Application of novel small molecule compound spermidine in promoting spermatogenesis and improving dyszoospermia

Abstract

The invention discloses and provides application of a novel small-molecule compound spermidine in promoting spermatogenesis and improving dyszoospermia, compared with traditional small-molecule compounds GDNF, EGF, bFGF and LIF, spermidine in the invention can be supplemented in vitro and promote spermatogenesis and recovery of animals with dyszoospermia, so that spermatogenesis and dyszoospermia of animals with dyszoospermia are improved, and spermatogenesis and dyszoospermia of animals with dyszoospermia are improved. A new small molecule compound target is provided for in-vitro amplification of spermatogonial stem cells, and a new scheme is provided for fertility remodeling of NOA patients.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to use of a new small molecule compound, spermidine, for promoting spermatogenesis and improving spermatogenesis disorders. Background Art

[0002] Currently, the fertility of the childbearing population is showing an overall downward trend worldwide. The WHO Special Program for Human Reproduction reports that the infertility rate of the global population is at least 15%, and there are about 60-80 million infertile couples worldwide. Infertility, along with tumors and cardiovascular diseases, has become one of the three major diseases affecting human health today. The decline in human sperm quantity and quality is worrying, and the incidence of azoospermia is about 1%.

[0003] Spermatogenesis is the process of spermatogonial stem cells constantly self-renewing and proliferating and differentiating into sperm, including the self-renewal and proliferation differentiation of spermatogonial stem cells, spermatocyte meiosis and sperm deformation. Any abnormality in any of these processes leads to spermatogenesis disorders. Severe cases can lead to non-obstructive azoospermia (NOA). The self-renewal and proliferation differentiation ability of spermatogonial stem cells in NOA patients is impaired. At present, the current treatment options for NOA mainly include drug therapy and surgical treatment. Drug therapy mainly includes aromatase inhibitors, L-carnitine and related Chinese patent medicines. Drugs can cause 10% of NOA patients to spontaneously ejaculate sperm in their semen. Most patients cannot spontaneously ejaculate through drug treatment and can only obtain sperm through surgical treatment. Surgical treatment mainly uses testicular sperm retrieval under a microscope, and its sperm retrieval rate is about 30-40%. More than half of them cannot obtain sperm and cannot give birth to their own offspring. However, most of these patients still have a small amount of spermatogonia in their testicular tissue. In order to preserve the fertility of these patients, their testicular tissue can be cryopreserved. The most promising methods for restoring fertility in such patients are to expand spermatogonial stem cells in vitro and induce their differentiation into functional sperm or to expand spermatogonial cells in vitro and then infuse them into the body.

[0004] The proliferation of spermatogonial stem cells is regulated by the spermatogenic microenvironment. The culture medium currently used contains small molecule compounds such as GDNF (10 ng / mL), LIF (10 ng / mL), EGF (20 ng / mL) and 10 ng / ml recombinant bFGF. The in vitro culture and expansion technology of human spermatogonial stem cells needs to be improved. At present, spermatogonial stem cells can be cultured in vitro for a maximum of more than 2 months and cannot be cultured and expanded for a long time. Based on this problem, we constructed a single-cell transcriptome map of the human testis throughout its life cycle and identified that the polyamine metabolic enzyme (spermine synthase SpermineSynthase, SMS) is specifically localized in spermatogonia. Then, by adding spermidine to spermatogonia in vitro, it was found that spermidine can promote the proliferation of spermatogonia, and through model animal studies, it was found that exogenous spermidine can promote the proliferation of endogenous spermatogonial stem cells. The present invention provides a new small molecule compound target for the in vitro expansion of spermatogonial stem cells and provides a new solution for the fertility reconstruction of NOA patients. Summary of the invention

[0005] The purpose of the present invention is to provide a new small molecule compound spermidine for promoting spermatogenesis and improving spermatogenesis disorders, and to provide a new therapeutic target for fertility reconstruction in NOA patients.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] On the one hand, the present invention provides a new small molecule compound spermidine for use in promoting spermatogenesis and improving spermatogenic disorders. The inventors found that spermidine can promote the proliferation of spermatogonia, and through model animal studies, they found that exogenous spermidine can promote the proliferation of endogenous spermatogonial stem cells.

[0008] As a further solution of the present invention: it includes a method for preparing a drug for promoting spermatogenesis and improving spermatogenesis disorders.

[0009] As a further embodiment of the present invention: the drug is a tablet, a powder, a granule, an oral solution or an injection.

[0010] Another aspect of the present invention provides a preparation method for preparing the above-mentioned small molecule compound spermidine (SPD-3 pathway), comprising the following steps:

[0011] Step 1: Knockout of OAZ1;

[0012] Step 2: Overexpression of ODC1;

[0013] Step 3: Increase ornithine production;

[0014] Step 4: Increase dSAM production and overexpress spermidine synthase (SPDS);

[0015] Step 5: Reduce spermidine bypass metabolism;

[0016] Step 6: Promote the conversion of MTA into dSAM;

[0017] Step 7: Promote spermidine to be excreted from cells or transferred into intracellular vesicles, thereby reducing its cytotoxic effects;

[0018] Step 8: further overexpression of AdoMetDC and SPDS;

[0019] Step nine: Fed-batch fermentation of JQ55 strain.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a use of a new small molecule compound, spermidine, in promoting spermatogenesis and improving spermatogenic disorders. Compared with traditional small molecule compounds GDNF, EGF, bFGF and LIF, the spermidine in the present invention can be supplemented in vitro and promote the recovery of spermatogenesis in spermatogenic disorder model animals, providing a new small molecule compound target for the in vitro expansion of spermatogonial stem cells and a new solution for the fertility restoration of NOA patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Chromatograms for polyamine detection of mouse testis tissue and cell line samples.

[0023] Figure 2 The figure shows the specific contents of putrescine, spermidine and spermine in mouse testicular tissue and cell line samples.

[0024] Figure 3 Figure 2 shows the spermidine / spermine ratio in mouse testicular tissue and cell line samples.

[0025] Figure 4 In the experiment to promote GC-1 cell proliferation by knocking out SMS, cell fluorescence images were observed after GC-1 was infected with lentivirus.

[0026] Figure 5 This is the detection diagram of SMS transcripts after GC-1 lentivirus infection in the experiment of knocking out SMS to promote GC-1 cell proliferation.

[0027] Figure 6 This is the detection diagram of SMS protein after GC-1 lentivirus infection in the experiment of knocking out SMS to promote GC-1 cell proliferation.

[0028] Figure 7 This is a graph showing the changes in the contents of spermidine, spermine, and putrescine after SMS was knocked out in GC-1 in the experiment of promoting GC-1 cell proliferation by knocking out SMS.

[0029] Figure 8This is a line graph of the CCK-8 proliferation experiment in which SMS knockout promoted GC-1 cell proliferation.

[0030] Fig. 9 This is the fluorescence image of EDU-stained cells in the experiment of SMS knockout promoting GC-1 cell proliferation.

[0031] Fig.10 This is a bar graph showing that knocking out SMS promotes cell proliferation in the experiment of promoting GC-1 cell proliferation.

[0032] Fig.11 This is a diagram showing the changes after SMS knockout in the experiment of promoting GC-1 cell proliferation (scale bar = 10 μm).

[0033] Fig.12 This is one of the line graphs of the CCK-8 proliferation experiment in the spermidine-promoting GC-1 cell proliferation experiment.

[0034] Fig.13 This is one of the fluorescence images of stained cells in the experiment of spermidine promoting GC-1 cell proliferation.

[0035] Fig.14 This is a bar graph showing the amount of cell proliferation in the spermidine-promoting GC-1 cell proliferation experiment.

[0036] Fig.15 This is a graph showing the changes in the contents of spermidine, spermine, and putrescine in the experiment in which spermidine promotes GC-1 cell proliferation.

[0037] Fig.16 This is the second line graph of the CCK-8 proliferation experiment in the spermidine-promoting GC-1 cell proliferation experiment.

[0038] Fig.17 This is the second fluorescence image of stained cells in the experiment of spermidine promoting GC-1 cell proliferation.

[0039] Fig.18 This is a bar graph showing the cell proliferation in the spermidine-promoting GC-1 cell proliferation experiment (scale bar = 10 μm).

[0040] Fig.19 This is a graph showing the growth changes of cell clones within five days after GS cells were treated with spermidine (100 μM) and spermine (200 μM) in the spermidine-promoting GS cell proliferation experiment.

[0041] Fig. 20 This is a bar graph showing the amount of cell proliferation in the spermidine-promoting GS cell proliferation experiment.

[0042] Fig.21 Flow chart for the design of an experiment to promote spermatogenesis restoration in azoospermia model mice using exogenous polyamines.

[0043] Fig. 22 This is the HE cell staining image of the testis and epididymis 4 weeks after busulfan establishment in the experiment of exogenous polyamines promoting spermatogenesis recovery in azoospermia model mice.

[0044] Fig.23 This is a statistical chart of the testicular mass index of each group of mice after the experiment of exogenous polyamines promoting spermatogenesis recovery in azoospermia model mice.

[0045] Fig.24 These are the HE cell staining images of the testis and epididymis of each group of mice after the exogenous polyamine promoted spermatogenesis recovery experiment in azoospermia model mice.

[0046] Fig.25 This is a statistical chart of damaged seminiferous tubules in each group of mice after the exogenous polyamine promoted spermatogenesis recovery experiment in azoospermia model mice (scale bar = 100 μm). DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] See also Figures 1 to 25 The present invention provides a novel small molecule compound - a polyamine metabolism regulator - that promotes the proliferation of spermatogonial stem cells.

[0049] Polyamines in mammals mainly include putrescine (PUT), spermidine (SPD) and spermine (SPM), which are widely distributed in various organs in the body. There are three main ways to produce them:

[0050] 1. De novo synthesis from amino acids, which is also the main pathway;

[0051] 2. Diet acquisition;

[0052] 3. Microbial synthesis in the body.

[0053] Arginine is converted into ornithine by the action of arginase1 (ARG1), and then catalyzed by ornithine decarboxylase1 (ODC1) to produce putrescine. Putrescine is catalyzed by spermidine synthase (SRM) and spermine synthase (Spermine Synthase, SMS) to produce spermidine and spermine, respectively. The amino group required for the synthesis process is provided by S-adenosylmethionine (SAM). Spermidine and spermine can be converted into precursors by enzymes.

[0054] First, the single-cell transcriptome characteristics of human testis throughout the life cycle were analyzed, and spermine synthase (SMS) was identified as being specifically expressed in spermatogonia. The inventors detected the expression of polyamines in testicular tissue, male germ stem cells (GS cells) and spermatogonia cell lines (GC-1 cells) by liquid chromatography, and found that these cells contained high concentrations of spermidine and spermine, and the spermidine / spermine ratio was higher than that of testicular tissue (specifically combined with Figure 1 , 3 ).

[0055] Specific combination Figure 4-11 , SMS knockout promotes GC-1 cell proliferation experiment:

[0056] In order to determine the effect of spermidine on the proliferation of spermatogonial stem cells, the inventors used the lentiviral vector pGMLV-ZsGreen1-PGK-Puro to construct three SMS knockdown shRNAs, which were co-transfected with lentiviral packaging plasmids pMD2.G and psPAX2 into 293T cells. After 48 hours, the virus solution was collected and concentrated, and the mouse spermatogonial cell line GC-1 was infected after the titer was determined. After 72 hours of infection, green fluorescence was observed, and puromycin with a final concentration of 2 μg / ml was added for drug screening to obtain a GC-1 cell line with stable SMS knockdown. GC-1 cell RNA and protein were extracted, and SMS expression was detected by Q-PCR and Western blot. It was found that shRNA-2 had the best knockdown effect (knockdown of about 78%), so it was selected for subsequent experiments ( Figure 4-6 );

[0057] GC-1 cells in the control group and shRNA-2 group were collected, and the intracellular polyamine concentrations were detected by high performance liquid chromatography (HPLC). The results showed that after SMS knockdown, the spermine level in GC-1 cells was significantly reduced, the spermidine content was significantly increased, and putrescine did not change significantly ( Figure 7 ). CCK-8 proliferation assay showed that the proliferation of GC-1 cells was significantly accelerated after SMS knockdown ( Figure 8), and EDU proliferation experiments also showed that knocking down SMS could increase the proliferation rate of GC-1 cells from 32% to 51% ( Fig. 9 , 10 ). Pathway analysis showed that knockdown of SMS activated the ERK and AKT pathways in GC-1 cells and increased spermidine-mediated modification of the transcription factor eIF5Ahypusine ( Fig.11 ).

[0058] Specific combination Figure 12-18 , Experiment on spermidine promoting GC-1 cell proliferation:

[0059] The inventors treated GC-1 cells with inhibitors of three key enzymes in the polyamine metabolic pathway to explore their effects on cell proliferation. The CCK-8 proliferation assay showed that 200 μM of the ODC1 inhibitor DFMO and 100 μM of the SRM inhibitor MCHA both significantly inhibited GC-1 cell proliferation, and the inhibition degree was comparable, while 20 μM of the SMS inhibitor SIBA significantly inhibited GC-1 cell proliferation ( Fig.12 ). EDU proliferation experiments showed that 20μM SIBA increased the proliferation rate of GC-1 cells from about 30% to about 50%, while 200μM DFMO and 100μM MCHA reduced the proliferation rate of GC-1 cells to about 10% ( Fig.13 , 14 HPLC showed that after 20 μM SIBA inhibited SMS activity, the spermine level in GC-1 cells was significantly reduced, the spermidine content was significantly increased, and putrescine did not change significantly ( Fig.15 ).

[0060] We further explored the effects of exogenous polyamines on the proliferation of GC-1 cells and mouse primary germline stem cells (GSCs). We directly added spermidine and spermine at different final concentrations to the GC-1 cell culture medium. The results showed that high concentrations of exogenous spermidine (>20μM) and spermine (>10μM) damaged cell activity, while low concentrations of spermidine (1μM) significantly promoted cell proliferation ( Fig.16 ), which can increase the proliferation rate of GC-1 cells from about 30% to 43% ( Figure 17-18 ). For GSCs, 200 μM spermidine can promote GSC proliferation, while spermine has no such effect ( Figure 19-20 ).

[0061] Specific combination Figure 21-25 , Experiment on the recovery of spermatogenesis in azoospermia model mice promoted by exogenous polyamines:

[0062] In the animal experiment, we first used busulfan to establish a mouse model. After 4 weeks, we took one mouse to evaluate the modeling effect, which proved that the mouse had formed an azoospermia phenotype ( Fig.21 , 22 ). The mice with successful modeling were then divided into three groups: one group was the control group, which was fed normally; one group was the spermine group, which added 3M spermine to the drinking water; and one group was the spermidine group, which added 3M spermidine to the drinking water. Samples were taken after 4 weeks to analyze the spermatogenesis of the three groups of mice. The results showed that both spermidine and spermine could promote the recovery of spermatogenesis, and the effect of spermidine was more significant, mainly manifested in an increase in testicular volume and a decrease in the number of damaged seminiferous tubules ( Figure 23-25 ).

[0063] It can be seen from this that

[0064] 1. Traditional spermatogonial stem cell culture conditions include: basic culture medium (DMEM-F12) + multiple growth factors, including GDNF, EGF, bFGF and LIF. However, the in vitro culture system of human spermatogonial stem cells is still not perfect. The present invention identifies a new small molecule compound, spermidine, that promotes the in vitro expansion of spermatogonial stem cells.

[0065] 2. Compared with traditional small molecule compounds GDNF, EGF, bFGF and LIF, spermidine in the present invention can be supplemented in vitro to promote the recovery of spermatogenesis in spermatogenic disorder model animals.

[0066] In summary, this patent identifies a new small molecule compound that promotes the proliferation of spermatogonial stem cells. This small molecule compound, spermidine, has broad prospects in the preparation of drugs for promoting spermatogenesis and improving spermatogenic disorders, and the drug types include but are not limited to tablets, powders, granules, oral solutions or injections.

[0067] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a new small molecule compound spermidine in promoting spermatogenesis and improving spermatogenesis disorders.

2. The use according to claim 1, characterized in that: Including drugs used to prepare drugs for promoting spermatogenesis and improving spermatogenesis disorders.

3. The use according to claim 2, characterized in that: The drug type is one of tablets, powders, granules, oral solutions or injections.

4. A preparation method for preparing the small molecule compound spermidine as claimed in claim 1, characterized in that: The following steps are involved: Step 1: Knockout of OAZ1; Step 2: Overexpression of ODC1; Step 3: Increase ornithine production; Step 4: Increase dSAM production and overexpress spermidine synthase (SPDS); Step 5: Reduce spermidine bypass metabolism; Step 6: Promote the conversion of MTA into dSAM; Step 7: Promote spermidine to be excreted from cells or transferred into intracellular vesicles, thereby reducing its cytotoxic effects; Step 8: further overexpression of AdoMetDC and SPDS; Step nine: Fed-batch fermentation of JQ55 strain.

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