Sperm Storage Organs Research Articles

Genomics of a sexually selected sperm ornament and female preference in Drosophila.

Our understanding of animal ornaments and the mating preferences driving their exaggeration is limited by knowledge of their genetics. Post-copulatory sexual selection is credited with the rapid evolution of female sperm-storage organ morphology and corresponding sperm quality traits across diverse taxa. In Drosophila, the mechanisms by which longer flagella convey an advantage in the competition among sperm for limited storage space in the female, and by which female sperm-storage organ morphology biases fertilization in favour of longer sperm have been resolved. However, the evolutionary genetics underlying this model post-copulatory ornament and preference system have remained elusive. Here we combined comparative analyses of 149 Drosophila species, a genome-wide association study in Drosophila melanogaster and molecular evolutionary analysis of ~9,400 genes to elucidate how sperm and female sperm-storage organ length co-evolved into one of nature's most extreme ornaments and preferences. Our results reveal a diverse repertoire of pleiotropic genes linking sperm length and seminal receptacle length expression to central nervous system development and sensory biology. Sperm length development appears condition-dependent and is governed by conserved hormonal (insulin/insulin-like growth factor) and developmental (including Notch and Fruitless) pathways. Central developmental pathway genes, including Notch, also comprised the majority of a restricted set of genes contributing to both intraspecific and interspecific variation in sperm length. Our findings support 'good genes' models of female preference evolution.

Regional specialization, polyploidy, and seminal fluid transcripts in the Drosophila female reproductive tract

Sexual reproduction requires the choreographed interaction of female cells and molecules with sperm and seminal fluid. In internally fertilizing animals, these interactions are managed by specialized tissues within the female reproductive tract (FRT), such as a uterus, glands, and sperm storage organs. However, female somatic reproductive tissues remain understudied, hindering insight into the molecular interactions that support fertility. Here, we report the identification, molecular characterization, and analysis of cell types throughout the somatic FRT in the premier Drosophila melanogaster model system. We find that the uterine epithelia is composed of 11 distinct cell types with well-delineated spatial domains, likely corresponding to functionally specialized surfaces that interact with gametes and reproductive fluids. Polyploidy is pervasive: More than half of lower reproductive tract cells are ≥4C. While seminal fluid proteins (SFPs) are typically thought of as male products that are transferred to females, we find that specialized cell types in the sperm storage organs heavily invest in expressing SFP genes. Rates of amino acid divergence between closely related species indicate heterogeneous evolutionary processes acting on male-limited versus female-expressed seminal fluid genes. Together, our results emphasize that more than 40% of annotated seminal fluid genes are better described as shared components of reproductive transcriptomes, which may function cooperatively to support spermatozoa. More broadly, our work provides the molecular foundation for improved technologies to catalyze the functional characterization of the FRT.

Females drive postmating reproductive trait evolution across Drosophila species, but not via remating rate.

While traits that contribute to premating sexual interactions are known to be wildly diverse, much less is known about the diversity of postmating (especially female) reproductive traits and the mechanisms shaping this diversity. To assess the rate, pattern, and potential drivers of postmating reproductive trait evolution, we analyzed male and female traits across up to 30 Drosophila species within a phylogenetic comparative framework. In addition to postmating reproductive morphology (e.g., sperm length, reproductive tract length and mass), we also quantified mating behaviors including female remating rate-a common proxy for the strength of postmating sexual selection. We found evidence for strong coevolution between male and female postmating traits (specifically sperm length and sperm storage organ size). However, remating rate was not associated with the rate of evolution or exaggeration of either male or female postmating reproductive morphology, once phylogenetic relatedness was accounted for. We infer that female-mediated and intersexual selection predominantly drive the evolution of our postmating morphological traits, including via divergent male and female interests in controlling paternity. In comparison, remating rate has a complex and likely secondary role in shaping this evolution, in part because this trait can be both a driver and a product of postmating selection.

Drosophila melanogaster sperm turn more oxidative in the female.

Males and females of many species store sperm for extended periods. During storage, sperm are predicted to undergo cellular and functional changes, especially towards glycolytic energy metabolism because oxygen radicals derived from oxidative phosphorylation can affect sperm motility and fertilisation ability. However, not all species can use both major energy metabolism pathways. Here, we examined the fruit fly Drosophila melanogaster and asked whether sperm metabolism can be fuelled by both glycolysis and oxidative phosphorylation, and to what extent metabolism changes during storage. Inhibiting glycolysis in vitro led to a more oxidative state of sperm; inhibiting oxidative phosphorylation increased the glycolytic component, assessed by multi-photon autofluorescence lifetime imaging (FLIM) of NAD(P)H. We further examined sperm in male and female sperm storage organs using FLIM of NAD(P)H and FAD. In intact storage organs, we found that, unexpectedly, (i) sperm were more oxidative in females than in males, and (ii) oxidative phosphorylation increased with storage duration in females. Our observation that the relative contribution of the two major energy metabolic pathways in D. melanogaster sperm differs in males and females and over storage time has important evolutionary implications.

Sperm storage organs change with female age and mating history in the Golden Silk spider Trichonephila clavipes (Araneae: Araneidae)

AbstractFemale sperm storage organs in arthropods are used as taxonomic characters since it is assumed that they do not change after maturation. However, in the Golden Silk spider, Trichonephila clavipes, the shape and sclerotization of the spermathecae change with a mating event. Moreover, mating in T. clavipes consists of several hundred copulations, but the male transfers all sperm during the first insertion of each pedipalp. Given this puzzling mating behaviour, we hypothesize that multiple copulations serve purposes other than sperm transfer and uptake, such as further altering the morphology of the storage site and/or transferring substances other than sperm. We compared virgin female T. clavipes with females after two copulations, one for each spermatheca, and after ad‐libitum mating. Each treatment consisted of two sub‐treatments in which the state of the sperm storage organs was inspected after a short (1–3 days) or a long (11 days) time interval after the female's final moult. The size of the spermathecae increased twofold in all mating treatments compared to virgin females. The thickness of the spermathecal wall increased significantly with time in all treatments similarly. The spermathecal epithelium, which releases secretion into the lumen of the spermathecae, seems most active in virgin females during the time when mating would normally occur and turns less active after mating and with age. The spermathecae contain secretion produced by the female before mating; and after mating, sperm and another secretion transferred by the male. The numerous sperm‐less matings in the ad‐libitum treatment resulted in male secretion in the copulatory ducts of the female genitalia, likely impeding sperm transfer by subsequent males. Out study shows that female genitalia can change considerably after having reached maturity and suggests that sperm‐less matings evolved in the context of sperm competition.

Three-dimensional correlative microscopy of the Drosophila female reproductive tract reveals modes of communication in seminal receptacle sperm storage

In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. We address this using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, we were able to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. Our findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues.

Proteomic analysis of spermathecal fluid reveals factors related to long‐term sperm storage in ant queens

AbstractAnt queens can maintain a large number of sperm cells for over a decade after mating at the beginning of their adult lives until they die. This sperm storage ability is prominent; however, the cellular mechanisms involved remain unclear. Sperm cells are maintained in the female sperm storage organ—the spermatheca—which supplies a suitable environment for sperm cells. To reveal the molecular basis of the long‐term sperm storage mechanisms in ant queens, protein profiles enriched in the spermathecal fluid relative to the hemolymph were identified in Lasius japonicus using data‐independent acquisition‐based quantitative proteomics technology. Proteins related to the extracellular matrix, antioxidants, metabolic pathways, proteases, chaperones, and with uncharacterized functions were especially abundant with higher log ratio values in the spermathecal fluid relative to the hemolymph. These enriched proteins were shared with highly expressed genes previously detected by transcriptome analyses of the spermatheca in queens of Crematogaster osakensis that belong to a different subfamily than L. japonicus. It is likely that the ability for long‐term sperm storage evolved early in the ant lineage. Therefore, the common proteins identified in these two ant species are possibly crucial for this ability.

Expression and potential regulatory functions of Drosophila octopamine receptors in the female reproductive tract.

Aminergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. In Drosophila, the ortholog of noradrenaline, octopamine, is required for ovulation as well as several other female reproductive processes. Two octopamine receptors have already been shown to be expressed in the Drosophila reproductive tract and to be required for egg-laying: OAMB and Octβ2R. The Drosophila genome contains 4 additional octopamine receptors-Octα2R, Octβ1R, Octβ3R, and Oct-TyrR-but their cellular patterns of expression in the reproductive tract and potential contribution(s) to egg-laying are not known. In addition, the mechanisms by which OAMB and Octβ2R regulate reproduction are incompletely understood. Using a panel of MiMIC Gal4 lines, we show that Octα2R, Octβ1R, Octβ3R, and Oct-TyrR receptors are not detectable in either epithelium or muscle but are clearly expressed in neurons within the female fly reproductive tract. Optogenetic activation of neurons that express at least 3 types of octopamine receptors stimulates contractions in the lateral oviduct. We also find that octopamine stimulates calcium transients in the sperm storage organs and that its effects in spermathecal, secretory cells, can be blocked by knock-down of OAMB. These data extend our understanding of the pathways by which octopamine regulates egg-laying in Drosophila and raise the possibility that multiple octopamine receptor subtypes could play a role in this process.

The Drosophila melanogaster Y-linked gene, WDY, is required for sperm to swim in the female reproductive tract

Unique patterns of inheritance and selection on Y chromosomes have led to the evolution of specialized gene functions. We report CRISPR mutants in Drosophila of the Y-linked gene, WDY, which is required for male fertility. We demonstrate that the sperm tails of WDY mutants beat approximately half as fast as those of wild-type and that mutant sperm do not propel themselves within the male ejaculatory duct or female reproductive tract. Therefore, although mature sperm are produced by WDY mutant males, and are transferred to females, those sperm fail to enter the female sperm storage organs. We report genotype-dependent and regional differences in sperm motility that appear to break the correlation between sperm tail beating and propulsion. Furthermore, we identify a significant change in hydrophobicity at a residue at a putative calcium-binding site in WDY orthologs at the split between the melanogaster and obscura species groups, when WDY first became Y-linked. This suggests that a major functional change in WDY coincided with its appearance on the Y chromosome. Finally, we show that mutants for another Y-linked gene, PRY, also show a sperm storage defect that may explain their subfertility. Overall, we provide direct evidence for the long-held presumption that protein-coding genes on the Drosophila Y regulate sperm motility.

Morphology of the Female and Male Reproductive Tracts and More Data on the Spermatostyle in the Brazilian Gyretes sp. (Coleoptera, Adephaga, Gyrinidae).

We investigated the male and female reproductive tracts of Gyretes sp. with light and transmission electron microscopies. The male has a pair of testes with a single coiled follicle, followed by short efferent ducts, which have a similar shape and diameter to the testes. Long ducts (epididymides) with differential epithelium open in a pair of long vasa deferentia that lead to the accessory glands. Glycoprotein secretions from the vas deferens epithelium constitute the spermatostyle for spermatozoa aggregation. The female has numerous ovarioles per ovary, a coiled fertilization duct, an accessory gland, and an elongated vagina. Spermatozoa are stored as unaggregated cells in the fertilization duct. In Gyrinidae, the testes and accessory glands show diverse shapes, and the female sperm storage organs vary in shape, size, and type and may play a role in the interaction with sperm aggregates. Testes with a single follicle and vasa deferentia opening in the accessory glands of Gyretes sp. are features shared with other Gyrinidae and other Adephaga. We proposed adding this latter trait to characterize this suborder of beetles. The morphology of the reproductive organs in both sexes contributes to comparative analyses and knowledge of the reproductive biology of Gyretes and may provide additional features for systematics.

Male-derived transcripts isolated from the mated female reproductive tract in Drosophila melanogaster.

In species with internal fertilization, sperm, and seminal fluid are transferred from male to female during mating. While both sperm and seminal fluid contain various types of molecules, including RNA, the role of most of these molecules in the coordination of fertilization or in other possible functions is poorly understood. In Drosophila, exosomes from the accessory gland, which produces seminal fluid, are transferred to females, but their potential cargoes have not been described. Moreover, while the RNA composition of sperm has been described in several mammalian species, little work on this problem has occurred in Drosophila. Here we use single nucleotide polymorphism differences between males and females from a set of highly inbred lines of D. melanogaster, and transcriptome data from the female reproductive tract, sperm, testis, and accessory gland, to investigate the potential origin, male vs female, RNA molecules isolated from 3 female reproductive tract organs, the seminal receptacle and spermatheca, which store sperm, and the parovaria, which does not. We find that mated females carry male-derived transcripts from many genes, including those that are markers of the accessory gland and known seminal fluid proteins. Our observations also support the idea that intact sperm transcripts can be isolated from the female sperm storage organs.

The reproductive tract of the black soldier fly (Hermetia illucens) is highly differentiated and suggests adaptations to sexual selection

AbstractThe reproductive biology of the black soldier fly (BSF), Hermetia illucens (L.) (Diptera: Stratiomyidae), was investigated in males and females with a focus on how sperm is handled by males before – and by females after – copulation. As this insect has great economic importance for protein production from agri‐food wastes, its reproduction needs to be precisely known. A detailed description of male and female reproductive organs was made, and sperm were counted in male and female storage organs. Measures of testis and sperm counts were performed in males of various ages and their dynamics was compared to the ovaries of females. Both male and female tracts were long, and consisted of various successive parts. In males, testes fill the vas deferens with mature sperm, that are tangled as ‘windrows’, and sperm pass successively through a ‘studded pipe’ and three phallic ‘wands’ before being transferred to females. In virgin males, mean (± SEM) sperm number increased from approximately 9700 (± 1400, n = 5) to 39600 (± 3470, n = 13) in 20 days, and testes gradually decreased in size, indicating continuous spermatogenesis. Sperm cells are long, more than 3 mm. During copulation, sperm are deposited by phallic ‘wands’ at the base of each spermatheca in a smooth tube followed by a ‘fishnet canal’ at the top of which they accumulate before complete storage. They then pass through a ringed canal, an elbow, and a rigid rod strewn with glands, before reaching the spermathecal reservoir. Females store approximately 680 spermatozoa after one mating in each spermathecal reservoir. Such complex sperm storage organs of females may indicate strong female post‐mating selection, and reflect competition for long and numerous sperm in this species, in which female multiple matings are suspected. Besides its economical interest, BSF is an appealing model for further investigations on sexual selection.

Copulatory behaviour and genital mechanics suggest sperm allocation by a non-intromittent sclerite in a pholcid spider.

The male genitalia of pholcid spiders, which is one of the most species-rich spider families, are characterized by a procursus, which is a morphologically diverse projection of the copulatory organ. It has been shown that the procursus interacts with the female genitalia during copulation. Here, we investigate the function of the procursus in Gertschiola neuquena, a species belonging to the early branched and understudied subfamily Ninetinae, using behavioural and morphological data. Although many aspects of the copulatory behaviour of G. neuquena follow the general pattern described for the family, males use only one pedipalp during each copulation. Based on our micro-CT analysis of cryofixed mating pairs using virgin females, we can show that the long and filiform procursus is inserted deeply into the unpaired convoluted female spermatheca, and the intromittent sclerite, the embolus, is rather short and stout only reaching the most distal part of the female sperm storage organ. Histological data revealed that sperm are present in the most proximal part of the spermatheca, suggesting that the procursus is used to allocate sperm deeply into the female sperm storage organ. This represents the first case of a replacement of the sperm allocation function of the intromittent sclerite in spiders.

No evidence for asymmetric sperm deposition in a species with asymmetric male genitalia.

Asymmetric genitalia have repeatedly evolved in animals, yet the underlying causes for their evolution are mostly unknown. The fruit fly Drosophila pachea has asymmetric external genitalia and an asymmetric phallus with a right-sided phallotrema (opening for sperm release). The complex of female and male genitalia is asymmetrically twisted during copulation and males adopt a right-sided copulation posture on top of the female. We wished to investigate if asymmetric male genital morphology and a twisted gentitalia complex may be associated with differential allocation of sperm into female sperm storage organs. We examined the internal complex of female and male reproductive organs by micro-computed tomography and synchrotron X-ray tomography before, during and after copulation. In addition, we monitored sperm aggregation states and timing of sperm transfer during copulation by premature interruption of copulation at different time-points. The asymmetric phallus is located at the most caudal end of the female abdomen during copulation. The female reproductive tract, in particular the oviduct, re-arranges during copulation. It is narrow in virgin females and forms a broad vesicle at 20 min after the start of copulation. Sperm transfer into female sperm storage organs (spermathecae) was only in a minority of examined copulation trials (13/64). Also, we found that sperm was mainly transferred early, at 2-4 min after the start of copulation. We did not detect a particular pattern of sperm allocation in the left or right spermathecae. Sperm adopted a granular or filamentous aggregation state in the female uterus and spermathecae, respectively. No evidence for asymmetric sperm deposition was identified that could be associated with asymmetric genital morphology or twisted complexing of genitalia. Male genital asymmetry may potentially have evolved as a consequence of a complex internal alignment of reproductive organs during copulation in order to optimize low sperm transfer rates.

Selection on sperm size in response to promiscuity and variation in female sperm storage organs.

Sperm cells are exceptionally morphologically diverse across taxa. However, morphology can be quite uniform within species, particularly for species where females copulate with many males per reproductive bout. Strong sexual selection in these promiscuous species is widely hypothesized to reduce intraspecific sperm variation. Conversely, we hypothesize that intraspecific sperm size variation may be maintained by high among-female variation in the size of sperm storage organs, assuming that paternity success improves when sperm are compatible in size with the sperm storage organ. We use individual-based simulations and an analytical model to evaluate how selection on sperm size depends on promiscuity level and variation in sperm storage organ size (hereafter, female preference variation). Simulations of high promiscuity (10 mates per female) showed stabilizing selection on sperm when female preference variation was low, and disruptive selection when female preference variation was high, consistent with the analytical model results. With low promiscuity (2-3 mates per female), selection on sperm was stabilizing for all levels of female preference variation in the simulations, contrasting with the analytical model. Promiscuity level, or mate sampling, thus has a strong impact on the selection resulting from female preferences. Furthermore, when promiscuity is low, disruptive selection on male traits will occur under much more limited circumstances (i.e. only with higher among-female variation) than many previous models suggest. Variation in female sperm storage organs likely has strong implications for intraspecific sperm variation in highly promiscuous species, but likely does not explain differences in intraspecific sperm variation for less promiscuous taxa.

Limitations of sperm transfer in the complex reproductive system of spiders

Abstract In spiders, sperm transfer from the male to the female is indirect via secondary copulatory structures, the pedipalps. At the time of transfer the sperm are not mobile and the ejaculate needs to move through narrow male and female ducts to the female sperm storage organ. In addition, copulation duration can be very short, often limited to just a few seconds. Finally, sexual cannibalism and genital damage limits male life-time mating opportunities. These features of the reproductive biology in spiders are likely to result in sperm transfer constraints. Here we review the intrinsic and extrinsic sperm transfer limitations and conduct a meta-analysis on sperm transfer data from published data. Most of the information available relates to orb-web spiders, but our meta-analysis also includes non-orb-web spiders. Our review identifies some of the behavioural factors that have been shown to influence sperm transfer, and lists several morphological and physiological traits where we do not yet know how they might affect sperm transfer.

Drought disrupts year-round breeding readiness in a tropical songbird

Climate change is expected to increase the prevalence and severity of extreme weather events like drought. For many opportunistically breeding tropical bird species, precipitation serves as a primary cue for onset of breeding, and sustained drought can have major impacts on reproductive fitness. The physiological effects of drought are poorly understood, but understanding physiological responses can help resolve the mechanisms underlying effects of drought on population demography. We used physiological data collected on a year-round breeding New Guinea endemic passerine, the White-shouldered Fairywren (<em>Malurus alboscapulatus</em>), during years with persistent rainfall and during the 2015–2016 El Niño drought event to assess patterns of breeding readiness, body condition, and molt. Many males lost their cloacal protuberances, a sperm storage organ they typically maintain year-round, during the drought period. Body molt, which also occurs year-round in this species, was higher in males during the drought period. Using a sliding-window statistical model approach, we identified a critical precipitation window of 0–40 days prior to capture for cloacal protuberance volume and a window of 8–53 days for molt, with lower precipitation associated with smaller cloacal protuberances and greater molt. Plasma androgens were predictive of variation in male cloacal protuberance volume and molt, thus potentially mediating transitions between life-history stages in response to environmental conditions. Female molt, androgens, and male mass varied with photoperiod, with longer but decreasing day lengths characteristic of the austral summer associated with increasing molt and decreasing androgens. Collectively, our results indicate the potential for photoperiod to cue some life-history stages, whereas reduced rainfall impacts male but apparently not female reproductive physiology and readiness to breed. Improving our understanding of environmental regulation of life-history transitions in tropical taxa is essential for identifying which taxa are most at risk under a changing climate.

Number and Relative Proportions of Eusperm and Parasperm in Various Lepidoptera

Lepidoptera produce two kinds of sperm, eusperm, which are conventional nucleated spermatozoa, and parasperm, which lack nuclei and are usually smaller. Both travel in the female to the sperm storage organ, the spermatheca, but there the parasperm become inactive or disappear without taking any direct role in fertilization of the eggs. There are widespread accounts in the literature of an excess of parasperm over eusperm, though relatively few actual counts, and those counts are from various locations in the lifetime of the sperm: from testes, seminal vesicles, ejaculates, or spermathecae. With phylogenetic comparisons in mind, we counted sperm in the final sperm storage organs of males, the seminal vesicles (ductus ejaculatorius duplex), to explore the prevalence of these two types of sperm over a wide range of species and sizes. We found parasperm in all species, including the most phylogenetically basal (non-ditrysian) ones, and a preponderance of parasperm over eusperm in almost all, including a majority of the smaller species with small total numbers of sperm. Total sperm number of both types increased with wingspan (our proxy for size) as did the ratio of parasperm to eusperm. The ubiquity of parasperm and their preponderance even in very small species indicates an important role in lepidopteran reproduction. Further, the large numbers of both sperm types in larger species of Lepidoptera, apparently vastly more than are needed to fertilize eggs, suggests a role in sperm competition.

Sperm Cyst "Looping": A Developmental Novelty Enabling Extreme Male Ornament Evolution.

Postcopulatory sexual selection is credited as a principal force behind the rapid evolution of reproductive characters, often generating a pattern of correlated evolution between interacting, sex-specific traits. Because the female reproductive tract is the selective environment for sperm, one taxonomically widespread example of this pattern is the co-diversification of sperm length and female sperm-storage organ dimension. In Drosophila, having testes that are longer than the sperm they manufacture was believed to be a universal physiological constraint. Further, the energetic and time costs of developing long testes have been credited with underlying the steep evolutionary allometry of sperm length and constraining sperm length evolution in Drosophila. Here, we report on the discovery of a novel spermatogenic mechanism—sperm cyst looping—that enables males to produce relatively long sperm in short testis. This phenomenon (restricted to members of the saltans and willistoni species groups) begins early during spermatogenesis and is potentially attributable to heterochronic evolution, resulting in growth asynchrony between spermatid tails and the surrounding spermatid and somatic cyst cell membranes. By removing the allometric constraint on sperm length, this evolutionary innovation appears to have enabled males to evolve extremely long sperm for their body mass while evading delays in reproductive maturation time. On the other hand, sperm cyst looping was found to exact a cost by requiring greater total energetic investment in testes and a pronounced reduction in male lifespan. We speculate on the ecological selection pressures underlying the evolutionary origin and maintenance of this unique adaptation.

The Drosophila Neprilysin 4 gene is essential for sperm function following sperm transfer to females.

Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. Here, we focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. We examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, we induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, we conclude that the Nep4 gene is essential for sperm function following sperm transfer to females.