Proteins within the seminal fluid are crucial to keep sperm viable in the honeybee Apis mellifera

Seminal fluid is a biochemically complex mixture of glandular secretions that is transferred to the females sexual tract as part of the ejaculate. Seminal fluid has received increasing scientific interest in the fields of evolutionary and reproductive biology, as it seems a major determinant of male fertility/infertility and reproductive success. Here we used the honeybee Apis mellifera, where seminal fluid can be collected as part of a male's ejaculate, and performed a series of experiments to investigate the effects of seminal fluid and its components on sperm viability. We show that honeybee seminal fluid is highly potent in keeping sperm alive and this positive effect is present over a 24 h time span, comparable to the timing of the sperm storage process in the queen. We furthermore show that the presence of proteins within the seminal fluid and their structural integrity are crucial for this effect. Finally, we activated sperm using fructose and provide evidence that the positive effect of seminal fluid proteins on sperm survival cannot be replicated using generic protein substitutes. Our data provide experimental insights into the complex molecular interplay between sperm and seminal fluid defining male fertility and reproductive success.     

Genetic Disruption of the Copulatory Plug in Mice Leads to Severely Reduced Fertility

Seminal fluid proteins affect fertility at multiple stages in reproduction. In many species, a male''s ejaculate coagulates to form a copulatory plug. Although taxonomically widespread, the molecular details of plug formation remain poorly understood, limiting our ability to manipulate the structure and understand its role in reproduction. Here I show that male mice knockouts for transglutaminase IV (Tgm4) fail to form a copulatory plug, demonstrating that this gene is necessary for plug formation and lending a powerful new genetic tool to begin characterizing plug function. Tgm4 knockout males show normal sperm count, sperm motility, and reproductive morphology. However, very little of their ejaculate migrates into the female''s reproductive tract, suggesting the plug prevents ejaculate leakage. Poor ejaculate migration leads to a reduction in the proportion of oocytes fertilized. However, Tgm4 knockout males fertilized between 3–11 oocytes, which should be adequate for a normal litter. Nevertheless, females mated to Tgm4 knockout males for approximately 14 days were significantly less likely to give birth to a litter compared to females mated to wild-type males. Therefore, it appears that the plug also affects post-fertilization events such as implantation and/or gestation. This study shows that a gene influencing the viscosity of seminal fluid has a major influence on male fertility.     

Sperm competition promotes the exploitation of rival ejaculates

In many polyandrous species, the second male to mate with a female has fertilization priority. Strategic mechanisms generating this pattern tend to involve facultative increases in ejaculate size by informed males. Here we suggest a novel mechanism to generate second male precedence in internal fertilizers, based on the established fact that the female site of insemination is hostile to sperm, and that non-sperm components of the ejaculate frequently act to ameliorate these hostile conditions. We suggest that in species where female remating is frequent and rapid, second male sperm precedence may be due to the prior buffering of the female tract by previous males' ejaculates. In this scenario, second male costs are lower due to reduced sperm mortality and/or the redundancy of seminal fluid. Contrary to many classical predictions, second males may gain a paternity advantage despite investing less in an ejaculate.     

Male mating rate is constrained by seminal fluid availability in bedbugs, Cimex lectularius

Sexual selection, differences in reproductive success between individuals, continues beyond acquiring a mating partner and affects ejaculate size and composition (sperm competition). Sperm and seminal fluid have very different roles in sperm competition but both components encompass production costs for the male. Theoretical models predict that males should spend ejaculate components prudently and differently for sperm and seminal fluid but empirical evidence for independent variation of sperm number and seminal fluid volume is scarce. It is also largely unknown how sperm and seminal fluid variation affect future mating rate. In bedbugs we developed a protocol to examine the role of seminal fluids in ejaculate allocation and its effect on future male mating rate. Using age-related changes in sperm and seminal fluid volume we estimated the lowest capacity at which mating activity started. We then showed that sexually active males allocate 12% of their sperm and 19% of their seminal fluid volume per mating and predicted that males would be depleted of seminal fluid but not of sperm. We tested (and confirmed) this prediction empirically. Finally, the slightly faster replenishment of seminal fluid compared to sperm did not outweigh the faster decrease during mating. Our results suggest that male mating rate can be constrained by the availability of seminal fluids. Our protocol might be applicable to a range of other organisms. We discuss the idea that economic considerations in sexual conflict research might benefit from distinguishing between costs and benefits that are ejaculate dose-dependent and those that are frequency-dependent on the mating rate per se.     

Condition‐dependent ejaculate production affects male mating behavior in the common bedbug Cimex lectularius

Food availability in the environment is often low and variable, constraining organisms in their resource allocation to different life‐history traits. For example, variation in food availability is likely to induce condition‐dependent investment in reproduction. Further, diet has been shown to affect ejaculate size, composition and quality. How these effects translate into male reproductive success or change male mating behavior is still largely unknown. Here, we concentrated on the effect of meal size on ejaculate production, male reproductive success and mating behavior in the common bedbug Cimex lectularius. We analyzed the production of sperm and seminal fluid within three different feeding regimes in six different populations. Males receiving large meals produced significantly more sperm and seminal fluid than males receiving small meals or no meals at all. While such condition‐dependent ejaculate production did not affect the number of offspring produced after a single mating, food‐restricted males could perform significantly fewer matings than fully fed males. Therefore, in a multiple mating context food‐restricted males paid a fitness cost and might have to adjust their mating strategy according to the ejaculate available to them. Our results indicate that meal size has no direct effect on ejaculate quality, but food availability forces a condition‐dependent mating rate on males. Environmental variation translating into variation in male reproductive traits reveals that natural selection can interact with sexual selection and shape reproductive traits. As males can modulate their ejaculate size depending on the mating situation, future studies are needed to elucidate whether environmental variation affecting the amount of ejaculate available might induce different mating strategies.     

Secondary sex traits,parasites, immunity and ejaculate quality in the Arctic charr

Ejaculate quality may limit male reproductive success. Sperm cells are immunologically perceived as non-self in the male reproductive tract and may therefore be attacked by the immune system. Males may consequently have to suppress their immune system in order to produce high-quality ejaculates. This suppression may be influenced by the current level of parasite infections, suggesting that only parasite-resistant males are able to produce high-quality ejaculates. In a study of naturally infected male Arctic charr sampled during their spawning period, we found that the density of circulating granulocytes, spleen mass and the intensity of infection by one nematode species located outside the testes were negatively associated with ejaculate quality. This suggests that a male''s extra-testicular immune environment may affect the production of high-quality ejaculates and that parasite infections located in the extra-testicular soma may influence ejaculate quality, a trait most likely under directional selection. Moreover, male fertilization potential was negatively correlated with their red spawning coloration. In conclusion, these results emphasize the importance of parasites and immunity as factors generating variability in sperm quality, suggesting that parasite resistance may be of importance for maintaining variance in reproductive success even after copulation.     

Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system

Seminal plasma is a fluid that originates from the testis, epididymis,prostate, and seminal vesicles, and hence, proteomic studies may identify potential markers of infertility and other diseases of the genito-urinary tract. We profiled the proteomes of pooled seminal plasma from fertile Control and post-vasectomy (PV) men. PV seminal plasma samples are void of proteins originating from the testis and the epididymis due to ligation of the vas deferens, and hence, comparative analysis of Control and PV data sets allows for identification of proteins originating from these tissues. Utilizing offline MudPIT and high-resolution mass spectrometry, we were able to identify over 2000 proteins in Control and PV pools each and over 2300 proteins all together. With semiquantitative analysis using spectral counting, we catalogued 32 proteins unique to Control, 49 at lower abundance in PV, 3 unique to PV, and 25 at higher abundance in PV. We believe that proteins unique to Control or at lower abundance in PV have their origin in the testis and the epididymis. Public databases have confirmed that many of these proteins originate from the testis and epididymis and are linked to the reproductive tract. These proteins may serve as candidate biomarkers for future studies of infertility and urogenital diseases.     

Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme

Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.     

Approche clinique d'une OAT

The clinical approach of the male partner of infertile couples seems to be still a problem. Indeed, clinical history and examination are too often lacking. Such an attitude induces that the treatment of male infertility tends to be based for most part on the examination of seminal fluid alone. With the consequence that the treatment of the semen rather the man is the focus of attention. Oligozoospermia (OAT) is a common finding among infertile men. This OAT can be due to factors that may be found through anamnesis, clinical examination and semen analysis. Anamnesis indicates history of cryptorchidism, urogenital infections, surgery for inguinal hernia. One of the most studied factors that can be found during clinical examination is varicocele, and recent data indicate that the surgical treatment of varicocele when associated with OAT could be beneficial to fertility of infertile men. Semen infection, a difficult diagnosis mainly based on bacteriospermia and leukospermia, could benefit from other diagnosis tools such as echography of the male reproductive tract and biochemical markers of the seminal plasma. Finally, when no potential factors responsible for an OAT are found, such an OAT is named “idiopathic”. However, some of these “idiopathic OAT” are false idiopathic OAT, as shown through three examples, such as unilateral testicular obstruction, partial ejaculatory duct obstruction and thermal pathologies of the testes, when anamnesis and clinical examination are adequately associated with semen analysis.     

Seminal exosomes – An important biological marker for various disorders and syndrome in human reproduction

BackgroundExosomes are nano-sized membrane vesicles, secreted by different types of cells into the body’s biological fluids. They are found in abundance in semen as compared to other fluids. Exosomes contain a cargo of lipid molecules, proteins, phospholipids, cholesterol, mRNAs, and miRNAs. Each molecule of seminal exosomes (SE) has a potential role in male reproduction for childbirth. Many potential candidates are available within the seminal exosomes that can be used as diagnostic markers for various diseases or syndromes associated with male reproduction. Also these seminal exospmes play a major role in female reproductive tract for effective fertilization.AimThe aim of this review is to focus on the advancement of human seminal exosomal research and its various properties.MethodsWe used many databases like Scopus, Google scholar, NCBI-NLM and other sources to filter the articles of interest published in exosomes. We used phrases like “Exosomes in human semen”, “Composition of exosomes in human semen” and other relevant words to filter the best articles.ResultsSeminal exosomes play a major role in sperm functions like cell-to-cell communication, motility of the sperm cells, maintaining survival capacity for the sperm in the female reproductive tract and spermatogenesis. Also, seminal exosomes are used as a carrier for many regulatory elements using small RNA molecules. miRNAs of the seminal exosomes can be used as a diagnostic marker for prostate cancer instead of prostate specific antigen (PSA). Epididymosomes can be used as a biomarker for reproductive diseases and male infertility.ConclusionSeminal exosomes could be used as biological markers for various reproductive disorders, male infertility diagnosis, and it can be used in anti-retroviral research for the identification of novel therapeutics for HIV-1 infection and transmission.     

Rat dorsal prostate is necessary for vaginal adhesion of the seminal plug and sperm motility in the uterine horns

The rat prostate comprises dorsal, ventral and lateral lobes that are morphologically and biochemically distinct. Lesions to these structures are expected to affect the quality of the ejaculate and male fertility. In experiment 1, we analyzed ejaculate parameters of males that had chemical lesions of the dorsal or ventral lobes. At pre-lesion and at 5 and 20 days post-lesion males were mated, and after ejaculation, seminal fluid and seminal plug were obtained from the mated females. In experiment 2, the ventral lobes were ablated, and the ejaculate was analyzed. In experiment 3, the fertility of males with chemically-lesioned dorsal lobes or ablation of the ventral lobes was evaluated. Chemical lesion of the dorsal lobe prevented the adhesion of the seminal plug to vaginal walls. When these males were tested at 5-days postlesion, no sperm were found in uterus, and at 20-days post-lesion, the few sperm encountered showed slow progressive motility. None of the females that mated with dorsal lobe-lesioned males became pregnant. However, chemical lesion or ablation of the ventral lobes did not affect ejaculate or fertility. Our results indicate that the dorsal prostatic lobes are indispensable for reproductive success in males, and define parameters of ejaculate with which fertility can be estimated.     

Can paternal effects via seminal fluid contribute to the evolution of polyandry?

Genetic benefits from mating with multiple males are thought to favour the evolution of polyandry. However, recent evidence suggests that non-genetic paternal effects via seminal fluid might contribute to the observed effects of polyandry on offspring performance. Here, we test this hypothesis using the field cricket Teleogryllus oceanicus. Using interference RNA, we first show that at least one seminal fluid protein is essential for embryo survival. We then show that polyandrous females mated to three different males produced embryos with higher pre-hatching viability than did monandrous females mated with the same male three times. Pseudo-polyandrous females that obtained sperm and seminal fluid from a single male and seminal fluid from two additional males had embryos with viabilities intermediate between monandrous and polyandrous females. Our results suggest either that ejaculate mediated paternal effects on embryo viability have both genetic and non-genetic components, or that seminal fluids transferred by castrated males provide only a subset of proteins contained within the normal ejaculate, and are unable to exert their full effect on embryo viability.     

解脲脲原体感染与不孕不育

解脲脲原体是人类泌尿生殖道常见的寄生菌之一,它可以引起男女泌尿生殖道感染,其中最严重的后果认为可导致男女不孕不育。支原体感染是否与男性和女性的不孕不育有关,其致病的主要机制是什么,探讨这些问题对于防治解脲脲原体感染,减少及避免不孕不育疾病的发生有重要意义。本文对近年来关于解脲脲原体感染导致女性不孕和男性不育的机制进行了综合概述。     

Female life span and fertility are increased by the ejaculates of preferred males

In animals with internal fertilization, sperm competition among males can favor the evolution of male ejaculate traits that are detrimental to females. Female mating preferences, in contrast, often favor traits in males that are beneficial to females, yet little is known about the effect of these preferences on the evolution of male ejaculates. A necessary condition for female preferences to affect the evolution of male ejaculate characteristics is that females select mates based on a trait correlated with ejaculate quality. Previous work has shown that females of the variable field cricket, Gryllus lineaticeps, prefer males that produce calling songs containing faster and longer chirps. In this study, we tested the hypothesis that females receive more beneficial ejaculates from preferred males. Females were placed on either a high- or a reduced-nutrition diet then mated twice to a male of known song phenotype. Females received only sperm and seminal fluid from males during these matings. There was no effect of male song phenotype on any fitness component for females on the high-nutrition diet. Reduced-nutrition females mated to males that produced preferred song types, however, lived longer, produced more eggs, produced more fertile eggs, and had a higher proportion of their eggs fertilized than those mated to other males. The life-span benefit was positively associated with male chirp duration, and the reproductive benefits were positively associated with male chirp rate. We explored two possible mechanisms for the life span and reproductive benefits. First, a path analysis suggested that part of the effect of male chirp duration on female life span may have been indirect; females mated to males that produced longer chirps showed delayed oviposition, and females that delayed oviposition lived longer. Males that produce longer chirps may thus transfer fewer or less potent oviposition stimulants to females in their seminal fluid. Second, there was a positive correlation between male chirp rate and the number of sperm transferred to females. The fertility benefit may thus have resulted from females receiving more sperm from males that produce faster chirps. Finally, there was a negative phenotypic correlation between male chirp rate and chirp duration, suggesting that females may have to trade off the life span and reproduction benefits when selecting a mate.     

Alterations des fonctions de reproduction chez les souris males deficientes en recepteur aux estrogenes

In male mice, ER disruption causes infertility due to deficiencies in both gametogenesis and sexual behavior. An age dependent development of testicular dysmorphogenesis results in a progressive loss of germ cells. Circulating gonadotropins and testosterone are not appreciably higher in ERKO males, indicating that testicular androgens can effectively exert feedback on gonadotropins in the absence of aromatization and activation of ER. Infertility in male mice ERKOs does not appear to be a result of compromised androgen hormone action since the morphology of male reproductive tract organs is largely unaffected. Mounting behavior of ERKO males is normal whereas the ability to intromit and ejaculate is severely compromised.     

Copulation, the dynamics of sperm transfer and female refractoriness in the leafhopper Balclutha incisa (Hemiptera: Cicadellidae: Deltocephalinae)

Abstract.  Mature sperm of the leafhopper Balclutha incisa (Matsumara) (Cicadellidae: Auchenorrhyncha: Hemiptera) are stored as a series of sperm bundles within seminal vesicles prior to ejaculation. During transfer, sperm are pumped from the vesicles into the ejaculatory duct to the complex aedeagus. Sperm transfer is marked by a c . 30-fold expansion of the spermatheca to accommodate both sperm and seminal fluid. Sperm number increases exponentially with male age, reaching a maximum of 700 000 after 14 days, while the number of sperm available on days 2–5 is between 70 000 and 100 000. During mating, maximum sperm transfer occurs after 7 min and mating is complete after about 10 min. Ejaculate size, defined by both sperm and associated accessory gland fluid, is influenced by male mating status and the interval since the previous mating. There is a positive correlation between duration of copulation and both ejaculate and the time to subsequent mating. Sperm are more likely to be retained in the testes during mating by males of 2–5 days post-emergence than older males. The number of sperm received by the female can be manipulated experimentally by mating males once (medium ejaculate) or twice (small ejaculate) immediately after their first mating. Females that receive small ejaculates from sperm-depleted males have a far shorter refractory period than females receiving medium to large ejaculates. Both ejaculate size and the time after males have mated influence the female post-mating refractory period as measured by the female's responsiveness to male sexual signalling.     

Sperm competition and the evolution of ejaculate composition

We present a model of sperm competition that incorporates both sperm and nonsperm parts of the ejaculate. Our primary focus is on determining how ejaculate composition and size evolves as a function of the effects of seminal fluid on male reproductive success and as a function of asymmetry in sperm usage by females. The model predicts that different patterns of investment in sperm and seminal products are expected to evolve as a function of the bias in sperm usage by females. It also predicts the evolution of distinct patterns in ejaculate composition depending on the function of seminal fluid. In the discussion, we highlight a number of potential approaches for testing the theory that we develop.     

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.     

Drosophila seminal fluid proteins enter the circulatory system of the mated female fly by crossing the posterior vaginal wall

Seminal fluid proteins from males of many insect species affect the behavior and physiology of their mates. In some cases, these effects result from entry of the proteins into the female's circulatory system. In the fruit fly Drosophila melanogaster, some seminal fluid proteins enter the female's circulatory system after transfer from the male while others remain confined within the reproductive tract. To address where and how seminal fluid proteins enter the hemolymph of the mated female, we compared the kinetics of transfer and localization in mated females of two seminal fluid proteins that enter the hemolymph (Acp26Aa and Acp62F) and one that does not (Acp36DE). We also generated transgenic flies that produce Acp26Aa tagged with Aequorea victoria green fluorescent protein (GFP) to monitor its transfer in vivo. We report that Acps enter the female circulatory system from the posterior vagina immediately after insemination. The ability of Acps to enter the female hemolymph correlates with their ability to cross the intima that lines the posterior vagina. The ventral posterior vagina is structurally unlike other parts of the female reproductive tract in that it lacks muscles. We hypothesize that it has higher permeability thus affording access to the female's circulatory system.