A method for rapid detection of calcium signals in undifferentiated and differentiated spermatogonia.

Abstract

This invention discloses a rapid method for detecting calcium signals in undifferentiated and differentiated spermatogonia. The method uses a mild enzymatic digestion method to prepare a testicular single-cell suspension. Indo-1 AM, Thy-APC, and c-kit-PE are used for staining and labeling. The cell population to be analyzed is determined based on the difference between the lateral and forward scattering areas of the cells in the testicular cell sample. Then, Thy-APC and c-kit-PE are used to label undifferentiated and differentiated spermatogonia, respectively, to further select specific cell groups. Finally, the fluorescence intensity changes of indo-1 AM in each specific cell subpopulation are detected, thereby obtaining the changes in calcium ion concentration in undifferentiated, undifferentiated-to-differentiated, and differentiated spermatogonia. This method can rapidly analyze the calcium ion concentration in undifferentiated, undifferentiated-to-differentiated, and differentiated spermatogonia. The method is simple and convenient, and provides a foundation for identifying spermatogonial dysfunction caused by calcium signal imbalance.

Description

Technical Field

[0001] This invention belongs to the field of reproductive medicine technology, specifically relating to a method for rapidly detecting calcium signals in undifferentiated and differentiated spermatogonia. Background Technology

[0002] Calcium signaling is a second messenger molecule mediating cell signaling, widely involved in various physiological and pathological processes, and closely related to the occurrence of many human diseases. Current research indicates that calcium signaling imbalance in mature sperm is a significant factor leading to male infertility. Calcium ions are known to play important roles in sperm motility, capacitation, hyperactivation, and fertilization. Because mature sperm are single and easily isolated cells, methods for studying calcium signaling are well-established, and various commercially available fluorescent probes for calcium signal detection are available. However, research on the role and mechanism of calcium signaling during spermatogenesis is very limited, and there is no clear evidence that its abnormalities lead to infertility.

[0003] Spermatogenesis involves the morphological differentiation of various cells, including the proliferation and differentiation of spermatogonia, meiosis of spermatocytes, spermatocyte transformation, and spermatogenesis and maturation. Abnormalities in any of these processes can lead to male infertility. The development from spermatogonia to spermatocyte involves the proliferation and differentiation of undifferentiated spermatogonia, but the regulatory mechanisms of spermatogonia proliferation and differentiation are not fully understood. Whether calcium ions play an important role in spermatogonia proliferation and differentiation remains unclear. The testes contain a diverse range of cells, and studying calcium signals in specific cell populations typically requires isolating target cells and then detecting them using fluorescent calcium ion probes. However, this process is cumbersome, causes significant cell damage, and leads to cell death, severely affecting the accuracy of the results. The calcium ion concentration in isolated cells increases significantly during cell death, meaning that the calcium ion level detected in the target cells does not accurately reflect the in vivo situation. Therefore, there is an urgent need to develop a rapid, efficient, and simple method for separating differentiated and undifferentiated spermatogonia and then studying their calcium ion levels to elucidate the regulatory mechanisms of calcium signals in spermatogonia proliferation and differentiation. Summary of the Invention

[0004] In view of the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide a method for rapid detection of calcium signals in undifferentiated and differentiated spermatogonia, applicable to mammals such as mice and humans where spermatogonia surface proteins are conserved.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for rapid detection of calcium signals in undifferentiated and differentiated spermatogonia includes the following steps:

[0007] Step 1, Processing of testicular cell samples: After cleaning the isolated testes, the testicular tissue was broken up and placed in HBSS solution containing collagenase II and dispersin for digestion. Fetal bovine serum was then added to stop the digestion. Cell clumps were removed by cell filtration. The resulting cell suspension was centrifuged, the supernatant was removed, and the suspension was resuspended in HBSS solution. Staining was performed separately, including staining with indo-1AM, c-kit-PE, and Thy-APC individually, as well as staining with a combination of the three drugs.

[0008] Step 2: Flow cytometry analysis of undifferentiated and differentiated spermatogonia:

[0009] a. Use flow cytometry to detect unstained samples, find the main population of testicular cells in the FSC-A / SSC-A scatter plot and draw the P1 phylum, and mark the quadrants of different cell distributions in the PE-A / APC-A scatter plot to obtain P1-1;

[0010] b. Cell samples stained with indo-1AM, c-kit-PE, and Thy-APC individually and in combination were analyzed using an instrument. The gate and quadrant distribution in a were applied to the three single-stained and triple-stained samples to obtain P2, P3, P4, and P5. The correctness of the quadrants was checked again in the APC-A / PE-A scatter plot to obtain P2-1, P3-1, P4-1, and P5-1.

[0011] c. Flow cytometry was used to detect triple-stained testicular cell samples. The above gate and quadrant distribution methods were applied. The voltage was adjusted according to the parameters in steps a and b to locate the cells. The samples were divided into three groups based on the differences in fluorescence signals of undifferentiated, undifferentiated to differentiated, and differentiated testicular cells (Thy-APC and c-kit-PE).

[0012] d. Select the three groups of cells in the first, second and third quadrants of P5-1 separately, and under the blue light scattering area of ​​SSC-A and indo-1, Indo-1(Blue)-A yields three gates: P6, P7 and P8, which are the three groups of cells separated: undifferentiated, undifferentiated to differentiated, and differentiated.

[0013] Step 5: Using software, add Indo-1(Blue)-A and Indo-1(Violet)-A to cells in different sections of the triple staining tube. Analyze the mean values ​​and obtain the relative calcium ion concentration in each cell population by the ratio of the corresponding mean values ​​of Indo-1(Violet)-A and Indo-1(Blue)-A.

[0014] Further, in step 1, each adult testis is digested with 3 mL of HBSS solution containing 200-250 U / mL collagenase II and 2 U / mL dispersant enzyme.

[0015] Furthermore, in step 1, the digestion conditions are 37°C for 30 minutes; the amount of fetal bovine serum used is 10% of the digestion liquid volume.

[0016] Furthermore, in step 1, the three drugs are stained together by first adding indo-1AM staining, followed by c-kit-PE and Thy-APC staining.

[0017] Compared with existing technologies, this invention combines the dual-wavelength calcium ion indicator Indo-1 AM with Thy-APC and c-kit-PE, fluorescent direct-labeling antibodies that specifically express proteins in spermatogonia, to sort cells, linking the role of calcium ions in male reproduction with cell proliferation and differentiation. This is of great significance for future research on the role of calcium ions in spermatogenesis and even male infertility.

[0018] By combining calcium ion indicators with cell surface markers, the sorting method of this invention can more easily, quickly, and efficiently sort undifferentiated and differentiated spermatogonia and detect the relative calcium concentration within the cells; it is novel and pioneers a new application of calcium ions in the field of reproduction. Attached Figure Description

[0019] Figure 1 This refers to the gate-drawing strategy used in Example 1 for sorting.

[0020] Figure 2 This is the result of sorting the three-stain samples in Example 1.

[0021] Figure 3 This is the second IF verification in Example 1. Implementation

[0022] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] The HBSS (Hanks Balanced Salt Solution) formula used in this invention is as follows: 1.26 mM CaCl2-2H2O, 0.49 mM MgCl2, 0.41 mM MgSO4-7H2O, 5.33 mM KCl, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 137.93 mM NaCl, 0.34 mM NaHPO4-12H2O, 5.56 mM D-Glu, and 20 mM Hepes. Example 1

[0026] 1. Processing of mouse testicular cell samples

[0027] Dissect an adult male mouse. Place the cleaned testis in a 2 cm culture dish containing 2 mL of HBSS solution. Clean off any surface grease and blood. Then place it in a clean culture dish. Remove the membrane with forceps and tear the testicular tissue. Add 3 mL of HBSS, collagenase II (200-250 U / mL), and dispersin (2 U / mL) to a 15 mL centrifuge tube. Place the torn tissue into the centrifuge tube and incubate at 37°C for 30 min, gently pipetting 2-3 times with a 1 mL pipette during digestion. Then add 10% fetal bovine serum (FBS) to the centrifuge tube to stop digestion. Filter the cell suspension through a 40 μm sieve into a 50 mL centrifuge tube. Pipette the digested cell suspension, filter again to remove cell clumps, and transfer the filtered cell suspension to a new 15 mL centrifuge tube. Centrifuge in 1 mL centrifuge tubes at 900 rpm for 5 min, remove supernatant, resuspend in 3 mL HBSS, centrifuge again, and repeat twice; finally dilute to 2500 μL with HBSS; divide into five 500 μL portions, and stain with indo-1AM (AAT Bioquest, 112926-02-0; 1:1000), c-kit-PE (Proteintech, PE-65054, 0.2 mg / mL; 1:125), Thy-APC (Proteintech, APC-65088, 0.1 mg / mL; 1:125) individually, or in combination with all three drugs, and a blank control group, respectively. Incubate in a CO2 incubator at 37 °C; after one hour, aspirate a small amount of the indo-1-containing sample and observe under a fluorescence microscope. If the staining is good, centrifuge at 900 rpm for 5 min, remove supernatant, wash, and resuspend in 2 mL HBSS.

[0028] Specifically: the indo-1AM flow cytometer was selected with 350nm excitation light; c-kit-PE was a marker on the surface of differentiated spermatogonia, with the APC channel of the flow cytometer and 561nm excitation light; Thy-APC was a marker on the surface of undifferentiated spermatogonia, with the PE channel of the flow cytometer and 640nm excitation light.

[0029] The triple staining process involves first staining with indo-1AM for 30 minutes, followed by adding c-kit-PE and Thy-APC, and incubating for 30-45 minutes. After incubation, the dye can be removed by low-speed centrifugation, followed by washing twice with HBSS (containing 0.5 mM probenecid) (900 rpm, 5 minutes).

[0030] 2. Flow cytometry analysis of undifferentiated and differentiated spermatogonia

[0031] a. Blank testicular cell samples were analyzed using a BD LSR Fortessa flow cytometer. The voltage parameters were adjusted to exclude cell debris with very small values ​​of FSC-A (forward scattered light-area) and SSC-A (side scattered light-area). The main population of testicular cells was found in the FSC-A / SSC-A scatter plot, and the P1 gate was drawn, representing most of the intact testicular cells. This served as a reference for pre-adjusting voltage parameters and accurately drawing gates for normal testicular cells. The quadrants of different cell distributions were marked in the PE-A / APC-A scatter plot to obtain P1-1.

[0032] b. Perform on-machine analysis of cell samples stained with Indo-1, C-kit, and Thy (single and triple staining). Apply the gate and quadrant distribution from step a to the three single-stained and triple-stained samples to obtain P2, P3, P4, and P5. Re-examine the quadrant accuracy in the corresponding APC-A / PE-A scatter plot. Figure 1 As shown.

[0033] c. Flow cytometry was used to analyze triple-stained testicular cell samples, applying the gating and quadrant distribution methods described above. Cells were located by adjusting the voltage according to the parameters in steps a and b. The samples were then divided into three groups based on the differences in fluorescence signals between undifferentiated, undifferentiated-to-differentiated, and differentiated testicular cells (Thy-APC and c-kit-PE).

[0034] d. Select the three cell populations in quadrants I, II, and III of P5-1 separately. Under the side-scattered light area (SSC-A) and the blue light scattering area of ​​indo-1, Indo-1(Blue)-A yields three phyla: P6, P7, and P8. These represent the three cell populations: undifferentiated, undifferentiated-to-differentiated, and differentiated. Figure 2 As shown.

[0035] e. The method for calculating the average value using FlowJo-v10.8.1 software is as follows: Drag out any quadrant from the triple-stained cell sample, move the mouse over the quadrant, right-click to add an analysis, select Mean, and then add mean analyses for indo-1(Blue)-A and indo-1(Violet)-A sequentially. Select all analyses and drag them to the remaining three quadrants to obtain the average fluorescence intensity at the corresponding wavelength. Mean(indo-1(Violet)-A) / Mean(indo-1(Blue)-A) is F405 / F495. This ratio reflects the relative calcium ion concentration in each cell population. If existing methods are used to determine the fluorescence intensity Fmax corresponding to the highest intracellular calcium ion concentration and the fluorescence intensity Fmin corresponding to the lowest intracellular calcium ion concentration, and these values ​​are substituted into the formula [Ca2+]i = K d * β * (R – Rmin) / (Rmax−R) can also be used to calculate the absolute calcium ion content in the target cell, where K d denoted as the dissociation constant of indo-1, β is the ratio of the emission signal at 495 nm under extremely low [Ca2+]i levels to that under saturated [Ca2+]i levels, R is the fluorescence ratio, Rmin is the ratio recorded under extremely low [Ca2+]i levels, and Rmax is the ratio recorded under saturated [Ca2+]i levels.

[0036] f. To further verify the accuracy of the above grouping, the stained samples were smeared and photographed under a fluorescence microscope for observation. Figure 3 As shown in the figure. The results showed that the testicular suspension cells included cells stained with Thy-APC (undifferentiated spermatogonia) and c-kit-PE (differentiated spermatogonia), respectively, as well as cells stained with both colors (undifferentiated spermatogonia transitioning to differentiation).

Claims

1. A method for rapid detection of calcium signals in undifferentiated and differentiated spermatogonia, characterized in that, Includes the following steps: Step 1, Processing of testicular cell samples: After cleaning the isolated testes, the testicular tissue was broken up and placed in HBSS solution containing collagenase II and dispersin for digestion. Fetal bovine serum was then added to stop the digestion. Cell clumps were removed by cell filtration. The resulting cell suspension was centrifuged, the supernatant was removed, and the suspension was resuspended in HBSS solution. Staining was performed separately, including staining with indo-1AM, c-kit-PE, and Thy-APC individually, as well as staining with a combination of the three drugs. Step 2: Flow cytometry analysis of undifferentiated and differentiated spermatogonia: a. Use flow cytometry to detect unstained samples, find the main population of testicular cells in the FSC-A / SSC-A scatter plot and draw the P1 phylum, and mark the quadrants of different cell distributions in the PE-A / APC-A scatter plot to obtain P1-1; b. Cell samples stained with indo-1AM, c-kit-PE, and Thy-APC individually and in combination were analyzed using an instrument. The gate and quadrant distribution in a were applied to the three single-stained and triple-stained samples to obtain P2, P3, P4, and P5. The correctness of the quadrants was checked again in the APC-A / PE-A scatter plot to obtain P2-1, P3-1, P4-1, and P5-1. c. Flow cytometry was used to detect triple-stained testicular cell samples. The above gate and quadrant distribution methods were applied. The voltage was adjusted according to the parameters in steps a and b to locate the cells. The samples were divided into three groups based on the differences in fluorescence signals of undifferentiated, undifferentiated to differentiated, and differentiated testicular cells (Thy-APC and c-kit-PE). d. Select the three groups of cells in the first, second and third quadrants of P5-1 separately, and under the blue light scattering area of ​​SSC-A and indo-1, Indo-1(Blue)-A yields three gates: P6, P7 and P8, which are the three groups of cells separated: undifferentiated, undifferentiated to differentiated, and differentiated. Step 3: Using software, add Indo-1(Blue)-A and Indo-1(Violet)-A to cells in different sections of the triple staining tube. Analyze the mean values ​​and obtain the relative calcium ion concentration in each cell population by the ratio of the corresponding mean values ​​of Indo-1(Violet)-A and Indo-1(Blue)-A.

2. The method according to claim 1, characterized in that, In step 1, each adult testis was digested with 3 mL of HBSS solution containing 200-250 U / mL collagenase II and 2 U / mL dispersant enzyme.

3. The method according to claim 1, characterized in that, In step 1, the digestion conditions are 37°C for 30 minutes; the amount of fetal bovine serum used is 10% of the digestion liquid volume.

4. The method according to claim 1, characterized in that, In step 1, the three drugs are stained together by first adding indo-1AM staining, followed by c-kit-PE and Thy-APC staining.

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