[p53 regulates primordial follicle activation through the mTOR signaling pathway]

by Selleckchem | Oct 22 2023

This paper aimed to investigate the role and potential mechanism of p53 on primordial follicle activation. Firstly, the p53 mRNA expression in the ovary of neonatal mice at 3, 5, 7 and 9 days post-partum (dpp) and the subcellular localization of p53 were detected to confirm the expression pattern of p53. Secondly, 2 dpp and 3 dpp ovaries were cultured with p53 inhibitor Pifithrin-μ (PFT-μ, 5 μmol/L) or equal volume of dimethyl sulfoxide for 3 days. The function of p53 in primordial follicle activation was determined by hematoxylin staining and whole ovary follicle counting. The proliferation of cell was detected by immunohistochemistry. The relative mRNA levels and protein levels of the key molecules involved in the classical pathways associated with the growing follicles were examined by immunofluorescence staining, Western blot and real-time PCR, respectively. Finally, rapamycin (RAP) was used to intervene the mTOR signaling pathway, and ovaries were divided into four groups: Control, RAP (1 μmol/L), PFT-μ (5 μmol/L), PFT-μ (5 μmol/L) + RAP (1 μmol/L) groups. The number of follicles in each group was determined by hematoxylin staining and whole ovary follicle counting. The results showed that the expression of p53 mRNA was decreased with the activation of primordial follicles in physiological condition. p53 was expressed in granulosa cells and oocyte cytoplasm of the primordial follicles and growing follicles, and the expression of p53 in the primordial follicles was higher than that in the growing follicles. Inhibition of p53 promoted follicle activation and reduced the primordial follicle reserve. Inhibition of p53 promoted the proliferation of the granulosa cells and oocytes. The mRNA and protein expression levels of key molecules in the PI3K/AKT signaling pathway including AKT, PTEN, and FOXO3a were not significantly changed after PFT-μ treatment, while the expression of RPS6/p-RPS6, the downstream effectors of the mTOR signaling pathway, was upregulated. Inhibition of both p53 and mTOR blocked p53 inhibition-induced primordial follicle activation. Collectively, these findings suggest that p53 may inhibit primordial follicle activation through the mTOR signaling pathway to maintain the primordial follicle reserve.

Comments:

The purpose of this study was to investigate the role of p53 and its potential mechanism in the activation of primordial follicles, which are immature ovarian follicles. The researchers conducted several experiments using neonatal mice ovaries to explore the effects of p53 on follicle activation.

Initially, they examined the expression pattern of p53 mRNA in the ovaries of neonatal mice at different post-partum time points. The subcellular localization of p53 was also determined. This analysis confirmed that p53 expression decreased as primordial follicles underwent activation. The researchers observed that p53 was expressed in the granulosa cells (cells surrounding the oocyte) and the cytoplasm of the oocytes themselves. Furthermore, the expression of p53 was higher in primordial follicles compared to growing follicles.

To assess the function of p53 in primordial follicle activation, the researchers cultured 2-day-old and 3-day-old ovaries with a p53 inhibitor called Pifithrin-μ (PFT-μ) for three days. They performed hematoxylin staining and counted the number of follicles in the whole ovary to evaluate follicle activation. Immunohistochemistry was used to detect cell proliferation. The results showed that inhibiting p53 promoted follicle activation and reduced the number of primordial follicles. Additionally, the inhibition of p53 enhanced the proliferation of granulosa cells and oocytes.

To further investigate the potential mechanism of p53 in primordial follicle activation, the researchers examined the mRNA and protein levels of key molecules involved in the classical pathways associated with growing follicles. They used immunofluorescence staining, Western blot, and real-time PCR to assess the relative expression of these molecules. Interestingly, the expression levels of AKT, PTEN, and FOXO3a, which are key components of the PI3K/AKT signaling pathway, were not significantly affected by PFT-μ treatment. However, the expression of RPS6 and p-RPS6, which are downstream effectors of the mTOR signaling pathway, were upregulated. This suggests that p53 may inhibit primordial follicle activation through the mTOR signaling pathway rather than the PI3K/AKT pathway.

To further investigate the role of the mTOR signaling pathway, the researchers used rapamycin (RAP) to intervene in this pathway. They divided the ovaries into four groups: Control, RAP (1 μmol/L), PFT-μ (5 μmol/L), and PFT-μ (5 μmol/L) + RAP (1 μmol/L). Hematoxylin staining and follicle counting were performed to determine the number of follicles in each group. The results demonstrated that inhibition of both p53 and mTOR blocked the primordial follicle activation induced by p53 inhibition.

In summary, the findings of this study suggest that p53 may inhibit primordial follicle activation by regulating the mTOR signaling pathway, thus maintaining the primordial follicle reserve. These results provide insights into the molecular mechanisms underlying follicle development and could have implications for understanding and potentially treating conditions related to follicle activation and ovarian reserve.