Papel de Sox9, miR-17-92 y sus parálogos en la función testicular

Résumé

Role of Sox9, miR-17-92, and their paralogs in the testicular function Introduction Gonadal development and maintenance Infertility is a growing global problem that affects both men and women. It is estimated that approximately 15-20% of all couples experience infertility at some point in their reproductive lives and approximately half of the cases are of male origin. Factors that can affect a man’s reproductive capacity include sperm problems, hormonal disorders, genetic abnormalities, diseases and infections of the male reproductive system. Thus, the understanding of the physiological, endocrine, and genetic processes involved in the development and function of the gonads, and the regulation of sex hormones production will help to design therapeutict strategies to overcome these reproductive disorders. Since the discovery of the Y-linked, mammalian sex determining gene, SRY, the scientific community has made significant findings to uncover the genetic basis of sex determination, sex differentiation and sex function. Although a substantial progress has been made in this field, the current situation is still far from having a full understanding of this processes, and thus, additional effort is needed to elucidate new genetic mechanisms underlying gonadal processes. It is known that the presence of a Y chromosome determines the male sex in mammals, while its absence leads to female development. Initially, the gonad is bipotential, which means that can develop as either testis or ovary. The decision as to which fate to follow depends on the presence/absence of sex-specific factors. In the male, SRY upregulates SOX9 which triggers testis differentiation, whereas in the female, theWNT/β-catenin signaling pathway becomes active and induces ovarian development (Sekido y Lovell-Badge, 2008, 2013; Svingen y Koopman, 2013). Both pathways antagonize each other: the loss of either SRY or SOX9 leads to the formation of XY ovaries (Berta et al., 1990; Foster et al., 1994; Wagner et al., 1994) and the absence of WNT -signaling molecules such as WNT4 or RSPO1 causes XX sex reversal (Vainio et al., 1999; Parma et al., 2006). Similarly, gain-offunction experiments confirmed this antagonism, as either upregulation of the testis promoting genes Sox9 or Dmrt1 in the XX bipotential gonad (Bishop et al., 2000; Vidal et al., 2001; Zhao et al., 2015) or ectopic activation of the canonical WNT signaling pathway in the XY bipotential gonad (Maatouk et al., 2008a) leads to XX and XY sex reversal, respectively. Furthermore, Sertoli cell-specific conditional inactivation of Sox9 on a Sox8-/- background at embryonic day 13.5 (E13.5), two days after the sex determination stage, leads to Dmrt1 downregulation with upregulation of the ovarian-specific genes Wnt4, Rspo1 and Foxl2 (Barrionuevo et al., 2009; Georg et al., 2012). Similarly, Sertoli cell-specific ablation of Dmrt1 at the same stage (E13.5) results in ectopic expression of Foxl2 by postnatal day 14 (P14) and to Sox9 downregulation by P28, including male-to-female genetic reprogramming, as revealed by mRNA profiling (Matson et al., 2011a). Again, gain-of-function experiments confirmed the existence of sexual antagonism after the sex determination period, as conditional stabilization of β-catenin in differentiated embryonic Sertoli cells (E13.5, Amh-Cre) resulted in testis cord disruption (Chang et al., 2008). The male-vs-female genetic antagonism also persists in the adult ovary. The finding that in adult fertile females granulosa cells transdifferentiate into Sertoli-like cells after Foxl2 ablation revealed that terminally differentiated female somatic cells require permanent repression of the male-promoting factors to maintain correct identity and function (Uhlenhaut et al., 2009). Furthermore, transgenic expression of Dmrt1 in the adult ovary silenced Foxl2 and transdifferentiated granulosa cells into Sertoli-like, Sox9 -expressing cells (Lindeman et al., 2015). Regarding the adult testis, a similar phenomenon appears to occur in fully functional Sertoli cells after Dmrt1 ablation (Matson et al., 2011a). In addition to cells with a Sertoli cell morphology expressing both SOX9 and FOXL2, some cells with typical granulosa cell features were also observed, including the absence of SOX9 and the presence of FOXL2. However, Sertoli-togranulosa cell transdifferentiation was not unambiguously documented, as the authors used an inducible ubiquitous promoter (UBC-CreERT2 ) for Dmrt1 ablation in adult Sertoli cells and the possible existence of genetic reprogramming was not investigated as no mRNA profiling was performed in adult mutant testes. Sox genes encode an important group of transcription factors with relevant roles in many aspects of pre- and postnatal development of vertebrates and other animal taxa. There are 20 Sox genes in vertebrates, which are classified into 9 groups. Sox8, Sox9, and Sox10 (SoxE group) are involved in many developmental processes (Lefebvre et al., 2007). All three SoxE genes are expressed during testis development, Sox9 being essential for testis determination and Sox9/Sox8 necessary for subsequent embryonic differentiation (Chaboissier et al., 2004; Barrionuevo et al., 2006, 2009). Sox10 can substitute for Sox9 during testis determination (Polanco et al., 2010). Nothing is known on the role of SOX9 in the adult testis, where it is expressed by Sertoli cells in a spermatogenic stage-dependent manner in several mammalian species (Fröjdman et al., 2000; Dadhich et al., 2011; Massoud et al., 2014). Here we report the use of two Sertolicell-specific Cre lines (Wt1-CreERT2 and Sox9-CreERT2 ) to induce Sox9 ablation on a Sox8-/- background in the adult testis, starting at postnatal day 60 (P60). We show that Sox9/8 Sertoli cell-specific knockout (SC-DKO) testes undergo testis-to-ovary genetic reprogramming and Sertoli-to-granulosa cell transdifferentiation. The process is retinoic acid (RA)-mediated and occurs as a consequence of Dmrt1 downregulation. SOX9/8 are necessary to maintain Dmrt1 expression and thus to prevent Foxl2 expression in the adult testis. Furthermore, double mutant testes exhibited complete degeneration of the seminiferous tubules and increased apoptosis, indicating that SOX9/8 are continually required for the maintenance of testis integrity. Role of microsARNs in testicular differentiation and function Micro-RNAs (miRNAs) are a family of small non-coding RNAs (±22 nucleotides) that regulate gene function at the post-transcriptional level by either preventing protein translation and/or promoting mRNA degradation (Siomi y Siomi, 2010). The miR-17-92 cluster, also known as MirC1, is a polycistronic miRNA gene encoding six members (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92-1 ) which are highly conserved in vertebrates and expressed in practically all tissues analyzed during embryonic and postnatal stages (Ventura et al., 2008). In human and mouse, two paralog clusters exist, i.e. the miR-106a-363 cluster comprising 6 miRNAS (miR-106a, miR-18b, miR-20b, miR-19b-2, miR92a-2 and miR-363 ) and the miR-106b-25 one with 3 members (miR106b, miR-93 and miR-25 ). The miR-17-92 cluster is a potent oncogene and it has been associated to several types of both hematopoietic cancers such as B-cell lymphomas, B-cell chronic lymphocytic leukemia and Tcell lymphoma, and solid cancers including retinoblastoma, pancreatic cancer and breast cancer, among others (Mogilyansky y Rigoutsos, 2013). The miR-17-92 cluster plays also important roles during development. Hemizygous deletions of MIR17HG, the miR-17-92 cluster host gene, have been associated to the Feingold syndrome, an autosomal dominant condition characterized by multiple skeletal abnormalities. Homozygous miR-17-92 null mutant mice died perinatally due to lung defects and cardiac hypoplasia (Ventura et al., 2008) and partially phenocopied some skeletal abnormalities of the Feingold syndrome (de Pontual et al., 2011). This miRNA cluster was also shown to play a role in B- and T-cell development (Ventura et al., 2008), neural stem cell differentiation (Bian et al., 2013), and orofacial clefting (Wang et al., 2013a). Several studies have shown that the members of the miR-17-92 cluster are expressed in germ cells (GCs) of the testis and conditional inactivation of the cluster in either embryonic GCs or the whole adult testis resulted in spermatogenic defects (Tong et al., 2012; Xie et al., 2016). The Sertoli cell-specific deletion of the RNase III enzyme Dicer in mice revealed that miRNAs also play an important role in the development and survival of Sertoli cells (SCs) (Papaioannou et al., 2009; Kim et al., 2010). Several studies have provided evidence that the members of the miR-17-92 cluster are expressed in SCs. All the members of the cluster were cloned from purified P6 SCs (Papaioannou et al., 2009), in situ hybridization with LNA (Locked Nucleic Acid) on adult testes showed miR-17 and miR-20a expression in SCs (Tong et al., 2012), and ulterior analysis of the small RNA transcriptome of SCs purified from mice at postnatal day 6 revealed high levels of expression for miR-19a and miR-19b, intermediated levels for miR-17 and miR-20a and low levels for miR-18a and miR-92a (Ortogero et al., 2013). Overall, available data show that miRNAs are required for normal development of SCs and that miR-17-92 expression in GCs plays a role in spermatogenesis maintenance, but nothing is currently known on the role of this miRNAs cluster in postnatal and adult SCs. In order to overcome the postnatal lethality of miR-17-92 -/- mice and to uncover novel functions for miR-17-92 during SC development, we conditionally deleted the miR-17-92 cluster in embryonic SCs shortly after the sex determination stage using an Amh-Cre allele. We have also studied the role of the miR-106b-25 cluster on testis differentiation and function. miR-106b-25 Knockout mice are fertile and no testicular phenotype have been described (Ventura et al., 2008). The reason for this is probably because nobody has studied testis development in these mice. We have performed a detailed analysis of the testicular function of miR-106b-25 -/- and found that they show altered spermatogenesis and reduced number of spermatozoa. Objectives We have 3 major objectives: 1. To study of the role of Sox9 and Sox8 in adult testes 2. To identify the role of the miR-17-92 cluster in Sertoli cell development and function 3. To analize the development and function of miR-106b-25 -/- testes. Material and Methods Sox9 and Sox8 in adult testes Mice and histological methods Previously generated Sox9f/f ;Sox8-/mice (Barrionuevo et al., 2009; Kist et al., 2002; Sock et al., 2001) were bred to Wt1-CreERT2 mice (Zhou et al., 2008) and the resulting double heterozygous offspring harboring the Cre allele was backcrossed to Sox9f/f ;Sox8-/- mice to obtain heterozygous and homozygous compound Sox9/Sox8 conditional mutants. The same mating scheme was followed with the Sox9-CreERT2 mouse line (Kopp et al., 2011). To report CRE activity, the R26R-EYFP reporter allele (Srinivas et al., 2001) was crossed into Wt1-CreERT2 and Sox9-CreERT2 ;Sox9f/f ;Sox8-/- mice. For genotyping we performed PCR and qPCR with DNA purified from tail tips. Tamoxifen (Sigma, T5648) dissolved in corn oil (Sigma, C8267) at a concentration of 30 mg/ml and 0.16 mg of TX per gram of body weight was initially administered. Other animals were treated with a TXsupplemented diet (40 mg TX/100 g Harlan 2914 diet) for one month. Histoloical and immunohistological methods were performed according to standard protocols. Transcriptome analysis Testes were extracted from six P150 (90 days after tamoxifen treatment; datx) mutant males (three Wt1CreERT2;Sox9f/f ;Sox8-/- and three Sox9-CreERT2;Sox9f/f ;Sox8-/- ). As controls, both gonads were also extracted from two P150 (not treated) and two P150 (90 datx) Sox9f/f male mice as well as from two 4–5 months old normal females. All TX-treated mice were euthanized three months after the initiation of diet TX-treatment for one month. The two gonads of each individual were pooled and the total RNA were then individually purified from the twelve samples using the Qiagen RNeasy Midi kit following the manufacturer’s instructions. After successfully passing the Macrogen Inc. (Seoul, South Korea) quality control, the twelve RNA samples were paired-end sequenced separately in an Illumina HiSeq 2000 platform at Macrogen and the quality of the resulting sequencing reads was assessed using FastQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). Bioinformatics RNAseq data were processed with the Tuxedo tools (Trapnell et al., 2012). Alignments were done with Tophat/Bowtie2 against the mm10 UCSC annotated mouse genome. Differential expression analyses were done with Cuffdiff. Analysis of the resulting data were performed with the CummeRbund Bioconductor package. The quality of RNA-seq was checked as described in the package documentation. Briefly, by comparing FPKM scores across samples, and looking for outliers replicates, by analyzing squared coefficient of variation which allows visualization of cross-replicate variability between conditions and by analyzing the dispersion plots. miR-17-92 cluster in Sertoli cell development and function Mice and histological methods To induce SC-specific deletion of miR-17-92, we mated mice with a floxed allele of miR-17-92 (Ventura et al., 2008) to mice harbouring the SC-expressed transgene Amh-Cre (Holdcraft y Braun, 2004) obtained from the Jackson Laboratory (Bar Harbor, ME, USA; Stock No: 007915 and 008458, respectively). They were bred in our animal house facilities and the resulting double heterozygous offspring carrying the Cre allele was backcrossed to homozygous miR-17-92f lox mice to obtain Amh-Cre;miR17-92flox/flox mice. To evaluate Cre-activity, Amh-Cre and Amh-Cre;miR17-92flox/flox mice were bred with the Crereporter mouse line R26R-LacZ (Soriano, 1999) (Jackson Laboratory, Bar Harbor, ME, USA; Stock No: 003309). Primers and PCR conditions for miR-17-92f lox and for R26R-EYFP and Cre have been previously described. Mice with genotype miR17-92flox/flox lacking the Cre allele were used as controls. All animal experiments in this study were approved by the University of Granada Ethics Committee for Animal Experimentation (exp. No: 2011–341), and were performed in accordance with the relevant guidelines and regulations dictated by this Committee. Apoptotic cells were labelled using the Roche TUNEL kit (Fluorescent In Situ Cell Death Detection Kit) according to the manufacturer’s instructions. An in vivo test was performed to study the permeability of the bloodtestis barrier (BTB) in the testes of control and mutant mice. For this, we used a biotin-labelled tracer compound (EZ-Link Sulfo-NHS-LC-Biotin tracer, Thermo Scientific) as described (Dadhich et al., 2013). Transcriptome analysis Total RNA was isolated from three Amh-Cre;miR17-92flox/flox and three control (miR17-92flox/flox ) testes at postnatal day 60 (P60) using the Qiagen RNeasy Midi kit following the manufacturer’s instructions. Subsequently, the six RNA samples were sent to Macrogen Inc. RNA samples were checked for quality and libraries were prepared using TrueSeq RNA Sample Prep Kit V2 and they were paired-end sequenced separately in an Illumina HiSeq 4000 platform, generating between 120 and 135 million reads per sample. Bioinformatics The RNA-seq reads were mapped to the UCSC mm10 mouse genome using the STAR RNA-seq aligner (version 2.5)(Dobin et al., 2013), and subsequently they were counted with the featureCounts function from the R subread package. Only genes with 5 or more RPKM (reads per kilobase per million) in at least two of the samples were considered to be expressed and were used for further analysis. Analysis of differential gene expression was performed with edgeR. Development and function of miR-106b-25-/- testes We used previously generated miR-106b-25-/- mice (Ventura et al., 2008), and the same matting strategy and technical procedures described above. Results Sox8/9 in adult testes At the histolical level, TX-treated controls were similar to untreated males, except between P80 (20 datx) and P120 (60 datx) and mainly at P90 (30 datx), when they showed some degenerating seminiferous tubules, but recovered afterwards. Testes in Sox9/8 DKO (Sox9 ) mice were similar to the TX-treated controls at P70 (10 datx) except for a few testis tubules with enlarged lumen. At P80 (20datx), only few seminiferous tubules showed signs of degeneration (shrinkage and germ cell depletion), whereas this was more frequent by P90 (30 datx). In many cases, Sertoli cellonly tubules were observed. By P120 (60 datx), tubules had become solid testis cords whose diameter appeared even more reduced at P150 (90 datx). While some mice continued to exhibit this phenotype at P180 (120 datx), a subset of mice in this group was more affected. In these latter mice Sertoli and germ cells had disappeared completely. At later time points, all mice showed this severe testicular phenotype. This progressive degeneration of the testicular phenotype in Sox9/8 SC-DKO mice was evident when we analyzed the relative abundance of the most relevant testicular morphological features between P70 (10 datx) and P180 (120 datx). In contrast, Leydig cells appeared morphologically normal in mutant testes. Sox9/8 DKO (Wt1 ) mice exhibited a similar testicular phenotype. These results show that Sox8 and Sox9 alleles act redundantly in adult Sertoli cells and are necessary to maintain the integrity of the seminiferous tubules of functional testes. Regarding the expression of several somatic and germ cell markers, LAMININ, a principal component of the basement membrane persisted in both P150 (90 datx) and P180 (120 datx) testes of SC-DKO mice. Alpha smooth muscle actin (Acta2) expressed by both peritubular myoid (PM) cells and arterialmuscle fibers was detected in the testes of both TX-treated controls and P150 (90 datx) SC-DKO mice. In contrast, at P180 (120 datx), strong ACTA2 signal persisted in the arteries but that of PM cells was almost undetectable. This shows that acellular cords in severely affected SC-DKO testes have lost not only Sertoli and germ cells, but also PM cells. CLAUDIN 11 is a principal component of tight junctions, the main junctional structures forming the BTB. Cldn11 (the claudin11 gene) expression was similar between controls and double mutants before P150 (90 datx) (not shown), but it was severely reduced by P150 (90 datx) and completely absent in P180 (120 datx) Sox9/8 mutant testes, indicating that the BTB is not functional in these testes. We also performed immunofluorescence for both PCNA, which is expressed in mitotic spermatogonia as well as in zygotene and early pachytene, but not leptotene spermatocytes, and DMC1, a meiotic recombination protein marking zygotene-pachytene spermatocytes. At P60 (30 datx), most mutant seminiferous tubules exhibited a clear reduction of spermatogenic activity and some spermatocytes were abnormally located in the inner region of the tubules and not at the periphery, as seen in TX-treated control testes. In P120 (60 datx) testes, spermatocytes were scarce and only proliferating spermatogonia were seen in most testis tubules, while at P150 (90 datx), both spermatogonia and spermatocytes had disappeared in most tubules. Unlike other somatic cells, Leydig cells appear not to be seriously affected in testes from Sox9/8 SC-DKO mice. These cells do not transdifferentiate into theca cells, as they never express Foxl2 (as theca cells do), and maintain the steroidogenic function for a long time after Sox9 ablation, as deduced from the expression of P450scc, a cytochrome involved in the synthesis of testosterone. Consistently, the testosteroneproducing enzyme HSD17b3 and the marker for adult functional Leydig cells Insl3 are expressed at high levels in the mutant testes. Analysis of Foxl2 expression revealed that by P105 (45 datx), FOXL2positive cells were present in almost all testis cords, and by P150 (90 datx), the most severely affected mice showed many FOXL2-positive cells within almost all testis cords. Genome-wide transcriptome analysis of P150 untreated control testis, P150 (90 datx) control and mutant testis and control ovary showed that SC-DKO testes exhibit a striking feminization of the testicular transcriptome. We found 12 380 genes with significant differential expression between the five sample conditions. With the exception of a few gene clusters, most genes in mutant testes adopted an ovary-like expression pattern. Expression heat maps for selected 39 ovarian somatic cell-specific genes and 33 oocyte-specific genes selected using bioGPS (http://biogps.org) revealed that the cell reprogramming observed in the SC-DKO testes only affects somatic cells. Notably, bar plots for six genes known to be adult granulosa cell markers showed that these genes were upregulated in the mutant Sertoli cells, revealing an ovary-like expression pattern. In addition, within the seminiferous cords of SC-DKO testes we found a few FOXL2+ cells expressing the enzyme aromatase. miR-17-92 in Sertoli cells SC-miR-17-92 KO mice were fertile and provided litters of apparently similar size than controls. At P60 we found no statistically significant difference between the testis mass of SC-miR-17-92 KO and controls. Histologically, 2 months old mutant testes were similar to controls showing seminiferous tubules of the same size and completing the spermatogenic cycle. Consistently, the epididymal tube was full of spermatozoa and 1epididymal sperm counts showed no difference between both conditions. The same situation persisted in one year old mutant mice: 1) they were fertile, 2) showed no difference in testis mass when compared to controls, 3) their testes showed normal spermatogenesis, 4) their epididymal tubes were full of spermatozoa, and 5) their epididymal sperm counts were similar to those of controls. Next, we studied the expression of several somatic and GC markers by immunofluorescence. SOX9 and WT1, two transcription factors expressed in adult SCs, are necessary for testis function and maintenance. Both control and SC-miR-17-92 mutant testes exhibited the same expression pattern for the two proteins in SCs. PCNA is expressed in mitotic spermatogonia as well as in zygotene and early pachytene spermatocytes and the meiotic recombination protein, DMC1, is expressed in early spermatocytes (leptotene-pachytene). No difference between mutant and control testes was observed in the expression patterns of these two proteins at both P60 and P365 stages. Also, LAMININ, a component of the basement membrane, and α-smooth muscle actin (ACTA2), a marker of peritubular myoid cells and arterial muscle fibers, showed similar expression in the two study groups. Likewise, the expression of P450scc, a steroidogenic cytochrome expressed in Leydig cells, was also similar in both SC-miR-17-92 KO and control testes. In addition, CLAUDIN 11 (Cldn11 ), a major component of the tight junctions forming the BTB, did not show a different pattern of expression between the control and mutant condition. Consistently, injection of a biotin tracer showed that the BTB in mutant males was impermeable, as observed in control testes. Finally, TUNEL assay showed no increase in the frequency of apoptotic cells in SC-miR-17-92 KO testes compared to control ones. We also performed RNA-seq experiments on the adult testes of three SC-miR-17-92 KO and three control males at postnatal day 60. The differences between the expression profiles of these samples were examined using a hierarchical clustering analysis. Replicate samples from the same condition clustered together, indicating that the whole testis transcriptome is significantly and consistently altered when miR-17-92 is deleted in SCs. To confirm this, we checked the presence of differentially expressed (DE) genes when comparing the two experimental conditions, and found 809 genes deregulated at P adjusted (FDR) < 0.01 (418 upregulated and 1391 downregulated). Smear plot of the differential expression test showed that the vast majority of the DE genes were deregulated by less than 2 times, indicating that, despite a reproducible deregulation of many genes, the magnitude of the change was generally moderate. Gene ontology (GO) analysis of DE genes revealed a significant enrichment (P adj. < 0.05) in terms related to normal testicular functions including spermatogenesis, meiotic cell cycle, chromosome segregation, cell-cell adhesion, cellprojection, and DNA-repair, among others. Interestingly, although we deleted miR-17-92 specifically in SCs, several GO terms referred to processes normally occurring in GCs (e.g. spermatid development and meiotic cell cycle), indicating that the GC transcriptomer was also altered in the mutant mice. Development and function of miR-106b-25-/- testes We found a 30% reduction of the testis mass and a 40% reduction in the sperm epidydimal content of the Mir-C3 KO mice. We also studied the testis phenotype at the histological level. For this we used the Johnsen score, which is a standard method to assess spermatogenesis. In this system testis tubules are sort out according to their appearance. Thus, a normal tubule containing sperm is given a score of 10 and a Sertoli cell only tubule is given a score of 0. We counted more than 100 tubules in 3 testes of each condition and we obtained an average Johnsen score of 9.2 in control testes with most of the tubules with a JS between 8 and 10. In contrast in Mir-C3 KO we obtained an average JS of 7.5, with the vast majority of the tubules with a JS between 7 and 8. In the mutants we observed some degenerated tubules with a JS of 4, an observation not found in the controls. Sertoli cell markers including Sox9 and Wt1 had a normal expression pattern in mutant testes. We also checked DMC1, a marker of leptotene to pachytene spermatocytes that is expressed only in some stages of the spermatogenic cycle. In both control and mutants we found a similar pattern of positive and negative DMC1 tubules. However, we observed that the percentage of DMC1 positive tubules was almost two fold in the mutant when compared to the control. We also found a significant increased in the number of apoptotic cells in Mir-C3 deleted testes. To identify dying cells, we performed double immunofluorescence for DMC1 and TUNEL and observed that TUNEL1stained cells frequently exhibited a weak staining for DMC1. Next we perform a RNA-seq analysis of mutant and control testes, and after mapping the reads we made a clustering analysis of the individual samples. We saw that replicate samples of the same condition clustered together, indicating that there is a significant and consistent alteration of the testis transcriptome when MirC3 is deleted. We found 2 255 genes deregulated at a FDR < 10%. 1 068 were upregulated and 1 187 were downregulated. The smear plot comparing the fold change in expression with the average expression showed that the vast majority of the DE genes are deregulated by less than two times, indicating that although there is a reproducible deregulation of many genes, the magnitude of this change is quite modest. Next, we performed a Gene Ontology analysis of the differentially expresed genes and we saw that the 30 most significantly enriched GO classes could be grouped in three general categories: Cell cycle and reproduction, Microtubule process, and ubiquitination and catabolism. Discussion Sox8/9 in adult testes There is now compelling evidence that the bipotential nature of the genital ridge at the beginning of gonad development is not completely lost once either testes or ovaries acquire their final adult morphology and functionality. During embryonic development the newly formed Sertoli cells can transdifferentiate to their ovarian counterparts when the testis promoting factors Sox9 or Dmrt1 are lost (Georg et al., 2012; Matson et al., 2011a). The finding that Foxl2 in the adult ovary was necessary to prevent granulosa-to-Sertoli cell transdifferentation revealed that this antagonism also operates in the adult gonad. In the adult testis, the same antagonism also appears to exist, as FOXL2+ cells were observed when Dmrt1 was ubiquitously deleted (Matson et al., 2011a). Here we show that Sertoli-to-granulosa cell transdifferentiation can be induced as well in the adult mouse testis by just deleting two SoxE genes, Sox9 and Sox8. These results evidence that Sox9 has a crucial role, not only during sex determination and testis differentiation, but also in adult testis maintenance, where, together with Sox8 and coordinately with Dmrt1, it prevents male-to-female genetic reprogramming. 1The regulatory relationship between Dmrt1 and Sox9 requires further discussion. At the sex determination stage of the mouse (E11.5), both Sox9 and Dmrt1 are expressed in the early embryonic testis (Kent et al., 1996; Raymond et al., 1999), but whereas early embryonic Sox9 mutants show sex reversal (Chaboissier et al., 2004; Barrionuevo et al., 2006), early embryonic Dmrt1 KO mice have testes that express Sox9 and appear histologically normal until P7 (Raymond et al., 2000). Thus, Sox9 expression is independent of DMRT1 during the sex determination stage and some time thereafter. Similarly, Sertoli cell-specific inactivation of Sox9/8 at E13.5, shortly after the sex determination stage, leads to a rapid downregulation of Dmrt1 that becomes already visible four days later, at E17.5 (Georg et al., 2012). In contrast, Dmrt1 ablation at E13.5 results in a very delayed Sox9 downregulation, which is seen at P14 (one month later), coinciding with Foxl2 upregulation (Matson et al., 2011a). This suggests again that Sox9 expression is independent of Dmrt1 in newly differentiated Sertoli cells and that the loss of Sox9 after Dmrt1 ablation is a secondary consequence of the upregulation of ovarian genes(s), such as Foxl2, in the same cells. On the other hand, several observations suggest the transactivation of SOX9 by DMRT1: 1) DMRT1 binds near the Sox9 locus in P28 mouse testes (Matson et al., 2011a), 2) ectopic expression of Dmrt1 in embryonic XX gonads causes XX sex reversal with upregulation of Sox9 (Zhao et al., 2015) and 3) FOXL2-/- sex reversed polled goats undergo a process of transdifferentiation in which DMRT1 expression precedes the upregulation of SOX9 (Elzaiat et al., 2014). In the latter two cases, however, female-promoting genes, including FOXL2, are either downregulated or not expressed, and thus, SOX9 upregulation could be again an indirect consequence of the downregulation of femalepromoting genes. Here we provide evidence that in the adult gonad, mutant Sox9/8 Sertoli cells lose DMRT1, and that FOXL2 protein appears concomitant with the loss of DMRT1, consistent with the notion that Dmrt1 expression is SOX9/8-dependent and that DMTR1 represses Foxl2. Additional observations support this view: 1) nearly all the genes strongly affected by the loss of DMRT1 were also affected by the loss of SOX9/8; 2) Sertoli-to-granulosa cell transdifferentiation observed in the testes of our Sox9/8 mutant mice may be reduced by decreasing levels of RA, a signaling pathway known to be blocked by DMRT1 in Sertoli cells to 1prevent Foxl2 expression and transdifferentiation into granulosa-like cells (Minkina et al., 2014); 3) DMRT1 can silence Foxl2 in the absence of SOX9 and SOX8 (Lindeman et al., 2015); and 4) Sox9 is upregulated in the adult ovary after the ectopic expression of Dmrt1, coinciding with Foxl2 downregulation (Lindeman et al., 2015). Altogether, available data suggest that, as observed at earlier stages, a main role for SOX9/8 in adult male sex maintenance is to keep Dmrt1 actively expressed, this latter gene having a fundamental role in repressing female-specific genes. However, these observations do not rule out the possibility that DMRT1 is also necessary for the maintenance of Sox9 expression in the adult testis and that a feed-forward regulatory loop between Sox9/8 and Dmrt1 exists that ensures testis maintenance and antagonizes the feminizing action of Foxl2. Additional experiments (e.g. a time course of Sox9 expression in adult SC-DKO Dmrt1 mice) will help to clarify this issue. mirR17-92 in Sertoli cells The SC-miR-17-92 KO adult testes showed apparently normal development and function, even though RNA-seq analyses evidenced consistent deregulation of many testicular genes, indicating that expression of the mir-17-92 cluster in SCs is necessary to maintain the gene expression levels of the whole testis within normal values. Thus, the question arises as to why such an alteration of the transcriptome homeostasis does not result in an observable phenotype. In this regard, it should be noted that, despite the high number of deregulated genes in SC-miR-17-92 KO testes, the magnitude of the changes in their expression levels was generally modest (less than twofold in most cases). Such a scenario, implying many deregulated genes showing just moderate changes in their expression levels, has been previously described for miR-17-92, and it is consistent with the proposed notion that its members act as fine-tuners of large gene networks rather than as major genetic switches of specific pathways (Han et al., 2015). The phenotypic consequences of transcriptome alterations induced by the absence of miR-17-92 may be variable and difficult to predict. As an example, despite the fact that the number of deregulated genes in the embryonic tail buds of mice harboring a homozygous deletion for the miR17 seed family (sim500) was lower than the observed in the same tissue of homozygous mutants for the miR-19 seed family (sim700), skeletal mal1formations were observed in the former mice (defects in axial patterning regulation), but not in the latter ones. Hence, it appears that the severity of the phenotype derived from the absence the miR-17-92 cluster depends on the spatial and temporal cellular context. Most importantly, our result evidence that testis homeostasis must be strictly controlled as even when hundreds of genes were deregulated in all testicular cell types of miR-1792 KO testes, these showed no evident pathologic phenotype, conserving normal structure and function. Probably many other regulatory factors contribute to maintain testis homeostasis, as the simple ablation of miR17-92 is not enough to disturb it significantly. However, it is likely that mutant testes become more sensitive to stressful situations. Consistent with this idea, it was recently reported that hepatocyte-specific miR-1792 -deficient mice were fertile and apparently healthy, but their capacity of liver regeneration after tissue injury had resulted significantly reduced (Zhou et al., 2016). Hence, although no obvious testicular phenotype is observed, the transcriptome alteration we detected in our miR-17-92 mutants might cause a suboptimal testis functional status. Development and function of miR-106b-25-/- testes The detailed analysis that we have performed in the murine miR-106btestes have revealed that a 30% reduction in the testis mass was produced, evidencing that this miRNA cluster plays a role during testis development and/or function. This phenotype is similar to that described for its paralog cluster miR-17-92 (Tong et al., 2012; Xie et al., 2016). However, in both cases the mutant mice are fertile. This suggests that a redundant function may exist between both clusters. The generation of compound mutant mice will clarify this issue. We are currently generating such mice in our laboratory. To check for the functional status of miR106b-25 -/- testes we analized the Jonhsen’s index, and we found that these testes displayed a mild alteration of the spermatogenesis. In addition, cell death was increased in mutant testis at the spermatocyte level and the meiotic cycle seemed to be slowed down also at the same stage, as shown by DMC1 immunofluorescence. Altogether, these observations indicate that the miR-106b-25 cluster may have a function in controlling the spermatogenic cycle at the meiotic prophase I stage. We found that more than 2 000 genes were deregulated in the mutant testes. As observed in SC-miR-17-92 KO testes, the magnitude in the change of the expression for most of the genes was very modest, generally lower than twofold. This again support the notion that the members of this cluster act as finetuners of large gene networks rather than as major genetic switches of specific pathways (Han et al., 2015). The GO analysis of the differentially expressed showed that the 30 most significantly enriched GO classes could be grouped in three general categories: Cell cycle and reproduction, Microtubule process, and ubiquitination and catabolism. All of them are normal processes ocurring during testicular function. The analysis of individual genes in these categories will help to elucidate the molecular mechanisms underlying the alteration seen in miR-106b-25 -/- . One of the genes that could have a relevant function is Ubc, a putative target of miR106b and miR-93 that becomes upregulated in the mutant testes. Conclusions • As during the embryonic stages of development, the Sox9 and Sox8 genes are needed to maintain the structure and function of the adult testes. Both genes exert this action in a redundant manner, as the testicular phenotype becomes more severe as the number of mutated Sox alleles increases. • In the absence of Sox9 and Sox8 the seminiferous tubules disappear completely. Our results suggest that both genes maintain the integrity of these structures by controlling the expression of cell adhesion molecules and other structural elements such as the cytoskeleton components or the extracellular matrix. • In the testes of mice mutant for both Sox9 and Sox8, the peritubular myoid cells disappear completely, suggesting that, as obseerved during embryonic development, this cell type retains its original dependence on Sertoli cells. In contrast, Leydig cells have an apparently normal structure and function, indicating that in the adult testis the maintenance of Leydig cells is independent of Sertoli cells. • The loss of the Sox9 and Sox8 genes in the adult testis leads to the transdifferentiation of Sertoli cells into granulosa cells and testis-toovary genetic reprogramming. This transdifferentiation affects the somatic but not germ cells. • In adult Sertoli cells, SOX9 and SOX8 are necessary for the maintenance of Dmrt1 expression and these testicular promoting factors negatively regulate Foxl2. • Our results indicate that the transdifferentiation process of Sertoli cells into granulosa cells in the mutant testis for Sox9/8 is mediated by the high levels of retinoic acid produced as a result of the absence of DMRT1. • SOX9 and SOX8 act as anti-apoptotic factors in adult Sertoli cells. • The deletion of the miR-17-92 cluster specifically in embryonic Sertoli cells has no apparent phenotypic effects during testis development and function. • By comparing the transcriptome of testes of mice mutant for miR17-92 with that of control animals, we found that more than 800 genes were differently expressed. These genes are enriched in gene ontology categories associated with some essential testicular functions including: spermatogenesis, meiosis, microtubule formation, ubiquitination, and cell adhesion. • Genes that show differential expression do so at a moderate magnitude, usually less than two fold. This situation, in which many moderately deregulated genes exist, is consistent with the hypothesis that miRNAs act as fine regulators of large gene networks rather than regulating individual genes that are essential in controlling specific pathways. • The specific ablation of the miR-17-92 cluster in Sertoli cells results in the deregulation of cell adhesion molecules present in the cell contacts by which the “cross-talk” between Sertoli cells and germ cells is established, and this in turn can alter their transcriptomes. • Most of the target genes of miR-17-92 are not overexpressed in mutant testes. Since this cluster was conditionally deleted in embryonic Sertoli cells and the transcripts were analyzed in adult testes, this transcriptome probably do not reflect the primary effects derived from the deletion of this cluster, but other effects induced later by a chain reaction of genes deregulated over time. • Mice mutant for the miR-106b-25 cluster show a reduction in testis mass and oligozoospermia, although they are fully fertile. • The miR-106b-25 mutant testes showed an altered spermatogenic cycle. These abnormalities are accompanied by a slowing down of meiosis and an increase in apoptosis in the sperm cells. • Transcriptomic analysis of the mutant testes showed that 2 255 genes were deregulated. As observed in the miR-17-92 cluster, the magnitude of the expression level change is quite moderate. Again, our results support the hypothesis that these miRNAs exert a fine regulation of large gene networks. • Differentially expressed genes in the testes of mice mutant for miR106b-25 can be grouped into three general categories, according to their function: 1) cell cycle and reproduction, 2) microtubule processes and 3) ubiquitination and catabolism. Alterations in these testicular functions can explain the phenotype of the mutant testes. • Among the genes that are both overexpressed in the mutant testes and targets for the miR-106b and miR-93, we have identified the Ubc gene. Given the essential role of ubiquitination in many biological processes, and without rulling out the possible contribution of other target genes, we suggest that the phenotype shown by the mice mutant for miR-106b-25 may be due in part to the deregulation of Ubc.