McLaughlin E A, Ford W C
University of Bristol Department of Obstetrics and Gynaecology, St. Michael's Hospital, UK.
Mol Reprod Dev. 1994 Feb;37(2):241-6. doi: 10.1002/mrd.1080370216.
The intracellular free calcium concentration [Ca2+]i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 microM progesterone so that the regulation of [Ca2+]i in a dynamic situation could be studied. [Ca2+]i (nM) was 290 +/- 13 in fresh spermatozoa vs. 550 +/- 26 in cryopreserved samples (mean +/- S.E.M. P < 0.0001 paired t-test). Progesterone at a dose of 3.18 microM stimulated a large and rapid increase in [Ca2+]i to a peak value > 1 microM after 10-20 seconds. [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 calcium channel blocker verapamil (200 microM) completely inhibited the transient rise in [Ca2+]i produced by progesterone, but 100 microM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca2+]i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation.
使用荧光探针Fura - 2测量了23份精液中精子冷冻保存前后的细胞内游离钙浓度[Ca2+]i。精子用3.18微摩尔的孕酮处理，以便研究动态情况下[Ca2+]i的调节。新鲜精子中的[Ca2+]i（纳摩尔）为290±13，而冷冻保存样本中的为550±26（平均值±标准误，配对t检验，P<0.0001）。3.18微摩尔剂量的孕酮在10 - 20秒后刺激[Ca2+]i大幅快速升高至峰值>1微摩尔。然后在接下来的30 - 40秒内[Ca2+]i下降至略高于基础水平。这种现象在所有新鲜样本中都出现，但约一半的冻融样本没有反应。添加孕酮后有反应的冷冻样本达到的[Ca2+]峰值与新鲜精子中观察到的相似。钙通道阻滞剂维拉帕米（200微摩尔）完全抑制了孕酮引起的[Ca2+]i的短暂升高，但100微摩尔的维拉帕米只有部分作用。我们得出结论：（1）冷冻保存导致人类精子中[Ca2+]i大幅升高；（2）冷冻保存过程中质膜的损伤可能导致孕酮受体的丧失。这两个因素都可能导致冷冻保存后生育能力的丧失。
