Sperm motility is essential for male fertility and depends on the structural integrity of the sperm axoneme, which features a canonical ‘9 + 2’ microtubule arrangement. This structure comprises nine outer doublet microtubules (DMTs) that are associated with various macromolecular complexes. Among them, the nexin–dynein regulatory complex (N-DRC) forms crossbridges between adjacent DMTs, contributing to their stabilization and enabling flagellar bending. In this study, we investigated Ankyrin repeat and EF-hand domain containing 1 (ANKEF1, also known as ANKRD5), a protein highly expressed in the sperm axoneme. We found that ANKEF1 interacts with DRC5/TCTE1 and DRC4/GAS8, two key components of the N-DRC, and these interactions occur independently of calcium regulation. Male Ankef1−/− mice exhibited impaired sperm motility and infertility. Cryo-electron tomography revealed a typical ‘9 + 2’ axoneme structure with intact DMTs in Ankef1 null sperm; however, the DMTs showed pronounced morphological variability and increased structural heterogeneity. Notably, ANKEF1 deficiency did not alter ATP levels, reactive oxygen species levels, or mitochondrial membrane potential. These findings suggest that ANKEF1 may attenuate the N-DRC’s mechanical buffering—akin to a ‘car bumper’—between adjacent DMTs, thereby compromising axonemal stability under high mechanical stress during vigorous flagellar beating.

Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.

Abstract During spermiogenesis, the formation of the mitochondrial sheath is critical for male fertility. The molecular processes that govern the development of the mitochondrial sheath remain unknown. Whether TBC1D21 serves as a GTPase-activating protein (GAP) for GTP hydrolysis in the testis is unclear, despite recent findings indicating that it collaborates with numerous proteins to regulate the formation of the mitochondrial sheath. To thoroughly examine the property of TBC1D21 in spermiogenesis, we applied the CRISPR/Cas9 technology to generate the Tbc1d21–/– mice, Tbc1d21D125A R128K mice with mutation in the GAP catalytic residues (IxxDxxR), and Tbc1d21-3xFlag mice. Male Tbc1d21–/– mice were infertile due to the curved spermatozoa flagella. In vitro fertilization is ineffective for Tbc1d21–/– sperm, although healthy offspring were obtained by intracytoplasmic sperm injection. Electron microscopy revealed aberrant ultrastructural changes in the mitochondrial sheath. Thirty-four Rab vectors were constructed followed by co-immunoprecipitation, which identified RAB13 as a novel TBC1D21 binding protein. Interestingly, infertility was not observed in Tbc1d21D125A R128K mice harboring the catalytic residue, suggesting that TBC1D21 is not a typical GAP for Rab-GTP hydrolysis. Moreover, TBC1D21 was expressed in the sperm mitochondrial sheath in Tbc1d21-3xFlag mice. Immunoprecipitation-mass spectrometry demonstrated the interactions of TBC1D21 with ACTB, TPM3, SPATA19, and VDAC3 to regulate the architecture of the sperm midpiece. The collective findings suggest that TBC1D21 is a scaffold protein required for the organization and stabilization of the mitochondrial sheath morphology. Summary sentence The formation of the mitochondrial sheath is critical for male fertility, but the molecular processes that govern the development of the mitochondrial sheath remain unknown. By using multiple transgenic mouse models, we demonstrated that TBC1D21 is an essential protein for sperm mitochondrial sheath assembly and male fertility. The Tbc1d21-/- mice were infertile due to the malformation of the mitochondrial sheath assembly. Importantly, Tbc1d21D125A R128K mice were fertile, suggesting that TBC1D21 may not be a classic Rab-GTP hydrolysis protein. Tbc1d21-3XFlag mice elucidated that TBC1D21, ACTB, and TPM3 complex in the form of a double helix around the mitochondrial sheath to maintain its assembly structure. This research sheds light on the pivotal role of TBC1D21 in the organization and stabilization of the mitochondrial sheath morphology. Graphical Abstract

Meiosis is essential for fertility in sexually reproducing species, extensive studies tried to delineate this sophisticated process. Notwithstanding, the molecules involved in meiosis have not been fully characterized. In this study, we investigate the role of zinc finger protein 541 (ZFP541) and its interacting protein potassium channel tetramerization domain containing 19 (KCTD19) in mice. We demonstrate that they are indispensable for male fertility by regulating proper pachytene progression. ZFP541 is expressed starting from leptotene to round spermatids, and KCTD19 is initially expressed in pachytene. Depletion of Zfp541 or Kctd19 leads to infertility in male mice, and exhibits retarded progression from early to mid/late pachynema. In addition, Zfp541 -/ - spermatocytes show abnormal programmed DNA double-strand breaks (DSBs) repair, impaired crossover formation/resolution, and asynapsis of the XY chromosomes. Immunoprecipitation-mass spectrometry (IP-MS) and in vitro Co-IP reveal that ZFP541 interacts with KCTD19, histone deacetylase 1/2 (HDAC1), HDAC2 and deoxynucleotidyltransferase terminal-interacting protein 1 (DNTTIP1). Furthermore, RNA-seq and CUT&Tag analyses demonstrate that ZFP541 binds to the promoter regions of genes involved in meiosis and post-meiosis including Kctd19 , and activates their transcription. Taken together, our studies reveal a ZFP541- Kctd19 transcription regulatory axis and the crucial role of ZFP541 and KCTD19 for pachytene progression and fertility in male mice.

Successful regeneration of severed peripheral nerves requires the breakdown and subsequent clearance of myelin, tightly packed membrane sheaths of Schwann cells that protect nerve fibers and harbor nerve growth-inhibitory proteins. How Schwann cells initiate myelin breakdown in response to injury is still largely unknown. Here we report that, following sciatic nerve injury, MLKL, a pseudokinase known to rupture cell membranes during necroptotic cell death, is induced and targets the myelin sheath membrane of Schwann cells to promote myelin breakdown. The function of MLKL in disrupting myelin sheaths requires injury-induced phosphorylation of serine 441, an activation signal distinct from the necroptosis-inducing phosphorylation by RIP3 kinase. Mice with Mlkl specifically knocked out in Schwann cells showed delayed myelin sheath breakdown. Lack of MLKL reduced nerve regeneration following injury, whereas overexpression of MLKL accelerated myelin breakdown and promoted the regeneration of axons.