Uterosome-like vesicles prompt human sperm fertilizing capability.
Does the rapid transit through the uterine environment modulate the sperm physiological state?



The uterosome-like vesicles (ULVs) secreted by endometrial epithelial cells (EECs) in vitro are able to fuse with human spermatozoa, prompting their fertilizing capacity.



Early studies suggest that sperm capacitation begins in the uterus and ends in the oviduct, and that a synergistic effect of both female organs may accelerate this process. Although it has been reported that co-incubation of human spermatozoa with endometrial cell-conditioned medium (CM) stimulates sperm capacitation, the mechanism mediating this communication is unknown.



Human ULVs secreted by EECs were characterized and their effect on human sperm physiology was analysed. Spermatozoa were incubated with EEC-derived CM or ULV, after which sperm capacitation was evaluated at different time points. In addition, the interaction of spermatozoa with ULV was analysed.



ULVs were isolated by ultracentrifugation and identified using electron microscopy and Western blotting to assess the presence of specific protein markers. Following seminal plasma removal, human spermatozoa were incubated CM or ULV, after which sperm capacitation was evaluated as the ability of the sperm to undergo the induced acrosome reaction and the level of protein tyrosine phosphorylation (PY) determined by Western blot and immunocytochemistry. The interaction of spermatozoa with labelled ULV was analysed by fluorescence microscopy. In all cases, at least three biological replicates from different sperm donors were performed for each set of experiments. Significant differences between mean values were determined by one-way ANOVA followed by Tukey's post hoc test. Differences between treatments were considered statistically significant at P ≤ 0.05.



The level of capacitated spermatozoa and those recruited by chemotaxis increased 3- to 4-fold when spermatozoa were incubated in the presence of CM for 4 h. Even a 15 min incubation of spermatozoa with CM was also enough to increase the level of capacitated cells 3- to 4-fold (P < 0.05). Furthermore, a short co-incubation of spermatozoa with ULV stimulates sperm capacitation, as determined by the increase in the level of induced acrosome reaction and the induction of PY. In addition, after the co-incubation of spermatozoa with fluorescent labelled ULV, the sperm cells acquired the fluorescent staining which indicates that ULV might be transferred to the sperm surface by a fusion mechanism.



This is an in vitro study performed with human biological material, spermatozoa and endometrial derived cells; the latter being a cell line originally isolated from a uterine adenocarcinoma.



The capability of spermatozoa to briefly interact with ULVs supports the hypothesis that any step of sperm transport may have physiological consequences, despite the interaction lasting for only a limited period of time. This way of communication of spermatozoa with cell products of uterine origin opens new frontiers of investigation (e.g. the signalling molecules involved), shedding light on the sperm processes that prepare the male gamete for fertilization, which might have implications for human infertility treatment.



N/A.



The project was financially supported by SECyT-UNC. The authors declare no conflict of interest.
Centro de Biología Celular y Molecular (FCEFN-UNC) and Instituto de Investigaciones Biológicas y Tecnológicas (CONICET-UNC), Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, 5016 Córdoba, Argentina.
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Franchi
Centro de Biología Celular y Molecular (FCEFN-UNC) and Instituto de Investigaciones Biológicas y Tecnológicas (CONICET-UNC), Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, 5016 Córdoba, Argentina.
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Cubilla
Centro de Biología Celular y Molecular (FCEFN-UNC) and Instituto de Investigaciones Biológicas y Tecnológicas (CONICET-UNC), Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, 5016 Córdoba, Argentina.
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Guidobaldi
Centro de Biología Celular y Molecular (FCEFN-UNC) and Instituto de Investigaciones Biológicas y Tecnológicas (CONICET-UNC), Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, 5016 Córdoba, Argentina.
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Bravo
Centro de Biología Celular y Molecular (FCEFN-UNC) and Instituto de Investigaciones Biológicas y Tecnológicas (CONICET-UNC), Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, 5016 Córdoba, Argentina lgiojalas@unc.edu.ar.
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Giojalas
D015153:Blotting, Western; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004717:Endometrium / Q000166:cytology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D046529:Microscopy, Electron, Transmission; D013075:Sperm Capacitation / Q000187:drug effects; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*
D017077:Culture Media, Conditioned
capacitation; endometrial cells; human spermatozoa; uterine environment; uterosomes