Effects of polyamine biosynthesis inhibitors on the progesterone‐initiated increase in intracellular free Ca²⁺ and acrosome reactions in human sperm

Abstract

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca2+ ([Ca2+]i) and an increase in a Ca(2+)-requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca2+ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone-initiated increase in [Ca2+]i and AR in capacitated human sperm in vitro: DL-alpha-(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5'[[(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine (MDL 73811), an inhibitor of S-adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 microM MDL 73811 with or without various polyamines (245 microM). Progesterone (3.09 microM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein-labeled Con A lectin. DFMO strongly inhibited the AR, but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone-initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone.(ABSTRACT TRUNCATED AT 250 WORDS)

A Nicotinic Acetylcholine Receptor Is Involved in the Acrosome Reaction of Human Sperm Initiated by Recombinant Human ZP31

“…The remaining sperm were immediately fixed in 4% formaldehyde in PBS. The acrosomal status of the fixed sperm was then assessed using the ConA-FITC AR assay [22]. Following staining, sperm were mounted in fluoromount, and a minimum of 200 sperm from each treatment were scored in a blind fashion.…”

Nicotinic Acetylcholine Receptor Subunits and Associated Proteins inHuman Sperm

“…At the end of the reaction, 5-l aliquots were removed to check for motility (37). One-half of the sperm were stained with mAb306 antibody for localization of the receptor as described above, and the remaining sperm were assessed for acrosomal status using concanavalin A-FITC (45). We also compared ␣7 subunit localization before and after treatment with cytochalasin D. Capacitated sperm were treated with 20 M cytochalasin D an inhibitor of actin polymerization for 3 h as described previously (46), and sperm were then stained with mAb306 antibody for localization of the receptor as described above.…”

“…Aliquots of capacitated sperm (100 l each) were first preincubated with either lavendustin A or PP2 or with their inactive analogs lavendustin B or PP3, respectively, for 5 min followed by the addition of 250 M acetylcholine and further incubation for 10 min. The reaction was stopped by adding 4% formaldehyde, and at least 200 sperm per sample were assessed for acrosomal status in a blind fashion using concanavalin A-FITC (45). AR percentage data were transformed to the arcsine of their square roots.…”

Acrosome membrane associated protein kinase activity in hamster spermatozoa

“…Polyamines like spermine and spermidine are known to be present in the mammalian spermatozoa and have been implicated in sperm acrosome reaction [24] probably by activating a polyamine dependent protein kinase like casein kinase. However, in the present investigation, it was observed that spermine and spermidine which are stimulators of casein kinase II did not stimulate the overall phosphorylation of the acrosomal membrane proteins of caput and cauda epididymal spermatozoa of hamster, thus implying the absence of casein kinase II in both membranes.…”