The Role of Androgens in Testicular Development and Dysgenesis

Disorders of male reproductive health which manifest at birth (cryptorchidism, hypospadias) or in young adulthood (testicular germ cell cancer and low sperm counts), are common and may be increasing in incidence. These disorders have a common fetal origin and share risk factors; consequently they are hypothesized to comprise a testicular dysgenesis syndrome (TDS). TDS arises when maldevelopment (dysgenesis) of the fetal testis results in hormonal malfunctions and abnormal development and function of the somatic cells. It is thought that the suppressed intratesticular testosterone levels associated with TDS may account for subsequent low sperm counts, via a reduction in perinatal Sertoli cell proliferation/number. Sertoli cells do not express androgen receptors (AR) in fetal life in the human or rat, so it is hypothesised that any androgen effects on Sertoli cell number occur indirectly, via the AR positive peritubular myoid cells. Evidence from the di (n‐butyl) phthalate (DBP)‐treated rat model for TDS suggests that reduced androgen action may play a role in testicular dysgenesis as in patients with complete androgen insensitivity syndrome (CAIS; ‘testicular feminization’), in whom focal areas of testicular dysgenesis have been reported. The studies in this thesis sought to establish if reduced androgen levels/action in the fetal rat testis contribute to putative testicular dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes or abnormal aggregation of fetal Leydig cells, the precursor of focal dysgenesis. Pregnant rats were exposed to treatments or co‐treatments expected to manipulate testicular testosterone levels (DBP, testosterone propionate; TP) or action (flutamide, DMBA) or to induce intrauterine growth restriction (dexamethasone), another risk factor for TDS. The aforementioned endpoints were analysed in fetal testes and related to testicular testosterone levels and peripheral androgen action (anogenital distance). The same endpoints were evaluated in mice with inactivation of the androgen receptor (tfm or ARKO mice). As androgen action is assumed to be mediated indirectly, via the peritubular myoid cells, changes in peritubular myoid cell number and function were investigated in testes with suppressed androgens. In vitro studies were also used to investigate the role of androgens in Sertoli cell proliferation. Fetal rat testis explants were cultured with various chemicals designed to manipulate androgen action and Sertoli cell proliferation. Potential non‐androgen related mechanisms of DBP action were investigated using Taqman RT‐PCR to determine the mRNA expression of key developmental genes after exposure to DBP. Sertoli cell number was reduced after exposure to treatments that reduced testicular testosterone levels, i.e. DBP alone or as a co‐treatment, TP and dexamethasone. Sertoli cell numbers in ARKO mice were also significantly reduced. The occurrence of multinucleated gonocytes and large Leydig cell clusters were induced after exposure to DBP, alone or as a co‐treatment, but not after exposure to TP or dexamethasone, and these dysgenetic endpoints did not occur either in tfm or ARKO mice. Rats exposed in utero to DBP have reduced testicular testosterone levels, however peritubular myoid cell number was unaffected by DBP, though AR expression in the peritubular myoid cells was delayed, and laminin and vimentin expression in Sertoli cells was altered after DBP exposure. DMRT‐1 and DAX‐1 mRNA expression levels were significantly reduced after DBP exposure, but this reduction was no longer evident once mRNA expression was corrected for Sertoli cell number. In conclusion, these studies provide strong evidence that androgens play a role in regulation of Sertoli cell number/proliferation, and this is supported by a comparable reduction in Sertoli cell number in ARKO and tfm mice. However, since the treatments that reduce testicular testosterone in the rat, may also have a direct affect on the Sertoli cells, this alternate mechanism of action cannot be ruled out, and the administration of a treatment that reduces testicular testosterone without directly affecting Sertoli cells is required. These studies also show that reduced testicular testosterone levels are associated with multinucleated gonocyte formation and fetal Leydig cell aggregation, although this evidence it is not supported by parallel findings from the TP and dexamethasone exposed rats or the ARKO and tfm mice, as neither of these endpoints were identified as being affected in these animals. Aside from the delay in AR expression, there were no obvious changes in peritubular myoid cell number or the peritubular myoid cell markers examined in testes deprived of androgens, although there are other markers that could be investigated. mRNA analysis of the developmental genes investigated after DBP exposure, demonstrated no change in expression after correction for Sertoli cell number, suggesting that they do not play a role in the dysgenetic features observed in DBP exposed testes

Cellular and Hormonal Disruption of Fetal Testis Development in Sheep Reared on Pasture Treated with Sewage Sludge

The purpose of this study was to evaluate whether experimental exposure of pregnant sheep to a mixture of environmental chemicals added to pasture as sewage sludge (n = 9 treated animals) exerted effects on fetal testis development or function; application of sewage sludge was undertaken so as to maximize exposure of the ewes to its contents. Control ewes (n = 9) were reared on pasture treated with an equivalent amount of inorganic nitrogenous fertilizer. Treatment had no effect on body weight of ewes, but it reduced body weight by 12–15% in male (n = 12) and female (n = 8) fetuses on gestation day 110. In treated male fetuses (n = 11), testis weight was significantly reduced (32%), as were the numbers of Sertoli cells (34% reduction), Leydig cells (37% reduction), and gonocytes (44% reduction), compared with control fetuses (n = 8). Fetal blood levels of testosterone and inhibin A were also reduced (36% and 38%, respectively) in treated compared with control fetuses, whereas blood levels of luteinizing hormone and follicle-stimulating hormone were unchanged. Based on immunoexpression of anti-Müllerian hormone, cytochrome P450 side chain cleavage enzyme, and Leydig cell cytoplasmic volume, we conclude that the hormone changes in treated male fetuses probably result from the reduction in somatic cell numbers. This reduction could result from fetal growth restriction in male fetuses and/or from the lowered testosterone action; reduced immunoexpression of α-smooth muscle actin in peritubular cells and of androgen receptor in testes of treated animals supports the latter possibility. These findings indicate that exposure of the developing male sheep fetus to real-world mixtures of environmental chemicals can result in major attenuation of testicular development and hormonal function, which may have consequences in adulthood

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein–RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein–protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function

Use of artificial intelligence in the detection of primary prostate cancer in multiparametric MRI with its clinical outcomes: a protocol for a systematic review and meta-analysis

INTRODUCTION: Multiparametric MRI (mpMRI) has transformed the prostate cancer diagnostic pathway, allowing for improved risk stratification and more targeted subsequent management. However, concerns exist over the interobserver variability of images and the applicability of this model long term, especially considering the current shortage of radiologists and the growing ageing population. Artificial intelligence (AI) is being integrated into clinical practice to support diagnostic and therapeutic imaging analysis to overcome these concerns. The following report details a protocol for a systematic review and meta-analysis investigating the accuracy of AI in predicting primary prostate cancer on mpMRI. METHODS AND ANALYSIS: A systematic search will be performed using PubMed, MEDLINE, Embase and Cochrane databases. All relevant articles published between January 2016 and February 2023 will be eligible for inclusion. To be included, articles must use AI to study MRI prostate images to detect prostate cancer. All included articles will be in full-text, reporting original data and written in English. The protocol follows the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols 2015 checklist. The QUADAS-2 score will assess the quality and risk of bias across selected studies. ETHICS AND DISSEMINATION: Ethical approval will not be required for this systematic review. Findings will be disseminated through peer-reviewed publications and presentations at both national and international conferences. PROSPERO REGISTRATION NUMBER: CRD42021293745

Loss of N-WASP drives early progression in an Apc model of intestinal tumourigenesis

N‐WASP (WASL) is a widely expressed cytoskeletal signalling and scaffold protein also implicated in regulation of Wnt signalling and homeostatic maintenance of skin epithelial architecture. N‐WASP mediates invasion of cancer cells in vitro and its depletion reduces invasion and metastatic dissemination of breast cancer. Given this role in cancer invasion and universal expression in the gastrointestinal tract, we explored a role for N‐WASP in the initiation and progression of colorectal cancer. While deletion of N‐wasp is not detectably harmful in the murine intestinal tract, numbers of Paneth cells increased, indicating potential changes in the stem cell niche and migration up the crypt‐villus axis was enhanced. Loss of N‐wasp promoted adenoma formation in an adenomatous polyposis coli (Apc) deletion model of intestinal tumourigenesis. Thus, we establish a tumour suppressive role of N‐WASP in early intestinal carcinogenesis despite its later pro‐invasive role in other cancers. Our study highlights that while the actin cytoskeletal machinery promotes invasion of cancer cells, it also maintains normal epithelial tissue function and thus may have tumour suppressive roles in pre‐neoplastic tissues

Trace CO2 capture by an ultramicroporous physisorbent with low water affinity.

CO2 accumulation in confined spaces represents an increasing environmental and health problem. Trace CO2 capture remains an unmet challenge because human health risks can occur at 1000 parts per million (ppm), a level that challenges current generations of chemisorbents (high energy footprint and slow kinetics) and physisorbents (poor selectivity for CO2, especially versus water vapor, and/or poor hydrolytic stability). Here, dynamic breakthrough gas experiments conducted upon the ultramicroporous material SIFSIX-18-Ni-β reveal trace (1000 to 10,000 ppm) CO2 removal from humid air. We attribute the performance of SIFSIX-18-Ni-β to two factors that are usually mutually exclusive: a new type of strong CO2 binding site and hydrophobicity similar to ZIF-8. SIFSIX-18-Ni-β also offers fast sorption kinetics to enable selective capture of CO2 over both N2 (S CN) and H2O (S CW), making it prototypal for a previously unknown class of physisorbents that exhibit effective trace CO2 capture under both dry and humid conditions

Effects of Monobutyl and Di(n-butyl) Phthalate in Vitro on Steroidogenesis and Leydig Cell Aggregation in Fetal Testis Explants from the Rat: Comparison with Effects in Vivo in the Fetal Rat and Neonatal Marmoset and in Vitro in the Human

BACKGROUND: Certain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human. OBJECTIVES: Our aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset. METHODS: Fetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15–19 weeks of gestation) were cultured for 24–48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP. RESULTS: Exposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5–20.5) DBP treatment. In vitro, MBP (10(−3) M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10(−3) M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2–7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy. CONCLUSIONS: These findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro

Recurrent Tissue-Specific Mtdna Mutations are Common in Humans

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage

Decreased expression of the mitochondrial bcat protein correlates with improved patient survival in idh-wt gliomas

Background and research question: Gliomas represent 43% of all solid intracranial tumours, of which glioblastomas have the poorest prognosis. Recently, the human cytosolic branched-chain aminotransferase protein (hBCATc), which metabolises the branched-chain amino acids (BCAA), was identified as a biomarker and therapeutic target for glioblastomas carrying wild-type isocitrate dehydrogenase (IDH-WT) genes. However, the clinical utility of the mitochondrial isoform, hBCATm, which also metabolises BCAAs, was not determined nor its potential role in predicting patient survival.Methods: Glioblastomas, of grades II-IV, from 53 patients were graded by a neuropathologist, where the IDH and MGMT status were assessed. Tumours positive for hBCATm, hBCATc and BCKDC were characterised using immunohistochemistry and Western blot analysis using antibodies specific to these proteins.Results: Here, we report that in IDH-WT tumours, the expression of hBCATm is significantly increased (p=0.034) relative to IDH mutation gliomas, and significantly correlates with patient survival, on Kaplan-Meier analysis, where low hBCATm expression is a positive prognostic factor (p=0.003). Moreover, increased hBCATm expression in these glioblastomas correlated with tumour grade indicating their role as a predictive biomarker of glioma progression. Multiple banding was observed for the branched-chain α-keto acid dehydrogenase complex, which catalyses the committed step in BCAA metabolism, but a significant change in expression was absent (p=0.690). Conclusion: Until now, IDH-WT glioblastomas have a uniformly poor prognosis, however we demonstrate for the first time that relatively low hBCATm may select for a better performing subset within this group and may represent a therapeutic target in these hard to treat patients