JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Published: April 18th, 2021

DOI:

10.3791/62027

1College of Life Sciences, Northwest Normal University, 2State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, 3Department of Bioscience, Tokyo University of Agriculture

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.

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.

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 development1,2,3. Many features of ovarian development are much conserved during evolution from fish to mammals1,2. 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.....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This research work was supported by the National Natural Science Foundation of China [32060170, 31601205 and 31560334], visiting scholar project supported by China Scholarship Council and the fund of State Key Laboratory of Freshwater Ecology and Biotechnology [2020FB05].

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Clelland, E., Peng, C. Endocrine/paracrine control of zebrafish development. Molecular and Cellular Endocrinology. 312, 42-52 (2009).
  2. Ge, W. Intrafollicular paracrine communication in the zebrafish ovary: the state of the art of an emerging model for the study of vertebrate folliculogenesis. Molecular and Cellular Endocrinology. 237, 1-10 (2005).
  3. Li, J., Ge, W. Zebrafish as a model for studying ovarian development: Recent advances from targeted gene knockout studies. Molecular and Cellular Endocrinology. 507, 1-19 (2020).
  4. Selman, K., Wallace, R. A., Sarka, A., Qi, X. Stages of oocyte development in the zebrafish, Brachydanio rerio. Journal of Morphology. 218, 203-224 (1993).
  5. Matzuk, M. M., Burns, K. H., Viveiros, M. M., Eppig, J. J. Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science. 296, 2178-2180 (2002).
  6. Liu, L., Ge, W. Growth differentiation factor 9 and its spatiotemporal expression and regulation in the zebrafish ovary. Biology of Reproduction. 76, 294-302 (2007).
  7. Zhou, R., Tsang, A. H., Lau, S. W., Ge, W. Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the zebrafish ovary: evidence for potentially dual roles of PACAP in controlling final oocyte maturation. Biology of Reproduction. 85, 615-625 (2011).
  8. Li, J., Liu, Z., Wang, D., Cheng, C. H. K. Insulin-like growth factor 3 is involved in oocyte maturation in zebrafish. Biology of Reproduction. 84, 476-486 (2011).
  9. Pang, Y., Thomas, P. Role of G protein-coupled estrogen receptor 1, GPER, in inhibition of oocyte maturation by endogenous estrogens in zebrafish. Developmental Biology. 342, 194-206 (2010).
  10. Peyton, C., Thomas, P. Involvement of epidermal growth factor receptor signaling in estrogen inhibition of oocyte maturation mediated through the G protein-coupled estrogen receptor (Gper) in zebrafish (Danio rerio). Biology of Reproduction. 85, 42-50 (2011).
  11. Welch, E. L., Eno, C. C., Nair, S., Lindeman, R. E., F, P. Functional manipulation of maternal gene products using in vitro oocyte maturation in zebrafish. Journal of Visualized Experiments. (122), e55213 (2017).
  12. Nair, S., Lindeman, R. E., Pelegri, F. In vitro oocyte culture-based manipulation of zebrafish maternal genes. Developmental Dynamics. 242, 44-52 (2013).
  13. Seki, S., et al. Development of a reliable in vitro maturation system for zebrafish oocytes. Reproduction. 135, 285-292 (2008).
  14. Baars, D. L., Takle, K. A., Heier, J., Pelegri, F. Ploidy manipulation of zebrafish embryos with heat shock 2 treatment. Journal of Visualized Experiments. , e54492 (2016).
  15. Xie, S. L., et al. A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish. Scientific Reports. 6, 34555 (2016).
  16. Li, J., Bai, L., Liu, Z., Wang, W. Dual roles of PDE9a in meiotic maturation of zebrafish oocytes. Biochemical and Biophysical Research Communications. , (2020).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved