The uterus has a well-developed innate immune system that recognizes microbes and sperm. We have recently shown that sperm binding to bovine uterine epithelial cells (BUEC) induces acute inflammatory response. This raises the query of whether the uterus uses a common or similar mucosal immune response to sperm and microbes; however, immune cross-talk between sperm and endometrium is unclear. Our BUEC culture model showed that pro-inflammatory response induced by sperm binding involves TLRs signaling pathway via TLR2; however, immunological functions of the uterus in vivo involve interactions of tissues and mucus coating the endometrial surface could modulate responses. Thus, we developed an ex vivo explant model to investigate the sperm-uterine interaction in vivo. Uterine explants were co-incubated with 106/ml washed fresh sperm. JC-1 labeled sperm were used in fluorescence microscopy. Sperm glide over the surface epithelium until they encounter and enter uterine glands. SEM observations show that endogenous neutrophils appeared in uterine glands along with the clusters of sperm; they may initiate sperm clearance. Co-incubation for 2 h resulted in upregulation of TNFA and IL1B mRNA expression, but IL8 expression started to increase earlier at 0.5 h. TLR2 antagonist reduced the sperm numbers in the glands and inhibited the increase of TNFA and IL8 which suggest that the sperm-uterine inflammatory process is at least partly mediated by TLR2 signaling. Our observations suggest that uterine glands serve as a site where sperm interact with glandular epithelium to trigger the innate immune response to rapidly clear sperm from the uterus and thus prepare the endometrium for embryo implantation.
The role of oocytes on follicular antrum formation has not been elucidated yet. We examined the effect of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on antrum formation using cultured bovine oocyte-cumulus cell complexes (OCCs). OCCs containing growing oocyte (105–115 μm) were collected from early antral follicles (1.2–1.8 mm) and used to prepare oocytectomized complexes (OXCs) and cumulus cell complexes (CCs). The mRNAs of GDF9 and BMP15 were expressed only in oocytes, but not in cumulus cells. The complexes were cultured for 5 days with or without GDF9 and BMP15 either alone or in combination. OCCs maintained their complex integrity and developed antrum structure, while OXCs and CCs neither maintained the structure nor developed any antrum without growth factors. GDF9 or BMP15 alone increased integrity of these complexes and induced antrum structure in OXCs and CCs. Furthermore, combination of GDF9 and BMP15 was more effective on both phenomena in all types of complexes. Outgrowing cumulus cells from OXCs and CCs cultured without growth factors or with BMP15 alone showed extensive spread of differentiated fibroblast-like cells. Combination of GDF9 and BMP15 suppressed the appearance of fibroblast-like cells in OXCs and CCs during the culture. Instead, the cumulus cells showed rhomboid and pebble stone-like appearances which were similar to those in OCCs cultured without GDF9 and BMP15. These results suggest that the oocytes maintain complex integrity by preventing cumulus cell differentiation and participate in follicular antrum formation via GDF9 and BMP15.
Although organisms belonging to different subspecies sometimes produce fertile offspring, a hallmark of the speciation process is reproductive isolation, with hybrid sterility (HS) due to failure in gametogenesis. In mammals, HS is usually exhibited by males, the heterogametic sex. The phenotypic manifestations of HS are complex, but most frequently manifested as abnormalities during meiotic prophase, or aberrations in post-meiotic spermiogenesis, all leading to defective or absent gametes. The aim of this study was to determine the HS phenotypes in intersubspecific F1 mice produced by matings between Mus musculus molossinus and diverse Mus musculus domesticus classical inbred laboratory mouse strains. Most of these crosses produced fertile F1 offspring. However, when female BALB/cJ (domesticus) mice were mated to male JF1/MsJ (molossinus) mice, the (BALBdomxJF1mol)F1 males were sterile, while the (JF1molxBALBdom)F1 males produced by the reciprocal cross were fertile; thus the sterility phenotype is asymmetric. The sterile (BALBdomxJF1mol)F1 males exhibited a high rate of meiotic metaphase arrest with misaligned chromosomes, likely related to a high frequency of XY dissociation. Intriguingly, in the sterile (BALBdomxJF1mol)F1 males we observed aberrant expression of the Spo11 gene, encoding a meiosis-specific endonuclease playing a critical role in recombination and thereby affecting chromosome pairing. Together, these observations of an asymmetrical HS phenotype in intersubspecific F1 mice, causing misregulation of Spo11 gene expression and subsequent meiotic segregation defects of the XY chromosomes, provide new directions for understanding mechanisms leading to speciation in mammals.
Polymyxin B (PMB) has been reported to beneficial for boar semen storage by neutralizing endotoxin while PMB has also been shown to directly affect signalling molecules in somatic cells. This study aimed to examine the direct effect of PMB on boar spermatozoa. Ejaculated boar spermatozoa stored with BTS extender at 17°C were washed, incubated with various concentrations of PMB (0–100 µM) for 20 min, examined subjectively for percentages of total and progressive motillity, vigour grade (0–4) and agglutination score (0–4) and stained by propidium iodide for viability. In separate experiments, spermatozoa were preincubated with PMB (0–0.5 µM) for 10 min, stimulated for acrosomal exocytosis with calcium and calcium ionophore A23187 and fixed with glutaraldehyde at 5, 10 and 15 min. Per cent total motility and viability were not significantly different between 0 and 50 µM PMB with gradual decline at higher concentrations while progressive motility was significantly reduced at 25 µM or higher concentrations of PMB. Vigour grade was decreased at 75 µM or higher concentrations but agglutination scores were not affected. Preincubation with PMB resulted in a dose-dependent increase in per cent acrosomal exocytosis by 0.01–0.05 µM PMB at 10 min and by 0.05–0.5 µM at 15 min. These results show that PMB has direct effect on boar sperm motility, viability and acrosomal exocytosis.