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Unique patterns of inheritance and selection on Y chromosomes lead to the evolution of specialized gene functions. Yet characterizing the function of genes on Y chromosomes is notoriously difficult. We report CRISPR mutants in Drosophila of the Y-linked gene,WDY, which is required for male fertility.WDYmutants produce mature sperm with beating tails that can be transferred to females but fail to enter the female sperm storage organs. We demonstrate that the sperm tails ofWDYmutants beat approximately half as fast as wild-type sperm’s and that the mutant sperm do not propel themselves within the male ejaculatory duct or female reproductive tract (RT). These specific motility defects likely cause the sperm storage defect and sterility of the mutants. Regional and genotype-dependent differences in sperm motility suggest that sperm tail beating and propulsion do not always correlate. Furthermore, we find significant differences in the hydrophobicity of key residues of a putative calcium-binding domain between orthologs ofWDYthat are Y-linked and those that are autosomal. Given thatWDYappears to be evolving under positive selection, our results suggest thatWDY’s functional evolution coincides with its transition from autosomal to Y-linked inDrosophila melanogasterand its most closely related species. Finally, we show that mutants for another Y-linked gene,PRY, also show a sperm storage defect that may explain their subfertility. In contrast toWDY, PRYmutants do swim in the female RT, suggesting they are defective in yet another mode of motility, navigation, or a necessary interaction with the female RT. Overall, we provide direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.

Sex peptide (SP), a seminal fluid protein of Drosophila melanogaster males, has been described as driving a virgin-to-mated switch in females, through eliciting an array of responses including increased egg laying, activity, and food intake and a decreased remating rate. While it is known that SP achieves this, at least in part, by altering neuronal signaling in females, the genetic architecture and temporal dynamics of the female’s response to SP remain elusive. We used a high-resolution time series RNA-sequencing dataset of female heads at 10 time points within the first 24 h after mating to learn about the genetic architecture, at the gene and exon levels, of the female’s response to SP. We find that SP is not essential to trigger early aspects of a virgin-to-mated transcriptional switch, which includes changes in a metabolic gene regulatory network. However, SP is needed to maintain and diversify metabolic changes and to trigger changes in a neuronal gene regulatory network. We further find that SP alters rhythmic gene expression in females and suggests that SP’s disruption of the female’s circadian rhythm might be key to its widespread effects.

Interactions between sperm and the female reproductive tract (FRT) are complex, in many cases poorly understood, and likely to contribute to the mechanistic basis of idiopathic infertility. As such, it is not surprising that the FRT was often viewed historically as a "hostile" environment for sperm. The FRT has also been touted as a selective environment to ensure that only the highest quality sperm progress to the oocyte for the opportunity to participate in fertilization. Recent advances, however, are giving rise to a far more nuanced view in which supportive sperm x FRT interactions - in both directions - contribute to beneficial, even essential, effects on fertility. In this perspective article, we discuss several examples of positive sperm x FRT interactions. We believe that these examples, arising in part from studies of taxonomically diverse, non-mammalian systems, are useful to efforts to study mammalian sperm x FRT interactions and their relevance to fertility and the advancement of assisted reproductive technologies. This article is protected by copyright. All rights reserved.

In many species, female reproductive investment comes at a cost to immunity and resistance to infection. Mated Drosophila melanogaster females are more susceptible to bacterial infection than unmated females. Transfer of the male seminal fluid protein Sex Peptide reduces female post-mating immune defense. Sex Peptide is known to cause both short- and long-term changes to female physiology and behavior. While previous studies showed that females were less resistant to bacterial infection as soon as 2.5 h and as long as 26.5 h after mating, it is unknown whether this is a binary switch from mated to unmated state or whether females can recover to unmated levels of immunity. It is additionally unknown whether repeated mating causes progressive reduction in defense capacity. We compared the immune defense of mated females when infected at 2, 4, 7, or 10 days after mating to that of unmated females and saw no recovery of immune capacity regardless of the length of time between mating and infection. Because D. melanogaster females can mate multiply, we additionally tested whether a second mating, and therefore a second transfer of seminal fluids, caused deeper reduction in immune performance. We found that females mated either once or twice before infection survived at equal proportions, both with significantly lower probability than unmated females. We conclude that a single mating event is sufficient to persistently suppress the female immune system. Interestingly, we observed that induced levels of expression of genes encoding antimicrobial peptides (AMPs) decreased with age in both experiments, partially obscuring the effects of mating. Collectively, the data indicate that being reproductively active versus reproductively inactive are alternative binary states with respect to female D. melanogaster immunity. The establishment of a suppressed immune status in reproductively active females can inform our understanding of the regulation of immune defense and the mechanisms of physiological trade-offs.

Sex Peptide, a seminal fluid protein ofD. melanogastermales, has been described as driving a virgin-to-mated switch in females, through eliciting an array of responses, including increased egg laying, activity and food intake and a decreased re-mating rate. While it is known that Sex Peptide achieves this, at least in part, by altering neuronal signaling in females, the identity of key molecular regulators that act downstream of Sex Peptide is not known. Here, we used a high-resolution time series RNA-sequencing dataset of female heads at 10 time points within the first 24 hours after mating to investigate the genetic architecture, at the gene- and exon-level, of the female’s response to Sex Peptide. We find that Sex Peptide is not essential to trigger a virgin-to-mated transcriptional switch, which involves changes in a metabolic gene regulatory network. However, Sex Peptide is needed to maintain and diversify metabolic changes and to trigger changes in a neuronal gene regulatory network. We further find that Sex Peptide might interact with the female’s circadian clock to orchestrate transcriptional changes across different regulatory networks. That a male seminal fluid protein can alter a female’s rhythmic gene expression has implications for our understanding of both reproductive and circadian behaviors.

BackgroundMale-derived seminal fluid proteins (SFPs) enter and induce a myriad of physiological and behavioral changes in mated female flies optimizing fertility. Many post-mating changes in female Drosophila melanogaster persist for ∼10-14 days, because the seminal protein that induces them, Sex Peptide (SP), is retained long-term in females by binding to sperm, with gradual release of its active domain from sperm. Several other “long-term response SFPs” (LTR-SFPs) “prime” sperm to bind SP. Whether female factors play a role in this process is unknown, though it is important to study both sexes for a comprehensive physiological understanding of this reproductive process and for consideration in models of sexual conflict.ResultsWe report here that sperm in male ejaculates bind SP more weakly than sperm that have entered females, and that the amount of SP and other SFPs bound to sperm increases with time and transit of individual seminal proteins within the female reproductive tract. Thus, female contributions are needed for maximal and appropriate binding of SP, and other SFPs, to sperm. Towards understanding the molecular roles of the female, we found no dramatic change in pH of the female reproductive tract after mating and that initial binding of SP to sperm is normal in females with ablated or defective spermathecal secretory cells and/or parovaria, but that higher levels of SP (and sperm) are retained in the latter females.ConclusionThis study reveals that the SP pathway is not entirely male-biased and that females also contribute to regulating it.

SignificanceIn species with internal fertilization, sperm spend an important part of their lives within the female. To examine the life history of the sperm during this time, we used semiquantitative proteomics and sex-specific isotopic labeling in fruit flies to determine the extent of molecular continuity between male and female reproductive tracts and provide a global catalog of sperm-associated proteins. Multiple seminal fluid proteins and female proteins associate with sperm immediately after mating. Few seminal fluid proteins remain after long-term sperm storage, whereas female-derived proteins constitute one-fifth of the postmating sperm proteome by then. Our data reveal a molecular "hand-off" from males to females, which we postulate to be an important component of sperm-female interactions.

The model organism Drosophila melanogaster has become a focal system for investigations of rapidly evolving genital morphology as well as the development and functions of insect reproductive structures. To follow up on a previous paper outlining unifying terminology for the structures of the male terminalia in this species, we offer here a detailed description of the female terminalia of D. melanogaster. Informative diagrams and micrographs are presented to provide a comprehensive overview of the external and internal reproductive structures of females. We propose a collection of terms and definitions to standardize the terminology associated with the female terminalia in D. melanogaster and we provide a correspondence table with the terms previously used. Unifying terminology for both males and females in this species will help to facilitate communication between various disciplines, as well as aid in synthesizing research across publications within a discipline that has historically focused principally on male features. Our efforts to refine and standardize the terminology should expand the utility of this important model system for addressing questions related to the development and evolution of animal genitalia, and morphology in general.