In vivo evidence for the crucial role of SF1 in steroid-producing cells of the testis, ovary and adrenal gland.

Adrenal and gonadal steroids are essential for life and reproduction. The orphan nuclear receptor SF1 (NR5A1) has been shown to regulate the expression of enzymes involved in steroid production in vitro. However, the in vivo role of this transcription factor in steroidogenesis has not been elucidated. In this study, we have generated steroidogenic-specific Cre-expressing mice to lineage mark and delete Sf1 in differentiated steroid-producing cells of the testis, the ovary and the adrenal gland. Our data show that SF1 is a regulator of the expression of steroidogenic genes in all three organs. In addition, Sf1 deletion leads to a radical change in cell morphology and loss of identity. Surprisingly, sexual development and reproduction in mutant animals were not compromised owing, in part, to the presence of a small proportion of SF1-positive cells. In contrast to the testis and ovary, the mutant adult adrenal gland showed a lack of Sf1-deleted cells and our studies suggest that steroidogenic adrenal cells during foetal stages require Sf1 to give rise to the adult adrenal population. This study is the first to show the in vivo requirements of SF1 in steroidogenesis and provides novel data on the cellular consequences of the loss of this protein specifically within steroid-producing cells.

LIN28A marks the spermatogonial progenitor population and regulates its cyclic expansion.

One of the hallmarks of highly proliferative adult tissues is the presence of a stem cell population that produces progenitor cells bound for differentiation. Progenitor cells undergo multiple transit amplifying (TA) divisions before initiating terminal differentiation. In the adult male germline, daughter cells arising from the spermatogonial stem cells undergo multiple rounds of TA divisions to produce undifferentiated clones of interconnected 2, 4, 8, and 16 cells, collectively termed A(undifferentiated) (A(undiff)) spermatogonia, before entering a stereotypic differentiation cascade. Although the number of TA divisions markedly affects the tissue output both at steady state and during regeneration, mechanisms regulating the expansion of the TA cell population are poorly understood in mammals. Here, we show that mice with a conditional deletion of Lin28a in the adult male germline, display impaired clonal expansion of the progenitor TA A(undiff) spermatogonia. The in vivo proliferative activity of Au(ndiff) spermatogonial cells as indicated by BrdU incorporation during S-phase was reduced in the absence of LIN28A. Thus, contrary to the role of LIN28A as a key determinant of cell fate signals in multiple stem cell lineages, in the adult male germline it functions as an intrinsic regulator of proliferation in the population of A(undiff) TA spermatogonia. In addition, neither precocious differentiation nor diminished capacity for self-renewal potential as assessed by transplantation was observed, suggesting that neither LIN28A itself nor the pool of Aal progenitor cells substantially contribute to the functional stem cell compartment.

Plzf is required in adult male germ cells for stem cell self-renewal.

Adult germline stem cells are capable of self-renewal, tissue regeneration and production of large numbers of differentiated progeny. We show here that the classical mouse mutant luxoid affects adult germline stem cell self-renewal. Young homozygous luxoid mutant mice produce limited numbers of normal spermatozoa and then progressively lose their germ line after birth. Transplantation studies showed that germ cells from mutant mice did not colonize recipient testes, suggesting that the defect is intrinsic to the stem cells. We determined that the luxoid mutant contains a nonsense mutation in the gene encoding Plzf, a transcriptional repressor that regulates the epigenetic state of undifferentiated cells, and showed that Plzf is coexpressed with Oct4 in undifferentiated spermatogonia. This is the first gene shown to be required in germ cells for stem cell self-renewal in mammals.

A large CRISPR-induced bystander mutation causes immune dysregulation.

A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic "bystander" mutations that escape detection by routine targeted genotyping assays.

Deregulated sex chromosome gene expression with male germ cell-specific loss of Dicer1.

MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs that mediate post-transcriptional gene silencing by inhibiting mRNA translation and promoting mRNA decay. DICER1, an RNase III endonuclease encoded by Dicer1, is required for processing short 21-22 nucleotide miRNAs from longer double-stranded RNA precursors. Here, we investigate the loss of Dicer1 in mouse postnatal male germ cells to determine how disruptions in the miRNA biogenesis pathway may contribute to infertility. Reduced levels of Dicer1 transcripts and DICER1 were confirmed in germ cell knock-out (GCKO) testes by postnatal day 18 (P18). Compared to wild-type (WT) at 8 weeks, GCKO males had no change in body weight; yet showed significant reductions in testis mass and sperm number. Histology and fertility tests confirmed spermatogenic failure in GCKO males. Array analyses at P18 showed that in comparison to WT testes, 75% of miRNA genes and 37% of protein coding genes were differentially expressed in GCKO testes. Among these, 96% of miRNA genes were significantly down-regulated, while 4% miRNA genes were overexpressed. Interestingly, we observed preferential overexpression of genes encoded on the sex chromosomes in GCKO testes, including more than 80% of previously identified targets of meiotic sex chromosome inactivation (MSCI). Compared to WT, GCKO mice showed higher percentages of germ cells at early meiotic stages (leptotene and zygotene) but lower percentages at later stages (pachytene, diplotene and metaphase I) providing evidence that deletion of Dicer1 leads to disruptions in meiotic progression. Therefore, deleting Dicer1 in early postnatal germ cells resulted in deregulation of transcripts encoded by genes on the sex chromosomes, impaired meiotic progression and led to spermatogenic failure and infertility.