Optimizing the protocol for vitrification of individual spermatozoa by adjusting equilibration time.

Efficient cryopreservation of small numbers of human spermatozoa is essential in cases of severe male infertility, especially those requiring surgical sperm retrieval. Although vitrifying individual spermatozoa on sperm vitrification devices (SpermVD®) provided optimal cell retrieval upon warming, motility rates tended to be lower than with bulk-freezing. Post-warming motility is directly affected by cryoprotectant exposure; however, optimal cryoprotectant equilibration time is unknown. We evaluated several timeframes exposing individual spermatozoa to cryoprotectant before freezing and different cryoprotectants. A total of 2,925 spermatozoa from 20 patients ranging from normozoospermic to moderate oligoteratoasthenozoospermic were vitrified in small groups, on 60 SpermVD®, in 1 µl droplets of 1:1 v/v cryoprotectant/washing medium mixture. Each group was vitrified after 2-60 minutes equilibration time. Motility of each group was evaluated after warming. Leftover pellets were frozen in cryotubes in a mixture of 1:1 v/v cryoprotectant/washing medium after 10 minutes equilibration at room temperature and 10 minutes on liquid nitrogen vapors. Post-thaw motility correlated negatively with cryoprotectant exposure time. The highest post-warming motility rate (32.1%) was observed with 8-minutes equilibration. After 10 minutes, motility rate of vitrified sperm was lower than that of bulk-freezing (31.7% vs. 37.0%, p < 0.0001). Different cryoprotectants did not affect the results. Therefore, for vitrifying small numbers of spermatozoa, we suggest maximum equilibration time of 8-minutes to achieve maximum motility after warming.

Protein kinase A inhibition induces EPAC-dependent acrosomal exocytosis in human sperm.

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 µmol l-1), whereas higher concentrations (>5 µmol l-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.