Antioxidant defenses are modulated in the cow oviduct during the estrous cycle

The balanced presence of reactive oxygen species and antioxidants has a positive impact on sperm functions, oocyte maturation, fertilization, and embryo development in vitro. The mammalian oviduct is likely to provide an optimal environment for final gamete maturation, sperm-egg fusion, and early embryonic development. However, the expression and distribution of antioxidant enzymes in the bovine oviduct are poorly characterized. We analyzed the mRNA expression and enzymatic activities of major antioxidants glutathione peroxidase (GPx), superoxide dismutase (Cu,ZnSOD), and catalase in the bovine oviduct throughout the estrous cycle. The high levels of expression for GPx-3 in the isthmus were in contrast to expression of GPx-1 and GPx-2, which occurred mostly in the ampulla and infundibulum of the oviduct. The highest levels of mRNA expression for GPx-1 were observed toward the end of the estrous cycle before ovulation, whereas GPx-2 was mostly expressed at midcycle. Catalase and Cu,ZnSOD mRNA analyses revealed a homogenous expression along the oviduct. The highest levels of glutathione and enzymatic activities for GPx and catalase occurred at the middle (10-12 days) and end (18-20 days) of the estrous cycle, whereas total SOD activity remained constant throughout the estrous cycle in the oviductal fluids. These findings underscore the importance of hydrogen peroxide and hydroperoxide removal by GPx in the oviduct. The heterogeneous expression of antioxidants such as GPx along the oviduct is a possible indication of their physiological role in the events leading to successful fertilization and implantation in vivo.     

GRP78 expression and immunohistochemical localization in the female reproductive tract of mice

The 78-kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum chaperone, with multiple functional roles in protein processing and provision of cellular protection. However, the physiological role of GRP78 in embryo development is not clear. Localization of GRP78 and expression of its mRNA in the reproductive organs throughout the estrous cycle in mice were investigated by immunohistochemistry and real-time polymerase chain reaction, respectively. Whereas there was intense staining for GRP78 in the oviduct at estrus, the ciliated cells of isthmus had better staining than those of infundibulum and ampulla at all phases of the cycle. Furthermore, GRP78 was located in the uterine luminal and glandular epithelial cells throughout the estrous cycle, particularly during the estrus phase. However, levels of GRP78 mRNA in the oviduct and uterus varied during the cycle, with peaks at estrus. In conclusion, GRP78 expression varied with the phase of the murine estrous cycle; this might be related to gamete transport, fertilization and early development of the zygote/embryo.     

Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro

Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development.     

Fluctuation of Aquaporin 1, 5, and 9 Expression in the Pig Oviduct during the Estrous Cycle and Early Pregnancy

Thirteen mammalian aquaporin (AQPs) isoforms with a unique tissue-specific pattern of expression have been identified. To date, 11 isoforms of AQP have been reported to be expressed in female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig oviduct during different stages of the estrous cycle and early pregnancy. The results demonstrated that AQP1, 5, and 9 were clearly detected in all studied stages of the estrous cycle and pregnancy. AQP1 was localized within oviductal blood vessels. In cyclic gilts, the expression of AQP1 protein did not change significantly between days 10–12 and 14–16 but increased on days 2–4 and 18–20. AQP5 was localized in smooth muscle cells and oviductal epithelial cells. The expression of AQP5 protein did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. The anti-AQP9 antibody labeled epithelial cells of the oviduct. The expression of AQP9 did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. In pregnant gilts, expression of AQP1, 5, and 9 did not change significantly in comparison with the estrous cycle. Therefore, a functional and distinctive collaboration seems to exist among diverse AQPs in water handling during the different oviductal phases in the estrous cycle and early pregnancy.     

Regulation of the immunoexpression of aquaporin 9 by ovarian hormones in the rat oviductal epithelium

The volume of oviductal fluid fluctuates during the estrous cycle, suggesting that water availability is under hormonal control. It has been postulated that sex-steroid hormones may regulate aquaporin (AQP) channels involved in water movement across cell membranes. Using a functional assay (oocytes of Xenopus laevis), we demonstrated that the rat oviductal epithelium contains mRNAs coding for water channels, and we identified by RT-PCR the mRNAs for AQP5, -8, and -9, but not for AQP2 and -3. The immunoreactivity for AQP5, -8, and -9 was localized only in epithelial cells of the oviduct. The distribution of AQP5 and -8 was mainly cytoplasmic, whereas we confirmed, by confocal microscopy, that AQP9 localized to the apical plasma membrane. Staining of AQP5, -8, and -9 was lost after ovariectomy, and only AQP9 immunoreactivity was restored after estradiol and/or progesterone treatments. The recovery of AQP9 reactivity after ovariectomy correlated with increased mRNA and protein levels after treatment with estradiol alone or progesterone administration after estradiol priming. Interestingly, progesterone administration after progesterone priming also induced AQP9 expression but without a change in mRNA levels. Levels of AQP9 varied along the estrous cycle with their highest levels during proestrus and estrus. These results indicate that steroid hormones regulate AQP9 expression at the mRNA and protein level and that other ovarian signals are involved in the expression of AQP5 and -8. Thus hormonal regulation of the type and quantity of water channels in this epithelium might control water transport in the oviductal lumen. water channels; epithelial cells; estradiol; progesterone     

The concentrations of catecholamines and oxytocin receptors in the oviduct and its contractile activity in cows during the estrous cycle

In vitro experiments on oviducts of cyclic cows were undertaken to study: (1) the content of dopamine (DA), noradrenaline (NA) and adrenaline (A) in infundibulum, ampulla and isthmus, (2) the concentration of oxytocin receptors (OTR) in oviductal tissues and (3) the motility of ampulla and isthmus. Changes of DA content were observed in the infundibulum and the ampulla with maximal values occurring on Days 6-10 of the estrous cycle. The mean NA content was greatest in infundibulum相似文献   

Expression of nuclear progesterone receptor and progesterone receptor membrane components 1 and 2 in the oviduct of cyclic and pregnant cows during the post-ovulation period

ABSTRACT: BACKGROUND: Progesterone (P4) may modulate oviductal functions to promote early embryo development in cattle. In addition to its nuclear receptor (PR), P4 may mediate its actions through P4 receptor membrane component 1 (PGRMC1) and its relative, PGRMC2. Two successive experiments were undertaken to characterise the expression of PR, PGRMC1 and PGRMC2 in the bovine oviduct during the post-ovulation period, and to relate their expression to the presence of an embryo, the proximity of the CL and to the region of the oviduct. METHODS: In the first experiment (Exp. I), whole oviduct sections were collected from Holstein cows at Day 1.5, Day 4 and Day 5 post-ovulation (n = 2 cows per stage). The expression of PR, PGRMC1 and PGRMC2 was studied in the ampulla and isthmus by RT-PCR, western-blot and immunohistochemistry. In Exp. II, oviduct epithelial cells were collected from cyclic and pregnant Charolais cows (n = 4 cows per status) at Day 3.5 post-ovulation and mRNA expression of PR, PGRMC1 and PGRMC2 was examined in the ampulla and isthmus by real-time quantitative PCR. RESULTS: In Exp. I, PR, PGRMC1 and PGRMC2 were expressed in all oviduct samples. PGRMC1 was mainly localised in the luminal epithelium whereas PR and PGRMC2 were localised in the epithelium as well as in the muscle and stroma layers of the oviduct. The expression was primarily nuclear for PR, primarily cytoplasmic for PGRMC1 and both nuclear and cytoplasmic for PGRMC2. In Exp. II, mRNA levels for PR, PGRMC1 and PGRMC2 were not affected by either the pregnancy status or the side relative to the CL. However, the expression of PR and PGRMC2 varied significantly with the region of the oviduct: PR was more highly expressed in the isthmus whereas PGRMC2 was more highly expressed in the ampulla. CONCLUSIONS: This is the first evidence of PGRMC2 expression in the bovine oviduct. Our findings suggest that P4 regulates the functions of the bovine oviduct in a region-specific manner and through both classical and non classical pathways during the post-ovulation period.     

Immunohistochemical demonstration of S-100 protein in epithelial cells of bovine oviduct

The present study deals with an immunohistochemical localization of S-100 protein in the bovine oviduct. The epithelium of the infundibulum, ampulla and isthmus showed a positive staining for S-100 protein. The immunoreactivity for S-100 was observed both in the ciliated and nonciliated (secretory) cells of the oviductal epithelium at any stages of the estrous cycle. The immunoreactivity was also found in nervous elements and endothelial cells of blood vessels. No cell outside these cells showed any immunoreactivity for S-100. Although the functional significance of S-100 protein in the oviductal epithelium remains to be elucidated, the present results introduce new perspectives into the investigation of function and localization of S-100 protein.     

Monosaccharides are not detected in whole or isthmic bovine oviductal fluid collected throughout the estrous cycle, as analyzed by HPLC

In the bovine oviduct, monosaccharides may play a role in the preparation of gametes for fertilization. Sperm are sequestered in the isthmic region of the oviduct where capacitation, requisite biochemical changes in sperm membranes, may take place. Retention of spermatozoa in the oviductal isthmus is dependent on a carbohydrate recognition system between oviductal epithelium and sperm membrane lectins. The monosaccharide, fucose, has been found to be important to this recognition system. However, both gametes and epithelium are also bathed in oviductal fluid (ODF), and fucose or other monosaccharides may be constituents of ODF and so may be important to sperm binding to oviductal epithelium and subsequent preparation for fertilization. In this study, ODF from dairy cows was analyzed by HPLC for the presence of 5 monosaccharides (fucose, galactose, glucosamine, mannose and xylose). Both whole ODF, collected by cannulation of the entire oviduct of 1 cow over a complete estrous cycle, and regional staged ODF, collected and pooled from 13 cows from the isthmic region only at estrus, were analyzed. We report negligible concentrations of all 5 monosaccharides in both types of ODF analyzed. Because the detection limit of our assay was 10(8) times lower than fucose concentrations found to be physiologically important in earlier in vitro studies, we conclude that bovine ODF does not contain physiologically active levels of free fucose or other, similar monosaccharides at any time of the estrous cycle.     

Origin of oestrus-associated glycoproteins in bovine oviductal fluid.

The aim of the present study was to determine whether the synthesis of an oestrus-associated protein found in bovine oviductal fluid varies with oviductal region, stage of cycle or day of pregnancy. Explant culture was performed using oviducts recovered from naturally cycling animals either at oestrus or 12-14 days after oestrus. Three oviductal regions, the preampulla, ampulla and isthmus, were cultured individually in the presence of 20 muCi [35S]methionine in serum-free medium for 6 h at 37 degrees C. Synthesis of oestrus-associated protein was assessed by one-dimensional SDS-PAGE, fluorography and densitometry of radiolabelled bands. Significantly more oestrus-associated protein was synthesized by the ampullar region of the oviduct, although it was detected in explant culture media from both the isthmic and preampullar regions. A polyclonal antibody produced against oestrus-associated protein was used to localize the protein in paraffin-embedded sections of oviductal explant cultures and other bovine tissues. Localization of the protein in oviductal tissue sections varied with stage of cycle (oestrus > luteal > pregnant) and region of oviduct (ampulla > preampulla/isthmus). These findings indicate an effect of oviductal region and hormonal state (cycling versus pregnant) on the synthesis and secretion of the oestrus-associated protein. Lectin affinity studies indicated that galactosyl(beta 1,3)N-acetylgalactosamine and N-acetylglucosamine residues are associated with the oestrus-associated protein.     

The polyol pathway in the bovine oviduct

The oviducts likely provide optimized micro‐environments for the final maturation of gametes, fertilization, and early embryo development. Hexoses, including glucose, fructose, and sorbitol, are involved in these critical reproductive events. Monosaccharide production is controlled, in part, by the polyol pathway and requires two enzymes: an aldose reductase (AR) that reduces glucose into sorbitol, followed by its oxidation into fructose by sorbitol dehydrogenase (SDH). We analyzed the expression of AR and SDH in the isthmus and ampulla of the bovine oviduct at the proliferative, mid‐luteal, and late‐luteal phases of the estrous cycle by quantitative PCR and immunoblots. Immunochemistry and an assay of SDH activity were also performed. The quantity of hexoses in whole sections of isthmus and ampulla were determined by liquid chromatography coupled to mass spectrometry. In sum, AR expression was restricted to the isthmus, while SDH was mostly expressed in the isthmic–ampullary junction and the ampulla, specifically concentrated in the luminal epithelium of the oviduct. The estrous cycle had no impact on protein expression of AR and SDH. Instead, the levels of AR and SDH expression were associated with higher ratios of sorbitol to fructose in the isthmus (1.6) than in the ampulla (4.1; P = 0.005). These results are discussed in light of physiological events occurring in the oviduct. Mol. Reprod. Dev. 79: 603–612, 2012. © 2012 Wiley Periodicals, Inc.     

Fluctuations of lactoferrin protein and messenger ribonucleic acid in the reproductive tract of the mouse during the estrous cycle.

The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.     

Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig

Recent identification of plasminogen activator inhibitor-1 (PAI-1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy. To examine PAI-1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured. Culture media was collected and de novo synthesized PAI-1 analyzed by 2D-SDS-PAGE, fluorography, and densitometry. To determine hormonal regulation of PAI-1 synthesis and secretion, four groups of ovariectomized (OVX) cross-bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured. Steady-state mRNA levels of PAI-1 were evaluated throughout the estrous cycle in cross-bred gilts. To compare steady-state PAI-1 mRNA levels between cyclic and pregnant cross-bred gilts, tissue was collected on days 0, 2, and 12. Quantitative analysis of steady-state levels of PAI-1 mRNA were analyzed by dot-blot hybridization and densitometry. A greater (P < 0.01) synthesis and secretion of PAI-1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed. No difference could be detected for PAI-1 protein between breeds. The Large White had a greater (P < 0.05) secretion of PAI-1 on day 2 of early pregnancy relative to other days examined. Whole oviductal tissue from cross-bred gilts was found to have a significantly greater amount of PAI-1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12. A significant effect of day and segment was detected for levels of PAI-1 mRNA from cyclic and early pregnant cross-bred gilts. PAI-1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy. An interaction was detected for estrogen and progesterone on PAI-1 mRNA (P < 0.05) and protein (P = 0.09). Estrogen was found to inhibit PAI-1 protein synthesis and also inhibited progesterone-mediated stimulation of PAI-1 mRNA. Our results demonstrate expression of PAI-1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage-stage embryo.     

Detection of Fas ligand in the bovine oviduct

Presence of a Fas-Fas ligand (FasL) system defines the immune-privileged status of certain tissues such as placenta. This study examined the fluids and tissue(s) of the bovine oviduct, where both spermatozoa and early embryos escape elimination by the female immune system, for the presence and the distribution of Fas and FasL, which might provide an explanation for the immune-privileged site of this organ. In the present study, the immunolocalisation of FasL and Fas, as well as the gene expression of FasL, were determined in the uterotubal junction (UTJ), isthmic (I) and ampullar (A) segments of the oviduct during oestrus and the luteal phase of the oestrous cycle. The degree of apoptosis of oviductal epithelium was examined by the TUNEL method. Oviductal fluid (ODF), collected chronically via indwelling catheters from the I or A segments during both non-luteal and luteal phases of the cycle, was analysed for the presence of FasL. The Fas immunostaining was scattered along the epithelium of all regions of the oviduct and cycle stages investigated, whereas FasL immunolabelling was more conspicuous in oestrous samples. This staining disappeared during the luteal phase, which was particularly evident in the sperm reservoir (UTJ and I). There were fewer TUNEL-positive cells than Fas- or FasL-positive cells in the oviductal epithelium, suggesting that tubal Fas and FasL are not directly involved in epithelial apoptosis. Western blot analyses detected FasL in ODF collected from both I and A, most conspicuously as a 24-27kDa band but also at a 40-45kDa band level. FasL mRNA was expressed in the epithelial cells from the sperm reservoir and A during both non-luteal and luteal phases. However, the level of expression differed significantly between segments during the luteal phase. The results provide novel evidence that the Fas-FasL system is present in the bovine oviduct and could be involved in mediating survival of spermatozoa and early embryos.     

Expression of superoxide dismutases in the bovine oviduct during the estrous cycle

The superoxide dismutases (SODs) are first-line enzymatic antioxidants that dismute superoxide anion (O(2)(-)) to produce hydrogen peroxide (H(2)O(2)). The primary objective was to characterize, by western blot analysis, the expression of two SODs, the cytosolic (Cu,ZnSOD or SOD1) and the mitochondrial (MnSOD or SOD2) forms in three sections of the oviduct, i.e. isthmus (I), ishtmic-ampullary junction (IA), and ampulla (A), during the estrous cycle. The Cu,ZnSOD and MnSOD proteins were mostly expressed in the ampulla (I相似文献   

Sulphated glycosaminoglycans (S-GAGs) and syndecans in the bovine oviduct

In vivo, bull sperm capacitation seems to occur mainly in the oviduct. Capacitation of bull spermatozoa can be triggered in vitro by exposure to heparin, a heavily sulphated glycosaminoglycan (S-GAG). We determined the concentration of S-GAGs in oviductal fluid from dairy heifers, collected over the course of several oestrous cycles via surgically implanted intraluminal catheters. We also investigated the presence of syndecans, i.e. heparan sulphate proteoglycans, in the bovine oviductal epithelium of Swedish dairy cattle during standing oestrus and the luteal phase of the oestrous cycle, using immunohistochemistry for three different polyclonal antibodies raised against human syndecan-2 and rat syndecan-1 and syndecan-2, respectively. The concentration of S-GAGs in oviductal fluid obtained from the ampullar segment of the oviduct was significantly higher (P=0.0026) than it was in fluid from the isthmic segment during the functional period, i.e. from prooestrus to metaoestrus (73.5+/-10.49 mg/L in ampullar ODF, compared to 43.2+/-10.74 mg/L in isthmic ODF); least square mean (L.S.M.)+/-standard error of the mean (S.E.M.). There was also a significantly higher concentration of S-GAGs in the fluid from the oviduct ipsilateral to the ovulation side 73.5+/-10.54 mg/L on the ovulation side, compared to 43.1+/-10.71 mg/L in the oviduct on the contralateral side (L.S.M.+/-S.E.M., P=0.0026) during this period. Both syndecan-1 and syndecan-2 were present in the epithelial cells lining all studied segments of the bovine oviduct, i.e. the UTJ, isthmus and ampulla, during both standing oestrus and dioestrus. The syndecans and S-GAGs found may influence the gametes, while they reside in the oviduct; the amounts of S-GAGs found in the bovine oviduct seem sufficient to act as capacitating factors in vivo.     

Expression of prostaglandin E synthases in the bovine oviduct

The oviduct is a specialized organ responsible for the storage and the transport of male and female gametes. It also provides an optimal environment for final gamete maturation, fertilization, and early embryo development. Prostaglandin (PG) E₂ is involved in many female reproductive functions, including ovulation, fertilization, implantation, and parturition. However, the control of its synthesis in the oviduct is not fully understood. Cyclooxygenases (COXs) are involved in the first step of the transformation of arachidonic acid to PGH_2. The prostaglandin E synthases (PGESs) constitute a family of enzymes that catalyze the conversion of PGH₂ to PGE₂, the terminal step in the formation of this bioactive prostaglandin. Quantitative real-time PCR was used to determine the expression of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), and cytosolic prostaglandin E synthase (cPGES) mRNA in various sections of the oviduct, both ipsilateral and contralateral (to the ovary on which ovulation occurred) at various stages of the estrous cycle. Furthermore, protein expression and localization of cPGES, mPGES-1, and mPGES-2 were determined by Western blot and immunohistochemistry. All three PGESs were detected at both mRNA and protein levels in the oviduct. These PGESs were mostly concentrated in the oviductal epithelial layer and primarily expressed in the ampulla section of the oviduct and to a lesser extent in the isthmus and the isthmic-ampullary junction. The mPGES-1 protein was highly expressed in the contralateral oviduct, which contrasted with mPGES-2 mostly expressed in the ipsilateral oviduct. This is apparently the first report documenting that the three PGESs involved in PGE₂ production were present in the Bos taurus oviduct.