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In many animals, females respond to mating with changes in physiology and behavior that are triggered by molecules transferred by males during mating. In Drosophila melanogaster, proteins in the seminal fluid are responsible for important female postmating responses, including temporal changes in egg production, elevated feeding rates and activity levels, reduced sexual receptivity, and activation of the immune system. It is unclear to what extent these changes are mutually beneficial to females and males or instead represent male manipulation. Here we use an experimental evolution approach in which females are randomly paired with a single male each generation, eliminating any opportunity for competition for mates or mate choice and thereby aligning the evolutionary interests of the sexes. After >150 generations of evolution, males from monogamous populations elicited a weaker postmating stimulation of egg production and activity than males from control populations that evolved with a polygamous mating system. Males from monogamous populations did not differ from males from polygamous populations in their ability to induce refractoriness to remating in females, but they were inferior to polygamous males in sperm competition. Mating-responsive genes in both the female abdomen and head showed a dampened response to mating with males from monogamous populations. Males from monogamous populations also exhibited lower expression of genes encoding seminal fluid proteins, which mediate the female response to mating. Together, these results demonstrate that the female postmating response, and the male molecules involved in eliciting this response, are shaped by ongoing sexual conflict.

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks.

Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, between reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted tradeoffs, it remains unclear whether systemic lifespan-extending interventions could ameliorate the declining performance of the ejaculate as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration contributes to male reproductive decline via mating-dependent mechanisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that spermproduction declines chronologically with age, invariant tomating activity such that oldermultiplymated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproductive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling results in improved late-life ejaculate performance, indicating simultaneous amelioration of both somatic and reproductive aging.

Zinc-alpha2-glycoprotein (ZAG) is a soluble glycoprotein primarily produced in adipocytes and the liver, with key roles in lipid metabolism, including lipolysis and the browning of adipose tissue. Despite extensive studies on its role in rodents, the relationship between ZAG and insulin in humans remains unclear. Given the emerging interest in ZAG’s involvement in metabolic diseases such as metabolic-dysfunction-associated steatotic liver disease, this study aimed to investigate the acute effects of insulin on ZAG levels both in vivo and in vitro. We recruited 24 healthy, individuals who were non-obese and assessed the impact of oral glucose overload, a standardized liquid nutritional supplement, and intravenous glucagon on circulating ZAG levels. In parallel, we explored the effects of insulin on ZAG production in cultured HepG2 cells. Our findings revealed a consistent acute reduction in serum ZAG levels following all in vivo tests, coinciding with increased insulin levels. In vitro, insulin rapidly downregulated ZAG protein and mRNA levels in HepG2 cells, with significant reductions observed within 15 min, followed by partial recovery after 2 h. These results suggest a potential acute inhibitory effect of insulin on ZAG production, supporting its role in promoting energy storage by suppressing lipolysis postprandially. This study provides new insights into the complex interplay between insulin and ZAG in regulating energy balance and highlights the potential of ZAG as a therapeutic target in metabolic diseases.

Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction.

Seminal fluid proteins (SFPs) exert potent effects on male and female fitness. Rapidly evolving and molecularly diverse, they derive from multiple male secretory cells and tissues. In Drosophila melanogaster, most SFPs are produced in the accessory glands, which are composed of ∼1,000 fertility-enhancing “main cells” and ∼40 more functionally cryptic “secondary cells.” Inhibition of bone morphogenetic protein (BMP) signaling in secondary cells suppresses secretion, leading to a unique uncoupling of normal female postmating responses to the ejaculate: refractoriness stimulation is impaired, but offspring production is not. Secondary-cell secretions might therefore make highly specific contributions to the seminal proteome and ejaculate function; alternatively, they might regulate more global—but hitherto undiscovered—SFP functions and proteome composition. Here, we present data that support the latter model. We show that in addition to previously reported phenotypes, secondary-cell-specific BMP signaling inhibition compromises sperm storage and increases female sperm use efficiency. It also impacts second male sperm, tending to slow entry into storage and delay ejection. First male paternity is enhanced, which suggests a constraint on ejaculate evolution whereby high female refractoriness and sperm competitiveness are mutually exclusive. Using quantitative proteomics, we reveal changes to the seminal proteome that surprisingly encompass alterations to main-cell–derived proteins, indicating important cross-talk between classes of SFP-secreting cells. Our results demonstrate that ejaculate composition and function emerge from the integrated action of multiple secretory cell types, suggesting that modification to the cellular make-up of seminal-fluid-producing tissues is an important factor in ejaculate evolution.

Seminal fluid proteins (SFPs) are emerging as fundamental contributors to sexual selection given their role in post-mating reproductive events, particularly in polyandrous species where the ejaculates of different males compete for fertilisation. SFP identification however remains taxonomically limited and little is known about avian SFPs, despite extensive work on sexual selection in birds. We characterize the SF proteome of the polyandrous Red junglefowl, Gallus gallus , the wild species that gave rise to the domestic chicken. We identify 1,141 SFPs, including proteins involved in immunity and antimicrobial defences, sperm maturation, and fertilisation, revealing a functionally complex SF proteome. This includes a predominant contribution of blood plasma proteins that is conserved with human SF. By comparing the proteome of young and old males with fast or slow sperm velocity in a balanced design, we identify proteins associated with ageing and sperm velocity, and show that old males that retain high sperm velocity have distinct proteome characteristics. SFP comparisons with domestic chickens revealed both qualitative and quantitative differences likely associated with domestication and artificial selection. Collectively, these results shed light onto the functional complexity of avian SF, and provide a platform for molecular studies of fertility, reproductive ageing, and domestication.

In Drosophila melanogaster, seminal fluid regulates the reproductive and immune responses of mated females. Some seminal fluid proteins may provide protective functions to mated females, such as antimicrobial activity and/or stimulation of antimicrobial gene expression levels, while others appear to have negative effects, contributing to a “cost of mating.” To identify seminal proteins that could participate in these phenomena, we used a systemic ectopic expression screen to test the effects on unmated females of proteins normally produced by the male accessory gland (Acps). Of the 21 ectopically expressed Acps that we tested for ability to assist in the clearance of a bacterial infection with Serratia marcescens, 3 Acps significantly reduced the bacterial counts of infected females, suggesting a protective role. Of the 23 Acps that we tested for toxicity, 3 were toxic, including one that has been implicated in the cost of mating in another study. We also tested ectopic expression females for other Acp-induced effects, but found no additional Acps that affected egg laying or receptivity upon ectopic expression.

Three genes are known to be essential for gamete adhesion/fusion ( Cd9, Izumo1 and Juno ). Here, we confirmed that Spaca6 null males are infertile and showed that their sperm accumulate in the perivitelline space but are unable to fuse with oocyte. Like IZUMO1, SPACA6 which is expressed by human sperm, is remained on the equatorial segment after acrosomal reaction and is involved in human fertilization since an anti-SPACA6 antibody inhibited it. Despite the similarity of the phenotypes caused by Spaca6 and Izumo1 knockouts, these are not redundant and the essential relocation of IZUMO1 is not affected by the lack of SPACA6. We propose a model in which IZUMO1 and SPACA6 would be part of a molecular complex necessary for gamete fusion and that their concomitant presence would be required for the recruitment of another essential molecular actor, such as a fusogen, for the fusion to take place.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization.