Calcium is the major second messenger which has an important role in sperm physiology.
Decreased fertility potential of spermatozoa is closely associated with the disturbances of Ca2+-homeostasis. Sperm mitochondria may serve as intracellular Ca2+ stores, although their contribution to [Ca2+]i homeostasis and signalling cascades in spermatozoa is still unclear. Since mitochondria are important in the regulation of the [Ca2+]i in sperm, we set out to describe the progesterone-evoked intracellular Ca2+-signals in response to uncoupling agent CCCP in spermatozoa of fertile (normozoospermia) and infertile men (oligo- and asthenozoospermia).
Human semen was obtained from 10 healthy volunteers (control group) and 16 pathozoospermic men undergoing routine semen analysis for couple infertility. Human ejaculates were obtained from healthy volunteers and pathozoospermic men by masturbation after 3–4 days sexual abstinence and processed immediately upon liquefaction. For determination of cytosolic free calcium concentration ([Ca2+]i), spermatozoa were loaded with 2 μM fluorescent probe Fluo-4. In this study we have investigated the progesterone-evoked intracellular Ca2+ signal in response to uncoupling agent CCCP in ejaculated spermatozoa from infertile men with different form of pathospermia. To investigate the role of mitochondria in Ca2+ signaling, we used an uncoupling agents carbonyl cyanide m-chloro phenyl hydrazone (CCCP) at a concentration of 10 μM which leads to dissipation of the mitochondrial membrane potential and blocking of Ca2+-uptake. Preapplication of CCCP induced an increase in the amplitude of [Ca2+]i rise caused by progesterone (20 μM), which indicate that mitochondria regulate rapid Ca2+ uptake.
CCCP application after [Ca2+]i transient induced an additional [Ca2+]i rise in normozoospermic samples, which indicates significant Ca2+ release that was previously accumulated by mitochondria. Our results show that amplitude of CCCP-induced [Ca2+]i transient after progesterone- induced [Ca2+]i transient in oligo- and ashenozoospermic samples was much smaller compared to calcium signal obtained in spermatozoa from normozoospermic men. In asthenozoospermic samples CCCP application after progesterone-induced [Ca2+]i caused sustained (not transient) [Ca2+]i rise.
We presume that disrupted calcium mobilization in spermatozoa from pathozoospermic men might be associated with decrease in the Ca2+-accumulating properties of mitochondria.
Keywords: calcium, spermatozoa, mitochondria, male infertility, oligozoospermia, asthenozoospermia
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