Sperm penetration into the bovine pre-ovulatory uterine glands induces TNFA in glands and triggers the inflammatory cascade

Abstract

Sperm interaction with the uterus and subsequent immune responses by the endometrium to remove excess, defective and dead sperm are important to prepare the endometrium for embryo implantation. We have recently developed a bovine ex-vivo explant culture model to study the link between sperm interaction with endometrium and immune responses. It was shown that sperm entering bovine pre-ovulatory uterine glands induce an acute inflammatory response by upregulation of pro-inflammatory cytokines mRNA expression (SRD, 2018). We used the developed co-culture model to study the effect of sperm on protein expression of TNFA which is a strong pro-inflammatory marker. Further, sperm interaction and immune responses in the luteal phase were evaluated. Sections of bovine pre-ovulatory endometrial explants stained with hematoxylin and eosin showed normal tissue architecture with intact surface epithelial layer and simple tubular uterine glands that present throughout the extracted endometrium. Pre-ovulatory uterine explants were co-incubated with 10⁶/ml washed fresh sperm for 4 h and TNFA protein expression was detected using immunofluorescence and immunohistochemical staining. Both staining methods showed that sperm upregulate TNFA protein expression in uterine glands. Luteal phase uterine explants were co-incubated with 10⁶/ml sperm. JC-1 labeled sperm were used in fluorescence microscopy. Live, motile sperm did not appear to enter the luteal glands. Also, co-incubation for 2 h did not alter gene expression of TNFA, IL8 or IL1B in the luteal explants. Our observations reveal that sperm entering bovine pre-ovulatory uterine glands, trigger the uterine inflammatory cascade which begins in the glands.