The Female Response to Seminal Fluid.

Seminal fluid is often assumed to have just one function in mammalian reproduction, delivering sperm to fertilize oocytes. But seminal fluid also transmits signaling agents that interact with female reproductive tissues to facilitate conception and .pregnancy. Upon seminal fluid contact, female tissues initiate a controlled inflammatory response that affects several aspects of reproductive function to ultimately maximize the chances of a male producing healthy offspring. This effect is best characterized in mice, where the female response involves several steps. Initially, seminal fluid factors cause leukocytes to infiltrate the female reproductive tract, and to selectively target and eliminate excess sperm. Other signals stimulate ovulation, induce an altered transcriptional program in female tract tissues that modulates embryo developmental programming, and initiate immune adaptations to promote receptivity to implantation and placental development. A key result is expansion of the pool of regulatory T cells that assist implantation by suppressing inflammation, mediating tolerance to male transplantation antigens, and promoting uterine vascular adaptation and placental development. Principal signaling agents in seminal fluid include prostaglandins and transforming growth factor-ß. The balance of male signals affects the nature of the female response, providing a mechanism of ?cryptic female choiceË® that influences female reproductive investment. Male-female seminal fluid signaling is evident in all mammalian species investigated including human, and effects of seminal fluid in invertebrates indicate evolutionarily conserved mechanisms. Understanding the female response to seminal fluid will shed new light on infertility and pregnancy disorders and is critical to defining how events at conception influence offspring health.

Evolutionary Quantitative Proteomics of Reproductive Protein Divergence in Drosophila.

Reproductive traits often evolve rapidly between species. Understanding the causes and consequences of this rapid divergence requires characterization of female and male reproductive proteins and their effect on fertilization success. Species in the Drosophila virilis clade exhibit rampant interspecific reproductive incompatibilities, making them ideal for studies on diversification of reproductive proteins and their role in speciation. Importantly, the role of intraejaculate protein abundance and allocation in interspecific divergence is poorly understood. Here, we identify and quantify the transferred male ejaculate proteome using multiplexed isobaric labeling of the lower female reproductive tract before and immediately after mating using three species of the virilis group. We identified over 200 putative male ejaculate proteins, many of which show differential abundance between species, suggesting that males transfer a species-specific allocation of seminal fluid proteins during copulation. We also identified over 2000 female reproductive proteins, which contain female-specific serine-type endopeptidases that showed differential abundance between species and elevated rates of molecular evolution, similar to that of some male seminal fluid proteins. Our findings suggest that reproductive protein divergence can also manifest in terms of species-specific protein abundance patterns.

Metabolomics analysis of human spermatozoa reveals impaired metabolic pathways in asthenozoospermia.

BACKGROUND: Infertility is a major health issue, affecting 15% of reproductive-age couples with male factors contributing to 50% of cases. Asthenozoospermia (AS), or low sperm motility, is a common cause of male infertility with complex aetiology, involving genetic and metabolic alterations, inflammation and oxidative stress. However, the molecular mechanisms behind low motility are unclear. In this study, we used a metabolomics approach to identify metabolic biomarkers and pathways involved in sperm motility. METHODS: We compared the metabolome and lipidome of spermatozoa of men with normozoospermia (n = 44) and AS (n = 22) using untargeted LC-MS and the metabolome of seminal fluid using 1H-NMR. Additionally, we evaluated the seminal fluid redox status to assess the oxidative stress in the ejaculate. RESULTS: We identified 112 metabolites and 209 lipids in spermatozoa and 27 metabolites in the seminal fluid of normozoospermic and asthenozoospermic men. PCA analysis of the spermatozoa's metabolomics and lipidomics data showed a clear separation between groups. Spermatozoa of asthenozoospermic men presented lower levels of several amino acids, and increased levels of energetic substrates and lysophospholipids. However, the metabolome and redox status of the seminal fluid was not altered inAS. CONCLUSIONS: Our results indicate impaired metabolic pathways associated with redox homeostasis and amino acid, energy and lipid metabolism in AS. Taken together, these findings suggest that the metabolome and lipidome of human spermatozoa are key factors influencing their motility and that oxidative stress exposure during spermatogenesis or sperm maturation may be in the aetiology of decreased motility in AS.

Elucidation of ejaculatory bulb proteins in Bemisia tabaci Asia-1 and Asia II-1 and confirmation of their mating transfer via RNAi.

BACKGROUND: Bemisia tabaci, a significant agricultural pest in Asia, contains distinct genetic groups, Asia-1 and Asia II-1. Understanding its reproductive biology, particularly the role of ejaculatory bulb proteins (EBPs) in mating, is crucial. However, EBPs in B. tabaci were not well characterised until this study. METHODS AND RESULTS: The EBPs have been characterised in the Asia-1 and Asia II-1 genetic groups of the whitefly B. tabaci, prevalent in Asia. The transcriptomic analysis yielded over 40,000,000 and 30,000,000 annotated transcripts, respectively, from Asia II-1 and Asia-1. Differential gene expression revealed the presence of 270 upregulated and 198 downregulated genes, with significant differences between these two genetic groups. Orphan genes (1992 numbers) were identified in both genetic groups. We report, for the first time, full-length sequences of EBP genes from B. tabaci. The 10 EBPs each deduced in B. tabaci Asia-1 and Asia II-1 are structurally akin to chemosensory proteins having four conserved cysteine residues. Additionally, we did domain analysis, protein structure prediction, mapping of these EBPs in the chromosomes of B. tabaci, and phylogenetic analysis to track their evolutionary lineage. We have specifically demonstrated the transfer of EBPs from males to females during mating using qPCR and further validated the transfer of EBPs through RNAi. Specifically, we targeted the highly expressed EBPs (EBP-3, 7, and 8 in BtAsia1; EBP-8, 9, and 10 in BtAsia II-1) through feeding bioassays of dsRNAs. Tracking by qPCR revealed that the females, when mated with dsRNA-treated males, did not show expression of the specific EBP, suggesting that the silencing of these genes in males hinders the transfer of EBP to females during mating. CONCLUSION: Our findings provide novel insights into the genomic contours of EBPs in B. tabaci and underscore the potential of RNAi-based strategies for pest management by disrupting the reproductive processes.

Functional Diversity and Evolution of the Drosophila Sperm Proteome.

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and ß-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of 'sperm-associated Sfps' are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.

Glyphosate presence in human sperm: First report and positive correlation with oxidative stress in an infertile French population.

Environmental exposure to endocrine disruptors, such as pesticides, could contribute to a decline of human fertility. Glyphosate (GLY) is the main component of Glyphosate Based Herbicides (GBHs), which are the most commonly herbicides used in the world. Various animal model studies demonstrated its reprotoxicity. In Europe, GLY authorization in agriculture has been extended until 2034. Meanwhile the toxicity of GLY in humans is still in debate. The aims of our study were firstly to analyse the concentration of GLY and its main metabolite, amino-methyl-phosphonic acid (AMPA) by LC/MS-MS in the seminal and blood plasma in an infertile French men population (n=128). We secondly determined Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) using commercial colorimetric kits and some oxidative stress biomarkers including malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) by ELISA assays. We next analysed potential correlations between GLY and oxidative stress biomarkers concentration and sperm parameters (sperm concentration, progressive speed, anormal forms). Here, we detected for the first time GLY in the human seminal plasma in significant proportions and we showed that its concentration was four times higher than those observed in blood plasma. At the opposite, AMPA was undetectable. We also observed a strong positive correlation between plasma blood GLY concentrations and plasma seminal GLY and 8-OHdG concentrations, the latter reflecting DNA impact. In addition, TOS, Oxidative Stress Index (OSI) (TOS/TAS), MDA blood and seminal plasma concentrations were significantly higher in men with glyphosate in blood and seminal plasma, respectively. Taken together, our results suggest a negative impact of GLY on the human reproductive health and possibly on his progeny. A precaution principle should be applied at the time of the actual discussion of GLY and GBHs formulants uses in Europe by the authorities.

A predominant role of genotypic variation in both expression of sperm competition genes and paternity success in Drosophila melanogaster.

Sperm competition is a crucial aspect of male reproductive success in many species, including Drosophila melanogaster, and seminal fluid proteins (Sfps) can influence sperm competitiveness. However, the combined effect of environmental and genotypic variation on sperm competition gene expression remains poorly understood. Here, we used Drosophila Genetic Reference Panel (DGRP) inbred lines and manipulated developmental population density (i.e. larval density) to test the effects of genotype, environment and genotype-by-environment interactions (GEI) on the expression of the known sperm competition genes Sex Peptide, Acp36DE and CG9997. High larval density resulted in reduced adult body size, but expression of sperm competition genes remained unaffected. Furthermore, we found no significant GEI but genotypic effects in the expression of SP and Acp36DE. Our results also revealed GEI for relative competitive paternity success (second male paternity; P2), with genes' expression positively correlated with P2. Given the effect of genotype on the expression of genes, we conducted a genome-wide association study (GWAS) and identified polymorphisms in putative cis-regulatory elements as predominant factors regulating the expression of SP and Acp36DE. The association of genotypic variation with sperm competition outcomes, and the resilience of sperm competition genes' expression against environmental challenges, demonstrates the importance of genome variation background in reproductive fitness.

Low-polarity untargeted metabolomic profiling as a tool to gain insight into seminal fluid.

INTRODUCTION: A decrease in sperm cell count has been observed along the last several decades, especially in the most developed regions of the world. The use of metabolomics to study the composition of the seminal fluid is a promising approach to gain access to the molecular mechanisms underlying this fact. OBJECTIVES: In the present work, we aimed at relating metabolomic profiles of young healthy men to their semen quality parameters obtained from conventional microscopic analysis. METHODS: An untargeted metabolomics approach focusing on low- to mid-polarity compounds was used to analyze a subset of seminal fluid samples from a cohort of over 2700 young healthy men. RESULTS: Our results show that a broad metabolic profiling comprising several families of compounds (including acyl-carnitines, steroids, and other lipids) can contribute to effectively distinguish samples provided by individuals exhibiting low or high absolute sperm counts. CONCLUSION: A number of metabolites involved in sexual development and function, signaling, and energy metabolism were highlighted as being distinctive of samples coming from either group, proving untargeted metabolomics as a promising tool to better understand the pathophysiological processes responsible for male fertility impairment.

Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry.

Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes' urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033-substances listed under anabolic substances (S1) on the World Anti-Doping Agency's Prohibited List-in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02-0.40 ng/mL) and in sf (0.01-0.25 ng/mL) as well as of LGD-4033 in bp (0.21-2.00 ng/mL) and in sf (0.03-0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine.

Males can adjust offspring sex ratio in an adaptive fashion through different mechanisms.

Sex allocation research has primarily focused on offspring sex-ratio adjustment by mothers. Yet, fathers also benefit from producing more of the sex with greater fitness returns. Here, we review the state-of-the art in the study of male-driven sex allocation and, counter to the current paradigm, we propose that males can adaptively influence offspring sex ratio through a wide variety of mechanisms. This includes differential production and motility of X- versus Y-bearing sperms in mammals, variation in seminal fluid composition in haplo-diploid invertebrates, and epigenetic mechanisms in some fish and lizards exhibiting environmental sex determination. Conflicts of interest between mothers and fathers over offspring sex ratios can emerge, although many more studies are needed in this area. While many studies of sex allocation have focused on adaptive explanations with little attention to mechanisms, and vice versa, the integration of these two topics is essential for understanding male-driven sex allocation.

Male reproductive aging arises via multifaceted mating-dependent sperm and seminal proteome declines, but is postponable in Drosophila.

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 trade-offs, 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 sperm production declines chronologically with age, invariant to mating activity such that older multiply mated 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.

Mass Spectrometry-Based Untargeted Approaches to Reveal Diagnostic Signatures of Male Infertility in Seminal Plasma: A New Laboratory Perspective for the Clinical Management of Infertility?

Male infertility has been recognized as a global health problem. Semen analysis, although considered the golden standard, may not provide a confident male infertility diagnosis alone. Hence, there is the urgent request for an innovative and reliable platform to detect biomarkers of infertility. The rapid expansion of mass spectrometry (MS) technology in the field of the 'omics' disciplines, has incredibly proved the great potential of MS-based diagnostic tests to revolutionize the future of pathology, microbiology and laboratory medicine. Despite the increasing success in the microbiology area, MS-biomarkers of male infertility currently remain a proteomic challenge. In order to address this issue, this review encompasses proteomics investigations by untargeted approaches with a special focus on experimental designs and strategies (bottom-up and top-down) for seminal fluid proteome profiling. The studies reported here witness the efforts of the scientific community to address these investigations aimed at the discovery of MS-biomarkers of male infertility. Proteomics untargeted approaches, depending on the study design, might provide a great plethora of biomarkers not only for a male infertility diagnosis, but also to address a new MS-biomarkers classification of infertility subtypes. From the early detection to the evaluation of infertility grade, new MS-derived biomarkers might also predict long-term outcomes and clinical management of infertility.

A Multicenter Retrospective Cohort Study of the Profile of Seminal Fluid Analyses of Men Seeking Fertility Care at Different Hospitals.

BACKGROUND: Male infertility contributes 40 % of couple infertility. The prevalence of abnormal semen parameters has been on the increase. Age among other factors affects the fertility potential of males. This study analysed the pattern of seminal fluid parameters of males, seeking fertility treatment in hospitals and the relationship between age, volume and liquefaction time on these other semen parameters. METHODS: This is a multicentre retrospective cohort study conducted in eight secondary and tertiary hospitals in Nigeria. The case notes of couples that sort fertility care at the Gynaecology and Urology clinics of these hospitals from January 1st 2022 to December 31st 2022 were retrieved after receiving ethical approval. A purposeddesigned proforma based on the WHO manual for the examination of human semen was used for data collection. Outcome measures were time of semen collection and examination, volume of semen, sperm number, sperm concentration, PH, agglutination, liquefaction, motility,morphology, vitality, and white blood cell count. Data was analysed using SPSS version 23. Data were presented as means and proportions. P-value of < 0.05 was used as the level of significance. RESULTS: Overall, 1063 couples attended gynaecology and urology clinics with fertility-related concerns within the study period with a retrieval rate of 98.3%. The mean age of participants was 38.24 ± 8 years, while the mean semen volume and sperm concentrations were 2.62 ± 1.6 mls and 34.32 ± 7.4 million respectively. The age of participants significantly affected motility, volume and morphology (p-values of 0.001, 0.001 and 0.004 respectively). The total motility and sperm concentration have an inverse relationship with the age of the participants. CONCLUSION: This study shows that sperm motility decreases with the age of participants. It was also observed that the most common combined abnormality was oligoasthenozoospermia.

CONTEXTE: L'infertilité masculine représente 40 % de l'infertilité des couples. La prévalence des paramètres anormaux du sperme est en augmentation. L'âge, entre autres facteurs, affecte le potentiel de fertilité des hommes. Cette étude a analysé le profil des paramètres du liquide séminal des hommes cherchant un traitement de fertilité dans les hôpitaux et la relation entre l'âge, le volume et le temps de liquéfaction sur ces autres paramètres du sperme. MÉTHODES: Il s'agit d'une étude de cohorte rétrospective multicentrique menée dans huit hôpitaux secondaires et tertiaires au Nigeria. Les notes de cas des couples qui ont eu recours à des soins de fertilité dans les cliniques de gynécologie et d'urologie de ces hôpitaux entre le 1er janvier et le 31 décembre 2022 ont été récupérées après avoir reçu une approbation éthique. Un proforma conçu à dessein et basé sur le manuel de l'OMS pour l'examen du sperme humain a été utilisé pour la collecte des données. Les mesures des résultats étaient le temps de collecte et d'examen du sperme, le volume de sperme, le nombre de spermatozoïdes, la concentration en spermatozoïdes, le PH, l'agglutination, la liquéfaction, la motilité, la morphologie, la vitalité et la numération des globules blancs. Les données ont été analysées à l'aide de SPSS version 23. Les données ont été présentées sous forme de moyennes et de proportions. Une valeur P < 0,05 a été utilisée comme niveau de signification. RÉSULTATS: Dans l'ensemble, 1 063 couples ont fréquenté les cliniques de gynécologie et d'urologie pour des problèmes de fertilité au cours de la période d'étude, avec un taux de récupération de 98,3 %. L'âge moyen des participants était de 38,24 ± 8 ans, tandis que le volume moyen de sperme et les concentrations de spermatozoïdes étaient respectivement de 2,62 ± 1,6 ml et 34,32 ±7,4 millions. L'âge des participants a affecté de manière significative la motilité, le volume et la morphologie (valeurs p de 0,001, 0,001 et 0,004 respectivement). La motilité totale et la concentration en spermatozoïdes ont une relation inverse avec l'âge des participants. CONCLUSION: Cette étude montre que la mobilité des spermatozoïdes diminue avec l'âge des participants. Il a également été observé que l'anomalie combinée la plus fréquente était l'oligoasthénozoospermie. Mots-clés: Infertilité Masculine, Anomalies du Liquide séminal, Nigeria.

Infectious Toscana Virus in Seminal Fluid of Young Man Returning from Elba Island, Italy.

We report detecting infectious Toscana virus in the seminal fluid of a 25-year-old man from Italy returning from Elba Island. The presence of infectious virus in human semen adds Toscana virus to the long list of viruses detected in this genital fluid and indicates a potential for sexual transmission.

Characterization of reproductive proteins in the Mexican fruit fly points towards the evolution of novel functions.

Seminal fluid proteins (Sfps) modify female phenotypes and have wide-ranging evolutionary implications on fitness in many insects. However, in the Mexican fruit fly, Anastrepha ludens, a highly destructive agricultural pest, the functions of Sfps are still largely unknown. To gain insights into female phenotypes regulated by Sfps, we used nano-liquid chromatography mass spectrometry to conduct a proteomic analysis of the soluble proteins from reproductive organs of A. ludens. The proteins predicted to be transferred from males to females during copulation were 100 proteins from the accessory glands, 69 from the testes and 20 from the ejaculatory bulb, resulting in 141 unique proteins after accounting for redundancies from multiple tissues. These 141 included orthologues to Drosophila melanogaster proteins involved mainly in oogenesis, spermatogenesis, immune response, lifespan and fecundity. In particular, we found one protein associated with female olfactory response to repellent stimuli (Scribble), and two related to memory formation (aPKC and Shibire). Together, these results raise the possibility that A. ludens Sfps could play a role in regulating female olfactory responses and memory formation and could be indicative of novel evolutionary functions in this important agricultural pest.

On how to identify a seminal fluid protein: A response to Wigby et al.

The choice of criteria to delimit a group or class is a subjective matter, even though the reasoning, the objectives and the criteria themselves should always be clearly stated. This paper is part of a discussion about the criteria used to identify seminal fluid proteins (SFPs) in Drosophila species. SFPs are proteins that are transferred to females during copulation together with sperm. The only way to ascertain that a protein is an SFP is to prove that it is produced in a male reproductive organ and is found in the female reproductive tract after insemination. Nevertheless, the required methodology is labour-intensive and expensive, and therefore this kind of data is unlikely to be available for many species, precluding comparative and evolutionary studies on the subject. To conduct evolutionary analyses, in a previous study, we capitalized on the accumulated knowledge we have in the model species D. melanogaster to recommend a set of criteria for identifying candidate SFPs in other Drosophila species. Those criteria, based on transcriptomic evidence and in silico predictions from sequences, would allow a good balance between sensitivity (the inclusion of true SFPs) and specificity (the exclusion of false positives). In view of the criticism raised by another group, here we defend our criteria on one hand while accepting there is room for improvement on the other. The results are updated sets of criteria and SFPs that we believe can be useful in future evolutionary studies.

On how to identify a seminal fluid protein: A commentary on Hurtado et al.

Seminal fluid proteins (Sfps) have striking effects on the behaviour and physiology of females in many insects. Some Drosophila melanogaster Sfps are not highly or exclusively expressed in the accessory glands, but derive from, or are additionally expressed in other male reproductive tissues. The full suite of Sfps includes transferred proteins from all male reproductive tissues, regardless of expression level or presence of a signal peptide.

Research gaps and new insights in the evolution of Drosophila seminal fluid proteins.

While the striking effects of seminal fluid proteins (SFPs) on females are fairly conserved among Diptera, most SFPs lack detectable homologues among the SFP repertoires of phylogenetically distant species. How such a rapidly changing proteome conserves functions across taxa is a fascinating question. However, this and other pivotal aspects of SFPs' evolution remain elusive because discoveries on these proteins have been mainly restricted to the model Drosophila melanogaster. Here, we provide an overview of the current knowledge on the inter-specific divergence of the SFP repertoire in Drosophila and compile the increasing amount of relevant genomic information from multiple species. Capitalizing on the accumulated knowledge in D. melanogaster, we present novel sets of high-confidence SFP candidates and transcription factors presumptively involved in regulating the expression of SFPs. We also address open questions by performing comparative genomic analyses that failed to support the existence of many conserved SFPs shared by most dipterans and indicated that gene co-option is the most frequent mechanism accounting for the origin of Drosophila SFP-coding genes. We hope our update establishes a starting point to integrate further data and thus widen the understanding of the intricate evolution of these proteins.

Protective roles of seminal plasma exosomes and microvesicles during human sperm cryopreservation.

RESEARCH QUESTION: Do seminal plasma microvesicles and exosomes, as two subtypes of extracellular vesicles, exert cryoprotective properties in sperm cryopreservation? DESIGN: Microvesicles and exosomes isolated from normozoospermic semen samples (n = 10) by serial ultracentrifugation were determined using scanning electron microscopy, dynamic light scattering and western blot analysis. The interactions between extracellular vesicles and spermatozoa were detected using Dil labelling. Purified spermatozoa from different normozoospermic samples (n = 25) were then treated individually with exosomes or microvesicles for 1 h and subsequently cryopreserved. The effects of extracellular vesicles during cryopreservation were investigated by determining post-thaw sperm motility, morphology, viability, reactive oxygen species (ROS) generation, lipid peroxidation, total antioxidant capacity (TAC), mitochondrial membrane potential (MMP), DNA integrity, and apoptosis rate. RESULTS: Microvesicles and exosomes displayed a round-shape morphology, with about 70% of exosomes ranging from 43-144 nm, microvesicles ranging from 144.5-486 nm and both expressed tetraspanin markers. Fluorescence microscopy showed that exosomes and microvesicles absorbed mainly in the sperm head and less frequently in the neck and tail. The post-thawing results indicated that the diluent with exosomes or microvesicles had improved sperm motility (P = 0.007), morphology (P < 0.001) and viability (P < 0.001) compared with untreated samples. The ROS levels decreased significantly (P = 0.001), with a consequent decrease in DNA damage (P = 0.001). The TAC activity (P = 0.001) and MMP levels (P = 0.001) were also significantly improved; levels of malondialdehyde (MDA) (P = 0.62) and apoptosis rate (P = 1.000) remained unchanged. CONCLUSION: Seminal plasma microvesicles and exosomes could protect spermatozoa from cryopreservation chilling injuries.

Copulation Song in Drosophila: Do Females Sing to Change Male Ejaculate Allocation and Incite Postcopulatory Mate Choice?

Drosophila males sing a courtship song to achieve copulations with females. Females were recently found to sing a distinct song during copulation, which depends on male seminal fluid transfer and delays female remating. Here, it is hypothesized that female copulation song is a signal directed at the copulating male and changes ejaculate allocation. This may alter female remating and sperm usage, and thereby affect postcopulatory mate choice. Mechanisms of how female copulation song is elicited, how males respond to copulation song, and how remating is modulated, are considered. The potential adaptive value of female signaling during copulation is discussed with reference to vertebrate copulation calls and their proposed function in eliciting mate guarding. Female copulation song may be widespread within the Drosophila genus. This newly discovered behavior opens many interesting avenues for future research, including investigation of how sexually dimorphic neuronal circuits mediate communication between nervous system and reproductive organs.