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Sperm function is suggested to evolve by sexual selection but is also reduced by microbial damage. Here, we provide experimental evidence that male fertility can adapt to microbes. We found that in vivo, male fertility was reduced by one-fifth if sperm encountered microbes in the females that they had not previously been exposed to, compared to sperm from males that coevolved with these microbes. The female immune system activation reduced male fertility by an additional 13 percentage points. For noncoevolved males, fertility was larger if microbes were injected into females after they had stored away the sperm, indicating microbial protection as a previously unrecognized benefit of female sperm storage. Both medical and evolutionary research on reproductive health and fertility will benefit from considering our findings that the impact of microbes on sperm depends on their joint evolutionary history. Our results may assist in reconciling contradictory results of sexually transmitted disease effects on sperm and bring empirical realism to a recently proposed role of locally adapted reproductive microbiomes to speciation. [ABSTRACT FROM AUTHOR]

Theoretically, males should increase their ejaculate expenditure when the probability of sperm competition occurring (or risk) is high but decrease ejaculate expenditure as the number of competing ejaculates (or intensity) increases. Here we examine whether male decorated crickets (Gryllodes sigillatus) use cuticular hydrocarbons (CHCs) transferred to females by rival males at mating to assess the risk and intensity of sperm competition and adjust their ejaculate accordingly. Unmated females and those perfumed with CHCs extracted from one, three, or five males could be distinguished chemically, providing a reliable cue of the risk and intensity of sperm competition. In agreement with theory, males mating with these females increased sperm number with the risk of sperm competition and decreased sperm number with the intensity of sperm competition. Similarly, as the risk of sperm competition increased, males produced a larger and more attractive spermatophylax (an important non-sperm component of the ejaculate) but these traits did not vary with the intensity of sperm competition. Our results therefore demonstrate that both sperm and non-sperm components of the male ejaculate respond to the risk and intensity of sperm competition in different ways and that CHCs provide males with an important cue to strategically tailor their ejaculate. [ABSTRACT FROM AUTHOR]

Copulations are very brief in many species, sometimes taking only seconds, but in other species, they can be quite prolonged. Potential explanations for prolonged copulations include time requirements for the transfer of sperm and/or other ejaculate substances. Ejaculate substances could function to regulate female receptivity to subsequent matings, provide nutritional nuptial gifts, or hasten egg oviposition at a potential survival cost to the female. We investigated prolonged copulation in a member of the Enchenopa binotata complex of treehoppers (Hemiptera: Membracidae), in which females rarely remate and copulation can last several hours. We assigned females to treatments in which we interrupted copulation at different times. We also included a control where copulation was not interrupted. We found that females that experienced shorter copulations were more likely to be subsequently receptive to an attractive male. We also found that few females produced offspring when they engaged in short copulations compared to those with longer copulations. We did not find any differences in female survival. Our results support the sperm transfer and receptivity regulation hypotheses. We discuss potential reasons for why these processes should take so long in a species with low female remating. Graphical Abstract [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can illegal discharge of toxic waste into the environment induce a new condition of morpho-epigenetic pathozoospermia in normozoospermic young men? SUMMARY ANSWER Toxic environmental contaminants promote the onset of a new pathozoospermic condition in young normozoospermic men, consisting of morpho-functional defects and a sperm increase of low-quality circular RNA (circRNA) cargo, tightly linked to contaminant bioaccumulation in seminal plasma. WHAT IS KNOWN ALREADY Epidemiological findings have reported several reproductive anomalies depending on exposure to contaminants discharged into the environment, such as germ cell apoptosis, steroidogenesis defects, oxidative stress induction, blood–testis barrier dysfunctions, and poor sperm quality onset. In this scenario, a vast geographical area located in Campania, Italy, called the 'Land of Fires', has been associated with an excessive illegal discharge of toxic waste into the environment, negatively impacting human health, including male reproductive functions. STUDY DESIGN, SIZE, DURATION Semen samples were obtained from healthy normozoospermic men divided into two experimental groups, consisting of men living in the 'Land of Fires' (LF; n = 80) or not (CTRL; n = 80), with age ranging from 25 to 40 years. The study was carried out following World Health Organization guidelines. PARTICIPANTS/MATERIALS, SETTING, METHODS Quality parameters of semen from CTRL- and LF-normozoospermic men were evaluated by computer-assisted semen analysis; high-quality spermatozoa from CTRL and LF groups (n = 80 for each experimental group) were obtained using a 80–40% discontinuous centrifugation gradient. Seminal plasma was collected following centrifugation and used for the dosage of chemical elements, dioxins and steroid hormones by liquid chromatography with tandem mass spectrometry. Sperm morpho-functional investigations (cellular morphology, acrosome maturation, IZUMO1 fertility marker analysis, plasma membrane lipid state, oxidative stress) were assessed on the purified high-quality spermatozoa fraction by immunochemistry/immunofluorescence and western blot analyses. Sperm circRNA cargo was evaluated by quantitative RT-PCR, and the physical interaction among circRNAs and fused in sarcoma (FUS) protein was detected using an RNA-binding protein immunoprecipitation assay. Protein immunoprecipitation experiments were carried out to demonstrate FUS/p-300 protein interaction in sperm cells. Lastly, in vitro lead (Pb) treatment of high-quality spermatozoa collected from normozoospermic controls was used to investigate a correlation between Pb accumulation and onset of the morpho-epigenetic pathozoospermic phenotype. MAIN RESULTS AND THE ROLE OF CHANCE Several morphological defects were identified in LF-spermatozoa, including: a significant increase (P  < 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P  < 0.01) of peanut agglutinin and IZUMO1 null signal cells. In agreement with these data, abnormal steroid hormone levels in LF seminal plasma suggest a premature acrosome reaction onset in LF-spermatozoa. The abnormal immunofluorescence signals of plasma membrane cholesterol complexes/lipid rafts organization (Filipin III and Flotillin-1) and of oxidative stress markers [3-nitrotyrosine and 3-nitrotyrosine and 4-hydroxy-2-nonenal] observed in LF-spermatozoa and associated with a sperm motility reduction (P  < 0.01), demonstrated an affected membrane fluidity, potentially impacting sperm motility. Bioaccumulation of heavy metals and dioxins occurring in LF seminal plasma and a direct correlation between Pb and deregulated circRNAs related to high- and low-sperm quality was also revealed. In molecular terms, we demonstrated that Pb bioaccumulation promoted FUS hyperacetylation via physical interaction with p-300 and, in turn, its shuttling from sperm head to tail, significantly enhancing (P  < 0.01 versus CTRL) the endogenous backsplicing of sperm low-quality circRNAs in LF-spermatozoa. LIMITATIONS, REASONS FOR CAUTION Participants were interviewed to better understand their area of origin, their eating habits as well as their lifestyles, however any information incorrectly communicated or voluntarily omitted that could potentially compromise experimental group determination cannot be excluded. A possible association between seminal Pb content and other heavy metals in modulating sperm quality should be explored further. Future investigations will be performed in order to identify potential synergistic or anti-synergistic effects of heavy metals on male reproduction. WIDER IMPLICATIONS OF THE FINDINGS Our study provides new findings regarding the effects of environmental contaminants on male reproduction, highlighting how a sperm phenotype classified as normozoospermic may potentially not match with a healthy morpho-functional and epigenetic one. Overall, our results improve the knowledge to allow a proper assessment of sperm quality through circRNAs as biomarkers to select spermatozoa with high morpho-epigenetic quality to use for ART. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by 'Convenzione Azienda Sanitaria Locale (ASL) Caserta, Regione Campania' (ASL CE Prot. N. 1217885/DIR. GE). The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Sperm competition is a potent mechanism of postcopulatory sexual selection that has been found to shape reproductive morphologies and behaviours in promiscuous animals. Especially sperm size has been argued to evolve in response to sperm competition through its effect on sperm longevity, sperm motility, the ability to displace competing sperm, and ultimately fertilization success. Additionally, sperm size has been observed to co-evolve with female reproductive morphology. Theoretical work predicts that sperm competition may select for longer sperm but may also favour shorter sperm if sperm size trades-off with number. In this study, we studied the relationship between sperm size and postmating success in the free-living flatworm, Macrostomum lignano. Specifically, we used inbred isolines of M. lignano that varied in sperm size to investigate how sperm size translated into the ability of worms to transfer and deposit sperm in a mating partner. Our results revealed a hump-shaped relationship with individuals producing sperm of intermediate size having the highest sperm competitiveness. This finding broadens our understanding of the evolution of sperm morphology by providing empirical support for stabilizing selection on sperm size under sperm competition. Graphical Abstract [ABSTRACT FROM AUTHOR]

Males often strategically adjust the number of available sperm based on the social context (i.e. sperm priming response), but it remains unclear how environmental and genetic factors shape this adjustment. In freshwater ecosystems, high ambient temperatures often lead to isolated pools of hotter water in which inbreeding occurs. Higher water temperatures and inbreeding can impair fish development, potentially disrupting sperm production. We used guppies (Poecilia reticulata) to investigate how developmental temperature (26 °C, 30 °C) and male inbreeding status (inbred, outbred) influence their sperm priming response. We also tested if sperm priming was affected by whether the female was a relative (sister) and whether she was inbred or outbred. There was no effect of rearing temperature; male inbreeding status alone determined the number of available sperm in response to female presence, her inbreeding status, and her relatedness. Inbred males produced significantly more sperm in the presence of an unrelated, outbred female than when no female was present. Conversely, outbred males did not alter the number of sperm available in response to female presence or relatedness. Moreover, inbred males produced marginally more sperm when exposed to an unrelated female that was outbred rather than inbred, but there was no difference when exposed to an inbred female that was unrelated versus related. Together, a sperm priming response was only observed in inbred males when exposed to an outbred female. Outbred females in our study were larger than inbred females, suggesting that inbred males strategically allocated ejaculate resources toward females in better condition. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does the use of frozen sperm affect live birth rate (LBR) and cumulative LBR (CLBR) compared to fresh sperm samples in oocyte donation ICSI cycles? SUMMARY ANSWER Although there were slight decreases in pregnancy rates (PRs) and LBR, as well as CLBR per embryo replaced and per embryo transfer (ET), when frozen sperm samples were used compared to fresh ejaculates, their clinical impact was limited. WHAT IS KNOWN ALREADY Sperm cryopreservation is part of the daily routine in reproduction clinics worldwide because of its many advantages in cycle planning. Nonetheless, there is a lack of agreement in terms of its impact on the outcomes of ICSI cycles. Previous studies showed conflicting conclusions and focused on different populations, which makes reaching consensus on the impact of sperm freezing-thawing complicated. Moreover, classical parameters are used to assess cycle success: pregnancy, live birth and miscarriage rates per ET. This study reports those measurements plus CLBR, which more accurately reflects the impact of the technique on the likelihood of achieving a newborn. STUDY DESIGN, SIZE, DURATION A retrospective multicenter observational cohort study, including data from 37 041 couples and 44 423 ICSI procedures from January 2008 to June 2022, was carried out. The group using frozen sperm included 23 852 transferred embryos and 108 661 inseminated oocytes, whereas the fresh sample group comprised 73 953 embryos replaced and 381 509 injected oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS Outcomes measured per first ET and per ET were compared between groups using Fisher's exact test and Chi-squared test, as appropriate. Binary-logistics regression models were used to adjust the analyses according to clinically relevant co-variables. Kaplan–Meier curves plotted the CLBR per oocyte inseminated, per embryo replaced and per ET, and compared between groups using the Mantel–Cox test. Cox regressions were employed for the multivariate analyses of CLBR. MAIN RESULTS AND THE ROLE OF CHANCE The frozen sperm group showed a slightly lower biochemical (3.55% and 2.56%), clinical (3.68% and 3.54%) and ongoing (3.63% and 3.15%) PR compared to the cycles using fresh sperm, respectively, both per first ET and per ET. LBR was 4.57% lower per first ET and 3.95% lower per ET in the frozen sperm group than the fresh sperm group. There was also a subtle increase of 2.66% in biochemical miscarriage rate per ET when using frozen versus fresh sperm. All these differences remained statistically significant after the multivariate analysis (adjusted P  ≤ 0.001). There were statistically significant differences in CLBR per embryo replaced and per ET but not per oocyte used (adjusted P  = 0.071). Despite the statistical significance of the differences between the groups, those using frozen sperm required only 0.54 more oocytes injected, 0.45 more embryos transferred and 0.41 more ET procedures, on average, to achieve a live birth compared to the fresh samples. LIMITATIONS, REASONS FOR CAUTION The retrospective nature of the study subjects the data to biases or potential errors during annotation on the source clinical and cycle records. This study uses multivariate analyses to control biases as much as possible. Using the oocyte donation model also contributes to reducing heterogeneity in the oocyte quality factor. WIDER IMPLICATIONS OF THE FINDINGS The large sample sizes included in this study allowed for the detection of small changes in cycle success rates between groups. Although statistically significant, the decrease in PRs, LBR, and CLBR when using frozen sperm can be clinically overlooked in favor of the many benefits of sperm cryopreservation. STUDY FUNDING/COMPETING INTEREST(S) None declared. TRIAL REGISTRATION NUMBER Not applicable [ABSTRACT FROM AUTHOR]

STUDY QUESTION Are sperm phospholipase C zeta (PLCζ) profiles linked to the quality of embryogenesis and pregnancy? SUMMARY ANSWER Sperm PLCζ levels in both mouse and humans correlate with measures of ideal embryogenesis whereby minimal levels seem to be required to result in successful pregnancy. WHAT IS KNOWN ALREADY While causative factors underlying male infertility are multivariable, cases are increasingly associated with the efficacy of oocyte activation, which in mammals occurs in response to specific profiles of calcium (Ca2+) oscillations driven by sperm-specific PLCζ. Although sperm PLCζ abrogation is extensively linked with human male infertility where oocyte activation is deficient, less is clear as to whether sperm PLCζ levels or localization underlies cases of defective embryogenesis and failed pregnancy following fertility treatment. STUDY DESIGN, SIZE, DURATION A cohort of 54 couples undergoing fertility treatment were recruited at the assisted reproductive technology laboratory at the King Faisal Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia. The recruitment criteria for males was a minimum sperm concentration of 5×106 sperm/ml, while all female patients had to have at least five oocytes. Sperm PLCζ analysis was performed in research laboratories, while semen assessments were performed, and time-lapse morphokinetic data were obtained, in the fertility clinic as part of routine treatment. The CRISPR/Cas9 system was concurrently used to induce indels and single-nucleotide mutations within the Plcζ gene to generate strains of Plcζ mutant mice. Sperm PLCζ was evaluated using immunofluorescence and immunoblotting with an antibody of confirmed consistent specificity against PLCζ. PARTICIPANTS/MATERIALS, SETTING, METHODS We evaluated PLCζ profiles in sperm samples from 54 human couples undergoing fertility treatment in the context of time-lapse morphokinetic analysis of resultant embryos, correlating such profiles to pregnancy status. Concurrently, we generated two strains of mutant Plcζ mice using CRISPR/Cas9, and performed IVF with wild type (WT) oocytes and using WT or mutant Plcζ sperm to generate embryos. We also assessed PLCζ status in WT and mutant mice sperm in the context of time-lapse morphokinetic analysis and breeding outcomes. MAIN RESULTS AND THE ROLE OF CHANCE A significant (P  ≤   0.05) positive relationship was observed between both PLCζ relative fluorescence and relative density with the times taken for both the second cell division (CC2) (r  = 0.26 and r  = 0.43, respectively) and the third cell division (S2) (r  = 0.26). Examination of localization patterns also indicated significant correlations between the presence or absence of sperm PLCζ and CC2 (r  = 0.27 and r  = −0.27, respectively; P  ≤   0.025). Human sperm PLCζ levels were at their highest in the ideal times of CC2 (8–12 h) compared to time ranges outside the ideal timeframe (<8 and >12 h) where levels of human sperm PLCζ were lower. Following assignment of PLCζ level thresholds, quantification revealed a significantly higher (P  ≤   0.05) rate of successful pregnancy in values larger than the assigned cut-off for both relative fluorescence (19% vs 40%, respectively) and relative density (8% vs 54%, respectively). Immunoblotting indicated a single band for PLCζ at 74 kDa in sperm from WT mice, while a single band was also observed in sperm from heterozygous of Plcζ mutant mouse sperm, but at a diminished intensity. Immunofluorescent analysis indicated the previously reported (Kashir et al. , 2021) fluorescence patterns in WT sperm, while sperm from Plcζ mutant mice exhibited a significantly diminished and dispersed pattern at the acrosomal region of the sperm head. Breeding experiments indicated a significantly reduced litter size of mutant Plcζ male mice compared to WT mice, while IVF-generated embryos using sperm from mutant Plcζ mice exhibited high rates of polyspermy, and resulted in significantly reduced numbers of these embryos reaching developmental milestones. LIMITATIONS, REASONS FOR CAUTION The human population examined was relatively small, and should be expanded to examine a larger multi-centre cohort. Infertility conditions are often multivariable, and it was not possible to evaluate all these in human patients. However, our mutant Plcζ mouse experiments do suggest that PLCζ plays a significant role in early embryo development. WIDER IMPLICATIONS OF THE FINDINGS We found that minimal levels of PLCζ within a specific range were required for optimal early embryogenesis, correlating with increased pregnancy. Levels of sperm PLCζ below specific thresholds were associated with ineffective embryogenesis and lower pregnancy rates, despite eliciting successful fertilization in both mice and humans. To our knowledge, this represents the first time that PLCζ levels in sperm have been correlated to prognostic measures of embryogenic efficacy and pregnancy rates in humans. Our data suggest for the first time that the clinical utilization of PLCζ may stand to benefit not just a specific population of male infertility where oocyte activation is completely deficient (wherein PLCζ is completely defective/abrogated), but also perhaps the larger population of couples seeking fertility treatment. STUDY FUNDING/COMPETING INTEREST(S) J.K. is supported by a faculty start up grant awarded by Khalifa University (FSU-2023-015). This study was also supported by a Healthcare Research Fellowship Award (HF-14-16) from Health and Care Research Wales (HCRW) to J.K. alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST) for J.K. and A.M.A. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Sexual assault sample processing, despite recent funding and research efforts, remains time-consuming, labourious, and inefficient. These limitations, combined with the prevalence of sexual assaults, have prompted the need to develop a cheaper, quicker, and more robust method for separating victim and perpetrator contributions within sexual assault evidence so that analysts can keep pace with submissions and cases can be resolved in a timely manner. Thus, this study examined the use of a combined enzymatic and alkaline approach for differential cell lysis—with the goal of developing a quick, cheap, and more efficient DNA isolation method. Quantification results for this assay revealed that (72.0 ± 18.3)%, (15.8 ± 14.2)%, and (29.5 ±  23.7)% of total DNA were retained in sperm fractions for neat semen, neat vaginal, and semen–vaginal mixture eluates, respectively. Short tandem repeat (STR) analysis of mixture samples processed with this technique exhibited sperm fraction DNA profiles with mean male-to-female ratios of 1.74:1, which was a 3.01 ± 2.30-fold improvement in male-to-female ratios and led to the recovery of 5.90 ± 7.80 unshared male contributor alleles in sperm fractions that were otherwise undetected in unseparated controls. Overall, this study presented a modified differential lysis approach using prep GEM™ and sodium hydroxide treatments that can accomplish cell elution and fractional lysis within 25 min. Future studies should investigate alternative "non-sperm" cell lysis methods to enhance lysis efficiency and minimize the potential for inhibition, as well as the optimization and automation of this technique. Key points Traditional sexual assault sample processing methods are time-consuming and inefficient. This modified differential lysis method produces lysates with sufficient DNA yield and quality. A combined technique using enzymatic and alkaline lysis can accomplish fractional separation. Lysis with prep GEM and NaOH absent purification is compatible with downstream processes. [ABSTRACT FROM AUTHOR]

Auanema freiburgense is a nematode with males, females, and selfing hermaphrodites. When XO males mate with XX females, they typically produce a low proportion of XO offspring because they eliminate nullo-X spermatids. This process ensures that most sperm carry an X chromosome, increasing the likelihood of X chromosome transmission compared to random segregation. This occurs because of an unequal distribution of essential cellular organelles during sperm formation, likely dependent on the X chromosome. Some sperm components are selectively segregated into the X chromosome's daughter cell, while others are discarded with the nullo-X daughter cell. Intriguingly, the interbreeding of 2 A. freiburgense strains results in hybrid males capable of producing viable nullo-X sperm. Consequently, when these hybrid males mate with females, they yield a high percentage of male offspring. To uncover the genetic basis of nullo-spermatid elimination and X chromosome drive, we generated a genome assembly for A. freiburgense and genotyped the intercrossed lines. This analysis identified a quantitative trait locus spanning several X chromosome genes linked to the non-Mendelian inheritance patterns observed in A. freiburgense. This finding provides valuable clues to the underlying factors involved in asymmetric organelle partitioning during male meiotic division and thus non-Mendelian transmission of the X chromosome and sex ratios. [ABSTRACT FROM AUTHOR]

De novo evolved genes emerge from random parts of noncoding sequences and have, therefore, no homologs from which a function could be inferred. While expression analysis and knockout experiments can provide insights into the function, they do not directly test whether the gene is beneficial for its carrier. Here, we have used a seminatural environment experiment to test the fitness of the previously identified de novo evolved mouse gene Pldi , which has been implicated to have a role in sperm differentiation. We used a knockout mouse strain for this gene and competed it against its parental wildtype strain for several generations of free reproduction. We found that the knockout (ko) allele frequency decreased consistently across three replicates of the experiment. Using an approximate Bayesian computation framework that simulated the data under a demographic scenario mimicking the experiment's demography, we could estimate a selection coefficient ranging between 0.21 and 0.61 for the wildtype allele compared to the ko allele in males, under various models. This implies a relatively strong selective advantage, which would fix the new gene in less than hundred generations after its emergence. [ABSTRACT FROM AUTHOR]

Objectives: Oxidative stress and disruption of energy metabolism in the reproductive system, especially sperm, play a significant role in diabetes-related infertility. Zataria multiflora Boissis (ZMB), a medicinal plant containing various bioactive compounds, may have efficacy in treating metabolic diseases and reproductive disorders. Therefore, the aim of this study was to investigate the effects of different doses of ZMB extract on diabetes-induced reproductive dysfunction by assessing oxidative damage and the gene expression of insulin receptor substrate (IRS) and pyruvate kinase (PK) in male rats' sperm. Methods: Sixty adult male Wistar rats were randomly divided into six groups; control (C), diabetes (D), and diabetic animals treated with glibenclamide (G, 50 mg/kg) and thyme extract (T100, T200, and T400). Diabetes was induced by intraperitoneal injection of Streptozotocin (STZ) (50 mg/kg). Insulin, glucose, oxidative and pro-inflammatory markers in the serum, and gene expressions of IRS, and PK were measured in the stored sperms in the epididymis. Changes in the process of spermatogenesis were assessed through the histological evaluation of the testis. Gas chromatography–mass spectrometry (GC–MS) was used to analyze the quantity and quality of thyme extract. Key findings: The study results indicated that body weight, food intake, and sperm parameters significantly improved in a dose-dependent manner in the T200 group compared to the other groups. Additionally, in the same group, pro-inflammatory biomarkers, DNA fragmentation, and MDA levels decreased, while the levels of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzymes in the stored epididymal sperm significantly improved compared to the other groups. The expression of IRS and PK, along with the mean counts of spermatogenesis cell lines (especially Sertoli cells), significantly increased in the T200 group. Summary: In conclusion, thymol appears to alleviate diabetes-induced reproductive dysfunction by inhibiting oxidative damage, improving the metabolic state, and upregulating the expression of IRS and PK genes in the sperm of male rats. Graphical Abstract [ABSTRACT FROM AUTHOR]

STUDY QUESTION Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? SUMMARY ANSWER The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. WHAT IS KNOWN ALREADY To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. STUDY DESIGN, SIZE, DURATION The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023–2024. PARTICIPANTS/MATERIALS, SETTING, METHODS Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. MAIN RESULTS AND THE ROLE OF CHANCE In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P  < 0.05). LIMITATION, REASONS FOR CAUTION The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. WIDER IMPLICATIONS OF THE FINDINGS Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. STUDY FUNDING/COMPETING INTEREST(S) The study is funded by the Iran University of Medical Sciences and no competing interest exists. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility? SUMMARY ANSWER A 110-kb microdeletion of the X chromosome, only including the ACTRT1 gene, was identified as responsible for infertility in two Chinese males with sperm showing acrosomal ultrastructural defects and fertilization failure. WHAT IS KNOWN ALREADY The actin-related proteins (e.g. ACTRT1, ACTRT2, ACTL7A, and ACTL9) interact with each other to form a multimeric complex in the subacrosomal region of spermatids, which is crucial for the acrosome-nucleus junction. Actrt1 -knockout (KO) mice are severely subfertile owing to malformed sperm heads with detached acrosomes and partial fertilization failure. There are currently no reports on the association between ACTRT1 deletion and male infertility in humans. STUDY DESIGN, SIZE, DURATION We recruited a cohort of 120 infertile males with sperm head deformations at a large tertiary hospital from August 2019 to August 2023. Genomic DNA extracted from the affected individuals underwent whole exome sequencing (WES), and in silico analyses were performed to identify genetic variants. Morphological analysis, functional assays, and ART were performed in 2022 and 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS The ACTRT1 deficiency was identified by WES and confirmed by whole genome sequencing, PCR, and quantitative PCR. Genomic DNA of all family members was collected to define the hereditary mode. Papanicolaou staining and electronic microscopy were performed to reveal sperm morphological changes. Western blotting and immunostaining were performed to explore the pathological mechanism of ACTRT1 deficiency. ICSI combined with artificial oocyte activation (AOA) was applied for one proband. MAIN RESULTS AND THE ROLE OF CHANCE We identified a whole-gene deletion variant of ACTRT1 in two infertile males, which was inherited from their mothers, respectively. The probands exhibited sperm head deformations owing to acrosomal detachment, which is consistent with our previous observations on Actrt1 -KO mice. Decreased expression and ectopic distribution of ACTL7A and phospholipase C zeta were observed in sperm samples from the probands. ICSI combined with AOA effectively solved the fertilization problem in Actrt1 -KO mice and in one of the two probands. LIMITATIONS, REASONS FOR CAUTION Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility. WIDER IMPLICATIONS OF THE FINDINGS Our results reveal a gene–disease relation between the ACTRT1 deletion described here and human male infertility owing to acrosomal detachment and fertilization failure. This report also describes a good reproductive outcome of ART with ICSI-AOA for a proband. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM008 and 2023MSXM054). There are no competing interests to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N , N -dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3′-dipropylthiadicarbocyanine iodide and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. WHAT IS KNOWN ALREADY EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. STUDY DESIGN, SIZE, DURATION EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. MAIN RESULTS AND THE ROLE OF CHANCE EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

Hermaphrodites exhibit a wide variety of reproductive strategies, yet empirical studies lag behind theories about their evolution. Planorbella trivolvis , a simultaneous hermaphrodite capable of reciprocal copulation and rare self-fertilization, provides a unique model system for studying reproduction. This study assessed sex roles during mating, demonstrating that mating is primarily reciprocal, and investigated the relationship between physical coupling and reproductive output for single copulation events. Snails were isolated before sexual maturity and placed in 10-l aquaria in size-matched groups. Upon copulation, pairs were removed to small cups until they separated; they were then kept isolated and eggs counted until egg production ceased. The frequency of reciprocal copulation (both members of a pair produced egg masses following copulation) was 95% (35/37 productive matings), higher than previously reported. The duration of egg production ranged from 19 to 197 days (mean ± SE = 110.13 ± 6.3 days) with a sharp drop in production of egg masses and eggs per mass occurring between 15 and 20 weeks postmating, consistent with previous estimates of sperm storage. There was no relationship between mating duration and total egg production (a biomarker of sperm availability) or the duration of egg production (a marker of sperm longevity). Further investigation is needed to directly estimate sperm transfer and tease apart the duration of reciprocal physical contact from gamete and accessory protein transfer. [ABSTRACT FROM AUTHOR]

PRDM9-mediated reproductive isolation was first described in the progeny of Mus musculus musculus (MUS) PWD/Ph and Mus musculus domesticus (DOM) C57BL/6J inbred strains. These male F1 hybrids fail to complete chromosome synapsis and arrest meiosis at prophase I, due to incompatibilities between the Prdm9 gene and hybrid sterility locus Hstx2. We identified 14 alleles of Prdm9 in exon 12, encoding the DNA-binding domain of the PRDM9 protein in outcrossed wild mouse populations from Europe, Asia, and the Middle East, 8 of which are novel. The same allele was found in all mice bearing introgressed t -haplotypes encompassing Prdm9. We asked whether 7 novel Prdm9 alleles in MUS populations and the t -haplotype allele in 1 MUS and 3 DOM populations induce Prdm9 -mediated reproductive isolation. The results show that only combinations of the dom2 allele of DOM origin and the MUS msc1 allele ensure complete infertility of intersubspecific hybrids in outcrossed wild populations and inbred mouse strains examined so far. The results further indicate that MUS mice may share the erasure of PRDM9msc1 binding motifs in populations with different Prdm9 alleles, which implies that erased PRDM9 binding motifs may be uncoupled from their corresponding Prdm9 alleles at the population level. Our data corroborate the model of Prdm9-mediated hybrid sterility beyond inbred strains of mice and suggest that sterility alleles of Prdm9 may be rare. [ABSTRACT FROM AUTHOR]

Spermatogenesis is critical to sexual reproduction yet evolves rapidly in many organisms. High-throughput single-cell transcriptomics promises unparalleled insight into this important process but understanding can be impeded in nonmodel systems by a lack of known genes that can reliably demarcate biologically meaningful cell populations. Tribolium castaneum , the red flour beetle, lacks known markers for spermatogenesis found in insect species like Drosophila melanogaster. Using single-cell sequencing data collected from adult beetle testes, we implement a strategy for elucidating biologically meaningful cell populations by using transient expression stage identification markers, weighted principal component clustering, and SNP-based haploid/diploid phasing. We identify populations that correspond to observable points in sperm differentiation and find species specific markers for each stage. Our results indicate that molecular pathways underlying spermatogenesis in Coleoptera are substantially diverged from those in Diptera. We also show that most genes on the X chromosome experience meiotic sex chromosome inactivation. Temporal expression of Drosophila MSL complex homologs coupled with spatial analysis of potential chromatin entry sites further suggests that the dosage compensation machinery may mediate escape from meiotic sex chromosome inactivation and postmeiotic reactivation of the X chromosome. [ABSTRACT FROM AUTHOR]

Many entomopathogenic fungi cause infections that kill their insect host. Little is understood about changes in the reproductive investment that occurs during an infection by a lethal disease over the waning life of an insect. Life-history theory suggests the host will respond by investing resources into fighting the disease or increasing reproduction. Here, we investigate how the reproductive life of adult house flies, Musca domestica , is impacted by its host-specific fungal pathogen, Entomophthora muscae. Specifically, we test how the week-long infection alters the mating behavior of virgin adult male house flies. We find that the pathogen significantly decreases male libido, an effect which grows stronger over the course of the infection. Furthermore, females were significantly less likely to choose an infected male, reducing male mating success. Additionally, we assessed sperm viability to understand the reproductive costs for monandrous females to mate with infected males. Analyses revealed that sperm quality decreases as early as 3 days post-infection. These results show that E. muscae , which can have a prevalence near 100% in wild populations, causes severe lifetime reproductive costs to male house flies. Understanding how host–pathogen interactions affect host life history is crucial for elucidating all the negative effects pathogen virulence exerts on hosts. [ABSTRACT FROM AUTHOR]

Understanding of how selection can act on traits that improve competitiveness and subsequent paternity has advanced, including the idea that internal and external fertilization presents different environments that may select differentially on ejaculate traits. However, no studies have quantitatively synthesized the intra-specific relationships between these traits and paternity. Therefore, we conducted a meta-analysis across 52 papers to determine which ejaculate traits positively correlate with paternity share and how these correlations vary with fertilization mode. Overall, most ejaculate traits were positively associated with paternity, with the notable exception of sperm length. Sub-analyses on sperm number, sperm length, and sperm velocity revealed no statistical differences between fertilization modes in the relationship between traits and paternity when all effect sizes across species were combined. However, in a sub-analysis on fish species only, we found evidence that sperm velocity may be more important in external fertilizers. We also observed differences in the importance of phylogenetic relatedness and some species-specific differences. Our results suggest that while most ejaculate traits should be under positive directional selection in both internal and external fertilizers, sperm length may be subject to more nuanced selection pressures. Overall, we highlight important patterns of intra-specific relationships between ejaculate traits and competitive fertilization success. [ABSTRACT FROM AUTHOR]

Sexual traits may be selected during multiple consecutive episodes of selection, occurring before, during, or after copulation. The overall strength and form of selection acting on traits may thus be determined by how selection (co-)varies along different episodes. However, it is challenging to measure pre- and postcopulatory phenotypic traits alongside variation in fitness components at each different episode. Here, we used a transgenic line of the transparent flatworm Macrostomum lignano expressing green fluorescent protein (GFP) in all cell types, including sperm cells, enabling in vivo sperm tracking. We assessed the mating success, sperm-transfer efficiency, and sperm fertilizing efficiency of GFP(+) focal worms in which we measured 13 morphological traits. We found linear selection on sperm production rate arising from pre- and postcopulatory components and on copulatory organ shape arising from sperm fertilizing efficiency. We further found nonlinear (mostly concave) selection on combinations of copulatory organ and sperm morphology traits arising mostly from sperm-transfer efficiency and sperm fertilizing efficiency. Our study provides a fine-scale quantification of sexual selection, showing that both the form and strength of selection can change across fitness components. Quantifying how sexual selection builds up along episodes of selection allows us to better understand the evolution of sexually selected traits. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does sperm DNA recover from damage in all men after 2 years from the end of cytotoxic treatments? SUMMARY ANSWER The current indication of 2 years waiting time for seeking natural pregnancy after cytotoxic treatment may not be adequate for all men, since severe sperm DNA damage is present in a proportion of subjects even after this timeframe. WHAT IS KNOWN ALREADY Data in the literature on sperm DNA fragmentation (SDF) in lymphoma patients after cytotoxic treatments are scarce. The largest longitudinal study evaluated paired pre- and post-therapy (up to 24 months) semen samples from 34 patients while one study performed a longer follow-up (36 months) in 10 patients. The median/mean SDF values >24 months after therapy did not show significant differences but the studies did not explore the proportion of patients with severe DNA damage and the analysis was done on frozen-thawed samples. STUDY DESIGN, SIZE, DURATION In this study, 53 Hodgkin lymphoma (HL) and 25 non-Hodgkin lymphoma (NHL) post-pubertal patients were included over a recruitment period of 10 years (2012–2022). Among them, 18 subjects provided paired semen samples for SDF analysis at the three time points. SDF was evaluated in patients before (T0) and after 2 (T2) and 3 years (T3) from the end of, cytotoxic treatments (chemotherapy alone or in combination with radiotherapy). A cohort of 79 healthy, fertile, and normozoospermic men >18 years old served as controls (recruited between 2016 and 2019). PARTICIPANTS/MATERIALS, SETTING, METHODS SDF was evaluated on fresh semen samples (i.e. spermatozoa potentially involved in natural conception) from patients and controls using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled with flow cytometry. SDF median values were compared between groups: (i) HL and NHL patients versus controls at the three time points; (ii) HL versus NHL patients at baseline; and (iii) patients at T0 versus T2 and T3. Severe DNA damage (SDD) was defined for SDF levels above the 95th percentile of controls (50%) and the proportion of patients with SDD at all time points was established. MAIN RESULTS AND THE ROLE OF CHANCE At T0, patients displayed higher median SDF than controls, reaching statistical significance in the NHL group: 40.5% [IQR: 31.3–52.6%] versus 28% [IQR: 22–38%], P  < 0.05. Comparing SDF pre-treatment to that post-treatment, HL patients exhibited similar median values at the three time points, whereas NHL showed significantly lower values at T3 compared to T0: 29.2% [IQR: 22–38%] versus 40.5% [IQR: 31.3–52.6%], P  < 0.05. The proportion with SDD in the entire cohort at T2 was 11.6% and 13.3% among HL and NHL patients, respectively. At T3, only one in 16 NHL patients presented SDD. LIMITATIONS, REASONS FOR CAUTION TUNEL assay requires at least 5 million spermatozoa to be performed; hence, severe oligozoospermic men were not included in the study. Although our cohort represents the largest one in the literature, the relatively small number of patients does not allow us to establish precisely the frequency of SDD at T2 which in our study reached 11–13% of patients. WIDER IMPLICATIONS OF THE FINDINGS Our data provide further insights into the long-term effects of cytotoxic treatments on the sperm genome. The persistent severe DNA damage after 2 years post-treatment observed in some patients suggests that there is an interindividual variation in restoring DNA integrity. We propose the use of SDF as a biomarker to monitor the treatment-induced genotoxic effects on sperm DNA in order to better personalize pre-conceptional counseling on whether to use fresh or cryopreserved spermatozoa. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from the Istituto Toscano Tumori (ITT), Fondazione Ente Cassa di Risparmio di Firenze, the European Commission—Reproductive Biology Early Research Training (REPROTRAIN). C.K. G.F. V.R. and A.R.-E. belong to COST Action CA20119 (ANDRONET) which is supported by the European Cooperation in Science and Technology (www.cost.eu). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Although male infertility is currently diagnosed when abnormal sperm parameters are found, the poor predictive ability of sperm parameters on natural fecundity and medically assisted reproduction outcome poses the need for improved diagnostic techniques for male infertility. The accumulating evidence about the role played by the sperm epigenome in modulation of the early phases of embryonic development has led researchers to focus on the epigenetic mechanisms within the sperm epigenome to find new molecular markers of male infertility. Indeed, sperm epigenome abnormalities could explain some cases of unexplained male infertility in men showing normal sperm parameters and were found to be associated with poor embryo development in IVF cycles. The present mini-review summarizes the current knowledge about this interesting topic, starting from a description of the epigenetic mechanisms of gene expression regulation (i.e. DNA methylation, histone modifications, and non-coding RNAs' activity). We also discuss possible mechanisms by which environmental factors might cause epigenetic changes in the human germline and affect embryonic development, as well as subsequent generations' phenotypes. Studies demonstrating sperm epigenome abnormalities in men with male infertility are reviewed, with particular emphasis on those with the more severe form of spermatogenic dysfunction. Observations demonstrate that the diagnostic and prognostic efficacy of sperm epigenome evaluation will help facilitate the management of men with male factor infertility. [ABSTRACT FROM AUTHOR]

Polyploidy, a significant catalyst for speciation and evolutionary processes in both plant and animal kingdoms, has been recognized for a long time. However, the exact molecular mechanism that leads to polyploid formation, especially in vertebrates, is not fully understood. Our study aimed to elucidate this phenomenon using the zebrafish model. We successfully achieved an effective knockout of the cyclin N-terminal domain containing 1 (cntd1) using CRISPR/Cas9 technology. This resulted in impaired formation of meiotic crossovers, leading to cell-cycle arrest during meiotic metaphase and triggering apoptosis of spermatocytes in the testes. Despite these defects, the mutant (cntd1−/−) males were still able to produce a limited amount of sperm with normal ploidy and function. Interestingly, in the mutant females, it was the ploidy not the capacity of egg production that was altered. This resulted in the production of haploid, aneuploid, and unreduced gametes. This alteration enabled us to successfully obtain triploid and tetraploid zebrafish from cntd1−/− and cntd1−/−/− females, respectively. Furthermore, the tetraploid-heterozygous zebrafish produced reduced-diploid gametes and yielded all-triploid or all-tetraploid offspring when crossed with wild-type (WT) or tetraploid zebrafish, respectively. Collectively, our findings provide direct evidence supporting the crucial role of meiotic crossover defects in the process of polyploidization. This is particularly evident in the generation of unreduced eggs in fish and, potentially, other vertebrate species. [ABSTRACT FROM AUTHOR]

Harvestmen are a major arachnid order that has experienced a dramatic increase in biological knowledge in the 21st century. The publication of the book Harvestmen: The Biology of Opiliones in 2007 stimulated the development of many behavioral studies. Although the book is relatively recent, our understanding of the reproductive biology of harvestmen is already outdated due to the fast accumulation of new data. Our goal is to provide an updated review of the subject to serve as a benchmark for the following years. In the pre-copulatory phase, we explore the evolution of facultative parthenogenesis, the factors that may affect the types of mating system, and the role of nuptial gifts in courtship. Regarding the copulatory phase, harvestmen are unique arachnids because they have aflagellate spermatozoa and a penis with complex morphology. We discuss the implications of these two features for sperm competition and cryptic female choice. In the post-copulatory phase, we connect oviposition site selection and climate conditions to the widespread occurrence of resource defense polygyny, alternative reproductive tactics, and sexual dimorphism in several clades of tropical harvestmen. Finally, we present the different forms of parental care in the order, and discuss the benefits and costs of this behavior, which can be performed either by females or males. Throughout the review, we indicate gaps in our knowledge and subjects that deserve further studies. Hopefully, the information synthesized here will stimulate researchers worldwide to embrace harvestmen as a study system and to improve our effort to unravel the mysteries of their reproductive biology. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the mechanism behind cryoinjury in human sperm, particularly concerning the interplay between reactive oxygen species (ROS) and autophagy, and how does it subsequently affect sperm fate? SUMMARY ANSWER The freeze-thaw operation induces oxidative stress by generating abundant ROS, which impairs sperm motility and activates autophagy, ultimately guiding the sperm toward programmed cell death such as apoptosis and necrosis, as well as triggering premature capacitation. WHAT IS KNOWN ALREADY Both ROS-induced oxidative stress and autophagy are thought to exert an influence on the quality of frozen-thawed sperm. STUDY DESIGN, SIZE, DURATION Overall, 84 semen specimens were collected from young healthy fertile males, with careful quality evaluation. The specimens were split into three groups to investigate the ROS-induced cryoinjury: normal control without any treatment, sperm treated with 0.5 mM hydrogen peroxide (H2O2) for 1 h, and sperm thawed following cryopreservation. Samples from 48 individuals underwent computer-assisted human sperm analysis (CASA) to evaluate sperm quality in response to the treatments. Semen samples from three donors were analyzed for changes in the sperm proteome after H2O2 treatment, and another set of samples from three donors were analyzed for changes following the freeze-thaw process. The other 30 samples were used for fluorescence-staining and western blotting. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm motility parameters, including progressive motility (PR %) and total motility (PR + NP %), were evaluated using the CASA system on a minimum of 200 spermatozoa. The proteomic profiles were determined with label-free mass spectrometry (MS/MS) and protein identification was performed via ion search against the NCBI human database. Subsequently, comprehensive bioinformatics was applied to detect significant proteomic changes and functional enrichment. Fluorescence-staining and western blot analyses were also conducted to confirm the proteomic changes on selected key proteins. The ROS level was measured using 2′,7′-dichlorodihydrofluorescein diacetate labeling and the abundance of bioactive mitochondria was determined by evaluating the inner mitochondrial membrane potential (MMP) level. Molecular behaviors of sequestosome-1 (p62 or SQSTM1) and microtubule-associated proteins 1A/1B light chain 3 (LC3) were monitored to evaluate the state of apoptosis in human sperm. Fluorescent probes oxazole yellow (YO-PRO-1) and propidium iodide (PI) were utilized to monitor programmed cell death, namely apoptosis and necrosis. Additionally, gradient concentrations of antioxidant coenzyme Q10 (CoQ10) were introduced to suppress ROS impacts on sperm. MAIN RESULTS AND THE ROLE OF CHANCE The CASA analysis revealed a significant decrease in sperm motility for both the H2O2-treatment and freeze-thaw groups. Fluorescence staining showed that high ROS levels were produced in the treated sperm and the MMPs were largely reduced. The introduction of CoQ10 at concentrations of 20 and 30 μM resulted in a significant rescue of progressive motility (P  < 0.05). The result suggested that excessive ROS could be the major cause of sperm motility impairment, likely by damaging mitochondrial energy generation. Autophagy was significantly activated in sperm when they were under oxidative stress, as evidenced by the upregulation of p62 and the increased conversion of LC3 as well as the upregulation of several autophagy-related proteins, such as charged multivesicular body protein 2a, mitochondrial import receptor subunit TOM22 homolog, and WD repeat domain phosphoinositide-interacting protein 2. Additionally, fluorescent staining indicated the occurrence of apoptosis and necrosis in both H2O2-treated sperm and post-thaw sperm. The cell death process can be suppressed when CoQ10 is introduced, which consolidates the view that ROS could be the major contributor to sperm cryoinjury. The freeze-thaw process could also initiate sperm premature capacitation, demonstrated by the prominent increase in tyrosine phosphorylated proteins, verified with anti-phosphotyrosine antibody and immunofluorescence assays. The upregulation of capacitation-related proteins, such as hyaluronidase 3 and Folate receptor alpha, supported this finding. LARGE SCALE DATA The data underlying this article are available in the article and its online supplementary material. LIMITATIONS, REASONS FOR CAUTION The semen samples were obtained exclusively from young, healthy, and fertile males with progressive motility exceeding 60%, which might overemphasize the positive effects while possibly neglecting the negative impacts of cryoinjury. Additionally, the H2O2 treatment conditions in this study may not precisely mimic the oxidative stress experienced by sperm after thawing from cryopreservation, potentially resulting in the omission of certain molecular alterations. WIDER IMPLICATIONS OF THE FINDINGS This study provides substantial proteomic data for a comprehensive and deeper understanding of the impact of cryopreservation on sperm quality. It will facilitate the design of optimal protocols for utilizing cryopreserved sperm to improve applications, such as ART, and help resolve various adverse situations caused by chemotherapy, radiotherapy, and surgery. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from the Major Innovation Project of Research Institute of National Health Commission (#2022GJZD01-3) and the National Key R&D Program of China (#2018YFC1003600). All authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

The evolution of gonochorism from hermaphroditism is linked with the formation of sex chromosomes, as well as the evolution of sex-biased and sex-specific gene expression to allow both sexes to reach their fitness optimum. There is evidence that sexual selection drives the evolution of male-biased gene expression in particular. However, previous research in this area in animals comes from either theoretical models or comparative studies of already old sex chromosomes. We therefore investigated changes in gene expression under 3 different selection regimes for the simultaneous hermaphrodite Macrostomum lignano subjected to sex-limited experimental evolution (i.e. selection for fitness via eggs, sperm, or a control regime allowing both). After 21 and 22 generations of selection for male-specific or female-specific fitness, we characterized changes in whole-organism gene expression. We found that female-selected lines had changed the most in their gene expression. Although annotation for this species is limited, gene ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggest that metabolic changes (e.g. biosynthesis of amino acids and carbon metabolism) are an important adaptive component. As predicted, we found that the expression of genes previously identified as testis-biased candidates tended to be downregulated in the female-selected lines. We did not find any significant expression differences for previously identified candidates of other sex-specific organs, but this may simply reflect that few transcripts have been characterized in this way. In conclusion, our experiment suggests that changes in testis-biased gene expression are important in the early evolution of sex chromosomes and gonochorism. [ABSTRACT FROM AUTHOR]

Reprograming of chromatin structures and changes in gene expression are critical for plant male gamete development, and epigenetic marks play an important role in these processes. Histone variant H3.3 is abundant in euchromatin and is largely associated with transcriptional activation. The precise function of H3.3 in gamete development remains unclear in plants. Here, we report that H3.3 is abundantly expressed in Arabidopsis anthers and its knockout mutant h3.3–1 is sterile due to male sterility. Transcriptome analysis of young inflorescence has identified 2348 genes downregulated in h3.3–1 mutant, among which 1087 target genes are directly bound by H3.3, especially at their 3ʹ ends. As a group, this set of H3.3 targets is enriched in the reproduction-associated processes including male gamete generation, pollen sperm cell differentiation and pollen tube growth. The function of H3.3 in male gamete development is dependent on the Anti-Silencing Factor 1A/1B (ASF1A/1B)-Histone regulator A (HIRA)-mediated pathway. Our results suggest that ASF1A/1B-HIRA-mediated H3.3 deposition at its direct targets for transcription activation forms the regulatory networks responsible for male gamete development. [ABSTRACT FROM AUTHOR]

The reintroduced population of Mexican gray wolves (Canis lupus baileyi) continues to rely on a carefully managed captive breeding program. Although success of that program depends on detailed knowledge of reproductive processes, age of puberty has not been determined. This study assessed male puberty status (presence of sperm in ejaculates), maintained in nine US facilities, during their first breeding season. Variables possibly associated with puberty were also evaluated. There was a significant effect of body weight, testis size, facility latitude, and date of collection, whereas a statistical trend was found for age and inbreeding coefficient. Social factors, including being housed with sire, had no effect. Over half the males in their first breeding season were producing sperm, although in some cases sperm quality was poor, suggesting possible infertility. Although there was no minimum body weight associated with presence of sperm, likelihood increased with increasing body weight, highlighting a possible critical role for nutrition. The trend for sperm production in males collected later in the breeding season and at lower latitudes suggests that later collections, especially at higher latitudes, might reveal a higher percentage of pubertal males. These results have potential implications for breeding program management and introduce the possibility that more wild gray wolves than previously thought might produce sperm during their first year, even if they do not sire young. [ABSTRACT FROM AUTHOR]

Proteomic methodologies offer a robust approach to identify and quantify thousands of proteins from semen components in both fertile donors and infertile patients. These strategies provide an unprecedented discovery potential, which many research teams are currently exploiting. However, it is essential to follow a suitable experimental design to generate robust data, including proper purification of samples, appropriate technical procedures to increase identification throughput, and data analysis following quality criteria. More than 6000 proteins have been described so far through proteomic analyses in the mature sperm cell, increasing our knowledge on processes involved in sperm function, intercommunication between spermatozoa and seminal fluid, and the transcriptional origin of the proteins. These data have been complemented with comparative studies to ascertain the potential role of the identified proteins on sperm maturation and functionality, and its impact on infertility. By comparing sperm protein profiles, many proteins involved in the acquisition of fertilizing ability have been identified. Furthermore, altered abundance of specific protein groups has been observed in a wide range of infertile phenotypes, including asthenozoospermia, oligozoospermia, and normozoospermia with unsuccessful assisted reproductive techniques outcomes, leading to the identification of potential clinically useful protein biomarkers. Finally, proteomics has been used to evaluate alterations derived from semen sample processing, which might have an impact on fertility treatments. However, the intrinsic heterogeneity and inter-individual variability of the semen samples have resulted in a relatively low overlap among proteomic reports, highlighting the relevance of combining strategies for data validation and applying strict criteria for proteomic data analysis to obtain reliable results. This mini-review provides an overview of the most critical steps to conduct robust sperm proteomic studies, the most relevant results obtained so far, and potential next steps to increase the impact of sperm proteomic data. [ABSTRACT FROM AUTHOR]

We encountered unexpected transgene silencing in Arabidopsis thaliana sperm cells; transgenes encoding proteins with no specific intracellular localization (cytoplasmic proteins) were silenced transcriptionally or posttranscriptionally. The mRNA of cytoplasmic protein transgenes tagged with a fluorescent protein gene was significantly reduced, resulting in undetectable fluorescent protein signals in the sperm cell. Silencing of the cytoplasmic protein transgenes in the sperm cell did not affect the expression of either its endogenous homologous genes or cotransformed transgenes encoding a protein with targeted intracellular localization. This transgene silencing in the sperm cell persisted in mutants of the major gene silencing machinery including DNA methylation. The incomprehensible, yet real, transgene silencing phenotypes occurring in the sperm cell could mislead the interpretation of experimental results in plant reproduction, and this Commentary calls attention to that risk and highlights details of this novel cytoplasmic protein transgene silencing. [ABSTRACT FROM AUTHOR]

When populations become geographically isolated, they begin to diverge in various traits and at variable rates. The dynamics of such trait divergences are relevant for understanding evolutionary processes such as local adaptation and speciation. Here we examine divergences in sperm and body structures in a polygynandrous songbird, the alpine accentor (Prunella collaris) between two allopatric high-altitude populations, in Morocco and Spain. The populations diverged around 82,000 years ago, as estimated with a coalescence-based phylogenetic analysis of genome-wide single-nucleotide polymorphisms. We found that birds in the two areas had nonoverlapping sperm lengths, which suggests adaptation to divergent female reproductive tract environments. Sperm length also showed an exceptionally low coefficient of among-male variation, a signal of strong stabilizing selection imposed by sperm competition. The evolutionary rate of sperm length was almost twice the rates for the most divergent morphological traits and more than three times higher than expected from literature data over a similar generational timescale. This rapid evolution of a key reproductive trait has implications for reproductive isolation and ultimately for speciation. Strong selection for different sperm length optima in allopatry predicts conspecific sperm precedence and disruptive selection in sympatry, hence a possible postcopulatory prezygotic barrier to gene flow. [ABSTRACT FROM AUTHOR]

Spermatozoa vary in quantity, quality and design across species and individuals. Here, we reported the first detailed analysis of sperm traits in two primitively eusocial paper wasps, Polistes dominula and Polistes gallicus , which differ in their nuptial strategies and body size as follows: lek-territoriality of the large P. dominula males, defending exclusive territories at landmarks; scramble competition of the small P. gallicus , where serial mating may occur because males interfere with rivals and replace them during copulation. We compared the number, viability, and length of sperm, and its coefficient of variation, between these sympatric paper wasps patrolling and marking the same landmarks during the same summer: a good opportunity to analyse sperm across species while controlling for environmental variables. Spermatozoa were more numerous and longer in P. dominula than in P. gallicus males. Viability was similar in both species and positively covaried with male body size. Intra-male variation in sperm length was significantly reduced in P. gallicus , as predicted in the case of selection for an optimal sperm design. We hypothesized that precopulatory sexual selection (lek-territoriality vs. scramble competition) may affect sperm traits. [ABSTRACT FROM AUTHOR]

In Hymenoptera, arrhenotokous parthenogenesis (arrhenotoky) is a common reproductive mode. Thelytokous parthenogenesis (thelytoky), when virgin females produce only females, is less common and is found in several taxa. In our study, we assessed the efficacy of recombination and the effect of thelytoky on the genome structure of Diplolepis rosae , a gall wasp-producing bedeguars in dog roses. We assembled a high-quality reference genome using Oxford Nanopore long-read technology and sequenced 17 samples collected in France with high-coverage Illumina reads. We found two D. rosae peripatric lineages that differed in the level of recombination and homozygosity. One of the D. rosae lineages showed a recombination rate that was 13.2 times higher and per-individual heterozygosity that was 1.6 times higher. In the more recombining lineage, the genes enriched in functions related to male traits ('sperm competition", "insemination", and "copulation" gene ontology terms) showed signals of purifying selection, whereas in the less recombining lineage, the same genes showed traces pointing towards balancing or relaxed selection. Thus, although D. rosae reproduces mainly by thelytoky, selection may act to maintain sexual reproduction. [ABSTRACT FROM AUTHOR]

When females mate with more than one male, competition between rival ejaculates is expected to favor adaptations that promote fertilization success. There is now compelling evidence that sperm competition selects for increased production and allocation of sperm. However, sperm comes packaged in ejaculates that also contain protein-rich seminal fluids. Predicting how males should allocate individual seminal fluid proteins in response to sperm competition is hampered by our limited knowledge of their precise function. We use gene expression studies and interference RNA to ask how seminal fluid proteins in the ejaculate of a cricket, Teleogryllus oceanicus, affect a male's paternity share when in competition for fertilizations. We find that the relative expression of one seminal fluid gene, gagein, positively affects the paternity share of competing males and that knockdown of this and two other seminal fluid protein genes renders males mating in the offensive role of sperm competition incapable of fathering living offspring. Despite having a negative effect on offspring viability these seminal fluid genes have been found to be up regulated in response to rival males, consistent with a role in promoting competitive fertilization success. Our data contribute to a growing body of evidence that, like sperm, seminal fluid gene expression is subject to post-mating sexual selection via sperm competition. [ABSTRACT FROM AUTHOR]

Male X-linked meiotic drive systems, which cause the degeneration of Y-bearing sperm, are common in the Diptera. Sperm killing is typically associated with fitness costs that arise from the destruction of wildtype sperm and collateral damage to maturing drive sperm, resulting in poor success under sperm competition. We investigate X-linked meiotic drive fertility in the stalk-eyed fly, Teleopsis dalmanni. Drive male paternity was measured in double mating trials under sperm competition against a wildtype male. Drive males sired the same number of offspring as wildtype males, both when mated first or second. This is the first evidence that drive males can compete equally with non-drive males in double matings, challenging the assumption that drive males inevitably suffer reduced fertility. The finding is in accord with previous work showing that the number of sperm per ejaculate transferred to females during non-competitive single matings does not differ between drive and wildtype males, which is likely due to the adaptive evolution of enlarged testes in drive males. Future experiments will determine whether the competitive ability of drive males is maintained under higher rates of female remating likely to be experienced in nature. [ABSTRACT FROM AUTHOR]

Asexual reproduction is ancestral in prokaryotes; the switch to sexuality in eukaryotes is one of the major transitions in the history of life. The study of the maintenance of sex in eukaryotes has raised considerable interest for decades and is still one of evolutionary biology's most prominent question. The observation that many asexual species are of hybrid origin has led some to propose that asexuality in hybrids results from sexual processes being disturbed because of incompatibilities between the two parental species' genomes. However, in some cases, failure to produce asexual F1s in the lab may indicate that this mechanism is not the only road to asexuality in hybrid species. Here, we present a mathematical model and propose an alternative, adaptive route for the evolution of asexuality from previously sexual hybrids. Under some reproductive alterations, we show that asexuality can evolve to rescue hybrids' reproduction. Importantly, we highlight that when incompatibilities only affect the fusion of sperm and egg's genomes, the two traits that characterize asexuality, namely unreduced meiosis and the initiation of embryogenesis without the incorporation of the sperm's pronucleus, can evolve separately, greatly facilitating the overall evolutionary route. Taken together, our results provide an alternative, potentially complementary explanation for the link between asexuality and hybridization. [ABSTRACT FROM AUTHOR]

Background: Bisphenol A (BPA), an endocrine-disrupting chemical (EDC), is ubiquitous in our environment and poses a significant threat to male fertility. Date seeds (DSs) are used in folk medicine due to their antioxidant activity. Aim: The purpose of this study was to assess the beneficial effects of DSs, whether in powder or nanoparticle form, against BPA-induced testicular oxidative challenges and apoptosis, aided by inspection of specific genes linked to fertility, oxidative stress and intrinsic mitochondrial pathway of apoptosis. Methods: Thirty-five adult male albino rats were equally divided into 5 groups including control, BPA, BPA + date seeds powder "DSP", BPA + date seed nanoparticle 1/10 (DSNP 1/10) and BPA + DSNP 1/20 groups. Results: TEM showed that the ball-mill method was effective to form DSNP with an average size of 20 nm. BPA significantly impaired sperm motility, morphology, viability and concentration. It also reduced serum testosterone levels and evoked marked oxidative stress in the testes. Additionally, serum levels of triiodothyronine and thyroxine were extremely reduced. Moreover, testicular mRNA relative expression levels of CYP11A1 and Nrf-2 were markedly downregulated. Testicular apoptosis was also promoted whereas Bax/Bcl-2 ratio was profoundly elevated. Histological pictures of the testes, epididymis, seminal vesicles and prostate confirmed the unfavorable effects of BPA. Surprisingly, we first demonstrated that DSs, specifically the nanoparticle form, strongly alleviated all of BPA's negative effects, with DSNP 1/20 achieving the best results. Conclusion: Therefore, DSNP in both doses could be regarded as an ideal candidate for abating the male reproductive challenges caused by BPA. [ABSTRACT FROM AUTHOR]

Multiple morphological abnormalities of the sperm flagella (MMAF) are one of the major causes of male infertility and are characterized by multiple defects. In this study, we found that the coiled-coil domain-containing 189 (Ccdc189) gene was predominantly expressed in mouse testes and that inactivation of the Ccdc189 gene caused male infertility. Histological studies revealed that most sperm from Ccdc189-deficient mice carried coiled, curved or short flagella, which are typical MMAF phenotypes. Immunoelectron microscopy showed that the CCDC189 protein was located at the radial spoke of the first peripheral microtubule doublet in the sperm axoneme. A CCDC189-interacting protein, CABCOCO1 (ciliary-associated calcium-binding coiled-coil protein 1), was discovered via co-immunoprecipitation and mass spectrometry, and inactivation of Cabcoco1 caused malformation of sperm flagella, which was consistent with findings obtained with Ccdc189-deficient mice. Further studies revealed that inactivation of CCDC189 caused downregulation of CABCOCO1 protein expression and that both CCDC189 and CABCOCO1 interacted with the radial-spoke-specific protein RSPH1 and intraflagellar transport proteins. This study demonstrated that Ccdc189 is a radial-spoke-associated protein and is involved in sperm flagellum formation through its interactions with CABCOCO1 and intraflagellar transport proteins. [ABSTRACT FROM AUTHOR]

During IVF treatments, normal fertilization is generally evidenced by the appearance of two pronuclei, one arising from the oocyte and the other from the male gamete. Embryos derived from zygotes with a pronuclei number other than two are assumed to possess a ploidy abnormality and their transfer is usually avoided owing to increased risk of implantation failure, miscarriage, and molar pregnancies. Nonetheless, the inclusion of genotyping data in preimplantation genetic testing has revealed that a normal diploid configuration is possible in embryos deriving from zygotes with an abnormal pronuclei number such as tripronuclear and one pronucleus. Here, we present a one-of-a-kind transfer of a tetrapronuclear-derived embryo that was discovered to be diploid and negative for other whole chromosome or segmental aneuploidies during preimplantation genetic testing using a targeted next-generation sequencing approach. The transfer resulted in the live birth of a healthy infant who is now 4 years old and has no apparent health or developmental impairments. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) appears as a core of reproductive management in the hog industry. AI involves the use of chilled semen doses meeting routine thresholds for motility (≥ 70%) and morphology (≥ 80%) at collection. However, semen doses meeting these standards exhibit a gradual decline in sperm quality, with likely divergent profiles during preserva- tion that impact fertility outcomes. Hence, this study aims to unravel the sperm quality dynamics of boar semen doses during 7-d chilled storage at 17ºC. Semen doses (n = 25) from proven-fertile Duroc boars were provided by a commercial hog farm (Prestage Farm, MS) and transported to the laboratory within 30 min of collection. Each semen sample was aliquoted and analyzed on the day of collection (d 0) and after 7 d of storage (d 7) at 17°C. Sperm total motility (TM), progressive motility (PM), and normal morphology (NM) were assessed using a computer-assisted sperm analyzer (CASA; CEROS II, IMV Biotechnologies). Apoptosis (Annexin V/7-AAD), viability (Propidium iodide – PI), and intracellular reactive oxygen species (ROS; DCFH-DA) were evaluated using flow cytometry. Total antioxidant activity (TAOC) and lipid peroxidation (malondialdehyde; MDA) were assessed using commercial kits (Cayman Chemical). Data analysis was conducted using parametric statistics (SPSS), with P < 0.05 considered significant. Results were pre- sented as mean ± SEM. Data analysis confirmed the significant decrease of CASA parameters, particularly TM from d 0 to d 7 (78.87 ± 1.37 vs. 73.19 ± 1.98%), with extreme variance on d 7 allowing to discriminate high motility (HM, n = 6) vs low motility (LM, n = 6) semen doses. HM doses evidenced significantly greater (P < 0.05) TM (83.73 ± 1.17%), PM (36.07 ± 1.11%), and NM (89.45 ± 2.30%) compared with LM doses (TM: 59.40 ± 3.35%, PM: 16.57 ± 3.25%, NM: 79.93 ± 2.61%) on d 7. Unlike HM samples, which were comparable between d 0 and d 7, LM doses displayed sig- nificantly greater proportions of apoptotic and viable cells, and increased intracellular ROS levels on d 0. Likewise, LM samples revealed significantly greater (P < 0.05) proportions of apoptotic cells (3.69 ± 1.66%), dead cells (38.41 ± 3.71%), and cells with intracellular ROS (36.21 ± 3.62%) than HM samples (apoptotic cells: 0.03 ± 0.02%, dead cells: 4.73 ± 4.65%, ROS: 5.34 ± 4.91%) on d 0; however, only the proportions of dead cells displayed significant increase in LM on d7. MDA concentrations were comparable between HM and LM samples on d 0. However, on d 7, LM samples exhibited significantly greater MDA concentrations than HM samples. In contrast, TAOC was significantly greater in HM samples (2.53 ± 0.14 mM) compared with LM samples (2.17 ± 0.07 mM) on d 0. In conclusion, semen samples with similar gross characteristics at collection exhibited differential responses during storage. Their initial redox potential and apoptosis-like changes may contribute to their varying ability to with- stand prolonged storage. Work supported by USDAARS grant# 6066-31000-015-00D. [ABSTRACT FROM AUTHOR]

The pig industry relies heavily on artificial insemination as the primary breeding technique, using high-quality semen doses for genetic improvement. Semen consists of seminal plasma, a complex fluid that provides an optimal environment for sperm maturation, viability, and fertilization. Seminal plasma contains membranous structures called extracellular vesicles (EVs), facilitating intercellular communication. These seminal plasma-EVs (SP-EVs) contain microRNAs, which hold promise as predictive markers of semen quality. Thus, we hypothesized that the composition of SP-EVs might reflect the divergent quality status of corresponding spermatozoa. Therefore, this study aimed to explore the microRNA content of SP-EVs isolated from accepted (Passed) or rejected (Failed) boar semen for artificial insemination. Over 8 wk during the hot summer, raw semen was collected (n = 83) from sexually mature Duroc boars at a commercial boar stud (Prestage Farms, MS). Semen samples underwent sperm analysis to evaluate motility and morphology, classified as Passed or Failed quality based on predefined criteria (≥ 70% and < 70%, respectively). Seminal plasma was isolated through serial centrifugation, followed by SP-EV extraction using differential centrifugation. The isolated SP-EVs were characterized using transmission electron microscopy (TEM) for EV morphology and nanoparticle tracking analysis (NTA) for size and concentration. SP-EV miRNAs were extracted and identified via the Illumina NextSeq sequencing platform. Data were analyzed using SPSS software and significance was set at P < 0.05. Both TEM and NTA demonstrated that SP-EVs had a spherical structure with a diameter range of 50 to 300 nm. A total of 437 miRNAs were expressed in the Failed group and 443 miRNAs in the Passed group. MiRNA differential expression analysis revealed 28 downregulated and two upregulated miRNAs in the Passed group compared with the Failed group. Validated miRNA gene targets were associated with signaling pathways, including N-glycan biosynthesis, p53 signaling, pancreatic cancer, and apoptosis. These findings suggest that SP-EVs carry specific microRNAs potentially linked to boar semen quality. Thus, microRNAs from SP-EVs have the potential to serve as biomarkers to assess sperm quality, thereby providing insight into therapeutic approaches to address male infertility. Additionally, seminal plasma- EVs can be useful nanocarriers for further research interventions. [ABSTRACT FROM AUTHOR]

In sheep meat production, prolificity is one of the main factors influencing economic profitability of a farm. Belclare sheep are used for meat production and have passed through intense selection pressure to high prolificity, leading them to have large litter sizes. This study aimed to identify selection signature regions and carry out functional analyses related to reproduction traits in the Belclare breed in contrast to a group of non-prolific meat breeds. A total of 50,166 biallelic SNPs from the Illumina OvineSNP50 genotyped in the Belclare breed (n = 2,377) and four meat breeds (Beltex, n = 146; Charollais, n = 1,477; Suffolk, n = 1,231 and Texel, n = 3,330). SNP filtering and quality control were performed within each breed using PLINK v1.9. Only SNPs on autosomal chromosomes with defined positions according to the sheep (Ovis aries) genome assembly OAR3.1 were analyzed. Samples and SNPs with a call rate below 95% were discarded, as well as SNPs with MAF below 1% and deviation of Hardy-Weinberg equilibrium with P-value ≤ 10-6. Imputation of missing genotypes and haplotype phasing were performed using BEAGLE v.3.3.2. After quality control, an average of 30,249 SNPs were further analyzed. Selection signature detection was performed by Cross-Population Extended Haplotype Homozygosity (XP-EHH) and Ratio of Site-specific Extended Haplotype Homozygosity between populations (Rsb), with the REHH v.3.2.2 software in R, using 100 kb non-overlapping windows with at least two markers per window. The top 1% windows with the greatest average values were considered regions under positive selection. Gene and QTL annotation were conducted with the GALLO R package. Gene and metabolic pathways enrichment (FDR< 0.10) were performed with gprofiler2 software. A total of 67 genomic regions co-localized with 76 candidate genes and 65 QTLs were identified. The genes were enriched for 12 molecular function terms. Among the annotated genes, FSHR, INSL6, HSD17B12, and SGK1 were previously found to be related to reproduction traits. The enriched term follicle-stimulating hormone receptor activity was related to the FSHR gene. The follicle-stimulating hormone (FSH) has an important role in follicular development, and their concentrations are regulated by the FSH receptor (FSHR), which suggests that FSHR is also involved in prolificity of the ewe. The testes weight trait was detected in the reproductive QTL class, but no gene related to fertility was identified. The gene INSL6 is associated with spermatogenesis and sperm motility and, as Belclare rams are used to produce prolific replacements, it may have an important role in semen quality and prolificity, as increased semen quality might increase the chance of a successful fecundation. The identification of selection signatures highlights that the selection pressure left footprints in the Belclare breed genome related to reproduction, which may affect the litter sizes. [ABSTRACT FROM AUTHOR]

This article discusses a case study of a Chinese man with infertility and multiple morphological abnormalities of sperm flagella (MMAF). The study identified compound heterozygous mutations in the CFAP65 gene as the likely genetic cause of the patient's infertility. CFAP65 is essential for spermatogenesis and male fertility, and mutations in this gene have been linked to MMAF and ciliopathy. The findings of this study contribute to the understanding of the genetic basis of male infertility and expand the variant spectrum of the CFAP65 gene. [Extracted from the article]

There is growing evidence that the female reproductive fluid (FRF) plays an important role in cryptic female choice through its differential effect on the performance of sperm from different males. In a natural spawning event, the male(s) may release ejaculate closer or further away from the spawning female. If the relative spatial proximity of competing males reflects the female pre‐mating preference towards those males, then favoured males will encounter higher concentrations of FRF than unpreferred males. Despite this being a common situation in many external fertilizers, whether different concentrations of FRF can differentially influence the sperm performance of distinct male phenotypes (favoured and unfavoured by the female) remains to be elucidated. Here, we tested this hypothesis using the grass goby (Zosterisessor ophiocephalus), a fish with distinct territorial‐sneaker reproductive tactics and female pre‐mating preference towards territorial males, that consequently mate in an advantaged position and whose sperm experience higher concentrations of FRF. Our findings revealed a differential concentration‐dependent effect of FRF over sneaker and territorial sperm motility only at low concentrations (i.e. at the distance where sneakers typically ejaculate), with increasing FRF concentrations (i.e. close to the eggs) similarly boosting the sperm performance of both sneaker and territorial males. The ability to release sperm close to the eggs is a prerogative of territorials, but FRF can likewise advantage the sperm of those sneakers that are able to get closer, allowing flexibility in the direction of female post‐mating choice. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Is it possible to identify a reliable marker of successful sperm retrieval (+SR) in men with idiopathic non-obstructive azoospermia (iNOA) undergoing microdissection testicular sperm extraction (mTESE)? SUMMARY ANSWER A higher likelihood of +SR during mTESE is observed in men with iNOA and lower preoperative serum anti-Müllerian hormone (AMH) levels, with good predictive accuracy achieved using an AMH threshold of <4 ng/ml. WHAT IS KNOWN ALREADY AMH has been previously linked to +SR in men with iNOA undergoing mTESE prior to ART. STUDY DESIGN, SIZE, DURATION A multi-centre cross-sectional study was carried out with a cohort of 117 men with iNOA undergoing mTESE at three tertiary-referral centres. PARTICIPANTS/MATERIALS, SETTING, METHODS Data from 117 consecutive white-European men with iNOA presenting for primary couple's infertility associated with a pure male factor at three centres were analysed. Descriptive statistics was applied to compare patients with negative (−SR) versus +SR at mTESE. Multivariate logistic regression models were fitted to predict +SR at mTESE, after adjusting for possible confounders. Diagnostic accuracy of the factors associated with +SR was assessed. Decision curve analyses were used to display the clinical benefit. MAIN RESULTS AND THE ROLE OF CHANCE Overall, 60 (51.3%) men had an −SR and 57 (48.7%) had a +SR at mTESE. Patients with +SR had lower levels of baseline AMH (P  = 0.005) and higher levels of estradiol (E2) (P  = 0.01). At multivariate logistic regression analysis, lower levels of AMH (odds ratio: 0.79; 95% CI: 0.64–0.93, P  = 0.03) were associated with +SR at mTESE, after adjusting for possible confounders (e.g. age, mean testicular volume, FSH, and E2). A threshold of AMH <4 ng/ml achieved the highest accuracy for +SR at mTESE, with an AUC of 70.3% (95% CI: 59.8–80.7). Decision curve analysis displayed the net clinical benefit of using an AMH <4 ng/ml threshold. LIMITATIONS, REASONS FOR CAUTION There is a need for external validation in even larger cohorts, across different centres and ethnicities. Systematic reviews and meta-analysis to provide high level of evidence are lacking in the context of AMH and SR rates in men with iNOA. WIDER IMPLICATIONS OF THE FINDINGS Current findings suggest that slightly more than one in two men with iNOA had −SR at mTESE. Overall, men with iNOA with lower levels of AMH had a significantly higher percentage of successful SR at surgery. A threshold of <4 ng/ml for circulating AMH ensured satisfactory sensitivity, specificity, and positive predictive values in the context of +SR at mTESE. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by voluntary donations from the Urological Research Institute (URI). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A [ABSTRACT FROM AUTHOR]

Haplodiploidy and paternal genome elimination (PGE) are examples of asymmetric inheritance, where males transmit only maternally inherited chromosomes to their offspring. Under haplodiploidy, this results from males being haploid, whereas under PGE, males inherit but subsequently exclude paternally inherited chromosomes from sperm. Their evolution involves changes in the mechanisms of meiosis and sex determination and sometimes also dosage compensation. As a result, these systems are thought to be an evolutionary trap, meaning that once asymmetric chromosome transmission evolves, it is difficult to transition back to typical Mendelian transmission. We assess whether there is evidence for this idea in the scale insect family Eriococcidae, a lineage with PGE and the only clade with a suggestion that asymmetric inheritance has transitioned back to Mendelian inheritance. We conduct a cytological survey of 13 eriococcid species, and a cytological, genetic, and gene expression analysis of species in the genus Cystococcus, to investigate whether there is evidence for species in this family evolving Mendelian chromosome transmission. Although we find that all species we examined exhibit PGE, the mechanism is extremely variable within Eriococcidae. Within Cystococcus, in fact, we uncover a previously undiscovered type of PGE in scale insects that acts exclusively in meiosis, where paternally inherited chromosomes in males are present, uncondensed, and expressed in somatic cells but eliminated prior to meiosis. Broadly, we fail to find evidence for a reversion from PGE to Mendelian inheritance in Eriococcidae, supporting the idea that asymmetric inheritance systems such as PGE may be an evolutionary trap. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? SUMMARY ANSWER Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. WHAT IS KNOWN ALREADY The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. STUDY DESIGN, SIZE, DURATION Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn−/−) mouse model was generated by CRISPR–Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography–mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. MAIN RESULTS AND THE ROLE OF CHANCE Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn−/− male mice. VSL, VCL, and VAP in the sperm of Iqcn−/− male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn−/− male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. LIMITATIONS, REASONS FOR CAUTION More cases are needed to demonstrate the relation between IQCN variants and phenotypes. WIDER IMPLICATIONS OF THE FINDINGS Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]