P-586 3D micro-Computed Tomography imaging and reconstruction of the mouse ovary before and after gonadotropins treatment
Authors: Fiorentino G , Parrilli A , Cimadomo D , Vaiarelli A , Rienzi L , Ubaldi F.M , Garagna S , Zuccotti M 
Published: June 2022

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 &gt; (p &lt; 0.05).

Main results and the role of chance
Compared to CTR, 48hr after PMSG injection, the T3, T4 and T5 follicle classes remained numerically unchanged, suggesting that the FSH-dependent recruitment involving T4-5 follicles is balanced by an equivalent number of follicles growing from the preceding primordial pool. The most evident change in PMSG ovaries was a 5-fold decrease (p = 0.002) in the number of antral T7 follicles, at the stage of follicle selection. This observation was paralleled by the presence, in the medulla region, of many atretic-like follicles, characterized by a more intense microCT contrast associated with a collapsed antrum, de-structured granulosa-cell layers and fragmented oocytes.
In PMSG+hCG ovaries, the number of T3-7 follicles was analogous to that described for PMSG, although the atretic-like follicles almost disappeared, suggesting their elimination sometime prior to ovulation.
To understand whether follicle recruitment and selection have a territoriality within the 3D female gonad, ovaries were virtually divided into eight dorsal (D-I/II/III/IV) and ventral (V-I/II/III/IV) sectors. Compared to CTR, PMSG injection appeared to increase follicle selection in sectors V-I (p = 0.03), V-II (p = 0.01) and V-IV (p = 0.02), whereas PMSG+hCG displayed a higher follicle recruitment in sector V-IV (p = 0.0007). Overall, these data suggest a spatial differential effect of the gonadotropins treatment inside the 3D mouse ovary.

Limitations, reasons for caution
To strengthen the results and better account for the inter-individual variability in response to hormonal treatments, the number of ovaries/individuals should be increased. Also, important is extending the analysis to the time-interval after hCG injection, beyond 15 hr, to assess the effects of ovarian stimulation on subsequent cycles.

Wider implications of the findings
The method proposed allows to map all follicle stages and to reconstruct a 3D in-silico model of the mouse ovary. This approach could be extended to other Mammals, including human, in normal and pathological conditions, as well as in response to different hormonal stimuli (e.g., dose or type of gonadotropins).

Trial registration number
not applicable