SERPINE2 antiserum, at a dilution of 1:50, had a maximal effect on epididymal sperm capacitation, with a small increase (14%–19%), compared to that in the control group without protein supplementation in the medium, as demonstrated by CTC fluorescence staining and in vitro fertilization, respectively. However, this slight
effect on sperm capacitation was reversed by 0.2 mg/ml SERPINE. A similar effect was found in a study by Ni et al. [28]. The antisera against HongrES1, a caudal epididymis-specific protein, displayed a more significant increase in guinea pig sperm capacitation, and HongrES1 significantly inhibited the effect [28]. In fact, our preliminary data also showed that BSA capacitation of sperm could be reversed by the addition of 0.2 mg/ml SERPINE2, further supporting the role of SERPINE2 as a decapacitation factor.
Cholesterol efflux is one of the initiation events of sperm capacitation. BSA is a cholesterol acceptor, which induces cholesterol efflux, leading to sperm capacitation [12,77,78]. In this study, we revealed that SERPINE2 inhibits BSA-induced cholesterol efflux; the precise mechanism that enables SERPINE2 to retain the cholesterol on the sperm warrants further investigation.
SERPINE2 seems to have protective effects toward sperm cultured in vitro by preventing spontaneous capacitation and the acrosome reaction of sperm. As our study demonstrates, the addition of SERPINE2 effectively lowered protein tyrosine phosphorylation levels (Fig. 6A, lanes 1 and 3), thus yielding a lower percentage of capacitated and acrosome-reacted sperm (Fig. 6, B and C). Additional evidence for this tendency is the fact that in the presence of SERPINE2, there is a reduced number of sperm that bind to an egg and a lower fertilization rate than that of the control medium
group (Fig. 7).
Our SERPINE2 antiserum detected two major and one minor SERPINE2 isoforms in the male reproductive tract. A BLASTP search revealed the existence of three SERPINE2 isoforms in the protein database. Many alternatively spliced SERPINE2 gene products can also be seen at the NCBI AceView website (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/). Previous reports have also supported the existence of multiple forms of SERPINE2 protein among mouse tissues [79].
The Serpine2 mRNA and protein are predominantly expressed in seminal vesicles among mouse tissues [69]. The SERPINE2 protein was previously detected in the epithelium of bovine seminal vesicles and epididymides and in Leydig cells of the testes [45]. However, there is no comprehensive study that has specifically detected this protein in all male reproductive tissues. In this study, we found that if paraffin-embedded testicular sections were not treated with the antigen-retrieval solution, then SERPINE2 was immunostained only on Leydig cells. However, if the sections were retrieved to unmask the SERPINE2 antigen, staining was detected on spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa (Fig. 3). This demonstrates the existence of SERPINE2 protein and mRNA in germ cells of adult testes. The Serpine2 mRNA, in fact, exhibits a male-specific expression pattern in
developing gonads, suggesting its involvement in a testis-determining pathway [80].
Plasminogen activators and their cognate inhibitors, including SERPINE1, SERPINE2, SERPINB2, and SERPINA5, are all found in mouse testes. The level of Serpina5 mRNA is the highest among them, followed by, respectively, Serpine2,
Serpinb2, and Serpine1 mRNA levels [81]. However, murine testicular SERPINE2
seems to have more extensive localization than SERPINA5, as the latter is detected only in Leydig cells before birth and postnatally but is restricted to early spermatids within the acrosomal region in adult testes [82]. SERPINE2 and SERPINA5 are both members of the SERPIN family. Their gene expression levels are regulated by androgen [70,71], but they seem to work as counterparts in different species. A comprehensive proteomic study detected only the SERPINA5 protein but not the SERPINE2 protein in human seminal plasma [83]. However, only minor amounts of Serpina5 mRNA were found in other accessory glands of mouse reproductive tissues, although it was prominently expressed in testes. SERPINE2 was expressed predominantly in the SVS (Fig. 3) and contributes to mouse seminal plasma. SERPINE2 would be the major plasminogen activator inhibitor in murine seminal plasma. Interestingly, they are all sperm surface-binding proteins. SERPINA5 binds to human sperm and may influence sperm–oocyte interactions [84], while SERPINE2 binds to mouse sperm and modulates sperm capacitation (Figs. 6–8). Interestingly, like SERPINE2, PDC-109 is also a
heparin-binding protein [85].
Some protease inhibitors are found on the sperm surface. SERPINA5 is present on the acrosomal surface of human sperm and has been suggested to prevent sperm from prematurely undergoing an acrosomal reaction [84]. SPINK3 was shown to exist on the acrosomal cap of murine sperm and is able to inhibit Ca2+ uptake by epididymal sperm [35]. A proteinase inhibitor of seminal vesicle origin was shown to block sperm–zona binding and the acrosome reaction [86]. In addition, SPINKL, found mainly on the midpiece, is able to inhibit sperm capacitation [37]. In this study, we showed that SERPINE2, another sperm acrosome-binding protein, also exhibits the ability to inhibit sperm capacitation in vitro. Interestingly, these proteins are all predominantly expressed in seminal vesicles.
SERPINE2 may be like PEBP1 in that it has the dual functions of serving as a decapacitation factor [25] and a serine protease inhibitor [87]. HongrES1, another SERPIN family protein, also displayed similar functions [28,29]. The inhibitory activity of a serine protease inhibitor and its role as a decapacitation factor might not necessarily be related. SPINKL inhibits sperm capacitation but does not seem to have inhibitory activities against serine proteases [37]. SPINK3 has no effects on sperm capacitation [88] while exhibiting trypsin inhibitory activity.
Glycosaminoglycans, including heparin, are moieties of the ECM. Sperm were
found to possess a surface sialic acid moiety [89], which is an anionic glycan residue like heparin. Thus, the binding of SERPINA5 and SERPINE2 to sperm may be like the case of protein binding to the ECM to protect sperm from protease attack in the testes and the epididymis during sperm maturation or in the female reproductive tract during transit toward fertilization. Whether they bind to sperm via sialic acid binding awaits further investigation.