Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Milk provisioning in oviparous caecilian amphibians.

Among vertebrates, the yolk is commonly the only form of nutritional investment offered by the female to the embryo. Some species, however, have developed parental care behaviors associated with specialized food provisioning essential for offspring survival, such as the production of lipidic-rich parental milk in mammals. Here, we show that females of the egg-laying caecilian amphibian Siphonops annulatus provide similarly lipid-rich milk to altricial hatchlings during parental care. We observed that for 2 months, S. annulatus babies ingested milk released through the maternal vent seemingly in response to tactile and acoustic stimulation by the babies. The milk, composed mainly of lipids and carbohydrates, originates from the maternal oviduct epithelium's hypertrophied glands. Our data suggest lactation in this oviparous nonmammalian species and expand the knowledge of parental care and communication in caecilians.

Oviductal sperm storage in the Chinese pond turtle, Mauremys reevesii, depends on androgen-based promotion of the BCL 2 anti-apoptotic pathway.

CONTEXT: Sperm storage is a complex and highly coordinated process that is regulated by a variety of factors. The BCL 2 protein family plays a key role in regulating apoptosis, and determines sperm survival. AIMS: The objective of this study was to explore the correlation between sperm storage and the BCL 2 protein family in the oviduct of Mauremys reevesii . METHODS: Hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) techniques were used to investigate three parts of the reproductive tract (isthmus, uterus and vagina) of mated and unmated female M. reevesii . KEY RESULTS: Hematoxylin-eosin staining revealed many sperm stored in the oviduct. IHC showed positive immunostaining for the BCL 2 and BAX proteins in epithelial ciliated and glandular cells. RT-qPCR indicated that the mRNA expressions of anti-apoptotic genes (BCL 2 , MCL 1 , BCL- W , BCL-XL ) and the androgen receptor (AR) were significantly higher in mated turtles than unmated turtles. However, the expression of pro-apoptotic genes (BAX , BAD , BID and CASPASE 3 ) showed the opposite relationship. CONCLUSIONS: These results suggest that sperm entering the oviduct can promote the synthesis of anti-apoptotic genes to protect themselves from various degradation factors. IMPLICATIONS: These findings will help researchers understand the mechanisms of sperm storage.

Optical coherence tomography for dynamic investigation of mammalian reproductive processes.

The biological events associated with mammalian reproductive processes are highly dynamic and tightly regulated by molecular, genetic, and biomechanical factors. Implementation of live imaging in reproductive research is vital for the advancement of our understanding of normal reproductive physiology and for improving the management of reproductive disorders. Optical coherence tomography (OCT) is emerging as a promising tool for dynamic volumetric imaging of various reproductive processes in mice and other animal models. In this review, we summarize recent studies employing OCT-based approaches toward the investigation of reproductive processes in both, males and females. We describe how OCT can be applied to study structural features of the male reproductive system and sperm transport through the male reproductive tract. We review OCT applications for in vitro and dynamic in vivo imaging of the female reproductive system, staging and tracking of oocytes and embryos, and investigations of the oocyte/embryo transport through the oviduct. We describe how the functional OCT approach can be applied to the analysis of cilia dynamics within the male and female reproductive systems. We also discuss the areas of research, where OCT could find potential applications to progress our understanding of normal reproductive physiology and reproductive disorders.

Dynamics of gametes and embryos in the oviduct: what can in vivo imaging reveal?

In brief: In vivo imaging of gametes and embryos in the oviduct enables new studies of the native processes that lead to fertilization and pregnancy. This review article discusses recent advancements in the in vivo imaging methods and insights which contribute to understanding the oviductal function. Abstract: Understanding the physiological dynamics of gametes and embryos in the fallopian tube (oviduct) has significant implications for managing reproductive disorders and improving assisted reproductive technologies. Recent advancements in imaging of the mouse oviduct in vivo uncovered fascinating dynamics of gametes and embryos in their native states. These new imaging approaches and observations are bringing exciting momentum to uncover the otherwise-hidden processes orchestrating fertilization and pregnancy. For mechanistic investigations, in vivo imaging in genetic mouse models enables dynamic phenotyping of gene functions in the reproductive process. Here, we review these imaging methods, discuss insights recently revealed by in vivo imaging, and comment on emerging directions, aiming to stimulate new in vivo studies of reproductive dynamics.

The oocyte cumulus complex regulates mouse sperm migration in the oviduct.

As the time of ovulation draws near, mouse spermatozoa move out of the isthmic reservoir, which is a prerequisite for fertilization. However, the molecular mechanism remains unclear. The present study revealed that mouse cumulus cells of oocytes-cumulus complexes (OCCs) expressed transforming growth factor-ß ligand 1 (TGFB1), whereas ampullary epithelial cells expressed the TGF-ß receptors, TGFBR1 and TGFBR2, and all were upregulated by luteinizing hormone (LH)/human chorionic gonadotropin (hCG). OCCs and TGFB1 increased natriuretic peptide type C (NPPC) expression in cultured ampullae via TGF-ß signaling, and NPPC treatment promoted spermatozoa moving out of the isthmic reservoir of the preovulatory oviducts. Deletion of Tgfb1 in cumulus cells and Tgfbr2 in ampullary epithelial cells blocked OCC-induced NPPC expression and spermatozoa moving out of the isthmic reservoir, resulting in compromised fertilization and fertility. Oocyte-derived paracrine factors were required for promoting cumulus cell expression of TGFB1. Therefore, oocyte-dependent and cumulus cell-derived TGFB1 promotes the expression of NPPC in oviductal ampulla, which is critical for sperm migration in the oviduct and subsequent fertilization.

Histology, ultrastructure, and differential gene expression in relation to seasonal sperm storage in the oviduct of the Chinese alligator, Alligator sinensis.

Although oviductal sperm storage are essential steps in reproduction for female animals with internal fertilisation, no systematic study on the identification of genes involving sperm storage has been performed in crocodilian species. In the present research, the relationship between morphological variation related to sperm storage in the oviduct and gene expression patterns derived from RNA sequencing analyses between active period (AP), breeding period (BP), and hibernation period (HP) were investigated. The corresponding results indicated that sperm were observed not only in the ciliated cells within infundibulum and mucosal layer of uterus during BP, but also been detected in the spermatosperm storage tube (SST) in the anterior uterus at HP stage. The further transmission electron microscopy analysis indicated that the differences in the number and activity of the secretory cells likely to attributed to the seasonal variation of microenvironment related to the sperm storage. Based on the RNA-sequecing, 13147 DEGs related to the Peroxisome proliferator-activated receptors (PPARs) and FOXO signalling were identified, including these, the down-regulated ATG12 and BCL2L11 in the HP group may thus constitute an important point of convergence between autophagy and apoptosis involving the FOXO1 pathway. The genes involved in the PPARs pathway might modulate the immune response and thereby contribute to prolong the life span of stored spermatozoa in Alligator sinensis . The outcomes of this study provide fundamental insights into the mechanism of sperm storage in A. sinensis .

Transcriptome analysis of inseminated sperm storage tubules throughout the duration of fertility in the domestic turkey, Meleagris gallopavo.

Sperm storage tubules (SST) are specialized invaginations of the oviductal epithelium that permit avian species to store spermatozoa for extended periods of time, without compromising sperm fertilization capacity. The molecular and physiological mechanisms behind sperm storage tubule differentiation, sperm protection, and regression remain largely unknown, but most likely have potential implications for substantially improving hen fertility, sperm storage, and semen cryopreservation in commercial poultry species. RNA sequencing was performed on sperm storage tubules isolated from the epithelium of the uterovaginal junction (UVJ) from hens at d 1, 7, 30, 60, and 90 postinsemination (n = 4 per timepoint). Read mapping and differential expression analysis were performed using CLC Genomics Workbench. A total of 2,340 differentially expressed genes were subjected to pathway analysis through Ingenuity Pathway Analysis (IPA). Through functional annotation of differentially expressed genes during early, peak, and late egg production, novel insights regarding the role of innate and acquired immune response to sperm, lipid synthesis and transfer, steroid hormone signalling, cytoskeletal reorganization, and regulation of ion homeostasis in SST were obtained. Additionally, potential pathways were identified that could be involved with suppressing sperm motility while sperm reside within the SST. Upstream analysis identified potential regulatory roles for 18 upstream regulators that could modulate sperm storage tubule function, including suppression of sperm motility. Understanding sperm storage tubule function throughout the laying cycle, especially with regards to sperm preservation may allow for the development of industry-based protocols for semen storage and cryopreservation that mimic the sperm preservation capabilities of SST and improve fertility.

Integrated multiple transcriptomes in oviductal tissue across the porcine estrous cycle reveal functional roles in oocyte maturation and transport.

Understanding the changes in the swine female reproductive system is important for solving issues related to reproductive failure and litter size. Elucidating the regulatory mechanisms of the natural estrous cycle in the oviduct under non-fertilisation conditions can improve our understanding of its role in the reproductive system. Herein, whole transcriptome RNA sequencing of oviduct tissue samples was performed. The differentially expressed genes (DEGs) were identified for each time point relative to day 0 and classified into three clusters based on their expression patterns. Clusters 1 and 2 included genes involved in the physiological changes through the estrous cycle. Cluster 1 genes were mainly involved in PI3K-Akt signaling and steroid hormone biosynthesis pathways. Cluster 2 genes were involved in extracellular matrix-receptor interactions and protein digestion pathways. In Cluster 3, the DEGs were downregulated in the luteal phase; they were strongly associated with cell cycle, calcium signaling, and oocyte meiosis. The gene expression in the oviduct during the estrous cycle influenced oocyte transport and fertilization. Our findings provide a basis for successfully breeding pigs and elucidating the mechanisms underlying the changes in the pig oviduct during the estrous cycle.

Understanding the swine female reproductive system is important for solving issues related to reproductive failure and litter size. The oviduct is the site of fertilization. After fertilization, the fertilized egg moves to the uterus for implantation. Elucidating the regulatory mechanisms of the estrous cycle in the oviduct can improve our understanding of their roles. In this study, whole transcriptome RNA sequencing of oviduct tissue samples was performed throughout the estrous cycle to screen for differentially expressed genes (DEGs). The DEGs were classified into three clusters based on their expression patterns. Clusters 1 and 2 included genes involved in the physiological changes observed through the estrous cycle. The expression levels of Cluster 3 genes were downregulated specifically in the luteal phase; this was associated with calcium signalling and oocyte meiosis. In this study, we identified that the expression of genes in the oviduct influences oocyte transport and fertilization, which are the key functions of the oviduct. This study provides a basis for successful breeding in the pig industry and elucidating the mechanisms underlying the changes in the pig oviduct during the estrous cycle.

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.

Oviductal motile cilia are essential for oocyte pickup but dispensable for sperm and embryo transport.

Mammalian oviducts play an essential role in female fertility by picking up ovulated oocytes and transporting and nurturing gametes (sperm/oocytes) and early embryos. However, the relative contributions to these functions from various cell types within the oviduct remain controversial. The oviduct in mice deficient in two microRNA (miRNA) clusters (miR-34b/c and miR-449) lacks cilia, thus allowing us to define the physiological role of oviductal motile cilia. Here, we report that the infundibulum without functional motile cilia failed to pick up the ovulated oocytes. In the absence of functional motile cilia, sperm could still reach the ampulla region, and early embryos managed to migrate to the uterus, but the efficiency was reduced. Further transcriptomic analyses revealed that the five messenger ribonucleic acids (mRNAs) encoded by miR-34b/c and miR-449 function to stabilize a large number of mRNAs involved in cilium organization and assembly and that Tubb4b was one of their target genes. Our data demonstrate that motile cilia in the infundibulum are essential for oocyte pickup and thus, female fertility, whereas motile cilia in other parts of the oviduct facilitate gamete and embryo transport but are not absolutely required for female fertility.

Spatiotemporal transcriptional dynamics of the cycling mouse oviduct.

Female fertility in mammals requires iterative remodeling of the entire adult female reproductive tract across the menstrual/estrous cycle. However, while transcriptome dynamics across the estrous cycle have been reported in human and bovine models, no global analysis of gene expression across the estrous cycle has yet been reported for the mouse. Here, we examined the cellular composition and global transcriptional dynamics of the mouse oviduct along the anteroposterior axis and across the estrous cycle. We observed robust patterns of differential gene expression along the anteroposterior axis, but we found surprisingly few changes in gene expression across the estrous cycle. Notable gene expression differences along the anteroposterior axis included a surprising enrichment for genes related to embryonic development, such as Hox and Wnt genes. The relatively stable transcriptional dynamics across the estrous cycle differ markedly from other mammals, leading us to speculate that this is an evolutionarily derived state that may reflect the extremely rapid five-day mouse estrous cycle. This dataset fills a critical gap by providing an important genomic resource for a highly tractable genetic model of mammalian female reproduction.

Cell-type specific analysis of physiological action of estrogen in mouse oviducts.

One of the endogenous estrogens, 17ß-estradiol (E2 ) is a female steroid hormone secreted from the ovary. It is well established that E2 causes biochemical and histological changes in the uterus. However, it is not completely understood how E2 regulates the oviductal environment in vivo. In this study, we assessed the effect of E2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single-cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E2 did not drastically alter the transcriptomic profile from that of endogenous E2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E2 -target gene in the mouse oviduct and was also expressed in human fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types, including epithelial, stromal, and muscle cells, are differentially regulated by E2 and support gene expression changes, such as growth factors that are required for normal embryo development and transport in mouse models. Furthermore, we have identified cell-specific and region-specific gene markers for targeted studies and functional analysis in vivo.

Effect of sperm surface oligosaccharides in sperm passage into sperm storage tubules in Japanese quail (Coturnix japonica).

In birds, the ejaculated spermatozoa do not directly pass to the site of fertilization but rather are stored initially in specialized structures, referred to as sperm storage tubules (SSTs), located in the utero-vaginal junction (UVJ) of the oviduct. The fertilizing capacity of spermatozoa in the SSTs is maintained for an extended period (i.e., several days to months). Although many studies have been conducted to ascertain the mechanisms involved in sperm storage, the understanding of the phenomenon is limited. In this study, there was investigation of the effects of sperm surface oligosaccharides in sperm passage into SSTs in Japanese quail. Results from lectin staining of ejaculated spermatozoa indicated galactose/N-Acetylgalactosamine (Gal/GalNAc), N-Acetylglucosamine (GlcNAc) or mannose/glucose (Man/Glc) moieties were present on the sperm surface, indicating the presence of glycoproteins/glycolipids containing these oligosaccharides. When ejaculated spermatozoa were co-incubated with UVJ explants, the lectins derived from Agaricus bisporus and Canavalia ensiformis had marked inhibitory effects on sperm passage into SSTs. Preincubation of UVJ explants with these lectins, however, had no effect indicating there were no effects of UVJ oligosaccharides in this process. Furthermore, none of these lectin had effects on values of sperm motility variables. These results indicate that O-glycans with terminal ß-Gal or GalNAc and N-glycans with terminal α-D-Man or α-D-Glc may have functions in the process of sperm passage into SSTs.

Oviductal fluid counterbalances the negative effect of high temperature on sperm in an ectotherm model.

Global warming is affecting biodiversity; however, the extent to which animal reproductive processes respond to predicted temperature increments remains largely unexplored. The thermal environment has a pronounced impact on metabolic rates of ectotherms; therefore, an interesting question to assess is whether temperature increase might affect specific reproductive mechanisms like sperm performance in ectotherms. Moreover, in many species, oviductal fluid (OF) is known to regulate and maintain sperm quality; however, the role of OF in relation to the effects of high temperature on sperm remains unclear. Our aim was to experimentally test the effect of increased temperature on sperm velocity, swimming path and percentage of motility in neutral conditions at ejaculation (without OF) and in female's reproductive tract fluid (with OF), in a social ectotherm lizard model, Tropidurus spinulosus, which has specific thermal requirements for reproduction. Our results suggest that a rising temperature associated with global warming (+4°C) affects negatively sperm dynamics and survival. However, OF ameliorated the harmful effects of high temperature. This is an important point, as this study is the first to have tested the role of OF in preserving sperm from a warmer pre-fertilization environment. These results contribute to our understanding of how thermal environment changes might affect post-copulatory reproductive mechanisms. This article has an associated First Person interview with the first author of the paper.

Possibility of Undeveloped Egg Absorption during Embryogenesis: A Unique Phenomenon Observed in the Ovoviviparous Mayfly Cloeon dipterum.

Oviparous, ovoviviparous and viviparous reproduction are interesting subjects for understanding animals' evolutionary pathways and adaptation to their life history and habitat conditions. In this study, we examined the reproductive mode of the ovoviviparous mayfly Cloeon dipterum, particularly comparing embryogenesis between hand-pairing and unmated females' common oviduct. Our study suggested that the high developmental rate of C. dipterum observed in a recent study could be ascribed to their absorption of unfertilized eggs. The developmental rates of hand-paired females were almost 100%, while their egg-bearing numbers were lower than those of virgin females. Thus, such reduced egg numbers suggest the maternal absorption of unfertilized eggs. This trait is thought to have evolved with the ovoviviparous characteristics of C. dipterum. We identified the basis of the irregularity of this species exhibiting such a high (i.e., 100%) developmental rate in our previous recent study.

Progesterone induces porcine sperm release from oviduct glycans in a proteasome-dependent manner.

In mammals, the oviduct retains sperm, forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and secondly, if the ubiquitin-proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and channeled with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein, we found that the release of sperm from an immobilized oviduct glycan, a six-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis-X trisaccharide. Thus, CatSper may be required for release of sperm from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release requires both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.

Transcriptome analysis and identification of age-associated fertility decreased genes in hen uterovaginal junction.

Avian sperm storage tubules (SSTs), which are located in the uterovaginal junction (UVJ) of the oviduct, are primary sperm storage sites after mating or artificial insemination. The mechanism underlying reduced sperm storage efficiency of SSTs which is highly correlated with decreased fertility rates in aged laying breeders remains largely unclear. Here, comparative transcriptomic analysis between the aged and young White Leghorn hens (120 vs. 30 wk) was applied to identify gene expression changes of UVJs containing SSTs. Bioinformatics analysis revealed 567 upregulated and 1998 downregulated differentially expressed genes. Gene ontology analysis was highly enriched in terms of immune system, cell adhesion, and cytoskeleton proteins. Kyoto Encyclopedia of Genes and Genomes analysis revealed 5 significant (P < 0.05) pathways including inositol phosphate and glycerophospholipid metabolism. ß-Galactosidase staining of chicken UVJ sections suggested increased cell senescence via aging. Oil Red O staining and immunohistochemistry detection of ADFP both confirmed distribution of lipid droplets in SST cells with increased intensity in aged breeders. The lipid synthesis and metabolism-related genes represented by TFAP2 and PLD1 were differentially expressed in aged laying breeders. The upregulation of IL15 and downregulation of a large number of immune-related genes in aged breeders indicate altered immune homeostasis in UVJs and SSTs. The increased accumulation of lipids, and altered immunity homeostasis, combined with other factors (TJP1, MYL9, AFDN, and RPL13, etc.) are potentially dominant effectors to decrease the sperm storage efficiency and egg fertility in aged laying breeders.

Epididymal and ejaculated sperm functionality is regulated differently by periovulatory oviductal fluid in pigs.

BACKGROUND: The current results of in vitro reproduction techniques in pigs, such as in vitro fertilization (IVF) and embryo development, show high performance with both epididymal and ejaculated spermatozoa. However, the results using ejaculated spermatozoa are even better. Ejaculated spermatozoa are exposed to the secretions of the accessory seminal glands: the seminal plasma (SP). It has been reported that exposure of spermatozoa to reproductive fluids, such as SP or periovulatory oviductal fluid (pOF), modulates sperm functionality both in vivo and in vitro. But whether or not this modulating effect of pOF depends on the origin of the spermatozoa being epididymal or ejaculated, is still unknown. OBJECTIVES: To determine and compare the effect of pOF on epididymal and ejaculated sperm functionality. MATERIAL AND METHODS: The effects of incubating spermatozoa from the epididymis and ejaculate with pOF in capacitating conditions were investigated by analyzing sperm motility, phosphorylation of protein kinase A substrates and proteins in tyrosine (pPKAs and pTyr, respectively), the interaction of the spermatozoa with the oocyte in IVF and intracytoplasmic sperm injection (ICSI), and, finally, the spermatozoa chromatin condensation status. RESULTS: The pOF modified events related to capacitation in epididymal spermatozoa by decreasing motility, pPKAs and pTyr. In the interaction with the oocyte after sperm capacitation, pOF regulated the epididymal and ejaculated spermatozoa differently. While pOF decreased the number of spermatozoa bound to the zona pellucida (Spz/ZP) and increased oocyte activation after ICSI with epididymal spermatozoa, with the ejaculated spermatozoa, it decreased the mean number penetrating each oocyte (Spz/O). Additionally, pOF significantly increased the nuclear decondensation of the epididymal spermatozoa after the fertilization of the oocyte. CONCLUSION: The modulation of sperm functionality by pOF is conditioned by the origin of the spermatozoa.

Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting.

To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.