Oral exposure to bisphenol S is associated with alterations in the oviduct proteome of an ovine model, with aggravated effects in overfed females.

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.

Structure, function, and evolution of Gga-AvBD11, the archetype of the structural avian-double-ß-defensin family.

Out of the 14 avian ß-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian ß-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to ß-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed ß-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-ß-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

Genetic determinants of seed protein plasticity in response to the environment in Medicago truncatula.

As the frequency of extreme environmental events is expected to increase with climate change, identifying candidate genes for stabilizing the protein composition of legume seeds or optimizing this in a given environment is increasingly important. To elucidate the genetic determinants of seed protein plasticity, major seed proteins from 200 ecotypes of Medicago truncatula grown in four contrasting environments were quantified after one-dimensional electrophoresis. The plasticity index of these proteins was recorded for each genotype as the slope of Finlay and Wilkinson's regression and then used for genome-wide association studies (GWASs), enabling the identification of candidate genes for determining this plasticity. This list was enriched in genes related to transcription, DNA repair and signal transduction, with many of them being stress responsive. Other over-represented genes were related to sulfur and aspartate family pathways leading to the synthesis of the nutritionally essential amino acids methionine and lysine. By placing these genes in metabolic pathways, and using a M. truncatula mutant impaired in regenerating methionine from S-methylmethionine, we discovered that methionine recycling pathways are major contributors to globulin composition establishment and plasticity. These data provide a unique resource of genes that can be targeted to mitigate negative impacts of environmental stresses on seed protein composition.

The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo.

BACKGROUND: Despite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro-derived bovine embryos, there is no comparison at the protein expression level. RESULTS: A total of 38 pools of grade-1 quality bovine embryos at the 4-6 cell, 8-12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development. CONCLUSIONS: The maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4-6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

Avian eggshell formation reveals a new paradigm for vertebrate mineralization via vesicular amorphous calcium carbonate.

Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where eggshell mineralization takes place. Herein, we report further experimental support for this model. We confirmed the presence of extracellular vesicles (EVs) using transmission EM and showed high levels of mRNA of vesicular markers in the oviduct segments where eggshell mineralization occurs. We also demonstrate that EVs contain ACC in uterine fluid using spectroscopic analysis. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of bicarbonate ions (HCO[Formula: see text]), for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first-time experimental evidence for the components of vesicular transport to supply ACC in a vertebrate model of biomineralization.

Metabolic exchanges between the oocyte and its environment: focus on lipids.

Finely regulated fatty acid (FA) metabolism within ovarian follicles is crucial to follicular development and influences the quality of the enclosed oocyte, which relies on the surrounding intra-follicular environment for its growth and maturation. A growing number of studies have examined the association between the lipid composition of follicular compartments and oocyte quality. In this review, we focus on lipids, their possible exchanges between compartments within the ovarian follicle and their involvement in different pathways during oocyte final growth and maturation. Lipidomics provides a detailed snapshot of the global lipid profiles and identified lipids, clearly discriminating the cells or fluid from follicles at distinct physiological stages. Follicular fluid appears as a main mediator of lipid exchanges between follicular somatic cells and the oocyte, through vesicle-mediated and non-vesicular transport of esterified and free FA. A variety of expression data allowed the identification of common and cell-type-specific actors of lipid metabolism in theca cells, granulosa cells, cumulus cells and oocytes, including key regulators of FA uptake, FA transport, lipid transformation, lipoprotein synthesis and protein palmitoylation. They act in harmony to accompany follicular development, and maintain intra-follicular homeostasis to allow the oocyte to accumulate energy and membrane lipids for subsequent meiotic divisions and first embryo cleavages.

The Unique Features of Proteins Depicting the Chicken Amniotic Fluid.

In many amniotes, the amniotic fluid is depicted as a dynamic milieu that participates in the protection of the embryo (cushioning, hydration, and immunity). However, in birds, the protein profile of the amniotic fluid remains unexplored, even though its proteomic signature is predicted to differ compared with that of humans. In fact, unlike humans, chicken amniotic fluid does not collect excretory products and its protein composition strikingly changes at mid-development because of the massive inflow of egg white proteins, which are thereafter swallowed by the embryo to support its growth. Using GeLC-MS/MS and shotgun strategies, we identified 91 nonredundant proteins delineating the chicken amniotic fluid proteome at day 11 of development, before egg white transfer. These proteins were essentially associated with the metabolism of nutrients, immune response and developmental processes. Forty-eight proteins were common to both chicken and human amniotic fluids, including serum albumin, apolipoprotein A1 and alpha-fetoprotein. We further investigated the effective role of chicken amniotic fluid in innate defense and revealed that it exhibits significant antibacterial activity at day 11 of development. This antibacterial potential is drastically enhanced after egg white transfer, presumably due to lysozyme, avian beta-defensin 11, vitelline membrane outer layer protein 1, and beta-microseminoprotein-like as the most likely antibacterial candidates. Interestingly, several proteins recovered in the chicken amniotic fluid prior and after egg white transfer are uniquely found in birds (ovalbumin and related proteins X and Y, avian beta-defensin 11) or oviparous species (vitellogenins 1 and 2, riboflavin-binding protein). This study provides an integrative overview of the chicken amniotic fluid proteome and opens stimulating perspectives in deciphering the role of avian egg-specific proteins in embryonic development, including innate immunity. These proteins may constitute valuable biomarkers for poultry production to detect hazardous situations (stress, infection, etc.), that may negatively affect the development of the chicken embryo.

The Defibrillation Conundrum: New Insights into the Mechanisms of Shock-Related Myocardial Injury Sustained from a Life-Saving Therapy.

Implantable cardiac defibrillators (ICDs) are recommended to prevent the risk of sudden cardiac death. However, shocks are associated with an increased mortality with a dose response effect, and a strategy of reducing electrical therapy burden improves the prognosis of implanted patients. We review the mechanisms of defibrillation and its consequences, including cell damage, metabolic remodeling, calcium metabolism anomalies, and inflammatory and pro-fibrotic remodeling. Electrical shocks do save lives, but also promote myocardial stunning, heart failure, and pro-arrhythmic effects as seen in electrical storms. Limiting unnecessary implantations and therapies and proposing new methods of defibrillation in the future are recommended.

Protein Palmitoylation in Bovine Ovarian Follicle.

Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.

Avian uterine fluid proteome: Exosomes and biological processes potentially involved in sperm survival.

Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom-up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.

Oviduct Fluid Extracellular Vesicles Change the Phospholipid Composition of Bovine Embryos Developed In Vitro.

Oviduct fluid extracellular vesicles (oEVs) have been proposed as bringing key molecules to the early developing embryo. In order to evaluate the changes induced by oEVs on embryo phospholipids, fresh bovine blastocysts developed in vitro in the presence or absence of oEVs were analyzed by intact cell MALDI-TOF (Matrix assisted laser desorption ionization-Time of flight) mass spectrometry (ICM-MS). The development rates, cryotolerance, and total cell number of blastocysts were also evaluated. The exposure to oEVs did not affect blastocyst yield or cryotolerance but modified the phospholipid content of blastocysts with specific changes before and after blastocoel expansion. The annotation of differential peaks due to oEV exposure evidenced a shift of embryo phospholipids toward more abundant phosphatidylcholines (PC), phosphatidylethanolamines (PE), and sphingomyelins (SM) with long-chain fatty acids. The lipidomic profiling of oEVs showed that 100% and 33% of the overabundant masses in blastocysts and expanded blastocysts, respectively, were also present in oEVs. In conclusion, this study provides the first analysis of the embryo lipidome regulated by oEVs. Exposure to oEVs induced significant changes in the phospholipid composition of resulting embryos, probably mediated by the incorporation of oEV-phospholipids into embryo membranes and by the modulation of the embryonic lipid metabolism by oEV molecular cargos.

MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Identification of 56 Proteins Involved in Embryo-Maternal Interactions in the Bovine Oviduct.

The bovine embryo develops in contact with the oviductal fluid (OF) during the first 4-5 days of pregnancy. The aim of this study was to decipher the protein interactions occurring between the developing embryo and surrounding OF. In-vitro produced 4-6 cell and morula embryos were incubated or not (controls) in post-ovulatory OF (OF-treated embryos) and proteins were then analyzed and quantified by high resolution mass spectrometry (MS) in both embryo groups and in OF. A comparative analysis of MS data allowed the identification and quantification of 56 embryo-interacting proteins originated from the OF, including oviductin (OVGP1) and several annexins (ANXA1, ANXA2, ANXA4) as the most abundant ones. Some embryo-interacting proteins were developmental stage-specific, showing a modulating role of the embryo in protein interactions. Three interacting proteins (OVGP1, ANXA1 and PYGL) were immunolocalized in the perivitelline space and in blastomeres, showing that OF proteins were able to cross the zona pellucida and be taken up by the embryo. Interacting proteins were involved in a wide range of functions, among which metabolism and cellular processes were predominant. This study identified for the first time a high number of oviductal embryo-interacting proteins, paving the way for further targeted studies of proteins potentially involved in the establishment of pregnancy in cattle.

Water stress combined with sulfur deficiency in pea affects yield components but mitigates the effect of deficiency on seed globulin composition.

Water stress and sulfur (S) deficiency are two constraints increasingly faced by crops due to climate change and low-input agricultural practices. To investigate their interaction in the grain legume pea (Pisum sativum), sulfate was depleted at the mid-vegetative stage and a moderate 9-d water stress period was imposed during the early reproductive phase. The combination of the stresses impeded reproductive processes in a synergistic manner, reducing seed weight and seed number, and inducing seed abortion, which highlighted the paramount importance of sulfur for maintaining seed yield components under water stress. On the other hand, the moderate water stress mitigated the negative effect of sulfur deficiency on the accumulation of S-rich globulins (11S) in seeds, probably due to a lower seed sink strength for nitrogen, enabling a readjustment of the ratio of S-poor (7S) to 11S globulins. Transcriptome analysis of developing seeds at the end of the combined stress period indicated that similar biological processes were regulated in response to sulfur deficiency and to the combined stress, but that the extent of the transcriptional regulation was greater under sulfur deficiency. Seeds from plants subjected to the combined stresses showed a specific up-regulation of a set of transcription factor and SUMO ligase genes, indicating the establishment of unique regulatory processes when sulfur deficiency is combined with water stress.

Proteomic analysis of uterine fluid of fertile and subfertile hens before and after insemination.

Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve the understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen's genital tract and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F-) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F- hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage.

Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos.

The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification-warming, exposure to P4 improved the proportion of embryos that re-expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI-TOF mass spectrometry, a total of 242 phospholipid masses of 400-1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.

Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells.

Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.

Binder of Sperm Proteins 1 and 5 have contrasting effects on the capacitation of ram spermatozoa.

Binder of Sperm Proteins (BSPs) are the most abundant seminal plasma protein family in the ram and bull. They have been extensively studied in the bull but less is known about their function in ovine seminal plasma and current knowledge suggests that BSPs may have different effects in these two species. In the bull, they facilitate capacitation and destabilize the sperm membrane during in vitro handling, whereas in the ram, they appear to stabilize the sperm membrane and prevent cryopreservation-induced capacitation-like changes. Further investigation into the effects of BSPs on ram spermatozoa under capacitating conditions is required to further clarify their physiological roles in the ram. We investigated the effects of Binder of Sperm Proteins 1 and 5 on epididymal ram spermatozoa in conditions of low, moderate, and high cAMP. BSPs had minimal effects on sperm function in low-cAMP conditions, but caused significant changes under cAMP upregulation. BSP1 stabilized the membrane and qualitatively reduced protein tyrosine phosphorylation, but significantly increased cholesterol efflux and induced spontaneous acrosome reactions. BSP5 slightly increased spontaneous acrosome reactions and caused sperm necrosis. However, BSP5 had minimal effects on membrane lipid order and cholesterol efflux and did not inhibit protein tyrosine phosphorylation. These findings demonstrate that under maximal cAMP upregulation, BSP1 affected ram spermatozoa in a manner comparable to bull spermatozoa, while BSP5 did not.

Identification by proteomics of oviductal sperm-interacting proteins.

The interactions between oviductal fluid (OF) proteins and spermatozoa play major roles in sperm selection, storage and capacitation before fertilization. However, only a few sperm-interacting proteins in the OF has been identified and very little is known about the regulation of sperm-oviduct interactions across the estrous cycle. Samples of bovine frozen-thawed sperm from three bulls were incubated with OF at pre-, post-ovulatory stages (Pre-/Post-ov) or luteal phase (LP) of the estrous cycle (7 mg/mL proteins, treated groups) or with a protein-free media (control). The proteomes of sperm cells were assessed by nanoLC-MS/MS and quantified by label-free methods. A total of 27 sperm-interacting proteins originating in the OF were identified. Among those, 14 were detected at all stages, eight at Post-ov and LP and five only at LP. The sperm-interacting proteins detected at all stages or at LP and Post-ov were on average more abundant at LP than at other stages (P < 0.05). At Pre-ov, OVGP1 was the most abundant sperm-interacting protein while at Post-ov, ACTB, HSP27, MYH9, MYH14 and OVGP1 were predominant. Different patterns of abundance of sperm-interacting proteins related to the stage were evidenced, which greatly differed from those previously reported in the bovine OF. In conclusion, this study highlights the important regulations of sperm-oviduct interactions across the estrous cycle and provides new protein candidates that may modulate sperm functions.