Three-Dimensional Micro-Computed Tomography of the Adult Mouse Ovary.

Giulia Fiorentino,Silvia Garagna,Annapaola Parrilli,Maurizio Zuccotti

doi:10.3389/fcell.2020.566152

Abstract

In the mouse ovary, folliculogenesis proceeds through eight main growth stages, from small primordial type 1 (T1) to fully grown antral T8 follicles. Most of our understanding of this process was obtained with approaches that disrupted the ovary three-dimensional (3D) integrity. Micro-Computed Tomography (microCT) allows the maintenance of the organ structure and a true in-silico 3D reconstruction, with cubic voxels and isotropic resolution, giving a precise spatial mapping of its functional units. Here, we developed a robust method that, by combining an optimized contrast procedure with microCT imaging of the tiny adult mouse ovary, allowed 3D mapping and counting of follicles, from pre-antral secondary T4 (53.2 ± 12.7 μm in diameter) to antral T8 (321.0 ± 21.3 μm) and corpora lutea, together with the major vasculature branches. Primordial and primary follicles (T1–T3) could not be observed. Our procedure highlighted, with unprecedent details, the main functional compartments of the growing follicle: granulosa, antrum, cumulus cells, zona pellucida, and oocyte with its nucleus. The results describe a homogeneous distribution of all follicle types between the ovary dorsal and ventral regions. Also, they show that each of the eight sectors, virtually segmented along the dorsal-ventral axis, houses an equal number of each follicle type. Altogether, these data suggest that follicle recruitment is homogeneously distributed all-over the ovarian surface. This topographic reconstruction builds sound bases for modeling follicles position and, prospectively, could contribute to our understanding of folliculogenesis dynamics, not only under normal conditions, but, importantly, during aging, in the presence of pathologies or after hormones or drugs administration.

Highlights

In the adult ovary, folliculogenesis is a hormonally regulated process of growth and maturation from small primordial to fully grown antral follicles
We describe a method that, by combining an optimized contrast procedure with Micro-Computed Tomography (microCT) imaging, allowed, for the first time, the 3D mapping and counting of follicles, from pre-antral secondary type 4 (T4) to fully grown antral type 8 (T8), together with the identification of the major vasculature branches inside the adult mouse ovary, one of the smallest among Mammals
Contrast was homogeneous throughout the whole organ volume, highlighting, on microCT images acquired at 5 μm/pixel resolution, 3D MicroCT Adult Mouse Ovary distinctly marked follicles

Summary

Introduction

Folliculogenesis is a hormonally regulated process of growth and maturation from small primordial to fully grown antral follicles. The gonadotropin-independent initial recruitment of primordial follicles is followed, at puberty, by Follicle Stimulating Hormone (FSH) cyclic recruitment of early antral follicles (Hsueh et al, 2015), the latter enveloped within a calyx of capillaries arising from a branch of the ovarian artery (Fraser, 2006). Of these follicles, only a few, in polytocic species like the mouse, or one, in monotocic species like humans, complete their growth. A 3D reconstruction of the mammalian ovary would further improve our understanding of its tissue architecture, but, when combined with specific functional markers, it would help to reveal the multi-layered flow of molecular information that contributes to its biological function

Methods

Results

Conclusion