Esperanza Mata-Martínez1, Ana Alicia Sánchez-Tusie2, Alberto Darszon3, Luis S Mayorga1,4, Claudia L Treviño3, Gerardo A De Blas1,5. 1. Laboratorio de Fusión de Membranas y Exocitosis Acrosomal, Instituto de Histología y Embriología Dr. Mario H. Burgos (IHEM), Universidad Nacional de Cuyo, CONICET, Mendoza, Argentina. 2. Laboratorio de Fisiología Celular y Molecular, Departamento de Investigación Biomédica, Facultad de Medicina, Universidad Autónoma de Querétaro, México. 3. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, México. 4. Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, Mendoza, Argentina. 5. Laboratorio de Teleanálisis e Investigación Traslacional, Área Farmacología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.
Abstract
BACKGROUND: The signaling pathways of the intracellular second messengers cAMP and Ca2+ play a crucial role in numerous physiological processes in human spermatozoa. One such process is the acrosome reaction (AR), which is necessary for spermatozoa to traverse the egg envelope and to expose a fusogenic membrane allowing the egg-sperm fusion. Progesterone and zona pellucida elicit an intracellular Ca2+ increase that is needed for the AR in the mammalian spermatozoa. This increase is mediated by an initial Ca2+ influx but also by a release from intracellular Ca2+ stores. It is known that intracellular Ca2+ stores play a central role in the regulation of [Ca2+ ]i and in the generation of complex Ca2+ signals such as oscillations and waves. In the human spermatozoa, it has been proposed that the cAMP analog and specific agonist of Epac 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (2'-O-Me-cAMP) elicits an intracellular Ca2+ release involved in the AR. OBJECTIVE: To identify the molecular entities involved in the Ca2+ mobilization triggered by 2'-O-Me-cAMP in human spermatozoa. MATERIALS AND METHODS: In capacitated human spermatozoa, we monitored Ca2+ dynamics and the occurrence of the AR in real time using Fluo 3-AM and FM4-64 in a Ca2+ -free medium. RESULTS: Epac activation by 2'-O-Me-cAMP induced a Ca2+ wave that started in the midpiece and propagated to the acrosome region. This Ca2+ response was sensitive to rotenone, CGP, xestospongin, NED-19, and thapsigargin, suggesting the participation of different ion transporters (mitochondrial complex I and Na+ /Ca2+ exchanger, inositol 3-phosphate receptors, two-pore channels and internal store Ca2+ -ATPases). DISCUSSION: Our results suggest that Epac activation promotes a dynamic crosstalk between three different intracellular Ca2+ stores: the mitochondria, the redundant nuclear envelope, and the acrosome. CONCLUSION: The Ca2+ wave triggered by Epac activation is necessary to induce the AR and to enhance the flagellar beat.
BACKGROUND: The signaling pathways of the intracellular second messengers cAMP and Ca2+ play a crucial role in numerous physiological processes in human spermatozoa. One such process is the acrosome reaction (AR), which is necessary for spermatozoa to traverse the egg envelope and to expose a fusogenic membrane allowing the egg-sperm fusion. Progesterone and zona pellucida elicit an intracellular Ca2+ increase that is needed for the AR in the mammalian spermatozoa. This increase is mediated by an initial Ca2+ influx but also by a release from intracellular Ca2+ stores. It is known that intracellular Ca2+ stores play a central role in the regulation of [Ca2+ ]i and in the generation of complex Ca2+ signals such as oscillations and waves. In the human spermatozoa, it has been proposed that the cAMP analog and specific agonist of Epac 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (2'-O-Me-cAMP) elicits an intracellular Ca2+ release involved in the AR. OBJECTIVE: To identify the molecular entities involved in the Ca2+ mobilization triggered by 2'-O-Me-cAMP in human spermatozoa. MATERIALS AND METHODS: In capacitated human spermatozoa, we monitored Ca2+ dynamics and the occurrence of the AR in real time using Fluo 3-AM and FM4-64 in a Ca2+ -free medium. RESULTS: Epac activation by 2'-O-Me-cAMP induced a Ca2+ wave that started in the midpiece and propagated to the acrosome region. This Ca2+ response was sensitive to rotenone, CGP, xestospongin, NED-19, and thapsigargin, suggesting the participation of different ion transporters (mitochondrial complex I and Na+ /Ca2+ exchanger, inositol 3-phosphate receptors, two-pore channels and internal store Ca2+ -ATPases). DISCUSSION: Our results suggest that Epac activation promotes a dynamic crosstalk between three different intracellular Ca2+ stores: the mitochondria, the redundant nuclear envelope, and the acrosome. CONCLUSION: The Ca2+ wave triggered by Epac activation is necessary to induce the AR and to enhance the flagellar beat.