Success in the acquisition of Bombyx mori sperm motility is influenced by the extracellular production of nitric oxide (NO) in the presence of seminal fluid nitric oxide synthase (NOS)

Abstract

A trypsin-like protease called initiatorin is known to initiate sperm motility in the silkworm, Bombyx mori, but little is known about the signaling events leading to sperm flagellar beating. The aim of this study was to investigate whether this mechanism of sperm motility activation involves the signaling transmitter nitric oxide (NO). NO is produced from the amino acid L-arginine by the enzyme action of nitric oxide synthase (NOS; EC 1.14.13.39). Simple treatment of quiescent sperm with an NO donor (SNAP or NOC7) in vitro did not lead to activation of motility. Nevertheless, initiatorin- or trypsin-induced motility was blocked by pretreatment of sperm with either the NOS inhibitor L-NAME or NO scavenger carboxy-PTIO. These observations suggested that NO may play important physiological roles in the acquisition of sperm motility under the in vitro condition used here. Then, we investigated whether NO synthesis would occur in the spermatophore, a capsule containing spermatozoa that is created by the contents of various male reproductive glands and is the site of sperm maturation. The amounts of NO₂^- and NO₃^-, stable metabolites of NO, reached maximum values after enclosure in the spermatophore, a time when apyrene spermatozoa acquire vigorous motility. Moreover, RT-PCR and Western blotting analyses of NOS indicated that it is abundantly expressed in glandula (g.) lacteola of the virgin male ejaculatory duct, from which it is secreted to the seminal fluid and transferred to the female during mating. Previous studies demonstrated that free L-arginine is supplied de novo by a specific proteolytic reaction in which initiatorin participates during spermatophore formation (Osanai et al., 1987c). Based on these results, it can be presumed that the mixing of seminal fluid contents from each male reproductive organ during ejaculation induced NO production outside of the spermatid, and exogenous NO stimulated a signaling pathway involved in the activation of silkworm apyrene sperm.

Keywords: Bombyx mori; Nitric oxide (NO); Nitric oxide synthase (NOS); Seminal fluid proteins; Sperm activation; Trypsin-like protease.