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 brine shrimp.</li>
	</ol>
	</li>
	<li>Pulled glass capillary
	<ol>
		<li>Use a 15 cm glass capillary and place its head in an alcohol burner to heat it. Hold one end of glass capillary by hand and hold the other end with forceps.</li>
		<li>After heating 5-10 seconds, the glass on the fire of alcohol burner become red and soft. Stretch the head of the glass capillary with forceps to make the capillary opening with required diameter. 
		NOTE: The diameter depends on the follicle diameters: previtellogenic (PV; about 0.30 mm in diameter), early vitellogenic (EV; about 0.40 mm in diameter), midvitellogenic (MV; about 0.50 mm in diameter), late vitellogenic (LV; about 0.60 mm in diameter) and full grown but immature (FG; about 0.65 mm in diameter).</li>
		<li>Stretch the opening of glass capillary to the appropriate diameter, which is slightly smaller the size of separated follicles. The glass capillary with an opening much bigger than the size of ovarian follicles cannot perform separation, but the much smaller ones would break the follicles during separation. Practice the stretching of the glass capillary several times. 
		NOTE: Heating time depends on the size of glass capillary. Do not directly stretch the glass capillary on the fire of alcohol burner. It will break the glass capillary.</li>
		<li>Cut the glass capillary with an ampoule cutter and break the glass capillary with forceps to obtain a smooth incision. 
		NOTE: A sharp or broken opening of the glass capillary would damage the follicles.</li>
		<li>Using a 30 cm plastic tube, insert a 1 mL pipette tip with a filter element at one end, insert the pulled glass capillary at the end of the pipette, and connect a 200 &#181;L pipette tip at another end.</li>
	</ol>
	</li>
</ol>
2. Separation of zebrafish oocytes and follicular cells at different stages
<ol>
	<li>Dissection of the zebrafish ovary
	<ol>
		<li>Fill an ice bucket to 4/5 full with slurry ice and add sufficient fish water to let slurry ice float. Wait 2-5 minutes, check the water temperature, and make sure the temperature is between 2-4 &#176;C.</li>
		<li>Anesthetize adult females by placing fish in the ice water for at least 2-5 minutes, until fish stop gill movement. Decapitate the fish by severing the spinal cord using a sharp scissors to ensure death.</li>
		<li>Place the fish on the dissecting plate with forceps.</li>
		<li>Use dissecting scissors to cut as follows. Dissect from the cloaca to the gills along the midline of the abdomen. Cut from the back of the cloaca. Cut from the anterior tip to the dorsal side. Gently remove the skin and muscles on one side of the body. Expose the internal organs and take out the entire ovary with forceps.</li>
		<li>Immediately, place the ovaries gently into a 100 mm culture dish containing 60% Leibovitz&#39;s L-15 (L-15) medium.</li>
	</ol>
	</li>
	<li>Isolation of ovarian follicles 
	NOTE: The staging system that we have adopted is based on the original definition of Selman et al.4.
	<ol>
		<li>Manually isolate follicles of different stages using a pair of fine forceps as described previously8.</li>
		<li>Group the ovarian follicles into the following stages: PV, EV, MV, LV and FG stages.</li>
	</ol>
	</li>
	<li>Separation of follicular cells and oocyte from ovarian follicles
	<ol>
		<li>Put the ovarian follicles at different stages into a clean Petri dish containing 60% L-15 medium.</li>
		<li>Using the pulled glass capillary from step 1.2, suck the follicles into the glass capillary 2-3 cm and blow them out. 
		NOTE: When the opening diameter of the glass capillaries is appropriate, the follicle can be separated from the oocyte by inhalation once.
		<ol>
			<li>If the follicular cell layer is attached to the oocyte firmly, repeat 2-3 times to accomplish a complete separation. Avoid multiple attempts to force a follicle through a smaller capillary, which would damage the follicle. 
			NOTE: In the process of inhalation and blowing out, the follicular layer falls off and separates from the oocyte, and finally, follicular cells and denuded oocytes are separated (Figure 1).</li>
		</ol>
		</li>
		<li>Pool the denuded but intact oocytes and collect the surviving denuded oocytes for further assays.</li>
		<li>To investigate whether the follicular cells were separated from intact follicles, stain the intact follicles and separated oocytes with 4&#39;,6-diamidino-2-phenylindole (DAPI).
		<ol>
			<li>Fix the samples in 4% buffered paraformaldehyde at room temperature (RT) for 1 h. Wash several times by PBS.</li>
			<li>Stain by DAPI at RT for 30 min. Washed several times with PBS. View and photograph with a fluorescence microscope (e.g., Leica DFC7000 T).</li>
		</ol>
		</li>
		<li>To confirm the completed separation, fix the intact follicles and separated oocytes in 4% buffered paraformaldehyde overnight at 4 &#176;C, and embed intissue-freezing medium (e.g., Leica) at -25 &#176;C.
		<ol>
			<li>Cut the fixed tissues on a microtome, and mount onto glass slides.</li>
			<li>Wash the sections in PBS and visualize the cell nuclei with DAPI. View and photograph on a fluorescence microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. In vitro maturation (IVM) and in vitro fertilization (IVF)
NOTE: The procedure of IVM of IVF was followed as described previously with minor modification 11,12,13.
<ol>
	<li>Prepare fresh maturation medium (+DHP medium) before ovary dissection from adult zebrafish female. To prepare the fresh maturation medium, add 9 mL of Leibovitz&#39;s L-15 medium, pH 9.0, to 15 mL conical tubes. Add 10 &#181;L of 17&#945;-20&#946;-dihydroxy-4 pregnen-3-one (DHP) (5 mg/mL), 490 &#181;L of dH2O, and 500 &#181;L of 10% bovine serum albumin (BSA).</li>
	<li>Prepare Hank&#39;s solution and E3 solution in advance. Prepare Hanks&#39; solution as described by Baars et al.14. Prepare E3 medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 1-5% Methylene Blue.</li>
	<li>Perform dissection of zebrafish ovary and isolation of ovarian follicles as in steps step 2.</li>
	<li>Collect full grown stage follicles and use as immature oocytes. For each adult female zebrafish, at least 150-200 full grown stage follicles can be isolated. Select the intact and healthy follicles for IVM.</li>
	<li>Incubate the full-grown stage follicles in +DHP medium in 12-well plates, checking periodically to ensure that the oocytes remain intact. Remove any lysing oocytes with a Pasteur pipette. 
	NOTE: During the oocyte maturation, the oocytes will become progressively translucent. A majority of the oocytes become translucent after treatment of DHP for 2 h. This can be observed under a dissecting microscope with transmitted light optics.</li>
	<li>Remove the outermost follicular cells from each matured oocyte as described in step 2.3.</li>
	<li>Transfer the denuded oocytes to a Petri dish with a few drops of culture medium and proceed to fertilization.</li>
	<li>Prepare the fresh sperm solution using testes dissected from at least three males in 500 &#181;L of Hanks&#39; solution. Put the sperm solution on ice, where it can keep its potency of fertilization for up to 2-3 h.</li>
	<li>Add 100 &#181;L of sperm solution to denuded &#38; matured oocytes on a Petri dish. Gently add the sperm solution among the eggs, and swirl the sperm and eggs together using a pipette tip.</li>
	<li>Immediately add 1 mL of E3 medium solution to activate the eggs and again gently swirl eggs and sperm using a pipette tip.</li>
	<li>After around 1 minute post-fertilization (mpf), flood the plate with E3 medium solution. Full chorion expansion can be observed within 10-15 mpf.</li>
	<li>Between 35-45 mpf, select the embryos that are undergoing symmetrical cleavage into the 2-cell stage, and which are therefore fertilized. Remove embryos that are not undergoing cell cleavage.</li>
	<li>Allow embryos to develop in the Petri dish at 28 &#176;C, with a limit of 80 embryos per 10 cm plate. View and photograph the embryo development.</li>
</ol>

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The authors have nothing to disclose.

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 cells and oocytes from a fully grown stage follicle within 5-10 s using the glass capillary. However, at least 30-60 s is needed to do this separation using forceps. More importantly, usually at least 20% of the denuded oocytes separated by the glass capillary can undergo spontaneous oocyte maturation. In contrast, not beyond 1% of the denuded oocytes can undergo spontaneous oocyte maturation using forceps. Thus, the efficiency and effectiveness of the new method using s glass capillary is better than the previously established method using forceps. Second, this method can be used to separate follicular celld and oocyted of ovarian follicles at early developmental stages such as the PV, EV, and MV stages. Such separation cannot be performed at early-stage follicles using current methodologies. 
There are also a few of limitations using this method. For example, the appropriate diameter of glass capillary opening is crucial for separation of follicular cells and oocytes in this method. An opening much bigger or smaller than the size of ovarian follicles would lead to the failure of separation. Thus, the opening diameter of glass capillary should be adjusted depending on the size of ovarian follicles, which needs some practice. 
Separation of follicular cells and oocytes have several applications in studying ovarian development. Follicular cells and oocytes at different stages of ovarian follicles obtained by this method can be used to analyze gene expression. Follicular cells separated can be used for primary cell culture. Denuded oocytes separated are useful for investigating the crosstalk between follicular cells and oocytes, and supply a good model for studying the oocyte maturation. In addition, this method can be used to remove the follicular cells in some assays such as in vitro oocyte maturation or in vitro fertilization. The availability of a method that greatly increases the ease and speed of separation should facilitate wider exploitation of zebrafish ovarian follicles in the long-term goal of understanding the intricacies of ovarian development. In addition, the structure of ovarian follicles is similar among different fish species; thus, it is interesting to test whether this method can also be applied in other fishes.

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><tbody><tr><td>17&amp;alpha;,20&amp;beta;-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 Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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&amp;nbsp;</td><td></td></tr><tr><td>Fluorescence Stereomicroscope&amp;nbsp;</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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Kaixin Chemical</td><td>500 g</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 medium</td><td>Gibco</td><td>41300-039 (10&amp;times;1L)</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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 stained with DAPI (50 &#181;g/mL) at 37 &#176;C for 30 minutes. The nucleus of follicular cells can be stained by DAPI. The blue signal of DAPI was observed in intact follicles but not denuded oocytes (Figure 2).This indicates that the oocytes and follicular cells of the follicle can be separated cleanly by this method. To further confirm the clear separation, the intact follicles and separated oocytes were cut into the histological sections. DAPI staining results showed that follicular cells were clearly removed in denuded oocytes (Figure 3). All these results suggest that this method can be used to separate follicular cells and oocytes in different stage follicles of zebrafish.
This method can be used for studying the oocyte maturation of zebrafish. To study the bidirectional communication between oocytes and follicular cells during the oocyte maturation, the denuded oocytes must be obtained for various assays. Using a pulled glass capillary, the denuded oocytes separated from full grown stage zebrafish follicles were found to undergo the spontaneous oocyte maturation without any treatment after 4 hours incubation, usually at least 30% separated oocytes survived and the rate of maturity of these survived oocytes could be up to 80% (Figure 4). These matured oocytes could undergo the full chorion expansion after placement into the water, suggesting that the follicular cells were completely removed (Figure 4). Thus, this method can be used to obtain the denuded oocytes for studying the oocyte maturation of zebrafish.
This method can be used for IVF in zebrafish. In vitro maturation (IVM) methods have been well established in zebrafish. Using these methods, the fully grown stage follicles can be induced into matured stage11,12,13,15,16. To further perform in vitro fertilization (IVF), the follicular layer of matured follicles still need to be manually removed. Here, IVM of zebrafish was induced by treatment of DHP. The follicular layer of matured follicles was removed by a capillary glass tube. These defolliculated oocytes could be fertilized and developed into the hatching stage (Figure 5). The result suggests that this method can be used for IVF in zebrafish. Usually around 10% of healthy embryos can be obtained from the matured oocytes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62027/62027fig01.jpg" /> 
Figure 1. Separation of follicular cells and oocytes from zebrafish ovarian follicles using a pulled glass capillary. 1) The morphology of the intact follicle at FG stage. 2)Inhalation of the intact follicle into the pulled glass capillary. 3)The follicular cell is detached from the oocyte when the follicle is blown out from the pulled glass capillary. 4) Successful separation of follicular cells and oocyte. Scale bars: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62027/62027fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62027/62027fig02.jpg" /> 
Figure 2. The morphology of intact follicles and denuded oocytes separated from different stages of follicles after DAPI staining. The intact follicles and separated oocytes from different stages of follicles from PV stage to FG stage were stained by DAPI. The blue signals of DAPI were observed in intact follicles but not denuded oocyte.Scale bars: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62027/62027fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62027/62027fig03.jpg" /> 
Figure 3. Histological sections of intact follicles and isolated oocytes after DAPI staining. Cell nuclei of follicular cells was visualized with DAPI staining. The blue signal can be observed. Follicular cells were clearly removed in denuded oocytes.Scale bars: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62027/62027fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62027/62027fig04.jpg" /> 
Figure 4. The maturation and full chorion expansion of denuded oocytes separated from full grown stage follicles using a pulled glass capillary. (A) The morphology of intact follicles at full grown stage follicles. (B) The denuded oocytes separated from full grown stage follicles can undergo spontaneous oocyte maturation without any treatment after 4 hours incubation. (C) The matured oocytes can undergo full chorion expansion after activation by water.Scale bar: 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62027/62027fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62027/62027fig05.jpg" /> 
Figure 5. The morphology of early embryos obtained by IVM and IVF. IVM was induced by treatment of DHP (5 &#181;g/mL) for 2 h. After IVM, the follicular cells were removed from the matured oocytes by a pulled glass capillary. The matured oocytes were fertilized by IVF. The development of embryos at different stages was viewed and photographed. Scale bars: (A-I) 200 &#181;m, (J) 1 mm.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62027/62027fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

7.4K Views.  College of Life Sciences, Northwest Normal University. 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.

Video: Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

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 within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between 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 membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

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 described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

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

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

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 reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

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

Dissection of Larval Zebrafish Gonadal Tissue

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.

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

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Improved Procedure to Obtain Granulosa Cell-Free Stage I Zebrafish Oocytes

Rapid Isolation of Stage I Oocytes in Zebrafish Devoid of Granulosa Cells

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model organism to investigate early developmental processes from oocyte to embryo. In zebrafish, oocytes are surrounded by a single layer of somatic granulosa cells. However, separating granulosa cells from oocytes poses a challenge, as achieving pure oocytes is crucial for precise analysis. Although various methods have been proposed to isolate zebrafish oocytes at different developmental stages, current techniques fall short in removing granulosa cells completely, limiting the accuracy of genome analysis focused solely on oocytes. In this study, we successfully developed a rapid and efficient process for isolating pure stage I oocytes in zebrafish while eliminating granulosa cell contamination. This technique facilitates biochemical and molecular analysis, particularly in exploring epigenetic and genome structure aspects specific to oocytes. Notably, the method is user-friendly, minimizes oocyte damage, and provides a practical solution for subsequent research and analysis.

Author Spotlight: Enhanced Method for Isolating Pure Zebrafish Stage I Oocytes

This protocol describes a modified procedure for rapidly isolating clean stage I oocytes in zebrafish devoid of granulosa cells, thereby providing a convenient method for oocyte-specific research.

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model ...

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 &#181;m cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 &#181;m and 40 &#181;m cell strainers. The contents retained in the 40 &#181;m strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and ...

Biology

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

Separation of Hen Ovarian Follicle Layers for Reproductive Research Applications

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable source of protein. They are also an emerging animal model for the study of human reproductive disease. As the field of poultry research develops, the health and function of the layer hen and broiler breeder ovary will be an important point of study for both agricultural and biomedical researchers. One of the challenges presented by this emerging interest is the need for replicable techniques that all researchers can employ in ovarian specimen collection. In particular, a detailed visual process must be established to define the proper separation of the specialized granulosa and theca cell layers from hen follicles to achieve agreement and consistency among researchers. 
This study describes the extraction of preovulatory follicles and ovary tissue in white leghorn hens of prime reproductive age. The separation of these follicles is performed under cold, liquid conditions to congeal the yolk for easier manipulation and to prevent the follicle's own weight from tearing apart cell layers during the separation process. Once the separation is complete, the desired cell layers can be further digested for tissue culture approaches or can be cryopreserved for genomic and proteomic analyses.

Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights.

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

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Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Isolation and Characterization of Single Cells from Zebrafish Embryos

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

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

Dissection of Larval Zebrafish Gonadal Tissue

Rapid Isolation of Stage I Oocytes in Zebrafish Devoid of Granulosa Cells

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications