Biphasic elevation of [Ca2+](i) in individual human spermatozoa exposed toprogesterone

Fluorimetric studies on progesterone-induced [Ca2+](i) signalling in mammal ian spermatozoa how both the well-characterised [Ca2+](i) transient and a s ubsequent sustained phase. However, the sustained phase is thought to refle ct release of the fluorochrome during the acrosome reaction and has not bee n subject to critical investigation, We have used single-cell imaging of [C a2+](i) to analyse the progesterone-induced [Ca2+](i) response in large num bers (>2000) of capacitated, human spermatozoa. In 70% of cells, treatment with progesterone induced a transient increase, which typically peaked with in 1 min and decayed with a similar time course. Upon rapid application of progesterone this response peaked within 5-20 s. In 35% of progesterone-tre ated spermatozoa a sustained elevation of [Ca2+](i) occurred, which became discernible during the falling phase of the transient response and persiste d for at least 20 min. Both [Ca2+](i) responses were localised to the posta crosomal region. Averaging of large numbers of single cell responses genera ted traces similar to these seen in fluorimetric studies. Although the sust ained response was strongly associated with the initial, transient response , a few spermatozoa generated sustained responses that were not preceded by a significant transient response (5% of cells). It is concluded that a gen uine biphasic [Ca2+](i) signal is activated by progesterone and that the su stained response is a discrete signalling event with biological significanc e. (C) 2000 Academic Press.