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Series GSE23119 Query DataSets for GSE23119
Status Public on Aug 03, 2010
Title Effect of vitamin A deficiency (VAD) on mouse spermatogonial transcriptome profiles
Organism Mus musculus
Experiment type Expression profiling by array
Summary The objective of this study was to understand the genetic mechanisms of Vitamin-A-Deficiency (VAD)-induced arrest of spermatogonial stem-cell differentiation. Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that, in the postnatal testis, leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. In this study, we investigated the molecular basis of VAD on spermatogenesis in mice. We used adult Balb/C mice fed with a Control or VAD diet for an extended period of time (8-28 weeks) and selected two time points (18 and 25 weeks) for microarray analysis.

To understand the effect of VAD on the spermatogonial stem cell transcriptome, we studied isolated pure populations of spermatogonia from control and vitamin-A-deficient mice from two representative time points (18 and 25 weeks) using Affymetrix GeneChip microarrays. We identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis. Our results establish a better understanding of the chronology and magnitude of the consequences of VAD on mouse testes and add to the current knowledge of the molecular regulatory mechanisms of germ cell development.
 
Overall design Spermatogonia were isolated by the STATPUT procedure with minor modifications. VAD mice were sacrificed at 18 and 25 weeks of VAD treatment for spermatogonia isolation. Decapsulated testes from 2 mice were suspended in RPMI medium containing collagenase (0.6 mg/ml), Hyaluronidase (0.12 mg/ml) and DNAse I (1.25 mg/ml) and incubated at 37ºC for 30 minutes in a shaking water bath. The tissues were then allowed to come down the tube and the supernatant (containing interstitial cells) was removed. The pellet was incubated with 3 ml of 0.25% Trypsin /EDTA (GIBCO, Invitrogen, USA), in the presence of DNAse I (0.2 mg/ml) at 37ºC for 15 minutes in a shaking water bath. The dispersed cells were washed twice with RPMI medium containing 10% heat inactivated FBS to neutralize the protease activity, and filtered through a sterile 0.22 nylon to remove any undigested fragments. Cells of the dissociated seminiferous epithelium were then plated overnight in a 34ºC 3% CO2 incubator. The following day, germ cells, in suspension, were separated by sedimentation with use of a 2-4% BSA gradient. The cells were allowed to sediment for a standard period of 2.5 h, and fractions of 2-ml volume were collected. The cells of each fraction were examined under a phase contrast microscope, and fractions containing cells of similar size and morphology were pooled and spun down by low-speed centrifugation. Purity of spermatogonia was estimated and was routinely higher than 90%. Total RNA was extracted from the isolated germ cells using TRIzol ® Reagent, and cleaned with RNeasy minicolumns. RNA content was determined by measurement of optical density at 260 nm. Only the RNA samples showing an OD 260/280 ratio higher than 1.8 were used for microarray hybridization. Raw expression values in Affymetrix CEL file format were generated by GeneChip Operating Software.
 
Contributor(s) Lee T
Citation(s) 23253600
Submission date Jul 23, 2010
Last update date Feb 11, 2019
Contact name TIN-LAP LEE
Organization name National Institutes of Health
Department NICHD
Lab Laboratory of Clincal Genomics
Street address 2C08 Building 49
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platforms (1)
GPL1261 [Mouse430_2] Affymetrix Mouse Genome 430 2.0 Array
Samples (9)
GSM573579 Normal spermatogonia control, rep1
GSM573580 Normal spermatogonia control, rep2
GSM573581 Normal spermatogonia control, rep3
Relations
BioProject PRJNA131717

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Supplementary file Size Download File type/resource
GSE23119_RAW.tar 32.3 Mb (http)(custom) TAR (of CEL, CHP)
Raw data provided as supplementary file
Processed data included within Sample table
Processed data provided as supplementary file

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