Method for efficiently differentiating monkey spermatogonial stem cells in vitro and application

Abstract

The invention discloses a method for efficiently differentiating monkey spermatogonial stem cells in vitro and application. The invention belongs to the technical field of biology, and particularly relates to a method for efficiently differentiating monkey spermatogonial stem cells in vitro and application. The active ingredients of the composition for promoting in-vitro differentiation of the monkey spermatogonial stem cells are retinoic acid, BMP4, testosterone and a basic differentiation medium. The concentration of the retinoic acid in the composition is 1 [mu] mol / L, the concentration of the BMP4 is 50 [mu] g / L, and the concentration of the testosterone is 0.1 mmol / L. According to the method, a culture system for in-vitro differentiation of the monkey spermatogonial stem cells (SSCs) into the functional haploid sperms is successfully established, the haploid differentiation efficiency can be obviously improved (the proportion exceeds 8%) after 96 hours of continuous treatment, and the blank of research on in-vitro differentiation systems and molecular mechanisms of the monkey SSCs is filled.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a method for in vitro differentiation of monkey spermatogonial stem cells and application thereof. BACKGROUND

[0002] At present, 70-90% of the causes of male infertility are due to spermatogenic disorders. Spermatogonial stem cells (SSCs) have the potential of self-renewal and differentiation, and are the only cells in male mammals that can transmit genetic information to the next generation. In vitro induction and differentiation of SSCs can provide theoretical support for the treatment of some genetic diseases, endogenous SSC damage in humans and the study of male infertility, and has important theoretical and practical significance. How to make SSCs differentiate into functional sperm cells in vitro has become a difficult and hot topic in the world. At present, methods such as culture of seminiferous tubule fragments, co-culture of spermatogenic cells and Sertoli cells, co-culture with Vero cells, and 3D culture of testicular tissue can induce human SSCs to differentiate in vitro and obtain haploid cells. Although a variety of methods for differentiation of SSCs have emerged in recent years, the differentiation efficiency of SSCs is low and the differentiation system is imperfect, and the practical application value is limited.

[0003] Spermatogonial stem cells (SSCs) are located in the basement membrane of the seminiferous tubules of the testis. They maintain the stem cell pool stable through asymmetric division of type A dark spermatogonia (resting state) and type A pale spermatogonia (proliferative state), and then start and enter differentiation under the induction of retinoic acid (RA), BMP4 and other signals, forming type A1-A4 spermatogonia, type B spermatogonia, and type B spermatogonia further differentiating into primary spermatocytes and entering meiosis to produce haploid sperm cells. Sperm cells develop into mature sperm through morphological remodeling such as acrosome formation, flagellum assembly and chromatin condensation. In the differentiation of spermatogonia, retinoic acid (RA) is a key signal molecule, and its core role is reflected in multidimensional regulation: activating downstream differentiation genes through RAR / RXR heterodimers, inhibiting NANOS2, PLZF and other stemness genes, and breaking the balance of spermatogonial stem cells; driving cell fate conversion and promoting undifferentiated spermatogonia to type A1 and subsequent types. Human spermatogonial stem cells cultured in vitro are prone to epigenetic abnormalities (such as Stra8 promoter methylation), and exogenous RA is difficult to rebuild the differentiation microenvironment when the function of Sertoli cells is impaired. In addition, there are problems such as large individual differences in clinical efficacy (some infertile patients do not respond), high-dose RA causing testicular degeneration / liver toxicity, and lack of individualized dosing regimens based on RA-related molecular expression, which ultimately restrict its application. The role of RA in spermatogonial differentiation is very important, but there is currently no effective use of RA to establish a method for efficient in vitro differentiation of spermatogonial stem cells. SUMMARY

[0004] The main problem to be solved by the present application is how to improve the in vitro differentiation ability of monkey spermatogonial stem cells.

[0005] To solve the above problem, the present application provides a composition for promoting the in vitro differentiation of monkey spermatogonial stem cells, and the active ingredients of the composition are retinoic acid, BMP4, testosterone and a basic differentiation medium.

[0006] Further, the concentration of retinoic acid in the composition is 1 μmol / L, the concentration of BMP4 is 50 μg / L, the concentration of testosterone is 0.1 mmol / L, and the basic differentiation medium is DMEM / F12 medium containing 5% KnockOut TM serum replacement (KSR), 10 ng / mL stem cell factor, 10 ng / mL fibroblast growth factor, 25 ng / mL epidermal growth factor, 10 ng / mL insulin-transferrin-selenium, 2 mM glutamine (G3126), 0.05 lU / mL FSH, 0.05 lU / mL LH and 1% penicillin-streptomycin.

[0007] The present application also provides a method for constructing an in vitro differentiation model of monkey SSCs, comprising the following steps: 1) Obtain fresh testicular tissue of a 2-month-old young monkey; 2) Perform tissue separation and enzymatic digestion to obtain monkey seminiferous tubule fragments and single cell mixtures, culture, count the haploid differentiation efficiency, and establish an SSC initial culture system; 3) Perform differentiation culture on the cells obtained from the SSC initial culture system of step 2) to obtain an in vitro differentiation model of monkey SSCs.

[0008] Further, the differentiation culture step of step 3) is to add 1 μmol / L retinoic acid, 50 μg / L BMP4 and 0.1 mmol / L testosterone to the basic medium for continuous treatment for 96 hours, and then replace it with the basic differentiation medium described above, and culture for 9 days.

[0009] The present application also provides the use of the composition described above in promoting the in vitro differentiation of monkey spermatogonial stem cells.

[0010] The present application also provides the use of the composition described above in the preparation of a product for promoting the in vitro differentiation of monkey spermatogonial stem cells.

[0011] The present application also provides the use of the composition described above in constructing an in vitro differentiation model of monkey SSCs.

[0012] The present invention also provides the use of the composition described above in the preparation of products for in vitro induction of sperm production in subject SSCs for assisted reproduction.

[0013] This invention successfully established a culture system for the in vitro differentiation of monkey spermatogonial stem cells (SSCs) into functional haploid sperm, and clearly demonstrated that continuous treatment for 96 hours can significantly improve the haploid differentiation efficiency (more than 8%), filling the gap in the study of in vitro differentiation system and molecular mechanism of monkey SSCs.

[0014] Because monkeys and humans share highly similar reproductive anatomy and physiology, the established system provides an ideal alternative model for studying the differentiation mechanism of human SSCs. At the same time, it provides key technical support and experimental basis for the treatment of male infertility (such as in vitro induction of sperm from patient SSCs for assisted reproduction), reproductive toxicology screening (detecting the interference of external factors on spermatogenesis), and transgenic animal production (in vitro acquisition of gene-edited sperm). Attached Figure Description

[0015] Figure 1 Spermatogonia in the testes of young monkeys. A: HE staining of testicular tissue from 2-month-old monkeys. B: Purple indicates the immunohistochemical results of RARα. C: Western blotting results of RARα, with tublin as the internal reference gene.

[0016] Figure 2 Spermatogonia isolation and differentiation. A) Spermatogonia obtained by enzymatic digestion. B) Immunofluorescence results of Plzf and GFRα-1 in spermatogonia. C) The left image contains three peaks: haploid, diploid, and tetraploid, corresponding to differentiated sperm, spermatogonia, and spermatocytes in the mitotic stage, respectively.

[0017] Figure 3 To illustrate differentiation efficiency and sperm motility. A. The left image shows sperm cells cultured from the differentiation system, already exhibiting key features such as sperm head and tail. The right image is the control group, showing sperm in semen. B. Each of the three sub-images contains three peaks: haploid, diploid, and tetraploid, corresponding to differentiated sperm, spermatogonia, and spermatocytes in the mitotic stage, respectively. C. The left image shows the immunofluorescence results of sperm cells cultured from the differentiation system stained with acrocin and DAPI; the right image shows the corresponding bright field. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0020] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0021] The monkeys used in the following examples are described in: Yiming Yuan et al., Paternity Testing for Crab-eating Macaques and Rhesus Macaques by Using Microsatellite Markers. ANIMAL MODELS AND EXPERIMENTAL MEDICINE (2576-2095), Vol. 8, pp. 1710-1716. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0022] The retinoic acid used in the following examples was purchased from Solarbio (Catalog No. A9120); BMP4 was purchased from MedChemExpress (Catalog No. HY-P10710); and testosterone was purchased from MedChemExpress (Catalog No. HY-135794).

[0023] Example 1: Establishment of a method for in vitro differentiation of monkey spermatogonial stem cells 1. Identification and initial culture of monkey SSCs Fresh testicular tissue was obtained from 2-month-old juvenile monkeys. The tissue was divided in half, and one half was immersed in tissue fixative and then removed from the laboratory. Paraffin sections were prepared, dewaxed with xylene, hydrated with a gradient of ethanol to distilled water, and then the nuclei were stained with hematoxylin for 5-10 minutes. After differentiation with hydrochloric acid ethanol and warm water blueing, the cytoplasm was stained with eosin for 2-5 minutes. Subsequently, the sections were dehydrated with a gradient of ethanol, cleared with xylene, mounted with neutral resin, and air-dried. Microscopic observation revealed only undifferentiated spermatogonia in the seminiferous tubules, with no mature germ cells, ruling out differentiation interference. Figure 1 (A) Transport the sample to the laboratory using PBS buffer containing 100 mg / mL streptomycin and 100 IU / mL penicillin to avoid sample contamination and reduced activity.

[0024] 1) Detection of spermatogonia's responsiveness: Testicular tissue from juvenile monkeys was removed from fixative, cryo-embedded in an OCT complex, and then cut into 7 μm thick sections. Histological analysis of seminiferous tubules was performed using hematoxylin and eosin (H&E) staining. Immunohistochemistry was used to detect the expression patterns of UCHL1 and RARα. Briefly, after washing three times with PBS, slides were incubated at room temperature for 1 hour in PBS containing 1% BSA. Primary antibody (1:200 dilution) was added. After 4 hours of incubation, secondary antibody was added and incubated for 1 hour. Staining was visualized using a DAB substrate kit. Microscopic observation revealed the expression of retinoic acid (RA) receptor RARα in germ cells. Figure 1 (B) This demonstrates that the cells possess RA response capabilities.

[0025] A small portion of the tissue was lysed and preserved in PBS buffer to prepare and quantify protein samples. After denaturation with loading buffer, the samples were separated by SDS-PAGE electrophoresis. The samples were then transferred to nitrocellulose or PVDF membranes, blocked, and sequentially incubated with primary and secondary antibodies. Finally, qualitative and relative quantitative analysis was performed using ECL chemiluminescence and other colorimetric imaging techniques. RARα expression in the lysed tissue was determined to be (…). Figure 1 (C), which once again proves that it has RA response capability.

[0026] Figure 1 Image A shows the HE staining results of testicular tissue from a 2-month-old monkey. Numerous round spermatogonia were observed in the testes, with no other cell shapes, indicating that the spermatogonia were not differentiated. Image B (purple) shows the immunohistochemical results of RARα, confirming its expression in the testes. Image C shows the Western blot results of RARα, quantitatively demonstrating its expression in the testes; tublin is the internal reference gene.

[0027] 2) Tissue isolation and enzymatic digestion Under aseptic conditions, the tunica vaginalis was removed from the testis. The seminiferous tubules were carefully dissected and completely fragmented using fine-tipped forceps. After decoction, the seminiferous tubules were incubated at 37°C for 15 minutes with an enzyme mixture containing 0.1 mg / mL type IV collagenase and 1.0 μg / mL DNase, followed by neutralization with 10% fetal bovine serum. The mixture was filtered through a 40-mesh sieve to obtain single cells and fragments of the seminiferous tubules.

[0028] The SSCs were cultured using an initial culture system containing 20% ​​KnockOut. TM Serum substitute (KSR), 2 mmol / L L-glutamine, 1% non-essential amino acids, and 10 ng / mL fibroblast growth factor in DMEM / F12 medium.

[0029] Three days later, cell morphology was observed daily, and it was found that spermatogonia had already adhered to the bottom of the dish and grown. Figure 2 (A). After fixing and blocking spermatogonia, primary antibodies of Plzf and GFRα-1 were added and incubated overnight at 4°C. Unbound antibodies were washed away, followed by incubation with fluorescent secondary antibody. After washing in the dark, the nuclei were stained with DAPI, and finally observed under a fluorescence microscope. Figure 2 (B) Spermatogonia simultaneously express Plzf and GFRα-1. Plzf focuses on inhibiting differentiation and stabilizing stem cell characteristics, while GFRα-1 regulates proliferation and survival through signaling pathways. The two work together to ensure the homeostasis of the stem cell pool.

[0030] Using basal differentiation medium, haploid differentiation efficiency was calculated on day 9 to establish an initial culture system for SSCs. Figure 2 (C), laying the foundation for subsequent differentiation.

[0031] The basic differentiation medium contains 5% KnockOut TM Serum substitute (KSR), 10 ng / mL stem cell factor, 10 ng / mL fibroblast growth factor, 25 ng / mL epidermal growth factor, 10 ng / mL insulin-transferrin-selenium, 2 mM glutamine (G3126), 0.05 lU / mL FSH, 0.05 lU / mL LH and 1% penicillin-streptomycin in DMEM / F12 medium.

[0032] The results are as follows: the tissue fragments after enzymatic hydrolysis can be cultured into spermatogonia, indicating that the enzymatic hydrolysis method is feasible and effective. Figure 2 (A); Spermatogonia simultaneously express Plzf and GFRα-1 ( Figure 2 (B)

[0033] Depend on Figure 2 As shown in Figure C, the left image contains three peaks (the first peak is located at the leftmost 2.15%), representing haploid, diploid, and tetraploid cells, corresponding to differentiated spermatogonia, spermatogonia, and spermatocytes in the mitotic stage, respectively. It can be seen that 2.15% of the cells have differentiated into spermatogonia, indicating the successful construction of the SSC differentiation culture system. The right image is the control group, showing the semen analysis results, where 90.4% of the cells are spermatogonia.

[0034] 2. In vitro differentiation culture of monkey SSCs To investigate the effects of retinoic acid, BMP4, and testosterone on the differentiation of monkey SSCs, the following experiments were conducted: Group M (control group): cultured on basal differentiation medium for 9 days, with half of the medium replaced every 2 days.

[0035] M+RBT-96h group: 1 μmol / L retinoic acid, 50 μg / L BMP4, and 0.1 mmol / L testosterone were added to the basal differentiation medium and treated continuously for 96 hours. Subsequently, the medium was replaced with basal medium without retinoic acid, BMP4, and testosterone, and cultured for 9 days. Cell growth was observed daily, and the time and number of sperm-like cells (single-tailed structures) were recorded. Round sperm cells were clearly visible in the later stages of differentiation culture. Figure 3 (A)

[0036] On day 9, haploid differentiation efficiency was calculated. The sperm percentage in the M+RBT-96h group reached 8.07%, while that in the M group was 4.88%. Figure 3 (Middle B). This indicates that the addition of retinoic acid, BMP4, and testosterone improved the differentiation capacity of spermatogonia.

[0037] After fixing and blocking differentiated sperm cells, primary antibodies against acrocin were added and incubated overnight at 4°C. Unbound antibodies were washed away, followed by incubation with fluorescent secondary antibody. After washing in the dark, the nuclei were stained with DAPI, and finally observed under a fluorescence microscope. Figure 3 (C) Green represents acrosomal protease, and blue represents the cell nucleus. It can be seen that differentiated sperm can produce acrosomal protease, indicating reproductive capacity. This demonstrates that this differentiation system can differentiate into reproductively capable sperm.

[0038] The results are as follows Figure 3 As shown in Figures A and C: In the left image, 4.88% of the cells differentiated into sperm, and in the middle image, 8.07% of the cells differentiated into sperm, indicating that the culture system with retinoic acid, BMP4, and testosterone can improve differentiation efficiency. The right image is the control group, showing the results of semen analysis, where 90.4% of the cells were sperm. Figure C: The left image shows the immunofluorescence results of sperm cells cultured in the differentiation system stained with acrocin and DAPI, and the right image shows the corresponding bright field. This indicates that the differentiated sperm have reproductive capacity.

[0039] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition for promoting the in vitro differentiation of monkey spermatogonial stem cells, characterized in that, The active ingredients of the composition are retinoic acid, BMP4, testosterone, and basal differentiation medium.

2. The composition according to claim 1, characterized in that, The composition contains retinoic acid at a concentration of 1 μmol / L, BMP4 at a concentration of 50 μg / L, testosterone at a concentration of 0.1 mmol / L, and a basal differentiation medium containing 5% KnockOut. TM Serum substitute (KSR), 10 ng / mL stem cell factor, 10 ng / mL fibroblast growth factor, 25 ng / mL epidermal growth factor, 10 ng / mL insulin-transferrin-selenium, 2 mM glutamine, 0.05 lU / mL FSH, 0.05 lU / mL LHLH and 1% penicillin-streptomycin in DMEM / F12 medium.

3. A method for constructing an in vitro differentiation model of monkey SSCs, characterized in that, It includes the following steps: 1) Obtain fresh testicular tissue from 2-month-old juvenile monkeys; 2) Perform tissue isolation and enzymatic digestion to obtain a mixture of seminiferous tubule fragments and single cells, culture them, statistically analyze the haploid differentiation efficiency, and establish an initial culture system for SSCs; 3) The cells obtained from the initial SSC culture system in step 2) were cultured for differentiation to obtain an in vitro differentiation model of monkey SSCs.

4. The method according to claim 3, characterized in that, Step 3) The differentiation culture step involves adding 1 μmol / L retinoic acid, 50 μg / L BMP4 and 0.1 mmol / L testosterone to the basal culture medium and treating for 96 hours. Subsequently, the medium is replaced with the basal differentiation culture medium described in claim 2 and cultured for 9 days.

5. The use of the composition according to claim 1 in promoting the in vitro differentiation of monkey spermatogonial stem cells.

6. The use of the composition of claim 1 in the preparation of a product that promotes the in vitro differentiation of monkey spermatogonial stem cells.

7. The use of the composition of claim 1 in constructing an in vitro differentiation model of monkey SSCs.

8. The use of the composition of claim 1 in the preparation of a product for assisted reproduction by inducing sperm production in subjects (SSCs) in vitro.

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