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Luiz Cordeiro, Cindy Riou, Rustem Uzbekov, and Nadine Gérard

In birds, oviductal cells play a crucial role in the storage of sperm via cell-to-cell communication including extracellular vesicles (EV). We developed a culture of oviductal organoids enriched in sperm storage tubules (SSTorg) to demonstrate the release of EV. SSTorg were cultured for 24 h and added to live (LV), frozen (FZ) and lysed (LY) avian sperm, seminal plasma (SP), avian sperm conditioned medium (CM), or bovine sperm (BV). Western blot demonstrated that SSTorg contained EV protein markers, valosin-containing protein (VCP), heat shock proteins (HSP90AA1, HSPA8), and annexins (ANXA2, A4, A5). Co-culture with LV significantly decreased the intracellular level of all these proteins except HSPA8. Immunohistochemistry confirmed this result for VCP and ANXA4. LY, CM, SP and BV had no effect on the intracellular level of these proteins, whereas FZ induced a decrease in ANXA2, A4 and A5. In culture media, VCP and HSP90AA1 signals were detected in the presence of LV, FZ, BV, LY, CM and SP, but no ANXA4 signal was observed in the presence of FZ and SP. ANXA2 and A5 were only detected in the presence of LV. The most abundant EV were less than 150 nm in diameter. ANXA4 and A5 were more abundant in EV isolated from the SSTorg culture medium. This study provides a useful culture system for studying interactions between SST cells and sperm. We demonstrated the release of EV by SSTorg in vitro, and its regulation by sperm. This may be of crucial importance for sperm during storage in hens.

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Cindy Riou, Aurélien Brionne, Luiz Cordeiro, Grégoire Harichaux, Audrey Gargaros, Valérie Labas, Joël Gautron, and Nadine Gérard

Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve the understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen’s genital tract and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F−) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F− hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage.

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Luiz Cordeiro, Hsiu-Lien Herbie Lin, Anaïs Vitorino Carvalho, Isabelle Grasseau, Rustem Uzbekov, and Elisabeth Blesbois

Male subfertility causes are very varied and sometimes related to post-gonadic maturation disruption, involving seminal plasma constituents. Among them, extracellular vesicles are involved in key exchanges with sperm in mammals. However, in birds, the existence of seminal extracellular vesicles is still debated. The aim of the present work was first to clarify the putative presence of extracellular vesicles in the seminal plasma of chickens, secondly to characterize their size and protein markers in animals showing different fertility, and finally to make preliminary evaluations of their interactions with sperm. We successfully isolated extracellular vesicles from seminal plasma of males showing the highest differences in semen quality and fertility by using ultracentrifugation protocol (pool of 3 ejaculates/rooster, n =3/condition). Size characterization performed by electron microscopy revealed a high proportion of small extracellular vesicles (probably exosomes) in chicken seminal plasma. Smaller extracellular vesicles appeared more abundant in fertile than in subfertile roosters, with a mean diameter of 65.12 and 77.18 nm, respectively. Different protein markers of extracellular vesicles were found by western blotting (n = 6/condition). Among them, HSP90A was significantly more abundant in fertile than in subfertile males. In co-incubation experiments (n = 3/condition), extracellular vesicles enriched seminal fractions of fertile males showed a higher capacity to be incorporated into fertile than into subfertile sperm. Sperm viability and motility were impacted by the presence of extracellular vesicles from fertile males. In conclusion, we successfully demonstrated the presence of extracellular vesicles in chicken seminal plasma, with differential size, protein markers and putative incorporation capacity according to male fertility status.