A comparative analysis of genes differentially expressed between rete testis cells and Sertoli cells of the mouse testis

The rete testis (RT) is a region of the mammalian testis that plays an important role in testicular physiology. The RT epithelium consists of cells sharing some well-known gene markers with supporting Sertoli cells (SCs). However, little is known about the differences in gene expression between these two cell populations. Here, we used fluorescence-activated cell sorting (FACS) to obtain pure cultures of neonatal RT cells and SCs and identified differentially expressed genes (DEGs) between these cell types using RNA sequencing (RNA-seq). We then compared our data with the RNA-seq data of other studies that examined RT cells and SCs of mice of different ages and generated a list of DEGs permanently upregulated in RT cells throughout testis development and in culture, which included 86 genes, and a list of 79 DEGs permanently upregulated in SCs. The analysis of studies on DMRT1 function revealed that nearly half of the permanent DEGs could be regulated by this SC upregulated transcription factor. We suggest that useful cell lineage markers and candidate genes for the specification of both RT cells and SCs may be present among these permanent DEGs.


Establishment of a pure culture of RT cells by FACS
Pure populations of neonatal (5-6 dpp) RT cells and SCs were needed to accurately compare their transcriptomes using bulk RNA-seq.Pure SCs were obtained by the method developed in our previous study which was based on labeling peritubular myoid cells with PDGFRA antibody and then sorting them out of the cell suspension of seminiferous tubules by FACS 13 .PDGFRA -cell fraction was cultured for 3 days to eliminate germ cells.As a result, SCs identified by WT1 staining accounted for 93.3 ± 2.4% of cells in the culture (Supplementary Fig. S1).
To obtain a pure culture of RT epithelial cells we also applied FACS.It was shown in the study of Nagasawa et al. 14 and also in our previous study 12 that all RT cells expressed surface marker cadherin 1 (CDH1).So, we first verified that a CDH1 antibody for flow cytometry stained cells of the RT (Fig. 1a,b).Next, we used this antibody to sort CDH1 + cell fraction from a neonatal testicular cell suspension (Fig. 1c).The cells were maintained in culture for 3 days to eliminate spermatogonial cells which also expressed CDH1 15 .
We also demonstrated that such an isolation procedure could be applied to adult RT cells.After cell staining and FACS (Fig. 1h), CDH1 + cell fraction was cultured for 8 days.RT cells formed large colonies (Fig. 1i) stained for PAX8 (Fig. 1j) and SOX9 (Fig. 1k), DMRT1 was present in some cells (Fig. 1j).The number of RT cells was 94.1 ± 1.5% according to PAX8 staining, while the number of SCs was negligible.All cells in the culture were positive for vimentin (Fig. 1j) and negative for DDX4 (Supplementary Fig. S2).RT qPCR data confirmed the purity of the culture (Fig. 1l).

Identification of DEGs between neonatal RT cells and SCs and their functional analysis
Bulk RNA-seq of three samples of neonatal RT cell cultures and three samples of neonatal SC cultures was performed.A heatmap of Pearson correlation coefficients between samples is shown in Fig. 2a.According to the principal component analysis (PCA), RT cells and SCs were greatly separated in principal component 1 (PC1) (Fig. 2b).Differential expression analysis of the RNA-seq data identified 20,666 unique genes (Supplementary Data).
We selected the genes with a minimum mean expression level of 5 RPKM in at least one of the cell populations and generated a volcano plot (Fig. 2c).DEGs with FDR (false discovery rate) < 0.05 and with an absolute value of log2 fold change (LFC) > 1 were chosen for the further analysis.Among these DEGs, 858 genes were upregulated in RT cells and 835 genes were upregulated in SCs (Supplementary Data).The volcano plot indicated a trend towards greater LFC absolute values of DEGs upregulated in RT cells (Fig. 2c).Indeed, among DEGs with an absolute value of LFC > 4, 144 genes were upregulated in RT cells and only 4 genes were upregulated in SCs (Fig. 2d).The expression of three genes upregulated in RT cells and four genes upregulated in SCs was validated by RT qPCR in independent samples.qPCR data correlated with RNA-seq data (Fig. 2e,f).
Panther-based Gene Ontology (GO) enrichment analysis 16,17 was performed to characterize RT cells and SCs.Selected GO terms are presented in Table 1, and complete results of the analysis with statistics can be found in Supplementary Data.The term collagen-containing extracellular matrix and terms associated with plasma membrane bounded cell projections were overrepresented in both cell populations.The latter group of terms might indicate cell migration and movements during the establishment of cell cultures.
DEGs upregulated in SCs exhibited an enrichment in categories associated with tight junctions, organelles from the endocytic pathway, mitochondria as well as with lipid metabolism and response to endogenous stimuli including hormones (Table 1).In contrast, DEGs upregulated in RT cells were enriched in terms associated with proliferation, heterochromatin, actin cytoskeleton, adherens junctions and cell polarity.They also showed an overrepresentation of genes involved in TGF beta, BMP, and Wnt signaling pathways and genes important for embryonic development (Table 1).

(c,h) FACS of cells isolated from RT regions of neonatal (c) and adult mice (h).
The cells were stained with CDH1 and isotype control antibodies labeled with PE.Debris and doublets were excluded from the analysis.The percentage of CDH1 + cells was presented as the mean ± SEM from three independent experiments.(d,i) Morphological appearance of neonatal (d) and adult (i) RT cell cultures on day 3 and day 8 respectively.(e,j) Triple immunofluorescent staining of neonatal (e) and adult (j) RT cell cultures for PAX8, DMRT1, and vimentin (Vim).The inset in (e) shows DMRT1 staining of a neonatal SC culture for comparison.(f,k) Immunofluorescent staining of neonatal (f) and adult (k) RT cultures for SOX9; the neonatal culture (f) was co-stained for ACTA2.Images in (e,f,j,k) were stitched from several adjacent fields of view.(g,l) mRNA levels of markers of different testicular cell populations in neonatal (g) and adult (l) RT cultures relative to that in adult testicular tissue (the zero line).Data are presented as the mean ± SEM from three independent experiments.**p < 0.01, ***p < 0.001.Scale bars: 50 μm (a,b), 100 μm (d-f,i-k).www.nature.com/scientificreports/ As we especially focused on the specification of RT cells, we used GO and Panther databases to identify DEGs with transcription factor (TF) activity (Table 2).Besides Pax8, RT cells exhibited increased expression of Sox17 described in the RT recently 2 .Emx2 and Pbx1 participating in embryonic gonad development 18,19 as well as Tbx1, Tbx2, and Tead2 contributing to formation of various organs 20,21 were all increased in an RT cell culture.Genes for three members of ID family of TFs were also upregulated in RT cells.It was previously reported that the upregulation of ID TFs dramatically increased SC proliferative activity 22 .
SCs exhibited increased expression of Dmrt1, Sox9, Stat1, and Thra (Table 2).Dmrt1 and Sox9 were shown to be crucial for maintenance of SC fate 23,24 , and Stat1 and Thra were reported to be important regulators of SC function 25,26 .
Considering the importance of intercellular communications between RT cells and SCs 1,2 , we also identified DEGs demonstrating receptor ligand activity (Table 2).Different members of TGFb, FGF and BMP families of signaling molecules were upregulated in both cell populations.RT cells showed increased expression of Gdf15 previously identified in adult RT cells 12 .Genes encoding activin and inhibin subunits were upregulated in SCs.

Comparative analysis of DEGs between RT cells and SCs of various ages
To examine transcriptomes of RT cells and SCs of different ages we used publicly available data obtained in other studies.First, we analyzed scRNA-seq data on juvenile (14 dpp) wild-type mice from the study by Uchida et al. 2 .We generated Uniform Manifold Approximation and Projection (UMAP) plot with 18 cell clusters www.nature.com/scientificreports/(Supplementary Fig. S3) and identified clusters of RT cells and SCs using the same criteria as in the original study 2 .Specifically, clusters 2, 3, 11, and 14 highly expressed Sox9.Cells from clusters 2, 3, and 11 were defined as SCs.Cluster 14 which also exhibited increased expression levels of Pax8, Cdh1 and Krt8, was defined as an RT cell cluster (Supplementary Fig. S3).Further analysis identified 1791 genes differentially expressed between RT cells and SCs with Padj value < 0.05 (Supplementary Data).
We also used scRNA-seq data on fetal mice from the study by Mayère et al. 11 .In this case, DEGs between fetal RT cells (XY late supporting-like cells) and fetal SCs had already been identified by the authors 11 .
Next, we performed pairwise comparisons of DEG lists obtained for fetal 11 , neonatal (the current study), and juvenile cells 2 .The results are presented as Venn diagrams (Fig. 2g,h).The lists of overlapped DEGs can be found in Supplementary Data.As it is shown in Fig. 2g,h, 86 DEGs upregulated in RT cells and 79 DEGs upregulated in SCs are common for all three datasets, which means that these DEGs are permanently differentially expressed between RT cells and SCs throughout testis development and in culture.
As we suggest that cell lineage markers and key genes for the specification of both RT cells and SCs may be among these DEGs, we examined the DEG products using the Panther Protein Class database (Table 3).A noncoding RNA Meg3 from the list of RT upregulated DEGs was excluded from the analysis.The same was done to Gm14226 from the SC list as it encoded a viral or transposable element protein.As many as 18 DEGs out of 85 DEGs in the RT list encoded TFs such as PAX8, EMX2, PBX1, ID, and TEAD2 (Table 3).However, only Dmrt1 and Mef2c encoded TFs in the SC list.Mef2c was not previously described in SCs whereas Dmrt1 was identified as a crucial factor in SC fate maintenance, as it was mentioned above.Our immunofluorescence data confirmed the higher level of DMRT1 expression in SCs compared with that in RT cells (Fig. 1e, inset).Genes of transcription cofactors Cited1 and Cited2 were also permanently upregulated in SCs with Cited2 known to be involved in embryonic testis development 34 .
As opposed to the situation with TFs, 23 out of 78 DEGs in the SC list encoded enzymes (PC00262 and PC00260) whereas only 8 DEGs in the RT list were enzyme genes (Table 3).SC enzymes included GSTM6 and GSTM7, CTSL, and 6 ubiquitin-protein ligases, whereas ALDH1A3, which was responsible for the synthesis of all-trans retinoic acid 35 , was present in the RT list.
A considerable number of DEGs permanently upregulated in RT cells encoded proteins involved in cell signaling such as growth factors TGFB2, 3 and BMP4 and transmembrane signal receptors FGFR2 and KDR.Genes encoding intercellular signal molecules SEMA6C, DHH and INHA and a transmembrane receptor AXL were permanently upregulated in SCs (Table 3, PC00207 and PC00197).In both DEG lists there were genes associated with intracellular signaling through small GTPases with Cdc42ep3, Arhgap42, Dlc1, and Slit3 upregulated in RT cells and Rab31, Rnd2, and Cdc42se1 upregulated in SCs (Table 3, PC00095, PC00226).Genes encoding specific microtubule-associated proteins TUBB3 and DYNLT1A (PC00085), calcium-binding proteins, chaperones (CLU, HSPB1), and proteins related to mitochondrion function were present in the SC list.Genes encoding proteins related to extracellular matrix and cell adhesion, such as BCAM, PODXL, and 5 protease inhibitors (PC00095), were present in the RT list (Table 3).
A manual search of published articles showed that 50 DEGs from the SC list and 17 DEGs from the RT list were previously described in SC lineage (Table 3, references for Table 3 can be found in Supplementary References), however the functional significance of many of them in the testis was not defined.The SC list from the current study also strongly corresponded to the gene list from the study utilizing the RiboTag approach to identify SC genes that were actively translated 36 .
Considering that a crucial TF DMRT1 was one of the only two TFs in the SC list, we conducted an additional comparative analysis using two recent studies on the mechanisms of DMRT1 action.One of them performed bulk RNA-seq to examine the changes in the transcriptome of cultured granulosa cells after DMRT1 activation 37 .The other study utilized the same method for identifying the differences in gene expression between neonatal SCs isolated from mice homozygous and heterozygous for a Dmrt1 null mutation 38 .We used the lists of DEGs

Expression of RT and SC genes in adult testicular cultures
To examine if the lists of RT and SC permanently upregulated genes were appropriate for the adult cells, we compared mRNA levels of selected genes in cultures of adult RT cells and SCs using RT qPCR.Establishment of a pure culture of adult RT cells was described above (Fig. 1h-l).For an adult SC culture, we used a method developed in the study by Saewu et al. 39 which was based on serial enzymatic digestions.In our hands, the SC number, identified by WT1, SOX9 and DMRT1 staining, was 64.8 ± 6.4% on day 8 (Fig. 3a), and the main admixture was peritubular myoid cells (Fig. 3b).Germ cells were present in the culture at first, but they were gradually removed during medium replacements.So, only a few germ cells, identified by DDX4 staining (Fig. 3c, arrow), and a small amount of germ cell debris (Fig. 3a,d) remained on day 8.According to RT qPCR, markers of germ, Table 3. Classification of DEGs permanently upregulated in RT cells and SCs according to the Panther Protein Class database.*Manually classified DEGs.The list of references is in Supplementary Data.The references cite studies that reported expression of a gene in the SC lineage or in seminiferous tubules/testis cords.Bold type labels DEGs in the RT list that could be downregulated by DMRT1 and DEGs in the SC list that could be upregulated by DMRT1 (according to 37,38 ).DEGs are listed in order of decreasing mean RPKM value.Leydig, and endothelial cells were expressed at lower levels compared with those in testicular tissue, whereas a myoid cell marker Myh11 was slightly upregulated (Fig. 3e).Despite the fact that the culture lengths of neonatal and adult cells were different, all of the selected genes from the RT list were strongly upregulated in an adult RT culture compared with an adult SC culture (Fig. 3f).We also measured the expressions of TF genes Tbx1 and Tbx2.They were not included in the RT list, but Tbx1 was upregulated in juvenile RT cells 2 , Tbx2 was elevated in fetal RT cells 11 , and both of them were increased in neonatal and adult RT cells.So, we suggested that they might be important regulators of RT function.Selected genes from the SC list were upregulated in an adult SC culture compared with an adult culture of RT cells, although for two of them, Basp1 and Tubb3, the increase was statistically insignificant (Fig. 3g).Similarly to RNA-seq data, the genes from the RT list exhibited higher expression fold changes than the genes from the SC list.
These results suggest that at least some of the genes from the lists of RT and SC permanently upregulated genes are valid for adult cells and also for prolonged culture.www.nature.com/scientificreports/

Discussion
In our previous report 12 , we used RT cell cultures that also contained peritubular myoid cells and SCs, with the numbers of these cells being especially high in neonatal RT cell cultures.That hindered the interpretation of the experiments and the culture propagation.In the current study, we developed a procedure for establishing a pure culture of RT cells.Specifically, we used FACS to sort CDH1 + cell fraction from a testicular cell suspension.Applying this approach, we verified our previous results, according to which a substantial number of RT cells express TF DMRT1, which was earlier observed only in SCs and germ cells 40 .
Next, we employed pure cultures of neonatal RT cells and SCs 13 to compare their gene expression by bulk RNA-seq.A general view of RNA-seq data (Fig. 2c,d) showed that a considerable number of DEGs upregulated in RT cells exhibited great values of expression fold change, which suggested that these genes were expressed in SCs at extremely low levels.As opposed to that, fold change values tended to be lower for DEGs upregulated in SCs.On the one hand, this could reflect the presence of at least two subpopulations of RT cells, one of which is more similar to SCs.On the other hand, this could indicate that RT cells express many SC genes at some basic level, while the phenotype and function of RT cells are mainly determined by specific RT genes.
Functional enrichment analysis of the DEGs showed that genes upregulated in neonatal SCs indicated enrichment in GO terms related to tight junctions, lipid and glutathione metabolism, and mitochondria, which were all consistent with SC functions in the testis 41 .Enrichment in terms associated with the endocytic pathway could point to the transformation of the cell membrane during SC differentiation that occurs postnatally 42 .DEGs upregulated in neonatal RT cells indicated enrichment in terms related to proliferation, cell polarity, and actin cytoskeleton, which characterized RT cells as highly proliferating epithelial cells.RT upregulated DEGs were also strongly enriched in terms of various signaling pathways and in the term embryo development, which could indicate that some morphogenetic processes occur in the RT cell population.Figure 4 shows a graphic interpretation of the results of the functional analysis.
It was shown earlier 12 that RT cells express WT1, SOX9, DMRT1, and AMH, which are all often used as SC markers.PAX8, CDH1, and KRT8 were reported to be RT cell markers 12,14 , although two of them, CDH1 and KRT8, were demonstrated to gradually disappear from cultured RT cells 12 .scRNA-seq analyses of fetal and juvenile testes revealed some additional genes upregulated in RT cells, such as Ncam1, Tbx2, and Ennp2 for fetal cells 11 and Sox17 for juvenile cells 2 .Microarray analysis of adult testes identified that various signaling factors, such as Fgf9, Bmp4, Notch1, and Wnt, are upregulated in the RT epithelium 1 .However, it was unclear if all these genes were constantly upregulated in the RT during fetal and postnatal development or if they remained in RT cells in culture.So, there was a shortage of reliable universal markers for distinguishing these two cell populations.
A comparative analysis of DEGs between RT cells and SCs of different ages, both in vivo and in vitro, could address the issue.There are studies examining only the SC transcriptome 25,36 , but they are not suitable for comparison to RT cells.There is a study that performed scRNAseq analysis on all testicular cells throughout the perinatal period 43 .However, it was not designed to specifically examine the RT cell population, which is

Figure 1 .
Figure 1.Characterization of RT cell cultures.(a) Whole-mount immunofluorescent staining of the neonatal RT with PAX8 antibody and CDH1 antibody for flow cytometry.(b) Staining with isotype control antibodies exhibited no specific signal.(c,h)FACS of cells isolated from RT regions of neonatal (c) and adult mice (h).The cells were stained with CDH1 and isotype control antibodies labeled with PE.Debris and doublets were excluded from the analysis.The percentage of CDH1 + cells was presented as the mean ± SEM from three independent experiments.(d,i) Morphological appearance of neonatal (d) and adult (i) RT cell cultures on day 3 and day 8 respectively.(e,j) Triple immunofluorescent staining of neonatal (e) and adult (j) RT cell cultures for PAX8, DMRT1, and vimentin (Vim).The inset in (e) shows DMRT1 staining of a neonatal SC culture for comparison.(f,k) Immunofluorescent staining of neonatal (f) and adult (k) RT cultures for SOX9; the neonatal culture (f) was co-stained for ACTA2.Images in (e,f,j,k) were stitched from several adjacent fields of view.(g,l) mRNA levels of markers of different testicular cell populations in neonatal (g) and adult (l) RT cultures relative to that in adult testicular tissue (the zero line).Data are presented as the mean ± SEM from three independent experiments.**p < 0.01, ***p < 0.001.Scale bars: 50 μm (a,b), 100 μm (d-f,i-k). https://doi.org/10.1038/s41598-023-48149-7

Figure 2 .
Figure 2. RNA-seq analysis of cultures of neonatal RT cells and SCs with the comparison with the data of the other studies.(a) A heatmap of Pearson correlation coefficients between RT and SC samples.(b) A plot of PCA analysis showing a cluster of RT cells and a cluster of SCs.(c) A volcano plot of the RNA-seq data.DEGs selected for the further analysis are highlighted in blue.(d) A histogram of LFC absolute values for genes differentially expressed between RT cells and SCs.(e,f) Validation of RNA-seq data by RT qPCR in cultures of neonatal RT cells (e) and SCs (f); the zero line represents mRNA levels in an SC culture (for e) and mRNA levels in an RT cell culture (for f).Data are presented as the mean ± SEM from three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(g) Comparison of DEGs identified for neonatal, fetal 11 , and juvenile 2 RT cells.(h) Comparison of DEGs identified for neonatal, fetal 11 , and juvenile 2 SCs.The total numbers of upregulated DEGs in each dataset are presented inside the circles; the numbers of overlapped DEGs are highlighted in bold; and the percentage of the overlap are presented in parentheses.The red type labels the numbers of DEGs that are common to all datasets.

Figure 3 .
Figure 3. Characterization of an adult SC culture and confirmation of the RT and SC gene lists using adult cell cultures.(a-c) Immunofluorescent staining of an adult SC culture on day 8 for WT1 (a), for ACTA2 and SOX9 (b), and for DDX4 and DMRT1 (c).Images were stitched from several adjacent fields of view.An arrow in (c) points to a germ cell.(d) Morphological appearance of an adult SC culture.(e) mRNA levels of markers of different testicular cell populations in an adult SC culture relative to that in adult testicular tissue (the zero line).(f) mRNA levels of genes from the RT list in an adult RT cell culture relative to that in an adult SC culture (the zero line).(g) mRNA levels of genes from the SC list in an adult SC culture relative to that in an adult RT cell culture (the zero line).Data in (e-g) are presented as the mean ± SEM from three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Scale bars: 100 μm.

Figure 4 .
Figure 4.A schematic picture illustrating the differences between RT cells and SCs.Numbers in circles refer to selected terms of the functional analysis of DEGs between neonatal RT cells and SCs.1-adherens junction, 2-DNA replication, 3-extracellular matrix, 4-actin cytoskeleton, 5-tight junction, 6-mitochondrion, 7-lipid metabolism, 8-endocytic pathway.Proteins encoded by some permanent DEGs from Table 3 are shown.

Table 1 .
Selected terms of GO enrichment analysis of DEGs between neonatal RT cells and neonatal SCs.*FDR < 0.05 for all GO terms.

Table 2 .
Selected DEGs between neonatal RT cells and neonatal SCs classified according to GO molecular function complete database and Panther Protein Class database.DEGs are listed in order of decreasing mean RPKM value.DEGs mentioned in the text are highlighted in bold.

Table 3 ,
DMRT1 candidate targets were highlighted in bold).