Metabolic signature of cervical mucus in ewe breeds with divergent cervical sperm transport: a focus on metabolites involved in amino acid metabolism.

INTRODUCTION: Cervical artificial insemination (AI) with frozen-thawed semen in sheep has yielded unacceptably low pregnancy rates. The exception is in Norway where vaginal AI yields non-return rates in excess of 60%, which has been attributed to the ewe breed used. OBJECTIVES AND METHODS: This study aimed to characterise, for the first time, the ovine follicular phase cervical mucus metabolome, with a focus on the amino acid profile. Cervical mucus was collected from four European ewe breeds with known differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk (low fertility), Belclare (medium fertility), Norwegian White Sheep (NWS) and Fur (both high fertility). RESULTS: A total of 689 metabolites were identified in the cervical mucus of all the four ewe breeds. Of these, 458 metabolites were altered by ewe breed, which had the greatest effect in the dataset (P < 0.05). We detected 194 metabolites involved in the amino acid pathway, of which 133, 56 and 63 were affected by ewe breed, type of cycle and their interaction, respectively (P < 0.05). N-methylhydantoin and N-carbamoylsarcosine (degradation products of creatinine pathway) exhibited the greatest fold change decrease in the Suffolk breed compared to Fur and NWS (P < 0.001). Oxidized metabolites were also decreased in Suffolk compared to high fertility breeds (P < 0.05). In contrast, other metabolites such as 3-indoxyl-sulfate, putrescine, cadaverine were significantly increased in Suffolk at the synchronised cycle. CONCLUSION: The suboptimal amino acid profile in the cervical mucus of the low fertility Suffolk breed may have negative consequences for sperm transport.

The Addition of Opuntia ficus-indica Ethanolic Extract to a Skimmed Milk-Based Extender Impacts Ram Sperm Quality.

Recently, researchers have focused on the use of natural antioxidants to improve semen quality as a key element for successful artificial insemination. In this context, the first aim of this study was to determine the antioxidant activity and composition (minerals, vitamins, and sugars) of Opuntia ficus-indica cladode ethanolic extract (ETHEX). A further purpose of the study was to investigate the effect of ETHEX supplementation on the quality of liquid ram semen extended with skim milk (SM) at 5°C. The antioxidant activity of ETHEX was studied using free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH•) assay. The mineral composition and the sugar and vitamin contents of ETHEX were determined using an inductively coupled plasma optical emission spectrometry (ICP-OES) and HPLC-DAD-RID analytical instruments. As a second part, semen was collected from five Boujaâd rams with an artificial vagina. The ejaculates with more than 70% motility were pooled, extended with skim milk (SM) extender without (control) or supplemented with 1-8% of ETHEX (37°C; 0.8 × 109 sperm/mL). Sperm quality parameters were assessed at 8, 24, 48, and 72 h. The results showed that ETHEX had a higher antioxidant activity compared to those of ascorbic acid and butylated hydroxytoluene (BHT). Furthermore, ETHEX contains a considerable amount of minerals, vitamins, and sugars. The inclusion of 1 or 2% ETHEX in SM increased the sperm motility, viability, and membrane integrity and decreased the abnormality of spontaneous and catalyzed lipids peroxidation (p < 0.05) up to 72 h. In addition, semen diluted with 1 and 2% ETHEX decreased the level of DNA fragmentation compared to the control group (p < 0.05). In conclusion, the ETHEX could be recommended to improve the quality of liquid ram spermatozoa. However, its effects on artificial insemination should be further studied.

Topical reinforcement of the cervical mucus barrier to sperm.

Close to half of the world's pregnancies are still unplanned, reflecting a clear unmet need in contraception. Ideally, a contraceptive would provide the high efficacy of hormonal treatments, without systemic side effects. Here, we studied topical reinforcement of the cervical mucus by chitosan mucoadhesive polymers as a form of female contraceptive. Chitosans larger than 7 kDa effectively cross-linked human ovulatory cervical mucus to prevent sperm penetration in vitro. We then demonstrated in vivo using the ewe as a model that vaginal gels containing chitosan could stop ram sperm at the entrance of the cervical canal and prevent them from reaching the uterus, whereas the same gels without chitosan did not substantially limit sperm migration. Chitosan did not affect sperm motility in vitro or in vivo, suggesting reinforcement of the mucus physical barrier as the primary mechanism of action. The chitosan formulations did not damage or irritate the ewe vaginal epithelium, in contrast to nonoxynol-9 spermicide. The demonstration that cervical mucus can be reinforced topically to create an effective barrier to sperm may therefore form the technological basis for muco-cervical barrier contraceptives with the potential to become an alternative to hormonal contraceptives.

Cervical immune activation during the luteal phase may compromise subsequent trans-cervical ram sperm transport†.

Worldwide, cervical artificial insemination using frozen-thawed semen yields low pregnancy rates. The only exception to this is in Norway, where vaginal insemination with frozen-thawed semen yields pregnancy rates in excess of 60% and which has been attributed to the specific ewe breed used. Our previous work demonstrated differences in cervical gene expression at the follicular phase of the estrous cycle in ewe breeds with known differences in pregnancy rates. In this study, we characterized the cervical transcriptome of the same ewe breeds [Suffolk, Belclare, Fur, and Norwegian White Sheep (NWS)] during the luteal phase, as an optimal environment at the luteal phase could better prepare the cervix for sperm migration through the cervix at the subsequent follicular phase. High-quality RNA extracted from postmortem cervical tissue was analyzed by RNA sequencing. After stringent filtering, 1051, 1924, and 611 differentially expressed genes (DEGs) were detected in the low-fertility Suffolk breed compared with Belclare, Fur, and NWS, respectively. Gene ontology analysis identified increased humoral adaptive immune response pathways in Suffolk. Increased expression of multiple immune genes supports the presence of an active immune response in the cervix of Suffolk ewes, which differentiates them significantly from the other three ewe breeds. Inflammatory pathways were upregulated in the Suffolk, resulting in higher expression of the potent pro-inflammatory cytokines. Therefore, higher levels of pro-inflammatory cytokines indicate unresolved inflammation in the cervix of the low-fertility Suffolk breed that could contribute to reduced cervical sperm transport in the next follicular phase.

Ewe breed differences in the cervical transcriptome at the follicular phase of a synchronised oestrous cycle.

BACKGROUND: Cervical artificial insemination (AI) with frozen-thawed semen results in unacceptably low pregnancy rates internationally. The exception is in Norway, where vaginal deposition of frozen-thawed semen to a natural oestrous routinely yields pregnancy rates in excess of 70%. Previous studies by our group has demonstrated that this is due to differences in cervical sperm transport. However, a potentially important contributory factor is that ewes are inseminated to a natural oestrous in Norway but to a synchronised oestrous across most of the rest of the world. In this study, we interrogated the gene expression of the sheep cervix of four ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen under the effect of exogenous hormones to synchronise the oestrous cycle. These four ewe breeds (n = 8 to 11 ewes per breed) are from two countries: Ireland (Belclare and Suffolk; medium and low fertility, respectively) and Norway (Norwegian White Sheep (NWS) and Fur; both with high fertility compared to the Irish ewe breeds). RESULTS: RNA extracted from cervical biopsies collected from these breeds was analysed by RNA-sequencing and differential gene expression analysis. Using the low-fertility Suffolk breed as a reference level; 27, 1827 and 2641 genes were differentially expressed in Belclare, Fur and NWS ewes, respectively (P <  0.05 and FC > 1.5). Gene ontology (GO) analysis revealed that Fur and NWS had an up-regulation of enriched pathways involved in muscle contraction and development compared to Suffolk. However, there was a down-regulation of the immune response pathway in NWS compared to Suffolk. In addition, GO analysis showed similar expression patterns involved in muscle contraction, extracellular matrix (ECM) development and cell-cell junction in both Norwegian ewe breeds, which differed to the Irish ewe breeds. CONCLUSIONS: This novel study has identified a number of conserved and breed-specific biological processes under the effect of oestrous synchronisation that may impact cervical sperm transport during the follicular phase of the reproductive cycle.

Biochemical and molecular characterization of sialylated cervical mucins in sheep†.

Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen-thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway. Expression of mucin and sialic acid related genes was quantified using RNA-sequencing in cervical tissue from Suffolk, Belclare, Fur, and NWS only. Cervical tissue was also assessed for the percentage of cervical epithelial populated by mucin secreting goblet cells in the same four ewe breeds. Biochemical analysis showed that there was an effect of ewe breed on sialic acid species, which was represented by Suffolk having higher levels of Neu5,9Ac2 compared with NWS (P < 0.05). Suffolk ewes had a lower percentage of goblet cells than Fur and NWS (P < 0.05). Gene expression analysis identified higher expression of MUC5AC, MUC5B, ST6GAL1, and ST6GAL2 and lower expression of ST3GAL3, ST3GAL4, and SIGLEC10 in Suffolk compared with high fertility ewe breeds (P < 0.05). Our results indicate that specific alterations in sialylated mucin composition may be related to impaired cervical sperm transport.

Identification and characterization of O-linked glycans in cervical mucus as biomarkers of sperm transport: A novel sheep model.

Cervical mucus plays an important role in female fertility, since it allows the entry of motile and morphological normal sperm while preventing the ascent of pathogens from the vagina. The function of cervical mucus is critically linked to its rheological properties that are in turn dictated by O-glycosylated proteins, called mucins. We aimed to characterize the O-glycan composition in the cervical mucus of six European ewe breeds with known differences in pregnancy rates following cervical/vaginal artificial insemination with frozen-thawed semen, which are due to reported differences in cervical sperm transport. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway (n = 28-30 ewes/breed). We identified 124 O-glycans, from which 51 were the major glycans with core 2 and fucosylated glycans as the most common structures. The use of exogenous hormones for synchronization did not affect the O-glycan composition in both high-fertility ewe breeds, but it did in the other four ewe breeds. There was a higher abundance of the sulfated glycan (Galß1-3[SO3-GlcNAcß1-6]GalNAc), fucosylated glycan (GlcNAcß1-3(Fucα1-2Galß1-3)GalNAc) and core 4 glycan (GlcNAcß1-3[GlcNAcß1-6]GalNAc) in the low-fertility Suffolk breed compared with NWS (high fertility). In addition, core 4 glycans were negatively correlated with mucus viscosity. This novel study has identified O-glycans that are important for cervical sperm transport and could have applications across a range of species including human.

Conserved and breed-specific differences in the cervical transcriptome of sheep with divergent fertility at the follicular phase of a natural oestrus cycle.

BACKGROUND: The outcome of cervical artificial insemination (AI) with frozen-thawed semen in sheep is limited by the inability of sperm to traverse the cervix of some ewe breeds. Previous research has demonstrated that cervical sperm transport is dependent on ewe breed, as sperm can traverse the cervix in greater numbers in some higher fertility ewe breeds. However, the molecular mechanisms underlying ewe breed differences in sperm transport through the cervix remain unknown. In this study, we aimed to characterise the cervical transcriptome of four European ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen at the follicular phase of a natural oestrous cycle. Cervical post mortem tissue samples were collected from two Irish ewe breeds (Belclare and Suffolk; medium and low fertility, respectively) and from two Norwegian ewe breeds (Norwegian White Sheep (NWS) and Fur; high fertility compared to both Irish breeds) at the follicular phase of a natural oestrous cycle (n = 8 to 10 ewes per breed). RESULTS: High-quality RNA extracted from biopsies of the mid-region of the cervix was analysed by RNA-sequencing and Gene Ontology (GO). After stringent filtering (P <  0.05 and FC > 1.5), a total of 11, 1539 and 748 differentially expressed genes (DEGs) were identified in Belclare, Fur and NWS compared to the low fertility Suffolk breed, respectively. Gene ontology analysis identified significantly enriched biological processes involved in muscle contraction, extracellular matrix (ECM) development and the immune response. Gene co-expression analysis revealed similar patterns in muscle contraction and ECM development modules in both Norwegian ewe breeds, which differed to the Irish ewe breeds. CONCLUSIONS: These breed-specific biological processes may account for impaired cervical sperm transport through the cervix in sheep during the follicular phase of the reproductive cycle. This novel and comprehensive dataset provides a rich foundation for future targeted initiatives to improve cervical AI in sheep.

Sperm migration, selection, survival, and fertilizing ability in the mammalian oviduct†.

In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival, and acquisition of fertilizing ability in the oviduct is being considered under six aspects: (1) the utero-tubal junction that selects a sperm sub-population entering the oviduct; (2) the presence of sperm binding sites on luminal epithelial cells in the oviduct, which prolong sperm viability and plays a role in limiting polyspermic fertilization; (3) the contractions of the oviduct, which promote sperm migration toward the site of fertilization in the ampulla; (4) the regions of the oviduct, which play different roles in regulating sperm physiology and interactions with oviduct epithelial cells; (5) the time of ovulation, and (6) the steroid hormonal environment which regulates sperm release from the luminal epithelial cells and facilitates capacitation in a finely orchestrated manner.

Beta-nerve growth factor stimulates spontaneous electrical activity of in vitro embryonic mouse GnRH neurons through a P75 mediated-mechanism.

The control of ovulation helps guarantee the success of reproduction and as such, contributes to the fitness of a species. In mammals, two types of ovulation are observed: induced and spontaneous ovulation. Recent work on camelids, that are induced ovulators, highlighted the role of a factor present in seminal plasma, beta Nerve Growth Factor (ß-NGF), as the factor that triggers ovulation in a GnRH dependent manner. In the present work, we characterized alpaca ß-NGF (aß-NGF) and its 3D structure and compared it with human recombinant ß-NGF (hß-NGF). We showed that the ß-NGF enriched fraction of alpaca semen and the human recombinant protein, both stimulated spontaneous electrical activity of primary GnRH neurons derived from mouse embryonic olfactory placodes. This effect was dose-dependent and mediated by p75 receptor signaling. P75 receptors were found expressed in vitro by olfactory ensheathing cells (OEC) in close association with GnRH neurons and in vivo by tanycytes in close vicinity to GnRH fibers in adult mouse. Altogether, these results suggested that ß-NGF induced ovulation through an increase in GnRH secretion provoked by a glial dependent P75 mediated mechanism.

Sperm interactions with the female reproductive tract: A key for successful fertilization in mammals.

Sperm migration through the female genital tract is not a quiet journey. Uterine contractions quickly operate a drastic selection, leading to a very restrictive number of sperm reaching the top of uterine horns and finally, provided the presence of key molecules on sperm, the oviduct, where fertilization takes place. During hours and sometimes days before fertilization, subpopulations of spermatozoa interact with dynamic and region-specific maternal components, including soluble proteins, extracellular vesicles and epithelial cells lining the lumen of the female tract. Interactions with uterine and oviductal cells play important roles for sperm survival as they modulate the maternal immune response and allow a transient storage before ovulation. The body of work reported here highlights the importance of sperm interactions with proteins originated from both the uterine and oviductal fluids, as well as hormonal signals around the time of ovulation for sperm acquisition of fertilizing competence.

Circuit-level analysis identifies target genes of sex steroids in ewe seasonal breeding.

Thyroid hormone (TH) and estradiol (E2) direct seasonal switches in ovine reproductive physiology. In sheep, as in other mammals and birds, control of thyrotropin (TSH) production by the pars tuberalis (PT) links photoperiod responsiveness to seasonal breeding. PT-derived TSH governs opposite seasonal patterns of the TH deiodinases Dio2/Dio3 expression in tanycytes of the neighboring medio-basal hypothalamus (MBH), which explain the key role of TH. We recently used RNA-Seq to identify seasonal markers in the MBH and define the impact of TH. This impact was found to be quite limited, in terms of number of target genes, and very restricted with regards to neuroanatomical location, as TH specifically impacts genes expressed in tanycytes and hypothalamus, not in the PT. Here we address the impact of E2 on these seasonal markers, which are specifically expressed in either PT, tanycytes or hypothalamus. We also investigate if progesterone (P4) may be involved in timing the seasonal transition to anestrus. Our analysis provides circuit-level insights into the impact of sex steroids on the ewe seasonal breeding cycle. First, seasonal gene expression in the PT is independent of the sex steroid status. The fact that seasonal gene expression in the PT is also TH-independent strengthens the view that the PT is a circannual timer. Second, select tanycytic markers display some level of responsiveness to E2 and P4, which indicates another potential level of feedback control by sex steroids. Third, Kiss1 neurons of the arcuate nucleus are responsive to both TH and E2, which places them at the crossroads of photoperiodic transduction pathway and sex steroid feedback. This provides strong support to the concept that these Kiss1 neurons are pivotal to the long-recognized "seasonal switch in the ability of E2 to exert negative feedback", which drives seasonal breeding.

Intact cell MALDI-TOF mass spectrometry, a promising proteomic profiling method in farm animal clinical and reproduction research.

The objective of this review is to provide new insights into the possible use of a proteomic method known as Intact Cell Matrix-Assisted Laser Desorption-ionization Time-Of-Flight Mass Spectrometry (ICM-MS) in animal clinical research. Here, we give an overview of the basics of this technique, its advantages and disadvantages compared with other proteomic approaches, past applications and future perspectives. A special emphasis on its implementation in animal reproduction science is given, including examples of the reliable use of ICM-MS on fertility screening. In mammals, the ICM-MS profiles from pig epididymal spermatozoa reflect the proteome changes that they undergo during epididymal maturation and could be associated with the acquisition of fertilizing ability. In chicken, using adequate pre-processing and bioinformatics analysis tools, sperm ICM-MS profiles showed characteristic spectral features that allowed their classification according to their actual fertilizing ability. The association of ICM-MS and Top-down proteomic strategies allowed the identification of chicken fertility biomarkers candidates such as protein vitelline membrane outer layer protein 1 (VMO-1) and avian beta-defensin 10 (AvBD10). In female reproduction, a similar approach on ovarian follicular cells allowed the identification of specific markers of oocyte maturation in the oocyte and surrounding cumulus cells. Altogether, these results indicate that ICM-MS profiling could be a suitable approach for molecular phenotyping of male and female gametes.

Bisphenol S Impaired In Vitro Ovine Early Developmental Oocyte Competence.

INTRODUCTION: Bisphenol A (BPA) is a widespread compound in the plastic industry that is especially used to produce baby bottles, food packaging and metal cans. BPA, an endocrine disruptor, leads to alterations in reproductive function and therefore has been banned from the food industry. Unregulated BPA analogues, particularly Bisphenol S (BPS), have emerged and are now used in the plastic industry. Thus, this study aimed to examine the acute effects of low and environmental doses of BPS on ewe oocyte quality and developmental competence, and its mechanism of action, during in vitro maturation. METHODS: Ewe cumulus-oocyte complexes underwent in vitro maturation in the presence or absence of BPS (1 nM, 10 nM, 100 nM, 1 µM or 10 µM). Oocytes were then subjected to in vitro fertilisation and development. RESULTS: 1 µM BPS induced a 12.7% decrease in the cleavage rate (p = 0.004) and a 42.6% decrease in the blastocyst rate (p = 0.017) compared to control. The blastocyst rate reduction was also observed with 10 nM BPS. Furthermore, 10 µM BPS reduced the oocyte maturation rate, and 1 µM BPS decreased cumulus cell progesterone secretion. PR and AMH gene expression were reduced in cumulus cells. BPS induced a 5-fold increase in MAPK 3/1 activation (p = 0.04). CONCLUSIONS: BPS impaired ewe oocyte developmental competence. The data suggest that BPS might not be a safe BPA analogue. Further studies are required to elucidate its detailed mechanism of action.

Progesterone induces sperm release from oviductal epithelial cells by modifying sperm proteomics, lipidomics and membrane fluidity.

The sperm reservoir is formed after insemination in mammals, allowing sperm storage in the oviduct until their release. We previously showed that physiological concentrations of progesterone (P4) trigger in vitro the sperm release from bovine oviductal epithelial cells (BOECs), selecting a subpopulation of spermatozoa with a higher fertilizing competence. Here, by using Western-Blot, confocal microscopy and Intact Cell MALDI-TOF-Mass Spectrometry strategies, we elucidated the changes derived by the P4-induced release on sperm cells (BOEC-P4 spz). Our findings show that, compared to controls, BOEC-P4 spz presented a decrease in the abundance of Binder of Sperm Proteins (BSP) -3 and -5, suggesting one mechanism by which spermatozoa may detach from BOECs, and thus triggering the membrane remodeling with an increase of the sperm membrane fluidity. Furthermore, an interesting number of membrane lipids and proteins were differentially abundant in BOEC-P4 spz compared with controls.

Seminal plasma proteins as markers of sperm fertility.

During ejaculation and deposition in the female genital tract, spermatozoa are exposed to seminal plasma, a mix of secretions primarily from the accessory sex glands. Proteins, which make up the largest contribution to seminal plasma by weight, have been the focus of much interest, in particular the identification of specific proteins both in the plasma and/or found bound to the sperm surface post ejaculation. Global proteomic studies of seminal plasma originating from a range of species over the last 15 years have revealed their hitherto unknown diversity and complexity. Seminal plasma is generally known to aid sperm survival and fertility in a range of species and studies have begun to reveal its link with sperm function and identification, as markers of fertility. This review summarises recent data on proteins found on the sperm surface that originate from seminal plasma and have subsequently been shown to correlate with fertility, with a focus on the pig.

Seminal plasma differentially alters the resistance of dog, ram and boar spermatozoa to hypotonic stress.

During ejaculation and the deposition in the female genital tract, spermatozoa undergo hypo-osmotic stress and need to withstand it for optimal fertility. Resistance to hypo-osmotic stress may be affected by the interaction of the spermatozoa with seminal fluid components. The hypo-osmotic resistance of epididymal and ejaculated spermatozoa from dogs, rams and boars was assessed by flow cytometric measurement of sperm viability after incubation in NaCl solutions with osmolalities ranging from 0 to 300 mmol/kg. The hypotonic resistance of epididymal spermatozoa was greater than those of ejaculated spermatozoa in all three species. Among species comparison revealed that ejaculated spermatozoa from dogs were much more resistant than those from rams and boars as 80.4 ±â€¯5.3%, 56.7 ±â€¯4.7 and 9.6 ±â€¯3.6% of live spermatozoa were observed following exposure to an osmolality of 90 mmol/kg in dogs, rams and boars respectively. This can be explained by the fact that dog, ram and boar differ markedly in composition of the seminal plasma owing to the presence (ram, boar) or absence (dog) of seminal vesicles. Hypotonic resistance of epididymal and ejaculated dog spermatozoa was similar whereas ram and boar spermatozoa showed a marked drop in resistance after ejaculation. The in vitro incubation of boar epididymal spermatozoa with raw seminal plasma or the seminal plasma protein fraction induced a similar loss of resistance, suggesting that seminal proteins are involved in the lack of resistance to hypotonic stress of boar ejaculated spermatozoa.

Seminal plasma proteomes and sperm fertility.

During ejaculation, the spermatozoa are transported by the seminal plasma, a fluid resulting from secretions originating mainly from the prostate and the seminal vesicles in mammals. The interaction of the seminal plasma with spermatozoa induces binding of seminal proteins onto the sperm surface and membrane remodeling potentially impacting the sperm transport, survival and fertilizing ability in the female genital tract. The seminal plasma also contains peptides and proteins involved in the inflammatory and immune response of the female tract. Therefore the seminal plasma proteome has been investigated in a large range of taxa, including mammals, birds, fishes and insect species. The association of the seminal plasma with semen preservation or fertility identified proteic markers of seminal plasma function in domestic species. This review summarizes the current knowledge in seminal plasma proteomes and proteic markers of sperm preservation in animal species.

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage.

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

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.