DMRT1 repression using a novel approach to genetic manipulation induces testicular dysgenesis in human fetal gonads

Abstract

STUDY QUESTIONDoes loss of DMRT1 in human fetal testis alter testicular development and result in testicular dysgenesis?SUMMARY ANSWERDMRT1 repression in human fetal testis alters the expression of key testicular and ovarian determining genes, and leads to focal testicular dysgenesis.WHAT IS KNOWN ALREADYTesticular dysgenesis syndrome (TDS) is associated with common testicular disorders in young men, but its etiology is unknown. DMRT1 has been shown to play a role in the regulation of sex differentiation in the vertebrate gonad. Downregulation of DMRT1 in male mice results in trans-differentiation of Sertoli cells into granulosa (FOXL2+) cells resulting in an ovarian gonadal phenotype.STUDY DESIGN, SIZE, DURATIONTo determine the effect of DMRT1 repression on human fetal testes, we developed a novel system for genetic manipulation, which utilizes a Lentivral delivered miRNA during short-term in vitro culture (2 weeks). A long-term (4–6 weeks) ex vivo xenograft model was used to determine the subsequent effects of DMRT1 repression on testicular development and maintenance. We included first and second-trimester testis tissue (8–20 weeks gestation; n = 12) in the study.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman fetal testes were cultured in vitro and exposed to either of two DMRT1 miRNAs (miR536, miR641), or to scrambled control miRNA, for 24 h. This was followed by a further 14 days of culture (n = 3–4), or xenografting (n = 5) into immunocompromised mice for 4–6 weeks. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence and quantitative RT-PCR. Endpoints included histological evaluation of seminiferous cord integrity, mRNA expression of testicular, ovarian and germ cell genes, and assessment of cell number and protein expression for proliferation, apoptosis and pluripotency factors. Statistical analysis was performed using a linear mixed effect model.MAIN RESULTS AND THE ROLE OF CHANCEDMRT1 repression (miR536/miR641) resulted in a loss of DMRT1 protein expression in a sub-population of Sertoli cells of first trimester (8–11 weeks gestation) human fetal testis; however, this did not affect the completion of seminiferous cord formation or morphological appearance. In second-trimester testis (12–20 weeks gestation), DMRT1 repression (miR536/miR641) resulted in disruption of seminiferous cords with absence of DMRT1 protein expression in Sertoli (SOX9+) cells. No differences in proliferation (Ki67+) were observed and apoptotic cells (CC3+) were rare. Expression of the Sertoli cell associated gene, SOX8, was significantly reduced (miR536, 34% reduction, P = 0.031; miR641 36% reduction, P = 0.026), whilst SOX9 expression was unaffected. Changes in expression of AMH (miR536, 100% increase, P = 0.033), CYP26B1 (miR641, 38% reduction, P = 0.05) and PTGDS (miR642, 30% reduction, P = 0.0076) were also observed. Amongst granulosa cell associated genes, there was a significant downregulation in R-spondin 1 expression (miR536, 76% reduction, P < 0.0001; miR641, 49% reduction, P = 0.046); howev...

Disorders of Sex Development—Novel Regulators, Impacts on Fertility, and Options for Fertility Preservation

“…Whilst it is likely that cases of 46,XY DSD in individuals with deletions of chromosome 9p24 are due to loss of DMRT1, it cannot be excluded that other genes in this region contribute, or indeed cause, the observed gonadal phenotypes [121][122][123]. In accordance with the former hypothesis, knockdown of DMRT1 expression in a human fetal testis ex vivo model induced focal testicular dysgenesis and expression of FOXL2 in a small sub-population of supporting cells in which SOX9 expression was lost [124].…”

Sequence analysis of 37 candidate genes for male infertility: challenges in variant assessment and validating genes

“…In patient M1816, who presented SCO and increased FSH levels, we identified potentially damaging variants in two different genes (DMRT1 and KLHL10), hampering our ability to define the causal factor of azoospermia. DOUBLESEX-AND MAB3-RELATED TRANSCRIPTION FACTOR 1 (DMRT1; OMIM 602424) is a conserved sex-determination transcription factor that regulates genes in Sertoli cells and pre-meiotic germ cells during postnatal testis differentiation (Macdonald et al, 2018). KELCH HOMOLOG 10 (KLHL10; OMIM 615081) is a highly evolutionarily conserved gene in mammals, exclusively expressed in the cytoplasm of spermatids.…”

Role of Nodal signalling in testis development and initiation of testicular cancer

“…Interestingly, a recent study in which DMRT1 expression was repressed in human fetal testes using a gene-editing approach, focal testicular dysgenesis was observed as well as increased expression of OCT4 (Macdonald et al 2018). However, germ cells (especially gonocytes) were rarely observed in areas with focal testicular dysgenesis (Macdonald et al 2018), indicating that these cells were lost most likely due to insufficient support from the somatic niche. These observations emphasise that it would be highly relevant to further examine the effects on gonocyte to pre-spermatogonia transition in a fetal testis model with focal testicular dysgenesis to better address this in future studies.…”