CN110724725A - Method for evaluating influence of smoking on oocyte quality - Google Patents

Method for evaluating influence of smoking on oocyte quality Download PDF

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CN110724725A
CN110724725A CN201911042740.0A CN201911042740A CN110724725A CN 110724725 A CN110724725 A CN 110724725A CN 201911042740 A CN201911042740 A CN 201911042740A CN 110724725 A CN110724725 A CN 110724725A
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CN110724725B (en
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程金妹
孙斐
陈爱春
范晓博
米盼盼
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Abstract

The invention discloses an evaluation method for the influence of smoking on oocyte quality, and particularly relates to mature culture of oocytes in culture solutions containing cotinine with different concentrations. And finally, evaluating the influence of smoking on the quality of the oocyte by detecting the indexes of the oocyte maturation rate, the chromosome aneuploidy, the actin distribution, the mitochondrial membrane potential, the intracellular reactive oxygen level, the spindle body shape, the chromosome arrangement shape of the mature oocyte, the combined number of sperms after in vitro fertilization, the pronucleus formation rate after parthenogenetic activation and the like. According to the invention, the influence of smoking on the quality of the oocyte is evaluated through a large number of objective tests, and a reference of theoretical data is provided for maintaining the reproductive performance of smoking women.

Description

Method for evaluating influence of smoking on oocyte quality
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an evaluation method for influence of smoking on oocyte quality.
Background
According to the report of "sunshine journal": of 11 hundred million smokers worldwide, our country accounts for 3.3 hundred million, and our country dies about 100 million people who suffer from smoking-related diseases each year, and the rest smokers are in sub-health status. It has been reported that smoking can reduce female reproductive capacity. In addition, if a woman smokes during pregnancy, the fetus is exposed to a higher risk of spontaneous abortion and perinatal death, significantly reducing the fetal birth weight. However, no relevant research has been reported on whether smoking has an effect on reproductive systems, particularly on oocyte quality.
The main cause of cigarette addiction is its nicotine component. Cotinine (cotinine) is reported to be a product of nicotine in tobacco after being metabolized by cytochrome oxidase 2a6(CYP2a6) in vivo, i.e., a main metabolite and a metabolite precursor of nicotine in vivo, and exists in plasma mainly in the form of plasma. Cotinine has the function of promoting nervous system excitation, and reflects certain anti-inflammatory and pulmonary edema-relieving effects in certain murine experiments. Cotinine has a long half-life (3-4 days) and is stable, and therefore, cotinine is a main biomarker for measuring smoking amount of smokers and passive smokers, and is generally evaluated by the concentration of cotinine in serum. Recent research results show that the cotinine concentration in plasma and the cotinine concentration in serum have consistency and also have detection significance. Kendall V et al found that when 8mM cotinine was added to the mouse early embryo culture medium, the rate of blastocyst formation in mice was significantly reduced; however, when less than 8mM cotinine was added to the mouse embryo culture solution, there was no significant difference in the blastocyst formation rate compared to the normal control group. There is currently no relevant study on the effect of cotinine on oocyte quality.
In the prior art, the correlation research of cotinine and oocyte quality is not carried out, the cotinine with different concentrations is added into an oocyte in-vitro maturation culture solution to directly evaluate the influence of smoking on the oocyte quality, and further evaluate the smoking amount of smokers and passive smokers, which causes the oocyte quality to be reduced, and finally leads to the reproductive capacity to be weakened. The invention provides data reference for the smoking female to maintain the oocyte quality and reproductive capacity.
Disclosure of Invention
Aiming at the research that the correlation between cotinine and oocyte quality does not exist in the prior art, the invention provides an evaluation method for evaluating the influence of smoking on the oocyte quality, which directly evaluates the influence of smoking on the oocyte quality by adding cotinine with different concentrations into an oocyte in-vitro maturation culture solution, and further evaluates the reduction of the oocyte quality and finally the reduction of reproductive capacity caused by the reduction of smoking amount of smokers and passive smokers.
In order to achieve the purpose, the invention adopts the following technical scheme:
an evaluation method for the influence of smoking on oocyte quality specifically comprises the steps of adding cotinine solutions with different concentrations into an oocyte in-vitro maturation culture solution, and detecting the quality of in-vitro maturation oocytes.
Further, the oocytes were taken from CD1 mice.
Further, cotinine is (-) -cotinine.
Further, the cotinine solution is prepared by using DMSO (dimethyl sulfoxide).
Further, the parameters for evaluating the quality of the in vitro matured oocyte comprise: oocyte maturation rate, mature oocyte Reactive Oxygen Species (ROS) level, mitochondrial membrane potential, chromosome aneuploidy, actin (alpha-actin) distribution expression, spindle body morphology, pronucleus rate after parthenogenetic activation, number of in vitro fertilization combined sperm and arrangement of chromosomes.
Further, the concentration of cotinine in the solution with different cotinine concentrations is 0-1000 mu mol/L.
Further, the concentration of cotinine in the solution with different cotinine concentrations is 0 mu mol/L, 100 mu mol/L, 500 mu mol/L, 800 mu mol/L and 1000 mu mol/L.
The evaluation method of the invention can be used for obtaining: (1) when cotinine is added into the in vitro maturation culture solution to reach 500 mu mol/L, the ROS level, chromosome aneuploidy and chromosome misarrangement in the mature oocyte can be obviously increased, and the mitochondrial membrane potential is obviously reduced. However, the oocyte maturation rate, the MII stage oocyte actin, the spindle body shape, the pronucleus rate after parthenogenetic activation and the number of sperms combined with in vitro fertilization have no significant difference from the normal control group.
(2) When the cotinine concentration in the in vitro maturation culture solution is 800 mu mol/L and 1000 mu mol/L, compared with a normal control group, the oocyte maturation rate, the mitochondrial membrane potential and the pronucleus rate after parthenogenetic activation are obviously reduced, and actin distribution disorder and spindle morphology abnormality around the mature oocytes are realized. In addition, there was a significant increase in ROS levels, chromosomal aneuploidies and chromosomal misalignments in oocytes after maturation.
Has the advantages that:
animal experiments prove that the method can be used for evaluating the influence of smoking on the quality of the oocyte and providing data reference of smoking amount for active smoking and passive smoking women when the women want to maintain the quality and reproductive capacity of the oocyte.
Drawings
FIG. 1 is a graph showing the effect of cotinine at different concentrations in example 1 on the distribution of α -actin protein and spindle morphology in mature oocytes in vitro.
FIG. 2 is a graph showing the effect of different concentrations of cotinine in example 1 on mitochondrial membrane potential and ROS levels in mature oocytes in vitro.
FIG. 3 is a graph showing the effect of various concentrations of cotinine on the number of sperm bound to mature oocytes in vitro in example 2.
FIG. 4 is a graph showing the effect of different concentrations of cotinine on the expression of different genes in mature oocytes in vitro as in example 3.
The significance plotted in figures 1-4 is the significance compared to the 0 μ M group; in fig. 1-4, different lower case letters indicate significant difference (P <0.05) and different upper case letters indicate significant difference (P < 0.01).
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The reagents and materials used in the following examples are all commercially available. Wherein, the purity of cotinine is required to reach 99.8 percent of concentration.
The animals used in the examples below were CD1 female mice 8-10 weeks old, purchased from the university of Nantong, laboratory animal center, and were fed and drunk freely. Controlling light (illumination for 12h/d) and controlling the indoor temperature to be 20-22 ℃.
The method for collecting oocytes in the germinal vesicle stage (GV stage) was as follows:
8-10 weeks old CD1 female mice were injected intraperitoneally with PMSG (10IU) at intervals of 46h, cervical dislocation was performed for sacrifice, and ovarian tissue was removed. The ovaries were placed in a pre-warmed M2 solution containing 50. mu. mol/L IBMX at 37 ℃ and the ovaries were shredded using a 1mL syringe needle and 90-120 μ M morphologically normal GV oocytes were collected using a pipette and glass needle. The operation was carried out at room temperature of 25 ℃ on a thermostatic table at 37 ℃.
Example 1
Effect of cotinine on oocyte quality
Drops of in vitro maturation medium containing 0, 100, 500, 800 and 1000. mu. mol/L cotinine were first prepared and incubated at 37 ℃ with 5% CO2The cell incubator was pre-equilibrated for 2 hours. Then 30-35 collected GV-stage oocytes were placed in each droplet and cultured in an incubator. After culturing for 16h, the oocyte maturation rate, i.e. the first polar body expulsion rate, was counted. Results are shown in table 1:
TABLE 1 Effect of different concentrations of cotinine on oocyte in vitro development and quality
Figure BDA0002253306220000031
Note: the difference is significant when the table is shouldered with different lower case letters (P <0.05) and the difference is significant when the table is marked with different upper case letters (P < 0.01). All groups were compared in the 0 μ M group to give significance.
As can be seen from the above table, when cotinine is added to the in vitro maturation solution at a concentration of less than 800. mu.M, the maturation rate of the oocytes in mice is not significantly affected as compared to the normal control group (0. mu.M). However, when the in vitro mature cotinine concentration reaches 800. mu. mol/L and 1000. mu. mol/L, the mouse oocyte maturation rate is significantly reduced.
The first polar body-depleted mouse oocytes were collected and transferred to freshly prepared 2% paraformaldehyde for 20min, then infiltrated with 0.15% Triton for 15min, blocked with 0.3% BSA for 15min, incubated primary antibody (α -actin and β -tubulin) for 1 hour at 37 ℃, washed 3 times with phosphate buffer for 5min each, then incubated secondary antibody for 1 hour at room temperature, washed 3 times with phosphate buffer for 5min each, DAPI stained, and placed under a fluorescence microscope for image acquisition. As shown in FIG. 1, when cotinine concentration in the in vitro maturation solution reached 800. mu. mol/L, the α -actin distribution (FIG. 1B) and β -tubulin morphology (FIG. 1C) of the in vitro matured oocytes were abnormal. Wherein, FIG. 1A is a representative picture of the expression level of alpha-actin in vitro mature oocytes in the 0. mu. mol/L cotinine and 800. mu. mol/Lcotinine groups, and a representative picture of the form of a spindle body after beta-tubulin staining in the 0. mu. mol/Lcotinine group, respectively.
Chromosome aneuploidy, mitochondrial membrane potential, metaphase alignment of chromosomes, and intracellular ROS level are one of the important indicators for oocyte quality evaluation. Through test methods such as a chromosome spreading technology, JC1 dye, an immunofluorescence staining technology, an ROS kit and the like, the chromosome aneuploidy (table 1), the metaphase chromosome alignment error ratio (table 1) and the intracellular ROS level of a mature oocyte are remarkably increased (figure 2B) and the mitochondrial membrane potential is remarkably reduced (figure 2A) when the concentration of cotinine in-vitro cell maturation liquid reaches 500 mu mol/L. And as the concentration of cotinine in the in vitro maturation solution increased (at 800. mu. mol/L and 1000. mu. mol/L), the chromosomal aneuploidy (Table 1), metaphase chromosomal misarrangement ratio (Table 1) and intracellular ROS levels gradually increased (FIG. 2B) and mitochondrial membrane potential gradually decreased (FIG. 2A) in the mature oocytes.
Example 2
Effect of cotinine on the in vitro fertilization Capacity of mature oocytes
In order to visually evaluate the quality of the in vitro matured oocytes, the matured oocytes were first subjected to in vitro fertilization with mouse sperm and tested for how many sperm the matured oocytes could bind to.
The specific method comprises the following steps: (1) killing 12-13 weeks old male mice by dislocation of cervical vertebrae, taking out epididymis tail with small scissors, cutting several cracks, releasing sperm in vitro insemination solution, placing at 37 deg.C with 5% CO2Culturing in cell culture box, taking 10 after 30 minutes6Placing sperm into drop containing mature oocyte at 37 deg.C and 5% CO2Culturing in a cell culture box for 1 hour, fixing, permeating, DAPI staining, and counting the number of sperms on the combined mature oocytes under a fluorescence microscope.
(2) Mature oocytes are placed into parthenogenetic activating solution A (in-vitro fertilization fluid containing 100 mu M strontium chloride and 5mg/L cytochalasin D) for 2.5h, the mature oocytes are transferred into parthenogenetic activating solution B (in-vitro fertilization fluid containing 5mg/L cytochalasin D) by using a glass needle for 3.5h, and then the pronucleus rate is counted.
The results show that the ability of the mature oocytes to bind sperm is not significantly reduced with the increase of cotinine concentration in the in vitro maturation solution (fig. 3), but the rate of pronuclei formation after parthenogenetic activation is significantly reduced (table 1).
Example 3
Effect of cotinine on different levels of Gene expression in mature oocytes
In order to explore the molecular mechanism of cotinine causing in-vitro mature oocyte quality reduction, the fluorescent quantitative PCR technology is adopted, 50 mature oocytes in each group are subjected to RNA extraction and are subjected to reverse transcription to form CDNA, and the relative expression quantity of antioxidant-related genes (sod1, sod2, glrx2 and GPX1) and apoptosis-related genes (p53, BAX and BCL-1) in each group is detected through SYBR Green fluorescent dye, wherein GADPH serves as an internal reference gene.
The results are shown in figure 4, the expression quantity of the anti-oxidation related genes is gradually reduced along with the increase of the cotinine concentration in the in vitro maturation liquid, and when the cotinine concentration reaches 800 mu mol/L and 1000 mu mol/L, the genes are significantly different and extremely significantly reduced compared with the cotinine-free group. Meanwhile, the expression quantity of the genes related to apoptosis is gradually increased along with the increase of cotinine concentration in-vitro maturation liquid, wherein when the cotinine concentration reaches 800 mu mol/L and 1000 mu mol/L, the genes are remarkably different and remarkably increased compared with a cotinine group without adding cotinine.
Although only the effect of smoking on the quality of mouse oocytes is evaluated in the above embodiments, the process and mechanism of maturation of human oocytes are very similar to those of mice, and the method is also applicable to the evaluation of the effect of smoking on the quality of human oocytes, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should be considered as the protection scope of the present invention.

Claims (7)

1. A method for evaluating the influence of smoking on oocyte quality is characterized in that: and adding cotinine solutions with different concentrations into the oocyte in-vitro maturation culture solution, and detecting the quality of the in-vitro maturation oocyte.
2. The evaluation method according to claim 1, wherein: the oocytes were taken from CD1 mice.
3. The evaluation method according to claim 1, wherein: the cotinine is (-) -cotinine.
4. The evaluation method according to claim 1, wherein: the cotinine solution is prepared by DMSO.
5. The evaluation method according to claim 1, wherein: the concentration of cotinine in the cotinine solution with different concentrations is 0-1000 mu mol/L.
6. The evaluation method according to claim 5, wherein: the concentration of cotinine in the cotinine solution with different concentrations is 0 mu mol/L, 100 mu mol/L, 500 mu mol/L, 800 mu mol/L and 1000 mu mol/L.
7. The evaluation method according to claim 1, wherein: the evaluation parameters of the in vitro mature oocyte quality comprise: oocyte maturation rate, mature oocyte active oxygen level, mitochondrial membrane potential, chromosome aneuploidy, actin distribution expression, spindle body morphology, pronucleus rate after parthenogenetic activation, number of sperm fertilized in vitro and chromosome arrangement.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111289424A (en) * 2020-03-04 2020-06-16 浙江星博生物科技股份有限公司 Method for detecting sperm mitochondrial membrane potential and active oxygen by double-standard method
CN113881623A (en) * 2021-10-08 2022-01-04 南开大学 Method for forming ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenetic embryo

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
XIAO-YU ZHANG ET AL: "Disruption of retinal pigment epithelial cell properties under the exposure of cotinine", 《SCIENTIFIC REPORTS》 *
唐媛媛: "香烟烟雾对雄性小鼠生殖能力的影响及男性不育的相关生物信息学研究", 《中国优秀硕士学位论文全文数据库（电子期刊）》 *
闫学花等: "黄芪多糖对小鼠卵母细胞体外成熟的影响", 《动物医学进展》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111289424A (en) * 2020-03-04 2020-06-16 浙江星博生物科技股份有限公司 Method for detecting sperm mitochondrial membrane potential and active oxygen by double-standard method
CN113881623A (en) * 2021-10-08 2022-01-04 南开大学 Method for forming ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenetic embryo
CN113881623B (en) * 2021-10-08 2022-11-22 南开大学 Method for forming ovum by in-vitro differentiation of parthenogenetic embryonic stem cells activated by parthenogenetic embryo

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