EFFECTS OF CRYOPRESERVATION ON THE INTRACELLULAR CALCIUM-CONCENTRATION OF HUMAN SPERMATOZOA AND ITS RESPONSE TO PROGESTERONE

The intracellular free calcium concentration [Ca2+](i) of sperm from 2 3 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 mu M prog esterone so that the regulation of [Ca2+](i) in a dynamic situation co uld be studied. [Ca2+](i) (nM) was 290 +/- 13 in fresh spermatozoa vs. 550 +/- 26 in cryopreserved samples (mean +/- S.E.M. P < 0.0001 paire d t-test). Progesterone at a dose of 3.18 mu M stimulated a large and rapid increase in [Ca2+](i) to a peak value > 1 mu M after 10-20 secon ds. [Ca2+](i) then declined to a slightly raised basal level over the next 30-40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca2+] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The ca lcium channel blocker verapamil (200 mu M) completely inhibited the tr ansient rise in [Ca2+](i) produced by progesterone, but 100 mu M verap amil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca2+](i) in human spermatozoa a nd (2) damage to the plasma membrane during cryopreservation may resul t in the loss of the progesterone receptor. Both factors may contribut e to the loss of fertility after cryopreservation. (C) 1994 Wiley-Liss , Inc.