Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of rat seminiferous tubules, purifed germ cells and sertoli cells

ABSTRACT: Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes and spermatids suggests the existence of precise programs of gene expression in these cells as well as in their neighboring somatic Sertoli cells. The objective of this study was to elucidate genes encoding the proteins that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, XIII-XIV of the cycle were isolated by microdissection and Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes and spermatids were purified from enzymatically-dispersed testes. Microarray analysis using Rat Genome 230 2.0 arrays identified a total of 16,971 probe sets that recognized transcripts. A comparison with the transcriptome of other tissues identified 398 testis-specific probe sets, which therefore are potential targets for the development of new contraceptives. Sequential waves of cell and stage-specific gene expression are associated with progression of germ cells through the stages of the cycle of the seminiferous epithelium and 1612 probe sets recognized transcripts whose expressions varied at least 4-fold across the stages of the cycle. Pathway analyses reveal that entire biological processes are regulated cyclically in testicular cells. Important among these are cell cycle and DNA repair. Thus, stage-specific gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells. Experiment Overall Design: Seminiferous tubules at the following stages or groups of stages were isolated by transillumination-assisted microdissection: I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, XIII-XIV. Twenty-five segments of tubules, 2 m in length, were collected at each stage or group of stages from each rat. Tubules from two separate animals were pooled to form one sample from each stage or groups of stages. A total of 5 independent samples per stage or groups of stages were analyzed. Mature Sertoli cells were isolated to about 85% purity (n=4). Three groups of germ cells were isolated by centrifugal elutriation from enzyme-dispersed testes: round spermatids (n=4), pachytene spermatocytes (n=4) and spermatogonia plus early spermatocytes (n=3) . Purity of these cells has been shown to vary between 85% to 95%

ORGANISM(S): Rattus norvegicus

SUBMITTER: Scott Jelinsky

PROVIDER: E-GEOD-8978 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:TSLC1/IGSF4, an immunoglobulin superfamily molecule, is predominantly expressed in the brain, lung, and testis and plays important roles in epithelial cell adhesion, cancer invasion, and synapse formation. We generated Tslc1/Igsf4-deficient mice by disrupting exon 1 of the gene and found that Tslc1-/- mice were born with the expected Mendelian ratio but Tslc1-/- male mice were infertile. In adult Tslc1-/- mice of 11-week of age, the weight of the testis was 90% of that of the Tslc1+/+ mice, and the number of sperm in the semen was approximately 0.01% of it. Histological analysis revealed that the round spermatids and the pachytene spermatocytes failed to attach to the Sertoli cells in the seminiferous tubules and sloughed off into the lumen with apoptosis in the Tslc1-/- mice. On the other hand, the spermatogonia and the Sertoli cells, as well as the interstitial cells, were essentially unaffected. In the Tslc1+/+ mice, TSLC1/IGSF4 expression was observed in the spermatogenic cells from the intermediate spermatogonia to the early pachyten spermatocytes and from step-7 or later spermatids. These findings suggest that TSLC1/IGSF4 expression is indispensable for the adhesion of spermatocytes and spermatids to Sertoli cells and for their normal differentiation to mature spermatozoa. Experiment Overall Design: The protocol used for the sample preparation and microarray processing is available from Affymetrix (Santa Clara, CA, USA). Briefly, 3 ïg purified RNA, extracted from the testis from Tslc1+/+ and Tslc1-/- mice of 16-week-old, was reverse-transcribed with Superscript II reverse transcriptase (Invitrogen) using the primer T7-dT24 containing a T7 RNA polymerase promoter. After a second strand of cDNA was synthesized using RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase, in vitro transcription was carried out on the cDNA to produce biotin-labeled cRNA with a MEGAscript High Yield Transcription Kit (Ambion, Austin, TX, USA) as recommended by the manufacturer. After the cRNA was linearly amplified with T7 polymerase, the biotinylated cRNA was cleaned with an RNeasy Mini Column (Qiagen), fragmented to 50 to 200 nucleotides, and then hybridized to Mouse Genome U74A ver.2 arrays (Affymetrix). The stained microarray was scanned with a GeneArray Scanner (Affymetrix), and the signal was calculated with Affymetrix software, Microarray Suite 5.0. All of the data were scaled with the global scaling method to adjust the target intensity to 1,000.

Project description:Sry is sufficient to induce testis formation and subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth due to germ cell autonomous defects. Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here we demonstrated that the testicular somatic environment of XX/Sry males is defective in the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, entering meiosis and differentiating into the round spermatid stage. XY donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, thereby preventing further progress beyond the elongated spermatid stage. In contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed missing expression of several Y-linked genes and alterations in the expression profile of genes associated with spermatogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, supporting our hypothesis that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice. Experiment Overall Design: Whole testes and seminiferous tubules of XX/Sry and W/Wv males were used for microarray expression analysis using the Affymetrix GeneChip system (Affymetrix, CA). In order to isolate the seminiferous tubules, the tunica was carefully removed from the testes which were then incubated in the medium with 5 mg/ml collagnease at 37oC for 40 min. The remaining seminiferous tubules were washed several times with PBS using a 70-Âºm cell strainer to remove interstitial cells. After total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD), double-stranded cDNA and biotin-labeled cRNA were synthesized using One-Cycle cDNA Synthesis and IVT Labeling kits (Affymetrix, CA), respectively. Twenty micrograms of fragmented biotin-labeled cRNA was hybridized to the Affymetrix Mouse Expression Array MOE 430A for 16 hr at 45oC. The chips were washed, stained, and then scanned with the GeneArray Scanner (Hewlett Packard, CA) in accordance with the manufacturer's standard protocols. Finally, the microarray data were analyzed using Microarray Suite ver. 5.0 (Affymetrix). Differential expression was defined as a difference of 2-fold or more in both whole testis and seminiferous tubule samples between two recipient males. Mouse 430A Affymetrix Genome Array IDs were used to query the NetAffx data mining tool for gene annotations.

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Transcription profiling of rat seminiferous tubules, purifed germ cells and sertoli cells

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