Human Oviduct-specific Glycoprotein Research Articles

Recombinant human OVGP1 increases intracellular calcium and further potentiates the effects of progesterone on human sperm

PurposeTo investigate the effects of recombinant human oviduct–specific glycoprotein (rHuOVGP1) alone and in combination with progesterone (P4) on intracellular Ca2+ concentration [Ca2+]i and to investigate if rHuOVGP1 in combination with P4 can further enhance tyrosine phosphorylation (pY) of sperm proteins during human sperm capacitation.MethodsFluorometric flow cytometry was performed to examine the effects of rHuOVGP1 on [Ca2+]i in human sperm during capacitation. Confocal microscopy was used in conjunction with live cell imaging to analyze the influence of rHuOVGP1 and P4 on [Ca2+]i in the sperm tail and to examine the involvement of CatSper channels in their effect on [Ca2+]i. Western blot analysis was performed to assess the protein levels of p105, a major tyrosine-phosphorylated sperm protein.ResultsrHuOVGP1 increases [Ca2+]i in human sperm at the beginning of capacitation and further increases and sustains the level of [Ca2+]i in the sperm tail following the addition of P4. Inhibition of CatSper channels impedes the effects of rHuOVGP1 on [Ca2+]i in the sperm tail. P4 alone can increase pY of a major human sperm protein, p105, yet yields a further increase when used in combination with rHuOVGP1.ConclusionThe present study revealed that rHuOVGP1 may work with P4 to upregulate [Ca2+]i at the beginning of capacitation in part through CatSper channels which, in turn, leads to the downstream event of pY of sperm proteins and enhancement of sperm capacitation.

Human OVGP1 enhances tyrosine phosphorylation of proteins in the fibrous sheath involving AKAP3 and increases sperm-zona binding

PurposeTo investigate if the recombinant human oviduct-specific glycoprotein (rHuOVGP1)–enhanced tyrosine-phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding.MethodsImmunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3 (AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemizona assay.ResultspY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida (ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm-egg binding.ConclusionsThe present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.

Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells

Oviduct-specific glycoprotein (OVGP1) is a major mucin-like glycoprotein synthesized and secreted exclusively by non-ciliated secretory cells of mammalian oviduct. In vitro functional studies showed that OVGP1 plays important roles during fertilization and early embryo development. We have recently produced recombinant human oviduct-specific glycoprotein (rhOVGP1) in human embryonic kidney 293 (HEK293) cells. The present study was undertaken to characterize the structures and determine the biosynthetic pathways of the N- and O-glycans of rhOVGP1. Treatment of the stable rhOVGP1-expressing HEK293 cells with either GalNAcα-Bn to block O-glycan extension, tunicamycin to block N-glycosylation, or neuraminidase increased the electrophoretic mobility of rhOVGP1. A detailed analysis of O- and N-linked glycans of rhOVGP1 by mass spectrometry showed a broad range of many simple and complex glycan structures. In order to identify the enzymes involved in the glycosylation of rhOVGP1, we assayed glycosyltransferase activities involved in the assembly of O- and N-glycans in HEK293 cells, and compared these to those from the immortalized human oviductal cells (OE-E6/E7). Our results demonstrate that HEK293 and OE-E6/E7 cells exhibit a similar spectrum of glycosyltransferase activities that can synthesize elongated and sialylated O-glycans with core 1 and 2 structures, as well as complex multiantennary N-glycans. It is anticipated that the knowledge gained from the present study will facilitate future studies of the role of the glycans of human OVGP1 in fertilization and early embryo development.

Production and Purification of Recombinant Human Oviduct-Specific Glycoprotein (rhOGP) and the Role of rhOGP in Sperm Capacitation and Acrosome Reaction.

Production and Purification of Recombinant Human Oviduct-Specific Glycoprotein (rhOGP) and the Role of rhOGP in Sperm Capacitation and Acrosome Reaction.

Expression of human oviductin in an immortalized human oviductal cell line

To determine whether OE-E6/E7, an immortalized human oviductal epithelial cell line, expresses oviductin messenger RNA (mRNA) and its translated protein. Transmission electron microscopy was employed to characterize the morphology of OE-E6/E7 cells followed by reverse-transcription polymerase chain reaction (PCR) analysis of oviductin mRNA and sequencing of the nested-PCR product. Confocal microscopy was used, using a polyclonal antibody against human oviductin and Con A as a marker for mannose residues, to reveal the colocalization of human oviduct-specific glycoprotein with the endoplasmic reticulum and Golgi compartments. University-based anatomy and cell biology department. Women undergoing laparoscopy for tubal ligation or hysterectomy due to uterine fibroma. An immortalized OE-E6/E7 cell line was previously established using human oviductal epithelial cells. Electron microscopy, RT-PCR, sequencing, immunohistochemistry and confocal microscopy were performed. The presence of human oviductin mRNA and protein in OE-E6/E7 cells. OE-E6/E7 cells retain morphological features characteristic of secretory cells and express human oviductin mRNA and its translated protein. OE-E6/E7 cells were characterized for the first time by electron microscopy and shown to exhibit histological features typical of secretory cells. Reverse-transcription PCR with sequencing and confocal microscopy showed, respectively, that human oviductin mRNA and protein are expressed in OE-E6/E7 cells. Our results suggest that OE-E6/E7 could be a useful tool for future studies of the function of human oviductin.

Cloning and characterization of the human oviduct-specific glycoprotein (HuOGP) gene promoter.

Oviduct-specific glycoprotein (OGP) is a high molecular weight glycoprotein belonging to the chitinase protein family. OGPs are found to associate with the zona pellucida and plasma membrane of the oocyte and developing embryo. Recent studies have shown that OGP plays an important role in pre-fertilization reproductive events (i.e. sperm capacitation, sperm-zona binding and zona penetration). To have a better understanding of human OGP (HuOGP) gene expression, a 3.3 kb DNA fragment containing the 5'-flanking and the intron I region of the HuOGP gene was isolated. DNA sequence analysis of the HuOGP putative promoter revealed little homology with its hamster and mouse ogp gene counterparts. One transcription initiation site was found 12 nucleotides upstream of the first ATG codon of the HuOGP gene. The HuOGP gene promoter lacked typical CAAT or GC boxes, but contained eight half estrogen-responsive elements (ERE) and an imperfect ERE (iERE; 5'-GGTCANNNTGACT-3') site. Deletion mutants of the 3.3 kb DNA fragment were generated and fused to a promoterless beta-galactosidase (beta-Gal) gene. Transfection studies revealed that in the presence of 100 nmol/l estradiol-17 beta (E(2)), a minimal 0.3 kb promoter construct (pH-298/+25 beta Gal) mediated a high level of beta-Gal expression in immortalized human oviductal epithelial OE-E6/E7 cells, but not in MCF-7 and CHO-K1 cells. By electromobility shift assay and specific estrogen receptor antibodies, we demonstrated that estrogen receptor beta present in the OE-E6/E7 cells binds to the iERE. These findings allow a better understanding of the regulation of OGP gene expression in the human oviduct.

Establishment and characterization of an immortalized human oviductal cell line.

Human oviductal cells stimulate embryo development in vitro partly by the production of embryotrophic glycoproteins. The identity of these glycoproteins is not yet known mainly because oviductal samples are limited and that the cultured parental oviductal cells cannot produce sufficient amount of embryotrophic factors for characterization. In this study, human oviductal epithelial cells (OE) were immortalized by HPV 16 E6/E7 open reading frame (ORF) by retroviral expression. The characteristics of this immortalized cell line (OE-E6/E7) were compared to the parental OE. HPV 16 E6/E7 DNA was found only in OE-E6/E7 but not in OE. Human oviduct-specific glycoprotein, estrogen receptors, and cytokeratin were found in both cell types. Both OE and OE-E6/E7 possessed telomerase activities but the former had much lower activity. OE-E6/E7 also produced glycoproteins with chromatographic behavior similar to the embryotrophic glycoproteins derived from OE. These results showed that OE-E6/E7 retained a number of characteristics of OE. The development of preimplantation mouse embryo was significantly better after coculture with OE-E6/E7 when compared to medium alone culture in term of blastulation rates (52% vs. 32%) and blastocyst diameter (113.0 +/- 2.07 microm vs. 83.9 +/- 5.23 microm). This immortalized cell line can be used as a continuous and stable in vitro system for the study of the oviductal embryotrophic activity. Mol. Reprod. Dev. 59: 400-409, 2001.

A human oviduct-specific glycoprotein: synthesis, secretion, and localization during the menstrual cycle.

The major objective of this study was to examine the hormonal regulation of a human oviduct-specific glycoprotein (huOGP) throughout the menstrual cycle and in all regions of the human oviduct. Regulation of synthesis and secretion was examined at both the protein (Western immunoblots and immunocytochemistry) and mRNA (Northern and slot blots) levels and correlated with changes in the morphological features of the oviductal epithelial cells throughout the cycle. Immunoblot analysis of oviductal fluid and explant culture media from all regions of the oviduct demonstrated that huOGP is primarily found during the follicular stage of the cycle and is not present in serum, follicular fluid, or uterine endometrium. Moreover, two-dimensional (2-D) immunoblots showed that all major isoelectric variants of huOGP observed on 2-D fluorographs are immunologically related. Light microscopic immunocytochemistry localized huOGP to oviductal secretory cells in both ampulla and isthmic regions, with the most intense immunoperoxidase staining seen in midcycle samples. Using an indirect immunogold technique at the electron microscopic level, huOGP was specifically localized to secretory granules of the ampullary and isthmic nonciliated epithelial cells. The ultrastructural characteristics of these secretory cells during the mid to late follicular phase of the cycle suggested elevated protein synthetic activity. In addition, mRNA expression for huOGP was elevated in all regions of the oviduct in midcycle specimens. Collectively, these data indicate that huOGP is a major tissue-specific, stage-specific secretory product of the human oviduct during the periovulatory stage of the cycle and support the hypothesis that huOGP synthesis and secretion may be regulated by fluctuations in the levels of estrogen and progesterone.

In vitro incubation of golden (syrian) hamster ovarian oocytes and human sperm with a human oviduct specific glycoprotein

The objective of this study was to determine if human oviduct specific glycoprotein (huOGP) would associate with hamster ovarian oocytes and human sperm during in vitro incubation. The huOGP used in these studies was partially purified from human hydrosalpinx fluid. Hamster ovarian oocytes and human sperm samples were incubated in culture medium with and without huOGP. Association of huOGP was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against huOGP. Intense fluorescence of the zona pellucida, and bright but uneven fluorescence of the perivitelline space, were observed in hamster ovarian oocytes following incubation in the presence of huOGP. A similar but more uniform pattern of fluorescence was observed when hamster oviductal oocytes (positive controls) were incubated in culture medium alone. Fluorescence was absent when oocytes were assayed with preimmune serum. The association of huOGP with the zona pellucida and perivitelline space appeared to be specific since thyroglobulin, a large molecular weight glycoprotein, and human serum albumin, the major protein in oviduct fluid, did not associate with the hamster oocytes nor inhibit huOGP association when included in the culture medium. Fluorescence was absent when human sperm incubated with huOGP were assayed with antiserum to huOGP. However, human sperm fluoresced when incubated with a uterine glycoprotein, CUPED, which had previously been shown to bind to cat sperm during in vitro incubation. Sperm also fluoresced brightly when human sperm antibody was used as a positive control. Solubilization of sperm membrane proteins postincubation and analysis of these proteins by 1-D SDS-PAGE followed by immunoblotting also failed to show an association of huOGP with human sperm. Electron microscopy of sperm both pre- and postsolubilization confirmed that the sperm membranes were removed by this process. In conclusion, the association of huOGP with hamster oocytes in vitro suggests that huOGP may associate with human oocytes in vivo, whereas that may not be true for human sperm in vivo. The association of huOGP with oocytes may serve to facilitate the process of fertilization and early embryonic development within the oviduct.