This work was supported by the National Natural Science Foundation of China (32170813 and 31871449) and Science and Technology Department of Sichuan (2024NSFSC0651), and 1&#183;3&#183;5 project for disciplines of excellence&#8211;Clinical Research Fund, West China Hospital, Sichuan University (2024HXFH035). The authors would like to thank Zhao Wang and Yanqiu Gao of the Laboratory of Pediatric Surgery for breeding of zebrafish related to this work. The authors would also like to thank all the reviewers who participated in the review, as well as MJEditor (www.mjeditor.com) for providing English editing services during the preparation of this manuscript.

All zebrafish were handled following stringent animal care guidelines outlined by the relevant national and/or local animal welfare bodies. The maintenance and handling of fish received approval from both local authorities and the animal ethics committee of the West China Hospital of Sichuan University (approval No. 20220422003). Zebrafish ovaries contain a mixture of multi-stage oocytes, with each developmental stage being present in adult zebrafish ovaries. However, juvenile zebrafish were selected for this study due to the dominance of late-stage oocytes (stage II-III) in the ovaries of adult fish, which are large and opaque. In contrast, juveniles have flat and elongated ovaries primarily consisting of stage I oocytes (Figure 1B) (Figure 1B). Previous research has demonstrated that the morphological features of early oogenesis in adult and juvenile zebrafish are highly consistent, supporting the feasibility of utilizing juvenile zebrafish13. The procedures described below, outlined in Figure 2, provide a detailed description of the isolation process, from dissection to obtaining clean stage I oocytes. For further reference, the reagents, buffers, and equipment utilized in the study are listed in the Table of Materials.
1. Ovary dissection
<ol>
	<li>Select several female zebrafish with a standard length (SL) ranging from 10 mm to 15 mm. 
	NOTE: The SL, measured from the snout to the tail base in millimeters, serves as a reliable criterion for selecting appropriate fish. It is recommended to use juvenile fish between 5 and 7 weeks post-fertilization (wpf) with an SL ranging from 10 mm to 15 mm for this procedure. The ovaries of juvenile zebrafish are predominantly occupied by early oocytes, providing optimal conditions for easily obtaining stage I oocytes (Figure 1B).</li>
	<li>Euthanize the juvenile female fish by placing them in ice-cold water (0-4 &#176;C). 
	NOTE: The following procedures must be carried out to ensure euthanasia and minimize the pain experienced by zebrafish during euthanasia: confirm that the temperature of the ice-cold water is within the 0-4 &#176;C range using a thermometer, swiftly transfer the zebrafish to the ice-cold water, and maintain them in the ice-cold water for at least 10 min after cessation of opercular movement16,17,18.</li>
	<li>Dry excess water on the fish&#39;s surface using absorbent paper.</li>
	<li>Dissect the zebrafish under a stereomicroscope by following these steps:
	<ol>
		<li>Cut off the head using micro-scissors along the posterior margin of the gill cover; cut off the tail along the cloaca. Transfer the middle trunk to a 35 mm dish containing 2 mL of L-15 medium (with L-glutamine).</li>
		<li>Dissect the middle trunk in the L-15 medium. Cut along the central axis of the abdomen and remove the viscera and swim bladder. 
		NOTE: The viscera of zebrafish are connected. Therefore, pinch the hindgut near the anal region and lift it to easily remove the entire visceral mass.</li>
		<li>Detach the bilateral ovaries from the abdomen using tweezers. Remove the adipose tissue, scales, and other body tissues attached to the ovary. 
		NOTE: The ovaries are covered with adipose tissue. Therefore, it is best to remove them together to avoid mechanical damage to the oocytes. 
		CAUTION: Handle tweezers with care to avoid injury.</li>
	</ol>
	</li>
</ol>
2. Digestion
<ol>
	<li>Transfer the ovaries to a 6-well plate containing 2 mL of Kinger&#39;s cell dissociation solution and incubate for 2-3 h&#160;at 28.5 &#176;C. Shake the 6-well plate every 30 min to promote dispersion. 
	NOTE: Use tweezers to separate the ovaries into small tissue blocks to enhance digestion efficacy. The digestion time is variable; regularly monitor and terminate the process when a significant number of stage I oocytes are scattered under the stereomicroscope. The Kinger&#39;s cell dissociation solution is the working solution and can be used directly. It can be stored stably at &#8722;20 &#176;C after packaging and thawed at a low temperature (4 &#176;C) before use.</li>
	<li>Add L-15 medium preheated at 28.5 &#176;C to the digestion buffer to stop digestion.</li>
</ol>
3. Filtration
<ol>
	<li>Place a 100 &#181;m cell strainer into another well of the 6-well plate, add L-15 medium, and ensure that the medium level is higher than the filter. 
	NOTE: The theoretical diameter of stage I oocytes is less than 140 &#181;m. However, oocytes with larger diameters may still pass through the cell strainer. Therefore, a cell strainer with a smaller diameter than the target was selected at this step. Mesh sizes can be adjusted depending on the specific experimental needs.</li>
	<li>Draw the digestive medium through the cell strainer using a pipette. Wait 1-2 min until all the stage I oocytes have passed through the strainer, then remove it. Ensure that the oocytes remain in the liquid throughout the transfer process. 
	NOTE: Keeping the oocytes in the medium helps maintain their integrity and optimal condition. If the oocytes are exposed to air for an extended period, they could dry out and become damaged, affecting the final separation efficiency. Keep the pipette below the liquid surface when transferring the oocytes through the cell strainer rather than adding them in a suspended manner.</li>
</ol>
4. Washing
<ol>
	<li>Remove excess medium using a pipette. Add 4 mL of fresh L-15 medium and gently resuspend the oocytes. Wait 1-2 min, then remove the supernatant. 
	NOTE: Use instruments with low adhesion properties to prevent damage to the oocytes.</li>
	<li>Repeat step 4.1 5-6 times until no other impurities are present. 
	NOTE: Gentle handling during the washing process is crucial to avoid oocyte damage. Despite obtaining an abundance of stage I oocytes (approximately 200-400 stage I oocytes from one zebrafish) following the previous washing steps, the oocytes may still be mixed with cell fragments, oocytes from other stages, or small granules formed by broken late-stage oocytes. Additionally, some granulosa cells may still adhere to the surface of several oocytes due to incomplete enzyme digestion. Therefore, for applications demanding a higher level of purity, such as genome sequencing, additional steps are necessary to select completely clean oocytes. To address this, proceed to steps 5 and 6.</li>
</ol>
5. Selection for quality control
<ol>
	<li>Transfer the washed stage I oocytes to a 35 mm dish containing fresh L-15 medium.</li>
	<li>Under a microscope with a magnification equal to or exceeding 10x, remove cell fragments, other-stage oocytes, and stage I oocytes adhered by granulosa cells using a tool that can be manipulated with precision, such as a blunt injection needle. 
	NOTE: This step requires patience from the experimenter, but it ensures the isolation of clean and intact oocytes. 
	CAUTION: Handle injection needles with care to avoid injury.</li>
</ol>
6. Confirmation by nuclear staining
NOTE: For further refinement of the isolated oocytes, staining with Hoechst 33342 was employed to identify and remove individual oocytes adhered with granulosa cells that may have been missed in the previous step.
<ol>
	<li>Add a final concentration of 5 &#181;g/mL Hoechst 33342 to the L-15 medium and incubate at room temperature for 10 min.</li>
	<li>Observe the Hoechst fluorescence through a fluorescence microscope under UV laser excitation. 
	NOTE: Hoechst is typically excited in the ultraviolet region, usually at wavelengths between 310 and 360 nm. It is recommended to use a magnification between 80x and 100x during this process. Employ the automatic exposure mode. The excitation wavelength and exposure time can be adjusted based on the specific microscope model and individual requirements.</li>
	<li>Using a needle, carefully pick out the oocytes that do not meet the desired criteria. 
	NOTE: When visualizing the cells, a large nuclear chromatin is present in the center of the oocyte. If granulosa cells were adhered to the oocyte, small and bright nuclei would be visible, clinging to the oocyte&#39;s edge (Figure 3). Pick them out under the microscope.</li>
	<li>Thoroughly and repeatedly wash the oocytes with L-15 to remove excess Hoechst.</li>
	<li>Use the identified stage I oocytes directly for subsequent extraction or store them at &#8722;80 &#176;C after flash-freezing in liquid nitrogen. 
	CAUTION: Wear a protective face mask and cryogenic gloves when handling liquid nitrogen and ultra-low temperature freezers.</li>
</ol>

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

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E., Schleiffer, A., Mechtler, K., Pauli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+ski7+tunes+RNA+levels+during+the+oocyte-to-embryo+transition.">Zebrafish ski7 tunes RNA levels during the oocyte-to-embryo transition.</a> PLoS Genet. 17 (2), e1009390 (2021).</li><li>Liu, Y., 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=Single-cell+transcriptome+reveals+insights+into+the+development+and+function+of+the+zebrafish+ovary.">Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary.</a> Elife. 11, 76014 (2022).</li><li>Li, C. 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=Spatiotemporal+expression+of+bone+morphogenetic+protein+family+ligands+and+receptors+in+the+zebrafish+ovary:+A+potential+paracrine-signaling+mechanism+for+oocyte-follicle+cell+communication.">Spatiotemporal expression of bone morphogenetic protein family ligands and receptors in the zebrafish ovary: A potential paracrine-signaling mechanism for oocyte-follicle cell communication.</a> Biol Reprod. 85 (5), 977-986 (2011).</li><li>Zampolla, T., Spikings, E., Rawson, D., Zhang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeleton+proteins+F-actin+and+tubulin+distribution+and+interaction+with+mitochondria+in+the+granulosa+cells+surrounding+stage+III+zebrafish+(Danio+rerio)+oocytes.">Cytoskeleton proteins F-actin and tubulin distribution and interaction with mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes.</a> Theriogenology. 76 (6), 1110-1119 (2011).</li><li>Lubzens, E., Young, G., Bobe, J., Cerda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+teleosts:+How+eggs+are+formed.">Oogenesis in teleosts: How eggs are formed.</a> Gen Comp Endocrinol. 165 (3), 367-389 (2010).</li><li>Song, Y., Hu, 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=Establishment+of+transgenic+zebrafish+(Danio+rerio)+models+expressing+fluorescence+proteins+in+the+oocytes+and+somatic+supporting+cells.">Establishment of transgenic zebrafish (Danio rerio) models expressing fluorescence proteins in the oocytes and somatic supporting cells.</a> Gen Comp Endocrinol. 314, 113907 (2021).</li><li>Sousa, M. 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=Reproductive+hormones+affect+follicular+cells+and+ooplasm+of+stage+I+and+II+oocytes+in+zebrafish.">Reproductive hormones affect follicular cells and ooplasm of stage I and II oocytes in zebrafish.</a> Reprod Fertil Dev. 28 (12), 1945-1952 (2016).</li><li>Yan, Y. 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=Gonadal+soma+controls+ovarian+follicle+proliferation+through+Gsdf+in+zebrafish.">Gonadal soma controls ovarian follicle proliferation through Gsdf in zebrafish.</a> Dev Dyn. 246 (11), 925-945 (2017).</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> J Morphol. 218 (2), 203-224 (1993).</li><li>Wallace, R. A., Selman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+aspects+of+oogenesis+and+oocyte+growth+in+fish+and+amphibians.">Ultrastructural aspects of oogenesis and oocyte growth in fish and amphibians.</a> J Electron Microsc Tech. 16 (3), 175-201 (1990).</li><li>Elkouby, Y. M., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+analysis+of+early+oogenesis+in+zebrafish.">Methods for the analysis of early oogenesis in zebrafish.</a> Dev Biol. 430 (2), 310-324 (2017).</li><li>Ai, N., Liu, L., Lau, E. S., Tse, A. C., 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=Separation+of+oocyte+and+follicle+layer+for+gene+expression+analysis+in+zebrafish.">Separation of oocyte and follicle layer for gene expression analysis in zebrafish.</a> Methods Mol Biol. 2218, 1-9 (2021).</li><li>Zhan, C., 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=Explorations+of+the+optimal+method+for+isolating+oocytes+from+zebrafish+(Danio+rerio)+ovary.">Explorations of the optimal method for isolating oocytes from zebrafish (Danio rerio) ovary.</a> J Exp Zool B Mol Dev Evol. 330 (8), 417-426 (2018).</li><li>Avma guidelines for the euthanasia of animals: 2020 edition. Avma Available from: <a target="_blank" href="https://www.avma.org/resources-tools/avma-policies/avma-guidelines-euthanasia-animals">https://www.avma.org/resources-tools/avma-policies/avma-guidelines-euthanasia-animals</a> (2020)</li><li>Phs policy on humane care and use of laboratory animals. Olaw Available from: <a target="_blank" href="https://olaw.nih.gov/policies-laws/phs-policy.htm#Introduction">https://olaw.nih.gov/policies-laws/phs-policy.htm#Introduction</a> (2015)</li><li>Matthews, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+euthanasia+in+zebrafish.">Anesthesia and euthanasia in zebrafish.</a> ILAR J. 53 (2), 192-204 (2012).</li></ol>

The authors have nothing to disclose.

In this study, we developed a method for isolating pure and clean stage I oocytes, excluding granulosa cells, for downstream analysis (particularly genomic analyses). Comparing this modified method with the referenced method13, the stage I oocytes obtained using this method are morphologically intact, sufficient in number, and free from contamination with other somatic cells, making them suitable for various subsequent studies and analyses. Furthermore, compared with other mechanical separation methods14,15, the improved method is simpler to operate, facilitates the acquisition of a larger number of oocytes, and prevents oocyte damage.
Moreover, in addition to effectively isolating stage I zebrafish oocytes, this method can also be applied to pond loaches, indicating its broad applicability among a variety of fish species. To investigate the impact of granulosa cell contamination on gene expression and epigenetic analysis, we conducted RNA sequencing (RNA-seq) and CUT&amp;Tag analysis on oocytes obtained using the reference and modified methods. Although a few differentially expressed genes were identified by RNA-seq, some genes that were specifically expressed in granulosa cells were still identified (data not shown). The analysis also revealed substantial differences in histone modifications between oocytes isolated with different methods (data not shown). These results underscore the importance of this method in completely separating oocytes and granulosa cells for accurate gene expression and epigenetic analysis.
Several aspects require attention when implementing the proposed method: (1) Selecting the appropriate size of zebrafish is crucial. This ensures that enough oocytes are obtained and reduces subsequent washing steps. The ovaries of zebrafish with an SL &lt;10 mm do not provide sufficient oocytes, thus requiring a larger number of fish to meet sampling requirements and increasing breeding pressure. Conversely, zebrafish with an SL greater than 15 mm exhibit the gradual formation of stage II-III oocytes, the membranes of which are easily broken during the digestion reaction. (2) When larger fish are used, the leakage of cell contents resulting from damaged oocytes may lead to contamination, thereby inhibiting the digestion reaction to some extent. In such cases, removing the old digestive buffer, washing it with L-15, and then adding a new Kinger's cell dissociation solution for further digestion is recommended. (3) If appropriate equipment is available, shaking during digestion can enhance separation.
In step 3.1, smaller granulosa cells may accompany oocytes through the cell strainer. However, practical observations indicate that the final collected oocyte sample does not contain granulosa cells. Except in cases where incomplete digestion resulted in the presence of residual granulosa cells in some oocytes, no other Hoechst-labeled granulosa cells were detected in the system. Here, we hypothesized that granulosa cells, being smaller and lighter, might remain suspended in the culture medium and were subsequently removed during the multiple washing steps. The isolation efficiency was also quantified by calculating the proportion of oocytes with granulosa cell attachment and clean oocytes after Hoechst staining. Clean oocyte rates of up to 98% were observed in samples containing 1000 oocytes.
Despite its many advantages, the proposed approach also had some noteworthy limitations. Firstly, this method was validated for DNA or RNA extraction, sequencing, and immediate detection of intact live cells, but the feasibility and reliability of extending to other assay areas are yet to be validated. Another limitation is that the proposed method can only be applied for isolating stage I oocytes, as the isolation efficiency for other stages is unsatisfactory. Therefore, additional efforts are required to improve the efficiency and applicability of the proposed method. Additionally, future studies should focus on exploring whether the proposed method can be applied to efficiently isolate oocytes from other stages, thereby broadening its applicability.

The zebrafish is among the most important model systems in developmental biology. In recent years, numerous studies have utilized the zebrafish as a model to study important biological events and regulatory processes from oocyte to embryo. These encompass the intricate processes of oocyte development and maturation1, the functionality of maternal genes2, the regulation of maternal-zygotic transitions3, and extensive omics analyses4.
Granulosa cells, the somatic cells enveloping and nurturing the developing oocyte within the ovarian follicle5,6, play a pivotal role in this developmental process. As primordial germ cells (PGCs) evolve into oogonia, they become surrounded by a monolayer of granulosa cells7. Together with the external thecal cells, the oocyte and its surrounding granulosa cells constitute a mature follicle8. Given the fundamental distinction between germ cells and somatic cells, obtaining a pure oocyte sample is imperative, especially for genome-related analyses.
Within the follicular structure of zebrafish, granulosa cells typically exhibit a diameter of only a few microns8, emphasizing the intimate interconnection between granulosa cells and oocytes9. This close association presents a challenge in achieving complete separation due to the considerable difference in both the number and volume of granulosa cells versus oocytes (hundreds of granulosa cells compared to a single oocyte)10,11. Even minimal contamination with a single granulosa cell can impede downstream analyses specifically targeting oocytes. Therefore, for studies focusing on genomic and epigenetic characteristics, the elimination of granulosa cells is essential.
Benefiting from well-characterized morphological criteria, oocytes at each stage can be distinguished based on diameter11. The oogenesis process in zebrafish is categorized into five stages according to morphology and karyotype11. Stage I oocytes (7-140 &#181;m diameter) encompass oocytes from the onset of meiosis to the early stage of meiosis I. Crucially, these oocytes are transparent, allowing for the observation of the central nucleus through transmitted light (Figure 1Ai). Stage II oocytes (140-340 &#181;m diameter) gradually become foamy and translucent. With the enlargement of follicles and the proliferation of cortical alveoli, the germinal vesicles in the center become difficult to distinguish12 (Figure 1Aii). Stage III oocytes (340-690 &#181;m diameter) progressively accumulate vitellogenin, and fresh follicles become increasingly opaque (Figure 1Aiii). Meiosis continues in stage IV oocytes (690-730 &#181;m diameter) as chromosomes enter the middle of meiosis II, where they stagnate (Figure 1Aiv). Stage V oocytes (730-750 &#181;m diameter) have matured and are ready for ovulation7,11(Figure 1Av).
Based on the unique characteristics of each of the aforementioned stages, a method has been proposed to isolate oocytes from stages I to III by digesting zebrafish ovaries using a digestive solution containing collagenase I, collagenase II, and hyaluronidase, followed by filtration through a specific-sized cell strainer13. However, while this method allows for obtaining oocytes at different developmental stages, it fails to completely separate oocytes and granulosa cells. Other researchers have also suggested methods to separate granulosa cells from oocytes. However, these methods primarily rely on mechanical approaches, which can cause oocyte damage, are time-consuming, and are inadequate for obtaining a substantial number of oocytes for analysis14,15.
Given the limitations of existing methods and specific research requirements, this study aims to establish a procedure to thoroughly separate oocytes and granulosa cells and obtain a sufficient number of clean stage I oocytes for analysis. Expanding upon the referenced method13, we employ an improved digestion buffer (see Table of Materials) that is gentler and facilitates the dispersion of oocytes and dissociation of granulosa cells. Subsequently, the oocytes are passed through a cell strainer, followed by washing and further microscopic selection, enabling the acquisition of a large number of clean stage I oocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Kinger&#39;s cell dissociation solution</td>
			<td>PlantChemMed</td>
			<td>PC-33689</td>
			<td>Kinger&#39;s cell dissociation solution can be stored stably at -20 &#176;C after packaging and can be used after thawing at low temperature (4 &#176;C). It can be used directly for dissociating zebrafish ovaries. The optimal temperature is 28.5 &#176;C, for approximately 2-3 hours. The duration can be adjusted according to the specific dissociation conditions, either shortened or extended (https://www.plantchemmed.com/chanpin?productNo=PC-33689).</td>
		</tr>
		<tr>
			<td>Cell strainers (100 &#956;m )</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Zeiss</td>
			<td>Axio Zoom.V16</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>#5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary needle</td>
			<td>/</td>
			<td>/</td>
			<td>Blunted by burning with lighter</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Yesen</td>
			<td>40732ES03</td>
			<td></td>
		</tr>
		<tr>
			<td>Low adsorption pipette tips (10 &#956;l )</td>
			<td>Labsellect</td>
			<td>T-0010-LR-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s L-15 medium medium (with L-glutamine)</td>
			<td>Hyclone</td>
			<td>SH30525.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>/</td>
			<td>/</td>
			<td>Ice-cold water is used to euthanize zebrafish</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>WIGGENS</td>
			<td>WH-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Juvenile fish</td>
			<td>/</td>
			<td>/</td>
			<td>5&#8211;6 weeks post-fertilization, standard length [SL] of 10&#8211;15 mm</td>
		</tr>
		<tr>
			<td>Plastic dish (35 mm )</td>
			<td>SORFA</td>
			<td>230101</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Motic</td>
			<td>SMZ-161</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Plate (6-wells)</td>
			<td>SORFA</td>
			<td>0110006</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>Fine Science Toosl</td>
			<td>#15000-00</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 illustrates the ovarian morphology observed in both adult and juvenile zebrafish, showcasing oocytes at different developmental stages to serve as a reference. Figure 1A provides a graphical representation of oocyte morphology and size at each developmental stage, beginning with the primary growth stage (stage I) and ending with the ovulated egg (stage V). Figures 1Ai-v illustrate the diameters corresponding to stages I to V of the oocytes. Figure 1B (left panel) displays ovarian images in juvenile fish (6 wpf with SL = 1.5 cm). Notably, the juvenile ovary exhibits a distinct lobular appearance, primarily characterized by an abundance of transparent stage I oocytes, accompanied by a smaller population of stage II oocytes. This prevalence of stage I oocytes greatly facilitates their observation and retrieval. The right panel depicts the morphology of the adult fish ovary (6 months), where oocytes at various developmental stages can be observed. However, opaque late-stage oocytes (stage II-III) are predominant in the ovary.
Figure 2 illustrates the representative processes from dissection to obtaining clean stage I oocytes. Figure 3 presents stage I oocytes obtained using both the reference method and the modified method. Following Hoechst staining, the oocyte and granulosa cell nuclei exhibited blue fluorescence under UV light. In the images obtained using the reference method (Figure 3A), the oocytes&#39; surface was densely surrounded by numerous stained granulosa cell nuclei. In contrast, stage I oocytes separated by the modified method (Figure 3B) exhibit complete exposure and cleanliness, with no granulosa cell nuclei staining present.
Our findings demonstrated the effectiveness of the proposed method for isolating stage I oocytes. The developmental process of oocytes in teleost fish exhibits several common characteristics7. To assess its cross-species applicability, further analyses were conducted on other fish species. Female pond loaches were acquired from a local market, and early-stage oocytes (diameter &#60;100 &#181;m) were isolated following the same procedure. The ovarian composition of the pond loach also displayed a mixture of oocytes at multiple developmental stages, characterized by a striking resemblance in oocyte morphology and size to that of zebrafish during the same stage. As illustrated in Figure 4A, the ovaries of adult female pond loach were dominated by opaque late oocytes interspersed with transparent early oocytes. A clear monolayer of granulosa cells was observed on the surface of oocytes. Consistent with our observations in zebrafish, the loach oocytes measuring less than 100 &#181;m in diameter corresponded to the stage prior to the diplotene stage. Figure 4B depicts the clean pond loach (Misgurnus anguillicaudatus) oocytes obtained by the modified method. Compared to zebrafish ovaries, pond loach ovaries are more challenging to dissociate. After 6-7 h&#160;of digestion, more early-stage oocytes dissociate. Nonetheless, this adjusted approach yielded the desired clean and naked oocytes. Zebrafish belong to the Cyprinidae family, whereas pond loaches are classified within the Cobitidae family. These two families exhibit a distant taxonomic relationship, but their ovarian development stage and morphology are remarkably similar, indicating that this method can generally be utilized for analyzing oocytes of various fish species.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66458/66458fig01v2.jpg" /> 
Figure 1: Representative images of zebrafish ovaries and oocytes at different stages. (A) Morphology and size of oocytes at each developmental stage (St). i-v represents the images and diameter of the oocytes from stage I to V. Scale bar = 200 &#181;m. (B) Ovarian morphology of adult and juvenile zebrafish. Scale bars = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66458/66458fig01largev2.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/66458/66458fig02v2.jpg" /> 
Figure 2: Representative images of the isolation procedure, from dissection to the acquisition of clean stage I oocytes. <a href="https://www.jove.com/files/ftp_upload/66458/66458fig02largev2.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/66458/66458fig03.jpg" /> 
Figure 3: Stage I oocytes obtained through the two methods. (A) Stage (St) I oocytes isolated by the referenced method. (B) Stage I oocytes isolated through the modified method. The oocyte and granulosa cell nuclei were stained with Hoechst and excited with UV light. Scale bars = 100 &#181;m. BF = bright field. <a href="https://www.jove.com/files/ftp_upload/66458/66458fig03large.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/66458/66458fig04.jpg" /> 
Figure 4: Early-stage oocytes from pond loach obtained by the modified method. (A) Representative images of ovary and oocytes of pond loach. i, ii represent the ovaries of pond loaches under different magnifications, and iii-iv represents the early-stage oocytes of pond loaches. Scale bars: i (500 &#181;m); ii (200 &#181;m); iii, iv (100 &#181;m). (B) Early-stage oocytes isolated from pond loach. The oocytes and granulosa cell nuclei were stained with Hoechst and excited with UV light. Scale bars = 100 &#181;m. BF = bright field. <a href="https://www.jove.com/files/ftp_upload/66458/66458fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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 ...

rapid-isolation-stage-i-oocytes-zebrafish-devoid-granulosa

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

571 Views.  Sichuan University. 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.

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

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

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 ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

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 disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

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 ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...