A network of interactions among seminal underlies the long-term postmating response in Drosophila

K. Ravi Ram1 and Mariana F. Wolfner2

Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853

Edited by Gene E. Robinson, University of Illinois, Urbana, IL, and approved June 23, 2009 (received for review March 17, 2009) Despite the importance of seminal proteins in fertility and their persists in mated females’ -storage organs for Ն5 days capacity to alter mated females’ physiology, the molecular path- postmating (14). Sex peptide’s gradual release from stored ways and networks through which they act have not been well sperm is suggested to allow it to access its target cells to induce characterized. Drosophila seminal fluid includes proteins that fall postmating responses over an extended period. Specifically, SP into biochemical classes conserved from to , mak- has been shown to regulate egg-laying and receptivity behaviors ing it an excellent model with which to address this question. through the action of its G--coupled receptor in a group Drosophila seminal fluid also contains a ‘‘sex peptide’’ (SP, Acp70A) of ppkϩfruϩ neurons associated with the female reproductive that plays a major role in regulating egg production and mating tract (19–21). behavior in females for several days after mating. This long-term Recently, four additional Acps [lectins CG1652 and CG1656, postmating response (LTR) initially requires the association of SP protease CG9997, and cysteine-rich secretory protein (CRISP) with sperm. The LTR also requires members of the conserved CG17575] were shown to also be required for manifestation of seminal protein classes (two lectins, a protease, and a cysteine-rich the LTR (4, 15). All of these Acps tested were undetectable in secretory protein). Here, we show that these seminal proteins female by 24 h postmating (22). This suggests the hypothesis that function interdependently, regulating a three-step cascade (first, the four short-lived Acps effect the LTR by allowing SP to be at the level of seminal protein transfer to the female; second, at the retained within the female, for example, by promoting SP’s level of stability; and third, at the level of localization within localization to sperm-storage organs. To test this hypothesis, we females), leading to the normal localization of SP to sperm-storage analyzed protein levels and localization of each of these Acps in organs. This localization is, in turn, necessary for successful induc- the absence or knockdown of each of the remaining four Acps. tion of the LTR. The requirements for manifestation of the LTR in We show that CG1652/CG1656, CG9997, and CG17575 interact Drosophila establish the paradigm that multiple seminal proteins to mediate the normal localization of SP to sperm and sperm- can exert their actions through a multistep, multicomponent net- storage organs. work of interactions. Results and Discussion ͉ ͉ ͉ ͉ mating response reproduction sperm sperm storage seminal fluid Long-Term Persistence of SP in Mated Females Requires CG1652/ CG1656, CG9997, and CG17575. To test whether CG1652/CG1656, n sexually reproducing organisms, molecular contributions CG9997, or CG17575 are required for the transfer of SP to the Ifrom both males and females are essential for successful female reproductive tract, we compared the accumulation and reproduction. Males provide sperm and seminal fluid to the levels of SP in females mated to males deficient in each of these female. These contributions induce physiological and behavioral Acps. As parallel controls, we tested SP persistence in females changes in mated females. Seminal fluid proteins have been mated to the normal siblings of the experimental males or to identified in a range of animals (reviewed in refs. 1–3), and loss males deficient in CG33943, an Acp that does not affect long- or reduction in levels of some of these molecules can impair term postmating responses (15). We did not observe detectable fertility (examples reviewed in refs. 1–3). Thus, unraveling their differences in SP transfer or levels in females mated to control mechanisms of action will offer insights of potential relevance to or any of the knockdown males at2hafterthestartofmating understanding some causes of human infertility and to designing (ASM) (Fig. 1A). That knockdown males transfer normal pest management strategies. Yet despite their importance, amounts of SP to females implies that they had made normal the molecular actions of seminal proteins, even those in bio- amounts of this Acp. From this, we infer that synthesis of SP in chemical classes conserved from insects to mammals (4), are males and SP transfer to females are independent of CG9997, poorly understood. An exception is that a few seminal proteases CG1652/CG1656, or CG17575. mediate proteolytic pathways [e.g., semenogelin processing in Upon transfer to females, SP binds to sperm and persists in (5–7) and ovulin processing in Drosophila (8, 9)]. We mated females’ seminal receptacles (SRs) for Ն5 days postmat- show here that Drosophila seminal fluid proteins falling into ing (14). We tested whether the four short-lived LTR Acps affect several conserved biochemical classes interact to regulate the the accumulation of SP in SRs (see Materials and Methods for persistence of postmating responses. details) of mated females. Although SP normally persists Ն5 In Drosophila, an important subset of seminal proteins is made in the male’s accessory glands. These accessory gland proteins (Acps) induce postmating changes in the physiology and behav- Author contributions: K.R.R. and M.F.W. designed research; K.R.R. performed research; ior of mated females (reviewed in refs. 3 and 10). Long-term K.R.R. and M.F.W. analyzed data; and K.R.R. and M.F.W. wrote the paper. persistence of these postmating responses [the long-term post- The authors declare no conflict of interest. mating response (LTR) (11–15)] requires both Acps and stored This article is a PNAS Direct Submission. sperm in the female. A critical component of the LTR is an Acp 1Present address: Embryotoxicology Division, Indian Institute of Toxicology Research, P.O. called sex peptide [SP, Acp70A (16, 17)]. On its own, SP can Box 80, M. G. Marg, Lucknow, UP, 226001, India. induce increased in egg production and can reduce in receptivity 2To whom correspondence should be addressed. E-mail: [email protected]. to remating, but these effects are transient (18). Upon transfer This article contains supporting information online at www.pnas.org/cgi/content/full/ to females during a normal mating, SP binds to sperm and 0902923106/DCSupplemental.

15384–15389 ͉ PNAS ͉ September 8, 2009 ͉ vol. 106 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902923106 Downloaded by guest on September 30, 2021 Fig. 2. Localization of sex peptide (SP) within seminal receptacles (SRs) of females mated to males knocked down (lane, Ϫ) for CG1652/CG1656, CG9997, CG17575, or CG33943 or mated to their control males (lane, ϩ). Seminal receptacles from mated female reproductive tracts were dissected at 1 h after the start of mating (ASM), and sperm were pulled out after puncturing the middle of the SRs. Protein was extracted from sperm (lane, sperm) or SRs minus sperm [lane, SR (no sperm)] and processed for Western blot analysis with anti-SP (or anti-␣-tubulin as a loading control). To confirm that SR (no sperm) samples contain no or few sperm, sperm were counted (see Materials and Methods for details) in those SRs after pulling out the sperm; we found only Ϸ25 sperm in those SRs (n ϭ 5), whereas normally SRs contain 250–300 sperm at this time point. Each lane contains protein equivalents of 20 females’ stored sperm or SRs lacking sperm.

Fig. 1. Levels of different accessory gland proteins (Acps) in mates of control or Acp-deficient males. Samples are: reproductive tracts (RTs) (A) or levels of SP in seminal receptacles (SRs) (B) of females mated to CG1652, CG1656, Fig. S2). Because very little SP accumulates in SRs in the absence CG9997, CG17575, or CG33943 control (ϩ) or knockdown (Ϫ) males at 2 h after of CG1652/CG1656, CG9997, or CG17575 (Fig. 1B), we tested the start of mating (ASM). Female RTs (minus ovaries, here and in remaining whether SP binding to sperm depends on the presence of these blots) or SRs were dissected at 2 h ASM. Protein equivalents of four mated Acps. For this purpose, we dissected sperm out of the SRs of female RTs or 10 SRs were loaded in each lane. The blot was probed with females mated to CG1652/CG1656, CG9997, and CG17575 anti-sex peptide (SP row) or with CG1652, CG1656, CG9997, or CG17575 antibodies, as indicated in successive rows. (C) Levels of SP in SRs at 24 h ASM. control or knockdown males at 2 h ASM and probed both the Although we represented CG1652 and CG1656 independently, knockdown of sperm sample and the remaining empty SR samples (see caption either one knocks down both (ref. 15; see Materials and Methods for details), to Fig. 2 for details) with anti-SP antibody. We detected sperm- because CG1652 and CG1656 are gene duplicates (32). The AG lane contained bound SP in controls (as expected from ref. 14). Interestingly, we protein equivalents of one pair of accessory glands from Canton-S males in this detected high levels of SP in SRs from controls, even after we blot (and the remaining figures in this article). The blots were probed with had removed sperm by dissection [Fig. 2, controls (ϩ), also anti-␣-tubulin as a loading control (tubulin row). The AG samples typically ϩ Ϫ show low signal with tubulin antibody. CG9997 is processed into a smaller control ( ) and knockdown ( ) in CG33943 row], suggesting form in the mated female; arrows indicate the full-length 45-kDa and pro- that SP also binds to SRs. In contrast, we found only traces of SP cessed 36-kDa forms in this figure (and in the remaining figures in this article). in samples of sperm or of SRs lacking sperm from females mated All of the samples were resolved on 7.5–15% gradient SDS/PAGE in this figure to CG1652/CG1656, CG9997, or CG17575 knockdown males (and in the remaining Western blots in this article). [Fig. 2, knockdown (Ϫ)]. These results indicate that in normal matings SP binds to sperm as well as to the SR and that such association requires days in SRs (14, 17), we observed less or no SP in SRs of females mated to CG1652/CG1656, CG9997, or CG17575 knockdown CG1652/CG1656, CG9997, and CG17575. Our finding that males (as compared with controls) even at 2 h ASM (Fig. 1B). association of SP with sperm requires these other Acps explains By 24 h ASM, we no longer detect SP in SRs of CG1652/CG1656, why recapitulating the normal binding of SP (i.e., specific to CG9997, or CG17575 knockdown mates, in contrast with con- sperm tails) by incubating pure SP with sperm dissected from trols or parallel controls (CG33943) (Fig. 1C). Reduced SP males’ seminal vesicles has not been possible (23); the additional localization to SRs in the absence of each of these four Acps does Acps needed to mediate the specific binding were not present in not reflect a lack of sperm in storage, because mates of knock- those incubations. The need for several Acps to mediate SP down males contain sperm in their SRs at levels similar to or association with sperm also suggests the intriguing possibility higher than those of their controls (15). Therefore, CG1652/ that these Acps’ action might act as a sort of timer controlling CG1656, CG9997, and CG17575 are required for localization or SP’s localization. If SP entered the female unbound to sperm, retention of SP in the SR. In contrast, SP is not required for the then it therefore initially would be free to cross a permeable part transfer of CG1652, CG1656, CG9997, and CG17575 to females of the female’s reproductive tract to enter the female’s hemo- and/or for subsequent accumulation of the former three to SRs lymph and access targets such as the corpora allata, where it (SI Text and Fig. S1). could modulate juvenile hormone synthesis (24–26). However, Sex peptide binds to sperm and accumulates in SRs (11, 14). later, once the actions of the Acps described here caused the SP EVOLUTION Sperm are necessary for SP accumulation in the SR (SI Text and to associate with sperm, SP would be present in the sperm-

Ravi Ram and Wolfner PNAS ͉ September 8, 2009 ͉ vol. 106 ͉ no. 36 ͉ 15385 Downloaded by guest on September 30, 2021 Fig. 4. Transfer and localization of CG1652, CG1656, and CG9997 to seminal receptacles (SRs) in females mated to CG17575 control or knockdown males. Samples prepared from SRs dissected from reproductive tracts and the re- maining reproductive tracts (RTs) at 1 h after the start of mating were Fig. 3. CG1652 and CG1656 in females mated to CG9997 control (ϩ)or processed for Western blot analysis and probed with anti-CG1656, CG1652, knockdown (Ϫ) males at 10 min and 1 h after the start of mating (ASM). CG9997, and ␣-tubulin antibodies. Protein equivalents of 20 SRs and 10 female Protein equivalents of four reproductive tracts (RTs) were loaded into each RTs were loaded. lane and processed for Western blot analysis with anti-CG1652 (CG1652 row), and CG1656 (CG1656 row), CG9997 (negative control), Acp29AB (positive control for the normal transfer of other Acps/lectins), and ␣-tubulin (loading samples collected from females during and after mating to control) antibodies as indicated. CG1652/CG1656 knockdown or control males showed that females mated to CG1652/CG1656 knockdown males had the storage organs, from where it could be slowly released to 36-kDa band but little to no 45-kDa band of CG9997 from 15 min continue maintaining postmating responses. to 2 h ASM (Fig. S4) unlike their controls, which had both (Fig. S4). This suggests that CG1652, CG1656, or both are required for CG9997 Is Required for the Normal Transfer of CG1652 and CG1656. the molecular stability of CG9997; whether effects of CG1652/ Because we observed similar effects on SP upon knockdown of CG1656 on stability and on processing of CG9997 are indepen- CG1652/CG1656, CG9997, or CG17575, we hypothesized that dent is not presently clear. Nevertheless, it suggests that CG9997 these Acps could interact in a network. To test this, we first (protease) and CG1652/CG1656 (lectins) function interdepen- analyzed the requirement of each Acp for the transfer of the dently and also the possibility of regulatory interactions between others to females by comparing females mated to Acp-deficient seminal proteases and seminal lectins in reproduction. males to their controls. CG9997, CG1656, and CG1652 do not require CG17575 for synthesis or transfer, because their levels CG17575 Is Required for CG1652 and CG1656 to Localize to the Seminal did not differ between mates of CG17575 knockdown and Receptacle. Above, we reported that CG1652/CG1656, CG9997, control males and vice versa (Fig. 1A). In contrast, females and CG17575 are required for the accumulation of SP in the SRs mated to CG9997 knockdown males had greatly reduced levels of mated females. We wished to determine whether any Acp in of CG1652 and CG1656 in their uteri compared with those of this group was required for the accumulation of any other Acp(s) controls at 2 h ASM [Fig. 1A, control (ϩ) and RNAi (Ϫ) lanes in this group within the SR. Because of technical limitations in of CG1652 and CG1656 in 9997 column]. These females received detection of some Acps (see SI Text), we could only analyze the other Acps tested at levels similar to controls (ref. 15; Fig. 1A, requirement for CG17575 in the accumulation of CG1652, SP and CG17575 rows). Thus, CG9997 affects either the transfer CG1656, and CG9997 in the mated female’s SR. of CG1652 and CG1656 or their retention in mated females. We detected CG9997 in SRs of females mated to CG17575 Subsequent analysis of reproductive tract samples from CG9997 knockdown males at levels similar to those of controls (Fig. 4, SR control and knockdown mates at 10 min and 1 h ASM indicated lanes in CG9997 row), suggesting that CG17575 is not necessary that CG9997 is required for the normal transfer of CG1652 and for transfer, stability, and accumulation of CG9997 and vice CG1656: Females mated to CG9997 knockdown males had less versa (also see Fig. 1A). In contrast, we detected CG1652 and CG1652 and CG1656, even at early times during mating (Fig. 3 CG1656 only in SRs of females mated to CG17575 control males and see SI Text). [Fig. 4, SR control (ϩ) lanes in CG1652 and CG1656 rows] but not in those of mates of CG17575 knockdown males [Fig. 4, SR CG1652, CG1656, or Both Are Required for the Stability of CG9997 in RNAi (Ϫ) lanes in CG1652 and CG1656 rows]. Sperm are Mated Females. To further define the interactions among these required for the accumulation of CG1652, CG1656, and CG9997 Acps, we tested whether CG9997 requires the other Acps for in sperm-storage organs (SI Text and Fig. S2). However, differ- transfer and tissue targeting. CG9997 is normally detected as a ences in levels of CG1652 and CG1656 in SRs of mates of 45-kDa protein in males and undergoes processing in the male CG17575 knockdown versus control males do not reflect dif- during transit to the female at the time of mating. Both full length ferences in sperm storage given the normal sperm counts at 2 h (45 kDa) and processed (36 kDa) CG9997 are detected in mated ASM (15) and at 1 h ASM (control mates, 486 Ϯ 12.46; females’ reproductive tracts (see Fig. 1A, Fig. S3, and SI Text for knockdown mates, 514 Ϯ 30.59; P ϭ 0.43). Rather, CG1652 and details). Females mated to CG17575 control or knockdown CG1656 require CG17575 for their localization to SRs. males had 45- and 36-kDa products of CG9997 at 2 h ASM (Fig. Above, we showed that CG9997 is required for transfer of 1A, CG9997 in 17575 column), showing that transfer and CG1652 and CG1656 to females. Here, we show that CG17575 processing of CG9997 are independent of CG17575. In contrast, controls accumulation of the same two Acps in SRs. Given these females mated to CG1652/CG1656 knockdown males contained effects, the lack of SP localization to sperm-storage organs in relatively lower levels of CG9997 than controls and had only the females mated to CG9997 or CG17575 knockdown males pos- 36-kDa form of CG9997 [Fig. 1A, CG9997 in RNAi (Ϫ) lanes of sibly reflects a role for CG1652 and CG1656 and that CG9997 1652 and 1656 columns]. A time course analysis of CG9997 on and CG17575’s effects on SP localization are thus indirect

15386 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902923106 Ravi Ram and Wolfner Downloaded by guest on September 30, 2021 (through CG1652/CG1656). However, we cannot rule out at present a direct effect of CG9997 and CG17575 on the accu- mulation of SP to sperm-storage organs.

CG1656 Binds to Sperm, and This Binding Requires CG17575. We showed, above, that CG17575 is required for the accumulation of SP, CG1652, and CG1656 in the SR and for the normal association of SP with sperm (Fig. 2). However, we did not detect CG17575 in mated females’ SRs upon transfer (ref. 22; Fig. S1 and Fig. S2). These observations suggested that for CG17575 to AB exert its effects, it and the other LTR-promoting Acps might interact with one another, with other male- or female-derived molecules, or with both, before entry of sperm and Acps into SRs, for example, when Acps and sperm are still in the uteri of mated females (refs. 14, 17, and 22; Figs. S1 and S2). Because SP association with sperm requires CG17575, we wished to explore whether the LTR-promoting Acps CG1652 and CG1656 also associated with stored sperm and whether this association re- quires CG17575. We tested this by immunofluorescence on sperm dissected out of SRs at 1 h ASM. Due to the cross- CD reactivity of anti-CG1652 with other proteins, we could only perform these experiments with anti-CG1656. We detected CG1656 on the tails, but not heads, of sperm from SRs of mated females in all controls (including knockdown of CG33943, which does not affect the LTR) or in mates of SP knockout males (no effect on CG1656 localization, see above) (Fig. 5 A, C–G, and I). In contrast, we did not detect CG1656 on sperm from females mated to CG1656, CG9997, or CG17575 knockdown males (Fig. 5 B, H, and J). These results indicate that CG1656 associates with sperm tails and that this association requires CG17575. The lack EF of detectable CG1656 on sperm tails from CG9997 knockdown males is likely a consequence of the requirement for CG9997 for the normal transfer of CG1656 into the female (see above, Fig. 3). At present, whether CG17575 plays a direct or indirect role in associating CG1656 with sperm (or in allowing it to enter the SR) cannot be determined. Thus, we find that a Drosophila seminal CRISP (CG17575) plays an important role in mediating the association of a lectin (CG1656) with sperm and with a sperm-storage organ. This is also interesting given that CRISPs and lectins in mammalian seminal fluid are GH suggested to be involved in sperm function (reviewed in refs. 27 and 28).

Model for Acp Interactions That Establish the Long-Term Response. On the basis of our results (summarized in Table S1), we propose the existence of a regulatory ‘‘LTR network’’ of Acps (Fig. 6) that mediates the localization of SP to sperm and SR. That SP then can be gradually released from those sperm to access targets that will allow it to mediate the LTR (14). Importantly, 138 seminal proteins have been identified so far in Drosophila IJ melanogaster (reviewed in refs. 1 and 3; see also ref. 29). Thus, future functional analyses of additional seminal proteins are Fig. 5. Immunofluorescence detection of CG1656 on sperm from seminal likely to add more players, and perhaps more steps, to this LTR receptacles (SRs) of females mated to control (Left) or knockdown (Right) network. males at 1 h after the start of mating. (A) CG1656 is a control for the specificity Most Acps in the LTR network are in conserved protein of the antibody. In these overlay images, anti-CG1656 staining (green) is seen families found in seminal fluid; our results therefore provide a on sperm tails [but not on sperm heads (stained red with propidium iodide)] in the control mating and is undetectable in mates of CG1656 knockdown model for dissecting seminal fluid regulatory cascades in other males (B), as expected because they do not receive CG1656. Anti-CG1656 animals. Our results also have implications for evolutionary staining of sperm from SRs of females mated to control males (C, E, G, I)or biology. Some seminal proteins evolve rapidly (30–35), which has knockdown males (D, F, H, J) lacking the indicated Acps also is shown. Staining been suggested to be the consequence of sexually antagonistic was as in (A, B). coevolution (36) or intrasexual competition. That some seminal proteins function interdependently as shown here could con- strain their ability to evolve rapidly or might imply that only targets for genetic association studies in Drosophila (37). Despite certain members of the network are in a position to do so. the conservation of SP and its receptor across Drosophila [and Consistent with the former notion, CG17575 is highly conserved beyond in the case of the receptor (21)], outbred males show (31, 32), whereas CG9997 shows evidence for rapid evolution genetic variation in their defensive sperm competitive abilities EVOLUTION (31, 35). Further, our results also suggest that LTR candidates and in their ability to cause female refractoriness to remating listed here, particularly the rapidly evolving ones, may be useful (38, 39). Therefore, studying whether these phenotypic differ-

Ravi Ram and Wolfner PNAS ͉ September 8, 2009 ͉ vol. 106 ͉ no. 36 ͉ 15387 Downloaded by guest on September 30, 2021 seminal protein interactions such as the ones we report here. Dissection of such interaction networks is also important for consideration in strategies to diagnose or alleviate certain in- fertilities, to control the reproduction of insect pests, or both.

Materials and Methods . We used transgenic fly lines carrying sympUAST-Acp (UAS-Acp-UAS) constructs for CG1652/CG1656, CG9997, and CG17575 to knock down mRNA and protein levels of these Acps (9, 15). The nonknockdown siblings of these flies were controls. As an additional control for the strain background, we carried out parallel crosses and experiments with flies carrying a sympUAST construct for knockdown of CG33943, which is an Acp that does not affect LTR (15). As described in refs. 9 and 15, we crossed the sympUAST-Acp lines to tubulin-GAL4/TM3, Sb flies (41) to generate the knockdown (experimental) males (tubulin-GAL4;UAS-Acp-UAS) and control males (TM3, Sb/UAS-Acp- UAS). Two independent insertion lines were analyzed for each Acp knock- down. The RNAi lines tested here knocked down the targeted Acp to Ͻ2.5% of the wild-type levels (9, 15). CG1652 and CG1656 are gene duplicates (4, 32) with Ϸ80% identity at the nucleotide level; RNAi of either one knocks down both (15). Hence, we cannot at present determine whether the observed phenotypes with their knockdown lines are due to both of these genes or to one in particular. Therefore, for the ease of discussion pertaining to results from these knockdown lines, we represent them as CG1652/CG1656 (also see the note below in Sample Preparation and Western Blot Analysis section). For SP, we used an SP null mutant (17) kindly provided by Eric Kubli (University of Zu¨rich, Switzerland). Sex peptide null mutant males and their sibling control males were generated by crossing the SP knockout line (0325/TM3, Sb ry)toa deficiency line (⌬130/TM3, Sb ry), as described in refs. 14 and 17. Matings were carried out by crossing 3- to 5-day-old unmated SP mutant or Acp knockdown or control males to 3- to 5-day-old virgin females of the Canton-S strain of D. melanogaster. To test for the requirement of sperm for Fig. 6. A model for the network of interactions among accessory gland the entry of Acps into sperm-storage organs, spermless males were generated proteins (Acps) and events that set up the long-term postmating response by crossing tudor (tud1 bw sp/tud1 bw sp; ref. 42) females to Canton-S males. (LTR) in mated Drosophila females. Highlighting denotes Acps that function at Isogenic control males for this experiment were the Cyϩ sons of tud1 bw sp/CyO those particular steps in the LTR interaction network; question marks denote females crossed with Canton-S males; we verified that these flies made sperm. uncertainties, such as whether action of CG17575 on CG1656 localization is direct or whether or how sex peptide (SP) is released from its seminal recep- All flies were maintained on standard yeast–glucose medium at room tem- Ϯ tacle (SR) binding. CG9997 assists in the transfer of CG1652 and CG1656 perature (22 1 °C) and 12:12 light/dark cycle. (CG17575 and SP are transferred without its assistance). The protease CG9997 appears to be the most upstream member in the LTR network (thus far). Sample Preparation and Western Blot Analysis. To determine the efficiency of (Transfer of CG9997 does not depend on any other Acps in the network.) After transfer of Acps, we used Western blots to do a time course analysis using entry into the female, the lectins CG1652 and CG1656 regulate stability, protein samples prepared from reproductive tracts of mated females as in Ravi processing, or both of CG9997. In the female’s uterus, the cysteine-rich secre- Ram et al. (22). Three- to 5-day-old Canton-S virgin females were mated to 3- tory protein CG17575 action allows, or is a precondition for, the binding of to 5-day-old unmated control or knockdown males or SP null males. The CG1656 to the sperm and accumulation of CG1652 and CG1656 in SRs. Sex mating pairs (mating normally lasts for Ϸ18–25 min in these flies) were frozen peptide appears to be the most downstream molecule in the LTR network, at 15 min ASM, or matings were allowed to complete without interruption, because it requires CG1652/CG1656, CG9997, and CG17575 for its accumula- and mated females were frozen at 30 min, 1, and 2 h ASM. tion in the SR. Gradual release of SP from sperm tails and SRs is proposed to Localization of Acps to SRs in the absence of CG1652/CG1656, CG9997, allow it to access its targets and cause the LTR (17–19). CG17575, or SP was analyzed using protein samples containing 18–20 SRs dissected from the mates of control, Acp knockdown, or SP null males at 1 h ASM. The localization of SP to the SRs in the absence of CG1652/CG1656, ences are attributable to LTR candidates mediating SP’s func- CG9997, or CG17575 was analyzed at 2, 24 h, and 4 days ASM. Although tions in mated females will be interesting. Drosophila has two types of sperm-storage organs (SR and spermathecae), we To conclude, we have shown that interdependent interactions analyzed only the samples from SRs, for three reasons. First, females mated to among a group of seminal proteins result in the localization of males knocked down for CG1652/CG1656, CG9997, or CG17575 had signifi- a long-term mediator, the SP, within the sperm-storage organs, cantly more sperm in their SRs compared with controls, but levels of sperm in thus leading to the manifestation of the LTR. Previous work in their spermathecae were similar to those of controls (15). Second, sperm are Drosophila and in primates has identified specific enzymatic easy to retrieve from SRs but difficult to retrieve intact from spermathecae effects of seminal proteins on one another (5–8). Here, using during dissections for sample preparation. Third, only SRs had been analyzed Drosophila as a model because of its tractable genetics, we show in the previous study that showed SP binding to the sperm (14). that interactions among seminal proteins are even more multi- Sample preparation and Western blot analysis were carried out as de- faceted: Seminal proteins work in multistep interactive networks scribed in ref. 22, except that the samples in the current study were separated that go beyond simple enzymatic interactions to regulate each using 7.5–15% gradient SDS/PAGE. We used affinity-purified anti-Acp anti- bodies [CG1656, CG1652, and CG17575 (22)], partially purified anti-serum [for others’ localization, sperm association, and stability or transfer SP (17); gift of Eric Kubli], or anti-serum (for CG9997; see SI Text). We have in females. Given that these molecules fall into conserved antibodies specific to CG1652 or CG1656; therefore, we represent their results seminal fluid protein classes (4), we believe that the sort of independently, even though both are knocked down simultaneously by RNAi. interactions defined in the present study are also likely to be a model for seminal protein action in animals beyond Drosophila. Sperm Counts. Sperm counts were done as in ref. 43. Slides were coded before Similar to the requirement of females’ environment for proteo- counting and decoded afterward to avoid bias. Each slide was counted twice; lytic cleavage of some seminal proteins (9, 40), opportunities repeatability was Ͼ91%. Five replicates were counted per treatment, and data might exist for female modulation of, and participation in, were analyzed using a two-tailed Student’s t test.

15388 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902923106 Ravi Ram and Wolfner Downloaded by guest on September 30, 2021 Immunofluorescence. Immunofluorescence to detect sperm-bound Acps was our past and present laboratory members for help and suggestions, especially carried out following the protocols of refs. 14 and 44, with minor modifica- N. A. Buehner for assistance with Western blot analysis. We are also grateful to tions. Please see SI Text for details. our Seminal Peptides group colleagues for helpful discussion of the timer hy- pothesis. We also thank Dr. Eric Kubli (University of Zu¨rich, Switzerland) for kindly ACKNOWLEDGMENTS. We thank Drs. M. C. Bloch Qazi, V. L. Horner, F. W. Avila, providing the biochemical and genetic reagents for SP and for personal commu- L. A. McGraw, J. L. Mueller, A. Saunders, L. K. Sirot, A. Wong, Ms. B.A. LaFlamme, nication about the results of the in vitro binding experiments. This work was and anonymous reviewers for helpful comments on the manuscript. We thank supported by National Institutes of Health Grant HD038921 (to M.F.W.).

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Ravi Ram and Wolfner PNAS ͉ September 8, 2009 ͉ vol. 106 ͉ no. 36 ͉ 15389 Downloaded by guest on September 30, 2021