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 t...

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. 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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. 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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 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.

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

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

This research is focused on establishing process for rapidly and efficiently isolating pure stage one ooyctes in zebrafish while eliminating granulosa cell contamination. The primary aim is to develop a method that facilities more precise analysis in oocyte specific research areas, particularly in genome and epigenetic research. The oocytes of zebrafish are enveloped by a mono-layer of granule cells.It's difficult to swiftly separate granule cells from oocytes due to the significant disparity in both number and volume, making it challenging to obtain pure oocyte samples for specific analysis, especially in genome related studies. This method has survival advantage over mechanical methods and the previous detection approaches. First, the improved detection buffer is gentler, helping to isolate the granulosa cells while minimizing damage to the oocytes.Plus, it allows us to get plenty of clean stage one oocytes. We hope as research helps scientists get a better look at the detail and growth of the phage oocytes, including cell stack, epigenetics and genome structure. This method work just as well for pompanos, and we think it could be applied to study a lot of different kind of fish.

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...

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

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

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

Research

JoVE Journal

Developmental Biology

296 ビュー.  Sichuan University. この研究は、ゼブラフィッシュの純粋なステージ1のooyctesを迅速かつ効率的に分離し、顆粒膜細胞の汚染を排除するプロセスを確立することに焦点を当てています。主な目的は、卵子固有の研究領域、特にゲノムおよびエピジェネティックな研究において、より正確な解析を行う方法を開発することです。ゼブラフィッシュの卵母細胞は、単層?...

ビデオ: 著者スポットライト:純粋なゼブラフィッシュステージI卵子を単離するための強化された方法(英語)

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...

https://cloudfront.jove.com/CDNSource/teasers/66458_b.jpg

zebrafish ovary excision for obtaining granulosa cell-free stage i oocytes

Zebrafish Ovary Excision for Obtaining Granulosa Cell-Free Stage I Oocytes

isolation of granulosa cell-free stage i oocytes from zebrafish ovaries

Isolation of Granulosa Cell-Free Stage I Oocytes from Zebrafish Ovaries

an intraperitoneal injection technique in adult zebrafish that minimizes body damage and associated mortality

Author Spotlight: Novel Intraperitoneal Injection Method for Zebrafish Cardiotoxicity Models

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish...

Optimized Intraperitoneal Injection for Compound Delivery in Adult Zebrafish

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separation of follicular cells and oocytes in ovarian follicles of zebrafish

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

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

techniques for imaging prometaphase and metaphase of meiosis i in fixed drosophila oocytes

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

novel quantification protocol for cardiovascular calcification progression using longitudinal micropet/microct images

Author Spotlight: Enhanced Quantification of Cardiovascular Calcification Progression for Longitudinal Micro PET/CT Studies in Small Research Animals

This novel protocol entails the quantification of cardiovascular calcification progression from serial micro positron emission tomography (PET)/micro computed tomography (CT) images in small research animals.

Longitudinal Quantification of Cardiovascular Calcification Using PET/CT Imaging

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fertilization of xenopus oocytes using the host transfer method

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer...

Fertilization of Xenopus oocytes using the Host Transfer Method

three-dimensional reconstruction for the whole lung with early multiple pulmonary nodules

Author Spotlight: Advancing 3D Modeling for Enhanced Diagnosis and Treatment of Pulmonary Nodules in Early-Stage Lung Cancer 

This study introduces a three-dimensional (3D) reconstruction method for the entire lung in patients with early multiple pulmonary nodules. It offers a comprehensive visualization of nodule distribution and their interplay with lung tissue, simplifying the assessment of diagnosis and prognosis for these...

Enhancing Pulmonary Nodule Diagnosis Using Whole-Lung 3D Reconstruction

using mouse oocytes to assess human gene function during meiosis i

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse...

gut isolation from zebrafish larvae for single-cell rna sequencing

Author Spotlight: Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing...

Processing Gut Cells from Zebrafish Larvae for Single-Cell RNA Sequencing

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in vitro culturing technique for studying cellular dynamics in zebrafish scales

Author Spotlight: Advancing Bone Cell Activity Research with Zebrafish Scales 

This protocol offers a method to study cellular dynamics using a simple in vitro culture technique. It provides an opportunity for zebrafish researchers and educators to study cellular processes, such as those related to bone homeostasis and basic cell biology, by visualizing fluorescent nuclei and apoptotic cells within the...

Removing Zebrafish Scales for Culturing and Bone Biology Studies

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enhancing density maps by removing the majority of particles in single particle cryogenic electron microscopy final stacks

Author Spotlight: Optimizing Cryo-EM Analysis with CryoSieve for Enhanced Particle Selection Efficiency

An advanced particle selection method for cryo-EM, namely CryoSieve, improves density map resolution by removing a majority of particles in final stacks, as demonstrated through its application on a real-world...

Enhancing Cryo-EM Map Quality with CryoSieve Particle Selection

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two-photon calcium imaging of forebrain activity in behaving adult zebrafish

Author Spotlight: Advancements in Adult Zebrafish Brain Research 

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult...

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

capturing cytoskeleton-based agitation of the mouse oocyte nucleus across spatial scales

Author Spotlight: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and...

Impact of Cytoskeleton on Nuclear Shape and Condensate Dynamics in Mouse Oocytes

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isolating malignant and non-malignant b cells from lck:egfp zebrafish

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP...

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

enhanced electron injection and exciton confinement for pure blue quantum-dot light-emitting diodes by introducing partially oxidized aluminum cathode

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

A protocol is presented for fabricating high-performance, pure blue ZnCdS/ZnS-based quantum dots light-emitting diodes by employing an autoxidized aluminum...

isolation and culture of primary human mammary epithelial cells

Author Spotlight: Integrating Eastern and Western Medicine for Treatment of Granulomatous Mastitis

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical...

Protocol for Isolating Mammary Epithelial Cells from Human Tissue

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microscopic varicocelectomy under local anesthesia as the treatment of varicocele

Author Spotlight: Advancing Varicocele Treatment Through Enhanced Surgical Techniques

Varicocele accounts for 35%-40% of infertility cases among men of reproductive age. The international standard treatment method is microscopic varicocelectomy, typically performed under general anesthesia. This study proposes and implements microscopic varicocelectomy under local anesthesia, which provides more humane care for patients and reduces the economic...

Microsurgical Varicocele Removal Under Local Anesthesia

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To begin, place a euthanized female zebrafish on a table. Dab the excess water on the fish's surface with absorbent paper. With a pair of micro-scissors, cut off the head of the fish along the posterior margin of the gill cover.Then cut off the tail with the cloaca. Transfer the middle trunk to a 35-millimeter wide dish with 2 milliliters of L-15 medium. Now, cut the central axis of the abdomen and remove the viscera and swim bladder.Use a pair of tweezers to detach the bilateral ovaries from the abdomen. Remove the adipose tissue, scales, and other body tissues attached to the ovary.

To begin, transfer the excised ovaries of a zebrafish to a six-well plate containing two milliliters of cell dissociation solution. Incubate the ovaries at 28.5 degrees Celsius for two to three hours. Add two to three milliliters of pre-warmed L15 medium to the buffer to stop digestion.Next, place a 70 micrometer cell strainer into another well of the six-well plate. Add L15 medium to the well, ensuring the medium level is higher than the strainer. Now, use a pipette to draw the digestive medium through the cell strainer.Remove the excess medium with a pipette. Add four milliliters of fresh L15 medium into the well, and gently resuspend the oocytes. After a couple of minutes, pipette out the supernatant.To select the oocytes for quality control, transfer them into a 35 millimeter wide dish containing fresh L15 medium. Observe the oocytes under a light microscope using a magnification of 10X or more. Use a blunt injection tool to remove any cell fragments, stage one oocytes, or any other stage oocytes.For confirmation of the stage of growth, add Hoechst 33342 to the L15 medium containing the oocytes and incubate. Observe the oocytes with a fluorescence microscope under UV laser excitation. Carefully pick out the oocytes that do not meet the desired criteria with a needle.The juvenile ovary showed an abundance of transparent stage one oocytes accompanied by a smaller population of stage two oocytes. Adult fish ovaries showed a predominance of opaque late stage two to three oocytes. The reference method resulted in numerous stained granulosa cell nuclei on the surface of the oocytes densely enveloping the oocytes.In contrast, the modified method allowed for the separation of stage one oocytes devoid of granulosa cell nuclei staining.