The progesterone (P)-induced [Ca2+]i response in human spermatozoa is biphasic, consisting of an initial transient, which peaks and decays within 3 min, and a subsequent sustained elevation of [Ca2+]i that can be maintained for 20 min or more. Both responses are dependent upon Ca2+ influx (Blackmore et al. 1990). We have used fura-2 fluorimetry of human sperm populations to investigate the potential role of capacitative calcium entry (CCE) in the sustained component of P-induced Ca2+ influx.
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 1 h. 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 fura-2 AM were centrifuged to remove excess dye (500 g, 5 min) and resuspended in the same media, with a further 10 min incubation to allow dye hydrolysis to complete. Readings were taken with 340, 380 nm fast-filter excitation pairs in a Perkin Elmer LS50B system, with emission at 510 nm.
Addition of thapsigargin to human sperm has previously been demonstrated to induce an increase in [Ca2+]i, largely due to Ca2+ influx, providing evidence for a calcium store and capacitative calcium entry (CCE) (Blackmore, 1993; Rossato et al. 2001). Application of 10 µM thapsigargin to capacitated or uncapacitated human sperm induced a sustained elevation of [Ca2+]i, which was dependent upon [Ca2+]o.
When thapsigargin was applied > 180 s after P (during development of the sustained [Ca2+]i signal) it induced a transient (100 s) increase in [Ca2+]i (n = 7). When cells were pre-treated with 2-aminoethyl diphenylborate (2 µM) the sustained component of the response to P was significantly inhibited (from 126 ± 10 to 88 ± 14 nM, P < 0.01, paired t test, n = 9) but not abolished.
These data provide evidence for the role of CCE in the sustained component of the P-induced [Ca2+]i signal, as previously proposed (Kirkman-Brown et al. 2000).
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.
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