Oviduct-specific glycoprotein (OGP) displays estrus-associated regional and temporal differences in expression and localizes to the zona pellucida, perivitelline space, and plasma membrane of oviductal oocytes and embryos, suggesting that it may have a role in regulation of fertilization and/or early embryonic development. The aims of this study were to evaluate the effect of exogenous OGP on in vitro fertilization (IVF) and embryo development in the pig using a defined serum-free culture system. In vitro-matured porcine oocytes were incubated with homologous OGP (0, 1, 10, 20, and 40 μg/ml) for 3 h and then washed prior to IVF. Exposure of oocytes to 10 or 20 μg/ml porcine OGP (pOGP) significantly reduced the incidence of polyspermy compared with the control (P < 0.01) while maintaining high penetration rates. When oocytes, spermatozoa, or both were preincubated with 10 μg/ml pOGP prior to IVF, the incidence of polyspermy was similarly reduced (P < 0.01) by all three treatments without affecting penetration rates. The ability of spermatozoa to undergo calcium ionophore-induced acrosome reaction was similar with or without exposure to pOGP. However, significantly fewer spermatozoa (P < 0.01) bound to the zona pellucida when oocytes were preincubated with pOGP. To evaluate the effect of pOGP on embryo development, embryos were cultured in pOGP-supplemented medium for 48 h or 144 h. Both transient and continuous exposure to pOGP significantly enhanced cleavage and blastocyst formation rate compared with the control (P < 0.01). These data demonstrate that exposure of either in vitro-matured oocytes or spermatozoa to pOGP decreased polyspermy and spermatozoa binding while maintaining high penetration rates of pig oocytes fertilized in vitro. Furthermore, pOGP exerted an embryotrophic effect independent of effects demonstrated on spermatozoa and oocytes at fertilization.

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Oviduct-Specific Glycoprotein Modulates Sperm-Zona Binding and Improves Efficiency of Porcine Fertilization In Vitro

Endometrioid carcinoma (EC) is a relatively indolent ovarian carcinoma subtype that is nonetheless deadly if detected late. Existing genetically engineered mouse models (GEMMs) of the disease, based on transformation of the ovarian surface epithelium (OSE), take advantage of known ovarian EC driver gene lesions, but do not fully recapitulate the disease features seen in patients. An EC model in which the Apc and Pten tumour suppressor genes are conditionally deleted in murine OSE yields tumours that are biologically more aggressive and significantly less differentiated than human ECs. Importantly, OSE is not currently thought to be the tissue of origin of most ovarian cancers, including ECs, suggesting that tumour initiation in Mullerian epithelium may produce tumours that more closely resemble their human tumour counterparts. We have developed Ovgp1-iCreERT2 mice in which the Ovgp1 promoter controls expression of tamoxifen (TAM)-regulated Cre recombinase in oviductal epithelium - the murine equivalent of human Fallopian tube epithelium. Ovgp1-iCreERT2 ;Apcfl/fl ;Ptenfl/fl mice treated with TAM or injected with adenovirus expressing Cre into the ovarian bursa uniformly develop oviductal or ovarian ECs, respectively. On the basis of their morphology and global gene expression profiles, the oviduct-derived tumours more closely resemble human ovarian ECs than do OSE-derived tumours. Furthermore, mice with oviductal tumours survive much longer than their counterparts with ovarian tumours. The slow progression and late metastasis of oviductal tumours resembles the relatively indolent behaviour characteristic of so-called Type I ovarian carcinomas in humans, for which EC is a prototype. Our studies demonstrate the utility of Ovgp1-iCreERT2 mice for manipulating genes of interest specifically in the oviductal epithelium, and establish that the cell of origin is an important consideration in mouse ovarian cancer GEMMs. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/path.4783

Impact of oviductal versus ovarian epithelial cell of origin on ovarian endometrioid carcinoma phenotype in the mouse.

The mammalian estrogen induced oviductal glycoprotein (OGP) has been known to associate with capacitated sperm, oocytes and developing embryos. This study aimed to identify the putative binding partner of OGP on gametes using N-terminal peptide of bonnet monkey (Macaca radiata) OGP, Nmon, as bait. A protein(s) of molecular size ~54 kDa was detected by farwestern blot analysis of detergent solubilized human sperm proteins. MALDI-TOF mass spectra analysis of ~54 kDa tryptic peptides gave a significant hit to non-muscle myosin heavy chain. Biochemical characterization of ~54 kDa was done with antibodies specific to non-muscle myosin IIA, MYH9. The ~54 kDa protein, possible breakdown product of MYH9, immunoreacted with MYH9 antibody in western blot analysis. OGP binding to ~54 kDa could also be demonstrated in far-western blot analysis of detergent solubilized human sperm proteins and nuclear matrix intermediate filament (NM-IF) preparations from human sperm and mouse oocytes. Far-western blot analysis of MYH9 enriched by immunoprecipitation identified the native ~220 kDa protein as OGP-binding partner. The identical and characteristic immunogold localization pattern of Nmon and MYH9 on sperm NM-IF preparation substantiated these findings. The results suggest that OGP binds to both gametes through its interaction with MYH9 through the non-glycosylated N-terminal conserved region of OGP, spanning the residues 11–137.

Identification and characterization of oviductal glycoprotein-binding protein partner on gametes: epitopic similarity to non-muscle myosin IIA, MYH 9.

The mammalian oviduct synthesizes and secretes a major glycoprotein known as oviductin (OVGP1), which has been shown to interact with gametes and early embryos. Here we report the use of recombinant DNA technology to produce, for the first time, the secretory form of human OVGP1 in HEK293 cells. HEK293 colonies stably expressing recombinant human OVGP1 (rHuOVGP1) were established by transfecting cells with an expression vector pCMV6-Entry constructed with OVGP1 cDNA. Large quantities of rHuOVGP1 were obtained from the stably transfected cells using the CELLSPIN cell cultivation system. A two-step purification system was carried out to yield rHuOVGP1 with a purity of >95%. Upon gel electrophoresis, purified rHuOVGP1 showed a single band corresponding to the 120-150 kDa size range of human OVGP1. Mass spectrometric analysis of the purified rHuOVGP1 revealed its identity as human oviductin. Immunofluorescence showed the binding of rHuOVGP1 to different regions of human sperm cell surfaces in various degrees of intensity. Prior treatment of sperm with 1% Triton X-100 altered the immunostaining pattern of rHuOVGP1 with an intense immunostaining over the equatorial segment and post-acrosomal region as well as along the length of the tail. Addition of rHuOVGP1 in the capacitating medium further enhanced tyrosine phosphorylation of sperm proteins in a time-dependent manner. After 4-h incubation in the presence of rHuOVGP1, the number of acrosome-reacted sperm induced by calcium ionophore significantly increased. The successful production of rHuOVGP1 can now facilitate the study of the role of human OVGP1 in fertilization and early embryo development.

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Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction.

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.

Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP). Molecular cDNA cloning of most of the mammalian OGP has been accomplished. The nucleotide and deduced amino acid sequences show a remarkable homology across species and also to chitinase protein. Even though OGP has been shown to interact with gametes and the early embryo, the protein’s direct function has not yet been established. A prerequisite for such studies is the availability of well-characterized protein in bulk. We used recombinant DNA technology to obtain OGP (rOGP). An authentic partial cDNA clone encoding bonnet monkey (Macaca radiata) OGP (accession number AF132 215) was recloned into expression vector pET20b. Overexpression of the protein could be demonstrated after induction with isopropylthio-b-galactopyranoside. Recombinant protein was purified by gel filtration of Escherichia coli lysate through Sephadex G75. The protein migrated with a molecular weight of ;14 kDa on SDS-PAGE. The molecular weight as assessed by matrix-assisted laser adsorption time-of-flight was 14 439 daltons. With Western blot procedures the protein could be immunostained with antibodies to human OGP, baboon OGP, and antipeptide antibodies generated against a well-conserved region of mammalian OGP. The monospecificity of rabbit antibodies generated against rOGP was established by its ability to immunostain human OGP (100‐110 kDa) isolated from hydrosalpinx by Western blot analysis, and the antibody immunostained epithelial cells that secrete OGP in human fallopian tubes. OGP binding sites on the head and tail region of monkey sperm could be demonstrated by using antibody against rOGP.

https://bioone.org/journals/Biology-of-Reproduction/volume-67/issue-6/biolreprod67.6.1897/Overexpression-of-Monkey-Oviductal-Protein--Purification-and-Characterization-of/10.1095/biolreprod67.6.1897.pdf

S.B. Moodbidri

Papers

Overexpression of Monkey Oviductal Protein: Purification and Characterization of Recombinant Protein and Its Antibodies