Name: separation of follicular cells and oocytes in ovarian follicles of zebrafish
Uploaded: 2021-04-18T14:00:00
Description: Watch this Scientific Journal Video about Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish at JoVE.com

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].

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
<ol>
	<li>Animals
	<ol>
		<li>Use adult female zebrafish with a body length of 4-6 cm. 
		NOTE: We used zebrafish from a local market.</li>
		<li>Keep the zebrafish in a circulated water system with a 14 h light and 10 h dark cycle at about 28 &#176;C.</li>
		<li>Feed fish twice daily with newly hatched ...

<ol>
	<li>Clelland, E., Peng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine/paracrine+control+of+zebrafish+development.">Endocrine/paracrine control of zebrafish development.</a> Molecular and Cellular Endocrinology. 312, 42-52 (2009).</li><li>Ge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrafollicular+paracrine+communication+in+the+zebrafish+ovary:+the+state+of+the+art+of+an+emerging+model+for+the+study+of+vertebrate+folliculogenesis.">Intrafollicular paracrine communication in the zebrafish ovary: the state of the art of an emerging model for the study of vertebrate folliculogenesis.</a> Molecular and Cellular Endocrinology. 237, 1-10 (2005).</li><li>Li, J., Ge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+studying+ovarian+development:+Recent+advances+from+targeted+gene+knockout+studies.">Zebrafish as a model for studying ovarian development: Recent advances from targeted gene knockout studies.</a> Molecular and Cellular Endocrinology. 507, 1-19 (2020).</li><li>Selman, K., Wallace, R. A., Sarka, A., Qi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+oocyte+development+in+the+zebrafish,+Brachydanio+rerio.">Stages of oocyte development in the zebrafish, Brachydanio rerio.</a> Journal of Morphology. 218, 203-224 (1993).</li><li>Matzuk, M. M., Burns, K. H., Viveiros, M. M., Eppig, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+communication+in+the+mammalian+ovary:+oocytes+carry+the+conversation.">Intercellular communication in the mammalian ovary: oocytes carry the conversation.</a> Science. 296, 2178-2180 (2002).</li><li>Liu, L., Ge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+differentiation+factor+9+and+its+spatiotemporal+expression+and+regulation+in+the+zebrafish+ovary.">Growth differentiation factor 9 and its spatiotemporal expression and regulation in the zebrafish ovary.</a> Biology of Reproduction. 76, 294-302 (2007).</li><li>Zhou, R., Tsang, A. H., Lau, S. W., Ge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Biology of Reproduction. 85, 615-625 (2011).</li><li>Li, J., Liu, Z., Wang, D., Cheng, C. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor+3+is+involved+in+oocyte+maturation+in+zebrafish.">Insulin-like growth factor 3 is involved in oocyte maturation in zebrafish.</a> Biology of Reproduction. 84, 476-486 (2011).</li><li>Pang, Y., Thomas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+G+protein-coupled+estrogen+receptor+1,+GPER,+in+inhibition+of+oocyte+maturation+by+endogenous+estrogens+in+zebrafish.">Role of G protein-coupled estrogen receptor 1, GPER, in inhibition of oocyte maturation by endogenous estrogens in zebrafish.</a> Developmental Biology. 342, 194-206 (2010).</li><li>Peyton, C., Thomas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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).">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).</a> Biology of Reproduction. 85, 42-50 (2011).</li><li>Welch, E. L., Eno, C. C., Nair, S., Lindeman, R. E., F, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+manipulation+of+maternal+gene+products+using+in+vitro+oocyte+maturation+in+zebrafish.">Functional manipulation of maternal gene products using in vitro oocyte maturation in zebrafish.</a> Journal of Visualized Experiments. (122), e55213 (2017).</li><li>Nair, S., Lindeman, R. E., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+oocyte+culture-based+manipulation+of+zebrafish+maternal+genes.">In vitro oocyte culture-based manipulation of zebrafish maternal genes.</a> Developmental Dynamics. 242, 44-52 (2013).</li><li>Seki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+reliable+in+vitro+maturation+system+for+zebrafish+oocytes.">Development of a reliable in vitro maturation system for zebrafish oocytes.</a> Reproduction. 135, 285-292 (2008).</li><li>Baars, D. L., Takle, K. A., Heier, J., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ploidy+manipulation+of+zebrafish+embryos+with+heat+shock+2+treatment.">Ploidy manipulation of zebrafish embryos with heat shock 2 treatment.</a> Journal of Visualized Experiments. , e54492 (2016).</li><li>Xie, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+based+on+in+vitro+oocyte+injection+to+improve+CRISPR/Cas9+gene+editing+in+zebrafish.">A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish.</a> Scientific Reports. 6, 34555 (2016).</li><li>Li, J., Bai, L., Liu, Z., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+roles+of+PDE9a+in+meiotic+maturation+of+zebrafish+oocytes.">Dual roles of PDE9a in meiotic maturation of zebrafish oocytes.</a> Biochemical and Biophysical Research Communications. , (2020).</li></ol>

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 ...

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. The follicle consists of the oocyte that is surrounded by one or several layers of somatic cells called follicular cells. The development of follicles depends on the bidirectional communication between oocytes and follicular cells5. It is vital to separate follicular cells and oocytes from ovarian follicles for different research purposes such as follicular cell primary culture, gene expression analysis, oocyte maturation, and in vitro fertilization.
Traditional separation methods include mechanical separation by forceps and enzymatic digestion6,7,8,9,10. However, the mechanical separation by forceps is time-consuming and laborious. It will also cause the high damage to the oocyte during separation. Although the enzyme digestion method is simple to operate and requires a short time, the treatment time and enzyme concentration should be validated, and the integrity and survival rate of the isolated oocytes are not ideal.Therefore, we have established a simple method to separate both compartments at different developmental stages using pulled glass capillary tubes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >17&#945;,20&#946;-DHP</td>
			<td >Cayman</td>
			<td >16146-5 (5 mg)</td>
			<td ></td>
		</tr>
		<tr >
			<td >24-well plate</td>
			<td >Corning</td>
			<td >3524</td>
			<td></td>
		</tr>
		<tr >
			<td >Ampoule cutter</td>
			<td >AS ONE</td>
			<td >5-124-22 1 bag (100 pieces)</td>
			<td></td>
		</tr>
		<tr >
			<td >Anhydrous Na2HPO4</td>
			<td >Kaixin Chemical</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Brine shrimp</td>
			<td >Hongjie</td>
			<td >250 g</td>
			<td></td>
		</tr>
		<tr >
			<td >CaCl2</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Culture dish</td>
			<td >Biosharp</td>
			<td >BS-90-D (10PCS/PK)</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Solarbio</td>
			<td >S2110 (25mL)</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting Microscope</td>
			<td >ZEISS</td>
			<td >Stemi 305</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection forcep</td>
			<td >VETUS</td>
			<td >HRC30</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection scissor</td>
			<td >Kefu</td>
			<td >160 mm&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence Stereomicroscope&#160;</td>
			<td >Leica</td>
			<td >M205C</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass capillary</td>
			<td >IWAKI</td>
			<td >IK-PAS-5P (200 pcs/PACK)</td>
			<td></td>
		</tr>
		<tr >
			<td >Hoechst 33342</td>
			<td >Solarbio</td>
			<td >C0031 (1 mg)</td>
			<td></td>
		</tr>
		<tr >
			<td >KCl</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >KH2PO4</td>
			<td >Kaixin Chemical</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Leibovitz&#8217;s L-15 medium</td>
			<td >Gibco</td>
			<td >41300-039 (10&#215;1L)</td>
			<td></td>
		</tr>
		<tr >
			<td >MgSO4&#8226;7H2O</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette tips</td>
			<td >Axygen</td>
			<td >MCT-150-C</td>
			<td></td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >NaHCO3</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicilia-streptomycia</td>
			<td >Gibco</td>
			<td >#15140122 (100 mL)</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereomicroscope</td>
			<td >ZEISS</td>
			<td >Discover.v20</td>
			<td></td>
		</tr>
	</tbody>
</table>

separation of follicular cells and oocytes in ovarian follicles of 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.

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 ...

Watch this Scientific Journal Video about Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish at JoVE.com

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

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 ...

Research

JoVE Journal

Developmental Biology

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...