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. The mammalian oviduct is the venue of important events leading to the establishment of pregnancy. These events include final maturation and transport of the female and male gametes, fertilization, cleavage-stage embryonic development, and transport of the embryo to the uterus. In mammals, the physiological interaction between gametes, embryos, and oviductal epithelia involves intimate and specific contact between the two cell types (1-6). During the estrous or menstrual cycle, the mammalian oviduct undergoes significant endocrine-induced morphological, biochemical, and physiological changes. These changes establish an essential microenvironment within the oviduct. Oviductal fluid is a crucial part of this milieu and consists of transudate from serum together with specific compounds synthesized by the luminal epithelium. The current dogma states that the oviductal environment and the composition of oviductal fluid are solely under the influence of the hormonal changes in the oviduct (7-9). However, in recent years, several investigations from our laboratory and others have challenged this view by providing evidence of transcriptional changes in the oviduct in response to gametes irrespective of oviductal hormonal status (10 -12).Although these data provide strong evidence in relation to the modulation of the oviductal environment by gametes, they lack information regarding changes to the oviductal proteomic profile, for example the secretory profile. In mammals, not all the changes in the transcriptome are translated into proteomic alterations due to post-translational modifications. Ellington et al. (13) and Thomas et al. (14) provide the only evidence that at least spermatozoa can influence the (secretory) proteomic profile of oviductal epithelial cells. These investigations have reported de novo protein synthesis...

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.

Previous studies have shown that a soluble protein fraction derived from preparations of apical plasma membrane (APM) of the oviductal epithelium enhances the in vitro survival of mammalian spermatozoa. Here, we show that the survival enhancing property of the soluble protein fraction seems to depend significantly upon heat shock 70 kDa protein 8 (HSPA8 previously known as HSPA10). The following findings in the present study enabled us to draw this conclusion: first, using proteomic analysis, we identified a subset of 70 kDa oviductal surface proteins that bound to spermatozoa, one of which was HSPA8. Second, pre-treatment of the soluble protein fraction with anti-HSPA8 antibody reduced the 24 h (at 39 8C) sperm survival enhancement effect normally induced by the presence of 200 mg/ml soluble APM proteins. Third, complementary experiments showed that substituting the soluble protein fraction with bovine recombinant HSPA8 (0.5-2 mg/ml) also elicited the sperm survival effect. Finally, we also tested the effect of bovine recombinant HSPA8 on bull spermatozoa and found similar, dose-responsive, sperm survival promoting effects. The conserved nature of HSPA8 between mammalian species suggests that this protein may represent a common biological mechanism for the maintenance of sperm survival in the oviduct.

BackgroundThe epigenetic regulator Histone Deacetylase 1 (Hdac1) is required for specification and patterning of neurones and myelinating glia during development of the vertebrate central nervous system (CNS). This co-ordinating function for Hdac1 is evolutionarily conserved in zebrafish and mouse, but the mechanism of action of Hdac1 in the developing CNS is not well-understood.ResultsA genome-wide comparative analysis of the transcriptomes of Hdac1-deficient and wild-type zebrafish embryos was performed, which identified an extensive programme of gene expression that is regulated by Hdac1 in the developing embryo. Using time-resolved expression profiling of embryos, we then identified a small subset of 54 genes within the Hdac1-regulated transcriptome that specifically exhibit robust and sustained Hdac1-dependent expression from early neurogenesis onwards. 18 of these 54 stringently Hdac1-regulated genes encode DNA-binding transcription factors that are implicated in promoting neuronal specification and CNS patterning, including the proneural bHLH proteins Ascl1a and Ascl1b, as well as Neurod4 and Neurod. Relatively few genes are strongly repressed by Hdac1 but expression of the Notch target gene her6 is attenuated by Hdac1 in specific sub-regions of the developing CNS, from early stages of neurogenesis onwards. Selected members of the stringently Hdac1-regulated group of genes were tested for Hdac1 binding to their promoter-proximal cis-regulatory elements. Surprisingly, we found that Hdac1 is specifically and stably associated with DNA sequences within the promoter region of ascl1b during neurogenesis, and that this Hdac1-ascl1b interaction is abolished in hdac1 mutant embryos.ConclusionsWe conclude that Hdac1 regulates histone acetylation and methylation in the developing zebrafish embryo and promotes the sustained, co-ordinate transcription of a small set of transcription factor genes that control expansion and diversification of cell fates within the developing CNS. Our in vivo chromatin immunoprecipitation results also suggest a specific function for Hdac1 in directly regulating transcription of a key member of this group of genes, ascl1b, from the beginning of neurogenesis onwards. Taken together, our observations indicate a novel role for Hdac1 as a positive regulator of gene transcription during development of the vertebrate CNS, in addition to its more well-established function in transcriptional repression.

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.

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.

SUMMARYThe global increase of the human parasite, the common bed bug Cimex lectularius, calls for specific pest control target sites. The bed bug is also a model species for sexual conflict theory which suggests seminal fluids may be highly diverse. The species has a highly unusual sperm biology and seminal proteins may have unique functions. 1-D PAGE gels showed 40 to 50% band sharing between C. lectularius and another cimicid species, Afrocimex constrictus. However, adult, sexually rested C. lectularius males were found to store 5 to 7μg of seminal protein and with only 60μg of protein we obtained informative 2-D PAGE gels. These showed 79% shared protein spots between two laboratory populations, and more than half of the shared protein spots were detected in the mated female. Further analysis using liquid chromatography electrospray ionisation tandem mass spectrometry revealed that 26.5% of the proteins had matches among arthropods in data bases and 14.5% matched Drosophila proteins. These included ubiquitous proteins but also those more closely associated with reproduction such as moj 29, ubiquitin, the stress-related elongation factor EF-1alpha, a protein disulfide isomerase and an antioxidant, Peroxiredoxin 6.

Complement component 3 (C3) has well-established roles within immune system, but its roles outside of immune system are less characterized. The extensive presence of C3 throughout the female reproductive tract, and its temporal, and gamete-specific regulation of expression suggest a potential role for C3 in reproduction. In the present investigation, the effects of C3, C3b and iC3b on porcine oocyte maturation, fertilization and embryonic development were examined. We identified the ability of iC3b to positively influence oocyte maturation. No effects on fertilization efficiency, penetration rates, polyspermy and blastocyst formation were observed. However, C3, C3b and iC3b presence in embryo culture medium resulted in fewer total cells in test blastocysts compared to control blastocysts. The results of this study indicate a potential function for iC3b in oocyte maturation. Furthermore, it was demonstrated that the presence of either C3, C3b or iC3b has a negative influence on early embryonic development in the porcine species.