Alteration in porcine testicular gene expression in response to reduced testicular estradiol synthesis
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PROVIDER: PRJNA647900 | ENA |
REPOSITORIES: ENA
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Project description:ITo derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine, we developed a method to produce a cytocompatible decellularized testicular matrix (DTM) while maintaining the native tissue-specific characteristics and components. The potential benefits of tissue-specific scaffolds consisting of naturally-derived extracellular matrix (ECM) have been demonstrated using a wide variety of animal and human tissue sources. However, so far, testis scaffolds have never been considered for constructive remodelling purposes. We have therefore developed a protocol for the preparation of cell-free extracellular matrix and characterized the material extensively using a combination of proteomics, immunohistochemsitry, and cell population assays. To prepare cell-free matrix, human cadaveric testicular tissue was exposed for 24 h or 48 h to 1% Triton X-100 and/or 1% sodium dodecyl sulfate (SDS). The extent of decellularization was evaluated by histology. Confirmation of cell removal in DTM was done by a DNA quantification technique. The protein composition was analysed by LC-MS/MS. The retention of testicular tissue-specific characteristics was evaluated by immunohistochemistry, Alcian blue staining and scanning electron microscopy. Soluble toxicity and testicular cell attachment was assessed to check the cytocompatibility of DTM scaffolds. Histological analysis showed that DTM could be obtained by mechanical agitation in 1% SDS for 24 h. The resulting DTM was found to be clear of cells while retaining the typical three-dimensional structure. Proteomics analysis revelaed the presence of the major components of the native tissue scaffold, including collagen type I and IV, fibronectin, laminin and glycosaminoglycans. In addition, numerous additional ECM proteins in DTM were detected, indicating its complex nature. Importantly, we demonstrated that DTM scaffolds are not cytotoxic, as evidenced by MTT assay showing a normal fibroblast proliferation activity after indirect exposure, and support testicular cell attachment and infiltration.
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Project description:Adult male marine fish were exposed to 100ng/L of ethinyl estradiol (EE2) for 7 days in 1.8L vessels, dimethyl sulfoxide (DMSO) was used as carrier never reaching concentrations > 0.001% v/v. The same volume of DMSO was added to the solvent control vessels. Culture conditions, monitored daily, were as follows: temperature 18.8 ± 0.2°C, pH 7.94±0.03, dissolved oxygen saturation 99.3% ± 1.1 and 16:8 hours of light:dark photoperiod. 90% of the water was renewed daily and EE2 and DMSO concentrations were adjusted after that.
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Project description:Testicular germ cell tumors are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse testicular germ cell tumor model in which germ cell-specific oncogenic Kras activation and tumor suppressor Pten inactivation was driven by CRE-mediated recombination. The resulting mice rapidly developed malignant, metastatic testicular cancers composed of both teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. As part of our analysis of mouse gPAK testicular tumors, as well as comparison to benign 129-Dnd1Ter/Ter testicular teratomas, we used NimbleGen Mouse CGH 3x720k Whole-Genome Tiling Arrays to assess copy number variations in this novel genetically engineered mouse model of malignant, metastatic testicular cancer.
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Project description:This study assessed the implications of a 14 day sub-chronic exposure of ethinylestradiol (EE2; 1.0 or 10.0 M-BM-5g/L EE2) on male medaka fertility, testicular histology and testicular gene expression. The findings demonstrate that a 14 day exposure to EE2 induced impaired male reproductive capacity and time- and dose-dependent alterations in testicular morphology and gene expression. The average fertilization rate/day following the exposure for control, 1.0 and 10.0 M-BM-5g/L EE2 was 91.3% (M-BM-14.4), 62.8% (M-BM-18.3) and 28.8% (M-BM-15.8), respectively. The testicular morphologic alterations observed include increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. The morphologic changes observed were highly associated with gene expression changes observed using a medaka-specific microarray. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell cycle and proliferation, collagen production/extracellular matrix organization, hormone signaling, male reproduction and protein ubiquitination among others. Six month old male medaka were exposed to ethinyl estradiol (EE2) for a 14 day time period. Treatment exposures were completed in triplicate including DMSO (vehicle control), 1.0 M-BM-5g/L EE2, and 10.0 M-BM-5g/L EE2. Fish were sampled for gene expression on days 1, 7 and 14 of exposure. Five male fish were placed in 2-liter beaker replicates for each treatment and sampling time point.
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Project description:In addition to germ cell population, testes contain several types of somatic cells, including Sertoli cells, Leydig cells, and peritubular myoid (PTM) cells. PTM cells are a type of smooth muscle-like cells and have contraction ability. To identify a marker of PTM cells, we isolated primary cells and subsequently performed miRNA-Seq of testicular peritubular myoid cells (PTM_1, PTM_2, PTM_3), testicular Sertoli cells (Sertoli_1, Sertoli_2, Sertoli_3), testicular Leydig cells (Leydig_1, Leydig_2, Leydig_3), testicular germ cells (Germ_1, Germ_2, Germ_3), vascular smooth muscle cells (VSMC_1, VSMC_2, VSMC_3), intestinal smooth muscle cells (ISMC_1, ISMC_2, ISMC_3), and tracheal smooth muscle cells (TSMC_1, TSMC_2, TSMC_3) using BGISEQ-500 platform (BGI, China).
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Project description:In addition to germ cell population, testes contain several types of somatic cells, including Sertoli cells, Leydig cells, and peritubular myoid (PTM) cells. PTM cells are a type of smooth muscle-like cells and have contraction ability. To identify a marker of PTM cells, we isolated primary cells and subsequently performed RNA-Seq of testicular peritubular myoid cells (PTM_1, PTM_2, PTM_3), testicular Sertoli cells (Sertoli_1, Sertoli_2, Sertoli_3), testicular Leydig cells (Leydig_1, Leydig_2, Leydig_3), testicular germ cells (Germ_1, Germ_2, Germ_3), vascular smooth muscle cells (VSMC_1, VSMC_2, VSMC_3), intestinal smooth muscle cells (ISMC_1, ISMC_2, ISMC_3), and tracheal smooth muscle cells (TSMC_1, TSMC_2, TSMC_3) using BGISEQ-500 platform (BGI, China).
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Project description:We have evaluated the transcriptome of different testicular cell types, including germ cells, at single cell level in a testicular biopsy of a man with Klinefelter syndrome (XXY karyotype)
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