Etablierung eines in vitro-Systems zur Analyse der Differenzierung und Apoptose immaturer testikulärer Keimzellen

Diese Arbeit beschreibt die Etablierung eines in vitro-Systems für primäre männliche Keimzellen, das die Analyse von Differenzierung und Apoptose der Keimzellen ermöglicht. Es wurde Hodengewebe 20 Tage alter Wistar Ratten verwendet. Mittels mechanischer Dissoziation wurden daraus testikuläre Zellen und Gewebefragmente definierter Größe gewonnen. Die Differenzierung der Keimzellen wurde in verschiedenen Kultursystemen untersucht: der Organkultur, Kultur von Tubulusfragmenten und Kultur vereinzelter Zellen. Im System der Tubulusfragmente differenzierten sich pachytäne Spermatocyten zu runden Spermatiden. Dieser Entwicklungsschritt vollzog sich unter dem Einfluss von Testosteron und Follikel stimulierendem Hormon innerhalb von zwei Wochen. Der Differenzierungsgrad der Keimzellen wurde anhand der Expression stadienspezifischer Marker, wie Transition Protein 1, Transition Protein 2 und Protamin 1, bestimmt. Die Keimzellapoptose wurde nach Stimulation durch die Transforming Growth Factors-beta (beta-1 und beta-2) anhand der Aktivierung der Caspase-3 gemessen. Es gab Hinweise dafür, dass die TGF-betas die Keimzellapoptose induzieren, jedoch konnte dies nicht definitiv gezeigt werden

Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells

<p>Abstract</p> <p>Background</p> <p>The transforming growth factors (TGF)-β, TGF-β1, TGF-β2 and TGF-β3, and their receptors [TβRI, TβRII, TβRIII (betaglycan)] elicit pleiotropic functions in the prostate. Although expression of the ligands and receptors have been investigated, the splice variants have never been analyzed. We therefore have analyzed all ligands, the receptors and the splice variants TβRIB, TβRIIB and TGF-β2B in human prostatic cells.</p> <p>Results</p> <p>Interestingly, a novel human receptor transcript TβRIIC was identified, encoding additional 36 amino acids in the extracellular domain, that is expressed in the prostatic cancer cells PC-3, stromal hPCPs, and other human tissues. Furthermore, the receptor variant TβRIB with four additional amino acids was identified also in human. Expression of the variant TβRIIB was found in all prostate cell lines studied with a preferential localization in epithelial cells in some human prostatic glands. Similarly, we observed localization of TβRIIC and TGF-β2B mainly in the epithelial cells with a preferential localization of TGF-β2B in the apical cell compartment. Whereas in the androgen-independent hPCPs and PC-3 cells all TGF-β ligands and receptors are expressed, the androgen-dependent LNCaP cells failed to express all ligands. Additionally, stimulation of PC-3 cells with TGF-β2 resulted in a significant and strong increase in secretion of plasminogen activator inhibitor-1 (PAI-1) with a major participation of TβRII.</p> <p>Conclusion</p> <p>In general, expression of the splice variants was more heterogeneous in contrast to the well-known isoforms. The identification of the splice variants TβRIB and the novel isoform TβRIIC in man clearly contributes to the growing complexity of the TGF-β family.</p

Etablierung eines in vitro-Systems zur Analyse der Differenzierung und Apoptose immaturer testikulärer Keimzellen

Diese Arbeit beschreibt die Etablierung eines in vitro-Systems für primäre männliche Keimzellen, das die Analyse von Differenzierung und Apoptose der Keimzellen ermöglicht. Es wurde Hodengewebe 20 Tage alter Wistar Ratten verwendet. Mittels mechanischer Dissoziation wurden daraus testikuläre Zellen und Gewebefragmente definierter Größe gewonnen. Die Differenzierung der Keimzellen wurde in verschiedenen Kultursystemen untersucht: der Organkultur, Kultur von Tubulusfragmenten und Kultur vereinzelter Zellen. Im System der Tubulusfragmente differenzierten sich pachytäne Spermatocyten zu runden Spermatiden. Dieser Entwicklungsschritt vollzog sich unter dem Einfluss von Testosteron und Follikel stimulierendem Hormon innerhalb von zwei Wochen. Der Differenzierungsgrad der Keimzellen wurde anhand der Expression stadienspezifischer Marker, wie Transition Protein 1, Transition Protein 2 und Protamin 1, bestimmt. Die Keimzellapoptose wurde nach Stimulation durch die Transforming Growth Factors-beta (beta-1 und beta-2) anhand der Aktivierung der Caspase-3 gemessen. Es gab Hinweise dafür, dass die TGF-betas die Keimzellapoptose induzieren, jedoch konnte dies nicht definitiv gezeigt werden

() Comparison of the exon structure of the human TβRII mRNA with the truncated sequence provided by Yang et al

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> [35]. Lines depict the 5'-UTR, 3'-UTR and ESTs with additional exons. () Expression pattern of both transcript variants of the TβRII gene in human prostatic cells (upper panel, 5-HTBR2B/3-HTBR2B). Expression of the novel splice variant TβRIIC in human prostatic cells (lower panel, nested PCR first round 5-HTBR2E3/3-HTBR2E4, second round 5-HTBR2Z/3-HTBR2E4) and normal human tissues (5-HTBR2E3/3-HTBR2CD) is shown. Additionally, GAPDH expression is also provided. () Fluorescence detection of TβRIICΔ4 (5-HTBR2E3/3-HTBR2CD, arrows) and TβRIIC is demonstrated. Caco, Caco-2; ctrl, control; g, gland; m, muscle; mu, mucosa; s, small; ma, marrow

Schematic drawing of the TGF-β2 protein (LAP, latency-associated peptide) with the alternatively spliced exon 2B

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> Nucleotide and amino acid sequence of exon 2B (underlined capital letters) of the variant TGF-β2B are shown. Additionally, the partial sequence of TGF-β2 without exon 2B is shown. The sequence of TGF-β2 was not available for oryctolagus cuniculus. Underlined amino acids indicate amino acid exchange at the splice site junction due to the alternative splicing. Bold letters mark the amino acid and nucleotide exchanges with respect to the human sequence. Arrows indicate the exon boundaries. The accession numbers are also given. (hs, homo sapiens; pt, pan troglodytes; mmu, macaca mulatta; cf, canis familiaris; oc, oryctolagus cuniculus; mm, mus musculus; rn, rattus norvegicus)

() Schematic drawing of the TβRII protein (EC, extracellular domain; TM, transmembrane domain; Kinase, Ser/Thr kinase domain) with the two alternatively spliced exons 2B and 4B

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> () Nucleotide sequence of the cDNA and deduced amino acid sequence of exon 2B (underlined capital letters) and splice site junctions (lower case letters) of the variant TβRIIB are shown. () Additionally, the partial nucleotide and amino acid sequence of TβRII without exon 2B is shown. Underlined amino acids indicate amino acid exchange at the splice site junction due to the alternative splicing. Bold letters mark the amino acid and nucleotide exchanges with respect to the human sequence. Arrows indicate the exon boundaries. (hs, homo sapiens; pt, pan troglodytes; mmu, macaca mulatta; mm, mus musculus)

() Comparison of the exon structure of the human TGF-β2 mRNA with the ESTs BP214137 and BF752669 containing the additional alternative exon 2B

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of TGF-betas and their high-affinity receptors TβRI, TβRII and TβRIII (betaglycan) reveal new variants in human prostatic cells"</p><p>http://www.biomedcentral.com/1471-2164/8/318</p><p>BMC Genomics 2007;8():318-318.</p><p>Published online 11 Sep 2007</p><p>PMCID:PMC2075524.</p><p></p> Lines depict the 5'-UTR, 3'-UTR and introns. () Expression of both transcript variants (upper panel, 5-TGFB2E1B/3-TGFB2E2B) and expression of the splice variant TGF-β2B (lower panel; 5-HTB2CP/3-HTB2CP) is shown. () Exon structure of the human TGF-β3 mRNA. Lines depict the 5'-UTR and 3'-UTR. () Expression pattern of the TGF-β3 gene in human prostatic cells (left panel, 5-TGFB3E1/3-TGFB3E2). Additionally, GAPDH expression of all cell lines studied is shown. Ctrl, control