A sperm-activating trypsin-like protease from the male reproductive tract of Spodoptera litura: Proteomic identification, sequence characterization, gene expression profile, RNAi and the effects of ionizing radiation

• Published in: Journal of insect physiology 2024-07, Vol.156, p.104664, Article 104664
• Main Authors: Yadav, Priya, Seth, Rakesh K., Reynolds, Stuart E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Gene expression; Genitalia, Male - metabolism; Genitalia, Male - radiation effects; Insect Proteins - genetics; Insect Proteins - metabolism; Ionizing radiation; Male; Proteome profiling; Proteomics; Radiation, Ionizing; RNA Interference; RNAi; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Serine Proteases - genetics; Serine Proteases - metabolism; Sperm activation; Spermatozoa - radiation effects; Spodoptera - enzymology; Spodoptera - genetics; Spodoptera litura; Transcriptome
• ISSN: 0022-1910; 1879-1611; 1879-1611
• DOI: 10.1016/j.jinsphys.2024.104664

γ-irradiation of adults reduces trypsin-like protease expression in both the irradiated and the F1 generation. [Display omitted] •Like other moths, male Spodoptera litura have 2 types of sperm (apyrene, eupyrene).•Apyrene and eupyrene sperm are stored in the reproductive tract’s duplex region.•Extracts of the prostatic region of the male tract cause activation of motility in apyrene sperm.•Extracts contain a trypsin-family protease, TLSP identical in sequence to proteins encoded in 8 other noctuid moth genomes.•S. litura TLSP is expressed in the prostatic region and in accessory glands.•RNAi knockdown of tlsp expression significantly reduces male fertility. Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region. Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage. cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLS