Prenatal Bisphenol A Exposure Alters Epithelial Cell Composition in the Rhesus Macaque Fetal Oviduct.

Bisphenol A (BPA) is an endocrine disrupting compound that is a pervasive environmental contaminant. Although it has been reported to affect the development of a variety of fetal reproductive tissues, data on the effect of fetal BPA exposure on oviducts were extremely limited and were only available in mice. To determine if there are adverse effects of gestational BPA exposure on fetal oviduct, we exposed pregnant rhesus macaques with female fetuses to oral or nonoral BPA during the last trimester of gestation (day 100 to term). After the treatment, fetal oviducts were collected for morphology evaluation. BPA exposure altered the percentages of different cell types (ciliated, nonciliated, and secretory) in the fetal oviduct and resulted in a significant high ciliated cell population in the BPA-exposed fetal oviduct. The distribution of ciliated cells on the epithelium in the BPA-exposed fetal oviduct was also altered. Gestational BPA exposure reduced the expression of mucosubstance and uteroglobin in secretory cells in the fetal oviduct. A comparison of the outcome of the fetal oviduct studies with similar outcomes previously reported in the lung from the same fetuses demonstrates that BPA exhibits opposite effects in these two organs. In conclusion, the BPA-associated alterations in the fetal oviduct could potentially affect the oviduct morphology and function later in life with a negative impact on fertility. The mechanisms of action of the differential response in the oviduct and the lung to BPA exposure require further investigation.

The contraceptive efficacy of intravas injection of Vasalgel™ for adult male rhesus monkeys.

BACKGROUND: Options for male contraception are limited. The purpose of this study was to use a nonhuman primate model to evaluate Vasalgel™, a high molecular weight polymer being developed as a contraceptive device for men. METHODS: Sixteen adult male rhesus monkeys received intravas injections of Vasalgel, consisting of 25% styrene maleic acid in dimethyl sulfoxide. After a one-week recovery, males were returned to outdoor group housing, which included at least 3 and up to 9 intact, breeding females with a successful reproductive history. RESULTS: Treated males have had no conceptions since Vasalgel injections. All males were housed with intact females for at least one breeding season and seven have been almost continually housed with females for 2 years. Complications were minor and included one incident of incorrect placement of Vasalgel into the vas deferens and the development of a sperm granuloma in one animal. Three unilateral vasectomies were performed, one was necessary for incorrect placement of Vasalgel, the other two were elective. CONCLUSIONS: Intravas injection of Vasalgel in sexually mature adult male rhesus monkeys was effective in preventing conception in a free-living, group environment. Complications were few and similar to those associated with traditional vasectomy.

Bisphenol A exposure alters developmental gene expression in the fetal rhesus macaque uterus.

Bisphenol A (BPA) exposure results in numerous developmental and functional abnormalities in reproductive organs in rodent models, but limited data are available regarding BPA effects in the primate uterus. To determine if maternal oral BPA exposure affects fetal uterine development in a non-human primate model, pregnant rhesus macaques carrying female fetuses were exposed orally to 400 µg/kg BPA or vehicle control daily from gestation day (GD) 50-100 or GD100-165. Fetal uteri were collected at the completion of treatment (GD100 or GD165); tissue histology, cell proliferation, and expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) were compared to that of controls. Gene expression analysis was conducted using rhesus macaque microarrays. There were no significant differences in histology or in the percentage of cells expressing the proliferation marker Ki-67, ERα, or PR in BPA-exposed uteri compared to controls at GD100 or GD165. Minimal differences in gene expression were observed between BPA-exposed and control GD100 uteri. However, at GD165, BPA-exposed uteri had significant differences in gene expression compared to controls. Several of the altered genes, including HOXA13, WNT4, and WNT5A, are critical for reproductive organ development and/or adult function. We conclude that second or third trimester BPA exposure does not significantly affect fetal uterus development based on morphological, proliferation, and steroid hormone receptor assessments. However, differences in expression of key developmental genes after third trimester exposure suggest that BPA could alter transcriptional signals influencing uterine function later in life.

EGF-like ligands mediate progesterone's anti-apoptotic action on macaque granulosa cells.

A local autocrine/paracrine role for progesterone is an absolute requirement for corpus luteum formation in primates. Despite this, the mechanism(s) remain obscure, although existing data suggest an anti-apoptotic action to be central. There are a limited number of progestin-regulated gene targets identified in the luteinizing primate follicle, suggesting that a small number of important genes may mediate progesterone action. Possible gene targets could be the epidermal growth factor (EGF) family members amphiregulin (AREG) and epiregulin (EREG). Using macaques undergoing controlled ovarian stimulation cycles, we show that the phosphorylation of EGF receptor (EGFR), ERK 1/2, and AKT increases 6 h after an ovulatory human chorionic gonadotropin (hCG) stimulus and remains activate through 24 h. Immunoreactive EREG and AREG ligands in the follicular fluid both increased in a time frame commensurate with EGFR phosphorylation. The mRNA expression of AREG and EREG in nonluteinized granulosa cells (NLGC) was induced in culture with hCG, an effect blocked by progesterone receptor (PGR) antagonists. Overexpression of PGR B in NLGC and treatment with a nonmetabolizable progestin did not increase either gene, indicating both progesterone and luteinizing hormone/CG are necessary. Addition of EGF and EGF-like ligands did not promote steroidogenesis in vitro by granulosa cells in the presence of gonadotropin, but were able to partially reverse RU486-induced cell death. These data suggest that progesterone promotes the expression of AREG and EREG, which in turn maintain viability of luteinizing granulosa cells, representing one possible mechanism whereby progesterone promotes corpus luteum formation in the primate.

Bisphenol A alters the development of the rhesus monkey mammary gland.

The xenoestrogen bisphenol A (BPA) used in the manufacturing of various plastics and resins for food packaging and consumer products has been shown to produce numerous endocrine and developmental effects in rodents. Exposure to low doses of BPA during fetal mammary gland development resulted in significant alterations in the gland's morphology that varied from subtle ones observed during the exposure period to precancerous and cancerous lesions manifested in adulthood. This study assessed the effects of BPA on fetal mammary gland development in nonhuman primates. Pregnant rhesus monkeys were fed 400 µg of BPA per kg of body weight daily from gestational day 100 to term, which resulted in 0.68 ± 0.312 ng of unconjugated BPA per mL of maternal serum, a level comparable to that found in humans. At birth, the mammary glands of female offspring were removed for morphological analysis. Morphological parameters similar to those shown to be affected in rodents exposed prenatally to BPA were measured in whole-mounted glands; estrogen receptor (ER) α and ß expression were assessed in paraffin sections. Student's t tests for equality of means were used to assess differences between exposed and unexposed groups. The density of mammary buds was significantly increased in BPA-exposed monkeys, and the overall development of their mammary gland was more advanced compared with unexposed monkeys. No significant differences were observed in ER expression. Altogether, gestational exposure to the estrogen-mimic BPA altered the developing mammary glands of female nonhuman primates in a comparable manner to that observed in rodents.

Ethanol, acetaldehyde, and estradiol affect growth and differentiation of rhesus monkey embryonic stem cells.

BACKGROUND: The timing of the origins of fetal alcohol syndrome has been difficult to determine, in part because of the challenge associated with in vivo studies of the peri-implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development. METHODS: Rhesus monkey ESC lines (ORMES-6 and ORMES-7) were treated with 0, 0.01, 0.1, or 1.0% ethanol, 1.0% ethanol with estradiol, or 0.00025% acetaldehyde with or without estradiol for 4 weeks. RESULTS: Although control ESCs remained unchanged, abnormal morphology of ESCs in the ethanol and acetaldehyde treatment groups was observed before 2 weeks of treatment. Immunofluorescence staining of key pluripotency markers (TRA-1-81 and alkaline phosphatase) indicated a loss of ESC pluripotency in the 1.0% ethanol group. ORMES-7 was more sensitive to effects of ethanol than ORMES-6. CONCLUSIONS: Estradiol appeared to increase sensitivity to ethanol in the ORMES-6 and ORMES-7 cell line. The morphological changes and labeling for pluripotency, proliferation, and apoptosis demonstrated that how ethanol affects these early cells that develop in culture, their differentiation state in particular. The effects of ethanol may be mediated in part through metabolic pathways regulating acetaldehyde formation, and while potentially accentuated by estradiol in some individuals, how remains to be determined.

Ontological aspects of pluripotency and stemness gene expression pattern in the rhesus monkey.

Two essential aspects of mammalian development are the progressive specialization of cells toward different lineages, and the maintenance of progenitor cells that will give rise to the differentiated components of each tissue and also contribute new cells as older cells die or become injured. The transition from totipotentiality to pluripotentiality, to multipotentiality, to monopotentiality, and then to differentiation is a continuous process during development. The ontological relationship between these different stages is not well understood. We report for the first time an ontological survey of expression of 45 putative "stemness" and "pluripotency" genes in rhesus monkey oocytes and preimplantation stage embryos, and comparison to the expression in the inner cell mass, trophoblast stem cells, and a rhesus monkey (ORMES6) embryonic stem cell line. Our results reveal that some of these genes are not highly expressed in all totipotent or pluripotent cell types. Some are predominantly maternal mRNAs present in oocytes and embryos before transcriptional activation, and diminishing before the blastocyst stage. Others are well expressed in morulae or early blastocysts, but are poorly expressed in later blastocysts or ICMs. Also, some of the genes employed to induce pluripotent stem cells from somatic cells (iPS genes) appear unlikely to play major roles as stemness or pluripotency genes in normal embryos.

Frozen-thawed rhesus sperm retain normal morphology and highly progressive motility but exhibit sharply reduced efficiency in penetrating cervical mucus and hyaluronic acid gel.

The preservation of the genetic diversity of captive populations of rhesus monkeys is critical to the future of biomedical research. Cryopreservation of rhesus macaque sperm is relatively simple to perform, yields high post-thaw motility, and theoretically, provides via artificial insemination (AI) a way to easily transfer genetics among colonies of animals. In the interest of optimizing semen cryopreservation methods for use with vaginal AI, we evaluated the ability of frozen-thawed rhesus sperm to penetrate periovulatory cervical mucus (CM). Motile sperm concentration of pre-freeze ("fresh") and post-thawed ("thawed") samples from five different males were normalized for both computer assisted sperm motion analysis and CM penetration experiments. Sperm samples were deposited into slide chambers containing CM or gel composed of hyaluronic acid (HA) as a surrogate for CM and numbers of sperm were recorded as they entered a video field a preset distance from the sperm suspension-CM (or HA) interface. Fresh and thawed sperm were dried on glass slides, "Pap"-stained, and assessed for changes in head dimensions and head and flagellar shape. While retaining better than 80% of fresh sperm progressive motility, thawed sperm from the same ejaculate retained on average only 18.6% of the CM penetration ability. Experiments using HA gel yielded similar results only with reduced experimental error and thus improved detection of treatment differences. Neither the percentage of abnormal forms nor head dimensions differed between fresh and thawed sperm. While findings suggests that sperm-CM interaction is a prominent factor in previous failures of vaginal AI with cryopreserved macaque sperm, neither sperm motility nor morphology appears to account for changes in the ability of cryopreserved sperm to penetrate CM. Our data points to a previously unidentified manifestation of cryodamage which may have implications for assessment of sperm function beyond the cervix and across mammalian species.

1H NMR to investigate metabolism and energy supply in rhesus macaque sperm.

Sperm ATP is derived primarily from either glycolysis or mitochondrial oxidative phosphorylation. In the present studies, (1)H NMR spectroscopy was used to characterize the metabolite profile in primate sperm treated either with alpha-chlorohydrin (ACH), a known inhibitor of sperm glycolysis or pentachlorophenol (PCP), an uncoupler of oxidative phosphorylation. Sperm were collected from monkeys in the fall and spring, washed and incubated with either the media control, ACH (0.5mM) or PCP (50 microM). Using principal components analysis, PC1 scores plot indicated that the greatest level of variance was found between fall and spring samples and not chemical-treated samples. However, PC4 scores plot did show a consistent effect of ACH treatment. From the PC1 loadings plot, metabolites contributing to the seasonal differences were higher levels of formate in the fall and higher levels of carnitine and acetylcarnitine in the spring as well as possible differences in lipoprotein content. The PC4 loadings plot indicated that ACH treatment decreased lactate and ATP consistent with inhibition of glycolysis. Carnitine also was decreased and acetylcarnitine increased although the latter was not statistically significant. With PCP-treated sperm, no difference between control and treated samples could be discerned suggesting either that primate sperm are insensitive to uncoupling agents or that glycolysis played the more important role in maintaining sperm ATP levels. Overall, NMR studies may prove useful in the development of metabolomic markers that signal sperm metabolic impairments and have the potential to provide useful biomarkers for reproductive health.

Association of luteinizing hormone receptor gene expression with cell cycle progression in granulosa cells.

During hormonally induced ovarian follicle growth, granulosa cell proliferation increases and returns to baseline prior to the administration of an ovulatory stimulus. Several key genes appear to follow a similar pattern, including the luteinizing hormone receptor (LHCGR), suggesting an association between cell cycle progression and gene expression. The expression of LHCGR mRNA in granulosa cells isolated from immature rats and treated in culture with FSH increased in a time-dependent manner, whereas administration of the cell cycle inhibitor mimosine completely suppressed expression. Although forskolin was able to induce luteinization in cells treated with mimosine, human chorionic gonadotropin had no effect, indicating the functional loss of LHCGR. The effects of mimosine on cell cycle progression and LHCGR mRNA expression were reversible within 24 h of mimosine removal. Cell cycle inhibition did not alter the stability of LHCGR mRNA, indicating that the primary effect was at the transcriptional level. To determine whether the relationship between LHCGR expression and cell cycle were relevant in vivo, immature rats were given a bolus of PMSG, followed by a second injection of either saline or PMSG 24 h later to augment levels of proliferation. The expression of LHCGR mRNA was elevated in the ovaries of animals receiving a supplement of PMSG. Mimosine also blocked cell cycle progression and LHCGR mRNA expression in macaque granulosa cells isolated following controlled ovarian stimulation cycles and in two different mouse Leydig tumor lines. These data collectively indicate that LHCGR mRNA is expressed as a function of the passage of cells across the G1-S phase boundary.

Role for cumulus cell-produced EGF-like ligands during primate oocyte maturation in vitro.

The developmental competence of in vitro-matured (IVM) rhesus macaque cumulus oocyte complexes (COCs) is deficient compared with in vivo-matured (IVM) oocytes. To improve oocyte quality and subsequent embryo development following IVM, culture conditions must be optimized. A series of experiments was undertaken to determine the role of epidermal growth factor (EGF) during IVM of rhesus macaque COCs. The addition of Tyrphostin AG-1478 (a selective inhibitor of the EGF receptor EGFR) to the IVM medium yielded fewer oocytes maturing to metaphase II of meiosis II (MII), decreased cumulus expansion, and a lower percentage of embryos that developed to the blastocyst stage compared with untreated IVM controls, indicating that EGFR activation is important for IVM maturation in the rhesus macaque. However, the addition of recombinant human EGF (r-hEGF) to the IVM medium did not enhance outcome. The expression of mRNAs encoding the EGF-like factors amphiregulin, epiregulin, and betacellulin in cumulus cells indicates that these factors produced by cumulus cells may be responsible for maximal EGFR activation during oocyte maturation, precluding any further effect of exogenous r-hEGF. Additionally, these results illustrate the potential futility of exogenous supplementation of IVM medium without prior knowledge of pathway activity.

Effects of environmental tobacco smoke in vivo on rhesus monkey semen quality, sperm function, and sperm metabolism.

The objective of this study was to use a non-human primate model to examine the effect of environmental tobacco smoke (ETS) in vivo on semen quality, sperm function, and sperm metabolism. Four adult rhesus macaques (Macaca mulatta) were exposed to ETS for six months, and semen samples were collected every week for evaluation. ETS exposure in vivo did not affect semen quality and sperm function. The sperm X:Y chromosome ratio remained unchanged after ETS exposure. The sex ratio of the embryos fertilized by ETS-exposed males was not different from the control male. However, sperm showed changes in metabolome detected by NMR during the ETS exposure. We concluded that with the duration and level of ETS exposure in this study, semen quality and sperm function were not affected, whereas sperm did undergo metabolic changes with ETS exposure in vivo.

Expression of scavenger receptor-BI and low-density lipoprotein receptor and differential use of lipoproteins to support early steroidogenesis in luteinizing macaque granulosa cells.

An ovulatory hCG stimulus to rhesus macaques undergoing controlled ovarian stimulation protocols results in a rapid and sustained increase in progesterone synthesis. The use of lipoproteins as a substrate for progesterone synthesis remains unclear, and the expression of lipoprotein receptors [very-low-density lipoprotein receptor (VLDLR), low-density lipoprotein receptor (LDLR), and scavenger receptor-BI (SR-BI)] soon after human chorionic gonadotropin (hCG) (<12 h) has not been characterized. This study investigated lipoprotein receptor expression and lipoprotein (VLDL, LDL, and HDL) support of steroidogenesis during luteinization of macaque granulosa cells. Granulosa cells were aspirated from rhesus monkeys undergoing controlled ovarian stimulation before or up to 24 h after an ovulatory hCG stimulus. The expression of VLDLR decreased within 3 h of hCG, whereas LDLR and SR-BI increased at 3 and 12 h, respectively. Granulosa cells isolated before hCG were cultured for 24 h in the presence of FSH or FSH plus hCG with or without VLDL, LDL, or HDL. Progesterone levels increased in the presence of hCG regardless of lipoprotein addition, although LDL, but not HDL, further augmented hCG-induced progesterone. Other cells were cultured with FSH or FSH plus hCG without an exogenous source of lipoprotein for 24 h, followed by an additional 24 h culture with or without lipoproteins. Cells treated with hCG in the absence of any lipoprotein were unable to maintain progesterone levels through 48 h, whereas LDL (but not HDL) sustained progesterone synthesis. These data suggest that an ovulatory stimulus rapidly mobilizes stored cholesterol esters for use as a progesterone substrate and that as these are depleted, new cholesterol esters are obtained through an LDLR- and/or SR-BI-mediated mechanism.

Sperm mitochondrial integrity is not required for hyperactivated motility, zona binding, or acrosome reaction in the rhesus macaque.

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 microM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.

Blastocyst-derived trophoblast stem cells from the rhesus monkey.

Although trophoblast stem cells can be obtained directly from blastocyst outgrowths in the mouse, this has never been described in primates. In human and non-human primates, trophoblast cells have been obtained from embryonic stem (ES) cells or embryoid bodies (EBs). The results reported here show for the first time that cells with the characteristics of trophoblast stem cells can be derived directly from rhesus monkey blastocyst outgrowths. The cells expressed trophoblast markers and were maintained for multiple passages in the absence of feeder layers or growth factors. The cells could be maintained as adherent, mononuclear cells by regular passaging, but they formed syncytial-like structures if maintained in culture for prolonged periods or if incubated in the presence of 17beta-estradiol. The cells also demonstrated invasive behavior similar to extravillous trophoblasts. The availability of these lines provides a useful experimental system for studying trophoblast differentiation and for developing novel intervention strategies to treat placental dysfunction.

Therapeutic cloning: status and prospects.

Embryonic stem cells (ESCs) offer a new and remarkable potential for treating and curing a wide range of genetic diseases such as diabetes and muscular dystrophy, degenerative diseases such as Parkinson's disease, renal disease and heart disease, and traumatic injury such as spinal cord injury. Therapeutic cloning, wherein patient-specific ESCs can be derived from pre-implantation stage embryos produced by somatic cell nuclear transfer, constitutes one approach of obtaining histocompatible cells for engraftment. Recent improvements in the production of cloned embryos in non-human primate models, combined with advances in the ability to establish human ESC lines and direct their differentiation along specific pathways support the notion that therapeutic cloning may soon be feasible. This review summarizes the status and current feasibility of the approach and the technical hurdles that must be addressed, and discusses the ethical issues that have arisen as a result.

Effects of environmental tobacco smoke in vitro on rhesus monkey sperm function.

The objective of this study was to use a non-human primate model to examine the effect of ETS on sperm function. Sperm samples were collected from adult rhesus monkeys (Macaca mulatta) and treated with different levels of ETS exposed medium. ETS treatment decreased the percentage of motile sperm and motion parameters. Sperm treated with ETS exposed medium showed a limited response to the activators and exhibited decreased binding to the zonae pellucida after activation. The mitochondrial integrity of the ETS-treated sperm was disrupted; however, there was no decrease in viability compared to control groups. Sperm acrosomal status was similar in the control and treatment groups. The results imply that the exposure of primate sperm to ETS could impair sperm transport in vivo.

In vitro exposure to environmental tobacco smoke induces CYP1B1 expression in human luteinized granulosa cells.

Women smokers and women exposed to environmental tobacco smoke (ETS) have reduced ovarian function as evidenced by an earlier menopause, reduced follicular numbers, decreased levels of circulating estradiol, and decreased conception rates; however, the mechanism of action of altered ovarian function by ETS is poorly understood. The direct effects of ETS were evaluated using human luteinized granulosa cells (HLGCs) exposed to ETS in primary cell culture. Exposure to ETS caused a decrease in both estradiol and progesterone production. There was a concentration dependent increase in CYP1B1 gene and protein expression without a change in catechol-O-methyltransferase (COMT) expression. This is the first report of CYP1B1 induction secondary to ETS exposure in cells from the human ovary. CYP1B1 metabolizes both endogenous estrogens and polyaromatic hydrocarbons in ETS to a variety of reactive species and may contribute to the complex effects of ETS on ovarian function.

Interactions of macaque blastocysts with epithelial cells in vitro.

BACKGROUND: Early in vitro studies of blastocyst formation in several primate species have demonstrated the feasibility of such studies. Initial studies of in vitro-fertilized oocytes cultured with buffalo rat liver cells suggested that other epithelial cells might be used to assess blastocyst adherence and penetration in vitro. METHODS: Macaque blastocysts were incubated with different epithelial cell lines or with Matrigel. The interaction was studied using light and transmission electron microscopy. RESULTS: In general, zona-free blastocysts attached 2 days after placing on the substrates. MDCK cells provided optimal conditions for blastocyst development. The best preparations showed some development of an amniotic cavity and distribution of cytotrophoblast and syncytial trophoblast. Distribution of syncytial trophoblast at the margin of the site and cytotrophoblast centrally was similar to that seen at the trophoblastic plate stage in this species. However, there was less syncytial trophoblast than is normally found at this stage, and total time from fertilization to the trophoblastic plate stage was delayed 2 days. CONCLUSIONS: While in vitro studies with blastocysts cannot completely mimic the intrauterine environment, they can illustrate some of the potential interactions and provide a situation in which parameters may be manipulated.

Effect of cooling and exposure to ethylene glycol on in vitro maturation and embryo development of rhesus oocytes.

The meiotic spindle of metaphase II-stage oocytes is damaged when mature oocytes are cooled to temperatures close to 0 degrees C, as occurs during cryopreservation by equilibrium cooling. Since a spindle has not yet formed within a germinal vesicle-stage oocyte, it has been suggested that immature oocytes may be more resistant than metaphase II oocytes to cryopreservation by equilibrium cooling. To test this proposition, we examined the effects on rhesus macaque oocytes of chilling and exposure to ethylene glycol (EG) on their maturation and embryo development. A total of 202 cumulus-intact oocytes was collected from adult female rhesus monkeys that had been given follicle stimulating hormone for controlled ovarian hyperstimulation. Within two hours of their having been aspirated and prior to germinal vesicle breakdown, oocytes were either cooled to 0 degrees C for 10 minutes or were exposed for 15 minutes at 35 degrees C to 1.5 M EG to be tested as a possible cryoprotectant. After being exposed, oocytes were cultured in maturation medium, fertilized in vitro with rhesus spermatozoa, and cultured. The maturation rate and subsequent development into blastocysts of those oocytes that had been exposed to EG or cooled to 0 degrees C did not differ significantly from untreated control oocytes. Additional germinal vesicle oocytes were exposed to 1.5 M EG at 35 degrees C for 3 minutes and then supercooled to -7 degrees C or frozen at -7 degrees C or frozen at 0.5 degrees C to -35 degrees C. Rates of maturation and embryo development of oocytes cooled to or frozen at -7 degrees C were significantly lower than rates for control oocytes; none of those frozen to -35 degrees C even underwent maturation. These results suggest that germinal vesicle-stage oocytes may be less susceptible to injury resulting from chilling or exposure to ethylene glycol, but are still damaged by freezing.