Manjunatha K. Nanjappa

The Industrial Chemical Bisphenol A (BPA) Interferes with Proliferative Activity and Development of Steroidogenic Capacity in Rat Leydig Cells

ABSTRACT 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.

Estrogens in Male Physiology

Estrogens have historically been associated with female reproduction, but work over the last two decades established that estrogens and their main nuclear receptors (ESR1 and ESR2) and G protein-coupled estrogen receptor (GPER) also regulate male reproductive and nonreproductive organs. 17β-Estradiol (E2) is measureable in blood of men and males of other species, but in rete testis fluids, E2 reaches concentrations normally found only in females and in some species nanomolar concentrations of estrone sulfate are found in semen. Aromatase, which converts androgens to estrogens, is expressed in Leydig cells, seminiferous epithelium, and other male organs. Early studies showed E2 binding in numerous male tissues, and ESR1 and ESR2 each show unique distributions and actions in males. Exogenous estrogen treatment produced male reproductive pathologies in laboratory animals and men, especially during development, and studies with transgenic mice with compromised estrogen signaling demonstrated an E2 role in normal male physiology. Efferent ductules and epididymal functions are dependent on estrogen signaling through ESR1, whose loss impaired ion transport and water reabsorption, resulting in abnormal sperm. Loss of ESR1 or aromatase also produces effects on nonreproductive targets such as brain, adipose, skeletal muscle, bone, cardiovascular, and immune tissues. Expression of GPER is extensive in male tracts, suggesting a possible role for E2 signaling through this receptor in male reproduction. Recent evidence also indicates that membrane ESR1 has critical roles in male reproduction. Thus estrogens are important physiological regulators in males, and future studies may reveal additional roles for estrogen signaling in various target tissues.

Therapeutic effects of progesterone and its metabolites in traumatic brain injury may involve non-classical signaling mechanisms

Traumatic brain injury (TBI) is an important and costly medical problem for which no clinically proven treatment currently exists. Studies in rodents and humans have shown beneficial effects of progesterone (P4) on both mortality and functional outcomes following TBI. Neuroprotective effects of P4 in TBI likely involve the classical nuclear progesterone receptors (Pgr) that are widely distributed in both glial cells and neurons of the brain. However, P4 may have critical effects not mediated through Pgr. In the brain, P4 is converted to a metabolite, allopregnanolone (ALLO), whose beneficial effects equal or exceed those of P4 in TBI. ALLO does not bind Pgr, suggesting it acts through non-classical pathways. ALLO has effects on GABAA and pregnane X receptors, as well as on the mitochondrial permeability transition pore. In addition, ALLO is metabolized to another compound, 5alpha-dihydroprogesterone, which binds Pgr, suggesting ALLO actions may involve signaling through Pgr as well as the aforementioned mechanisms of action. P4 and ALLO also signal through a number of membrane receptors (progesterone receptor membrane component 1, and membrane progesterone receptors (mPRs) alpha, beta, gamma, delta, and epsilon) in the brain that are distinct from Pgr, although the role of these receptors in the normal brain and in the therapeutic response to P4 and ALLO following TBI is unclear. In summary, P4 has the potential to become the first clinically effective treatment for TBI, and the effects of P4 and its metabolite ALLO in TBI may involve Pgr, mPRs, and other signaling pathways. Elucidating these mechanisms will more clearly reveal the potential of classical and non-classical pathways to mediate important effects of P4 and its metabolites, and potentially offer new therapeutic approaches to TBI.

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 [(3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-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.

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.

Membrane-Localized Estrogen Receptor 1 Is Required for Normal Male Reproductive Development and Function in Mice

Estrogen receptor 1 (ESR1) mediates major reproductive functions of 17β-estradiol (E2). Male Esr1 knockout (Esr1KO) mice are infertile due to efferent ductule and epididymal abnormalities. The majority of ESR1 is nuclear/cytoplasmic; however, a small fraction is palmitoylated at cysteine 451 in mice and localized to cell membranes, in which it mediates rapid E2 actions. This study used an Esr1 knock-in mouse containing an altered palmitoylation site (C451A) in ESR1 that prevented cell membrane localization, although nuclear ESR1 was expressed. These nuclear-only estrogen receptor 1 (NOER) mice were used to determine the roles of membrane ESR1 in males. Epididymal sperm motility was reduced 85% in 8-month-old NOER mice compared with wild-type controls. The NOER mice had decreased epididymal sperm viability and greater than 95% of sperm had abnormalities, including coiled midpieces and tails, absent heads, and folded tails; this was comparable to 4-month Esr1KO males. At 8 months, daily sperm production in NOER males was reduced 62% compared with controls. The NOER mice had histological changes in the rete testes, efferent ductules, and seminiferous tubules that were comparable with those previously observed in Esr1KO males. Serum T was increased in NOER males, but FSH, LH, and E2 were unchanged. Critically, NOER males were initially subfertile, becoming infertile with advancing age. These findings identify a previously unknown role for membrane ESR1 in the development of normal sperm and providing an adequate environment for spermatogenesis.

Neonatal Uterine and Vaginal Cell Proliferation and Adenogenesis Are Independent of Estrogen Receptor 1 (ESR1) in the Mouse

ABSTRACT Neonatal uterus and vagina express estrogen receptor 1 (ESR1) and respond mitogenically to exogenous estrogens. However, neonatal ovariectomy does not inhibit preweaning uterine cell proliferation, indicating that this process is estrogen independent. Extensive literature suggests that ESR1 can be activated by growth factors in a ligand-independent manner and drive uterine cell proliferation. Alternatively, neonatal uterine cell proliferation could be ESR1 independent despite its obligatory role in adult luminal epithelial proliferation. To determine ESR1s role in uterine and vaginal development, we analyzed cell proliferation, apoptosis, and uterine gland development (adenogenesis) in wild-type (WT) and Esr1 knockout (Esr1KO) mice from Postnatal Day 2 to Postnatal Day 60. Uterine and vaginal cell proliferation, apoptosis, and uterine adenogenesis were comparable in WT and Esr1KO mice before weaning. By Days 29–60, glands had regressed, and uterine cell proliferation was reduced in Esr1KO mice in contrast to continued adenogenesis and proliferation in WT. Apoptosis in Esr1KO uterine epithelium was not increased compared to WT at any age, indicating that differences in cell proliferation, rather than apoptosis, cause divergence of uterine size in these two groups at puberty. Similarly, vaginal epithelial proliferation was reduced, and the epithelium became atrophic in Esr1KO mice by 29 days of age and later in Esr1KO mice. These results indicate that preweaning uterine and vaginal development is ESR1 independent but becomes dependent on ESR1 by Day 29 on. It is not yet clear what mechanisms drive preweaning vaginal and uterine development, but ligand-independent activation of ESR1 is not involved.

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.

Plasticity of spermatogonial stem cells

There have been significant breakthroughs over the past decade in the development and use of pluripotent stem cells as a potential source of cells for applications in regenerative medicine. It is likely that this methodology will begin to play an important role in human clinical medicine in the years to come. This review describes the plasticity of one type of pluripotent cell, spermatogonial stem cells (SSCs), and their potential therapeutic applications in regenerative medicine and male infertility. Normally, SSCs give rise to sperm when in the testis. However, both human and murine SSCs can give rise to cells with embryonic stem (ES) cell-like characteristics that can be directed to differentiate into tissues of all three embryonic germ layers when placed in an appropriate inductive microenvironment, which is in contrast to other postnatal stem cells. Previous studies have reported that SSCs expressed an intermediate pluripotent phenotype before differentiating into a specific cell type and that extended culture was necessary for this to occur. However, recent studies from our group using a tissue recombination model demonstrated that SSCs differentiated rapidly into another tissue, in this case, prostatic epithelium, without expression of pluripotent ES cell markers before differentiation. These results suggest that SSCs are capable of directly differentiating into other cell types without going through an intermediate ES cell-like stage. Because SSCs do not require reprogramming to achieve a pluripotent state, they are an attractive source of pluripotent cells for use in regenerative medicine.

Maximal Dexamethasone Inhibition of Luminal Epithelial Proliferation Involves Progesterone Receptor (PR)- and Non-PR-Mediated Mechanisms in Neonatal Mouse Uterus

ABSTRACT Progesterone (P4) and the synthetic glucocorticoid dexamethasone (Dex) inhibit luminal epithelial (LE) proliferation in neonatal mouse uteri. This study determined the roles of progesterone receptor and estrogen receptor 1 (PR and ESR1, respectively) in P4- and Dex-induced inhibition of LE proliferation using PR knockout (PRKO) and Esr1 knockout (Esr1KO) mice. Wild-type (WT), heterozygous, and homozygous PRKO female pups were injected with vehicle, P4 (40 μg/g body weight), or Dex (4 or 40 μg/g body weight) on Postnatal Day 5, then 24 h later immunostained for markers of cell proliferation. In WT and heterozygous mice, P4 sharply reduced LE proliferation, and Dex produced dose-responsive decreases equaling those of P4 at the higher dose. Critically, although both doses of Dex similarly decreased proliferation compared to vehicle-treated PRKOs, treatment of PRKO pups with the high Dex dose (40 μg/g) did not inhibit LE as much as treatments of WT mice with this Dex dose or with P4. Stromal proliferation was stimulated by P4 in WT but not PRKO mice, and Dex did not alter stromal proliferation. Uteri of all genotypes strongly expressed glucocorticoid receptor (GR), demonstrating that impaired Dex effects in PRKOs did not reflect GR deficiency. Furthermore, inhibition of LE proliferation by Dex (40 μg/g body weight) in Esr1KO mice was normal, so this process does not involve ESR1. In summary, inhibitory Dex effects on LE proliferation occur partially through non-PR-mediated mechanisms, presumably GR, as indicated by Dex inhibition of LE proliferation in PRKOs. However, maximal inhibitory Dex effects on uterine LE proliferation are not seen in PRKO mice with even high Dex, indicating that maximal Dex effects in WT mice also involve PR.