Culex pipiens sperm motility is initiated by a trypsin‐like protease from male accessory glands

Abstract

In most animals, sperm are stored in a quiescent state in the male reproductive tract and only initiate motility when released into either the female reproductive tract, or, in the case of broadcast spawners, the external environment. Male accessory gland secretions transferred into the female reproductive tract may provide factors that modulate sperm viability and storage, or aid in sperm competition, as well as activate sperm motility. In several insects, serine proteases have been implicated in activating sperm motility. Our previous studies have shown that, in Culex quinquefasciatus, either a male accessory gland extract or purified trypsin is sufficient to initiate sperm motility in vitro. The objective of this study was to identify and characterize trypsin-like enzymes produced in the Culex male accessory glands. Mass spectrometry was used to analyze accessory gland proteins and this preliminary proteomic analysis identified 4 trypsin-like proteases (trypsin, trypsin4, and two trypsin7 isoforms). When measured with the chromogenic trypsin substrate N -benzoyl-L-arginine-ethyl-ester-hydrochloride (BAEE), trypsin-like protease activity in the accessory glands was robust, with a pH optimum of 8. The pH range for the Culex trypsin activity was substantially narrower than a mammalian homologue (porcine pancreatic trypsin). A soybean trypsin inhibitor (SBTI) -agarose affinity column was used to independently identify trypsin-like accessory gland proteins. Several proteins were enriched in the eluate, as detected by silver staining of SDS-PAGE gels. Taken together, these data demonstrate the presence of trypsin-like activity and several trypsin-like proteins in the Culex male accessory glands.

Proteins, Transcripts, and Genetic Architecture of Seminal Fluid and Sperm in the Mosquito Aedes aegypti

“…albopictus (28), Cx. quinquefasciatus (105), and several nonmosquito taxa (21, 22, reviewed in 69, 106), and are a common function of many insects' seminal fluid. Based on studies in other insects, functions of these enzymes may include the activation of sperm motility or the cleavage of propeptides into their active forms (107).…”

A Comparative Protein Profile of Virgin and Mated Male of Leucinodes Orbonalis Accessory Gland

“…These proteins have also been detected in the female sperm storage organs of Crematogaster osakensis (Gotoh et al, 2017b) and D. melanogaster (Allen & Spradling, 2008;Prokupek et al, 2008). In MAG of Culex pipiens, serine protease inhibitors were reported to play a role in downregulating protease activity before the transfer of sperm and semen to the female reproductive tract (Stephens et al, 2018). While others have also suggested their role in stimulating ovulation (Heifetz & Rivlin, 2010;LaFlamme et al, 2014), egglaying (Marshall et al, 2009), decreasing receptivity to remating (Peng et al, 2005) and controlling the release of sperms (Avila et al, 2010).…”

Physiological and Environmental Factors Affecting the Composition of the Ejaculate in Mosquitoes and Other Insects

“…Sperm motility in Culex mosquitoes can be activated by adding trypsin, which likely acts via the MAPK pathway to activate sperm [55]. Recent work by Stephens et al [56] identified four trypsin-like proteases that are expressed in the accessory glands of males of Cx. quinquefasciatus .…”

“…aegypti and An. gambiae , suggesting that sperm motility might be similarly regulated by trypsin proteases across mosquito species [56]. Additionally, Rogers et al [57] have demonstrated a critical role for transglutaminase, TG3, in cross-linking the seminal fluid protein, Plugin, to create the mating plug in An.…”