Early-onset ovarian insufficiency animal model and construction method thereof

Abstract

The invention provides a premature ovarian insufficiency (POI) animal model and a construction method thereof, and particularly, the method utilizes chronic unpredictable pressure stress to construct different pathological stage models of mouse POI, and comprises the following steps: screening experimental animals with normal ovarian functions, adaptively feeding, applying various pressure stresses to the experimental animals, and obtaining the POI animal model. The POI model has a compensatory period, and is more in line with the clinical pathological process of patients suffering from premature ovarian insufficiency caused by common pressure in modern society.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. More specifically, the present invention relates to an animal model of premature ovarian insufficiency and a method for constructing the same. Background Art

[0002] The dynamic change of female ovarian function is an important indicator of reproductive health. Diminished ovarian reserve (DOR) is characterized by a decrease in the number and quality of oocytes and is a pre-state of ovarian failure. It is worth noting that when this pathological process occurs in women under 40 years old, it may develop into premature ovarian insufficiency (POI), which is more clinically challenging.

[0003] POI is not only typically manifested by menstrual abnormalities (amenorrhea or oligomenorrhea), but also accompanied by a significant increase in FSH (>25 U / L) and a fluctuating decrease in estrogen, with a prevalence rate of 1%-3.8%. Although both are related to a decline in ovarian reserve, the pathological process of POI is more severe, with characteristics of age limitation, hormone level threshold, and a sharp decline in fertility. Moreover, due to its irreversibility and long-term effects on systemic metabolism, cardiovascular, and bone health, it has become the focus of reproductive medicine research.

[0004] With the accelerating pace of modern social life, chronic mental stress has become an important environmental inducer for the occurrence of POI in modern women. It is worth noting that the physiological characteristics of women aged 35-40, who are the peak population for POI onset (corresponding to 8-11-month-old mice), are significantly different from those of the commonly used 2-month-old chemically induced models in existing research. The latter not only fails to truly simulate the pathological process of stress-induced diseases, but also, due to the essential differences in the developmental stages of the reproductive endocrine system, leads to a generational shift in its molecular regulatory network compared to the clinical reality. Establishing a POI animal model that can simulate the real pathological process is the key basis for promoting relevant research, especially the need to construct an animal model system that conforms to the characteristics of social stress and matches the clinical age characteristics.

[0005] Therefore, developing an age-appropriate POI animal model based on chronic stress induction and systematically analyzing the biological characteristics and molecular regulatory network in the early stage of POI has important scientific value for revealing the molecular mechanism of stress-induced ovarian function decline, establishing early warning indicators, and formulating precise intervention strategies based on the pathological process. Summary of the Invention

[0006] The purpose of the present invention is to provide an age-appropriate POI animal model that is closer to clinical POI.

[0007] The present invention provides a method for establishing a mouse model of different pathological stages of premature ovarian insufficiency (POI) using chronic unpredictable stress (CUS) to simulate the pathological process of POI patients caused by stress factors in modern society and provide a theoretical basis for the development of early intervention diagnosis and treatment strategies.

[0008] In a first aspect of the present invention, there is provided a premature ovarian insufficiency (POI) animal model, and the POI animal model has a compensatory stage.

[0009] The compensatory stage includes characteristics selected from the group consisting of:

[0010] (a) Disordered estrous cycle;

[0011] (b) Increased sex hormone levels;

[0012] (c) No decrease in the number of follicles; and

[0013] (d) No ovarian atrophy.

[0014] In another preferred embodiment, the disordered estrous cycle is selected from the group consisting of: prolonged estrous cycle and / or disordered proportion of each period in the estrous cycle.

[0015] In another preferred embodiment, the estrous cycle includes proestrus, estrus, metestrus, and diestrus.

[0016] In another preferred embodiment, the sex hormones are selected from the group consisting of: anti-Müllerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), or a combination thereof.

[0017] In another preferred embodiment, the ovarian atrophy is selected from the group consisting of: decreased ovarian weight and / or atrophy of ovarian morphological structure.

[0018] In another preferred embodiment, the POI includes characteristics selected from the group consisting of:

[0019] (A) Disordered estrous cycle;

[0020] (B) Decreased sex hormone levels;

[0021] (C) Decrease in the number of follicles; and / or

[0022] (D) Ovarian atrophy.

[0023] In another preferred embodiment, the animal model is a mouse animal model.

[0024] In another preferred embodiment, the mouse is a C57BL / 6 mouse.

[0025] In another preferred embodiment, the modeling duration of the POI animal model is 8 - 12 weeks.

[0026] In another preferred embodiment, the compensatory period appears at 8 ± 1 week.

[0027] In another preferred embodiment, the POI animal model is used to analyze the biological characteristics and molecular regulatory network in the early stage of POI, study the molecular mechanism of stress-induced ovarian function decline, establish early warning indicators, and / or develop precise intervention strategies based on the pathological process.

[0028] In the first aspect of the present invention, there is provided a method for constructing a POI animal model as described in claim 1, the method comprising the steps of:

[0029] (i) Screening experimental animals for adaptive feeding;

[0030] (ii) Subjecting the experimental animals to chronic unpredictable stress stimuli; and

[0031] (iii) Obtaining a POI animal model.

[0032] In another preferred embodiment, the stress sources for the chronic unpredictable stress stimuli are selected from the group consisting of: acoustic, optical, and electrical stimuli, food deprivation, water deprivation, social crowding, social isolation, dirty cages, wet cages, empty bedding, tail suspension, restraint stress, or a combination thereof.

[0033] In another preferred embodiment, the acoustic, optical, and electrical stimuli are selected from the group consisting of: noise interference, nocturnal lighting, stroboscopic lighting, foot shock, or a combination thereof.

[0034] In another preferred embodiment, the experimental animals are 6-month-old mice.

[0035] In another preferred embodiment, the modeling duration of the method is 8 - 12 weeks.

[0036] In another preferred embodiment, step (ii) further comprises: randomly using 1 - 3 stress sources for stimulation every day.

[0037] In another preferred embodiment, step (iii) of the method further comprises: performing relevant structure / function detection of POI.

[0038] In another preferred embodiment, the structure / function detection is selected from the group consisting of: performing vaginal smear detection in the last two weeks of modeling, obtaining blood samples after the end of modeling for sex hormone level detection, performing HE staining on the ovaries after the end of modeling, recording the ovarian weight after the end of modeling, or a combination thereof.

[0039] In another preferred embodiment, the method is used to construct a POI animal model.

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Schematic diagram of estrous cycle detection (10 times under light microscope), where A, B, C, and D are, in sequence: proestrus, estrus, metestrus, and diestrus; E, F, and G are statistical schematic diagrams of the proportion of estrous cycle disorders, cycle duration, and the proportion of each period in mice. ** indicates P < 0.01, and *** indicates P < 0.001.

[0042] Figure 2 : Schematic diagram of the detection results of AMH and E2 levels. *** indicates P < 0.001.

[0043] Figure 3 : Schematic diagram of the effect of CUS modeling on follicle morphology and number observed by HE staining (4 times under light microscope) and statistical schematic diagram of the number of follicles at each level. ** indicates P < 0.01.

[0044] Figure 4 : Statistical schematic diagram of the detection results of mouse body weight, ovarian weight, and ovarian index. * indicates P < 0.05, and ** indicates P < 0.01.

[0045] Figure 5 : Schematic diagram of the detection results of AMH, E2, FSH, and LH levels. ** indicates P < 0.01, and *** indicates P < 0.001.

[0046] (Note: Con is the control group, and CUS is the modeling group) DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] The inventor of the present invention has conducted extensive and in-depth research and developed an animal model of premature ovarian insufficiency (POI) and a method for constructing the same. The method includes: screening 6-month-old mice with normal ovarian function, adaptively raising them, and applying various stress stimuli to the mice to obtain a POI animal model. On this basis, the present invention has been completed.

[0048] TERMINOLOGY

[0049] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used in this article have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary.

[0050] As used herein, the terms "comprising", "including", and "containing" are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of" and "consisting essentially of".

[0051] In a specific embodiment of the present invention, to construct an animal model in the POI compensatory period, the intensity of chronic unpredictable stress used is milder than that in previous studies, and the specific methods of the stressors are shown in Table 1.

[0052] Table 1

[0053]

[0054] During the entire modeling period, each stressor is randomly repeated, and at the same time, in order to avoid the test animals from developing tolerance, the intensity of stressors such as tail suspension, restraint stress, and acoustic, optical, and electrical stimulation is increased weekly. And the test animals are weighed every week. Therefore, stressors that are likely to cause weight changes, such as fasting, water deprivation, and wet bedding, are not arranged on the 7th day of each week to prevent deviation of the mouse body weight. The specific CUS administration schedule is shown in Table 2.

[0055]

[0056]

[0057]

[0058] In some embodiments, vaginal smear tests are performed on the mice for two weeks in the last two weeks after the modeling is completed. Preferably, vaginal smears are taken from the mice at 9:00 am every day to observe the changes in the estrous cycle. It is used to judge whether the stress has successfully caused ovarian function disorder in the mice.

[0059] Furthermore, the judgment criteria for the vaginal smear test can adopt the conventional criteria in the technical field, for example: the estrous cycle of normal mice is 4 - 5 days. Proestrus: Round or tadpole-shaped nucleated epithelial cells account for the vast majority in the smear, and leukocytes and keratinized epithelial cells are very few. Estrus: Keratinized anucleated epithelial cells account for the vast majority in the smear, and leukocytes and nucleated epithelial cells are very few. Metestrus: Flaky keratinized epithelial cells, nucleated epithelial cells, and leukocytes are all present in the smear, with no significant difference. Diestrus: Leukocytes account for the vast majority in the smear, and nucleated epithelial cells and keratinized epithelial cells are very few.

[0060] In some embodiments, after the mice are weighed for the last time, blood is taken from their eyes. The collected blood samples are left standing at room temperature for 12 hours and then centrifuged at 4°C. The levels of anti-Müllerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in the blood are detected.

[0061] In some embodiments, after blood was taken from the eyeballs, the mice were sacrificed, and their ovaries were removed for HE staining. The obtained ovaries were subjected to HE staining to observe the morphological structure of the mouse ovaries and the number of follicles at all levels. The staining results showed that the cell nuclei were blue, and the cytoplasm, muscle, connective tissue, red blood cells, and eosinophilic granules were red to varying degrees. Calcium salts and various microorganisms were stained blue or blue-violet.

[0062] Furthermore, the judgment characteristics of follicles at all levels can adopt the conventional standards in the technical field, for example: primordial follicle: a layer of squamous flat granulosa cells can be observed outside the oocyte; primary follicle: the oocyte is surrounded by a single layer of cubic granulosa cells; secondary follicle: the oocyte is surrounded by multiple layers of cubic granulosa cells, and there is no obvious cavity; antral follicle: an obvious antrum can be observed in the oocyte; atretic follicle: the oocyte shows karyopyknosis, and the cells present irregular shapes.

[0063] In some embodiments, it further includes: recording the ovarian weights of the mouse control group and the model group, and calculating the ovarian index. Whether the modeling causes ovarian atrophy can be judged according to the ovarian index.

[0064] Compared with the prior art, the main advantages of the present invention are as follows:

[0065] 1. The modeling time of the POI animal model of the present invention is up to twelve weeks, including the POI compensatory period, which is more in line with the clinical pathological process of patients with premature ovarian insufficiency caused by stress commonly seen in modern society.

[0066] 2. Compared with the existing modeling methods, the POI modeling method of the present invention is milder, causes less harm to animals, and the obtained POI animal model is closer to clinical POI.

[0067] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0068] Example 1. Chronic unpredictable stress for 8 weeks caused estrous cycle disorders in C57BL / 6 mice, but did not cause POI, and they were still in the compensatory period.

[0069] After adaptively raising 26 six-month-old female C57BL / 6 mice, they were randomly divided into two groups, namely the control group (normal raising without stress) and the CUS group. In the CUS group, 1 - 2 stressors were applied daily for 8 weeks of modeling. In the last two weeks of modeling (2 - 3 estrous cycles), vaginal cell smears were observed daily, and the estrous cycle of the mice was judged by observing the proportion of the number of white blood cells, nucleated epithelial cells, and anucleated squamous epithelial cells in the field of view through HE staining. Figure 1 A, 1B, 1C, and 1D are respectively the example diagrams of the stained vaginal cell smears of mice in the proestrus, estrus, metestrus, and diestrus phases. The estrous cycle changes of the mice in each group were statistically analyzed for 14 days. It was found that the estrous cycle of the mice in the CUS group was prolonged, and the proportion of disorders was significantly higher than that of the control group. The proportion of each period in the estrous cycle was also significantly disordered ( Figure 1 E, 1F, and 1G). However, the increase in AMH level indicates an increase in the number of antral follicles, and the increase in E2 level also indicates that the ovary compensatorily stimulates follicle development by secreting more E2 at this time ( Figure 2 A, 2B).

[0070] The above results show that 8 weeks of chronic unpredictable stress did not directly induce POI in mice, and they were still in the compensatory period.

[0071] The average number of days of the estrous cycle in each group is shown in Table 3.

[0072] Table 3

[0073]

[0074] The concentrations of AMH and E2 are shown in Table 4.

[0075] Table 4

[0076]

[0077] Example 2: 12 weeks of chronic unpredictable stress led to a significant decline in ovarian reserve in C57BL / 6 mice, and they were in the POI period.

[0078] After adaptively raising 40 six-month-old female C57BL / 6 mice, they were randomly divided into two groups, namely the control group (normal raising without stress) and the CUS group. In the modeling group, 1 - 2 stressors were applied daily. The mice were weighed once every 7 days during the modeling period, and the modeling duration was 12 weeks. After sacrificing the mice, the ovaries were dissected, and the number of follicles at all levels in the ovaries was counted through HE staining and serial tissue sectioning. The results are as Figure 3 shown in A, 3B, and 3C. The number of growing follicles in the CUS group was significantly lower than that in the control group, indicating that 12 weeks of chronic unpredictable stress had led to a decrease in the number of antral follicles, presenting an obvious POI phenotype.

[0079] The number of follicles at each level in each group is shown in Table 5.

[0080] Table 5

[0081]

[0082] Example 3: 12 weeks of chronic unpredictable stress led to ovarian atrophy in mice, a decrease in sex hormone levels, and the mice were in the POI period.

[0083] The results of body weight and ovarian weight changes in the control group and the CUS group are as Figure 4 shown in Figures A, 4B, 4C. The body weight of the mice in the model group was significantly lower than that of the control group, and the ovarian weight and ovarian index of the mice in the model group were also significantly decreased compared with those of the control group. This indicates that 12 weeks of chronic unpredictable stress led to obvious ovarian atrophy in mice.

[0084] The body weight changes are shown in Table 6.

[0085] Table 6

[0086]

[0087]

[0088] The ovarian weight changes are shown in Table 7.

[0089] Table 7

[0090]

[0091] Mouse hormone detection method

[0092] Centrifuge the mouse blood samples at 1500 r / min for 20 min, take the upper serum, and store it in a -20°C refrigerator. According to the instructions of the hormone ELISA kit, measure the concentrations of AMH, E2, FSH, and LH. The detection sensitivities are 10 pg / mL, 1.0 pmol / L, 1.0 mIU / mL, and 1.0 IU / L respectively.

[0093] The hormone detection results are as Figure 5 shown in Figures A, 5B, 5C, 5D. Compared with the control group, the levels of AMH and E2 in the CUS group were significantly decreased, indicating insufficient ovarian reserve. The decrease in the levels of FSH and LH suggested that stress might lead to abnormal function of the hypothalamic-pituitary axis. Overall, it was reflected that 12 weeks of chronic unpredictable stress led to ovarian insufficiency in mice.

[0094] The concentrations of AMH, E2, FSH, and LH in each group are shown in Table 8.

[0095] Table 8

[0096]

[0097] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An animal model of premature ovarian insufficiency (POI), characterized in that: The POI animal model has a compensatory period. The compensatory phase includes characteristics selected from the group consisting of: (a) Disorders of estrus cycle; (b) Increased levels of sex hormones; (c) No decrease in follicle number; and (d) No ovarian atrophy occurs.

2. The POI animal model according to claim 1, characterized in that: The estrus cycle disorder is selected from the following group: prolonged estrus cycle and / or disordered proportions of various periods in the estrus cycle.

3. The POI animal model according to claim 1, wherein: The sex hormone is selected from the group consisting of anti-Mullerian hormone (AMH), estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), or a combination thereof.

4. The POI animal model according to claim 1, wherein: The ovarian atrophy is selected from the group consisting of decreased ovarian weight and / or atrophy of ovarian morphology and structure.

5. The POI animal model according to claim 1, wherein: The POI comprises a feature selected from the group consisting of: (A) Disordered estrous cycle; (B) Decreased levels of sex hormones; (C) Decreased number of ovarian follicles; and / or (D) Ovarian atrophy.

6. The POI animal model according to claim 1, wherein: The POI animal model is used to analyze the biological characteristics and molecular regulatory networks of the early stages of POI, study the molecular mechanisms of stress-induced ovarian dysfunction, establish early warning indicators, and / or formulate precise intervention strategies based on pathological processes.

7. A method for constructing a POI animal model as claimed in claim 1, characterized in that: The method comprises the steps of: (i) Screening experimental animals for adaptive breeding; (ii) subjecting experimental animals to chronic unpredictable stress; and (iii) Obtaining POI animal model.

8. The method according to claim 7, characterized in that The source of chronic unpredictable stress is selected from the group consisting of acoustic and optical stimulation, food deprivation, water deprivation, social crowding, social isolation, dirty cages, wet cages, empty bedding, tail suspension, restraint stress, or a combination thereof.

9. The method according to claim 7, characterized in that The acoustic-optical-electric stimulation is selected from the group consisting of noise disturbance, night lighting, strobe lighting, foot shock, or a combination thereof.

10. The method according to claim 7, characterized in that The step (ii) further comprises: randomly using 1 to 3 stress sources for stimulation every day.

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