Patricia B. Hoyer

Reproductive toxicology: current and future directions

During the 20th century, there has been an increased risk from environmental by-products that may be harmful to reproductive function in humans. Therefore, as the 21st century begins, it is appropriate to evaluate future directions within the field of reproductive toxicology. This commentary identifies several approaches and developing technologies that would help research continue in a meaningful direction. Four areas for development are suggested, and selected examples of research involved in those areas are discussed: (1) Translational applications: workplace exposures thought to cause infertility in men (1,2-dibromo-3-chloropropane, DBCP) and menstrual disturbances in women (2-bromopropane, 2BP) are given as examples of human effects that have prompted animal studies. (2) Exposure paradigms: extrapolating dosing in animals to exposures in humans becomes complex. Two examples of surprising findings using lower doses are cited: ovotoxicity caused by polycyclic aromatic hydrocarbons (PAHs), and disrupted sexual differentiation caused by the fungicide vinclozolin. (3) Gender differences: predicting variable risk between women and men requires investigation of the effects of reproductive toxicants in both genders. The phthalates provide a good example for this comparison. Whereas di-(2-ethylhexyl)phthalate (DEHP) is a reproductive toxicant working by similar mechanisms in males and females, di-n-butyl phthalate (DBP) produces developmental effects in males and reproductive tract effects in females. (4) Endocrine disruptors: recent research has identified environmental chemicals that disrupt reproductive processes by altering the actions of endogenous steroid hormones. The endocrine disruptor issue is discussed in terms of evaluation of the actual risk these chemicals may pose in humans.

The Follicle-Deplete Mouse Ovary Produces Androgen

Abstract The follicle-depleted postmenopausal ovary is enriched in interstitial cells that produce androgens. This study was designed to cause follicle depletion in mice using the industrial chemical, 4-vinylcyclohexene diepoxide (VCD), and characterize the steroidogenic capacity of cells in the residual ovarian tissue. From a dose-finding study, the optimal daily concentration of VCD was determined to be 160 mg/kg. Female B6C3F1 immature mice were treated daily with vehicle control or VCD (160 mg kg−1 day−1, 15 days, i.p.). Ovaries were removed and processed for histological evaluation. On Day 15 following onset of treatment, primordial follicles were depleted and primary follicles were reduced to about 10% of controls. On Day 46, primary follicles were depleted and secondary and antral follicles were reduced to 0.7% and 2.6% of control, respectively. Seventy-five percent of treated mice displayed disruptions in estrous cyclicity. All treated mice were in persistent diestrus (acyclic) by Day 58. Plasma FSH levels were increased (P < 0.05) relative to controls on Day 37 and had plateaued by Day 100. Relative to age-matched cyclic controls, by Day 127, the significant differences in VCD-treated mice included reduced ovarian and uterine weights, elevated plasma LH and FSH, and reduced plasma progesterone and androstenedione. Furthermore, plasma 17β-estradiol levels were nondetectable. Unlike controls, immunostaining for LH receptor, and the high density lipoprotein receptor (SR-BI), was diffuse in ovarian sections from VCD-treated animals. Ovaries from Day 120 control and VCD-treated animals were dissociated and dispersed cells were placed in culture. Cultured cells from ovaries of VCD-treated animals produced less LH-stimulated progesterone than control cells. Androstenedione production was nondetectable in cells from cyclic control animals. Conversely, cells from VCD-treated animals produced androstenedione that was doubled in the presence of insulin and LH (1 and 3 ng/ml). Collectively, these data demonstrate that VCD-mediated follicle depletion results in residual ovarian tissue that may be analogous to the follicle-deplete postmenopausal ovary. This may serve as a useful animal model to examine the dynamics of follicle loss in women as ovarian senescence ensues.

Cloning of a Carboxyl-terminal Isoform of the Prostanoid FP Receptor

An FP prostanoid receptor isoform, which appears to arise from alternative mRNA splicing, has been cloned from a mid-cycle ovine large cell corpus luteum library. The isoform, named the FPB receptor, is identical to the original isoform, the FPA, throughout the seven transmembrane domains, but diverges nine amino acids into the carboxyl terminus. In contrast to FPA, whose carboxyl terminus continues for another 46 amino acids beyond the nine shared residues, the FPB terminates after only one amino acid. The FPA isoform appears to arise by the failure to utilize a potential splice site, while a 3.2-kilobase pair intron is spliced out from the FP gene to generate the FPB isoform mRNA. The two isoforms have indistinguishable radioligand binding properties, but seem to differ in functional coupling to phosphatidylinositol hydrolysis. Thus, in COS-7 cells transiently transfected with either the FPA or the FPB receptor cDNAs, prostaglandin F2α stimulates inositol phosphate accumulation to the same absolute maximum, but the basal level of inositol phosphate accumulation is approximately 1.3-fold higher in cells transfected with the FPB as compared with cells transfected with the FPA isoform. Using the polymerase chain reaction, mRNA encoding the FPB isoform was identified in the ovine corpus luteum.

Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide

4-vinyl-1-cyclohexene diepoxide (VCD) is known to destroy oocytes in ovaries of immature rats. Since ovaries functionally differ between immature and adult animals, we examined the effect of VCD on oocytes in adult rats. Adult (58 days) and immature (28 days) rats were injected daily (30 days) with vehicle or VCD. Each group contained 10 rats. During this time, cyclicity was determined daily by vaginal cytology. Animals were terminated on day 31 and tissues were collected. Oocytes were counted; livers, spleens, and uteri were weighed. VCD reduced the number of regular estrous cycles/30 days in adults, but not immature rats (n = 20, P < 0.05). VCD reduced the number of oocytes in adult and immature rats (n = 20, P < 0.05). Liver, spleen, or ovarian weights were not affected by VCD in either group. VCD reduced uterine weight in adult (n = 20, P < 0.05) but not in immature rats. These results demonstrate that VCD decreases uterine weight in adult rats and as with immature rats, selectively destroys oocytes in ovaries of adults.

Long-term effects of ovarian follicular depletion in rats by 4-vinylcyclohexene diepoxide

4-Vinylcyclohexene diepoxide (VCD) destroys preantral ovarian follicles in rats. Female 28-day Fisher 344 (F344) rats were dosed (30 days) with VCD (80 mg/kg per day, i.p.) or vehicle, and animals were evaluated for reproductive function at subsequent time points for up to 360 days. At each time point animals were killed, and ovaries and plasma collected. VCD reduced (P<0.05) the number of preantral follicles by day 30 relative to control. There were no ultrastructural differences in morphology between VCD-treated and control ovaries. Circulating FSH levels in VCD-treated animals were greater (days 120, 240, and 360, P<0.05) than in controls. Cyclicity was disrupted in the VCD-treated group by day 360. These results show that VCD-induced follicular destruction in rats is associated with a sequence of events (loss of preantral follicles, increased plasma FSH, and cyclic disruption) preceding premature ovarian senescence that is similar to events that occur during the onset of menopause in women.

Expression and Redistribution of Cellular Bad, Bax, and Bcl-xL Protein Is Associated with VCD-Induced Ovotoxicity in Rats

Abstract Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25–100 μm) and large (100–250 μm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 ± 0.16; mitochondria, 2.20 ± 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-xL protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 ± 0.08; cytosol, 1.39 ± 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-xL in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 ± 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage 1 primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, redistribution of Bcl-xL protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/Bcl-xL ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.

Apoptosis Induced in Rats by 4-Vinylcyclohexene Diepoxide Is Associated with Activation of the Caspase Cascades

Abstract Previous studies have shown that ovotoxicity induced in rats by dosing with 4-vinylcyclohexene diepoxide (VCD) is likely via acceleration of the normal rate of atresia (apoptosis). The present study was designed to investigate the apoptosis-related caspase cascades as a component of this phenomenon in isolated ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., on Day 1; a time when ovotoxicity has not been initiated), or dosed daily for 15 days (80 mg/kg, i.p., on Day 15; a time when significant ovotoxicity is underway). Ovaries were collected after the final dose. Small preantral follicles (25–100 μm in diameter) were isolated, cellular fractions were prepared, and cleavage activity or protein expression levels of caspases-3, -8, and -9 were measured. Cytosolic caspase-3 activity was increased in small follicles (P < 0.01) by VCD treatment (Day 1, 2.86 ± 0.23; Day 15, 3.25 ± 0.64, VCD/control, n = 3). This activation was not seen in large or antral follicles (not targeted by VCD). Procaspase-3 protein was increased(P < 0.05) by VCD treatment 212% over controls in small ovarian follicles in Day 15, but not Day 1-dosed rats. Immunofluorescence staining intensity was evaluated by confocal microscopy. Caspase-3 protein, located in the cytosolic compartment of oocytes and granulosa cells of preantral follicles in various stages of development, was selectively increased (P < 0.05) in primordial and small primary follicles from Day 15 VCD-dosed rats. Caspase-8 activity was increased in small follicles in Day 15, but not in Day 1-treated rats; whereas caspase-9 activity was increased by VCD on Day 1 in the mitochondrial fraction. Thus, these data provide evidence that accelerated atresia induced in small ovarian follicles in rats by VCD is associated with activation of a caspase-mediated cascade.

Early effects of ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats and mice.

The industrial chemical 4-vinylcyclohexene diepoxide (VCD) causes specific destruction of oocyte-containing small preantral follicles (primordial and primary) in ovaries of rats and mice. The mouse seems more susceptible to ovotoxic effects of VCD than the rat. The purpose of this study was to better understand these species differences in susceptibility to VCD by comparing the initial rates of VCD-induced follicle damage and loss in response to dosing in both species. Female Fischer 344 rats and B6C3F1 mice (age, Day 28) were dosed daily (vehicle or 80 mg/kg, i.p.) for 6, 8, 10, or 12 d. Ovaries collected after the final dose were prepared for histologic evaluation. Primordial and primary follicles in ovarian slices were counted and classified as healthy or atretic. A VCD-dependent increase (P < 0.05) in percent atretic primordial follicles was first observed 4 h after the final dose in mice on Day 8 (VCD-treated, 44.4 +/- 3.1% vs. control, 26.9 +/- 5.4%). Conversely, in rats, this significant increase was not seen until Day 10 (VCD-treated, 44.3 +/- 1.3% vs. control, 23.1 +/- 4.0%). A VCD-dependent increase in percent atretic primary follicles was not observed in either species before Day 12. There was no significant effect on growing or preantral follicles on any day in either species. Significant loss of primordial and primary follicles (P < 0.05) was first measured on day 12 in both rats and mice. However, when compared with controls, follicle loss on that day was greater (P < 0.05) in mice (64.2 +/- 4.5%) than in rats (34.7 +/- 4.9%). Once VCD-dependent follicle loss was observed, the rate of follicle damage was similar in rats and mice, and was fairly constant in response to each dose. VCD-induced follicle damage in mice, as with rats, also displayed morphologic characteristics of atresia (apoptosis). In summary, follicle destruction seems to be similar in rats and mice; however, follicle damage is initiated earlier and to a greater extent in mice than in rats. Additionally, ovotoxic effects of VCD seem to initially directly target primordial follicles. These results provide temporal evidence that mice are more susceptible to VCD-induced ovotoxicity than rats.

Expression of superoxide dismutase, catalase and glutathione peroxidase in the bovine corpus luteum: evidence supporting a role for oxidative stress in luteolysis.

Apoptosis, a type of physiological or active cell death, has been implicated as a mechanism underlying regression of the corpus luteum (CL) in the rat, bovine, rabbit and ovine ovary. Previousin vitro studies of cultured luteal cells have also provided evidence which suggests that reactive oxygen species play an important role in luteolysis in the rodent ovary. To further evaluate the potential role of oxidative stress in luteal cell demise, changes in the expression of several enzymes known to protect cells from oxidative stress were investigated using bovine CL collected from ovaries of non-pregnant (day 21 of the estrous cycle; regressed CL) and pregnant (day 21 of pregnancy; functional CL) animals. Biochemical analysis of genomic DNA extracted from these two pools of CL demonstrated the presence of extensive levels of internucleosomal DNA cleavage characteristic of cell death via apoptosis in regressed, but not in functional, CL. Northern blot analysis of total RNA indicated that functional CL expressed significantly higher levels of mRNA encoding secreted superoxide dismutase (SEC-SOD, 1.9 kb) and manganese-containing or mitochondrial SOD (Mn-SOD, multiple transcripts) as compared to regressed CL. Similarly, levels of mRNA encoding catalase (2.1 kb), an enzyme responsible for detoxification of peroxides to water, were significantly higher in functional versus regressed CL. From these data, we conclude that a decline in expression of specific oxidative response genes occurs during luteolysis, and that maintained expression of these genes in the CL during pregnancy may prevent oxidative damage and delay regression.

Ovarian Toxicity of 4-Vinylcyclohexene Diepoxide: A Mechanistic Model

Female mammals are born with a fi nite number of ovarian primordial follicles that cannot be regenerated; thus, chemicals that destroy oocytes contained in these follicles can produce premature ovarian failure (early menopuase in women). Exposure of women to known ovotoxicants, such as contaminants in cigarette smoke, is associated with early menopause. Thus, the potential risks posed by ovotoxic chemicals is of concern. Our studies have focused on the environmental chemical 4-vinylcyclohexene (VCH), which is produced during the manufacture of rubber tires, flame retardants, insecticides, plasticizers, and antioxidants. Dosing of female rats and mice with the ovotoxic diepoxide metabolite of VCH, 4-vinylcyclohexene diepoxide (VCD), for 30 days destroyed the majority of ovarian primordial follicles. Using VCD in rats as a generalized model for ovotoxicity, we determined that 1) repeated daily dosing is required, 2) cell death is via apoptosis, and 3) altered expression of specific genes is involved. An integrated approach at the morphologic, biochemical, and molecular level was used to support these conclusions. Studies in isolated rat small preantral follicles (targeted for VCD-induced ovotoxicity) focused on the role of cell death genes, mitochondrion-associated events, and VCD metabolism. We also evaluated how this information relates to human risk for early menopause. These animal research results provide a better understanding of the potential risk of human exposure to environmental ovarian toxicants and greater insight as to the impact of these toxicants on reproductive health in women.