Hamster Oviductin Research Articles

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

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.

Hamster Oviductin Regulates Tyrosine Phosphorylation of Sperm Proteins During In Vitro Capacitation1

Oviductin or OVGP1, also known as oviduct-specific glycoprotein, has been shown to enhance sperm capacitation in addition to its other beneficial effects on fertilization and early embryo development. We hypothesized that estrus stage-specific hamster oviductin (eHamOVGP1) can potentiate the enhancement of tyrosine phosphorylation of sperm proteins during capacitation. Immunofluorescent staining and confocal microscopy as well as immunocytochemistry and surface replica technique localized tyrosine-phosphorylated proteins to the equatorial segment and midpiece after incubation of hamster sperm in capacitation medium in the presence or absence of eHamOVGP1. Increase of tyrosine phosphorylation level in the equatorial segment occurred as early as 5 min after incubation in the presence of eHamOVGP1. Immunostaining for eHamOVGP1 further increased upon prolonged incubation of sperm in medium containing the glycoprotein. Regardless of the presence or absence of eHamOVGP1, phosphotyrosine expression was observed along the tail, particularly at the midpiece. Western blotting of NP40-extracted sperm proteins (25, 37, and 44 kDa) and NP40-non-extractable sperm proteins (70, 83, 90 kDa) showed increased immunolabeling intensity after 5, 60, 120, and 180 min of capacitation in the presence of eHamOVGP1. Mass spectrometric analysis identified several proteins of functions known to be involved in metabolic pathways responsible for enhancement of tyrosine phosphorylation in its presence. The present investigation provides evidence that eHamOVGP1 regulates the expression of protein tyrosine phosphorylation in sperm capacitated in vitro, further supporting an important role of the presence of OVGP1 in the oviductal milieu during the process of fertilization.

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

Detection of glycosyltransferases in the golden hamster (Mesocricetus auratus) oviduct and evidence for the regulation of O-glycan biosynthesis during the estrous cycle

Recently, we provided evidence that the glycosylation of hamster oviductin, a member of the mucin family of glycoproteins, is regulated during the estrous cycle. In order to further elucidate the glycosylation process of oviductal glycoproteins, we identified biosynthetic pathways involved in the assembly of mucin-type O-linked oligosaccharide (O-glycan) chains in the hamster oviduct. Our results demonstrated that the hamster oviduct has high activities of glycosyltransferases that synthesize O-glycans with core 1, 2, 3 and 4 structures as well as elongated structures. Oviduct therefore represents a typical mucin-secreting tissue. Our results also showed that specific glycosyltransferase activities are regulated during the estrous cycle. Mucin-type core 2 beta6-GlcNAc-transferase (C2GnT2) is responsible for synthesizing core 2 and core 4 structures in the oviduct. Specific assays for C2GnT2 revealed a cyclical pattern throughout the estrous cycle with high activity at the stages of proestrus and estrus and low activity at diestrus 1. Using semiquantitative RT-PCR, the mRNA levels for C2GnT2 in the estrous cycle stages could be correlated with the enzyme activities. An increase in glycosyltransferase activity in the hamster oviduct at the time of ovulation suggests that glycosylation of oviductal glycoproteins may be necessary for these proteins to exert their functions during the process of fertilization.

Detection of nascent and/or mature forms of oviductin in the female reproductive tract and post-ovulatory oocytes by use of a polyclonal antibody against recombinant hamster oviductin.

Oviductins belong to a family of glycoproteins that have been suggested to play several roles during the early processes of reproduction. Recently, a polyclonal antibody was raised against recombinant hamster oviductin (rhaOv(m)). Here the anti-rhaOv(m) antibody was used to investigate the sites of localization of oviductin in the female golden hamster. In the hamster oviduct, immunolabeling was restricted to the content of the Golgi saccules and secretory granules of the non-ciliated oviduct cells. After its release into the lumen, oviductin becomes associated with the zona pellucida of post-ovulatory oocytes. In unfertilized oocytes, oviductin was also detected in membrane invaginations along the oolemma and in some vesicles within the ooplasm. Furthermore, oviductin was detected over the microvilli and within multivesicular bodies of uterine epithelial cells. Western blotting analysis revealed the presence of oviductin in the hamster oviduct but not in the uterus or ovary. In the oviduct, the anti-rhaOv(m) antibody detected a polydispersed band corresponding to native oviductin (160-350 kD) and several lower molecular weight bands (<100 kD) corresponding to nascent and partially glycosylated forms of oviductin. The anti-rhaOv(m) antibody provides an additional tool for investigation into the cytochemical and biochemical properties of different forms of hamster oviductin in the female reproductive tract.

Evidence for the regulation of glycosylation of golden hamster (Mesocricetus auratus) oviductin during the estrous cycle.

The oviduct contributes to the reproductive environment by secreting various factors, including a family of glycoproteins termed oviductins. Although many studies have demonstrated that ovarian hormones modulate oviductin gene expression in several mammalian species, there has been controversy surrounding the regulation of golden hamster oviductin. The current study was undertaken to investigate the transcriptional and translational modifications of hamster oviductin during the estrous cycle. First, we verified that hamster oviductin mRNA expression remains constant throughout the estrous cycle by semiquantitative reverse transcription polymerase chain reaction. We then developed a polyclonal antibody against recombinant hamster oviductin (rhaOvm). The anti-rhaOvm antibody was subsequently used in conjunction with quantitative immunocytochemistry to investigate the oviductin levels in the hamster oviduct during the estrous cycle. Quantification of immunolabeling revealed a high, consistent level of glycoprotein throughout the estrous cycle. Therefore, it appears that the production of oviductin is not regulated differentially during the estrous cycle. Size variations in hamster oviductin expression were also investigated by Western blot analysis. The oviduct contains several forms of oviductin at each stage of the estrous cycle, the native glycosylated form(s) of 160-350 kDa, and several precursor forms of 70-100 kDa. Although variations in the intensities of the polydispersed band were not evident during the estrous cycle, additional bands ranging from 90 to 100 kDa were detected in the estrus, metestrus, and diestrus 1 stages. The results from the present investigations suggest that whereas ovarian hormones do not appear to influence the hamster oviductin mRNA and protein expressions, glycosylation of hamster oviductin appears to be differentially regulated during the estrous cycle.

Oviductin (Muc9) is expressed in rabbit endocervix.

While isolating and characterizing cervical mucin glycoproteins, oviductin (Muc9) was identified in the rabbit endocervix. Following tissue homogenization, endocervical proteins were fractionated by exclusion chromatography (Sepharose CL-4B). High molecular weight components of the void volume were resolved by density gradient centrifugation using cesium bromide and dissociative conditions (4 M guanidinium chloride). High density fractions (rho = 1.40 - 1.56 g/ml) were deglycosylated with anhydrous trifluoromethane sulfonic acid and sent to Harvard Microchemistry where in situ digestion and tryptic peptide separation were performed. Out of an HPLC map, microsequence (KLIMGFPTYGR) from peak 51 was 100% identical to mouse oviductin, and microsequence (KSTGHNFPLP) from peak 70 was 90% identical to hamster oviductin. Temporal expression of oviductin transcripts (2.4-kilobase) was negligible during the first three months of postnatal cervical differentiation. Transcripts were minimally detectable in the cervices of 4-month-old juveniles. Strong expression in the endocervices of adults was eliminated by ovariectomy and restored by estrogen treatment. The presence of oviductin in the rabbit endocervix indicates this glycoprotein may have multiple functions, and it can no longer be considered oviduct-specific.

Organization of a gene coding for an oviduct-specific glycoprotein (oviductin) in the hamster.

Hamster oviductin, a high molecular weight glycoprotein secreted by the oviducts, is believed to participate in fertilization and protection of the tubal epithelium. Expression of the oviductin gene is confined strictly to nonciliated secretory cells of the oviduct and is regulated by hormones. The objective of this study was to characterize the genomic organization and to identify potential regulatory elements implicated in the control of transcription of the oviductin gene. Polymerase chain reaction was performed on hamster genomic DNA, yielding 2.2 kb of the 5' flanking region as well as 13.6 kb of genomic sequence comprising the entire coding sequence of the oviductin gene distributed in 11 exons. Sequencing of the 5' flanking region revealed, among other elements, an almost perfect estrogen-responsive element (GGTCACTGTGACT), an atypical TATA box (TATTAA), and a perfect inverted Sp1 site located between the transcription start site and the atypical TATA box. Primer extension analyses indicated that the hamster oviductin transcript possesses an unusually short 5' untranslated region of only 14 nucleotides. The distinct organization of the hamster oviductin gene in the vicinity of the transcription start site provides an interesting ground for further functional studies.

Fate of hamster oviductin in the oviduct and uterus during early gestation.

Oviductins are a family of glycoproteins which are synthesized and secreted by oviductal secretory cells and which, upon their secretion in the lumen of the oviduct, become associated with postovulatory oocytes and developing embryos. Recently, we showed that hamster oviductin is maximally secreted in the oviduct at the time of ovulation and is later associated with a certain population of uterine epithelial cells, where it is subsequently endocytosed and degraded. In light of these results, this study was conducted to follow the fate of hamster oviductin in the oviduct and uterus during early gestation. Using a monoclonal antibody against hamster oviductin, immunofluorescence and immunogold labeling revealed that during early gestation, immunoreactivity to oviductin in the uterus gradually diminished to an almost total disappearance at time of implantation. However, the strong labeling intensity remained unchanged in the oviduct. Biochemical analyses demonstrated that a degradation of oviductin occurs in the uterus, and a loss of immunoreactivity was also observed as gestation progressed, so that by the time of implantation, immunoreactivity to oviductin was barely detectable. The decrease of oviductin along the uterine epithelium at the time of blastocyst attachment and its final disappearance at implantation suggest that this glycoprotein could be a potential modulator of uterine receptivity.

Allelic polymorphism in the hamster oviductin gene is due to a variable number of mucin-like tandem repeats.

Oviductins are high-molecular-weight glycoproteins specifically secreted by the oviduct. These proteins bind to the zona pellucida of the ovulated oocyte and remain associated with the embryo during its transit in the oviduct. They may be involved in fertilization and early embryonic development. In order to explore their putative biological function, the cDNA sequence corresponding to oviductin in the golden hamster was determined. We found that the deduced amino acid sequence of this heavily O-glycosylated protein presents characteristics typical of mucins, including serine- or threonine-rich tandem repeats. Analysis of several cDNA clones and of genomic DNA revealed the presence of a single copy gene with two frequent alleles differing in the number of repeats. Comparison with oviductin sequences from other mammals indicates a high degree of conservation amongst species, except for the repeat region which shows divergence, notably in the number of repeats. Based on its biochemical and genetic properties, hamster oviductin can now be classified as a secretory mucin. This concept provides a new insight in the elucidation of its biological role: oviductin could possibly provide the oviduct and the oocyte with a protective coating ensuring normal tubal function and embryonic development.

Hormonal control of the biosynthesis of hamster oviductin

In several mammalian species, the epithelial secretory cells of the oviduct synthesize and secrete specific glycoproteins that become associated with the zona pellucida of the ovulated oocyte. These glycoproteins are collectively designated as oviductins. A monoclonal antibody directed against hamster oviductin was used to study the ontogeny of this glycoprotein. Indirect immunofluorescence experiments performed on sections of hamster oviduct revealed that the glycoprotein begins to be secreted in 10-day-old females and that all of the oviductal secretory cells showed fluorescent staining by day 14. The intensity of the immunofluorescence reaction reached a maximum in the 28-day-old females. The oviducts of the 7-day-old hamster incorporated [35S]methionine in vitro into several proteins; however, the production and secretion of detectable amounts of radiolabeled oviductin only began at 14 days of age and reached a maximum at day 28 of age. It appears that the ontogeny of oviductin parallels the hormone dependent changes leading to sexual maturation and that its maximum secretion is already established at the time of the first ovulatory cycle. These results are substantiated by the fact that the production of oviductin is induced in estradiol-treated, but not progesterone or non-treated prepubertal animals, as determined by indirect immunofluorescence experiments.

Oviductins possess chitinase‐ and mucin‐like domains: A lead in the search for the biological function of these oviduct‐specific ZP‐associating glycoproteins

Over the last 10 years considerable progress has been made in the immunological and biochemical characterization of oviduct-specific glycoproteins. It is now well established that a subclass of these secretory products, designated as oviductins, associate with the zona pellucida of the ovulated oocyte and with the early embryo. Recent reports on the cloning of cDNAs of oviductins from various species, including that of golden hamster (Mesocricetus auratus) oviductin by our laboratory, allowed us to compare their deduced amino acid sequences with those of other proteins. Optimal alignment analysis showed that oviductins contain regions of significant similarity with catalytically inactive mammalian members of the bacterial and microfilarial chitinase protein family. Most importantly, a close examination of the hamster and human deduced amino acid sequences revealed that both glycoproteins possess contiguous Ser/Thr rich repeated units, clustered in their carboxy-terminal portions. These mucin-type motifs are similar in the hamster and human glycoprotein, although hamster oviductin contains more of these complete units. This striking feature might indicate that these molecules play a similar role to mucin-type glycoproteins, e.g., in protecting the oocyte and early embryo against attacks from their environment. We propose a model whereby oviductins are targeted to the oocyte via the interaction of their chitinase-like domains with specific oligosaccharide moieties of the zona pellucida. Once localized to this structure, oviductin molecules would act as a protective shield around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains.

Affinity binding of hamster oviductin to spermatozoa and its influence on in vitro fertilization

The aim of this study was to determine the influence of hamster oviductal glycoprotein (oviductin) on in vitro gamete interaction. Oviductin was purified from the oviducts using lithium 3,5-diiodosalicylate, followed by phenol extraction. Immunocytochemistry using indirect fluorescence staining revealed that oviductin binds to the sperm anterior acrosomal region. The specific binding of oviductin resulted in inhibition of in vitro fertilization in studies using cumulus-free oocytes. The inhibitory effect was dependent on the concentration of oviductin and occurred in both ovarian and oviductal oocytes but not zona-free oocytes, indicating that sperm-zona interaction was interferred by oviduction. However, the inhibitory effect of oviductin in sperm-zona interaction was reduced when cumulus-enclosed oocytes from ovaries and oviducts were used, indicating that the egg investment including cumulus oophorus has some effect on oviductin-sperm complex and maintaining the fertilizing ability.

Complementary Deoxyribonucleic Acid Cloning and Molecular Characterization of an Estrogen-Dependent Human Oviductal Glycoprotein1

A 120-kDa oviduct-specific glycoprotein is synthesized and secreted into the oviductal lumen during estrogen dominance in the human. The objective of this investigation was to clone, sequence, and characterize the cDNA to this core protein. Rapid amplification of cDNA ends was used to clone a contiguous 3' CDNA end and 5' cDNA end. The total length of the cDNA was determined to be 2.2 kb by sequence analysis and exhibited a 92% sequence identity with the comparable overlapping baboon cDNA (1.2 kb). A high degree of homology was found to the N-terminal sequence of hamster oviductin and the partial sequence of a homologous baboon and bovine oviduct glycoprotein. Northern blots revealed a single mRNA species of 2.4 kb. Using RNA from various tissues of an estrogen-treated baboon, we found that the mRNA for the oviductal glycoprotein was present only in the oviduct. Hybridization was detected to an mRNA of similar size from oviductal tissue of the baboon, hamster, and mouse and to an mRNA of slightly smaller size in the rabbit, cow, and cat but not to any mRNA species from rat oviductal RNA. Slot-blot analysis showed that the message was present in significantly greater (p < 0.05) concentrations in RNA from oviductal tissue from the late follicular stage than from the early follicular, early or late luteal, or postpartum stages. In conclusion, we have isolated the complete cDNA for a human oviductal glycoprotein. The presence of significantly greater amounts of the mRNA during the late follicular phase of the menstrual cycle is consistent with the proposed estrogen control. The mRNA for the oviductal glycoprotein is present only in the oviduct of an estrogen-treated baboon, and a cross-hybridizing RNA is found in oviductal RNA from various mammals.

Biochemical characterization of hamster oviductin as a sulphated zona pellucida-binding glycoprotein.

Oviductins are a family of glycoproteins, synthesized and released by oviductal secretory cells, which bind to the zona pellucida of the oocyte after ovulation. Hamster oviductin migrates as diffuse species of 160-350 kDa during SDS/PAGE under reducing as well as non-reducing conditions. In this report, we describe the one-step purification of hamster oviductin using either immuno- or lectin-affinity chromatography. Probing with specific lectins showed that the glycoprotein contains terminal alpha-D-GalNAc, and either terminal alpha-D-NeuAc or non-terminal beta-D-(GlcNAc)2 residues, but fails to react with concanavalin A and Ulex Europeus A-1 lectins which are specific for branched alpha-D-mannose and alpha-L-fucose moieties respectively. Intraovarian oocytes do not contain this glycoprotein and we demonstrate here that the immunoaffinity-purified oviductin readily binds to their zonae pellucidae in vitro, thus mimicking the in vivo phenomenon. Two major immunologically related forms of hamster oviductin (named alpha and beta) were characterized using one- and two-dimensional gel electrophoresis. The alpha-form (160-210 kDa) has an acidic pI of 3.5-4.5 and the beta-form (approx. 210-350 kDa) is localized at the cathodic site in the isoelectric focusing dimension; in between these two major forms lies a smear of minor-charge isomers. Peptide mapping of both major forms with papain and Staphylococcus aureus V8 protease yielded fragments of identical size. Moreover, the two forms share the same N-terminal sequence which display no significant homology with other reported proteins. Treatment with trifluoromethanesulphonic acid showed that a protein with the size and pI of the alpha-form can be generated from the beta-form. Both the alpha- and beta-forms are sulphated on O-linked oligosaccharide side chains but are not phosphorylated. Collectively, these results suggest that the hamster oviductin polymorphism observed in two-dimensional PAGE is a consequence of different glycosylation patterns and not the polypeptide chain itself. Hamster oviductin is mostly O-glycosylated and contains a few N-linked oligosaccharide side chains (approx. 10 kDa). We propose that hamster oviductin is a mucin-type glycoprotein which might act as a protective secretion influencing the first steps of the reproductive process necessary for the normal triggering of fertilization and early embryonic development.

S8.22 The binding of hamster oviductin to spermatozoa and its inhibitory effect toin vitro fertilization

S8.22 The binding of hamster oviductin to spermatozoa and its inhibitory effect toin vitro fertilization

Demonstration by lectin‐gold cytochemistry of transfer of glycoconjugates of oviductal origin to the zona pellucida of oocytes after ovulation in hamsters

We have previously localized an antigen of oviductal origin to the zona pellucida of superovulated hamster oocytes (Kan et al.: Journal of Histochemistry and Cytochemistry 36:1441-1447, 1988) and described the intracellular distribution of this antigen in the oviductal epithelium (Kan et al.: Biology of Reproduction 40:585-598, 1989). These results led to our hypothesis that the oviduct is a bona fide site of origin of certain components of the zona pellucida. In this report, using the high resolution lectin-gold approach with Helix pomatia lectin (HPL)-colloidal gold complex, we present cytochemical evidence to show that glycoconjugates containing terminal N-acetyl-D-galactosamine residues are absent from the zona pellucida of ovarian oocytes but are synthesized and secreted by the nonciliated secretory cells of the oviduct and later become associated with well-defined structural elements of the zona matrix of oocytes during passage through the oviduct. The nature of the HPL-binding glycoconjugates was determined by biochemical analyses. Electrophoretic and immunological experiments demonstrated that the glycoconjugates correspond to the high molecular weight polydispersed glycoprotein that we have previously described. We have designated this glycoprotein "hamster oviductin 1" (Hm OV-1). Our results further substantiate the belief that the oviduct is a source of origin of zona pellucida constituents.