Primordial Germ Cells (Spermatogonial Stem Cells) of Bullfrogs Express Sex Hormone-Binding Globulin and Steroid Receptors during Seasonal Spermatogenesis

Abstract

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers – alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor) – and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)β was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERβ expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERβ, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus.

Immunoexpression of aromatase and estrogen receptors β in stem spermatogonia of bullfrogs indicates a role of estrogen in the seasonal spermatogonial mitotic activity

“…Moreover, in the sections of calvariae bone, the osteoblasts, another site of high aromatase expression [45], showed enhanced and specific CYP19 immunolabeling. Regarding ERb immunoblotting, as demonstrated in our previous study, the bullfrog testicular protein ex- tracts yielded two immunoreactive bands of approximately 55 kDa which are also coincident to other species [4].…”

“…A strong testosterone immunolabeling has been observed in the cytoplasm of bullfrogs primordial germ cells -PGCs (stem spermatogonia) during winter [39]. Moreover, androgen receptors (ARs) and sex-hormone binding globulin (SHBG) have also been immunohistochemically detected in these cells during this seasonal period, confirming that testosterone is maintained by AR and/or SHBG and plays a role in PGCs [4].…”

“…In non-mammalian vertebrates, as frogs, testosterone also seems to inhibit the mitotic activity of spermatogonia [47]. Thus, it has been speculated that testosterone plays a role in the PGCs proliferative control avoiding the formation of new spermatogonial cysts in winter when the number of germ cells reach a maximal limit to be supported within the large seminiferous lobules [4,39].…”

Effect of estradiol on apoptosis, proliferation and steroidogenic enzymes in the testes of the toad Rhinella arenarum (Amphibia, Anura)

“…For instance, gonadotropins may play an important role, since they induce spermatogonial proliferation in newts (Abé and Ji, 1994;Ito and Abé, 1999;Ji et al, 1992). In P. esculentus, besides the role of estradiol on proliferation previously described, spermatogonial proliferation is also associated with seasonal changes in testosterone (Raucci and Di Fiore, 2007) while in L. catesbeianus both testosterone and E 2 control proliferation (Caneguim et al, 2013a). Melatonin is another hormone that has also been suggested to regulate proliferation by inhibiting estradiol-stimulated mitotic activity of spermatogonia in P. esculentus (d Istria et al, 2003).…”

“…In Lithobates catesbeianus estrogens play an important role in the control of primordial germ cells during the seasonal spermatogenesis (Caneguim et al, 2013a(Caneguim et al, , 2013b. Moreover, in juveniles Xenopus laevis estradiol (E 2 ) induces an acceleration of spermatogenesis (Hu et al, 2008).…”