Identification of genes associated with sperm storage capacity in hens at different times after insemination by RNA-seq and Ribo-seq.

BACKGROUND: Sperm storage capacity (SSC) determines the duration of fertility in hens and is an important reproduction trait that cannot be ignored in production. Currently, the genetic mechanism of SSC is still unclear in hens. Therefore, to explore the genetic basis of SSC, we analyzed the uterus-vagina junction (UVJ) of hens with different SSC at different times after insemination by RNA-seq and Ribo-seq. RESULTS: Our results showed that 589, 596, and 527 differentially expressed genes (DEGs), 730, 783, and 324 differentially translated genes (DTGs), and 804, 625, and 467 differential translation efficiency genes (DTEGs) were detected on the 5th, 10th, and 15th days after insemination, respectively. In transcription levels, we found that the differences of SSC at different times after insemination were mainly reflected in the transmission of information between cells, the composition of intercellular adhesion complexes, the regulation of ion channels, the regulation of cellular physiological activities, the composition of cells, and the composition of cell membranes. In translation efficiency (TE) levels, the differences of SSC were mainly related to the physiological and metabolic activities in the cell, the composition of the organelle membrane, the physiological activities of oxidation, cell components, and cell growth processes. According to pathway analysis, SSC was related to neuroactive ligand-receptor interaction, histidine metabolism, and PPAR signaling pathway at the transcriptional level and glutathione metabolism, oxidative phosphorylation, calcium signaling pathway, cell adhesion molecules, galactose metabolism, and Wnt signaling pathway at the TE level. We screened candidate genes affecting SSC at transcriptional levels (COL4A4, MUC6, MCHR2, TACR1, AVPR1A, COL1A1, HK2, RB1, VIPR2, HMGCS2) and TE levels(COL4A4, MUC6, CYCS, NDUFA13, CYTB, RRM2, CAMK4, HRH2, LCT, GCK, GALT). Among them, COL4A4 and MUC6 were the key candidate genes differing in transcription, translation, and translation efficiency. CONCLUSIONS: Our study used the combined analysis of RNA-seq and Ribo-seq for the first time to investigate the SSC and reveal the physiological processes associated with SSC. The key candidate genes affecting SSC were screened, and the theoretical basis was provided for the analysis of the molecular regulation mechanism of SSC.

Sperm replacement in sperm-storage tubules causes last-male sperm precedence in chickens.

1. This study elucidated the last-male sperm precedence (LMSP) mechanism in chickens by examining replacement in storage tubules (SSTs) after multiple artificial inseminations (AI) and the effects of seminal plasma (SP) and male breed on sperm replacement in SSTs.2. Hens were artificially inseminated with fluorescent dye-labelled spermatozoa from White Leghorn (WL) chickens. Secondary AI was conducted 3 d later with sperm labelled with different nuclear fluorescent dye. Percentage of first and second inseminated sperm in SSTs and their logarithmic odds were calculated. The effect of SP on LMSP was examined using (1) Lake's solution-washed sperm before second insemination, and (2) SP injected continuously after first insemination. Effect of breed difference on sperm replacement was investigated using Barred Plymouth Rock (BP) sperm.3. Successive WL-sperm inseminations at three-day intervals caused > 70% stored sperm replacement in SSTs. Although SP removal from sperm from second insemination significantly decreased replacement, its intra-vaginal injection did not affect release. Secondary insemination using BP sperm significantly increased replacement.4. Sperm replacement is a major factor favouring LMSP in domestic chickens. Two fluorescent staining of sperm, and intra-vaginal multiple AI technique have enabled visualisation, differentiation, and quantification of multiple inseminated sperm stored in the SSTs.

Loss of Avian Intromittent Organs as a Sperm Competition Strategy: A Race to Be Last.

AbstractThe loss of the intromittent organ (IO) in the majority of birds remains unexplained. Here, I propose that IO loss results from sperm competition in the context of the unique avian sperm storage system. The first stage of fertilization is the movement of sperm through the vagina from the site of ejaculation to the sperm storage tubules (SSTs) at the uterovaginal junction. In a second stage, sperm are released from the SSTs and move through the uterus to the site of fertilization in the infundibulum. Last-male fertilization precedence can occur if sperm that arrive later at the uterovaginal junction occupy uterus-side SSTs, which then have a head start in the race to the infundibulum with each ovulation. Under such "last-in, first-out" conditions, there is strong sperm competition to arrive later at the SSTs. Consequently, the optimal male strategy would be placement of the ejaculate at the cloacal opening to allow any other sperm in the vagina to reach the SSTs first. Cloacal placement is effectively achieved by loss of the IO. The evolution of altricial development in Neoaves, the largest clade that lacks IOs, created conditions that favor IO loss. Specifically, the smaller clutch sizes and hatching asynchrony of altricial birds increase the intensity of sperm competition for fertilization of early eggs in the laying sequence and thus the selective advantage of later arrival at the SSTs. The rarity of IO loss among all animals suggests that the complex mechanism of avian fertilization creates unique conditions for sperm competition.

Participation of apoptotic markers in the process of maturation and elimination of spermatozoa in the epididymis of the Corynorhinus mexicanus bat.

The Corynorhinus mexicanus bat is characterized by a specific form of reproductive asynchrony between males and females. After mating, some sperm remain in the male's epididymis, the organ where the sperm had matured. It has not yet been determined if apoptotic markers participate in the process of the maturation and/or elimination of these cells, so studying this topic is essential for our understanding of this species. Male bats were collected during three stages: Before mating; during the Mating phase; After mating and the final phase, which we call, Storage. Their epididymides were removed, weighed and measured. Sperm were extracted and the following sperm parameters were evaluated: active caspases, phosphatidylserine externalization, and mitochondrial membrane potential. Sperm from the testes enter the epididymis during Before mating, causing the organ to grow. During Mating phase, spermatozoa present a large amount of active caspases with externalization of phosphatidyl serine, even while still alive. This suggests that these two markers could participate in maturation and elimination, respectively.

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.

Despite genetic isolation in sympatry, post-copulatory reproductive barriers have not evolved between bat- and human-associated common bedbugs (Cimex lectularius L.).

BACKGROUND: The common bedbug Cimex lectularius is a widespread ectoparasite on humans and bats. Two genetically isolated lineages, parasitizing either human (HL) or bat (BL) hosts, have been suggested to differentiate because of their distinct ecology. The distribution range of BL is within that of HL and bedbugs live mostly on synanthropic bat hosts. This sympatric co-occurrence predicts strong reproductive isolation at the post-copulatory level. RESULTS: We tested the post-copulatory barrier in three BL and three HL populations in reciprocal crosses, using a common-garden blood diet that was novel to both lineages. We excluded pre-copulation isolation mechanisms and studied egg-laying rates after a single mating until the depletion of sperm, and the fitness of the resulting offspring. We found a higher sperm storage capability in BL, likely reflecting the different seasonal availability of HL and BL hosts. We also observed a notable variation in sperm function at the population level within lineages and significant differences in fecundity and offspring fitness between lineages. However, no difference in egg numbers or offspring fitness was observed between within- and between-lineage crosses. CONCLUSIONS: Differences in sperm storage or egg-laying rates between HL and BL that we found did not affect reproductive isolation. Neither did the population-specific variation in sperm function. Overall, our results show no post-copulatory reproductive isolation between the lineages. How genetic differentiation in sympatry is maintained in the absence of a post-copulatory barrier between BL and HL remains to be investigated.

Sperm calcium flux and membrane potential hyperpolarization observed in the Mexican big-eared bat Corynorhinus mexicanus.

Mammalian sperm capacitation involves biochemical and physiological changes, such as an increase in intracellular calcium ion concentration ([Ca2+]i), hyperpolarization of the plasma membrane potential and sperm hyperactivation, among others. These changes provide sperm with the ability to fertilize. In the bat Corynorhinus mexicanus, there is an asynchrony between spermatogenesis and sperm storage in the male with the receptivity of the female. For instance, in C. mexicanus, spermatogenesis occurs before the reproductive season. During the reproductive period, sperm are stored in the epididymis for a few months and the testis undergoes a regression, indicating low or almost null sperm production. Therefore, it is unclear whether the elements necessary for sperm fertilization success undergo maturation or preparation during epididymis storage. Here, we characterized pH-sensitive motility hyperactivation and Ca2+ influx in sperm, regulated by alkalinization and progesterone. In addition, by electrophysiological recordings, we registered currents that were stimulated by alkalinization and inhibited by RU1968 (a CatSper-specific inhibitor), strongly suggesting that these currents were evoked via CatSper, a sperm Ca2+-specific channel indispensable for mammalian fertilization. We also found hyperpolarization of the membrane potential, such as in other mammalian species, which increased according to the month of capture, reaching the biggest hyperpolarization during the mating season. In conclusion, our results suggest that C. mexicanus sperm have functional CatSper and undergo a capacitation-like process such as in other mammals, particularly Ca2+ influx and membrane potential hyperpolarization.

Female factors modulate Sex Peptide's association with sperm in Drosophila melanogaster.

BACKGROUND: Male-derived seminal fluid proteins (SFPs) that enter female fruitflies during mating induce a myriad of physiological and behavioral changes, optimizing fertility of the mating pair. Some post-mating changes in female Drosophila melanogaster persist for ~10-14 days. Their long-term persistence is because the seminal protein that induces these particular changes, the Sex Peptide (SP), is retained long term in females by binding to sperm, with gradual release of its active domain from sperm. Several other "long-term response SFPs" (LTR-SFPs) "prime" the binding of SP to sperm. Whether female factors play a role in this process is unknown, though it is important to study both sexes for a comprehensive physiological understanding of SFP/sperm interactions and for consideration in models of sexual conflict. RESULTS: We report here that sperm in male ejaculates bind SP more weakly than sperm that have entered females. Moreover, we show that the amount of SP, and other SFPs, bound to sperm increases with time and transit of individual seminal proteins within the female reproductive tract (FRT). Thus, female contributions are needed for maximal and appropriate binding of SP, and other SFPs, to sperm. Towards understanding the source of female molecular contributions, we ablated spermathecal secretory cells (SSCs) and/or parovaria (female accessory glands), which contribute secretory proteins to the FRT. We found no dramatic change in the initial levels of SP bound to sperm stored in mated females with ablated or defective SSCs and/or parovaria, indicating that female molecules that facilitate the binding of SP to sperm are not uniquely derived from SSCs and parovaria. However, we observed higher levels of SP (and sperm) retention long term in females whose SSCs and parovaria had been ablated, indicating secretions from these female tissues are necessary for the gradual release of Sex Peptide's active region from stored sperm. CONCLUSION: This study reveals that the SP-sperm binding pathway is not entirely male-derived and that female contributions are needed to regulate the levels of SP associated with sperm stored in their storage sites.

Multiple-brooding rockfishes (Sebastes spp.) can utilize stored sperm from individual sires to fertilize consecutive broods.

Viviparous rockfishes (Sebastes spp., family Scorpaenidae) mate and store sperm in the ovaries for several months prior to fertilization, as oocytes develop for the parturition season. Although multiple paternity has been documented in single-brooding rockfishes, paternity in consecutive broods of multiple-brooding species has not been studied. Analyses of multilocus microsatellite genotypes in both residual larvae left in the ovary from a previous parturition and upcoming fertilized broods in the same ovary demonstrated evidence of the same sires in consecutive broods in chilipepper (Sebastes goodei) and speckled (Sebastes ovalis) rockfishes. One S. goodei mother showed evidence of multiple paternity from the same two sires in both consecutive broods. The ability to retain sperm, even after a parturition event, for use in subsequent broods, confers an advantage to ensure fertilization and allows for extension of the parturition season. This life-history strategy provides a bet-hedging advantage in the California Current system, an environmentally dynamic ecosystem where larval survivorship and subsequent recruitment to adult populations can vary temporally by orders of magnitude.

Transcriptome analysis and identification of genes associated with individual fertilisation rate differences in hen infundibulum.

1. Fertilisation rate is closely related to the reproductive performance and economic status of chicken laying breeders. In this study, two flocks (n = 1,029 in population I and n = 358 in population II) in the later laying period were used for investigating the individual differences among population fertilisation rates (FRs).2. The funnel and distal parts of the infundibulum were collected from nine individuals (five with low FR and four with high FR from population II) and RNA-sequencing (RNA-seq) method was used to investigate the transcriptome differences in fertilisation. Differences in fertilisation regulation were investigated by comparing the different parts (funnel and distal) of the infundibulum between the low FR and high FR groups.3. There were notable individual FR differences in both categories. Some individuals had a relative high FR (≥90%) for a long time (>5 days after AI), contrarily, some individuals lost the ability to fertilise eggs in a very short time.4. Differentially expressed genes (DEGs), such as DUSP7, PPP1R3B, FYB, OVA, OVALX and OVALY may be associated with sperm functional regulation, fertilisation and early-stage fertilised ovum development processes. DEGs such as AVBD1, AVBD2, AVBD6, NFATC2 and BANK1 indicated a severe immune response to sperm survival and fertilisation in the oviduct.5. The results suggested that individual differences should be considered in the breeding and reproduction process. The DEGs identified in this study may promote our understanding of different fertilisation regulation in laying breeders.

Male Mediterranean fruit flies prefer warmer temperatures that improve sexual performance.

Females and males have divergent strategies of energy investment, so the thermal preference of each sex in insects may differ because energetic conversion of metabolic reserves is dependent on temperature. We determined the thermal preference of virgin, sexually mature Mediterranean fruit flies, Ceratitis capitata, and found that males preferred a significantly higher temperature (23.8 ± 0.3 °C) than that of females (22.1 ± 0.3 °C). We then tested predictions for the difference in thermal preference related to the energetic demands of reproduction over a range of temperatures. The frequency and duration of calling bouts by male C. capitata were optimal at 26 °C. Mating propensity and latency, and copula duration, were optimal over the range of 22-28 °C. When mating occurred, temperature had little effect on the incidence of sperm storage by females, but there was a notable decline in the number stored at 28 °C. Female lifespan was highest at 18 °C, but lifetime egg production was optimal at 24 °C. These results illustrate temperature-related differences in the reproductive fitness of the sexes in C. capitata, although the optima for male traits align best with their thermal preference. They also support the theoretical prediction that insect thermal preference should be lower than the optimum for fitness.

Female fruit flies cannot protect stored sperm from high temperature damage.

Recently, it has been demonstrated that heat-induced male sterility is likely to shape population persistence as climate change progresses. However, an under-explored possibility is that females may be able to successfully store and preserve sperm at temperatures that sterilise males, which could ameliorate the impact of male infertility on populations. Here, we test whether females from two fruit fly species can protect stored sperm from a high temperature stress. We find that sperm carried by female Drosophila virilis are almost completely sterilised by high temperatures, whereas sperm carried by female Zaprionus indianus show only slightly reduced fertility. Heat-shocked D. virilis females can recover fertility when allowed to remate, suggesting that the delivered heat-shock is damaging stored sperm and not directly damaging females in this species. The temperatures required to reduce fertility of mated females are substantially lower than the temperatures required to damage mature sperm in males, suggesting that females are worse than males at protecting mature sperm. This suggests that female sperm storage is unlikely to ameliorate the impacts of high temperature fertility losses in males, and instead exacerbates fertility costs of high temperatures, representing an important determinant of population persistence during climate change.

Effects of reliance on stored sperm on reproduction in the sailfin molly Poecilia latipinna.

The impacts of relying on stored sperm were evaluated in the sailfin molly, Poecilia latipinna. Females reliant on stored sperm had fewer offspring compared to remated females, but offspring size and short-term growth rate did not differ. Thus, females may use stored sperm in cases such as previous mating with a preferred male, lack of access to mating opportunities during a reproductive cycle, or to maximize egg fertilization. Females do not compensate for producing fewer offspring however, by allocating more resources to offspring relative to their size or initial growth.

Competitive Sperm-Marked Beetles for Monitoring Approaches in Genetic Biocontrol and Studies in Reproductive Biology.

Sperm marking provides a key tool for reproductive biology studies, but it also represents a valuable monitoring tool for genetic pest control strategies such as the sterile insect technique. Sperm-marked lines can be generated by introducing transgenes that mediate the expression of fluorescent proteins during spermatogenesis. The homozygous lines established by transgenesis approaches are going through a genetic bottleneck that can lead to reduced fitness. Transgenic SIT approaches have mostly focused on Dipteran and Lepidopteran pests so far. With this study, we provide sperm-marked lines for the Coleopteran pest model organism, the red flour beetle Tribolium castaneum, based on the ß2-tubulin promoter/enhancer driving red (DsRed) or green (EGFP) fluorescence. The obtained lines are reasonably competitive and were thus used for our studies on reproductive biology, confirming the phenomenon of 'last-male sperm precedence' and that the spermathecae are deployed for long-term sperm storage, enabling the use of sperm from first mating events even after secondary mating events for a long period of time. The homozygosity and competitiveness of the lines will enable future studies to analyze the controlled process of sperm movement into the long-term storage organ as part of a post-mating cryptic female choice mechanism of this extremely promiscuous species.

Transcriptome Analysis Reveals Key Gene Expression Changes in Blue Catfish Sperm in Response to Cryopreservation.

The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality.

In vivo and in vitro aging of common carp Cyprinus carpio sperm after multiple hormonal application and stripping of males.

The present study was designed to evaluate sperm phenotypic variables during in vivo and in vitro storage following multiple sperm stripping in common carp (Cyprinus carpio L.). Each male was injected 3 times with carp pituitary 3 days apart. Sperm was stored in vivo in the body cavity for 0.5 days (Fresh sperm) and 3 days (Old sperm) after hormonal stimulation. Then sperm was collected and diluted with a carp extender at a ratio of 1:1, and stored in vitro on ice for 0, 3, and 6 days. The phenotypic parameters, including the number of total motile spermatozoa, number of fast motile spermatozoa, number of motile spermatozoa, percentage of fast motile spermatozoa, and percentage of spermatozoa motility were the major components of principal component analysis (PCA). In general, Fresh sperm from the first stripping showed slightly better quality than Old sperm from the second and third stripping, especially in the phenotypic parameters of a number of total spermatozoa and a number of total motile spermatozoa (P < 0.05). The highest kinetic and quantitative spermatozoa variables were obtained in Fresh and Old sperm just after sperm collection (0-day storage in vitro), and then they were decreased during the period of in vitro storage up to 6 days (P < 0.05). However, the fertilization, hatching, and malformation rates from Fresh sperm were similar compared with the Old sperm. Sperm could be stripped 0.5 days post hormonal treatment and stored in vitro up to 6 days with good fertilization performance (fertility, hatching, and malformation rates were 92.5%, 91.5%, and 1.3%, respectively). Therefore, our results suggested that multiple hormonal treatments with multiple stripping could be used in artificial reproduction in common carp.

Real-time PCR-based Y-specific sperm quantification assay in Queensland fruit fly: Insights to patterns of sperm storage.

Studies of reproductive biology in insects often require quantification of sperm production, transfer or storage. Here, we develop a quantitative real-time PCR-based assay using a Y-specific marker for quantification of sperm from spermathecae of female Queensland fruit fly ('Q-fly'), overcoming constraints typical of traditional sperm quantification methods. The assay enables accurate and reliable quantification of as few as 50 sperms and provides a means to analyse large numbers of samples with flexible timing. The real-time PCR method enables revised understanding of how many sperms are stored by female Q-flies, the distribution of storage between the two spermathecae and the relationship between copula duration and sperm storage. Real-time PCR assays based on Y-specific markers provide an effective solution for sperm quantification in tephritid flies, as well as in other insects and potentially other animals with sperm storage organs.

Female sperm storage in the bonnethead Sphyrna tiburo oviducal gland: Immunolocalization of steroid hormone receptors in sperm storage tubules.

Female sperm storage (FSS) has been demonstrated to occur in representatives from all major vertebrate groups and has been hypothesized to have several possible adaptive benefits that may maximize reproductive success of its practitioners. However, while the range of taxa that exhibit FSS and its possible evolutionary benefits have received significant attention in past years, the physiological mechanisms by which FSS occurs in vertebrates have only recently been explored. In this study, we examined the potential role of gonadal steroid hormones in regulating FSS in the bonnethead Sphyrna tiburo, a small hammerhead species in which females have been shown to be capable of storing male spermatozoa for up to 6 - 7 months following copulation. Like past studies on this species, we observed associations between plasma concentrations of the gonadal steroids 17ß-estradiol, testosterone, and progesterone with FSS in female bonnetheads, suggesting roles for these hormones in regulating this process. Using immunohistochemistry, we also observed presence of androgen receptor, estrogen receptor alpha (ERα), and progesterone receptor in epithelial cells of sperm storage tubules in the bonnethead oviducal gland, as well as occurrence of ERα in stored spermatozoa, specifically during the sperm storage period. These results suggest that E2, T, and P4 may regulate certain aspects of FSS in bonnethead indirectly through actions on the female reproductive tract, whereas E2 may also have direct effects on sperm function. This is the first study on the regulation of FSS in sharks and has formed a basis for future work geared towards improving our understanding of this process in chondrichthyans.

Protein-Level Interactions as Mediators of Sexual Conflict in Ants.

All social insects with obligate reproductive division of labor evolved from strictly monogamous ancestors, but multiple queen-mating (polyandry) arose de novo, in several evolutionarily derived lineages. Polyandrous ant queens are inseminated soon after hatching and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their lifetime reproductive success is ultimately sperm limited. In the leaf-cutting ant Atta colombica,, the survival of newly inseminated sperm is known to be compromised by seminal fluid of rival males and to be protected by secretions of the queen sperm storage organ (spermatheca). Here we investigate the main protein-level interactions that appear to mediate sperm competition dynamics and sperm preservation. We conducted an artificial insemination experiment and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid followed by a mass spectrometry analysis of both secretions that allowed us to identify the sex-specific origins of the proteins that had changed in abundance. We found that spermathecal fluid targets only seven (2%) of the identified seminal fluid proteins for degradation, including two proteolytic serine proteases, a SERPIN inhibitor, and a semen-liquefying acid phosphatase. In vitro, and in vivo, experiments provided further confirmation that these proteins are key molecules mediating sexual conflict over sperm competition and viability preservation during sperm storage. In vitro, exposure to spermathecal fluid reduced the capacity of seminal fluid to compromise survival of rival sperm in a matter of hours and biochemical inhibition of these seminal fluid proteins largely eliminated that adverse effect. Our findings indicate that A. colombica, queens are in control of sperm competition and sperm storage, a capacity that has not been documented in other animals but is predicted to have independently evolved in other polyandrous social insects.

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio.

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.