CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions

Abstract

Optimal distribution of heterogeneous organelles and cell types within an organ is essential for physiological processes. Unique for the ovary, hormonally regulated folliculogenesis, ovulation, luteal formation/regression and associated vasculature changes lead to tissue remodeling during each reproductive cycle. Using the CLARITY approach and marker immunostaining, we identified individual follicles and corpora lutea in intact ovaries. Monitoring lifetime changes in follicle populations showed age-dependent decreases in total follicles and percentages of advanced follicles. Follicle development from primordial to preovulatory stage was characterized by 3 × 105-fold increases in volume, decreases in roundness, and decreased clustering of same stage follicles. Construction of follicle-vasculature relationship maps indicated age- and gonadotropin-dependent increases in vasculature and branching surrounding follicles. Heterozygous mutant mice with deletion of hypoxia-response element in the vascular endothelial growth factor A (VEGFA) promoter showed defective ovarian vasculature and decreased ovulatory responses. Unilateral intrabursal injection of axitinib, an inhibitor of VEGF receptors, retarded neo-angiogenesis that was associated with defective ovulation in treated ovaries. Our approach uncovers unique features of ovarian architecture and essential roles of vasculature in organizing follicles to allow future studies on normal and diseased human ovaries. Similar approaches could also reveal roles of neo-angiogenesis during embryonic development and tumorigenesis.

Vasculature-staining with lipophilic dyes in tissue-cleared brains assessed by deep learning

“…The vasculature of the brain is vital for the survival of neurons and investigating the structure in 3D can provide crucial insight into the origin of different pathological states. The 3D structure can be achieved post-mortem by tissue clearing, where the vessels have been perfusion-stained with fluorescent probes (Ertürk et al, 2012;Qi et al, 2019;Hsu et al, 2017;Cai et al, 2019) and serial z-stack microscopy using light sheet or confocal microscopes (Carrillo et al, 2018;Liebmann et al, 2016;Feng et al, 2017). The tissue clearing removes primarily lipids but also other molecules, including the dye for staining the vasculature, which depends on the type of dye and how well it integrates into the fixed protein fabric.…”

Advances in Ex Situ Tissue Optical Clearing

“…Up to date, every chemically distinct TOC method was applied to study organs of female reproductive system and everyone made clear contribution to better understanding of either female reproductive general physiology or pregnancy . Study by Feng et al . allowed for the first time to count the total number of five various follicles types (primordial, primary, secondary, antral and preovulatory) at five different time points throughout mice life (3, 10, 21, 60, and 360 days) and assess their spatial organization and clustering characteristics ( Figure a) .…”

“…Study by Feng et al . allowed for the first time to count the total number of five various follicles types (primordial, primary, secondary, antral and preovulatory) at five different time points throughout mice life (3, 10, 21, 60, and 360 days) and assess their spatial organization and clustering characteristics ( Figure a) . Using PECAM‐1 (CD31) staining, the group also examined ovarian vascular network surrounding follicles and corpora lutea throughout mouse lifetime and upon treatment with human/equine chorionic gonadotropin or axitinib (type 1–3 VEGF, c‐Kit, and PDGF receptor antagonist), which increased the vasculature size and number of blood vessel branches and significantly decreased the number of ovulated oocytes, respectively.…”

Advances and perspectives in tissue clearing using CLARITY

“…Saponin reportedly shortens the incubation time required in the hydrogel monomer infusion process. However, saponin may cause bubbles, and therefore it is not recommended and red has only been reported utilised in a study of ovarian follicles (Feng et al, 2017). In zebrafish adding dimethyl sulfoxide (DMSO) improves monomer penetration.…”

“…CLARITY-based protocols have been used with little, e.g. lower acrylamide concentrations, or no modifications to clear most other organs such as lever (Lee et al, 2014;Font-Burgada et al, 2015), kidney (Yang et al, 2014;Lee et al, 2014;Unnersjö-Jess et al, 2015), pancreas (Lee et al, 2014;Muzumdar et al, 2016), adrenal gland (Epp et al, 2015), spleen (Epp et al, 2015;Kie↵er et al, 2017), lymphoid tissues (Kie↵er et al, 2017), intestine (Yang et al, 2014;Lee et al, 2014;Epp et al, 2015;Neckel et al, 2016;Kie↵er et al, 2017), testes (Epp et al, 2015;Frétaud et al, 2017), ovaries (Feng et al, 2017), and lung tissue (Yang et al, 2014;Lee et al, 2014;Epp et al, 2015;Saboor et al, 2016). However, given that organs have di↵erent densities and ratios of connective tissue, lipids, and protein the clearing times and the average RI of the tissue-hydrogel vary (table 2).…”