Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Summary

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Abstract

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Introduction

Female mouse ovaries contain several thousand follicles¹, and periodic ovulation matures approximately ten oocytes at each estrus cycle. Follicles are classified into several developmental stages: primordial, primary, secondary, antral, and Graafian follicles, depending on the form of the granulosa cellular layer surrounding each oocyte. Most primordial follicles are dormant, and some of them are activated and grow into primary follicles at each estrus cycle². After the secondary follicular stage, follicle development is mainly regulated by gonadotropins, follicular stimulating hormone (FSH), and luteinizing hormone (LH)....

Protocol

Mice were housed in an environmentally controlled room at 23 ±1 °C with a 12 h light/12 h dark cycle. Animal care protocols and experiments were conducted in accordance with the Guidelines for Animal Experimentation at Aichi Medical University and were approved by the incumbent Animal Care and Use Committee.

1. Preparation of Culture Medium and Dishes

1. To prepare basic culture medium, add fetal bovine serum (FBS, 5% v/v), FSH (100 mIU/mL), LH (10 mU/mL), and penicillin-streptomycin (penicillin, 100 U/mL; streptomycin, 100 mg/mL) to minimum essential medium alpha (MEM-alpha) and mix in 50 mL tubes.
   NOTE: Total vol....

Results

Figure 1 shows the protocol for changes in media during ovarian tissue culture. Following this program, 4-week-old ICR mice ovaries were cultured and imaged at 24-h intervals using confocal microscopy (Figure 2). During culture of ovary tissues for 3 weeks, most antral and secondary follicles were degenerated by follicle atresia and some were ovulated (Figure 2D and Table 2

Discussion

In this study, we developed two new methods for studying follicle development in mouse ovaries. The first method involves culture of sliced ovarian adult mice tissues followed by analyses of follicle development, and the second involves the use of time-lapse imaging to visualize early follicle development during the gonadotropin-independent stage. Previously, we used the present ovary tissue culture method to assess the effect of leukemia inhibitory factor and progesterone on follicle development⁸.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Follicle stimulating hormone from human pituitary	SIGMA	F4021
Lutenizing hormone from equine pituitary	SIGMA	L9773
Penicillin-streptomycin solution	Wako Pure Chemical Industries	168-23191
MEM a, GlutaMax, no nucleotides	Thermo Fisher	32561037
Glass bottom dish	MatTek	P35G-0-10-C	35mm dish, No. 0 coverslip, 10mm glass diameter
Millicell cell culture insert	Merck Millipore	PICM0RG50	Diameter: 315 mm, pore size: 0.4 mm, material: hydrophilic PTTE
3.5cm cell culture dishes	greiner bio-one	627160
50ml / centrifuge tube with triple seal cap	IWAKI	2345-050
Low-profile disposable blades 819	Leica	14035838925
LSM 710	Carl Zeiss		Confocal microscope
CellVoyager, CV1000	Yokogawa Electric Corporation		Time-lapse imaging
BZ-X700	KEYENCE		Time-lapse imaging