E Martínez-León1, C Osycka-Salut2, J Signorelli1, P Pozo1, B Pérez1, M Kong1, P Morales3, S Pérez-Martínez2, E S Díaz4. 1. Laboratory of Biology of Reproduction, Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, 1240000 Antofagasta, Chile. 2. Laboratory of Biology of Reproduction in Mammals, Center for Pharmacological and Botanicals Studies (National Council of Scientific and Techniques Research), University of Buenos Aires, 1121 Buenos Aires, Argentina. 3. Laboratory of Biology of Reproduction, Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, 1240000 Antofagasta, Chile Antofagasta Institute, University of Antofagasta, 1240000 Antofagasta, Chile. 4. Laboratory of Biology of Reproduction, Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, 1240000 Antofagasta, Chile emilce.diaz@uantof.cl.
Abstract
STUDY QUESTION: Does fibronectin (Fn) stimulate the sperm capacitation process in humans? SUMMARY ANSWER: Fibronectin stimulates human sperm capacitation. WHAT IS KNOWN ALREADY: Capacitation is a process that occurs in the oviduct. It has been suggested that some molecules present in the oviductal fluid and cells as well as proteins present in the cumulus oophorus could be involved in the modulation of sperm function and their acquisition of fertilizing capacity. Fibronectin is a glycoprotein that is present in the fluid and the oviduct epithelium, and its receptor (alpha 5 beta 1 integrin) is present in human sperm. When alpha 5 beta 1 (α5β1) integrin binds to fibronectin, intracellular signals similar to the process of sperm capacitation are activated. STUDY DESIGN, SIZE, DURATION: Human sperm were selected via a percoll gradient and were then incubated in non-capacitated medium (NCM) or reconstituted capacitated medium (RCM), in the presence or absence of fibronectin for different time periods. A total of 39 donors were used during the study, which lasted 3 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Freshly ejaculated sperm from healthy volunteers were obtained by masturbation. All semen samples were normal according to the World Health Organization parameters. Six approaches were used to determine the effects of fibronectin on sperm capacitation: chlortetracycline (CTC) assay, heterologous co-culture of human sperm with bovine oviductal epithelial cells (BOEC), measurement of cyclic (c) AMP levels, activity of protein kinase A (PKA), phosphorylation of proteins in tyrosine (Tyr) residues, and induction of acrosome reaction with progesterone. MAIN RESULTS AND THE ROLE OF CHANCE: When sperm were incubated in RCM in the presence of Fn, we observed differences with respect to sperm incubated in RCM without Fn (control): (i) a 10% increase in the percentage of sperm with the B pattern (capacitated sperm) of CTC fluorescence from the beginning of capacitation (P < 0.001); (ii) an effect on both the concentration of cAMP (P < 0.05) and PKA activity (P < 0.05) during early capacitation; (iii) an increase in the degree of phosphorylation of proteins on tyrosine residues after 60 min of capacitation (P < 0.01); (iv) an increase in the percentage of acrosome-reacted sperm in response to progesterone (P < 0.05); and (v) a decrease in the percentage of sperm attached to BOEC (P < 0.05). Moreover, we noted that the effect of Fn was specific and mediated by alpha 5 beta 1 integrin (P < 0.001). Fn by itself had no effect on sperm capacitation. LIMITATIONS, REASONS FOR CAUTION: This study was carried out with sperm from young adult men. Men with abnormal semen samples were excluded. The results cannot be directly extrapolated to other mammalian species. WIDER IMPLICATIONS OF THE FINDINGS: Currently, male subfertility has become a huge public health problem, which makes it imperative to develop new treatments. This is a novel discovery that extends our current knowledge concerning normal and pathological sperm physiology as well as events that regulate the process of fertilization. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from FONDECYT (1130341, E.S.D. and 1120056, P.M.) and FONCYT (PIP 2011-0496, S.P.-M). The authors have no conflicts of interest.
STUDY QUESTION: Does fibronectin (Fn) stimulate the sperm capacitation process in humans? SUMMARY ANSWER: Fibronectin stimulates human sperm capacitation. WHAT IS KNOWN ALREADY: Capacitation is a process that occurs in the oviduct. It has been suggested that some molecules present in the oviductal fluid and cells as well as proteins present in the cumulus oophorus could be involved in the modulation of sperm function and their acquisition of fertilizing capacity. Fibronectin is a glycoprotein that is present in the fluid and the oviduct epithelium, and its receptor (alpha 5 beta 1 integrin) is present in human sperm. When alpha 5 beta 1 (α5β1) integrin binds to fibronectin, intracellular signals similar to the process of sperm capacitation are activated. STUDY DESIGN, SIZE, DURATION: Human sperm were selected via a percoll gradient and were then incubated in non-capacitated medium (NCM) or reconstituted capacitated medium (RCM), in the presence or absence of fibronectin for different time periods. A total of 39 donors were used during the study, which lasted 3 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Freshly ejaculated sperm from healthy volunteers were obtained by masturbation. All semen samples were normal according to the World Health Organization parameters. Six approaches were used to determine the effects of fibronectin on sperm capacitation: chlortetracycline (CTC) assay, heterologous co-culture of human sperm with bovine oviductal epithelial cells (BOEC), measurement of cyclic (c) AMP levels, activity of protein kinase A (PKA), phosphorylation of proteins in tyrosine (Tyr) residues, and induction of acrosome reaction with progesterone. MAIN RESULTS AND THE ROLE OF CHANCE: When sperm were incubated in RCM in the presence of Fn, we observed differences with respect to sperm incubated in RCM without Fn (control): (i) a 10% increase in the percentage of sperm with the B pattern (capacitated sperm) of CTC fluorescence from the beginning of capacitation (P < 0.001); (ii) an effect on both the concentration of cAMP (P < 0.05) and PKA activity (P < 0.05) during early capacitation; (iii) an increase in the degree of phosphorylation of proteins on tyrosine residues after 60 min of capacitation (P < 0.01); (iv) an increase in the percentage of acrosome-reacted sperm in response to progesterone (P < 0.05); and (v) a decrease in the percentage of sperm attached to BOEC (P < 0.05). Moreover, we noted that the effect of Fn was specific and mediated by alpha 5 beta 1 integrin (P < 0.001). Fn by itself had no effect on sperm capacitation. LIMITATIONS, REASONS FOR CAUTION: This study was carried out with sperm from young adult men. Men with abnormal semen samples were excluded. The results cannot be directly extrapolated to other mammalian species. WIDER IMPLICATIONS OF THE FINDINGS: Currently, male subfertility has become a huge public health problem, which makes it imperative to develop new treatments. This is a novel discovery that extends our current knowledge concerning normal and pathological sperm physiology as well as events that regulate the process of fertilization. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from FONDECYT (1130341, E.S.D. and 1120056, P.M.) and FONCYT (PIP 2011-0496, S.P.-M). The authors have no conflicts of interest.