to occurrence of symptoms and of dietary factors
to bone density have been published as have data on changes in
DHEAS with changes in menopausal status. One doctoral student has
completed a dissertation examining the relation of occupational
status, activity and exposures to symptom reporting, and another
student has received supplement funds to evaluate the use of complementary
and alternative therapies in relation to symptoms and to use of
conventional health care. Two additional grants were recently received
to evaluate factors affecting sleep in these perimenopausal women
and to evaluate risk factors for breast density as measured by
mammography.
Sacramento Community Women’s Health Study (Ellen Gold, Bill Lasley). This project will recruit 750 women in the Sacramento area, 250
from each of 2 areas near a Superfund site and one comparison area,
and examine general and reproductive health status of women to
determine the effects of lifestyle and environmental exposures
on adverse health outcomes. To date, 124 women have been recruited.
Data are collected by interview, serum assays for thyroid hormones,
daily diaries and daily urine collection or assessment of metabolites
of estrogen and progesterone.
Women’s Healthy Eating and Living (Ellen Gold). This
is a randomized trial of a plant-based dietary intervention for
women with early
stage breast cancer. The dietary intervention is high in fiber
and low in fat and is compared to the National Cancer Institute’s
5-a-day diet. We have recruited 500 women at this site as part
of this 7-site trial and are following the women for up to
8 years with annual assessments for recurrence. Doctoral students
have
completed dissertations related to factors affecting quality
of life and affecting physical activity and body mass index
in these
women with a recent history of breast cancer.
Risk Factors for Primary Biliary Cirrhosis (Ellen Gold
and Eric Gershwin). This is a nationwide case-control study of lifestyle,
reproductive and environmental risk factors for this rare autoimmune
disease that primarily affects middle-aged women. The goal is to
recruit 2,000 cases and matched, random-digit-dialed controls,
and we are approximately 25% complete in this effort.
Physical Activity and Smoking in Relation to Ovarian
Function (Ellen Gold and Bill Lasley). This project involves analyses from
a prior prospective study of 400 women from the semiconductor industry
who provided interview and daily diary and daily urine collections.
A doctoral student is analyzing these data to assess menstrual
cycle patterns, anovulation and daily urinary hormone metabolite
patterns in relation to smoking and physical activity while controlling
for race/ethnicity, body mass index, and age.
Risk Factors for Premenstrual Symptoms (Ellen Gold). This is
a prospective study of the relation of smoking and dietary factors
to physical and emotional symptoms in the premenstrual period.
Four hundred women were recruited, interviewed and followed with
daily diaries. Analyses of the data by a doctoral student are nearing
completion.
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