Abstract Sox30 has recently been demonstrated to be a key regulator of spermatogenesis. However, the precise roles of Sox30 in the testis remain largely unclear. Here, the specific functions of Sox30 in testicular cells were determined by single-cell sequencing and confirmed via pathological analyses. Sox30 loss appears to damage all testicular cells to different extents. Sox30 chiefly drives the differentiation of primary spermatocytes. Sox30 deficiency causes spermatocyte arrest at the early phase of meiosis I, with nearly no normally developing second spermatocytes and three new spermatocyte -subclusters emerging. In addition, Sox30 seems to play important roles in the mature phenotypes of Sertoli and Leydig cells, and the proliferation and differentiation of spermatogonia. The developmental trajectory of germ cells begins with spermatogonia and splits into two different spermatocyte branches, with Sox30-null spermatocytes and wild-type spermatocytes placed at divergent ends. An opposite developmental trajectory of spermatocyte subclusters is observed, followed by incomplete development of spermatid subclusters in Sox30-null mice. Sox30 deficiency clearly alters the intercellular cross-talk of major testicular cells and dysregulates the transcription factor networks primarily involved in cell proliferation and differentiation. Mechanistically, Sox30 appears to have similar terminal functions that are involved mainly in spermatogenic development and differentiation among major testicular cells, and Sox30 performs these similar crucial roles through preferential regulation of different signalling pathways. Our study describes the exact functions of Sox30 in testicular cell development and differentiation and highlights the primary roles of Sox30 in the early meiotic phase of germ cells. Subject terms: Differentiation, Meiosis, Infertility Introduction Reproduction is a vital characteristic of life, because any form of life can occur and continue in various modes via reproduction [[36]1, [37]2]. To achieve fertility, a male requires a set of perfect and wholesome capabilities for sperm production [[38]3], which primarily depend on the normal development of both testicular germ cells and somatic cells, and the maintenance of fertility hinges on the continuous production of sperm [[39]4, [40]5]. In mammals, the testis develops in utero when the Y chromosome gene sex-determining region Y (SRY) is expressed. SRY initiates a genetic program, involving Sox9 that directs a subset of bipotential progenitor cells differentiate into Sertoli cells, which orchestrate further testis development [[41]4, [42]6, [43]7]. Testicular somatic cells contribute to morphogenesis of the foetal testis, and the dynamic regulation of different types of testicular somatic cells provides a highly specialized microenvironment, the testis niche, to support the normal development and differentiation of germ cells [[44]5]. Substained-steady spermatogenesis is driven by the balance between the self-renewal and differentiation of spermatogonial stem cells (SSCs), alongside highly complex germ cell–niche interactions [[45]8–[46]10]. On the basis of the balance of their self-renewal and differentiation, SSCs then undergo niche-guided transitions between multiple cell states and cellular processes, including a commitment to mitosis, meiosis, and the subsequent stage of sperm maturation accompanied by chromatin repackaging and morphological changes [[47]8, [48]11, [49]12]. Although considerable progress in considering the biological process of gametogenesis and germline-niche communication has been achieved [[50]5, [51]8, [52]9, [53]13], the finely programmed and developmental molecular events of gametogenesis remain largely unknown. SRY-box (Sox) proteins are a family of transcription factors that contain a highly conserved high mobility group (HMG)-box for DNA-binding [[54]7, [55]14]. The mammalian Sox family is composed of approximately 20 members, including the testis-determining factors Sry and Sox9, and is widely involved in diverse developmental processes and tissue systems, including preimplantation development, endoderm induction, cell fate commitment and reprogramming, haematopoiesis, and sex determination and differentiation [[56]7, [57]14–[58]16]. Sox factors may be closely associated with developmental disorders and various diseases, including cancer. An increasing number of studies have confirmed the potential of Sox family members in the diagnosis and treatment of developmental diseases and tumours [[59]17, [60]18] Among Sox family members, Sox30, the only member of subfamily H, is a relatively unkonwn member. In recent years, SOX30 has been shown to play important roles in tumorigenesis, including in lung cancer, ovarian cancer, and other types of cancer [[61]19–[62]22]. However, although Sox30 is a male-specific gene that is highly expressed in adult testes but not in ovaries [[63]23, [64]24], the major roles of Sox30 in male development and reproduction seem to have been ignored for a long time. On the basis of the positive expression pattern of Sox30 in testicular development, Sox30 has been proposed to be involved in spermatogenic differentiation and spermatogenesis in mice [[65]24–[66]26]. Studies have demonstrated that Sox30 is a key regulator of spermiogenesis [[67]27]. Sox30-knockout mice exhibit specific testicular pathological defects with lower testicular size and weight concomitant with sterility without spermatozoa, whereas fertile spermatozoa are observed after Sox30 is re-expression in the knockout mice [[68]27–[69]29]. Although the importance of Sox30 in regulating spermatogenesis has gradually become understood, the specific stage at which Sox30 predominantly functions in spermatogenic cells is still controversial. Sox30 is considered to have a role work in later meiotic cells and postmeiotic haploids, as spermiogenesis is arrested at the early round spermatid stage in mutant males [[70]27, [71]30]. Mechanistically, Sox30 controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development [[72]31]. However, the rich expression of Sox30 seems to be restricted meiotic spermatocytes and round spermatids, and Sox30 expression is clearly greater in meiotic spermatocytes than in round spermatids [[73]29, [74]31, [75]32], suggesting that Sox30 may act before round spermatid formation. Our previous study revealed that the germ cells of Sox30-null male mice are arrested during the meiotic process, markedly impaired in the zygotene-to-pachytene transition, and concomitant abnormal proliferation of Leydig cells [[76]29]. Mechanistically, Sox30 directly targets the meiotic genes Stra8, Rec8 and Cyp26b1, which are involved in the retinoic acid (RA) signalling pathway, and it directly or indirectly regulates a group of sex differentiation genes (including Sox9, Wnt4, Rspo1 and Ctnnb1) [[77]29]. These data imply that Sox30 likely plays a role in germ cell differentiation and even sex determination in early developmental stages. However, the precise roles of Sox30 in testicular cell development and differentiation remain largely unexplored. In this study, we conducted single-cell RNA sequencing on testicular tissue from a successfully constructed Sox30-null mouse model. Combined with transcriptional profiles of testicular cells, 10 testicular cell clusters and their proportional change were identified in Sox30-null mice. The major results of the cell proportional changes were further confirmed by pathological phenotype analyses. The subclusters of the major testicular cells and their proportional variation were further analysed in Sox30-null mice. Loss of Sox30 resulted in spermatocytes arrest at meiosis I, suggesting that Sox30 primarily drives the development and differentiation of spermatocytes. Moreover, the aberrant germ cell differentiation of Sox30-null mice likely begins as early as spermatogonial process, and Sox30 plays a role in regulating the mature phenotypes of Sertoli cells and Leydig cells. The developmental pseudotime trajectory of testicular cells was subsequently constructed, which revealed that the development of spermatocytes was influenced mainly by Sox30. In addition, intercellular communication and transcription factor regulatory network analyses were performed, and revealed that Sox30 plays essential roles in the cross-talk of testicular cells and maintaining the spermatogenic microenvironment. Our present study preliminarily presents the functional features of Sox30, and reveals the specific roles of Sox30, in testicular cell development and differentiation. Materials and methods Generating Sox30-null mice Sox30-null mice were generated by the Model Animal Research Center at Nanjing University, and the construction strategy has been described in our previously reports [[78]29, [79]33]. Briefly, the targeted gene including homologous arms was retrieved from a BAC vector, and a LoxP-SA-IRES-GFP-NEO-STOP-PPS-LoxP cassette was inserted between Exon1 and Exon2 of Sox30 via homologous recombination. The targeting vector was validated by PCR, enzyme digestion, and sequencing, then linearized with AsiSI and electroporated into C57BL/6 embryonic stem (ES) cells. Recombinants were selected under G418 and ganciclovir (Ganc). Targeted ES cells were screened by PCR and Southern blot, and positive clones were chosen for microinjection to generate chimeras. The chimeras were crossed with C57BL/6 mice to produce heterozygous offspring. The mice were maintained in a specific pathogen-free (SPF) facility under a 12-h light/12-h dark cycle with ad libitum access to water and food. The genotype of the offspring was identified by PCR. All mouse experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Chongqing Medical University and Army Medical University, China. Hematoxylin-eosin staining analysis Testes were dissected from mice of different genotypes and immediately fixed in Bouin’s fluid (Scientific Phygene Inc, China). The fixed testes were dehydrated, embedded in paraffin, and sectioned into 5 μm thick slices. These sections were de-waxed, rehydrated, and stained with hematoxylin and eosin (H&E; Beyotime, Shanghai, China). Cell proliferation analysis The proliferation of testicular cells was assessed using 5-ethynyl-2’-deoxyuridine (EdU). Mice of different genotypes were intraperitoneally injected with 100 µg of EdU. Seventy-two hours after injection, the proliferation assay of testicular cells was performed using an EdU detection kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. Immunofluorescence analysis Testis tissues from mice of different genotypes were harvested into ice-cold PBS, fixed with freshly prepared 4% paraformaldehyde for 30 min, and punctured with a needle. After overnight fixation, the tissues were incubated in 15% sucrose for one day and then in 30% sucrose for another day. The tissues were frozen in liquid-cooled isopentane. Sections (5 µm thick) were obtained and placed onto Fisherbrand Colorfrost Plus slides, then air-dried. The section slides were washed with PBS and permeabilized with 0.1% Triton X-100 for 1 h. These sections were quenched with 0.1 M glycine, washed with PBS, and blocked with 10% foetal bovine serum. They were then incubated with the primary antibody, Sycp3(Scp3) mouse monoclonal antibody (1:100, sc-74569, Santa Cruz Biotechnology), followed by the fluorescent secondary antibody, DyLight 488 Goat Anti-Mouse IgG (1:100, A23210, Abbkine). The slides were observed under a fluorescence microscope (Super-Resolution Mocroscope system, C2/SIME, Nikon, Japan). Immunohistochemistry (IHC) analysis Paraffin-embedded testis tissue sections (5 µm thick) were baked at 60 °C for 2 h, deparaffinized in xylene, and rehydrated through graded alcohol series to water. The sections were immersed in citrate buffer and subjected to antigen retrieval for 15 min at 95 °C. The tissue sections were then blocked with 0.3% H[2]O[2] and, treated with 10% normal goat serum for 15 min. They were incubated with the primary antibody, Cyp26b1 (1:100, 21555-1-AP, proteintech) overnight at 4 °C, followed by incubation with a biotinylated secondary antibody (1:2000, Abbkine) for 2 h at room temperature. The slides were sealed with neutral resin (Solarbio, Beijing, China), and photographs were taken under a light microscope (Olympus, Japan). Single-cell solution preparation Fresh testicular tissues were removed from sacrificed and dissected mice of different genotypes randomly, and immediately stored in MACS Tissue Storage Solution (Miltenyi Biotec, Germany) on ice. To generate sufficient material, testicular tissues from three 10-week-old mice of the same genotype were pooled. The testicular tissues were cut into about 1 mm^3 pieces, washed in PBS, and digested using the Solo^TM Dissociation Kit (Sinotech Genomics, JZ-SC-58201) for 45 min at 37 °C. The enzymatic digestion was stopped by adding excess RPMI-1640 medium, and the cell solution was filtered through a 40 μm cell strainer. The cells were then sorted using a MACS Dead Cell Removal Kit (Miltenyi Biotec) to remove dead cells. Living cells were resuspended, and the prepared single cell suspension was kept on ice until loaded onto the Chromium controller instrument (10× Genomics) for single-cell transcriptome. Single-cell transcriptome capturation, library construction and sequencing The prepared cells were loaded into Chromium microfluidic chips and barcoded using a 10× Chromium Controller (10× Genomics). Single-cell transcriptomes were reverse-transcribed into cDNA libraries containing 10× cell barcodes and unique molecular identifiers (UMIs). All procedures were performed using reagents from the Chromium Next GEM Single Cell 5’ Library & Gel Bead Kit v1.1 (10× Genomics, Cat. No. 1000165) according to the manufacturers’ protocol. The libraries were sequenced in PE150 mode (pair-end for 150 bp reads) on the NovaSeq platform (Illumina). Raw sequencing reads from the cDNA libraries were mapped to the reference genome using 10× Genomics Cell Ranger (v3.1.0). Sequencing data processing Raw sequencing reads of the cDNA library were mapped to the reference genome using 10× Genomics Cell Ranger (v3.1.0). Genome Reference Consortium Mouse Build 38 (GRCm38) was used as the reference genome for the 10× Cell Ranger. Contamination removal of single-cell sequencing When visualizing the expression levels of specific cell marker genes using FeaturePlot in the Seurat package v3.1.2, ambient RNA contamination was detected. To address this issue, ambient RNA removal was performed using the decontx algorithm in the celda package v.1.3.8 [[80]34]. The decontx algorithm assumes the presence of K cell populations and employs Bayesian variational inference to estimate the ambient RNA contamination as a weighted combination of the specific cell population distributions. This algorithm requires the raw UMI counts and several cell populations, identified using the Find Clusters function in the Seurat package v3.1.2 as input. As a result, a decontaminated count matrix was derived from the raw data. A default random seed was used throughout the analyses to ensure reproducibility. Quality control, dimension-reduction and clustering of single-cell sequencing Cells were filtered based on gene counts ranging from 200 to 5,000 and UMI counts below 30,000. Cells with more than over 20% mitochondrial content were removed. The Seurat v3.1.2 package [[81]35] was used for dimensionality reduction and clustering. Gene expression data were normalized and scaled using the Normalize Data and Scale Data functions, respectively. The top 2000 variable genes identified using the Find Variable Features function and were selected for principal component analysis (PCA). Based on the top 20 principle components, cells were clustered using the Find Clusters function. Batch effect between samples were corrected using Harnomy [[82]36]. Finally, uniform manifold approximation and projection (UMAP) algorithm was applied to visualize cells in a two-dimensional space. Cell type annotation of single-cell sequencing The cell type for each cluster was identified based on the expression of canonical markers found among the DEGs using the SynEcoSys database. Heatmaps and dot plots displaying the expression of these markers were generated using the DoHeatmap, DotPlot, and Vlnplot functions in Seurat v3.1.2. Differentially expressed genes (DEGs) analysis of single-cell sequencing To identify differentially expressed genes (DEGs), the Find Markers function in Seurat, based on the Wilcox likelihood-ratio test with default parameters, was used. Genes were selected as DEGs if they were expressed in more than 10% of the cells within a cluster and had an average log fold change (FC) value greater than 0.25 or less than -0.25. For the annotation of cell types in each cluster, the expression of canonical markers found among the DEGs was evaluated using literature knowledge. The expression of markers for each cell type was visualized using heatmaps, dot plots, and violin plots generated with the DoHeatmap, DotPlot, and Vlnplot functions in Seurat. Cells identified as doublet, expressing markers for different cell types, were manually removed. Function and pathway enrichment analysis of single-cell sequencing To investigate the potential functions and pathways of DEGs, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the “clusterProfiler” R package (version 3.16.1). Functions and pathways with an adjusted p-value(p-adj) less than 0.05 were considered significantly enriched. GO gene sets including molecular function (MF), biological process (BP), and cellular component (CC) categories were used as references.