InteractiveFly: GeneBrief

Accessory gland protein 26Aa/Ovulin: Biological Overview | References

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, the increase was studied in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg's release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. Tests were performed to see whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover a mating- dependent relaxation of oviduct musculature was identified, for which ovulin is a necessary and sufficient male contribution. It is further reported that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin's increasing of OA neuronal signaling. Finally, it was shown that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction (Rubinstein, 2013).

Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating. Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology (Rubinstein, 2013).

In Drosophila, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (Sirot, 2009; Avila, 2011). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (Herndon, 1995: Heifetz, 2000), and the seminal protein Sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating. However, although a receptor for SP has been identified, along with elements of the neural circuit in which it is required, SP's mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses (Rubinstein, 2013)?

This question was addressed by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics. In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine. Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates. Noradrenaline, the vertebrate structural and functional equivalent to OA important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders. This paper investigated the role of neurons that release OA and tyramine in ovulin's action. For simplicity, these neurons are referred as 'OA neurons' to reflect the well-established role of OA in ovulation behavior (Rubinstein, 2013).

This study investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology. Ovulin was shown to increase ovulation and egg laying through OA neuronal signaling. Ovulin was found to relax oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects an ovulin-dependent increase was detected in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin's stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology (Rubinstein, 2013).

To investigate whether ovulin acts through neurons that release OA, tests were performed to see whether ectopically increasing the activity of those neurons could rescue the ovulation defect that is observed when females are mated to males without ovulin. This phenotype was tested by examining the number of eggs in the lateral oviducts 3 h after the start of mating; at this early time, ovulation rate can still be measured without being confounded by the influence of increased postmating oogenesis. If ovulin acts through OA neuronal signaling, females with ectopically hyperactive OA neurons should not show an ovulin-dependent increase in ovulation behavior. If ovulin acts independent or downstream of OA neuronal signaling, females with hyperactive OA neurons should still require ovulin for their postmating increase in ovulation rate. The tdc2-GAL4 driver was used to express a dominant-negative ether-a-gogo potassium channel subunit (UAS-eagDN) in OA neurons (loss of function of eag is known to induce increased neuronal activity) (Rubinstein, 2013).

As expected, control tdc2-GAL4 females had significantly more eggs in their lateral oviducts after mating with control males compared with mates of ovulin-deficient males; as expected, this increased ovulation also affected egg-laying rate. However, tdc2-GAL4/UAS-eagDN experimental females, which would have elevated activity of OA neurons, showed no difference in postmating ovulation rate regardless of whether or not they received ovulin from their mates. These results indicate that increasing OA neuronal activity in females compensates for a lack of ovulin received during mating. Thus, ovulin acts through OA neuronal signaling (Rubinstein, 2013).

No difference was detected in ovulation rates between control females and females with elevated OA neuronal activity after either type of female had mated to control males. This suggests that the normal increase in a Drosophila female's ovulation rate after mating reflects OA neurons' maximal possible contribution to ovulation rate. In this scenario, ovulin's role would be to increase ovulation to OA neuron's maximal contribution. Although this study focused on the principal known ovulation regulator, OA, it is likely that other signaling factors could further increase ovulation and that variation among the females could also result in differences in timing for the initiation of ovulation; all of these factors may dynamically control ovulation rates after mating. Consistent with this idea, many females in these experiments did not show the maximum possible number of eggs in their lateral oviducts (Rubinstein, 2013).

The tdc2-GAL4 driver was used to manipulate neurons that produce and release OA because tdc2-GAL4 expression strongly overlaps with immunoreactivity for OA and for TβH, the metabolic enzyme producing OA. These neurons also produce the OA metabolic precursor, tyramine, which can also affect behaviors. Although this study has shown that ovulin acts through OA neuronal signaling, the possibility cannot be excluded that ovulin might act through tyramine signaling in OA neurons. However, given the documented role of OA and the OA receptor OAMB in Drosophila ovulation, OA signaling is the most likely pathway for ovulin's action via OA neurons (Rubinstein, 2013).

OA increases ovarian muscle contractions, while blocking contractions of oviduct musculature in Drosophila and other insects. This effect of OA is proposed to facilitate ovulation of an egg from the ovary into the relaxed oviduct. Because ovulin stimulates ovulation, it was of interest to determine whether ovulin contributes to the relaxation of the oviduct that occurs after mating. Ovulin could relax oviduct musculature either through decreasing rates or amplitudes of phasic muscle contraction twitches, or it could induce a more basally relaxed state by modulating muscle tonus. Because OA blocks contractions of the oviduct, it was reasoned that ovulin's action on the oviduct might be observable by examining muscle tonus (Rubinstein, 2013).

Oviduct muscle tonus was assayed by measuring the average sarcomere length of oviduct myofibrils. To do this, females were used expressing Myosin heavy-chain fused to GFP. In these flies, GFP labels the A band of the sarcomere units. Thus, the distance between each GFP band represents the sarcomere length. Sarcomere lengths were measured in these females after mating to control or ovulin-deficient males and in virgin females. Although ovulin's most direct effect is to increase the release of an egg from the ovary to the lateral oviduct, it was not possible to obtain reliable measurements of sarcomere length in lateral oviducts, because sarcomere lengths varied along the length and circumference of the lateral oviduct (Rubinstein, 2013).

Therefore, myofibril sarcomere lengths were measured at the adjacent upper (anterior) common oviduct, where it is possible to reproducibly measure from a standardized position in the reproductive tract across individuals (Rubinstein, 2013).

A significant increase was observed in oviduct muscle sarcomere length between virgin females and control-mated females at 1.5 h after the start of mating, indicating that the oviduct relaxes after mating. Then, whether ovulin contributes to this relaxation was tested by measuring the sarcomere lengths of females mated to ovulin-null males. Sarcomere lengths of ovulin-null–mated females were significantly shorter than those of wild type-mated femalesa and were indistinguishable from those of virgin females. These data suggest that ovulin is necessary for the postmating relaxation of oviduct musculature (Rubinstein, 2013).

Then whether ovulin is sufficient to increase oviduct sarcomere lengths in females, in the absence of anything else male-derived, was tested. Virgin females were generated that could ectopically express ovulin (which is normally a male-expressed gene; UAS-ovulin) under control of a heat shock promoter (hs-GAL4) and control females that could not (they only carried hs-GAL4). Both types of females were subjected to heat shock to induce ovulin expression in experimental females and then oviduct sarcomere lengths were measured. Females that ectopically expressed ovulin showed significantly increased sarcomere lengths compared with control females (Rubinstein, 2013).

These results indicate that although male-derived ovulin is necessary for maximal ovulation and is sufficient to increase ovulation rates, ovulin is also a necessary and sufficient male contribution for relaxing the oviduct musculature. This suggests that ovulin's relaxation of oviduct musculature underlies its role in increasing ovulation rate (Rubinstein, 2013).

Because OA is critical for proper ovulation and blocks oviduct muscle contraction and because ovulin works through OA neuronal signaling, the results above suggested that OA might also be required for postmating oviduct muscle relaxation. This hypothesis was tested by examining oviduct sarcomere lengths in tbh^M18 mutant females, which are unable to produce OA and instead accumulate tyramine. In tbh^M18 heterozygous control females that also carried the MHC-GFP construct, a mating-dependent increase in sarcomere length was detected, like that seen with wild-type females. However, in tbh^M18 homozygous females, there was no detectable mating-dependent increase in sarcomere length. To determine whether tbh^M18 homozygous females do not exhibit an increase in sarcomere length after mating because they lack OA, tbh^M18 homozygous females were fed OA at 7.5 mg/mL and subsequently sarcomere lengths of virgin and mated females was measured. After tbh^M18 homozygous females were fed OA, there was an increase in sarcomere length in females mated to wild-type males compared with virgin controls. Although other signaling molecules (i.e., tyramine and glutamate) likely also play a role in oviduct muscle contraction, this result indicates that OA plays a critical role in the postmating increase in oviduct sarcomere length (Rubinstein, 2013).

To determine if oviduct sarcomere lengths depend on OA neuronal activity, thermally activated TrpA channels (UAS-TrpA) under control of tdc2-GAL4 were used to conditionally increase activity of OA neurons. At the nonactivating temperature of 22 ^oC, no difference in sarcomere lengths was seen between control virgin females that carried only the tdc2-GAL4 construct vs. experimental virgin females that carried tdc2-GAL4 and UAS-TrpA. However, at the TrpA-activating temperature of 32 ^oC, the experimental virgin females that carried both constructs had longer oviduct sarcomere lengths than control females. These data indicate that increasing the activity of OA neurons induces relaxation of oviduct muscle (Rubinstein, 2013).

These results suggested that ovulin increases OA neuronal activity to increase ovulation rates and that such increased activity induces a relaxation of oviduct muscle. To test this directly, the UAS-eagDN transgene was used to determine whether ovulin affects oviduct sarcomere length through OA neuronal signaling. The same experimental design measured oviduct sarcomere lengths instead of egg numbers. A significant difference was observed between tdc2- GAL4/+ control females when they were mated to males with (+) or without (-) ovulin. There was no significant difference in oviduct sarcomere length between experimental tdc2-GAL4/UAS-eagDN females that did or did not receive ovulin from their mates. This indicates that ectopically increasing OA neuronal activity can compensate for a lack of ovulin. Taken together, these data indicate that ovulin increases oviduct sarcomere length through OA neuronal signaling and support the model that ovulin works through OA neurons to maximize ovulation rates (Rubinstein, 2013).

To confirm whether increased oviduct sarcomere lengths confer an increased ability to accommodate an egg ovulated from the ovary, the circumference of oviduct musculature was measured through 3D reconstruction of the datasets used to determine sarcomere lengths, using a subset of control females (tbh^M18 heterozygotes). Sarcomere length and musculature circumference showed a significant, positive, and linear relationship, suggesting that increased sarcomere lengths also indicate an increase in oviduct circumference and therefore an increased ability to accommodate an egg. Further, mating resulted in a 19.4% increase in sarcomere length (comparing virgins and females mated to control male), which corresponds to a 42.6% increase of oviduct cross-sectional area, reflecting a sizable increase in the oviduct's activity to accommodate an egg after mating (Rubinstein, 2013).

By 6h after mating, increased synaptic development is detected for axons with type II boutons at the female reproductive tract (Kapelnikov, 2008). Type II synaptic boutons characterize OA neuronal release sites (Middleton, 2006; Monastirioti, 1995), and Kapelnikov (2008) reported an increased number of axonal boutons, anatomical swellings indicative of neurotransmitter release sites, in these synapses by 6 h postmating. An increase in synaptic bouton number is often attributed to increased activity at that synapse, a form of synaptic plasticity whereby a heavily used synapse grows to meet signaling demands. Because the results suggest that ovulin increases OA neuronal signaling and because type II synaptic boutons characterize OA neuronal release sites, tests were performed to see whether ovulin could increase the synaptic development of OA neuronal– reproductive tract neuromuscular junctions (NMJs) after mating. This was assessed by labeling OA neurons with GFP and counting synaptic boutons at OA neuronal termini at the common oviduct after the start of mating (Rubinstein, 2013).

Because it can take ∼2 h for changes in bouton number to become visible after neuronal stimulation, even though ovulin's effects on ovulation behavior are detectable by 1.5 h after the start of mating, initially 6 h postmating was chosen to examine ovulin effects on bouton numbers. At this time point, a significant increase was detected in the number of GFP-labeled boutons between unmated females and females mated to control males, suggesting that the type II boutons whose increase was reported by Kapelnikov (2008) are in fact from OA neurons. To determine whether ovulin affected the bouton number, the number of OA neuron boutons in females mated to control vs. ovulin-null males, was tested at 6 h after the start of mating. It was observed that females that had mated to ovulin-null males had significantly fewer GFP-labeled boutons than females mated to control males. Indeed, the numbers of GFP-labeled boutons in females that had mated to ovulin-null males were not significantly different from virgin females. These results support the conclusion that ovulin acts to up-regulate OA neuronal signaling (Rubinstein, 2013).

It was then asked whether the increase in the number of boutons might be evident at 1.5 h after the start of mating, when ovulin's function is detectable. It was reasoned that if ovulin induces the increase in synaptic boutons indirectly, i.e., by increasing activity of OA neurons, this process would involve biochemical transduction of increased activity; contributions from retrograde signaling; signal transduction of those signals; and finally mobilization of scaffolding, cell adhesion, and vesicle release machinery. Therefore, fully developed boutons would likely not be evident at 1.5 h after the start of mating, before the ∼2 h time window previously reported for the visible appearance of type II boutons. However, if ovulin directly affects cell-signaling or protein-mobilization pathways within OA neurons to induce an increase in bouton numbers, the process referred to above would not be required, and the ovulin-dependent increase in bouton counts might be visible at this early time point. No differences were detected in the number of GFP-labeled boutons between unmated females and females mated to control males at 1.5 h after the start of mating. Thus, neither mating nor any seminal protein (including ovulin) causes a detectable increase in bouton counts this early after mating. Because there is an ovulin-dependent increase in bouton number but it is only evident at a later time point, the increase in OA bouton number likely represents synaptic plasticity as an indirect response to ovulin-induced increase in OA neuronal activity, rather than a causative effect of ovulin directly on OA synaptic boutons. The data cannot distinguish whether the increased bouton number is the consequence of a direct effect of ovulin on OA neurons that leads to the ovulation increase or is caused by the passage of additional eggs through the oviducts (Rubinstein, 2013).

Future studies to measure sarcomere length in eggless females will resolve this question, and additional further measures, such as measurement of bouton numbers in tdc-GAL4/UAS-eagDN females, may also help to define the precise mechanism by which ovulin increases the number of type II boutons after mating (Rubinstein, 2013).

This study has reported that the Drosophila seminal protein, ovulin, acts through an endogenous signaling pathway in the female to affect reproductive physiology. Further, the relaxation of oviduct musculature by ovulin and its downstream regulator, OA neurons, provides a potential mechanism to explain how ovulin increases a female's ovulation rate after mating. The data also indicate that ovulin, likely indirectly, underlies the subsequent increase in the number of OA synaptic sites at the reproductive tract after mating. This provides another indication of ovulin's interaction with the OA neuronal signaling system. Moreover, the data suggest that the increased type II bouton counts reported after mating reflect, specifically, OA neurons and that the changes in mating-dependent synaptic regulation that had been measured by bouton counts and GFP-labeled vesicle contents can be caused by the action of specific seminal proteins such as ovulin. Further, these findings demonstrate a link between a seminal protein's postmating effect and a mechanism of action through known female regulators of reproductive physiology and behavior. It will be useful for future studies to determine whether ovulin acts centrally in the nervous system, on presynaptic terminals of the reproductive tract to increase OA synaptic output, or on nonneuronal tissue in the reproductive tract which signals back to the nervous system (Rubinstein, 2013).

Although OA's regulation of ovulation and egg-laying behavior is conserved in many arthropods, the ovulin gene is evolving rapidly under strong positive selection. An evolutionarily labile regulator sitting atop an evolutionarily conserved regulatory 'core' (in this case, the OA signaling pathway) allows selection to act on innovations enhancing the core's output without disrupting the core itself; a similar idea has been suggested for sex determination mechanisms in Diptera (Pomiankowski, 2004). Applying this logic to the model of ovulin and OA neuronal signaling, the conserved control by OA of ovulation could ensure that this essential process occurs unfettered, whereas frequent changes in ovulin sequence could allow for small, yet selectively advantageous, increases in ovulation rate (Rubinstein, 2013).

Because ovulation rates increase partially (but not to the full extent) after matings in which females do not receive ovulin, other signaling factors apart from ovulin-dependent OA neuronal signaling must also contribute to the postmating increase in ovulation. For example, in Drosophila and other insects, other signaling factors (e.g., females' glutamate, proctolin, and ILP7) are known to be important in ovulation and egg laying (Lange, 2009; Rodríguez-Valentín, 2006; Yang, 2008). However, two post-mating phenotypes described in this study, oviduct muscle tonus and synaptic plasticity, can be largely attributed to ovulin and its interaction with OA signaling. These results imply that other mating-related stimuli that increase ovulation might work through independent mechanisms, perhaps distinct from OA neuronal signaling (Rubinstein, 2013).

Observing effects of ovulin on synaptic strength at 6 h postmating was intriguing because by this time, ovulin is nearly undetectable in mated females and because ovulin has been reported to affect egg laying only within the first 24 h after mating. It is possible that the increased synaptic strength seen at 6 h is simply a visible, but indirect, consequence of ovulin's earlier effects and/or that the synaptic growth/plasticity that ovulin induced is somehow transient. Alternatively, ovulin might be able to affect egg production even after it has disappeared by having induced a longer-lasting developmental effect at the oviduct NMJ. Such consequences of ovulin's earlier actions may not be detectable after the first postmating day if they are masked by later, larger effects from other sources. For example, ovulin's primary action has been proposed to be to stimulate release of the mature oocytes that had built up in females before mating; in such a model, once all those accumulated eggs have been released, effects of ovulin may not be apparent in the context of Sex Peptide's large stimulation of egg production rate (Rubinstein, 2013).

This study found that a male seminal protein, ovulin, controls reproductive behavior in female Drosophila through neurons that release a canonical neuromodulator, OA. Understanding the female modulators of reproductive physiology thus provides a useful framework for addressing how male seminal proteins influence females. These findings provide support for the idea that seminal proteins modulate conserved endogenous physiological mechanisms and circuits (Rubinstein, 2013).

A novel function for the Hox gene Abd-B in the male accessory gland regulates the long-term female post-mating response in Drosophila

In insects, products of the male reproductive tract are essential for initiating and maintaining the female post-mating response (PMR). The PMR includes changes in egg laying, receptivity to courting males, and sperm storage. In Drosophila, previous studies have determined that the main cells of the male accessory gland produce some of the products required for these processes. However, nothing was known about the contribution of the gland's other secretory cell type, the secondary cells. In the course of investigating the late functions of the homeotic gene, Abdominal-B (Abd-B), it was discovered that Abd-B is specifically expressed in the secondary cells of the Drosophila male accessory gland. Using an Abd-B BAC reporter coupled with a collection of genetic deletions, an enhancer from the iab-6 regulatory domain was discovered that is responsible for Abd-B expression in these cells and that apparently works independently from the segmentally regulated chromatin domains of the bithorax complex. Removal of this enhancer results in visible morphological defects in the secondary cells. It was determined that mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity, and that products of the secondary cells are influential during sperm competition. Many of these phenotypes seem to be caused by a defect in the storage and gradual release of Sex peptide in female mates of iab-6 mutant males. It was also found that Abd-B expression in the secondary cells contributes to glycosylation of at least three accessory gland proteins: ovulin (Acp26Aa), CG1656, and CG1652. The results demonstrate that long-term post-mating changes observed in mated females are not solely induced by main cell secretions, as previously believed, but that secondary cells also play an important role in male fertility by extending the female PMR. Overall, these discoveries provide new insights into how these two cell types cooperate to produce and maintain a robust female PMR (Gligorov, 2013).

The AG synthesizes seminal proteins that are essential for male fertility. These >180 accessory gland proteins ('Acps') are transferred to females during mating and cause post-mating changes in the females known collectively as the post-mating response (PMR). The PMR includes increased rates of egg-laying and ovulation, sperm storage, decreased receptivity to courting males, as well as changes in longevity, feeding, and sleep patterns. The PMR is divided into two phases. The short term response (STR) refers to changes in the above behaviors during the first ~24 hours post-mating. It requires Acps, but not the receipt of sperm. Persistence of the PMR after 24 hr (and for up to ~10 days) is known as the long-term response (LTR). The LTR requires Acps and stored sperm. Many of the roles of Acps were initially discovered by experiments in which whole AG extracts or purified Acps were injected into unmated females, or by whole-tissue ablation in males (Gligorov, 2013).

Each lobe of the AG is composed of a monolayer of approximately 1000 secretory cells comprised of two morphologically distinct cell types. Roughly 96% of these cells are flat, polygonally shaped 'main cells'. The remaining 4% of the cells are large, spherical, vacuole filled 'secondary cells'; these are dispersed among the main cells at the distal tip of the gland. Enhancer trapping and other studies have shown that, in addition to their morphological differences, these two secretory cell types are biochemically distinct. Ablation of the main cells only showed that products of these cells are essential for the PMR. These products include ovulin (Acp26Aa), an Acp that acts in the STR to stimulate ovulation, and the sex peptide (SP, Acp70A), which is the ultimate regulator of most other PMR effects. SP binds to sperm within the mated female, and its active portion is gradually released from the sperm. This binding and release allows SP to affect the female for as long as she contains stored sperm. A network of five other Acps is necessary for SP to bind to sperm and enter storage. The predicted protease CG10586 (Seminase) appears to be necessary for both STR and LTR related events, while the predicted protease CG9997, the predicted cysteine-rich secretory protein (CRISP) CG17575, and the predicted lectins CG1656/1652 appear to be LTR specific. The cellular source of each of these proteins is currently unknown (Gligorov, 2013).

In spite of the detailed characterization of the main cells and several specific Acps, the role of the secondary cells has remained mysterious. No PMR-associated Acps were known to be expressed exclusively in the secondary cells, and no tools have been available to specifically target those cells. This study identified the secondary cells of the male AG as a novel location of Abd-B expression in the adult fly. By screening an extensive collection of cis-regulatory deletions, discovered a 2.8 kb enhancer from the iab-6 cis-regulatory domain was discovered, whose removal completely abolishes Abd-B expression in the secondary cells. Loss of Abd-B expression in the secondary cells causes those cells to develop aberrantly. Moreover, these mutant males provide their mates with substances that initiate the PMR, but are insufficient to maintain it. The results indicate that Abd-B expression in the secondary cells is essential for their proper development and for the production of proteins important for long-term changes in female post-mating responses (Gligorov, 2013).

Protein-specific manipulation of ejaculate composition in response to female mating status in Drosophila melanogaster

Female promiscuity can generate postcopulatory competition among males, but it also provides the opportunity for exploitation of rival male ejaculates. For example, in many insect species, male seminal fluid proteins (Sfps) transferred in a female's first mating stimulate increased fecundity and decreased receptivity to remating. Subsequent mates of females could potentially take advantage of the effects of the first male's Sfps and strategically reduce investment in their own ejaculate. This study compared postmating responses (fecundity and sexual receptivity) of Drosophila melanogaster females after their first (virgin) matings (V), to the responses of females remating (M) 24 h after their first mating. The results show that M matings fail to boost fecundity and, thus, males are unlikely to gain fitness from transferring Sfps whose sole function-in V matings-is fecundity-stimulation. However, males can protect their likelihood of paternity in M matings through the transfer of receptivity-inhibiting Sfps. The levels of a fecundity-stimulating Sfp (ovulin) were significantly lower in M females relative to V females, at the same time point shortly after the end of mating. In contrast, the levels of a key receptivity-inhibiting Sfp (sex peptide) were the same in M and V females. These results support the hypothesis that males can adaptively tailor the composition of proteins in the ejaculate, allowing a male to take advantage of the fecundity-stimulating effects of the previous male's ovulin, yet maintaining investment in sex peptide. Furthermore, the results demonstrate sophisticated protein-specific ejaculate manipulation (Sirot, 2011).

Although mating duration was not the focus of this study, it is related to male reproductive success in D. melanogaster: the duration of a female's first mating is positively associated with her latency until remating but not with the number of sperm received. Thus, longer first matings are associated with higher male reproductive success, presumably because of greater receptivity inhibition. However, the results demonstrate that the relationship between mating duration and receptivity inhibition is not a result of a general increase in SP transfer in longer matings, because mating duration changes independently of the amount of SP transferred. It was also found that the mating duration was longer for a female's first mating than for a female's second mating, results that are broadly consistent with some studies on D. melanogaster but at odds with two others. The differences between studies in mating duration patterns may be because of differences in D. melanogaster strains, experimental methods, or housing conditions before the assays. For example, the duration of a female's first mating is influenced both by exposure of the male to rival males before mating and exposure of the pair to extrapair males during mating. It appears that in this species the relationship between female mating status and mating duration may be specific to genetic background and the particular mating environment. However, a recent cross-taxa meta-analysis showed that proxies for ejaculate investment, such as mating duration and ejaculate mass, are greater in matings with virgins than with mated females, a pattern consistent with the finding that mating duration and allocation of some components of the ejaculate may be reduced in M matings. Intriguingly, the same study also found no evidence for increased sperm numbers transferred to virgin relative to mated females, suggesting that ejaculate investment in response to mating status may often specifically involve changes in nonsperm components, such as Sfps (Sirot, 2011).

Across a wide range of taxa, studies have established unequivocally that males can strategically allocate sperm based on the relative risk or intensity of sperm competition. However, only recently have researchers begun to investigate nonsperm aspects of ejaculate allocation theoretically and empirically. Moreover, the idea that males can potentially exploit the ejaculates of rival males is a recent one that has previously received only theoretical attention. The current results, together with previous studies, show that male D. melanogaster have the opportunity to exploit rival ejaculates. Furthermore, the results indicate that males may do so by tailoring the Sfp composition of their ejaculate in a protein-specific manner, suggesting an extraordinary level of sophistication in ejaculate strategies. It will now be important to determine whether such protein-specific allocation strategies are taxonomically widespread. More theoretical and empirical studies are required to determine the evolutionary consequences of such strategies for intersexual and intrasexual interactions (Sirot, 2011).

Seminal fluid protein allocation and male reproductive success

Postcopulatory sexual selection can select for sperm allocation strategies in males, but males should also strategically allocate nonsperm components of the ejaculate, such as seminal fluid proteins (Sfps). Sfps can influence the extent of postcopulatory sexual selection, but little is known of the causes or consequences of quantitative variation in Sfp production and transfer. Using Drosophila melanogaster, this study demonstrates that Sfps are strategically allocated to females in response to the potential level of sperm competition. Males who can produce and transfer larger quantities of specific Sfps have a significant competitive advantage. When males were exposed to a competitor male, matings were longer and more of two key Sfps, sex peptide and ovulin, were transferred, indicating strategic allocation of Sfps. Males selected for large accessory glands (a major site of Sfp synthesis) produced and transferred significantly more sex peptide, but not more ovulin. Males with large accessory glands also had significantly increased competitive reproductive success. These results show that quantitative variation in specific Sfps is likely to play an important role in postcopulatory sexual selection and that investment in Sfp production is essential for male fitness in a competitive environment (Wigby, 2009).

Although there are clear male reproductive benefits associated with the ability to transfer large quantities of some Sfps, AG size was close to a minimum level in the starting lab population: selection for smaller AGs was unsuccessful. AG size might be subject to truncation selection if a minimal investment is required to avoid too much depletion of Sfps from small AGs over successive matings. Sfp depletion leads to dramatically decreased male fertility and paternity assurance, thus there is likely to be strong selection on males to avoid depletion. However, AGs could be costly to develop, maintain or fill with Sfps, in which case AG size could trade off against other life history traits. So far there is no evidence of any such trade-offs in terms of development time or virgin male survival in the AG selection lines. However, trade-offs could have been minimised in the selection lines by rearing conditions that reduced competition for resources, with low densities of flies and excess food. This may have permitted the evolution of larger AGs in the L lines without the costs that would usually inhibit such investment under natural or standard lab-cage conditions (Wigby, 2009).

The receipt of Sfps such as SP can be costly to females and can potentially mediate sexually antagonistic coevolution. In certain experimental evolution studies, rapid changes in female resistance to male-induced harm have been observed. In these studies male-male interaction was eliminated or reduced, through enforced monogamy or female biased sex-ratios respectively. The results suggest that under such conditions males would plastically (i.e. immediately) reduce the level of Sfp transfer, which would reduce mating costs to females. Selection on female resistance would therefore immediately be relaxed even before evolutionary changes in males occurred. Thus, plastic Sfp allocation could potentially select for rapid intersexual coevolution (Wigby, 2009).

The results show that, in D. melanogaster, Sfp allocation is plastic, can evolve rapidly under selection and is more complex than has hitherto been considered. It will be important to determine whether Sfp allocation is as taxonomically widespread as sperm allocation. Testing this should be possible, because the functions of specific Sfps are known in species ranging from arthropods to mammals, antibodies have been developed to Sfps in several species (e.g, fruit flies; carp; bulls; humans, and bioinformatic, proteomic and RNA-interference tools to aid the discovery and characterisation of new Sfps are becoming increasingly available. More theory is also needed to generate predictions of how males should invest in, and allocate, the Sfps that play important roles in post-copulatory sexual selection, and crucially how females should evolve in response to Sfp allocation. A potential application of this work is the manipulation of males used in biological and genetic insect-pest management. Males released for pest control are often poor in both acquiring mates and inducing the post-mating behavioural changes that are stimulated by Sfps. The results demonstrate the potential for increasing the reproductive competitiveness of mass-reared males by selecting on AG size or by selecting on the ability of males to induce female post-mating responses (Wigby, 2009).

Post-mating gene expression profiles of female Drosophila melanogaster in response to time and to four male accessory gland proteins

In Drosophila, the genetic and molecular bases of post-mating changes in the female's behavior and physiology are poorly understood. However, DNA microarray studies have demonstrated that, shortly after mating, transcript abundance of >1700 genes is altered in the female's reproductive tract as well as in other tissues. Many of these changes are elicited by sperm and seminal fluid proteins (Acps) that males transfer to females. To further dissect the transcript-level changes that occur following mating, gene expression profiles of whole female flies were examined at four time points following copulation. It was found that, soon after copulation ends, a large number of small-magnitude transcriptional changes occurred in the mated female. At later time points, larger magnitude changes were seen, although these occurred in a smaller number of genes. Then how four individual Acps (ovulin, Acp36DE, Acp29AB, and Acp62F) with unique functions independently affected gene expression in females shortly after mating was examined. Consistent with their early and possibly local action within the female, ovulin and Acp36DE caused relatively few gene expression changes in whole bodies of mated females. In contrast, Acp29AB and Acp62F modulated a large number of transcriptional changes shortly after mating (McGraw, 2008).

During mating, males provide females with seminal fluids that include proteins affecting female physiology and, in some cases, reproductive behavior. In several species these male-derived modulators of reproduction are processed upon transfer to the female, suggesting molecular interaction between the sexes. Males could increase their reproductive success by contributing to regulation of this processing; consistent with this hypothesis, seminal fluids are rich in proteolysis regulators. However, whether these molecules carry out processing of male-derived reproductive modulators is unknown. Tested were performed for this role using RNAi to knock down individually 11 Drosophila seminal fluid proteases and protease inhibitors. It was found that CG11864, a predicted astacin-type metalloprotease in seminal fluid, is necessary to process two other seminal proteins: the ovulation hormone ovulin (Acp26Aa) and the sperm storage protein Acp36DE. This processing occurs only after all three proteins have entered the female. Moreover, CG11864 itself is processed inside males while en route to the female and before its action in processing ovulin and Acp36DE. Thus, processing of seminal proteins is stepwise in Drosophila, beginning in the male after the proteins leave their site of synthesis and continuing within another organism, the mated female, and the male-donated protease CG11864 is an agent of this latter processing (Ravi Ram, 2006).

Immortal coils: conserved dimerization motifs of the Drosophila ovulation prohormone ovulin

Dimerization is an important feature of the function of some proteins, including prohormones. For proteins whose amino acid sequences evolve rapidly, it is unclear how such structural characteristics are retained biochemically. This study addresses this question by focusing on ovulin, a prohormone that induces ovulation in Drosophila melanogaster females after mating. Ovulin is known to dimerize, and is one of the most rapidly evolving proteins encoded by the Drosophila genome. Residues within a previously hypothesized conserved dimerization domain (a coiled-coil) and a newly identified conserved dimerization domain (YxxxY) within ovulin are necessary for the formation of ovulin dimers. Moreover, dimerization is conserved in ovulin proteins from non-melanogaster species of Drosophila despite up to 80% sequence divergence. Heterospecific ovulin dimers can be formed in interspecies hybrid animals and in two-hybrid assays between ovulin proteins that are 15% diverged, indicating conservation of tertiary structure amidst a background of rapid sequence evolution. The results suggest that because ovulin's self-interaction requires only small conserved domains, the rest of the molecule can be relatively tolerant to mutations. Consistent with this view, in comparisons of 8510 proteins across 6 species of Drosophila it was found that rates of amino acid divergence are higher for proteins with coiled-coil protein-interaction domains than for non-coiled-coil proteins (Wong, 2010).

Characterizing male-female interactions using natural genetic variation in Drosophila melanogaster

Drosophila melanogaster females commonly mate with multiple males establishing the opportunity for pre- and postcopulatory sexual selection. Traits impacting sexual selection can be affected by a complex interplay of the genotypes of the competing males, the genotype of the female, and compatibilities between the males and females. This study scored males from 96 2nd and 94 3rd chromosome substitution lines for traits affecting reproductive success when mated with females from 3 different genetic backgrounds. The traits included male-induced female refractoriness, male remating ability, the proportion of offspring sired under competitive conditions and male-induced female fecundity. Significant effects of male line, female genetic background, and strong male by female interactions. Some males appeared to be 'generalists' and performed consistently across the different females; other males appeared to be 'specialists' and performed very well with a particular female and poorly with others. 'Specialist' males did not, however, prefer to court those females with whom they had the highest reproductive fitness. Using 143 polymorphisms in male reproductive genes, several genes were mapped that had consistent effects across the different females including a derived, high fitness allele in Acp26Aa that may be the target of adaptive evolution. Also, a polymorphism upstream of Protein ejaculatory bulb II (PebII) was identified that may interact with the female genetic background to affect male-induced refractoriness to remating. These results suggest that natural variation in PebII might contribute to the observed male-female interactions (Reinhart, 2014).

Reproductive hacking; A male seminal protein acts through intact reproductive pathways in female Drosophila

Seminal proteins are critical for reproductive success in all animals that have been studied. Although seminal proteins have been identified in many taxa, and female reproductive responses to receipt of these proteins have been documented in several, little is understood about the mechanisms by which seminal proteins affect female reproductive physiology. To explore this topic, this study investigated how a Drosophila seminal protein, ovulin, increases ovulation rate in mated females. Ovulation is a relatively simple physiological process, with known female regulators: previous studies have shown that ovulation rate is promoted by the neuromodulator octopamine (OA) in D. melanogaster and other insects. Ovulin was found to stimulate ovulation by increasing OA signaling in the female. This finding supports a model in which a male seminal protein acts through 'hacking' a well-conserved, regulatory system females use to adjust reproductive output, rather than acting downstream of female mechanisms of control or in parallel pathways altogether. Similarities are discussed between two forms of intersexual control of behavior through chemical communication: seminal proteins and pheromones (Dustin Rubinstein, 2014).

Evidence for structural constraint on ovulin, a rapidly evolving Drosophila melanogaster seminal protein

The egg-laying hormone ovulin (Acp26Aa) is among the most rapidly evolving proteins in the Drosophila genome. Against the background of ovulin's high sequence variability within and between species, highly conserved motifs were identified that may play an important structural role. Using yeast two-hybrid and GST-pull-down assays, it was shown that ovulin interacts with itself. The C terminus of ovulin is necessary and sufficient for self-interaction, with its C-terminal 45 aa playing a major role. Under nonreducing conditions, ovulin participates in a high-molecular-mass complex, suggesting that it occurs in an oligomeric form. One or more of three predicted coiled-coil domains in the C terminus of ovulin may be involved in its self-interaction. These structural elements are conserved between species despite an overall rapid pace of evolution in ovulin's primary sequence. It is therefore suggested that domains involved in ovulin's self-interaction form a conserved structural backbone for the protein, resulting in greater evolutionary flexibility at other sites (Wong, 2006).

Two cleavage products of the Drosophila accessory gland protein ovulin can independently induce ovulation

Proteins and peptides in Drosophila melanogaster seminal fluid induce mated females to increase their rates of egg deposition. One seminal-fluid protein, ovulin (Acp26Aa), stimulates an early step in the egg-laying process, the release of oocytes by the ovary. Ovulin, upon transfer to females, is cleaved sequentially within the mated female's reproductive tract. This study shows that systemic ectopic expression of ovulin is sufficient to stimulate ovulation in unmated females. By using this assay to assess the functionality of ovulin's cleavage products, it was found that two of the four cleavage products of ovulin can stimulate ovulation independently. Thus, ovulin's cleavage in mated females is not destructive and instead may liberate additional functional products with potential to modulate ovulation independently (Heifetz, 2005).

The sequence of ovulin suggests that it is a prohormone. The primary translation product is 264 aa long, including a predicted signal sequence of 18 aa that is expected to be cleaved to allow secretion of ovulin. Ovulin contains amino acid sequences that resemble prohormone cleavage sites in other organisms (e.g., egg laying hormone ELH, Aplysia; oxytocin, cattle; and tachykinin-related peptide, Drosophila). Shortly after it enters the female's reproductive tract, ovulin is proteolytically processed (Monsma, 1990; Monsma, 1988, Park 1995). A map of cleavage products (CPs) of ovulin suggests that three sequential cleavages occur at ovulin residues that match prohormone cleavage consensus. The first cleavage is after Lys-48, the second is after Lys-68, and the third is after either Lys-115 or Lys-117. The C-terminal CP of ovulin contains two small regions (residues 120-137 and 154-188) of similarity (46% amino acid identity) to Aplysia californica egg-laying hormones that regulate egg deposition. These mollusk hormones, ELH and califin C, are also processed from prohormone precursors. The cleavage products of ELH and califin C are released into the Aplysia reproductive tract, where they stimulate the release of oocytes (Heifetz, 2005 and references therein).

One possible mechanism for regulating the activity of seminal peptides within the female reproductive tract is to process them from an inactive to an active form (as occurs for the Aplysia hormones ELH and califin C). Alternatively, processing could be inactivational, particularly given that some Acps are harmful to the female. This study tested whether full-length ovulin or its predicted cleavage products and its ELH- and califin-similar regions are sufficient to induce ovulation. It was found that ectopic expression of full-length ovulin and at least two of its cleavage products stimulate ovulation. Thus, the cleavage products of ovulin can act independently, and the cleavage seen in mated females is not degradative (Heifetz, 2005(.

The Acp26Aa seminal fluid protein is a modulator of early egg hatchability in Drosophila melanogaster

Drosophila melanogaster male accessory gland proteins (Acps) that are transferred in the ejaculate with sperm mediate post-mating competition for fertilizations between males. The actions of Acps include effects on oviposition and ovulation, receptivity and sperm storage. Two Acps that modulate egg production are Acp26Aa (ovulin) and Acp70A (the sex peptide). Acp26Aa acts specifically on the process of ovulation (the release of mature eggs from the ovaries), which is initiated 1.5 h after mating. In contrast, sperm storage can take as long as 6-9 h to complete. Initial ovulations after matings by virgin females will therefore occur before all sperm are fully stored and the extra eggs initially laid as a result of Acp26Aa transfer are expected to be inefficiently fertilized. Acp26Aa-mediated release of existing eggs should not cause a significant energetic cost or lead to a decrease in female lifespan assuming, as seems likely, that the energetic cost of egg laying comes from de novo egg synthesis (oogenesis) rather than from ovulation. These predictions were tested using Acp26Aa¹ mutant males that lack Acp26Aa but are normal for other Acps and Acp26Aa² males that transfer a truncated but fully functional Acp26Aa protein. Females mating with Acp26Aa² (truncation) males that received functional Acp26Aa produced significantly more eggs following their first matings than did mates of Acp26Aa¹ (null) males. However, as predicted above, these extra eggs, which were laid as a result of Acp26Aa transfer to virgin females, showed significantly lower egg hatchability. Control experiments indicated that this lower hatchability was due to lower rates of fertilization at early post-mating times. There was no drop in egg hatchability in subsequent non-virgin matings. In addition, as predicted above, females that did or did not receive Acp26Aa did not differ in survival, lifetime fecundity or lifetime progeny, indicating that Acp26Aa transfer does not represent a significant energetic cost for females and does not contribute to the survival cost of mating. Acp26Aa appears to remove a block to oogenesis by causing the clearing out of existing mature eggs and, thus, indirectly allowing oogenesis to be initiated immediately after mating. The results show that subtle processes coordinate the stimulation of egg production and sperm storage in mating pairs (Chapman, 2001).

The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary

Mating stimulates the rate of egg-laying by female insects. In Drosophila melanogaster this stimulation is initially caused by seminal fluid molecules transferred from the male (Acps or accessory gland proteins. Egg-laying is a multi-step process. It begins with oocyte release by the ovaries, followed by egg movement down the oviducts and the deposition of eggs onto the substratum. Although two Acps are known to stimulate egg-laying, they were detected by assays that do not discriminate between the steps of this process or allow examination of its earliest changes. To determine how egg-laying is regulated, a generally applicable assay was developed to separate the process into quantifiable steps, allowing assessment of the ovulation pattern and rate of egg movement. As the steps are interdependent yet potentially subject to independent controls, the contribution of each step and effector was determined independent of the others. A statistical method was uded that separately considers and quantifies each 'path' to a common end. It was found that the prohormone-like molecule Acp26Aa stimulates the first step in egg-laying - release of oocytes by the ovary. During mating, Acp26Aa begins to accumulate at the base of the ovaries, a position consistent with action on the ovarian musculature to mediate oocyte release. Understanding how individual Acps regulate egg-laying in fruitflies will help provide a full molecular picture of insects' prodigious fertility, of reproductive hormones, and of the roles of these rapidly evolving proteins (Heifetz, 2000).

A Drosophila seminal fluid protein, Acp26Aa, stimulates egg laying in females for 1 day after mating

Mating triggers behavioral and physiological changes in the Drosophila melanogaster female, including an elevation of egg laying. Seminal fluid molecules from the male accessory gland are responsible for initial behavioral changes, but persistence of these changes requires stored sperm. Using genetic analysis, this study has identified a seminal fluid protein that is responsible for an initial elevation of egg laying. This molecule, Acp26Aa, has structural features of a prohormone and contains a region with amino acid similarity to the egg-laying hormone of Aplysia. Acp26Aa is transferred to the female during mating, where it undergoes processing. This study reports the generation and analysis of mutants, including a null, in Acp26Aa. Females mated to male flies that lack Acp26Aa lay fewer eggs than do mates of normal males. This effect is apparent only on the first day after mating. The null mutation has no other detectable physiological or behavioral effects on the male or the mated female (Herndon, 1995).

Search PubMed for articles about Ovulin

Avila, F. W., Sirot, L. K., LaFlamme, B. A., Rubinstein, C. D. and Wolfner, M. F. (2011). Insect seminal fluid proteins: identification and function. Annu Rev Entomol 56: 21-40. PubMed ID: 20868282

Chapman, T., Herndon, L. A., Heifetz, Y., Partridge, L. and Wolfner, M. F. (2001). The Acp26Aa seminal fluid protein is a modulator of early egg hatchability in Drosophila melanogaster. Proc Biol Sci 268: 1647-1654. PubMed ID: 11506676

Dustin Rubinstein, C. and Wolfner, M. F. (2014). Reproductive hacking; A male seminal protein acts through intact reproductive pathways in female Drosophila. Fly (Austin) 8: 80-85. PubMed ID: 25483253

Gligorov, D., Sitnik, J. L., Maeda, R. K., Wolfner, M. F. and Karch, F. (2013). A novel function for the Hox gene Abd-B in the male accessory gland regulates the long-term female post-mating response in Drosophila. PLoS Genet 9: e1003395. PubMed ID: 23555301

Heifetz, Y., Lung, O., Frongillo, E. A., Jr. and Wolfner, M. F. (2000). The Drosophila seminal fluid protein Acp26Aa stimulates release of oocytes by the ovary. Curr Biol 10: 99-102. PubMed ID: 10662669

Heifetz, Y., Vandenberg, L. N., Cohn, H. I. and Wolfner, M. F. (2005). Two cleavage products of the Drosophila accessory gland protein ovulin can independently induce ovulation. Proc Natl Acad Sci U S A 102: 743-748. PubMed ID: 15640356

Herndon, L. A. and Wolfner, M. F. (1995). A Drosophila seminal fluid protein, Acp26Aa, stimulates egg laying in females for 1 day after mating. Proc Natl Acad Sci U S A 92: 10114-10118. PubMed ID: 7479736

Kapelnikov, A., Rivlin, P. K., Hoy, R. R. and Heifetz, Y. (2008). Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct. BMC Dev Biol 8: 114. PubMed ID: 19063748

Lange, A. B. (2009). Neural mechanisms coordinating the female reproductive system in the locust. Front Biosci (Landmark Ed) 14: 4401-4415. PubMed ID: 19273358

McGraw, L. A., Clark, A. G. and Wolfner, M. F. (2008). Post-mating gene expression profiles of female Drosophila melanogaster in response to time and to four male accessory gland proteins. Genetics 179: 1395-1408. PubMed ID: 18562649

Middleton, C. A., Nongthomba, U., Parry, K., Sweeney, S. T., Sparrow, J. C. and Elliott, C. J. (2006). Neuromuscular organization and aminergic modulation of contractions in the Drosophila ovary. BMC Biol 4: 17. PubMed ID: 16768790

Monastirioti, M., Gorczyca, M., Rapus, J., Eckert, M., White, K. and Budnik, V. (1995). Octopamine immunoreactivity in the fruit fly Drosophila melanogaster. J Comp Neurol 356: 275-287. PubMed ID: 7629319

Monsma, S. A. and Wolfner, M. F. (1988). Structure and expression of a Drosophila male accessory gland gene whose product resembles a peptide pheromone precursor. Genes Dev 2: 1063-1073. PubMed ID: 3142802

Monsma, S. A., Harada, H. A. and Wolfner, M. F. (1990). Synthesis of two Drosophila male accessory gland proteins and their fate after transfer to the female during mating. Dev Biol 142: 465-475. PubMed ID: 2257979

Park, M. and Wolfner, M. F. (1995). Male and female cooperate in the prohormone-like processing of a Drosophila melanogaster seminal fluid protein. Dev Biol 171: 694-702. PubMed ID: 7556947

Pomiankowski, A., Nöthiger, R. and Wilkins, A. (2004). The evolution of the Drosophila sex-determination pathway. Genetics 166: 1761-1773. PubMed ID: 15126396

Ravi Ram, K., Sirot, L. K. and Wolfner, M. F. (2006). Predicted seminal astacin-like protease is required for processing of reproductive proteins in Drosophila melanogaster. Proc Natl Acad Sci U S A 103: 18674-18679. PubMed ID: 17116868

Reinhart, M., Carney, T., Clark, A. G. and Fiumera, A. C. (2014). Characterizing male-female interactions using natural genetic variation in Drosophila melanogaster. J Hered 106(1):67-79. PubMed ID: 25425680

Rodríguez-Valentín, R., Lopez-Gonzalez, I., Jorquera, R., Labarca, P., Zurita, M. and Reynaud, E. (2006). Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic. J Cell Physiol 209: 183-198. PubMed ID: 16826564

Rubinstein, C. D. and Wolfner, M. F. (2013). Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling. Proc Natl Acad Sci U S A. PubMed ID: 24101486

Sirot, L. K., LaFlamme, B. A., Sitnik, J. L., Rubinstein, C. D., Avila, F. W., Chow, C. Y. and Wolfner, M. F. (2009). Molecular social interactions: Drosophila melanogaster seminal fluid proteins as a case study. Adv Genet 68: 23-56. PubMed ID: 20109658

Sirot, L. K., Wolfner, M. F. and Wigby, S. (2011). Protein-specific manipulation of ejaculate composition in response to female mating status in Drosophila melanogaster. Proc Natl Acad Sci U S A 108: 9922-9926. PubMed ID: 21628597

Wigby, S., Sirot, L. K., Linklater, J. R., Buehner, N., Calboli, F. C., Bretman, A., Wolfner, M. F. and Chapman, T. (2009). Seminal fluid protein allocation and male reproductive success. Curr Biol 19: 751-757. PubMed ID: 19361995

Wong, A., Albright, S. N. and Wolfner, M. F. (2006). Evidence for structural constraint on ovulin, a rapidly evolving Drosophila melanogaster seminal protein. Proc Natl Acad Sci U S A 103: 18644-18649. PubMed ID: 17130459

Wong, A., Christopher, A. B., Buehner, N. A. and Wolfner, M. F. (2010). Immortal coils: conserved dimerization motifs of the Drosophila ovulation prohormone ovulin. Insect Biochem Mol Biol 40: 303-310. PubMed ID: 20138215

Yang, C. H., Belawat, P., Hafen, E., Jan, L. Y. and Jan, Y. N. (2008). Drosophila egg-laying site selection as a system to study simple decision-making processes. Science 319: 1679-1683. PubMed ID: 18356529

date revised: 3 November 2013

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