SPINKL, a Kazal-type serine protease inhibitor-like protein purified from mouse seminal vesicle fluid, is able to inhibit sperm capacitation

Abstract

We report a secreted serine protease inhibitor Kazal-type-like (SPINKL) protein. The SPINKL protein was purified from mouse seminal vesicle secretions through a series of steps, including ion-exchange chromatography on a diethylaminoethyl-Sephacel column, gel filtration on a Sephadex G-75 column, and ion-exchange HPLC on a Q strong anion exchange column. Further analysis identified several SPINKL proteins with various N-linked carbohydrates. The SPINKL protein has six conserved cysteine residues that are nearly identical to those of members of the SPINK protein family. It was noted that the SPINKL protein showed no inhibitory activities against common serine proteases such as trypsin, chymotrypsin, subtilisin, or elastase. Spinkl mRNA and SPINKL proteins were found to be primarily expressed in seminal vesicles. Immunohistochemistry revealed that the SPINKL protein occurred in the luminal fluid and mucosal epithelium of the seminal vesicles and was regulated by testosterone. The SPINKL protein was able to bind onto sperm and enhance sperm motility. Also, it was able to suppress BSAstimulated sperm capacitation and block sperm-oocyte interactions in vitro, suggesting that SPINKL may be a decapacitation factor.

SERPINE2, a Serine Protease Inhibitor Extensively Expressed in Adult Male Mouse Reproductive Tissues, May Serve as a Murine Sperm Decapacitation Factor1

“…A proteinase inhibitor of seminal vesicle origin was shown to block sperm-zona binding and the acrosome reaction [53]. In addition, SPINKL, found mainly on the midpiece, is able to inhibit sperm capacitation [20]. In this study, we showed that SERPINE2, another sperm acrosome-binding protein, also exhibits the ability to inhibit sperm capacitation in vitro.…”

“…Protein bands on the SDS gel were excised and subjected to in-gel digestion with trypsin following our previously described method [20]. In brief, the gel was washed in a solution of 50% (v/v) acetonitrile and 100 mM NH 4 HCO 3 and digested by trypsin overnight at 378C.…”

“…In humans, some potential decapacitation factors that have been reported are glycodelin-S [7], semenogelin I [8], a 130-kDa glycoprotein [9], and some mannosyl glycopeptides [10]. Several potential decapacitation factors of rodents were also identified, including a 40-kDa glycoprotein [11], phosphatidylethanolamine-binding protein 1 (PEBP1) [12]; three epididymal proteins, a cysteine-rich secretory protein 1 [13], an acrosome-stabilizing factor [14], and an epididymis-specific secretory protein, HongrES1 (symbol not official) [15,16]; and three secreted seminal vesicle proteins, SVA [17], SVS2 [18,19], and SPINKL [20].…”

Impaired Spermatogenesis and Fertility in Mice Carrying a Mutation in the Spink2 Gene Expressed Predominantly in Testes

“…The first member of the SPINK family, human SPINK1 (mouse Spink3), also known as pancreatic secretory trypsin inhibitor was discovered by Kazal (12). Previous studies and our in silico searches indicate that at least 13 SPINK family members are expressed in diverse tissues (13)(14)(15)(16)(17)(18). SPINK2, the only SPINK family member expressed in testes, was first identified in humans (11,19); however, informa-tion on mouse Spink2 has not yet been reported.…”

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: Comparative analysis of structure and activities

“…SPINKL, a Kazal-type serine protease inhibitor-like protein found in mouse seminal vesicle fluid, binds to sperm and enhances motility. In addition, SPINKL also suppresses BSA-stimulated sperm capacitation and block spermeoocyte interactions in vitro [18].…”