Abstract
Reactive oxygen species (ROS) are fundamental for intracellular signaling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes.
Estrous sheep serum (ESS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of ESS on ram sperm physiology, especially on ROS production, during in vitro capacitation.
Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: Control (CTL), 10% ESS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 μM), and EES (10%) plus caspase inhibitor (I+E).
Sperm samples were incubated for 30 min at 38.5 oC and 5% CO2 and evaluated: motility and kinetic parameters by computer-assisted semen analysis (CASA), and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-H2DCFDA) by flow cytometry. ESS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (P<0.05).
Moreover, ESS increased the proportion of M540+ viable spermatozoa, YO-PRO-1+ and acrosome reacted spermatozoa (P<0.05). After incubation, ESS and I+E achieved lower ROS levels (P < 0.05).
Ca2+ levels did not change with the incubation, but were slightly higher (P<0.05) when both ESS and the inhibitor were present. We suggest that ESS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.