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Alterations in alternative RNA splicing in Srsf3-flox/flox hepatocytes with AAV-CRE mediated deletion of SRSF3 vs hepatocytes with AAV-GFP

ABSTRACT: Hepatocytes were isolated and infected in vitro, then RNA extracted and subjected to RNAseq

ORGANISM(S): Mus musculus

PROVIDER: GSE133398 | GEO | 2020/04/13

REPOSITORIES: GEO

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Alterations in alternative RNA splicing in Srsf3-flox/flox hepatocytes with AAV-CRE mediated deletion of SRSF3 vs hepatocytes with AAV-GFP

Project description:Alterations in alternative RNA splicing in Srsf3-flox/flox hepatocytes with AAV-CRE mediated deletion of SRSF3 vs hepatocytes with AAV-GFP

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The acceleration of reproductive aging in Nrg1<sup>flox/flox</sup> ;Cyp19-Cre female mice.

Project description:Irregular menstrual cycles, reduced responses to exogenous hormonal treatments, and altered endocrine profiles (high FSH/high LH/low AMH) are observed in women with increasing age before menopause. In this study, because the granulosa cell-specific Nrg1 knockout mice (gcNrg1KO) presented ovarian and endocrine phenotypes similar to older women, we sought to understand the mechanisms of ovarian aging and to develop a new strategy for improving fertility in older women prior to menopause. In the ovary of 6-month-old gcNrg1KO mice, follicular development was blocked in bilayer secondary follicles and heterogeneous cells accumulated in ovarian stroma. The heterogeneous cells in ovarian stroma were distinguished as two different types: (i) the LH receptor-positive endocrine cells and (ii) actin-rich fibrotic cells expressing collagen. Both the endocrine and fibrotic cells disappeared following long-term treatment with a GnRH antagonist, indicating that the high levels of serum LH induced the survival of both cell types and the abnormal endocrine profile to reduce fertility. Moreover, follicular development to the antral stages was observed with reduced LH and the disappearance of the abnormal stromal cells. Mice treated with the GnRH antagonist regained normal, recurrent estrous cycles and continuously delivered pups for at least for 3 months. We conclude that endocrine and matrix alternations occur within the ovarian stroma with increasing age and that abolishing these alternations resets the cyclical release of LH. Thus, GnRH antagonist treatments might provide a new, noninvasive strategy for improving fertility in a subset of aging women before menopause.

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Differential CRE Expression in Lhrh-cre and GnRH-cre Alleles and the Impact on Fertility in Otx2-Flox Mice.

Project description:There is an increasing trend in studies utilizing cell-specific deletion of genes through conditional gene deletion by CRE recombination. Despite numerous advantages, this strategy also has limitations such as ectopic CRE-expression and germline recombination. Two commonly used gonadotropin-releasing hormone (Gnrh)-driven CRE-expressing mice both target GnRH neurons. However, a direct comparison of the cells targeted and their phenotypic outcome have not yet been presented. To compare where recombination takes place, we crossed the Gnrh-cre and Lhrh-cre lines with the Rosa26-LacZ reporter mouse. Lhrh-cre allowed recombination of the Rosa26-LacZ gene in ∼700 cells, which is comparable to the GnRH neuronal population. Surprisingly, there were > 20 times more LacZ expressing cells in the adult Gnrh-cre:Rosa26-LacZ than the Lhrh-cre:Rosa26-LacZ brain. The greatest differences in targeting of the Gnrh-cre and Lhrh-cre lines were found in the septum, the suprachiasmatic nucleus, and the septohypothalamic area. This difference in cells targeted was present from embryonic day 12. A prior study using the Gnrh-cre to delete the transcription factor Otx2 found fewer GnRH neurons, leading to male and female subfertility. To recapitulate this study, we performed a fertility assay in Otx2:Lhrh-cre mice. We confirmed the requirement for Otx2 in GnRH neuron development, fertility and correct gonadotropin hormone release in Otx2:Lhrh-cre males, but the subfertility was more modest than in Otx2:Gnrh-cre and absent in female Otx2:Lhrh-cre. This suggests that ectopic expression of Gnrh-cre contributes to the reproductive phenotype observed. Finally, the Cre alleles caused germline recombination of the flox allele when transmitted from either parent, generating embryonic lethal knock-out offspring, producing smaller live litters.

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Project description:C57BL/6J male mice were fed a 60% HFD for 10 weeks, then hepatocytes were isolated, RNA extracted and subjected to RNAseq

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Gene expression profile of Dnmt1 flox/flox, Dnmt3a flox/flox, Dnmt3b flox/flox, cre negative (Wild type) and Dnmt1 flox/flox, Dnmt3a flox/flox, Dnmt3b flox/flox, Rx-cre (Triple mutant) murine retina transcriptomes

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