Project description:Spermatogenesis is a recurring differentiation process that results in the production of male gametes within the testes. During this process, spermatogonial stem cells differentiate to form spermatocytes, which undergo two rounds of meiotic division to form haploid spermatids. Throughout spermiogenesis, round spermatids elongate to form mature sperm. To profile maturing cell types, we generated bulk RNA-seq data from whole testis undergoing the first round of spermatogenesis between post-natal day P6 and P35. We also compared these libraries to adult samples.
Project description:Temporal study of the first wave of spermatogenesis in juvenile mouse testis and analysis of four germ cell deficient mouse models.<br> A series of purified testis cell-types (McCarrey libraries)constituting a focussed gene set was exploited to examine gene expression in prepubertal mouse testis. Testis RNA from C57/BL6J mice at various ages post partum was compared to a common control consisting of adult (8 week) testis RNA from the same strain(also corresponding to the last time-point).This generates a time course showing the activation of genes on the array across the first wave of spermatogenesis, which can then be correlated with the appearance of specific germ cell types, allowing assignation of unknown genes to differing cell types. <br> <br> Adult testis RNA from four different genetic models of infertility (XXSxrb, mshi, Bax -/-, bs) were compared to age- and strain-matched normal control testis RNA. These models possess different cellular complements within the testis, which can be interpreted within the framework established by the first wave analysis and used to refine the assignment of genes to cell types.
Project description:Adenosine-to-inosine RNA editing, a fundamental RNA modification, is regulated by adenosine deaminase (AD) domain containing proteins. Within the testis, RNA editing is catalyzed by ADARB1 and is regulated in a cell-type dependent manner. This study examined the role of two testis-specific AD domain proteins, ADAD1 and ADAD2, on testis RNA editing and male germ cell differentiation. ADAD1, previously shown to localize to round spermatids, and ADAD2 had distinct localization patterns with ADAD2 expressed predominantly in mid- to late-pachytene spermatocytes suggesting a role for both in meiotic and post-meiotic germ cell RNA editing. AD domain analysis showed the AD domain of both ADADs was likely catalytically inactive, similar to known negative regulators of RNA editing. To assess the impact of Adad mutation on male germ cell RNA editing, CRISPR-induced alleles of each were generated in mouse. Mutation of either Adad resulted in complete male sterility with Adad1 mutants displaying severe teratospermia and Adad2 mutant germ cells unable to progress beyond round spermatid. However, mutation of neither Adad1 nor Adad2 impacted RNA editing efficiency or site selection. Taken together, these results demonstrate ADAD1 and ADAD2 are essential regulators of male germ cell differentiation with molecular functions unrelated to A-to-I RNA editing.
Project description:Using bulk RNAseq, we explored transcriptomic landscape of three reward areas - PrL, NAc, VTA and two sensory areas -S1, VBT in perinatal fentanyl exposed juvenile mice
Project description:We precipitated dsRNA using the monoclonal J2 antibody and deep-sequenced the enriched samples from testis of juvenile dicer knock-out mice, age matched controls and adult animals. The dsRNA transcriptome is significantly less complex in juvenile mice as compared to adult controls and, possibly as a consequence, the knock-out of Dicer had only a minor effect of the total number of transcribed regions associated with dsRNA. The genes that potentially generate dsRNA are significantly expressed in isolated sperm cells with particular enrichment in pachytene spermatocytes.
Project description:To identify differentially expressed genes in Boris KO testis, gene expression profilings were performed with NIAID mouse customized arrays. Gene expressions of five biological replicates of Boris KO testis were compared to those of wild type testis.
Project description:An ex vivo protocol was optimized to expose testis tissues from juvenile Silurana tropicalis to 5 uM finasteride. Testis tissues were incubated in L-15 media with (n = 8) and without (n = 8) finasteride for 6 h at 26 °C on top of an orbital shaker. Afterwards, the tissues were flash frozen on dry ice and RNA was isolated using Trizol. The isolated RNA of four control tissue samples and four finasteride exposed tissue samples were then analyzed using a custom microarray for S. tropicalis containing 44,000 probes .