Mechanisms Underlying the Inhibition of Murine Sperm Capacitation by the Seminal Protein, SPINKL Huan-Chin Tseng, 1 Robert Kuo-Kuang Lee, 1,2,3 Yuh-Ming Hwu, 1,2,4 Chung-Hao Lu, 1 Ming-Huei Lin, 1,2,4 and Sheng-Hsiang Li 1,4 * 1 Department of Medical Research, Mackay Memorial Hospital, Tamshui, New Taipei City, Taiwan 2 Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan 3 Department of Obstetrics and Gynecology, Taipei Medical University, Taipei, Taiwan 4 Mackay Medicine, Nursing and Management College, Taipei, Taiwan ABSTRACT SPINKL, a serine protease inhibitor kazal-type-like protein initially found in mouse seminal vesicle secretions, possesses structurally conserved six-cysteine residues of the kazal-type serine protease inhibitor family. However, it has no inhibitory activity against serine proteases. Previously, it was found to have the ability to suppress murine sperm capacitation in vitro. Herein, we investigated the mechanisms underlying the suppressive effect of SPINKL on sperm capacitation. Three in vitro capacitation-enhancing agents, including bovine serum albumin (BSA), methyl-beta-cyclodextrin (MBCD), and dibutyryl cyclic AMP (dbcAMP), coupled with 3-isobutyl-1-methylxanthine (IBMX), were used to evaluate the influence of SPINKL on capacitation signaling. Preincubation of sperm with SPINKL suppressed BSA- and MBCD- induced sperm capacitation by blocking three upstream signals of capacitation that is the cholesterol efflux from sperm plasma membranes, extracellular calcium ion influx into sperm, and increases in intracellular cAMP. Moreover, SPINKL also inhibited downstream signal transduction of capacitation since it suppressed dbcAMP/IBMX and N 6 -phenyl cAMP (6-Phe-cAMP)-activated cAMP-dependent protein kinase-associated protein tyrosine phosphorylation. Such inhibition is probably mediated by attenuation of SRC tyrosine kinase activity. Furthermore, SPINKL could not reverse capacitation once sperm had been capacitated by capacitation-enhancing agents or capacitated in vivo in the oviduct. SPINKL bound to sperm existed in the uterus but had disappeared from sperm in the oviduct during the sperm’s transit through the female reproductive tract. Therefore, SPINKL may serve as an uncapacitation factor in the uterus to prevent sperm from precocious capacitation and the subsequent acrosome reaction and thus preserve the fertilization ability of sperm. J. Cell. Biochem. 114: 888–898, 2013. ß 2012 Wiley Periodicals, Inc. KEY WORDS: SERINE PROTEASE INHIBITOR; SPERM; CAPACITATION; cAMP; CHOLESTEROL; CALCIUM C apacitation is a complex process first independently described and defined by Chang [1951] and Austin [1952]. It is a physiological change in sperm that occurs in the oviduct of some mammals to acquire the ability to fertilize an egg [Suarez, 2008]. It can be mimicked in vitro in specifically defined medium [Chang, 1951; Austin, 1952]. Our current knowledge of capacitation largely originates from in vitro studies [Visconti et al., 1995a; Cross, 1998; Visconti and Kopf, 1998; Naz and Rajesh, 2004; de Jonge, 2005]. Capacitation is initiated by removal of cholesterol from the sperm plasma membrane [Go and Wolf, 1985; Choi and Toyoda, 1998; Cross, 1998; Visconti et al., 1999a1999b; Shadan et al., 2004]. Cholesterol efflux leads to changes in the membrane structure and fluidity and increases in the permeability of sperm to calcium (Ca 2þ ) and bicarbonate (HCO  3 ) ions, thus raising levels of sperm intracellular calcium ions ([Ca 2þ ] i ) and the pH. Elevated levels of sperm intracellular Ca 2þ and HCO  3 can activate adenyl cyclase leading to increases in intracellular levels of cyclic(c)AMP, Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 114:888–898 (2013) 888 Huan-Chin Tseng and Robert Kuo-Kuang Lee contributed equally to this work. Grant sponsor: National Sciences Council, Taipei, Taiwan; Grant numbers: NSC 99-2314-B-195-006-MY3, NSC 100- 2811-B-195-001; Grant sponsor: Mackay Memorial Hospital, Taipei, Taiwan; Grant numbers: MMH 10029, 10105, 10116. *Correspondence to: Dr. Sheng-Hsiang Li, Department of Medical Research, Mackay Memorial Hospital, Tamshui 251, New Taipei City, Taiwan. E-mail: lsh@ms1.mmh.org.tw Manuscript Received: 9 April 2012; Manuscript Accepted: 15 October 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 23 October 2012 DOI 10.1002/jcb.24428  ß 2012 Wiley Periodicals, Inc.