Use of minocycline or its derivatives in promoting in vitro maturation of oocytes

Abstract

The present invention relates to the field of assisted reproductive technology, and particularly relates to the use of minocycline or its derivatives in promoting in vitro maturation of oocytes. Since it is important to promote nuclear and cytoplasmic maturation during the process of oocytes resuming meiosis in vitro in the IVM technology, cytoplasmic maturation is the key factor affecting embryonic developmental potential. The research of the present invention has found that the application of MiH in the IVM culture medium not only facilitates nuclear maturation and helps improve the extrusion of the first polar body of oocytes, but also balances the damage of ROS to cytoplasmic organelles and the nucleus, which is beneficial to the maturation of oocyte cytoplasm. At the same time, it improves the blastocyst formation rate of fertilized eggs of aged mouse oocytes. Adding MiH to the IVM culture medium may become a new method to improve and optimize the IVM technology, which is of great significance for improving the fertility of elderly women and the development of clinical reproductive medicine.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproductive technology, and in particular to use of minocycline or its derivatives in promoting in vitro maturation of oocytes. Background Art

[0002] At present, the proportion of assisted pregnancy in elderly women has increased, but elderly female patients often have problems such as decreased ovarian function, decreased responsiveness, small number of eggs obtained, and high rate of chromosomal abnormalities in embryos, which leads to lower pregnancy rates and increased miscarriage rates. This is often related to the decline in egg quality. Therefore, how to improve the quality of eggs in elderly women is a clinical problem that needs to be solved urgently.

[0003] Assisted reproduction technology (ART) has become an important means of treating infertility, including artificial insemination and in vitro fertilization-embryo transfer. On this basis, new technologies have been developed, including intracytoplasmic sperm injection (ICSI), in vitro maturation of oocytes (IVM) and preimplantation genetic diagnosis (PGD). Assisted reproductive technology has been used in the world for more than 30 years. In the process of application, its various technologies have solved various causes of infertility, but even today, test-tube babies are not 100% successful. According to the data of the Chinese Medical Association Reproductive Society, in 2017, the clinical pregnancy rate and live birth rate of artificial insemination with husband's sperm in China were 26.46% and 22.89% respectively, and the clinical pregnancy rate and live birth rate of conventional in vitro fertilization (IVF) were 53.45% and 43.55% respectively. A successful test-tube baby is often born after 2 to 3 cultivation cycles.

[0004] Currently, the maturation level of in vitro cultured oocytes and the embryonic development ability after fertilization are both lower than those of in vivo matured oocytes. The maturation rate from immature oocytes to mature oocytes is about 50%, the fertilization rate is about 80%, and the clinical pregnancy rate after transplantation is about 15%-20%. Therefore, effectively optimizing the oocyte culture system is of great significance for improving the clinical pregnancy rate. The IVM technology began in the 1960s. By simulating the in vivo oocyte maturation environment, immature germinal vesicle (GV) oocytes are directly obtained from the ovaries without drug stimulation or with low-dose drug stimulation, and are cultured in vitro to mature into the MⅡ phase for in vitro fertilization-embryo transfer (IVF-ET). In 1991, the world's first clinical pregnancy was achieved using the IVM technology. In 2000, the first clinical pregnancy in China was obtained by Chinese scholars using the IVM technology. The application of the IVM technology can enable the obtained immature oocytes to also develop into high-quality embryos. While reducing the drug dosage, it solves the problem of long medication cycles, makes more full use of the eggs obtained each time of egg retrieval, helps to improve the cumulative pregnancy rate, and also reduces the economic burden on patients. IVM can also reduce the incidence risk of long-term diseases after ovulation induction; at the same time, it can effectively avoid the occurrence of ovarian hyperstimulation syndrome (OHSS). However, although the IVM technology has been applied to clinical treatment for a long time, the maturation rate of oocytes of elderly women by IVM is relatively low, and even if the oocytes of elderly women mature after IVM, their blastocyst formation rate is still relatively low.

[0005] Minocycline hydrochloride (MiH) is a semi-synthetic tetracycline derivative with a long half-life and high tissue penetrability, and is widely used clinically to treat infectious diseases. Recently, minocycline has shown anti-inflammatory and immunomodulatory effects in many inflammatory diseases and has received wide attention. However, there is no application of MiH in the in vitro culture of aging oocytes, especially the impact on the developmental potential of aging oocytes. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide the use of minocycline or its derivative in promoting the in vitro maturation of oocytes.

[0007] For this purpose, the present invention provides the following technical solutions:

[0008] The embodiments of the present invention disclose the use of minocycline or its derivative in promoting the in vitro culture and maturation of oocytes or in preparing a product for promoting the in vitro culture and maturation of oocytes.

[0009] In some embodiments, the minocycline or its derivatives can improve the maturation level and quality of in vitro cultured oocytes. On the one hand, the obtained oocytes with improved maturation level can be used in the field of assisted reproductive technology for humans or non-human mammals, such as in vitro fertilization of oocytes to cultivate high-quality embryos, promoting embryo development, and can improve the clinical pregnancy rate and live birth rate of conventional IVF. On the other hand, the obtained oocytes with improved maturation level can also be used for experimental research and observation of the development and function of oocytes. In addition, it can also help obtain more fertilized oocytes for reproductive toxicology experiments.

[0010] In some embodiments, the use of the minocycline or its derivatives in improving the developmental potential of embryos after in vitro fertilization of oocytes or in preparing a product for improving the developmental potential of embryos after in vitro fertilization of oocytes. The minocycline or its derivatives can improve the developmental potential of embryos obtained after in vitro fertilization of in vitro cultured oocytes, and the obtained embryos can be used in the field of assisted reproductive technology for humans or non-human mammals, such as it can improve the clinical pregnancy rate and live birth rate of conventional IVF. On the other hand, the obtained embryos can also be used for experimental research and observation of the development and function of embryos.

[0011] In some embodiments, the oocytes are oocytes of humans or non-human mammals. In some embodiments, the non-human mammals include non-human mammalian models for construction. The animals of the non-human mammalian models include rodents, felines, canines or primates. In a preferred embodiment, the animals used for constructing the non-human mammalian models are selected from at least one of the rodents in the family Muridae; more preferably, the animals are at least one of the genus Rat (Rat; Rattus norregicus), and / or at least one of the genus Mouse (Mouse; Mus culus); even more preferably, the animals are the genus Mouse (Mouse; Mus culus).

[0012] In some embodiments, the oocytes are oocytes in the germinal vesicle stage.

[0013] In some embodiments, the oocytes are aged oocytes; optionally, compared with the oocytes in the normal group, the aged oocytes have a higher chromosome abnormality rate, a higher spindle abnormality rate, and / or a lower first polar body extrusion rate. For example, in a non-human animal model, when comparing the oocytes of 8-month-old aged female mice and 6-8-week-old young female mice, the aged female mice have a higher chromosome abnormality rate, a higher spindle abnormality rate, and / or a lower first polar body extrusion rate in their oocytes. For humans, the aged oocytes can be the oocytes of elderly women or oocytes of poor quality. The oocytes of poor quality can be the oocytes of young women (under 35 years old), which are of poor quality and aged due to personal constitution, taking medications, or other factors. The age range of the elderly women is 35 years old or above, such as 35 years old, 40 years old, 45 years old or above, etc. Compared with the oocytes of normal young women, the aged oocytes have a lower first polar body extrusion rate, a higher chromosome abnormality rate, and / or a higher spindle abnormality rate.

[0014] Furthermore, the chromosome abnormality rate, spindle abnormality rate, and / or first polar body extrusion rate of the oocytes are selected as evaluation indicators for the aged oocytes, and the reasons are as follows: As is well known, poor oocyte quality is an important cause of female or female animal infertility and birth defects. Precise regulation of spindle formation and chromosome assembly during oocyte meiosis is a key factor in oocyte quality. Mammalian oocytes are arrested at the diplotene stage of the first meiotic prophase, also known as the germinal vesicle stage (GV stage). Under the stimulation of LH hormone, fully grown oocytes undergo germinal vesicle breakdown (GVBD), which marks the restoration of meiosis. In the first meiotic prophase I, chromosomes begin to recombine, and all or most of the proteins required for fertilized egg or embryo development are synthesized. The chromosomes condense into short rod-shaped structures, the spindle begins to assemble, the centrioles disappear during development, the spindle enters the nucleus, and at the same time the chromosomes move towards the middle position of the spindle, completing the process of transformation from the first meiotic prophase to metaphase. The neat arrangement of chromosomes on the equatorial plate, the complete formation of the bipolar spindle, and the microfilament cap enriched in the cortical region together constitute the typical morphological characteristics of metaphase I (MI) of the first meiosis.

[0015] Before and after birth, oocytes enter the diplotene stage of the first meiotic prophase and remain arrested at this stage for a relatively long time. It is not until puberty or adulthood that, under the action of luteinizing hormone (LH) in the mid-menstrual cycle, the oocyte extrudes the first polar body and is arrested again at metaphase II (MII) awaiting fertilization. Precise regulation of spindle assembly and chromosome formation during meiosis is crucial for the production of healthy gametes. Errors in chromosome segregation during meiosis can lead to uneven chromosomal distribution in gametes, resulting in aneuploid gametes, which can cause developmental arrest, miscarriage, or offspring defects after fertilization. Poor-quality or aged oocytes show abnormal spindle assembly, chromosome formation, and abnormal extrusion of the first polar body during meiosis.

[0016] In some embodiments, the promotion of in vitro maturation of oocytes means that after treatment with minocycline or its derivatives, the oocytes have a low rate of chromosomal abnormalities, a low rate of spindle abnormalities, and / or a high rate of extrusion of the first polar body.

[0017] In some embodiments, compared with aged oocytes, the promotion of in vitro maturation of oocytes means that after treatment with minocycline or its derivatives, the aged oocytes have a reduced rate of chromosomal abnormalities, a reduced rate of spindle abnormalities, and / or an increased rate of extrusion of the first polar body.

[0018] In some embodiments, the improvement of the developmental potential of embryos after in vitro fertilization of oocytes includes that after in vitro fertilization of oocytes treated with minocycline or its derivatives, the proportion of at least one of the 2-cell stage, 4-cell stage, 8-cell stage, morula, early blastocyst formation, and late blastocyst formation of the fertilized eggs obtained increases in the total fertilized eggs. Since mature oocytes complete their second meiotic division after binding to sperm, forming a fertilized egg containing a haploid paternal pronucleus and a haploid maternal pronucleus, and then the two pronuclei begin to fuse, and the embryo undergoes several cleavages to form 2-cell, 4-cell, 8-cell embryos and morulas, and finally forms a blastocyst. During oocyte maturation, the oocyte accumulates a large amount of maternal RNA and proteins necessary for fertilization, maternal-to-zygotic transition, and pre-implantation embryo development. Therefore, oocyte quality determines the developmental potential of early embryos. Thus, the developmental potential of embryos formed after in vitro fertilization of mature oocytes can be evaluated by the proportion of at least one of the 2-cell stage, 4-cell stage, 8-cell stage, morula, early blastocyst formation, and late blastocyst formation in the total fertilized eggs.

[0019] In some embodiments, compared with the developmental potential of embryos after in vitro fertilization of oocytes in the normal group, the low developmental potential of embryos after in vitro fertilization of aged oocytes includes a decrease in the proportion of at least one of the 2-cell stage, 4-cell stage, 8-cell stage, morula, early blastocyst formation, and late blastocyst formation of the fertilized eggs after in vitro fertilization of the maternal cells in the total fertilized eggs.

[0020] In some embodiments, the derivative of minocycline includes minocycline hydrochloride;

[0021] An oocyte culture medium disclosed in an embodiment of the present invention contains the minocycline or its derivative.

[0022] In some embodiments, the concentration of the derivative of minocycline is 500 nM to 5 μM, such as any value among 500 nM, 1 μM, 2 μM, 5 μM or a range value between any two values. nM is expressed as nmol / L, and μM is expressed as μmol / L.

[0023] Optionally, the oocyte culture medium further includes an IVM culture medium.

[0024] The technical solution of the present invention has the following advantages:

[0025] 1. The use of minocycline or its derivative provided by the present invention in promoting in vitro maturation of oocytes or preparing a product for promoting in vitro maturation of oocytes; in view of the importance of promoting nuclear and cytoplasmic maturation during the process of oocytes resuming meiosis in vitro in the IVM technology, cytoplasmic maturation is a key factor affecting embryonic development potential. The research of the present invention finds that the application of MiH in the IVM culture medium promotes nuclear maturation, helps to improve the extrusion of the first polar body of oocytes, reduces the chromosomal abnormality rate and spindle abnormality rate; at the same time, it also balances the damage of ROS to cytoplasmic organelles and the nucleus, which is beneficial to the maturation of oocyte cytoplasm. In addition, it also improves the fertilized egg blastocyst formation rate of aged mouse oocytes. Therefore, adding MiH to the IVM culture medium may become a new method for improving and optimizing the IVM technology, which is of great significance for improving the fertility of elderly women and promoting the development of clinical reproductive medicine. Description of the Drawings

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 Histogram of the germinal vesicle breakdown rate and the first polar body extrusion rate of oocytes in the young group (NC), the aged group (crtl), and the MiH group in the experiment of MiH promoting in vitro maturation of aged mouse oocytes in Experimental Example 1;

[0028] Figure 2In Experiment Example 1, the experiment of MiH promoting the in vitro maturation of oocytes from aged mice. Bright-field images of oocyte in vitro maturation in the young group (NC), the aged group (crtl), and the MiH (1 μM) group; Scale bar: 50 μm;

[0029] Figure 3 In Experiment Example 1, MiH improves meiotic abnormalities in oocytes from aged mice; A Immunofluorescence staining images of oocytes in the young group, the aged group, and the MiH group; Green represents the spindle, and red represents chromosomes; B Histogram of the abnormal rate of spindles and chromosomes in oocytes in the young group, the aged group, and the MiH group; Scale bar: 20 μm;

[0030] Figure 4 In Experiment Example 1, MiH reduces the ROS level in oocytes from aged mice; A Immunofluorescence staining images of oocytes in the young group, the aged group, and the MiH group; Green represents reactive oxygen species; B Fluorescence quantification graph in the young group, the aged group, and the MiH group; Scale bar: 20 μm;

[0031] Figure 5 In Experiment Example 2, MiH promotes the embryonic development of aged mice; A Histogram of the number of cells at different embryonic development stages after in vitro culture of oocytes in the aged group and the MiH group after fertilization; B Histogram of the number of inner cell mass cells of embryos at the blastocyst stage in the aged group and the MiH group;

[0032] In the above figures, p < 0.05, p < 0.01. Detailed implementation manners

[0033] The following embodiments are provided to better further understand the present invention, which are not limited to the described optimal implementation manner, and do not constitute a limitation to the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0034] For those embodiments in which specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0035] In the following embodiments

[0036] ART, assisted reproduction technology, assisted reproductive technology;

[0037] IVF-ET, in vitro fertilization and embryo Transfer, in vitro fertilization-embryo transfer;

[0038] ICSI, intracytoplasmic sperm injection, intracytoplasmic sperm injection;

[0039] IVM, in-vitro maturation of oocyte, in vitro maturation of immature oocytes;

[0040] PGD, preimplantation genetic diagnosis, preimplantation genetic diagnosis;

[0041] OHSS, ovarian hyperstimulation syndrome, ovarian hyperstimulation syndrome;

[0042] MiH, minocycline hydrochloride, minocycline hydrochloride;

[0043] PARP1 poly ADP-ribose polymerase1, poly ADP-ribose polymerase 1;

[0044] GV, germinal vesicle, germinal vesicle;

[0045] GVBD, germinal vesicle breakdown, germinal vesicle breakdown;

[0046] MI, metaphase I, metaphase I of the first meiosis;

[0047] MII, metaphase II, metaphase II of the second meiosis;

[0048] PB1, polar body, first polar body;

[0049] PMSG, pregnant mare serum gonadotropin, pregnant mare serum gonadotropin;

[0050] hCG, human chorionic gonadotropin, human chorionic gonadotropin.

[0051] Minocycline hydrochloride (MiH) was purchased from Selleck, USA, with the catalog number S4226.

[0052] Experimental Example 1 In vitro maturation culture of GV-stage oocytes

[0053] As is well known, poor oocyte quality is an important cause of female infertility and birth defects. Precise regulation of spindle formation and chromosome assembly during oocyte meiosis is a key factor in oocyte quality. Mammalian oocytes arrest at the diplotene stage of the first meiotic prophase, also known as the germinal vesicle stage (GV stage). Under the stimulation of LH hormone, fully grown oocytes undergo germinal vesicle breakdown (GVBD), which marks the resumption of meiosis. During the first meiotic prophase I, chromosomes begin to recombine, and all or most of the proteins required for fertilized egg or embryonic development are synthesized. Chromosomes condense into short rod-like shapes, the spindle begins to assemble, the centrioles disappear during development, the spindle enters the nucleus, and at the same time the chromosomes move towards the middle position of the spindle, completing the process of transitioning from the first meiotic prophase to metaphase. The neat arrangement of chromosomes on the equatorial plate, the complete formation of the bipolar spindle, and the microfilament cap enriched in the cortical region together constitute the typical morphological characteristics of metaphase I (MI) of the first meiosis.

[0054] Before and after birth, oocytes enter the diplotene stage of the first meiotic prophase and remain arrested at this stage for a long time. Until puberty or adulthood, under the action of the luteinizing hormone (LH) peak in the middle of the menstrual cycle, oocytes extrude the first polar body and are arrested again at metaphase II (MII) waiting for fertilization. Precise regulation of spindle assembly and chromosome formation during meiosis is the key to producing healthy gametes. Errors in chromosome segregation during meiosis can lead to uneven distribution of gamete chromosomes, resulting in aneuploid gametes, causing developmental arrest, miscarriage, or offspring defects after fertilization. Literature reports that the quality of oocytes from aging mice decreases, the maturation rate decreases, and the abnormal rate of spindles increases.

[0055] Thirty aging female mice (ICR mice, 8 months old) and ten young female mice (ICR mice, 6 - 8 weeks old) were respectively intraperitoneally injected with 6 IU of pregnant mare serum gonadotropin (PMSG). After 48 h of injection, the mice were sacrificed by cervical dislocation, and bilateral ovaries were removed. Cumulus-oocyte complexes were aspirated under a stereomicroscope using a mouth pipette, and denuded oocytes without granulosa cells were obtained by repeated pipetting, which were GV-stage oocytes.

[0056] NC group: The GV-stage oocytes (35) of young female mice were placed in 50 μL of IVM culture medium (M2 culture medium, the formula can be referred to the M7167 product of sigma aldrich company);

[0057] Ctrl group: 35 GV-stage oocytes from aged female mice were placed in 50 μL of IVM medium (M2 medium, the formula can be referred to the M7167 product of sigma aldrich company).

[0058] MiH group: 35 GV-stage oocytes from aged female mice were placed in 50 μL of IVM medium supplemented with MiH (M2 medium, the formula can be referred to the M7167 product of sigma aldrich company); the final concentration of MiH was 500 nM, 1 μM or 2 μM.

[0059] Each of the above groups was cultured at a constant temperature of 37 °C with 5% CO 2 for 14 hours. By observing and calculating the germinal vesicle breakdown rate and the first polar body extrusion rate, the IVM maturation of oocytes was evaluated. Germinal vesicle breakdown rate = number of oocytes with germinal vesicle breakdown (GVBD) / total number of cultured oocytes × 100%. First polar body extrusion rate = number of first polar bodies / total number of cultured oocytes × 100%. Then the oocytes in the young group, aged group and MiH group were continued to be cultured. After 14 hours, MII-stage oocytes were collected, fixed with 4% paraformaldehyde fixative (PFA), permeabilized with 0.5% Triton, blocked with 1% BSA, the spindle was labeled with β-tubulin, and the DNA was labeled with 4',6-diamidino-2-phenylindole (DAPI) to observe the meiosis of the three groups of oocytes, as well as the abnormal rate of oocyte spindles. Oocyte spindle abnormality rate = number of oocytes with abnormal spindles / total number of cultured oocytes × 100%.

[0060] Data statistical method: Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Unless otherwise stated, all experiments were repeated at least three times, and each experimental group included at least 30 oocytes. Data were expressed as mean ± standard error; p < 0.05, p < 0.01, Differences with p < 0.001 were considered statistically significant.

[0061] The results of the germinal vesicle breakdown rate and the first polar body extrusion rate are as Figure 1 shown. It can be seen from Figure 1 that the germinal vesicle breakdown rate of the aged group was lower than that of the young group (84.96 ± 1.44% v.s 94.12 ± 2.44%). After adding MiH, the germinal vesicle breakdown rate could be increased, but the effects were different at different concentrations (500 nm, 1 μM, 2 μM) (88.40 ± 5.07%v.s 91.49±0.72% v.s 88.12±6.70%). Meanwhile, it can be seen that the young group has a higher first polar body extrusion rate than the aging group (75.83±3.58% vs 45.90 ±9.99%), and there is a significant difference p < 0.05, indicating that the first polar body extrusion rate of oocytes in the aging group is low. Indeed, as reported in the literature, the quality of oocytes in aging mice decreases and the maturation rate decreases. However, after using the IVM culture medium containing MiH, the MiH group has a higher first polar body extrusion rate than the aging group (the three concentration groups are 64.71±3.57% v.s 69.50±6.54% v.s 69.34±6.64%), and there is a significant difference p < 0.05. At the same time, there is no significant difference in the first polar body extrusion rate between the MiH group and the young group. This shows that during IVM, adding 500 nM - 2 μM of MiH to the culture medium can significantly increase the first polar body extrusion rate of oocytes and promote the in vitro maturation of aging oocytes, as Figure 2 shown.

[0062] The meiotic situation of oocytes is as shown in Figures A and B in Figure 3 . The abnormal spindle rate and the proportion of chromosomal abnormalities of oocytes in the MiH group (corresponding to a MiH concentration of 1 μM) are significantly lower than those in the aging group, indicating that MiH participates in the regulation of oocyte meiosis and promotes the in vitro maturation of aging mouse oocytes.

[0063] High ROS levels are presented in aging mouse oocytes, as shown in Figures A and B in Figure 4 . After adding MiH (corresponding to a MiH concentration of 1 μM), the ROS level in aging mouse oocytes can be significantly reduced.

[0064] Experimental Example 2 Fertilization of Oocytes and Subsequent Embryo Development Ability

[0065] After mature oocytes bind to sperm, they complete their second meiosis to form a fertilized egg containing a haploid paternal pronucleus and a haploid maternal pronucleus. Then, the two pronuclei begin to fuse, and the embryo undergoes several cleavages to form 2-cell, 4-cell, 8-cell embryos and morulas, and finally forms blastocysts. During oocyte maturation, oocytes accumulate a large amount of maternal RNAs and proteins necessary for fertilization, maternal-to-zygotic transition, and preimplantation embryo development. Therefore, oocyte quality determines the potential for early embryo development. In this experimental example, oocytes were fertilized through an IVF experiment, and the embryonic development potential of oocytes after fertilization in the control group (IVM) and the experimental group (IVM + MiH) was tested. The method is as follows:

[0066] Five fertile male mice (ICR mice, 10 weeks old) were sacrificed by cervical dislocation. They were fixed on the dissecting table, and the epididymal ducts were removed by opening the abdominal cavity and placed in a 35-mm cell culture dish. Under a stereomicroscope, the epididymal ducts were punctured with sharp forceps to release the sperm into pre-warmed HTF fertilization medium (purchased from Nanjing Aibei Biotechnology Co., Ltd.) and placed in an incubator at 37°C with 5% CO 2 for 1 h for capacitation.

[0067] MII-stage oocytes from the aging group and the MiH group (corresponding MiH concentration was 1 μM) in Experimental Example 1 were taken respectively. Forty oocytes (about 20 - 25 oocytes were taken from one mouse, generally 2 - 3 female mice corresponding to 1 male mouse) were added to HTF fertilization medium (volume 50 μl), and then capacitated sperm (2 μl, concentration 10,000 - 200,000 / mL) (the upper-layer capacitated sperm was aspirated) was added. It was placed in an incubator at 37°C with 5% CO 2 for 6 h for fertilization. The fertilized eggs were transferred to KSOM embryo culture medium (purchased from Nanjing Aibei Biotechnology Co., Ltd.) and washed 3 times, and subsequent culture was completed in KSOM embryo culture medium. The following time points (after fertilization) were selected in sequence to observe the corresponding stages of embryo development: 2-cell (16 h), 4-cell (40 h), 8-cell (53 h), morula (70 h), early blastocyst formation (85 h), late blastocyst formation (100 h).

[0068] Data statistical method: Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Unless otherwise stated, all experiments were repeated at least three times, and each experimental group included at least 40 oocytes. Data were expressed as mean ± standard error; p < 0.05, p < 0.01, A difference with p < 0.001 was considered statistically significant.

[0069] Embryo ratio = number of embryos at the corresponding stage / total number of fertilized eggs × 100%.

[0070] The results are as Figure 5 shown. The embryo ratios of each group are in Figure A, and the number of inner cell mass cells is in Figure B. From Figure 5As can be seen from Figure A, compared with the aging group, the fertilization rate and 2-cell rate of oocytes in the MiH group were significantly increased. At the fertilized egg stage, the proportions of embryos developed to 2-cell, 4-cell, 8-cell, morula, and blastocyst among the total fertilized eggs (the total number of fertilized eggs in each group) were higher than those in the aging group, indicating that MiH can significantly improve the in vitro maturation rate of oocytes in aged mice and enhance the quality of their oocytes, thereby enhancing the developmental potential of embryos after fertilization. From Figure 5 As can be seen from Figure B, compared with the aging group, the average number of inner cell mass cells in embryos at the blastocyst stage in the MiH group was significantly higher than that in the blastocysts of the aging group, indicating that the embryo quality in the MiH group was significantly better than that in the aging group.

[0071] Adding MiH to the culture medium can significantly improve the in vitro maturation rate of oocytes in aged mice, enhance the quality and developmental potential of oocytes, and promote the early embryonic development of mice. The present invention can promote the in vitro maturation of oocytes in aged mice, improve the in vitro maturation technology of mouse oocytes, and is of great significance for the wide application of the in vitro maturation technology of oocytes.

[0072] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

1. Use of minocycline or a pharmaceutically acceptable salt thereof in promoting in vitro maturation of oocytes or in preparing a product for promoting in vitro maturation of oocytes; the oocytes are oocytes in the germinal vesicle stage.

2. Use of minocycline or a pharmaceutically acceptable salt thereof in the preparation of a product for improving the embryonic development potential of oocytes after in vitro fertilization; the oocytes are oocytes in the germinal vesicle stage.

3. The use according to claim 1 or 2, characterized in that The oocyte is an oocyte of a human or non-human mammal.

4. Use of minocycline or a pharmaceutically acceptable salt thereof in promoting in vitro maturation of oocytes or in preparing a product for promoting in vitro maturation of oocytes, characterized in that: The oocyte is an aged oocyte.

5. The use according to claim 1, characterized in that: The promoting oocyte in vitro culture maturation refers to promoting a decrease in the chromosome abnormality rate, a decrease in the spindle abnormality rate and / or an increase in the first polar body extrusion rate after the oocyte is treated with minocycline or a pharmaceutically acceptable salt thereof.

6. The use according to claim 2, characterized in that: The method of improving the embryonic development potential of oocytes after in vitro culture and fertilization includes increasing the proportion of at least one of the 2-cell stage, 4-cell stage, 8-cell stage, morula, early blastocyst formation, and late blastocyst formation stages of the fertilized eggs obtained after in vitro culture and fertilization of oocytes treated with minocycline or a pharmaceutically acceptable salt thereof in the total fertilized eggs.

7. The use according to claim 1 or 2, characterized in that: The pharmaceutically acceptable salt of minocycline includes minocycline hydrochloride; And / or, the product comprises culture fluid.

8. The use according to claim 7, characterized in that The culture medium includes oocyte culture medium, and the oocyte culture medium contains the minocycline or a pharmaceutically acceptable salt thereof.

9. The use according to claim 8, characterized in that In the oocyte culture medium, the concentration of minocycline or a pharmaceutically acceptable salt thereof is 500 nM to 5 μM; And / or, the oocyte culture medium also includes IVM culture medium.

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