No abstract

Hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate is thought to be intimately involved in agonist-induced changes in intracellular Ca2+ levels. Recently we have shown that human preovulatory follicular fluid, which induces exocytosis in human sperm, can stimulate a rapid, transient increase in sperm cytosolic [Ca2+] [Thomas & Meizel (1988) Gamete Res. 20, 397-411]. We report here that both a Sephadex G-75 column fraction, derived from follicular fluid, and progesterone (a component of both the G-75 fraction and whole follicular fluid) stimulate rapid hydrolysis of PtdIns(4,5)P2 and PtdIns4P in human sperm. We also report that progesterone stimulates a rapid influx of Ca2+ in human sperm. Human spermatozoa were labelled for 24 h with myo-[3H]inositol and then treated with either the G-75 fraction or progesterone. A 30-65% loss of label was detected in PtdIns(4,5)P2 and PtdIns4P within 15 s of stimulus addition; no changes were observed in PtdIns during 2 min of treatment. The loss of label from both lipids was accompanied by an increase in water-soluble inositol phosphates. Production of both InsP3 and InsP2 was seen within 10 s; however, InsP3 was rapidly removed and had reached control levels by 1 min. Similarly, formation of InsP2 reached a peak by 30 s and then began a decline accompanied by a corresponding increase in InsP. No increases in InsP4 were seen in sperm treated in this fashion. Stimulated hydrolysis of the phosphoinositides and release of inositol phosphates were both blocked by the Ca2+ antagonist La3+. Likewise, the progesterone-induced increase in intracellular Ca2+ was inhibited by La3+, and phosphoinositide hydrolysis stimulated by this hormone was dependent upon the presence of extracellular Ca2+.

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G‐75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G‐75 fraction containing the peak of acrosome reaction‐inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona‐free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.

Progesterone initiation of te human sperm acrosome reaction (AR) includes stimulation of a transient Ca2+ influx and a transient Cl- efflux. A role for one or more plasma membrane receptors has been suggested, but, except for evidence supporting the involvement of a sperm GABAA-like receptor/Cl- channel, there is little information about possible receptor identity. Here, we attempt to identify plasma membrane progesterone receptors in human sperm using a mouse monoclonal antibody (mAb-C262) raised against the C-terminal steroid-binding domain of the human intracellular progesterone receptor. C-262 inhibited the progesterone-initiated AR in a dose-dependent manner. Maximum inhibition was 77% as detected by fluorescein isothiocyanate (FITC)-concanavalin A (conA). Motility was unaffected. A control mouse mAb (h-151) raised against the human estrogen receptor did not inhibit the progesterone-initiated AR. Western blotting with C-262, but not with h-151, detected a major sperm protein band of 50-52 kDa. In indirect immunofluorescence localization studies, live and ethanol-fixed uncapacitated sperm and fixed capacitated sperm incubated with C-262, but not with h-151, displayed fluorescence at the equatorial segment region of the sperm head plasma membrane. In spectrofluorometric studies using capacitated sperm loaded with the Ca2+ probe Fura-2 or the Cl- probe MEQ, C-262 but not h-151 inhibited both Ca2+ influx and Cl- efflux. These ion fluxes could be due to the binding of progesterone to two different receptor/channels or to its binding to one and cross talk with the other. Our results strongly support the involvement of sperm plasma membrane receptors in the progesterone-initiated AR and provide a candidate for one such receptor.

The role of Ca2+ in the human sperm acrosome reaction was investigated using the fluorescent calcium indicator fura-2. Previous experiments have shown that a Sephadex G-75 column fraction of human follicular fluid can stimulate the human sperm acrosome reaction [Suarez SS, Wolf DP, Meizel S (1986): Gamete Res 14:107-121]. Using fura-2, we demonstrated that this Sephadex G-75 fraction also stimulates a rapid, transient increase in intracellular free Ca2+. This Ca2+ transient is blocked either by chelation of extracellular calcium or by addition of the Ca2+ antagonist La3+. We have also been able to stimulate the acrosome reaction in human sperm without significant loss of motility, using the divalent cation ionophore ionomycin. Acrosome reactions stimulated by whole follicular fluid, the G-75 fraction, or ionomycin are all blocked by removal of extracellular Ca2+. These results strongly suggest that an influx of extracellular Ca2+ is responsible for initiating the acrosome reaction in human sperm treated with human follicular fluid. This is the first demonstration in mammalian sperm that a potentially physiological stimulus can cause an increase in intracellular Ca2+ concomitant with the acrosome reaction.