Fish germ cell subcellular structure separation method

Abstract

The invention relates to the technical field of information, and particularly provides a fish germ cell subcellular structure separation method, which comprises the following steps: obtaining fish gonad tissues in an environment without RNA enzyme pollution, washing with a precooling buffer solution, directly quick-freezing, embedding in an embedding medium, and storing at low temperature; freezing and slicing the quick-frozen and embedded sample to obtain a thin-layer slice, and attaching the thin-layer slice to a membrane subjected to adhesion enhancement treatment and enzyme-free treatment; the attached section is subjected to visual treatment by adopting a specific dyeing combination and then is stored at low temperature; the visualized diaphragm is placed in a humidity-controlled windless enzyme-free environment, and a target membraneless subcellular structure is recognized by combining phase difference or differential interference contrast imaging and three-dimensional stack scanning through a multiple objective lens; the laser microdissection system adopts laser parameters optimized for a small-area target to complete cutting; and adsorbing the cut membraneless subcellular structure into a tube cover of a collecting tube, immediately adding a lysis solution, mixing, and storing at low temperature for nucleic acid extraction.

Description

Technical Field

[0001] This invention belongs to the field of information technology, and in particular relates to a method for isolating subcellular structures of fish reproductive cells. Background Technology

[0002] Research in reproductive biology and epigenetics relies heavily on isolating specific subcellular structures from fish germ cells. These structures are often not enclosed by lipid membranes and are directly exposed to the cytoplasmic environment. They carry crucial genetic and epigenetic information and play an irreplaceable role in understanding reproductive processes, developmental mechanisms, and genetic diseases.

[0003] Existing separation methods often struggle to completely eliminate ribonuclease contamination throughout the entire process. These enzymes are widely present in the experimental environment and inside cells, and upon contact with non-membrane structures, they rapidly degrade the nucleic acid components, leading to a significant decline in the quality of the separated samples. Furthermore, because these structures are tiny and have indistinct boundaries, they are difficult to identify clearly under a microscope, easily causing deviations during localization and cutting, further exacerbating the degradation risk due to prolonged nucleic acid exposure time.

[0004] Membrane-free subcellular structures are highly susceptible to contamination and degradation by ribonucleases during separation. The lack of physical protection from a lipid membrane places nucleic acid components in a high-risk state immediately after cell wall disruption. The tiny size and indistinct boundaries of these structures further prolong identification and localization, inevitably extending the time the structures are exposed to potential contamination. Thermal effects or mechanical damage also accelerate nucleic acid instability. This contamination risk and identification difficulty reinforce each other, creating a chain reaction: attempting to more precisely locate and cut target structures inevitably increases the number of steps and time required, significantly increasing the chance of nucleic acid exposure to ribonucleases; conversely, shortening the operation time to reduce degradation sacrifices localization accuracy, resulting in the inability to accurately acquire the target structure.

[0005] Therefore, how to achieve precise manipulation of tiny and poorly defined membrane-free subcellular structures while minimizing the risks of ribonuclease contamination and nucleic acid degradation throughout the entire process from identification and localization to cutting and collection has become a key issue in ensuring the quality of separated samples and the reliability of research. Summary of the Invention

[0006] In view of this, the present invention aims to provide a method for separating subcellular structures of fish reproductive cells, in order to solve the problem of balancing the precise operation of tiny and poorly defined membrane-free subcellular structures with the reduction of ribonuclease contamination and nucleic acid degradation risks throughout the entire process from identification and positioning to cutting and collection.

[0007] To achieve the above objectives, the technical solution created by this invention is implemented as follows: Fish gonadal tissue was obtained in an RNase-free environment, rinsed with pre-cooled buffer, and directly flash-frozen and embedded in an embedding medium for low-temperature storage. The flash-embedded samples were then cryosectioned to obtain thin-layer sections, which were then attached to membranes that had undergone adhesion enhancement and enzyme-free treatment to obtain attached sections. These attached sections were visualized using a specific staining combination and then stored at low temperature. The visualized membranes were placed in a humidity-controlled, windless, enzyme-free environment, and the target non-membrane subcellular structures were identified using multi-magnification objectives combined with phase-contrast or differential interference contrast imaging and 3D stack scanning. Based on laser parameters optimized for small-area targets, a pre-set laser microdissection system was used to cut the identified target non-membrane subcellular structures, obtaining the cut non-membrane subcellular structures. The cut non-membrane subcellular structures were adsorbed into the cap of a collection tube, immediately mixed with lysis buffer, and stored at low temperature for nucleic acid extraction.

[0008] Furthermore, the process of obtaining fish gonadal tissue in an RNase-free environment, rinsing it with pre-cooled buffer, and directly flash-freezing and embedding it in an embedding medium for low-temperature storage includes: treating the fish with eugenol at a concentration lower than the normal anesthetic dosage, then obtaining intact gonadal tissue using autoclaved instruments in a laminar flow hood sprayed with RNase remover and disinfected with ultraviolet light; briefly rinsing the gonadal tissue with pre-cooled 1× phosphate buffer and placing it directly into an embedding cassette treated with RNase remover to avoid air contact; adding a portion of the optimal cutting temperature embedding agent to the embedding cassette, pre-freezing it with dry ice, then placing the tissue inside and completely covering it with the optimal cutting temperature embedding agent, followed by flash freezing with dry ice and storage at -80 degrees Celsius.

[0009] Furthermore, the quick-frozen embedded sample is cryosectioned to obtain thin slices, which are then attached to a membrane that has undergone adhesion enhancement treatment and enzyme-free treatment to obtain attached slices. This process includes: the membrane is irradiated with ultraviolet light, coated with poly-L-lysine, left to stand, rinsed with sterile water treated with diethyl pyrocarbonate, dried, and sealed; a pre-designed cryostat for cryosectioning the quick-frozen embedded sample and its contact area are treated with an RNase remover to equilibrate and fix the sample in a low-temperature slicing environment; the slice thickness is controlled within the thin layer range, and flat slices are selected and attached to the pre-treated membrane.

[0010] Furthermore, the attached sections are visualized using a specific staining combination and then stored at low temperature, including: the sections are first placed in a pre-cooled paraformaldehyde solution for a predetermined time, then stained with tar violet for 45 seconds and rinsed multiple times with sterile water treated with diethyl pyrocarbonate to obtain rinsed sections; the rinsed sections are then stained with eosin for 10 seconds and rinsed multiple times with sterile water treated with diethyl pyrocarbonate to obtain stained sections; the stained sections are then stored for a short period at -80 degrees Celsius after absorbing excess moisture with silica gel desiccant.

[0011] Furthermore, the visualized membrane is placed in a humidity-controlled, windless, enzyme-free environment. The target non-membranous subcellular structure is identified using multiple objectives combined with phase-contrast or differential interference contrast imaging and three-dimensional stacked scanning. This includes: the environment is disinfected with alcohol and sprayed with RNase remover, maintaining a humidity of 50% and a windless state; the imaging first uses a low-power objective with bright-field localization, then switches to a high-power objective to adjust phase-contrast or differential interference contrast for clear contrast; the identification involves establishing a three-dimensional stacked scan covering the full thickness of the cell, including any excess, with finer scanning steps to observe multiple layers of continuously dense granular clusters and accompanying mitochondrial-related small dot clusters, edge contrast effects, or uneven brightness / darkness clumps, thus confirming the target non-membranous subcellular structure.

[0012] Furthermore, based on laser parameters optimized for small-area targets, a preset laser micro-cutting system is used to cut the identified target membraneless subcellular structure to obtain the cut membraneless subcellular structure. The laser parameters are set according to the cutting area of ​​400 to 1600 square micrometers, with an intensity of 50 to 54, an aperture of 1 to 2, a speed of 8 to 10, a pulse of 16 to 25, a current of 70% to 90%, and a frequency of 4500 to 4800. Before cutting, a trial cut is performed in a blank area to verify trajectory alignment, and then the target separation is completed under a high-magnification field of view with a limited single-membrane cutting time.

[0013] Furthermore, the process of adsorbing the cut non-membrane subcellular structure into the cap of the collection tube, immediately adding lysis buffer, mixing, and storing at low temperature for nucleic acid extraction includes: the collection uses an enzyme-free collection tube to adsorb the cut structure into the cap; immediately after cutting, adding TPK cell lysis buffer and diethyl pyrocarbonate-treated sterile water to the cap for nucleic acid extraction of the subcellular structure; after adding TPK cell lysis buffer and diethyl pyrocarbonate-treated sterile water, inverting the collection tube and shaking it to dissolve the cut non-membrane subcellular structure, then centrifuging, collecting the liquid, labeling, and storing at -80 degrees Celsius.

[0014] Compared with the prior art, the present invention can achieve the following beneficial effects: This invention discloses an innovative method for isolating non-membrane subcellular structures in fish germ cells. It focuses on addressing the challenges of ribonuclease contamination and degradation during non-membrane structure isolation due to the lack of lipid membrane protection, while also tackling the complex challenges of small structures with indistinct boundaries, difficult identification, and maintaining nucleic acid integrity. This invention employs end-to-end ribonuclease contamination control, combined with rapid cryopreservation to preserve cellular ultrastructure, thin-layer frozen sections and mild staining to enhance structural visualization, advanced optical imaging and 3D stack scanning for precise target structure localization, and optimized laser microdissection parameters to minimize thermal damage. Finally, immediate lysis and cryogenic storage ensure nucleic acid integrity. This systematic solution significantly improves the accuracy and reliability of non-membrane subcellular structure isolation, providing a high-quality sample preparation method for reproductive biology and epigenetics research, demonstrating significant technological value and application prospects. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a flowchart of a method for isolating subcellular structures of fish reproductive cells according to the present invention; Figure 2 These are morphological images of samples before, during, and after laser micro-dissection of subcellular structures according to the present invention. Figure 3 This is a quality diagram of RNA in subcellular structures obtained by laser microdissection according to the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and do not constitute a limitation thereof. Similar elements in different embodiments are referred to by associated similar element reference numerals. In the following embodiments, many details are described to facilitate a better understanding of the invention. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other elements, materials, or methods. In some cases, some operations related to the invention are not shown or described in the specification. This is to avoid obscuring the core parts of the invention with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; they can fully understand the related operations based on the description in the specification and general technical knowledge in the art.

[0017] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined to form various implementations. Furthermore, the order of the steps or actions in the method description can be changed or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various orders in the specification and drawings are merely for the clear description of a particular embodiment and do not imply a mandatory order, unless otherwise stated that a particular order must be followed.

[0018] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this invention. The term "based on" should be understood as "at least partially based on." Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, features defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more, and the term "including" means "including but not limited to." Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the range referred to.

[0019] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0020] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0021] like Figure 1 This embodiment of a method for isolating subcellular structures of fish reproductive cells may specifically include: This invention provides a method for isolating subcellular structures of fish germ cells. This method is particularly suitable for the precise isolation and collection of membrane-free subcellular structures highly sensitive to RNases from fish gonadal tissue, such as Barr's bodies (BBs) and perinuclear dense bodies (PDBs) in germ cells—organelles or functional complexes without membrane boundaries. Because these structures lack lipid membrane protection, they are highly susceptible to degradation or contamination during conventional processing. Therefore, the entire process is conducted under strict RNase-free contamination control to ensure the integrity of subsequent nucleic acid extraction and downstream molecular analysis.

[0022] Step S1: After obtaining fish gonadal tissue in an RNase-free environment, it is rinsed with pre-cooled buffer and directly quick-frozen and embedded in an embedding medium for low-temperature storage.

[0023] Specifically, obtaining fish gonadal tissue is the starting point of the entire process, directly determining the integrity of RNA molecules in subsequent samples. In one embodiment, the experimental fish are first treated with eugenol at a concentration lower than the normal anesthetic dosage to reduce the impact of stress on the tissue. After anesthesia, the fish are placed in a clean bench thoroughly sprayed with an RNase scavenger and sterilized by ultraviolet irradiation. Intact gonadal tissue, such as the testes of male fish or the ovaries of female fish, is rapidly separated using autoclaved dissecting instruments. The entire procedure must be completed within a short time to avoid prolonged exposure of the tissue to air, which could lead to RNase contamination or autolysis.

[0024] Immediately after obtaining the tissue, perform a brief rinse with 1×PBS buffer. This buffer should be pre-cooled at 4°C to reduce tissue metabolic activity. The purpose of rinsing is to remove blood, mucus, or other substances that may carry exogenous RNases from the tissue surface. The rinsing time should be strictly controlled to 10 seconds to avoid over-rinsing, which could lead to cell damage or the release of endogenous RNases. After rinsing, avoid prolonged contact between the tissue and air to prevent drying or oxidation.

[0025] Subsequently, the rinsed gonadal tissue was directly placed into an embedding cassette treated with an RNase remover. In one embodiment, the embedding cassette was first filled with 1 / 4 volume of OCT (optimal cutting temperature embedding medium), and pre-cooled on dry ice to solidify the bottom medium and form a support layer. The tissue was then placed flat on top, and finally, OCT was added to completely cover the tissue. During embedding, it is essential to ensure that the tissue is completely submerged and free of air bubbles to guarantee the flatness of subsequent sections. After embedding, the embedding cassette was placed in a foam box and completely encased in dry ice for rapid freezing, allowing the tissue to quickly cool to a vitrified state, thereby maximizing the preservation of cellular ultrastructure and RNA molecule activity. After rapid freezing, the sample was transferred to a -80°C freezer for long-term storage.

[0026] It should be noted that this direct rapid freezing and embedding method avoids the introduction of chemical reagents such as formaldehyde or ethanol that may be introduced in the traditional fixation-dehydration-paraffin embedding process. These reagents can severely damage the integrity of membrane-free subcellular structures. Rapid low-temperature treatment effectively inhibits endogenous RNase activity, providing a high-quality sample basis for subsequent accurate separation.

[0027] In a preferred embodiment, for the gonadal tissue of the leopard gill spiny perch, the rinsing time after acquisition is controlled at 10 seconds, the embedding medium is an OCT compound, and the quick-freezing process is carried out by wrapping with dry ice to further shorten the freezing time.

[0028] Step S2: The quick-frozen embedded sample is frozen and sectioned to obtain thin slices, which are then attached to a membrane that has undergone adhesion enhancement treatment and enzyme-free treatment.

[0029] Specifically, frozen sectioning is a crucial step in obtaining thin-layer tissue, directly affecting the accuracy of subsequent microscopic observation and laser cutting. In one embodiment, the membrane is first pretreated to enhance tissue adhesion and remove potential RNases. LCM-specific metal membranes are used. The membranes are placed flat in a clean bench with the back side facing up, irradiated with ultraviolet light for 10 minutes, and then 1 ml of 0.1 mg / ml poly-L-lysine is added to each membrane. After evenly spreading, the membranes are allowed to stand for 5 minutes, then rinsed twice with sterile water treated with 0.1% DEPC. After drying, the membranes are sealed and stored in an enzyme-free environment.

[0030] Before sectioning, the cryostat and its surrounding contact areas were thoroughly wiped with RNase remover and sterilized with UV irradiation. After removing the samples stored at -80℃, they were equilibrated in the cryostat at -25℃ for 30 minutes to avoid ice crystal damage caused by temperature differences. The samples were then fixed on the stage, and the section thickness was adjusted to 5 μm.

[0031] During sectioning, select sections of tissue that are flat and free of fragments, and gently attach them to the pretreated membrane. Ensure a tight, air-free fit between the section and the membrane, while avoiding excessive stretching that could deform the tissue. The attached membrane sections can be stored briefly at -80°C, but should be processed as soon as possible.

[0032] It should be noted that the poly-L-lysine coating on the membrane significantly enhances the adhesion strength of frozen tissue, preventing tissue detachment during subsequent staining or laser cutting. The rinsing step with 0.1% DEPC-treated water completely eliminates any potential residual RNase, ensuring enzyme-free slicing throughout the entire process.

[0033] In another embodiment, for the ovarian tissue of the leopard gill spiny perch, since the follicle structure is relatively large, the slice thickness is still maintained at 5μm. When attaching, a light pressure method is used to ensure that the oocyte area is attached flat, so as to avoid follicle rupture or structural deformation.

[0034] Step S3: The attached sections are visualized using a specific staining combination and then stored at low temperature.

[0035] Specifically, visualization is a crucial step in clearly revealing membraneless subcellular structures under an optical microscope, and one of the core innovations of this invention is the staining method and time control. Since Bartholin's bodies and perinuclear compact bodies lack natural contrast, a tar purple-eosin staining combination with minimal RNA interference is required.

[0036] In one embodiment, the metal membrane with the sample attached is first fixed in a 50ml centrifuge tube containing 4% paraformaldehyde and pre-cooled for 10 minutes. After removing the membrane, it is rinsed 2-3 times with sterile water treated with 0.1% DEPC. Approximately 5 drops of tar purple dye are added to each membrane, and after staining for 45 seconds, it is rinsed 2-3 times with sterile water treated with 0.1% DEPC. Then, 2-3 drops of eosin dye are added to each membrane, and after staining for 10 seconds, it is rinsed 2-3 times with sterile water treated with 0.1% DEPC. The entire staining process requires strict control of the time duration, while avoiding RNase contamination.

[0037] After staining, absorb excess moisture with silica gel desiccant, and then transfer to a -80℃ freezer for short-term storage for 1 to 2 days to avoid RNA degradation or structural deformation caused by long-term storage.

[0038] It should be noted that the combination staining of tar violet 45s and eosin 10s can accurately highlight the morphological boundaries of Bartholin's bodies and perinuclear compact bodies, and the staining time is short and the damage to RNA is minimal. Multiple rinsing with 0.1% DEPC water is the core measure to prevent RNA degradation and avoid dye residue affecting observation.

[0039] In another embodiment, to further enhance the contrast effect, the sample can be quickly rinsed once with 0.1% DEPC-treated water before eosin staining to ensure uniform staining. However, no other staining reagents should be added to avoid interfering with the identification of the target structure. Low-temperature storage after staining ensures the stability of the sample while awaiting laser cutting, providing a reliable guarantee for subsequent accurate identification.

[0040] For example, in actual operation, Bartholin's bodies and perinuclear compact bodies in the germ cells can be clearly observed under a phase contrast microscope in testicular sections of the leopard gill spiny perch treated with the above staining, that is, the target non-membrane subcellular structures, while sections without this combination of staining are difficult to distinguish from the surrounding cytoplasm.

[0041] Step S4: The visualized membrane is placed in a windless, humidity-controlled, enzyme-free environment and the target membrane-free subcellular structure is identified by using multiple objectives combined with phase contrast or differential interference contrast imaging and three-dimensional stack scanning.

[0042] Specifically, the identification of target membraneless subcellular structures is one of the most critical steps in the entire method. Due to the small size and blurred boundaries of Bartholin's bodies and perinuclear compact bodies, precise positioning requires strict environmental control and advanced optical imaging technology.

[0043] In one implementation, the observation environment is first prepared. After disinfecting the laser micro-cutting instrument room with alcohol and spraying it with RNase remover, the humidity is maintained at 50% and there is no wind to prevent airflow or static electricity from causing the cut sample to fall off or become contaminated.

[0044] After staining, the membrane sections were removed from storage at -80℃ and equilibrated in an enzyme-free environment at room temperature for 5-10 minutes, then fixed onto the stage of a laser microdissection system. Imaging was performed by first using a 10× objective lens (NA 0.25) for rapid bright-field scanning to locate regions containing target germ cells, such as the seminiferous tubules in the testes or the follicles in the ovary; then switching to a 20× objective lens for observation, adjusting the phase contrast ring or DIC gradient to obtain the clearest internal structural contrast.

[0045] It should be noted that three-dimensional stack scanning is the core technology for identifying target structures. By establishing a z-stack scan, with the Z-start and end points from the top (Z_top) to the bottom (Z_bottom) of the cell, ±5–10 µm margin, and a Z-step size of 0.5 µm, the cytoplasm is scanned layer by layer to look for clusters of high-contrast granules. Visual cues during identification include: the continuous presence of dense granule clusters in multiple z-layers, obvious contrast / "shading" effects at the edges under DIC, unevenly bright / dark clumps under phase contrast, and the presence of other mitochondrial dot clusters, etc., which are used to identify Bartholin's bodies and perinuclear dense bodies.

[0046] For example, in testicular sections of leopard gill spiny perch, the perinuclear dense bodies in germ cells appear as dense spheres or irregular clumps of a specific diameter in a three-dimensional stack, maintaining a consistent morphology across multiple consecutive focal planes; Bartholin's bodies appear as ultrastructural complexes formed by the aggregation of membranous organelles, accompanied by clusters of mitochondrial dots, which can be accurately distinguished from the surrounding mitochondria or lipid droplets.

[0047] In another implementation, Bartholin's bodies in the ovarian tissue of the leopard-gill sea bass are unique ultrastructural complexes of the oocyte. During scanning, the oocyte region can be preferentially located, and their relative position to the nuclear membrane can be observed through three-dimensional stacked scanning, further improving recognition efficiency. The entire recognition process is typically completed within one hour for a single membrane, avoiding prolonged exposure that increases the risk of RNA degradation.

[0048] Step S5: Based on the laser parameters optimized for small-area targets, a preset laser micro-cutting system for cutting the identified target non-membrane subcellular structures is used to complete the cutting.

[0049] Specifically, laser microdissection is a core step in achieving the physical separation of membrane-free subcellular structures, and one of the core innovations of this invention is the optimized range of laser parameters. Since the cutting area of ​​Bartholin's bodies and perinuclear compact bodies is typically 400-1600 μm², specially optimized parameters are required to achieve precise cutting while minimizing thermal damage.

[0050] In one implementation, the laser parameters are dynamically adjusted according to the cutting area: for a cutting area of ​​400-1600 μm², the laser intensity is 50-54, aperture is 1-2, speed is 8-10, pulse rate is 16-25, current is 70%-90%, and pulse frequency is 4500-4800 Hz. The cutting path is drawn as a closed trajectory 0.5-1 micrometer along the outer edge of the target structure to ensure complete separation while preserving surrounding tissue as support. Figure 2 As shown, the positional relationship between the target subcellular structure and the surrounding tissue can be clearly observed before laser micro-cutting. During the cutting process, the laser trajectory precisely surrounds the target structure. After cutting, the target structure is completely separated from the surrounding tissue, and there is no damage to the edges.

[0051] Before cutting, a trial cut was performed in a blank area of ​​the membrane to verify that the laser trajectory was aligned with the microscope field of view without any deviation. Cutting was carried out under a 20x objective lens, and the cutting time for each membrane was controlled within 2 hours to avoid environmental RNase contamination or sample degradation due to prolonged operation.

[0052] In one embodiment, Bartholin's bodies in the germ cells of the leopard gill spiny perch are cut using parameters of intensity 51, aperture 1.5, speed 9, pulse 18, current 80%, and frequency 4600Hz. The resulting structure has smooth edges without thermal denaturation, thus preserving the integrity of RNA and protein to the greatest extent.

[0053] For target structures with a large area, such as a dense cluster of perinuclear bodies with a diameter of 1600 μm², parameters such as intensity 54, aperture 2, speed 8, pulse 25, current 90%, and frequency 4800 Hz can be used to ensure thorough cutting while avoiding the spread of thermal damage.

[0054] Step S6: The cut non-membrane subcellular structure is adsorbed into the cap of the collection tube, lysis buffer is immediately added, mixed, and then stored at low temperature for nucleic acid extraction.

[0055] Specifically, collection and immediate lysis are the final critical steps in preserving the integrity of the RNA in the isolated structures. After cleavage, the isolated Bartholin's bodies and perinuclear compact bodies are adsorbed onto the cap of the enzyme-free collection tube using a laser microdissection system.

[0056] In one embodiment, the collection tube is pretreated with an RNase remover and sterilized with UV light, and the inner surface of the tube cap is kept clean and free of impurities. After each membrane is cut, 1 μL of 10×TPK cell lysis buffer and 9 μL of 0.1% DEPC-treated sterile water are immediately added to the collection tube cap to ensure complete coverage of the target structure.

[0057] After adding the lysis buffer, quickly invert the collection tube and gently shake to fully dissolve the sample in the liquid. Then, briefly centrifuge to allow the liquid to flow into the body of the collection tube. After labeling, immediately store the collection tube at -80°C for the extraction of trace RNA.

[0058] It should be noted that immediate addition of TPK cell lysis buffer rapidly inactivates any residual RNases, and sterile water treated with 0.1% DEPC further inhibits potential contamination, ensuring RNA protection throughout the entire process from isolation to storage. Samples can be stored long-term at -80°C without affecting subsequent transcriptome and proteome sequencing.

[0059] In one embodiment, for precious leopard gill spiny perch ovarian tissue samples, a small amount of RNA stabilizer can be added to the lysis buffer after collection to further enhance protection, and nucleic acid extraction can be performed within 1 week to obtain more complete RNA molecules.

[0060] For example, Bartholin's bodies and perinuclear dense bodies isolated from the germ cells of the leopard gill spiny perch using this method can be used to obtain intact long-chain RNA in subsequent extractions, see [link to relevant documentation]. Figure 3 The RNA quality map shows that the RNA integrity index (RIN value) obtained by this method meets the requirements of transcriptome sequencing, with no obvious degradation bands. After passing the Agilent 2100 test, it can be used for transcriptome and proteome sequencing analysis, and the sequencing data has high reliability. In contrast, traditional methods often only yield fragmented products due to degradation.

[0061] Furthermore, the entire process from tissue acquisition to structure collection is strictly controlled without RNase. Combined with multiple technologies such as quick freezing, 5μm thin-layer sectioning, precise tar purple-eosin staining, advanced imaging, optimized laser cutting, and instant lysis, the method makes the acquisition of non-membrane subcellular structures in fish reproductive cells a reproducible and highly intact operation, providing a reliable sample preparation method for reproductive biology and epigenetics research.

[0062] In one embodiment, the basic process is the same for gonadal tissues of other non-model fish such as grouper. Only the laser parameters need to be adjusted appropriately according to the tissue size to achieve the same precise separation.

[0063] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0064] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for isolating subcellular structures of fish reproductive cells, characterized in that, The method comprises the following steps: The fish gonadal tissue is obtained in a RNAse-free environment, washed with pre-cooled buffer solution, directly frozen and embedded in embedding medium, and stored at low temperature; The frozen and embedded sample is subjected to frozen sectioning to obtain thin sections, which are attached to a film piece subjected to adhesion enhancement treatment and enzyme-free treatment to obtain attached sections; The attached sections are subjected to visualization treatment with a specific staining combination, and then stored at low temperature; The visualized film piece is placed in a RNAse-free environment with controlled humidity and no wind, and target membrane-free subcellular structures are identified through multiple objective lenses combined with phase contrast or differential interference contrast imaging and three-dimensional stack scanning; According to laser parameters optimized for a small area target, a preset laser microdissection system for cutting the identified target membrane-free subcellular structure is used to complete the cutting, and the cut membrane-free subcellular structure is obtained; The cut membrane-free subcellular structure is adsorbed into a collection tube cap, and a lysis solution is immediately added for mixing, and then stored at low temperature for nucleic acid extraction.

2. The method for isolating subcellular structures of fish reproductive cells as described in claim 1, characterized in that, The method comprises the following steps: The fish is treated with eugenol at a concentration less than the normal anesthetic dosage, and the gonadal tissue is obtained in an ultraclean table subjected to RNAse scavenger spraying and ultraviolet disinfection using an autoclave device; The gonadal tissue is briefly washed with pre-cooled 1× phosphate buffer solution, and then directly placed in an embedding box subjected to RNAse scavenger treatment; The embedding box is first added with part of the optimal cutting temperature embedding agent, and then the tissue is placed in the embedding box after dry ice pre-cooling and solidification, and the optimal cutting temperature embedding agent is completely covered, and then the embedding box is frozen with dry ice and transferred to negative 80 degrees for storage.

3. The method for isolating subcellular structures of fish reproductive cells as described in claim 1, characterized in that, The method comprises the following steps: The film piece is subjected to ultraviolet irradiation, coated with polylysine, rinsed with diethyl pyrocarbonate treated sterile water, dried, sealed, and then used as a substrate for the thin sections obtained by frozen sectioning of the frozen and embedded sample. The film piece is subjected to ultraviolet irradiation, coated with polylysine, rinsed with diethyl pyrocarbonate treated sterile water, dried, sealed, and then used as a substrate for the thin sections obtained by frozen sectioning of the frozen and embedded sample. The method comprises the following steps:

4. The method for isolating subcellular structures of fish reproductive cells as described in claim 1, characterized in that, The method comprises the following steps: The method comprises the following steps: The method comprises the following steps: The method comprises the following steps:

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6. The method for isolating subcellular structures of fish reproductive cells as described in claim 1, characterized in that, According to the laser parameters optimized for small area targets, a preset laser microdissection system for cutting the identified target subcellular structure without membrane is used to complete the cutting, and the cut subcellular structure without membrane is obtained, including: The laser parameters are set as intensity 50-54, aperture 1-2, speed 8-10, pulse 16-25, current percentage 70-90, and frequency 4,500-4,800 according to the cutting area of 400-1,600 square microns; Before the cutting, a test cut is performed in a blank area to verify the alignment of the track, and then the cutting time of a single membrane piece is limited under high-power field of view to complete the target separation.

7. The method for isolating subcellular structures of fish reproductive cells as described in claim 1, characterized in that, The cut subcellular structure without membrane is adsorbed into the cap of the collection tube, and then a lysis solution is immediately added for mixing, followed by low-temperature storage for nucleic acid extraction, including: The collection is performed by using an enzyme-free collection tube to adsorb the cut structure into the cap of the tube; Immediately after the cutting is completed, TPK cell lysis solution and diethyl pyrocarbonate treated sterile water for nucleic acid extraction of subcellular structure are added to the cap of the tube; After the TPK cell lysis solution and diethyl pyrocarbonate treated sterile water for nucleic acid extraction of subcellular structure are added, the collection tube is inverted and shaken to dissolve the cut subcellular structure without membrane, and then centrifugation is performed to collect the liquid marker, followed by storage at -80 degrees.

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