https://ogma.newcastle.edu.au/vital/access/ /manager/Index ${session.getAttribute("locale")} 5 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.]]> Wed 24 Nov 2021 15:52:22 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:39598 Wed 15 Jun 2022 12:54:07 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:52821 Wed 07 Feb 2024 14:59:02 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:48760 Wed 05 Apr 2023 13:49:19 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:38140 Wed 04 Aug 2021 15:49:43 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:53286 Tue 21 Nov 2023 10:30:35 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:54323 Tue 20 Feb 2024 15:59:02 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:27 Thu 25 Jul 2013 09:10:14 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:23094 Thu 14 Apr 2022 11:02:28 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:31198 Thu 13 Jan 2022 10:30:44 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:8358 Sat 24 Mar 2018 08:39:51 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:9572 Sat 24 Mar 2018 08:34:47 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:9576 Sat 24 Mar 2018 08:34:47 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:1630 Sat 24 Mar 2018 08:30:38 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:17174 Sat 24 Mar 2018 08:06:30 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:18843 50 unselected donors and at least three independent samples were used for each component of the analysis. Participants/Materials, Setting, Methods: The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computeraided sperm analysis to measure spermmovement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media. Main results and the role of chance: Oxidative DNA damage is induced in spermatozoa prepared on PureSperm discontinuous colloidal silicon gradients (P < 0.001 versus repeated centrifugation) because this medium contains metals, particularly Fe, Al and Cu, which are known to promote free radical generation in the immediate vicinity of DNA. This damage can be significantly accentuated by reducing agents, such as ascorbate (P < 0.001) and inhibited by selective chelation (P < 0.001). This problem is not confined to PureSperm®; analysis of additional commercial sperm preparation media revealed that metal contamination is a relatively constant feature of such products. Limitations, reasons for caution: While the presence of metals, particularly transition metals, may exacerbate the levels of oxidative DNA damage seen in human spermatozoa, the significance of such damage has not yet been tested in suitably powered clinical trials. Wider implications of the findings: The results explain why the preparation of spermatozoa on discontinuous colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes.]]> Sat 24 Mar 2018 08:03:19 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:17909 Sat 24 Mar 2018 07:56:41 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:5481 Sat 24 Mar 2018 07:47:04 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:25242 Sat 24 Mar 2018 07:38:18 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:46738 Mon 10 Jul 2023 13:53:20 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:45506 Fri 28 Oct 2022 16:14:22 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:42487 Chlamydia be found in the testes of infertile men? Summary Answer: Chlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men. What is known already: Male chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility. Study Design, Size, Duration: We collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling. Participants/Materials, Setting, Methods: The diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques. Main Results and the Role of Chance: Chlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia. Limitations, Reasons for Caution: No reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples. Wider Implications for the Findings: Application of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure.]]> Fri 26 Aug 2022 09:47:49 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:51929 Fri 22 Sep 2023 11:07:45 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:34517 Fri 22 Mar 2019 13:04:05 AEDT ]]>