Purification and Identification of SERPINE2 from Mouse SVS
To prepare SERPINE2 for functional analyses and antibody production, we prefractionated SVS by ion exchange and gel filtration chromatography. The possible SERPINE2-containing fraction, based on the molecular mass, was further purified to homogeneity using a heparin Sepharose column (Fig. 1A, peak II), as SERPINE2 is a heparin-binding protein [75]. The purity of the resulting protein was shown with SDS-PAGE (Fig. 1B). To identify this protein, the bands on the gel were excised and digested in-gel with trypsin, and the resulting tryptic peptides were subjected to LC-MS/MS analysis. Results showed that the purified protein had significant homology to SERPINE2, with the tryptic peptides matching 41%–43% of the protein sequences (Fig. 1C). The purified SERPINE2 protein showed potent inhibitory activity against PLAU (Fig. 1D), indicating that the purification procedures were not harmful to its protease-inhibitory activity. The SERPINE2 protein concentration in the SVS was estimated to be 0.6–0.8 mg/ml by the ELISA method.
Distribution of SERPINE2 Protein in Adult Male Mouse Reproductive Tissues
To study the tissue distribution of SERPINE2 protein in male reproductive tissues, we examined tissue homogenates, including the seminal vesicle, epididymis, testis, coagulating gland, vas deferens, and prostate, by Western blotting. The antibody against SERPINE2 recognized the purified 45-kDa band and at least three forms, 40-, 42-, and 45-kDa proteins, from thousands of protein components in the tissue extract Fig. 2). In addition, high-molecular-weight proteins were also detected in protein extract from the testes. These proteins may be aggregated forms as no signal was seen when the antiserum was removed from the blots and samples were reprobed with the antiserum that was pretreated with SERPINE2-conjugated beads (control antiserum), indicating the high specificity of the antibody. When protein database searching was conducted using basic Local Alignment Search Tool (BLAST) algorithms http://www.ncbi.nlm.nih.gov/BLAST) against a nonredundant database, using the SERPINE2 protein sequence (Swiss-Prot 07235) as the query, three isoforms were revealed, with accession numbers gb j EDL16269.1 j , gb j EDL16267.1 j , and gb j EDL16268.1 j. The theoretical molecular masses of the three isoforms were 30.812, 35.668, and 44.206 kDa, respectively. This was not processed by a signal peptidase.
Thus, the mature protein would have the smaller molecular mass. However, the three
proteins recognized by the anti-SERPINE2 antiserum had greater molecular masses, indicating they might be the glycosylated forms. In fact, SERPINE2 expression was demonstrated as two forms of glycoproteins [76]. However, we treated the protein extract from seminal vesicles with -glycosidase F, only the 45-kDa protein was found to be deglycosylated. To reveal the cell types and subcellular compartments among male reproductive tissues that expressed the SERPINE2 protein, an immunolocalization study was conducted using the specific anti-SERPINE2 antiserum. The SERPINE2 protein was immunolocalized to epithelial cells of seminal vesicles, coagulating glands, vas deferens, and caput or caudal epididymides (Fig. 3, B–E and G). However, when slides were immunostained with control antiserum, no signal was detected (Fig. 3A). Signals on corpus epididymides and the rostate were relatively weaker (Fig. 3, F and H). The most prominent expression was found in the luminal fluid of seminal vesicles of adult mice. A signal on smooth muscle cells of seminal vesicles was also visible (Fig. 3B).
Interestingly, ERPINE2 protein was identified on spermatogonia, spermatocytes, spermatids, Leydig cells, and spermatozoa (Fig. 3I), as revealed by the control slide treated with control antiserum (Fig. 3J).
Binding of the SERPINE2 Protein to Spermatozoa
A visible SERPINE2 protein signal was prominently present on sperm in the
lumen of the vas deferens and caudal epididymides (Fig. 3, D and G). To verify binding of the SERPINE2 protein onto sperm, sperm isolated from testes or epididymides were smeared on slides, as shown in the phasecontrast image for comparison (Fig. 4A, a).
When slides were immunostained by control antiserum and an FITC-conjugated secondary antibody, no fluorescent signal was detected (Fig. 4A, b). In contrast, SERPINE2 was detected on the acrosomal caps of caput, corpus, and caudal epididymal sperm by using the anti-SERPINE2 antiserum (Fig. 4A, c–e). Likewise, apparent SERPINE2 protein signals were also visualized on the acrosomal cap of testicular sperm (Fig. 4A, f). These results suggested that the SERPINE2 protein is an intrinsic surface protein of sperm during spermiogenesis and sperm maturation. Exogenous SERPINE2 can apparently bind to caudal epididymal sperm, as demonstrated by incubation of the epididymal sperm with purified SERPINE2. The signal from this binding was so strong that it was prominently detected by a more-dilute anti-SERPINE2 antiserum (1:1000) (Fig. 4B, b). The binding was strong on the acrosomal cap and on the tails of living epididymal sperm. Under the same detection conditions, epididymal sperm showed only a very faint intrinsic SERPINE2 signal (Fig. 4B, a). Although faint, the intrinsic signal, as mentioned above, was detected using more highly concentrated antiserum (1:100) (Fig. 4A, e). The SERPINE2 protein derived from seminal plasma was also detected on ejaculated and oviductal sperm (Fig. 4C, b and d, respectively) as
demonstrated by control slides stained with control antiserum (Fig. 4C, a and c, respectively). The binding was strong on the acrosomal cap but weaker on the tail.
These findings indicate that the exogenous SERPINE2 may be a sperm surface protein in vivo.
Removal of SERPINE2 from Capacitated Sperm in the Oviduct
To determine whether capacitated or uncapacitated sperm have a SERPINE2-binding zone, oviductal sperm were immunostained with anti-SERPINE2 antiserum, and then the same sperm were fluorescently stained with CTC. As shown in Figure 5, four staining types (Fig. 5, A–D) of sperm were found. Staining type A was defined as capacitated sperm without SERPINE2 on the acrosome; type B was uncapacitated sperm with SERPINE2 on the head; type C was capacitated sperm with less SERPINE2 on the head; and type D was uncapacitated sperm with no SERPINE2 on the sperm surface. About 40% of the SERPINE2-bound sperm were the uncapacitated B type, but only about 10% of the capacitated type C sperm were seen in the oviduct. In addition, about 50% of the sperm in the oviduct were capacitated and not bound by SERPINE2. Interestingly, SERPINE2 was prominently present on uncapacitated sperm. It seems that SERPINE2 was released from the acrosomal region when the sperm underwent capacitation.
Effects of SERPINE2 on Sperm Function In Vitro
To examine the effects of SERPINE2 on epididymal sperm capacitation, we assessed the protein tyrosine phosphorylation pattern of epididymal sperm after incubation with BSA and/or SERPINE2. As shown in Figure 6A, only a few sperm proteins were phosphorylated in control medium without supplementation with BSA or SERPINE2 (Fig. 6A, lane 1). However, BSA induced sperm capacitation accompanied by tyrosine phosphorylation of a group of proteins with a pattern similar to that found in previous studies (Fig. 6A, lane 2) [7]. The SERPINE2 protein prominently decreased the phosphorylation of the control medium (Fig. 6A, lane 3). In addition, the extent of BSA-induced protein tyrosine phosphorylation was successively suppressed by the increased concentration of SERPINE2 (Fig. 6A, lanes 4–7). Clearly, the characteristic capacitation specific protein tyrosine phosphorylation pattern induced by BSA was inhibited by SERPINE2. CTC fluorescence staining is often used to assess capacitation, as judged by the morphology of fluorescently stained sperm. In the control medium without BSA or SERPINE2, sperm showed a spontaneous capacitation rate of approximately 22%. The addition of SERPINE2 significantly decreased the spontaneous capacitation rate to 11%. The population of capacitated sperm remarkably increased (64%) after the control medium was supplemented with 3 mg/ml BSA. However,
SERPINE2 inhibited BSA-induced sperm capacitation significantly after 0.05, 0.1, or 0.2 mg/ml of SERPINE2 was added to BSA-containing medium (Fig. 6B). These observations are in accordance with the inhibition of BSA-induced tyrosine phosphorylation by SERPINE2 (Fig. 6A). Next, we examined the acrosome reaction induced by the calcium ionophore A23187. A spontaneous acrosome reaction was found (15%–26%) with in vitro-capacitated sperm in the incubation medium with or without BSA and/or SERPINE2 (Fig. 6C, white bars). The concentration of the vehicle used, 0.2% DMSO, did not increase the percentage of acrosome-reacted sperm.
SERPINE2-treated sperm did not show an increased acrosome reaction compared to that of control medium. However, BSA-treated sperm showed remarkable enhancement of the acrosome reaction after A23187 induction. In contrast, the acrosome reaction was significantly inhibited when sperm were incubated with BSA and SERPINE2. An 85%
reduction was observed after treatment with 0.2 mg/ml of SERPINE2 (Fig. 6C). Only capacitated sperm can be induced to undergo an acrosome reaction [16]. Thus, these results further indicated that SERPINE2 is able to inhibit sperm capacitation induced by BSA. Capacitated sperm can bind to the zona pellucida of oocytes and are induced to undergo an acrosome reaction. If SERPINE2 can inhibit sperm capacitation, this would affect sperm–egg binding and subsequent fertilization. As shown in Figure 7, epididymal sperm with no treatment had a low capacity to bind to oocytes, with a
fertilization rate of approximately 20% in vitro, which may have resulted from fertilization by sperm that reacted to acrosome spontaneously. In contrast, BSA-treated sperm showed strong binding to oocytes and had a higher fertilization rate (55%–66%).
However, supplementation with 0.2 mg/ml SERPINE2 significantly reduced oocyte binding and the fertilization rates of BSA-capacitated epididymal spermatozoa by 82%
and 64%, respectively. These findings further demonstrated that SERPINE2 can inhibit BSA-induced sperm capacitation and lead to failure of in vitro fertilization. BSA is used in most media for sperm capacitation. It is able to promote sperm membrane cholesterol efflux [12,77,78]. Cholesterol was released into the medium from BSA-treated sperm, while that release was significantly inhibited by SERPINE2 (Fig. 8A).
SERPINE2-treated sperm retained an amount of cholesterol that was similar to that of untreated sperm (Fig. 8B).