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. 2015 Jun 9;200(4):1171–1179. doi: 10.1534/genetics.115.176669

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

Frank W Avila *,1, Allie B Cohen *, Fatima S Ameerudeen *,2, David Duneau †, Shruthi Suresh *,2, Alexandra L Mattei *,3, Mariana F Wolfner *,1
PMCID: PMC4574237  PMID: 26058847

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

IN 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), suggesting that the mouse MP is important for proper sperm function.

MPs are common in insects [reviewed in Avila et al. (2011)]. Insect MPs have a variety of functions that affect fertility, from altering female receptivity postmating to being required for sperm storage in mated females. For example, Drosophila hibisci and D. melanogaster MPs reduce female receptivity in the short term (Polak et al. 1998; Bretman et al. 2010). In bumblebees, MPs physically switch off receptivity (Baer et al. 2001; Sauter et al. 2001) and have functions related to sperm competition (Duvoisin et al. 1999). In the malaria mosquito Anopheles gambiae, the MP is necessary for sperm storage (Rogers et al. 2009) and has been proposed to contribute to immunity and Plasmodium transmission (Mitchell et al. 2015).

In species whose MP biochemistry has been examined, MPs are generally composed of male-derived seminal fluid proteins (SFPs) (Lung and Wolfner 2001; Kawano and Yoshida 2007; Rogers et al. 2009; Bretman et al. 2010; Dottorini et al. 2012; Dean 2013). Upon transfer, SFPs effect numerous physiological and behavioral changes in mated females [reviewed in Poiani (2006) and Avila et al. (2011)]. However, female-derived proteins also have been found in MPs (Rogers et al. 2009). We know little about the role of MPs, and most of the SFPs that comprise them, in reproduction. Here we used the model organism D. melanogaster to dissect the role of MPs in postmating events in this species.

D. melanogaster MPs form shortly after mating begins (Neubaum and Wolfner 1999; Lung and Wolfner 2001). They have two distinct regions comprised of SFPs originating from different male reproductive tissues. A dense posterior mating plug (PMP) composed of SFPs from the ejaculatory bulb (EB) (Lung and Wolfner 2001; Bretman et al. 2010) forms ∼5 min after the start of mating (ASM). A gelatinous anterior mating plug (AMP) primarily composed of SFPs from the male accessory gland (Bertram et al. 1996; Lung and Wolfner 2001) forms ∼20 min ASM. The MP remains in the uterus for several hours until females eject the structure along with unstored sperm (e.g., excess sperm from the recent mate and displaced sperm from previous mates) (Manier et al. 2010; Lüpold et al. 2013; Lee et al. 2015). A recent study identified female innervation that modulates MP ejection and showed that this behavior has an impact on fertility (Lee et al. 2015). MP ejection may be important in postcopulatory sexual selection because the timing of ejection influences the fertilization success of competing males (Lüpold et al. 2013).

Some functions of the D. melanogaster AMP are known. Females that do not receive the SFP Acp36DE during mating do not form an AMP (Bertram et al. 1996) and fail to store sperm at optimal levels (Neubaum and Wolfner 1999; Bloch Qazi and Wolfner 2003). Further, Acp36DE induces conformational changes in the female's uterus that are important for efficient sperm storage (Avila and Wolfner 2009). Whether the presence of Acp36DE in the AMP or its localization to sperm and/or other female tissues (Bertram et al. 1996) is required for its sperm storage function is unknown.

Less is known about the PMP. Two PMP proteins have been identified previously: protein of the ejaculatory bulb of melanogaster (PEBme) (Ludwig et al. 1991; Lung and Wolfner 2001) and protein of the ejaculatory bulb II (PEBII) (Bretman et al. 2010). While the function of PEBme is unknown, PEBII reduces the likelihood of female remating in the first 4 hr postmating (Bretman et al. 2010). However, additional functions of the PMP in general, and of PEBme specifically, are not well understood.

We report that PEBme is essential for complete male fertility: it is required for PMP integrity and retention of the ejaculate in mated females. RNA interference (RNAi) knockdown of PEBme causes defects in early sperm storage events, suppressing the number of sperm in storage by 24 hr ASM, and also seriously impairs other postmating responses. Further, when PEBme knockdown males disengage from females at the end of mating, the ejaculate adheres to their genitalia, resulting in ejaculate loss from the female reproductive tract. Our results show that PEBme is necessary for proper PMP coagulation and to retain ejaculate within the uteri of mated females to ensure maximal sperm storage. Finally, we identified 60 additional MP proteins by mass spectrometry and showed that two of the most abundant are also required for full fertility. The results reported here show that the D. melanogaster MP is integral for optimal fertility.

Materials and Methods

Fly stocks

We used the UAS;GAL4 system (Brand and Perrimon 1993) to express transgenic constructs (under UAS control) in the specified tissue. CrebA-GAL4 [Bloomington Drosophila Stock Center (BDSC) #49409] was used to express mCD8::GFP (BDSC #5137), Rh1G69D (Ryoo et al. 2007), and PEBme double-stranded RNA (dsRNA) [two independent Vienna Drosophila RNAi Center (VDRC) lines: #100183 and #18973] in male EBs. Also, ovulin-GAL4 (Chapman et al. 2003) was used to express CG8626 and CG15616 dsRNA (VDRC #103960 and #105778, respectively) in male accessory glands. All males were mated to Canton-S females. In our Rh1G69D experiments, CyO balancer siblings were used as controls. In our PEBme, CG15616, and CG8626 experiments, control males were generated by crossing the CrebA-GAL4 driver to attP2 females (VDRC #60100). Knockdowns were quantified by RT-PCR (Supporting Information, Figure S1); the data illustrated that CrebA-GAL4 is an effective driver for RNAi in the EB. Flies were raised at 23° on standard yeast-glucose medium and a 12:12-hr light:dark cycle and aged 3–5 days before use in each experiment.

Microscopy

Male reproductive tract expression of CrebA-GAL4 was determined by driving membrane-bound mCD8::GFP (Lee and Luo 1999). Reproductive tracts were dissected from UAS-mCD8::GFP/+; CrebA-GAL4/+ males and visualized for GFP expression. To examine MP formation, lower reproductive tracts (i.e., uterus, sperm storage organs, and common oviduct) of females mated to experimental or control males were dissected and visualized under ultraviolet (UV) illumination to take advantage of PEBme’s autofluorescence (Lung and Wolfner 2001). Tissues were dissected in 1× PBS and visualized using a Leica DM 500B fluorescence microscope (Leica Microsystems).

Fertility assays and interrupted matings

Fertility (progeny) and fecundity (eggs laid) assays were performed as in Herndon and Wolfner (1995). Females used in our fertility assays were all mated on the same day, and at the same time. We counted the number of eggs and resulting progeny for each female for each day of the experiment. Comparisons of egg and progeny production of the 5-day assays were performed using a repeated-measures ANOVA in JMP 9.02. This allows for the study of a single individual over a longitudinal period (i.e., a single female and how her egg laying/progeny production changes over time). To interrupt matings, a vial containing the mating pair was lightly flicked with a finger at 13 min ASM; such agitation caused control as well as experimental pairs to uncouple. Males then were immediately removed.

Uterine conformation assays

Uterine conformation assays were performed as in Avila and Wolfner (2009). Females mated to experimental or control males were frozen in liquid nitrogen at 35 min ASM. To obtain sufficiently large sample sizes, females from two to three independent mating events were used. Uteri were dissected in 1× PBS, visualized with an Olympus SZ61 dissection microscope, and staged as in Adams and Wolfner (2007). For each female, the stage of uterine conformation was determined, and the distribution of stages in females mated to experimental males, compared to mates of control males, was analyzed using a Wilcoxon test (rank sum) in JMP 9.02.

Sperm counts

Females were mated to experimental or control males (matings were observed), frozen in liquid nitrogen at 35 min or 24 hr ASM, and stored at −80° until reproductive tracts were prepared for sperm counts. To obtain sufficiently large sample sizes, females from two to three independent mating events were used. Sperm cells were stained and counted as in Avila et al. (2010) using a Zeiss 47 30 11-9901 stereomicroscope. Samples were blind coded before counting. Counts were analyzed using Wilcoxon tests (rank sums) in JMP 9.02.

Receptivity assays

Receptivity assays were done as in Ravi Ram and Wolfner (2007). Females singly mated to experimental or control males were assessed at 24 hr and 4 days ASM for their willingness to remate with a Canton-S male in a 1 hr window. Remating rate was analyzed using Wilcoxon tests (rank sums) in JMP 9.02.

Mass spectrometry

Mated females were frozen in liquid nitrogen at 1 hr ASM to ensure complete MP formation. One-hundred whole PMPs and the posterior half of the AMP (to minimize sperm contamination) were dissected in 1× PBS and then placed in 100 µl 6 M guanidine, 50 mM Tris, pH 7.8, and 0.5% Triton X-100. MPs were solubilized by three rounds of sonication for 30 sec using a Bioruptor (Diagenode) and boiling for 5 min. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed at the Cornell Biotechnology Resource Center Proteomics and Mass Spectrometry Facility. Mass spectra were searched against the annotated D. melanogaster genome. Identified proteins were ranked by calculating the mean normalized spectral abundance factor as in Kelleher et al. (2009).

RNA extraction, video recording, and mating duration methods can be found in the Supporting Information, File S5.

Results and Discussion

CrebA-GAL4 allows knockdown of PEBme

To determine the role of the PMP in D. melanogaster reproduction, we targeted the major EB protein PEBme (Ludwig et al. 1991; Lung and Wolfner 2001). Numerous male reproductive tract GAL4 drivers were available, but none drove specific expression in the EB. Ubiquitous RNAi-mediated knockdown of PEBme using a tubulin-GAL4 driver resulted in lethality. Therefore, we screened through Janelia Farm Fly Light GAL4 lines (Pfeiffer et al. 2008; Jenett et al. 2012) for EB expression. We identified one line that drove expression in the EB and ejaculatory duct of the male reproductive tract (Figure S2). This CrebA-GAL4 driver contains sequence fragments from flanking noncoding regions of cyclic-AMP response element binding protein A (CrebA), a gene that regulates secretory capacity in Drosophila (Fox et al. 2010).

We used CrebA-GAL4 to induce PEBme knockdown in males and examined PMP formation in their mates. Results described here and in the following section are for PEBme knockdown with VDRC line #100183; we obtained similar results with an independent VDRC line (#18973) (Figure S3). At 35 min ASM, when the PMP is typically fully formed, many mates of CrebA > PEBmeRNAi males had uncoagulated PMPs (Figure 1C) or PMPs that were reduced in size (Figure 1C′). In cases where the PMP did not coagulate, we observed fluorescent particles in the uterus, suggesting that CrebA > PEBmeRNAi males transferred some PEBme to females, possibly due to incomplete knockdown. However, these particles spread out under the weight of a coverslip (Figure 1C), indicating that the PMPs had not solidified. These findings suggest that PEBme is an essential component for PMP coagulation within the female reproductive tract.

Figure 1.

Figure 1

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PEBme is required for proper PMP formation. PMPs in mates of control males are fully formed (A and B) at 35 min ASM. PMPs in mates of CrebA > PEBmeRNAi males are not coagulated (C) or are reduced (C′) at this time. In an additional experiment to obtain quantitation, we observed that among 15 females mated to CrebA > PEBmeRNAi males, ∼33% had uncoagulated MPs, ∼33% had reduced MPs, and ∼33% had MPs that looked normal (to this resolution). To see the PMP in the context of the uterus, the reproductive tract in A was not placed under a coverslip (the dotted box highlights the PMP). To better visualize the PMP, uteri in B and C′ were placed under a coverslip. B and C′ are at the same magnification. PMPs are visualized under UV illumination.

The role of PEBme in MP coagulation is intriguing given PEBme’s sequence (Lung and Wolfner 2001). PEBme contains repetitive PGG motifs, similar to those that facilitate self-interaction in proteins that form homopolymers, such as preCol-D of mussel byssal threads (Qin et al. 1997) and the flagelliform gland silk protein of spiders (Hayashi and Lewis 1998; Hayashi and Lewis 2001). This suggests a possible mechanism for the role of PEBme in MP coagulation that will be a fruitful avenue for future investigation.

PEBme knockdown reduces the fertility of mating pairs

We assessed egg laying and progeny production when PMP formation was impaired by PEBme knockdown. Mates of CrebA > PEBmeRNAi males laid significantly fewer eggs (Figure 2A) and generated significantly fewer progeny (Figure 2B) than mates of control males. Thus, impairing PMP formation by knocking down PEBme adversely affects reproductive success.

Figure 2.

Figure 2

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PEBme knockdown decreases fertility. Mates of PEBme knockdown males (A) lay significantly fewer eggs (NPEB = 18, NCont = 19, d.f. = 1, F = 7.42, P = 0.01) and (B) produce significantly fewer progeny than mates of control males in the first 5 days after mating (NPEB = 18, NCont = 19, d.f. = 1, F = 9.12, P = 0.0038).

PEBme knockdown affects sperm storage and female willingness to remate

MP formation influences sperm storage in D. hibisci and A. gambiae (Polak et al. 1998; Rogers et al. 2009). Because PMP formation requires PEBme, we asked whether PEBme is also required for sperm storage in D. melanogaster females. Uteri of D. melanogaster females undergo a series of conformational changes upon SFP receipt—from a closed conformation to completely open (Adams and Wolfner 2007). These changes are necessary for sperm to be stored at maximal levels (Avila and Wolfner 2009). We found that disrupting PMP formation affected postmating uterine conformation. By 35 min ASM, uteri are typically in the final conformational stages (Adams and Wolfner 2007; Avila and Wolfner 2009). In mates of CrebA > PEBmeRNAi males, however, a large proportion of uteri remained closed or failed to open completely (Figure 3A).

Figure 3.

Figure 3

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PEBme knockdown affects sperm storage processes and female remating. (A) Progression of the uterine conformational changes in mates of PEBme knockdown males differs significantly from that of controls (NPEB = 28, NCont = 25, Z = 4.99, P < 0.0001). Stages 1–4 (closed) are placed into a single group for simplicity. (B) In mates of PEBme knockdown males, sperm accumulation into storage did not differ from that of controls at 35 min ASM (SR: NPEB = 21, NCont = 16, Z = −1.61, P = 0.11; ST: NPEB = 14, NCont = 15, Z = −0.79, P = 0.43; total: NPEB = 14, NCont = 15, Z = 0.72, P = 0.46). (C) By 24 hr ASM, sperm in storage was reduced in mates of PEBme knockdown males compared to controls (SR: NPEB = 22, NCont = 17, Z = 3.26, P = 0.001; ST: NPEB = 16, NCont = 16, Z = 3.11, P = 0.0017; total: NPEB = 16, NCont = 16, Z = 3.77, P = 0.0002). (D) Female remating rate is significantly higher by 4 days ASM after mating with a PEBme knockdown male rather than with a control male (24 hr: NPEB = 26, NCont = 22, Z = −1.88, P = 0.06; 4 days: NPEB = 26, NCont = 27, Z = 3.04, P = 0.0023). For the box plots in B and C, the middle horizontal line represents the median, the lower and upper margins of the box represent the 25th and 75th quartiles, and the whiskers extend to the minimum and maximum of the data (excluding potential outliers, which are shown as points outside the whiskers). SR, seminal receptacle; ST, spermathecae; total, SR + ST.

Because a significant proportion of mates of PEBme knockdown males were defective in progression through the uterine shape changes, they were not expected to accumulate sperm into storage at wild-type levels. We examined sperm numbers in both types of storage organ (the seminal receptacle and the spermathecae) in these females. At 35 min ASM, shortly after females begin active sperm storage (Manier et al. 2010), mates of CrebA > PEBmeRNAi males did not differ significantly from controls in sperm accumulation into the seminal receptacle (Figure 3B) or spermathecae (Figure 3B), having similar levels of sperm accumulation overall (Figure 3B). However, we observed high variation in the number of sperm cells stored at 35 min ASM in mates of CrebA > PEBmeRNAi males. Since this time point may not have been sufficient to observe the totality of sperm storage defects, we also examined sperm storage at 24 hr ASM, when defects from PEBme knockdown should be readily detected. At 24 hr ASM, mates of CrebA > PEBmeRNAi males contained significantly fewer stored sperm cells than mates of controls in the seminal receptacle (Figure 3C) and spermathecae (Figure 3C); mates of CrebA > PEBmeRNAi males had stored only ∼43% as much sperm compared as did controls (Figure 3C). These results suggest that PEBme and, by extension, the PMP is required for sperm to be stored at wild-type levels.

Female refractoriness is affected by knockdown of the PMP protein PEBII (Bretman et al. 2010) and can influence male success in competitive situations. Female receptivity decreases after a single mating due to the action of the SFP sex peptide. Sex peptide is retained in females by binding to sperm and is slowly released into the female’s circulation, altering her behavior (Liu and Kubli 2003; Peng et al. 2005; Ravi Ram and Wolfner 2009). Thus, decreased receptivity persists when females contain stored sperm. Since female refractoriness can affect progeny outcomes, particularly in situations of sperm competition, we tested whether knockdown of PEBme affected female refractoriness. At 24 hr ASM, we observed a slight, but not significant, rise in female remating after initially mating to CrebA > PEBmeRNAi males compared to controls (Figure 3D). However, by 4 days ASM, females were significantly more likely to remate (relative to controls) if they did not receive PEBme (Figure 3D). The decrease in sexual refractoriness after mating with PEBme knockdown males likely results from the reduced amount of stored sperm in these females. Thus, PEBme is required for normal sperm storage and to reduce female receptivity after mating.

PEBme is required to maintain the ejaculate within the female reproductive tract

The severe and varied fertility effects caused by knockdown of the single MP protein PEBme contrast with the more limited phenotype reported for knockdown of PEBII (the only other MP protein tested in this way to date). PEBII knockdown affects remating but causes no observable defects in egg laying and progeny production by the mates of knockdown males (Bretman et al. 2010). This comparison suggested that the breadth of effects on PEBme knockdown might reflect loss of functions of many proteins, perhaps by the presence of less ejaculate remaining in the female.

We therefore examined the fate of the ejaculate in mates of PEBme knockdown males. We observed that CrebA > PEBmeRNAi males often had difficulty uncoupling from females at the end of mating and that these males remained physically attached to females via the ejaculate for ∼10–20 sec (Figure 4A and File S1 and File S2). Often, when CrebA > PEBmeRNAi males detached from females, the ejaculate protruded from the female’s posterior (Figure 4B and File S2). When documenting this phenotype, we observed a male incidentally pulling the ejaculate from the female without remaining attached to her (File S3), suggesting that the phenotype may have occurred even when males did not appear to be “stuck” to females following mating. We never observed these phenotypes with control males (File S4). Thus, the decreased fertility of CrebA > PEBmeRNAi males is likely due to the inability of their mates to retain the ejaculate within their uteri when the males uncouple. These observations, in conjunction with the data in Figure 1, suggest that PEBme is needed for the ejaculate to coagulate and thereby to allow a “clean” uncoupling at the end of mating and for female ejaculate retention.

Figure 4.

Figure 4

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PEBme is required to retain the ejaculate in the female reproductive tract. Video screen captures of PEBme knockdown and control males disengaging from females after mating ends: (A) PEBme reduction results in mating males and females becoming “stuck” together via the ejaculate (yellow arrow), (B) often resulting in the ejaculate protruding from the female when males eventually uncouple. NPEB = 8, NCont = 4.

Physical agitation exacerbates the fertility defects of CrebA > PEBmeRNAi males

Given that ejaculate was incidentally pulled from females when they and their CrebA > PEBmeRNAi mates uncoupled, we wondered whether physically agitating mating pairs would increase the occurrence of this effect and further reduce fertility. To test this, we agitated mating pairs by interrupting mating at 13 min ASM, after sperm and SFPs are transferred to females; SFP and sperm transfers occur at 3 and 8–10 min ASM, respectively (Gilchrist and Partridge 2000; Lung and Wolfner 2001; Manier et al. 2010). After interrupted matings, mates of CrebA > PEBmeRNAi males laid significantly fewer eggs (Figure 5A) and generated significantly fewer progeny (Figure 5B) than when mating was uninterrupted (Figure 2, A and B).

Figure 5.

Figure 5

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Interrupting mating exacerbates the fertility defects of PEBme knockdown. Interrupting mating after sperm and SFP transfer further reduces (A) the number of eggs laid (NPEB = 13, NCont = 15, d.f. = 1, F = 51.86, P < 0.0001) and (B) the progeny produced (NPEB = 13, NCont = 15, d.f. = 1, F = 66.55, P < 0.0001) of females mated to PEBme knockdown males compared to controls. Interrupting mating (C) perturbs progression of the uterine conformational changes (NPEB-int = 27, NCont-int = 25, Z = 5.77, P < 0.0001) and (D) exacerbates the effect seen in mates of PEBme knockdown males when mating was uninterrupted (NPEB-int = 27, NPEB = 28, Z = −2.07, P = 0.038), (E) leading to a significant reduction in sperm accumulation into storage at 35 min ASM compared to controls (represented as box plots; SR: NPEB-int = 17, NCont-int = 17, Z = 2.10, P = 0.035; ST: NPEB-int = 17, NCont-int = 17, Z = 3.54, P = 0.004; total: NPEB-int = 17, NCont-int = 17, Z = 2.38, P = 0.017). SR, seminal receptacle; ST, spermathecae; total, SR + ST.

Interrupting mating did not affect the progression of the uterine conformational changes in mates of control males (Figure S4), indicating that these changes occur normally on SFP transfer during mating. As in uninterrupted matings, progression of the uterine changes significantly differed between mates of CrebA > PEBmeRNAi males and mates of controls when mating was interrupted (Figure 5C). However, interrupting mating significantly exacerbated this effect—progression of the uterine changes in mates of CrebA > PEBmeRNAi males was significantly worse than when mating was uninterrupted (Figure 5D). Finally, interrupting mating affected sperm accumulation in the seminal receptacle and spermathecae at 35 min ASM (Figure 5E), leading to only ∼50% of sperm in storage in mates of CrebA > PEBmeRNAi males compared to controls (Figure 5E). Our results show that interrupting mating after sperm and SFP transfer exacerbates the fertility defects associated with PEBme knockdown.

Blocking translation of EB proteins affects PMP formation and fertility

Our results show that removal of one EB protein, PEBme, decreases fertility. However, the EB secretes additional PMP proteins (Bretman et al. 2010). To determine whether blocking translation of all EB proteins gave more severe fertility defects compared to those observed after knocking down PEBme, we drove expression of misfolded rhodopsin (Rh1G69D) in the EB. Expressing Rh1G69D in secretory tissues causes high levels of endoplasmic reticulum (ER) stress, which blocks translation by inducing the unfolded protein response (Ryoo et al. 2007). Driving expression of Rh1G69D in the male accessory gland—the major site of Drosophila SFP synthesis—blocks translation and secretion of SFPs from this tissue (Chow et al. 2015). Expressing Rh1G69D in the EB similarly blocks the function of this tissue.

In mates of CrebA > Rh1G69D males, the PMP did not coagulate or was reduced in size (Figure S5). Mates of CrebA > Rh1G69D males laid significantly fewer eggs (Figure S6A) and produced significantly fewer progeny (Figure S6B) compared to mates of control males. Additionally, in mates of CrebA > Rh1G69D males, progression of the uterine stages lagged significantly behind that of controls (Figure S6C), and significantly less sperm was stored in the seminal receptacle (Figure S6D) and spermathecae (Figure S5D) at 24 hr ASM, leading to an overall reduction in total sperm stored (Figure S6D).

Surprisingly, the magnitude of the fertility defects in mates of CrebA > Rh1G69D males was not as severe as that in mates of CrebA > PEBmeRNAi males. We noted that, in contrast to CrebA > PEBmeRNAi males, the mating duration of CrebA > Rh1G69D males was significantly longer than that of control males (Figure S7, A and B), with 15% of males mating an abnormally long time (>38 min) (Figure S7A). CrebA-GAL4 drives expression in some non-EB tissues (Jenett et al. 2012), making it possible that inducing ER stress in nonreproductive tissues affected the mating duration of CrebA > Rh1G69D males. We hypothesize that the additional time that these males mated attenuated the fertility defects associated with impairing PMP formation. These experiments confirm that EB proteins are required for proper PMP formation. The reduced fertility observed in mates of CrebA > PEBmeRNAi and CrebA > Rh1G69D males likely stems from perturbing PMP formation in their mates, suggesting that proper PMP formation is required for optimal Drosophila fertility.

Identification of additional MP proteins

To date, only three D. melanogaster MP proteins have been identified: the PMP proteins PEBme and PEBII (Ludwig et al. 1991; Lung and Wolfner 2001; Bretman et al. 2010) and the AMP protein Acp36DE (Bertram et al. 1996). Of these, Acp36DE (Neubaum and Wolfner 1999; Bloch Qazi and Wolfner 2003; Avila and Wolfner 2009) and PEBme (this report) have an impact on female fertility. Because >200 SFPs are transferred to females during mating (Findlay et al. 2008, 2009; Yamamoto and Takemori 2010), we wished to identify additional MP proteins that may be important in Drosophila reproduction. We analyzed the MP by LC-MS/MS and identified 60 annotated D. melanogaster MP proteins (after eliminating four sperm-specific proteins) (Wasbrough et al. 2010); the 25 most abundant MP proteins are shown in Table S1. A sizable portion of the MP proteins identified are products of the male accessory glands (flyatlas.org). That PMP formation occurs after genetic ablation of the accessory glands (Xue and Noll 2000) or their products (Chow et al. 2015) suggests that the accessory gland proteins identified are part of the AMP. SFPs with functions unrelated to the MP have been observed in the AMP: in experiments using sex-peptide::GFP fusions, GFP signal is detected throughout the AMP (but not the PMP) after MP coagulation has occurred [see Figure 3 in Minami et al. (2012)].

To determine whether the identified MP proteins are important for Drosophila fertility, we knocked down two additional abundant MP proteins in males and examined egg laying in mates of those knockdown males. We found that knocking down either CG15616 (Acp53C14a) or CG8626 (Acp53C14b) significantly impaired egg laying (Figure S8). This analysis further demonstrates the importance of the MP and the proteins that comprise it in Drosophila fertility.

Conclusions

We have shown the importance of PEBme, and, by extension, the PMP, in Drosophila reproduction. PMP formation occurs quickly in the female reproductive tract after mating begins and is retained for several hours, until the structure is expelled (Manier et al. 2010; Lee et al. 2015). When PEBme was knocked down in males, PMP coagulation was severely reduced or absent in their mates. This caused removal of the ejaculate from the uterus as mating pairs uncoupled. Extraction of ejaculate during uncoupling is a unique phenotype that, to our knowledge, has not been described previously in MP studies of other species.

One would expect the inability to cleanly uncouple to negatively affect fertility due to sperm and SFP loss. Indeed, we observed defects in uterine conformation, sperm storage, and egg laying in mates of PEBme knockdown males. This shows that PEBme (and proper PMP formation) is required to prevent ejaculate loss when males disengage from females at the end of mating. However, this effect was not absolute. We also observed some females with high levels of sperm in storage shortly after mating ended (35 min ASM), but when sperm storage was assessed at 24 hr ASM, we detected a significant reduction of sperm in storage in mates of PEBme knockdown males. This suggests that even when the ejaculate was maintained within some females at the termination of copulation, ejaculate loss occurred soon thereafter. Thus, an additional function of the PMP might be to prevent MP ejection after mating, providing time for sperm to be stored at maximal levels and for SFPs to exert their effects on mated females (e.g., inducing ovulation and promoting sperm storage).

Our results show that MP formation and ejection are important aspects of Drosophila reproduction. This analysis has begun to elucidate the male molecules required for MP formation, but female molecular contributions to MP formation and ejection are largely unknown. MP dynamics may influence postcopulatory sexual selection. In doubly -mated Drosophila females, the timing of MP ejection influences paternity—the later that MP ejection occurs, the more progeny are sired by a second mating male (Lüpold et al. 2013). Female molecular contributions to MP formation and/or ejection may be found among secreted components of the female reproductive tract (Allen and Spradling 2008; Prokupek et al. 2009; Schnakenberg et al. 2011; Wong et al. 2012; Sun and Spradling 2013), neuromodulators (Avila et al. 2012; Heifetz et al. 2014), and/or innervation (Middleton et al. 2006; Rubinstein and Wolfner 2013) required for fertility. In particular, a recent study showed that the activity of the neuropeptide diuretic hormone 44 in a subset of doublesex-expressing neurons is required for MP ejection (Lee et al. 2015). Future investigations into these female molecules and/or neural components also will greatly add to our understanding of Drosophila reproduction.

Supplementary Material

Supporting Information
supp_200_4_1171__index.html (3.4KB, html)

Acknowledgments

We thank Hyung Don Ryoo (New York University) for generously providing the UAS-Rh1G69D line, the BDSC for the CrebA-GAL4 driver, and the VDRC for the PEBme, CG8626, and CG15616 dsRNAi lines. We thank Dan Barbash for microscope access, Erin Kelleher for helpful discussions, Jessica Sitnik for assistance with statistics and with the CrebA-GAL4 driver identification, and Laura Harrington, Kristin Hook, and anonymous reviewers for helpful comments on the manuscript. We are grateful to the National Institutes of Health/National Institute of Child Health and Human Development for grant R01-HD038921 (to M.F.W.), the Rawlings Cornell Presidential Research Scholars Program (A.B.C.), the Weill Cornell Medical College in Qatar and the Qatar Foundation (F.S.A. and S.S.), the Swiss National Foundation (D.D.), and the NSF Biology Research Scholars (A.L.M.; National Science Foundation grant 0933921 to M. Shulman and R. Harris-Warrick) for support of this work.

Footnotes

Communicating editor: L. Cooley

Supporting information is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.115.176669/-/DC1

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Supporting Information
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