We identified a testis-specific protease-like protein tentatively named TESPL and a pancreatic trypsinogen Prss2 from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. The enzymatic motifs and the cysteine patterns in serine proteases are highly conserved in these two proteins. Based on the phylogenetic analysis, Prss2 duplicated recently and TESPL underwent distant evolution without gene duplication from the progenitor of trypsin-like and chymotrypsin-like proteases. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. In contrast, Prss2 was constitutively expressed in many tissues including testis. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region. Further, orthology group for mouse TESPL was identified in the conserved gene family of eutherian testis serine protease 5.
Spink, serine protease inhibitor Kazal; HUSI-II, human acrosinâtrypsin inhibitor; BUSI-IIA, bull semen inhibitor type IIA; POSI, porcine semen inhibitor; SVS, seminal vesicle secretions; TESPL, testis-specific protease-like protein; Prss2, anionic trypsinogen-2; TESP, testicular serine protease; PI-PLC, phosphatidylinositol-specific phospholipase C; RT, reverse transcription; GPI, glycosylphosphatidylinositol; ARE, androgen response element.
Protease inhibitors are ubiquitous in the reproductive tract [Fink and Fritz, 1976; Fritz et al., 1976; Meloun et al., 1983]. It is believed that they are important in balancing protease activities to protect against genital tract epithelium damage by proteolysis [Tschesche et al., 1982]. In addition, they also play a role in regulating the fertilization process [CechovÃ¡ and JonÃ¡kovÃ¡, 1981; Huhtala, 1984]. There are 48 distinct families of protease inhibitors, one of which is the serine protease inhibitor Kazal (Spink) family [Rawlings et al., 2004]. Spinks have been identified in seminal plasma of different mammalian species, for instance, human acrosinâtrypsin inhibitor (HUSI-II) (Spink2) from human seminal plasma [Fink et al., 1990], bull semen inhibitor type IIA (BUSI-IIA) (Spink6) from bull semen [Meloun et al., 1983], porcine semen inhibitor (POSI) from porcine semen [Tschesche et al., 1982], and the protease inhibitors from guinea pig seminal vesicle [Fink and Fritz, 1976] and rabbit seminal plasma [Zaneveld et al., 1971]. Although a mouse seminal vesicle protease inhibitor possessing many similarities to bovine pancreatic trypsin inhibitor has been identified [Haendle et al., 1965; Fritz et al., 1968; Poirier and Jackson, 1981], its molecular structure remains obscure. Previously, we purified a Kazal-type trypsin inhibitor from mouse seminal vesicle secretions (SVS) and determined its primary structure, consisting of 57 amino acid residues [Lai et al., 1991]. We found that the open reading frame that encodes this protein corresponds to that of P12 cDNA, which had been cloned from mouse ventral prostate by Mills et al. 1987; therefore, it was tentatively named P12. Since then, the Mouse Genome Informatics nomenclature committee has referred P12 to mouse Spink3 (NCBI reference sequences NP_033284). The seminal vesicle inhibitor mentioned above may be identical to Spink3, considering that they are secreted from the same organ and have similar molecular size.
Spink3 is exclusively expressed in the male accessory sexual glands and it binds to postmeiotic cells such as spermatides and spermatozoa in the seminiferous tubules of testis [Chen et al., 1998]. Yet, the nature of Spink3-binding site on sperm cells is still not understood. In this study, we searched for serine protease-like proteins that interact with Spink3 in the testis. We identified two potential candidates, a membrane-bound testis-specific protease-like protein TESPL devoid of proteolytic activity and a pancreatic trypsinogen Prss2. Moreover, we discuss the significance of our study in molecular reproduction of mammals.