Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme

Abstract

Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.KEYWORDS mating plug; sperm storage; PEBme; Drosophila reproduction I N numerous species comprising diverse taxa, a solidified structure forms inside the female reproductive tract during (or shortly after) mating that is referred to as the mating plug (MP; also called the copulatory plug; we will refer to these structures collectively as MPs). MPs are largely a coagulation of male seminal fluid components. In species that produce a MP, its role in reproduction varies. In some species, MP formation is thought to guard against sperm competition. For example, in primates, MPs are seen most often in species whose females mate multiply (Dixson and Anderson 2002). Primate MPs have been suggested to prevent remating (Dorus et al. 2004), thus acting as a form of passive mate guarding (Dunham and Rudolf 2009). In the mouse, perturbing (Murer et al. 2001) or preventing (Dean 2013) MP formation reduces male fertility; in the absence of MP formation, sperm migration to the sites of fertilization is impaired (Dean 2013) receptivity in the short term (Polak et al. 1998;Bretman et al. 2010). In bumblebees, MPs physically switch off receptivity Sauter et al. 2001) and have functions related to sperm competition (Duvo...

The molecular basis and reproductive function(s) of copulatory plugs

“…The sperm‐transport hypothesis has received considerable support. When male anopheline mosquitoes were prevented from forming a plug through RNA interference, their sperm failed to reach the female's sperm storage organ and therefore did not fertilize any eggs (Rogers et al, ); a similar effect was observed in Drosophila (Avila et al, ). The sperm of male rats surgically prevented from forming a copulatory plug or who formed incomplete plugs generally failed to migrate past the vagina (Blandau, ,; Matthews and Adler, ; Toner et al, ; Cukierski et al, ; Carballada and Esponda, ).…”

A mosquito sperm's journey from male ejaculate to egg: Mechanisms, molecules, and methods for exploration

“…They demonstrated that the mating plug ensures successful sperm storage, and sperm storage was severely inhibited without the plug‐forming enzyme transglutaminase. Similarly, the seminal fluid protein PEBme is required for effective mating plug coagulation and sperm storage in D. melanogaster (Avila et al, ).…”

Cleavage of the Drosophila seminal protein Acp36DE in mated females enhances its sperm storage activity

“…3). Further, the number of sperm in the seminal receptacle at 35min when females received Acp36DE FL was similar to females who received endogenous Acp36DE at the same time point (Avila et al 2015b). Females mated to Acp36DE CP1 males stored the most sperm, although they did not contain significantly more sperm than those mated to Acp36DE FL males.…”

“…We focused on sperm accumulation into the seminal receptacle at 35min ASM. At this time, sperm are rapidly entering the seminal receptacle (Manier et al 2010) and defects in sperm storage there are readily detected (Avila et al 2015b). …”

“…After transfer to the female RT, sperm must travel to and enter these storage organs, be maintained viably within storage, and ultimately be released to fertilize eggs (Bloch Qazi et al 2003). Individual SFPs act at each of these steps, working collectively to ensure sperm storage occurs efficiently (Ravi Ram and Wolfner 2007, Wong et al 2008, Avila and Wolfner 2009, Ravi Ram and Wolfner 2009a, Avila et al 2010, LaFlamme et al 2012, Avila et al 2015b). …”