Mammalian target of rapamycin/eukaryotic initiation factor 4F pathway regulates follicle growth and development of theca cells in mice.

The aim of the present study was to clarify the roles of the mammalian target of rapamycin (mTOR) signalling pathway in follicular growth and development of thecal cells. Using in vivo-grown and in vitro-cultured ovaries, histological changes were evaluated using haematoxylin and eosin (HE) staining. Differentially expressed genes (DEGs) from 0 day post partum (d.p.p.) to 8 d.p.p. ovaries were screened by microarray and verified by quantitative real-time polymerase chain reaction. Forty-two DEGs related to cell proliferation and differentiation were screened out, with most DEGs being related to the to mTOR signalling pathway. Then, 3 d.p.p. ovaries were retrieved and used to verify the role of mTOR signalling in follicle and thecal cell development using its activators (Ras homologue enriched in brain (Rheb) and GTP) and inhibitor (rapamycin). The development of follicles and thecal cells was significantly impaired in ovaries cultured in vitro Day 3 to Day 8. In in vitro-cultured ovaries, Rheb and GTP (is 100ngmL-1 Rheb and 500ngmL-1 GTP for 48h) significantly increased follicle diameter, the percentage of primary and secondary follicles and the umber of thecal cells, and upregulated expression of mTOR, phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), eukaryotic initiation factor (eIF) 4F and cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1). Rapamycin (10nM rapamycin for 24h) had opposite effects to those of Rheb and GTP, and partly abrogated (significant) the effects of Rheb and GTP when added to the culture in combination with these drugs. Thus, mTOR signalling plays an important role in follicle growth and thecal cell development.

The Anti-Apoptotic Role of Berberine in Preimplantation Embryo In Vitro Development through Regulation of miRNA-21.

Traditional Chinese medicinal herbs containing berberine have been historically used to prevent miscarriage. Here, we investigated whether the anti-apoptotic effects of berberine on pre-implantation embryonic development are regulated by miRNA-21. Mouse pronuclear embryos were cultured in medium with or without berberine, and some were then microinjected with a miRNA-21 inhibitor. The in vitro developmental rates of 2- and 4-cell embryos and blastocysts, blastocyst cell numbers, apoptotic rates, and apoptotic cell numbers were measured in each group. Furthermore, we examined the transcription levels of miRNA-21 and its target genes (caspase-3, PTEN, and Bcl-2) and their translation levels. Comparisons were made with in vivo-developed and untreated embryos. We found that berberine significantly increased the developmental rates and cell numbers of mouse blastocysts and decreased apoptotic cell rates in vitro. Berberine also significantly increased miRNA-21 and Bcl-2 transcription levels and significantly decreased caspase-3 and PTEN transcription levels. In embryos treated with a miRNA-21 inhibitor, the results followed the opposite trend; PTEN and caspase-3 transcription levels increased significantly, while the transcription level of Bcl-2 decreased significantly. Additionally, berberine treatment significantly increased the Bcl-2 protein level and significantly decreased the caspase-3 and PTEN protein levels in blastocysts, but there were no significant differences observed in the levels of these proteins in 2- and 4-cell embryos. This study revealed that miRNA-21 is important for pre-implantation embryonic development, especially blastocyst development in vitro. Berberine elevates miRNA-21 expression, decreases PTEN and caspase-3 levels, increases Bcl-2 levels, and exerts anti-apoptotic and pro-growth effects.

The regulatory role of icariin on apoptosis in mouse preimplantation embryos with reduced microRNA-21.

We constructed a model of apoptosis in mouse preimplantation embryos and investigated the effect of the flavonol icariin on embryonic development in vitro in embryos with reduced microRNA-21 (miR-21). The model was generated by microinjecting an miR-21 inhibitor into the cytoplasm of mouse pronuclear embryos, which were cultured in vitro using modified CZB (mCZB) basal medium (model group), or using mCZB medium with 0.6 µg/mL icariin as an experimental group (model-Ica). These were compared with embryos collected in vivo (vivo group) or not microinjected (control group). Developmental rates in vitro of two- and four-cell embryos and blastocysts were observed, and Hoechst 33342 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were used to count blastocyst cell numbers and apoptotic cell numbers and percentages. The transcriptional levels of miR-21, the apoptotic genes caspase 3 and phosphatase and tensin homolog deleted on chromosome ten (PTEN), and the antiapoptotic gene Bcl-2 were detected by quantitative polymerase chain reaction (qPCR). Western immunoblotting was used to detect the protein levels of caspase 3, PTEN, and Bcl-2. Compared with the model group, icariin treatment significantly improved blastocyst development in vitro (58.43 ± 7.53% vs. 37.85 ± 6.47%; P < 0.01), whereas it was not significantly different to the control group (60.34 ± 9.86%). Icariin treatment significantly increased the blastocyst cell numbers (47.02 ± 4.93 vs. 37.70 ± 5.80; P < 0.01), and reduced the rates of apoptosis (5.51 ± 2.35% vs. 10.11 ± 4.21%; P < 0.01), whereas the blastocyst cell numbers and apoptotic rates revealed no significant differences between the vivo (46.06 ± 6.50, 5.95 ± 2.56%) and control groups (45.77 ± 4.09, 6.18 ± 2.41%). Icariin treatment significantly improved miR-21 expression in all embryo stages, reduced the transcriptional levels of caspase 3 and PTEN, and increased the levels of Bcl-2. The protein expression levels of caspase 3 and PTEN were decreased in blastocysts and the level of Bcl-2 was increased (P < 0.01). These had no significant differences with the vivo and control groups, and the protein levels revealed no significant differences between two- and four-cell embryos. Thus, miR-21 was necessary for preimplantation embryonic development, and embryo quality was closely associated with the apoptosis-related protein expression levels regulated by miR-21. Icariin upregulated miR-21 expression and reduced apoptosis in embryos with reduced miR-21. It also improved embryonic developmental quality in vitro, indicating an important regulatory role for miR-21 in blastocyst development in vitro.