https://ogma.newcastle.edu.au/vital/access/ /manager/Index ${session.getAttribute("locale")} 5 Prepubertal mouse testis growth and maturation and androgen production are acutely sensitive to di-n-butyl phthalate https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:13743 Wed 11 Apr 2018 14:31:53 AEST ]]> DMRT1 repression using a novel approach to genetic manipulation induces testicular dysgenesis in human fetal gonads https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:34420 +) cells resulting in an ovarian gonadal phenotype. STUDY DESIGN, SIZE, DURATION: To 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, METHODS: Human 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 CHANCE: DMRT1 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); however, there were no changes in expression of the granulosa cell marker, FOXL2. Analysis of germ cell associated genes demonstrated a significant increase in the expression of the pluripotency gene OCT4 (miR536, 233%, P < 0.001). We used the xenograft system to investigate the longer-term effects of seminiferous cord disruption via DMRT1 repression. As was evident in vitro for second-trimester samples, DMRT1 repression resulted in focal testicular dysgenesis similar to that described in adults with TDS. These dysgenetic areas were devoid of germ cells, whilst expression of FOXL2 within the dysgenetic areas, indicated trans-differentiation from a male (Sertoli cell) to female (granulosa cell) phenotype. LIMITATIONS, REASONS FOR CAUTION: Human fetal testis tissue is a limited resource; however, we were able to demonstrate significant effects of DMRT1 repression on the expression of germ and somatic cell genes, in addition to the induction of focal testicular dysgenesis, using these limited samples. In vitro culture may not reflect all aspects of human fetal testis development and function; however, the concurrent use of the xenograft model which represents a more physiological system supports the validity of the in vitro findings. WIDER IMPLICATIONS OF THE FINDINGS: Our findings have important implications for understanding the role of DMRT1 in human testis development and in the origin of testicular dysgenesis. In addition, we provide validation of a novel system that can be used to determine the effects of repression of genes that have been implicated in gonadal development and associated human reproductive disorders.]]> Tue 03 Sep 2019 17:59:48 AEST ]]>