Accurate Follicle Enumeration in Adult Mouse Ovaries

Summary

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

Abstract

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Introduction

The immature, meiotically-arrested oocytes stored within primordial follicles in the ovary are the storage unit for the female germline and comprise an individual’s lifetime ovarian reserve. Primordial follicle numbers decline naturally with age¹, or alternatively, can be prematurely depleted following exposure to exogenous chemicals, including some pharmaceuticals and environmental toxicants in air, food and water². Given that the primordial follicle number is finite, the quantity and quality of follicles present within the ovary largely determine female fertility and offspring health. Thus, accurate quantificatio....

Protocol

Ovaries were collected from female C57BL6J mice. All animal procedures and experiments were performed in accordance with the NHMRC Australian Code of Practice for the Care and Use of Animals and approved by the Monash Animal Research Platform Animal Ethics Committee.

NOTE: A chemotherapy agent shown to deplete primordial follicle oocytes, as determined using stereology¹¹ and direct counts^12,13 was used in this r.......

Discussion

This article provides a step-by-step protocol for the gold standard technique for enumerating mouse primordial follicles, stereology, and the more commonly employed method of direct follicle counting. Chemotherapy treatment was used to compare and contrast the results obtained from these two different methods within the left and right ovary from the same animal. Both methods revealed high inter-animal variability in primordial follicle numbers. A significant depletion of the ovarian reserve was recorded using stereology,.......

Materials

Name	Company	Catalog Number	Comments
1-Butanol (HPLC)	Fisher Chemical	#A383-1
Acid alcohol	Amber Scientific	#ACDL
Bouin’s fixative	Sigma-Aldrich	#HT10132	Picric acid 0.9% (w/v), formaldehyde 9% (v/v), acetic acid 5% (w/v)
Cyclophosphamide	Sigma-Aldrich	#C0768-5G
Dibutylphthalate Polystyrene Xylene (DPX)	Sigma-Aldrich	#06522
Ethanol	Amber Scientific	#ETH	Ethanol 100%
Micro Feather opthalmic scalpel with aluminium handle	Designs for Vision	#FEA-745-SR	Feather blade for dissections (seen in Figure 1)
Formalin fixative	Australian Biostain	#ANBFC
Glass coverslip	Thermo Scientific	#MENCS22501GP	22 mm x 50 mm
Glycomethacrylate resin RM2165 microtome	Leica Microsystems	#RM2165
Glycolmethacrylate DPX	*made in house		*Mix 1.5 L Xylene; 800 g polystyrene pellets; 100mL Dibutyl phthalate for 3 weeks
Histolene	Trajan	#11031
Mayer’s haematoxylin	Amber Scientific	#MH
Olympus BX50 microscope	Olympus	#BX50	Brightfield microscope fitted with 10x dry & 100x oil immersion objective (numerical aperture 1.3)
Olympus immersion oil type-F	Olympus	#IMMOIL-F30CC
Olympus TH4-200 light source	Olympus	#TH4-200
Paraffin wax	Sigma-Aldrich	#03987
Periodic acid	Trajan	#PERI1%	Periodic acid 1%
Rotary Microtome CUT 4060	MicroTec	#4060R/F	Used to cut paraffin sections
Schiff’s reagent	Trajan	#SCHF
Scott's tap water	Amber Scientific	#SCOT	Potassium carbonate, magnesium sulphate, water
StereoInvestigator Stereological System	MBF Bioscience		Includes StereoInvestigator software, multi-control unit, automatic stage and joystick
Superfrost microscope slides	Thermo Scientific	#MENSF41296SP	1 mm, 72 pcs
Technovit 7100 Plastic embedding system	Emgrid Australia	#64709003	500 mL/5 x 1 g/40 mL
Technovit 3040 yellow	Emgrid Australia	#64708805	100 g/80 mL