Micro-Computed Tomography Of The Adult Mouse Ovary: An In-Silico 3d Reconstruction Of Folliculogenesis

Abstract Study question Which are the spatial dynamics of follicles recruitment and growth inside the ovary? Summary answer 3D micro-Computed Tomography (microCT) shows a simultaneous and homogeneous distribution of follicle recruitment all-over the cortex,and subsequent growth within the same ovarian region. What is known already In the mouse ovary, folliculogenesis progresses from the primordial type 1 (T1) to the fully-grown T8 follicle. Most of our knowledge of the folliculogenetic process has been obtained by disaggregating the ovary into its functional units (i.e., follicles and oocytes), thus losing the complexity of the whole histo-functional context. To date, few studies employed 3D imaging approaches to gain information on the inside 3D ovary organisation. MicroCT is the only technique that combines a high spatial resolution (down to ∼1 µm) with the production of a true 3D organ reconstruction, with cubic voxels and isotropic resolution. Study design, size, duration Three ovaries of three different adult mice were treated with the contrast agent and then imaged with microCT. A typical experiment required a total of 35 man/h from ovaries isolation to completion of X-ray scanning, and 24 man/h for follicles classification and mapping. Participants/materials, setting, methods Three ovaries of three different 8-week-old CD1 mice were fixed in 4% Paraformaldehyde and treated with Lugol’s solution for 3 hr at RT. Ovaries were scanned with Skyscan 1172 (Bruker) using a 1.5 µm/pixel resolution. MicroCT sections were processed with Fiji ImageJ (NIH), and 3D rendering of follicles and blood vessels were obtained with Avizo-9 (Thermo Fisher Scientific). ANOVA and Bonferroni post-­hocstatistical analyses were performed with RStudio, considering data significantly different when p < 0.05. Main results and the role of chance Using microCT we built the first in silico3D reconstruction of the tiny mouse ovary, identifying, mapping and counting follicles,from pre-antral secondary T4 (53.2 + 12.7 µm in diameter) to fully-grown antral T8 (321.0 + 21.3 µm), and the corpora lutea.MicroCTbrought up the main functional compartments of the growing follicle, i.e., granulosa and cumulus cells, the antrum, the zona pellucida, and the oocyte with its nucleus. Instead, primordial and primary follicles (T1–T3) could not be observed, perhaps due to the reduced size of their enclosed oocyte and to the absence of a well-formed zona pellucida around the germ cell. In addition, our analysis allowed the visualisation and 3D modelling of the main ovarian vasculature, from the largest vessel that enters the organ at the hilum site (∼150 µm size in diameter)to smaller branches present in the medulla region (∼35 µm). These results show that each of the eight ovarian sectors, virtually segmented along the dorsal-ventral axis,houses an equal number of each follicle type, suggesting a simultaneous and homogeneous distribution of follicle recruitment all-over the cortex,and subsequent growth within the same ovarian region. Limitations, reasons for caution To strengthen the results, the number of ovaries/individuals analysed should be increased. Wider implications of the findings This 3D mapping of follicles and vessels could contribute our understanding of folliculogenesis dynamics, not only under normal conditions, but also during ageing, after hormones or drugs administration, or in the presence of ovarian pathologies. Trial registration number not applicable