ABSTRACT: WIN 18,446/RA treatment of neonatal male mice was used to synchronize spermatogenesis to 2-3 different stages of the cycle of the seminiferous epithelium in the adult testis 6 different groups of synchronized adult mouse testis samples (N = 2 per group), each representing only 2-3 stages of the cycle, were collected and analyzed using GeneChip Mouse Gene ST 1.0 Arrays
Project description:WIN 18,446/RA treatment of neonatal mice was used to synchronize the initial wave of spermatogenesis and identify novel messages expressed within either germ or Sertoli cells as spermatogonia enter meiosis. germ cell-specific (Stra8-cre: RiboTag; or Ngn3-cre:RiboTag) and Sertoli cell-specific (Amh-Cre: RiboTag)
Project description:Analysis of 2dpp whole testes cultured for 24h in the presence of WIN 18,446, a BDAD compound. BDADs (Bis-(dichloroacetyl)-diamines) have been shown previously to inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully described. Results provide insight into the ability of WIN 18,446 to inhibit retinoic acid synthesis in the murine testis. Testes were removed from 2dpp 129 mice and cultured for 24h in an organ culture mold in the presence of WIN 18,446. Total RNA was extracted and hybridized to Affymetrix Mouse Genome 430 2.0 arrays.
Project description:This experiment was performed to investigate the effect of the manipulation of social rank on gene expression. Fire ants newly mated queens were paired and placed in nesting chambers. After emergence of workers, queens behavior was monitored. Once the behavioral observation revealed the social rank of the two cofoundresses (winners and losers), queens were weighed again and re-paired with a different partner. We created the following three groups of queens: a) winner + winner (similar weight), b) loser + loser (similar weight), and c) winner + loser (different weights). Again, we monitored the behavior until the social rank of the newly coupled specimens was evident and we collected 4 new behavioral phenotypes in the same way as above: a) winners switched into losers (win/los), b) losers switched into winners (los/win), c) continuing winners (win/win) and d) continuing losers (los/los).
Project description:This study describes a temporal profile of gene expression from normal human fetal testes and ovaries. Gonads from 34 fetuses between 9 weeks and 20 weeks of gestation were obtained from the Department of Pathology and the Birth Defects Research Laboratory at the University of Washington. Relative transcript levels were determined using the Affymetrix Human Genome U133A Plus 2.0 arrays. Sex determination occurs in the human gonad at approximately 6 weeks; gestation with development of the testis driven by expression of SRY. In this study, SRY transcript was present and elevated at 9 weeks gestation in the testis but absent in the ovary. The transcript levels of other testis-specific factors SOX9, AMH, and the steroidogenic genes CYP17a1, CYP11a1, STAR and HSD17β3 were all significantly higher in the testis. In contrast, transcripts known to be involved in meiosis including STRA8, SPO11, SYCP3, TEX11, TEX14 and STAG3 showed highest expression in the fetal ovary beginning at week 12. These gene expression profiles will be a resource for understanding and defining normal gonad development and provide the opportunity to dissect abnormal development. Experiment Overall Design: Gonads from 34 fetuses between 9 weeks and 20 weeks of gestation were obtained, total RNA was extracted and hybridized to Affymetrix microarrays.
Project description:PRDM9, a histone methyltransferase, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we compared activity of the Prdm9Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced. Comparing these two strains, we find that haplotype differences at hotspots leads to qualitative and quantitative changes in PRDM9 binding and activity. Most variants affecting PRDM9Cst binding arose and were fixed in M.m castaneus, suppressing hotspot activity. Furthermore, M.m castaneus x M.m domesticus F1 hybrids exhibit novel hotspots, representing sites of historic evolutionary erosion. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion. Identify position of meiotic H3K4me3 from various sub-species of mice and F1 hybrids from crosses between subspecies. In addition, perform ChIP-seq analysis on the meiosis-specific methyltransferase PRDM9.
Project description:Our testis transplantation data demonstrate that only Sox2-GFP+c-kit- cells contain testis-repopulating potential and we wondered whether a molecular comparison of the Sox2-GFP+c-kit+ and Sox2-GFP+c-kit- spermatogonial cells would uncover genes that could explain the exclusive repopulation potential of the latter cell population. To this end, we sorted individual testis cell populations and subjected extracted and amplified RNA to array analysis. Mouse testes of 2-week old Sox2GFP mice were isolated, and dissociated with collagenase. Single cell suspensions were generated and stained with FACS antibody for ckit. FACS analysis was done based on internal GFP signal and ckit antibody signal. PI was used to exclude dead cells. Sox2GFP+ckit- and Sox2GFP+ckit+ populations were sorted into Trizol and sent to ExpressionAnalysisR for processing and profiling. 20 testis were pooled into one sample, two biological replicates were analyzed.
Project description:The infant leukemia-associated gene, Ott1(Rbm15), has broad regulatory effects within the murine hematopoiesis. However, germline Ott1 deletion results in fetal demise prior to E10.5, indicating additional developmental requirements for Ott1. The spen gene family, to which Ott1 belongs, has a transcriptional activation/repression domain and RNA recognition motifs, and in Drosophila has a significant role in the development of the head and thorax. Early Ott1-deficient embryos show growth retardation and incomplete closure of the notochord. Further analysis demonstrated placental defects in the spongiotrophoblast and syncytiotrophoblast layers, resulting in an arrest of vascular branching morphogenesis. Rescue of the placental defect using a conditional allele with a trophoblast-sparing cre transgene allowed embryos to form a normal placenta and survive gestation. This result shows that the process of vascular branching morphogenesis in Ott1-deficient animals is regulated by the trophoblast compartment rather than the fetal vasculature. Mice surviving to term manifested hyposplenia and abnormal cardiac development. Analysis of global gene expression of Ott1-deficient embryonic hearts shows enrichment of hypoxia-related genes and significant alteration of several candidate genes critical for cardiac development. Thus, Ott1-dependent pathways in addition to being implicated in leukemogenesis, may also be important in the pathogenesis of placental insufficiency and cardiac malformations. Experiment Overall Design: We generated in triplicate mice containing the Ott1flox and Ott1null alleles. Tg-Sox2-cre animals were obtained from Jackson labs (Bar Harbor, ME). Genotyping was performed by PCR analysis of murine or embryonic tissue [Raffel, 2007 #218]. Timed matings were determined by presence of a vaginal plug at E0.5. Experiment Overall Design: RNA was extracted from 3 hearts each from either Ott1null/nullSox2-cre or Ott1null/wtSox2-cre E18.5 embryos using the RNeasy Micro kit (Qiagen, Valencia, CA) and treated with RNAse-free DNAse (Qiagen, Valencia, CA). 50 ng of purified total RNA was linearly amplified using the Ovation RNA amplification system V2 (Nugen, San Carlos, CA) and biotinylated with the FL-Ovation Biotin Module V2 (Nugen, San Carlos, CA) per the supplied protocol. cDNA was then hybridized to Affymetrix Mouse expression array 430A2.0 chips by the Dana Farber Microarray Core Facility (Boston, MA). The raw gene expression values were preprocessed with robust multi-array analysis (RMA) algorithm using BioConductor software.
Project description:Analysis of 2dpp whole testes cultured for 24h in the presence of WIN 18,446, a BDAD compound. BDADs (Bis-(dichloroacetyl)-diamines) have been shown previously to inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully described. Results provide insight into the ability of WIN 18,446 to inhibit retinoic acid synthesis in the murine testis. Overall design: Testes were removed from 2dpp 129 mice and cultured for 24h in an organ culture mold in the presence of WIN 18,446. Total RNA was extracted and hybridized to Affymetrix Mouse Genome 430 2.0 arrays.
Project description:Here we characterize the genome-wide chromatin modification by PRDM9, a histone H3 lysine 4 methyltransferase. In order to detect PRDM9 binding sites we created coisogenic strains of mice differing only in the zinc finger array of PRDM9. One strain is C57BL/6J, which carries the Prdm9Dom2 allele, the other strain was created using genomic replacement and named B6.PRDM9Cst (also called KI), and contains the Prdm9Cst allele originally found in CAST/EiJ mice. Many H3K4me3 positions are common between strains and represent other methyltransferase activity (such as promoters), sites that are unique to one mouse strain likely represent the binding position of that allele of PRDM9. Identify PRDM9-dependent H3K4me3 sites by comparing modified chromatin from mice coisogenic for Prdm9.