CN114223649A

CN114223649A - Application of anthocyanin in sperm cryopreservation and sperm cryopreservation liquid

Info

Publication number: CN114223649A
Application number: CN202111603229.0A
Authority: CN
Inventors: 白卫滨; 白顺; 李旭升; 陈嘉莉; 王晓涵; 蒋鑫炜; 蔡冬宝
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-03-25
Anticipated expiration: 2041-12-24
Also published as: CN114223649B

Abstract

The application belongs to the technical field of sperm preservation, and particularly relates to application of anthocyanin in sperm cryopreservation and sperm cryopreservation liquid. In a first aspect, the present application provides the use of an anthocyanin selected from one or more of cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargonidin-3-O-glucoside, malvidin-3-O-glucoside, peonidin-3-O-glucoside and morning gloin-3-O-glucoside in the cryopreservation of sperm in vitro. In a second aspect, the present application provides a sperm freezing medium comprising anthocyanin and a sperm freezing culture acceptable substance. The application provides the application of anthocyanin in sperm cryopreservation and the sperm cryopreservation liquid, and effectively solves the technical problems that the conventional frozen sperm is easy to generate oxidative stress in the unfreezing process to cause DNA fragmentation and activity reduction.

Description

Application of anthocyanin in sperm cryopreservation and sperm cryopreservation liquid

Technical Field

The application belongs to the technical field of sperm preservation, and particularly relates to application of anthocyanin in sperm cryopreservation and sperm cryopreservation liquid.

Background

Sperm cryopreservation began as early as half a century ago and developed after the discovery of glycerol as a supplement. Sperm cryopreservation has evolved over the past several decades as a routine practice for assisted reproductive technology therapy. Importantly, cryopreservation processes can lead to sperm damage, including excessive dehydration, plasma membrane disintegration, acrosome leakage, mitochondrial damage, and DNA fragmentation.

In human sperm freezing injury, rapid changes in temperature create chemical and physical stress on sperm during the freeze-thaw process and alter sperm membrane lipid composition, which is considered multifactorial. In addition, oxidative stress during cryopreservation of sperm can cause DNA damage. Physiological levels of sperm Reactive Oxygen Species (ROS) are required for normal sperm function, and excessive amounts of ROS can accumulate harmful substances, resulting in sperm damage. Therefore, reducing the oxidative stress of sperm plays an important role in preventing additional damage caused by cryopreservation.

Disclosure of Invention

In view of the above, the application provides an application of anthocyanin in sperm cryopreservation and a sperm cryopreservation liquid, and effectively solves the technical problems that the conventional frozen sperm is easy to generate oxidative stress in a thawing process to cause DNA fragmentation and activity reduction.

In a first aspect, the application provides the use of anthocyanins for the cryopreservation of sperm in vitro.

In another embodiment, the in vitro cryopreservation of sperm comprises: mixing the sperms with a sperm freezing culture medium, and then performing cryopreservation according to a sperm cryopreservation method; wherein the sperm freezing medium comprises anthocyanin and acceptable sperm freezing culture substances.

In another embodiment, the anthocyanin is one or more of cyanidin-3-O-glucoside (hereinafter referred to as C3G), delphinidin-3-O-glucoside, pelargonidin-3-O-glucoside, malvidin-3-O-glucoside, peonidin-3-O-glucoside and morning gloin-3-O-glucoside.

In another embodiment, the concentration of the anthocyanin in the sperm freezing medium is 50 to 200. mu.M/L.

In another embodiment, the concentration of the cyanidin-3-O-glucoside in the sperm freezing medium is in the range of 50 μ M/L to 200 μ M/L.

In another embodiment, the concentration of the anthocyanin in the sperm freezing medium is 50 to 100. mu.M/L.

In another embodiment, the concentration of the cyanidin-3-O-glucoside in the sperm freezing medium is between 50 μ M/L and 100 μ M/L.

In another embodiment, the concentration of the cyanidin-3-O-glucoside in the sperm freezing medium is 50 μ M/L.

In another embodiment, the sperm cell culture acceptable substance comprises one or more of human serum albumin, recombinant human insulin, and an antibiotic.

Specifically, the dosage of the human serum albumin, the recombinant human insulin and the antibiotic is the conventional dosage.

In another embodiment, the use comprises use of the anthocyanins for improving sperm survival and motility.

In another embodiment, said use comprises use of said anthocyanins in reducing the level of ROS in sperm, the rate of DNA fragmentation in sperm DFI and the sperm nucleus integrity HDS.

In a second aspect, the present application provides a sperm freezing medium comprising anthocyanin and a sperm freezing culture acceptable substance;

the anthocyanin is selected from one or more of cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargonidin-3-O-glucoside, malvidin-3-O-glucoside, Paeonin-3-O-glucoside and morning glory pigment-3-O-glucoside.

Sperm quality is negatively impacted due to processes of cryopreservation of sperm, including ice crystal formation, toxicity of cryoprotective media, and changes in sperm osmolarity, due to disruption of sperm membranes, reduction of mitochondrial oxidative phosphorylation, and excessive production of ROS. The release of ROS and the activity of antioxidant enzymes reduce sperm motility and induce sperm DNA damage. Aiming at the problems that the activity of sperms is reduced in the freezing and unfreezing processes in the prior art, the generation of sperm ROS and the defects of DNA fragments are increased, the new application of anthocyanin, especially C3G, in the frozen preservation of sperms is discovered, and C3G can be used as an antioxidant in the frozen preservation solution of the sperms, the C3G is added into a sperm freezing culture medium, the survival and forward movement proportion of the sperms can be effectively improved after the sperms are unfrozen, the ROS level of the sperms, the DNA fragment rate DFI of the sperms and the generation of HDS of the integrity of the sperms are reduced, and the C3G is an effective sperm cryoprotectant and can improve the oxidative stress in the low-temperature preservation process of the human sperms.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a graph showing a comparison of the in vitro survival rates of cryopreserved sperm cells at various concentrations of C3G; wherein the content of the first and second substances,^aP<0.05；^bP<0.05 compared to control (0. mu.M/L C3G);

FIG. 2 is a graph showing a comparison of the in vitro activity rates of various concentrations of C3G on cryopreserved sperm after thawing as provided in the examples herein; wherein the content of the first and second substances,^aP<0.05；^bP<0.05 compared to control (0. mu.M/L C3G);

FIG. 3 is a comparison of ROS in sperm cells after thawing cryopreserved sperm cells at various concentrations of C3G in vitro as provided in the examples herein; wherein the content of the first and second substances,^aP<0.05，^bP<0.05 and control (0. mu.M/L C3G);^cp in comparison with the addition of C3G (50. mu.M/L C3G), P<0.05；

FIG. 4 is a comparison of sperm DFI after thawing of cryopreserved sperm cells at various concentrations of C3G in vitro as provided in the examples herein; wherein the content of the first and second substances,^aP<0.05，^bP<0.05 and control (0. mu.M/L C3G);^cp in comparison with the addition of C3G (50. mu.M/L C3G), P<0.05；

FIG. 5 is a comparison of HDS of thawed sperm cells of different concentrations of C3G versus cryopreserved sperm cells in vitro, as provided in the examples herein; wherein the content of the first and second substances,^aP<0.05，^bP<0.05 and control (0. mu.M/L C3G);^cp in comparison with the addition of C3G (50. mu.M/L C3G), P<0.05。

Detailed Description

The application provides application of anthocyanin in sperm cryopreservation and sperm cryopreservation liquid, which are used for solving the technical defects that DNA fragmentation and activity reduction are caused by oxidative stress easily generated in the unfreezing process of the existing frozen sperm.

The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all 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 application.

The raw materials and reagents used in the following examples are commercially available or self-made.

In the following examples semen was obtained from 39 men seeking fertility diagnosis in the reproductive centre, the clinical characteristics and sperm quality of which are shown in table 1. Males who took any medication or antioxidant supplement 3 months prior to the trial were excluded. The experiments in the following examples were approved by the ethical committee of the first subsidiary hospital of the university of science and technology of china (2021-KY-040).

TABLE 1

Feature(s)	Mean. + -. standard deviation of
		Age (year)	31.2±5.5
Sexual desire (Tian)	5.2±4.9
		Semen volume (ml)	3.4±1.1
Sperm cell concentration (10)⁶mL^-1)	94.1±57.9
		Survival rate (%)	70.4±8.5
Forward motion (%)	44.6±8.3
		Normal form (%)	6.3±2.1
Sperm ROS (%)	2.6±2.8
		Sperm DNA Debris (DFI) (%)	7.8±6.2
Sperm nucleus integrity (HDS) (%)	7.7±2.6

In the following examples C3G was extracted from black bean skin according to the literature method of Jiang et al. The structure and purity of the product are identified by liquid chromatography-mass spectrometry (LC-MS).

Sperm ROS levels in the following examples were measured by ROS detection kit (Beyotime Biotechnology, Shanghai, China) according to manufacturer's instructions. The samples were detected using a flow cytometer (CytoPOC, Gaugene, Beijing, China).

Sperm DNA fragment evaluation in the following examples sperm DNA fragments were evaluated by flow cytometry (Becton Dickinson, Franklin Lakes, USA) for analysis of sperm chromatin structure, at least 5000 sperm per sample were collected and data were collected and analyzed using BDAccuri software (Becton Dickinson, Franklin Lakes, USA).

Example 1

The embodiment of the application provides an analytical test of semen, specifically includes:

semen is derived from 39 men seeking fertility diagnosis in the reproductive center, semen samples are collected by masturbation 2-7 days after abstinence, liquefied at 37 ℃ for at least 30 minutes and evaluated. Semen analysis was performed according to the WHO fifth guideline (world health organization: Manual of human semen examination and processing laboratories, fifth edition, Rinewa: world health organization; 2010). Sperm concentration and motility analyses were performed using computer-assisted semen analysis (CASA) under a microscope (CX43, Olympus, Tokyo, Japan) and a SAS-II system (SAS Medical, Beijing, China). Sperm viability was determined using the eosin-nigrosine staining method (Ankebio, Hefei, China). The morphology of 200 sperm per sample was analyzed under a light microscope (UB100i, UOP, Chongqing, China) using dff-quick staining (Ankebio, Hefei, China) at 100-fold magnification.

Fresh semen parameters were measured and recorded and the results are given in table 1. The mean age of 39 subjects was 31.2 ± 5.5 years. The semen sample is evaluated to be normal spermatozoa according to WHO 2010 guideline (semen volume is more than or equal to 1.5ml, forward movement is more than or equal to 32%, sperm concentration is more than or equal to 1500 ten thousand/ml, and sperm motility is more than or equal to 59%). The average level of ROS, DFI and HDI in the sperm was 2.6 + -2.8%, 7.8 + -6.2% and 7.7 + -2.6%, respectively.

Sperm were then separated from the seminal plasma by density gradient centrifugation according to the instructions of the instrument (PureCention, Cooper Surgical, Trumball, United States). 2.0mL of an 80% gradient solution (PureCeptionwith 80% gradient) was transferred to a conical centrifuge tube. Another 2.0mL of 40% gradient solution (PureCeptionwith 40% gradient) was pipetted slowly onto the lower phase liquid. Then, 2.5mL of fresh semen was carefully placed on the upper phase and centrifuged at 400g for 20 minutes. The sperm were washed and resuspended in the SpermRinse (Vitroffe, San Diego, USA).

Example 2

The embodiment of the application measures the influence of cyanidin-3-O-glucoside (C3G) on the cryopreservation of sperms, and the specific method comprises the following steps:

C3G was added to the freezing medium, the freezing medium containing C3G was divided into 4 groups, and equal concentrations of sperm suspension were added to each group, and the concentrations of C3G in the freezing medium were 0. mu.M/L (control), 50. mu.M/L, 100. mu.M/L, and 200. mu.M/L, respectively. The sperm samples were equilibrated at room temperature and placed into a pipette, which was placed 10cm above liquid nitrogen vapor for suspension freezing for 30min, and then placed into liquid nitrogen for 2 weeks. Thawed samples were soaked in warm water (37 ℃) for 10min and sperm cells were analyzed for correlation with post-freezing correlation parameters (including sperm viability, sperm motility, sperm ROS, sperm DNA fragmentation rate DFI, and sperm nucleus integrity HDS) prior to low temperature freezing, while sperm ROS, sperm DNA fragmentation rate DFI were analyzed for correlation. The results of the assay were statistically analyzed using SPSS 23.0 statistical software (SPSS inc., Chicago, IL, USA). All data in this example are expressed as mean ± SD. The normality of the data was analyzed by the Shapiro-Wilk test. Parameters were compared between different groups using one-way anova, Tukey test and Kruskal-Wallis test. Two comparisons were performed using paired t-tests. The correlation of C3G addition concentration with ROS, DFI levels of sperm after freezing was analyzed using Spearman correlation test. P <0.05 is statistically significant for the differences.

Wherein the freezing medium is obtained from Sperm frozen stock solution Sperm FreezingMedium (product No. 1067) of ORIGIO corporation (Denmark), and its components comprise human albumin, recombinant human insulin, gentamicin sulfate, etc.

The results of the sperm survival rate, the sperm motility rate, the ROS, the DFI and the HDS are shown in FIGS. 1 to 5, and the correlation analysis between the ROS and DFI is shown in Table 2. Fig. 1 is a graph showing a comparison of survival rates of cryopreserved sperm at different concentrations C3G in vitro according to an embodiment of the present application after thawing, fig. 2 is a graph showing a comparison of motility rates of cryopreserved sperm at different concentrations C3G in vitro according to an embodiment of the present application after thawing, fig. 3 is a graph showing a comparison of sperm ROS of cryopreserved sperm at different concentrations C3G in vitro according to an embodiment of the present application after thawing, fig. 4 is a graph showing a comparison of sperm DFI of cryopreserved sperm at different concentrations C3G in vitro according to an embodiment of the present application after thawing, and fig. 5 is a graph showing a comparison of sperm HDS of cryopreserved sperm at different concentrations C3G in vitro according to an embodiment of the present application after thawing.

Beforeing freezing in FIGS. 1 and 2 indicates the survival rate and activity rate of sperm Before cryofreezing, and C3G at 0. mu.M/L, 50. mu.M/L, 100. mu.M/L and 200. mu.M/L after cryofreezing, respectively. As can be seen from the data in FIGS. 1 and 2, sperm motility and progressive motility decreased after freezing, especially in the control group (0. mu.M/L). The addition of low concentrations of C3G (50. mu.M/L) to the cryopreservation media increased sperm motility and progressive motility. In addition, the sperm motility and viability of the group added with 100 or 200 μ M/L C3G were higher than those of the control group.

Beforeing freezing in FIGS. 3-5 is sperm ROS, sperm DFI and sperm HDS Before sperm cryo-freezing, respectively, and 0. mu.M, 50. mu.M, 100. mu.M and 200. mu.M are sperm ROS, sperm DFI and sperm HDS of C3G at 0. mu.M/L, 50. mu.M/L, 100. mu.M/L and 200. mu.M/L after sperm cryo-freezing, respectively. As can be seen from the data in fig. 3-5, the ROS levels were significantly increased after thawing the sperm as compared to fresh sperm (fig. 3). While the addition of lower concentrations of C3G (50. mu.M/L to 100. mu.M/L) reduced the rise in sperm ROS levels due to cryopreservation. After cryopreservation, sperm DFI and HDS were lower in the C3G-added group than in the control group.

Correlation analysis from the data in table 2 shows that the sperm ROS levels after freezing are correlated with lower supplemented C3G (50 μ M/L) (r ═ 0.2, P ═ 0.03).

TABLE 2

The cryopreservation process of sperm in the prior art, including the formation of ice crystals, the toxicity of cryoprotective media and the change of sperm osmotic pressure, has negative effects on sperm quality due to the destruction of sperm membranes, the reduction of mitochondrial oxidative phosphorylation and the excessive generation of ROS. The release of ROS and the activity of antioxidant enzymes reduce sperm motility and induce sperm DNA damage. The data of the above embodiment shows that: addition of C3G to the freezing medium, especially at low concentrations of C3G, increased sperm quality by controlling intracellular ROS levels. Namely, the antioxidant C3G is added into a low-temperature culture medium, and during the process of thawing after sperm freezing, C3G can protect the sperm from freeze-thaw damage by controlling the generation of ROS.

The above experimental data demonstrate that the addition of C3G to the freezing medium reduces intracellular ROS production in a concentration-dependent manner. Supplementation of C3G in the freezing medium improved the motility and vitality of the thawed sperm. The addition of low concentration of C3G to the freezing medium more effectively reduced the ROS level after sperm thawing than the addition of high concentration of C3G. That is, the addition of 50 μ M/L of C3G during freeze-thawing was likely to induce sperm motility and reduced motility protection more effectively than the addition of two additional high doses of C3G (100 and 200 μ M/L). It was shown that the antioxidant capacity of C3G at low doses was sufficient to overcome low temperature damage.

In conclusion, compared with the control group (without the addition of C3G), the low dose (50. mu.M/L or 100. mu.M/L) of C3G can improve sperm motility and activity, reduce sperm Reactive Oxygen Species (ROS), and the DNA Fragmentation Index (DFI) and the sperm nucleus integrity HDS after freezing are higher than those of the C3G addition group. Supplementation of low concentrations of C3G (50 μ M/L) in freezing medium was negatively correlated with sperm ROS levels (r ═ 0.2, P ═ 0.03). Low dose supplementation with C3G can improve sperm motility and reduce DNA damage by reducing ROS levels after sperm thawing. Therefore, C3G is an effective sperm cryoprotectant and can improve oxidative stress in the process of preserving human sperm at low temperature.

The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. Application of anthocyanin in vitro cryopreservation of sperm is provided.

2. The use of claim 1, wherein said in vitro cryopreservation of sperm comprises: mixing the sperms with a sperm freezing culture medium, and then performing cryopreservation according to a sperm cryopreservation method; wherein the sperm freezing medium comprises anthocyanin and acceptable sperm freezing culture substances.

3. Use according to claim 1, wherein the anthocyanins are selected from one or more of cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargonidin-3-O-glucoside, malvidin-3-O-glucoside, peonidin-3-O-glucoside and morning gloin-3-O-glucoside.

4. The use of claim 3, wherein the concentration of anthocyanin in the sperm freezing medium is 50 μ M/L to 200 μ M/L.

5. The use of claim 3, wherein the concentration of anthocyanin in the sperm freezing medium is 50 to 100 μ M/L.

6. The use of claim 1, wherein the sperm cell culture acceptable substance comprises one or more of human serum albumin, recombinant human insulin, and an antibiotic.

7. The use of claim 1, wherein said use comprises use of said anthocyanins for improving sperm survival and motility.

8. The use of claim 1, wherein said use comprises use of said anthocyanins in reducing the level of ROS in sperm, the rate of DNA fragmentation in sperm DFI and the integrity of sperm nucleus HDS.

9. A sperm freezing medium, which is characterized by comprising anthocyanin and acceptable substances for sperm freezing culture;

10. A sperm freezing medium according to claim 9, wherein the concentration of the anthocyanins in the sperm freezing medium is between 50 μ M/L and 200 μ M/L.