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.

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.

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.

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.

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.

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.

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.

Bisphenol A regulation of testicular endocrine function in male rats is affected by diet.

There is concern that early-life exposure to bisphenol A (BPA) may alter developmental programming and predispose individuals to obesity and reproductive anomalies. The present study was designed to determine if a high fat diet at sexual maturation moderates testicular toxicity occasioned by exposure to BPA during reproductive development. Therefore, male rats were exposed to BPA by maternal gavage (0, 2.5 or 25 µg/kg body weight/day) from gestational day 12 to postnatal day 21. At weaning, control and BPA-exposed animals were placed on a regular normal fat diet (NFD) until 70 days of age when they were continued on the NFD or were maintained on a high fat diet (HFD) until euthanasia at 98 days. Adult male rats maintained on HFD were generally heavier than NFD animals due to greater energy intake but energy intake per unit body weight gain was similar in all animals. However, perinatal exposure to BPA decreased (P<0.05) serum adiponectin as well as adiponectin and AdipoR2 protein expression levels in Leydig cells. Importantly, the combination of BPA exposure and HFD consumption promoted lipid peroxidation evidenced by elevated serum thiobarbituric acid reactive substances and glutathione concentrations. These findings imply that interaction between BPA and HFD potentially causes testicular dysfunction to a greater degree than would be due to BPA exposure or HFD consumption. Given the relationship that exists between energy homeostasis and reproductive activity, additional studies are warranted to investigate the consequences of BPA-diet interactions on testicular function.

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.

Effects of methoxychlor and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on human and rat 17α-hydroxylase/17,20-lyase activity.

Exposure to methoxychlor, an agricultural pesticide, has been associated with reduced testicular androgen secretion. However, methoxychlor is converted to 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) in the liver, which then acts as its biologically active metabolite. Both methoxychlor and HPTE have been credited with estrogenic properties and have a weak anti-androgenic activity. However, the exact mechanisms of steroidogenic enzyme inhibition remain to be clarified. In the present study, human and rat testis microsomes were employed to investigate the inhibitory activities of methoxychlor and HPTE on 17α-hydroxylase/17,20-lyase (CYP17A1). The CYP17A1 enzyme is critical for androgen biosynthesis and catalyzes conversion of progesterone into androstenedione. The results demonstrated that HPTE directly inhibited human and rat CYP17A1 activities, while methoxychlor had no effects on this enzyme activity even at a concentration of 100 µM. The IC50 values of HPTE were 1.13±0.10 (human) and 6.87±0.13 µM (rat), respectively. When HPTE was incubated with rat immature Leydig cells, it also inhibited CYP17A1 activity with an IC50 value of 6.29±0.1 µM. Results of enzyme inhibition were supported by the observation that HPTE inhibited luteinizing hormone-stimulated 5α-androstane-3α,17ß-diol and testosterone secretion by immature Leydig cells with IC50 values of 6.61±0.03 and 3.78±0.003 µM, respectively. The mode of action of HPTE on CYP17A1 activity was determined to be uncompetitive with the substrate progesterone. In conclusion, HPTE, the metabolite of MXC, directly inhibited human and rat testis CYP17A1 activities.

Aflatoxin B1 disrupts the androgen biosynthetic pathway in rat Leydig cells.

The present study investigated if Aflatoxin B1 (AFB1), a potent and naturally occurring mycotoxin, interferes with the steroidogenic pathway in rat Leydig cells. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone (T) that maintains the male phenotype and support fertility. Leydig cells, isolated from 35-day-old male Long-Evans rats (Rattus norvegicus), were incubated with AFB1 at 0, 0.01, 0.1, 1, 10µM followed by measurement of T secretion by radioimmunoassay and analysis of protein expression in western blots. Results demonstrated that AFB1 suppressed testosterone secretion in a dose-dependent manner and inhibited expression of cholesterol transporter steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes [(3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 17ß-hydroxysteroid dehydrogenase enzyme (HSD17B3)]. Protein expression analysis showed that AFB1 treatment increased ERK phosphorylation but suppressed p38 MAPK and JNK activation in Leydig cells. AFB1-induced inhibition of Leydig cells was alleviated by co-treatment with the ERK inhibitor UO 126, implying that ERK mediates, at least in part, the inhibitory effects of AFB1 in Leydig cells. The findings highlight potential extra-hepatic effects of aflatoxin exposure and indicate that exposure to AFB1 has significant reproductive health implications for consumers of contaminated products even under conditions of low dietary toxin levels.

Adiponectin receptors in energy homeostasis and obesity pathogenesis.

Adipokines, that is factors secreted by adipose tissue, act through a network of autocrine, paracrine, and endocrine pathways to regulate several aspects of physiology, including glucose and lipid metabolism, neuroendocrine function, reproduction, and cardiovascular function. In particular, adiponectin, a 30-kDa protein, is associated with the regulation of insulin sensitivity, and its levels in serum are affected by altered metabolic homeostasis. Adiponectin effects are mediated by adiponectin receptors, which occur as two isoforms (AdipoR1 and AdipoR2). Transcriptional regulation of adiponectin is by the peroxisome proliferator-activated receptor-gamma (PPAR-γ). However, acting through AdipoR1 and AdipoR2, adiponectin enhances 5' adenosine monophosphate-activated protein kinase (AMPK) and the PPARα-mediated pathways in the liver and skeletal muscles. Adiponectin receptors mediate a wide spectrum of metabolic reactions, including gluconeogenesis and fatty-acid oxidation. Altogether, adiponectin deficiency and/or decreased adiponectin receptor-mediated activity possibly contribute to insulin resistance in metabolic syndromes, coronary heart disease, and liver disease.

Sex hormone regulation of collagen concentrations in cranial cruciate ligaments of sexually immature male rabbits.

OBJECTIVE: To investigate the effects of gonadectomy on collagen homeostasis in cranial cruciate ligaments of male rabbits. ANIMALS: 30 sexually immature (16-week-old) male New Zealand White rabbits. PROCEDURES: Rabbits were randomly assigned to 5 groups of 6 rabbits each: sexually intact, placebo (control group); castrated, placebo; castrated, testosterone; castrated, dihydrotestosterone; and castrated, 17ß-estradiol (E(2)). Control rabbits underwent a sham operation, and all other rabbits underwent gonadectomy. At the time of gonadectomy, the placebo and sex hormones were administered via slow-release pellets implanted subcutaneously as assigned. After 21 days of hormone supplementation, measurements were obtained of serum testosterone and E(2) concentrations, ligament collagen characteristics, and androgen receptor, estrogen receoptor α, and matrix metalloproteinase expression. RESULTS: Following gonadectomy and hormone supplementation, the treatment groups differed in serum testosterone and E(2) concentrations to various degrees. Collagen concentrations were lower and fiber diameters higher in the absence of sex hormones, in association with the degrees of estrogen receptor a and androgen receptor expression. Although differences were detected among the groups in matrix metalloproteinase expression, these differences were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Sex hormones appeared to play a role in cranial cruciate ligament homeostasis in male rabbits. Physiologic changes triggered by the lack of sex hormones following gonadectomy in sexually immature rabbits may potentially predispose those rabbits to orthopedic injuries.

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.

Effects of phthalates on 3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase 3 activities in human and rat testes.

The 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3ß-HSD and 17ß-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5µM for human 3ß-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7µM for rat 3ß-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2µM) and rat (IC(50)s, 30.24 and 9.1µM) 17ß-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3ß-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17ß-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3ß-HSD and 17ß-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.

Regulation of adiponectin secretion by soy isoflavones has implication for endocrine function of the testis.

Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone (T), which is required to maintain male fertility. There is now growing evidence that environmental stressors, including chemicals present in food, air and water, may affect energy balance. A relationship between energy balance and reproductive capacity has been proposed for a long time. In the present study, developmental exposures of male rats to soy isoflavones in the maternal diet from gestational day 12 to day 21 post-partum enhanced adiponectin expression in adipose tissue and increased serum adiponectin concentrations in adulthood. However, exposure to soy isoflavones caused a decrease in T production and expression of adiponectin and its receptor (adipoR2) in Leydig cells. In separate experiments, incubation of Leydig cells with recombinant adiponectin in the absence of isoflavones caused a decrease in T biosynthesis associated with diminished expression of the cholesterol transporter steroidogenic acute regulatory protein (StAR). Thus, chemical-induced alterations in serum adiponectin concentrations have implication for steroid hormone secretion. The results also imply that changes in adipose tissue metabolism occasioned by exposure to dietary estrogens, and perhaps other estrogenic agents, possibly contribute to deficiencies in reproductive capacity attributed to these compounds.