The Role of Androgens in Testicular Development and Dysgenesis
Scott, Hayley M
Disorders of male reproductive health which manifest at birth (cryptorchidism, hypospadias) or in young adulthood (testicular germ cell cancer and low sperm counts), are common and may be increasing in incidence. These disorders have a common fetal origin and share risk factors; consequently they are hypothesized to comprise a testicular dysgenesis syndrome (TDS). TDS arises when maldevelopment (dysgenesis) of the fetal testis results in hormonal malfunctions and abnormal development and function of the somatic cells. It is thought that the suppressed intratesticular testosterone levels associated with TDS may account for subsequent low sperm counts, via a reduction in perinatal Sertoli cell proliferation/number. Sertoli cells do not express androgen receptors (AR) in fetal life in the human or rat, so it is hypothesised that any androgen effects on Sertoli cell number occur indirectly, via the AR positive peritubular myoid cells. Evidence from the di (n‐butyl) phthalate (DBP)‐treated rat model for TDS suggests that reduced androgen action may play a role in testicular dysgenesis as in patients with complete androgen insensitivity syndrome (CAIS; ‘testicular feminization’), in whom focal areas of testicular dysgenesis have been reported. The studies in this thesis sought to establish if reduced androgen levels/action in the fetal rat testis contribute to putative testicular dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes or abnormal aggregation of fetal Leydig cells, the precursor of focal dysgenesis. Pregnant rats were exposed to treatments or co‐treatments expected to manipulate testicular testosterone levels (DBP, testosterone propionate; TP) or action (flutamide, DMBA) or to induce intrauterine growth restriction (dexamethasone), another risk factor for TDS. The aforementioned endpoints were analysed in fetal testes and related to testicular testosterone levels and peripheral androgen action (anogenital distance). The same endpoints were evaluated in mice with inactivation of the androgen receptor (tfm or ARKO mice). As androgen action is assumed to be mediated indirectly, via the peritubular myoid cells, changes in peritubular myoid cell number and function were investigated in testes with suppressed androgens. In vitro studies were also used to investigate the role of androgens in Sertoli cell proliferation. Fetal rat testis explants were cultured with various chemicals designed to manipulate androgen action and Sertoli cell proliferation. Potential non‐androgen related mechanisms of DBP action were investigated using Taqman RT‐PCR to determine the mRNA expression of key developmental genes after exposure to DBP. Sertoli cell number was reduced after exposure to treatments that reduced testicular testosterone levels, i.e. DBP alone or as a co‐treatment, TP and dexamethasone. Sertoli cell numbers in ARKO mice were also significantly reduced. The occurrence of multinucleated gonocytes and large Leydig cell clusters were induced after exposure to DBP, alone or as a co‐treatment, but not after exposure to TP or dexamethasone, and these dysgenetic endpoints did not occur either in tfm or ARKO mice. Rats exposed in utero to DBP have reduced testicular testosterone levels, however peritubular myoid cell number was unaffected by DBP, though AR expression in the peritubular myoid cells was delayed, and laminin and vimentin expression in Sertoli cells was altered after DBP exposure. DMRT‐1 and DAX‐1 mRNA expression levels were significantly reduced after DBP exposure, but this reduction was no longer evident once mRNA expression was corrected for Sertoli cell number. In conclusion, these studies provide strong evidence that androgens play a role in regulation of Sertoli cell number/proliferation, and this is supported by a comparable reduction in Sertoli cell number in ARKO and tfm mice. However, since the treatments that reduce testicular testosterone in the rat, may also have a direct affect on the Sertoli cells, this alternate mechanism of action cannot be ruled out, and the administration of a treatment that reduces testicular testosterone without directly affecting Sertoli cells is required. These studies also show that reduced testicular testosterone levels are associated with multinucleated gonocyte formation and fetal Leydig cell aggregation, although this evidence it is not supported by parallel findings from the TP and dexamethasone exposed rats or the ARKO and tfm mice, as neither of these endpoints were identified as being affected in these animals. Aside from the delay in AR expression, there were no obvious changes in peritubular myoid cell number or the peritubular myoid cell markers examined in testes deprived of androgens, although there are other markers that could be investigated. mRNA analysis of the developmental genes investigated after DBP exposure, demonstrated no change in expression after correction for Sertoli cell number, suggesting that they do not play a role in the dysgenetic features observed in DBP exposed testes.