Reproduction Abstracts

Ca²⁺ signaling is crucial for regulation of sperm motility and [Ca²⁺]_i oscillations may underlie ‘switching’ of sperm behaviors in human spermatozoa, which is believed to be important for sperm progression in the female tract. We have investigated the contribution of membrane potential (Vm) and the sperm Ca²⁺ channel CatSper to [Ca²⁺]_i oscillations induced by progesterone (P4). Manipulation of Vm was performed using the K⁺ ionophore valinomycin (VLN-1 μM), alone or with high K⁺ media (100 mM) and confirmed by whole-cell patch clamping. [Ca²⁺]_i signaling was assessed by loading sperm cells with the Ca²⁺-indicator fluo-4-AM and stimulating them with 3 μM P4. 29.8+4.53% of cells exhibited [Ca²⁺]_i oscillations after P4 stimulation (n=7366 cells). [Ca²⁺]_i rose first in the flagellum, consistent with activation of CatSper, then spread actively to the head, apparently triggering Ca²⁺ store release. Two oscillations patterns were observed: ‘rapid’ transients resembling the initial progesterone response and slower transients with lower amplitude. Pre- or post-treatment with VLN to ‘clamp’ Vm at −66.4 mV abolished ‘rapid’ [Ca²⁺]_i oscillations in 95% of cells but ‘slow’ transients were resistant. Both Ca²⁺ oscillations patterns completely recovered upon VLN washout. Both ‘rapid’ and ‘slow’ oscillations were more resistant to depolarized clamp of Vm (−4.6 mV with VLN/100 mM K⁺) and recovery of oscillations was slower after VLN/K⁺ removal. Treatment with CatSper inhibitor abolished ‘rapid’ and ‘slow’ [Ca²⁺]_i oscillations and both recovered rapidly when the inhibitor was removed. Tracking of fluo-4-labelled, free-swimming cells showed switching of sperm behaviour during ‘rapid’ [Ca²⁺]_i oscillations. Our results show that Vm contributes to generation of high amplitude [Ca²⁺]_i oscillations and sperm behaviour (probably by regulating CatSper) but low amplitude transients may be regulated differently. Financial Support: CAPES foundation.