A switch in Sertoli cell responsiveness to FSH may be responsible for robust onset of germ cell differentiation during prepubartal testicular maturation in rats

Abstract

FSH and Testosterone (T) regulate spermatogenesis via testicular Sertoli cells (Sc), which bear receptors for these hormones. Despite sufficient circulating levels of FSH and T postnatally, predominant appearance of spermatogonia B and spermatocytes is not discernible until 11 and 18 days of postnatal age, respectively, in rat testes. In an attempt to explore the underlying causes, we cultured Sc from neonatal (5- and 9-day-old) and prepubertal (12- and 19-day-old) rat testes and compared the status of FSH receptor (FSH-R) and androgen receptor (AR) signaling. Protein and mRNA levels of FSH-R and AR remained uniform in cultured Sc from all age groups. Androgen binding ability of AR was similar, and T-induced nuclear localization of AR was discernible in Sc from all age groups. Binding of FSH to FSH-R, subsequent production of cAMP, and mRNA of stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF), known to be essential for the robust differentiation of repopulating spermatogonia, were significantly augmented in prepubertal Sc compared with those in neonatal Sc. However, treatment of neonatal Sc with cholera toxin or forskolin, which stimulate cAMP production bypassing FSH-R, demonstrated a concomitant rise in SCF and GDNF mRNA expression, which was similar to the FSH-mediated rise observed in prepubertal Sc. These observations suggested that, during prepubertal Sc maturation, the ability of FSH-R to respond to FSH is significantly augmented and is associated with the robust differentiation of repopulating spermatogonia, and such a switch in Sc from FSH-resistant to FSH-responsive mode during prepubertal development may underlie the initiation of robust spermatogenesis.

Advantages of pulsatile hormone treatment for assessing hormone-induced gene expression by cultured rat Sertoli cells

“…Primary cultures of Sc provide an excellent opportunity for investigating their ability to express various genes under endocrine (FSH and T together or alone) or paracrine control (Fix et al 2004;Walker and Cheng 2005;Devi et al 2006;Cheng et al 2007;Viswanathan et al 2009;Shupe et al 2011;Bhattacharya et al 2012;Mishra et al 2012). Such information provides vital clues for determining the role of Sc in regulating fertility in males.…”

“…Pulsatile and constant hormonal treatments of Sc culture were run in parallel and gene expression was evaluated at various time points. Since pre-pubertal rat Sc of around 18 ± 2 days of age are reported to be the most hormone-responsive in culture, as found by us earlier (Devi et al 2006;Bhattacharya et al 2012Bhattacharya et al , 2015b and others (Fix et al 2004;Cheng et al 2007;Viswanathan et al 2009;Shupe et al 2011), we used Sc from this age group of rats. FSH and T were used in combination to stimulate cultured Sc, thereby mimicking the in vivo condition.…”

“…The expression of three important Sc genes, namely, Scf (only responsive to FSH), Claudin11 (only responsive to T) and Transferrin (both FSH-and T-responsive) and the respective hormone-receptors, e.g., Fshr and Ar, were evaluated as readouts (Walker and Cheng 2005;Bhattacharya et al 2012).…”

“…Although the use of primary cultures of Sc is routinely employed for the investigation of the mode of action of FSH and T (Fix et al 2004;Walker and Cheng 2005;Cheng et al 2007;Viswanathan et al 2009;Shupe et al 2011;Bhattacharya et al 2012), the behavior of Sc in culture and its relevance to in vivo situations have been contested (Russell and Steinberger 1989). Therefore, FrSc as reported by us earlier (Bhattacharya et al , 2015b and others Wood et al 2011) were also used in this study.…”

“…The synergistic effect of FSH and T promotes the secretion of various growth factors and metabolites by Sc to support the initiation and maintenance of male germ cell (Gc) division and differentiation (Walker and Cheng 2005). In order to understand the role of Sc in the regulation of Gc maturation and fertility, neonatal, pubertal and adult Sc have been extensively studied upon their isolation from testes of rodent and primate species (Majumdar et al 1995(Majumdar et al , 1998Walker and Cheng 2005;Devi et al 2006;Bhattacharya et al 2012Bhattacharya et al , 2015a. However, Sc cultured from rats aged 18 ± 2 days are predominantly used for in vitro studies (Steinberger et al 1978;Shupe et al 2011;Matsushita et al 2016).…”