Exposure of human spermatozoa to micromolar levels of progesterone evokes a biphasic increase in [Ca2+]i; an initial transient, which peaks and decays within 3-4 min, and a secondary, sustained [Ca2+]i response (Kirkman-Brown et al. 2000). Nifedipine, an inhibitor of L- and T-type voltage-operated Ca2+ channels, is reported to inhibit progesterone-induced acrosome reaction in mammalian spermatozoa (O’Toole et al. 1996). We have carried out a detailed study on the effects of nifedipine (10 µM; which strongly inhibited progesterone-induced acrosome reaction; P ▓le│ 0.01, Student’s paired t test) on [Ca2+]i signalling in large numbers of individual human spermatozoa loaded with Calcium Green-1 AM.
Spermatozoa from donors of proven fertility were prepared by under-layering 2 ml of supplemented Earle’s balanced salts with 1 ml of semen and incubating for an hour. The top 1.5 ml of media containing the experimental population of highly motile spermatozoa was then removed and incubated for a further 6 h at 6 million cells ml-1. Sperm labelled with Calcium Green-1 AM were adhered to coverslips in a perfusion chamber and imaged on a confocal microscope. Dye retention and movement of the flagellum confirmed sperm viability.
We report that the drug has two discrete effects. Firstly, the mean proportion of cells in which progesterone induced a response was significantly decreased (77.2 to 61.7% P ▓le│ 0.014, t test) after pretreatment with nifedipine. This effect occurred disproportionately among cells in which nifedipine-pretreatment caused a significant fall in fluorescence (45.3 ± 11.7 % compared with 72.0 ± 7.4 %) and may therefore reflect the importance of resting [Ca2+]i in the ability of cells to respond to progesterone. Secondly, nifedipine significantly inhibited the [Ca2+]i signal in those cells that did respond to progesterone. The amplitude of the [Ca2+]i transient in responsive cells was not affected but the mean duration was significantly shorter (P ▓le│ 0.000025) and the amplitude of the sustained [Ca2+]i response was significantly reduced (49.9 to 34.1% P ▓le│ 0.038). These findings are consistent with the existence of a previously undetected nifedipine-sensitive component of the progesterone-induced transient [Ca2+]i signal that becomes activated approximately 1 min after exposure to the hormone and is crucial for induction of acrosome reaction.
The research was carried out according to local ethical guidelines in collaboration with Birmingham Women’s Hospital (HFEA no. 0119). Donors gave informed consent.This work was supported by BBSRC and The Wellcome Trust.
- Kirkman-Brown, J.C., Bray, C.M., Stewart, P., Barratt, C.L. & Publicover, S.J. (2000). Develop. Biol. 222, 326-335.
O’Toole, C.M.B., Roldan, E.R.S. & Fraser, L.R. (1996). Mol. Reprod. Devel. 45, 204-211.