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

<ol><li>Qin, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unraveling+the+mechanism+of+long-term+bisphenol+S+exposure+disrupted+ovarian+lipids+metabolism%2C+oocytes+maturation%2C+and+offspring+development+of+zebrafish%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">Unraveling the mechanism of long-term bisphenol S exposure disrupted ovarian lipids metabolism, oocytes maturation, and offspring development of zebrafish.</a> Chemosphere. 277, 130304(2021).</li>
<li>Hau, H. T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+Larp6+controls+oocyte+development%2C+chorion+formation+and+elevation%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Maternal Larp6 controls oocyte development, chorion formation and elevation.</a> Development. 147 (4), 187385(2020).</li>
<li>Cabrera-Quio, L. E., Schleiffer, A., Mechtler, K., Pauli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+ski7+tunes+RNA+levels+during+the+oocyte-to-embryo+transition%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">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/pubmed?term=((Single-cell+transcriptome+reveals+insights+into+the+development+and+function+of+the+zebrafish+ovary%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">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/pubmed?term=((Spatiotemporal+expression+of+bone+morphogenetic+protein+family+ligands+and+receptors+in+the+zebrafish+ovary%3A+A+potential+paracrine-signaling+mechanism+for+oocyte-follicle+cell+communication%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">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/pubmed?term=((Cytoskeleton+proteins+F-actin+and+tubulin+distribution+and+interaction+with+mitochondria+in+the+granulosa+cells+surrounding+stage+III+zebrafish+%28Danio+rerio%29+oocytes%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">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/pubmed?term=((Oogenesis+in+teleosts%3A+How+eggs+are+formed%5BTitle%5D)+AND+%22Gen+Comp+Endocrinol%22%5BJournal%5D)">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/pubmed?term=((Establishment+of+transgenic+zebrafish+%28Danio+rerio%29+models+expressing+fluorescence+proteins+in+the+oocytes+and+somatic+supporting+cells%5BTitle%5D)+AND+%22Gen+Comp+Endocrinol%22%5BJournal%5D)">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/pubmed?term=((Reproductive+hormones+affect+follicular+cells+and+ooplasm+of+stage+I+and+II+oocytes+in+zebrafish%5BTitle%5D)+AND+%22Reprod+Fertil+Dev%22%5BJournal%5D)">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/pubmed?term=((Gonadal+soma+controls+ovarian+follicle+proliferation+through+Gsdf+in+zebrafish%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">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/pubmed?term=((Stages+of+oocyte+development+in+the+zebrafish%2C+Brachydanio+rerio%5BTitle%5D)+AND+%22J+Morphol%22%5BJournal%5D)">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/pubmed?term=((Ultrastructural+aspects+of+oogenesis+and+oocyte+growth+in+fish+and+amphibians%5BTitle%5D)+AND+%22J+Electron+Microsc+Tech%22%5BJournal%5D)">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/pubmed?term=((Methods+for+the+analysis+of+early+oogenesis+in+zebrafish%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Separation+of+oocyte+and+follicle+layer+for+gene+expression+analysis+in+zebrafish%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Explorations+of+the+optimal+method+for+isolating+oocytes+from+zebrafish+%28Danio+rerio%29+ovary%5BTitle%5D)+AND+%22J+Exp+Zool+B+Mol+Dev+Evol%22%5BJournal%5D)">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>
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</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 me...

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><tbody><tr><td>Kinger&#039;s cell dissociation solution</td><td>PlantChemMed</td><td>PC-33689</td><td>Kinger&#039;s cell dissociation solution can be stored stably at -20 &amp;deg;C after packaging and can be used after thawing at low temperature (4 &amp;deg;C). It can be used directly for dissociating zebrafish ovaries. The optimal temperature is 28.5 &amp;deg;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 &amp;mu;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 &amp;mu;l )</td><td>Labsellect</td><td>T-0010-LR-R-S</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;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&amp;ndash;6 weeks post-fertilization, standard length [SL] of 10&amp;ndash;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 oocy...

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

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717 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 ...

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 ...

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 ...

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.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

Biology

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

Improving IVF Outcomes with Partial Granulosa Cell Removal Post-Retrieval

The Immediate Partial Removal of Cumulus-Oocyte Complexes: A Refined Approach for Rapid Observation of In Vitro Fertilization

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients continue to experience fertilization disorders, leading to low fertilization rates and the production of nonviable embryos. Short-term fertilization involves the early removal of granulosa cells to observe the extrusion of the second polar body, enabling the assessment of fertilization and early remedial measures to address low fertilization rates and complete fertilization failure. However, the observation of short-term fertilization in IVF is impeded by challenges such as excessively large unprocessed clusters of cumulus-oocyte complexes and adhesion between eggs, necessitating complex external procedures for granulosa cell removal, and prolonged observation duration. To tackle this issue, this study proposes a method of immediate partial removal of granulosa cells post egg retrieval. This approach streamlines subsequent stages of short-term fertilization and traditional fertilization monitoring, reduces the time needed for oocyte extrusion, minimizes the likelihood of external environmental impacts on the oocytes, and decreases the risk of missing the critical fertilization observation window. Consequently, the study yields novel clinical evidence aimed at enhancing the operational efficiency of embryonic laboratories in IVF.

Author Spotlight: Evaluating the Impact of Immediate Partial Removal of Cumulus-Oocyte Complexes on Fertilization Efficiency and Embryo Quality

Here, we present an optimized method for promptly removing a portion of cumulus-oocyte complexes following egg retrieval to expedite the IVF observation process, reducing the time required for granulosa cell removal, minimizing oocyte exposure to external elements, and does not hinder embryo development, ultimately improving the efficiency of IVF laboratory procedures.

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients ...

Medicine

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 ...

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 ...

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

Summary

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

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