The Drosophila melanogaster Seminal Fluid Protease “Seminase” Regulates Proteolytic and Post-Mating Reproductive Processes

Abstract

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.

A Drosophila Protease Cascade Member, Seminal Metalloprotease-1, Is Activated Stepwise by Male Factors and Requires Female Factors for Full Activity

“…Given how little is known about the regulation of seminal fluid proteases, a model system for studying these proteins is particularly useful. We recently demonstrated that a Drosophila melanogaster seminal fluid proteolysis pathway that begins in the male during copulation, and is completed in the female following copulation, is regulated by at least two proteases (LaFlamme et al 2012). This pathway lends itself well to genetic manipulation and is therefore a useful model in which to study the regulation seminal fluid proteases.…”

“…In D. melanogaster, as previously described, a proteolytic pathway is initiated in the male genital tract during ejaculation whereby the trypsin-like serine protease seminase is activated, allowing for the rapid cleavage and activation of the astacin metalloprotease CG11864 (LaFlamme et al 2012), which we rename here Seminal metalloprotease-1 (gene symbol: semp1). Semp1, in turn, is required for the proteolytic cleavage of at least two downstream Sfps: ovulin (Acp26Aa) and Acp36DE (LaFlamme et al 2012).…”

“…Semp1, in turn, is required for the proteolytic cleavage of at least two downstream Sfps: ovulin (Acp26Aa) and Acp36DE (LaFlamme et al 2012). Ovulin stimulates the release of eggs from the ovary during the first 24 hr after mating and contains a short region of sequence similarity to the egg-laying hormone of Aplysia californica (Monsma and Wolfner 1988;Monsma et al 1990).…”

“…Acp36DE undergoes one cleavage, beginning at 8 min ASM, to produce cleavage products of 68 and 50 kDa from the 122-kDa full-length protein LaFlamme et al 2012) in the female reproductive tract (RT) (Bertram et al 1996;Bloch Qazi and Wolfner 2003). As with ovulin, the role of Acp36DE cleavage in its activity is unknown.…”

BARD workshop: “Insect reproductive molecules: From model systems to agricultural applications”

“…Finally, on the male side of the SP/SPR paradigm, Mariana Wolfner (Cornell University) described how secreted products of the male's accessory gland are required for extended SP function within mated females. She reported results of collaborative studies with François Karch's lab (University of Geneva) that show that products from each cell type in this gland are needed for long term SP persistence in mated females, and that a seminal protease is important for this persistence of SP (LaFlamme et al, 2012).…”