Catalysis-Mediated Male Contraception through Black Phosphorus Nanosheets.

Nanocontraception has been proposed and received extensive attention in recent years for population control. However, currently developed methods for nanocontraception still face problems in efficacy and safety. Here, we propose catalysis-mediated oxidation as a new strategy for nanocontraception. With the catalytic production of highly oxidative species, male contraception was successfully achieved after the administration of black phosphorus nanosheets into the testes of male mice. Further mechanistic studies revealed that contraception was induced by oxidative stress and apoptosis of spermatogenesis cells. Meanwhile, the apoptosis of germ cells released testis antigen and induced immune cell infiltration, which enhanced reproductive damage. Notably, the introduced black phosphorus nanosheets naturally degraded during the catalytic oxidation process and ultimately converted to harmless phosphates, indicating the safety of the strategy. Furthermore, the catalysis-mediated strategy avoids utilizing additional inducers, such as near-infrared irradiation, magnetic fields, or ultrasound, which may cause severe pain. In summary, the proposed catalysis-mediated contraception can be a self-cleared, convenient, and safe strategy for controlling male fertility.

Chemotherapeutic-caused liver toxicity hinders nanomedicine development.

Few nanomedicines are approved for clinical cancer treatment as only about 0.7% (median) of nanoparticles enter solid tumors. Nanomedicine as the second medication is usually used in cancer treatment after chemotherapy, immunotherapy surgery, or radiotherapy treatment. However, it is currently unpredictable whether the priority treatment enhances or reduces the therapeutic effect of nanomedicine. Here, by considering prior chemotherapy (5-FU or cisplatin treatment), immunotherapy (IL-2, IL-6, or IL-21-treatment), or phosphate-buffered saline (PBS treatment), we compared the biodistribution of AuNPs in the liver, spleen, kidney, and tumor. We found that the accumulation of AuNPs in the liver and spleen increased in cisplatin pretreatment compared to the PBS treatment, while there was no significant effect on the accumulation of AuNPs in the tumor due to cisplatin-induced significant liver damage while other treatments did not change the biodistribution of AuNPs in the liver, spleen, kidney, and tumor. These results indicated that cisplatin pretreatment is not suitable for subsequent nanomedical cancer therapy. Our work opens a new insight to design low-toxicity chemotherapy to be applied before nanomedicine.

BET bromodomain inhibitor JQ1 regulates spermatid development by changing chromatin conformation in mouse spermatogenesis.

As a BET bromodomain inhibitor, JQ1 has been proven have efficacy against a number of different cancers. In terms of male reproduction, JQ1 may be used as a new type of contraceptive, since JQ1 treatment in male mice could lead to germ cell defects and a decrease of sperm motility, moreover, this effect is reversible. However, the mechanism of JQ1 acting on gene regulation in spermatogenesis remains unclear. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse testes treated with JQ1 or vehicle control to determine the transcriptional regulatory function of JQ1 in spermatogenesis at the single cell resolution. We confirmed that JQ1 treatment could increase the numbers of somatic cells and spermatocytes and decrease the numbers of spermatid cells. Gene Ontology (GO) analysis demonstrated that differentially expressed genes which were down-regulated after JQ1 injection were mainly enriched in "DNA conformation change" biological process in early developmental germ cells and "spermatid development" biological process in spermatid cells. ATAC-seq data further confirmed that JQ1 injection could change the open state of chromatin. In addition, JQ1 could change the numbers of accessible meiotic DNA double-stranded break sites and the types of transcription factor motif that functioned in pachytene spermatocytes and round spermatids. The multi-omics analysis revealed that JQ1 had the ability to regulate gene transcription by changing chromatin conformation in mouse spermatogenesis, which would potentiate the availability of JQ1 in male contraceptive.

Sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress via evading offspring embryonic reprogramming.

Paternal life experiences impact offspring health via germline, and epigenetic inheritance provides a potential mechanism. However, global reprogramming during offspring embryogenesis and gametogenesis represents the largest hurdle to conceptualize it. Yet, detailed characterization of how sperm epigenetic alterations carrying "environmental memory" can evade offspring embryonic reprogramming remains elusive. Here, mice exposed to long-term restraint stress were employed to study the mechanisms underlying inter- and transgenerational effects of paternal exposure to a long-term psychological stress. We found that stress could induce paternal inheritance of reproductive, behavioral, and metabolic disorders. Bisulfite methylation profiling of 18 sperm and 12 embryo samples of three consecutive generations identified inter- and transgenerational inheritance of paternal Differential DNA Methylation Regions (DMRs) at frequencies ~11.36% and 0.48%, respectively. These DMRs related to genes with functional implications for psychological stress response, and tissue inheritance of these DMRs passed paternal disorders epigenetically to offspring. More importantly, these DMRs evaded offspring embryonic reprogramming through erasure and subsequent reestablishment, but not via un-erasure way. Nonetheless, their reestablishment proportions in the primitive streak (E7.5) stage were altered. Furthermore, sncRNA-seq revealed that stress-induced tsRNA, miRNA and rsRNA dysregulation in paternal sperm might play important roles in DMRs occurrence and paternal inheritance. These finding implied that sperm epigenetic alterations contribute to inter- and transgenerational effects of paternal exposure to long-term psychological stress, and highlighted the possible underlying molecular mechanism.

Thy1-Positive Spermatogonia Suppress the Proliferation of Spermatogonial Stem Cells by Extracellular Vesicles In Vitro.

The self-renewal of mammalian spermatogonial stem cells (SSCs) supports spermatogenesis to produce spermatozoa, and this is precisely controlled in a stem niche microenvironment in the seminiferous tubules. Although studies have revealed the role of the surrounding factors in SSCs, little is known about whether the division of SSCs is controlled by extracellular vesicles. Here, extracellular vesicles were found in the basal compartment of seminiferous tubules in mouse, rat, rabbit and human testes. In the mice, the testicular extracellular vesicles are secreted by spermatogonia and are taken up by SSCs. Further, the extracellular vesicles from thy1-positive spermatogonia were purified by anti-Thy1-coupled magnetic beads, which suppress their proliferation of SSCs but do not lead to the apoptosis in vitro.

[RHNO1: A novel noncoding RNA highly expressed in the testis and involved in DNA double-strand break repair].

OBJECTIVE: To investigate the role of a long noncoding RNA (lncRNA) transcribed from the RHNO1 gene we newly identified in DNA double-strand break (DSB) repair. METHODS: The transcription and translation of the RHNO1 gene were validated by Western blot, real-time PCR and liquid chromatography-tandem mass spectrometry (LC-MS/MS) based on the overexpressed RHNO1 plasmid. The transcription level of RHNO1 in the mouse tissue was detected by real-time PCR and its expression in the spermatogenic cycle determined by in situ hybridization. The role of RHNO1 in the DNA DSB repair was further verified using the DSB model established by exposing the germ cells to ultraviolent radiation. RESULTS: The full-length RHNO1 gene could be transcribed as a novel lncRNA in vitro, highly expressed in the mouse testis tissue, and mainly located in spermatocytes and round spermatids. RHNO1 was involved in DNA DSB repair in the spermatogenic cells. CONCLUSIONS: We identified a novel lncRNA, RHNO1, which is highly expressed in the mouse testis and participates in DNA damage repair in the germ cell line.

Tethering of Telomeres to the Nuclear Envelope Is Mediated by SUN1-MAJIN and Possibly Promoted by SPDYA-CDK2 During Meiosis.

During meiosis, telomeres attach to the nuclear envelope (NE) to promote homologous chromosome moving, pairing, synapsis, and recombination. The telomere-NE attachment is mediated by SUN1, TERB1-TERB2-MAJIN (TTM complex), and TRF1. The interaction of the TTM complex with shelterin is mediated by TERB1 and TRF1, but how SUN1 interacts with the TTM complex is not yet fully understood. In this study, we found that SUN1 not only interacted with TERB1 but also interacted with MAJIN, and the interaction of SUN1 with MAJIN is stronger than TERB1. We also found that SUN1 interacted with SPDYA, an activator of CDK2. The binding sites of MAJIN and SPDYA at SUN1 were mapped, and both MAJIN and SPDYA bound to the N-terminal domain of SUN1 and the two binding sites were close to each other. Furthermore, SPDYA bound to SUN1 via the Ringo domain and recruited CDK2 to SUN1. Then, we found that the interaction of SUN1 with MAJIN was decreased by the CDK2 inhibitors. Taken together, our results provide the possible mechanism of SUN1, MAJIN, and SPDYA-CDK2 in promoting the telomere-NE attachment during meiosis.

Autism-like behaviors and abnormality of glucose metabolism in offspring derived from aging males with epigenetically modified sperm.

Accumulating evidence from epidemiological studies of humans and genetic models in rodents has shown that offspring from males of advanced paternal age (APA) are susceptible to metabolic and neurological disorders. However, knowledge of molecular mechanism(s) underlying these metabolic and behavioral changes at the intergeneration and trans-generation levels from APA is limited. Here, we characterized changes on glucose and cholesterol metabolism, and also autism spectrum disorders (ASD)-like behaviors in 1st and 2nd generations from 12- and 18-month-old male mice, respectively. Whole Genome Bisulfite Sequencing (WGBS) of sperm from APA mice identified differentially methylated regions (DMRs) within the whole genome, and DMRs within promoter regions, suggesting that specific genes and relevant pathways might be associated with autism and aberrant glucose metabolism in the offspring from APA males. These results strongly suggest that epigenetic reprogramming induced by aging in male sperm may lead to high risks of aberrant glucose metabolism and the development of ASD behaviors in intergenerational and transgenerational offspring.

Expression and localization of retinoid receptors in the testis of normal and infertile men.

Retinoic acid (RA), the active metabolite of vitamin A, is one of the most important factors regulating spermatogenesis. RA activates downstream pathways through its receptors (retinoic acid receptor alpha [RARA], retinoic acid receptor beta, and retinoic acid receptor gamma [RARG]) and retinoid X receptors (retinoid X receptor alpha [RXRA], retinoid X receptor beta [RXRB], and retinoid X receptor gamma [RXRG]). These receptors may serve as therapeutic targets for infertile men. However, the localization and expression of retinoid receptors in normal and infertile men were unknown. In this study, we found RARA and RARG were mostly localized in spermatocytes and round spermatids, RXRB was mainly expressed in Sertoli cells, and RXRG was expressed in most cell types in the fertile human testis. The localization of RARA, RARG, RXRB, and RXRG in men with hypospermatogenesis (HYPO) was similar to that of men with normal fertility. In addition, the messenger RNA expression levels of RARA, RARG, RXRA, RXRB, and RXRG were significantly decreased in men with Sertoli cell-only syndrome (SCOS) and maturational arrest (MA), but not in men with HYPO. These results suggest that reduced levels of RARA, RARG, RXRB, RXRA, and RXRG are more closely associated with SCOS and MA spermatogenetic failure. These results could contribute to the development of new molecular indicators of spermatogenic dysfunction and might provide novel therapeutic targets for treating male infertility.

Whole-exome sequencing of a large Chinese azoospermia and severe oligospermia cohort identifies novel infertility causative variants and genes.

Rare coding variants have been proven to be one of the significant factors contributing to spermatogenic failure in patients with non-obstructive azoospermia (NOA) and severe oligospermia (SO). To delineate the molecular characteristics of idiopathic NOA and SO, we performed whole-exome sequencing of 314 unrelated patients of Chinese Han origin and verified our findings by comparing to 400 fertile controls. We detected six pathogenic/likely pathogenic variants and four variants of unknown significance, in genes known to cause NOA/SO, and 9 of which had not been earlier reported. Additionally, we identified 20 novel NOA candidate genes affecting 25 patients. Among them, five (BRDT, CHD5, MCM9, MLH3 and ZFX) were considered as strong candidates based on the evidence obtained from murine functional studies and human single-cell (sc)RNA-sequencing data. These genetic findings provide insight into the aetiology of human NOA/SO and pave the way for further functional analysis and molecular diagnosis of male infertility.

Overexpression of human-derived DNMT3A induced intergenerational inheritance of DNA methylation and gene expression variations in rat brain and testis.

In mammals, DNA methylation patterns are established by various types of DNA methyltransferases and can be stably passed on during cell division, thus creating a paradigm for epigenetic regulation that can mediate long-lasting changes in gene expression even when the initial triggering signal has disappeared. Although functional deficiency of DNMT3A, one of the methyltransferases, leads to abnormal DNA methylation patterns that result in developmental deficits in mammals, the impacts of its overexpression on tissue gene expression and DNA methylation patterns remain unclear. Here, our previously established hDNMT3A transgenic rat model and mRNA sequencing and bisulphite sequencing PCR were used to analyse the impact of hDNMT3A overexpression on tissue transcriptome and methylome, and whether the impact could be inherited intergenerationally was subsequently investigated. Our results revealed that the overexpression of hDNMT3A could induce notable gene expression variations in rat testis and brain. More importantly, 36.02% and 38.89% of these variations could be intergenerationally inherited to offspring without the transmission of the initial endogenic trigger in the brain and testis, respectively. Furthermore, we found that intergenerationally inherited DNA methylation variations in their promoters and exons could be the underlying mechanism. Compared with inheritable variations that were passively induced by environmental factors, these variations were actively induced by endogenous epigenetic modifiers. This study provided evidence for the epigenetic inheritance of endogenous factors that actively induce gene expression and DNA methylation variations; however, more studies are needed to determine the number of generations that these variations can be stably inherited.

The testis-specific gene 1700102P08Rik is essential for male fertility.

Male infertility is a rising problem around the world. Often the cause of male infertility is unclear, and this hampers diagnosis and treatment. Spermatogenesis is a complex process under sophisticated regulation by many testis-specific genes. Here, we report the testis-specific gene 1700102P08Rik is conserved in both the human and mouse and highly expressed in spermatocytes. To investigate the role of 1700102P08Rik in male fertility, knockout mice were generated by CRISPR-Cas9. 1700102P08Rik knockout male mice were infertile with smaller testis and epididymis, but female knockout mice retained normal fertility. Spermatogenesis in the 1700102P08Rik knockout male mouse was arrested at the spermatocyte stage, and no sperm were found in the epididymis. The deletion of 1700102P08Rik causes apoptosis in the testis but did not affect the serum concentration of testosterone, luteinizing hormone, and follicle-stimulating hormone or the synapsis and recombination of homologous chromosomes. We also found that 1700102P08Rik is downregulated in spermatocyte arrest in men. Together, these results indicate that the 1700102P08Rik gene is essential for spermatogenesis and its dysfunction leads to male infertility.

The novel testicular enrichment protein Cfap58 is required for Notch-associated ciliogenesis.

Cilia and flagella are critical organelles with conserved internal structures and diverse developmental and physiological processes according to cell type. Although the core components of structures are shared with thousands of associated proteins involved in cilia or flagella formation, we hypothesized that some unknown proteins, such as outer dense fiber 2 (Odf2/Cenexin) perform distinct functions in these organelles. In the present study, we identified several uncharacterized proteins through mass spectrometry interactome analysis of Odf2/Cenexin proteins. We further examined the expression patterns and functions of a protein named cilia and flagella associated protein 58 (Cfap58) in cultured astrocytes and sperm flagella. The results of a combination of biochemical analyses and drug administration studies reveal that Cfap58 is a testis-enrichment protein that exhibits similar localization to Odf2/Cenexin proteins and is required for the elongation of the primary cilium and sperm midpiece via modulation of the Notch signaling pathway. However, the cell cycle-related functions and localization of Odf2/Cenexin in the mother centriole were not altered in Cfap58 knockdown cells. These findings indicate that Cfap58 may be partially recruited by Odf2/Cenexin proteins and is indispensable for the cilia and flagellar assembly. These data provide us with a better understanding of ciliogenesis and flagellar elongation and may aid in identifying new targets for diseases caused by Notch-mediated ciliopathies and flagellar abnormalities.

Comparative analysis of mammalian sperm ultrastructure reveals relationships between sperm morphology, mitochondrial functions and motility.

BACKGROUND: Sperm morphology mainly refers to the shape of the head, the length of the flagellar segments, including the midpiece, principal piece and end piece, and the size of the accessory structures, including axonemes, outer dense fibers (ODFs), mitochondrial sheath (MS) and fibrous sheath (FS). Across species, there is considerable diversity in morphology. An established theory posits that the length of the sperm flagellum, especially the length of the midpiece, is a critical factor influencing sperm metabolism and velocity. However, our understanding of the relationships between sperm ultrastructures and the sperm flagellar length is incomplete. METHODS: The morphologies of sperm from 10 mammalian species, human, mouse, rat, dog, rabbit, goat, pig, bull, guinea pig and golden hamster, were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). According to the SEM and TME images, the length of sperm heads and flagellar segments, the cross-sectional areas of the accessory structures and flagella and the width of sperm heads were measured using Image J software. The variation tendencies (referred to as slope) of the accessory structures along flagella were calculated by the linear regression method. Mitochondrial functions were measured using commercial kits. The velocities of sperm were measured using CASA software. RESULTS: The three-dimensional morphologies of sperm from 10 species and the slopes of internal accessory structures along flagella were obtained. The width of the axoneme tapered slightly from the base to the tip of the sperm flagellum, and slopes of the axonemes correlated negatively with the variability in flagellar length across species. Additionally, the cross-sectional areas of the ODFs and/or the MS were positively correlated with the lengths of the midpiece, principal piece, and total flagellum, as well as with sperm velocities. Mitochondrial volumes were positively correlated with ATP content and sperm swimming velocities. CONCLUSIONS: Our results not only show the relationship between sperm internal structures, flagellar length and sperm physiology but also provide sizes of mitochondria and ODFs as new targets with which to study the regulation of sperm length and velocity.

Functional role of GKAP1 in the regulation of male germ cell spontaneous apoptosis and sperm number.

G kinase-anchoring protein 1 (GKAP1) is a G kinase-associated protein that is conserved in many eutherians and is mainly expressed in the testis, especially in spermatocytes and round spermatids. The function of GKAP1 in the testis is largely unknown. Here, we revealed that deletion of GKAP1 led to an increase in sperm production with swollen epididymis, and germ cell apoptosis was found to decrease in GKAP1 knock-out mice. Further investigations showed that a deficiency of GKAP1 could partly change the cellular location of cGK-Iα and increase the amount of active cAMP response element-binding protein (CREB) in the nucleus. Therefore, the expression of a particular inhibitor of apoptosis proteins (IAPs) was upregulated because of the activation of CREB, and this increase in IAPs was associated with a decrease in the level of activated caspase-3. These results suggest that a deficiency of GKAP1 in mouse testis could increase sperm production through a reduction of the spontaneous apoptosis of germ cells in the testis, possibly because of a change in the activity of the cGK-Iα pathway.

Proteomic analysis of seminal extracellular vesicle proteins involved in asthenozoospermia by iTRAQ.

Asthenozoospermia is a common cause of male infertility, but in most cases its etiology is unknown. The exocytic cell vesicles called seminal extracellular vesicles in the human seminal fluid have been reported to play a pivotal role in promoting the motility of spermatozoa, and functional disorder of seminal extracellular vesicles may cause male infertility. To determine whether abnormal seminal extracellular vesicles are involved in asthenozoospermia, the differential abundance proteins between normozoospermic (NSEV) and asthenozoospermic seminal extracellular vesicles (ASEV) samples were analyzed by iTRAQ coupled with two-dimensional liquid chromatography-tandem mass spectrometry. A total of 3,699 proteins were identified in the seminal extracellular vesicles (false discovery rate <0.01). Overall, 11 proteins were significantly upregulated (>1.2) in ASEV and 80 were significantly downregulated (<0.833). Functional bioinformatic analysis showed that these proteins with differential abundance were mainly associated with transport, metabolism, and signal pathways. The changes of OPTN, SMYD2, EIF2B2, TRPV6, ACE, PRSS8, and PPAP2A in ASEV were verified by western blot analysis, and we found that the abundance of TRPV6 markedly reduced in the seminal extracellular vesicles and ejaculated spermatozoa of asthenozoospermic patients, which indicated trpv6 was important in sperm motility. This study provides deeper insight into the involvement of seminal extracellular vesicles in asthenozoospermia and should aid the search for novel biomarkers of male infertility.

Murine PAIP1 stimulates translation of spermiogenic mRNAs stored by YBX2 via its interaction with YBX2†.

The expression of many genes during the postmeiotic stages of spermatogenesis is largely regulated by germ cell-specific RNA-binding proteins at the level of posttranscription. One of these RNA-binding proteins, YBX2, participates in mRNA storage and regulation of translation in haploid spermatids. How YBX2-stored mRNAs become translationally competent during spermiogenesis remains unknown. In the present study, we report for the first time that YBX2 interacts with PAIP1, a protein translation enhancer, in vitro and in vivo. In murine testes, PAIP1 is highly expressed and colocalizes with YBX2 in round spermatids. Using sequential RNA immunoprecipitation and sequence analysis, we identified a group of spermiogenic mRNAs indirectly bound by PAIP1 through YBX2. Translation of mRNAs bearing the YBX2 target sequence was significantly blocked by YBX2 protein, but was reinitiated when YBX2 was coexpressed with PAIP1 in vitro. Taken together, these results indicate that PAIP1 regulates the translation of YBX2-masked mRNAs during spermiogenesis, and we propose this YBX2-PAIP1 interaction plays an important role in male germ cell development.

Imsnc761 and DDX6 synergistically suppress cell proliferation and promote apoptosis via p53 in testicular embryonal carcinoma cells.

Intermediate-sized non-coding RNAs (imsncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. In the present research, we selected imsncRNA 761 (imsnc761) as a research target. Expression analyses in a previous study showed that imsnc761 was down-regulated in maturation-arrested testis tissues as compared with the level in normal controls. In the present study, we found that imsnc761 could interact with DEAD-box helicase 6 (DDX6) to induce NTERA-2 (NT2 (testicular embryonal carcinoma cell)) cell apoptosis and proliferation inhibition via the p53 pathway. This interaction between imsnc761 and DDX6 also inhibited mitochondrial function and specific gene transcription and translation. To facilitate further research, we used label-free quantitation method to analyze the associated differences in Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways and biological processes. This confirmed the changes in several specific pathways, which matched our molecular experimental results.

The expression of the new epididymal luminal protein of PDZ domain containing 1 is decreased in asthenozoospermia.

Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus, and cauda, and the luminal proteins of the epididymis play important roles in the above modifications. However, the proteins with differential enrichment between the caput and cauda are still largely unknown. To reveal the functions of the caput and cauda during sperm maturation, luminal proteins from caput and cauda of mice were analyzed by isobaric tag for relative and absolute quantitation (iTRAQ). Overall, 128 differentially enriched proteins were found, of which 46 were caput enriched and 82 were cauda enriched. Bioinformatic analysis showed that lipid metabolism was active in the caput; while anion- and cation-binding activity and phosphorus and organophosphate metabolism were active in the cauda. A new epididymal luminal protein, the caput-enriched PDZ domain containing 1 (Pdzk1), also named Na+/H+ exchange regulatory cofactor 3 (NHERF3), which plays a critical role in cholesterol metabolism and carnitine transport, was found in the lipid metabolism. Western blotting and immunofluorescence analyses showed that Pdzk1 was expressed in the epididymis but not in the testis, and localized at the middle piece of the sperm tail. Pdzk1 protein level was also reduced in the spermatozoa in case of asthenozoospermic patients compared with that in normozoospermic men, suggesting that Pdzk1 may participate in sperm maturation regulation and may be associated with male infertility. These results may provide new insights into the mechanisms of sperm maturation and male infertility.

Outer dense fibers stabilize the axoneme to maintain sperm motility.

Outer dense fibers (ODFs), as unique accessory structures in mammalian sperm, are considered to play a role in the protection of the sperm tail against shear forces. However, the role and relevant mechanisms of ODFs in modulating sperm motility and its pathological involvement in asthenozoospermia were unknown. Here, we found that the percentage of ODF defects was higher in asthenozoospermic samples than that in control samples and was significantly correlated with the percentage of axoneme defects and non-motile sperm. Furthermore, the expression levels of ODF major components (Odf1, 2, 3, 4) were frequently down-regulated in asthenozoospermic samples. Intriguingly, the positive relationship between ODF size and sperm motility existed across species. The conditional disruption of Odf2 expression in mice led to reduced sperm motility and the characteristics of asthenozoospermia. Meanwhile, the expression of acetylated α-tubulin was decreased in sperm from both Odf2 conditional knockout (cKO) mice and asthenozoospermic men. Immunofluorescence and biochemistry analyses showed that Odf2 could bind to acetylated α-tubulin and protect the acetylation level of α-tubulin in HEK293T cells in a cold environment. Finally, we found that lithium elevated the expression levels of Odf family proteins and acetylated α-tubulin, elongated the midpiece length and increased the percentage of rapidly moving sperm in mice. Our results demonstrate that ODFs are beneficial for sperm motility via stabilization of the axoneme and that hypo-expression of Odf family proteins is involved in the pathogenesis of asthenozoospermia. The lithium administration assay will provide valuable insights into the development of new treatments for asthenozoospermia.