Application of epigallocatechin gallate in oocyte protection and oocyte protective agent

Abstract

The invention discloses application of epigallocatechin gallate in protection of oocytes and an oocyte protective agent, and relates to the technical field of biology. The invention finds that epigallocatechin gallate can play a role in protecting the oocytes through at least one way of protecting the meiosis process of the oocytes, reducing the oxidative stress level of the oocytes, relieving mitochondrial dysfunction of the oocytes, reducing apoptosis of the oocytes and improving the sperm binding capacity of the oocytes; therefore, the problem of damage of malathion exposure to reproductive systems of animals or human bodies is solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of epigallocatechin gallate in the protection of oocytes and an oocyte protectant. Background Technology

[0002] Malathion is a widely used organophosphate insecticide. Its mechanism of action is mainly through inhibiting acetylcholinesterase activity, leading to excessive accumulation of acetylcholine in nerve synapses, thereby causing dysfunction of the insect's nervous system and death.

[0003] However, malathion has potential reproductive toxicity, meaning that long-term or high-dose exposure to malathion in animals and humans can lead to damage to the reproductive system.

[0004] Currently, intervention strategies and remediation methods for reproductive toxicity caused by malathion are still very limited, and there is a lack of highly efficient and specific antidotes or bioremediation technologies. Summary of the Invention

[0005] The main objective of this invention is to propose the application of epigallocatechin gallate in the protection of oocytes and an oocyte protectant, aiming to solve the problem that exposure to malathion can cause damage to the reproductive system of animals or humans in the prior art.

[0006] To achieve the above objectives, this invention proposes the application of epigallocatechin gallate in the protection of oocytes.

[0007] In one embodiment, the epigallocatechin gallate exerts its protective effect on oocytes through at least one of the following pathways: A. Protects the meiotic process of oocytes; B. Reduce the level of oxidative stress in oocytes; C. Reduces mitochondrial dysfunction in oocytes; D. Reduces oocyte apoptosis; E. Improves the ability of oocytes to combine with sperm.

[0008] In one embodiment, reducing the level of oxidative stress in oocytes includes reducing the level of reactive oxygen species in oocytes.

[0009] In one embodiment, the oocyte is exposed to malathion.

[0010] In one embodiment, the concentration of malathion is 0.1~300 μM.

[0011] The present invention also provides an oocyte protectant, the oocyte protectant comprising epigallocatechin gallate.

[0012] In one embodiment, the concentration of epigallocatechin gallate in the oocyte protectant is 40-60 μM.

[0013] This invention discovers that epigallocatechin gallate can protect oocytes through at least one of the following pathways: protecting the oocyte meiotic process, reducing oocyte oxidative stress levels, alleviating oocyte mitochondrial dysfunction, reducing oocyte apoptosis, and improving oocyte spermatogenesis ability, thereby improving the damage to the reproductive system of animals or humans caused by malathion exposure. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0015] Figure 1 (A) is a micrograph of mouse cumulus-oocyte complex in Example 1 after being exposed to different concentrations of malathion and cultured for 14 hours, after the granulosa cells were removed; Figure 1 (B) is a graph showing the statistical results of the first polar body expulsion rate (PBE) of mouse cumulus-oocyte complexes exposed to different concentrations of malathion and cultured for 14 hours in Example 1. Figure 1 (C) is a micrograph of the cumulus-oocyte complex in Example 1 after exposure to 200 μM malathion and cultured for 14 hours with or without the addition of 50 μM epigallocatechin gallate, and after the removal of granulosa cells. Figure 1 (D) is a graph showing the statistical results of the first polar body discharge rate in different treatment groups in Example 1; Figure 2 Image (A) shows the staining of reactive oxygen species in oocytes from different treatment groups in Example 1; Figure 2 (B) shows the relative fluorescence intensity of reactive oxygen species in oocytes from different treatment groups in Example 1; Figure 3 (A) is a staining diagram of the mitochondrial membrane potential of oocytes from different treatment groups in Example 1; Figure 3 (B) represents the ratio of red to green fluorescence intensity of oocytes from different treatment groups in Example 1; Figure 4 (A) is a staining diagram of early apoptosis of oocytes in different treatment groups in Example 1; Figure 4(B) is a fluorescence intensity diagram of early apoptosis of oocytes in different treatment groups in Example 1; Figure 5 Image (A) shows the staining of the zona pellucida of oocytes in different treatment groups in Example 1, where sperm binds to the oocytes. Figure 5 (B) is a graph showing the average number of sperm-binding cells in oocytes of different treatment groups in Example 1.

[0016] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Malathion is a widely used organophosphate insecticide. Its mechanism of action is mainly through inhibiting acetylcholinesterase activity, leading to excessive accumulation of acetylcholine in nerve synapses, thereby causing dysfunction of the insect's nervous system and death.

[0019] However, malathion has potential reproductive toxicity, meaning that long-term or high-dose exposure to malathion in animals and humans can lead to damage to the reproductive system.

[0020] Currently, intervention strategies and remediation methods for reproductive toxicity caused by malathion are still very limited, and there is a lack of highly efficient and specific antidotes or bioremediation technologies.

[0021] In view of this, the present invention provides the application of epigallocatechin gallate in the protection of oocytes.

[0022] This invention discovers that epigallocatechin gallate can protect oocytes through at least one of the following pathways: protecting the oocyte meiotic process, reducing oocyte oxidative stress levels, alleviating oocyte mitochondrial dysfunction, reducing oocyte apoptosis, and improving oocyte spermatogenesis ability, thereby improving the damage to the reproductive system of animals or humans caused by malathion exposure.

[0023] In some embodiments, the epigallocatechin gallate exerts its protective effect on oocytes through at least one of the following pathways: A. protecting the oocyte meiotic process; B. reducing oocyte oxidative stress levels; C. alleviating oocyte mitochondrial dysfunction; D. reducing oocyte apoptosis; E. improving oocyte spermatogenesis. That is, when animals or humans are exposed to malathion, epigallocatechin gallate can be used to protect the normal functioning of oocytes, and this protective effect is exerted through at least one of pathways A, B, C, D, and E.

[0024] It is understood that, in some embodiments, reducing oocyte oxidative stress levels includes reducing the level of reactive oxygen species (ROS) in oocytes. That is, epigallocatechin gallate can reduce the oxidative stress level of oocytes by lowering the level of ROS, thereby protecting oocytes.

[0025] In some embodiments, the oocytes are exposed to malathion. That is, when oocytes are exposed to malathion, their reproductive functions, such as the expulsion of the first polar body, are impaired. In this case, epigallocatechin gallate can be used to protect the oocytes and restore some of their reproductive functions.

[0026] In some embodiments, the concentration of malathion is 0.1–300 μM. It is understood that the concentration of malathion can be 0.1 μM, 100 μM, 200 μM, or 300 μM, and within this concentration range, the functional impairment of oocytes caused by malathion can be partially or completely restored by intervention with epigallocatechin gallate.

[0027] This invention also provides an oocyte protectant comprising epigallocatechin gallate. Preferably, the concentration of epigallocatechin gallate in the oocyte protectant is 40-60 μM. That is, the concentration of epigallocatechin gallate in the oocyte protectant can be 40 μM, 50 μM, or 60 μM, and concentrations within this range can effectively protect oocytes.

[0028] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0029] Example 1 1. Epigallocatechin gallate (EGCG) has a restorative effect on meiotic maturation of mouse oocytes exposed to malathion (Mal). (1) First, the toxic effects of malathion exposure on mouse cumulus-oocyte complexes (COCs) were investigated: Cumulus-oocyte complexes were cultured for 14 hours in 0 μM (Control group), 100 μM, 200 μM, and 300 μM malathion medium, respectively. Granulocytes were then removed, and COCs were observed under a microscope (scale bar = 20 μm). The results are as follows: Figure 1 As shown in (A), the first polar body expulsion rate (PBE) was then observed and statistically analyzed, and the results are as follows. Figure 1 As shown in (B), Figure 1 Each data point in (B) is expressed as the mean ± SEM of at least three independent experiments, with significant differences in the mean between different letter labels (P<0.05). Granulosa cells here refer to cells with abnormal cell states; their removal is to facilitate observation of the oocyte maturation status.

[0030] It should be noted that extrusion of the first polar body is a marker of the oocyte completing its first meiotic division and is often used to assess its in vitro maturation capacity. Figure 1 (A) and Figure 1 Results (B) showed that the number of oocytes extruding the first polar body decreased with increasing malathion concentration, indicating that the meiotic maturation process was dose-dependently interfered with. Except for the control group, all other groups were malathion-exposed. The percentages of PBE in each group were: 84.26%±1.742% (Control group), 65.62%±2.453% (100μM Mal group), 56.31%±3.311% (200μM Mal group), and 48.77%±1.895% (300μM Mal group). The results showed that the first polar body extrusion rate was significantly lower in the malathion-exposed groups compared to the control group.

[0031] (2) Subsequently, a protection experiment was conducted using EGCG. 200 μM Mal was selected as the exposure group. To investigate whether EGCG could salvage meiotic failure induced by Mal, COCs exposed to 200 μM Mal were simultaneously cultured with 50 μM EGCG for 14 h. Granulosa cells were removed, and the expulsion rate of the first polar body was observed. The results are as follows: Figure 1 (C) and Figure 1 As shown in (D).

[0032] Figure 1 (C) and Figure 1 The results (D) showed that the PBE rate in the control group was 83.72% ± 1.901%; the PBE rate in the Mal group was 60.75% ± 3.951%; and the PBE rate in the "Mal + EGCG" group was 77.47% ± 2.518%. Analysis revealed that the PBE rate in the "Mal + EGCG" group was significantly higher than that in the 200 μM Mal exposure group (i.e.,... Figure 1 (C) and Figure 1 The results in the Mal group (D) were similar to those in the control group. Therefore, an appropriate amount of EGCG can effectively protect the meiotic process of Mal-damaged mouse oocytes.

[0033] 2. EGCG reduces ROS production in oocytes exposed to Mal. Mitochondrial dysfunction is accompanied by elevated intracellular oxidative stress levels. Therefore, DCFH fluorescent probes were used to stain oocytes from the Control group, Mal group (i.e., 200 μM Mal exposure group), and Mal+EGCG group. Green fluorescence was the signal of reactive oxygen species. The results are as follows: Figure 2 As shown in (1), the level of reactive oxygen species (ROS) in oocytes was assessed by relative fluorescence intensity, and the results are as follows. Figure 2 As shown in (B).

[0034] Depend on Figure 2 (A) and Figure 2 As shown in Figure (B), the green fluorescence intensity of oocytes in the Control group was weak, indicating a low level of ROS in the oocytes, with a relative fluorescence intensity of 14.731% ± 1.825%. However, the green fluorescence intensity of oocytes in the Mal group was significantly increased, indicating a high level of ROS in the oocytes, with a relative fluorescence intensity of 44.458% ± 3.174%. In contrast, the green fluorescence intensity of oocytes in the Mal+EGCG group was significantly decreased, indicating a significant reduction in the high ROS levels exposed to Mal oocytes, with a relative fluorescence intensity of 18.651% ± 1.914%. In conclusion, EGCG can reduce the oxidative stress level of aging oocytes.

[0035] 3. EGCG can restore mitochondrial dysfunction in oocytes exposed to Mal cells. To verify the effect of EGCG on mitochondrial function in oocytes exposed to Mal, the mitochondrial membrane potential (ΔΨm) of oocytes in the Control group, Mal group (i.e., 200 μM Mal exposure group), and Mal+EGCG group was detected using JC-1 staining, and microscopic observation was performed. The results are as follows: Figure 3As shown in (A), scale bar = 100 μm. A higher mitochondrial membrane potential indicates a stronger mitochondrial function, while a lower membrane potential indicates a weaker function. Mitochondria with high membrane potential typically exhibit red fluorescence, while those with low membrane potential exhibit green fluorescence. The ratio of red to green fluorescence was used to assess the mitochondrial membrane potential level in oocytes, and the results are shown in Figure 1. Figure 3 As shown in (B).

[0036] Depend on Figure 3 (A) and Figure 3 As shown in Figure (B), oocytes in the Control group exhibited red fluorescence with a high ratio of red to green fluorescence intensity (1.3904% ± 0.194%). Oocytes in the Mal group showed strong green fluorescence with a significantly decreased ratio of red to green fluorescence intensity (0.6471 ± 0.082%). After EGCG addition, oocytes showed weaker green fluorescence with an increased ratio of red to green fluorescence intensity (1.25% ± 0.137%). In conclusion, these results indicate that EGCG addition can alleviate mitochondrial dysfunction caused by oocyte aging.

[0037] 4. EGCG can reduce apoptosis in oocytes exposed to malathion. Since increased ROS levels in oocytes exposed to Mal accelerate oocyte apoptosis, oocyte apoptosis in the Control group, Mal group (i.e., 200 μM Mal exposure group), and Mal+EGCG group was assessed by Annexin V staining. Microscopic observation images are shown below. Figure 4 As shown in (A), the scale bar is 50 μm, and green represents the Annexin V signal; the fluorescence intensity was analyzed, and the results are as follows. Figure 4 As shown in (B). It should be noted that Annexin V staining is achieved by Annexin V specifically binding to phosphatidylserine residues that have everted to the cell membrane surface of apoptotic cells. Annexin V can also bind to dyes, thereby exposing apoptotic oocytes by detecting fluorescence intensity.

[0038] Depend on Figure 4 (A) and Figure 4As shown in Figure (B), a weak green fluorescence signal was detected on the oocyte membrane in the Control group, with a fluorescence intensity of 9.381% ± 0.82%. The green fluorescence signal on the oocyte membrane in the Mal group was significantly enhanced, with a fluorescence intensity of 20.227% ± 3.891%. However, the green fluorescence signal on the oocyte membrane in the Mal+EGCG group was significantly reduced, with a fluorescence intensity of 20.227% ± 3.891%. In conclusion, the addition of EGCG can reduce oocyte apoptosis by clearing excess ROS accumulated in exposed oocytes.

[0039] 5. EGCG enhances sperm binding ability in oocytes exposed to Mal. The ability of sperm to bind to the zona pellucida (ZP) is an important indicator for assessing the fertilization potential of oocytes. To investigate whether EGCG can improve the sperm-oocyte binding ability of oocytes exposed to Mal (maleic acid), sperm-oocyte binding assays were performed on oocytes from the Control group, Mal group (i.e., the 200 μM Mal exposure group), and Mal+EGCG group. DNA in the sperm head was stained with DAPI and observed under a microscope. The results are as follows: Figure 5 As shown in (A), scale bar = 25 μm; and the average number of sperm cells bound to the oocyte was counted, the results are as follows. Figure 5 As shown in (B).

[0040] Depend on Figure 5 (A) and Figure 5 As shown in Figure (B), in the Control group oocytes, ZP supported the binding of many sperm, with a sperm binding rate of 183.531% ± 5.481%. However, in the Mal group oocytes, the number of sperm bound by ZP was significantly reduced, with a sperm binding rate of 35.667% ± 4.935%. But in the Mal+EGCG group oocytes, the number of sperm bound by ZP was significantly increased, with a sperm binding rate of 144.67% ± 5.367%. In conclusion, adding EGCG can salvage the sperm binding capacity damaged in Mal oocytes.

[0041] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. Application of an epigallocatechin gallate in the protection of oocytes.

2. The application as described in claim 1, characterized in that, The epigallocatechin gallate exerts its protective effect on oocytes through at least one of the following pathways: A. Protects the meiotic process of oocytes; B. Reduce the level of oxidative stress in oocytes; C. Reduces mitochondrial dysfunction in oocytes; D. Reduces oocyte apoptosis; E. Improves the ability of oocytes to combine with sperm.

3. The application as described in claim 2, characterized in that, The reduction of oocyte oxidative stress levels includes reducing the level of reactive oxygen species in oocytes.

4. The application as described in claim 1, characterized in that, The oocytes were exposed to malathion.

5. The application as described in claim 4, characterized in that, The concentration of malathion is 0.1~300 μM.

6. An oocyte protectant, characterized in that, The oocyte protectant includes epigallocatechin gallate.

7. The oocyte protectant as described in claim 6, characterized in that, In the oocyte protectant, the concentration of epigallocatechin gallate is 40-60 μM.

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