Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding

Abstract

Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.

Fig 1. Immunochemical identification of rHamOVGP1.

Culture supernatant from control (lane 1) and from rHamOVGP1-transduced HEK293 cells (lane 2) were subjected to SDS-PAGE (6.0%) followed by transfer to PVDF membranes. Western blot analysis was performed as described in Materials and Methods.

Fig 2. Purification and analysis of rHamOVGP1.

(A) The samples were analyzed using 6% SDS-PAGE under reducing conditions followed by silver staining. Lane 1: protein ladder; lane 2: purified rHamOVGP1 (1 μg/lane) using HPA-agarose; (B) Samples were subjected to 6% SDS-PAGE followed by immunoblot analysis as described in Materials and Methods. Lane 1: purified rHamOVGP1 (100 ng/lane) using HPA-agarose; lane 2: purified HamOVGP1 (100 ng/lane) from homogenates of estrus-stage hamster oviducts using HPA-agarose; (C) Complete sequence of HamOVGP1 with the coverage of peptides (bold red) identified from the trypsin digest by MS.

Fig 3. Confocal microscope imaging of rHamOVGP1 binding sites on hamster sperm after capacitation.

Epididymal sperm were incubated in capacitating medium in the absence (-) or presence (+) of 20 μg/ml rHamOVGP1 for 0-, 1- and 3-h, respectively. Immunofluorescent signal was practically absent at all three time intervals in the absence of rHamOVGP1. In the presence of rHamOVGP1, immunostaining started to appear at 1 h mainly over the equatorial segment (ES) region of the sperm head (see insert for a higher magnification of the same sperm head indicated by double-head arrow in red) and the mid-piece (MP) of the sperm tail with a relatively weaker immunostaining over the principle piece (not shown). The pattern of immunostaining of the mid-piece (MP) and principle piece (PP) persisted at the 3 h time interval except that the previously seen immunofluorescent signal over the equatorial segment region now diminished or disappeared. Scale bars = 50 μm, Scale bar of insert = 140 μm

Fig 4. Dose-dependent effect of rHamOVGP1 on tyrosine phosphorylation of hamster sperm proteins.

Hamster caudal epididymal sperm, cultured for 4 h in modified TALP medium alone or supplemented with different concentration of rHamOVGP1 (10, 20, 40, and 60 μg/ml), were processed for SDS-PAGE, transferred to PVDF membrane and probed with anti-phosphotyrosine antibody. A: representative blot; B: summary of data. Asterisks (*) indicate values that are significantly different (p<0.05) from that obtained with untreated sperm.

Fig 5. Time course of protein tyrosine phosphorylation in caudal epididymal hamster sperm.

Sperm were incubated in modified TALP medium in the absence or presence of 20 μg/ml rHamOVGP1 for different time intervals (0–6 h). Equal numbers of sperm were solubilized for SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody.

Fig 6. Western blot analysis of tyrosine phosphorylation of hamster sperm proteins after demembranation.

(A) Hamster caudal epididymal sperm, cultured for 4 h in modified TALP medium alone (lane 1) or supplemented with 20 μg/ml of rHamOVGP1 (lane 2), were solubilized in SDS-loading buffer and subjected to immunoblot analysis with anti-phosphotyrosine antibody. Lanes 3 and 4 corresponded to lanes 1 and 2, respectively, except that sperm were demembranated with DTT-Triton X as described in Materials and Methods prior to immunoblotting. (B) The immunoblot was stripped and re-probed with anti-AKAP82 antibody as described in Materials and Methods.

Fig 7. Immunolocalization of rHamOVGP1-enhanced tyrosine-phosphorylated proteins in hamster sperm.

Sperm, after being cultured in modified TALP medium alone (A, C, E) or supplemented with 20 μg/ml rHamOVGP1 (B, D, F) for 0 h (A, B), 2 h (C, D) and 4 h (E, F), were subjected to indirect immunofluorescence as described in Materials and Methods. Immunofluorescent staining of tyrosine phosphorylated proteins was observed in the mid-piece (MP) and principal piece (PP). (A1-F1) are corresponding bright field photomicrographs of (A2-F2) respectively. (G) shows intense immunostaining over the principle piece (PP) with a weaker staining intensity over the mid-piece (MP) after sperm were demembranated with 2% Triton X-100 for 10 min following incubation for 4 h in modified TALP medium supplemented with 20 μg/ml rHamOVGP1. Scale bars = 50 μm

Fig 8. Effect of rHamOVGP1 on acrosome reaction of hamster sperm.

Hamster caudal epididymal sperm were incubated in modified TALP medium alone or supplemented with 20 μg/ml rHamOVGP1 for 0 to 6 h. Results are expressed as mean ± SEM as compared to the control group (n = 4 different experiments for each group carried out under the same conditions). * p<0.05; ** p<0.01; *** p<0.001.

Fig 9. Immunolocalization of rHamOVGP1 in hamster ovarian oocytes.

Confocal microscope imaging of rHamOVGP1 binding sites in hamster ovarian oocytes following 3 h of incubation in the absence or presence of 20 μg/ml rHamOVGP1. Column 1: immunofluorescent confocal microscope images; column 2: phase contrast confocal microscope images; column 3: merge of phase contrast and immunofluoresent confocal microscope images. ZP: zona pellucida; O: oocyte proper. Scale bars = 30 μm.

Fig 10. Effect of rHamOVGP1 (20 μg/ml) on sperm-oocyte binding.

Group A: control (medium without additive). Group B: hamster ovarian oocytes were pretreated with rHamOVGP1 for 1 h prior to sperm-oocyte binding assay in the absence of rHamOVGP1. Group C: hamster caudal epididymal sperm were pretreated with rHamOVGP1 for 1 h prior to sperm-oocyte binding assay in the absence of rHamOVGP1. Group D: Sperm-oocyte binding assay was performed in the presence of rHamOVGP1 without prior pretreatment of either oocytes or sperm. Group E: Sperm-oocyte binding assay was performed in the presence of rHamOVGP1 with pretreated oocytes and untreated sperm. F: Sperm-oocyte binding assay was performed in the presence of rHamOVGP1 with pretreated oocytes and untreated oocytes. Groups G to K correspond to Group B to F, respectively, except that monoclonal antibody against hamster OVGP1 was added to the culture medium during sperm-oocyte binding. Data are presented as Mean ± SEM of the number of sperm bound per oocyte. A total of 165 oocytes were obtained from six animals. Fifteen ovarian oocytes were present in each group. Group B to F are compared to Group A; Group G to K are compared to Group B to F, respectively. * p<0.05; ** p<0.001.