Spermiation, the release of late spermatids from the Sertoli cell, is disrupted by a number of toxicants. Control of the spermiation process, and the proteins that interact to adhere mature spermatids to Sertoli cells, is poorly understood. In these studies we used immunohistochemistry, coimmunoprecipitation/Western blotting, and mass spectrometry to refine an earlier model of sperm adhesion proposed by our laboratory. We have identified specific proteins linked together as part of a multiprotein complex, as well as several additional proteins (cortactin, ERK1/2, and 14-3-3 zeta) that may be functioning in both structural and signal transduction roles. The current and prior data suggest that protein phosphorylation is central to the control of spermiation. We also present and characterize an in vitro tubule culture system that allowed functional testing of the spermiation model by pharmacologic manipulation, and yielded data consistent with the importance of protein phosphorylation in spermiation.

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In order to address data gaps identified by the NAS report Pesticides in the Diets of Infants and Children, a study was performed using methoxychlor (MXC). Female rats were gavaged with MXC at 0, 5, 50, or 150 mg/kg/day for the week before and the week after birth, whereupon the pups were directly dosed with MXC from postnatal day (pnd) 7. Some dams were killed pnd7 and milk and plasma were assayed for MXC and metabolites. For one cohort of juveniles, treatment stopped at pnd21; a modified functional observational battery was used to assess neurobehavioral changes. Other cohorts of juveniles were dosed until pnd42 and evaluated for changes to the immune system and for reproductive toxicity. Dose-dependent amounts of MXC and metabolites were present in milk and plasma of dams and pups. The high dose of MXC reduced litter size by approximately 17%. Ano-genital distance was unchanged, although vaginal opening was accelerated in all treated groups, and male prepuce separation was delayed at the middle and high doses by 8 and 34 days, respectively. In the neurobehavioral evaluation, high-dose males were more excitable, but other changes were inconsistent and insubstantial. A decrease in the antibody plaque-forming cell response was seen in males only. Adult estrous cyclicity was disrupted at 50 and 150 MXC, doses which also showed reduced rates of pregnancy and delivery. Uterine weights (corrected for pregnancy) were reduced in all treated pregnant females. High-dose males impregnated fewer untreated females; epididymal sperm count and testis weight were reduced at the high, or top two, doses, respectively. All groups of treated females showed uterine dysplasias and less mammary alveolar development; estrous levels of follicle stimulating hormone were lower in all treated groups, and estrus progesterone levels were lower at 50 and 150 MXC, attributed to fewer corpora lutea secondary to ovulation defects. These data collectively show that the primary adult effects of early exposure to MXC are reproductive, show that 5 mg/kg/day is not a NO(A)EL in rats with this exposure paradigm (based on changes in day of vaginal opening, pubertal ovary weights, adult uterine and seminal vesicle weights, and female hormone data) and imply that the sites of action are both central and peripheral.

Previous studies have indicated that the androgen receptor antagonist, flutamide, can produce a suite of reproductive malformations in the male rat when orally administered daily on gestation days (GD) 12-21. The objective of this study was to investigate the gestation time dependence for the induction of these malformations to establish a robust animal model for future studies of gene expression related to specific malformations. Groups of timed-pregnant Sprague-Dawley rats (GD 0 = day of mating) were administered flutamide as a single gavage dose (50 mg/kg) on GD 16, 17, 18, or 19 with 10 dams per group. Control animals (5 dams per time per group) were administered corn oil vehicle (2 ml/kg). Dams were allowed to litter, and their adult male offspring were killed at postnatal day (PND) 100 +/- 10. Anogenital distance was measured at PND 1 and 100. Areolae were scored at PND 13, and permanent nipples evaluated at PND 100. No reproductive tract malformations were found in control male offspring. In the treated groups, malformations were noted following exposure at every GD, although the incidence of specific malformations varied by GD. At GD 16, the highest incidence was noted for permanent nipples (46% pups, 60% litters), epispadias (12% pups, 30% litters), and missing epididymal components (5% pups, 20% litters). The highest incidences for hypospadias (58% pups, 80% litters), vaginal pouch (49% pups, 70% litters), cleft prepuce (29% pups, 60% litters), and missing prostate lobes (12% pups, 60% litters) were noted at GD 17. At GD 18 the highest incidence of malformations noted were epispadias (5% pups, 30% litters), reduced prostate size (32% pups, 90% litters), and abnormal kidneys (3% pups, 30% litters) and bladders (7% pups, 30% litters), while on GD 19 70% of the litters had animals with abnormal seminal vesicles. Testicular and epididymal morphological changes were noted at all GDs and were consistent with the gross observations and peaked in incidence and severity on GD17. The major discrepancy between this study and previous multiple-dose studies was in the very few numbers of animals presenting with cryptorchidism (only one each on GDs 16 and 17), suggesting that exposure over multiple days may be required to induce this malformation. Thus, a single gestational exposure of flutamide induced numerous reproductive tract malformations consistent with previously reports following multiple exposures, with the timing of the exposure producing marked tissue selectivity in the response noted in adult offspring.

Retinoic acid (RA) has been shown to be teratogenic in many species, and 13-cis-RA is teratogenic in humans. Exposure to RA during embryonic morphogenesis produced a variety of malformations including limb defects and cleft palate. The type and severity of malformation depended on the stage of development exposed. The purpose of this study was to compare the effects of RA exposure in vivo on different stages of palate development. These results were compared to effects observed after exposure in organ culture. The vehicle used in RA dosing was also shown to be a major factor in the incidence of RA-induced cleft palate. For the in vivo studies, RA (100 mg/kg) in 10 ml corn oil/kg was given p.o. on gestation day (GD) 10 or 12, and the embryos were examined on GD 14 and 16. Exposure to RA in an oil:DMSO vehicle resulted in much higher incidences of cleft palate than were observed after dosing with RA in oil only. After exposure on GD 10, to RA, small palatal shelves formed which did not make contact and fuse on GD 14. The medial cells did not undergo programmed cell death. Instead, the medial cells differentiated into a stratified, squamous, oral-like epithelium. The RA-exposed medial cells did not incorporate 3H-TdR on GD 14 or 16, but the cells expressed EGF receptors and bound 125I-EGF. In contrast, RA-induced clefting after exposure on GD 12 did not involve growth inhibition. Shelves of normal size formed and made contact, but because of altered medial cell differentiation did not fuse. Medial cells differentiated into a pseudostratified, ciliated, nasal-like epithelium. This response was produced in vivo at exposure levels which produced cleft palate, and after exposure of palatal shelves to RA in vitro from GD 12-15. The medial cells exposed on GD 12 incorporated 3H-TdR on GD 14, expressed EGF receptors, and bound 125I-EGF. The responses to RA which lead to cleft palate differed after exposure on GD 10 or 12, and the pathways of differentiation which the medial cells followed depended on the developmental stage exposed.

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