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

<meta charset="utf8"></head><body>Granulosa cell-free Stage I 제브라피시 난모세포를 얻기 위한 개선된 절차

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>저자 스포트라이트: 순수 제브라피시 Stage I 난모세포를 분리하기 위한 향상된 방법

과립 세포가 없는 제브라피시에서 Stage I 난모세포의 신속한 분리

Granulosa cell-free Stage I 난모세포를 얻기 위한 제브라피시 난소 절제

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

Research

JoVE Journal

Developmental Biology

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

70 조회수. 시작하려면 안락사된 암컷 제브라피시를 테이블 위에 놓습니다. 흡수성 종이로 물고기 표면의 과도한 물을 두드려 두십시오. 마이크로 가위로 아가미 덮개의 뒤쪽 가장자리를 따라 물고기의 머리를 자릅니다.그런 다음 배설물로 꼬리를 자릅니다. 중간 줄기를 35ml의 L-2 매체가 있는 15mm 너비의 접시에 옮깁니다. 이제 복부의 중심축을 자르고 내?...

비디오: Granulosa cell-free Stage I 난모세포를 얻기 위한 제브라피시 난소 절제

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.

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 ...