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Hyperglycemia, the hallmark of diabetes mellitus (DM), is the main cause of DM-related systemic complications, including reproductive issues. Furthermore, the incidence of DM in males of reproductive ages is becoming an increasing concern, as the complexity of sperm capacitation (an essential process for fertilizing the egg) extends beyond conventional sperm parameters such as count, viability, and motility. Capacitation defects cause male infertility, and DM-related hyperglycemia may affect this process. We explore the effects of uncontrolled hyperglycemia on sperm using alloxan-induced hyperglycemic Wistar rats. In addition to assessing conventional sperm parameters, we also evaluated functional indicators, including hyperactivation (HA) with a pharmacological approach and assessed its effects with a computer-assisted sperm analysis (CASA); fluorescence indicators to monitor membrane potential (Em R , DiSC 3 (5)) and mitochondrial membrane potential (Ψ, JC-1); CatSper activity, using its ability to permeate Na + ions, and ATP levels with the luciferin-luciferase reaction. We confirmed previous findings with our hyperglycemic model, which replicated the typical reduction on conventional sperm parameters. In sperm from hyperglycemic rats, we observed increased motility and HA levels after pharmacological treatment. Additionally, CatSper activity was unaffected by hyperglycemia, while Em R was hyperpolarized under non-capacitating condition. Finally, we noted a low percentage of hyperpolarized Ψ and reduced ATP content. This study highlights the significance of impact of hyperglycemia on sperm physiology and capacitation. We proposed that low ATP levels perturb energy state, signaling pathways, ion channels activity, motility, and HA. Our findings offer insight into DM-associated infertility and potential treatment strategies., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aims: Many studies demonstrated reproductive damage in men residing in plains who are exposed to hypoxia at high altitudes. However, little is known about mechanisms between male reproductive impairment and hypobaric hypoxia. Hypoxia is one of the reasons for the imbalance of cellular redox system. Ferroptosis, involved in many pathophysiological progresses, is an oxidative damage-related, iron-dependent regulated cell death, which needs exogenous inducer. In our study, we explored the mechanism between hypoxia and male reproductive dysfunction., Materials and Methods: Here, we established animal model simulating hypobaric hypoxia at an altitude of 5000 m and used ELISA, WB, qPCR, flow cytometry and etc. to obtain different results., Key Findings: The results demonstrated decrease of plasma testosterone (T) and free testosterone (FT) levels under hypoxia, meanwhile there's decline in sperm counts and sperm motility, coupled with increase in sperm malformation rates. Flow cytometry confirmed significant reduction in Leydig cell numbers. Prussian blue staining showed iron depositions in interstitial testis. Features of ferroptosis such as increased MDA (malondialdehyde) levels, reduced solute carrier family 7 member 11 (SLC7A11, xCT) and glutathione peroxidase 4 (GPX4) expression were observed in testis after hypoxic exposure. Further in vitro experiments, we observed that hypoxia suppressed xCT-GPX4 pathway and enhanced cellular ROS accumulation to lead Leydig cell proliferation activity decline., Significance: Our findings firstly indicated that hypoxia leads to male reproductive dysfunction via inducing Leydig cell ferroptosis. This discovery may offer a potential intervention target for addressing male reproductive injuries under hypoxic conditions., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Axonemal protein complexes, including the outer and inner dynein arms (ODA/IDA), are highly ordered structures of the sperm flagella that drive sperm motility. Deficiencies in several axonemal proteins have been associated with male infertility, which is characterized by asthenozoospermia or asthenoteratozoospermia. Dynein axonemal heavy chain 3 (DNAH3) resides in the IDA and is highly expressed in the testis. However, the relationship between DNAH3 and male infertility is still unclear. Herein, we identified biallelic variants of DNAH3 in four unrelated Han Chinese infertile men with asthenoteratozoospermia through whole-exome sequencing (WES). These variants contributed to deficient DNAH3 expression in the patients' sperm flagella. Importantly, the patients represented the anomalous sperm flagellar morphology, and the flagellar ultrastructure was severely disrupted. Intriguingly, Dnah3 knockout (KO) male mice were also infertile, especially showing the severe reduction in sperm movement with the abnormal IDA and mitochondrion structure. Mechanically, nonfunctional DNAH3 expression resulted in decreased expression of IDA-associated proteins in the spermatozoa flagella of patients and KO mice, including DNAH1, DNAH6, and DNALI1, the deletion of which has been involved in disruption of sperm motility. Moreover, the infertility of patients with DNAH3 variants and Dnah3 KO mice could be rescued by intracytoplasmic sperm injection (ICSI) treatment. Our findings indicated that DNAH3 is a novel pathogenic gene for asthenoteratozoospermia and may further contribute to the diagnosis, genetic counseling, and prognosis of male infertility., Competing Interests: XW, GS, YY, CJ, TR, XY, LZ, YZ, YO, XZ, SL, XT, TL, YS No competing interests declared, (© 2024, Wang, Shen, Yang et al.)

Germ cell development and gamete production in animals require small RNA pathways. While studies indicate that microRNAs (miRNAs) are necessary for normal sperm production and function, the specific roles for individual miRNAs are largely unknown. Here, we use small RNA sequencing (RNA-seq) of dissected gonads and functional analysis of new loss-of-function alleles to identify functions for miRNAs in the control of fecundity and sperm production in Caenorhabditis elegans males and hermaphrodites. We describe a set of 29 male gonad-enriched miRNAs and identify a set of individual miRNAs (mir-58.1 and mir-235) and a miRNA cluster (mir-4807-4810.1) that are required for optimal sperm production at 20°C and a set of miRNAs (mir-49, mir-57, mir-83, mir-261, and mir-357/358) that are required for sperm production at 25°C. We observed defects in meiotic progression in mutants missing mir-58.1, mir-83, mir-235, and mir-4807-4810.1, which may contribute to the observed defects in sperm production. Further, analysis of multiple mutants of these miRNAs suggested genetic interactions between these miRNAs. This study provides insights on the regulatory roles of miRNAs that promote optimal sperm production and fecundity in males and hermaphrodites., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Adhesion G protein-coupled receptors are unique molecules. They are able to transmit classical signals via G protein activation as well as mediate functions solely through their extracellular N termini, completely independently of the seven transmembrane helices domain and the C terminus. This dual mode of action is highly unusual for G protein-coupled receptors and allows for a plethora of possible cellular consequences. However, the physiological implications and molecular details of this N terminus-mediated signaling are poorly understood. Here, we show that several distinct seven transmembrane helices domain-independent/trans functions of the adhesion G protein-coupled receptor latrophilin homolog latrophilin-1 in the nematode Caenorhabditis elegans together regulate reproduction: sperm guidance, ovulation, and germ cell apoptosis. In these contexts, the receptor elicits its functions in a noncell autonomous manner. The functions might be realized through alternative splicing of the receptor specifically generating N terminus-only variants. Thus, our findings shed light on the versatility of seven transmembrane helices domain-independent/N terminus-only/trans functions of adhesion G protein-coupled receptor and discuss possible molecular details., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Sperm motility is a crucial factor in male fertility. Photobiomodulation (PBM) has been reported to increase sperm motility, but a consistent approach suitable for identifying standardizable protocols is lacking. We collected asthenozoospermic (n = 70) and normozoospermic (n = 20) semen. The asthenozoospermic samples were irradiated with an 810 nm diode laser, in continuous wave mode, at 0.25 W, 0.5 W, 1 W and 2 W for 60 s on a circular area of 1 cm 2  through a novel handpiece with an innovative flat-top profile. Sperm motility was assessed immediately, after 30 and 60 min. A sample size calculator, unpaired t-test and one-way ANOVA with post-hoc Tukey HSD tests were used for statistics. One and 2 W were the most effective outputs in increasing progressive motility compared to control (p < 0.001). The maximum effect was immediately after 1 W-PBM (p < 0.001) and decreased after 60 min (p < 0.001). Time physiologically decreased vitality (p < 0.001), but less in the 1 W-PBM samples (p < 0.05). 1 W-PBM did not affect chromatin condensation. Asthenozoospermic samples displayed an impairment of 80% in oxygen consumption and ATP production and a slight inefficiency of oxidative phosphorylation compared to normozoospermic samples (p < 0.001). 1 W-PBM partially restored the functionality of aerobic metabolism (p < 0.001) by recovery of oxidative phosphorylation efficiency. PBM did not affect lactate dehydrogenase (glycolysis pathway). No irradiated samples increased accumulated malondialdehyde, a marker of lipidic peroxidation. In conclusion, PBM improves progressive motility in asthenozoospermia through increased mitochondrial energetic metabolism without harmful oxidative stress., (© 2024. The Author(s).)

Male infertility presents a substantial challenge in reproductive medicine, often attributed to impaired sperm motility. The present study investigates the role of CCDC28A, a protein expressed specifically in male germ cells, whose paralog CCDC28B has been implicated in ciliogenesis. We identify unique expression patterns for CCDC28A and CCDC28B within the mouse testes, where CCDC28A is expressed in germ cells, whereas CCDC28B is expressed in supporting somatic cells. Through knockout mouse models and histological analyses, we reveal that CCDC28A deficiency results in diminished sperm motility and structural aberrations in sperm tails, notably affecting the head-tail coupling apparatus (HTCA), thereby causing male infertility. Fine structural analyses by transmission electron microscopy reveal disruptions at the capitulum-basal plate junction of the HTCA in the CCDC28A mutants. This results in the bending of the head within the neck region, often accompanied by thickening of the tail midpiece. Our discovery demonstrates that CCDC28A plays an essential role in male fertility and sperm tail morphogenesis through the formation of HTCA., (© 2024. The Author(s).)

Spermatogenesis starts with the onset of puberty within the seminiferous epithelium of the testes. It is a complex process under intricate control of the endocrine system. Physiological regulations by steroid hormones in general and by estrogens in particular are due to their chemical nature prone to be disrupted by exogenous factors acting as endocrine disruptors (EDs). 17α-Ethynylestradiol (EE2) is an environmental pollutant with a confirmed ED activity and a well-known effect on spermatogenesis and chromatin remodeling in haploid germ cells. The aim of our study was to assess possible effects of two doses (2.5ng/ml; 2.5 μg/ml) of EE2 on both histone-to-protamine exchange and epigenetic profiles during spermatogenesis performing a multi/transgenerational study in mice. Our results demonstrated an impaired histone-to-protamine exchange with a significantly higher histone retention in sperm nuclei of exposed animals, when this process was accompanied by the changes of histone post-translational modifications (PTMs) abundancies with a prominent effect on H3K9Ac and partial changes in protamine 1 promoter methylation status. Furthermore, individual changes in molecular phenotypes were partially transmitted to subsequent generations, when no direct trans-generational effect was observed. Finally, the uncovered specific localization of the histone retention in sperm nuclei and their specific PTMs profile after EE2 exposure may indicate an estrogenic effect on sperm motility and early embryonic development via epigenetic mechanisms., (© 2024. The Author(s).)

Background: Chickens play a crucial role as the primary global source of eggs and poultry, and the quality of rooster semen significantly impacts poultry reproductive efficiency. Therefore, it is imperative to comprehend the regulatory mechanisms underlying sperm development., Results: In this study, we established transcriptome profiles of lncRNAs, miRNAs, and mRNAs in 3 testis tissues and 3 epididymis tissues from "Jing Hong No.1" roosters at 24, 35, and 64 weeks of age. Using the data, we conducted whole transcriptome analysis and constructed a ceRNA network. We detected 10 differentially expressed mRNAs (DEmRNAs), 33 differentially expressed lncRNAs (DElncRNAs), and 10 differentially expressed miRNAs (DEmiRNAs) in the testis, as well as 149 DEmRNAs, 12 DElncRNAs, and 10 DEmiRNAs in the epididymis. These genes were found to be involved in cell differentiation and development, as well as various signaling pathways such as GnRH, MAPK, TGF-β, mTOR, VEGF, and calcium ion pathways. Subsequently, we constructed two competing endogenous RNA (ceRNA) networks comprising DEmRNAs, DElncRNAs, and DEmiRNAs. Furthermore, we identified four crucial lncRNA-mRNA-miRNA interactions that govern specific biological processes in the chicken reproductive system: MSTRG.2423.1-gga-miR-1563-PPP3CA and MSTRG.10064.2-gga-miR-32-5p-GPR12 regulating sperm motility in the testis; MSTRG.152556.1-gga-miR-9-3p-GREM1/THYN1 governing immunomodulation in the epididymis; and MSTRG.124708.1-gga-miR-375-NDUFB9/YBX1 controlling epididymal sperm maturation and motility., Conclusions: Whole transcriptome sequencing of chicken testis and epididymis screened several key genes and ceRNA regulatory networks, which may be involved in the regulation of epididymal immunity, spermatogenesis and sperm viability through the pathways of MAPK, TGF-β, mTOR, and calcium ion. These findings contribute to our comprehensive understanding of the intricate molecular processes underlying rooster spermatogenesis, maturation and motility., (© 2024. The Author(s).)

Characterization of long non-coding telomeric repeat-containing RNAs in sperm of normozoospermic and oligoasthenozoospermic men as new biomarker of idiopathic male infertility. We conducted an observational prospective study with two groups of men with normal or orligoasthenozoospermic spermiogram, aged 40 and above. Fertility parameters were analyzed in men undergoing intracytoplasmic sperm injection with donor oocytes, to avoid the female factor. Telomeric RNAs and telomere length were measured by quantitative fluorescent in situ hybridization. Data from seminal parameters and in-vitro fertilization were assessed according to IVIRMA protocols. Patients with oligoasthenozoospermia, who had worse seminal parameters, also obtained embryos with lower inner-cell-mass quality (p = 0.04), despite using donor oocytes. While mean levels of telomeric RNAs were similar for both groups, the percentage of spermatozoa with more than 3 foci was higher in oligoasthenozoospermic men (p = 0.02). Regarding telomere length, oligoasthenozoospermic men had shorter mean, a higher accumulation of short telomeres (15th percentile; p = 0.03) and a lower percentage of very-long telomeres (85th percentile; p = 0.01). Finally, a positive correlation was found between telomeric-RNAs intensity and total progressive motility in the spermatozoa of normozoospermic patients (r = 0.5; p = 0.03). Telomeric parameters were altered in the spermatozoa of the oligoasthenozoospermic group, which also showed lower quality embryos. Interestingly, in the normozoospermic group, a correlation was found between progressive motility and telomeric RNA levels, suggesting that they could be a good biomarker of sperm quality. Further studies are required to confirm these results and translate them into the clinical practice.Trial registration number: 1711-MAD-109-CB, 07/07/2021., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Study Question: Do biallelic deleterious variants of Calreticulin 3 (CALR3) cause fertilization failure (FF), resulting in male infertility in humans?, Summary Answer: Biallelic mutations in CALR3 were identified in two infertile men from unrelated families and were shown to cause FF associated with failed sperm-zona pellucida (ZP) binding., What Is Known Already: In male mice, the Calr3-knockout has been reported to cause male infertility and FF. However, the mechanism behind this remains unclear in humans., Study Design, Size, Duration: Sequencing studies were conducted in a research hospital on samples from Han Chinese families with primary infertility and sperm head deformations to identify the underlying genetic causes., Participants/materials, Setting, Methods: Data from two infertile probands characterized by sperm head deformation were collected through in silico analysis. Sperm cells from the probands were characterized using light and electron microscopy and used to verify the pathogenicity of genetic factors through functional assays. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of FF. ICSI were administered to overcome CALR3-affected male infertility., Main Results and the Role of Chance: Novel biallelic deleterious mutations in CALR3 were identified in two infertile men from unrelated families. We found one homozygous frameshift CALR3 mutation (M1: c.17&#95;27del, p.V6Gfs*34) and one compound heterozygous CALR3 mutation (M2: c.943A>G, p.N315D; M3: c.544T>C, p.Y182H). These mutations are rare in the general population and cause acrosomal ultrastructural defects in affected sperm. Furthermore, spermatozoa from patients harbouring the CALR3 mutations were unable to bind to the sperm-ZP or they disrupted gamete fusion or prevented oocyte activation. Molecular assays have revealed that CALR3 is crucial for the maturation of the ZP binding protein in humans. Notably, the successful fertilization via SUZI and ICSI attempts for two patients, as well as the normal expression of PLCζ in the mutant sperm, suggests that ICSI is an optimal treatment for CALR3-deficient FF., Limitations, Reasons for Caution: The results are based on sperm-related findings from two patients. Further studies are required to gain insight into the developmental stage and function of CALR3 in human testis., Wider Implications of the Findings: Our findings highlight the underlying risk of FF associated with sperm defects and provide a valuable reference for personalized genetic counselling and clinical treatment of these patients., Study Funding/competing Interest(s): This study was supported by the National Key R&D Program of China (2021YFC2700901), Hefei Comprehensive National Science Center Medical-Industrial Integration Medical Equipment Innovation Research Platform Project (4801001202), the National Natural Science Foundation of China (82201803, 82371621, 82271639), Foundation of the Education Department of Anhui Province (gxgwfx2022007), Key Project of Natural Science Research of Anhui Educational Committee (2023AH053287), and the Clinical Medical Research Transformation Project of Anhui Province (202204295107020037). The authors declare no competing interests., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Gut microbiota symbiosis faces enormous challenge with increasing exposure to drugs such as environmental poisons and antibiotics. The gut microbiota is an important component of the host microbiota and has been proven to be involved in regulating spermatogenesis, but the molecular mechanism is still unclear. A male mouse model with gut microbiota depletion/dysbiosis was constructed by adding combined antibiotics to free drinking water, and reproductive parameters such as epididymal sperm count, testicular weight and paraffin sections were measured. Testicular transcriptomic and serum metabolomic analyses were performed to reveal the molecular mechanism of reproductive dysfunction induced by gut microbiota dysbiosis in male mice.This study confirms that antibiotic induced depletion of gut microbiota reduces sperm count in the epididymis and reduces germ cells in the seminiferous tubules in male mice. Further study showed that exosomes isolated from microbiota-depleted mice led to abnormally high levels of retinoic acid and decrease in the number of germ cells in the seminiferous tubules and sperm in the epididymis. Finally, abnormally high levels of retinoic acid was confirmed to disrupted meiotic processes, resulting in spermatogenesis disorders. This study proposed the concept of the gut microbiota-exosome-retinoic acid-testicular axis and demonstrated that depletion of the gut microbiota caused changes in the function of exosomes, which led to abnormal retinoic acid metabolism in the testis, thereby impairing meiosis and spermatogenesis processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media., Material and Methods: Processed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca 2 + concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca 2+ were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population., Results: After 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; P < 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % (P < 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (P < 0.01). Over time Ca 2+ increased in all treatment groups compared to time 0h., Discussion and Conclusion: Viable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters., Competing Interests: Declaration of competing interest All authors declare no potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Increasing fertility rates have become one of the factors that concern all people in the world. Therefore, the study aims to use two mutated strains of probiotics enriched with selenium (PSe40/60/1 and BSe50/20/1) to improve fertility. Thirty Swiss albino male mice were divided into three groups; control, LP + S was given Lactobacillus plantarum PSe40/60/1 plus selenium, and BL + S was given Bifidobacterium longum BSe50/20/1 plus selenium. Free testosterone, LH, and FSH were measured in serum by biochemical analysis. Testicular tissues were examined by histopathological analysis. The count and motility of sperm, and sperm abnormalities were determined by microscopic examination. The method of qRT-PCR was used to detect gene expression of Tspyl1, Hsd3b6, and Star genes. The biochemical results showed that serum content of free testosterone (FT) hormone had significantly increase in the BL + S and LP + S groups compared with control. Levels of LH and FSH hormones were the highest in the BL + S group. The treated groups showed all developmental stages of spermatogenesis, including spermatogenesis, spermatocytes, and seminiferous tubule spermatids, as well as intact Sertoli cells and Leydig cells without changes. When compared to the control group, sperm count and motility increased in the BL + S group, while sperm abnormalities decreased. The expression of Tspyl1 gene in testicular tissues decreased in the LP + S and BL + S groups, while the expression of Star and Hsd3b6 genes was higher in the BL + S group and lower in the LP + S group compared with the control group. Therefore, Bifidobacterium longum BSe50/20/1 enriched with selenium could be useful in enhancing male fertility., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Infertility is a major health issue, affecting 15% of reproductive-age couples with male factors contributing to 50% of cases. Asthenozoospermia (AS), or low sperm motility, is a common cause of male infertility with complex aetiology, involving genetic and metabolic alterations, inflammation and oxidative stress. However, the molecular mechanisms behind low motility are unclear. In this study, we used a metabolomics approach to identify metabolic biomarkers and pathways involved in sperm motility., Methods: We compared the metabolome and lipidome of spermatozoa of men with normozoospermia (n = 44) and AS (n = 22) using untargeted LC-MS and the metabolome of seminal fluid using 1 H-NMR. Additionally, we evaluated the seminal fluid redox status to assess the oxidative stress in the ejaculate., Results: We identified 112 metabolites and 209 lipids in spermatozoa and 27 metabolites in the seminal fluid of normozoospermic and asthenozoospermic men. PCA analysis of the spermatozoa's metabolomics and lipidomics data showed a clear separation between groups. Spermatozoa of asthenozoospermic men presented lower levels of several amino acids, and increased levels of energetic substrates and lysophospholipids. However, the metabolome and redox status of the seminal fluid was not altered inAS., Conclusions: Our results indicate impaired metabolic pathways associated with redox homeostasis and amino acid, energy and lipid metabolism in AS. Taken together, these findings suggest that the metabolome and lipidome of human spermatozoa are key factors influencing their motility and that oxidative stress exposure during spermatogenesis or sperm maturation may be in the aetiology of decreased motility in AS., (© 2024 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Bisphenol AF (BPAF) is increasingly used and now found in products intended for human consumption. The protective effect of 1,8-cineole (CIN) against BPAF-induced reproductive toxicity was investigated. Four groups were created, with each group consisting of eight rats: control, BPAF (200 mg/kg), CIN (200 mg/kg), and BPAF + CIN groups. The results demonstrated that the BPAF group exhibited a decline in testosterone levels and a decrease in sperm parameters compared with the control. Additionally, higher levels of MDA were observed, along with lower levels of GSH and GPx activity. CAT activity also decreased slightly. Tnf-α, Nf-κB levels were significantly higher, and caspase-3 expression was elevated, while PCNA expression decreased. BPAF significantly increased tissue degeneration compared with the control. However, the BPAF + CIN group showed statistically significant improvements in sperm parameters, except for concentration. They also exhibited an increase in testosterone levels and an improvement in MDA and GSH levels compared with the BPAF group. However, GPx activity partially enhanced. Tnf-α and Nf-κB levels were significantly reduced, and caspase-3 levels declined while PCNA and Bcl-2 levels increased. The Johnsen Testicular Biopsy score showed a substantial increase. Overall, these results suggest that CIN co-treatment in rats enhanced reproductive health and exhibited antioxidant, antiapoptotic, and anti-inflammatory properties against BPAF-induced testicular damage., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Background: Approximately 7% of the males exhibit reduced fertility; however, the regulatory genes and pathways involved remain largely unknown. TBC1 domain family member 21 (TBC1D21) contains a conserved RabGAP catalytic domain that induces GDP/GTP exchange to inactivate Rabs by interacting with microtubules. We previously reported that Tbc1d21-null mice exhibit severe sperm tail defects with a disrupted axoneme, and that TBC1D21 interacts with RAB10. However, the pathological mechanisms underlying the Tbc1d21 loss-induced sperm tail defects remain unknown., Results: Murine sperm from wild-type and Tbc1d21-null mice were comparatively analyzed using proteomic assays. Over 1600 proteins were identified, of which 15 were significantly up-regulated in Tbc1d21-null sperm. Notably, several tektin (TEKT) family proteins, belonging to a type of intermediate filament critical for stabilizing the microtubular structure of cilia and flagella, were significantly up-regulated in Tbc1d21 -/- sperm. We also found that TBC1D21 interacts with TEKT1. In addition, TEKT1 co-localized with RAB10 during sperm tail formation. Finally, we found Tbc1d21-null sperm exhibited abnormal accumulation of TEKT1 in the midpiece region, accompanied by disrupted axonemal structures., Conclusions: These results reveal that TBC1D21 modulates TEKTs protein localization in the axonemal transport system during sperm tail formation., (© 2024 American Association for Anatomy.)

Retinoic acid (RA) regulates all four major events in spermatogenesis; spermatogonial differentiation, meiotic entry, spermiogenesis, and spermiation. For the pre-meiotic phase, Sertoli cells are the source of RA and for the post-meiotic phase, pachytene spermatocytes are the source of RA. While the entire spermatogenic process is regulated by RA, how each of these phases is regulated by RA remains completely unknown. Homeobox B1 (Hoxb1) has two retinoic acid response elements (RARE) upstream and downstream of the gene. In this study, we investigated if RA facilitates spermatogenesis by its action on Hoxb1. The expressions of the Hoxb1 and Sonic hedgehog (Shh) genes were analyzed in the post-natal mouse testes and the testicular localizations of Hoxb1, Shh and Gli1 were analyzed by immunohistochemistry in the adult rat testis. To delineate the signaling mechanisms, Hoxb1 expression was altered in vitro and in vivo using retinoic acid and miR-361-3p. Finally, the levels of miR-361-3p and HOXB1 were analyzed in infertile human sperm samples. Hoxb1 and Shh gene expressions were found to be low in the testis of post-natal Swiss mice of 7, 14, 28, 35, and 60 days, after which the expressions of both spiked. Immunohistochemistry in the adult mouse testis showed the expressions of Hoxb1, Shh, and Gli1 in the elongating spermatids. Exposure of GC2 cells to RA and in vivo IP RA injection upregulated Hoxb1 and Shh signaling in the testis with increased expressions of Shh, Gli1, and Hdac1. Retinoic acid administration in Swiss mice compromised sperm production and reduced epididymal sperm count. The analysis of infertile human semen samples revealed an increased level of HOXB1 and a decreased level of miR-361-3p as compared to fertile controls. We conclude that retinoic acid regulates late stage of spermatogenesis (spermiogenesis) by affecting Hoxb1 and Shh signaling., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Phosducin-like proteins (PhLP) are thioredoxin domain-containing proteins that are highly conserved across unicellular and multicellular organisms. PhLP family proteins are hypothesized to function as co-chaperones in the folding of cytoskeletal proteins. Here, we present the initial molecular, biochemical, and functional characterization of CG4511 as Drosophila melanogaster PhLP3. We cloned the gene into a bacterial expression vector and produced enzymatically active recombinant PhLP3, which showed similar kinetics to previously characterized orthologues. A fly strain homozygous for a P-element insertion in the 5' UTR of the PhLP3 gene exhibited significant downregulation of PhLP3 expression. We found these male flies to be sterile. Microscopic analysis revealed altered testes morphology and impairment of spermiogenesis, leading to a lack of mature sperm. Among the most significant observations was the lack of actin cones during sperm maturation. Excision of the P-element insertion in PhLP3 restored male fertility, spermiogenesis, and seminal vesicle size. Given the high level of conservation of PhLP3, our data suggests PhLP3 may be an important regulator of sperm development across species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Petit et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Numerous variables that regulate the metabolism of Sertoli cells and sperm have been identified, one of which is sex steroid hormones. These hormones play a vital role in maintaining energy homeostasis, influencing the overall metabolic balance of the human body. The proper functioning of the reproductive system is closely linked to energy status, as the reproductive axis responds to metabolic signals. The aim of this study was to investigate the gene expression patterns of metabolite interconversion enzymes in testicular cells (Sertoli cells and spermatogonia) of non-obstructive azoospermia (NOA) patients, as compared to normal controls, to understand the molecular mechanisms contributing to NOA. We used microarray and bioinformatics techniques to analyze 2912 genes encoding metabolite interconversion enzymes, including methyltransferase, monooxygenase, transmembrane reductase, and phosphohydrolase, in both testicular cells and normal samples. In sperm, the upregulation of MOXD1, ACAD10, PCYT1A, ARG1, METTL6, GPLD1, MAOA, and CYP46A1 was observed, while ENTPD2, CPT1C, ADC, and CYB5B were downregulated. Similarly, in the Sertoli cells of three NOA patients, RPIA, PIK3C3, LYPLA2, CA11, MBOAT7, and HDHD2 were upregulated, while NAA25, MAN2A1, CYB561, PNPLA5, RRM2, and other genes were downregulated. Using STRING and Cytoscape, we predicted the functional and molecular interactions of these proteins and identified key hub genes. Pathway enrichment analysis highlighted significant roles for G1/S-specific transcription, pyruvate metabolism, and citric acid metabolism in sperm, and the p53 signaling pathway and folate metabolism in Sertoli cells. Additionally, Weighted Gene Co-expression Network Analysis (WGCNA) and single-cell RNA sequencing (scRNA-seq) were performed to validate these findings, revealing significant alterations in gene expression and cellular distribution in NOA patients. Together, these results provide new insights into the molecular mechanisms underlying NOA and identify potential therapeutic targets., (© 2024. The Author(s).)

Mammalian sperm are characterized as specialized cells, as their transcriptional and translational processes are largely inactive. Emerging researches indicate that Ca 2+ serves as a crucial second messenger in the modulation of various sperm physiological processes, such as capacitation, hyperactivation, and the acrosome reaction. Specifically, sperm-specific calcium channels, including CatSper, voltage-gated calcium channels (VGCCs), store-operated calcium channels (SOCCs), and cyclic nucleotide-gated (CNG) channels, are implicated in the regulation of calcium signaling in mammalian sperm. Calcium stores located in the sperm acrosomes, along with the IP3 receptors in the neck of the redundant nuclear envelope and the mitochondria in the tail, play significant roles in modulating intracellular Ca 2+ levels in sperm. However, the functions and mechanisms of these calcium channels in modulating mammalian sperm physiological functions have not yet been well elucidated. Therefore, by focusing on humans and mice, this study aims to provide a comprehensive review of the current advancements in research regarding the roles of calcium signaling and associated calcium channels in regulating sperm function. This endeavor seeks to enhance the understanding of calcium signaling in sperm regulation and to facilitate the development of drugs for the treatment of infertility or as non-hormonal male contraceptives., Competing Interests: The authors declare that they have no competing interests., (© 2024 Yang et al.)

Protein malnutrition during critical periods poses significant risks to reproductive health. Thus, this study aims to evaluate the immediate and delayed effects of a 30-day low-protein diet on the postnatal development of the male reproductive system. For so, male rats were fed a protein-deficient diet from postnatal day 30-60, followed by evaluations of testis, epididymis, and spermatozoa both at the end of the diet and after a 60-day recovery period. Testicular and epididymal weight was lowered in pubertal animals. Several histological alterations were found in the testis, such as acidophilic cells and vacuoles in the seminiferous epithelium, and sperm production was compromised. In the epididymis, the luminal compartment was diminished, and the stroma was enlarged both in the caput and cauda; in the cauda, the epithelial compartment was enlarged; the transit time of spermatozoa through this organ was diminished. Testosterone production was lowered. Spermatozoa's motility, mitochondrial activation, and acrosomal integrity were impaired, and several alterations in morphology were observed. After the recovery period, testicular and epididymal weight was restored. Tissue remodulation was observed in the epididymis, but the spermatozoa's transit time in this organ was not altered. Sperm and testosterone production, spermatozoa motility, mitochondrial activation, and acrosomal integrity were also restored. However, testicular histological alterations and spermatic morphological abnormalities were maintained in protein-restricted animals. Protein restriction during peripuberty impairs the reproductive maturation of pubertal Wistar rats, impairing testicular and epididymal function, with lasting effects even after dietary correction.

Pre-implantation genetic testing (PGT) is a crucial process for selecting embryos created through assisted reproductive technology (ART). Couples with chromosomal rearrangements, infertility, recurrent miscarriages, advanced maternal age, known single-gene disorders, a family history of genetic conditions, previously affected pregnancies, poor embryo quality, or congenital anomalies may be candidates for PGT. Preimplantation genetic testing for aneuploidies (PGT-A) enables the selection and transfer of euploid embryos, significantly enhancing implantation rates in assisted reproduction. Fluorescence in situ hybridization (FISH) is the preferred method for analyzing biopsied cells to identify these abnormalities. While FISH is a well-established method for identifying sperm aneuploidy, NGS offers a more comprehensive assessment of genetic material, potentially enhancing our understanding of male infertility. Chromosomal abnormalities, arising during meiosis, can lead to aneuploid sperm, which may hinder embryo implantation and increase miscarriage rates. This review provides a comparative analysis of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) in sperm evaluations, focusing on their implications for preimplantation genetic testing. This analysis explores the strengths and limitations of FISH and NGS, aiming to elucidate their roles in improving ART outcomes and reducing the risk of genetic disorders in offspring. Ultimately, the findings will inform best practices in sperm evaluations and preimplantation genetic testing strategies.

Oxidative stress (OS) is believed to be a significant factor in the decline of semen quality, with mitochondrial DNA copy number (mtDNAcn) serving as a sensitive biomarker for both semen quality and mitochondrial dysfunction resulting from oxidative stress. While glutathione S-transferases (GSTs) are commonly known as 'antioxidant' enzymes, there is ongoing debate regarding the relationship between GST genotypes and semen quality. In a study involving 568 male volunteers from the outpatient department of Puyang Reproductive Medicine Center, sperm mtDNAcn, semen quality, and GSTM1/GSTT1 genotypes were analyzed to investigate the potential link between GSTM1/GSTT1 gene variations and semen quality, as well as the impact of GSTs gene variations on the connection between sperm mtDNAcn and semen quality. Adjusting for variables such as age, BMI, smoking, and alcohol consumption, it was found that mtDNAcn was significantly correlated with decreased sperm concentration and total sperm count (b = - 0.109, - 0.128, respectively; P = 0.002, 0.001, respectively). GSTM1 was associated with progressive motility (OR 0.390, 95% CI 0.218, 0.697), Straight line velocity (VSL) (OR = 0.606, 95% CI 0.385, 0.953), and Straightness (STR) (OR 0.604, 95% CI 0.367, 0.994), while GSTT1 was linked to progressive motility (OR 0.554, 95% CI 0.324, 0.944) and Beat crossover frequency (OR 0.624, 95% CI 0.397, 0.982). The GSTT1 was found to moderate the relationship between mtDNAcn and sperm motility parameters linearity (LIN), STR, and Wobble (WOB), with additive interaction effects observed between GSTT1 and mtDNAcn on LIN, STR, and WOB (P for interaction = 0.008, 0.034, 0.010, respectively). Overall, this study suggests that GSTT1 and GSTM1 gene variations may play a role in sperm motility, with GSTT1 potentially influencing the impact of oxidative stress on sperm motility., (© 2024. The Author(s).)

The nuclear transport of proteins is mediated by karyopherins and has been implicated to be crucial for germ cell and embryonic development. Deletion of distinct members of the karyopherin alpha family has been shown to cause male and female infertility in mice. Using a genetrap approach, we established mice deficient for KPNA2 (KPNA2 KO) and investigated the role of this protein in male germ cell development and fertility. Breeding of male KPNA2 KO mice leads to healthy offsprings in all cases albeit the absence of KPNA2 resulted in a reduction in sperm number by 60%. Analyses of the KPNA2 expression in wild-type mice revealed a strong KPNA2 presence in meiotic germ cells of all stages while a rapid decline is found in round spermatids. The high KPNA2 expression throughout all meiotic stages of sperm development suggests a possible function of KPNA2 during this phase, hence in its absence the spermatogenesis is not completely blocked. In KPNA2 KO mice, a higher portion of sperms presented with morphological abnormalities in the head and neck region, but a severe spermiogenesis defect was not found. Thus, we conclude that the function of KPNA2 in round spermatids is dispensable, as our mice do not show any signs of infertility. Our data provide evidence that KPNA2 is not crucial for male germ cell development and fertility., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rother et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Theobromine (TBR) is a methylxanthine known for its bronchodilatory and stimulatory effects. This research evaluated the vitality, capacitation patterns, oxidative characteristics, microbial profile and expression of capacitation-associated proteins (CatSper1/2, sodium bicarbonate cotransporter [NBC], protein kinases A [PKA] and C [PKC] and adenylate cyclase 10 [ADCY10]) in cryopreserved bovine spermatozoa (n = 30) in the absence (cryopreserved control [Ctrl C ]) or presence of different TBR concentrations (12.5, 25, and 50 µM) in egg yolk extender. Fresh ejaculate served as a negative control (Ctrl N ). Significant post-thaw maintenance of the sperm motility, membrane and DNA integrity and mitochondrial activity ( p < 0.001) were recorded following the administration of 25 μM and 50 μM TBR, then compared to Ctrl C . All groups supplemented with TBR exhibited a significantly lower percentage of prematurely capacitated spermatozoa ( p < 0.001) than Ctrl C . Significantly decreased levels of global reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals were observed in the presence of 25 μM and 50 μM TBR ( p < 0.01). Western blot analysis revealed that supplementation with 50 μM TBR significantly prevented the loss of NBC and ADCY10 ( p < 0.01), while all TBR doses stabilized the levels of PKC ( p < 0.05 at 50 μM TBR; p < 0.001 at 12.5 μM and 25 μM TBR). In summary, we suggest that TBR is effective in protecting the spermatozoa during the cryopreservation process through its potential to stimulate energy synthesis while preventing ROS overproduction and the loss of proteins involved in the sperm activation process.

This study evaluates silver-doped carbon dots (AgCDs) as a novel agent for chemical castration using a rat model. Six groups of rats (five males and ten females each, except for the surgical group which had only males) were utilized to compare the effects of different concentrations of AgCDs. The groups included control, sham, and three experimental groups injected with 1.25, 50, and 200 µg/mL AgCDs, respectively, along with a surgical castration group. Testosterone levels, sperm parameters, fertility index, oxidative damage, histopathological parameters, and gene expression of P53, Bax, Bcl-2, caspase-3, AKT, and PI3K were analyzed. Results demonstrated that the high-dose AgCDs group significantly reduced testosterone levels, sperm concentration, and motility, resulting in a decreased fertility index. MDA and NO significantly increased, while CAT, SOD, GPx, and TAC significantly reduced in the chemically castrated groups. Histological and genes expression analysis also revealed apoptosis and testicular damage in the AgCDs groups, indicated by significant increases in P53, Bax, and Caspase-3 levels, and significant reductions in AKT, PI3K, and Bcl-2. Based on these findings, AgCDs could be considered a potent and efficient agent for chemical castration, offering a less invasive, cost-effective solution with potential applications for population control., (© 2024. The Author(s).)

Mammalian sperm glycans directly mediate several key life events. However, previous studies have not focused on two key factors that regulate these processes, the terminal glycan pattern and the anchoring sites. Herein, we group the capping monosaccharide sialic acid (Sia) and its capping substrates galactose/ N -acetylgalactosamine (Gal/GalNAc) into a "correlated terminal glycan pair" (glycopair) and, for the first time, reveal the differences in the aglycone pattern of this pair on spermatozoa using glyco-selective in situ covalent labeling techniques. Sia is mainly found in glycoproteins, whereas terminal Gal/GalNAc is mainly found in glycolipids. We quantitatively track the dynamic changes of the glycopair during sperm epididymal migration and find that the Sia capping ratio decreases with the increased expression of the glycopair; caudal upswim spermatozoa also show a lower Sia capping ratio than down spermatozoa. We thus propose two new parameters reflecting the terminal glycoforms of spermatozoa, which can well distinguish the maturity of spermatozoa. By fluorescence imaging of the glycopair in different regions of the sperm, we find that different parts of the sperm contribute differently to the overall glycan changes.

The ovarian microenvironment plays a crucial role in ensuring the reproductive success of viviparous teleosts. However, the molecular mechanism underlying the interaction between spermatozoa and the ovarian microenvironment has remained elusive. This study aimed to contribute to a better understanding of this process in black rockfish (Sebastes schlegelii) using integrated multi-omics approaches. The results demonstrated significant upregulation of ovarian complement-related proteins and pattern recognition receptors, along with remodeling of glycans on the surface of spermatozoa at the early spermatozoa-storage stage (1 month after mating). As spermatozoa were stored over time, ovarian complement proteins were progressively repressed by tryptophan and hippurate, indicating a remarkable adaptation of spermatozoa to the ovarian microenvironment. Before fertilization, a notable upregulation of cellular junction proteins was observed. The study revealed that spermatozoa bind to ZPB2a protein through GSTM3 and that ZPB2a promotes spermatozoa survival and movement in a GSTM3-dependent manner. These findings shed light on a key mechanism that influences the dynamics of spermatozoa in the female reproductive tract, providing valuable insights into the molecular networks regulating spermatozoa adaptation and survival in species with internal fertilization., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

In male reproductive system, proteins containing the coiled-coil domain (CCDC) are predominantly expressed in specific regions including the testis, epididymis, seminal vesicle, and prostate. They play a vital role in centriole formation, sperm motility and flagellar development in male gametes. Despite being highly expressed in the testis, the exact physiological function of the coiled-coil domain-containing 189 (Ccdc189) gene remain largely unclear. Our research provides a comprehensive and detailed investigation into the localization of CCDC189 protein within the testis seminiferous tubules. CCDC189 specifically expressed in spermatocytes, round spermatids, and elongating spermatids in mouse testis. The deletion of Ccdc189 in mouse leads to male infertility, characterized by significantly reduced sperm counts and motility. Abnormally shaped spermatozoa with irregular tails, exhibiting shortened and twisted morphology, were observed in the seminiferous tubules. Electron microscopy revealed disordered and missing peripheral microtubule doublets (MTD) and outer dense fibers (ODF) in the sperm flagella, accompanied by a consistent absence of central pairs (CP). The knockout of Ccdc189 resulted in oligo-astheno-teratozoospermia, which is characterized by low sperm count and reduced sperm motility and abnormal morphology. Furthermore, we identified poly(A)-binding protein cytoplasmic 1 (PABPC1) and PABPC2 as interacting proteins with CCDC189. These proteins belong to the PABP family and are involved in regulating mRNA translational activity in spermatogenic cells by specifically binding to poly(A) tails at the 3' ends of mRNAs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Male infertility affects nearly 50% of infertile couples, with various underlying causes, including endocrine disorders, testicular defects, and environmental factors. Spermatozoa rely on mitochondrial oxidative metabolism for motility and fertilization, with mitochondria playing a crucial role in sperm energy production, calcium regulation, and redox balance. Voltage-dependent anion channels (VDACs), located on the outer mitochondrial membrane, regulate energy and metabolite exchange, which are essential for sperm function. This review offers an updated analysis of VDACs in the male reproductive system, summarizing recent advances in understanding their expression patterns, molecular functions, and regulatory mechanisms. Although VDACs have been widely studied in other tissues, their specific roles in male reproductive physiology still remain underexplored. Special attention is given to the involvement of VDAC2/3 isoforms, which may influence mitochondrial function in sperm cells and could be implicated in male fertility disorders. This update provides a comprehensive framework for future research in reproductive biology, underscoring the significance of VDACs as a molecular link between mitochondrial function and male fertility.

Purpose: Chromosome 7 open reading frame 61 (C7orf 61) was a testis-specific gene, and may be involved in the process of spermatogenesis. This study aimed to investigate the expression of C7orf61 in the testis and determine its role in spermatogenesis., Materials and Methods: Reverse transcription-quantitative polymerase chain reaction, Western blot and immunofluorescence were performed to evaluate the expression characteristics of C7orf61 in mice and humans. In vitro fertilization assay was used to determine the role of the C7ORF61 protein in sperm-egg fusion., Results: The results demonstrated that C7orf61 was a testis-specific gene; the C7ofr61 mRNA expression level sharply increased in the fourth postnatal week and gradually increased until the adult stage. The C7ORF61 protein was located throughout the subacrosomal area and close to the nucleus in both mouse and human sperm. The incubation with the C7ORF61 antibody significantly decreased the fertilization rate of mouse eggs., Conclusion: The present findings suggested that the C7ORF61 protein might be involved in sperm-egg fusion, and could serve as a useful target for contraceptives. However, further research is still needed to know the detailed molecularmechanismofitsrole.

Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein-coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross-species analytical pipeline that can be repurposed to other cell types and pathologies., Competing Interests: RB, JA, MW, IJ, DS, CM, SD, LG, HS, NS, PP, AN, AL, JB, SL, MM, SK, MM, LR, FT, JB, PN No competing interests declared, (© 2024, Brattig-Correia, Almeida, Wyrwoll et al.)

Microfluidics provides unique opportunities for the high throughput selection of motile sperm with improved DNA integrity for assisted reproductive technologies (ARTs). Here, through a parametric study on dimensions and geometrical angles, a butterfly-shaped chip (BSC) is presented to isolate sperm with high progressive motility and intact DNA at a separation rate of 1125 sperm per minute. Using finite element simulations, the flow field and shear rates in the device were optimized to leverage the inherent motility characteristics of sperm for maximum selection throughput. The device incorporates a triple selection mechanism in series, initially activating sperm rheotaxis by rotation against the semen flow, penetrating the counter buffer flow and swimming against the direction of the buffer flow, leaving dead cells and debris behind, and subsequently leveraging boundary-following behavior to direct progressively motile sperm to swim along the walls and reach the device outlet. The device selects over 4.1 million sperm per mL within 20 minutes, with 29.2%, 68.2%, and 57.3% improvement in total motility, DNA integrity, and velocity parameter (VCL), as compared with the conventional swim-up method, respectively. Overall, the performance of the device to separate sperm with approximately 95.9% total motility, 97.8% viability, and 96.6% DNA integrity at high concentrations demonstrates its potential for enhancing the efficiency of conventional treatment methods.

De novo mutations (DNMs) are drivers of genetic disorders. However, the study of DNMs is hampered by technological limitations preventing accurate quantification of ultra-rare mutations. Duplex Sequencing (DS) theoretically has < 1 error/billion base-pairs (bp). To determine the DS utility to quantify and characterize DNMs, we analyzed DNA from blood and spermatozoa from six healthy, 18-year-old Swedish men using the TwinStrand DS mutagenesis panel (48 kb spanning 20 genic and intergenic loci). The mean single nucleotide variant mutation frequency (MF) was 1.2 × 10 - 7 per bp in blood and 2.5 × 10 - 8 per bp in sperm, with the most common base substitution being C > T. Blood MF and substitution spectrum were similar to those reported in blood cells with an orthogonal method. The sperm MF was in the same order of magnitude and had a strikingly similar spectrum to DNMs from publicly available whole genome sequencing data from human pedigrees (1.2 × 10 - 8 per bp). DS revealed much larger numbers of insertions and deletions in sperm over blood, driven by an abundance of putative extra-chromosomal circular DNAs. The study indicates the strong potential of DS to characterize human DNMs to inform factors that contribute to disease susceptibility and heritable genetic risks., (© 2024. The Author(s).)

Background: While polystyrene microplastics (PS-MPs) are emerging as potentially significant health threats, linked to cancer and reproductive dysfunction, their precise effects on human health remain largely unknown. We aimed to investigate the underlying mechanisms promoting microplastic-induced damage in the reproductive system., Methods: Thirty C57BL/6 male mice were randomly allocated into six equal-sized groups. Mice were exposed to fluorescent PS-MPs (5 µm, < 18%, green) at a dose of 1 and 3 mg/dL via oral gavage for 28 and 56 days, respectively (control, 0 mg/dL). The presence of antibodies and inflammatory and oxidative stress markers were evaluated using western blotting. Sperm analysis was also performed. Mouse testis Sertoli TM4 cells were divided into two groups: control (medium only) and PS-MPs (medium containing, 1,000 μg/mL) groups and cultured in vitro for 1, 24, 48, or 72 hours. The cells were cultured in a Ham's F12: Dulbecco's Modified Eagle Medium medium with 0.25% fetal bovine serum at 37°C with humidified atmosphere of 5% carbon dioxide in the air. Protein analyses for interleukin (IL)-6, IL-10, NADPH-oxidase (NOX)-2, NOX-4, hypoxia-inducible transcription factor (HIF)-2α, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β were performed using western blotting., Results: The testes were evaluated after 28 and 56 days of exposure. Varying sizes of PS-MPs were detected in the testes (ranging from 5.870 to 7.768 µm). Significant differences in sperm concentration, motility, and the proportion of normal sperm were observed between the two groups. An increase in TGF-β, HIF-2α, and NOX-4 levels was observed using western blot analysis. However, no dose-dependent correlations were observed between the two groups. In vitro evaluation of the PS-MPs group displayed PS-MP penetration of the lumen of Sertoli cells after 1 hour. Further PS-MP aggregation within Sertoli cells was observed at 24, 48, and 72 hours. A significant increase in inflammatory protein expressions (IL-10, TGF-β, MCP-1, IL-6, TNF-α, and HIF-2α) was observed through western blotting, although oxidative agents did not show a significant increase., Conclusion: PS-MPs induced reproductive dysfunction in male mice provide new insights into PS-MPs-associated toxicity in mammals., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2024 The Korean Academy of Medical Sciences.)

Cisplatin (CP), a chemotherapy drug commonly used in cancers treatment, causes serious reproductive toxicity. With younger cancer patients and increasing survival rates, it is important to preserve their reproductive capacity. NME8 is highly expressed in testis and contains thioredoxin and NDPK domains, suggesting it may be a target against the CP-induced reproductive toxicity. We deleted exons 6-7 of the Nme8 in mice based on human mutation sites and observed impaired transcript splicing. In mice, Nme8 was not essential for spermatogenesis, possibly due to functional compensation by its paralog, Nme5. Nme8 expression was elevated and translocated to the nucleus in response to two weeks of CP treatment. Under CP treatment, Nme8 deficiency further impaired antioxidant capacity, induced lipid peroxidation and increased ROS level, and failed to activate autophagy, resulting in aggravated DNA damage in testes and sperm. Consequently, the proliferation and differentiation of spermatogonia and the meiosis of spermatocyte were almost completely halted, and sperm motility was impaired. Our research indicates that NME8 protects against CP-induced testis and sperm damage. This may provide new insights into the physiological functions of the Nme family and potential targets for preserving fertility in young male cancer patients., (© 2024. The Author(s).)

Abstract: Seminal fluid extracellular vesicles (SFEVs) have previously been shown to interact with spermatozoa and influence their fertilisation capacity. Here, we sought to extend these studies by exploring the functional consequences of SFEV interactions with human spermatozoa. SFEVs were isolated from the seminal fluid of normozoospermic donors prior to assessing the kinetics of sperm-SFEV binding in vitro, as well as the effects of these interactions on sperm capacitation, acrosomal exocytosis, and motility profile. Biotin-labelled SFEV proteins were transferred primarily to the flagellum of spermatozoa within minutes of co-incubation, although additional foci of SFEV biotinylated proteins also labelled the mid-piece and head domain. Functional analyses of high-quality spermatozoa collected following liquefaction revealed that SFEVs did not influence sperm motility during incubation at pH 5, yet SFEVs induced subtle increases in total and progressive motility in sperm incubated with SFEVs at pH 7. Additional investigation of sperm motility kinematic parameters revealed that SFEVs significantly decreased beat cross frequency and increased distance straight line, linearity, straightness, straight line velocity, and wobble. SFEVs did not influence sperm capacitation status or the ability of sperm to undergo acrosomal exocytosis. Functional assessment of both high- and low-quality spermatozoa collected prior to liquefaction showed limited SFEV influence, with these vesicles inducing only subtle decreases in beat cross frequency in spermatozoa of both groups. These findings raise the prospect that, aside from subtle effects on sperm motility, the encapsulated SFEV cargo may be destined for physiological targets other than the male germline, notably the female reproductive tract., Lay Summary: A male's influence over the biological processes of pregnancy extends beyond the provision of sperm. Molecular signals present in the ejaculate can influence the likelihood of pregnancy and healthy pregnancy progression, but the identity and function of these signals remain unclear. In this study, we wanted to understand if nano-sized particles present in the male ejaculate, called seminal fluid extracellular vesicles, can assist sperm in traversing the female reproductive tract to access the egg. To explore this, we isolated seminal fluid extracellular vesicles from human semen and incubated them with sperm. Our data showed that seminal fluid extracellular vesicles act to transfer molecular information to sperm, but this resulted in only subtle changes to the movement of sperm.

Despite the numerous sequencing methods available, the diversity in RNA size and chemical modification makes it difficult to capture all RNAs in a cell. We developed a method that combines quasi-random priming with template switching to construct sequencing libraries from RNA molecules of any length and with any type of 3' modifications, allowing for the sequencing of virtually all RNA species. Our ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to identify and quantify all classes of coding and non-coding RNAs. With LIDAR, we comprehensively characterized the transcriptomes of mouse embryonic stem cells, neural progenitor cells, mouse tissues, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared with traditional ligation-dependent sequencing methods and uncovered tDRs with blocked 3' ends that had previously escaped detection. Therefore, LIDAR can capture all RNAs in a sample and uncover RNA species with potential regulatory functions., Competing Interests: Declaration of interests J.E.W. serves as a consultant for Laronde., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The low resistance of boar sperm to cryopreservation dictates that addition antioxidants and energetic substances to the diluent to improve sperm quality is necessary. This study evaluated the effect of spermidine and phosphocreatine in combination on the quality, antioxidant capacity, and antiapoptotic-like changes capacity of cryopreserved boar sperm based on previous reports. The results showed that the combined application of spermidine and phosphocreatine significantly enhanced the motility, average path velocity, straight-line velocity, curvilinear velocity, beat cross frequency, acrosome integrity, plasma membrane integrity, mitochondrial activity, and DNA integrity compared with the control group (p < 0.05). In addition, the combined application of spermidine and phosphocreatine significantly enhanced the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, and catalase activity while significantly decreasing malondialdehyde content and hydrogen peroxide content (p < 0.05). Western Blot analysis further showed that spermidine and phosphocreatine significantly decreased the expression of CASP3 and BAX and significantly enhanced the expression of BCL2 (p < 0.05); therefore, the combination of spermidine and phosphocreatine has potentially positive implications for improving the quality of cryopreserved boar sperm., (© 2024 Wiley Periodicals LLC.)

This review focuses on the contribution of the late David Garbers to chemotaxis of sperm, in particular from sea urchin. We will describe his discovery of chemotactic peptides and their cognate receptors, his discovery of a sperm-specific, unique Na + /H + exchanger that represents a chimera between a solute carrier (SLC) and an ion channel. Finally, we will discuss his contributions to the understanding of cAMP signaling in sperm via soluble adenylyl cyclase (sAC) and its control by Ca 2+ ions., (© 2024 The Author(s). Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Background: Rab25 is a member of the Rab family, functioning as a regulatory molecule in intracellular transport. Although its involvement in cellular functions and disease development is well-established, its precise roles in male reproductive physiology remain elusive., Methods: To explore the specific roles of Rab25 in testicular development and spermatogenesis, we established the Rab25 -/- mouse model and Rab25 knockdown germ cell line (GC-2). We compared the fertility, sperm analysis, and testicular tissues between Rab25 -/- and wild-type male mice. To delve deeper into potential mechanisms, we employed immunohistochemistry, TUNEL assay, Western Blotting, CCK-8 assay, etc. to evaluate cell proliferation and apoptosis in testicular tissues and GC-2 cells., Results: Our findings indicated that Rab25 was expressed in germ cells and Leydig cells in the testes. Although the weight of Rab25 -/- mice testes exhibited no significant changes, fertility was compromised, with a decrease in sperm quantity and reduced motility. HE staining revealed a disorganized arrangement of germ cells and vacuolization. Additionally, chromatin marginalization and nuclear pyknosis were observed in the Rab25 -/- mice. In both Rab25 -/- mice testes and Rab25 knockdown GC-2 cells, we found that germ cell proliferation was reduced, while apoptosis was increased., Conclusions: In conclusion, our study proposes that Rab25 plays a vital role in spermatogenesis by regulating the proliferation and apoptosis of germ cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) are highly heterogeneous, and different EV subpopulations from various origins mediate different biological effects. The separation of different subpopulations of EVs from mixtures is critical but challenging. Epididymosomes are secreted by the epididymal epithelium and play a key role in sperm maturation and function. However, limited access to human epididymal tissue and epididymal fluid has hampered further study of epididymosomes and their potential clinical applications. Here, we established a novel strategy based on flow cytometry sorting to isolate human CD63-positive epididymosomes from ejaculate. We identified CD52, a membrane-located protein expressed exclusively in the epididymis, as the sorting marker for human epididymosomes. Then, CD63-positive epididymosomes were isolated from human semen using a flow cytometry sorting instrument and concentrated. Additionally, we observed that isolated CD63-positive epididymosomes improved sperm function more than other CD63-positive seminal EV subpopulations did, demonstrating the successful isolation of a subpopulation of epididymosomes from human semen and their potential application in the clinic., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Bioenergetics plays a crucial role in sperm functions, including motility, capacitation-related protein modifications, oocyte recognition and interaction, all of which are essential for fertilization. Sperm metabolism is recognized as flexible, responding to environmental cues and energetic demands during ejaculation, the journey along the female tract, and until fertilization. Recent studies suggest that sperm metabolic functions are relevant beyond fertilization and may influence zygote and embryo development, impacting paternal-derived effects on offspring development and health. In recent years, sperm metabolic functions and homeostasis have gained increasing interest in male reproduction research. Given the crucial implications of sperm metabolism on fertility-related processes, this field is of interest not only in human male fertility but also in livestock research, semen conservation, and assisted reproductive techniques. Newly developed assessment tools are allowing a better understanding of sperm metabolism under different conditions and identifying species-specific peculiarities. This review aims to discuss the current knowledge of mammalian sperm metabolism, focusing on species-specific features, changes during the sperm journey, and potential contributions to translational research and reproductive biotechnologies. Furthermore, we propose future perspectives on sperm bioenergetics research., (© 2024 The Author(s). Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Phthalates are ubiquitous pollutants in the environment; however, the mechanisms of phthalate-associated reproductive disorders in men are not fully understood. The aim of this study is to investigate associations between urinary phthalate metabolite concentrations and sperm DNA methylation. The study was conducted on 697 men from three prospective pregnancy cohorts: Longitudinal Investigation of Fertility and the Environment (LIFE) Study, Sperm Environmental Epigenetics and Development Study (SEEDS), and Environment and Reproductive Health (EARTH) Study. Eighteen phthalate and two phthalate alternative metabolites were quantified by mass spectrometry in preconception urinary samples and sperm DNA methylation was measured via Illumina EPIC Array (v1). Regional methylation analyses were conducted to identify cohort-specific loci associated with urinary phthalate metabolites. Models were adjusted for age, body mass index (BMI), race, smoking status, urinary creatinine/specific gravity, and analytical batch for phthalate measurements. The cohort-specific results were meta-analyzed using METAL. Participants had an average age of 30 years, most (79.6 %) of whom had BMI>25 kg/m 2 and were non-smokers (90.1 %). A total of 7,979 differentially methylated regions (DMRs; 7,979 LIFE-specific DMRs, 72 SEEDS-specific DMRs, and 23 EARTH-specific DMRs) were associated with urinary MBzP, MiBP, MMP, MCNP, MCPP, MBP, and MCOCH. Meta-analysis identified fewer DMRs than cohort-specific models: 946 DMRs were associated with MBzP, 27 DMRs associated with MiBP, and 1 DMR associated with MEHP. The majority of cohort-specific and meta-analysis-derived DMRs displayed a positive association with phthalate metabolite concentrations and were enriched in genes associated with spermatogenesis, response to hormones and their metabolism, embryonic organ development and developmental growth. In conclusion, several preconception urinary phthalate metabolites were associated with increased DNA methylation patterns in sperm. These findings provide an epigenetic pathway by which environmental phthalate exposures can impact couples' reproductive outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Dr. David Garbers made many impactful contributions to science and vastly improved our understanding of sperm biology. In this review, we focus on his identification of a key role for the second messenger cAMP in mammalian sperm. As a graduate student David discovered that sperm motility, which is essential for sperm to fertilize the egg, is under the control of the (at the time) recently identified, prototypical second messenger cAMP. Fast-forwarding to the present, agents which turn off sperm's ability to generate cAMP and block sperm motility are being investigated as potential nonhormonal contraceptives for men and women. Should these efforts prove successful, Dave's discoveries will prove to be the spark which ignited a revolution in human health., (© 2024 Wiley Periodicals LLC.)

Seminal fluid is rich in sugars, but their role beyond supporting sperm motility is unknown. In this study, we found Drosophila melanogaster males transfer a substantial amount of a phospho-galactoside to females during mating, but only half as much when undernourished. This seminal substance, which we named venerose, induces an increase in germline stem cells (GSCs) and promotes sperm storage in females, especially undernourished ones. Venerose enters the hemolymph and directly activates nutrient-sensing Dh44 + neurons in the brain. Food deprivation directs the nutrient-sensing neurons to secrete more of the neuropeptide Dh44 in response to infused venerose. The secreted Dh44 then enhances the local niche signal, stimulating GSC proliferation. It also extends the retention of ejaculate by females, resulting in greater venerose absorption and increased sperm storage. In this study, we uncovered the role of a sugar-like seminal substance produced by males that coordinates reproductive responses to nutritional challenges in females., (© 2024. The Author(s).)

Diabetes mellitus is known as the "epidemic of the century" due to its global prevalence. Several pre-clinical and clinical studies have shown that male germ cell toxicity is one of the major consequences of diabetes mellitus. Although β-aminoisobutyric acid (BAIBA) has been shown to be advantageous in the diabetic nephropathy and cardiomyopathy, its specific role in the diabetes-induced testicular toxicity remains unknown. In this study, an attempt was made to elucidate the molecular mechanisms of BAIBA-mediated germ cell protection in diabetic rats. Adult male Sprague-dawley rats were subjected to either no treatment (control) or BAIBA (100 mg/kg; BAIBA control) or Streptozotocin (50 mg/kg; diabetic control) or low (25 mg/kg), medium (50 mg/kg) and high (100 mg/kg) doses of BAIBA in diabetic conditions. Significant alterations in sperm related parameters, oxidative stress and apoptotic biomarkers, pancreatic and testicular histology, DNA damage and changes in expression of proteins in testes were found in the diabetic rats. 100 mg/kg of BAIBA significantly reduced the elevated blood glucose levels (P ≤ 0.05), increased body weight (P ≤ 0.01 in the 4th week), lowered malondialdehyde (P ≤ 0.05) and nitrite levels (P ≤ 0.01), elevated testosterone (P ≤ 0.05) and FSH levels (P ≤ 0.05), increased sperm count and motility (P ≤ 0.01), decreased testicular DNA damage (P ≤ 0.001), improved histological features of pancreas and testes, decreased TUNEL positive cells (P ≤ 0.01), decreased RAGE (P ≤ 0.01) and Bax (P ≤ 0.05) expressions and increased SIRT1 (P ≤ 0.05) and Atg 12 (P ≤ 0.05) expressions in the testes. 50 mg/kg of BAIBA partially restored the above-mentioned parameters whereas 25 mg/kg of BAIBA was found to be insignificant in counteracting the toxicity. It is interesting to note that BAIBA protects male germ cell damage in diabetic rats by regulating the IGF-1/AMPK/SIRT-1 signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bisphenol A (BPA) is one of the most prevalent endocrine disrupting chemicals (EDCs) and there is widespread concern about the adverse effects of EDCs on human health. However, the exact mechanism of these toxicities has still not been fully deciphered. Additionally, studies have reported the toxicological effects at far low doses to the generally considered no-observed-adverse-effect level (NOAEL) dose. The present study investigates the effects of a sub-acute (28 days) exposure to BPA (10, 50 and 100 mg/kg/day) in adult male mice on various hormones levels, sperm motility, sperm count, functional integrity of sperm plasma membrane, testicular histological changes, oxidative stress markers and DNA damage. The key proteome signatures were quantified by LC-MS/MS analysis using Orbitrap Fusion Lumos Tribrid Mass Spectrometer equipped with nano-LC Easy-nLC 1200. Data suggest that the BPA exposure in all doses (below/above NOAEL dose) have greatly impacted the hormone levels, sperm parameters (sperm count, motility and membrane integrity) and testicular histology. Mass spectrometry-based proteomics data suggested for 1352 differentially expressed proteins (DEPs; 368 upregulated, 984 downregulated) affecting biological process, cellular component, and molecular functions. Specifically searched male reproductive function related proteins suggested a complex network where 46 potential proteins regulating spermatogenesis, sperm structure, activity and membrane integrity while tackling oxidative stress responses were downregulated. These potential biomarkers could shed some more light on our current understanding of the reproductive toxicological effects of BPA and may lead to exploration of novel interventions strategies against these targets for male infertility., (© 2024 Wiley Periodicals LLC.)

Chronic stress caused by prolonged emotional pressure can lead to various physiological issues, including reproductive dysfunction. Although reproductive problems can also induce chronic stress, the impact of chronic stress-induced reproductive dysfunction remains contentious. This study investigates the effects of chronic unpredictable stress (CUS) on reproductive neuropeptides, sperm quality, and testicular morphology. Sixteen twelve-week-old Sprague Dawley rats were divided into two groups: a non-stress control group and a CUS-induced group. The CUS regimen involved various stressors over 28 days, with both groups undergoing behavioural assessments through sucrose-preference and forced-swim tests. Hypothalamic gene expression levels of CRH, PNX, GPR173, kisspeptin, GnRH, GnIH, and spexin neuropeptides were measured via qPCR, while plasma cortisol, luteinizing hormone (LH), and testosterone concentrations were quantified using ELISA. Seminal fluid and testis samples were collected for sperm analysis and histopathological evaluation, respectively. Results showed altered behaviours in CUS-induced rats, reflecting stress impacts. Hypothalamic corticotropin-releasing hormone (CRH) expression and plasma cortisol levels were significantly higher in CUS-induced rats compared to controls (p < 0.05). Conversely, phoenixin (PNX) expression decreased in the CUS group (p < 0.05), while kisspeptin, spexin, and gonadotropin-inhibitory hormone (GnIH) levels showed no significant differences between groups. Despite a significant increase in GnRH expression (p < 0.05), plasma LH and testosterone concentrations were significantly lower (p < 0.05) in CUS-induced rats. Histopathological analysis revealed abnormal testis morphology in CUS-induced rats, including disrupted architecture, visible interstitial spaces between seminiferous tubules, and absence of spermatogenesis. In conclusion, CUS affects reproductive function by modulating PNX and GnRH expression, influencing cortisol levels, and subsequently reducing plasma LH and testosterone concentrations. This study highlights the complex interplay between chronic stress and reproductive health, emphasizing the significant impact of stress on reproductive functions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)