Method for detecting reproductive toxicity of environmental pollutants based on mouse in-vitro fertilization
By adding different concentrations of triclosan to the mouse in vitro fertilization system, the development of fertilized eggs was observed, and the problem of difficulty in simulating the long-term effects of environmental pollutants in the existing technology was solved, and effective detection of reproductive toxicity was achieved.
Patent Information
- Application Number
- CN202411510512.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art is difficult to simulate the long-term effects of environmental pollutants on the reproductive system, and the strict requirements of the in vitro fertilization operating environment and the cumbersome experiments have led to increased detection difficulty.
Using a method based on in vitro fertilization in mice, the development and morphological changes of fertilized eggs were observed by adding different concentrations of environmental pollutant triclosan to the in vitro fertilization culture medium, and the fertilization rate, cell development rate and blastocyst development rate were counted to detect reproductive toxicity.
It can effectively simulate long-term effects, significantly reduce fertilization and development rates by changing the impact of triclosan concentration on the fertilization system, thereby providing a new method to study the reproductive toxicity of environmental pollutants.
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Figure CN119506388A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of reproductive toxicity detection methods, in particular to a method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization. Background Art
[0002] As society develops, the additives in food, medicine and cleaning products have increased, environmental pollution has intensified, and pollutants are transmitted through the food chain, posing a threat to human health. People are exposed to particulate pollutants, heavy metals, food packaging compounds and nanomaterials every day. After these substances are absorbed by the intestines, they affect the whole body, including the reproductive system, through blood circulation. The impact of pollutants is particularly significant for men preparing for pregnancy and women during pregnancy.
[0003] In vitro fertilization (IVF) is an assisted reproductive technology in which sperm or eggs are removed from the body, processed or cultured into embryos, and then implanted into the mother's body. In vivo, the embryo is in a low-oxygen, light-free, constant temperature and humidity microenvironment, and develops in an environment protected by the mother. However, in vitro fertilization is an operation that places the embryo in an in vitro environment. When the embryo responds to changes in temperature, humidity, pH and osmotic pressure, its own developmental potential is weakened. Therefore, the requirements for the in vitro fertilization operating environment must be strict. At present, environmental pollutant toxicity detection experiments generally use mouse embryo experiments. Mouse embryo experiments not only require the breeding of mice, but also require ovulation induction. The process is cumbersome, especially for reproductive centers that do not have animal rooms.
[0004] The models currently used to study the toxicity of substances include 2D cell models, animal models, and organoid models. However, 2D cell models have single cells, animal models are subject to many restrictions such as ethics and time, and organoids lack integrity: organoid models can usually only simulate the structure and function of a specific organ or tissue. Environmental pollutants may undergo metabolic transformation, distribution, and excretion in real organisms, and organoid models are often unable to simulate these systemic effects. In addition, different organoid models differ in terms of cell sources and culture conditions, which affects the repeatability and comparability of experimental results; lack of long-term effects, organoid research often focuses on toxic effects under short-term exposure, while environmental pollutants may have long-term and chronic effects on organisms, and organoid models are difficult to simulate such long-term effects.
[0005] Reproductive organs contain key reproductive cell types, including sperm and oocytes, which can reproduce the in vivo reproductive process in vitro. Active sperm and oocytes located in reproductive organs can be fertilized and developed in vitro, and can reproduce the function of in vivo fertilization. The application of reproductive organs in toxicology is mainly to study the toxicity of small molecule compounds and drugs. Fertilizing sperm and oocytes in vitro and directly exposing them to the environmental pollutant triclosan makes it easier to study the toxicity of triclosan using in vitro fertilization. Summary of the invention
[0006] The present invention provides a method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization, so as to solve the problem that it is difficult to simulate long-term effects in the prior art methods for detecting reproductive toxicity of environmental pollutants.
[0007] In order to achieve the above object, the technical solution adopted by the present invention is: A method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization, comprising the following steps: Step 1, obtaining oocytes of female mice and sperms of male mice, preparing multiple in vitro fertilization culture dishes, and adding different concentrations of environmental pollutants to the culture solution in each in vitro fertilization culture dish; Step 2, placing the oocyte in the culture medium of each IVF culture dish after adding the environmental pollutant, and then adding sperm to the culture medium of each IVF culture dish with the oocyte, thereby fertilizing in each IVF culture dish; Step 3: Cultivate the fertilized eggs in each IVF culture dish, observe the development of the fertilized eggs in each IVF culture dish and the morphological changes of the fertilized eggs, and count the fertilization rate, cell development rate, and blastocyst development rate of the fertilized eggs in each IVF culture dish, thereby obtaining the reproductive toxicity test results.
[0008] In the further step 1, the oocyte is an oocyte isolated from a female mouse after superovulation.
[0009] In the further step 1, the sperm is obtained from the tail of the epididymis of male mice.
[0010] In the further step 1, the culture medium in each IVF culture dish is HTF covered with mineral oil.
[0011] In the further step 1, the contaminant is triclosan.
[0012] In a further step 2, the sperm is activated and capacitated before being added to the culture medium of the in vitro fertilization culture dish containing the oocytes.
[0013] In the further step 3, the statistical cell development rates include 4-cell development rate and 8-cell development rate.
[0014] Compared with the prior art, the present invention has the following advantages: The invention uses triclosan, an environmental pollutant, to be fully mixed with an in vitro fertilization medium, and then performs fertilization and culture of sperm and oocytes, so that triclosan directly acts on the fertilization of sperm and oocytes; the fertilization and culture methods of sperm and oocytes are determined by setting different concentrations of triclosan, and as the concentration of the environmental pollutant triclosan increases, the morphology and activity of sperm in the fertilization system begin to decrease, so that the fertilization rate, the 4-cell development rate, the 8-cell development rate, and the blastocyst development rate are seriously decreased, and the long-term effect can be simulated, thereby providing a new method for studying the toxicity of the environmental pollutant triclosan in vitro. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 1 is a diagram of the process of embryo development from 2 cells to blastocyst according to an embodiment of the present invention, wherein (a) shows the embryo developing to the pronuclear state, (b) shows the embryo developing to 2 cells, (c) shows the embryo developing to 4 cells, (d) shows the embryo developing to 8 cells, and (e) shows the embryo developing to blastocyst.
[0016] Figure 2 1 is the experimental operation process of the embodiment of the present invention, wherein (a) is the Untreated group, (b) is the Treated group, Figure 3 Graph showing the embryo development rates before and after TCS treatment according to an embodiment of the present invention.
[0017] Figure 4 Figure 2 is a graph showing embryonic development without and after TCS treatment according to an embodiment of the present invention. DETAILED DESCRIPTION
[0018] The present invention is further described below in conjunction with the accompanying drawings and embodiments.
[0019] This embodiment discloses a method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization, such as Figure 2 As shown, the following steps are included: Step 1: Obtain oocytes from female mice and sperm from male mice, prepare multiple in vitro fertilization culture dishes, and add different concentrations of environmental pollutants to the culture medium in each in vitro fertilization culture dish.
[0020] In this embodiment, a fallopian tube collection dish, a sperm capacitation dish, and four in vitro fertilization culture dishes were prepared. 200 µl of M2 covered with mineral oil was added to the fallopian tube collection dish as a culture medium; 200 µl of c-TYH covered with mineral oil was added to the sperm capacitation dish as a culture medium; and 200 µl of HTF covered with mineral oil was added to each in vitro fertilization culture dish as a culture medium. After the preparation, the fallopian tube collection dish, sperm capacitation dish, and in vitro fertilization culture dish were placed in a 37o C. Equilibrate in an incubator with 5% CO2 for at least 2 hours.
[0021] After equilibration, no environmental pollutants were added to the culture medium of the first IVF dish, 2µM environmental pollutants were added to the culture medium of the second IVF dish, 20µM environmental pollutants were added to the culture medium of the third IVF dish, and 200µM environmental pollutants were added to the culture medium of the fourth IVF dish. The environmental pollutants in the four IVF dishes were all triclosan.
[0022] In this embodiment, 4-8 weeks old female mice of C57BL / 6 strain were taken for superovulation, and 5-10 IU of PMSG was injected at 6 pm on the first day; and male mice of C57BL / 6 strain over 10 weeks were taken for co-cage. On the next morning, the male mice were taken out and raised in a single cage. And after 48 hours, the female mice injected with PMSG were injected with 5-10 IU of hCG for superovulation. Then, the oviduct of the female mouse after superovulation was taken and placed in a balanced oviduct collection dish, and the oocytes of the enlarged part were taken out under a microscope.
[0023] In this example, the cauda epididymis of a male mouse raised in a single cage was taken, and a hole was pierced in the cauda epididymis with an injection needle to allow sperm to flow out. The sperm that flowed out was placed in a equilibrated sperm capacitation dish and placed in a 37 o C. Activate and obtain energy in an incubator with 5% CO2 for 0.5-1h.
[0024] Step 2: Place the oocytes obtained in step 1 into four in vitro fertilization culture dishes that have been balanced and added with triclosan at corresponding concentrations. Also, add the capacitated sperm into each of the four in vitro fertilization culture dishes that already have oocytes and added with triclosan at corresponding concentrations. Thus, fertilization is performed in the four in vitro fertilization culture dishes.
[0025] Step 3: Add four IVF culture dishes to a 37 o C. Cultivate the fertilized eggs in an incubator with 5% CO2.
[0026] For each fertilized egg in the in vitro fertilization culture dish, they were cultured until E0.5 when male and female pronuclear fusion occurred, E1.5 when they grew into 2 cells, E2.0 when they grew into 4 cells, E2.5 when they grew into 8 cells, and E4.5 when they grew into mature blastocysts.
[0027] like Figure 1 As shown, the fertilization rate, 4-cell development rate, 8-cell development rate, and blastocyst development rate of the fertilized eggs in each in vitro fertilization culture dish were observed and counted, thereby obtaining the reproductive toxicity test results of triclosan at different concentrations.
[0028] Test results such as Figure 3 , 4 shown.
[0029] from Figure 3 It can be seen that both untreated and DMSO-treated fertilized eggs can develop normally to the 2-cell to blastocyst stage, with the development rates of 83.1% and 74.6% at the 2-cell stage and 62.8% and 49.9% at the blastocyst stage, respectively. There was no significant difference between the two groups. However, with the increase in TCS concentration, the blastocyst formation rate showed a downward trend, especially when the final TCS concentration was 200µM, the formation rate of 8cell and blastocyst was 0%. At 2μM, 20μM and 200μM doses, the 2-cell stage formation rate was significantly reduced, and the fertilization rates were 57.4%, 17.9% and 3.9%, respectively.
[0030] from Figure 4 It can be seen that different concentrations of TCS treatment affect the morphological changes of 2 cells. Fertilization is completed at E0.5, and sperm and oocytes form 2 cells at E1.5. Fertilized eggs treated with 20µM TCS show embryonic malformations (indicated by the arrows in the figure), and fertilized eggs treated with 200µM TCS show more death and embryonic malformations (indicated by the arrows in the figure).
[0031] The preferred embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. The embodiments described in the present invention are merely descriptions of the preferred embodiments of the present invention, and do not limit the concept and scope of the present invention. The various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction, and such combinations should also be regarded as the contents disclosed in the present disclosure as long as they do not violate the concept of the present invention. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.
[0032] The present invention is not limited to the specific details of the above-mentioned embodiments. Within the technical concept of the present invention and without departing from the design concept of the present invention, various modifications and improvements made to the technical solution of the present invention by technical personnel in this field should fall within the protection scope of the present invention. The technical contents for which protection is sought in the present invention have been fully recorded in the claims.
Claims
1. A method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization, characterized in that: The following steps are involved: Step 1, obtaining oocytes of female mice and sperms of male mice, preparing multiple in vitro fertilization culture dishes, and adding different concentrations of environmental pollutants to the culture solution in each in vitro fertilization culture dish; Step 2, placing the oocyte in the culture medium of each IVF culture dish after adding the environmental pollutant, and then adding sperm to the culture medium of each IVF culture dish with the oocyte, thereby fertilizing in each IVF culture dish; Step 3: Cultivate the fertilized eggs in each IVF culture dish, observe the development of the fertilized eggs in each IVF culture dish and the morphological changes of the fertilized eggs, and count the fertilization rate, cell development rate, and blastocyst development rate of the fertilized eggs in each IVF culture dish, thereby obtaining the reproductive toxicity test results.
2. The method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization according to claim 1, characterized in that: In step 1, the oocytes are oocytes separated from female mice after superovulation.
3. The method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization according to claim 1, characterized in that: In step 1, the sperm is obtained from the tail of the epididymis of male mice.
4. The method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization according to claim 1, characterized in that: In step 1, the culture medium in each IVF culture dish is HTF covered with mineral oil.
5. The method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization according to claim 1, characterized in that: In step 1, the contaminant is triclosan.
6. The method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization according to claim 1, characterized in that: In step 2, the sperm is activated and capacitated before being added to the culture medium of the in vitro fertilization culture dish containing the oocytes.
7. The method for detecting reproductive toxicity of environmental pollutants based on mouse in vitro fertilization according to claim 1, characterized in that: In step 3, the statistical cell development rates include 4-cell development rate and 8-cell development rate.