INVESTIGATING THE IMPACT OF mTOR INHIBITOR TREATMENT ON TRANSGENERATIONAL OFFSPRING HEALTH AND FERTILITY

Abstract

We previously showed that mTOR inhibitors are effective fertoprotectants during gonadotoxic chemotherapy and prolong reproductive lifespan in a murine model. We investigated the impact of mTOR inhibitor treatment on transgenerational offspring health and fertility. Female mice (C57BL/6, 8 weeks, n=6-7/group, F0) were treated for 4 weeks (oral gavage) with Everolimus (mTORC1 inhibitor, RAD001), DMSO (RAD Vehicle), Sapanisertib (mTORC1/2 inhibitor, INK128), and/or PVP/NMP (INK Vehicle). Estrous cyclicity was evaluated by vaginal lavage. Following treatment, 13 mice were sacrificed for organ harvest; remaining mice (n=12) were bred at 20 weeks with proven breeders to generate two litters. F1 pups were maintained until 12 weeks: male offspring underwent semen analysis and F1 females were sacrificed for ovarian harvest or bred to produce one litter of F2 pups. F2 pups were sacrificed on day 3 and weight, crown-rump length, and survival (%) were recorded. The following data were recorded for each birth: pup number, weight, anomalies, survival, sex, and days from male interaction to birth. Data are presented as mean +/- SEM; significance P < 0.05. Data analyzed using GraphPad Prism Software (8.0, La Jolla, California). There were no differences in estrous cyclicity between groups (P > 0.05). Mice treated with RAD001 and INK128 trended toward higher primordial follicle counts (NS, P > 0.05). Days from male interaction to birth did not differ between groups for both the first and second litter of F1 pups (P > 0.05). Notably, significantly more cumulative F1 pups were noted in INK-treated mice compared to all other groups (P < 0.05). During the first breeding trial, INK128 pups had significantly higher survival rates than INK Vehicle ( P < 0.005) and RAD001 had significantly lower survival rates than RAD Vehicle (P < 0.001). Pup survival rates normalized in the second litter (P > 0.05). F1 pups showed appropriate weekly weight gain from 2 weeks until 12 weeks of age. F1 males demonstrated normal sperm counts across treatment groups (P > 0.05). In F1 female breeding trials, there was no difference in time from breeding to birth, F2 pups per litter, or F2 pup survival (P > 0.05). At the time of sacrifice, F2 pups showed similar weights across groups. Of note, F2 male pups from RAD-treated F0 mice demonstrated significantly smaller crown-rump lengths compared to RAD vehicle (** P < .005) and INK-treated groups (* P < 0.05). Sex ratios were similar across all treatment groups and generations. Female mice treated with mTORC1/2 inhibitors had significantly more pups over the breeding period and higher pup survival rates, consistent with our previous findings. Treatment with mTOR inhibitors resulted in reassuring offspring health and fertility, but isolated findings among offspring of RAD-treated mice warrant further investigation.