A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Summary

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Abstract

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

Introduction

Given the lack of an in vitro system representative of spermatogenesis progression, and the presence of great cellular heterogeneity in testis, studies of male germ cell biology require robust techniques to isolate enriched populations of specific cell types. Fluorescence-activated cell sorting (FACS) has been widely used for this purpose ^1,2,3,4,5, as it provides high yield and purity, and surpasses other isolation methods in the number of germ cell types that it can identify and select

Protocol

All procedures described below complied with regulations of the Animal Studies Committee at Washington University in St. Louis.

1. Preparations for Mechanical Dissociation Protocol

1. Pre-wet a 50 µm disposable tissue disaggregation cartridge for dissociation of a whole murine testis.
   NOTE: This disposable tissue disaggregation cartridge contains microblades designed for cutting of tissues and an immobile steel mesh with approximately 100 hexagonal holes. Different sizes of mesh for tissue disaggregation cartridge are available to adapt this protocol based on species and desired cell types. 50 µm c....

Results

Single cell suspensions from mechanical dissociation of testicular tissue

Figure 2 compares single cell suspensions obtained by mechanical dissociation of mouse testicular tissue under different conditions. Samples obtained by processing fresh tissue, unstained (Figure 2A) or stained with Hoechst (Figure 2B), show the presence of single cells .......

Discussion

Considering the highly-conserved chromatin dynamics during spermatogenesis in mammals, the goal of this work was to develop a protocol to isolate male germ cells in distinct stages of differentiation from different mammalian testicular tissues (Figure 1). One of the major obstacles in the application of a single workflow to different animal models is the need of species-specific adjustments, especially in regard to tissue dissociation protocols. Current methods mostly rely on enzymatic tissu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% paraformaldehyde	VWR	#15710	4% PFA, For fixing sorted cells
Medimachine	BD Biosciences	#340588;	System for testis dissociation
1X Dulbecco modified Eagle medium	Life Technologies	#31053	DMEM, for testis dissociation
Medicon	BD Biosciences	#340591	Disaggregation cartridge for testis dissociation
Fetal bovine serum	Thermo Scientific	#10082139	FBS, for FACS collection
Hoechst 33342	Invitrogen	#H3570	Hoecsht, For FACS staining
40 µm cell strainer	GREINER BIO-ONE	#89508-342	For testis dissociation
100x 15 mm petri dish	Falcon	#351029	For testis dissection
5 mL polypropylene round-bottom tubes	Falcon	#352063	For FACS collection
1.5 mL Eppendorf tubes	VWR	#20170-650	For FACS collection
3 mL needless syringe	BD Biosciences	#309657	For testis dissociation
50 mL conical tube	MIDSCI	#C50R	For testis dissociation
Propidium Iodide	Invitrogen	#L7011	PI, For FACS staining
MoFlo Legacy cell sorter	Beckman Coulter	#ML99030	For FACS
Summit Cell Sorting software	Beckman Coulter	#ML99030	For FACS
Phosphate buffered saline	Thermo Scientific	#AM9625	PBS, For microscopy
Microscopic glass slide	Fisher Scientific	#12-544-4	For microscopy
Glass coverslip	Fisher Scientific	#12-548-87	For microscopy
Disposable transfer pipette (3 mL)	Samco Scientific	#225	For testis dissociation
DNase I	Roche	#10104159001	For testis dissociation
Carbon steel surgical blade	Miltex	#4-111	For testis dissection
Countess automated cell counter	Invitrogen	#C10227	For automatic cell counting
Trypan blue solution (0.4%)	Sigma	#T8154	For viability staining