P-586 3D micro-Computed Tomography imaging and reconstruction of the mouse ovary before and after gonadotropins treatment

Abstract Study question How does gonadotropins treatment impact on the number of the different follicle types, their 3D localization, and their recruitment and selection dynamics inside the ovary? Summary answer Treatment with gonadotropins modifies the dynamics of follicles recruitment and selection within the 3D context of the ovary. What is known already Gonadotropins regulate mouse folliculogenesis during follicles recruitment (type 4-5, T4-5) and selection for growth or elimination (T6-7). To further our understanding of the impact of gonadotropins treatment on folliculogenetic dynamics inside the ovary, we used Computed Tomography (CT). 3D imaging of the ovary provides information on its inner spatial organization, and microCT is the only technique that combines high resolution with cubic voxels and allows the organ 3D in-silico reconstruction. We recently published the method for microCT analysis of the adult mouse ovary demonstrating that follicle recruitment occurs simultaneously all-over the cortex, and folliculogenesis is completed within the same region. Study design, size, duration 25-day-old CD1-mice were used since this age rightly precedes the first ovulation: follicles, from the primordial T1 to the pre-ovulatory T8, are all present, but corpora lutea (CL) are yet not formed. Ovaries from different individuals were analyzed by microCT: three not-treated controls (CTR); three injected with 3.5 I.U. PMSG and sacrificed 48hr later (PMSG); three treated first with PMSG, 48hr later injected with 3.5 I.U. hCG and then sacrificed after 15hr upon ovulation (PMSG+hCG). Participants/materials, setting, methods Ovaries were fixed in 4% Paraformaldehyde and treated with Lugol’s solution for 3hr at RT. Then, they were imaged with the EasyTom XL (RX Solutions) tomographic system, using a 1-1.2 µm/pixel resolution. Following microCT imaging, follicles classification (Fiji ImageJ, NIH), 3D mapping (XnConvert, XnSoft) and ovary in-silico reconstruction (Avizo-9, Thermo Fisher Scientific) were performed. ANOVA and Bonferroni post-hoc statistical analyses were done with RStudio > (p