Neonatal Mouse Ovary Culture and Whole-mount Follicle Quantification: An Efficient In Vitro Approach for Studying Primordial Follicle Regulation

Summary

This study presents protocols for culturing postnatal mouse ovaries on membrane inserts and for whole-mount follicle quantification in an entire ovary. Results demonstrated that technical handling during ovary culture significantly affects primordial follicle counts, highlighting the importance of maintaining consistency to minimize non-biological variation.

Abstract

In female mammals, primordial follicles form during fetal ovarian development and serve as the sole source for sustaining adult ovarian function. Mechanisms underlying how primordial follicles assemble, maintain dormancy, activate for follicular development, and undergo cell death are important for understanding ovarian physiology and pathological conditions. This study presents a protocol for culturing postnatal mouse ovaries on membrane inserts, an approach that enables the culture and pharmaceutical treatment of intact ovaries for up to 10 days, depending on the developmental stage of the ovary. Changes in the culture conditions can be achieved by transferring inserts containing cultured ovaries between wells on a plate, thereby avoiding physical interference with the tissues during culture. P5 ovaries were isolated and cultured on a 12 mm insert in a 24-well plate as an example. Each ovary was separated within a droplet of DMEM/F12 medium supplemented with 10% FBS, 3 mg/mL BSA, 10 mIU/mL FSH, and Antibiotic-Antimycotic, and gently stabilized on the membrane insert. The medium was changed every two days, and the culture was maintained for a total of five days. Following the culture, ovaries were fixed in 4% paraformaldehyde for 2 h and processed for whole-mount antibody staining of the oocyte marker DDX4. Follicles were staged and quantified based on oocyte size and the nuclear morphology of somatic follicle cells. The results showed that the number of primordial follicles in each ovary was significantly affected by the proper placement of tissues on the membrane insert. In addition, differences in the number of ovaries on each insert may introduce non-biological variations and should be avoided.

Introduction

Oogenesis is a highly regulated and complex process that results in the production of mature oocytes. In mammals, oogenesis initiates in fetal ovaries, where primordial germ cells (PGCs) differentiate into primary oocytes. Each primary oocyte is enclosed by a layer of squamous pregranulosa cells, forming a primordial follicle. Most of these primordial follicles become dormant after formation, serving as the ovarian reserve^1,2,3. In the adult ovary, periodic activation of primordial follicles for follicular development, known as folliculogenesis, is essential for sustaining th....

Protocol

CD-1 male and female mice were housed together at a 1:1 ratio as breeding pairs. The date of birth of new pups was designated as postnatal day 0. All animal experiments were approved by the Institutional Animal Care & Use Committee (IACUC) at the University of Missouri (protocol number: 36647).

1. Reagents and Culture Media

1. All reagents used in this study are listed in the Table of Materials. Prepare all materials and media before ovary culture.
2. Culture media: Make ~5 mL of DMEM/F-12 media supplemented with 3 mg/mL bovine serum albumin (BSA), 10% fetal bovine serum (FBS), and 10 mI....

Results

In this article, we demonstrate the methods of neonatal ovary culture, whole-mount antibody staining, confocal imaging, and follicle quantification to assess whether the number and position of the ovaries in the insert during culture affect the experimental outcome, specifically, follicle counts in the ovary. We cultured ovaries with the following setups: one, two, or three ovaries per insert for stabilized culture, and three ovaries per insert for floating culture. The ovaries were cultured under two conditions: a contr.......

Discussion

In this study, we demonstrated methods for neonatal mouse ovary culture and for whole-mount ovarian follicle quantification. The culture approach can sustain primordial and early developing follicles in vitro. The whole-mount follicle quantification method requires minimal tissue processing, which saves time and preserves tissue integrity, ensuring accurate follicle quantification.

The number of primordial follicles observed in the control group suggests that, while there is a slight reduction.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well cell culture plates	Fisher scientific	09-761-146
Alexa Fluor 488 Donkey Anti-Rabbit	Jackson ImmunoResearch Laboratories	711-546-152
Anti-Anti (100X)	Gibco	15240-062
Bovine Serum Albumin	Sigma-Aldrich	A1470-25G
CD-1 IGS Mouse	Charles River Laboratories	022	Mice
Cell Culture Inserts	Millipore	PICM01250
Comfort Point Insulin Syringe With Needle 1cc 28G x 1/2	Exel	26027
DAPI	Thermo Scientific	62248
DDX4 Rabbit mAb	abcam	ab13840
DMEM/F12 (1:1) (1X)	Gibco	11320-033
Doxorubicine hydrochloride	Sigma-Aldrich	PHR1789
DPBS (1X)	Gibco	14190-144
Fetal Bovine Serum	Sigma-Aldrich	F0926-500ML
Fiji-ImageJ	NIH, USA (https://imagej.net/ij/)	ImageJ 1.52p
Fisherbrand Nutating Mixers - Variable Speed	Fisher scientific	88-861-043	Shaker
Follicle Stimulating Hormone	Sigma-Aldrich	F4021-10UG
Forma Steri-Cycle i160	Thermo Scientific™	i160	CO2 Incubator
Glycine	Fisher scientific	BP381-500
Imaris	Oxford Instruments (https://imaris.oxinst.com/)	Imaris 10.2
Leica TCS SP8	Leica Microsystems	SN 8100001409	Confocal
Normal Donkey Serum	Jackson ImmunoResearch Laboratories	017-000-121
Olympus SZ61	Olympus Microscopes	SZ61	Stereoscope
Paraformaldehyde	Fisher scientific	15714
SecureSeal imaging spacer	Grace Bio-Labs	654002
Triton X-100	Sigma-Aldrich	X100-500ML
TWEEN 20	Sigma-Aldrich	P1379-500ML
VECTASHIELD Antifade Mounting Medium	Vector Laboratories	H-1000