Effects of chronic dichlorodiphenyldichloroethylene exposure on testosterone secretion and steroidogenic pathway in the male gonad.

Endocrine disrupting chemicals are present in the environment and/or in consumer products. These agents have the capacity to mimic and/or antagonize endogenous hormones and thus perturb the endocrine axis. The male reproductive tract expresses steroid hormone (androgen and estrogen) receptors at high levels and is a major target for endocrine disrupting chemicals. In this study, Long-Evans male rats were exposed to dichlorodiphenyldichloroethylene, a metabolite of dichlorodiphenyltrichloroethane and a chemical present in the environment, in drinking water at 0.1 and 10 µg/L for 4 weeks. At the end of exposure, we measured steroid hormone secretion and analyzed steroidogenic proteins, including 17ß-hydroxysteroid dehydrogenase, 3ß-hydroxysteroid dehydrogenase, steroidogenic acute regulatory protein, aromatase, and the LH receptor. We also analyzed Leydig cell apoptosis (poly-(ADP-ribose) polymerase) and caspase-3 in the testes. Testicular testosterone (T) and 17ß-estradiol (E2) were both affected by exposure to dichlorodiphenyldichloroethylene by displaying altered steroidogenic enzyme expression. Dichlorodiphenyldichloroethylene exposure also increased the expression of enzymes mediating the pathway for programmed cell death, including caspase 3, pro-caspase 3, PARP, and cleaved PARP. Altogether, the present results demonstrate that dichlorodiphenyldichloroethylene directly and/or indirectly can target specific proteins involved in steroid hormone production in the male gonad and suggest that exposure to environmentally relevant dichlorodiphenyldichloroethylene levels has implications for male reproductive development and function.

Effects of different DDE exposure paradigms on testicular steroid hormone secretion and hepatic oxidative stress in male Long-Evans rats.

Chronic exposure to low doses of anthropogenic chemicals in the environment continues to be a major health issue. Due to concerns about the effects in humans and wildlife, use of persistent organic pollutants, such as dichlorodiphenyltrichloroethane (DDT), is prohibited. However, their ubiquitous nature and persistence allows them to remain in the environment at low levels for decades. Dichlorodiphenyldichloroethylene (DDE) is the most persistent metabolite of DDT and has been shown to cause hepatotoxicity, nephrotoxicity, hormonal disorders, and induce oxidative stress in many organisms. Although the effects of acute exposure to DDT and its metabolite DDE have been extensively studied, the chronic effects of sub-lethal DDE exposure at levels comparable to those found in the environment have not been well documented. Long-Evans male rats were used to determine the effect of relatively chronic and short term DDE (doses ranged from 0.001 to 100 µg/L) exposure on endocrine function and oxidative stress at different developmental time points. We found that circulating serum testosterone (T) levels were significantly decreased and T secretion in testicular explants were significantly influenced in a dose dependent manner in both pre-pubertal and pubertal male rats after DDE exposure, with pubertal rats being the most affected contrary to our original prediction. Additionally, exposure to DDE increased expression of protein oxidation indicating a possible increase in cellular damage caused by oxidative stress. This study suggests that chronic exposures to environmentally relevant levels of DDE affected testicular function and decreased T secretion with implications for reproductive capacity.

Regulation of the neuroendocrine axis in male rats by soy-based diets is independent of age and due specifically to isoflavone action†.

Soy-based foods are consumed for their health beneficial effects, implying that the population is exposed to soy isoflavones in the diet. Herein, male rats at 21, 35, and 75 days of age were maintained either on a casein control diet, soybean meal (SBM), or control diet supplemented with daidzin and genistin (G + D) for 14 days. Feeding of SBM and G + D diets decreased testicular testosterone (T) secretion regardless of age. Altered androgen secretion was due to decreased (P < 0.05) Star and Hsd17ß protein in the testes and was associated with increased (P < 0.05) Lhß and Fshß subunit protein expression in pituitary glands. Second, male rats were fed either a casein control diet, control diet + daidzin, control diet + genistin, or control diet + genistin + daidzin (G + D). Compared to control, feeding of all isoflavone-containing diets decreased (P < 0.05) testicular T concentrations, and more so in the G + D diet group. Interestingly, Esr1 and androgen receptor protein and pituitary Fshß with Lhß subunit protein were increased (P < 0.05) by feeding of genistin and G + D diets, but not the daidzin diet. However, daidzein and genistein both caused a concentration dependent inhibition (P < 0.05) of T secretion by Leydig cells in vitro with IC50 of 184 ηM and 36 ηM, respectively. Results demonstrated that altered testicular steroidogenic capacity and pituitary FSHß and LHß subunit expression due to soy-based diets result from specific actions by genistein and daidzein. Experiments to assess effects of isoflavone regulation of intratesticular androgen concentrations on male fertility are warranted.

Androgen and mineralocorticoid receptors are present on the germinal disc region in laying hens: Potential mediators of sex ratio adjustment in birds?

Female birds skew offspring sex ratios based on environmental and social stimuli; however, the mechanism mediating this phenomenon remains unknown. Growing evidence suggests that testosterone and corticosterone may influence meiosis, as they skew sex ratios when given immediately before chromosomal segregation. It is unclear if these hormones act on the germinal disc (GD) or through a downstream mediator. It is also unknown whether the GD contains receptors for these hormones. If testosterone and/or corticosterone act on the GD to skew sex ratios, then the GD should have receptors for them and that receptor levels should be higher in the GD regions compared to other follicular regions. Furthermore, fluctuations of receptor levels should occur near meiotic segregation. We collected ovarian follicles at 5 h pre-ovulation (just before meiotic segregation) and 20 h pre-ovulation (when sex chromosomes are arrested), and measured androgen receptor (AR) and mineralocorticoid receptor (MR) protein levels via Western blot. ARs and MRs were on the follicle in the GD and non-GD regions, and at 5 h and 20 h pre-ovulation. Both AR and MR protein levels were higher in the GD region than the non-GD region at both time points, but did not differ between time points. These results suggest that hen ovarian follicles have receptors for testosterone and corticosterone, and that the ability for testosterone to respond may be specifically higher in the GD-region, providing further support for the role of testosterone in the alteration of meiotic segregation.

Butylated Hydroxyanisole Potently Inhibits Rat and Human 11ß-Hydroxysteroid Dehydrogenase Type 2.

Butylated hydroxyanisole (BHA) is a widely used antioxidant for food preservation. 11ß-hydroxysteroid dehydrogenases, isoforms 1 (HSD11B1) and 2 (HSD11B2) have been demonstrated to be the regulators of the local level of active glucocorticoid, which has a broad range of physiological actions. In this study, the potency of BHA was tested for the inhibition of HSD11B1 and HSD11B2 in rat and human tissues. BHA showed potent inhibition of HSD11B2 with the half maximal inhibitory concentration calculated at 13.99 and 69.25 µmol/l for the rat and human, respectively. Results showed that BHA competitively inhibited HSD11B2 when a steroid substrate was used. However, it served as a mixed inhibition factor when the cofactor NAD+ was used. In contrast, the potency of BHA to inhibit both rat and human HSD11B1 was diminished, with the concentration of 100 µmol/l causing no inhibitory effect on the isoform. In conclusion, we observed that BHA is a selective inhibitor of HSD11B2, implying that this agent may cause excessive glucocorticoid action in local tissues such as kidney and placentas.

Testicular development in male rats is sensitive to a soy-based diet in the neonatal period.

Approximately 30% of infants in the United States are exposed to high doses of isoflavones resulting from soy infant formula consumption. Soybeans contain the isoflavones genistin and daidzin, which are hydrolyzed in the gastrointestinal tract to their genistein and daidzein aglycones. Both aglycones possess hormonal activity and may interfere with male reproductive development. Testosterone, which supports male fertility, is mainly produced by testicular Leydig cells. Our previous studies indicated that perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells and increased testosterone concentrations into adulthood. However, the relevance of the neonatal period as part of the perinatal window of isoflavone exposure remains to be established. The present study examined the effects of exposure to isoflavones on male offspring of dams maintained on a casein-based control or whole soybean diet in the neonatal period, that is, Days 2 to 21 postpartum. The results showed that the soybean diet stimulated proliferative activity in developing Leydig cells while suppressing their steroidogenic capacity in adulthood. In addition, isoflavone exposure decreased production of anti-Müllerian hormone by Sertoli cells. Similar to our previous in vitro studies of genistein action in Leydig cells, daidzein induced proliferation and interfered with signaling pathways to suppress steroidogenic activity. Overall, the data showed that the neonatal period is a sensitive window of exposure to isoflavones and support the view that both genistein and daidzein are responsible for biological effects associated with soy-based diets.

Legacy and Emerging Perfluoroalkyl and Polyfluoroalkyl Substances Regulate Steroidogenesis in the Male Gonad.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are widely used in a variety of industrial processes and manufacturing of consumer products. Current efforts by the manufacturing industry will limit use of long-chain or legacy PFAS represented by perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) and replace with short-chain or emerging PFAS such as perfluorobutanoic acid (PFBA) and perfluorobutane sulfonic acid (PFBS). However, there is little to no information on the toxicity of new and emerging PFAS. Therefore, we performed experiments in growing Long-Evans male rats to investigate effects of low-dose prepubertal and pubertal exposures to PFAS on gonadal steroid hormone secretion. The results demonstrated that both legacy and emerging PFAS have the capacity to regulate testicular steroidogenesis. For instance, prepubertal exposures to PFOS, PFBA, and PFBS increased serum and testicular testosterone concentrations. Exposure to PFBA increased testicular 17ß-estradiol (E2) concentrations, and PFOS and PFBS both decreased serum E2 concentrations while stimulating testicular E2 secretion. The data also demonstrated additive effects due to legacy and emerging PFAS mixtures compared with the individual chemicals. The gonadal effects due to PFAS exposures occurred at nanomolar concentrations, which approximate PFAS levels in the environment. Taken together, the present study supports the need for development of cost-effective and sustainable filtration media for different processes to remove PFAS from water and other sources of exposure. Current action by regulatory agencies such as the US Environmental Protection Agency to limit use of PFAS in the manufacture of consumer products will protect public health.

Effects of developmental exposures to Bisphenol-A and Bisphenol-S on hepatocellular function in male Long-Evans rats.

Bisphenol-S (BPS) is a current substitute for Bisphenol-A (BPA) in various commercial products (paper, plastics, protective can-coatings, etc.) used by all age groups globally. The current literature indicates that a drastic surge in pro-oxidants, pro-apoptotic, and pro-inflammatory biomarkers in combination with diminished mitochondrial activity can potentially decrease hepatic function leading to morbidity and mortality. Consequently, there are increasing public health concerns that substantial Bisphenol-mediated effects may impact hepatocellular functions, particularly in newborns exposed to BPA and BPS postnatally. However, the acute postnatal impact of BPA and BPS and the molecular mechanisms affecting hepatocellular functions are unknown. Therefore, the current study investigated the acute postnatal effect of BPA and BPS on the biomarkers of hepatocellular functions, including oxidative stress, inflammation, apoptosis, and mitochondrial activity in male Long-Evans rats. BPA and BPS (5 and 20 microgram/Liter (µg/L) of drinking water) were administered to 21-day-old male rats for 14 days. BPS had no significant effect on apoptosis, inflammation, and mitochondrial function but significantly reduced the reactive oxygen species (51-60 %, **p < 0.01) and nitrite content (36 %, *p < 0.05), exhibiting hepatoprotective effects. As expected, based on the current scientific literature, BPA induced significant hepatoxicity, as seen by significant glutathione depletion (50 %, *p < 0.05). The in-silico analysis indicated that BPS is effectively absorbed in the gastrointestinal tract without crossing the blood-brain barrier (whereas BPA crosses the blood-brain barrier) and is not a substrate of p-Glycoprotein and Cytochrome P450 enzymes. Thus, the current in-silico and in vivo findings revealed that acute postnatal exposure to BPS had no significant hepatotoxicity.

The industrial chemical bisphenol A (BPA) interferes with proliferative activity and development of steroidogenic capacity in rat Leydig cells.

The presence of bisphenol A (BPA) in consumer products has raised concerns about potential adverse effects on reproductive health. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone, which supports the male phenotype. The present report describes the effects of developmental exposure of male rats to BPA by gavage of pregnant and lactating Long-Evans dams at 2.5 and 25 µg/kg body weight from Gestational Day 12 to Day 21 postpartum. This exposure paradigm stimulated Leydig cell division in the prepubertal period and increased Leydig cell numbers in the testes of adult male rats at 90 days. Observations from in vitro experiments confirmed that BPA acts directly as a mitogen in Leydig cells. However, BPA-induced proliferative activity in vivo is possibly mediated by several factors, such as 1) protein kinases (e.g., mitogen-activated protein kinases or MAPK), 2) growth factor receptors (e.g., insulin-like growth factor 1 receptor-beta and epidermal growth factor receptors), and 3) the Sertoli cell-secreted anti-Mullerian hormone (also called Mullerian inhibiting substance). On the other hand, BPA suppressed protein expression of the luteinizing hormone receptor (LHCGR) and the 17beta-hydroxysteroid dehydrogenase enzyme (HSD17B3), thereby decreasing androgen secretion by Leydig cells. We interpret these findings to mean that the likely impact of deficits in androgen secretion on serum androgen levels following developmental exposure to BPA is alleviated by increased Leydig cell numbers. Nevertheless, the present results reinforce the view that BPA causes biological effects at environmentally relevant exposure levels and its presence in consumer products potentially has implication for public health.

Toxidromes for Working Dogs.

Terrorist attacks with biological and chemical warfare agents are increasing in frequency worldwide. Additionally, hazardous chemical accidents, illicit drug laboratories and intentional poisonings are potential sites for exposure to working dogs. Working dogs play a crucial role in law enforcement, military and search and rescue teams. Their intelligence, agility and strength make them ideal partners to be deployed to these natural disaster sites, terrorist attacks and industrial accidents. This, unfortunately, leads to increasing exposure to chemical and biological weapons and other hazardous substances. First responders have little to no training in emergency care of working dogs and veterinarians have very little training on recognition of the clinical signs of many of these agents. In order to ensure a rapid medical response at the scene first responders and veterinarians need a primer on these agents. Identifying a specific agent amidst the chaos of a mass casualty event is challenging. Toxidromes are a constellation of clinical and/or laboratory findings that allow for rapid identification of the clinical signs associated with a class of toxin and have been helpful in human medical triage. Focusing on a class of agents rather than on each individual toxin, allows for more expedient administration of antidotes and appropriate supportive care. This article reviews toxidromes for the most common chemical weapons with a special emphasis on clinical signs that are specific (and different) for canines as well as appropriate antidotes for working canines. To our knowledge, there are no publications describing toxidromes for working dogs.

Developmental exposures of male rats to soy isoflavones impact Leydig cell differentiation.

Testicular Leydig cells, which are the predominant source of the male sex steroid hormone testosterone, express estrogen receptors (ESRs) and are subject to regulation by estrogen. Following ingestion, the two major isoflavones in soybeans, genistin and daidzin, are hydrolyzed by gut microflora to form genistein and daidzein, which have the capacity to bind ESRs and affect gene expression. Thus, the increasing use of soy-based products as nondairy sources of protein has raised concerns about the potential of these products to cause reproductive toxicity. In the present study, perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells. Isoflavones have the capacity to act directly as mitogens in Leydig cells, because genistein treatment induced Leydig cell division in vitro. Genistein action regulating Leydig cell division involved ESRs, acting in concert with signaling molecules in the transduction pathway mediated by protein kinase B (AKT) and mitogen-activated protein kinase (MAPK). Enhanced proliferative activity in the prepubertal period increased Leydig cell numbers, which alleviated deficits in androgen biosynthesis and/or augmented serum and testicular testosterone concentrations in adulthood. Together, these observations indicate that the perinatal exposures of male rats to isoflavones affected Leydig cell differentiation, and they imply that including soy products in the diets of neonates has potential implications for testis function.

Genistein decreases androgen biosynthesis in rat Leydig cells by interference with luteinizing hormone-dependent signaling.

Testicular Leydig cells express estrogen receptors and are the predominant source of the male sex steroid hormone testosterone (T). Previous studies demonstrated that genistein acts through estrogen receptors in Leydig cells. In the present study, pre-treatment of Leydig cells isolated from 35 day-old male Long Evans rats with the epidermal growth factor receptor (EGFR) kinase inhibitor AG 1478 abrogated genistein inhibition of T biosynthesis. Also, incubation of Leydig cells in culture medium containing epidermal growth factor (EGF) decreased T secretion (control: 255+/-16; EGF: 190+/-17ng/10(6) cells, 24h) (P<0.05). However, T secretion by genistein-treated Leydig cells (0.1nM, 10muM; 24h) was rescued by post-treatment incubation with forskolin (control: 275+/-28 versus 325+/-35; 780+/-85; ng/10(6) cells, 3h) and dibutyryl cyclic adenosine 3'-5'-monophosphate (dbcAMP) (control: 370+/-65 versus 580+/-75; 2500+/-200; ng/10(6) cells, 3h) (P>0.05). Furthermore, post-treatment incubation with cholera toxin, an activator of G proteins, caused genistein-treated Leydig cells to produce similar T amounts as untreated control (control: 55+/-5 versus 52+/-2 and 47+/-4; ng/10(6) cells, 3h) (P>0.05). These observations imply that genistein action interferes with coupling of transmembrane luteinizing hormone receptors (LHR) with G proteins. Uncoupling of LHR from G proteins adversely affects adenylate cyclase function and impacts LH-dependent stimulation of Leydig cells. These findings have implications for testicular steroidogenesis in individuals exposed to genistein and soy-based products.

Prenatal exposures to bisphenol A and di (2-ethylhexyl) phthalate disrupted seminiferous tubular development in growing male rats.

Endocrine-disrupting compounds (EDCs) are found in the environment due to their use in industrial and manufacturing activities. Exposure of the population to bisphenol A (BPA) and di (2-ethylhexyl) phthalate (DEHP) is significant because they are present in many consumer products. EDCs target the reproductive tract because they express high levels of steroid hormone receptors, which act as transcriptional factors to regulate reproductive development. In the present study, timed-pregnant Long-Evans female rats (n = 8-10) were administered BPA and DEHP by oral gavage at 2.5 or 25 µg/kg body weight and 5 or 50 µg/kg body weight, respectively. Exposures to chemicals were limited to the period between gestational days 12 and 21 followed by assessment of testicular development in male offspring in the postnatal period. Leydig cells and Sertoli cells are the two major somatic cells present in the testis. The 17ß-hydroxysteroid dehydrogenase (17ß-HSD) steroidogenic enzyme is a marker for Leydig cell maturation, whereas transferrin is a marker for Sertoli cell differentiation. At day 10 post-partum, testes were obtained from cohorts of control and chemical-exposed male rats and processed to measure 17ß-HSD and transferrin expression levels in western blots. Compared to control, 17ßHSD enzyme protein was increased in BPA-treated rats but levels were decreased in animals exposed to DEHP (P < 0.05). Transferrin protein was decreased in male rats exposed to both BPA and DEHP compared to control animals (P < 0.05). To assess qualitative cellular changes within the spermatogenic epithelium, testes were obtained from separate cohorts of male rats at 35 days of age and processed for histopathological analysis. Results showed that prenatal exposures of male rats to BPA and DEHP caused disruption of the spermatogenic epithelium evident as disorganization and atrophy of seminiferous tubules as well as desquamation of germ cells into the tubular lumen. Together, results from the present study support the view that developmental exposures to environmentally relevant levels of BPA and DEHP are associated with disruptions of testicular cell development, which have implications for endocrine and exocrine functions of testis.

Gestational exposure to tetrabutyltin blocks rat fetal Leydig cell development.

Tetrabutyltin is a stable organotin and may exhibit endocrine disrupting properties. Herein, we investigated effects of tetrabutyltin on the development of rat fetal Leydig cells, which support differentiation of the male reproductive tract in late gestation. Female pregnant Sprague Dawley rats were gavaged with tetrabutyltin (0, 100, 200, and 500 mg/kg) from gestational day (GD) 12 to GD 21. Tetrabutyltin dose-dependently decreased testicular testosterone levels (0.756 ±â€¯0.208 and 0.813 ±â€¯0.277 ng/testis at the 200 and 500 mg/kg doses, respectively) compared to control (1.692 ±â€¯0.218 ng/testis) at GD 21. Furthermore, tetrabutyltin induced fetal Leydig cell aggregation, decreased fetal Leydig cell size and cytoplasmic size at the ≥100 mg/kg doses, and downregulated the expression levels of Scarb1, Cyp17a1, and Insl3 at doses ≥100 mg/kg and Star expression at 200 mg/kg. Taking together, the present results indicated that prenatal exposure of male rats to tetrabutyltin affected fetal Leydig cell development.

Exposure to phytoestrogens in the perinatal period affects androgen secretion by testicular Leydig cells in the adult rat.

The use of soy-based products in the diet of infants has raised concerns regarding the reproductive toxicity of genistein and daidzein, the predominant isoflavones in soybeans with estrogenic activity. Time-bred Long-Evans dams were fed diets containing 0, 5, 50, 500, or 1000 ppm of soy isoflavones from gestational d 12 until weaning at d 21 postpartum. Male rats in all groups were fed soy-free diets from postnatal d 21 until 90 d of age. The mean +/- SD concentration of unconjugated (i.e. biologically active) genistein and daidzein in serum from the group of dams maintained on the diet containing the highest amount of isoflavones (1000 ppm) were 17 +/- 27 and 56 +/- 30 nM, respectively, at d 21 postpartum. The concentrations were considerably greater in male offspring (genistein: 73 +/- 46 nM; daidzein: 106 +/- 53 nM). Although steroidogenesis was decreased in individual Leydig cells, male rats from the highest exposure group (1000 ppm diet) exhibited elevated serum levels of the sex steroid hormones androsterone at 21 d (control: 15 +/- 1.5 vs.28 +/- 3.5 ng/ml; P < 0.05) and testosterone at 90 d of age (control: 7.5 +/- 1 vs.17 +/- 2 ng/ml; P < 0.05). Testosterone secretion by immature Leydig cells, isolated from 35-d-old male rats, decreased on exposure to 0.1 nm genistein in vitro (control: 175 +/- 5 vs. 117 +/- 3 ng/10(6) cells per 24 h; P < 0.05), indicative of direct phytoestrogen action. Thus, phytoestrogens have the ability to regulate Leydig cells, and additional studies to assess potential adverse effects of dietary soy-based products on reproductive tract development in neonates are warranted.

In vitro molecular mechanisms of bisphenol A action.

Bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane; CAS# 80-05-7) is a chemical used primarily in the manufacture of polycarbonate plastic, epoxy resins and as a non-polymer additive to other plastics. Recent evidence has demonstrated that human and wildlife populations are exposed to levels of BPA which cause adverse reproductive and developmental effects in a number of different wildlife species and laboratory animal models. However, there are major uncertainties surrounding the spectrum of BPA's mechanisms of action, the tissue-specific impacts of exposures, and the critical windows of susceptibility during which target tissues are sensitive to BPA exposures. As a foundation to address some of those uncertainties, this review was prepared by the "In vitro" expert sub-panel assembled during the "Bisphenol A: An Examination of the Relevance of Ecological, In vitro and Laboratory Animal Studies for Assessing Risks to Human Health" workshop held in Chapel Hill, NC, Nov 28-29, 2006. The specific charge of this expert panel was to review and assess the strength of the published literature pertaining to the mechanisms of BPA action. The resulting document is a detailed review of published studies that have focused on the mechanistic basis of BPA action in diverse experimental models and an assessment of the strength of the evidence regarding the published BPA research.

Estrogen regulation of testicular function.

Evidence supporting a role for estrogen in male reproductive tract development and function has been collected from rodents and humans. These studies fall into three categories: i) localization of aromatase and the target protein for estrogen (ER-alpha and ER-beta) in tissues of the reproductive tract; ii) analysis of testicular phenotypes in transgenic mice deficient in aromatase, ER-alpha and/or ER-beta gene; and, iii) investigation of the effects of environmental chemicals on male reproduction. Estrogen is thought to have a regulatory role in the testis because estrogen biosynthesis occurs in testicular cells and the absence of ERs caused adverse effects on spermatogenesis and steroidogenesis. Moreover, several chemicals that are present in the environment, designated xenoestrogens because they have the ability to bind and activate ERs, are known to affect testicular gene expression. However, studies of estrogen action are confounded by a number of factors, including the inability to dissociate estrogen-induced activity in the hypothalamus and pituitary from action occurring directly in the testis and expression of more than one ER subtype in estrogen-sensitive tissues. Use of tissue-specific knockout animals and administration of antiestrogens and/or aromatase inhibitors in vivo may generate additional data to advance our understanding of estrogen and estrogen receptor biology in the developing and mature testis.

Prenatal Exposures of Male Rats to the Environmental Chemicals Bisphenol A and Di(2-Ethylhexyl) Phthalate Impact the Sexual Differentiation Process.

The increasing incidence of reproductive anomalies, described as testicular dysgenesis syndrome, is thought to be related to the exposure of the population to chemicals in the environment. Bisphenol A (BPA) and di(2-ethylhexyl)phthalate (DEHP), which have hormonal and antihormonal activity, have attracted public attention due to their presence in consumer products. The present study investigated the effects of BPA and DEHP on reproductive development. Timed-pregnant female rats were exposed to BPA and DEHP by gavage from gestational days 12 to 21. Results showed that prenatal exposures to test chemicals exerted variable effects on steroidogenic factor 1 and GATA binding protein 4 protein expression and increased (P < .05) sex-determining region Y-box 9 and antimüllerian hormone protein in the infantile rat testis compared with levels in the control unexposed animals. Pituitary LHß and FSHß subunit protein expression was increased (P < .05) in BPA- and DEHP-exposed prepubertal male rats but were decreased (P < .05) in adult animals relative to control. Exposure to both BPA and DEHP in utero inhibited (P < .05) global DNA hydroxymethylation in the adult testis in association with altered DNA methyltransferase protein expression. Together the present data suggest that altered developmental programming in the testes associated with chemical exposures are related to the disruption of sexual differentiation events and DNA methylation patterns. The chemical-induced effects impact the development of steroidogenic capacity in the adult testis.

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.

Estrogen receptor-alpha gene deficiency enhances androgen biosynthesis in the mouse Leydig cell.

Leydig cells, which produce the primary male steroid hormone testosterone (T), express the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, and have the capacity to convert testosterone to the natural estrogen 17beta-estradiol. Thus, Leydig cells are subject to estrogen action. The development of transgenic mice that are homozygous for targeted deletion of genes encoding the ER subtypes provides an opportunity to examine the role of estrogen in Leydig cell function. In this study androgen biosynthesis was analyzed in Leydig cells from mice that were homozygous for targeted deletion of the ERalpha gene (alphaERKO). T production by alphaERKO Leydig cells was 2-fold higher than that in wild-type (WT) cells. Serum T levels were accordingly higher in alphaERKO compared with WT mice (5.1 +/- 1.1 vs. 2.2 +/- 0.4 ng/ml; P