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Collection of oviductal cell secretions. Oviducts were dissected and removed from the mesosalphinx and fluid was collected either by direct aspiration (ex vivo) or after the culture of the oviductal epithelium (in vitro). Media used for culturing oviductal epithelial cells (OECs) were collected after passive conditioning for 48 hours or after a 4-hour stimulation with phorbol myristate acetate (PMA), ionomycin and dibutyryl cyclic adenosine monophosphate (Bt 2 cAMP). (1) Extruded oviductal mucosa containing intact epithelial sheets. (2) Representative image showing attachment and growth of the oviductal epithelial cells after 2-3 days in culture (Scale bar 300 μm). Immunohistochemistry for cytokeratin as a marker for OECs showed that the cultures established by this method were of high purity (Scale bar 200 μm).
Source publication
The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified...
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Citations
... Proteins are major components in the oviduct fluid and have been shown to play critical roles in sperm survival, gamete interaction, and embryo quality [4][5][6][7][8]. In addition, the proteomic composition of the oviduct fluid greatly varies according to the systemic and topical concentrations of estradiol and progesterone across the cycle in mammalian females [9][10][11]. Although poorly explored, there is growing evidence that the mammalian oviduct is also highly sensitive to environmental factors such as diet habits [12][13][14], heat stress [3,15], or chemical compounds commonly found in our daily life [16,17]. ...
Background
Bisphenol S (BPS) is a substitute for bisphenol A in plastic manufacturing and, as a potential endocrine disruptor, may alter the physiology of the oviduct, in which fertilization and early embryo development take place in mammals. The objective of this study was to assess the effect of a daily dietary exposure to BPS combined with a contrasted diet on the oviduct fluid proteome using an ovine model.
Results
Eighty adult cyclic ewes were allotted to four groups (20/group): overfed (OF) consuming 50 µg/kg/day of BPS in their diet, underfed (UF) consuming 50 µg/kg/day of BPS, and non-exposed controls in each diet group. After three months, the mean body condition score, plasma levels of glucose and non-esterified fatty acids were significantly higher in OF than in UF females. The proteins in collected OF samples (50 µg) were analyzed by nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). Overall, 1563 proteins were identified, among which 848 were quantified. Principal component analysis of the data revealed a clear discrimination of samples according to the diet and a segregation between BPS-exposed and non-exposed females in overfed ewes. Hierarchical clustering of differentially abundant proteins (DAPs) identified two clusters of 101 and 78 DAPs according to the diet. Pairwise comparisons between groups revealed a stronger effect of BPS in OF than in UF females (70 vs. 24 DAPs) and a stronger effect of the diet in BPS-exposed than non-exposed females (56 vs. 36 DAPs). Functional analysis of DAPs showed an enrichment in metabolic processes, immune system, cell response to stress, and reproductive processes.
Conclusions
This work highlights for the first time the important impact of BPS on the oviduct proteome, with larger effects seen in OF than UF females. These results, together with previous ones, raise health concerns for everyone and call for a greater regulation of BPS in the food industry.
... Proteins are major components in the oviduct uid and have been shown to play critical roles in sperm survival, gamete interaction, and embryo quality [4][5][6][7][8]. In addition, the proteomic composition of the oviduct uid greatly varies according to the systemic and topical concentrations of estradiol and progesterone across the cycle in mammalian females [9][10][11]. Although poorly explored, there is growing evidence that the mammalian oviduct is also highly sensitive to environmental factors such as diet habits [12][13][14], heat stress [3,15], or chemical compounds commonly found in our daily life [16,17]. ...
Background
Bisphenol S (BPS) is a substitute for bisphenol A in plastic manufacturing and, as a potential endocrine disruptor, may alter the physiology of the oviduct, in which fertilization and early embryo development take place in mammals. The objective of this study was to assess the effect of a daily dietary exposure to BPS combined with a contrasted diet on the oviduct fluid proteome using an ovine model.
Results
Eighty adult cyclic ewes were allotted to four groups (20/group): overfed (OF) consuming 50 µg/kg/day of BPS in their diet, underfed (UF) consuming 50 µg/kg/day of BPS, and non-exposed controls in each diet group. After three months, the mean body condition score and plasma levels of glucose and non-esterified fatty acids were significantly higher in over- than in underfed females. The proteins in collected OF samples (50 µg) were analyzed by nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). Overall, 1563 proteins were identified, among which 848 were quantified. Principal component analysis of the data revealed a clear discrimination of samples according to the diet and a segregation between BPS-exposed and non-exposed females in overfed ewes. Hierarchical clustering of differentially abundant proteins (DAPs) identified two clusters of 101 and 78 DAPs according to the diet. Pairwise comparisons between groups revealed a stronger effect of BPS in OF than in UF females (70 vs. 24 DAPs) and a stronger effect of the diet in BPS-exposed than non-exposed females (56 vs. 36 DAPs). Functional analysis of DAPs showed an enrichment in metabolic processes, immune system, cell response to stress, and reproductive processes.
Conclusions
This work highlights for the first time the important impact of BPS on the oviduct proteome, with larger effects seen in OF than UF females. These results, together with previous ones, raise health concerns for everyone and call for a greater regulation of BPS in the food industry.
... In another study, Pillai et al. used secretions from in vitro models of bovine oviductal epithelial cells (OEC) and ex vivo OF; a total of 2087 proteins were identified, of which 266 were secretory in nature [99]. In terms of functions, proteins were active in immune homeostasis, gamete maturation, fertilization, and early embryonic development. ...
Infertility is a major problem in farm animals, which has a negative economic effect on farm industries. Infertility can be defined as the inability of animals to achieve a successful pregnancy. Early pregnancy is crucial to establish a successful pregnancy, and it is reported that 70-80% and 20-30% of total embryonic loss occur in cattle and pigs, respectively, during the first month of pregnancy. The advanced high-throughput proteomics techniques provide valuable tools for in-depth understanding of the implantation process in farm animals. In the present review, our goal was to compile, assess, and integrate the latest proteomic research on farm animals, specifically focused on female reproduction, which involves endometrial tissues, uterine fluids, oviductal fluids, and mi-croRNAs. The series of studies has provided in-depth insights into the events of the implantation process by unfolding the molecular landscape of the uterine tract. The discussed data are related to pregnant vs. non-pregnant animals, pregnancy vs. oestrous cycle, different days of the early pregnancy phase, and animals with uterine infections affecting reproduction health. Some of the studies have utilized non-invasive methods and in vitro models to decipher the molecular events of embryo maternal interaction. The proteomics data are valuable sources for discovering biomarkers for infertility in ruminants and new regulatory pathways governing embryo-uterine interaction, endo-metrium receptivity, and embryonic development. Here, we envisage that the identified protein signatures can serve as potential therapeutic targets and biomarkers to develop new therapeutics against pregnancy diseases.
... In particular, the two most abundant proteins identified are the oviduct-specific glycoprotein (OVGP1) and albumin (Mondéjar et al., 2012;Canha-Gouveia et al., 2019), with important roles mainly for sperm capacitation and embryo development. Growth factors are also key proteins of the OF, which exert an activity of cell division control and contribute to the efficient development of early embryos (Pillai et al., 2017). Other components of the OF include low molecular weight hormones such as steroids (progesterone and estradiol) and prostaglandins, which are secreted by the ovarian follicles and the corpus luteus and can reach the OF via a local countercurrent transfer (Einer-Jensen and Hunter, 2005). ...
MicroRNAs are small non-coding molecules that control several cellular functions and act as negative post-transcriptional regulators of the mRNA. While their implication in several biological functions is already known, an important role as regulators of different physiological and pathological processes in fertilization and embryo development is currently emerging. Indeed, miRNAs have been found in the oviductal fluid packaged within the extracellular vesicles, which might act as natural nanoshuttles by transporting lipids, proteins, RNA molecules and miRNAs from the oviduct to the gametes or embryos. Here, an exhaustive bibliography search was carried out, followed by the construction of a computational model based on the networks theory in an attempt to recreate and elucidate the pathways potentially activated by the oviductal miRNA. The omics data published to date were gathered to create the Oviductal MiRNome, in which the miRNA target genes and their interactions are represented by using stringApp and the Network analyzer from Cytoscape 3.7.2. Then, the hyperlinked nodes were identified to investigate the pathways in which they are involved using the gene ontology enrichment analysis. To study the phenotypical effects after the removal of key genes on the reproductive system and embryo, knockout mouse lines for every protein-coding gene were investigated by using the International Mouse Phenotyping Consortium database. The creation of the Oviductal MiRNome revealed the presence of important genes and their interactions within the network. The functional enrichment analysis revealed that the hyperlinked nodes are involved in fundamental cellular functions, both structural and regulatory/signaling, suggesting their implication in fertilization and early embryo development. This fact was as well evidenced by the effects of the gene deletion in KO mice on the reproductive system and embryo development. The present study highlights the importance of studying the miRNA profiles and their enormous potential as tools to improve the assisted reproductive techniques currently used in human and animal reproduction.
... Angiogenesis plays an important role in cyclical mechanisms during the estrous cycle, such as follicular growth and corpus luteum formation, which are dependent on the development of a dense capillary net [55], and transudation from plasma contributes to the defining characteristics of the luminal microenvironment in cattle, such as uterine and oviductal fluid [56]. VEGFA is a member of the platelet-derived VEGF family that encodes a heparin-binding protein, which promotes vascular endothelial cell proliferation and migration [57]. ...
... The C3a peptide, also known as C3a anaphylatoxin, is a complement component that regulates inflammation and has antimicrobial effects [67]. Complement proteins have been suggested to mediate functional homeostasis, which has been linked to physiological aspects of cattle oviduct and fertility [56], sperm-oocyte interaction [68], and embryo development [69]. In rodents, C3 is secreted by OECs and increases trophectoderm development, blastocyst size, and hatching rates [70,71]. ...
Background and Aim: The oviduct environment is of particular importance because it is the site of fertilization and early
embryo development. The oviduct, as a component of the reproductive system, responds to ovarian hormone (estradiol [E2]
and progesterone [P4]) stimuli depending on the estrous cycle phase. This study aimed to elucidate the effect of estrous cycle
phases (follicular and early and late luteal phases) on gene expression patterns in bovine oviduct epithelial cells (BOECs).
Materials and Methods: Oviducts were obtained from healthy slaughterhouse animals, corresponding to ipsilateral
ovaries with dominant follicles or corpus luteum during early and late luteal phases. BOECs were recovered from the
isthmus (IST) and ampulla (AMP), and the expression patterns of genes related to cytokinesis and mitosis mechanisms
(rho-associated coiled-coil containing protein kinase and cellular communication network factor 2 [CCN2]), growth factors
(insulin-like growth factor-binding protein 3, epidermal growth factor receptor [EGFR], vascular endothelial growth factor
A, and EGFR), antioxidant mechanisms (glutathione peroxidase 4 [GPX4]), apoptosis (B-cell lymphoma 2), complement
component (C3), energy metabolism (aldose reductase gene family 1-member b1 [AKRIB1] and solute carrier family 2),
hormone receptors (estrogen receptor 1 and luteinizing hormone/choriogonadotropin receptor), and specific glycoproteins
(oviductal glycoprotein 1) were analyzed.
Results: High P4 levels (late luteal phase) affected the expression of important genes related to antioxidant mechanisms
(GPX4), energy metabolism (AKRIB1), growth factors (IGBP3 and EGFR), and cell growth regulation (CCN2) in the AMP.
Low P4 levels (early luteal phase) affected the expression of AKR1B1, IGBP3, and CCN2. In addition, estrogen likely had
an effect on OVPGP expression in the cattle oviduct.
Conclusion: Differential gene expression patterns of BOECs in the AMP during the luteal phase (antioxidant mechanisms,
energy metabolism, growth factors, and immunological regulators) and in the IST during the follicular phase (glycoproteins)
may influence their renewal and population proportions, modulating the oviduct environment as well as gamete and embryo
physiology.
... LC separation was done on a Proxeon Easy-nLC II HPLC (Thermo Scientific) with a Proxeon nanospray source and mass spectra were collected on an Orbitrap Q Exactive mass spectrometer (Thermo Scientific) as previously described (Pillai et al. 2017). Tandem mass spectra were extracted by Proteome Discoverer v1.2. ...
MA-10 cells, established four decades ago from a murine Leydig cell tumor, has served as a key model system for studying steroidogenesis. Despite a precipitous loss in their innate ability to respond to luteinizing hormone (LH), use of a cell permeable cAMP analog for induction ensured their continued use. In parallel, a paradigm that serum-free conditions are essential for trophic steroidogenic stimulation was rationalized. Through selection of LH-responsive single-cell MA-10 Slip clones, we uncovered that Leydig cells remain responsive in the presence of serum in vitro , and that exogenous cholesterol delivery by lipoproteins provided a significantly elevated steroid biosynthetic response (>2-fold). In scrutinizing the underlying regulation, systems biology of the MA-10 cell proteome identified multiple Rho-GTPase signaling pathways as highly enriched. Testing Rho function in steroidogenesis revealed that its modulation can negate the specific elevation in steroid biosynthesis observed in the presence of lipoproteins/serum. This signaling modality primarily linked to regulation of endocytic traffic, is evident only in the presence of exogenous cholesterol. Inhibiting Rho function in vivo also decreased hCG-induced testosterone production in mice. Collectively, our findings dispel a long-held view that use of serum could confound or interfere with trophic stimulation, and underscore the need for exogenous lipoproteins when dissecting physiological signaling and cholesterol trafficking for steroid biosynthesis in vitro . The LH-responsive MA-10 Slip clones derived in this study present a reformed platform enabling biomimicry to study the cellular and molecular basis of mammalian steroidogenesis.
... The differences in OEC gene expression between regions and stages of the cycle can explain, in part, the known/ detected spatiotemporal changes in OF composition. In fact, although the protein content of the OF is derived from both blood plasma transudation and OEC secretions (Aguilar and Reyley 2005), 89% of secreted proteins are detected as oviductal transcripts (Pillai et al. 2017). Despite this, in a study comparing the ipsi-and contralateral OF proteome at different stages of the cycle, >70% OF proteins could be detected throughout the cycle and >80% were common between sides; while the proportion of differentially abundant proteins between sides was 10% and 24% for the pre-and post-ovulatory stage respectively (Lamy et al. 2016). ...
... It is important to note that this study did not distinguish between secreted proteins and non-secreted intracellular proteins, which constituted a high proportion of the proteins identified. Considering that only 8% of the proteins identified by Pillai et al. (2017) could be classified as secreted, the results presented by Lamy et al. (2016) become difficult to interpret. The presence of intracellular elements detected in OF in their study are likely the result of induced cell death and/or damage during sample collection. ...
The coordinated interaction between the developing embryo and the maternal reproductive tract is essential for the establishment and maintenance of pregnancy in mammals. An early cross-talk is established between the oviduct/uterus and the gametes and embryo. This dialogue will shape the microenvironment in which gamete transport, fertilisation, and early embryonic development occur. Due to the small size of the gametes and the early embryo relative to the volume of the oviductal and uterine lumina, collection of tissue and fluid adjacent to these cells is challenging in cattle. Thus, the combination of in vivo and in vitro models seems to be the most appropriate approach to better understand this fine dialogue. In this respect, the aim of this review is to summarise the recent findings in relation to gamete/embryo–maternal interaction during the pre-elongation period.
... From the total of protein identi ed in our study, 37% were predicted as potentially secreted and 10% contained a signal peptide. These proportions of secreted proteins are comparable to those previously reported in the OF in cattle 23,24 and sheep 27 . It is unclear how intracellular proteins could be exported in high proportions to the oviductal lumen. ...
Unraveling the oviduct fluid (OF) and its regulation is crucial to understand the microenvironment in which sperm capacitation, fertilization and early embryo development take place. Therefore, our aim was to determine the spatiotemporal changes in the OF proteome according to the anatomical region of the oviduct (ampulla vs. isthmus), the proximity of the ovulating ovary (ipsilateral vs . contralateral side) and the peri-ovulatory stage (pre-ovulatory or Pre-ov vs . post-ovulatory or Post-ov). Oviducts from adult cyclic cows were collected at a local slaughterhouse and pools of OF were analyzed by nanoLC-MS/MS and label-free protein quantification (n=32 OF pools for all region ´ stage ´ side conditions). A total of 3,760 proteins were identified in the OF, of which 37% were predicted to be secreted. The oviduct region was the major source of variation in protein abundance, followed by the proximity of the ovulating ovary and finally the peri-ovulatory stage. Differentially abundant proteins between regions, stages and sides were involved in a broad variety of biological functions, including protein binding, response to stress, cell-to-cell adhesion, calcium homeostasis and the immune system. This work highlights the dynamic regulation of oviduct secretions and provides new protein candidates for interactions between the maternal environment, the gametes and the early embryo.
... To assess the likely origin of proteins, especially those actively secreted from the endometrium to ULF, a multi-tool analysis was performed as previously explained [37], using a combination of software applications with algorithms optimized for different uses. All these tools also feature a function to predict secretion via the classical secretion pathway, via Golgi apparatus and granular endoplasmic reticule, and thus may also expand the list of secreted proteins. ...
Dairy cow subfertility is a worldwide issue arising from multiple factors. It manifests in >30% early pregnancy losses in seasonal pasture-grazed herds, especially when cows are inseminated in the early post-partum period. Most losses occur before implantation, when embryo growth depends on factors present in maternal tract fluids. Here we examined the proteomic composition of early and mid-postpartum uterine luminal fluid in crossbred lactating dairy cows to identify molecular determinants of fertility. We also explored changes in uterine luminal fluid from first to third estrus cycles postpartum in individual cows, linking those changes with divergent embryo development. For this, we flushed uteri of 87 cows at day 7 of pregnancy at first and third estrus postpartum, recovering and grading their embryos. Out of 1563 proteins detected, 472 had not been previously reported in this fluid, and 408 were predicted to be actively secreted by bioinformatic analysis. The abundance of 18 proteins with roles in immune regulation and metabolic function (e.g. cystatin B, pyruvate kinase M2) was associated with contrasting embryo quality. Matched-paired pathway analysis indicated that, from first to third estrus postpartum, upregulation of metabolic (e.g. creatine and carbohydrate) and immune (e.g. complement regulation, antiviral defense) processes were related to poorer quality embryos in the third estrus cycle postpartum. Conversely, upregulated signal transduction and protein trafficking appeared related to improved embryo quality in third estrus. These results advance the characterization of the molecular environment of bovine uterine luminal fluid and may aid understanding fertility issues in other mammals, including humans.
... Previous studies demonstrated the alterations in the secretory oviductal proteome in response to the presence of oocytes and spermatozoa [12]. Recently, Pillai et al. [13] analyzed the proteomic profiling of bovine oviduct and revealed diverse functions of this luminal microenvironment. However, the functional involvement of secretory oviductal proteins on fertilization and embryonic development is poorly understood. ...
The oviduct is a site for early reproductive events including gamete maturation, fertilization, and early embryo development. Secretory cells lining the oviduct lumen synthesize and secrete proteins that interact with gametes and developing embryos. Although previous studies have identified some of the secretory proteins in the oviduct, however, knowledge and their precise specific functions in the oviduct are poorly understood. In this study, by using proteomic approach, we identified a secretory protein, Peroxiredoxin 6 (PRDX6), and evaluated its role in mediating early pregnancy events, fertilization, and embryo development in rabbit oviduct. The expression of PRDX6 was significantly higher in ampulla and isthmus sections of the oviduct in mated animal groups compared to non-mated controls. Furthermore, significant reduction in number of embryos recovered from PRDX6 siRNA-transfected oviductal horn was observed compared to the control contralateral horn. Moreover, in animals receiving PRDX6 siRNA in their oviductal horn, the number of implanted blastocysts was significantly less in the uterus as observed on day 9 post-coital (p.c.). Further, during embryo-rabbit oviduct epithelial cell (ROEC) co-culture, siRNA-mediated PRDX6 silencing attenuated the early embryonic development. Mechanistically, increased levels of ROS and expression of oxidative stress-and inflammation-related proteins were found in PRDX6 siRNA-treated ROEC cells as compared to control cells, implicating that ablation of PRDX6 in the oviduct creates a stress-induced micro-environment detrimental to early embry-onic development in oviduct. Taken together, our data suggest that PRDX6 maintains an optimal micro-environment conducive to successful embryo development and can be considered as a candidate to evaluate its therapeutic potential in IVF strategies.
