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 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.
Zebrafish is a major model organism for the study of vertebrate development and physiology. The zebrafish can serve as a good model for studying the molecular mechanisms of ovarian development1,2,3. Many features of ovarian development are much conserved during evolution from fish to mammals1,2. Similar to the other vertebrates,zebrafish adults have asynchronous ovaries, containing ovarian follicles of all developmental stages4. The follicle is the fundamental reproductive element of the ovary. The follicle consists of the oocyte that is surrounded by one or several layers of somatic cells called follicular cells. The development of follicles depends on the bidirectional communication between oocytes and follicular cells5. It is vital to separate follicular cells and oocytes from ovarian follicles for different research purposes such as follicular cell primary culture, gene expression analysis, oocyte maturation, and in vitro fertilization.
Traditional separation methods include mechanical separation by forceps and enzymatic digestion6,7,8,9,10. However, the mechanical separation by forceps is time-consuming and laborious. It will also cause the high damage to the oocyte during separation. Although the enzyme digestion method is simple to operate and requires a short time, the treatment time and enzyme concentration should be validated, and the integrity and survival rate of the isolated oocytes are not ideal.Therefore, we have established a simple method to separate both compartments at different developmental stages using pulled glass capillary tubes.
All of the procedures performed in fish experiments are in accordance with the regulations of the Animal Experimentation Ethics Committee of Northwest Normal University.
1. Preparations
2. Separation of zebrafish oocytes and follicular cells at different stages
3. In vitro maturation (IVM) and in vitro fertilization (IVF)
NOTE: The procedure of IVM of IVF was followed as described previously with minor modification 11,12,13.
This method can be used to separate follicular cells and oocytes at different stages of ovarian follicle development in zebrafish. Figure 1 shows the separation of zebrafish oocytes and follicular cells from ovarian follicles using a capillary glass tube (Figure 1). To investigate whether the follicular cells were separated from intact follicles, the intact follicles and separated oocytes from different stages of follicles from the PV stage to the FG stage were stained with DAPI (50 µg/mL) at 37 °C for 30 minutes. The nucleus of follicular cells can be stained by DAPI. The blue signal of DAPI was observed in intact follicles but not denuded oocytes (Figure 2).This indicates that the oocytes and follicular cells of the follicle can be separated cleanly by this method. To further confirm the clear separation, the intact follicles and separated oocytes were cut into the histological sections. DAPI staining results showed that follicular cells were clearly removed in denuded oocytes (Figure 3). All these results suggest that this method can be used to separate follicular cells and oocytes in different stage follicles of zebrafish.
This method can be used for studying the oocyte maturation of zebrafish. To study the bidirectional communication between oocytes and follicular cells during the oocyte maturation, the denuded oocytes must be obtained for various assays. Using a pulled glass capillary, the denuded oocytes separated from full grown stage zebrafish follicles were found to undergo the spontaneous oocyte maturation without any treatment after 4 hours incubation, usually at least 30% separated oocytes survived and the rate of maturity of these survived oocytes could be up to 80% (Figure 4). These matured oocytes could undergo the full chorion expansion after placement into the water, suggesting that the follicular cells were completely removed (Figure 4). Thus, this method can be used to obtain the denuded oocytes for studying the oocyte maturation of zebrafish.
This method can be used for IVF in zebrafish. In vitro maturation (IVM) methods have been well established in zebrafish. Using these methods, the fully grown stage follicles can be induced into matured stage11,12,13,15,16. To further perform in vitro fertilization (IVF), the follicular layer of matured follicles still need to be manually removed. Here, IVM of zebrafish was induced by treatment of DHP. The follicular layer of matured follicles was removed by a capillary glass tube. These defolliculated oocytes could be fertilized and developed into the hatching stage (Figure 5). The result suggests that this method can be used for IVF in zebrafish. Usually around 10% of healthy embryos can be obtained from the matured oocytes.
Figure 1. Separation of follicular cells and oocytes from zebrafish ovarian follicles using a pulled glass capillary. 1) The morphology of the intact follicle at FG stage. 2)Inhalation of the intact follicle into the pulled glass capillary. 3)The follicular cell is detached from the oocyte when the follicle is blown out from the pulled glass capillary. 4) Successful separation of follicular cells and oocyte. Scale bars: 200 µm. Please click here to view a larger version of this figure.
Figure 2. The morphology of intact follicles and denuded oocytes separated from different stages of follicles after DAPI staining. The intact follicles and separated oocytes from different stages of follicles from PV stage to FG stage were stained by DAPI. The blue signals of DAPI were observed in intact follicles but not denuded oocyte.Scale bars: 200 µm. Please click here to view a larger version of this figure.
Figure 3. Histological sections of intact follicles and isolated oocytes after DAPI staining. Cell nuclei of follicular cells was visualized with DAPI staining. The blue signal can be observed. Follicular cells were clearly removed in denuded oocytes.Scale bars: 100 µm. Please click here to view a larger version of this figure.
Figure 4. The maturation and full chorion expansion of denuded oocytes separated from full grown stage follicles using a pulled glass capillary. (A) The morphology of intact follicles at full grown stage follicles. (B) The denuded oocytes separated from full grown stage follicles can undergo spontaneous oocyte maturation without any treatment after 4 hours incubation. (C) The matured oocytes can undergo full chorion expansion after activation by water.Scale bar: 500 µm. Please click here to view a larger version of this figure.
Figure 5. The morphology of early embryos obtained by IVM and IVF. IVM was induced by treatment of DHP (5 µg/mL) for 2 h. After IVM, the follicular cells were removed from the matured oocytes by a pulled glass capillary. The matured oocytes were fertilized by IVF. The development of embryos at different stages was viewed and photographed. Scale bars: (A-I) 200 µm, (J) 1 mm. Please click here to view a larger version of this figure.
We describe here a novel method for the simple and rapid separation of follicular cells and oocytes from zebrafish ovarian follicles. This method has several advantages over the conventional method. Primary amongst these is the greatly increased ease of separation with high efficiency and effectiveness, as only a single and external manipulation is required. This point increases the applicability to researchers who are not good at microscopic anatomy. According to our experience, one can successfully separate follicular cells and oocytes from a fully grown stage follicle within 5-10 s using the glass capillary. However, at least 30-60 s is needed to do this separation using forceps. More importantly, usually at least 20% of the denuded oocytes separated by the glass capillary can undergo spontaneous oocyte maturation. In contrast, not beyond 1% of the denuded oocytes can undergo spontaneous oocyte maturation using forceps. Thus, the efficiency and effectiveness of the new method using s glass capillary is better than the previously established method using forceps. Second, this method can be used to separate follicular celld and oocyted of ovarian follicles at early developmental stages such as the PV, EV, and MV stages. Such separation cannot be performed at early-stage follicles using current methodologies.
There are also a few of limitations using this method. For example, the appropriate diameter of glass capillary opening is crucial for separation of follicular cells and oocytes in this method. An opening much bigger or smaller than the size of ovarian follicles would lead to the failure of separation. Thus, the opening diameter of glass capillary should be adjusted depending on the size of ovarian follicles, which needs some practice.
Separation of follicular cells and oocytes have several applications in studying ovarian development. Follicular cells and oocytes at different stages of ovarian follicles obtained by this method can be used to analyze gene expression. Follicular cells separated can be used for primary cell culture. Denuded oocytes separated are useful for investigating the crosstalk between follicular cells and oocytes, and supply a good model for studying the oocyte maturation. In addition, this method can be used to remove the follicular cells in some assays such as in vitro oocyte maturation or in vitro fertilization. The availability of a method that greatly increases the ease and speed of separation should facilitate wider exploitation of zebrafish ovarian follicles in the long-term goal of understanding the intricacies of ovarian development. In addition, the structure of ovarian follicles is similar among different fish species; thus, it is interesting to test whether this method can also be applied in other fishes.
The authors have nothing to disclose.
This research work was supported by the National Natural Science Foundation of China [32060170, 31601205 and 31560334], visiting scholar project supported by China Scholarship Council and the fund of State Key Laboratory of Freshwater Ecology and Biotechnology [2020FB05].
17α,20β-DHP | Cayman | 16146-5 (5 mg) | |
24-well plate | Corning | 3524 | |
Ampoule cutter | AS ONE | 5-124-22 1 bag (100 pieces) | |
Anhydrous Na2HPO4 | Kaixin Chemical | 500 g | |
Brine shrimp | Hongjie | 250 g | |
CaCl2 | Beichen Fangzheng | 500 g | |
Culture dish | Biosharp | BS-90-D (10PCS/PK) | |
DAPI | Solarbio | S2110 (25mL) | |
Dissecting Microscope | ZEISS | Stemi 305 | |
Dissection forcep | VETUS | HRC30 | |
Dissection scissor | Kefu | 160 mm | |
Fluorescence Stereomicroscope | Leica | M205C | |
Glass capillary | IWAKI | IK-PAS-5P (200 pcs/PACK) | |
Hoechst 33342 | Solarbio | C0031 (1 mg) | |
KCl | Beichen Fangzheng | 500 g | |
KH2PO4 | Kaixin Chemical | 500 g | |
Leibovitz’s L-15 medium | Gibco | 41300-039 (10×1L) | |
MgSO4•7H2O | Beichen Fangzheng | 500 g | |
Micropipette tips | Axygen | MCT-150-C | |
NaCl | Beichen Fangzheng | 500 g | |
NaHCO3 | Beichen Fangzheng | 500 g | |
Penicilia-streptomycia | Gibco | #15140122 (100 mL) | |
Stereomicroscope | ZEISS | Discover.v20 |