Male reproductive glands like the mammalian prostate and the paired Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate, stimulated by environmentally regulated endocrine and local androgens, grows throughout adult life. We previously showed that in fly accessory glands, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here, we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid ecdysone, whose concentration is reported to reflect sociosexual experience in adults. BMP signalling appears to regulate ecdysone receptor (EcR) levels via one or more mechanisms involving the EcR’s N terminus or the RNA sequence that encodes it. Nuclear growth in virgin males is dependent on ecdysone, some of which is synthesised in SCs. However, mating induces additional BMP-mediated nuclear growth via a cell type–specific form of hormone-independent EcR signalling, which drives genome endoreplication in a subset of adult SCs. Switching to hormone-independent endoreplication after mating allows growth and secretion to be hyperactivated independently of ecdysone levels in SCs, permitting more rapid replenishment of the accessory gland luminal contents. Our data suggest mechanistic parallels between this physiological, behaviour-induced signalling switch and altered pathological signalling associated with prostate cancer progression.

Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behavior. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterized protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term postmating responses, including increased ovulation, elevated feeding, and reduced receptivity to remating, primarily signaling through the SP receptor (SPR). Here, we demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Its absence leads to major changes in the seminal proteome transferred to females upon mating. Males expressing nonfunctional SP mutant proteins that affect SP’s binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread abnormalities in ejaculate function. Our data therefore reveal a role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species that lack the signaling functions seen in D. melanogaster.

20 Seminal fluid plays an essential role in promoting male reproductive success and modulating 21 female physiology and behaviour. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) 22 40 41 Significance Statement 42 Seminal fluid plays a critical role in reprogramming female physiology and behaviour to 43 promote male reproductive success. We show in the fruit fly that specific seminal proteins, 44 including the archetypal 'female-reprogramming' molecule Sex Peptide, are stored in male 45 seminal secretions in association with large neutral lipid-containing microcarriers, which 46 rapidly disperse in females. Related structures are also observed in other Sex Peptide-47 expressing Drosophila species. Males lacking Sex Peptide have structurally defective 48 microcarriers, leading to abnormal cargo loading and transfer to females. Our data reveal 49 that this key signalling molecule in Drosophila seminal fluid is also a microcarrier assembly 50 factor that controls transfer of other seminal factors, and that this may be a more 51 evolutionarily ancient role of this protein. 52 53 54

In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process.

Dysregulation of cell cycle components results in the development and progression of several cancer types. Unusually, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 have been linked to late-stage tumour progression in prostate cancer, rather than early-stage events. This change is associated with an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG) share several functional and signalling similarities with human prostate epithelial cells. Upon mating, SC growth regulation switches from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control that induces genome endoreplication. Here, we demonstrate that the Drosophila Rb homologue, Rbf, and E2F1, as well as cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key mediators of SC growth and endoreplication both in virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and associated growth in SCs after mating, mirroring changes in CRPC. We also demonstrate that excess Rbf activity reversibly suppresses binucleation in adult SCs. Overall, our work reveals mechanistic parallels between the physiological switch to hormone-independent EcR signalling in SCs, and the pathological switch seen in CRPC, and suggests that the latter may represent the dysregulation of a currently unidentified physiological process, which permits AR signalling when androgen levels are low.