Application of melatonin in promotion of primordial germ cell proliferation

Abstract

The invention discloses application of melatonin in promotion of primordial germ cell proliferation, explores a mechanism of melatonin in promotion of PGCs proliferation, and improves the proliferation speed and quality of PGCs by optimizing use conditions of melatonin, thereby solving the problem of low proliferation efficiency. By applying melatonin and optimizing culture conditions of PGCs, a more efficient and stable PGCs proliferation method can be provided, and related research and application in the field of reproductive biology are promoted; the accurate use of the growth factor MLT can be determined, and a new tool is provided for the research of reproductive biology, especially the application in animal breeding, reproductive biology and related fields; through more efficient PGCs proliferation, the genetic improvement efficiency of animal populations can be improved, and the development of the fields of biological pharmacy and the like is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and specifically relates to the application of melatonin in promoting the proliferation of primordial germ cells. Background Art

[0002] In the field of genetic breeding, primordial germ cells (PGCs) are a special type of cells differentiated at the early stage of embryonic development and have the potential to form gametes. During embryonic development, PGCs migrate from specific regions of the embryo to the gonads and further differentiate and mature in the gonads. Research shows that the proliferation, differentiation, and genetic stability of PGCs are crucial for the reproductive ability of animals. PGCs have a high proliferation ability during early embryonic development and maintain a stem cell state under certain physiological or experimental conditions, which makes them have important research and application values.

[0003] However, the technical problems of in vitro culture and proliferation of PGCs have always hindered the research progress. Under traditional PGC culture conditions, problems such as slow growth rate, low proliferation efficiency, and poor cell survival limit the application of this research in genetic breeding. Therefore, it is crucial to explore a new in vitro culture condition that helps to improve the growth rate, proliferation efficiency, and survival efficiency of PGCs. Summary of the Invention

[0004] Technical problems to be solved: Aiming at the above technical problems, the present invention provides the application of melatonin in promoting the proliferation of primordial germ cells, which can effectively solve the problems such as slow growth rate, low proliferation efficiency, and poor cell survival under traditional PGC culture conditions.

[0005] Technical solution: The present invention provides the application of melatonin in promoting the proliferation of primordial germ cells.

[0006] Preferably, melatonin is added to the in vitro culture medium of primordial germ cells, and the added concentration of melatonin does not exceed 400 μM / mL.

[0007] Furthermore, the added concentration of melatonin is 50 - 150 μM / mL.

[0008] Even further, the added concentration of melatonin is 100 μM / mL.

[0009] Preferably, the primordial germ cells are primordial germ cells of chickens or mammals.

[0010] Beneficial effects: By adding melatonin to the culture medium, the present invention improves the efficiency of in vitro culture of PGCs, provides a new tool for reproductive biology research, especially in the applications in animal breeding, reproductive biology, and related fields; through more efficient proliferation of PGCs, the genetic improvement efficiency of animal populations can be improved, and the development of fields such as biopharmaceuticals can be promoted. Description of the Drawings

[0011] Figure 1 It is the microscopic examination diagram (A) and growth curve diagram (B) of PGCs after treatment with melatonin at different concentrations. The scale in the lower right corner of Figure A is 200 times;

[0012] Figure 2 It is the detection result diagram of the proliferation ability of PGCs after treatment with melatonin. Among them, A is the fluorescence diagram of Edu detecting the cell proliferation ability, and B is the statistical chart of the difference in cell proliferation efficiency;

[0013] Figure 3 It is the detection result diagram of the ROS level in cells after treatment of PGCs with melatonin. Among them, A is the detection diagram of the ROS level in two groups of cells, and B is the result diagram of the difference analysis;

[0014] Figure 4 It is the detection result diagram of the mitochondrial membrane potential in cells after treatment of PGCs with melatonin. Among them, A is the staining diagram of the red mitochondrial membrane potential probe, and B is the difference analysis diagram;

[0015] Figure 5 It is the bar chart of the change differences of lipid peroxides, GSH and Fe in PGCs after treatment of PGCs with melatonin 2+ ;

[0016] Figure 6 It is the detection result diagram of the antioxidant gene level in PGCs after treatment of PGCs with melatonin;

[0017] Figure 7 It is the bar chart of the expression of genes related to the cell characteristics of PGCs after treatment of PGCs with melatonin. Among them, A is the PGC marker gene, B is the reproductive marker gene, and C is the migration-related gene;

[0018] Figure 8 It is the indirect immunofluorescence diagram of the expression of proteins related to the cell characteristics of PGCs after treatment of PGCs with melatonin. Among them, A is the CVH protein and B is the SSEA-1 protein. Detailed Embodiments

[0019] The present invention will be described in detail below with reference to the drawings and specific embodiments:

[0020] Example 1: Morphological Observation and Growth Trend Change of PGCs after Treatment with Different Concentrations of MLT

[0021] The sources of primary PGCs used in this experiment are as follows: Fresh fertilized eggs of Rugao yellow chickens (Poultry Research Institute, Chinese Academy of Agricultural Sciences, Jiangsu) were incubated in an incubator at 37°C until 7.5 days to obtain chicken embryos at stage 32 of Hamburger and Hamilton (HH). The gonads of each dissected chicken embryo were washed with 300 μL of phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin BSA (Signa, CAS: 9045-46-8). After centrifugation at 1000 rpm / min for 2 minutes using a centrifuge, the supernatant was removed. Subsequently, the gonads were incubated in 200 μL of a solution containing 0.25% trypsin-EDTA for 1 minute, and the gonadal tissue blocks were mashed using a pipette tip. The tissue block suspension was centrifuged at 1000 rpm / min for 2 minutes, and after centrifugation, the supernatant was removed. The remaining cell pellets and tissues were resuspended in PGC medium and transferred to a 48-well plate for culture. The culture conditions for PGCs were: 37°C, 5% CO2, and maximum humidity environment. The primary PGC cells were successfully established after 40 - 60 days of continuous culture. The established cells were stored at low temperature in liquid nitrogen and revived and cultured when needed.

[0022] The experiment was divided into 4 groups. The Control group was the basic culture system, and the experimental groups were supplemented with 50, 100, and 150 μM MLT respectively. The PGCs were cultured in a cell incubator at 37°C for 72 h, and the cell number and morphological changes were observed, and the cell growth curve was plotted. The results are as Figure 1 shown: Figure 1 A in the figure shows the treatment group under the condition of adding 100 μM MLT, with the highest cell density; at the same time, through a cell counter, the cell number was calculated and the cell curve was plotted, as Figure 1 shown in B in the figure: When 100 μM MLT was added to the treatment group, the cell growth curve was significantly higher than that of other groups, and it could significantly promote the proliferation of PGCs. Subsequently, the cells in the 100 μM treatment group were continuously detected.

[0023] Example 2: Detection of cell proliferation after treatment with 100 μM MLT by Edu

[0024] To accurately investigate the proliferation of PGCs by MLT, in this example, cells in the 100 μM treatment group were detected using an EdU detection kit (Beyotime, C0071S). The experiment was divided into two groups. The control group was the original culture system, and the experimental group was the treatment group with 100 μM MLT added. They were cultured in a 37 °C cell incubator for 72 h, and EdU was used to detect PGC proliferation. The PGCs were plated and passaged in a 24-well plate. The EdU solution was diluted with the complete medium for PGCs at a ratio of 1000:1 to prepare a 50 μM EdU medium; 100 μL of the 50 μM EdU medium was added to each well and incubated for 2 hours, and then the medium was discarded; the cells were washed with PBS 1 - 2 times, 5 minutes each time; centrifuged at 1400 rpm for 6 min, the supernatant was discarded, and 20 μL was left. The PGCs were pipetted and mixed well and then dropped onto a glass slide; 20 μL of cell fixative (PBS containing 4% paraformaldehyde) was added and incubated at room temperature for 30 minutes, and then the fixative was discarded; 20 μL of 2 mg / mL glycine was added and incubated for 5 minutes, and then the glycine solution was discarded; 20 μL of PBS was added and washed for 5 minutes, and then the PBS was discarded; 20 μL of permeabilizing agent (PBS containing 0.5% TritonX-100) was added and incubated for 10 minutes; washed with PBS once, 5 minutes; 20 μL of 1× Apollo staining reaction solution was added and incubated in the dark at room temperature for 30 minutes, and then the staining reaction solution was discarded; the permeabilizing agent was washed 2 - 3 times with 20 μL each time, 10 minutes each time, and then the permeabilizing agent was discarded; washed with PBS once, 5 minutes; 20 μL of Hoechst 33342 staining solution was added and incubated in the dark at room temperature for 30 minutes, and then the staining solution was discarded; 20 μL of permeabilizing agent was added and incubated for 10 minutes, washed with PBS once, 5 minutes; sealed with neutral balsam and observed under a fluorescence microscope using different channels. Figure 2 In A, the left, middle, and right show the bright field of the cells, Apollo staining (red), and Hoechst 33342 staining (blue) results respectively; cell number statistics were performed, and the results are as Figure 2 shown in B: The number of cells in the treatment group was significantly higher than that in the control group. This example shows that the treatment group with 100 μM MLT added promotes the proliferation of PGCs.

[0025] Example 3: Flow cytometry to detect the ROS level in cells after treatment with 100 μM MLT

[0026] To further explore the effect of MLT on the growth of PGCs, in this example, the reactive oxygen species (ROS) level of the cells was detected using a ROS detection kit (Beyotime, S0033S), which is an important indicator of normal cell growth. ROS detection: The collected cells were suspended in the diluted DCFH-DA drug. The dilution of DCFH-DA was prepared by diluting it 1:1000 with serum-free culture medium. The cell suspension was placed in a 37 °C cell culture incubator and incubated in the dark for 20 minutes to make the final concentration 10 μM. The cell pellet was collected by centrifugation, and the probe was added to ensure a cell density of 1.0×10 6 ~2.0×10 7 . Incubate in a 37 °C cell culture incubator for 20 minutes, and invert and mix gently every 5 minutes to allow the probe to fully contact the cells. Wash the cells 3 times with serum-free cell culture medium to thoroughly remove the DCFH-DA that did not enter the cells, and then detect with a flow cytometer.

[0027] Next, flow cytometry was used to detect cell apoptosis. PGCs were plated and passaged in a 24-well plate, and flow cytometry was used to detect PGCs. The method steps are as follows: Centrifuge to collect cells at 300×g and 4 °C; Wash the cells 2 times with pre-cooled PBS, each time centrifuging at 300×g and 4 °C for 5 min. Collect 1~5×10 5 cells; Discard the upper layer of PBS, add 100 μL of 1×Binding Buffer to resuspend the cells; Add 5 μL of Annexin V-FITC and 10 μL of PI Staining Solution, and mix gently; React in the dark at room temperature for 10 - 15 min; Add 400 μL of 1×Binding Buffer, mix well and place on ice, and the sample was detected with a flow cytometer within 1 hour. The results are as Figure 3 shown: Figure A is the detection chart of the ROS content in cells. The red color represents the control group, and the blue color represents the 100 μM MLT treatment group. The ROS content in the control group cells was significantly lower than that in the experimental group; Figure B is the statistical analysis chart, indicating that 100 μM MLT significantly reduced the ROS content in PGCs (p < 0.05), which can protect cells from oxidative stress damage.

[0028] Example 4: Detection of the mitochondrial membrane potential level of cells after treatment with 100 μM MLT using MitoTracker

[0029] The mitochondrial membrane potential level of cells is an important indicator for cells to maintain redox balance. To explore the effect of MLT on the oxidative stress level of PGCs, the mitochondrial membrane potential level of the MLT treatment group was detected in this example. By the MitoTracker probe method (Thermofisher, M7510), in the culture system, the experimental group was the treatment group with 100 μM added. The cells were cultured in a 37 °C cell incubator for 72 h, and the cell precipitate was obtained by centrifugation. The supernatant was aspirated, and the cells were gently resuspended in the staining solution (containing the MitoTracker probe) preheated to 37 °C. Staining was carried out by incubating for 30 min under the culture conditions of 37 °C and 5% carbon dioxide concentration. After staining, the cell precipitate was obtained by centrifugation again, and then the cells were resuspended in fresh culture medium and analyzed under a fluorescence microscope.

[0030] The results are as Figure 4 shown: The addition of 100 μM MLT significantly increased the mitochondrial membrane potential level of PGCs (p < 0.001), indicating enhanced mitochondrial activity of cells and being beneficial to maintaining the redox balance of cells.

[0031] Example 5: Determination of lipid peroxides, GSH and Fe in PGCs after treatment with MLT by microplate reader 2+ changes

[0032] To further explore the effect of MLT on the oxidative stress level of PGCs, lipid peroxides, GSH and Fe 2+ were detected. The steps are as follows: The treated cells were collected, and samples or standards were added to a 96-well plate in sequence and mixed evenly. After adding 150 μL of the total glutathione detection working solution, it was mixed evenly and incubated at 25 °C or room temperature for 5 minutes; 50 μL of 0.5 mg / mL NADPH solution was added and mixed evenly; immediately, the absorbance value at 405 nm was measured with a microplate reader. The results are as Figure 5 shown: The MLT treatment group reduced the oxidative stress level of PGCs and improved the ability of cells to metabolize reactive oxygen species.

[0033] Example 6: To explore the effect of MLT on the antioxidant stress ability of cells, qPCR quantitative verification of related genes was carried out

[0034] To further detect the effect of MLT on the antioxidant stress ability of cells and detect the expression levels of related genes of antioxidant stress, this example carried out:

[0035] ① Total RNA extraction: Collect the PGCs cultured in the control group and the MLT treatment group into a centrifuge tube, centrifuge at 1400 rpm for 6 min, and discard the supernatant; add 1 mL of TRIZOL reagent, pipette and mix well, and let it stand at 4 °C for 5 min; let the sample stand at room temperature for 5 min to fully dissociate the protein; add 0.2 mL of chloroform, tighten the lid, shake vigorously for 15 s and let it stand at room temperature for 2 - 3 min; centrifuge at 4 °C, 12000×g for 15 min. After centrifugation, the sample is layered, with RNA in the upper aqueous phase and protein and DNA in the lower organic phase; take 500 μL of the supernatant, add 0.5 mL of isopropanol, mix gently, and let it stand at room temperature for 10 min. A gelatinous precipitate will appear at the bottom of the tube, which is RNA; centrifuge at 4 °C, 12000×g for 10 min, and discard the supernatant; add 1 mL of 75% ethanol to the precipitate, mix gently; centrifuge at 4 °C, 7500×g for 5 min, and discard the supernatant; air-dry the RNA sample, add an appropriate amount of enzyme-free water to dissolve it (it can be promoted to dissolve at 55 - 60 °C for 10 min); measure the RNA concentration, detect with a UV spectrophotometer and calculate the OD 260 / OD 280 ratio, and a ratio of 1.9 - 2.0 indicates good results.

[0036] ② cDNA synthesis: Thaw the template RNA on ice; thaw 5×FastKing-RT SuperMix and RNase-Free ddH2O at room temperature and immediately place them on ice. Before use, vortex and mix each solution, and centrifuge briefly to collect the liquid remaining on the tube wall; prepare the reverse transcription reaction system: 4 μL of 5×FastKing-RT SuperMix, 50 ng - 2 μg of Total RNA (according to the RNA concentration), and make up to 20 μL with RNase-Free ddH2O; reverse transcription reaction: remove genomic DNA and reverse transcription reaction: 42 °C, 15 min; enzyme inactivation process: 95 °C, 3 min; dilute the cDNA concentration by adding RNase-Free ddH2O according to the RNA content used.

[0037] ③ Use qRT-PCR technology to detect the mRNA expression levels of antioxidant stress-related genes in PGCs after MLT treatment: The 10 μL qRT-PCR reaction system consists of 5 μL of 2×Universal SYBR Green Fast qPCR Mix, 0.4 μL of upstream primer (10 μM), 0.4 μL of downstream primer (10 μM), 2 μL of cDNA, and make up to 10 μL with RNase-Free ddH2O. Reaction process: 95 °C for 3 minutes; 95 °C for 5 seconds, 60 °C for 30 seconds, for a total of 40 cycles. The upstream and downstream primer sequences of the detected genes are shown in Table 1 below:

[0038] Table 1 Primer sequences for qPCR quantification of antioxidant genes in PGCs after MLT treatment

[0039]

[0040] Three biological replicates were set for PGCs culture, and three technical replicates were set for each sample. The 2 -ΔΔCT method was used for quantitative analysis of the expression level, and the results were as Figure 6 shown: Superoxide dismutase SOD1 showed a significant up-regulation trend after MLT treatment of PGCs, indicating that MLT can promote the antioxidant stress ability of cells.

[0041] Example 7: Explore whether MLT will affect the cell characteristics of PGCs, and perform qPCR quantitative verification on related genes

[0042] To explore whether MLT will affect the cell characteristics of PGCs such as pluripotency, migration ability, and cell stemness, the expression levels of related genes were detected. The steps were the same as in Example 6, and the primers used were as shown in Table 2 below:

[0043] Table 2 Primer sequences for qPCR quantification of genes related to cell characteristics of PGCs after MLT treatment

[0044]

[0045]

[0046] The results were as Figure 7 shown: There were no significant changes in POUV, SOX2, and NANOG among the PGCs marker genes, while CVH was significantly up-regulated; among the germ cell marker genes, DAZL was significantly up-regulated (p < 0.01), and PRDM1 showed no significant change; the migration-related gene CXCR4 was extremely significantly up-regulated. This example shows that MLT treatment will not reduce the expression of PGC-related genes and will not affect normal cell functions.

[0047] Example 8: Explore whether MLT will affect the cell characteristics of PGCs, and detect related proteins

[0048] For PGCs treated with melatonin (MLT), indirect immunofluorescence was used to detect whether both the pluripotency SSEA1 protein and the germ cell ability CVH protein of PGCs were expressed, and whether melatonin (MLT) had an impact on PGCs. The steps were as follows:

[0049] 1) Cell / tissue preparation

[0050] Cultured cells: PGCs were cultured in an appropriate medium to ensure that the cells were in the logarithmic growth phase;

[0051] Sampling and fixation: For tissue sections (such as embryonic tissue or other organ tissue), use an appropriate fixation method (4% paraformaldehyde) PFA to fix the tissue. The PGCs cell fixation method includes: first fix with 4% paraformaldehyde (PFA) for 15 minutes, and then wash the fixed PGCs cells with 0.1% PBS-T (containing 0.1% Tween 20), repeat 3 times, 5 minutes each time;

[0052] If necessary, use pre-cooled 4°C methanol to fix again for 15 minutes. Methanol has a better fixation effect, especially when studying membrane proteins.

[0053] 2) Cell permeabilization

[0054] Permeabilization: Use 0.1% Triton X-100 PBS diluent to permeabilize the cells for 15 minutes, so that the antibodies in subsequent experiments can penetrate the cell membrane and enter the cells to bind to the intracellular antigens. After permeabilization, use 0.1% PBS-T to wash the permeabilized cells.

[0055] 3) Blocking non-specific binding sites

[0056] Blocking treatment: Use PBS diluent containing 10% goat serum (Solarbio) and blocking solution to block nonspecific binding sites on cells. The blocking process needs to be blocked for 2 hours at room temperature. After blocking, the PGCs samples are washed with 0.1% PBS-T, and repeated 3 times, 5 minutes each time.

[0057] 4) Primary antibody incubation (specific antibody)

[0058] Select specific primary antibodies: Select antibodies targeting PGCs surface markers and endogenous proteins (including specific transcription factors, cell markers, etc.) according to the purpose of the study. SSEA-1 (Abcam, MC480), CVH (DDX4, Abcam, AB27591), incubation conditions are as follows: thaw the primary antibody stored at -20°C on ice at 4°C, dilute the required primary antibody at a concentration of 1:1000 using a universal antibody diluent and place on ice for later use, add the prepared primary antibody dilution dropwise to the PGCs slide sample after blocking and incubate at 4°C overnight (8 hours) to ensure that the antibody is fully bound to the target protein.

[0059] 5) Washing

[0060] After incubation, wash the PGCs samples on the slides with 0.1% PBS-T to remove the unbound primary antibody. Repeat 3 times, 5 minutes each time.

[0061] 6) Secondary antibody incubation (fluorescently labeled secondary antibody)

[0062] Select a secondary antibody conjugated with a fluorescent label corresponding to the primary antibody used: Select a secondary antibody conjugated with a fluorescent label corresponding to the species of the primary antibody used in the above experimental procedure. Rabbit anti- (Abclonal, AS039) corresponds to the CVH primary antibody in the above experimental procedure, and mouse anti- (Boster, BM2012) corresponds to the SSEA-1 primary antibody in the above experimental procedure. Incubation conditions: Thaw the fluorescently labeled secondary antibody stored at -20 °C on ice at 4 °C. Dilute the required secondary antibody to a concentration of 1:200 with a universal antibody diluent and place it on ice for later use (the entire process of using the secondary antibody needs to be carried out in the dark). Drop the prepared secondary antibody dilution onto the PGCs sample that has completed the primary antibody incubation and incubate for 2 hours. The incubation process should be carried out at room temperature. During the secondary antibody incubation, the fluorescently labeled secondary antibody will bind to the primary antibody, amplifying the signal.

[0063] 7) Washing

[0064] The PGCs sample after completing the secondary antibody incubation is washed with 0.1% PBS-T. The washing process can remove the unbound secondary antibody. Repeat the washing process 3 times, 5 minutes each time.

[0065] 8) DAPI staining

[0066] Nuclear staining: DAPI staining is mainly used for fluorescent labeling of the nucleus, which can label the nucleus for easy observation of its position and morphology. Drop DAPI onto the PGCs sample that has completed the secondary antibody incubation and incubate.

[0067] Incubation conditions: Incubate with DAPI at room temperature for 5 minutes. The PGCs sample after completing the DAPI incubation is washed again with 0.1% PBS-T, repeating 3 times, 5 minutes each time.

[0068] 9) Mounting and observation

[0069] Mounting: Use a mounting medium glycerol-PBS containing an anti-fluorescence quenching component to cover the PGCs sample evenly by dropping it onto the sample area. Be careful not to generate bubbles when dropping. The mounting medium helps reduce the attenuation of the fluorescent signal and maintain the stability of the fluorescence;

[0070] Observation under a fluorescence microscope: Observe the sample using an inverted fluorescence microscope (Leica). Select the corresponding fluorescence filter, excitation light, and observe the signal of the fluorescent label, observe the cell-labeled fluorescence and take pictures for recording.

[0071] 10) Data analysis

[0072] Image acquisition: Obtain images under an inverted fluorescence microscope, record the signals in different fluorescence channels, and perform quantitative and qualitative analysis.

[0073] Signal intensity analysis: Use image processing software (such as ImageJ or other professional software) to analyze the intensity and distribution of fluorescence signals, and evaluate the distribution, morphology or proliferation of PGCs in the sample;

[0074] The results are as Figure 8 shown: CVH in red ( Figure 8 in A) and SSEA-1 in green ( Figure 8 in B) are both expressed in PGCs. DAPI staining of cell nuclei shows that the addition of 100 μM MLT to PGCs has no effect on the cell characteristics of PGCs, and they still have the same characteristics as normal PGCs.

[0075] Example 9

[0076] Regarding melatonin protecting H2O2-induced oxidative damage of embryonic stem cells (ESCs). The process of oxidative stress refers to the excessive accumulation of reactive oxygen species (ROS), resulting in impaired proliferation, DNA damage, mitochondrial dysfunction and apoptosis. H2O2 (hydrogen peroxide) can induce oxidative stress in ESCs, ultimately affecting proliferation and survival. Melatonin (MLT) is an endogenous antioxidant and free radical scavenger, which plays an important role in scavenging ROS, increasing the activity of oxidase, inhibiting apoptosis, etc. The research results show that MLT can be used as an additive in the culture system to improve the survival rate of ESCs. And it is proposed that during the differentiation of ESCs into PGCs, MLT may help improve the survival and development of PGCs; and MLT can be used as an antioxidant drug for stem cell therapy and anti-aging research.

[0077] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of melatonin in promoting the proliferation of primordial germ cells.

2. The use of melatonin according to claim 1 in promoting the proliferation of primordial germ cells, characterized in that: Melatonin is added to the in vitro culture medium of primordial germ cells, and the added concentration of melatonin does not exceed 400 μM / mL.

3. The use of melatonin according to claim 2 in promoting the proliferation of primordial germ cells, characterized in that: The added concentration of melatonin is 50 - 150 μM / mL.

4. Use of melatonin according to claim 3 in promoting the proliferation of primordial germ cells, characterized in that: The added concentration of melatonin is 100 μM / mL.

5. Use of melatonin according to claim 1 in promoting the proliferation of primordial germ cells, characterized in that: The primordial germ cells are primordial germ cells of chickens or mammals.

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