Trypsin is a multifunctional factor in spermatogenesis

Abstract

Trypsin is well known as a pancreatic enzyme that is typically secreted into the intestine to digest proteins. We show in our current study, however, that trypsin is also a key factor in the control of spermatogenesis. A progestin in teleost fish, 17␣, 20␤-dihydroxy-4-pregnen-3-one (DHP), is an essential component of the spermatogenesis pathway, particularly during the initiation of the first meiotic division. In the course of our investigations into the mechanisms underlying progestin-stimulated spermatogenesis, we identified that eel trypsinogen is upregulated in eel testis by DHP treatment. Trypsinogen is expressed in the Sertoli cells surrounding spermatogonia and in the membranes of spermatids and spermatozoa. Using an in vitro eel testicular culture system, we further analyzed the roles of trypsin in spermatogenesis. The inhibition of trypsin using specific antibodies or serine protease inhibitors was found to compromise DHP-induced spermatogenesis. A low dose of trypsin induces DNA synthesis and the expression of Spo11, a molecular marker of meiosis, in germ cells. By comparison, a higher dose of trypsin partially induced spermiogenesis. Furthermore, trypsin was detectable in the membranes of the spermatozoa and found to be associated with fertilization in fish. Our results thus demonstrate that trypsin and/or a trypsin-like protease is an essential and multifunctional factor in spermatogenesis.fish ͉ germ cell ͉ in vitro culture ͉ teleost ͉ testis S permatogenesis is a complex developmental process that begins with the mitotic proliferation of spermatogonia and proceeds through extensive morphological changes that convert haploid spermatids into functional spermatozoa. Since the progression of the cellular stages of spermatogenesis is similar across the vertebrate kingdom, it is very possible that common control mechanisms exist in these species. However, there are known differences in this process between each class of vertebrate. For example, androgens play a major role in spermatogonial pathways in fish, but in mammals, the spermatogonia remain largely unaffected by a loss of androgen production. Moreover, the structures of the testes also differ among vertebrates. In mammals, the seminiferous tubules contain several successive generations of germ cells, whereas fish exhibit a cystic type of spermatogenesis.Various reproductive styles and gametogenic patterns exist in different teleost species. The Japanese eel, for example, has a specific spermatogenetic pattern and under fresh-water culture conditions, the males of this species show immature testes containing only non-proliferating type A and early type B spermatogonia. It has been reported, however, that injection with human chorionic gonadotropin (hCG) can induce all stages of spermatogenesis in the Japanese eel in vivo (1). Furthermore, the Japanese eel is the only animal in which complete spermatogenesis has been induced by hormonal treatments using an in vitro organ culture system and a germ cell/somatic cell coculture system (2-4). Hence, the ...

The synthesis and role of taurine in the Japanese eel testis

“…However, the results of long-term (15 days) organ culture showed that taurine cannot induce complete spermatogenesis. Consistent with this finding, we reported previously that trypsin is also up-regulated in eel testis by DHP and that it induces the expression of Spo11 in germ cells, but not all aspects of spermatogenesis (Miura et al 2009). These results collectively suggest that meiosis is controlled by the conglomeration of various factors including taurine and trypsin.…”

“…Several cDNA clones that are regulated by DHP were thereby identified (Ozaki et al 2006a, b;Miura et al 2009), one of which showed a high homology to cysteine dioxygenase 1 (CDO1) in other animals. CDO is one of the enzymes that catalyze the conversion of L-cysteine to cysteine sulfinate (cysteine sulfinic acid).…”

Cloning, pharmacological characterization, and expression analysis of Atlantic salmon (Salmo salar L.) nuclear progesterone receptor

“…late type B spermatogonia) were less intensely/not stained. In Japanese eel, the molecular mechanism underlying DHPmediated stimulation of spermatogenesis involved elevated expression of 11b-hydroxysteroid dehydrogenase and trypsinogen in Leydig and Sertoli cells respectively , Miura et al 2009). The Sertoli cell expression that we report is consistent with the presence of Pgr in testicular somatic cells in vertebrates, but we could not detect pgr mRNA in Leydig cells.…”

Cloning, pharmacological characterization and expression analysis of Atlantic cod (Gadus morhua, L.) nuclear progesterone receptor

“…Recent evidence indicates that progestins exert regulatory functions at early stages of fish spermatogenesis. For example, in Japanese eel (Anguilla japonica) DHP stimulated spermatogonial DNA synthesis and the initiation of meiosis, by inducing expression of the short form of 11b-hydroxysteroid dehydrogenase (Hsd11b) in Leydig cells and of trypsinogen in Sertoli cells [18,19].…”