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sex peptide receptor: Biological Overview | References

Mating in many species induces a dramatic switch in female reproductive behaviour. In most insects, this switch is triggered by factors present in the male's seminal fluid. How these factors exert such profound effects in females is unknown. Identified here is a receptor for the Drosophila melanogaster Sex peptide (SP, also known as Acp70A), the primary trigger of post-mating responses in this species. Females that lack the sex peptide receptor (SPR, also known as CG16752), either entirely or only in the nervous system, fail to respond to SP and continue to show virgin behaviours even after mating. SPR is expressed in the female's reproductive tract and central nervous system. The behavioural functions of SPR map to the subset of neurons that also express the fruitless gene, a key determinant of sex-specific reproductive behaviour. SPR is highly conserved across insects, opening up the prospect of new strategies to control the reproductive and host-seeking behaviours of agricultural pests and human disease vectors (Yapici, 2008).

At various stages in their lifespan, animals can undergo marked switches in their innate behavioural patterns. Such behavioural switches are attractive models to explore the genetic and neural control of innate behaviours more generally, and are particularly apparent in the dimorphic behaviours involved in mating and reproduction. For example, males and females of most species have distinct mating behaviours that are usually specified during development, but in some species these can also be switched in the adult. In Drosophila melanogaster, the switch that specifies male or female mating behaviour is thought to be set during development by the sex-specific transcripts of the fruitless (fru) gene (Yapici, 2008).

Another type of behavioural switch found in many species is the marked change in female behaviour that occurs on mating. For example, in many insect species, virgin females are receptive to courting males and retain their eggs, whereas those that have recently mated are unreceptive and lay eggs. These changes in female behaviour are induced by factors present in the male seminal fluid. In Drosophila, the primary trigger of this behavioural switch is the sex peptide (SP), a 36-amino-acid peptide produced in the male accessory gland (Chen, 1988; Chapman, 2003; Liu, 2003). How SP exerts its effects on female behaviour is unknown, although it has been suggested that the SP might act in part by modulating the activity of neurons that express fru (Kvitsiani, 2006). This study has identified a SP receptor, SPR, and shows that it is specifically required in the fru neurons for the post-mating switch in female reproductive behaviour (Yapici, 2008).

The gene CG16752, henceforth referred to as SPR, was identified in a genome-wide transgenic RNA interference (RNAi) screen for genes required in the female nervous system for post-mating reproductive behaviour. Specifically, it was found that pan-neuronal expression of an RNAi transgene targeting SPR (elav-GAL4 UAS-SPR-IR1) led to a marked reduction in egg laying. To examine this egg-laying phenotype more carefully, and to assess other reproductive behaviours, a protocol was used in which individual virgin females were first tested for receptivity towards a naive male. Those females that mated were then allowed to lay eggs for 48 h before being retested for receptivity with a second naive male. In the initial mating assays, virgin SPR RNAi females were as receptive as the control females. However, in contrast to control females, SPR RNAi females laid very few eggs after mating, mated again at high frequency, and did not actively reject the second male. In all these assays, mated SPR RNAi females behaved indistinguishably from wild-type virgin females, as well as from females previously mated to SP null males (Yapici, 2008).

To control for potential off-targeting effects of the initial RNAi transgene, a second independent line, UAS-SPR-IR2, was generated that targets a different region of the SPR gene. In all four assays, this new RNAi line gave results similar to those obtained with the original line from the genome-wide library. A molecularly defined deficiency, Df(1)Exel6234, was identified that removes 88 kilobases (kb) from the X-chromosomal region that includes SPR and four other annotated genes. The molecular breakpoints of this deficiency were verified and it was confirmed to delete the entire SPR gene. Females homozygous for this deficiency were fully viable and had no obvious defects in the gross anatomy of their nervous system or reproductive organs. When tested in parallel in the same series of receptivity and egg-laying assays, Df(1)Exel6234 homozygous females showed the same post-mating defects as observed on RNAi knockdown of SPR (Yapici, 2008).

By mating SPR RNAi or deficiency females to males with sperm labelled by GFP (green fluorescent protein), it was confirmed that sperm were transferred and stored normally in these animals. The few eggs laid by these females are also fertilized and develop normally. It is therefore postulated that the abnormal post-mating behaviours of these females could be due to a lack of sensitivity to SP, rather than due to a more general defect in reproductive physiology. To test this, SP was injected into the haemolymph of SPR RNAi or deficiency virgin females. The receptivity of these females was then tested 5 h later in pairings with naive wild-type males. As controls, it was confirmed that wild-type virgins injected with SP were unreceptive, whereas those injected with buffer alone were just as receptive as uninjected virgins. In contrast, SPR RNAi and deficiency virgins remained receptive even after injection with SP. These genetic data demonstrate that SPR is required in the nervous system for the behavioural switch triggered by SP (Yapici, 2008).

The SPR gene is predicted to encode a G-protein-coupled receptor (GPCR). To test whether this GPCR might be the SP receptor itself, SPR complementary DNA was expressed in mammalian Chinese hamster ovary (CHO) cells together with the Ca²⁺ reporter aequorin. In this assay, ligand-mediated GPCR activation triggers a luminescent flash by means of the Gα_q- or Gα ₁₁-dependent Ca²⁺ pathway. Only a very weak response to SP was detected in these cells, even at concentrations as high as 10 microM. It has been suggested that SP responses might involve the cAMP rather than the Ca²⁺ pathway (Harshman, 1999), and so one reason for this poor response might be that SPR normally couples to G proteins other than Gα_q/11. Accordingly, these cells were cotransfected with constructs encoding one of three different chimaeric G proteins (Gα_qs, Gα_qi or Gα_qo) designed to divert Gα_s-, Gα_i- or Gα_o-dependent signals, respectively, from the cAMP pathway into the Ca²⁺ pathway. Indeed, co-expression of Gα_qi or Gα_qo, but not Gα_qs, resulted in robust Ca²⁺ responses to SP (Yapici, 2008).

The response to SP is highly specific, because comparable levels of activation by any of eight other Drosophila peptides, even at 10 microM, were not detected. Among the closest relatives of SPR in Drosophila are CG2114 (also known as Fmrf Receptor) and CG8784, receptors for FMRFamides and hugin-gamma, respectively. Neither FMRFamide nor hugin-gamma activated SPR, and, conversely, expression of CG2114 or CG8784 in CHO cells conferred sensitivity to their respective ligands, but not to SP. In a dose-response assay, it was determined that SP activates SPR with an effector concentration for half-maximum response (EC₅₀) of 1.3 nM. The closely related peptide, DUP99B, which induces the same post-mating responses as SP in injection assays (Saudan, 2002), activates SPR with an EC₅₀ of 7.3 nM. Thus, both SP and DUP99B specifically activate SPR at physiological concentrations, with EC₅₀ values in the low nanomolar range typical for such peptide-GPCR interactions. It is concluded that SPR encodes a functional receptor for SP that couples to Gα_i and/or Gα_o to regulate cAMP levels (Yapici, 2008).

To define the cellular targets of SP, antisera were generated against an amino-terminal region of SPR. These antisera revealed high levels of SPR expression in the female reproductive organs, in particular in the spermathecae, the primary sites for long-term sperm storage, and the lower oviduct. Staining with the anti-SPR antisera was restricted to the cell membrane and was absent in Df(1)Exel6234 homozygous females, confirming the specificity of the antisera. SPR could not be detected in the male reproductive organs (Yapici, 2008).

SP is thought to pass from the reproductive tract into the haemolymph, and ultimately to act directly on targets in the central nervous system (CNS) (Kubli, 2003; Peng, 2005). Indeed, staining the adult female CNS with anti-SPR revealed a broad expression on the surface regions of both the brain and the CNS. This staining was absent or greatly reduced in SPR deficiency or RNAi females. Expression was most prominent in ventral regions of the suboesophageal ganglion (SOG), the cervical connective and many nerve roots in the brain and VNC. The restricted staining on the surface of the CNS is consistent with SPR detecting a ligand that circulates in the haemolymph and crosses the blood-brain barrier. It is unlikely to be an artefact caused by poor antibody penetration, because SPR was reliably detected in central brain regions on ectopic expression of a UAS-SPR transgene. Overall, the distribution of SPR concords remarkably well with the reported binding sites of radiolabelled SP applied to whole-female-tissue sections in vitro (Ottiger, 2000). Intriguingly, a very similar distribution was also observed in the male CNS, although at this point no function can be ascribed to SPR in males (Yapici, 2008).

Post-mating responses can be induced in virgin females not only by injection of SP but also by blocking synaptic transmission of neurons that express the sex-specific transcripts of the fru gene. It was also found that some of the central neurons that express SPR are also positive for fru, as revealed by the fru^GAL4 driver. In particular, SPR seemed to be expressed in many fru^GAL4-positive neurons in the SOG and throughout the VNC. To test whether SPR function is specifically required in fru neurons, the fru^GAL4 driver and UAS-SPR-IR1 were used to knockdown SPR only in these cells. Just like the SPR-deficiency mutants, these females showed normal receptivity as virgins, but then laid very few eggs and re-mated at high frequency (Yapici, 2008).

To test whether expression in fru neurons is also sufficient for the post-mating switch, fru^GAL4 and UAS-SPR were introduced into SPR-deficient females. In these females, SPR is expressed only in the fru neurons, yet complete rescue of the re-mating phenotype and partial but significant rescue of the egg-laying phenotype were observed. Together, these RNAi and rescue experiments strongly support the notion that SP triggers the post-mating behavioural switch primarily by modulating the activity of a subset of the fru neurons (Yapici, 2008).

The post-mating switch in female behaviour is not unique to D. melanogaster, but is common to most insect species. Although SP genes are difficult to identify outside the Drosophilidae, perhaps because of their small size, putative SPR orthologues in most sequenced insect genomes were readily identifiable, including Drosophila pseudoobscura, the mosquitos Aedes aegypti and Anopheles gambiae, the moth Bombyx mori and the beetle Tribolium castaneum. More distant relatives can also be detected in Caenorhabditis elegans, but potential vertebrate orthologues are less apparent (Yapici, 2008).

To test for functional conservation of the insect SPR family, SPR cDNAs were isolated from each of these five other insect species and tested for responses to D. melanogaster SP in the CHO cell assay. SP was shown to be a potent activator of the D. pseudoobscura, A. aegypti and B. mori receptors, with EC₅₀ values of 4.3 nM, 167 nM and 63 nM, respectively. These receptors also responded to DUP99B, but not to any of the eight control peptides, including FMRFamide and hugin-gamma. The A. gambiae and T. castaneum receptors were not activated by either SP or DUP99B, possibly because they do not bind the Drosophila ligands or were not functionally expressed in CHO cells. Nonetheless, the structural and functional conservation of SPR genes from Drosophila, Aedes and Bombyx, together with the observation that D. melanogaster SP can induce post-mating responses in the moth Helicoverpa armigera, indicates that the family of receptors identified are likely to mediate post-mating changes in female reproductive behaviour in many different insect orders (Yapici, 2008).

These data provide strong evidence that SPR is a receptor for SP, and that activation of SPR in fru neurons induces the switch to post-mating reproductive behaviour. The identification of SPR is the critical first step in explaining this behavioural switch at the molecular, cellular and circuit levels. Furthermore, because SPR is highly conserved across insect species, it provides the basis for cellular assays to identify SP-like activities in other species, and to develop new approaches for controlling the reproductive or host-seeking behaviours of various agricultural pests and human disease vectors (Yapici, 2008).

Sensory neurons in the Drosophila genital tract regulate female reproductive behavior

Females of many animal species behave very differently before and after mating. In Drosophila, changes in female behavior upon mating are triggered by the sex peptide (SP), a small peptide present in the male's seminal fluid. SP activates a specific receptor, the sex peptide receptor (SPR), which is broadly expressed in the female reproductive tract and nervous system. This study pinpoints the action of SPR to a small subset of internal sensory neurons that innervate the female uterus and oviduct. These neurons express both fruitless (fru), a marker for neurons likely to have sex-specific functions, and pickpocket (ppk), a marker for proprioceptive neurons. SPR expression in these fru⁺ ppk⁺ neurons is both necessary and sufficient for behavioral changes induced by mating. These neurons project to regions of the central nervous system that have been implicated in the control of reproductive behaviors in Drosophila and other insects (Häsemeyer, 2009).

SPR was initially identified in a genome-wide pan-neuronal RNAi screen. In this screen, the panneuronal elav-GAL4 driver was crossed to a genome-wide collection of RNAi transgenes, and female progeny were scored for egg-laying defects. Mated elav-GAL4 UAS-SPR-IR females lay very few eggs and remain sexually receptive, and thus, like SPR null mutants, behave as though they were still virgins. To define the cellular requirement for SPR function, the logic of this screen was inverted, crossing the UAS-SPR-IR transgene to a collection of 998 GAL4 lines and scoring the female progeny for egg-laying defects in the same fashion. In each of these lines, the GAL4 transcriptional activator is expressed in a random but stereotyped subset of cells, in which SPR function should now be inhibited by the UAS-SPR-IR transgene (Häsemeyer, 2009).

Fifty-nine lines were identified that resulted in a strong and reproducible egg-laying defect. Many of these lines were found to be broadly expressed, as revealed with a UAS-mCD8-GFP reporter. These lines were not examined further. More restricted neuronal expression was observed in seven lines, and for each of these a series of secondary assays was performed to confirm the egg-laying defect and to assess the receptivity of both virgin and mated females. For all seven GAL4 lines, SPR knockdown resulted in reduced egg laying and increased remating of mated females, but little if any change in the receptivity of virgin females. These defects were indistinguishable from those observed upon panneuronal SPR knockdown with the elav-GAL4 driver, or in SPR null mutant females. For the most restricted of the positive GAL4 lines, ppk-GAL4, it was confirmed that these defects can indeed be attributed to a diminished response to SP. It was then determined that SPR is required in ppk⁺ sensory neurons in the female reproductive tract (Häsemeyer, 2009).

This study describes a set of internal ppk⁺ fru⁺ sensory neurons in the female reproductive tract and provides evidence that SPR functions in these neurons to trigger the behavioral changes induced by SP upon mating. This conclusion rests on two complementary sets of observations. First, SPR is required in both ppk⁺ and fru⁺ cells, because postmating responses are eliminated upon knockdown of SPR in either cell population. Second, SPR is sufficient in either ppk⁺ or fru⁺ cells alone, as expression in either restores the postmating response in SPR null mutant females. This forces the conclusion that SPR acts exclusively in cells that are both ppk⁺ and fru⁺. The sensory neurons innervating the uterus are the only cells that were identified that express both of these markers. There are typically four to six such cells, and it is not yet known if they are functionally equivalent, or if egg laying and receptivity are regulated by two distinct cell subtypes (Häsemeyer, 2009).

Silencing synaptic transmission of ppk⁺ fru⁺ neurons mimics the activity of SP, in that they both cause virgin females to become unreceptive and initiate egg laying. Thus, an attractive hypothesis is that activation of SPR by SP reduces the synaptic output of these neurons. Like other ppk⁺ neurons, the ppk⁺ fru⁺ uterus neurons are probably mechanosensory. They may therefore have an important function as uterus stretch receptors in the coordination of sperm transfer, fertilization, and egg release. They may have two distinct functional states, depending on the presence or absence of SP. Because receptivity can be genetically uncoupled from egg production and egg laying, it is inferred that SP can also act independently of any stretch signal in the uterus. Modulation of receptivity and egg laying might be mediated through either distinct ppk⁺ fru⁺ subtypes or distinct central synapses (Häsemeyer, 2009).

How might SP regulate these sensory neurons? Two possibilities are envisioned. First, the ppk⁺ fru⁺ neurons may detect SP in the reproductive tract and alter their firing rate accordingly. In this model, passage of SP into the hemolymph would not be required to induce the postmating response. A second possibility is that SP enters the circulatory system and acts presynaptically to modulate the release of these neurons at their central targets. The fact that SP can indeed be detected in the hemolymph of mated females does not in itself exclude the former possibility. At least some effects of SP, such as stimulating juvenile hormone synthesis in the corpus allatum, probably do require SP to enter the hemolymph. Similarly, the fact that SP triggers a postmating response even when injected directly into the hemolymph is also consistent with either model. The somata and some processes of the ppk⁺ fru⁺ neurons lie outside the uterus and would be readily accessible to factors in the hemolymph. A neural rather than a circulatory route has been proposed to mediate postmating responses in several species of moths. However, this conclusion is based upon the loss of this response upon nerve cord transection, a result predicted by both of these models. Thus, both models are consistent with currently available evidence from studies in Drosophila and other species, and distinguishing between them will require detailed studies of the physiological properties of the ppk⁺ fru⁺ neurons in response to SP (Häsemeyer, 2009).

The central targets of the ppk⁺ fru⁺ sensory neurons include the abdominal and/or subesophageal ganglia -- regions of the CNS likely to contain circuits that mediate behavioral responses to mating. The abdominal ganglion houses the octopaminergic neurons that are believed to regulate the release and passage of mature eggs from the ovary to the uterus. It is suspected that these neurons are direct or indirect targets of the ppk⁺ fru⁺ sensory neurons and that these circuits serve to ensure that ovulation and oviposition are coordinated with the presence of sperm (Häsemeyer, 2009).

Some ppk⁺ fibers project from the abdominal trunk nerve right through to the SOG, potentially forming a direct neural connection from the reproductive tract to the brain. It is suspected that these projections may feed into circuits that regulate female receptivity and other postmating behaviors. Virgin females are enticed to mate by the male's courtship song. Most auditory sensory neurons project to the mechanosensory neuropil in the lateral SOG, close to the terminal arborizations of the ppk⁺ neurons. The proximity of the auditory processing centers and the ascending ppk⁺ projections raises the attractive possibility that mating modulates an early step in song processing. The SOG also contains processes of the Ilp7 neurons, which function in egg-laying site selection after mating. Direct evidence for mating-induced changes in SOG circuit function is lacking in flies but has been obtained in other insects. In some species of moth, mating induces a long-term inhibition of the SOG neurosecretory cells that regulate female pheromone biosynthesis, making mated females less attractive to other males (Häsemeyer, 2009).

Having identified sensory neurons that detect SP in the reproductive tract, it will now be important to characterize the central pathways that process these signals to regulate female behavior. In the olfactory system, sensory neurons that detect pheromones are fru⁺, as are their postsynaptic partners in the brain. Given that the sensory neurons that detect SP are also fru⁺, and many fru⁺ neurons are also located in both the abdominal and subesophageal ganglia, it is enticing to think that a similar logic may apply in these pathways too. Elucidating the operation of these circuits should reveal how the female CNS integrates both external and internal information to switch between two very different behavioral patterns (Häsemeyer, 2009).

Search PubMed for articles about Drosophila Sex peptide receptor

Chapman, T., et al. (2003). The Sex Peptide of Drosophila melanogaster: investigation of post-mating responses of females using RNA interference. Proc. Natl. Acad. Sci. 100: 9923-9928. PubMed citation: 12893873

Chen, P. S. et al. (1988). A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster. Cell 54: 291-29. PubMed citation: 3135120

Harshman, L. G., Loeb, A. M., Johnson, B. A. (1999). Ecdysteroid titers in mated and unmated Drosophila melanogaster females. J. Insect Physiol. 45: 571-577 PubMed citation: 12770342

Häsemeyer, M., Yapici, N., Heberlein, U. and Dickson, B. J. (2009). Sensory neurons in the Drosophila genital tract regulate female reproductive behavior. Neuron 61(4): 511-8. PubMed Citation: 19249272

Kubli, E. (2003). Sex-peptides: seminal peptides of the Drosophila male. Cell. Mol. Life Sci. 60: 1689-1704. PubMed citation: 14504657

Kvitsiani, D. and Dickson, B. J. (2006). Shared neural circuitry for female and male sexual behaviours in Drosophila. Curr. Biol. 16: R355-R356. PubMed citation: 16713940

Liu, H. and Kubli, E. (2003). Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster. Proc. Natl. Acad. Sci. 100: 9929-9933. PubMed citation: 12897240

Ottiger, M., Soller, M., Stocker, R. F. and Kubli, E. (2000). Binding sites of Drosophila melanogaster sex peptide pheromones. J. Neurobiol. 44: 57-71. PubMed citation: 10880132

Peng, J., et al. (2005a). Gradual release of sperm bound sex-Peptide controls female postmating behavior in Drosophila. Curr. Biol. 15: 207-213. PubMed citation: 15694303

Saudan, P., et al. (2002). Ductus ejaculatorius peptide 99B (DUP99B), a novel Drosophila melanogaster sex-peptide pheromone. Eur. J. Biochem. 269: 989-997. PubMed citation: 11846801

Yapici, N., Kim, Y. J., Ribeiro, C. and Dickson, B. J. (2008). A receptor that mediates the post-mating switch in Drosophila reproductive behaviour. Nature 451(7174): 33-7. PubMed citation: 18066048

date revised: 10 August 2010

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