Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Summary

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Abstract

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Introduction

In order to study the cellular composition and morphological features of mammalian ovaries, scientists often rely on in vivo experiments followed by immunohistological staining of paraffin embedded ovaries. More recently though, whole ovary organ culture has proven to be an effective alternative to study ovarian function^1,2,3,4 because the technique can be coupled with better visualization and quantification tools. Traditionally, analysis of ovarian morphology depends on reconstructing three-dimensional ovarian architecture from par....

Protocol

Cornell University's Institutional Animal Care and Use Committee (IACUC) has approved all the methods described here, under protocol 2004-0038 to JCS.

1. Preparation of Instruments and Culture Media

1. Wipe the working area clean with 10% bleach and allow the bleach to remain on the work area surface for at least 5 min. After 5 min, remove the excess bleach with clean paper towels and 70% ethanol (EtOH). Clean the dissecting microscope thoroughly with 70% EtOH.
   NOTE: It is important that the work area be at least semi-sterile to avoid contamination of organ cultures.
2. Wrap clean forceps and scissors with a paper....

Results

This protocol includes 6 major steps following dissection of the ovaries, as outlined in Figure 1. Figure 2, Figure 3, Figure 4 highlight the most novel features of this protocol, which include optimization of tissue clearing for ovaries and whole tissue oocyte quantification using FIJI-ImageJ. Figur.......

Discussion

The study of mammalian reproduction requires using and quantifying specialized cells that are not routinely amenable to cell culture. However, ex vivo culture systems are effective at maintaining ovary and follicle viability^1,15. During ovary culture, the tissue requires a larger surface area for exchange of nutrients through diffusion. Therefore, 5-day old mouse ovaries are ideal in size and shape for organ culture. This protocol was optimized for ovari.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Micro dissecting scissor 4.5", straight, sharp points	Roboz	RS-5912	Micro dissecting scissor
Jewelers style forceps 4-3/8", style 5	Integra Miltex	17-305X	Fine tip forceps
Micro Iris Scissors 4", straight, sharp points	Integra Miltex	18-1618	Micro dissecting iris scissor or micro dissecting spring scissor
1X Minimal Essential Media (MEM)	ThermoFisher Scientific	11090-081
Fetal Bovine Serium	Corning	35011CV
HEPES (1M)	Gibco	15630080
Antibiotic-Antimycotic (100X)	Gibco	15240062	Used in Step 1.3.1
35mm Tissue Culture Treated Dish	Corning	430165
Nunc™ 24-Well Carrier Plate for Cell Culture Inserts	ThermoFisher Scientific	141006	Pore size: 8μm
Paraformaldehyde (PFA)	Electron Microscopy Sciences	15710	16% solution
1X Phosphate-Buffered Saline (PBS)	Gibco	10010023
Nutator mixer GyroTwister™	Labnet	S1000-A-B	three dimensional shaker
Normal Goat Serum	VWR	103219-578
Bovine Serum Albumen (BSA)	VWR	97061-416
Sodium Azide (NaN3)	Sigma-Aldrich	S2002-25G
Sodium borohydride solution (NaBH4)	Sigma-Aldrich	452904-25ML	Use the solution, rather than the tablet or powder form
Polyvinyl Alcohol (PVA)	Sigma-Aldrich	P8136-250G	Cold water soluble
Triton™ X-100	Sigma-Aldrich	93443-100ML	polyethylene glycol tert-octylphenyl ether
Syringe filters	ThermoFisher Scientific	725-2520	25mm
10mL Syringes	BD	309695
Mouse anti-p63 antibody (4A4)	Biocare Medical	CM 163A	Dilution 1:500
Rabbit anti-MVH antibody	Abcam	ab13840	Dilution 1:600
Alexa Fluor® goat anti-mouse 594	ThermoFisher Scientific	A-11032	Dilution 1:1000
Alexa Fluor® goat anti-rabbit 488	ThermoFisher Scientific	A-11034	Dilution 1:1000
4,6-Diamidino-2-phenylindole (DAPI)	ThermoFisher Scientific	62248
D-(-)sorbitol	Sigma-Aldrich	240850-100G
Glycerol	Sigma-Aldrich	G9012-100ML
Urea	Sigma-Aldrich	U5378-500G
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650-5X10ML
FIJI-ImageJ			Image processing software
Disposable plastic transfer pipettes	VWR	414044-034