<meta charset="utf8"></head><body>Procedimento aprimorado para obter oócitos de peixe-zebra estágio I livres de células da granulosa

<ol>
	<li>Qin, J. 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=Unraveling+the+mechanism+of+long-term+bisphenol+S+exposure+disrupted+ovarian+lipids+metabolism,+oocytes+maturation,+and+offspring+development+of+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Larp6+controls+oocyte+development,+chorion+formation+and+elevation.">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/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>

<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 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.

<meta charset="utf8"></head><body>Autor em destaque: Método aprimorado para isolar oócitos puros de peixe-zebra estágio I

Isolamento rápido de oócitos estágio I em peixe-zebra desprovido de células da granulosa

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

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

410 visualizações.  Sichuan University. Esta pesquisa está focada em estabelecer um processo para isolar de forma rápida e eficiente ooyctes puros em estágio um em peixe-zebra, eliminando a contaminação das células da granulosa. O objetivo principal é desenvolver um método que facilite uma análise mais precisa em áreas de pesquisa específicas de oócitos, particularmente em pesquisa genômica e epigenética. Os oócitos do peixe-zebra são envolvidos por uma monocamada de células granul...

Vídeo: Autor em destaque: Método aprimorado para isolar oócitos puros de peixe-zebra estágio I

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.

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.