Sperm heteromorphism, the production multiple discrete sperm types (or "morphs"), occurs in diverse animal and plant taxa. This phenomenon represents a division of labour by male gametes, with one morph participating in fertilization and the other morph(s) specialized to perform some other reproductive function(s). One of the most intensively investigated systems is the Drosophila obscura species group, for which sperm heteromorphism evolved once in the common ancestor to the group, with all examined descendent species exhibiting sperm length heteromorphism. The absence of additional sperm heteromorphism origins among drosophilids has precluded comparative evolutionary analyses. Here, we report the discovery of a second origin within the Drosophilidae, withChymomyza procnemis exhibiting the most extreme sperm length heteromorphism known (>15-fold difference). Males produced and transferred to females approximately twice as many short sperm as long sperm. Shortly after insemination, females ejected from their reproductive tracts most of the short morph sperm, yet relatively few of the long morph sperm, resulting in a preponderance of long morph sperm occupying the females' sperm-storage organs. By the start of egg laying, few-to-no short morph sperm were detectable in females. Although C. procnemis have very long eusperm and the highest relative investment (gonadosomatic index; GSI) in testes for any drosophilid species, a comparative analysis of 149 Drosophila species revealed no significant coevolutionary relationship between sperm heteromorphism and either sperm length or GSI. Continued investigations with Chymomyza will advance our understanding of how sperm heteromorphism arises, persists, and diversifies across lineages.

One of the leading causes of human subfertility is the continuous decline in semen quality, contributing to a global fertility crisis. Over half of subfertile men suffer from asthenozoospermia and teratozoospermia, with mechanisms still largely unknown. Traditional small noncoding RNA sequencing (sncRNA-seq) primarily targets miRNAs, failing to capture the broader spectrum of small noncoding RNAs (sncRNAs), including abundant transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs). These sncRNAs possess complex RNA modifications and non-canonical terminal structures, impeding their accurate profiling. In this prospective cohort study, we addressed these limitations by combining our state-of-the-art PANDORA-seq with traditional sncRNA-seq, which generated the most comprehensive sncRNA landscape of human sperm from 25 participants with asthenozoospermia, teratozoospermia, or normozoospermia. PANDORA-seq significantly improved the annotation efficiency of sncRNAs and delivered a more detailed characterization for tsRNAs and rsRNAs, which were strongly correlated with key clinical indicators of sperm quality, thereby enhancing our understanding of the landscape of human sperm sncRNAome and its association with male subfertility. Importantly, machine learning with Lasso regression identified specific tsRNA/rsRNA signatures as highly effective clinical biomarkers (AUC ≥ 0.83) for predicting sperm abnormalities, offering significant improvements over World Health Organization-based semen quality assessments and novel insights for clinical diagnosis.

Male infertility, often caused by structural and functional sperm defects, remains genetically unexplained in a substantial proportion of cases. ATP1A4 encodes a testis-specific isoform of the Na+, K+-ATPase, a membrane enzyme crucial for maintaining cellular ionic homeostasis. Previous studies on Atp1a4 knockout mice have demonstrated severe defects in sperm motility and flagellar architecture; however, the contribution of ATP1A4 variants to human male reproduction remains to be elucidated. In this study, we identified compound biallelic variants in ATP1A4, a missense variant (c.2578 T>A, p.Tyr860Asn) and a frameshift variant (c.2582del, p.Gly861Aspfs*5), in a patient presenting with severe oligoasthenoteratozoospermia. Both variants markedly affected ATP1A4 protein expression. Morphological analyses revealed coiled and folded flagella, disrupted mitochondrial sheaths, and irregular head morphology in the patient's spermatozoa. Expression profiling revealed that ATP1A4 was highly enriched in post-meiotic spermatids and localized along the entire flagellum of mature sperm in both humans and mice, indicating a critical role in flagellar assembly and structural integrity. Notably, intracytoplasmic sperm injection (ICSI) in this patient resulted in low fertilization efficiency and failed implantation, suggesting a potential adverse impact of ATP1A4 deficiency on sperm functional competence beyond motility. These findings broaden the genetic spectrum of oligoasthenoteratozoospermia and highlight ATP1A4 as a potential gene associated with human male infertility.

Sperm cryopreservation is a fundamental technique in assisted reproductive technologies. While human sperm freezing is clinically routine, preserving nonhuman primate sperm remains challenging due to species-specific characteristics and low post-thaw viability. This study aims to investigate the proteomic changes in rhesus macaque sperm before and after cryopreservation, focusing on identifying the molecular mechanisms behind cryoinjury. Semen was collected from six sexually mature male rhesus macaques (6-10 years) using rectal electroejaculation. Motility parameters were quantified pre- and postcryopreservation using computer-assisted semen analysis (CASA) with vapor-phase liquid nitrogen protocols. Data-independent acquisition (DIA) quantitative proteomics, combined with LC-MS/MS, was used to profile global protein expression changes. Differential protein analysis was performed using multibioinformatics tools for functional annotation and network modeling. Papanicolaou and acridine orange (AO) staining were used to assess sperm morphology and DNA integrity, while reactive oxygen species (ROS) assay and immunofluorescence were employed to validate the proteomic analysis results. We identified 512 differentially expressed proteins (32 upregulated, 480 downregulated), which are predominantly enriched in pathways related to metabolic reprogramming, oxidative stress response, and protein homeostasis. Immunofluorescence staining further confirmed markedly reduced levels of SOD1, PGK2, PKC‑alpha, and phospho‑tyrosine in sperm after freeze-thaw cycling. These molecular hubs are associated with sperm motility and functional stability and may be essential for regulating energy metabolism, oxidative balance, and proteotoxicity during cryopreservation-induced stress in rhesus macaque sperm. Cryopreservation induces profound proteomic remodeling in rhesus macaque sperm, mitochondrial electron transport, and metabolic homeostasis. Our findings reveal conserved and species-specific cryoinjury mechanisms, offering molecular targets for optimizing cryopreservation protocols in nonhuman primates to advance reproductive conservation strategies.

Pregnancy outcomes after TAI with cooled versus frozen bovine semen: A meta-analysis.

The aim of the study was to assess the effect of the sperm concentration in boar ejaculates on the integrity of the sperm cell membrane during storage of liquid semen at 17C. The study was carried out on 16 Landrace boars used for artificial insemination, at the age of 18–24 months. Boars were selected for the study on the basis of measurements of the sperm concentration in all ejaculates collected from the start of their use for artificial insemination, collected by the gloved-hand method every 4–5 days. Two groups of boars were distinguished: boars producing ejaculates with a sperm concentration of at least 500 x 106/mL (group I) and boars producing ejaculates with a sperm concentration below 500 x 106/mL (group II). Ejaculate testing was carried out five times: on the day of collection and then after 24 hours, 48 hours, 96 hours and 168 hours. Sperm viability was assessed by two methods: the eosin-nigrosin technique and SYBR-14/PI fluorescence microscopy. Statistical analysis of the results was carried out by analysis of variance (ANOVA) using STATISTICA software v. 3.21. The results showed that greater changes in the cell membrane permeability of the sperm head take place in semen with a sperm concentration of ≥500 x 106/mL than in semen with a sperm concentration of <500 x 106/mL. This was observed in the case of both the fluorescence method and the eosin-nigrosin technique. Sperm from ejaculates with a higher sperm count are more sensitive to the conditions associated with storage at 17C than sperm from ejaculates with a sperm count below 500 x 106/mL.

Protective effects of seminal exosomes on cryopreserved sperm via inhibiting oxidative damage.

The 24,25(OH)2D3-TLCD3B signaling complex and reproductive function in mice and men.

Immediate and delayed impact of heat stress on sperm quality in a key pollinator, the buff-tailed bumblebee (Bombus terrestris).

We report the design, synthesis, and characterization of a series of targeted degraders directed against testis-specific serine/threonine kinases TSSK1 and TSSK2. A stable CHO-K1 cell line expressing HiBiT-tagged TSSK1 enabled quantitative cellular profiling and identified compound 5.1 as a potent TSSK1 degrader with a DC50 of 10 nM and a Dmax of 68% after 48 h. In contrast, ex vivo incubation of CD1 mouse sperm with degrader 5.4 induced up to an 80% reduction of the TSSK2 isoform within 4 h and translated into profound functional loss, resulting in a 97% reduction in sperm motility and near-complete loss of in vitro fertilization. Both alkyl- and PEG-linked degraders displayed metabolic N-dealkylation and high efflux, and therefore will require optimization. These data provide proof of concept that targeted degradation can occur within sperm cells, leading to a disruption of sperm function and encouraging further optimization toward in vivo evaluation for male contraception.

Haploid embryo seed production has been applied to accelerate plant breeding efficiency. Several genes, including DUF679 domain membrane protein (DMP) and phospholipase D3 (PLD3), are involved in maternal haploid induction in maize (Zea mays L.) and other plant species. However, how these gene variants trigger haploid induction and how they functionally interact remain largely unknown. Here, we generated CRISPR-induced DMP-knockout and examined the effects of DMP loss on the lipid composition of pollen and sperm cells in maize. Disruption of DMP led to a pronounced increase in phosphatidic acid (PA) accompanied by a decrease in phosphatidylcholine in sperm cells, with similar but weaker effects in pollen. Consistently, dmp mutants exhibited elevated transcript and protein levels of ZmPLDs, enzymes that hydrolyze phospholipids to produce PA, in both pollen and sperm cells. Immunoblot and PLD activity assays using isolated sperm cell proteins demonstrated the presence of active PLD enzymes in sperm cells and that DMP suppresses PLD activity. Manipulating sperm cell lipid composition further showed that increased PA levels, as well as the addition of DMP, enhance membrane fusogenicity. Structurally, DMP contains an N-terminal intrinsically disordered region and C-terminal transmembrane domains, and the full-length protein is required to suppress PLD activity and to promote membrane fusion. Together, these results indicate that DMP and PA have additive and compensatory effects on membrane fusion, while DMP suppresses PLD expression and PA production. These findings reveal a previously unrecognized role of DMP and its regulatory interplay with PLD in maintaining lipid homeostasis and modulating membrane fusion, providing mechanistic insights into maternal haploid induction.

Equilibration, a critical phase in cryopreservation, allows sperm cells to adapt to cryoprotective agents (CPAs) before freezing. This study investigated the impact of extended equilibration durations on the quality of frozen-thawed ram spermatozoa. Semen samples from five healthy Merino rams were pooled, extended with a Tris-based extender, and equilibrated for 3h (control), 24, 48, or 72h. post-equilibration and post-thaw sperm quality parameters, including motility, plasma membrane and acrosome integrity (PMAI), high mitochondrial membrane potential (HMMP), mitochondrial reactive oxygen species (MROS) levels, lipid peroxidation (LPO), and intracellular calcium levels (ICL) were analysed. Short equilibration durations (3-24h) preserved motility, PMAI, and HMMP while minimizing MROS production, lipid peroxidation, and calcium influx. Conversely, prolonged equilibration (48-72h) leads to CPA toxicity, oxidative stress, and premature capacitation-like changes, impairing sperm viability. Notably, 3-hour equilibration maintained post-thawed sperm with the highest motility (83.96 ± 1.87%) and lowest lipid peroxidation levels (11.46 ± 2.12%). These findings emphasize that shorter equilibration times optimize CPA penetration and minimize cryodamage, thereby enhancing post-thaw sperm function. These results provide valuable insights into refining cryopreservation protocols to improve freezing -thawing outcomes in ram sperm freezing centres.

Backgrounds Varicocele is a leading cause of male infertility, primarily manifesting as asthenozoospermia. The underlying molecular mechanisms remain unclear, and the potential role of protein lactylation in this condition is unknown. Methods We performed a comprehensive lactylome analysis using data-independent acquisition (DIA) mass spectrometry on paired sperm samples collected from patients with varicocele-associated asthenozoospermia before and after treatment. Differential lactylation analysis and bioinformatic enrichment analyses were conducted. Results Disrupted lactate metabolism was observed in asthenozoospermic sperm, with decreased intracellular L-lactate and elevated seminal plasma L-lactate. This was concomitant with a global reduction in protein lactylation, particularly in the sperm midpiece and tail. Lactylome profiling identified 2,699 lactylation sites on 1,216 proteins. Comparison of pre- and post-treatment samples revealed 133 differentially lactylated proteins, which were significantly enriched in pathways critical for sperm motility, including glycolysis/gluconeogenesis, microtubule-based movement, and flagellar assembly. Notably, lactylation abundance of key proteins (TEKT3, AKAP4, TUBA1A, AKAP3, TUBB4B) were significantly downregulated. Conclusions This study presents an initial characterization of the protein lactylation landscape in human sperm, revealing​ its alteration in the context of varicocele-associated asthenozoospermia. The findings suggest that dysregulation of the lactate-lactylation axis correlates with impaired sperm motility, potentially through concurrent alterations in energy metabolism and flagellar structure. This correlation underscores specific lactylation events that warrant future mechanistic investigation.

Sperm cryopreservation induces intracellular oxidative stress. Sesamol (SE), as an antioxidant, mitigates cryodamage caused by spermatozoa cryopreservation. This study was designed to evaluate the protective effects of SE-loaded nanostructured lipid carriers (SE-loaded NLCs) on frozen sperm quality. SE-loaded NLCs were synthesized using hot homogenization, followed by characterization for particle size, morphology, and encapsulation efficiency (EE). Normozoospermic semen samples were collected from 20 men and prepared according to the WHO 2021 guidelines. The samples were allocated into experimental groups of fresh, frozen-thawed control, SE-treated, blank NLCs, and SE-loaded NLCs. Following semen cryopreservation and thawing protocols, sperm quality was evaluated regarding motility, vitality, morphology, membrane and acrosome integrity, mitochondrial membrane potential (MMP), and DNA fragmentation using established staining and microscopy techniques. Statistical analysis was also performed at the significance level of 0.05. NLCs were successfully prepared with a mean particle size of 154 nm, zeta potential of -17.6 mV, and EE of 72 ± 2.64%. Fluorescence microscopy confirmed the efficient uptake of NLCs by spermatozoa within 60 minutes. The optimal SE dose was determined as 50 µM for cryoprotection. Sperm treated with SE-loaded NLCs showed improvements (p < 0.05) in motility, viability, membrane and acrosome integrity, MMP, and reduced (p < 0.05) DNA fragmentation than other groups. Application of SE-loaded NLCs showed accelerated protective effects compared with free SE, highlighting the associated potential in the enhancement of sperm quality during cryopreservation. SE-loaded NLCs effectively enhanced sperm quality indices during cryopreservation. Thus, SE-loaded NLCs represent a promising strategy to mitigate cryoinjury and improve fertility preservation outcomes.

Faithful chromosome segregation relies on precise kinetochore-microtubule interactions and checkpoint surveillance, yet the molecular basis of these processes varies widely across eukaryotes and is only beginning to be defined in apicomplexan parasites. In the malaria parasite Plasmodium berghei, chromosome segregation is especially critical during transmission from host to mosquito: rapid mitoses generate male gametes, and meiosis in the zygote seeds the next round of infection. Here, we identify Aurora-related kinase 1 (ARK1) as a central regulator of chromosome segregation in both mitotic and meiotic contexts. ARK1 localises to spindle poles, spindles, and kinetochores, and its depletion results in short and multipolar spindles, kinetochore misalignment, and failed chromosome partitioning. ARK1 forms a minimal Chromosomal Passenger Complex (CPC) with INCENP-A during male gametogenesis, but associates with additional components, including INCENP-B, kinetochores, centromeric histones, and spindle assembly checkpoint proteins, during meiosis. This stage-specific modularity supports efficient male gamete formation while safeguarding faithful chromosome inheritance during zygote development, thereby ensuring parasite transmission to the mosquito. Together, our findings indicate that P. berghei deploys distinct CPC states across sexual development, revealing developmental plasticity in chromosome-segregation control and a potential vulnerability for blocking transmission.

This study compares three different methods of sperm isolation using a novel laboratory test that permits the parallel assessment of vitality and DNA integrity in the same sperm cell. Three different sperm isolation procedures were compared including the electrophoretic Felix System, swim-up and density gradient centrifugation (DGC), each of which utilized a different separation principle comprising net charge, intrinsic motility and isopycnic density, respectively. These sperm isolation methods were also challenged with the more difficult task of isolating spermatozoa following cryopreservation, which compromises sperm vitality, motility and DNA integrity. To facilitate this comparison, a novel assessment technique was developed wherein a live/dead stain that formed covalent bonds with intracellular amines in non-viable spermatozoa was combined with the Sperm Chromatin Dispersal (SCD) assay for DNA integrity. Overall, Felix generated populations of spermatozoa that were superior to DGC (p < 0.001), and equivalent to swim-up, in terms of optimizing the proportion of 'live/DNA intact' cells and rejecting the 'dead/DNA intact' and 'dead/DNA damaged' subpopulations. The development of a novel method for the parallel assessment of vitality and DNA damage facilitated comparison of different sperm isolation techniques and may find applications in other areas of diagnostic andrology.

Nutrition of honeybees is constrained by the ratios of essential amino acids in pollen protein.

Liposomal delivery of gallic acid enhances human spermatozoa protection during freeze-thaw cycles.

Commercially available disposable products such as cell culture utensils and catheters do not necessarily possess sufficient quality for in vitro fertilization (IVF), from the perspective of pyrogen contamination. We aimed to comprehensively analyze the pyrogen contamination status, including bacterial endotoxins, of the products used for IVF in assisted reproductive technology (ART) and improve their cleanliness using a new sterilization technology. Pyrogen contamination levels were evaluated using a direct human cell-based pyrogen test that is not affected by the recovery ratio, unlike bacterial endotoxin tests. Pyrogen inactivation tests were performed using low-temperature ozone/hydrogen peroxide gas treatment. The residual hydrogen peroxide was colorimetrically quantified, and the effectiveness of its removal by drying treatment was evaluated using germ cell viability as an indicator. Significant amounts of pyrogen, from 0.014 to 1.110 EU/product, were detected in seven of the twenty products. Pyrogen contamination levels were reduced below the detection limit by ozone/hydrogen peroxide gas sterilization. Hydrogen peroxide remained on the surface of the GPS dish but was reduced to a level that did not affect human sperm viability and embryo development after drying at 80°C for 24 h following sterilization. These products may carry a potential risk of reducing ART success rates, and pyrogen contamination levels may exceed the previously reported allowable level of 0.01–0.02 EU/mL in human IVF during actual use. Our study suggests that the manufacturing of products free from pyrogens and without adverse effects on germ cells is possible using ozone/hydrogen peroxide gas sterilization and subsequent drying technologies.

Competition among related males, male gametes, or pollen grains to fertilize a limited number of females, eggs, or ovules (local mate competition) is expected to favor female-biased progeny sex ratios in dioecious species. In hermaphrodites, local mate competition should similarly promote female-biased sex allocation, with reduced investment in sperm or pollen relative to eggs, ovules, or seeds. Because inbreeding, especially self-fertilization, tends to generate local mate competition, female-biased sex allocation should be associated with positive inbreeding, for example as measured by FIS. Although inbreeding may result from mating among relatives within local populations, it can also be generated by population turnover in metapopulations subject to frequent local extinction and recolonization. This effect of population turnover has previously been considered for species with separate sexes. Here, we use formal analysis and individual-based simulations to show that population turnover can select for strongly female-biased sex allocation in hermaphroditic metapopulations, particularly when among-deme dispersal is low. When mating within established populations is random, so that FIS is zero, female-biased sex allocation is instead associated with positive FST, a measure of inbreeding at the metapopulation level caused by population structure. Our results thus extend local mate competition theory to hermaphroditic metapopulations and identify FST as a key predictor of sex allocation when inbreeding is generated by population turnover rather than by non-random mating within demes.