The role of the oviduct and extracellular vesicles during early embryo development in bovine

The oviduct is an important reproductive structure that connects the ovary to the uterus and takes place to important events such as oocyte final maturation, fertilization and early embryonic development. Thus, gametes and embryo can be directly influenced by the oviductal microenvironment composed by epithelial cells such secretory and ciliated cells and oviductal fluid. The oviduct composition is anatomically dynamic and is under ovarian hormones control. The oviductal fluid provides protection, nourishment and transport to gametes and embryo and allows interaction to oviductal epithelial cells. All these functions together allows the oviduct to provides the ideal environment to the early reproductive events. Extracellular vesicles (EVs) are biological nanoparticles that mediates cell communication and are present at oviductal fluid and plays an important role in gametes/embryo - oviductal cells communication. This review will present the ability of the oviducts based on its dynamic and systemic changes during reproductive events, as well as the contribution of EVs in this process.     

Gametes alter the oviductal secretory proteome

The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes.     

Inhalation of mainstream and sidestream cigarette smoke retards embryo transport and slows muscle contraction in oviducts of hamsters (Mesocricetus auratus).

Prior experiments have shown that the functioning of hamster oviducts is impaired by in vitro exposure to cigarette smoke. To determine if cigarette smoke affects oviductal functioning in vivo, an inhalation experiment was done in which hamsters were exposed to doses of smoke similar to those received by human smokers. The effects of mainstream smoke (the bolus of smoke inhaled by active smokers) and sidestream smoke (the main component in environmental tobacco smoke) were compared. Transport of preimplantation embryos through the hamster oviduct was retarded in females inhaling doses of mainstream or sidestream smoke that produced serum cotinine levels within the range reported for women who actively or passively smoke during pregnancy. In addition, hamster oviductal muscle contraction rate decreased significantly during a single exposure of animals to either mainstream or sidestream smoke, and contraction rate failed to return to initial control values during a 25-min recovery period. Both preimplantation embryo transport and muscle contraction were more sensitive to sidestream than mainstream smoke. These data demonstrate that inhalation of doses of mainstream and sidestream cigarette similar to those received by active and passive human smokers adversely affects functioning of the oviduct and may explain the increased incidence of ectopic pregnancies reported in women who smoke.     

Oviductal cell proteome alterations during the reproductive cycle in pigs

The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. During the reproductive cycle, the reproductive system undergoes various changes, including alterations in the number of different cell types in the oviductal epithelium and changes in the height of oviductal cells. Maintaining the unique oviductal environment required for the fertilization and early embryonic development comes with an energy cost to the organism. Therefore, it is hypothesized that structural and functional changes to the oviduct during the reproductive cycle represent vital preparations for the development of suitable environments for conception and embryo support. Here, we aimed to identify the changes in protein expression profile that occur during the follicular and luteal stages of the reproductive cycle in oviductal epithelial cells. The porcine oviductal epithelial cell proteomes from the follicular and luteal stages of the reproductive cycle were contrasted after separation by 2-D gel electrophoresis. Several oviductal epithelial cell proteins were up- or down-regulated during the reproductive cycle. We checked the quantitative changes of two of these molecules during different stages of the reproductive cycle using Western blot analysis. Finally, a number of these proteins were identified using tandem mass spectrometry. The results demonstrated distinctive differences in the proteomic profiles of the oviduct between follicular and luteal phases of the reproductive cycle.     

Anandamide Levels Fluctuate in the Bovine Oviduct during the Oestrous Cycle

Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA) regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

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.     

Neural and hormonal control of muscular activity of the spermatheca in the locust, Locusta migratoria

The spermatheca in insects is a tubular structure within the female that acts as a repository for spermatozoa deposited by the male during copulation. The spermatozoa remain viable within the spermatheca for extended periods of time, and are then delivered to the site of fertilization during oviposition (egg-laying). Thus, the production of viable offspring is dependent upon the coordination of events associated with fertilization, including the passage of the egg through the lateral and common oviducts and the passage of spermatozoa along the spermathecal duct. The egg and the spermatozoa are propelled along their respective tracts by contractions of the visceral muscles intrinsic to the oviduct and spermatheca. The neural and hormonal control of muscular activity of the locust oviducts has been well reviewed, with more recent studies examining the control over the spermatheca. This review highlights more recent literature, including new data, for neural and hormonal control of muscular activity of the spermatheca of the locust, Locusta migratoria, making reference to examples in other insects where relevant. A variety of neuronal types project to the spermatheca in L. migratoria, and a variety of neuroactive chemicals, including neuropeptides and amines, influence contraction. A comparison is made between the control of oviducts and spermatheca in L. migratoria with regard to their neural substrate and the composition of neuroactive chemicals.     

LEFTY2 expression and localization in rat oviduct during early pregnancy

In mammals, fertilization and preimplantation embryo development occurs in the oviduct. Cross-talk between the developing embryos and the maternal reproductive tract has been described in such a way as to show that the embryos modulate the physiology and gene expression of the oviduct. Different studies have indicated that transforming growth factor beta (TGF-β) can modulate the oviductal microenvironment and act as an autocrine/paracrine factor on embryo development. LEFTY2, a novel member of the TGF-β superfamily is involved in the negative regulation of other cytokines in this family such as nodal, activin, BMPs, TGF-β1 and Vg1. In previous studies, we have reported that LEFTY2 is differentially expressed in the rat oviduct during pregnancy. In this study, we describe the temporal pattern of LEFTY2 in pregnant and non-pregnant rat oviduct by western blotting, which showed higher levels of LEFTY2 on day 4 of pregnancy, a time at which the embryos are ending their journey along the oviduct. The cellular location of LEFTY2 was assessed by immunohistochemistry, which showed immunolabelling in the cytoplasm and at the apical surface of the oviductal epithelial cells. The oviductal fluid also presented a 26 kDa band, which corresponds to the biologically active form of this protein, at the preimplantation period of pregnancy, indicating LEFTY2 secretion to the lumen. As LEFTY2 is expressed at a high level just before the embryos pass to the uterus, its biological effect might be relevant and significant for the preimplantation stage of embryo development in the oviduct. The fact that embryos do not express LEFTY2 at this stage of development supports this hypothesis.     

Effect of steroids and oviductal cells, from the different parts of the oviduct, on the incidence of monospermy in porcine in vitro fertilization

A high incidence of polyspermy occurs in porcine in vitro fertilization. It is also known that in vivo, the oviductal cells and their secretions play an important role in fertilization and early development. Vesicles from oviductal cells from different parts of the oviduct (isthmus or ampulla) pretreated with estradiol or progesterone or ethanol were used to assess their role in the fertilization process. Oviductal cells were co-cultured with 0.5 million motile sperm/ml for 30 min. A 10-microl sample (spermatozoa bound with the cells) was added to 40-microl droplets of fertilization medium containing 5 oocytes. After 15 to 18 h, oocytes were examined for penetration and monospermy. The results show a lower penetration rate with oviductal cells than that of the control. The use of oviductal cells from the isthmus treated with estradiol significantly decreased the percentage of polyspermy compared with that of ampulla treated with the estradiol or with the control. When the isthmus cells were treated with progesterone, an increase in the incidence of polyspermy was observed. Therefore, it is possible to use oviductal cells to increase the incidence of monospermy in porcine in vitro fertilization; moreover, estradiol increases the proportion of monospermy when added to isthmus-derived oviductal cells.     

Global profiling of surface plasma membrane proteome of oviductal epithelial cells

In mammalian reproduction, many important events occur within the female reproductive tract, especially within the oviduct. These include transport and final maturation of the female and male gametes, fertilization, embryonic development, and transport of the embryo to the uterus. The plasma membrane molecules of oviductal epithelia that are in direct contact with gametes and embryo(s) and potentially mediate these processes are poorly characterized, and their function is poorly understood. Defining the oviductal cell surface proteome could provide a better understanding of the basis of reproductive processes taking place within the oviduct. We aimed to provide a detailed profile of the surface plasma membrane proteome of the oviductal epithelium by biotinylation of proteins at the cell surface, followed by highly specific purification of these proteins using avidin. This approach for enrichment of oviductal cell surface proteome was validated by immunohistochemistry, gel electrophoresis, and western blot analysis experiments. The enriched molecules were identified using two different technologies: (i) the combination of 2D gel electrophoresis with mass spectrometry and (ii) 1D gel electrophoresis with mass spectrometry (a modified multidimensional protein identification technology (MudPIT) technique). The number of proteins identified using the MudPIT approach was approximately 7 times the number of proteins identified by 2D gel electrophoresis using the same samples (40 versus 276, respectively). Some of the proteins found at the surface of oviductal cells had previously been reported as present in the oviduct and to have known functions in relation to reproductive processes. The other category of proteins that were highly represented in the oviductal surface proteome were various members of the family of heat-shock proteins. To the best of our knowledge, this is the first comprehensive study to identify and characterize proteins at the surface of the epithelium of the mammalian oviduct.     

Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 μM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 μM L-NAME, a NO synthase inhibitor, or 30 μg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.     

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.     

Pyrazine derivatives in cigarette smoke inhibit hamster oviductal functioning

Background  

Our past studies have shown that cigarette smoke inhibits oviductal functioning in vivo and in vitro. The goals in this study were to identify pyrazine derivatives in cigarette smoke solutions that inhibit ciliary beat frequency, oocyte pickup rate, and infundibular smooth muscle contraction in the hamster oviduct and to determine their lowest observable adverse effect levels (LOAELs) using in vitro bioassays.     

Effect of human oviductal epithelial cell cultural medium on cryopreserved human sperm survival

The human oviduct is known as a functional site for gamete transportation, retention, fertilization and zygote development. Previous studies have shown that human oviductal epithelial cell cultural medium (OECCM) has a positive effect on prolongation of sperm motility for some cryopreserved human sperm without cryodamage. However, for most cryopreserved sperm, OECCM could not improve their survival prolongation. In this study, we assessed the influence of human OECCM on the motility longevity of cryopreserved human sperm with an in vitro incubation method.     

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.     

In vitro-cultured bovine oviductal cells bind acrosome-intact sperm and retain this ability upon sperm release

The mammalian oviduct plays a key role in sperm storage, capacitation, and selection. Specific oviduct secretions and/or binding to oviductal cells are thought to be responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm-oviductal cell adhesion and release in the bovine species. Distribution and acrosomal status of sperm bound to in vitro-cultured ampullary and isthmic cell monolayers were followed until the time of sperm release by means of fluorescence labeling techniques. In order to understand whether release is due to surface changes of sperm or oviductal cells, double incubation experiments with unlabeled and Hoechst-labeled sperm have been performed. Main findings demonstrate that (1) only acrosome-intact sperm bind specific bovine oviductal epithelial cells; (2) acrosomes of bound sperm are preserved intact over time; and (3) release of unreacted sperm is likely to be due to changes of the sperm surface, probably triggered by capacitation. These findings support the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.     

The effect of cigarette smoke on fertilization and pre-implantation development: assessment using animal models, clinical data, and stem cells

Numerous studies have repeatedly shown that women who smoke experience problems establishing and maintaining pregnancies, and recent work has further demonstrated that the in utero effects of smoke may not be manifested until months or even years after birth. The purpose of this review is to examine the recent literature dealing with the effects of cigarette smoke on the earliest stages of human prenatal development. Studies in this area have included the use of animal models, patients undergoing in vitro fertilization, and embryonic stem cell models. Events leading to fertilization, such as cumulus expansion, hyperactivation of sperm motility, and oocyte pick-up by the oviduct are all impaired by smoke exposure in animal models. Steps crucial to fertilization such as the acrosome reaction and sperm binding to the zona pellucida are likewise inhibited by cigarette smoke. Preimplantation embryos and stem cells that model embryos show a number of adverse responses to smoke exposure, including poor adhesion to extracellular matrices, diminished survival and proliferation, and increased apoptosis. The current literature demonstrates that the earliest stages of prenatal development are sensitive to smoke exposure and indicates that pregnant women should be advised not to smoke during this time.     

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.     

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.