2. Hormonal Control of Spermatogenesis

The role of follicle-stimulating hormone (FSH) in the modulation of spermatogenesis has been a matter of debate since a study on five men with inactivating mutation of the FSH receptor (FSHR) gene showed that none was azoospermic and that two had children [3]. This finding prompted some researchers to hypothesize that FSH was not necessary for spermatogenesis, but the finding that men with inactivating mutations in FSH beta subunit were completely azoospermic [4] challenged that hypothesis. Further studies clarified that the mutant FSHR is not completely inactive [5], so that a residual FSH action could be able to promote spermatogenesis and that mutations in the FSH gene are more severe than those of the FSHR [6].

Studies in mice lacking FSH (FSHKO) or FSHR (FSHRKO) clearly demonstrated that FSH is required to increase the number of spermatogonia and spermatocytes [7] and that FSH treatment was found to increase spermatogonial and spermatocyte number in hypophysectomized or gonadotropin-releasing hormone (GnRH)-immunized adult rats [8]. FSH acts also as a survival factor for spermatogonia, since acute FSH suppression induces spermatogonial apoptosis [9] and is required to stimulate the prenatal and prepubertal proliferation of Sertoli cell, an effect which is totally independent from luteinizing hormone (LH) action, as demonstrated in hypogonadal LH receptor null mice [10], as well as from testosterone action, as demonstrated in mice lacking Sertoli cell androgen receptor (SCARKO) and FSHR, which had a Sertoli cell number comparable to that of FSHRKO mice [7]. In the absence of FSH or FSHR, the Sertoli cell number is decreased by about 30–45% in comparison to normal testicular development: since the Sertoli cell is able to support a certain number of germ cells, the number of Sertoli cells determines the quantity of sperm produced. This may explain why FSHRKO mice present with complete spermatogenesis, but the amount of germ cells is lower than in wild-type animals [7].

Studies in men with congenital hypogonadotropic hypogonadism suggest that pretreatment with FSH alone prior to combined gonadotropin treatment enhances spermatogenesis [11]. However, FSH alone is not able to promote spermatogenesis beyond the pachytene spermatocytes: a recent study on SCARKO mice demonstrated that Sertoli cell androgen receptor (AR) signaling is required for the survival of meiotic prophase spermatocytes, since SCARKO mice exhibited loss of meiotic germ cells and failure of surviving spermatocytes to progress. Early meiotic prophase events are not dependent upon androgen signaling, therefore, chromosome synapsis and recombination occurred normally in surviving spermatocytes that entered meiotic prophase; however, SCARKO pachytene spermatocytes were found to acquire aberrant transcriptomic attributes (leptotene or zygotene transcriptome state) and failed to progress to subsequent transcriptomic signatures [12].

FSH alone has been also found to maintain spermatogenesis independently from testosterone; this is the case of transgenic male mice with activating FSHR mutation that enabled strong FSH activation (cAMP response > 10-fold above basal). Use of the antiandrogen flutamide to interfere the binding of androgens to the AR had no effect on spermatogenesis [13].

In normal conditions, however, testosterone signaling is required for spermatogenesis to proceed beyond meiosis. Testosterone signaling contributes also to maintaining tight junctions between adjacent Sertoli cells (essential for the blood-testis barrier) and a specialized environment for germ cells, mainly through its modulation of micro-RNAs that target genes essential for cell junction restructuring and Sertoli-germ cell adhesion. The absence of T results in disruption of blood–testis barrier, premature detachment of developing spermatid germ cells from Sertoli cells, and block of the release of mature spermatozoa from Sertoli cells, with consequent germ cells phagocytosis by Sertoli cells [14].

Testosterone (T) is produced by Leydig cells in response to LH, and mediates its effects by the AR expressed by the Sertoli cells via classical and nonclassical pathways. In the classical (genomic) pathway, T diffuses through the plasma membrane and interacts with AR and the complex T/AR translocates to the nucleus to bind to androgen response elements (AREs) in gene promoter regions and regulates gene transcription, while in the nonclassical (nongenomic) pathway, T/AR rapidly phosphorylates the SRC kinase, resulting in the stimulation of the epithelial growth factor (EGF) receptor and the fast (within 1 min) activation of MAP-kinase cascade and the CREB transcription factor, with a resulting sustained (for at least 12 h) increased protein phosphorylation and long-term gene expression changes that are mediated by increased kinase activity [15]. Both pathways are essential for spermatogenesis: a study performed on testis explants of male Sprague Dawley rats containing intact seminiferous tubules and accompanying interstitial cells, using inhibitors to specifically block each pathway in vitro, demonstrated that both pathways are able to activate transcription of the Sertoli cell-specific Rhox5 mRNA, which is dramatically upregulated in the presence of T in vivo, and that activation of either T signaling pathway in Sertoli cells can differentially modulate germ cells gene expression [15].

It has been classically demonstrated that intratesticular testosterone (ITT) concentration are much higher (50–100-fold) than circulating levels, however, spermatogenesis may be maintained by very low ITT concentration: mice with inactivation of the LH receptor (LuRKO mice) had intact spermatogenesis despite very low ITT levels (2% of control level), but administration of the antiandrogen flutamide halted sperm maturation at the round spermatid stage [16]. In addition, a more recent study demonstrated that spermatogenesis in LuRKO mice could be normalized with exogenous testosterone that achieved a serum T concentration comparable to that of WT mice, but an ITT level less than 1.5% of the WT concentration [17]. The relationship between serum and intratesticular T levels is, therefore, far to have been clearly established, so that further studies are needed.

It has been proposed that testosterone alone could induce complete spermatogenesis without the need of FSH action; indeed, subcutaneous testosterone supplementation in male mice with hypogonadotropic hypogonadism due to Kiss1 knockout was able to restore serum and intratesticular testosterone levels, promote testicular descent, and induce complete spermatogenesis from spermatocytes to elongated spermatids, but the resultant testicular weight reached only 40% of wild-type controls, similarly to what was found in hypogonadal or GnRH KO mice treated with testosterone supplementation. [18]. Such a quantitative deficit of spermatogenesis is likely to be due to the lack of FSH.

Both FSH and testosterone are, therefore, required to promote full spermatogenesis; in addition, both hormones have synergistic effects upon spermatogenesis. FSH regulates transcripts required for normal testicular function, including StAR gene, which is essential for steroid synthesis [14], and stimulates the Sertoli cell production of androgen binding globulin, which helps maintain a high T concentration within the testes. On the other hand, testosterone is thought to modulate the oligosaccharide complexity of pituitary FSH; castration induces changes in the oligosaccharide composition of pituitary FSH both in prepubertal and adult animals, and administration of flutamide, able to interfere the binding of androgens to the AR both peripherally and at hypothalamic-pituitary level, lead to a predominance of circulating FSH glycosylation variants bearing incomplete oligosaccharides [19]. Administration of testosterone enanthate to pubertal patients does not modify the serum FSH levels, but lead to a significant increase in the proportion of FSH bearing complex oligosaccharides [20].