Loss of NR5A1 in Sertoli cells after sex determination changes their cellular identity and induces their death by anoikis

NR5A1 is an orphan nuclear receptor crucial for gonadal development in mammals. In the mouse testis it is expressed both in Sertoli cells (SC) and Leydig cells (LC). To investigate its role posteriorly to sex determination, we have generated and analysed mice lacking NR5A1 in SC from embryonic day (E) 13.5 onwards (Nr5a1SC−/− mutants). Ablation of Nr5a1 impairs the expression of genes characteristic of SC identity (e.g., Sox9, Amh), makes SC to progressively die from E14.5 by a Trp53-independent mechanism, and induces disorganization of the testis cords, which, together, yields germ cells (GC) to prematurely enter meiosis and die, instead of becoming quiescent. Single-cell RNA-sequencing experiments revealed that Nr5a1-deficient SC acquire a pre-granulosa cell-like identity, and profoundly modify the landscape of the adhesion molecules and extracellular matrix they express. We propose therefore that SC lacking NR5A1 transdifferentiate and die by anoikis. Fetal LC do not display major changes in their transcriptome, indicating that SC are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LC were missing in Nr5a1SC−/− postnatal testes. In addition, adult males display Müllerian duct derivatives (i.e., uterus, vagina), as well as a decreased anogenital distance and a shorter penis that can be explained by loss of AMH production and defective HSD17B1- and HSD17B3-mediated synthesis of testosterone in SC during fetal life. Together, our findings indicate that Nr5a1 expressed in SC after the period of sex determination safeguards SC identity, which maintains proper seminiferous cord organization and prevents GC to enter meiosis.


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Together our findings indicate that NR5A1-deficient SC lost their appropriate cell-cell In parallel, they gain expression of genes specifying pGrC differentiation (e.g., Wnt4, 2 8 6 Rspo1) and their transcriptome projected to a gonad atlas clearly shows that they 2 8 7 display an identity close to that of pGrC. Nonetheless, these "pGrC-like" cannot reach has shown that SC lacking Nr5a1 from E12.5 reach a functional, FOXL2-positive, indicates that SC devoid of NR5A1 from E12.5 can transdifferentiate into functional 2 9 2 pGrC, while those loosing NR5A1 from E13.5 are no longer licensed to do so. We further show that these "pGrC-like" subsequently die through a Trp53-independent 2 9 4 mechanism since concomitantly removing Trp53 and Nr5a1 does not prevent death. Quite interestingly, SC loosing Nr5a1 later than E14.5 either die by MDM2/TRP53- identity over time. In addition, SC die by distinct mechanisms, depending on the 3 0 0 stage at which NR5A1 is lost. It is accepted that cell adhesion/junction molecules and ECM components play genes coding for ECM components such as Col18a1, Fn1, Nid2 and Dcn ( independently of TRP53. As to the GC fate, the data show that most of them become meiotic and die in is sufficient to explain why GC entered meiosis (Fig.8). Actually, CYP26B1 is As to FLC, their specification and/or development rely on SC, notably thanks to Furthermore, these FLC appear functional since testicular descent is occurring in Nr5a1 SC-/mutants, suggesting normal INSL3 production [53].

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As to peritubular myoid cell (PTMC), it is admitted that SC are required during the fetal period to support their differentiation, notably by producing DHH and ECM.

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Death of the NR5A1-deficient SC affects genital tract development Müllerian duct regression in males proceeds rostro-caudally, and is dependent Nr5a1 SC-/mutants is lost only from E14.5, AMH production starts normally and then 3 6 3 gradually decreases to zero after E14.5. It is therefore not surprising that the rostral In addition, Nr5a1 SC-/mutants display normal Wolffian duct-derived genitalia presence indicates therefore that Nr5a1 SC-/mutants were exposed to testosterone 3 7 3 during fetal development. This is surprising since SC, converting FLC-produced 3 7 4 androstenedione into testosterone thanks to expression of Hsd17b1 and Hsd17b3 3 7 5 [65], are progressively lost in Nr5a1 SC-/mutants. Thus, another enzyme necessarily testosterone synthesis in a cell-type other than FLC. This production is nevertheless 3 8 4 Our study shows that SC lacking NR5A1 from E13.5 transiently 3 8 5 transdifferentiate to acquire a pre-granulosa cell-like identity, profoundly modify the 3 8 6 landscape of the adhesion molecules and ECM they express and die by a TRP53-3 8 7 independent mechanism related to anoikis. This yields a disorganization of the testis 3 8 8 cords, making GC to prematurely enter meiosis instead of becoming quiescent.

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Interestingly, the maintenance of fetal LC is not altered in the Nr5a1 SC-/mutant, but to understand the reason for the absence of adult LC in the postnatal testis. They were on a mixed C57BL/6 (50%)/129/SvPass (50%) genetic background.

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The Nr5a1 conditional mutant mouse line was established at the Institut Clinique de 3 9 6 la Souris (iCS, Illkirch, France), in the context of the French National Infrastructure for 3 9 7 Mouse Phenogenomics PHENOMIN (http://www.phenomin.fr). To construct the 3 9 8 targeting vector a 1.9 kb-long DNA fragment encompassing exon 7 3 9 9 (ENSMUSE00000 693512) was amplified by PCR using 129/SvPass genomic DNA with Gt(ROSA)26Sor tm1 (FLP1) Then, primers 5'-TGAGCCCTGGCACATCCCTCC-3' and 5'-  After anesthesia with a lethal dose of Xylasin and Ketamine as described 4 4 4 above, the blood of 9-11-week-old adult mice was collected into heparinized 4 4 5 Microvette tubes (Sarstedt, Nümbrecht, Germany) by intra-cardiac sampling. The 4 4 6 tubes were then centrifuged at 5000 g for 5 minutes and the resulting plasma Nord, Nordhorn, Germany). Statistical significance was further assessed by using and organ weight measurement. They were next embedded in paraffin and 5 µm-  For lHC, antigens were retrieved for 1 hour at 95°C either in 10 mM sodium The surface area occupied by YFP-positive cells was measured using a macro  Two hours later, fetuses were collected (E13.5-E15.5), fixed and embedded as   genotype. Data were expressed as the ratio between the number of TUNEL-positive 4 9 5 cells quantified on entire sections and the surface areas of the testis sections (μm 2 ).

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Statistical significance was assessed by using two tail Student's t-tests.

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Real-time RT-qPCR analyses of RNA extracted from whole gonad 4 9 8 Fetal testes were dissected, isolated from mesonephros, snap frozen in liquid 4 9 9 nitrogen and stored at -80°C until use. Whole testis total RNA was extracted using  and normalized to Hprt whose expression is not affected by ablation of Nr5a1. To dissociate cells, the testes of controls and mutants additionally bearing the suspensions, centrifuged at 3000g and suspended in 300 µl PBS as described [70].

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The YFP-positive and -negative cells were sorted separately by FACS using an buffer, DNA was extracted and genotyped by PCR using standard protocols and 5 1 6 primers as indicated above.

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Single cell RNA sequencing and data processing 5 1 8 Gonads from E13.5 and E14.5 control and mutant fetuses were dissected out in 5 1 9 PBS, sexed by their appearance under the microscope and cell suspensions were 5 2 0 prepared as described above. Cell number and viability were determined by a Trypan Reagent Kits User Guide (v3 Chemistry) from 10X Genomics. Briefly, GEMs were 100 bases paired-end reads, using standard protocols.

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Fastq files were processed with CellRanger (v6.0) on a chimeric genome composed Data were normalized using the NormalizeData and the SCT function implemented Seurat. Cells were then clustered by using Seurat graph-based clustering issue, the reference atlas was first processed by using the same pipeline.  18. Malki S, Nef S, Notarnicola C, Thevenet L, Gasca S, Méjean C et al.  Contribution of Leydig and Sertoli cells to testosterone production in mouse fetal  control, normalization and visualization of single-cell RNA-seq data in R. Sox8 and Ptgds mRNAs in whole testis RNA from control (n=4) and Nr5a1 SC-/- Penis bones dissected from control and Nr5a1 SC-/males at PND60 and stained with Rabbit