Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting
ABSTRACT: 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?
Project description:<h4>Background</h4>Sex allocation of offspring in mammals is usually considered as a matter of chance, being dependent on whether an X- or a Y-chromosome-bearing spermatozoon reaches the oocyte first. Here we investigated the alternative possibility, namely that the oviducts can recognise X- and Y- spermatozoa, and may thus be able to bias the offspring sex ratio.<h4>Results</h4>By introducing X- or Y-sperm populations into the two separate oviducts of single female pigs using bilateral laparoscopic insemination we found that the spermatozoa did indeed elicit sex-specific transcriptomic responses. Microarray analysis revealed that 501 were consistently altered (P-value < 0.05) in the oviduct in the presence of Y-chromosome-bearing spermatozoa compared to the presence of X-chromosome-bearing spermatozoa. From these 501 transcripts, 271 transcripts (54.1%) were down-regulated and 230 transcripts (45.9%) were up-regulated when the Y- chromosome-bearing spermatozoa was present in the oviduct. Our data showed that local immune responses specific to each sperm type were elicited within the oviduct. In addition, either type of spermatozoa elicits sex-specific signal transduction signalling by oviductal cells.<h4>Conclusions</h4>Our data suggest that the oviduct functions as a biological sensor that screens the spermatozoon, and then responds by modifying the oviductal environment. We hypothesize that there might exist a gender biasing mechanism controlled by the female.
Project description:Mating or cervical deposition of spermatozoa or seminal plasma (SP) modifies the expression of genes affecting local immune defense processes at the oviductal sperm reservoir in animals with internal fertilization, frequently by down-regulation. Such responses may occur alongside sperm transport to or even beyond the reservoir. Here, immune-related gene expression was explored with cDNA microarrays on porcine cervix-to-infundibulum tissues, pre-/peri-ovulation. Samples were collected 24 h post-mating or cervical deposition of sperm-peak spermatozoa or SP (from the sperm-peak fraction or the whole ejaculate). All treatments of this interventional study affected gene expression. The concerted action of spermatozoa and SP down-regulated chemokine and cytokine (P00031), interferon-gamma signaling (P00035), and JAK/STAT (P00038) pathways in segments up to the sperm reservoir (utero-tubal junction (UTJ)/isthmus). Spermatozoa in the vanguard sperm-peak fraction (P1-AI), uniquely displayed an up-regulatory effect on these pathways in the ampulla and infundibulum. Sperm-free SP, on the other hand, did not lead to major effects on gene expression, despite the clinical notion that SP mitigates reactivity by the female immune system after mating or artificial insemination.
Project description:Sperms being foreign to the female are to be promptly eliminated by the female local immune defense. However, avoiding the local immune defense sperm can be stored for lenthy period in the oviductal sperm reservoir. It is currently unknown whether oviductal sperm reservoirs changes their gene expression to tolerate the spermatozoa after mating or sperm free seminal plasma infusion. Therefore this was tested using Swedish Landrace pigs in this study using cDNA microarray. We used 12 sows seperated into three groups- either oestrus sows were inseminated with 50 ml BTS (control, n=4) or mated with boars (treatment 1, n=4) or inseminated with sperm-free seminal plasma (treatment 2, n=4). The utero-tubal junction was retrieved within 24 h of treatment by operation.
Project description:In order to better understand how sperm movement is regulated in the oviduct, we mated wild-type female mice with Acr-EGFP males that produce sperm with fluorescent acrosomes. The fluorescence improved our ability to detect sperm within the oviduct. Oviducts were removed shortly before or after ovulation and placed in chambers on a warm microscope stage for video recording. Hyperactivated sperm in the isthmic reservoir detached frequently from the epithelium and then reattached. Unexpectedly, most sperm found in the ampulla remained bound to epithelium throughout the observation period of several minutes. In both regions, most sperm produced deep flagellar bends in the direction opposite the hook of the sperm head. This was unexpected, because mouse sperm incubated under capacitating conditions in vitro primarily hyperactivate by producing deep flagellar bends in the same direction as the hook of the head. In vitro, sperm that are treated with thimerosal to release Ca(2+) from internal stores produce deep anti-hook bends; however, physical factors such as viscous oviduct fluid could also have influenced bending in oviductal sperm. Some sperm detached from epithelium in both the ampulla and isthmus during strong contractions of the oviduct. Blockage of oviduct contractions with nicardipine, however, did not stop sperm from forming a storage reservoir in the isthmus or prevent sperm from reaching the ampulla. These observations indicate that sperm continue to bind to oviductal epithelium after they leave the isthmic reservoir and that sperm motility is crucial in the transport of sperm to the fertilization site.
Project description:Mammalian spermatozoa undergo selective movement along the isthmus of the oviduct to the ampulla during ovulation, which is a prerequisite for fertilization. The factor(s) that involves in selective spermatozoa movement is still unknown. In this study, we found that the oviductal epithelium in mouse ampulla expressed high levels of natriuretic peptide type C (NPPC) in the presence of ovulated oocyte-cumulus complexes (OCCs). Spermatozoa expressed NPPC receptor natriuretic peptide receptor 2 (NPR2, a guanylyl cyclase) on the midpiece of flagellum. NPPC increased intracellular levels of cGMP and Ca<sup>2+</sup> of spermatozoa, and induced sperm accumulation in the capillary by attraction. Importantly, spermatozoa from Npr2 mutant mice were not attracted by NPPC, preventing fertilization in vivo. Oocyte-derived paracrine factors promoted the expression of Nppc mRNA in the ampulla. Therefore, NPPC secreted by oviductal ampulla attracts spermatozoa towards oocytes, which is essential for fertilization.
Project description:Gene Expression Microarray technology was used to compare oviduct transcriptome between inseminated and non-inseminated pigs during spontaneous oestrus. We used an in vivo model approaching the study from a physiological point of view in which no hormonal treatment (animals were in natural oestrus) and no artificial sperm selection (selection was performed within the female genital) were imposed. It is therefore emphasised that no surgical introduction of spermatozoa and no insemination at a site other than the physiological one were used. This approach revealed 17 genes that were two-fold or more up-regulated in oviducts exposed to spermatozoa and/or developing embryos and 9 genes that were two-fold or more down-regulated. These changes would indicate a modification of the environment preparing the oviduct for a successful fertilization and for an adequate embryo early development. The objective of this study was to investigate differences in oviductal transcriptome between inseminated and non-inseminated pigs during spontaneous oestrus in a specific area of the oviduct (ampullary-isthmic junction). We decided to analyse this specific part of the oviduct where spermatozoa released from sperm reservoir arrive close to the time of ovulation because it is where fertilization and zygote formation occurs. We used 5 artificially inseminated and 5 non-inseminated pigs during spontaneous oestrus. Inseminations were performed with seminal doses at a concentration of 3 x 107 spermatozoa/ml. Complete oviductal tissue samples from the ampullary-isthmic junction (sample size >1cm including the end of the ampulla) were taken from each animal and immediately frozen in liquid nitrogen for mRNA extraction.
Project description:Sperm storage in vivo extends the time window for fertilisation in several animal species, from a few days to several years. The underlying storage mechanisms, however, are largely unknown. In this study, spermatozoa from the epididymis and oviduct of Chinese soft-shelled turtles were investigated to identify potentially relevant morphological features and transformations at different stages of sperm storage. Large cytoplasmic droplets (CDs) containing lipid droplets (LDs) were attached to the midpiece of most spermatozoa in the epididymis, without migrating down the sperm tail. However, they were absent from the oviductal spermatozoa, suggesting that CDs with LDs may be a source of endogenous energy for epididymal spermatozoa. The onion-like mitochondria recovered their double-membrane morphology, with typical cristae, within the oviduct at a later stage of storage, thus implying that mitochondrial metabolism undergoes alterations during storage. Furthermore, a well developed fibrous sheath on the long principal piece was the integrating ultrastructure for glycolytic enzymes and substrates. These novel morphological characteristics may allow turtle spermatozoa to use diverse energy metabolism pathways at different stages of storage.
Project description:Spermatozoa need to conduct a series of biochemical changes termed capacitation in order to fertilize. In vivo, capacitation is sequentially achieved during sperm transport and interaction with the female genital tract, by mechanisms yet undisclosed in detail. However, when boar spermatozoa are stored in the tubal reservoir pre-ovulation, most appear to be in a non-capacitated state. This study aimed at deciphering the transcriptomics of capacitation-related genes in the pig pre-ovulatory oviduct, following the entry of semen or of sperm-free seminal plasma (SP). Ex-vivo samples of the utero-tubal junction (UTJ) and isthmus were examined with a microarray chip (GeneChip® Porcine Gene 1.0 ST Array, Thermo Fisher Scientific) followed by bioinformatics for enriched analysis of functional categories (GO terms) and restrictive statistics. The results confirmed that entry of semen or of relative amounts of sperm-free SP modifies gene expression of these segments, pre-ovulation. It further shows that enriched genes are differentially associated with pathways relating to sperm motility, acrosome reaction, single fertilization, and the regulation of signal transduction GO terms. In particular, the pre-ovulation oviduct stimulates the Catsper channels for sperm Ca2+ influx, with AKAPs, CATSPERs, and CABYR genes being positive regulators while PKIs and CRISP1 genes appear to be inhibitors of the process. We postulate that the stimulation of PKIs and CRISP1 genes in the pre-ovulation sperm reservoir/adjacent isthmus, mediated by SP, act to prevent premature massive capacitation prior to ovulation.
Project description:A recent study has demonstrated that porcine spermatozoa recognize with high affinity carbohydrate structures containing Lewis X motifs. Sperm adhesion to Lewis X is proposed to mediate sperm binding to the oviduct epithelium to form a reservoir. The objective of this study was to identify Lewis X-binding proteins from porcine spermatozoa as candidate receptors for oviduct glycans. To identify low-abundance proteins typically masked by proteins originating from seminal fluid, Lewis X candidate receptors were enriched from cauda epididymal boar spermatozoa. Plasma membrane preparations from cauda epididymal spermatozoa were subjected to RP-HPLC and glycan blotting assays to isolate and detect proteins that bind Lewis X. Following bottom-up LC-MS/MS analysis, among the two bands that bound sulfated Lewis X, ADAM5, which spermatozoa, was confidently identified. ADAM family members have been established as contributors to sperm entry into the oviduct. A second sulfated Lewis X-binding protein identified was the peripheral membrane protein lactadherin (also known as P47, SED1 and MFG-E8 in different species). The interaction between Lewis X and lactadherin was functionally important because competitive inhibition by soluble recombinant lactadherin reduced sperm binding to the oviduct epithelium. Furthermore, far-western blotting demonstrated that purified lactadherin could bind oviduct cells. In summary, these findings reveal that, in addition to the previously reported glycan affinity of accessory gland proteins that adhere to spermatozoa, multiple proteins intrinsic to spermatozoa have affinity for a specific oviduct glycan. Further, in addition to binding to the zona pellucida, lactadherin is now implicated in binding to oviduct glycans to promote formation of the sperm reservoir.
Project description:In sea urchins, spermatozoan motility is altered by chemotactic peptides, giving rise to the assumption that mammalian eggs also emit chemotactic agents that guide spermatozoa through the female reproductive tract to the mature oocyte. Mammalian spermatozoa indeed undergo complex adaptations within the female (the process of capacitation) that are initiated by agents ranging from pH to progesterone, but these factors are not necessarily taxic. Currently, chemotaxis, thermotaxis, and rheotaxis have not been definitively established in mammals.Here, we show that positive rheotaxis, the ability of organisms to orient and swim against the flow of surrounding fluid, is a major taxic factor for mouse and human sperm. This flow is generated within 4 hr of sexual stimulation and coitus in female mice; prolactin-triggered oviductal fluid secretion clears the oviduct of debris, lowers viscosity, and generates the stream that guides sperm migration in the oviduct. Rheotaxic movement is demonstrated in capacitated and uncapacitated spermatozoa in low- and high-viscosity media. Finally, we show that a unique sperm motion, which we quantify using the sperm head's rolling rate, reflects sperm rotation that generates essential force for positioning the sperm in the stream. Rotation requires CatSper channels, presumably by enabling Ca(2+) influx.We propose that rheotaxis is a major determinant of sperm guidance over long distances in the mammalian female reproductive tract. Coitus induces fluid flow to guide sperm in the oviduct. Sperm rheotaxis requires rotational motion during CatSper channel-dependent hyperactivated motility.