Reproductive Biology, Toxicology and Epidemiology
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.
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.
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.
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.
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.