Follicle inhibition at the primordial stage without increasing apoptosis, with a combination of everolimus, verapamil
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ORIGINAL ARTICLE
Follicle inhibition at the primordial stage without increasing apoptosis, with a combination of everolimus, verapamil Michail Pargianas1 · Ioannis Kosmas2 · Kyriaki Papageorgiou4,5 · Chrysoula Kitsou7 · Alexandra Papoudou‑Bai3 · Anna Batistatou3 · Sofia Markoula7 · Styliani Salta7 · Alexandros Dalkalitsis7 · Stratis Kolibianakis6 · Basil C. Tarlatzis6 · Ioannis Georgiou7 · Theologos M. Michaelidis4,5 Received: 23 December 2019 / Accepted: 12 October 2020 © Springer Nature B.V. 2020
Abstract The aim of the present study was to test whether inhibition of ovarian primordial follicles and subsequent activation can be achieved by transient mTOR inhibition. In this preclinical investigation, forty-five female immature Wistar rats were randomized in 5 groups. The control group received subcutaneous saline injections. The other groups received Everolimus, Everolimus plus Verapamil, Everolimus plus Fisetin, and Fisetin alone. Primary and secondary outcomes were measured in the left ovary after a treatment period of 8 weeks. Ten days later, animals received 35 IU FSH for 4 days and 35 IU of hCG on the 5th day. The same parameters were examined in the right ovary. AMH, estradiol, and progesterone levels were assessed at the end of both interventions. Significantly, more primordial and less atretic follicles were observed in the Everolimus plus Verapamil group. AMH and progesterone levels were substantially lower in the Everolimus group. Interestingly, after ovarian stimulation higher levels of AMH and progesterone were observed in the Everolimus plus Verapamil group. Immunoblot analysis of ovarian extracts revealed that the administration of Everolimus led to a significant reduction in the mTORC1-mediated phosphorylation of the 70-kDa ribosomal protein S6 kinase 1. This decrease was reversed in the presence of FSH after stopping drug administration. The expression of the anti-apoptotic molecule Bcl2 as well as of LC3-II and ATG12 was increased after removal of the Everolimus plus Verapamil combination, indicating reduced apoptosis and increased autophagy, whereas the levels of the proliferation marker PCNA in the granulosa cells were elevated, consistent with initiation of follicular growth.Thus, the combination of Everolimus plus Verapamil is capable of increasing the number of competent primordial follicles while reducing atresia. Keywords Primordial follicles · mTORC1 · mTORC2 · Everolimus plus verapamil · Apoptosis · Autophagy
Introduction Ovarian follicle activation and atresia results in follicular loss and takes place at menopause, premature ovarian failure, and after cancer treatment. Depletion of follicles is a common characteristic of these three conditions. The mammalian target of rapamycin (mTOR) is an essential regulator of cell growth. The activity of mTOR is negatively regulated by the heterodimeric TSC1/TSC2 complex Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11033-020-05917-2) contains supplementary material, which is a
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