A method for isolating a stage i oocyte of zebrafish

Abstract

The application provides a method for separating zebrafish I-stage oocytes, and belongs to the field of cell separation and preparation. The application first provides a mixed digestive enzyme solution, which comprises component II collagenase, component IV collagenase and an Accutase digestive solution. The application also provides a method for separating zebrafish I-stage oocytes by using the mixed digestive enzyme solution. The application effectively digests the ovarian connective tissue by using the mixed digestive enzyme solution containing the Accutase digestive solution and the component II and IV collagenases, disperses the oocytes, separates the granulosa cells attached to the surface of the oocytes, solves the problem of residual granulosa cells, and maintains the integrity of the oocytes and the cell viability. The I-stage oocytes obtained by the separation method have high purity and good cell viability, the proportion of the I-stage oocytes without granulosa cell nuclear pollution is about 98%, the proportion of the living cells is close to 100%, the body cell pollution can be controlled through quality control, and the I-stage oocytes are suitable for subsequent extraction, analysis and detection and various research purposes.

Description

Technical Field

[0001] This invention belongs to the field of cell separation and preparation, specifically relating to a method for isolating zebrafish stage I oocytes. Background Technology

[0002] Oocyte development and differentiation are crucial areas of research in developmental biology. Understanding their mechanisms can significantly advance research in embryogenesis, female reproduction, oncology, and congenital diseases. Maternal factors stored in the oocyte play a vital role in embryonic axis formation and early development. Oocyte genome transcription products provide essential substances for early zygote development, particularly during the zygote gene transcription arrest phase. Abnormalities in maternal gene function are often closely associated with infertility disorders and malformations. Therefore, isolating oocytes at different developmental stages is of great significance for studying oocyte development and maternal genes.

[0003] Zebrafish are one of the most important model organisms in developmental biology. Their embryos are transparent, and fertilized eggs develop in vitro, making them widely used in research on cell fate tracking, tissue and organ development, and genetic screening. Despite the many advantages of the zebrafish model, its application in studying early oocyte differentiation and ovarian development remains to be fully utilized. This is partly because the adult zebrafish ovary has a certain thickness, and the oocytes within are a mixture of multiple stages, mostly opaque late-stage oocytes. Furthermore, the significant size difference between late-stage and early-stage oocytes (8–730 μm) makes obtaining and observing early-stage oocytes difficult.

[0004] Furthermore, with the development of sequencing and omics technologies, more and more studies are focusing on the state of the genome during oocyte development, including epigenetic modifications such as DNA methylation, histone modifications, and chromatin openness. However, because the surface of the oocyte is surrounded by a layer of tiny granulosa cells, which are somatic cells, failure to effectively remove these cells during isolation will significantly affect the results of genome analysis.

[0005] Yaniv M et al. (Elkouby, YMand MCMullins, Methods for the analysis of early oogenesis in Zebrafish. Dev Biol, 2017, 430(2):p.310-324.) provided a method for isolating early oocytes from zebrafish. This method involves digesting zebrafish ovaries with type I and type II collagenase at concentrations of 3 mg / mL and hyaluronidase at a concentration of 1.6 mg / mL, respectively. After digestion, oocytes at different stages are sorted using cell sieves of different sizes. While this method can obtain early follicles (including oocytes and a large number of granulosa cells attached to their surface), it cannot obtain clean, naked oocytes. Somatic cell contamination prevents the obtained oocytes from undergoing genomic and other related detection and analysis. Furthermore, this method has a weak separation and dispersion effect, requiring a long time to isolate a large number of oocytes from the ovarian tissue. The high concentration of enzymes, acting for a prolonged period, leads to some cell breakage and reduced viability.

[0006] Currently, there is a lack of an effective method for separating clean, intact, and high-quality early zebrafish oocytes. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for isolating zebrafish stage I oocytes. The specific technical solution of this invention is as follows:

[0008] The present invention provides a mixed digestive enzyme solution comprising components type II collagenase, type IV collagenase, and Accutase digestive solution.

[0009] Furthermore, the aforementioned mixed digestive enzyme solution is composed of type II collagenase, type IV collagenase, Accutase digestive solution, and solvent.

[0010] Furthermore, in the mixed digestive enzyme solution, the concentrations of type II collagenase and type IV collagenase are 0.2–2 mg / mL, respectively; the volume percentage of Accutase digestive solution is 30–90%; and the remainder is solvent.

[0011] Furthermore, in the mixed digestive enzyme solution, the concentrations of type II collagenase and type IV collagenase are 0.5–1.5 mg / mL, respectively; the volume percentage of Accutase digestive solution is 50–70%; and the remainder is solvent.

[0012] Preferably, in the mixed digestive enzyme solution, the concentrations of type II collagenase and type IV collagenase are 1 mg / mL, respectively; the volume percentage of Accutase digestive solution is 50%; and the remainder is solvent.

[0013] Furthermore, the solvent is cell culture medium, PBS, or Hank's buffer;

[0014] Preferably, the cell culture medium is L-15 medium.

[0015] The present invention also provides a method for preparing the aforementioned mixed digestive enzyme solution, which includes the following steps:

[0016] Type II and Type IV collagenases are dissolved in a solvent and then mixed evenly with Accutase digestion solution to obtain the final product.

[0017] The present invention also provides the use of the aforementioned mixed digestive enzyme solution in the preparation of zebrafish stage I oocytes.

[0018] The present invention also provides a method for preparing zebrafish stage I oocytes, which is obtained by digesting zebrafish ovaries with the aforementioned mixed digestive enzyme solution;

[0019] Preferably, it includes the following steps:

[0020] (1) Remove the ovaries of zebrafish and digest them using the aforementioned mixed digestive enzyme solution;

[0021] (2) After digestion was terminated, the cells were filtered through a cell filter to separate stage I oocytes;

[0022] (3) Stage I oocytes obtained by washing, filtering and separation.

[0023] Furthermore,

[0024] In step (1), the zebrafish is a female juvenile zebrafish that is 5 to 8 weeks old;

[0025] And / or, in step (1), the method of removing the ovaries of the zebrafish includes the following steps: euthanizing the zebrafish, cutting off the head and tail, cutting open the abdomen, and removing the ovaries;

[0026] And / or, in step (1), the digestion temperature is 26-30°C; and / or, the digestion time is 2-3 hours;

[0027] And / or, in step (2), the method for terminating digestion is to add a large amount of culture medium;

[0028] And / or, in step (2), the cell filter is a cell filter with a diameter of 40 to 100 μm;

[0029] And / or, in step (3), the washing method is washing with a culture medium.

[0030] Furthermore,

[0031] In step (1), the method of removing the ovaries of zebrafish includes the following steps: after euthanizing the zebrafish, cut off the head from the posterior edge of the gill cover, cut off the tail from the cloaca, cut open the abdomen along the central axis in the culture medium, pinch the hindgut and anal end to remove the internal organs, remove the swim bladder, and take out the ovaries on both sides of the abdomen intact.

[0032] And / or, in step (1), the digestion is agitated every half hour;

[0033] And / or, in step (1), the digestion temperature is 28.5°C;

[0034] And / or, in step (2), the cell mesh is a 100 μm cell mesh;

[0035] And / or, in step (3), the washing method is to wash the stage I oocytes obtained by filtration and separation with culture medium 5 to 6 times;

[0036] Preferably, in step (1), after the ovaries on both sides of the abdomen are completely removed, fat and other tissues are peeled off;

[0037] And / or, in steps (1) to (3), the culture medium is L-15 culture medium.

[0038] Compared with the prior art, the zebrafish stage I oocyte isolation method provided by the present invention has the following advantages:

[0039] (1) The method of the present invention utilizes the combination of collagenase and Accutase to achieve a better balance between dispersed oocytes and digested granulosa cells, resulting in clean and intact stage I oocytes with a high survival rate.

[0040] (2) The method of the present invention can isolate and prepare a large number of stage I oocytes, which can meet the needs of various research such as extraction and sequencing analysis.

[0041] In summary, through repeated experiments, this invention provides a novel digestive enzyme solution and separation method for zebrafish stage I oocytes. This invention utilizes a mixed digestive enzyme solution containing Accutase and type II and IV collagenases to effectively digest ovarian connective tissue, disperse oocytes, and separate granulosa cells attached to the oocyte surface, thus solving the problem of granulosa cell residue and maintaining the integrity and viability of the oocyte structure. The stage I oocytes obtained by this method have high purity and good cell viability; approximately 98% of the stage I oocytes are free from granulosa cell nuclear contamination, and the viable cell rate is close to 100%. Somatic cell contamination can be controlled through quality control, making it suitable for various research purposes such as subsequent extraction and analysis.

[0042] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0043] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0044] Figure 1 The ovary of a zebrafish.

[0045] Figure 2 Morphology and size (50×) of zebrafish oocytes at stages I-III.

[0046] Figure 3 The oocytes in stage I (112×) isolated in Example 1.

[0047] Figure 4 Hoechst staining (112×) was performed on stage I oocytes isolated in Example 1.

[0048] Figure 5 The first-stage oocytes (112×) isolated from Comparative Example 1.

[0049] Figure 6 Hoechst staining (112×) was performed on stage I oocytes isolated from Comparative Example 1.

[0050] Figure 7 The morphology of stage I oocytes isolated in Example 1 (112×).

[0051] Figure 8 The morphology of stage I oocytes isolated in Comparative Example 1 (112×). Detailed Implementation

[0052] Unless otherwise stated, all raw materials and equipment used in this invention are known products, obtained by purchasing commercially available products.

[0053] Some experimental materials:

[0054] L-15 medium (L-glutamine-free) (Hyclone); Type II collagenase (Solarbio, C8150); Type IV collagenase (Solarbio, C8160); Accutase digestion solution (Yeasen, 40506ES60); 70μm cell filter (Falcon, 352350); 100μm cell filter (Falcon, 352360); 6-well cell culture plate (SORFA, 221100); 35mm cell culture dish (SORFA, 230301); #5 dissecting forceps, 0.10×0.06mm tip (Zhongjing Keyi, EZ5889); high-precision forceps (VETUS, ST-14); miniature spring scissors (FST, 15000-00); stereomicroscope (Motic, SMZ-161); fluorescence microscope (Zeiss, Axio). Zoom.V16); constant temperature incubator (WIGGENS, WH-01); Hoechst dye (Yeasen, 40732ES03).

[0055] Example 1: Method for isolating zebrafish stage I oocytes according to the present invention

[0056] I. Methods for isolating zebrafish stage I oocytes

[0057] The method for isolating zebrafish stage I oocytes includes the following steps:

[0058] 1. Preparation of collagenase storage solution

[0059] Preparation of Type II and Type IV collagenase storage solutions: Type II and Type IV collagenase were prepared into storage solutions with a concentration of 10 mg / mL using L-15 medium; the prepared storage solutions were stored at -20℃.

[0060] 2. Preparation of mixed digestive enzyme solution

[0061] Taking a 2 mL volume of digestive enzyme solution as an example, take 1 mL of Accutase digestion solution, add 200 μL each of pre-thawed type II collagenase storage solution and type IV collagenase storage solution, and supplement the remaining volume with L-15 medium. This brings the working concentrations of type II and type IV collagenase to 1.0 mg / mL.

[0062] 3. Zebrafish ovary dissection

[0063] Select female zebrafish juveniles (5-8 weeks old) with a body length of 1.5-2 cm and euthanize them in an ice-water mixture. Pat the fish dry with absorbent paper. Under a stereomicroscope, use miniature dissecting scissors to cut off the head from the posterior edge of the gill cover and remove the tail from the cloaca. Add 2 mL of L-15 medium to a 35 mm cell culture dish beforehand. Transfer the midsection of the body to the L-15 medium, cut along the abdominal midline, and remove the viscera by holding the hindgut and anal end with forceps. Remove the swim bladder and carefully remove the ovaries from both sides of the abdomen. Remove the adipose tissue, scales, and other body tissues attached to the surface of the ovaries in the culture medium to obtain the zebrafish ovaries (see...). Figure 1 ).

[0064] 4. Ovarian digestion

[0065] The dissected ovaries were transferred to 6-well plates containing 2 mL of digestive enzyme solution. The ovaries were then separated into smaller tissue pieces using forceps to increase the contact area with the digestive enzyme solution. The 6-well plates were placed in a 28.5°C incubator for 2-3 hours of digestion, gently shaking the plates every half hour to aid dispersion. When a large number of dispersed, clean-surfaced stage I oocytes were observed under a stereomicroscope, the digestive enzyme solution was diluted with a large amount of preheated (28.5°C) L-15 medium to stop the digestion process.

[0066] 5. Filtering stage I oocytes

[0067] Place a 100μm cell filter in another well of a 6-well plate, and add L-15 medium until the medium level is above the filter. Use a pipette to aspirate the digestive enzyme solution (after digestion is terminated) into the cell filter. The digestive enzyme solution containing stage I oocytes will pass through the cell filter into the well plate, while larger oocytes from other stages and tissue clumps will be filtered out. Note that stage I oocytes should remain in the medium throughout the filtration process.

[0068] 6. Washing stage I oocytes

[0069] Remove the cell filter and allow the oocytes to settle to the bottom for 1-2 minutes. Use a pipette to remove excess culture medium from the top layer. Gently rotate the 6-well plate, using centripetal force to rotate all the oocytes to the center. Transfer the stage I oocytes to a 2mL EP tube using a pipette, allow to stand for 1 minute, remove the supernatant, and resuspend the stage I oocytes in fresh L-15 medium. Repeat this process 5-6 times, being gentle during transfer and resuspension. This will yield a large number of clean stage I oocytes. The prepared stage I oocytes are largely free of granulosa cells, broken cells, and oocytes from other stages. In other words, the prepared stage I oocytes are very clean and morphologically intact.

[0070] 7. Quality control of stage I oocytes

[0071] While step 6 yields stage I oocytes with virtually no granulosa cells attached, enzymatic digestion cannot guarantee 100% absence of granulosa cells on the cell surface. Even if only one or two stage I oocytes retain granulosa cells, it can still contaminate the genome of the stage I oocytes, leading to biased results. Therefore, quality control of stage I oocytes is necessary when performing genome sequencing, epigenetic modification, and other genome-related tests.

[0072] The stage I oocytes obtained after washing in step 6 can be transferred to a 35 mm culture dish containing L-15 medium. Under a stereomicroscope with magnification greater than 10x, individual stage I oocytes with residual granulosa cells can be removed (small granules can be seen adhering to the cell surface under a stereomicroscope) to ensure that all cells are morphologically intact and free of granulosa cells.

[0073] 8. Staining identification

[0074] Oocytes at different stages can be identified by their opacity and the eccentric position and presence of germinal vesicles (GV bodies), or by measuring their size (Welch, EL, et al., Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish. J Vis Exp, 2017(122).): Stage I oocytes are completely transparent, with a diameter of 7–140 μm; Stage II oocytes contain yolk vesicles of varying sizes, appearing as translucent foams, with a diameter of 140–340 μm; Stage III oocytes continue to darken in color, with clear GV bodies visible in the center of the cell, and a diameter of 340–690 μm; Stage IV oocytes have a diameter of 690–730 μm. Figure 2 ).

[0075] The first-stage oocytes prepared by this invention are as follows: Figure 3 As shown. Hoechst dye was added to L-15 medium containing the prepared stage I oocytes (stage I oocytes prepared in step 7) to a final concentration of 5 μg / mL, and incubated at room temperature for 5 min. Fluorescence was then observed under a fluorescence microscope using UV laser excitation. Qualified live stage I oocytes should have clearly defined nuclei with intact chromatin, an intact cell membrane, and no granulosa cell residue on the cell membrane. According to... Figure 4 As shown: The stage I oocytes prepared by this invention possess the above-mentioned characteristics, and are therefore qualified stage I oocytes. Qualified stage I oocytes can be used for subsequent analysis, extraction, or flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0076] Comparative Example 1: Isolation of Stage I oocytes using literature methods

[0077] I. Experimental Methods

[0078] 1. Preparation of mixed digestive enzyme solution

[0079] Taking a 2 mL volume as an example, take 600 μL of pre-thawed type I and II collagenase and 320 μL of pre-thawed hyaluronidase (all in L-15 medium), and make up the remaining volume with L-15 medium. This will bring the working concentrations of type I and II collagenase to 3 mg / mL and the working concentration of hyaluronidase to 1.6 mg / mL.

[0080] 2. Ovarian anatomy and digestion

[0081] The zebrafish ovary was dissected, following the same procedure as step 3 in Example 1. The cleaned ovary was transferred to a 6-well plate containing 2 mL of digestive enzyme solution. The ovary was then separated into smaller tissue pieces using forceps to increase the contact area with the digestive enzyme solution. The 6-well plate was placed in a 28.5°C incubator for 2-3 hours for digestion. During this time, the plate was gently shaken every half hour to aid dispersion. When a large number of dispersed, clean-surfaced stage I oocytes were observed under a stereomicroscope, a large amount of preheated (28.5°C) L-15 medium was added to the digestive solution to dilute the digestive enzyme solution, thus terminating the digestion.

[0082] 3. Filtering stage I oocytes

[0083] Place a 100μm cell filter in another well of a 6-well plate, and add L-15 medium until the medium level is above the filter. Use a pipette to aspirate the digestive enzyme solution (after digestion is terminated) into the cell filter. The digestive enzyme solution containing stage I oocytes will pass through the cell filter into the well plate, while larger oocytes from other stages and tissue clumps will be filtered out. Note that stage I oocytes should remain in the medium throughout the filtration process.

[0084] 4. Washing stage I oocytes

[0085] Remove the cell filter and allow the oocytes to settle to the bottom for 1-2 minutes. Use a pipette to remove excess culture medium from the top layer. Gently rotate the 6-well plate, using centripetal force to move all oocytes to the center. Transfer the stage I oocytes to a 2mL EP tube using a pipette, allow to stand for 1 minute, remove the supernatant, and resuspend the stage I oocytes in fresh L-15 medium. Repeat this process 5-6 times, being gentle during transfer and resuspension. This will yield the stage I oocytes.

[0086] 5. Staining identification

[0087] The prepared stage I oocytes, such as Figure 5As shown. Hoechst dye was added to L-15 medium containing the prepared stage I oocytes, and the mixture was incubated at room temperature for 5 min. Fluorescence was observed under a fluorescence microscope using UV laser excitation.

[0088] II. Experimental Results

[0089] Staining results as follows Figure 6 As shown, the surface of stage I oocytes obtained by the method in Comparative Example 1 shows a large number of Hoechst-stained granulosa cell nuclei, and the purity of the isolated cells is far inferior to that of the method of this invention. It is impossible to perform quality control on the prepared stage I oocytes to ensure that all cells are morphologically intact and free of granulosa cells. The key difference between Comparative Example 1 and the method of this invention is that this invention can effectively digest and separate the granulosa cells attached to stage I oocytes, while Comparative Example 1 cannot digest and separate the granulosa cells attached to stage I oocytes, thus failing to obtain completely clean stage I oocytes.

[0090] In conducting viability comparison experiments, this invention found that morphologically abnormal cells could not be stained with DAPI or trypan blue; therefore, viability comparison was performed based on morphological integrity. Comparing the morphological integrity of stage I oocytes obtained by the methods in Example 1 and Comparative Example 1 under a microscope, the separation method provided by this invention yields the following results: Figure 7 As shown: Stage I oocytes are morphologically intact, without damage or leakage of cellular contents; the results of Comparative Example 1 are as follows. Figure 8 As shown: the red arrows mark areas where the membranes of stage I oocytes are damaged or ruptured, resulting in leakage of cell contents. It is evident that the integrity of stage I oocytes prepared in Comparative Example 1 is inferior to that of stage I oocytes prepared by the method of this invention, indicating that the viable cell count of stage I oocytes obtained in Comparative Example 1 is significantly lower than that of stage I oocytes obtained by this invention.

[0091] The above experimental results demonstrate that the method for isolating and preparing stage I oocytes of the present invention can not only effectively separate granulosa cells attached to the surface of oocytes, but also effectively maintain the integrity and cell viability of stage I oocytes. Compared with Comparative Example 1, the method of the present invention can obtain clean, intact, and high-quality zebrafish stage I oocytes.

[0092] In summary, through repeated experiments, this invention provides a novel digestive enzyme solution and separation method for zebrafish stage I oocytes. This invention utilizes a mixed digestive enzyme solution containing Accutase and type II and IV collagenases to effectively digest ovarian connective tissue, disperse oocytes, and separate granulosa cells attached to the oocyte surface, thus solving the problem of granulosa cell residue and maintaining the integrity and viability of the oocyte structure. The stage I oocytes obtained by this method have high purity and good cell viability; approximately 98% of the stage I oocytes are free from granulosa cell nuclear contamination, and the viable cell rate is close to 100%. Somatic cell contamination can be controlled through quality control, making it suitable for various research purposes such as subsequent extraction and analysis.

Claims

1. A mixed digestive enzyme solution for preparing zebrafish stage I oocytes, characterized in that: It is composed of type II collagenase, type IV collagenase, Accutase digestion solution and solvent; In the mixed digestive enzyme solution, the concentrations of type II collagenase and type IV collagenase are 1 mg / mL, respectively; the volume percentage of Accutase digestion solution is 50%; and the remainder is solvent. The solvent is L-15 culture medium.

2. A method for preparing the mixed digestive enzyme solution according to claim 1, characterized in that: It includes the following steps: Type II and Type IV collagenases are dissolved in a solvent and then mixed evenly with Accutase digestion solution to obtain the final product.

3. The use of the mixed digestive enzyme solution according to claim 1 in the preparation of zebrafish stage I oocytes.

4. A method for preparing zebrafish stage I oocytes, characterized in that: It is obtained by digesting zebrafish ovaries using the mixed digestive enzyme solution described in claim 1.

5. The preparation method according to claim 4, characterized in that: It includes the following steps: (1) Remove the ovaries of zebrafish and digest the ovaries using the mixed digestive enzyme solution described in claim 1; (2) After digestion is terminated, the cells are filtered through a cell filter to separate stage I oocytes; (3) Stage I oocytes obtained by washing, filtering and separation.

6. The preparation method according to claim 5, characterized in that: In step (1), the zebrafish is a female juvenile zebrafish that is 5 to 8 weeks old; And / or, in step (1), the method of removing the ovaries of the zebrafish includes the following steps: euthanizing the zebrafish, cutting off the head and tail, cutting open the abdomen, and removing the ovaries; And / or, in step (1), the digestion temperature is 26~30℃; and / or, the digestion time is 2~3h; And / or, in step (2), the method for terminating digestion is to add a large amount of culture medium; And / or, in step (2), the cell filter is a cell filter with a diameter of 40~100μm; And / or, in step (3), the washing method is washing with a culture medium.

7. The preparation method according to claim 6, characterized in that: In step (1), the method of removing the ovaries of zebrafish includes the following steps: after euthanizing the zebrafish, cut off the head from the posterior edge of the gill cover, cut off the tail from the cloaca, cut open the abdomen along the central axis in the culture medium, pinch the hindgut and anal end to remove the internal organs, remove the swim bladder, and take out the ovaries on both sides of the abdomen intact. And / or, in step (1), the digestion is agitated every half hour; And / or, in step (1), the digestion temperature is 28.5°C; And / or, in step (2), the cell mesh is a 100 μm cell mesh; And / or, in step (3), the washing method is to wash the filtered and separated stage I oocytes 5-6 times with culture medium.

8. The preparation method according to claim 7, characterized in that: In step (1), after the ovaries on both sides of the abdomen are completely removed, the fat and other tissues are peeled off. And / or, in steps (1) to (3), the culture medium is L-15 culture medium.

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