Characteristics of Circular RNA Expression Profiles of Porcine Granulosa Cells in Healthy and Atretic Antral Follicles.

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions-circ_CBFA2T2 and circ_KIF16B-were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes-the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

Lithium causes G2 arrest of renal principal cells.

Vasopressin-regulated expression and insertion of aquaporin-2 channels in the luminal membrane of renal principal cells is essential for urine concentration. Lithium affects urine concentrating ability, and approximately 20% of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder characterized by polyuria and polydipsia. Lithium-induced NDI is caused by aquaporin-2 downregulation and a reduced ratio of principal/intercalated cells, yet lithium induces principal cell proliferation. Here, we studied how lithium-induced principal cell proliferation can lead to a reduced ratio of principal/intercalated cells using two-dimensional and three-dimensional polarized cultures of mouse renal collecting duct cells and mice treated with clinically relevant lithium concentrations. DNA image cytometry and immunoblotting revealed that lithium initiated proliferation of mouse renal collecting duct cells but also increased the G2/S ratio, indicating G2/M phase arrest. In mice, treatment with lithium for 4, 7, 10, or 13 days led to features of NDI and an increase in the number of principal cells expressing PCNA in the papilla. Remarkably, 30%-40% of the PCNA-positive principal cells also expressed pHistone-H3, a late G2/M phase marker detected in approximately 20% of cells during undisturbed proliferation. Our data reveal that lithium treatment initiates proliferation of renal principal cells but that a significant percentage of these cells are arrested in the late G2 phase, which explains the reduced principal/intercalated cell ratio and may identify the molecular pathway underlying the development of lithium-induced renal fibrosis.