Sertoli cell-specific gene expression in conditionally immortalized cell lines.
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Biomedical subjects
B Kascheike
Publications and source records attributed to B Kascheike.
Alterations in the chromatin structure of the distal promoter region of the bovine oxytocin gene correlate with ovarian expression.
The mechanisms regulating the expression of the neuropeptide hormone gene oxytocin have not yet been elucidated in detail. The binding of the orphan receptor Ad4BP, the bovine homolog of steroidogenic factor-1 (SF-1), which is correlated with in vivo oxytocin transcription in the luteinizing granulosa cells of the bovine corpus luteum, is not sufficient to explain the transcriptional up-regulation in these cells. Therefore, we started experiments to identify other regions of the oxytocin locus that are involved in gene activation. The study presented here is the very first investigation of DNA methylation and chromatin structure in the distal promoter region of the bovine oxytocin gene. We show that this region is tissue-specifically hypomethylated in bovine granulosa cells. Upon stimulation of the cells with the adenylate cyclase-activator forskolin, a DNase I-hypersensitive site is induced in the distal promoter region. Additionally, we find binding of a monomeric nuclear orphan receptor directly within the region of inducible DNase I sensitivity; this factor is not identical to Ad4BP/SF-1. This study identifies a region in the bovine oxytocin distal promoter where tissue-specific changes in DNA methylation and chromatin structure correlate with high induction of oxytocin gene transcription, and suggests that the binding of transcription factors to this region may be important for the up-regulation of oxytocin gene expression.
Adenylyl Cyclases↗
An autocrine progesterone positive feedback loop mediates oxytocin upregulation in bovine granulosa cells during luteinization.
During luteinization of bovine granulosa cells in vitro in the presence of insulin, or insulin plus forskolin, there is a massive upregulation not only of progesterone production, but also of the gene for the peptide hormone oxytocin, with secretion of the peptide into the medium. By using the progesterone receptor antagonists, RU486 or onapristone, we have shown that this upregulation of the oxytocin gene is mediated by an autocrine loop involving endogenous progesterone and the progesterone receptor in the granulosa cells. The inhibition of oxytocin gene expression by the anti-gestagens can be reversed by medroxyprogesterone acetate but not by dexamethasone, showing that the effect is due to the endogenous progesterone. Since oxytocin also appears to be involved in a positive feedback loop regulating steroid output, these results show that endogenous progesterone itself is a key feedback element in the cascade of events termed luteinization, and that possibly anti-gestagens can influence ovarian function in vivo by directly disrupting this process.
Animals↗
Sertoli cell lines established from H-2Kb-tsA58 transgenic mice differentially regulate the expression of cell-specific genes.
Sertoli cell lines have been established from H-2K(b)-tsA58 transgenic mice carrying an inducible temperature-sensitive SV40 T antigen in their germline. All cell lines tested for expression of Sertoli cell products by reverse transcription-polymerase chain reaction were shown to express mRNAs for alpha-inhibin, Steel factor, SGP-2, and transferrin as well as for androgen receptor and the orphan nuclear receptor SF-1. Selected cell lines were shown by immunocytochemistry to express the established Sertoli cell-specific pattern of cytoskeletal markers. The FSH receptor gene was also expressed, though downregulated by comparison with in vivo levels of expression. In some lines low expression of the luteinizing hormone receptor gene could also be detected. The gene for the transcription factor GATA-1, which is expressed specifically in Sertoli cells, was expressed only in a subset of the cell lines. Quantitative analysis of SGP-2 transcript levels by ribonuclease protection assays showed an increase at the nonpermissive temperature, whereas using a similar assay, Steel factor mRNA was shown to be expressed in amounts comparable to the in vivo situation only in two cell lines during permanent growth. In summary, cell lines that exhibit distinct Sertoli cell characteristics have been established, which may resemble different stages of phenotypic development.
Animals↗
Regulation of oxytocin expression in the bovine corpus luteum. Orphan receptors and the oxytocin promoter.
The bovine oxytocin gene is massively up-regulated during the early development of the corpus luteum. Oxytocin transcription is induced in a highly synchronous fashion in the granulosa cells of the dominant follicle at the time of ovulation. The possibility to isolate large numbers of differentiating granulosa-luteal cells from exactly defined stages of development allows the investigation of the factors controlling oxytocin expression in vivo by molecular and cell biology methods. Using primary cultures of bovine granulosa cells the synergistic activation of oxytocin transcription by the cAMP pathway and stimulation of IGF-I or insulin receptors could be established. Analysis of transcription factors isolated from the nuclei of bovine granulosa cells and corpus luteum led to the identification of the tissue-specific orphan receptor SF-1 binding to the promoter of the actively transcribed oxytocin gene. The luteinizing bovine granulosa cells provide the only easily accessible experimental system established so far in which the endogenous oxytocin gene is expressed. Although the link between increased cAMP level and receptor tyrosine kinase activation on the one hand and the induction of oxytocin transcription on the other has not been established yet, these experiments constitute one of the few direct approaches to investigate the complexity of events that regulate oxytocin expression in vivo.
Animals↗