Energetic sources required to maintain human sperm hyperactivation in vitro

MARIN BRIGGILER, CI; LUQUE GM; KRAPF D; VISCONTI PE; BUFFONE MG

Mar del Plata

Congreso; Reunión de Sociedades Científicas SAIC, SAFE, SAB, SAB; 2019

Energetic sources required to maintain human sperm hyperactivation in vitro. Marín-Briggiler CI1, Luque GM1, Krapf D2, Visconti PE3, Buffone MG1.1Instituto de Biología y Medicina Experimental (IBYME)-FIBYME-CONICET, Buenos Aires, Argentina. 2Instituto de Biología Molecular y Celular de Rosario (CONICET-UNR), Rosario, Santa Fe, Argentina.3Department of Veterinary and Animal Science, University of Massachusetts, Amherst MA, USA. E-mail: clarisamarin@gmail.comTo gain fertilization competence, mammalian sperm must undergo a complex process called capacitation, which is associated with the development of a distinct type of non-progressive motility (hyperactivation, HA). In spite of its relevance, the molecular mechanisms underlying HA, including its ionic and metabolic requirements remain elusive. The objective of the present work was to analyze the metabolic substrates necessary to maintain human sperm HA. Motile sperm obtained from healthy donors, were incubated for 4 h in BWW media containing glycolysable (Glucose, Glu) or non-glycolysable substrates (Pyruvate/Lactate, Pyr/Lact), all these nutrients (Complete medium, Comp), or none of them (Incomplete medium, Incomp). Motility parameters were measured by Computer-Assisted Sperm Analysis (SCA system). ATP content was analyzed by luminescence (kit by Cayman Chemical), and PKA-related phosphorylation (pPKA) and tyrosine phosphorylation (pY) were evaluated by Western blotting. Only sperm incubated in Comp medium showed maximum percentages of HA, and significantly lower HA levels were obtained in Incomp, Pyr/Lact (P < 0.01) or Glu media (P < 0.05, n=4). Reduced ATP and pY levels were observed in Incomp, Pyr/Lact or Glu compared to Comp condition (P < 0.05, n > 4); however, similar pPKA levels were obtained in all conditions. When incubated for 4 h in Incomp medium, the addition of Glu and Pyr/Lact for 1 min allowed the development of HA, which was accompanied by an increase in ATP content, but not in pY levels. This study indicates that both glycolysable and non-glycolysable substrates are required to support human sperm HA, and provides a model for the study of signaling mechanisms underlying this process.