Characterization of an oviductal glycoprotein associated with the ovulated hamster oocyte

Hamster oviducts in culture incorporate [35S]-methionine into secretory proteins. One of these proteins is immunoprecipitated by a monoclonal antibody specific to an antigen found in oviductal oocytes but not in ovarian oocytes. This antigen, called oviductin, is progressively added to the oocyte during its transit through the oviduct. Oviductin migrates as a diffuse band with a molecular mass between 160 and 250 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The electrophoretic behavior of this protein suggests the presence of polysaccharide side chains. Chemical deglycosylation causes a decrease in molecular mass and removes the antigenic determinant originally present on the glycoprotein. By using the radiation inactivation method, the molecular mass of the core protein has been found to be approximately 44 kDa. These results indicate that the oviduct is an actual site of synthesis of the oviductin. This glycoprotein contains a high proportion of sugar residues, which account for antigenic determinant recognized by the monoclonal antibody.     

The zona pellucida binds the mature form of an oviductal glycoprotein (oviductin).

The use of a monoclonal antibody (MAb) specific for the oviductal zona pellucida (ZP) of the hamster has demonstrated that a new antigen (oviductin) is acquired by the ZP during transit of the oocyte in the oviduct. The epitope that is recognized by the MAb bears a terminal N-acetyl-D-galactosamine residue. We conducted a study in order to determine whether this immunoreactivity of the oviductal ZP results from the addition of the terminal sugar residue to a preformed ZP protein or from the transfer of the mature glycoprotein produced by oviductal secretory cells. We measured the incorporation of [35S]methionine into proteins using four different incubation systems: cumulus oophorus (CO) alone, CO in the presence of oviductal fluid, CO co-incubated with empty oviducts, and CO within intact oviducts. At the end of the incubation period, the ZP, vitelli, dispersed cumulus without oocyte, oviducts, and culture medium were isolated and analyzed for their protein content by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiography, and immunodetection. The cumulus cells synthesized several proteins, independently of the oviductal environment; however, none of these proteins corresponded to oviductin. The ZP and the vitelli of cumulus oophorus that were incubated either alone or in the presence of oviductal fluid did not contain radioactive oviductin. When the oviduct (empty or intact) was present in the incubation system, radiolabeled oviductin was synthesized and secreted into the incubation medium. The ZP picked up a detectable amount of radioactive antigen only in the system in which intact oviducts were incubated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence analysis of feline oviductin and its expression during the estrous cycle in the domestic cat (Felis catus)

Oviductins belong to a family of oviduct-specific glycoproteins believed to play an important role in fertilization and/or early embryonic development. Oviductin cDNA between species is highly conserved and shares 58% to 98% similarity in the deduced amino acid sequences. Our objective in this study was to sequence the full open reading frame of the feline oviductin and to examine its expression during the estrous cycle on both mRNA and protein level. The obtained cDNA containing the full open reading frame was determined to be 1677 nucleotides coding for a deduced protein of 558 amino acids. Identities between species range from 74% (mouse) to 80% (human, baboon, and rhesus) within the N-terminal protein region. Major differences were localized in the carboxy terminal region, which corresponds to exon 11 of the gene. Feline oviductin contained one putative N-linked glycosylation site, six O-linked glycosylation sites, a potential heparin binding site, and two cholesterol recognition and/or interaction amino acid consensus (CRAC) domains. Oviductin expression was analyzed by real time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Both approaches revealed an estrous cycle-dependent expression in the ampulla and isthmus. Quantitative PCR showed highest oviductin mRNA copy numbers in the early and late follicular stage and reduced mRNA expression during all other stages. With the exception of the early follicular stage, feline oviductin mRNA abundance was not significantly different in the oviductal segments ampulla and isthmus. A prominent immunolabeling was seen in the early and late follicular stage which disappeared after ovulation, indicating a function of the protein during sperm storage and fertilization.     

Oviductin, the Xenopus laevis oviductal protease that processes egg envelope glycoprotein gp43, increases sperm binding to envelopes, and is translated as part of an unusual mosaic protein composed of two protease and several CUB domains

The glycoprotein envelope surrounding the Xenopus laevis egg is converted from an unfertilizable to a fertilizable form during transit through the pars recta portion of the oviduct. Envelope conversion involves the pars recta protease oviductin, which selectively hydrolyzes envelope glycoprotein gp43 to gp41. Oviductin cDNA was cloned, and sequence analysis revealed that the protease is translated as the N terminus of an unusual mosaic protein. In addition to the oviductin protease domain, a protease domain with low identity to oviductin was present, possessing an apparent nonfunctional catalytic site. Three CUB domains were also present, which are related to the mammalian spermadhesin molecules implicated in mediating sperm-envelope interactions. We propose that during post-translational proteolytic processing of the mosaic oviductin glycoprotein, the processed N-terminal protease domain is released coupled to two C-terminal CUB domains and constitutes the enzymatically active protease molecule. In functional studies, isolated coelomic egg envelopes treated with oviductin purified from the oviduct showed a dramatic increase in sperm binding. This observation established that oviductin alone was the oviductal factor responsible for converting the egg envelope to a sperm-penetrable form, via an increase in sperm binding. Trypsin mimicked oviductin's effect on envelope hydrolysis and sperm binding, demonstrating that gp43 processing is the only requirement for envelope conversion.     

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.     

HeLa细胞表达分泌重组eGFP-DPF-1在卵母细胞上的定位

将兔输卵管蛋白(DPF-1)基因连结于增强型绿色荧光蛋白(eGFP)基因5′端,构建了真核表达重组质粒(pEGFP-N1/DPF-1),转染HeLa细胞,获得稳定表达分泌融合蛋白eGFP-DPF-1的HeLa细胞株。该融合蛋白呈现的分子量达120 KD,提示经翻译后修饰。取兔卵母细胞-卵丘细胞复合物(COC)、去除卵丘细胞后的卵母细胞或输卵管内的卵母细胞,与该株细胞共培养或培养于该株细胞条件培液中,观察兔输卵管蛋白在兔卵母细胞上的分布。结果显示DPF-1大量结合于卵母细胞透明带,先结合于透明带内层,然后维持在内层多外层少的分布状态上;在卵母细胞质膜表面则呈点状均匀分布。DPF-1在卵母细胞上的分布不受其周围颗粒细胞的阻碍,且颗粒细胞上未见有DPF-1结合的痕迹。本实验首次证实体外真核细胞表达分泌的输卵管蛋白能与卵母细胞结合,并借助绿色荧光蛋白作为示踪信号体外直接观察到该表达产物在卵母细胞上的动态分布,为进一步深入分析输卵管蛋白的功能提供了线索,也为研究输卵管内其他蛋白在配子/早胚上定位提供了可行的办法。     

HeLa细胞表达分泌重组eGFP—DPF-1在卵母细胞上的定位

将兔输卵管蛋白(DPF-1)基因连结于增强型绿色荧光蛋白(eGFP)基因5′端,构建了真核表达重组质粒(pEGFP-N1／DPF-1),转染HeLa细胞,获得稳定表达分泌融合蛋白eGFP—DPF-1的HeLa细胞株。该融合蛋白呈现的分子量达120KD,提示经翻译后修饰。取兔卵母细胞-卵丘细胞复合物(COC)、去除卵丘细胞后的卵母细胞或输卵管内的卵母细胞,与该株细胞共培养或培养于该株细胞条件培液中,观察兔输卵管蛋白在兔卵母细胞上的分布。结果显示DPF-1大量结合于卵母细胞透明带,先结合于透明带内层,然后维持在内层多外层少的分布状态上;在卵母细胞质膜表面则呈点状均匀分布。DPF-1在卵母细胞上的分布不受其周围颗粒细胞的阻碍,且颗粒细胞上未见有DPF-1结合的痕迹。本实验首次证实体外真核细胞表达分泌的输卵管蛋白能与卵母细胞结合,并借助绿色荧光蛋白作为示踪信号体外直接观察到该表达产物在卵母细胞上的动态分布,为进一步深入分析输卵管蛋白的功能提供了线索,也为研究输卵管内其他蛋白在配子／早胚上定位提供了可行的办法。     

The influence of recombinant feline oviductin on different aspects of domestic cat (Felis catus) IVF and embryo quality

Oviductin is known to be a key player providing a convenient environment for the process of fertilization affecting this by direct interaction with oocytes and sperm. As in vitro embryo production in the context of assisted reproduction for endangered felids is still in the process of optimization, oviductin might be used to improve IVF results. Recombinant His-tagged feline oviductin was expressed by transformed Escherichia coli BL21DE3 cells. The protein was purified by immobilized metal ion affinity chromatography. The effect of the recombinant protein was characterized in three experiments: a hemizona assay for sperm binding analysis, the IVF outcome, and the relative mRNA expression levels in blastocysts after IVF. A significant higher number of bound sperm cells were found after incubation in oviductin. No significant effect on cleavage, morula, and blastocyst rates with or without oviductin incubation during IVF could be observed. However, the relative mRNA abundance of GJA1, a gene, whose expression level is known to be a marker of embryo quality, was significantly increased (P value less than 0.05) in blastocysts after oviductin treatment. In contrast to this, expression of OCT4, HSP70, DNMT1, DNMT3A, BAX, IGF1R, and GAPDH was not significantly affected. We assume that our recombinant oviductin in its current nonglycosylated form is able to enhance sperm binding. Despite of a missing significant effect on IVF outcome, embryo quality in terms of relative GJA1 expression is influenced positively. These promising results demonstrate the value of recombinant oviductin for the IVF in cats.     

小鼠子宫颈部位输卵管蛋白表达的初步研究

用RT-PCR及免疫印迹法检测证实小鼠子宫颈粘膜上皮细胞具表达输卵管蛋白的能力,宫颈部所获得的输卵管蛋白转录产物, 310到 714的405bp的cDNA片段经序列测定及blast比较表明与输卵管部位表达的输卵管蛋白完全一致(100%)。小鼠子宫颈输卵管蛋白在动情周期的表达波动情况与输卵管粘膜上皮的类似,其表达于小鼠动情期明显增加。Westernblot显示小鼠子宫颈提取物中存在两种不同分子量(60KD,30KD)的输卵管蛋白。原位杂交结果则提示子宫颈粘膜上皮细胞含丰富的输卵管蛋白mRNA信号。通过"原体中风"实验未发现输卵管蛋白在体外对精子活动有影响,其功能尚须进一步分析。     

In vivo versus in vitro oviductal glycoprotein (OGP) association with the zona pellucida (ZP) in the hamster and baboon

The goal of this study was to determine if differences exist between in vivo vs. in vitro OGP association with the ZP and to quantitate those differences. Ovarian oocytes were harvested 12.5 or 27 hr post-hCG from hyperstimulated hamsters or baboons, respectively. Hamster and baboon ovarian oocytes were incubated in vitro in media +/- homologous OGP (100 or 200 microg/100 microl) or in some studies with 100 microl oviductal fluid for 3, 6, or 24 hr at 37 degrees C. Some of the baboon ovarian oocytes were transferred immediately after harvesting to the ampulla of both oviducts using a tom cat catheter and retrieved after a 3 hr in situ incubation. Hamster oviductal oocytes were collected 3, 6, and 24 hr following ovulation. After incubation or oocyte retrieval from the oviduct, cumulus cells were removed, oocytes were washed extensively and binding of OGP to the ZP was examined by immunofluorescence. Fluorescence intensity was quantified using densitometric scanning of photographic negatives with the background of each negative as an internal control. In all studies, OGP association with the ZP was significantly greater in vivo than in vitro (P < 0.05). In vitro OGP association with the ZP did not significantly increase with incubation time or OGP concentration; however, a small nonsignificant increase in OGP association with the ZP in the oviduct was detected over time. Differences did not appear to be due to depletion of OGP from the in vitro incubation media, since Western blot analysis of the media showed that OGP was still present. Although OGP concentration in vivo is unknown, Western blots showed similar intensity comparing 100 microg of OGP media and oviductal fluid. Immunolocalization of OGP using laser confocal microscopy showed regional differences in OGP binding. The outer half of the zona pellucida had significantly more OGP bound than the inner half on oviductal oocytes. No regional differences were detected for in vitro incubated oocytes. In conclusion, OGP association with the ZP is greater in vivo vs. in vitro, suggesting that one must be cautious in designing and evaluating in vitro studies of OGP function.     

Cumulus cell dispersion induced by estradiol in mouse oviduct in vitro

Dispersion of cumulus cells in nonmated mice is completed in the oviduct 15–20 h after ovulation. Oviducts, isolated 1 h after ovulation (13 h post-human chorionic gonaditropin), were cultured in vitro for 40 h. In these oviducts, denuded oocytes were first seen at 30 h of culture, indicating that cumulus dispersion proceeded at a slower rate in vitro. Oocyte denudation was accelerated in a dose-dependent manner by the addition of estiadiol to the culture medium in which oviducts were incubated. The addition of progesterone or cycloheximide to the culture medium strongly inhibited oocyte denudation even in the presence of estradiol. When isolated cumuli were incubated in the absence of oviductal tissue, the rate of cell dispersion was slower than that of cumuli incubated inside the oviduct and the addition of estradiol to the culture failed to accelerate this process. On the basis of these data, we propose that cumulus cell dispersion is accelerated by an estrogen-dependent protein produced by the oviduct and that this effect of estrogen is antagonized by progesterone.     

Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.     

Inhibition of in vivo and in vitro fertilization in rodents by gonadotropin-releasing hormone antagonists

We have examined the effect of two GnRH antagonists, Ac-D-Nal(1)-Cl-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH (Nal-Glu) and Ac(3,4)-dehydro-Pro(1),-p-fluoro-D-Phe(2),D-Trp(3,6)-GnRH (4pF), on in vivo and in vitro fertilization in rodents. Female rats were treated in the afternoon of proestrus with 2 micro l of Nal-Glu or 4pF (0.5 and 5 mM) injected directly into one oviductal horn (experimental); saline was injected into the contralateral horn (control). Females were then mated and the oviducts were perfused for egg and sperm recovery. The results indicate that both antagonists inhibited in vivo fertilization. Thus, the percentage of fertilized eggs in control oviducts ranged from 92% +/- 5% to 100% +/- 0%, whereas in treated oviducts, fertilization ranged from 25% +/- 6% to 73% +/- 5%. GnRH antagonists did not interfere with the process of ovulation, sperm migration to the site of fertilization, or early embryo development. In additional experiments with mice, GnRH antagonists inhibited in vitro fertilization. One fertilization event that was specifically inhibited by GnRH antagonists was the process of sperm binding to the zona pellucida. This step was precisely monitored using the hemizona assay. GnRH antagonists did not affect sperm movement or acrosomal status. These observations indicated that local treatment with GnRH antagonists inhibit in vivo fertilization and give additional support to the idea that endogenous GnRH may play an important role during fertilization by increasing the efficiency of sperm-zona binding.     

S100 A9 is expressed and secreted by the oviduct epithelium,interacts with gametes and affects parameters of human sperm capacitation in vitro

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [³⁵S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [³⁵S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.     

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. Mol. Reprod. Dev. 46:306–317, 1997. © 1997 Wiley-Liss, Inc.     

Effects of delayed excision of oviducts/ovaries on mouse oocytes and embryos

To achieve the best and reproducible results of experiments, effects of delayed excision of oviducts/ovaries on mouse ovarian/ovulated oocytes and embryos have been studied. Oviducts/ovaries were excised at different times after death of mice and effects of the postmortem interval on ovarian/ovulated oocytes and embryos were analyzed. When oviduct excision was delayed 10 min, many ovulated oocytes lysed or underwent in vitro spontaneous activation, and this postmortem effect aggravated with the extension of postmortem interval and oocyte aging. Oocytes from different mouse strains responded differently to delayed oviduct removal. Delayed oviduct excision did not cause lysis of zygotes or embryos but compromised their developmental potential. When ovaries were excised at 30 min after death, percentages of atretic follicles increased while blastocyst cell number declined significantly after oocyte maturation in vitro. Preservation of oviducts in vitro, in intact or opened abdomen at different temperatures and histological analysis of oviducts from different treatments suggested that toxic substance(s) were secreted from the dying oviducts which induced oocyte lysis and spontaneous activation and both this effect itself and the sensitivity of oocytes to this effect was temperature dependent. It is concluded that a short delay of oviduct/ovary removal had marked detrimental effects on oocytes and embryos. This must be taken into account in experiments using oocytes or embryos from slaughtered animals. The data may also be important for estimation of the time of death in forensic medicine and for rescue of oocytes from deceased valuable or endangered mammals.     

Sperm binding activity of the zona pellucida of immature mouse oocytes

Immature oocytes taken from ovarian follicles are sometimes used in studies of sperm-zona interaction in species for which it is difficult to obtain ovulated eggs. As yet, however, there has been no quantitative comparison of the sperm binding capacities of immature and ovulated oocytes. We report here that in mice there is no significant difference in the numbers of sperm which bind to the zonae pellucidae of immature and ovulated oocytes in vitro. These results support the use of immature oocytes in studies of sperm-zona interaction. We have also analyzed the sources of variability in sperm binding assays, and we make suggestions for the most efficient design of experiments.     

Purification, sequence characterization and effect of goat oviduct-specific glycoprotein on in vitro embryo development

Oviduct-specific glycoprotein (oviductin) plays an important role during fertilization and early embryonic development. The oviductin cDNA was successfully cloned and sequenced in goat, which possessed an open reading frame of 1620 nucleotides representing 539 amino acids. Predicted amino acid sequence showed very high identity with sheep (97%) followed by cow (94%), porcine (77%), hamster (69%), human (66%), rabbit (65%), mouse (64%) and baboon (62%). The bioinformatics analysis of the sequences revealed the presence of a signal sequence of 21 amino acids, one potential N-linked glycosylation site at position 402, 21 potential O-linked glycosylation sites and 36 potential phosphorylation sites. The native oviductin was purified from the oviductal tissue, which showed three distinct bands on SDS-PAGE and western blot (MW ∼60-95 kDa). The predicted molecular weight of goat oviductin was 57.5 kDa, calculated from the amino acid sequences. The observed higher molecular weight has been attributed to the presence of large number of potential O-linked glycosylation sites. The lower concentration (10 μg/mL) of oviductin increased the cleavage rate, morula and blastocyst yield significantly (P < 0.05) as compared to higher concentration (100 μg/mL). Goat oviductin retarded the activity of pronase (0.1%) on zona solubility of oocytes significantly (P < 0.01).