Sphingosine Phosphate Enhances Primordial Follicle Activation via LATS Inhibition in the Hippo Signaling Pathway
Authors: Huang Zichao , Huang Xiaoyuan , Xu Tianyue , Weng Linglin , Cai Jieyu , Ge Hongshan 
Published: June 2024

Background
We investigated whether the large kinase tumor suppressor (LATS) blocker sphingosine phosphate (S1P) can influence the maintenance/activation of primordial follicle dormancy and examined the role of the Hippo-YAP pathway in the fine-tuned regulation of primordial follicle activation/maintenance of dormancy, to elucidate the potential mechanisms underlying ovarian aging and explore innovative approaches for in vitro follicle activation.
Methods
We used the ovaries of 3-day-old ICR female mice, randomly divided them into groups, and placed them in culture medium containing 0 µM, 10 µM, 25 µM, and 50 µM sphingosine phosphate (S1P) for in vitro cultivation. Samples were collected for testing after 4 days. Hematoxylin-eosin (HE) staining was used to observe and count the number of primordial and growing follicles (primary and secondary follicles) in each group and their proportion to the total number of follicles, to determine the most suitable S1P concentration. The following experiments were conducted in the experimental group with 0 µM S1P and the optimal culture concentration S1P. Western blotting was used to detect the expression changes of LATS, other related indicators of the Hippo-YAP signaling pathway, follicle activation-related indicators (PCNA), ovarian reserve function indicator (AMH), and apoptosis-related indicators (caspase-3).
Results
HE staining results showed the presence of primordial follicles, primary follicles, and secondary follicles in all the studied ovaries. As the concentration of S1P treatment increased, primordial follicles appeared in the ovarian medulla, indicating that S1P could promote the formation or activation of ovarian medullary primordial follicles, while promoting the activation of ovarian cortical primordial follicles. The 25 µM S1P group (33.0 ± 7.6%) had a higher proportion of growing follicles than the 0 µM S1P group (19.4 ± 2.8%) (P < 0.01). After S1P treatment, the expression level of LATS protein significantly increased compared to the treatment group (P < 0.001). After S1P treatment, the protein expression levels of other related factors YAP, P-YAP, and MST in the Hippo-YAP pathway in the ovaries increased compared to the untreated group (P < 0.05). PCNA is expressed in oocytes and granulosa cells of various levels of follicles. After treatment with S1P, the expression of PCNA increased in various levels of follicles (P < 0.001). After culturing the ovaries in S1P culture medium, ovarian AMH expression increased (P < 0.05). There was no significant difference in the ratio of cleaved caspase-3/caspase-3 in the ovaries between S1P treatment and non-S1P treatment, indicating that S1P treatment did not significantly affect the regulation of ovarian apoptosis.
Conclusion:
S1P treatment can affect the activation of primordial follicles, alter the composition of ovarian follicles, and increase cell proliferation. It can also significantly affect the expression of proteins related to the Hippo-YAP pathway in ovarian tissue. Regulation of this pathway may be the main mechanism by which S1P affects the development and activation of primordial follicles. S1P can serve as an alternative for in vitro activation of primordial follicles therapy.