International Journal of Molecular Sciences Article Serine Protease Inhibitor SERPINE2 Reversibly Modulates Murine Sperm Capacitation Sheng-Hsiang Li 1,2, Yuh-Ming Hwu 1,2,3,4, Chung-Hao Lu 3, Ming-Huei Lin 2,3, Ling-Yu Yeh 1 and Robert Kuo-Kuang Lee 1,3,5,* 1 Department of Medical Research, Mackay Memorial Hospital, Tamsui District, New Taipei City 251, Taiwan; [email protected] (S.-H.L.); [email protected] (Y.-M.H.); [email protected] (L.-Y.Y.) 2 Mackay Junior College of Medicine, Nursing, and Management, Beitou District, Taipei City 112, Taiwan; [email protected] 3 Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei City 104, Taiwan; [email protected] 4 Mackay Medical College, Sanzhi District, New Taipei City 252, Taiwan 5 Department of Obstetrics and Gynecology, Taipei Medical University, Taipei City 110, Taiwan * Correspondence: [email protected]; Tel.: +886-2-2543-3535 Received: 2 May 2018; Accepted: 17 May 2018; Published: 19 May 2018 Abstract: SERPINE2 (serpin peptidase inhibitor, clade E, member 2), predominantly expressed in the seminal vesicle, can inhibit murine sperm capacitation, suggesting its role as a sperm decapacitation factor (DF). A characteristic of DF is its ability to reverse the capacitation process. Here, we investigated whether SERPINE2 can reversibly modulate sperm capacitation. Immunocytochemical staining revealed that SERPINE2 was bound onto both capacitated and uncapacitated sperm. It reversed the increase in BSA-induced sperm protein tyrosine phosphorylation levels. The effective dose and incubation time were found to be >0.1 mg/mL and >60 min, respectively. Calcium ion levels in the capacitated sperm were reduced to a level similar to that in uncapacitated sperm after 90 min of incubation with SERPINE2. In addition, the acrosome reaction of capacitated sperm was inhibited after 90 min of incubation with SERPINE2. Oviductal sperm was readily induced to undergo the acrosome reaction using the A23187 ionophore; however, the acrosome reaction was significantly reduced after incubation with SERPINE2 for 60 and 120 min. These findings suggested that SERPINE2 prevented as well as reversed sperm capacitation in vitro. It also prevented the acrosome reaction in in vivo-capacitated sperm isolated from the oviduct. Thus, SERPINE2 could reversibly modulate murine sperm capacitation. Keywords: SERPINE2; sperm capacitation; decapacitation factor; acrosome reaction; seminal plasma 1. Introduction Capacitation, a physiological process in the sperm, was first described in the early 1950s [1,2], in which the sperm resides in the female reproductive tract until it gains the ability to fertilize an egg. It is a complex change and is believed to be initiated by the removal of cholesterol from the sperm plasma membrane [3–6], leading to changes in the membrane structure and fluidity and increase in the permeability of the sperm for calcium and bicarbonate ions. Subsequently, a series of downstream 2+ signaling occurs, including an increase in the levels of sperm intracellular calcium ion ([Ca ]i) and pH, thereby activating adenylyl cyclase and leading to an increase in the intracellular levels of cyclic AMP (cAMP). The activation of cAMP-dependent protein kinase then induces tyrosine phosphorylation of a subset of sperm proteins [7]. The seminal plasma is a mixture of secretions from the male accessory sexual glands, mainly containing seminal vesicle secretions. It has been shown to reverse sperm capacitation [8]. Int. J. Mol. Sci. 2018, 19, 1520; doi:10.3390/ijms19051520 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 13 The seminal plasma is a mixture of secretions from the male accessory sexual glands, mainly containing seminal vesicle secretions. It has been shown to reverse sperm capacitation [8]. DecapacitationInt. J. Mol. Sci. 2018 ,factors19, 1520 (DFs) present in the seminal plasma can reverse sperm capacitation,2 ofi.e., 13 change sperm from a capacitated to an uncapacitated status. These factors are removed from the spermDecapacitation surface factorsbefore (DFs)or during present the in thecapacitation seminal plasma process can [9]. reverse Several sperm potential capacitation, DFs have i.e., change been identifiedsperm from in mice a capacitated [10–14], including to an uncapacitated SERPINE2 (serpin status. peptidase These factors inhibitor, are removed clade E, member from the 2) sperm [13]. surfaceSERPINE2, before or also during known the as capacitation glia-derived process nexin or [9 protease]. Several nexin-1, potential has DFs broad have antiprotease been identified activity in specificmice [10 –to14 ],serine including proteases; SERPINE2 e.g., (serpintrypsin, peptidase thrombin, inhibitor, plasmin, clade and E, memberplasminogen 2) [13 ].activator [15]. SERPINE2SERPINE2, is widely also expressed known as in glia-derived various tissues, nexin including or protease the nexin-1, reproductive has broad tissues antiprotease [16–20], with activity the highestspecific levels to serine found proteases; in seminal e.g., trypsin,vesicles thrombin, [13,20]. It plasmin,can be purified and plasminogen from the seminal activator vesicle [15]. SERPINE2 secretion [13].is widely expressed in various tissues, including the reproductive tissues [16–20], with the highest levelsRecent found studies in seminal have vesicles demonstrated [13,20]. Itthe can ability be purified of SERPINE2 from the seminalto inhibit vesicle sperm secretion capacitation [13]. by blockingRecent the studiescholesterol have efflux demonstrated from sperm the plasma ability membranes of SERPINE2 and to suppressing inhibit sperm the capacitationincrease in the by levelblocking of sperm the cholesterol protein tyrosine efflux phosphorylation. from sperm plasma These membranes results suggested and suppressing that SERPINE2 the increase is a potential in the spermlevel of DF sperm [13]. proteinDF has tyrosinebeen reported phosphorylation. to have the These ability results of reversing suggested the thatsperm SERPINE2 capacitation is a potential process [8].sperm In DFthe [ 13present]. DF has study, been reportedwe investigate to have thewhether ability SERPINE2 of reversing can the spermreversibly capacitation modulate process sperm [8]. capacitation.In the present study, we investigate whether SERPINE2 can reversibly modulate sperm capacitation. 2.2. Results Results 2.1.2.1. SERPINE2 SERPINE2 Binds Binds to to Both Both Capacitated Capacitated and and Uncapacitated Sperm IncubationIncubation with with bovine bovine serum serum albumin albumin (BSA) (BSA) can can induce induce capacitation capacitation in in epididymal epididymal sperm sperm [21]. [21]. InIn the immunocytochemicalimmunocytochemical staining staining analyses, analyses, SERPINE2 SERPINE2 was was shown shown to bind to bind to the to acrosome,the acrosome, mid-piece, mid- piece,and principal and principal tail of tail the of sperm.the sperm. This This binding binding was was detected detected regardless regardless of of whetherwhether the samples were incubated with with SERPINE2 SERPINE2 before before (Figure (Figure 11a)a) or after (Figure1 1b)b) thethe BSA-inducedBSA-induced capacitation.capacitation. These results results indicated indicated that SERPINE2 SERPINE2 can can bind bind to to both both capacitated capacitated and and uncapacitated uncapacitated murine murine sperm. sperm. FigureFigure 1. BindingBinding of of SERPINE2 SERPINE2 on on the the sperm. sperm. Live Live epididymal epididymal spermatozoa spermatozoa were were first first cultured cultured with 0.20.2 mg/mL SERPINE2 SERPINE2 for 20 min andand thenthen incubatedincubated with with 3 3 mg/mL mg/mL BSA BSA for for an an additional additional 90 90 min min (a )(a or) orvice vice versa versa (b ().b). After After washing washing by by centrifugation, centrifugation, spermatozoa spermatozoa werewere transferred,transferred, air-dried, and fixed fixed ontoonto slides.slides. Slides Slides were incubatedwere incubated with anti-SERPINE2 with anti-SERPINE2 antiserum, treated antiserum, with tetramethylrhodamine treated with tetramethylrhodamineisothiocyanate (TRITC)-conjugated isothiocyanate goat (TRITC)-conjugated anti-rabbit IgG, and goat counterstained anti-rabbit IgG, with and Hoechst counterstained 33258 to withlocalize Hoechst the nuclei 33258 for to contrast.localize the nuclei for contrast. Int. J. Mol. Sci. 2018, 19, 1520 3 of 13 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 13 2.2. SERPINE2 Reverses BSA-Induced Protein Tyrosine PhosphorylationPhosphorylation inin SpermSperm To assessassess whether whether SERPINE2 SERPINE2 can reversecan reverse sperm capacitation,sperm capacitation, we first established we first theestablished characteristic the spermcharacteristic protein sperm tyrosine protein phosphorylation tyrosine phosphorylation pattern for capacitated pattern sperm.for capacitated As shown sperm. in Figure As 2shown, murine in spermFigure cultured2, murine in sperm modified cultured Krebs-Ringer in modified bicarbonate Krebs-Ringer (mKRB) bicarbonate medium without (mKRB) BSA medium showed without a basal levelBSA showed of sperm a proteinbasal level tyrosine of sperm phosphorylation protein tyrosine (Figure phosphorylation2a, lane 1). When (Figure cultured 2a, lane in medium1). When supplementedcultured in medium with BSA, supplemented the sperm proteinwith BSA, tyrosine the sperm phosphorylation protein tyrosine level phosphorylation was significantly increasedlevel was (lanesignificantly 3). However, increased tyrosine (lane phosphorylation