CN114164169B - Application of astaxanthin in promoting development of preantral follicles of mice and preparation - Google Patents
Application of astaxanthin in promoting development of preantral follicles of mice and preparation Download PDFInfo
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Abstract
The invention relates to application of astaxanthin, in particular to application of astaxanthin in promoting pre-luminal follicle development of a mouse and a preparation. The astaxanthin is added into the in-vitro culture solution of the follicle in front of the cavity of the mouse to explore the antioxidation and antioxidation mechanisms of the astaxanthin in the development process of the follicle, and the result shows that the astaxanthin reduces lipid peroxidation in the follicle by improving the expression of the antioxidation gene in the follicle, improves the development rate of the follicle and the secretion of estrogen, promotes the in-vitro development of the follicle in front of the cavity of the mouse, and provides reference for the application of the astaxanthin in-vitro culture of the follicle in other species.
Description
Technical Field
The invention relates to application of astaxanthin, in particular to application of astaxanthin in promoting pre-luminal follicle development of a mouse and a preparation.
Background
Follicular development is a key, complex biological process for maintaining reproductive reproduction in humans. At present, in vitro culture technology of primordial follicles and pre-luminal follicles and in vitro maturation technology of cumulus oocyte complexes (CoCs) become important research methods for researching follicular development and oocyte maturation mechanisms, and oocytes produced by the technology are also important sources of mature oocytes in clinical assisted reproduction technology. In order to improve the in vitro culture effect of the above-mentioned technical systems, hormones, growth factors, and some plant extracts have been added to the culture medium to promote follicular development and oocyte maturation in vitro. During the in vitro culture process of the follicle, the follicle is in a high-oxygen environment compared with the in vivo environment, so that ROS are continuously generated in the follicle, and excessive ROS can cause the release of cytochrome c in the mitochondria to activate caspase cascade reaction, so as to cause follicle apoptosis. Antioxidants can capture oxygen free radicals and mitigate intracellular lipid peroxidation and DNA fragmentation. Therefore, it is also studied to add some antioxidants during the in vitro culture of ovarian follicles to improve the in vitro culture environment of ovarian follicles.
Astaxanthin is a natural carotenoid with very strong antioxidant properties, and is mainly found in algae, starfish, crustaceans, salmon, and other marine organisms. Astaxanthin has been studied for many years in the fields of anti-tumor, anti-diabetes, anti-inflammation, anti-obesity, etc. by virtue of its antioxidant activity. Due to the special structure of astaxanthin, hydroxyl and ketone groups are arranged on the ionone ring, so that the astaxanthin has extremely strong oxidation resistance. Astaxanthin has 10 times higher antioxidant activity than other carotenoids (canthaxanthin, beta-carotene, lutein and zeaxanthin) and 100 times higher antioxidant activity than alpha-tocopherol. More recently the use of astaxanthin in the reproductive field has attracted increasing interest. Astaxanthin is used to retard bovine in vitro embryo damage caused by heat or oxidative stress, which promotes the in vitro development of bovine somatic cell nuclear transfer embryos. Astaxanthin has been found to increase the capacitation of human sperm and prevent the association of the papillomavirus L1 protein with the sperm plasma membrane. However, there have been few studies on the application of astaxanthin to in vitro culture systems of pre-luminal follicles to alleviate oxidative damage of follicles during in vitro development.
Disclosure of Invention
The invention aims to relieve oxidative stress injury of in vitro development of a follicle before a cavity of a mouse, improve the in vitro development rate of the follicle before the cavity of the mouse and discuss the antioxidation effect and antioxidation mechanism of astaxanthin on the follicle before the cavity of the mouse.
In order to achieve the purpose, the invention provides application of astaxanthin in promoting the development of preantral follicles of mice.
Further, the development is in vitro development.
Furthermore, the in vitro development of the follicle before the cavity of the mouse is promoted by improving the expression of the antioxidant gene in the follicle, reducing the lipid peroxidation in the follicle, improving the development rate of the follicle and the secretion of estrogen, so as to promote the cavitation rate and the maturation rate of the follicle before the cavity of the mouse.
Further, the antioxidant gene is GSH and SOD1 gene.
Further, the use comprises culturing the pre-luminal follicles isolated from female mice in a follicular medium comprising astaxanthin.
Furthermore, the follicle culture solution is an alpha-MEM culture solution containing r-FSH, FBS and insulin-transferrin-selenium, wherein the concentration of the r-FSH is 100mIU/ml, the concentration of the FBS is 5% by mass, and the concentration of the insulin-transferrin-selenium is 1% by mass.
Further, the concentration of astaxanthin in the follicular culture fluid was 2.5 nM.
The invention also provides a preparation for promoting development of preantral follicles of mice, which contains the astaxanthin.
Compared with the prior art, the invention has the following beneficial effects:
the in vitro culture system of the follicle before the cavity of the mouse is an ideal experimental system for researching the biological growth of the follicle before the cavity of the mouse, in order to further improve the in vitro development rate of the follicle before the cavity of the mouse and relieve the oxidative damage of the follicle before the cavity of the mouse, astaxanthin is added into the in vitro culture solution of the follicle before the cavity of the mouse to explore the antioxidation effect and the antioxidation mechanism of the follicle during the development process of the follicle.
Drawings
Fig. 1 is an in vitro developed pre-luminal follicle in a mouse (inverted microscope, scale 100 μm).
FIG. 2 shows the adherent area (. mu.m) of the mouse follicles 2 ，x±s，n＝8)。
Fig. 3 shows the estrogen secretion levels of the mouse follicles (ng/L, x ± s, n ═ 3).
Fig. 4 shows the MDA content in the follicles (μ M, x ± s, n ═ 3).
Fig. 5 shows GSH and SOD1 gene expression (μ M, x ± s, n ═ 3) in follicles.
FIG. 6 is a gray scale analysis chart of the expression of Nrf-2 and HO-1 proteins in follicles, wherein A is a protein band detected by Western Blotting and B is a detection result of Western Blotting.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
Examples
1 materials and methods
1.1 Experimental animals
60 female clean-grade Kunming mice from day 14 of birth were purchased from the Burkholderia experimental animal center. The mice are raised in an environment with room temperature of (22 +/-2) ° C and humidity of (50 +/-10)% and the light cycle of 14h of light plus 10h of darkness is maintained, and the mice eat and eat freely. The experiment is approved by the ethical committee of medical research of the Jilin pharmaceutical institute, and the experimental operation conforms to the regulations of animal experimental research management.
1.2 Pre-luminal ovarian follicle in vitro culture
Mice were sacrificed by cervical dislocation and both ovaries were quickly removed from L-15 working solution (Gibco, Inc., 11415064, USA) containing 10% Fetal Bovine Serum (FBS). Tissues surrounding the ovary were removed by separation under a scope, and follicles were isolated using a separation needle. According to the follicle classification standard of Pedersen and the like, a preantral follicle with the diameter of 110-130 mu m and 3 layers of granular cells is selected and placed in alpha-MEM (follicle culture fluid) culture drops (namely follicle culture fluid) containing r-FSH (recombinant FSH), FBS and Insulin-Transferrin-Selenium (ITS), wherein the concentration of the r-FSH in the follicle culture fluid is 100mIU/ml, the mass percent concentration of the FBS is 5 percent, and the mass percent concentration of the Insulin-Transferrin-Selenium is 1 percent. The follicles used as the control group were cultured in a culture medium containing 0.1% DMSO or 2.5nM Asta, 25nM Asta, and 250nM Asta, and the follicles used as the DMSO group, 2.5nM Asta group, 25nM Asta group, and 250nM Asta group, respectively. Placing at 37 ℃ with 5% CO 2 Continuously culturing for 10 days in the incubator (2), changing liquid in a semi-quantitative manner every other day, observing and recording the growth condition of each group of follicles, wherein the adherent follicles on the day (2) are survival follicles, and the follicles with follicular cavities on the day (8) are cavitary follicles, and counting the cavitation rate of the follicles. On day 10, 2.5U/ml Human Chorionic Gonadotropin (HCG) was added to the follicle culture medium and cultured for 16 hours. Granular cells around cumulus-ootheca complexes (CoCs) are removed by using a glass tube with a thinner caliber, oocytes with the 1 st polar body are mature oocytes, and the maturation rate of the oocytes is counted. And calculating the adherent areas of follicles at 4 th, 6 th, 8 th and 10 th days of culture by using Image J Image analysis software.
1.3 detection of estradiol (E) in culture by ELISA 2 ) Level of
The follicular culture fluid was collected at 3:00 pm on days 4, 6, 8, and 10 of follicular culture and stored at-20 ℃. Each group was cultured with 30 follicles, and the culture solution of follicles in each group was collected 3 times and examined with an ELISA kit (BPE 20376, Shanghai Lanton Biotech Co., Ltd.)Measurement of E in culture 2 And (4) horizontal.
1.4 MDA level detection in ovarian follicles
20 follicles from day 10 of culture were lysed in cell lysate (Biyunyan, P0013) each time, and centrifuged at 10,000g-12,000g for 10 minutes to collect the supernatant for subsequent assays. The principle that the red MDA-TBA adduct is generated by the reaction of MDA and thiobarbituric acid (TBA) is adopted by using an MDA detection kit (Biyun day, S0131S). Respectively adding 0.1ml of sample to be detected, standard substance or lysis solution into a centrifuge tube, then adding 0.2ml of MDA detection working solution, uniformly mixing, and heating for 15 minutes at 100 ℃ or in a boiling water bath. The water bath was cooled to room temperature and 1000g was centrifuged at room temperature for 10 min. 200. mu.l of the supernatant was added to a 96-well plate, followed by measurement of absorbance at 532nm with a microplate reader. And calculating the MDA content in the sample solution according to the standard curve. Experiments for each group were repeated 3 times.
1.5qRT-PCR detection of GSH and SOD1 Gene expression in ovarian follicles
Real-time PCR analysis was performed on day 10 follicles from 20 cultures each time. RNAs in the follicles were extracted using the Rneasy Micro Kit (Qiagen, Hilden, Germany) and the RNA was reverse transcribed to cDNA in a 20. mu.L reverse transcription system. Quantitative analysis of gene products was performed by quantitative Real-time PCR using iQ5 Multicolor Real-time PCR Detection System (Bio-RAD) using primers shown in Table 1. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 59 ℃ for 20s, and extension at 72 ℃ for 30s, and circulating for 40 times. Housekeeping gene beta-actin as an internal reference, use 2 -△△Ct The method calculates the relative expression level of the target gene.
TABLE 1 primer sequences for qRT-PCR
1.6Western blotting method for detecting expression level of follicle Nrf-2 and HO-1 proteins
80 follicles from 10 days of culture were obtained each time, and proteins were extracted using RIPA-rich lysate (containing 1% PMSF). Preparing gel, loading, performing 120V constant voltage electrophoresis for about 80min, performing 300mA wet membrane transfer for 60min, sealing with 5% skimmed milk powder for 1h, and incubating with rabbit Nrf-2 antibody (Abcam, ab92946), HO-1 antibody (Abcam, ab52947) or beta-actin antibody (Abcam, ab8226) at 4 deg.C overnight. Beta-actin is used as an internal reference. Day 2, goat anti-rabbit secondary antibody (Thermo Pierce,31210) labeled with Horseradish peroxidase (HRP) was incubated at room temperature for 1 h. Finally, the PVDF membrane having the protein transferred thereon was put into a Tanon 4600 Imaging System, and 200. mu.l of an enhancing developer was applied thereto, and the film was photographed by using Tanon Imaging System software. Each experimental group was repeated 3 times and the picture grey values were analyzed using the tannon GIS ID software.
1.7 statistical treatment
Statistical analysis of the experimental data using SPSS 17.0, data expressed as mean. + -. standard deviation
Comparisons of inter-group differences were made using one-way ANOVA LSD post hoc<A difference of 0.05 was significant.
2 results of
2.1 Effect of astaxanthin on Pre-luminal follicle in vitro development in mice
The pre-luminal follicles of the mice isolated in vitro are shown in figure 1. Follicles began adherent growth on day 2; on day 4, granulosa cells of each group of follicles proliferate to break through the basement membrane and start to grow outwards; on the 6 th day, the follicular granulosa cells continue to proliferate, the size of the follicles becomes large, and a small number of follicles begin to appear in the follicular chamber; on day 8, most follicles appeared in the follicular antrum as the follicular volume increased; on day 10 the follicular chamber continued to increase and the antral follicles were nearly mature (FIG. 1)
The survival rate, cavitation rate and maturation rate of the follicles were counted, and the results are shown in table 2. The survival of the follicles did not differ significantly between groups, but the luminal and maturation rates of the Asta group 2.5nM were significantly higher than those of the control, 25nM and 250nM (P <0.05), and significantly lower than those of the control (P < 0.05). There was no significant difference in the luminal rate of the 250nM Asta group follicles compared to the 25nM Asta group, but the maturation rate was significantly reduced (P < 0.05). 2.5nM Asta promotes follicular development.
TABLE 2 in vitro growth rates of Pre-luminal follicles in mice: (
n＝8)
Compared with the control group, the compound of the formula, * P<0.05; in comparison to the 2.5nM Asta group, # P<0.05; in comparison to the 25nM Asta group, ■ P<0.05。
the adherent areas of the follicles of the control group, the DMSO group and the 2.5nM Asta group were calculated, and the results are shown in fig. 2. The adherent area of the follicles in each group was not significantly different at day 2, the volume of the follicles in the group Asta was increased by 2.5nM from day 4 compared with the control group and DMSO group, and the adherent area of the follicles at day 4, day 6, day 8 and day 10 was significantly larger than that of the control group and DMSO group (P < 0.05). Asta significantly promoted in vitro growth of the follicles at 2.5nM from day 4.
2.2 Effect of astaxanthin on secretion of follicle Estrogen in mice
The estrogen levels in the follicular culture fluid at day 4, day 6, day 8 and day 10 were measured as shown in figure 3. The 2.5nM Asta group of follicles secreted estrogen at day 4, day 6, day 8 and day 10 at significantly higher levels than the control and DMSO groups (P <0.05), and 2.5nM Asta promoted estrogen secretion by the follicles.
2.3 Effect of astaxanthin on MDA content in follicles
The level of MDA in the follicles at day 10 was examined, as shown in fig. 4. The MDA content in the follicles of the 2.5nM Asta group was significantly lower than in the control and DMSO groups (P <0.05), 2.5nM Asta reduced the peroxidation level of lipids in the follicles.
2.4 Effect of astaxanthin on the expression of the GSH and SOD1 genes in follicles
The expression of GSH and SOD1 genes in follicles at day 10 was examined, and the results are shown in fig. 5. The expression of GSH and SOD1 genes in the follicle of the 2.5nM Asta group was significantly higher than that of the control group and DMSO group (P <0.05), and the expression of reducing agent and antioxidant enzyme in the follicle was promoted by the 2.5nM Asta.
2.5 Effect of astaxanthin on expression of Nrf-2 and HO-1 proteins in follicles
The expression of Nrf-2 and HO-1 proteins in follicles at day 10 was examined, and the results are shown in FIG. 6. The expression of Nrf-2 and HO-1 proteins in 2.5nM Asta follicles was significantly higher than control and DMSO groups (P < 0.05). 2.5nM Asta promotes the expression of antioxidant pathway Nrf-2/HO-1 protein in follicles.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
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Application of astaxanthin in promoting development of preantral follicles of mice and preparation
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Claims (7)
1. Application of astaxanthin in preparing preparation for promoting development of follicle before cavity of mouse.
2. Use according to claim 1, wherein the development is in vitro development.
3. The use of claim 2, wherein the promotion of the in vitro development of the pre-luminal follicle in the mouse is achieved by increasing the expression of an antioxidant gene in the follicle, decreasing lipid peroxidation in the follicle, increasing the rate of development of the follicle and increasing the secretion of estrogen, thereby promoting the rate of cavitation and maturation of the pre-luminal follicle in the mouse.
4. The use as claimed in claim 3, wherein the antioxidant genes are GSH and SOD1 genes.
5. The use according to claim 4, comprising: the pre-luminal follicles isolated from female mice were cultured in a follicular medium containing astaxanthin.
6. The use according to claim 5, wherein the follicular fluid is an α -MEM fluid comprising r-FSH, FBS and insulin-transferrin-selenium, wherein the concentration of r-FSH is 100mIU/ml, the concentration of FBS is 5% by mass and the concentration of insulin-transferrin-selenium is 1% by mass.
7. The use according to claim 6, wherein the concentration of astaxanthin in the follicular fluid is 2.5 nM.
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