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.     

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.     

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.     

First evidence of the interaction between deleted in malignant brain tumor 1 and galectin-3 in the mammalian oviduct

The oviduct supports the transport and final maturation of gametes, and harbors fertilization and early embryo development. The oviductal epithelium is responsible for providing the correct environment for these processes. Deleted in malignant brain tumor 1 (DMBT1) is expressed by multiple organisms and several cell types, and the interaction of the rabbit ortholog of DMBT1 with galectin-3 (gal-3) modulates the polarity of epithelial cells. This interaction has not yet been shown in locations other than rabbit kidney and human-cultured endothelial cells. DMBT1 and gal-3 also protect epithelial layers from pathogens and trauma, and are innate immunity components. DMBT1 has been detected in the porcine oviduct, and gal-3 has been reported in the Fallopian tube and in the cow oviduct. Interaction between both proteins would show a probable physiological function in the female reproductive tract. This work describes the presence and co-localization of DMBT1 and gal-3 mainly in the apical region of the epithelial cells of the Fallopian tube and the porcine oviduct, and co-immunoprecipitation in membrane-enriched epithelial cell extracts from the porcine oviduct. The findings strongly support a functional interaction in the mammalian oviduct, suggestive of a role on epithelial protection and homeostasis, which might be related to epithelium–gamete interaction.     

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.     

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.     

Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos

Previously, we identified a 215 kd glycoprotein, GP215, which is associated with postovulatory oocytes and embryos, but not with preovulatory oocytes (Kapur and Johnson, '85). In this paper a polyclonal antibody that specifically recognizes GP215 has been used to study the distribution of the molecule in association with ova and preimplantation embryos and in the female reproductive tract. GP215 is present in epithelial cells lining the cranial portions of the oviduct and in oviductal fluid, ovarian bursal fluid, and medium conditioned by oviductal tissue in vitro. Immunofluorescence assays of the ovum and early embryo show that GP215 is sequestered in the perivitelline space. Since preovulatory oocytes exposed to bursal fluid in vitro acquire GP215, we hypothesize that GP215 is synthesized and secreted by the oviductal epithelium and secondarily associates with the ovulated oocyte. Sequestration of GP215 within the perivitelline space is relatively specific since mouse serum albumin, a major constituent of oviductal fluid, and other high molecular weight proteins are not similarly retained. These observations indicate that the composition of the perivitelline space may be significantly different from the greater environment external to the zona pellucida such that fertilization and early development of mammalian ova potentially take place in a distinct perivitelline microenvironment.     

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.     

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre‐Fertilization Processes by Quantitative Proteomics

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable‐isotope dimethyl labeling prior to nanoLC‐MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N‐glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.     

Smoking and reproduction: The oviduct as a target of cigarette smoke

The oviduct is an exquisitely designed organ that functions in picking-up ovulated oocytes, transporting gametes in opposite directions to the site of fertilization, providing a suitable environment for fertilization and early development, and transporting preimplantation embryos to the uterus. A variety of biological processes can be studied in oviducts making them an excellent model for toxicological studies. This review considers the role of the oviduct in oocyte pick-up and embryo transport and the evidence that chemicals in both mainstream and sidestream cigarette smoke impair these oviductal functions. Epidemiological data have repeatedly shown that women who smoke are at increased risk for a variety of reproductive problems, including ectopic pregnancy, delay to conception, and infertility. In vivo and in vitro studies indicate the oviduct is targeted by smoke components in a manner that could explain some of the epidemiological data. Comparisons between the toxicity of smoke from different types of cigarettes, including harm reduction cigarettes, are discussed, and the chemicals in smoke that impair oviductal functioning are reviewed.     

A dataset of human cornea proteins identified by Peptide mass fingerprinting and tandem mass spectrometry

Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.     

Modulation of the oviductal environment by gametes

The notion of a gamete recognition system that alerts females to the presence of gametes in their reproductive tract profoundly influences our understanding of the physiology of events leading to conception and the bearing of offspring. Here, we show that the female responds to gametes within her tract by modulating the environment in which pregnancy is initially established. We found distinct alterations in oviductal gene expression as a result of sperm and oocyte arrival in the oviduct, which led directly to distinct alterations to the composition of oviductal fluid in vivo. This suggests that either gamete activates a cell-type-specific signal transduction pathway within the oviduct. This gamete recognition system presents a mechanism for immediate and local control of the oviductal microenvironment in which sperm transport, sperm binding and release, capacitation, transport of oocytes, fertilization, and early cleavage-stage embryonic development occur. This may explain the mechanisms involved in postcopulatory sexual selection, where there is evidence suggesting that the female reproductive tract can bias spermatozoa from different males in the favour of the more biologically attractive male. In addition, the presence of a gamete recognition system explains the oviduct's ability to tolerate spermatozoa while remaining intolerant to pathogens.     

Glycobiology of fertilization in the pig

By adopting internal fertilization, the meeting of both gametes - the sperm and the egg - and thus the highly coordinated sequence of interactions leading to fertilization, occur in the female reproductive tract. In mammals, the oviduct has been shown to translate the requirements of the female, coordinating sperm activation (capacitation) and sperm transport with the arrival of the ovulated egg. A hierarchy of carbohydrate-based interactions accompanies these events ranging from the binding of uncapacitated sperm to the oviductal epithelium (establishment of the female sperm reservoir), to the primary and secondary binding processes contributing to gamete recognition and sperm penetration of the oocyte zona pellucida. The current perspective will focus on the carbohydrate-recognition systems in the binding events during fertilization in the pig. The roles of the major carbohydrate-binding proteins, the spermadhesins and the acrosomal serine proteinase, pro/acrosin are discussed under consideration of recent structural data. The glycans and the glycoproteins of the porcine oviduct with a focus on the candidate sperm receptors as well as the zona pellucida N-glycans of prepuberal pigs have been characterized by a mass spectrometric approach. Furthermore, some preliminary data supporting the hypothesis that the zona pellucida has to undergo a maturation process during oocyte development are presented.     

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.     

Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro

In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls.     

Immunolocalization of the hyaluronan receptor CD44 in the reproductive tract of the mare

Hyaluronan (HA), a glycosaminoglycan, is a major component of the pericellular matrix which envelopes mammalian cells. Binding of hyaluronan to one of its specific receptors, CD44, modulates transduction of intracellular signals which direct a variety of processes, including embryogenesis, wound healing, inflammation, and neoplasia. Since regulation of these processes is critical to equine reproductive success, localization of constitutive CD44 expression was evaluated by immunohistochemical methods in ovarian, oviductal, and uterine tissues from healthy mares. Ovarian stroma contained thecal cells with varying CD44 immunopositivity. Follicular and granulosa cells of some antral and atretic follicles were positive for CD44. In the oviduct, the luminal epithelium was variably positive for CD44, with overall decreasing intensity of immunostaining from the infundibulum to the isthmus. The CD44 molecule was expressed strongly by surface epithelial cells of the uterine endometrium, but was present only rarely among cells of uterine glands. In addition, CD44 was expressed by smooth muscle cells of vascular walls, oviduct, and uterus. Since CD44 is known to modulate cell movement and differentiation, and was present at multiple sites in the reproductive tract of normal mares, we inferred there may be an important role for the HA-CD44 signaling pathway in reproductive function and inflammation.     

A histological and ultrastructural investigation of the female reproductive system of the water snake (Erythrolamprus miliaris): Oviductal cycle and sperm storage

We studied the structural and cellular organisation of the oviduct of Erythrolamprus miliaris including its morphological variation during the reproductive cycle using light microscopy, scanning electron microscopy and transmission electron microscopy. Four anatomically distinct regions compose the oviduct of E. miliaris including the anterior and posterior infundibulum, glandular uterus, non-glandular uterus and pouch. The cells of the oviductal epithelium secrete material by apocrine and merocrine processes, which vary between the anatomical regions and according to each phase of the reproductive cycle. The infundibular epithelium secretes electron dense vacuoles, which suggests the production of lipids, whereas the epithelial secretion of the glandular uterus, non-glandular uterus and pouch creates lucent and slightly electron dense vacuoles, indicating the production of glycoproteins. The timing of mating, vitellogenesis and sperm storage directly influences the morphofunctional alterations in the oviducts of E. miliaris. Sperm storage occurs only in the infundibular receptacles with increased production of the neutral carbohydrates in the presence of male gametes. Sperm storage happens in vitellogenic, non-vitellogenic and pregnant females of E. miliaris. Thus, females may be able to produce multiple clutches at different seasons of the year regardless of mating during autumn.     

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)