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

Highlights

• Estradiol stimulates testicular apoptosis during the breeding season in male toads.

• Apoptosis occurs mostly in spermatocytes.

• Estradiol has no effect on testicular proliferation.

• Estradiol has no effect on the activity and expression of CypP450c17 in testes.

• Estradiol reduces the activity of 3β-HSD/I during the post reproductive season.

Abstract

Estrogens inhibit androgen production and this negative action on amphibian steroidogenesis could be related to the regulation of steroidogenic enzymes. Estrogens are also involved in the regulation of amphibian spermatogenesis by controlling testicular apoptosis and spermatogonial proliferation. The Bidder’s organ (BO) is a structure characteristic from the Bufonidae family and in adult males of Rhinella arenarum it is one of the main sources of plasma estradiol (E₂). The purpose of this study is to analyze the effect of E₂ on testicular steroidogenic enzymes, apoptosis and proliferation in the toad R. arenarum. For this purpose, testicular fragments were treated during 24 h with or without 2 or 20 nM of E₂. After treatments, the activities of cytochrome P450 17α-hydroxylase-C_17–20 lyase (CypP450c17) and 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD/I) were measured by the transformation of radioactive substrates into products, and CypP450c17 expression was determined by Western blot analysis. Apoptosis in testicular sections was detected with a commercial fluorescent kit based on TUNEL method, and proliferation was evaluated by BrdU incorporation. Results indicate that E₂ has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3β-HSD/I activity during the post reproductive season. Furthermore, although E₂ has no effect on apoptosis during the pre and the post reproductive seasons, it stimulates testicular apoptosis during the reproductive season, mostly in spermatocytes. Finally, E₂ has no effect on testicular proliferation all year long. Taken together, these results suggest that E₂ is involved in the regulation of testicular steroidogenesis and spermatogenesis.

Introduction

As in other vertebrates, amphibian testes are organized in two compartments, the interstitial and the germinal ones, with the seminiferous component organized in seminiferous lobules (Duellman and Trueb, 1994, Wake, 1969). In anamniotes, the unit of spermatogenesis is the spermatocyst or cyst that is formed when Sertoli cells enclose one spermatogonium with cytoplasmic processes (Duellman and Trueb, 1994, Lofts, 1974, Pudney, 1995, Wake, 1969). In the cysts, spermatogonia proliferate and differentiate into meiotic spermatocytes, spermatids and mature spermatozoa.

The dynamic balance between cell proliferation and apoptosis, mainly regulated by hormones and environmental factors, determines the rate of the spermatogenic process. Apoptosis is important because it determines the equilibrium between the number of germ cells and the supporting capacity of Sertoli cells. In this context, the study of the variations in amphibian spermatogenesis is intimately related not only to proliferation of the germ cells, but also to apoptosis.

Several authors have studied the hormonal regulation of spermatogenesis. In mammals, follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone regulate cell survival, and excess or deprivation of these hormones can lead to testicular apoptosis (see review by Schlatt and Ehmcke, 2014, Shaha, 2008). It is now accepted that estrogens are essential for spermatogenesis (Shaha et al., 2010) and in vitro and in vivo studies demonstrate that they induce apoptosis in germ cells via the extrinsic pathway through an upregulation of Fas/FasL (Mishra and Shaha, 2005, Nair and Shaha, 2003). In non-mammalian vertebrates, gonadotropins and steroids have also been proved to regulate spermatogenesis (see the review by Chianese et al., 2011, Meccariello et al., 2014). Particularly in amphibians, androgens increase testes size in Rhinella arenarum (Penhos, 1956) and spermatogenesis in Anaxyrus fowleri but suppress spermatogenesis at spermatogonia II stage in several species of the genus Rana and in Duttaphrynus melanostictus (for a review of evidences until 1980, see Rastogi and Iela, 1980). In Pelophylax esculentus, gonadotropins stimulate spermatogonial proliferation and also androgen production, while androgens induce spermatid formation and also stimulate spermatogonial mitosis (Minucci et al., 1992). Moreover, during the reproductive season of P. esculentus, there is a strong relationship between high levels of testosterone, the mitotic activity of the germinal epithelium and the expression of c-kit, which is indispensable for spermatogenesis and spermatogonial proliferation (Raucci and di Fiore, 2007). Furthermore, in Lithobates pipiens corticosterone induces a reduction in spermatid formation, while 5α-dihydrotestosterone reduces the amount of secondary spermatogonia and spermatids, and stimulates spermatocytes formation (Tsai et al., 2003).

Estrogens are also involved in the regulation of amphibian spermatogenesis by acting in the early spermatogenic process (Callard, 1992, Polzonetti-Magni et al., 1998). 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 (E₂) induces an acceleration of spermatogenesis (Hu et al., 2008). In L. pipiens, the action of chronic estradiol may be both inhibitory and stimulatory, depending on the stages of spermatogenesis, since chronic treatment retards the formation of spermatocytes and secondary spermatogonia, and accelerates the latter stages of spermatogenesis (Tsai et al., 2003). Besides, in P. esculentus there is a strong mitogenic activity of spermatogonia in the period of the year characterized by high concentrations of estradiol (Fasano et al., 1989, Rastogi et al., 1985, Varriale et al., 1986). In this species, estradiol promotes spermatogonial proliferation through the activity of Fos protein and the activation of mitogen-activated protein kinase in spermatogonia (Chieffi et al., 1995, Chieffi et al., 2000, Cobellis et al., 1999, Minucci et al., 1997) and proliferation is inhibited by melatonin (d́Istria et al., 2003). Moreover, estradiol regulates the expression of Fra-1, which is located in peritubular myoid cells, efferent ducts and blood vessels and may be involved in sperm transport. Estradiol is also involved in sperm release in this species, since impairment of estrogen activity affects the detachment of spermatozoa from the Sertoli cells (Cobellis et al., 2005, Cobellis et al., 2008). Taken together, these results suggest that anuran spermatogenesis is a complex process in which each stage can be independently regulated by different steroids.

It has also been suggested that estrogens inhibit androgen production in several amphibians, since estradiol treatment decreases plasma testosterone concentrations in X. laevis and P. esculentus (Fasano et al., 1991, Hecker et al., 2005, Pierantoni et al., 1986). The negative action of estradiol on amphibian steroidogenesis could be related to the regulation of steroidogenic enzymes. In the rainbow trout, E₂ treatment decreases the expression of cytochrome P450 17-hydroxylase-C_17–20-lyase (CypP450c17), 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD/I), and 11β-hydroxylase (Govoroun et al., 2001) while in P. esculentus it inhibits the activity of 17-hydroxylase (Fasano et al., 1991). Moreover, estradiol levels increase in males of P. esculentus in the early summer, following a decline in testosterone levels (Polzonetti-Magni et al., 1984) and authors speculated that estradiol inhibits GnRH release and LH secretion, leading to a decrease in androgen levels. It has been demonstrated that in P. esculentus the expression of testicular kisspeptin receptor is estradiol dependent and also that kisspeptins modulate the expression of testicular estrogen receptor alpha (Chianese et al., 2015) and upregulate the GnRH system and the expression of testicular estrogen receptor beta and PCNA (Chianese et al., 2015). These results suggest that kisspeptins and their receptors are involved in the regulation of estradiol-dependent testicular functions such as proliferative activity and spermatogenesis.

The toad R. arenarum is a species with an extensive reproductive season (Gallardo, 1974). Spermatogenic wave starts in October, when there is a great number of spermatogonia and scarce spermatocytes and spermatids (Burgos and Mancini, 1948). This is a species with a dissociated reproductive pattern, since the reproductive behavior in males is associated with low levels of plasma androgens due to a decrease in the activity of CypP450c17 (Canosa and Ceballos, 2002a, Canosa et al., 2003, Fernández Solari et al., 2002).

In species from the Ranidae family, it has been demonstrated that plasma estradiol is produced by the testes (Fasano et al., 1989, Varriale et al., 1986). However, in R. arenarum testes do not express aromatase activity and the main source of plasma estradiol in the toad is the Bidder organ (BO), a structure which is characteristic from the Bufonidae family (Scaia et al., 2011, Scaia et al., 2013). Plasma estradiol levels are low during the pre-reproductive season and they increase gradually during the reproductive season, reaching the highest values during the post-reproductive season (Scaia et al., 2013).

Although the effect of estradiol on testicular physiology has been investigated in frogs, the role of estradiol on testicular steroidogenesis and spermatogenesis in bufonids still remains unexplored. In the present study we analyze the relationship between estradiol and testicular steroidogenic enzymes, apoptosis and proliferation in R. arenarum.