Morphofunctional evaluation of stem spermatogonia during seasonal spermatogenesis of bullfrog

Abstract

Spermatogenesis and steroidogenesis in amphibians change according to the seasonal cycle. In Winter, the interstitial Leydig cells are scarce and quiescent. However, in the seminiferous lobules, the primordial germ cells (PG) are strongly testosterone-immunolabeled. In some toads, it has been demonstrated that testosterone is converted into estrogen by aromatase enzyme, and that estrogen play an important role on the spermatogenic process. The aim of the present study is to evaluate the serum testosterone levels as well as to localize testosterone in the PG cytoplasm and to verify how this hormone is maintained in these cells: via androgen receptor (AR) or androgen binding proteins (SHBG/ABP). Moreover, the mitotic activity (PCNA) of PG as well as a possible role of aromatase in these cells will also be evaluated. Adult male bullfrogs will be collected in Winter (WG; n=8) and summer (SG; n=8). The tests will be fixed and embedded in historesin and paraffin. In the paraffin sections, the following immunohistochemical reactions will be carried out: GFRalfa-1 (a stem cell marker for PG), SHBG/ABP, AR, aromatase and PCNA. The mitotic activity of PG will be evaluated by the quantification of the number of EP/mm2 of seminiferous lobules as well as by the number of PCNA-positive PG. With the aim to identify the localization of testosterone in PG, some testicular fragments will be prepared for analysis under transmission electron microscope (TEM). The ultrathin sections will be submitted to immunocytochemistry for detection of testosterone and analyzed under TEM. (AU)