Dissection of Larval Zebrafish Gonadal Tissue

Summary

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Abstract

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Introduction

The major female sex determinant in wild zebrafish chromosome 4 is lost or modified in the domesticated zebrafish (i.e. common lab strains)¹. Instead, they have a polygenic sex determination system accompanied by environmental factors such as temperature, hypoxia, food availability and population density. The detailed mechanisms of zebrafish sex development are not fully understood. Fundamental questions such as when zebrafish sex determination occurs, what the primary sex determination signal(s) is/are, and which genes regulate the first step of gonad transformation remain to be answered^2,

Protocol

Zebrafish experiments were approved by the Fudan University Institutional Animal Care and Use Committee. Zebrafish were raised and bred according to standard procedures²⁰.

1. Preparations

1. Cultivate 17 dpf and 25 dpf larval zebrafish
   1. Transfer 2 male and 2 female adult zebrafish (healthy, 3 to 6 months old, laboratory AB strain) to a crossing tank in the late afternoon before the crossing day. Separate the males and females with a barrier. The next morning, refresh the tank water and remove the barrier to allow them to mate. Collect eggs in 100 mm Petri dishes 1 to 2 h after fertilization.
   ....

Results

Dissections of the gonads were performed on AB strain larval zebrafish. Figure 1 shows typical gonadal tissue of larval zebrafish at 17 dpf and 25 dpf. Firstly, the skin and muscles of one side of the abdomen is cut to expose the internal organs. After removing the mass of internal organs, the swim bladder together with the gonad remain in the trunk. The gonad was attached to the ventral side of swim bladder (arrow in Figure 1B

Discussion

The zebrafish has become a powerful model and is extensively used in development and disease-related research. The methods for isolation of organs in adult zebrafish such as brain, heart, gonad, and kidney, have been well documented^23,24,25. Due to the small size and dynamic remodeling of the gonadal tissues in the larval zebrafish, isolation of gonadal tissue is a challenging task. Previous studies used whole dissected trunk ti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture dish 100 mm	Corning	430167	For embryo incubation
20x EM			For a 1 L needed: add 17.5 g NaCl, 0.75 g KCl and 2.9 g CaCl·2H2O; then add 0.41 g KH2PO4, 0.412 g Na2HPO4 anhydrous and 4.9 g MgSO4·7H2O.
1x EM			Dilute 20x EM in distilled water
AGAROSE G-10	Gene	121985	For preparing the 2% agar plates
Trizol Reagent	Invitrogen	15596-026	For RNA isolation
Meter glass	Shen Bo	250 ml	For preparing the 2% agar plates
Microwave Oven	Midea	M1-211A	For heating the AGAR
Tweezer DUMONT#5INOX	World Precision Instrument	500341	For dissection
Stereomicroscope	Motic	SMZ168	For dissection
Pure water equipment	Millipore
Ringer’s solution			For a 1 L needed: Add 6.78 g NaCl, 0.22 g KCl, 0.26 g CaCl2 and 1.19 g Hepes; then fill to 1 L; Adjust pH to 7.2. Sterilize by filtration and keep in an autoclaved clear polycarbonate container.
Transfer pipette	Samco	202, 204
Metal bath	QiLinbeier		Model GL-150
Microscope	Leica	M205 FA	For photomicrograph
Centrifuge	Eppendorf	5417R
Micro Scale RNA Isolation Kit	Ambion	AM1931	For RNA isolation from gonad tissues
Dnase I	Sigma	AMPD1-1KT	For DNA digestion in the RNA solution
RevertAid First Strand cDNA Synthesis Kit	Thermo Scientific	#K1631	For first-strand cDNA synthesis
Rnase H	Thermo Scientific	#EN0202	For digesting the residual RNA in the cDNA solution.
SYBR Green Realtime PCR Master Mix	TOYOBO	QPK-201	This product is a Taq DNA polymerase-based 2x master mix for real-time PCR and  applicable for intercalation assay with SYBR Green I.
Spectrophotometer	Ne Drop	OD-2000+	Measuring the concentration of the total RNA
Mastercycler	Eppendorf	AG 22331 Hamburg	gene expression profiling