Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Summary

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

Abstract

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Introduction

Zebrafish is a major model organism for the study of vertebrate development and physiology. The zebrafish can serve as a good model for studying the molecular mechanisms of ovarian development^1,2,3. Many features of ovarian development are much conserved during evolution from fish to mammals^1,2. Similar to the other vertebrates,zebrafish adults have asynchronous ovaries, containing ovarian follicles of all developmental stages⁴. The follicle is the fundamental reproductive element of the ovary.....

Protocol

All of the procedures performed in fish experiments are in accordance with the regulations of the Animal Experimentation Ethics Committee of Northwest Normal University.

1. Preparations

1. Animals
   1. Use adult female zebrafish with a body length of 4-6 cm.
      NOTE: We used zebrafish from a local market.
   2. Keep the zebrafish in a circulated water system with a 14 h light and 10 h dark cycle at about 28 °C.
   3. Feed fish twice daily with newly hatched brine shrimp.
2. Pulled glass capillary
   1. Use a 15 cm glass capillary and place its head in an alcohol burner to heat i....

Results

This method can be used to separate follicular cells and oocytes at different stages of ovarian follicle development in zebrafish. Figure 1 shows the separation of zebrafish oocytes and follicular cells from ovarian follicles using a capillary glass tube (Figure 1). To investigate whether the follicular cells were separated from intact follicles, the intact follicles and separated oocytes from different stages of follicles from the PV stage to the FG stage were .......

Discussion

We describe here a novel method for the simple and rapid separation of follicular cells and oocytes from zebrafish ovarian follicles. This method has several advantages over the conventional method. Primary amongst these is the greatly increased ease of separation with high efficiency and effectiveness, as only a single and external manipulation is required. This point increases the applicability to researchers who are not good at microscopic anatomy. According to our experience, one can successfully separate follicular .......

Materials

Name	Company	Catalog Number	Comments
17α,20β-DHP	Cayman	16146-5 (5 mg)
24-well plate	Corning	3524
Ampoule cutter	AS ONE	5-124-22 1 bag (100 pieces)
Anhydrous Na2HPO4	Kaixin Chemical	500 g
Brine shrimp	Hongjie	250 g
CaCl2	Beichen Fangzheng	500 g
Culture dish	Biosharp	BS-90-D (10PCS/PK)
DAPI	Solarbio	S2110 (25mL)
Dissecting Microscope	ZEISS	Stemi 305
Dissection forcep	VETUS	HRC30
Dissection scissor	Kefu	160 mm
Fluorescence Stereomicroscope	Leica	M205C
Glass capillary	IWAKI	IK-PAS-5P (200 pcs/PACK)
Hoechst 33342	Solarbio	C0031 (1 mg)
KCl	Beichen Fangzheng	500 g
KH2PO4	Kaixin Chemical	500 g
Leibovitz’s L-15 medium	Gibco	41300-039 (10×1L)
MgSO4•7H2O	Beichen Fangzheng	500 g
Micropipette tips	Axygen	MCT-150-C
NaCl	Beichen Fangzheng	500 g
NaHCO3	Beichen Fangzheng	500 g
Penicilia-streptomycia	Gibco	#15140122 (100 mL)
Stereomicroscope	ZEISS	Discover.v20