(338) Rapamycin Treatment Reduces Follicle Activation (In Vitro and In Vivo) and Attenuates Follicle Burnout After Subcutaneous Ovarian Autotransplantation

Paulo H. A. Campos1, Karine S. S. A. Dias¹, Luiza A. A. C. Pereira¹, Larissa A. Freitas1, Joao P. L. C. Castro1, Amanda P. Paz1, Michelly A. R. Silva2, Mateus Viana2, Luciola S. Barcelos2, Aantonio S. Varela Junior3

1. Laboratory for Research about Reproductive Biology, Federal University of São João del Rei, São João del Rei, Minas Gerais, Brazil

2. Federal University of Minas Gerais, Brazil.

3. Federal University of Rio Grande, Brazil.

Abstract Text:

Despite being promising, ovarian transplantation still requires improvements, especially to avoid excessive follicular activation (burnout). The PI3K-Akt-mTOR pathway is considered one of the main regulators of follicular activation and rapamycin treatment inhibits this pathway. Therefore, this study aimed to investigate in vitro the effect of treatment with different doses of rapamycin in preventing follicular activation and then evaluate the impact of treatment in vivo after ovarian transplantation in reducing burnout. Female C57BL/6 mice (n=42; #8896130223) were used. In vitro explants using PND3 ovaries (which present only primordial follicles) were cultured for 24h in supplemented DMEM/F12. The following concentrations of rapamycin 0,01 µM, 0,1 µM, 1 µM, 10 µM and 100 µM were evaluated and after treatment, these ovaries were directed to follicle counting. In the in vivo experiment, the animals were ovariectomized and the ovaries were autotransplanted to the dorsal subcutaneous region (at the base of the forelimbs). The animals were then treated (ip) with rapamycin (5 mg/kg) or vehicle, daily for 5 days. Euthanasia was performed 5 or 23 days after transplantation, and the ovaries were directed to follicular quantification. During the experimental period, vascular perfusion in the transplantation area was evaluated by laser Doppler, as well as body weight and estrous cycle. In the in vitro study, the percentage of primordial follicles was reduced (p=0.0370) in ovaries treated with rapamycin at 0.01 µM (61.06±3.41) compared to vehicle (48.08±3.75), while the primary follicular percentage was increased (p=0.0370) in the vehicle (51.92±3.75) compared to the 0.01 µM concentration (38.93±3.41). Regarding the in vivo study, body weight was not altered by treatment throughout the experiment at 5 days (p=0.2338, rapamycin 15.10±0.36, vehicle 15.61±0.23) and at 23 days (p=0.3188, rapamycin 18.00±0.62, vehicle 19.00±0.74). Ovarian weight was also not altered by treatment at 5 days (p=0.2204, rapamycin 0.0070±0.0016, vehicle 0.0096±0.0012) and at 23 days (p=0.8542, rapamycin 0.0092±0.0015, vehicle 0.0097±0.0021). The estrous cycle data showed that 100% of the animals in both groups returned to the post-transplant cycle, and the cycle duration was not altered by the treatment (p=0.8619, rapamycin 5.33±0.61, vehicle 5.16±0.70). Vascular perfusion in pixels/area was not altered throughout the evaluation days in the treatment (204.5±15.60) compared to the vehicle (192.8±19.78). At 5 days of treatment, the percentage of follicles in atresia was reduced (p=0.0131) in the treatment (37.33±5.85) compared to the vehicle (67.45±5.10). At 23 days, the primary follicular percentage of the treatment (85.50±9.23) was higher (*p=0.0125) than the vehicle (58.64±5.25). Our results showed that 0.01 µM is the dose that preserved a greater quantity of primordial follicles and the in vivo administration of rapamycin reduced follicular activation and consequently attenuated follicle burnout.
Finantial support: CAPES, CNPQ, FAPEMIG, FINEP, GOV. MG, UFSJ.