To investigate the role of the nuclear receptor NR5A1 in testis after sex determination, we have analyzed mice lacking NR5A1 in Sertoli cells (SC) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impairs the expression of genes characteristic of the SC identity (e.g., Sox9, Amh), causes SC death from E14.5 through a Trp53-independent mechanism related to anoikis, and induces disorganization of the testis cords. Together, these effects cause germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SC change their molecular identity: some acquire a “pre-granulosa-like” identity, while other revert to a “supporting progenitor-like” cell identity, most of them being “intersex” because they express both testicular and ovarian genes. Fetal Leydig cells (LC) do not display significant changes, indicating that SC are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LC were absent from the postnatal testes. In addition, adult mutant males display persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which can be explained by the loss of AMH and testosterone synthesis due to SC failure.
Overall design: Testes from E13.5 and E14.5 control and mutant fetuses were dissected out in PBS and cell suspensions were prepared as described above. Cell number and viability were determined by a Trypan Blue exclusion assay on a Neubauer Chamber. Samples consisting of > 90% viable cells were processed on the Chromium Controller from 10X Genomics (Leiden, The Netherlands). Ten thousand cells were loaded per well to yield approximately 5000 to 6000 captured cells into nanoliter-scale Gel Beads-in-Emulsion (GEMs). GEMs were generated by combining barcoded gel beads, a reverse transcription master mix containing cells, and partitioning oil onto Chromium Chip B. Following full length cDNA synthesis and barcoding from poly-adenylated mRNA, GEM were broken and pooled before cDNA amplification by PCR using 11 cycles. After enzymatic fragmentation and size selection, sequencing libraries were constructed by adding Illumina P5 and P7 primers (Illumina, Evry, France), as well as sample index via end repair, A tailing, adaptor ligation and PCR with 10 cycles. Library quantifications and quality controls were determined using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Libraries were then sequenced on Illumina HiSeq 4000TM as 100 bases paired-end reads, using standard protocols.
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