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:Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genomewide investigations of transcriptome complexities in major mammalian organs and their underlying cellular sources, transcriptional mechanisms, and functional relevance have been scarce. Here we first show, using extensive RNAseq data, that transcription of both functional and nonfunctional genomic elements is substantially more widespread in the testis than in other organs across representative mammals. By scrutinizing the transcriptomes of all main testicular cell types in the mouse, we then reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein coding genes and long noncoding RNA genes but also poorly conserved intergenic sequences, suggesting that much of it is not of immediate functional relevance. Rather, our analyses of genomewide epigenetic data show that this prevalent transcription, which apparently promoted the birth of new genes during evolution, results from a highly permissive chromatin state during and after meiosis that may ultimately facilitate the replacement of histones by protamines during late spermatogenesis. To study the cellular source and mechanisms of high transcriptome complexity in the mammalian testis, we generated strand-specific deep coverage RNASeq data for purified sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa as well as for brain, liver and the whole testis from the mouse. We prepared 8 sequencing libraries for the polyadenylated RNA fraction of each sample and sequenced each library in 3 lanes of the Illumina Genome Analyser IIx platform, yielding a total of >60 millions strand-specific reads of 76 base pairs per sample. In addition, we generated ChIP-Seq data for the H3K4me2 modification as well as RRBS data for brain, liver, testis, spermatocytes and spermatids. RNA-seq, ChIP-seq and RRBS data were generated from the same individual or pool of individuals, in the case of purified cells. RNASeq data for purified sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa

Project description: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%

Project description:Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome‐wide investigations of transcriptome complexities in major mammalian organs and their underlying cellular sources, transcriptional mechanisms, and functional relevance have been scarce. Here we first show, using extensive RNA‐seq data, that transcription of both functional and nonfunctional genomic elements is substantially more widespread in the testis than in other organs across representative mammals. By scrutinizing the transcriptomes of all main testicular cell types in the mouse, we then reveal that meiotic spermatocytes and especially post‐meiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein‐coding genes and long noncoding RNA genes but also poorly conserved intergenic sequences, suggesting that much of it is not of immediate functional relevance. Rather, our analyses of genome‐wide epigenetic data show that this prevalent transcription, which apparently promoted the birth of new genes during evolution, results from a highly permissive chromatin state during and after meiosis that may ultimately facilitate the replacement of histones by protamines during late spermatogenesis. To study the cellular source and mechanisms of high transcriptome complexity in the mammalian testis, we generated strand-specific deep coverage RNA‐Seq data for purified sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa as well as for brain, liver and the whole testis from the mouse. We prepared 8 sequencing libraries for the polyadenylated RNA fraction of each sample and sequenced each library in 3 lanes of the Illumina Genome Analyser IIx platform, yielding a total of >60 millions strand-specific reads of 76 base pairs per sample. In addition, we generated ChIP-Seq data for the H3K4me2 modification as well as RRBS data for brain, liver, testis, spermatocytes and spermatids. RNA-seq, ChIP-seq and RRBS data were generated from the same individual or pool of individuals, in the case of purified cells. ChIP-Seq data for the H3K4me2 modification as well as RRBS data for brain, liver, testis, spermatocytes and spermatids

Project description:Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome‐wide investigations of transcriptome complexities in major mammalian organs and their underlying cellular sources, transcriptional mechanisms, and functional relevance have been scarce. Here we first show, using extensive RNA‐seq data, that transcription of both functional and nonfunctional genomic elements is substantially more widespread in the testis than in other organs across representative mammals. By scrutinizing the transcriptomes of all main testicular cell types in the mouse, we then reveal that meiotic spermatocytes and especially post‐meiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein‐coding genes and long noncoding RNA genes but also poorly conserved intergenic sequences, suggesting that much of it is not of immediate functional relevance. Rather, our analyses of genome‐wide epigenetic data show that this prevalent transcription, which apparently promoted the birth of new genes during evolution, results from a highly permissive chromatin state during and after meiosis that may ultimately facilitate the replacement of histones by protamines during late spermatogenesis. To study the cellular source and mechanisms of high transcriptome complexity in the mammalian testis, we generated strand-specific deep coverage RNA‐Seq data for purified sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa as well as for brain, liver and the whole testis from the mouse. We prepared 8 sequencing libraries for the polyadenylated RNA fraction of each sample and sequenced each library in 3 lanes of the Illumina Genome Analyser IIx platform, yielding a total of >60 millions strand-specific reads of 76 base pairs per sample. In addition, we generated ChIP-Seq data for the H3K4me2 modification as well as RRBS data for brain, liver, testis, spermatocytes and spermatids. RNA-seq, ChIP-seq and RRBS data were generated from the same individual or pool of individuals, in the case of purified cells. RNA-Seq of brain, liver and the whole testis

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Expression profile of the testis from Tslc1-/- mice.

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