Two Spag6 genes control sperm formation and male fertility in mice

Liu, Y.; Li, W.; Li, T.; Zheng, C.; Niu, C.; Schmitt, A.; Yap, Y. T.; Abdulghani, M.; Yuan, S.; Melander, C.; Strauss, J. F.; Toure, A.; Zhang, L.; Zhang, Z.

doi:10.1101/2025.07.18.665465

bioRxiv · 10.1101/2025.07.18.665465

Two Spag6 genes control sperm formation and male fertility in mice

Liu, Y.·Li, W.·Li, T.·Zheng, C.·Niu, C.·Schmitt, A.·Yap, Y. T.·Abdulghani, M.·Yuan, S.·Melander, C.·Strauss, J. F.·Toure, A.·Zhang, L.·Zhang, Z.

View original source ↗Export metadata ↓

Abstract

Sperm-associated antigen 6 (SPAG6) is the mammalian orthologue of Chlamydomonas PF16, a central axonemal protein essential for flagellar motility. In mice, two homologous genes have been identified: the ancestral Spag6 on chromosome 2 and the evolutionary derived Spag6l on chromosome 16. Although Spag6 knockout mice (Spag6-/-) are phenotypically normal, the surviving Spag6l-/- males are infertile. To further investigate the roles of SPAG6 and SPAG6L, we generated compound mutants by crossing the two knockout lines. Compound heterozygous Spag6+/-; Spag6l+/- mice are fertile, while all Spag6-/-; Spag6l+/- males are infertile despite grossly normal appearance. Histological and ultrastructural analyses revealed defective spermiogenesis, including abnormal chromatin condensation, malformed acrosome and manchette, and disorganized mitochondrial and fibrous sheath. Both SPAG6 and SPAG6L bind to SPINK2, a key regulator of acrosome function, but SPAG6 has an approximately 10-fold higher binding affinity than SPAG6L. Moreover, SPAG6 modulates testicular AKAP4 and SPAG16L levels, which are critical components of the fibrous sheath and central apparatus respectively. Notably, SPAG6 suppresses tubulin acetylation, whereas SPAG6L enhances this post-translational modification, suggesting antagonistic roles in microtubule assembly. Overall, our findings demonstrate that SPAG6 and SPAG6L coordinately regulate sperm formation and male fertility during evolution.

Explore related subjects

developmental biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Liu, Y., Li, W., Li, T., Zheng, C., Niu, C., Schmitt, A., Yap, Y. T., Abdulghani, M., Yuan, S., Melander, C., Strauss, J. F., Toure, A., Zhang, L., Zhang, Z.. 2025-07-22. Two Spag6 genes control sperm formation and male fertility in mice. https://doi.org/10.1101/2025.07.18.665465

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A bicistronic Aldh1a3-P2A-TagBFP knock-in reporter mouse line for studying genitourinary tract development

Aldehyde dehydrogenase 1a3 (Aldh1a3) is an enzyme involved in retinoic acid synthesis with dynamic expression patterns during development, including in the urogenital system. Here, we generated a bicistronic Aldh1a3-P2A-TagBFP knock-in mouse using CRISPR/Cas9 genome editing, inserting TagBFP immediately upstream of the endogenous Aldh1a3 stop codon. Correct targeting was confirmed by Oxford Nanopore long-read sequencing, and heterozygous and homozygous mice were viable and fertile without overt morphological abnormalities. TagBFP fluorescence faithfully overlapped with endogenous Aldh1a3 immunoreactivity and reproduced established expression domains in the developing craniofacial region, intestine, kidney, and broader urogenital system. Extensive characterization of the urogenital system revealed dynamic, spatially restricted BFP reporter activity in Aldh1a3-expressing domains across several key structures, including the ureteric bud and collecting duct lineage, seminal vesicles, caput epididymis, and developing uterine horns. The Aldh1a3-P2A-TagBFP mouse provides a fluorescent resource for visualizing Aldh1a3 expression across development and in adult tissues, including for the characterization of Aldh1a3-expressing domains in the urogenital system. The relatively low fluorescence intensity of TagBFP should be considered when assessing low-level reporter expression.

developmental biology↗

bioRxiv2026

Translation of a small upstream open reading frame functions as a rheostat for the regulation of lin-41 by the Let-7 microRNA in Caenorhabditis elegans

MicroRNAs have been likened to the "dark matter" of eukaryotic genomes, reflecting their pervasive regulatory influence. MicroRNAs were first identified through genetic studies of developmental timing in the nematode Caenorhabditis elegans. Let-7 was the first microRNA recognized to be broadly conserved. The principal target of Let-7 in the developmental timing pathway is the TRIM-NHL RNA-binding protein LIN-41. During the L4 larval stage, Let-7 represses lin-41 translation by binding to two Let-7 complementary sites in the lin-41 3'UTR. Despite the importance of microRNA-based translational regulation, the underlying molecular mechanisms are incompletely understood. Through genetic analysis, we discovered an unrecognized feature of the mechanism by which Let-7 controls lin-41 translation. This mechanism requires a 5'-regulatory exon containing a seven-amino acid upstream open reading frame (uORF) and conserved sequence elements. Genome editing indicates that the specific uORF amino acid sequence itself is not important. Our data suggest that uORF translation and 5'UTR structure limit initiation at the downstream lin-41 start codon, enabling tight control by Let-7. Without this mechanism, the Let-7 microRNA is unable to properly regulate lin-41 to enable proper development.

developmental biology↗

bioRxiv2026

Ductal myofibroblasts reactivate contractile program to stabilize alveolar architecture during lung regeneration

The alveolar sac architecture is essential for efficient gas exchange and must be precisely maintained throughout life; however, how this delicate structure is preserved during adult regeneration remains poorly understood. Using a mouse pneumonectomy model, we found that Lgr6+ Hhip+ ductal myofibroblasts, a poorly characterized mesenchymal population, are indispensable for maintaining alveolar integrity during lung regrowth. Comprehensive characterization using single-cell transcriptomics, mouse genetics, and pharmacological assays demonstrated that these ductal myofibroblasts secrete myogenic factors, most notably CCN4, to reactivate a myogenic program that converts them into contractile PA-DMFs, thereby preserving alveolar architecture. Lineage-tracing further revealed that these ductal myofibroblasts originate from embryonic MCAM- SMA+ distal progenitors via subepithelial TGF-{beta} signaling, serving as a lifelong guardian of alveolar structural integrity. Notably, cross-species analysis identified an analogous population of LGR6+ fibromyocytes in human respiratory bronchioles. Together, these findings indicate ductal myofibroblasts as a developmentally programmed cell population that reactivate a contractile program to structurally support the regeneration of adult lungs.

developmental biology↗