Micromolar concentrations of the steroid hormone progesterone induce calcium influx and acrosome reaction (AR) in human sperm. Calcium influx occurs as an initial transient, lasting up to 4 min, followed by a sustained phase. The study aimed to analyse the effects of progesterone concentration on the calcium transient and subsequent AR.
Highly motile spermatozoa from human donors were harvested using direct swim-up with sEBSS media (0.3 % BSA), and were incubated for 6 h at 6 million cells ml-1. For single cell analysis, sperm were labelled with Oregon Green Bapta AM, adhered to coverslips in a perfusion chamber and imaged at 25 °C. For fluorimetric studies sperm were labelled with fura-2 AM and imaged at 37 °C; a Kd = 224 nM was assumed.
Progesterone sensitivity was measured across a series of concentrations ranging from 0.3 nM to 30 mM. AR was induced at a concentration of 3 nM (P < 0.025, n = 9, paired t test) and all higher concentrations. The amplitude of the initial calcium transient was analysed using fluorimetry. A doseÐresponse curve showed that a maximum [Ca2+]i was stimulated with 0.3 mM progesterone (730 ± 156 nM, S.E.M. n = 8); however, this was not significantly different from doses of 30 nM, 3 mM and 30 mM (P > 0.05, paired t test). Stimulation by 3 nM progesterone gave a [Ca2+]i transient of 70 ± 11 nM (n = 7), which was significantly lower than stimulation with 30 nM (P = 0.04, n = 5, paired t test). A slight but significant maintained elevation of resting [Ca2+]i (20 ± 3 nM, P = 0.01, n = 4, paired t test) was seen when cells were stimulated with 0.3 nM progesterone.
Aitken et al. (1996) reported that after treatment with 5 mM progesterone sperm became refractory to a second progesterone stimulus for 30Ð60 min. Populations of cells were stimulated sequentially with 3 nM, 30 nM and then 3 mM progesterone with a 7 min delay between additions, and were shown to re-evoke at each concentration in the fluorimeter. Single-cell analysis was then used to assess the number of cells responding to each progesterone concentration in the series. Analysis of 1209 cells revealed that 68 % were showing significant calcium transients at all three concentrations, indicating re-evokation of the progesterone transient [Ca2+]i pathway within individual cells. However, this re-evokation of the response does not occur if the initial concentration used is 0.3 mM or greater.
We conclude that progesterone exerts its full effect at doses much lower than those previously used and at these doses cells can respond to repeated stimuli.
The research was carried out according to local ethical guidelines with the Birmingham Women’s Hospital (HFEA no. 0119). Donors gave informed consent. This work was supported by BBSRC and The Wellcome Trust
All procedures accord with current local guidelines.