Combined iDISCO and CUBIC tissue clearing and lightsheet microscopy for in toto analysis of the adult mouse ovary†

Abstract

At any given time, the ovary contains a number of follicles in distinct growth stages, each with a set of identifying characteristics. Although follicle counting and staging using histological stains on paraffin-embedded ovary sections has been the gold standard in assessing ovarian health in fertility studies, the final counts rely on extrapolation factors that diverge greatly among studies. These methods also limit our ability to investigate spatial aspects of ovary organization. Recent advances in optical tissue clearing and lightsheet microscopy have permitted comprehensive analysis of intact tissues. In this study, we set out to determine the best clearing and imaging methods to generate 3D images of the complete adult mouse ovary that could be used for accurate assessments of ovarian follicles. We found that a combination of iDISCO and CUBIC was the best method to clear the immunostained ovary. Using lightsheet microscopy, we generated 3D images of the intact ovary and performed qualitative assessments of follicles at all stages of development. This study is an important step toward developing quantitative computational models that allow rapid and accurate assessments of growing and quiescent primordial follicles, and to investigate the integrity of extrinsic ovarian components including vascular and neuronal networks.

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

“…This study describes a microCT procedure which allows the observation of the inside of the tiny mouse ovary with its main ovarian artery branches, and the mapping of the 3D position of all the follicle types from T4 through T8 and corpora lutea, with unprecedented precision. This procedure is robust, has the advantage of being faster than other classical methods, like histology or tissue clearing (Cordeiro et al, 2015;Faire et al, 2015;Feng et al, 2017;Kagami et al, 2018;Oren et al, 2018;McKey et al, 2020), and gives a true isotropic 3D reconstruction (Flannery et al, 1987;Mizutani and Suzuki, 2012). A typical experiment requires a total of 35 man/h from ovaries isolation to completion of X-ray scanning, and 24 man/h for follicles classification and mapping.…”

“…Up to date, tissue clearing combined with optical imaging allowed the acquisition of molecular details on the inside 3D organization of the ovary (Cordeiro et al, 2015;Faire et al, 2015;Feng et al, 2017;Kagami et al, 2018;Oren et al, 2018;McKey et al, 2020). However, a major drawback of this procedure remains the production of a distorted 3D model of the organ, stretched out along the z-axis, due to the lack of an equal resolution on the three axes (Schermelleh et al, 2010;Cremer et al, 2017).…”

Local Accumulation of Axonal Mitochondria in the Optic Nerve Glial Lamina Precedes Myelination

“…The optic nerves underwent immunohistochemistry staining protocol adapted from McKey et al ( 2020 ). Adult mice were transcardially perfused with 1x PBS then 4% paraformaldehyde (PFA) and neonatal pups heads were drop fixed for 2 h in 4% PFA.…”

Local Accumulation of Axonal Mitochondria in the Optic Nerve Glial Lamina Precedes Myelination

“…The optic nerves underwent immunohistochemistry staining protocol adapted from McKey et al (2020) (McKey et al, 2020). Adult mice were transcardially perfused using with 1x PBS and 4% paraformaldehyde (PFA) and neonatal pups heads were drop fixed for 2 hours in 4% PFA.…”