Cyclin-dependent kinase 6 (CDK6) as a potent regulator of the ovarian primordial-to-primary follicle transition

Abstract

Introduction: Ovarian follicle development requires tight coordination between several factors to initiate folliculogenesis to generate a mature and fertile egg. Studies have shown that cell cycle factors might contribute to follicle development, hover specific knowledge on individual CDKs and follicle activation has not been investigated. Among cell cycle regulators, CDK6 is a key player through binding to cyclin D resulting DNA synthesis and genome duplication. Interestingly, the CDK6 gene is differentially expressed in oocytes and granulosa cells from human primordial and primary follicles, which suggest a potential role of CDK6 in the primordial-to-primary transition. In this study, we investigated the potential regulatory role of CDK6 in progression of primordial to primary follicle transition using BSJ-03-123 (BSJ), a CDK6-specific degrader.Methods: In mouse ovarian in vitro culture, BSJ reduced the activation of primordial follicles, and reduced follicle development. As a next step, we examined the egg maturation read-out and found that BSJ-treated follicles matured to competent MII eggs with resumption of first meiosis, comparable with the control group.Results: Noteworthy, it appears that inhibition of CDK6 did increase number of apotoptic cells, articular in the granulosa cells, but had no impact on ROS level of cultured ovaries compared to control group, indicating that the cells were not stressed. Oocyte quality thus appeared safe.Discussion: The results of this study indicate that CDK6 plays a role in the primordial-to-primary transition, suggesting that cell cycle regulation is an essential part of ovarian follicle development.

Dorsomorphin inhibits AMPK, upregulates Wnt and Foxo genes and promotes the activation of dormant follicles

“…Mice were sacrificed by cervical dislocation, and ovaries were removed and submitted to ovary in vitro culture 48 , 51 , 52 . Then, the ovaries were excised from the surrounding bursa and washed with α-minimal essential medium (α-MEM, Gibco, Scotland, Paisley, UK) supplemented with 10% fetal bovine serum (FBS, Gibco, Scotland, Paisley, UK).…”

“…Ovarian tissue pieces from mice (n = 3–16/group) and humans (n = 2 patients) were harvested at different time points and fixed in paraformaldehyde (PFA; 4%, Merck) overnight at 4 °C. Subsequently, the tissues were dehydrated in ascending concentrations of ethanol (70–99.9%), cleared in xylene, and finally embedded in paraffin wax 48 , 51 , 52 . This tissue was cut into five-micron serial sections and stained with haematoxylin and eosin.…”

“…For cytoplasmic and nuclear protein extraction, in vitro cultured ovaries were lysed by following the protocol for the CellLytic™ NuCLEAR™ Extraction Kit (NXTRACT, Sigma, St Louis, MO, USA) (n = 10/group). The protein concentration was quantified using BCA quantification and submitted to Western blotting 48 , 51 , 52 . For all assays, 20 µg of protein was separated by 4–12% SDS‒PAGE and electroblotted onto polyvinylidene difluoride membranes (0.45 µm pore size, Merck Millipore, Burlington, Massachusetts, USA).…”

“…For immunofluorescence assays, sectioned in vitro cultured ovaries (5 µm) (n = 3) were subjected to dehydration and deparaffinization 48 , 51 , 52 . Antigen retrieval using heat and citrate buffer (0.01 M) was performed, followed by permeabilization (0.5% Triton 100×) and blocking in donkey serum (10%) (Merck Millipore Chemicon, Burlington, Massachusetts, USA) for one hour at room temperature.…”

“…The TUNEL assay was performed to detect apoptosis in 5-day in vitro cultured ovaries ( n = 7–8) using the in-site Cell Death Detection Kit (Roche, Basel, Schweiz) 48 , 51 , 52 . In brief, sections were processed as described for immunofluorescence staining and labeled with TdT for two hours in a humidity chamber at 37 °C.…”