Dyeing method for fertilized ovum layers of mulinialateralis

Abstract

The invention provides a dyeing method of a dwarf fertilized ovum cortex, which effectively solves the technical problems that dyes are difficult to penetrate through a fertilized ovum zona pellucida and the dyeing effect is poor in a traditional method by optimally designing a streptavidin membrane removing liquid pretreatment system and combining a fluorescence labeling spanishneedles cyclic peptide dyeing technology. After the fertilized ovum of the dwarf is obtained by inducing parturition, the efficient coloring of the cortex layer of the fertilized ovum of the dwarf can be clearly observed under a laser confocal microscope through fixing, membrane removal, permeation and dyeing treatment, and the effect is obviously superior to that of a traditional trypsin treatment method. The method is easy and convenient to operate and high in dyeing efficiency, and the leather layer dyeing effect is remarkably enhanced while the integrity of the morphological structure of the leather layer is ensured. The invention provides a reliable technical means for researching the cortex change of the bivalve fertilized egg cells in the development process, and has important application value and popularization potential.

Description

Technical Field

[0001] The invention relates to the technical field of shellfish genetic breeding in marine agriculture, and in particular to a method for dyeing the cortex of fertilized eggs of dwarf clams. Background Art

[0002] Fertilization is a crucial step in biological reproduction, marking the beginning of new life and ensuring the accurate transmission of genetic information. The cortex plays a crucial role in fertilization by regulating chromosome positioning and preventing polyspermy (the cortical reaction), and is closely linked to the fertility and ploidy of the fertilized egg. Specifically, when the egg is activated by sperm, the contents of the cortical granules are released into the perivitelline space through exocytosis, altering its physical and chemical properties. Substances such as mucopolysaccharides within the cortical granules thicken the fluid in the perivitelline space, forming a physical barrier that prevents the reassociation and passage of excess sperm, thereby preventing polyspermy. Furthermore, components of the cortical granules induce the zona pellucida reaction, inactivating sperm receptors and hardening the zona pellucida, further ensuring successful fertilization by a single sperm. Studying the dynamic changes in cortical thickness is of great scientific significance for understanding the regulatory mechanisms of early embryonic development and the behavior of chromosomes.

[0003] Cortical granules are products of the Golgi complex and contain numerous important biomolecules. Their core component, filamentous actin (F-actin), plays a key role in cytoskeletal organization and maintenance of cell morphology. Microinjection of fluorescently labeled phalloidin dye enables the localization and quantitative analysis of intracellular F-actin, allowing precise observation of cortical position and morphological changes. This method has demonstrated promising results in various animal models, visually demonstrating changes in cortical thickness and structural dynamics, providing an important tool for studying the mechanisms of zygote development. However, this method faces significant technical barriers in the study of bivalve fertilized eggs: First, the diameter of bivalve fertilized eggs is small, typically only 50-60µm, and microinjection can easily cause irreversible cell damage, affecting the reliability of experimental results. Second, the microinjection technique itself is complex, time-consuming, and labor-intensive, with low throughput, making it difficult to meet the needs of large-scale experiments. Furthermore, the hardened zona pellucida surrounding bivalve fertilized eggs significantly blocks dye penetration in traditional fixation and staining methods, resulting in inefficient binding of phalloidin dye to cortical F-actin, ultimately leading to blurred imaging of cortical distribution. Therefore, given the characteristics of bivalve fertilized eggs, there is an urgent need to develop a new method that is simple to operate, has a significant staining effect, and causes less cell damage to address the shortcomings of existing technologies.

[0004] Dwarf Clam ( Mulinia lateralis) belongs to the Bivalvia family, Mollusca, and possesses both common bivalve characteristics and unique experimental advantages: 1) dioecious sexes, facilitating genetic manipulation; 2) miniature adults (up to 21 mm shell length), conserving culture space; 3) a short generation cycle, increasing experimental throughput; and 4) mild culture conditions and high survival rates, ensuring experimental reproducibility. Therefore, the dwarf clam has the potential to be developed as a model bivalve. This invention aims to develop an efficient staining method for the cortex of zygotes of dwarf clams. This method will not only help understand the structural changes in the cortex of zygotes during zygote development, but also has the potential to be extended to the entire bivalve family, providing a universal technical tool for research on developmental biology and genetic breeding of bivalves. Summary of the Invention

[0005] In view of this, the present invention provides a method for dyeing the cortex of fertilized eggs of dwarf clams to solve the above problems.

[0006] The technical solution of the present invention is achieved as follows: a method for dyeing the cortex of fertilized eggs of dwarf clams comprises the following steps: S1. Induction of spawning and fertilization of dwarf clams: Take out the dwarf clams with mature gonads from the aquaculture seawater, and gently wipe the surface of the clam with clean gauze to remove attachments. Screen sexually mature male and female individuals, use a strong flashlight to shine through the shell connection, the gonads are red for female individuals, and white or light yellow for male individuals. Separate the male and female individuals and place them near ice bags to dry in the shade to stimulate them to release sperm and eggs. Avoid direct sunlight and excessive drying during the drying process to avoid affecting the physiological state of the clam. Subsequently, transfer the clam body to filtered seawater for observation. Usually within 30 minutes to 1 hour, the dwarf clams will begin to release sperm and eggs. After release, quickly collect the sperm and eggs separately in sterilized glass crystallizing dishes; Slowly add the sperm to the egg suspension, carefully controlling the amount of sperm added. Observe under a microscope. Adequate amounts of sperm should be present around each egg to avoid polyspermy. Gently shake the crystallizing dish to thoroughly mix the sperm and eggs, completing the fertilization process.

[0007] S2. Fixation: Use a 500-mesh sieve to collect the fertilized eggs into a 2 mL centrifuge tube. Add a 4% w / v paraformaldehyde solution to fix the fertilized eggs. Place the centrifuge tube in the refrigerator overnight to ensure that the fertilized egg cells are fully fixed. After fixation, let it stand for 5 minutes and wait for the fertilized eggs to naturally settle to the bottom of the tube. Carefully aspirate the supernatant with a pipette to avoid disturbing the precipitated fertilized eggs and wash. S3. Demembranation: Add pronase demembranation solution to the cleaned fertilized eggs. Gently pipette to mix thoroughly, ensuring full contact between the fertilized eggs and the demembranation solution. Place the centrifuge tube at room temperature for ≥ 5 minutes. After demembranation, allow the eggs to settle and aspirate the demembranation solution. Once the eggs have settled, aspirate the supernatant and wash. S4. Staining and Observation: Add Triton X-100 solution to the membrane-removed zygotes and gently pipette to mix thoroughly, ensuring full contact between the solution and the zygotes. Permeabilize the centrifuge tube at room temperature to enhance cell membrane permeability and facilitate the entry of the stain into the cells.

[0008] After permeabilization is completed, the supernatant is aspirated and washed; Transfer the washed fertilized eggs to a 1.5 mL EP tube, add the dye, wrap the EP tube with aluminum foil to avoid light, and place it in the refrigerator for dyeing to ensure that the dye fully binds to F-actin; After staining, the dye was aspirated, the fertilized eggs were washed, 3×PBS was added to the EP tube, and the fertilized eggs were transferred to the confocal culture dish with a pipette. The cells were observed under a laser confocal microscope, and the microscope parameters were adjusted to observe the cortical structure of the fertilized eggs and calculate the cortical fluorescence intensity.

[0009] Furthermore, the shade drying method of S1 is to reduce the temperature to 4-8°C to promote drainage, the shade drying time is 1.2-2.5 hours, the filtered seawater temperature is 26-28°C, and the filtration accuracy is 0.22-0.45μm.

[0010] Furthermore, the temperature of S2 is fixed at 4° C. and the fixing time is 12-24 hours.

[0011] Furthermore, at least 2 mL of paraformaldehyde solution is added to S2 to ensure that the fertilized eggs are fully immersed.

[0012] Furthermore, the S3 membrane removal solution is a 0.05% w / v pronase membrane removal solution, each 10 mL containing: 1 mL of 0.5% w / v pronase E mother solution, 32 μL of 10M sodium hydroxide solution, 0.1 g of sodium thioglycolate, and filtered seawater is added to 10 mL.

[0013] Furthermore, the S3 membrane removal treatment is performed by repeatedly blowing and beating with a pipette 3-8 times to mix the fertilized eggs so that the fertilized eggs are fully in contact with the membrane removal solution.

[0014] Furthermore, the S4 stain is a fluorescently labeled phalloidin-derived dye, and the green fluorescence excitation wavelength is 490 nm.

[0015] Furthermore, the concentration of the Triton X-100 solution in the S4 permeabilization treatment is 0.1±0.02% v / v, the treatment temperature is 25-28° C., and the treatment time is 25-35 minutes.

[0016] Furthermore, the dyeing temperature of S4 is 4° C., and the dyeing time is 20-26 hours.

[0017] Furthermore, the washing solution in S2, S3, and S4 is 3×PBS phosphate buffer, and the washing is repeated 2-4 times, and each washing includes: (a) The amount added is 2 / 3 of the volume of the centrifuge tube; (b) Shake to mix for 3-8 minutes; (c) Let the tube stand for 3-8 minutes and then aspirate the supernatant.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a new staining method for the cortex of fertilized eggs of dwarf clams. By treating with 0.05% w / v pronase membrane removal solution and staining with fluorescent dye-labeled phalloidin, the cortical fluorescence signal is enhanced by 2.59-2.84 times ( Figure 1 ). Through membrane removal pretreatment, the zona pellucida outside the fertilized eggs was effectively removed, solving the problem of dye penetration difficulties and significantly improving the staining effect. Compared with the traditional microinjection method, this method breaks through the limitations of physical barriers and successfully analyzes the dynamic changes in the thickness of the cortex of fertilized eggs of dwarf clams. This method is simple to operate, has efficient staining, and does not affect the cortical structure. It is suitable for the study of fertilized eggs of dwarf clams. Using this technology, researchers can observe the structural changes of the cortex of fertilized eggs more clearly, providing a new technical means for the developmental biology and genetic breeding research of bivalves, avoiding complex operations such as microinjection, and reducing mechanical damage to cells. It is suitable for staining research of bivalve fertilized eggs and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Comparison of the cortical staining effects of fertilized eggs (developed 10 minutes after fertilization) without membrane removal solution and after membrane removal for 5 minutes according to Example 1. (A) Fertilized egg cortex under laser confocal microscopy; (B) Mean fluorescence intensity of the fertilized egg cortex. "**" indicates a significant difference between the groups ( P <0.01).

[0020] Figure 2 Comparison of the staining effects of the cortex of fertilized eggs of dwarf clams treated with pronase de-membrane solution and trypsin solution for different times. (A) Fertilized egg cortex under laser confocal microscope; (B) Average fluorescence intensity of fertilized egg cortex. "*" indicates significant difference from the control group ( P <0.05), “**” indicates extremely significant difference compared with the control group ( P <0.01).

[0021] Figure 3 The cortex of fertilized eggs of dwarf clams was stained after removal of membranes at different time points. (A) Treated with pronase removal solution; (B) Treated with trypsin solution; (C) Not treated with removal solution.

[0022] Figure 4 Statistics of the cortical thickness (A) and mean fluorescence intensity (B) of fertilized eggs of dwarf clams. Different letters indicate significant differences between groups ( P <0.05). DETAILED DESCRIPTION

[0023] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0024] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0026] Example 1 - Comparison of the staining effects of the cortex of fertilized eggs with and without membrane removal using different methods (1) Induction of spawning and fertilization of dwarf clams Sexually mature dwarf clams (12 females and 12 males) were removed from the seawater, separated, and placed in a cool, well-ventilated area near ice packs to dry for 1.5 hours. They were then transferred to filtered seawater at 27°C and observed. After about 30 minutes, the dwarf clams began to release sperm and eggs.

[0027] After the sperm and egg are released, they are quickly collected into sterilized glass crystallizing dishes; ensure that there are 3-5 sperm around each egg, and gently shake the crystallizing dish to fully mix the sperm and egg to complete the fertilization process.

[0028] (2) Fixed Ten minutes after fertilization, use a 500-mesh sieve to collect 2,000-3,000 zygotes in a 2 mL centrifuge tube. Add 4% w / v paraformaldehyde fixative to a volume of 2 mL. Place the tube in a 4°C refrigerator overnight (12 hours) to ensure adequate fixation of the zygotes.

[0029] The next day, the supernatant was removed with a pipette and the fertilized eggs were washed three times with 3× PBS.

[0030] (3) De-membrane The fertilized eggs were divided into 9 equal parts, and one part was not removed (control). The remaining 8 samples were treated with enzymes to remove the membranes: 1 mL of pre-prepared 0.05% w / v pronase stripping solution was added to four of the samples, and the treatment times were 5 minutes (strand-5 min), 10 minutes (strand-10 min), 20 minutes (strand-20 min), and 40 minutes (strand-40 min), respectively. Add 1 mL of pre-prepared 0.05% w / v trypsin solution to the remaining four aliquots. The treatment times are also 5 minutes (trypsin - 5 minutes), 10 minutes (trypsin - 10 minutes), 20 minutes (trypsin - 20 minutes), and 40 minutes (trypsin - 40 minutes). During the membrane removal process, gently pipette to mix the zygotes to ensure full contact with the membrane removal solution.

[0031] 0.05% w / v pronase membrane removal solution (10 mL) was prepared as follows: 1 mL of 0.5% w / v pronase E stock solution, 32 µL of 10 M sodium hydroxide solution, 0.1 g of sodium thioglycolate, and filtered seawater was added to make up to 10 mL.

[0032] Prepare 10 mL of 0.05% w / v trypsin solution as follows: 8 mL of filtered seawater and 2 mL of 0.25% w / v trypsin solution.

[0033] After the membrane removal, the membrane removal solution was aspirated and the fertilized eggs were washed three times with 3× PBS.

[0034] (4) Staining and observation Add 1 mL of 0.1% v / v Triton X-100 solution to the membrane-removed zygotes and gently pipette to mix thoroughly, ensuring full contact between the solution and the zygotes. Permeabilize the tube at 25°C for 30 minutes.

[0035] The supernatant was aspirated and the cells were washed three times with 3× PBS.

[0036] The washed fertilized eggs were transferred to a 1.5 mL EP tube, and 50 µL of Staining Solution (KTC4008, Abbkine) was added. The EP tube was wrapped with aluminum foil to avoid light and placed in a 4 °C refrigerator for staining for 24 h.

[0037] After staining, the dye was aspirated, the fertilized eggs were washed twice with 3× PBS, 50 μL of 3× PBS was added, and the cells were observed under a laser confocal microscope (excitation wavelength and emission wavelength were 490 nm and 515 nm, respectively, voltage 135 V, contrast 0) and photographed.

[0038] ImageJ software (v1.8.0) was used to measure the mean fluorescence intensity of the cortex of zygotes. The specific method is: grayscale the image (convert the color image to grayscale, 8-bit, grayscale value range: 0-255), select the green primary color channel, obtain the ROI (Region of Interest) of the cortex through the MorphoLibJ program, and calculate the average fluorescence intensity of the cortex.

[0039] Experimental results: The membrane removal pretreatment of the present invention effectively removed the zona pellucida around the fertilized egg, solved the problem of the difficulty of dye penetration, and significantly improved the staining effect; the fertilized egg treated by the method of the present invention showed a significant fluorescence enhancement effect under a laser confocal microscope ( Figure 2 A). The average fluorescence intensity of the cortex of fertilized eggs treated with 0.05% w / v pronase stripping solution for 5, 10, 20, and 40 minutes reached 47.76±4.08, 49.58±1.45, 50.52±7.77, and 46.00±4.55, respectively, which was significantly increased by 2.59-2.84 times compared with the control group (17.79±2.13) without stripping solution treatment ( P <0.01), and the dye staining effect was significantly better than that of the method using 0.05% w / v trypsin solution (the average fluorescence intensity of the cortex was 24.14-28.40, which was 1.37-1.62 times that of the control group, P <0.05), indicating that this method effectively improves the permeability and binding efficiency of the dye. Using this method to remove the membrane for at least 5 minutes can ensure the best effect of fertilized egg cortical staining ( Figure 2 B). Furthermore, throughout the treatment process, the fertilized eggs maintained intact cellular morphology, with no significant deformation or damage observed. This result confirms that the desmearing solution treatment effectively maintains the structural stability of the cells while enhancing staining.

[0040] Example 2 - Observation of cortical thickness and mean fluorescence intensity during zygote development (1) Induction of spawning and fertilization of dwarf clams Sexually mature dwarf clams (12 females and 12 males) were removed from the seawater, separated, and placed in a cool, well-ventilated area near ice packs to dry for 1.5 hours. They were then transferred to filtered seawater at 27°C and observed. After about 30 minutes, the dwarf clams began to release sperm and eggs.

[0041] After sperm and eggs are released, they are quickly collected into sterilized glass crystallizing dishes.

[0042] (2) Fixed Eggs were enriched in a sterilized 150 mm glass crystallizing dish and sperm was added. After fertilization, some fertilized eggs (2,000-3,000) were collected every 2 minutes in a 2 mL centrifuge tube. 2 mL of 4% w / v paraformaldehyde fixative was added and the tubes were fixed in a 4°C refrigerator overnight (about 12 hours). A total of 10 tubes of samples were collected, and the time points were 0 min (control group), 2 min, 4 min, 6 min, 8 min, 10 min, 12 min, 14 min, 16 min and 18 min after fertilization.

[0043] (3) De-membrane The fertilized eggs of dwarf clams fixed at different time points in step (2) were divided into three parts, one part was treated with 0.05% w / v pronase demembraning solution for 5 minutes, one part was treated with 0.05% w / v trypsin solution for 5 minutes, and the other part was not demembraned (untreated group).

[0044] (4) Staining and observation The operation is the same as in Example 1.

[0045] Experimental results: The vitelline membrane of fertilized dwarf clams treated with 0.05% w / v pronase was successfully eliminated, and there was no obvious change in cell morphology. The cortical staining effect ( Figure 3 A) was significantly better than the group treated with trypsin solution at the same concentration (0.05% w / v) ( Figure 3 B) and untreated group ( Figure 3 C).

[0046] Using the staining method of the present invention, no obvious nonspecific fluorescent labeling or fluorescent noise was observed, making the observation of cortical thickness more intuitive. In the dwarf clam egg cell (0 min), the vast majority of F-actin was located in the cortex, with a small amount present inside the cell. After fertilization, F-actin quickly gathered in the cortex, and the cortical thickness showed a trend of rapid thickening first and then recovery ( Figure 4 A).

[0047] Specifically, the fertilized eggs treated with the present invention can be clearly observed to have a rapid increase in cortical thickness after fertilization, reaching a maximum value (2.22±0.12µm) at 10 minutes, and then gradually recovering. At the same time, the average fluorescence intensity of the fertilized egg cortex after fertilization has a similar trend to the thickness, reaching a maximum value (58.56±10.58µm) at 10 minutes, and then gradually decreasing, reflecting the density change trend of the cortical particles in the fertilized egg cortex of the dwarf clam ( Figure 4 B). This change pattern is consistent with typical characteristics of cortical reactions, demonstrating the feasibility and accuracy of the present method. The changes in cortical thickness and density of zygotes treated with the present method can be visually observed, revealing the temporal changes in cortical thickness and mean fluorescence intensity after fertilization. This provides effective technical support for further research on cortical dynamics during the development of zygotes.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for dyeing the cortex of fertilized eggs of dwarf clams, characterized in that: The following steps are involved: S1. Induced spawning and fertilization of dwarf clams: Select sexually mature male and female individuals, dry them in the shade, and then transfer them to filtered seawater to induce the release of sperm and eggs. Separate and collect sperm and eggs, and control the amount of sperm added to achieve fertilization around 3-5 sperm per egg. S2, fixation: transfer the fertilized eggs to a centrifuge tube, add paraformaldehyde solution to fix the fertilized eggs, aspirate the supernatant after fixation, and wash; S3, membrane removal: use membrane removal solution to treat for ≥5 minutes, after membrane removal treatment, settle and aspirate the membrane removal solution, and wash; S4. Staining and observation: Use Triton X-100 solution for permeabilization, aspirate the supernatant, wash, add dye and stain in the dark. After staining, aspirate the dye, wash the fertilized eggs, and observe the cortical structure under a confocal microscope.

2. The method for dyeing the cortex of a fertilized egg of a dwarf clam as claimed in claim 1, wherein: The shade drying method of S1 is to reduce the temperature to 4-8°C to promote drainage, the shade drying time is 1.2-2.5 hours, the filtered seawater temperature is 26-28°C, and the filtration accuracy is 0.22-0.45 μm.

3. The method for dyeing the cortex of a fertilized egg of a dwarf clam as claimed in claim 1, wherein: The S2 is fixed at a temperature of 4° C. for a period of 12-24 hours.

4. The method for dyeing the cortex of a fertilized egg of a dwarf clam as claimed in claim 1, wherein: The ratio of fertilized eggs to membrane removal solution in S3 is 2000-3000 eggs / mL.

5. The method for dyeing the cortex of a fertilized egg of a dwarf clam as claimed in claim 2, wherein: The S3 membrane removal solution is a 0.05% w / v pronase membrane removal solution, and each 10 mL contains: 1-2 mL of 0.25-0.5% w / v pronase E stock solution, 25-40 µL of 8-12 M sodium hydroxide solution, and 0.08-0.12 g of sodium thioglycolate, and filtered seawater is added to 10 mL.

6. The method for dyeing the cortex of a fertilized egg of a dwarf clam according to claim 1, wherein: The S3 membrane removal treatment is performed by repeatedly blowing and beating with a pipette 3-8 times to mix the fertilized eggs so that the fertilized eggs are fully in contact with the membrane removal solution.

7. The method for dyeing the cortex of a fertilized egg of a dwarf clam according to claim 1, wherein: The S4 stain is a fluorescently labeled phalloidin-derived dye, and the green fluorescence excitation wavelength is 490 nm.

8. The method for dyeing the cortex of a fertilized egg of a dwarf clam according to claim 1, wherein: The concentration of the Triton X-100 solution in the S4 permeabilization treatment is 0.1±0.02% v / v, the treatment temperature is 25-28° C., and the treatment time is 25-35 minutes.

9. The method for dyeing the cortex of a fertilized egg of a dwarf clam according to claim 1, wherein: The S4 dyeing temperature is 4±0.5° C., and the dyeing time is 20-26 hours.

10. The method for dyeing the cortex of a fertilized egg of a dwarf clam according to claim 1, wherein: The cleaning solution in S2, S3, and S4 is 3×PBS phosphate buffer, and the cleaning is repeated 2-4 times. Each cleaning includes: (a) The amount added is 2 / 3 of the volume of the centrifuge tube; (b) Shake and mix for 3-8 minutes; (c) Let the tube stand for 3-8 minutes and then aspirate the supernatant.

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