Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

Summary

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Abstract

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P <0.01) between the length of the follicular capillaries and volume of the follicular wall.

Introduction

The follicle is the fundamental structural and functional unit of the ovary, and its development is highly related to the vasculature within the ovary. Blood vessels supply nutrition and hormones to the follicles and thus play important roles in the growth and maturation of follicles¹.

A combination of technologies, including selective blood vessel markers, transgenic mouse models, and pharmaceutical development, have increased our knowledge about ovarian vascular networks, angiogenesis, and the function of blood vessels in folliculogenesis. The ovary is known as an active organ because it remodels various tissues an....

Protocol

All procedures involving animal subjects followed the guidelines of the Animal Ethics Committee at Shanghai Medical College, Fudan University (Approval number 20160225-013).

1. Preparation of the Transparent Mouse Ovary

1. Preparation of the solutions
   1. Prepare phosphate-buffered saline (PBS) solution (1 M, pH 7.6) with 0.1% Triton X-100 (PBST). To make 1 L of 10x PBS stock solution, mix 87 g of NaCl, 3.1 g of NaH₂PO₄ · 2H₂

Discussion

In the current study, we present 3D imaging to evaluate the relationships between capillaries and individual growing follicles. In our previous work using the same protocol ⁹, we studied the roles of large vasculature, interactions between follicles, and the location of follicles in intact mouse ovaries. The passive CLARITY approach allowed us to study micro- and macro-vasculatures, folliculogenesis, and the interrelationships between the corpora lutea and follicles as well as to reconstruct the o.......

Materials

Name	Company	Catalog Number	Comments
Acrylamide	Vetec	v900845	http://www.sigmaaldrich.com/catalog/product/vetec/v900845
Alexa Flour 488 (Dilution 1:50)	Life Technologies	A11039	https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039
Alexa Flour 594 (Dilution 1:50)	Life Technologies	A11012	https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012
Bisacrylamide	Amresco	172	http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx
Black wall glass bottom dish (Willco-Dish)	Ted Pella	14032	http://www.tedpella.com/section_html/706dish.htm#black_wall
Boric acid	Sinopharm Chemical Reagent	10004818	http://en.reagent.com.cn/enshowproduct.jsp?id=10004818
Disodium hydrogen phosphate dodecahydrate (Na2HPO4 12H2O)	Sinopharm Chemical Reagent	10020318	http://en.reagent.com.cn/enshowproduct.jsp?id=10020318
FocusClear	Celexplorer	FC-102	http://www.celexplorer.com/product_list.asp?MainType=107&BRDarea=1
Parafilm	Bemis	PM996	http://www.parafilm.com/products
Paraformaldehyde	Sinopharm Chemical Reagent	80096618	http://en.reagent.com.cn/enshowproduct.jsp?id=80096618
PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)	Abcam	ab28364	http://www.abcam.com/cd31-antibody-ab28364.html
Photoinitiator VA044	Wako	va-044/225-02111	http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm
Sodium azide	Sigma	S2002	http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&region=US
Sodium chloride (NaCl)	Sinopharm Chemical Reagent	10019318	http://en.reagent.com.cn/enshowproduct.jsp?id=10019318
Sodium dihydrogen phosphate dihydrate (NaH2PO4 2H2O)	Sinopharm Chemical Reagent	20040718	http://en.reagent.com.cn/enshowproduct.jsp?id=20040718
Sodium dodecyl sulfate	Sinopharm Chemical Reagent	30166428	http://en.reagent.com.cn/enshowproduct.jsp?id=30166428
Sodium hydroxide (NaOH)	Sinopharm Chemical Reagent	10019718	http://en.reagent.com.cn/enshowproduct.jsp?id=10019718
Triton X-100	Sinopharm Chemical Reagent	30188928	http://en.reagent.com.cn/enshowproduct.jsp?id=30188928
Tyrosine hydroxylase (TH, Dilution 1:50)	Abcam	ab76442	http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html