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.     

Immunohistochemical localization of oviductin in the endometrial lining of the golden hamster (Mesocricetus auratus) during the estrous cycle and early gestation

Summary Oviductal non-ciliated secretory epithelial cells, under hormonal stimulation, synthesize and secrete a family of glycoproteins referred to as oviductins. These glycoproteins are found in oviductal fluid in several mammalian species, and have been localized in the oviduct, and in the zona pellucida of ovulated oocytes. In the golden hamster, this glycoprotein is named hamster oviductin-1. Recently, an immunofluorescent study on hamster uterine tissue has revealed the presence of the glycoprotein in luminal epithelial cells in a heterogeneous labelling pattern during the estrous cycle. The mechanism of endometrial epithelial cell receptivity to hamster oviductin-1 is not known.In this study, immunohistochemical studies were performed using a monoclonal antibody against the oviductin in conjunction with silver enhancement technique, in an attempt to determine further the factors playing a role in uterine receptivity to oviductin-1. Paraffin sections of hamster uterus obtained from different stages of the estrous cycle and from days 1–6 of gestation, and paraffin sections of hamster oviduct obtained from days 1–6 of gestation were used in this study. The results we obtained using the silver enhancement technique show that hamster uterus luminal epithelial cells exhibit a homogeneous, high intensity immunolabelling pattern throughout the estrous cycle, whereas, during gestation, labelling intensity decreases as the period for blastocyst implantation approaches. Oviduct epithelial cells revealed no definite fluctuating pattern in immunolabelling intensities during gestation, indicating no change in synthesis and secretion of the glycoprotein during this period.It is speculated that receptors for hamster oviductin-1 are present at the apical cell surface of endometrial cells and that implantation of the developing blastocyst into the uterine wall is possible only following downregulation of these receptors.The use of the silver enhancement technique proves to be an effective tool in immunohistochemical studies at the light microscope level, as seen through this study.     

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. © 1995 wiley-Liss, Inc.     

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.     

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)     

FSH促进牦牛输卵管上皮细胞分泌输卵管特异性糖蛋白的研究

为在胚胎共培养过程中添加相关激素提高哺乳动物胚胎发育的研究提供理论依据,本研究探讨了促卵泡生成素(FSH)对牦牛输卵管上皮细胞分泌特异性糖蛋白的影响。体外分离培养牦牛输卵管上皮细胞,并在细胞中添加不同浓度的FSH,作用6 h后运用荧光定量PCR分析输卵管特异性糖蛋白mRNA的表达水平,并用细胞免疫标记对其分泌输卵管蛋白的部位进行分析。结果显示,FSH的浓度为0.5-5.0μg/m L时,输卵管蛋白的表达量随着FSH浓度的上升而增加,在5.0μg/m L的FSH作用后,输卵管蛋白的mRNA表达量最高;浓度超过10.0μg/m L时,输卵管蛋白基因的表达量降低。结果表明,FSH具有促进输卵管上皮细胞分泌输卵管特异性糖蛋白的作用,且具有剂量依赖性,其最佳作用浓度为5.0μg/m L,输卵管蛋白主要由细胞质分泌。     

Antibodies against the C—terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2—cell stage

INTRODUCTIONUp to date, little is known about the func-tions of oviductins in promoting the developmentof mammalian early embryo. When the fertilizedeggs of mammals are cultured in composition definedmedium in vitro, they are often blocked at certaindevelopmental stage, called "early embryonic devel-opment block". The time of development block ofearly embryo is not consistent in different species.In mouse it happened at 2--cell stage, so called the2--cell block. In essence, the cause of t…     

Immunocytochemical evidence for the transfer of an oviductal antigen to the zona pellucida of hamster ova after ovulation

The zona pellucida (ZP), a glycoprotein layer that encloses the mammalian oocyte, is formed during follicular development in the ovary, persists at the time of fertilization within the oviduct, and then surrounds the embryo until implantation in the uterus. Although the structure and chemical properties of the ZP have been extensively studied, the precise site of origin of the ZP remains a matter of controversy. Moreover, the mechanism of synthesis and secretion of the ZP constituents is not fully elucidated. We have recently developed monoclonal antibodies (MAbs) against oviductal ZP of the golden hamster. We have used one of these MAbs (an immunoglobulin G) and the protein A-gold technique to study the localization of the corresponding antigenic sites, and we report here their distribution in the oviduct and within the cumulus oophorus complex of the superovulated hamster. In the oviductal epithelium, immunolabeling was observed in non-ciliated secretory cells in structures involved in protein secretion. In the cumulus masses collected from the oviduct, the sites of immunoreactivity were localized exclusively in the ZP encompassing the oocyte. Gold particles were evenly distributed throughout the entire thickness of the ZP. Treatment of the cumulus masses with hyaluronidase prior to preparation of isolated oocytes for immunocytochemistry did not affect this uniformity. The ZP of the preovulatory oocytes in ovarian follicles was not labeled. Our study provides immunocytochemical evidence for the secretion of an oviductal antigen that becomes intimately associated with the ZP of the oocytes during their passage through the oviduct.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).     

Immunolocalization of aquaporin 1, 5, and 9 in the female pig reproductive system

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been documented in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs are unknown in the female reproductive system of pigs. In this study, AQP1 immunoreactivity was detected in the capillary endothelium of the ovary. Distinct immunolabeling of capillary endothelium was also observed in the oviduct and uterus. AQP5 was expressed in flattened follicle cells of primordial follicles, granulosa cells of developing ovarian follicles, and muscle cells of the oviduct and uterus. Staining of AQP5 was also observed in the epithelial cells of the oviduct and uterine epithelium. AQP9 immunoreactivity was observed in granulosa cells of developing follicles. AQP9 was also localized in the luminal epithelial cells of the oviduct and uterine epithelia cells. This is, to our knowledge, the first study that shows tissue expression and cellular and subcellular localization of AQPs in the reproductive system of the female pig. Moreover, these results suggest that several subtypes of the AQPs (AQP1, 5, and 9) are involved in regulation of water homeostasis in the reproductive system of gilts.     

Possible expansion of "Window of Implantation" in pseudopregnant mice: time of implantation of embryos at different stages of development transferred into the same recipient

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and maternal environment. It is believed that the growth of transferred embryos of different ages is synchronized during preimplantation development and that such embryos are implanted in the uterus at the same time. To define the time of synchronization for developing embryos of different ages, embryos at two different stages of development were transferred separately into the oviducts of the same recipient. We then examined the subsequent development of the embryos at various time intervals after transfer. Pronucleus (PN) stage eggs were transferred separately to the right or left oviduct of recipients on Day 0, while eight-cell embryos (8C) were transferred to the other oviduct. For 8C, 5%, 63%, and 74% of transferred embryos were implanted in the uterus at 42, 66, and 90 h posttransfer, respectively. In contrast, none of the transferred PN was implanted until 90 h posttransfer. At 90 h posttransfer, 59% of the PN had successfully implanted. Histological examination revealed that developmental stage of the embryos in both groups synchronized around 162 h posttransfer, even though the implantation was accelerated in 8C compared with PN. Our results indicate that embryos of advanced stage transferred to the oviduct implant in the uterus in advance of younger embryos and that the uterine development is synchronized at the neural plate, presomite stage. Our results strongly suggest that uterine receptivity for implantation is expandable in pseudopregnant mice.     

Regional differences in expression of progesterone receptor in oviduct and uterus of rabbit during early pregnancy

We characterized the expression pattern of progesterone receptor (PR) in two regions of the oviduct (ampullae and isthmus), and the uterus (epithelium and stroma) of the rabbit (Oryctolagus cuniculus) during early pregnancy (1-4 days) by RT-PCR and immunohistochemistry. We observed a significant increase in the expression of PR at mRNA level in the uterus on days 1 and 2 of pregnancy, followed by a decrease on days 3 and 4. These changes were also observed at protein level in the uterine epithelium. Interestingly, PR immunoreactivity decreased in stromal cells in all days of pregnancy as compared with non-pregnant rabbits (NG). In the isthmus PR mRNA expression significantly increased on day 2 of pregnancy and diminished on days 3 and 4, whereas no significant changes were observed in the ampullae. In epithelial and stromal cells of the isthmus, PR immunostaining was reduced through pregnancy as compared with NG group. In contrast, a reduction in PR immunostaining was observed on days 1-3 with an increase on day 4 in epithelial and stromal cells of the ampullae. The overall results suggest that PR exhibit a differential expression pattern in the oviduct and the uterus during early pregnancy of the rabbit, and that these differences are related to different functions of PR in the reproductive tract during early pregnancy.     

Histology and functional morphology of the oviduct of an oviparous snake,Diadophis punctatus

Oviductal functional morphology remains poorly understood in oviparous snakes, particularly in regard to oviductal formation of albumen and the eggshell and to sperm storage. The oviduct of Diadophis punctatus was examined using histology and scanning electron microscopy to determine oviductal functional morphology throughout the reproductive cycle. The oviduct is composed of four morphologically distinct regions: infundibulum, uterine tube, uterus, and vagina. The infundibulum is thin, flaccid, and lined with simple ciliated cuboidal epithelial cells. The tube contains ciliated and secretory epithelial cells, which reach a maximum height and hypertrophy during early gravidity and produce glycosaminoglycans. The posterior portion of the tube contains temporary sperm storage receptacles. The uterus retains eggs throughout gestation and secretes the eggshell constituents. The endometrial glands of the uterus hypertrophy during vitellogenesis and become depleted of the secretory granules during gravidity. The functional morphology of the oviduct therefore shows cyclical changes that are correlated with eggshell formation. The vagina consists of thick longitudinal and circular smooth muscle layers, which may serve in retention of eggs during gestation. Furthermore, the vagina contains long furrows in the mucosa that serve as sperm storage receptacles. These receptacles store sperm following fall mating and overwintering, whereas the receptacles in the tube are utilized briefly during vitellogenesis just prior to ovulation. © 1996 Wiley-Liss, Inc.     

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.     

The influence of ovarian hormones on the rat oviductal and uterine concentration of noradrenaline and 5-hydroxytryptamine

This paper describes the effects of estradiol and progesterone on the concenirations of noradrenaline and 5-hydroxytryptamine in the Wistar rat oviduct and uterus. The levels of noradrenaline and 5-hydroxytryptamine are higher in the oviduct than in the uterus whereas p-tyrosine and tryptophan are similar in both tissues. Estradiol treatment reduced the oviductal concentration of noradrenaline but not 5-hydroxytryptamine in oviduct, while the concentrations of both noradrenaline and 5-hydroxytryptamine were reduced in uterine horn. The levels of noradrenaline in the oviduct and uterus in rats in estrus were lower than those of diestrous rats. Bilateral ovariectomy produced an increase in uterine noradrenaline and 5-hydroxytryptamine levels. These changes were reversed in the presence of ovarian hormones as indicated by experiments where unilateral ovariectomy was performed. Reserpine administration reduced noradrenaline concentration in both the oviduct and the uterus but did not change oviductal or uterine 5-hydroxytryptamine.These results indicate the existence of noradrenaline within postganglionic sympathetic nerve terminals and suggest that estrogens increase the utilization and the synthesis of noradrenaline in both the oviducts and the uterine horns. With respect to 5-hydroxytryptamine the data support the concept that it is mainly associated with mast cells.