CN113403263A - Method for improving oocyte in vitro and in vivo maturation quality and application - Google Patents

Abstract

The invention belongs to the technical field of biological medicine, and relates to a method and application for improving the in vitro and in vivo maturation quality of oocytes. The invention provides a method for improving the influence of oxybenzone on the quality of female animal oocytes, and has important significance for diagnosis and treatment of abnormal oocyte maturation patients and in-vitro production of animal embryos caused by clinical environmental factors.

Description

Method for improving oocyte in vitro and in vivo maturation quality and application

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a method for improving the in vitro and in vivo maturation quality of oocytes and application thereof.

Background

Melatonin (MT) is known as N-acetyl-5-methoxytryptamine, and is found in the bodies of mammals, including humans, and other higher animals, and is first found in the pineal gland. In addition, many organs, tissues and cells (ovary, testis, skin, red blood cells, etc.) may be involvedTo synthesize and secrete small amounts of MT (see: Rodri i guez C, pure-Moncada N, Reiter RJ, et al. Regulation of cancer Cell glucose metabolism for cancer Cell surface adjuvant. J. Cell physical. 2021; 236 (1): 27-40.). Melatonin not only has the function of directly removing free radicals, but also can regulate and control the expression of antioxidant enzyme, and plays an important role in maintaining the redox balance of organisms and improving the reproductive capacity of female animals. In recent years, many studies have shown that addition of MT to the culture medium can reduce ROS, promote maturation of ova, and Improve Oocyte quality, including Human, mouse, pig, cow, etc. (see: Rodri i rule-variable C, Labarta E.clinical application of antibiotics to Improve Human Octobyte Mitochlorine Function: A review. antibiotics (base) 2020; 9 (12): 1197; Zhang M, Lu Y, Chen Y, Zhang Y, Xiong B.Instructions of collagen in collagen yield gene conversion use for improved protein KT-related protein uptake in cells, 2. incision J. 28: 28. Yituch. J. Pat. No. 28. J. Yituch. J. 9. J. Shituch. 9. 1. 9. Zhang M. 9

JC, Lucas-Hahn A, Hadel KG, Aldag P, Niemann H.Melatonin industries in video level competition of culture-complete complexes collected by in vitro pick-up in prepubertal and adult day cat. Theriology.2020; 161: 285-293.). In addition, MT can also improve oocyte maturation damage and quality caused by environmental pollutants (see: Bahelka I, Stupka R,

J，

M.The impact of bisphenols on reproductive system and on offspring in pigs-A review 2011-2020.Chemosphere.2021；263：128203.)。

in recent years, the overuse of Oxybenzone (OBZ) has become a new type of environmental pollutant detected in oceans, lakes and even daily drinking water (see: Tsui MM, Leung HW, Wai TC, et al. Occurence, distribution and ecological ridge assessment of multiple classes of UV filters in surface waters from differences countries, Water Res. 2014; 67: 55-65.) as the amount of skin care products used increases. Oxybenzone can also be absorbed directly into the body through the skin and has been successfully detected in human blood, urine, semen, amniotic fluid, placenta, and breast milk (see: US Food and Drug Administration (FDA), sungreen Drug products for over-the-counter human use: disposed rule. fed register.2019; 84: 6204-6275. https:// www.govinfo.gov/content/pkg/FR-2019-02-26/pdf/2019-03019. pdf). In 2014, the journal of epidemiology in the United states reported that oxybenzone is closely associated with decreased human fertility (Buck Louis GM, Kannan K, Sapra KJ, Maisog J, Sundaram R. Urrinary concentrations of benzophenone-type ultrasound irradiation filters and couples' efficacy. am J epidemiology.2014; 180 (12): 1168-75.). In 2019, Matta et al found that the Concentration of oxybenzone in human Plasma was between 0.74. mu.M and 0.92. mu.M, however, the recommended amount by the U.S. Food and Drug Administration (FDA) was only no more than 2.2nM (see: Matta MK, Zusterzel R, Pilli NR, et al. Effect of sunlight Application Under maximum Use comparisons on plasmid Concentration of sunlight Active Ingredients: A random doped Clinical Trial. JAMA. 2019; 321: 2082-2091.). Recent studies have demonstrated that the widespread use of oxybenzone has a pathological effect on brain cells, particularly neuronal cells, in fetal and infant development, e.g., oxybenzone induces hirschsprong' disease by interfering with migration of neural crest cells (see: Wnuk A, Rzemieniec J, Laso)

W，Krzeptowski W，Kajta M.Benzophenone-3 Impairs Autophagy，Alters Epigenetic Status，and Disrupts RetinoidX Receptor Signaling in Apoptotic neurological cells, mol neurobiol.2018; 55(6): 5059-5074.). In 2019, the guidance rules issued by the FDA in the United states reported that high levels of oxybenzone in women's urine were associated with the birth weight and head circumference of offspring (see: US Food and Drug Administration (FDA). Sunscreen Drug products for over-the-counter human use: disposed rule. Fed Regist. 2019; 84: 6204-6275. https:// www.govinfo.gov/content/pkg/FR-2019-02-26/pdf/2019-03019. pdf). In addition, oxybenzone has toxic effects on the development of organs such as sperm, prostate and uterus (see: Frederiksen H, Krause M,

n, Rehfeld A, Skakkebaek NE, Andersson AM. UV filters in matched sealed fluidic-, urea-, and serum samples from Young men.J Expo Sci Environ epidemic.2020; 10.1038/s 41370-020-. In conclusion, although some studies on the reproductive toxicology of oxybenzone have been published, no report has been made on whether oxybenzone exposure affects meiotic maturation and ovum quality of mammalian oocytes.

At present, in the prior art, no correlation study on the meiotic maturation and the quality of oxybenzone and the oocyte is carried out, for example, a method for relieving the ovum maturation disorder caused by oxybenzone and improving the quality of the oocyte can be developed, and the method has important theoretical guidance significance on the safe use amount of oxybenzone and the treatment of female infertility caused by the safe use amount of oxybenzone.

Disclosure of Invention

The invention aims to provide a medicine for improving the in vitro and in vivo maturation quality of oocytes and an application method thereof, and the medicine and the application method can well improve the problems of ovum maturation disorder and oocyte quality reduction caused by environmental pollution, obesity, aging and the like.

The purpose of the invention can be realized by the following technical scheme:

a method of improving the quality of maturation of an oocyte in vitro and in vivo, the method comprising administering to the oocyte a drug a, said drug a comprising melatonin;

wherein, when the maturation quality of the oocyte is improved in vitro, the in vitro addition concentration of the melatonin is 10^-5-10^{- 9}M; when the maturation quality of the oocyte is improved in vivo, the gavage dosage of the melatonin is 13-17 mg/kg/day, and the gavage time is 26-30 days.

As a preferable technical scheme of the invention, the concentration of the melatonin added in vitro is 10^-7M。

As a preferable technical scheme of the invention, the gavage dosage of the melatonin is 15 mg/kg/day, and the gavage time is 28 days.

The method for improving the maturation quality of the oocytes in vitro and in vivo is applied to the fields of ovum maturation disorder and oocyte quality reduction.

The invention has the beneficial effects that:

(1) the method directly adds melatonin in the oocyte in vitro maturation culture solution and performs gastric lavage on a mouse, so that the melatonin is proved to be capable of relieving the problem of reduction of in vitro and in vivo maturation rates of the oocyte of the mouse caused by oxybenzone, improving the quality of the oocyte, being beneficial to relieving the problem of abnormal morphology of the spindle body of the oocyte caused by the oxybenzone, improving the function of mitochondria and reducing the damage of oxidative stress to the oocyte.

(2) The invention provides a method for improving the influence of oxybenzone on the in vivo and in vitro maturation and quality of female oocytes, and provides an effective method for improving female fertility.

Drawings

In order to facilitate understanding for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.

FIG. 1 is a schematic representation of the effect of oxybenzone on in vitro maturation of mouse oocytes;

FIG. 2 is a graph showing the effect of melatonin and oxybenzone addition on the in vitro maturation rate of oocytes in mice;

FIG. 3 is a schematic illustration of the effect of melatonin and oxybenzone addition on mouse embryo in vitro development;

FIG. 4 is a schematic illustration of the effect of melatonin and oxybenzone addition on spindle morphology and alpha-tubulin expression levels in mouse oocytes;

FIG. 5 is a schematic illustration of the effect of melatonin and oxybenzone addition on mouse oocyte oxidative stress, mitochondrial mass and early apoptosis;

FIG. 6 is a schematic illustration of the effect of gavage melatonin and oxybenzone on in vivo maturation of mouse oocytes and blastocyst rate;

figure 7 is a graph of the effects of gavage melatonin and oxybenzone on mature oocyte oxidative stress, early apoptosis, and mitochondrial quality in mice.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The melatonin relieves the expression that oxybenzone induces the in vitro maturation quality of the oocyte to be reduced:

step S1: preparing experimental animals and feeding management;

the animals used in the embodiment are ICR female mice and male mice, the female mice are 6-10 weeks old, the male mice are 10-14 weeks old, the ICR female mice and the male mice are purchased from the centers of medical experimental animals in Guangdong province, the ICR male mice are raised in a temperature-controlled and light-controlled environment with the temperature of 20-25 ℃ and the illumination of 12h light and shade intervals, the experimental mice can freely eat and drink water, the experiment is carried out after 1 week of adaptation, and the operation conforms to the relevant regulations of the welfare of the experimental animals;

step S2: preparing main reagents and instruments for experiments;

the main reagents and instruments include: PMSG, hCG, M2 culture solution, M16 culture solution, KSOM culture solution, paraffin oil, alpha-tubulin antibody, melatonin, oxybenzone, active oxygen kit, GSH kit, Annexin-V-FITC cell early apoptosis kit, MitoTracker Deep Red mitochondrial Red fluorescent Probe MitoProbe^TMJC-1 mitochondrial membrane potential detection kit, an inverted fluorescence microscope and a confocal microscope;

wherein: human chorionic gonadotropin hCG was obtained from Ningbo second hormone plant, melatonin was obtained from Sigma, USA, and oxybenzone was obtained from Selleck, MitoProbe^TMJC-1 mitochondrial membrane potential detection kit was purchased from Thermo Fisher Scientific, USA, an inverted fluorescence microscope was purchased from Nikon, Ti-U, Japan, and a confocal microscope was purchased from Nikon, Japan, A1R;

step S3: obtaining GV-stage oocytes and carrying out in-vitro culture on the oocytes;

injecting 10IU PMSG into ICR female mouse, removing neck after 46-48h, removing ovary, opening abdominal cavity, taking out bilateral ovaries, chopping the ovaries under a stereomicroscope with a blade, flushing with M2 culture solution, picking up normal GV stage oocytes with an oral pipette, cleaning with M2 culture solution to remove impurities, transferring to M16 culture solution containing melatonin and oxybenzone, and adding CO₂The culture box carries out in-vitro maturation culture, and the maturation rate is judged according to the discharge condition of the first polar body;

step S4: obtaining MII stage oocyte;

injecting 10IU PMSG into the abdominal cavity of a CR female mouse, injecting 10IU hCG into the abdominal cavity after 48 hours, removing the neck and killing the CR female mouse after 13-16 hours, opening the abdominal cavity, exposing and shearing off an oviduct, placing the oviduct into M2 culture solution, finding out and piercing the most part of the oviduct under a stereomicroscope, releasing a cumulus-oocyte complex, removing cumulus layer granulocytes by hyaluronidase, transferring the cumulus-removed oocytes in the MII stage into M2 culture solution, and judging the maturation rate according to the first polar body discharge condition;

step S5: preparing an M16 oocyte maturation culture solution, and counting the oocyte maturation rate, namely the first polar body discharge rate;

m16 oocyte maturation medium containing 2nM, 50nM, 250nM, 500nM, 1000nM oxybenzone was prepared in advance. Preparing 30 mu L of oocyte culture liquid drops in a 60mm culture dish, and pre-balancing for 6 hours in a CO2 incubator; adding 13-18 collected liquid drops into each liquid dropGV stage oocytes in CO₂Carrying out in-vitro maturation culture in an incubator; after culturing for 13h, the oocyte maturation rate, i.e. the first polar body expulsion rate, was counted.

FIG. 1A shows the morphology of oocytes treated with different concentrations of oxybenzone, at a scale bar of 100 μm; FIG. 1B shows the effect of different concentrations of oxybenzone on the first polar body discharge rate of mouse oocytes (control: 82.45. + -. 8.54%, n ═ 367; 2 nM: 76.84. + -. 12.84%, n ═ 360; 50 nM: 71.11. + -. 11.87%, n ═ 346,; 250 nM: 69.41. + -. 8.59%, n ═ 314; 500nM, 55.87. + -. 10.19%, n ═ 351; 1000 nM: 54.21. + -. 12.68%, n ═ 343). Control stands for "Control group"; OBZ stands for "oxybenzone group".^a-dRepresenting significant differences (P < 0.05), data are presented as Mean + -SEM of five replicates.

The results are shown in FIG. 1: the addition of 50nM, 250nM, 500nM and 1000nM oxybenzone can significantly reduce the oocyte maturation rate. In combination with the 2019 JAMA paper data, we chose 500nM of oxybenzone as the post-experimental treatment concentration.

Step S6: culturing fertilized eggs in vitro;

injecting 10IU PMSG into ICR female mouse abdominal cavity, injecting 10IU hCG into abdominal cavity after 48h, closing with ICR male mouse, checking vaginal suppository in the next morning, taking out neck of female mouse, killing, taking oviduct in M2 culture solution, cutting huge ampulla in oviduct under microscope to see that fertilized egg is dissociated, removing cumulus cell with hyaluronidase, cleaning, transferring fertilized egg into balanced 50 microliter KSOM culture drop, and adding CO₂Culturing in an incubator, and judging the blastocyst rate according to the blastocyst formation condition;

step S7: prepared in advance with 500nM oxybenzone and 10^-5mol/L、10^-7mol/L、10^-9M16 culture solution of melatonin in mol/L. Collecting GV stage oocytes, and counting the maturation rate of the oocytes after in vitro maturation culture.

Fig. 2A shows the morphology of oocytes of mice treated with oxybenzone and melatonin at a scale bar of 100 μm; FIG. 2B is a graph of the effect of different concentrations of melatonin on the first polar body discharge rate of 500nM oxybenzone-exposed oocytes in mice (control)Group (2): 80.72 + -10.76%, n is 262; 500nM oxybenzone group: 51.62 ± 11.59%, n is 245; 500nM oxybenzone +10^-9mol/L melatonin group: 69.09 +/-7.01%, and n is 220; 500nM oxybenzone +10^-7mol/L melatonin group: 76.33 ± 8.34%, n is 241; 500nM oxybenzone +10^-5mol/L melatonin group: 57.40 ± 13.95%, n ═ 254). Control stands for "Control group"; OBZ stands for "oxybenzone group"; OBZ + MT stands for the "oxybenzone + melatonin group".^a-cRepresenting significant differences (P < 0.05), data are presented as Mean + -SEM of five replicates.

The results are shown in FIG. 2: the influence of oxybenzone on oocyte first polar body expulsion can be remarkably relieved by adding melatonin. Wherein is added with 10^-7mol/L、10^-9The influence of the decrease of the in vitro maturation rate of the oocyte caused by the oxybenzone can be relieved by mol/L melatonin.

Step S8: prepared in advance with 500nM oxybenzone and 10^-7KSOM embryo culture solution of melatonin in mol/L. Making 30. mu.L embryo culture drops in a 60mm Petri dish, CO₂Pre-balancing for 10 hours in an incubator; adding 13-18 fertilized eggs obtained by collection into each drop, and placing in CO₂Carrying out in-vitro culture in an incubator; and after culturing for 72h, counting the blastocyst rate.

Fig. 3A is the blastocyst morphology of mice treated with oxybenzone and melatonin at a scale bar of 100 μm; fig. 3B shows the effect of melatonin on the rate of blastocyst formation in vitro in oxybenzone-treated embryos (control: 84.52 ± 7.08%, n ═ 184; oxybenzone: 41.25 ± 7.04%, n ═ 191; oxybenzone + melatonin: 76.12 ± 8.61%, n ═ 217). Control stands for "Control group"; OBZ stands for "oxybenzone group"; OBZ + MT stands for the "oxybenzone + melatonin group".^a-cRepresenting significant differences (P < 0.05), data are presented as Mean + -SEM of five replicates.

The results are shown in FIG. 3: addition 10^-7The influence of the oxybenzone on the blastocyst formation rate of the mice can be obviously relieved by mol/L melatonin.

Step S9: detecting spindle morphology and alpha-tubulin level;

dissolving the collected oocytes with acid table type liquid to remove zona pellucida, transferring the oocytes to 4% paraformaldehyde for fixing for 30min, permeating 1% Triton for 20min, sealing 2% BSA for 2h, incubating a primary antibody (diluting alpha-tubulin according to a ratio of 1: 300), cleaning the oocytes after overnight, incubating a secondary antibody, cleaning, carrying out DAPI staining, observing and photographing under a confocal microscope, and analyzing the average fluorescence intensity of the alpha-tubulin of the oocytes by using ImageJ software;

step S10: detecting the content of ROS and GSH;

MII stage oocytes were transferred to M2 medium containing 100. mu. mol/L DCHFDA or 20. mu. mol/L ThiolTracker TM Violet staining solution at 37 ℃ with 5% CO according to kit instructions₂Incubating in a dark place in the incubator for 30min, cleaning, placing under an inverted fluorescence microscope for observation and photographing, and analyzing the ROS and GSH average fluorescence intensity of the oocyte by ImageJ software;

step S11: detecting the mitochondrial distribution and membrane potential condition of the oocyte;

according to the kit instructions, the MII stage oocytes were transferred to M2 medium containing 200nmol/L MitoTracker probe or 4. mu. mol/L JC-1 at 37 ℃ with 5% CO₂Incubating in a dark place in an incubator for 30min, washing, tabletting, transferring to an inverted fluorescence microscope for observation and photographing, and analyzing JC-1 red and JC-1 green average fluorescence intensity of the oocytes by ImageJ software;

step S12: detecting early apoptosis of the oocyte;

according to the kit instruction, 10 mu L Annexin-V-FITC and 490 mu L Annexin-binding buffer are used for staining the oocyte for 15min at room temperature under the condition of keeping out of the light, M2 culture solution is washed, tabletting is carried out, the photograph is observed under an inverted fluorescence microscope, and the early apoptosis rate of the oocyte is counted.

FIG. 4A is a graph showing α -tubulin staining of oocytes in MI stage after oxybenzone and melatonin treatment; FIG. 4B shows the rate of morphological abnormality of spindle bodies of oocytes; FIG. 4C shows the fluorescence intensity of oocyte α -tubulin. Control stands for "Control group"; OBZ stands for "oxybenzone group"; OBZ + MT stands for the "oxybenzone + melatonin group".^a，bRepresenting significant differences (P < 0.05), data are presented as Mean + -SEM of 3 replicates.

FIG. 5A shows the fluorescence intensity of ROS in mouse oocytes after treatment with oxybenzone and melatonin; FIG. 5B is the fluorescence intensity of GSH within oocytes; FIG. 5C shows abnormal distribution rate of oocytes mitochondria; FIG. 5D is oocyte membrane potential level; FIG. 5E shows the early apoptosis rate of oocytes. Control stands for "Control group"; OBZ stands for "oxybenzone group"; OBZ + MT stands for the "oxybenzone + melatonin group".^a，bRepresenting significant differences (P < 0.05), data are presented as Mean + -SEM of 3 replicates.

Spindle morphology, intracellular ROS and GSH levels, mitochondrial function and early apoptosis level are important indexes for evaluating oocyte quality, and therefore, the detection results of the spindle morphology and alpha-tubulin expression level of the oocyte find that melatonin can obviously relieve oxybenzone-induced spindle morphology disorder and alpha-tubulin expression abnormality as shown in figure 4. In addition, as shown in fig. 5, melatonin was able to significantly improve the level of oxybenzone on mitochondrial function and early apoptosis, reduce ROS levels in MII-stage oocytes, and increase GSH levels.

Example 2

The melatonin relieves the expression that oxybenzone induces the in vivo maturation quality of the oocyte to be reduced:

step SS 1: taking an experimental mouse as a research object, and feeding and managing the experimental mouse;

the animals used in the embodiment are ICR female mice and male mice, the female mice are 6-10 weeks old, the male mice are 10-14 weeks old, the ICR female mice and the male mice are purchased from the centers of medical experimental animals in Guangdong province, the ICR male mice are raised in a temperature-controlled and light-controlled environment with the temperature of 20-25 ℃ and the illumination of 12h light and shade intervals, the experimental mice can freely eat and drink water, the experiment is carried out after 1 week of adaptation, and the operation conforms to the relevant regulations of the welfare of the experimental animals;

step SS 2: preparing a main reagent and an apparatus for an experiment, the main reagent and the apparatus comprising: PMSG, hCG, M2 culture solution, M16 culture solution, KSOM culture solution, paraffin oil, alpha-tubulin antibody, melatonin, oxybenzone, active oxygen kit, GSH kit, Annexin-V-FITC cell early apoptosis kit, MitoTracker Deep Red mitochondrial RedFluorescent probes, MitoProbe^TMJC-1 mitochondrial membrane potential detection kit, an inverted fluorescence microscope and a confocal microscope;

wherein: human chorionic gonadotropin hCG was obtained from Ningbo second hormone plant, melatonin was obtained from Sigma, USA, and oxybenzone was obtained from Selleck, MitoProbe^TMJC-1 mitochondrial membrane potential detection kit was purchased from Thermo Fisher Scientific, USA, an inverted fluorescence microscope was purchased from Nikon, Ti-U, Japan, and a confocal microscope was purchased from Nikon, Japan, A1R;

step SS 3: after melatonin (15 mg/kg/day) and oxybenzone (500 nM/day) were gavaged for 28 days, oocyte maturation rate and blastocyst rate were measured.

FIG. 6A shows the in vivo maturation rate of oocytes in mice in the control group, oxybenzone group, and oxybenzone + melatonin group (control group: 81.51. + -. 8.51%, n ═ 252; oxybenzone group: 59.58. + -. 9.92%, n ═ 301; oxybenzone + melatonin group: 71.16. + -. 12.01%, n ═ 267); fig. 6B shows the blastocyst ratios of mice in the control group, oxybenzone group, and oxybenzone + melatonin group (control group: 78.89 ± 6.29%, n ═ 186; oxybenzone group: 68.53 ± 26.25%, n ═ 180; oxybenzone + melatonin group: 84.10 ± 11.98%, n ═ 183). Control stands for "Control group"; OBZ stands for "oxybenzone group"; OBZ + MT stands for the "oxybenzone + melatonin group".^a，bRepresenting significant differences (P < 0.05), data are presented as Mean + -SEM of 3 replicates.

FIG. 7A is the fluorescence intensity of ROS in oocytes of mice of control group, oxybenzone + melatonin group; FIG. 7B is the fluorescence intensity of GSH within oocytes; FIG. 7C shows abnormal distribution rate of oocytes mitochondria; FIG. 7D is oocyte membrane potential level; FIG. 7E is the oocyte early apoptosis rate; FIG. 7F shows the rate of morphological abnormality of oocyte spindles. Control stands for "Control group"; OBZ stands for "oxybenzone group": OBZ + MT stands for the "oxybenzone + melatonin group".^a，bRepresenting significant differences (P < 0.05), data are presented as Mean + -SEM of 3 replicates.

The results are shown in FIG. 6: the melatonin can obviously reduce the harm of the decrease of the in vivo maturation rate and blastocyst rate of the oocyte of a mouse caused by the oxybenzone. The evaluation result of the oocyte quality shows that melatonin can obviously relieve the morphological abnormality of the oocyte spindle induced by oxybenzone, reduce the intracellular ROS level, improve the GSH level, inhibit the early apoptosis of the oocyte and improve the uniform distribution and the membrane potential level of mitochondria, as shown in figure 7.

The embodiment shows that melatonin can relieve the problem of the decrease of the in vitro and in vivo maturation rate of the mouse oocyte caused by oxybenzone and improve the quality of the oocyte; and the method is favorable for relieving the abnormal morphology problem of the oocyte spindle body caused by oxybenzone, improving the function of mitochondria and reducing the damage of oxidative stress to the oocyte.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims (4)

1. A method of improving the quality of oocyte maturation in vitro and in vivo, comprising: the method comprises administering a drug a to the oocyte, said drug a comprising melatonin;

wherein, when the maturation quality of the oocyte is improved in vitro, the in vitro addition concentration of the melatonin is 10^-5-10^-9M; when the maturation quality of the oocyte is improved in vivo, the gavage dosage of the melatonin is 13-17 mg/kg/day, and the gavage time is 26-30 days.

2. A method of improving the quality of oocyte maturation in vitro and in vivo according to claim 1, wherein: the melatonin is added in vitro at a concentration of 10^-7M。

3. A method of improving the quality of oocyte maturation in vitro and in vivo according to claim 1, wherein: the gavage dose of the melatonin is 15 mg/kg/day, and the gavage time is 28 days.

4. Use of a method according to claim 1 for improving the quality of oocyte maturation in vitro and in vivo in the field of egg maturation disorders and oocyte quality degradation.

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