The molecular mechanism associated with mammalian meiosis has yet to be fully explored, and one of the main reasons for this lack of exploration is that some meiosis-essential genes are still unknown. The profiling of gene expression during spermatogenesis has been performed in previous studies, yet few studies have aimed to find new functional genes. Since there is a huge gap between the number of genes that are able to be quantified and the number of genes that can be characterized by phenotype screening in one assay, an efficient method to rank quantified genes according to phenotypic relevance is of great importance. We proposed to rank genes by the probability of their function in mammalian meiosis based on global protein abundance using machine learning. Here, nine types of germ cells focusing on continual substages of meiosis prophase I were isolated, and the corresponding proteomes were quantified by high-resolution MS. By combining meiotic labels annotated from the mouse genomics informatics mouse knockout database and the spermatogenesis proteomics dataset, a supervised machine learning package, FuncProFinder (https://github.com/sjq111/FuncProFinder), was developed to rank meiosis-essential candidates. Of the candidates whose functions were unannotated, four of 10 genes with the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik, and Kctd19, were validated as meiosis-essential genes by knockout mouse models. Therefore, mammalian meiosis-essential genes could be efficiently predicted based on the protein abundance dataset, which provides a paradigm for other functional gene mining from a related abundance dataset.

The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles toward nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generatedSsh2knockout (KO) mice and HA-taggedSsh2knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. InSsh2KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes ofSsh2KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.

Sperm motility and structural integrity are essential for successful fertilization in vivo, and any hindrance of the correct assembly of the axoneme and peri-axonemal structures in the sperm flagellum can lead to fertility problems. While there has been considerable advancement in studying diseases related to the flagellum, the underlying mechanisms that control sperm movement are not yet fully understood. In this study, we reveal that the tetratricopeptide repeat protein 6 (Ttc6) gene, expressed mainly in the testes, plays a crucial role in maintaining male fertility in mice. We further demonstrate that the knockout of Ttc6 in mice results in decreased sperm motility and induces an abnormal circular swimming pattern, consequently leading to male subfertility. Morphological analysis showed an atypical hairpin-like appearance of the spermatozoa, and ultrastructural studies showed unsheathed flagella at the juncture between the midpiece and principal piece. Collectively, these findings suggest that TTC6 plays an essential role in maintaining the stability of the annulus region of the sperm flagellum, thus ensuring the swift and directed motion of sperm.

Conversion of astrocytes to neurons (AtN) is a promising potential strategy for the treatment of neurodegenerative diseases. Recent studies have reported that shRNA-, CasRx-, or ASO-mediated Ptbp1 suppression could reprogram resident astrocytes to neurons ^1–3 . However, some groups have disputed the data interpretation of the reported AtN conversion events ^4–7 . These controversies surrounding AtN conversion may due to differences in the astrocyte fate-mapping systems they applied from that in the original study, i . e ., recombinant mouse strains with astrocyte specific reporter constructs versus AAV-based labeling systems. Here, we applied AAV-based tracing systems to label astrocytes with GFAP-driven HA-tagged Cas13X (AAV-GFAP::Cas13X-NLS-HA-sgPtbp1). Compared to GFAP-driven tdTomato reporter (AAV-GFAP::tdTomato) system in previous studies, we found no AtN conversion in mouse striatum. Furthermore, no intermediate neurons and no neuron density increase suggested no AtN conversion. Our findings indicated that inconsistent AtN outcomes may arise from different fate-mapping systems between AAV and transgenic mice, as well as through use of different reporter proteins. Thus, the complexity of astrocyte labeling systems warrants careful attention when drawing conclusions about whether AtN conversion occurs.

Mammalian meiosis is a specific cell division process during sexual reproduction, whereas a comprehensive proteome of the different meiotic stages has not been systematically investigated. Here, we isolated different types of spermatocytes from the testes of spermatogenesis-synchronized mice and quantified the corresponding proteomes with high-resolution mass spectrometry. A total of 8,002 proteins were identified in nine types of germ cells, and the protein signatures of spermatogenesis were characterized using the dynamic proteomes. A supervised machine learning package, FuncProFinder, was developed to predict meiosis-essential candidate genes based on changes in their protein abundance. Of the candidates without functional annotation, four of the ten genes with the highest prediction scores,Zcwpw1, Tesmin, 1700102P08Rik, andKctd19, were validated as meiosis-essential genes using knockout mouse models. The proteomic analysis of spermatogenic cells provides a solid foundation for studying the mechanism of mammalian meiosis.