Analysis of xenoestrogen effects in the male rat gonad

Abstract

Naturally occurring estrogenic chemicals existing in food such as soybeans and synthetic xenoestrogens, termed Xenoestrogens, contaminating food and water sources have become issues of public health interest. The concern about these estrogenic compounds is particularly concerning to male individuals because of the association of estrogenic compounds with the decline in male reproductive health. This dissertation describes the adverse effects of developmental exposure to the dietary estrogenic compounds found in soybeans (genistein and daidzein) and the synthetic estrogenic compounds that commonly contaminate food and water sources (BPA, BPS, and EE2) on testicular cell function using the rat model. The Leydig cell is the primary source of the male sex steroid testosterone which supports the male phenotype, development of secondary sexual characteristics, and maintains fertility. The Sertoli cell is the principal link between germ cells and the regulatory hormones needed for spermatogenesis. The overall aim is to analyze the effect of xenoestrogens in testicular cells under the following specific objectives: I. Determine whether soy-based diets exposure are due to naturally occurring isoflavones and whether the effects are influenced by age at exposure II. Investigate whether isoflavone effects in the testis are due to daidzein, genistein, or both compounds III. Investigate the effect of synthetic estrogens, i.e., BPA, BPS, EE2, on steroid hormone secretion in the developing male rat gonad To achieve the first objective, 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 Hsd17β protein in the testes and was associated with increased (p<0.05) Lhβ and Fshβ subunit protein expression in pituitary glands. For the second objective, 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. Interestingly, Esr1 and androgen receptor (Ar) protein and pituitary Fshβ with Lhβ subunit protein were increased (p<0.05) by feeding genistin and G+D diets, but not the daidzin diet. Studies of primary Leydig cell culture showed that 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. Together, results demonstrated that altered testicular steroidogenic capacity and pituitary FSHβ and LHβ subunit expression due to soy-based diets result from specific actions by genistein and daidzein. Our data are relevant to public health due to increased consumption of soy-based products by all segments of the population and the increasing incidence of reproductive anomalies in the population. Additional studies are warranted to assess the impact of isoflavone-induced changes in testicular androgen concentrations on germ cell development, sperm production, and male fertility. To achieve the third objective two separate experiments were conducted in which prepubertal and pubertal male rats at 21 and 35 days of age were provided test chemicals in drinking water (5 µg/L) for 14 days. At the end of the exposure period, results showed that BPS and EE2 inhibited testicular testosterone (T) concentrations in a similar manner to BPA. Our results also showed that although BPA had no effect on gonadal estradiol (E2) secretion, both BPS and EE2 increased testicular E2 concentrations. Furthermore, protein expressions of Sertoli cell-produced factors were altered. For example, BPS and EE2 but not BPA decreased anti-Mullerian hormone (Amh) protein expression, while expression of Desert hedgehog (Dhh) protein was increased by all tested chemicals. Together, these observations not only demonstrated that BPA, BPS, and EE2 have similar inhibitory effects on gonadal steroidogenesis but have the potential to disrupt the functions of gonadal cells in the male. These findings on the effects of dietary isoflavones (genistein and daidzein) and the synthetic estrogenic compounds (BPA, BPS, and EE2) on testicular cells reinforce the view that exposures of the population to environmental chemicals have the potential to impair reproductive health. Additional studies are warranted to understand the effects of changes in intratesticular androgen concentrations on germ cell maturation and the mechanisms by which single and chemical mixtures impact testicular cells.