Episodic ozone exposure in Long-Evans rats has limited effects on cauda sperm motility and non-coding RNA populations.

Epidemiological evidence suggests the potential for air pollutants to induce male reproductive toxicity. In experimental studies, exposure to ozone during sensitive windows in the sperm lifecycle has been associated with impaired sperm motility. Subsequently, we sought to investigate the effects of episodic exposure to ozone during sperm maturation in the rat. Long-Evans rats were exposed to either filtered air or ozone (0.4 or 0.8 ppm) for five non-consecutive days over two weeks. Ozone exposure did not impact male reproductive organ weights or sperm motility ∼24 hours following the final exposure. Furthermore, circulating sex hormones remained unchanged despite increased T3 and T4 in the 0.8 ppm group. While there was indication of altered adrenergic signaling attributable to ozone exposure in the testis, there were minimal impacts on small non-coding RNAs detected in cauda sperm. Only two piwi-interacting RNAs (piRNAs) were altered in the mature sperm of ozone-exposed rats (piR-rno-346434 and piR-rno-227431). Data across all rats were next analyzed to identify any non-coding RNAs that may be correlated with reduced sperm motility. A total of 7 microRNAs (miRNAs), 8 RNA fragments, and 1682 piRNAs correlated well with sperm motility. Utilizing our exposure paradigm herein, we were unable to substantiate the relationship between ozone exposure during maturation with sperm motility. However, these approaches served to identify a suite of non-coding RNAs that were associated with sperm motility in rats. With additional investigation, these RNAs may prove to have functional roles in the acquisition of motility or be unique biomarkers for male reproductive toxicity.

The use of purified rat Leydig cells complements the H295R screen to detect chemical-induced alterations in testosterone production.

Exposure to endocrine disrupting chemicals has been associated with compromised testosterone production leading to abnormal male reproductive development and altered spermatogenesis. In vitro high-throughput screening (HTS) assays are needed to evaluate risk to testosterone production, yet the main steroidogenesis assay currently utilized is a human adrenocortical carcinoma cell line, H295R, which does not synthesize gonadal steroids at the same level as the gonads, thus limiting assay sensitivity. Here, we propose a complementary assay using a highly purified rat Leydig cell assay to evaluate the potential for chemical-induced alterations in testosterone production by the testis. We evaluated a subset of chemicals that failed to decrease testosterone production in the HTS H295R assay. The chemicals examined fit into one of two categories based on changes in substrates upstream of testosterone in the adrenal steroidogenic pathway (17α-hydroxyprogesterone and 11-deoxycorticosterone) that we predicted should have elicited a decrease in testosterone production. We found that 85% of 20 test chemicals examined inhibited Leydig cell testosterone production in our assay. Importantly, we adopted a 96-well format to increase throughput and efficiency of the Leydig cell assay. We identified a selection criterion based on the AC50 values for 17α-hydroxyprogesterone and 11-deoxycorticosterone generated from the HTS H295R assay that will help prioritize chemicals for further testing in the Leydig cell screen. We hypothesize that the greater dynamic range of testosterone production and sensitivity of the Leydig cell assay permits the detection of small, yet significant, chemical-induced changes not detected by the HTS H295R assay.

Method to assess component contribution to toxicity of complex mixtures: Assessment of puberty acquisition in rats exposed to disinfection byproducts.

A method based on regression modeling was developed to discern the contribution of component chemicals to the toxicity of highly complex, environmentally realistic mixtures of disinfection byproducts (DBPs). Chemical disinfection of drinking water forms DBP mixtures. Because of concerns about possible reproductive and developmental toxicity, a whole mixture (WM) of DBPs produced by chlorination of a water concentrate was administered as drinking water to Sprague-Dawley (S-D) rats in a multigenerational study. Age of puberty acquisition, i.e., preputial separation (PPS) and vaginal opening (VO), was examined in male and female offspring, respectively. When compared to controls, a slight, but statistically significant delay in puberty acquisition was observed in females but not in males. WM-induced differences in the age at puberty acquisition were compared to those reported in S-D rats administered either a defined mixture (DM) of nine regulated DBPs or individual DBPs. Regression models were developed using individual animal data on age at PPS or VO from the DM study. Puberty acquisition data reported in the WM and individual DBP studies were then compared with the DM models. The delay in puberty acquisition observed in the WM-treated female rats could not be distinguished from delays predicted by the DM regression model, suggesting that the nine regulated DBPs in the DM might account for much of the delay observed in the WM. This method is applicable to mixtures of other types of chemicals and other endpoints.

Systems Toxicology of Male Reproductive Development: Profiling 774 Chemicals for Molecular Targets and Adverse Outcomes.

BACKGROUND: Trends in male reproductive health have been reported for increased rates of testicular germ cell tumors, low semen quality, cryptorchidism, and hypospadias, which have been associated with prenatal environmental chemical exposure based on human and animal studies. OBJECTIVE: In the present study we aimed to identify significant correlations between environmental chemicals, molecular targets, and adverse outcomes across a broad chemical landscape with emphasis on developmental toxicity of the male reproductive system. METHODS: We used U.S. EPA's animal study database (ToxRefDB) and a comprehensive literature analysis to identify 774 chemicals that have been evaluated for adverse effects on male reproductive parameters, and then used U.S. EPA's in vitro high-throughput screening (HTS) database (ToxCastDB) to profile their bioactivity across approximately 800 molecular and cellular features. RESULTS: A phenotypic hierarchy of testicular atrophy, sperm effects, tumors, and malformations, a composite resembling the human testicular dysgenesis syndrome (TDS) hypothesis, was observed in 281 chemicals. A subset of 54 chemicals with male developmental consequences had in vitro bioactivity on molecular targets that could be condensed into 156 gene annotations in a bipartite network. CONCLUSION: Computational modeling of available in vivo and in vitro data for chemicals that produce adverse effects on male reproductive end points revealed a phenotypic hierarchy across animal studies consistent with the human TDS hypothesis. We confirmed the known role of estrogen and androgen signaling pathways in rodent TDS, and importantly, broadened the list of molecular targets to include retinoic acid signaling, vascular remodeling proteins, G-protein coupled receptors (GPCRs), and cytochrome P450s. CITATION: Leung MC, Phuong J, Baker NC, Sipes NS, Klinefelter GR, Martin MT, McLaurin KW, Setzer RW, Darney SP, Judson RS, Knudsen TB. 2016. Systems toxicology of male reproductive development: profiling 774 chemicals for molecular targets and adverse outcomes. Environ Health Perspect 124:1050-1061; http://dx.doi.org/10.1289/ehp.1510385.

Reproductive toxicity of a mixture of regulated drinking-water disinfection by-products in a multigenerational rat bioassay.

BACKGROUND: Trihalomethanes (THMs) and haloacetic acids (HAAs) are regulated disinfection by-products (DBPs); their joint reproductive toxicity in drinking water is unknown. OBJECTIVE: We aimed to evaluate a drinking water mixture of the four regulated THMs and five regulated HAAs in a multigenerational reproductive toxicity bioassay. METHODS: Sprague-Dawley rats were exposed (parental, F1, and F2 generations) from gestation day 0 of the parental generation to postnatal day (PND) 6 of the F2 generation to a realistically proportioned mixture of THMs and HAAs at 0, 500×, 1,000×, or 2,000× of the U.S. Environmental Protection Agency's maximum contaminant levels (MCLs). RESULTS: Maternal water consumption was reduced at ≥ 1,000×; body weights were reduced at 2,000×. Prenatal and postnatal survival were unaffected. F1 pup weights were unaffected at birth but reduced at 2,000× on PND6 and at ≥ 1,000× on PND21. Postweaning F1 body weights were reduced at 2,000×, and water consumption was reduced at ≥ 500×. Males at 2,000× had a small but significantly increased incidence of retained nipples and compromised sperm motility. Onset of puberty was delayed at 1,000× and 2,000×. F1 estrous cycles and fertility were unaffected, and F2 litters showed no effects on pup weight or survival. Histologically, P0 (parental) dams had nephropathy and adrenal cortical pathology at 2,000×. CONCLUSIONS: A mixture of regulated DBPs at up to 2,000× the MCLs had no adverse effects on fertility, pregnancy maintenance, prenatal survival, postnatal survival, or birth weights. Delayed puberty at ≥ 1,000× may have been secondary to reduced water consumption. Male nipple retention and compromised sperm motility at 2,000× may have been secondary to reduced body weights.

Novel molecular events associated with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat.

Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and testis (intact) proteomes in rats after 3 days of exposure. The adrenal accounted for most of the serum progesterone and all of the corticosterone increases in intact and castrated males. Serum luteinizing hormone, androstenedione, and testosterone in intact males shared a non-monotonic response suggesting transition from an acute stimulatory to a latent inhibitory response to exposure. Eight adrenal proteins were significantly altered with dose. There were unique proteomic changes between the adrenals of intact and castrated males. Six testis proteins in intact males had non-monotonic responses that significantly correlated with serum testosterone. Different dose-response curves for steroids and proteins in the adrenal and testis reveal novel adverse outcome pathways in intact and castrated male rats.

Phthalate-induced pathology in the foetal testis involves more than decreased testosterone production.

Foetal exposure to phthalates is known to adversely impact male reproductive development and function. Developmental anomalies of reproductive tract have been attributed to impaired testosterone synthesis. However, species differences in the ability to produce testosterone have been noted; e.g., following foetal exposure, abnormal clustering of Leydig cells or decreased production of testosterone that is manifested in rats does not occur in mice or humans. Nonetheless, other facets of testicular dysgenesis occur in both rats and mice as well as in some other species tested. We recently published a comprehensive evaluation of the foetal rat testis proteome, following in utero exposure to diethylhexyl phthalate (DEHP), which revealed changes in individual proteins that are known to be factors in cellular differentiation and migration or related to the capacity of the foetal Leydig cell to produce testosterone and fit a pathway network in which each is regulated directly or indirectly by oestradiol. Plasma oestradiol indeed was found to be elevated approximately twofold in 19-day-old DEHP-exposed foetal male rats. In this brief review, we discuss our new findings vis-à-vis 'oestrogen hypothesis' as a cause for testicular dysgenesis syndrome.

Comprehensive assessment of a chlorinated drinking water concentrate in a rat multigenerational reproductive toxicity study.

Some epidemiological studies report associations between drinking water disinfection byproducts (DBPs) and adverse reproductive/developmental effects, e.g., low birth weight, spontaneous abortion, stillbirth, and birth defects. Using a multigenerational rat bioassay, we evaluated an environmentally relevant "whole" mixture of DBPs representative of chlorinated drinking water, including unidentified DBPs as well as realistic proportions of known DBPs at low-toxicity concentrations. Source water from a water utility was concentrated 136-fold, chlorinated, and provided as drinking water to Sprague-Dawley rats. Timed-pregnant females (P0 generation) were exposed during gestation and lactation. Weanlings (F1 generation) continued exposures and were bred to produce an F2 generation. Large sample sizes enhanced statistical power, particularly for pup weight and prenatal loss. No adverse effects were observed for pup weight, prenatal loss, pregnancy rate, gestation length, puberty onset in males, growth, estrous cycles, hormone levels, immunological end points, and most neurobehavioral end points. Significant, albeit slight, effects included delayed puberty for F1 females, reduced caput epidydimal sperm counts in F1 adult males, and increased incidences of thyroid follicular cell hypertrophy in adult females. These results highlight areas for future research, while the largely negative findings, particularly for pup weight and prenatal loss, are notable.

Androgen deprivation from pre-puberty to peripuberty interferes in proteins expression in pubertal and adult rat epididymis.

Few studies have focused on experimental testosterone deprivation in immature animals. Therefore, this study used sexually immature rats aiming to evaluate the testes and epididymis histology and proteins expression in these organs on PND50 and 75, after premature antiandrogen exposure, from PND21 to 44. Although the androgen deprivation from pre-puberty up to peripuberty did not alter the histological organization of the testes and epididymis either at puberty or at adulthood, the treatment impaired the expression of specific proteins in epididymal tissue at puberty and adulthood (androgen receptor, calmodulin, Rab11A). These changes may be related to impaired epididymal function, sperm quality and fertility capacity as observed in a previous study. Further studies are necessary to better investigate the molecular mechanisms involved in the impairment on reproductive competence of male rats after precocious hormonal injury.

Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol.

Significant research has been focused on phthalate-induced alterations in male reproductive development. Studies on rodents have prompted the notion that a syndrome exists in the human male which includes phenotypic alterations such as hypospadias, cryptorchidism, poor semen quality, and even testicular cancer. Each phenotype in this 'testicular dysgenesis syndrome' is predicated on reduction in testosterone production by the fetal Leydig cell. We sought to examine the relationship between dysgenesis and steroidogenic capacity in the fetal rat testis more stringently by incorporating lower exposures than those typically used, conducting a comprehensive, non-targeted quantitative evaluation of the fetal testis proteome, and relating alterations in individual proteins to the capacity of the fetal Leydig cell to produce testosterone, and histopathology of the fetal testis. Pregnant dams were dosed orally from gestation day (GD) 13-19 with 0, 10, or 100 mg diethylhexyl phthalate (DEHP)/kg body weight per day. Each endpoint was represented by 16l. Clustering of Leydig cells occurred before any significant decrease in the capacity of the GD19 Leydig cell to produce testosterone. At 100 mg DEHP/kg, testosterone production was reduced significantly, Leydig cell clusters became quite large, and additional dysgenetic changes were observed in the fetal testis. Of 23 proteins whose expression was altered significantly at both DEHP exposure levels, seven were found to be correlated with and predictive of the quantified endpoints. None of these proteins have been previously implicated with DEHP exposure. Notably, pathway analysis revealed that these seven proteins fit a pathway network in which each is regulated directly or indirectly by estradiol.

Impairment on sperm quality and fertility of adult rats after antiandrogen exposure during prepuberty.

This study evaluated the effects of antiandrogen exposure during the prepubertal period on reproductive development and reproductive competence in adults. Male rats were divided into two groups: flutamide, receiving 25 mg/kg/day of flutamide by oral gavage and control, receiving vehicle daily. Dosing continued from PND 21 to 44, and animals were killed on PND 50 or PND 75-80. The epididymis, prostate, vas deferens and seminal vesicle weights were lower in Flutamide group on PND 50, while on PND 80 only seminal vesicle weight was reduced. Fertility assessed by IUI revealed a decrease in the fertility potential in the flutamide-treated adults. Flutamide accelerated sperm transit time through the epididymis, impairing sperm motility and storage. A quantitative analysis of the cauda sperm membrane proteome revealed a few significant changes in protein expression. Thus, exposure to flutamide during the prepubertal period compromises the function of the epididymis along with epididymal sperm quality at adulthood.

Gestational atrazine exposure: effects on male reproductive development and metabolite distribution in the dam, fetus, and neonate.

Few studies have investigated the long-term effects of atrazine (ATR) following in utero exposure. We evaluated the effects of gestational exposure of Sprague Dawley dams to ATR (0, 1, 5, 20, or 100mg/kg-d) on the reproductive development of male offspring. We also quantified the distribution of ATR and its chlorinated metabolites in maternal, fetal, and neonatal fluid and tissue samples following gestational and/or lactational exposure. Dose-dependent levels of chlorotriazines, primarily diamino-s-chlorotriazine, were present in most samples analyzed, including fetal tissue. In utero exposure to 1-20mg/kg-d ATR did not alter testosterone production, the timing of puberty, play behavior, or other androgen-dependent endpoints of male offspring. Significant maternal toxicity and postnatal mortality were observed at 100mg/kg-d. We conclude that, although levels of chlorotriazines within the fetus were considerable, gestational exposures of 1-20mg/kg-d do not lead to alterations in the measures of male development examined in this study.

Saga of a sperm fertility biomarker.

A decade ago a novel sperm protein associated with the fertility of sperm was discovered by quantifying individual proteins in the sperm membrane proteome of cauda epididymal sperm from rats exposed to epididymal toxicants that compromised the fertility of these sperm. Upon identification, this protein (SP22) was found to a ubiquitous, highly conserved protein never before observed in the male reproductive tract. The expression of SP22 in sperm appears driven by a testis specific mRNA transcript, and the molecule is translocated from the cytoplasmic droplet of rete testis sperm to the equatorial segment of epididymal and ejaculated sperm. The appearance of SP22 mRNA and protein coincide with the formation of pachytene spermatocytes and round spermatids, respectively, and given this testis ontogeny of SP22, we validated its use as a biomarker of fertility by extending our studies to toxicants that target spermiogenesis. Studies of both epididymal and testicular toxicants now have demonstrated that compromised SP22 gene expression is sensitive and correlated with fertility. Importantly, this applies to ejaculated sperm as well as epididymal sperm. With the goal of developing a user-friendly diagnostic assay for SP22 on epididymal and ejaculated sperm, we are attempting to identify exposed, functional domains of the protein. For this, we have generated antibodies to both full length and truncated SP22 recombinants, as well as antibodies to synthetic SP22 peptides. Each antibody has been characterized for its ability to inhibit fertilization both in utero and in vitro. Linear epitope mapping has been done for each antibody, and synthetic peptides corresponding to each epitope have been used in competition experiments designed to elucidate exposure on the sperm surface and function. Most of the linear epitopes identified appear to be exposed although there are relative differences in the degree of their exposure. Interestingly, one of the exposed epitopes does not appear to be functional, at least by itself. Many more domains of the molecule need to be studied, but based on our findings with the epitopes already identified, it seems a combinatorial targeting strategy may be beneficial. If one assumes that the protein's role in fertility resides in a single exposed epitope, or some combination of exposed epitopes, such targeting may also ultimately lead to successful modulation of the fertilizing potential of sperm.

Continuous exposure to dibromoacetic acid delays pubertal development and compromises sperm quality in the rat.

Previously our work on the haloacid by-products of drinking water disinfection focused on adult exposures. Herein we evaluate the consequence of continuous exposure to dibromoacetic acid (DBA) via drinking water through reproductive development into adulthood. An initial study in which offspring were exposed from gestation day (GD) 15 through adulthood revealed significant delays in preputial separation and vaginal opening, dose-related decreases in the fertility of cauda epididymal sperm, and dose-related diminutions in the sperm membrane protein SP22. Subsequent studies consisted of groups in which exposure ceased on postnatal day 21 (PND 21) versus adulthood. For each exposure, animals were evaluated after puberty (PND 56) as well as at adulthood (PND 120). Exposure to 4, 40, or 400 ppm DBA from GD 15 through PND 21 failed to result in any significant reproductive alterations. By contrast, continuous exposure until adulthood resulted in dose-related alterations consistent with those observed in the dose-finding study. Preputial separation and vaginal opening were delayed 4 and 3 days in males and females exposed to 400 ppm (76.3 mg/kg) DBA. This was associated with increased responsiveness of both the testis and ovary to hCG ex vivo; hCG-stimulated testosterone production by testicular parenchyma on PND 56 was increased at 4 ppm (0.6 mg/kg) DBA and higher. Finally, the quality of proximal cauda epididymal sperm was compromised by continuous exposure to DBA. The sperm membrane proteome was altered in a dose-related manner with SP22, and one of its charged variants, diminished at 40 ppm (3.6 mg/kg) DBA and higher. As more sensitive endpoints are evaluated, lower effect levels can be attributed to haloacid exposure. We are now extending our evaluations to epidemiology studies designed to evaluate sperm quality in men exposed to varying levels of disinfection by-products.

Haloacid induced alterations in fertility and the sperm biomarker SP22 in the rat are additive: validation of an ELISA.

Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water that produce defects in spermatogenesis and fertility in adult rats. Previously we demonstrated that BCA compromises the fertility of cauda epididymal rat sperm and SP22, a sperm membrane protein that is highly correlated with the fertility of these sperm. Herein, we administered DBA and BCA, individually and in combination, to determine whether fertility and levels of SP22 on sperm were diminished in an additive fashion. Moreover, we wished to validate an immunoassay for quantitation of SP22. In a dose finding study, animals were exposed by oral gavage daily for 14 days to: BCA alone at 1.6, 4, and 8 mg/kg; DBA at equimolar levels of 2, 5, and 10 mg/kg; and two binary mixtures of 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. The ED(50)s for the decrease in SP22 quantified by two-dimensional SDS-PAGE were 7.2 and 4.6 mg/kg for DBA and BCA. The ED(50)s for the decrease in SP22 quantified by ELISA were 8.1 and 5.9 mg/kg for DBA and BCA. The definitive study consisted of 2 and 4 mg/kg DBA, 1.6 and 3.2 mg/kg BCA, and a 2 mg/kg DBA + 1.6 mg/kg BCA mixture. The ED(50)s for decreases in fertility assessed by intrauterine insemination were 3.5 mg/kg and 2.7 mg/kg for DBA and BCA. Immunolocalization of SP22 in spermatocytes and spermatids, as well as on the cytoplasmic droplet and the equatorial segment of luminal sperm, was decreased by the DBA + BCA mixture. The decrease in SP22 in testicular parenchyma was comparable to that observed for sperm extracts. Based on 2D SDS-PAGE, ELISA, or fertility the haloacid-induced decreases in SP22 or fertility were additive or synergistic. The correlation between SP22 levels by ELISA and fertility was r(2) = 0.72 compared to 0.82 for SP22 levels by 2D SDS-PAGE and fertility, validating SP22 quantitation by ELISA.

Phthalate-induced Leydig cell hyperplasia is associated with multiple endocrine disturbances.

The possibility that exposures to environmental agents are associated with reproductive disorders in human populations has generated much public interest recently. Phthalate esters are used most commonly as plasticizers in the food and construction industry, and di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate in the environment. Daily human exposure to DEHP in the U.S. is significant, and occupational and clinical exposures from DEHP-plasticized medical devices, e.g., blood bags, hemodialysis tubing, and nasogastric feeding tubes, increase body burden levels. We investigated the effects of chronic exposures to low environmentally relevant DEHP levels on testicular function. Our data show that prolonged exposures to this agent induced high levels of the gonadotropin luteinizing hormone and increased the serum concentrations of sex hormones [testosterone and 17beta-estradiol (E2)] by >50%. Increased proliferative activity in Leydig cells was evidenced by enhanced expression of cell cycle proteins, as determined by RT-PCR. The numbers of Leydig cells in the testis of DEHP-treated rats were 40-60% higher than in control rats, indicating induction of Leydig cell hyperplasia. DEHP-induced elevations in serum testosterone and E2 levels suggest the possibility of multiple crosstalks between androgen, estrogen, and steroid hormone receptors, whereas the presence of estrogen receptors in nonreproductive tissues, e.g., cardiovascular system and bones, implies that the increases in serum E2 levels have implications beyond reproduction, including systemic physiology. Analysis of the effects of phthalate exposures on gonadotropin and steroid hormone levels should form part of overall risk assessment in human populations.

Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells.

Exposure of humans to bisphenol A (BPA), a monomer in polycarbonate plastics and a constituent of resins used in food packaging and dentistry, is significant. In this report exposure of rats to 2.4 microg/kg.d (a dose that approximates BPA levels in the environment) from postnatal d 21-35 suppressed serum LH (0.21 +/- 0.05 ng/ml; vs. control, 0.52 +/- 0.04; P < 0.01) and testosterone (T) levels (1.62 +/- 0.16 ng/ml; vs. control, 2.52 +/- 0.21; P < 0.05), in association with decreased LHbeta and increased estrogen receptor beta pituitary mRNA levels as measured by RT-PCR. Treatment of adult Leydig cells with 0.01 nm BPA decreased T biosynthesis by 25% as a result of decreased expression of the steroidogenic enzyme 17alpha-hydroxylase/17-20 lyase. BPA decreased serum 17beta-estradiol levels from 0.31 +/- 0.02 ng/ml (control) to 0.22 +/- 0.02, 0.19 +/- 0.02, and 0.23 +/- 0.03 ng/ml in rats exposed to 2.4 microg, 10 microg, or 100 mg/kg.d BPA, respectively, from 21-35 d of age (P < 0.05) due to its ability to inhibit Leydig cell aromatase activity. Exposures of pregnant and nursing dams, i.e. from gestation d 12 to postnatal d 21, decreased T levels in the testicular interstitial fluid from 420 +/- 34 (control) to 261 +/- 22 (P < 0.05) ng/ml in adulthood, implying that the perinatal period is a sensitive window of exposure to BPA. As BPA has been measured in several human populations, further studies are warranted to assess the effects of BPA on male fertility.

Effects of gestational exposure to ethane dimethanesulfonate in CD-1 mice: microtia and preliminary hearing tests.

BACKGROUND: Microtia is a reduction in pinna size, usually seen in humans in conjunction with other medical conditions. We report microtia in CD-1 mice after gestational exposure to ethane dimethanesulfonate (EDS), an alkylating agent and adult rat Leydig cell toxicant. METHODS: Time-pregnant CD-1 mice were administered 0, 80, or 160 mg EDS/kg on gestation days (GD) 11-17, or 0 or 160 mg EDS/kg on GD 11-13, GD 13-15 or GD 15-17. Pinnae were measured on postnatal days (PND) 4, 8, 18, and 28; and were observed for detachment from birth through PND 8. Branchial-arch derived skeletal structures and histology of the pinna was examined on PND 4 and 24. Brainstem auditory evoked response (BAER) tests were carried out at approximately PND 160 to determine possible effects on hearing. RESULTS: All offspring of EDS-treated dams exhibited bilateral, dose-related decreases in pinna size. Gestational exposure during GD 11-13 produced smaller ears than during GD 13-15 or 15-17, but not as small as the GD 11-17 regimen. Ossification of other pharyngeal arch derivatives was delayed whereas histology was unremarkable. BAER analysis showed a decrease in the proportion of adult offspring producing a quantifiable response to varied auditory stimuli among EDS-treated litters. CONCLUSIONS: Gestational exposure to EDS affects pinna development in the mouse, with a broad period of sensitivity during the second half of gestation. Microtia induced by EDS may be associated with hearing deficits, suggesting functional importance of pinna size or additional effects of EDS on ear development not detected by morphological examination.

L166P mutant DJ-1, causative for recessive Parkinson's disease, is degraded through the ubiquitin-proteasome system.

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.

Gestational exposure to ethane dimethanesulfonate permanently alters reproductive competence in the CD-1 mouse.

Although the adult mouse Leydig cell (LC) has been considered refractory to cytotoxic destruction by ethane dimethanesulfonate (EDS), the potential consequences of exposure during reproductive development in this species are unknown. Herein pregnant CD-1 mice were treated with 160 mg/kg on Gestation Days 11-17, and reproductive development in male offspring was evaluated. Prenatal administration of EDS compromised fetal testosterone (T) levels, compared with controls. EDS-exposed pups recovered their steroidogenic capacities after birth because T production by hCG-stimulated testis parenchyma from prepubertal male offspring was unchanged. However, prepubertal testes from prenatally exposed males contained seminiferous tubules (STs) devoid of germ cells, indicating a delay in spermatogenesis. In adults, some STs in exposed males still contained incomplete germ cell associations corroborating observed reductions in epididymal sperm reserves, fertility ratios, and litter size. Morphometry revealed an EDS-induced increase in interstitial area and a concomitant decrease in ST area, but stereology revealed an unexpected decrease in the number and size of the LCs per testis in exposed males. Paradoxically, there was an increase in both serum LH and T production by adult testis parenchyma, indicating that the LCs were hyperstimulated. These data demonstrate permanent lesions in LC development and spermatogenesis caused by prenatal exposure in mice. Thus, although adult mouse LCs are insensitive to EDS, EDS appears to have direct action on fetal LCs, resulting in abnormal testis development.