Actions of the endocrine disruptor methoxychlor and its estrogenic metabolite on in vitro embryonic rat seminiferous cord formation and perinatal testis growth

Abstract

The current study examines the actions of methoxychlor and its estrogenic metabolite, 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE), on seminiferous cord formation and growth of the developing rat testis. The developing testis in the embryonic and early postnatal period is likely more sensitive to hormonally active agents than at later stages of development. Embryonic day 13 (E13) testis organ cultures were treated with either 0.2, 2, or 20 μM methoxychlor or 1, 3, 6, 15, 30, or 60 μM HPTE to examine effects on cord formation. No concentration of methoxychlor completely inhibited cord formation. However, cord formation was abnormal with the presence of a reduced number of cords and appearance of “swollen” cords at the 2 and 20 μM concentrations of methoxychlor. The swollen cords were due to an increase in the number of cells in a cord cross section and reduction of interstitial cell numbers between cords. Treatment of embryonic day 13 (E13) testes with HPTE caused abnormal cord formation at the 3 μM and 6 μM concentrations, and completely inhibited cord formation at the 15, 30, and 60 μM concentrations. In addition to the estrogenic metabolite HTPE, methoxychlor can also be metabolized into anti-androgenic compounds. Therefore, to determine the spectrum of potential actions of methoxychlor on testis development, different concentrations of estradiol, testosterone, and an anti-androgen (flutamide) were utilized to determine their effects on E13 testis organ culture morphology. Estradiol (1 μM) and flutamide (0.1μM) both inhibited seminiferous cord formation in E13 testis organ cultures. Therefore, methoxychlor may be acting through the androgen and/or estrogen receptors to elicit its actions on seminiferous cord formation. Reverse transcription polymerase chain reaction (PCR) (RT-PCR) confirmed the presence of estrogen receptor alpha (ERα) mRNA from embryonic day 14 (E14) through postnatal day 5 (P5) while estrogen receptor beta (ERβ) mRNA did not appear until approximately E16 of testis development. Androgen receptor (AR) expression was present from E14 through P5 of testis development, but at apparently reduced levels at E14 and E16. Immunohistochemical analysis localized ERα to the cells of the seminiferous cords at E14 though P5 while ERβ was present in cells of the interstitium at E16 and P0. Androgen receptor was localized to germ and interstitial cells. The effects of methoxychlor, HPTE, estradiol, and testosterone on cell growth of perinatal testes was determined with a thymidine incorporation assay in postnatal day zero (P0) testis cell cultures. Methoxychlor (0.002, 0.02, and 0.2 μM) and HPTE (2 and 20 μM) stimulated thymidine incorporation in P0 testis cell cultures in a similar manner to estradiol (0.01, 0.1, and 1 μM). In addition, testosterone (0.1 μM) also stimulated thymidine incorporation in P0 testis cultures. Observations suggest that methoxychlor and its metabolite HPTE can alter normal embryonic testis development and growth. The actions of methoxychlor and HPTE are likely mediated in part through the steroid receptors confirmed to be present in the developing testis.

Introduction

Normal testis morphology and development is dependent upon critical somatic and germ cell differentiation and proliferation during the embryonic period. In the rat, crucial morphogenesis of the testis occurs at embryonic day 13.5 (E13.5; plug day considered to be E0) [1], [2] when pre-peritubular cells migrate from the adjacent mesonephros to enclose Sertoli and germ cell aggregates forming seminiferous cords [1], [2], [3], [4]. After seminiferous cord formation, somatic and germ cells undergo the highest levels of cellular proliferation that occurs during testis development [5]. Embryonic somatic and germ cell proliferation ensures adequate numbers of germ and somatic cells are present for adult testicular spermatogenesis [6]. Therefore, many critical developmental events occur from E13 to postnatal day zero (P0) during gestation in the rat that allow for normal testis development.

Exposure to agents that interfere or alter morphogenic or proliferative events during embryonic testis development will impair the ability of the testis to develop, proliferate, or produce normal numbers of viable sperm [7], [8], [9]. Accumulating evidence has suggested that increasing amounts of synthetic endocrine active chemicals in the environment are compromising normal sex determination and the ability of testes to produce normal numbers of sperm [7], [8], [9]. The degree this is an issue with the human population remains to be elucidated; however, a number of wildlife species clearly are affected. Environmental agents can act as weak estrogens or anti-androgens and may contribute to the increase in abnormal sex determination [10], reduced sperm counts [11], and overall reductions in the ability of individuals to produce viable offspring in many species [10].

Methoxychlor is a chlorinated hydrocarbon pesticide currently used in the United States as a replacement for DDT [10] and has been categorized as weakly estrogenic. Methoxychlor can be metabolized by the liver into two demethylated compounds {[(2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane, (HPTE) and 2,2-bis-(p-hydroxyphenyl)-1,1,1-dichloroethane, (HPDE)}] and two 0-ring methylated compounds. Each of these metabolites have been demonstrated to act differentially at the estrogen and androgen receptors. The most active estrogenic metabolite is HPTE [12], [13], [14]. HPTE has been determined in several experiments to be weakly estrogenic [15], [16], [17]. HPTE stimulates the expression of estrogen receptors [18], and has been determined to cause decreased testosterone concentrations and decreased seminal and prostate weights when administered neonatally in rodents [19]. There are conflicting reports on whether methoxychlor elicits its response through the estrogen receptors [18], [20]. Some of methoxychlor’s actions may be through a metabolite that acts in an anti-androgenic manner [21]. Therefore, exposure to methoxychlor in the environment may impair or alter reproductive function in many domesticated and wildlife species through actions on both estrogen and androgen receptors.

Previous studies have demonstrated the effects of methoxychlor on the female reproductive system that have resulted in reduced embryo implantation efficiency, increased abortion rate, and formation of smaller gonads with a higher incidence of germ cell apoptosis [22]. Exposure to methoxychlor either late embryonically or during the early postnatal period also has resulted in reproductive tract abnormalities [19] and altered reproductive behavior in males [23]. To date no experiments have been conducted to evaluate the effect of exposure of methoxychlor on early embryonic testis morphology (seminiferous cord formation) or growth. Therefore, the current study was conducted to determine if methoxychlor or its metabolite HPTE may alter morphologic sex determination (formation of seminiferous cords) and/or growth of the testis during the perinatal period in rats.