Application of minocycline or derivative thereof in promoting in-vitro maturation of oocytes

Abstract

The invention relates to the technical field of assisted reproduction, in particular to application of minocycline or a derivative thereof in promoting in-vitro maturation of oocytes. Due to the fact that in the IVM technology, it is very important to promote cell nucleus and cytoplasm maturation in the in-vitro meiosis recovery process of oocytes, cytoplasm maturation is the key to influence the embryonic development potential, and it is found through research that when MiH is applied to an IVM culture medium, cell nucleus maturation is facilitated, and discharging of first polar bodies of the oocytes is improved; moreover, the damage of ROS to organelles and cell nucleuses in cytoplasm is balanced, and maturation of ovum cytoplasm is facilitated. Meanwhile, the blastocyst formation rate of fertilized eggs of oocytes of aged mice is improved. The method for adding MiH into the IVM culture solution can become a new method for improving and optimizing the IVM technology, and is of great significance to improvement of fertility of elderly women and 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] At present, the maturation level of oocytes cultured in vitro and the embryonic development ability after fertilization are lower than those of mature oocytes in vivo. The maturation rate from immature eggs to mature eggs is about 50%, the fertilization rate is about 80%, and the clinical pregnancy rate of transplantation is about 15%-20%. Therefore, it is of great significance to optimize the oocyte culture system to improve the clinical pregnancy rate. IVM technology began in the 1960s. It simulates the oocyte maturation environment in vivo, directly obtains immature germinal follicle (GV) oocytes from ovaries that are not stimulated by drugs or low-dose drugs, and uses them for in vitro fertilization (IVF-ET) after maturation to MⅡ stage in vitro. In 1991, the world's first clinical pregnancy was obtained using IVM technology. In 2000, scholars in my country obtained the first clinical pregnancy in my country through IVM technology. The application of IVM technology can enable the obtained immature oocytes to develop into high-quality embryos. While reducing the drug dosage, it solves the problem of long medication cycle, makes more full use of the eggs obtained from each egg retrieval, helps to improve the cumulative pregnancy rate, and also reduces the economic burden for patients. IVM can also reduce the 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 IVM technology has been used in clinical treatment for a long time, the maturation rate of IVM oocytes in older women is low, and even if the oocytes of older women mature after IVM, their blastocyst formation rate is still low.

[0005] Minocycline hydrochloride (MiH) is a semisynthetic tetracycline derivative with a long half-life and high tissue penetration, which is widely used in the clinic to treat infectious diseases. Recently, minocycline has shown anti-inflammatory and immunomodulatory effects in many inflammatory diseases and has received widespread attention. However, there is no application of MiH in the in vitro culture of aged oocytes, especially the effect on the developmental potential of aged oocytes. Summary of the invention

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

[0007] To this end, the present invention provides the following technical solutions: The embodiments of the present invention disclose the use of minocycline or its derivatives in promoting the maturation of oocytes in vitro culture or in preparing products for promoting the maturation of oocytes in vitro culture.

[0008] In some embodiments, the minocycline or its derivatives can improve the maturation level and quality of oocytes cultured in vitro. On the one hand, the oocytes with improved maturation level obtained can be used in the field of assisted reproductive technology of humans or non-human mammals, such as in vitro fertilization of oocytes to cultivate high-quality embryos, promote the development of embryos, and improve the clinical pregnancy rate and live birth rate of conventional IVF. On the other hand, the oocytes with improved maturation level obtained 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.

[0009] In some embodiments, the minocycline or its derivatives are used to improve the embryonic development potential of oocytes after in vitro culture and fertilization or to prepare products that improve the embryonic development potential of oocytes after in vitro culture and fertilization. The minocycline or its derivatives can improve the developmental potential of embryos obtained after fertilization of oocytes cultured in vitro, and the obtained embryos can be used in the field of assisted reproductive technology for humans or non-human mammals, such as improving 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 embryonic development and function.

[0010] In some embodiments, the oocyte is an oocyte of a human or non-human mammal. In some embodiments, the non-human mammal includes a non-human mammal model for construction. The animal of the non-human mammal model includes a rodent, a feline, a canine, or a primate. In a preferred embodiment, the rodent selected for constructing the non-human mammal model is at least one of the family Muridae; preferably, the animal is at least one of the genus Rat (Rattus norregicus), and / or at least one of the genus Mouse (Mouse; Mus culus); more preferably, the animal is a genus rat Mouse (Mouse; Mus culus).

[0011] In some embodiments, the oocyte is an oocyte in the germinal vesicle stage.

[0012] In some embodiments, the oocyte is an aged oocyte; optionally, compared with the oocytes of the normal group, the aged oocyte has a high chromosomal abnormality rate, an increased spindle abnormality rate and / or a low first polar body extrusion rate. For example, in a non-human animal model, compared with the oocytes of an aged female mouse of 8 months old and a young female mouse of 6-8 weeks old, the oocytes of the aged female mouse have a high chromosomal abnormality rate, an increased spindle abnormality rate and / or a low first polar body extrusion rate. For humans, the aged oocyte can be an oocyte of an advanced age woman or an oocyte of poor quality. The poor quality oocyte can be an oocyte of a young woman (under 35 years old), and the oocyte is of poor quality and aging due to personal constitution, medication or other factors. The age stage of the advanced age woman is over 35 years old, such as 35 years old, 40 years old, 45 years old or above. Compared with oocytes from normal young women, the aged oocytes have a lower first polar body extrusion rate, a higher chromosome abnormality rate and / or an increased spindle abnormality rate.

[0013] Furthermore, the oocyte chromosome abnormality rate, spindle abnormality rate and / or first polar body extrusion rate were selected as evaluation indicators of aging oocytes for the following reasons: It is well known that poor oocyte quality is an important cause of infertility and birth defects in women or female animals. Accurate regulation of spindle formation and chromosome assembly during oocyte meiosis is a key factor in oocyte quality. Mammalian oocyte development is arrested in the diplotene stage of the first meiotic prophase, also known as the germinal vesicle stage (GV stage). Under the stimulation of LH hormone, the fully grown oocyte undergoes germinal vesicle breakdown (GVBD), marking the resumption of meiosis. In the prophase I of the first meiotic division, chromosomes begin to reorganize and synthesize all or most of the proteins required for the development of the fertilized egg or embryo. Chromosomes condense into short rods, the spindle begins to assemble, the centrioles disappear during the development process, the spindle enters the interior of the nucleus, and the chromosomes move to the middle position of the spindle, completing the process of transition from the prophase of the first meiotic division to metaphase. The chromosomes are neatly arranged at the equatorial plate, the bipolar spindle is fully formed, and the microfilament cap is enriched in the cortical region, which together constitute the typical morphological characteristics of the metaphase (metaphase I, MI) of the first meiotic division.

[0014] Before and after birth, the oocyte enters the diplotene phase of the first meiotic prophase and stagnates in this phase for a long time. It is not until puberty or adulthood that the oocyte expels the first polar body under the action of luteinizing hormone (LH) in the middle of the menstrual cycle and is arrested again in the second meiotic metaphase (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, produce aneuploid gametes, and cause developmental arrest after fertilization, miscarriage, or offspring defects. Poor quality or aged oocytes have abnormal spindle assembly and chromosome formation and abnormal first polar body expulsion during meiosis.

[0015] In some embodiments, the promoting oocyte maturation in vitro means that after the oocyte is treated with minocycline or its derivatives, the chromosome abnormality rate, spindle abnormality rate and / or the first polar body extrusion rate are low.

[0016] In some embodiments, compared with aged oocytes, the promoting oocyte in vitro maturation means that after the aged oocytes are treated with minocycline or its derivatives, the chromosome abnormality rate is reduced, the spindle abnormality rate is reduced and / or the first polar body extrusion rate is increased.

[0017] In some embodiments, the improvement of embryonic development potential after in vitro culture fertilization of oocytes includes in vitro culture fertilization of oocytes after minocycline or its derivatives, and the 2-cell stage of the fertilized egg obtained, 4-cell stage, 8-cell stage, morula, blastocyst formation early stage, blastocyst formation late stage at least one ratio of total fertilized eggs increases. Due to the completion of its second meiosis after mature oocytes and sperm combination, a fertilized egg containing haploid paternal pronucleus and haploid maternal pronucleus is formed, and two pronuclei begin to fuse afterwards, and the embryo undergoes several cleavages, forming 2 cells, 4 cells, 8 cells embryos and morula, and finally forming blastocysts. In the process of egg maturation, oocytes accumulate a large amount of maternal RNA and protein necessary for fertilization, maternal to zygote conversion and preimplantation embryonic development. Therefore, oocyte quality determines early embryonic development potential. Therefore, the developmental potential of embryos formed from mature oocytes after in vitro fertilization can be examined by measuring the proportion of at least one of the 2-cell stage, 4-cell stage, 8-cell stage, morula stage, early blastocyst stage, and late blastocyst stage of the fertilized eggs in the total fertilized eggs.

[0018] In some embodiments, compared with the embryonic development potential of oocytes in vitro cultured and fertilized in a normal group, the low embryonic development potential of the aged oocytes in vitro cultured and fertilized in vitro 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 mother cells in vitro cultured and fertilized in total fertilized eggs.

[0019] In some embodiments, the minocycline derivative comprises minocycline hydrochloride; The embodiment of the present invention discloses an oocyte culture medium containing the minocycline or its derivatives.

[0020] In some embodiments, the concentration of the minocycline derivative is 500 nM to 5 μM, such as any one of 500 nM, 1 μM, 2 μM, 5 μM, or a range between any two values. nM is represented by nmol / L, and μM is represented by μmol / L.

[0021] Optionally, the oocyte culture medium also includes IVM culture medium.

[0022] The technical solution of the present invention has the following advantages: 1. The use of minocycline or its derivatives provided by the present invention in promoting the maturation of oocytes in vitro culture or preparing products that promote the maturation of oocytes in vitro culture; in view of the importance of promoting the maturation of nuclei and cytoplasm in the process of in vitro recovery of meiosis in oocytes in IVM technology, the maturation of cytoplasm is a key factor affecting the potential of embryonic development. The study of the present invention found that the application of MiH in IVM culture medium promotes the maturation of nuclei, helps to improve the discharge of the first polar body of oocytes, and reduces the rate of chromosome abnormalities and spindle abnormalities; at the same time, it also balances the damage of ROS to organelles and nuclei in the cytoplasm, which is conducive to the maturation of egg cytoplasm. In addition, it also improves the blastocyst formation rate of fertilized eggs in aging mouse oocytes. Therefore, adding MiH to IVM culture fluid may become a new method to improve and optimize IVM technology, which is of great significance for improving the fertility of elderly women and promoting the development of clinical reproductive medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 In the experiment of MiH promoting in vitro maturation of aged mouse oocytes in Experimental Example 1, the histogram of germinal vesicle rupture rate and first polar body extrusion rate of oocytes in the young group (NC), aged group (crtl) and MiH group; Figure 2In the experiment of MiH promoting in vitro maturation of aged mouse oocytes in Experimental Example 1, bright field images of in vitro maturation of oocytes in the young group (NC), aged group (crtl) and MiH (1μM) group; scale bar: 50 μm; Figure 3 In Experimental Example 1, MiH improves meiotic abnormalities in oocytes of aged mice; A Immunofluorescence staining of oocytes in the young group, aged group and MiH group; green represents spindles, and red represents chromosomes; B Bar graph of spindle and chromosome abnormality rates in oocytes in the young group, aged group and MiH group; Scale bar: 20 μm; Figure 4 In Experimental Example 1, MiH attenuated the level of ROS in oocytes of aged mice; A Immunofluorescence staining of oocytes in the young group, aged group and MiH group; green represents reactive oxygen species; B Fluorescence quantitative graph of the young group, aged group and MiH group; scale: 20 μm; Figure 5 In Experimental Example 2, MiH promotes the embryonic development of aged mice; A. A bar graph showing the number of cells at different embryonic development stages in the aged group and the MiH group after fertilization and continued in vitro culture; B. A bar graph showing the number of inner cell mass cells in the blastocyst stage embryos in the aged group and the MiH group; In the above figure, p<0.05, p<0.01. DETAILED DESCRIPTION

[0025] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical 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 prior arts shall fall within the protection scope of the present invention.

[0026] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0027] In the following embodiments ART, assisted reproduction technology, assisted reproductive technology; IVF-ET, in vitro fertilization and embryo Transfer; ICSI, intracytoplasmic sperm injection; IVM, in-vitro maturation of oocyte, in vitro maturation of immature oocytes; PGD, preimplantation genetic diagnosis; OHSS, ovarian hyperstimulation syndrome; MiH, minocycline hydrochloride; PARP1 poly ADP-ribose polymerase1, poly ADP-ribose polymerase 1; GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I, first meiotic metaphase; MII, metaphase II, second meiotic metaphase; PB1, polar body, first polar body; PMSG, pregnant mare serum gonadotropin; hCG, human chorionic gonadotropin, human chorionic gonadotropin.

[0028] Minocycline hydrochloride (MiH) was purchased from Selleck, USA, catalog number S4226.

[0029] Experimental Example 1 In vitro maturation of GV-stage oocytes It is well known that poor oocyte quality is an important cause of female infertility and birth defects. Accurate regulation of spindle formation and chromosome assembly during oocyte meiosis is a key factor in oocyte quality. Mammalian oocyte development is arrested in the diplotene phase of the first meiotic prophase, also known as the Germinalvesicle (GV) phase. Under the stimulation of LH hormone, the fully grown oocyte undergoes Germinalvesicle breakdown (GVBD), marking the resumption of meiosis. In the first meiotic prophase I, chromosomes begin to recombine and synthesize all or most of the proteins required for fertilized egg or embryo development. Chromosomes condense into short rods, spindles begin to assemble, centrioles disappear during development, spindles enter the nucleus, and chromosomes move to the middle of the spindle, completing the transition from the first meiotic prophase to metaphase. The chromosomes are neatly arranged at the equatorial plate, the bipolar spindle is fully formed, and the microfilament cap enriched in the cortical region together constitute the typical morphological characteristics of the first meiotic metaphase (metaphase I, MI).

[0030] Before and after birth, the oocyte enters the diplotene phase of the first meiotic prophase and remains in this phase for a long time. It is not until puberty or adulthood that the oocyte expels the first polar body under the action of the luteinizing hormone (LH) peak in the middle of the menstrual cycle and is arrested again in the metaphase of the second meiotic division (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, resulting in developmental arrest after fertilization, miscarriage, or offspring defects. Literature reports that the quality of oocytes in aged mice decreases, the maturation rate decreases, and the rate of spindle abnormalities increases.

[0031] Thirty aged female mice (ICR mice, 8 months old) and ten young female mice (ICR mice, 6-8 weeks old) were intraperitoneally injected with 6 IU pregnant mare serum gonadotropin (PMSG). The mice were killed by cervical dislocation 48 hours after the injection, and the bilateral ovaries were removed. The cumulus-oocyte complex was removed by oral pipette under a stereoscope, and naked eggs without granulosa cells were obtained by repeated blowing, which were GV stage oocytes.

[0032] NC group: 35 oocytes from young female mice at the GV stage were placed in IVM culture medium (M2 culture medium, the formula can be found in the M7167 product of Sigma Aldrich) with a volume of 50 μL; Ctrl group: GV-stage oocytes (35) of aged female mice were placed in IVM culture medium (M2 culture medium, the formula can be found in M7167 product of Sigma Aldrich) with a volume of 50 μL; MiH group: GV-stage oocytes (35) of aged female mice were placed in 50 μL IVM culture medium (M2 culture medium, the formula can be found in the M7167 product of Sigma Aldrich) supplemented with MiH; the final concentration of MiH was 500 nM, 1 μM or 2 μM; All the above groups were cultured at 5% CO2 and 37℃ for 14 hours. The IVM maturation of oocytes was evaluated by observing and calculating the germinal vesicle rupture rate and the first polar body extrusion rate. The germinal vesicle rupture rate = oocytes with germinal vesicle rupture (GVBD) / total number of cultured oocytes × 100%. The first polar body extrusion rate = the number of first polar bodies / total number of cultured oocytes × 100%. Then the oocytes in the young group, the aged group and the MiH group were cultured continuously. After 14 hours, the MII stage oocytes were collected and fixed with 4% paraformaldehyde fixative (PFA), permeabilized with 0.5% Triton, blocked with 1% BSA, and the spindle was marked with β-tubulin and DNA was marked with 4',6-diamidino-2-phenylindole (DAPI). The meiosis of the oocytes in the three groups and the abnormality rate of the oocyte spindle were observed. The abnormality rate of the oocyte spindle = abnormal spindle oocytes / total number of cultured oocytes × 100%.

[0033] Statistical methods: Statistical data were analyzed 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 are expressed as mean ± standard error; p<0.05, p<0.01, Differences with p < 0.001 were considered statistically significant.

[0034] The results of germinal vesicle rupture rate and first polar body extrusion rate are as follows Figure 1 As shown by Figure 1 It can be seen that the rupture rate of hair vesicles in the aging group was lower than that in the young group (84.96±1.44%). vs 94.12±2.44%), and the addition of MiH increased the rate of germinal vesicle rupture, but the effects were different at different concentrations (500nm, 1μM, 2μM) (88.40±5.07%). vs 91.49±0.72% vs88.12±6.70%). At the same time, it can be seen that the young group has a higher first polar body excretion rate than the aging group (75.83±3.58% v.s45.90±9.99%), and the difference is significant. p<0.05, indicating that the first polar body extrusion rate of oocytes in the aging group was low. As reported in the literature, the quality and maturation rate of oocytes in aging mice decreased. However, after using the IVM culture medium containing MiH, the MiH group had a higher first polar body extrusion rate than the aging group (the three concentration groups were 64.71±3.57%, respectively). vs 69.50±6.54% vs 69.34±6.64%), and the difference was significant p<0.05. At the same time, there was no significant difference in the first polar body extrusion rate between the MiH group and the young group. This indicates that during IVM, adding 500nM~2μM MiH to the culture medium can significantly increase the first polar body extrusion rate of oocytes and promote the in vitro maturation of aged oocytes. Figure 2 as shown in .

[0035] Oocyte meiosis Figure 3 As shown in Figures A and B, the rate of spindle abnormalities and the proportion of chromosome abnormalities in the MiH group (corresponding to a MiH concentration of 1 μM) were significantly lower than those in the aging group, indicating that MiH is involved in regulating oocyte meiosis and promoting in vitro maturation of aged mouse oocytes.

[0036] Mouse aged oocytes show high ROS levels, such as Figure 4 As shown in Figures A and B, the addition of MiH (corresponding to a MiH concentration of 1 μM) can significantly reduce the level of ROS in mouse aged oocytes.

[0037] Experimental Example 2 Fertilization of oocytes and subsequent embryonic development After the mature oocyte combines with the sperm, it completes its second meiotic division to form a fertilized egg containing a haploid paternal pronucleus and a haploid maternal pronucleus. After that, 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 the egg maturation process, the oocyte accumulates a large amount of maternal RNA and protein necessary for fertilization, maternal-to-zygotic transition, and preimplantation embryo development. Therefore, the quality of the oocyte determines the potential for early embryonic development. In this experimental example, the oocyte was fertilized through IVF experiments, and the embryonic development potential of the oocytes after fertilization in the control group (IVM) and the experimental group (IVM+MiH) was tested. The method is as follows: Five fertile male mice (ICR mice, 10 weeks old) were killed suddenly by cervical dislocation, fixed on the dissection table, and the peritoneal cavity was opened to remove the epididymal duct and placed in a 35 mm cell culture dish. The epididymal duct was punctured with sharp forceps under a stereoscope, and the sperm was released into the pre-warmed HTF fertilization solution (purchased from Nanjing Aibei Biotechnology Co., Ltd.) and placed in a 37°C, 5% CO2 incubator for 1 h.

[0038] Take the MII stage oocytes from the aging group and the MiH group (corresponding to MiH concentration of 1 μM) in Experimental Example 1, add 40 oocytes (about 20-25 oocytes from one mouse, generally 2-3 female mice correspond to 1 male mouse) into HTF fertilization solution (volume 50 μl), then add capacitated sperm (2 μl, concentration of 10000-200000 sperm / mL) (absorb the capacitated sperm on the upper layer), and place in a 37°C, 5% CO2 incubator for fertilization for 6 hours. Transfer the fertilized eggs to KSOM embryo culture medium (purchased from Nanjing Aibei Biotechnology Co., Ltd.), wash 3 times, and complete subsequent culture in KSOM embryo culture medium. The following time points (after fertilization) were selected in sequence to observe the corresponding stages of embryonic development: 2-cell (16 h), 4-cell (40 h), 8-cell (53 h), morula (70 h), early blastocyst formation (85 h), and late blastocyst formation (100 h).

[0039] Statistical methods: Statistical data were analyzed 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 are expressed as mean ± standard error; p<0.05, p<0.01, Differences with p < 0.001 were considered statistically significant.

[0040] Embryo ratio = number of embryos in the corresponding period / total number of fertilized eggs × 100%.

[0041] The results are as follows Figure 5 Figure A shows the ratio of embryos in each group, and Figure B shows the number of cells in the inner cell mass. Figure 5As can be seen from Figure A, compared with the aged group, the fertilization rate and 2-cell rate of oocytes in the MiH group increased significantly. During the fertilized egg period, the proportion of 2-cell, 4-cell, 8-cell, morula and blastocyst embryos in the total fertilized eggs (the total number of fertilized eggs in each group) was higher than that in the aged group, indicating that MiH can significantly increase the in vitro maturation rate of oocytes in aged mice and improve the quality of their oocytes, thereby improving the developmental potential of embryos after fertilization. Figure 5 As can be seen from Figure B, compared with the aging group, the average number of inner cell mass cells in the embryos of the MiH group that reached the blastocyst stage 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.

[0042] Adding MiH to the culture medium can significantly increase the in vitro maturation rate of aged mouse oocytes, improve the quality and developmental potential of oocytes, and promote early embryonic development of mice. The present invention can promote the in vitro maturation of aged mouse oocytes, improve the in vitro maturation technology of mouse oocytes, and has important significance for the wide application of in vitro maturation technology of oocytes.

[0043] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of minocycline or its derivatives in promoting in vitro maturation of oocytes or in preparing products for promoting in vitro maturation of oocytes.

2. Use of minocycline or its derivatives in improving the embryonic development potential of oocytes after in vitro fertilization or in preparing products for improving the embryonic development potential of oocytes after in vitro fertilization.

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. The use according to claim 1 or 2, characterized in that: The oocyte is an oocyte in the germinal vesicle stage.

5. The use according to claim 1 or 2, characterized in that: The oocyte is an aged oocyte.

6. The use according to claim 1, characterized in that: The promoting of oocyte maturation in vitro refers to promoting a decrease in chromosome abnormality rate, a decrease in spindle abnormality rate and / or an increase in the first polar body extrusion rate after the oocyte is treated with minocycline or its derivatives.

7. 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 stage, and late blastocyst formation stage of fertilized eggs obtained after in vitro culture and fertilization of oocytes treated with minocycline or its derivatives in the total fertilized eggs.

8. The use according to claim 1 or 2, characterized in that: The minocycline derivatives include minocycline hydrochloride; And / or, the product comprises culture fluid.

9. An oocyte culture medium, characterized in that: Containing minocycline or its derivatives as described in any one of claims 1 to 8.

10. The oocyte culture medium according to claim 9, characterized in that: The concentration of minocycline or its derivatives is 500nM~5μM; And / or, the oocyte culture medium also includes IVM culture medium.

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