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>
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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. 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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 ...

The overall goal of this procedure is to separate follicular cells and Oocytes in ovarian follicular of Zebrafish. The follicular is the basic unit of the ovary, which consists of the Oocytes and the surrounding follicular cells. It is the wide tool to separate both the follicular cell and outside compartments for well-research policies including primary capture of follicular cells and rises on gene expression, all saturation and in vitro fertilization.Recently, the Zebrafish has become a powerful model of Nissan, which studies a control of already development. Here, we present a simple method, so separates follicular cells and oocytes of Zebrafish follicular which will facilitate investigations of already development in Zebrafish The commercial, no mass or the two separates about follicular cells and all sides of spins the are using post which has a labyrinth, time-consuming and had high damage to the outside. Here, we have established a simple method to two separate compartments using or part glasses Tamela rate under a pure reef.Our Metro scope, both outside and the follicular cells can be easily fabricated by typing the pulled glass capillary. Firstly, prepare a poet glass capillary. Hit the head of glass capillary on the alcohol burner.Use a four sides to stretch the glass capillary to the rave card diameter. Secondly, desect that overate from the adult female fish, isolate ovarian follicles from the ovary. Finally use a polled glass capillary to separate follicular cell and Oocyte from ovarian follicles.Prepare the glass capillary, sharps container, ample cutter, alcohol burner, and a forceps. Lie the alcohol burner, take home a glass of capillary and heat one end of glass capillary on the fire of alcohol burner. Use a forceps to hold another end of glass capillary, stress the glass capillary quickly and gently.Put the glass waste to the sharps container. Use the ample cutter Gretch on the pool with glass. Then break off by the forceps Gently to make a smooth opening of the port glance quickly burn the end of the glass capillary on alcohol burner several times and rub the open end by ample cutter carefully.Take how the fish tank from the cultivated water system. And now the ties adult females by placing fish on the ice water for at least two to five minutes until fish stop Gill movement. Take out the fish from the ice box and remove the remaining water fish body by towel, placed a fish on the D section plate with four sets.Fish were decapitated by serving Spang chord using their sharp scissors to ensure the death. Use dissecting scissors to cut as follows. Dissect from the Chloe cut to the gills along the midline of the abdomen, cut from the bank of the Chloe, cut from the anterior tip to the dorsal side.Gently removed the skin and muscles on one side of the body, expose the internal organs and take healthy entire ovary with four steps. The ovaries were gently placed immediately in the 100 millimeter in culture, dish container 60%L 50 median. Sent a dissecting microscope with transmitted light optics based on the size and morphology of ovarian follicles of different stages.Isolate these ovarian follicles manually using a beer of vying four sets. Collect the isolated ovarian follicles and put them in another cultured dish containing 60%L 50 medium, nice isolated ovarian follicles will be used to for separating follicular cells and Oocytes. Then set a device for separation.Use a 30 centimeters plastic tube. One end is connected with a one milliliter pipette tip with a filter element. Another end is connected with a 200 microliter pipette tip.Under the disecting microscope, use mouth to control the airflow to let the faulty goes moving to the pool with glass capillary then blow the folly goes out from the glass capillary. During this process, follicular cell and Oocytes of ovarian follicles can be separated. This is a separation process for a mature stage follicle.During the process of inhalation, the follicular cell layer is detached from the Oocyte, does after blowing out the follicular cell and Oocyte can be separated. This is a full ground stage follicle. We can see the follicular cell layer is detached from the Oocyte.This method can also be applied in the early stage ovarian follicles. This is a follicle at middle Latella Genesis stage. This is a follicle at early vatella Genesis stage.This is a follicle at a pre-vatalla Genesis stage. From this picture, you can see the process of separation. Panel one shows a intact follicle at full grown stage panel.Panel two shows the angulation of this follicle into pulled glass capillary. Panel three shows the follicular was blown out from the pulled glass capillary. Panel four indicates the successful separation F denuded Oocyte and follicular cell.Even this method, separation of follicular cell in Oocyte can be performed at different stage follicles. Investigate whether the follicular cells were separated from the intact follicles. Intact a follicle and is separated Oocyte from different stages of follicles from BV stage to FG stage were stained with DAPI.The nucleus of follicular cells can be stained by DAPI. The blue signal of DAPI was observed in intact follicles but not denuded Oocytes at these different stage follicles. To further confirm the clear separation, the intact follicles and separated Oocytes were gutting to the histological sections.DAPI stating results showed that follicular cells were clearly removed and denuded Oocytes. Using this method, denuded Oocytes separated from full grown stage follicles were found to undergo the spontaneous Oocyte maturation. without any treatment after four hours incubation.After activation by water, these matured Oocytes can undergo the full chorion expansion. This result indicates the low damage to the Oocyte using this method. Thus, this mustard can be used to obtain the denuded Oocyte for studying the Oocyte matured ration of Zebra fish.The follicular layer of material to follicles can be removed by a capillary glass tube. These can be fertilized and devolve into the hatching stage. The results suggest that this method can be used in for In Vitro fertilization in Zebra fish.We describe here a novel method for the rapid and easy separation of follicular cell and Oocyte from Zebra fish ovarian Follicles. This method has several significant advantages, overturned methodologies. Primary amongst these is to greatly increase the ease of separation as only a single and external manipulation is required.The increased ease of separation increases the availability of this method to those researchers not skilled in microdissection. Second, this method can be used to separate follicular cell and Oocyte of ovarian follicles and early developmental stages, including PV, EBV MV stage, such separation can not be performed at early stage follicle using current methodologies. Third, this new method leads to less damage to the Oocytes.The denuded Oocytes separated by this method can undergo the Oocyte maturation and can be fertilized. In addition, this method can also be applied in other fish.

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