Project description:Infertility observed in adult Sertoli cell (SC)-specific Connexin 43 Knockout-mice rather seems to be an effect of the disturbed SC-Germ cell (GC) crosstalk than a direct consequence of the loss of Cx43 protein in SC with important GC specific genes being mostly affected by this deletion.
Project description:Infertility observed in adult Sertoli cell (SC)-specific Connexin 43 Knockout-mice rather seems to be an effect of the disturbed SC-Germ cell (GC) crosstalk than a direct consequence of the loss of Cx43 protein in SC with important GC specific genes being mostly affected by this deletion. Identification of differentially expressed genes in testis of cx43 KO-mice at developmental stage 8 days post partum when compared with testis of WT-mice
Project description:Sertoli cells, which are the only somatic cells in the seminiferous tubules, facilitate the maintenance of testicular immune privilege through the formation of the blood-testis barrier (BTB) and the expression of immunoregulatory factors. Rho guanosine exchange factor 15 (ARHGEF15) is a member of guanosine exchange factors, which are involved in cell migration, cell polarity, and cell cycle progression via activation of Rho GTPases. This study investigated the functions of ARHGEF15 in Sertoli cells during spermatogenesis using Sertoli cell–specific Arhgef15 knockout mice. The results showed that Arhgef15 deficiency in Sertoli cells affected the localization of Sertoli cell nuclei, disrupted BTB integrity, and led to premature shedding of germ cells. In Arhgef15flox/flox/Amh-Cre+ mice, the ultrastructure of the round spermatids was impaired, accompanied by acrosome degeneration, acrosomal vesicle shedding, and atrophic nuclei. Consequently, the percentage of abnormal sperm in the Arhgef15flox/flox/Amh-Cre+ epididymis was markedly elevated. RNA-sequencing analysis revealed that most of the differentially expressed genes in Sertoli cells of Arhgef15flox/flox/Amh-Cre+ mice were related to immunity. Further study revealed that the sera of Arhgef15flox/flox/Amh-Cre+ mice showed immunoreactivity against testicular lysate of wild-type mice, indicating the production of antibodies against testicular autoantigens in Arhgef15flox/flox/Amh-Cre+ mice. In conclusion, the specific deletion of Arhgef15 in Sertoli cells leads to sperm abnormality, probably by disrupting the testicular immune homeostasis, in mice.
Project description:Gene expression profiling study of Sertoli cells (Sc)-specific Retinoblastoma (Rb) knockout (KO) mice. We generated a Sc-specific Rb knockout (Sc-RbKO) line and characterised its testicular phenotype. Sc-RbKO showed a progressive germ cell depletion leading to infertility and Sc dysfunction. We observed that up to 10 days old, Sc-RbKO mice present a phenotype similar to that of their WT siblings. This age was choosen to study the gene expression changes that would eventually lead to the described phenotypical changes. Sc-RbKO testis gene expression was compared to WT and to Sc-Rb+/- samples. Our Microarray results show changes of many genes involved in cell cycle control, DNA repair, apoptosis and misregulation of genes that may be important for proper Sertoli cell function such as tight junction formation, cell membrane and tissue remodeling. Total RNA extracted from 12 testicular samples, 5 controls, 3 hetorozygotes and 4 homozygotes.
Project description:Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, an established testicular toxicant. MAA induces the degradation of testicular germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and global gene expression was monitored by microarray analysis. A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes and 60 DNA-binding proteins that responded to MAA rapidly but transiently, and which may contribute to the downstream effects of MAA seen for large numbers of mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. These findings on the progressive changes in gene expression induced by MAA in Leydig cells may help elucidate the signaling pathways perturbed by this testicular toxicant and explain its mechanism of toxicity at the gene level.
Project description:To systematically investigate the expression patterns of all potential niche factors in testis, we performed single-cell RNA sequencing (scRNA-seq) of all testicular somatic cell types. To enrich somatic cells, we depleted Tomato+ cells from the testes of 2-month-old Ddx4-creER; R26tdTomato mice at 4 weeks after tamoxifen treatment. Then the cells were further captured with 10x Genomics platform. After analysis of the integrated data, we mapped the expression patterns of all known niche factors in testicular somatic cells. We also performed scRNA-seq of testicular cells from 6-week-old control and Amh-cre;Scf fl/fl mice to study the effect of Scf conditional knockout from Sertoli cells on spermatogenesis. By scRNA-seq data analysis, we found that conditional knockout of Scf from Sertoli cells blocks spermatogenesis by depleting differentiating spermatogonia
Project description:Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, an established testicular toxicant. MAA induces the degradation of testicular germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and global gene expression was monitored by microarray analysis. A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes and 60 DNA-binding proteins that responded to MAA rapidly but transiently, and which may contribute to the downstream effects of MAA seen for large numbers of mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. These findings on the progressive changes in gene expression induced by MAA in Leydig cells may help elucidate the signaling pathways perturbed by this testicular toxicant and explain its mechanism of toxicity at the gene level. Mouse TM3 Leydig cells (American Type Culture Collection, Manassas, VA) were grown in DMEM-F12 medium containing 5% horse serum and 2.5% FBS. Cells were grown to ~60% confluence and treated with culture medium alone, or with culture medium containing 1 mM or 5 mM MAA for either 3, 8 or 24 h. Total RNA was then isolated using TRIzol reagent, followed by incubation with RQ1 RNAse-free DNAse for 1 h at 37°C and then heating at 75°C for 5 min using the manufacturerâs protocol. A total of 6 cultures of TM3 cells were independently treated with MAA under each of the 6 treatment conditions specified above (i.e., 1 mM or 5 mM MAA for either 3, 8 or 24 h), and the corresponding 6 sets of RNA samples were validated by RNA integrity analysis (Agilent Bioanalyzer). Each RNA sample was also validated by qPCR analysis using SYBR Green I-based chemistry and primers specific for 3 genes known to respond to MAA (Cyp17a1, Shbg, and Igfbp3) to verify consistency of the MAA responses. The 6 RNA samples were then used to prepare two independent pools (n=3 RNA samples each) for microarray analysis with dye swaps. Sample labeling, hybridization to microarrays, scanning and calculation of normalized expression ratios were carried out at the Wayne State University Institute of Environmental Health Sciences microarray facility using Alexa 555 and Alexa 647 aminoallyl-aRNA samples
Project description:Pre-pubertal Holstein bull calves fed a higher plane of nutrition had larger testes, earlier puberty, higher serum LH, testosterone and greater sperm production potential than those fed a restricted diet. In addition, pre-pubertal calves fed a high-nutrition diet had higher IGF-I and more proliferating and differentiating Sertoli cells much earlier in life, compared to those fed normal or low-nutrition diets. The objective was to determine changes in mRNA expression of genes in the testes of bulls fed either a high or low pre-pubertal diet. Holstein bull calves maintained on either a high (20% crude protein (CP) and 71.6% Total Digestible Nutrients (TDN)) or low (12% CP and 64.4% TDN) diet from 2 wk of age, were castrated at 8, 16, 24 and 32 wk and testicular mRNA extracted and sequenced. Differential expression of genes mainly occurred at 16 and 24 wk, with minor changes detected at 32 wk. At 16 wks, functional analysis of DE mRNA with DAVID revealed the common biological processes enriched to be "cholesterol" and "fatty acid biosynthesis" with majority of the genes including HMGCR, HMGCS1, HSD17 being upregulated in high-diet bulls (P<0.05). Major pathways enriched at 16 wks were "cholesterol biosynthesis", "steroid metabolism" and "activation of gene expression by Sterol regulatory element-binding protein (SREBP)" (P<0.05). Mature Sertoli cell marker Connexin 43, was upregulated at 16 wk, whereas an immature Sertoli cell marker, AMH was downregulated at 24 wk, in the high-diet group. Network analysis using IPA, revealed an indirect interaction between insulin family receptor and most upregulated cholesterol biosynthesis genes, implying regulation of testicular function. Thus, enhanced pre-pubertal nutrition in Holstein bulls enhanced testicular cholesterol/steroid biosynthesis and Sertoli cell maturation to promote early reproductive development.