CN113005075A - Application of ROCK inhibitor in promotion of up-regulation of oocyte gene Bmp15 and Gdf9 expression level - Google Patents

Application of ROCK inhibitor in promotion of up-regulation of oocyte gene Bmp15 and Gdf9 expression level Download PDF

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CN113005075A
CN113005075A CN202110253455.4A CN202110253455A CN113005075A CN 113005075 A CN113005075 A CN 113005075A CN 202110253455 A CN202110253455 A CN 202110253455A CN 113005075 A CN113005075 A CN 113005075A
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裴秀英
俞晓丽
张樱馨
王燕蓉
常青
马会明
杨延周
马文智
付旭锋
何瑞
蒲静
刘心蕊
邱意开
张彦平
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Abstract

The invention discloses application of a ROCK inhibitor in promoting the up-regulation of oocyte genes Bmp15 and Gdf 9. Wherein, the ROCK inhibitor Y27632 can promote the up-regulation of the expression level of oocyte specific genes Bmp15 and Gdf9 and promote the proliferation and development of follicular cells. This application has adopted the serum-free culture system, simultaneously, in order to simulate the follicle in internal growing environment, this research has adopted the ultralow adsorption culture plate to cultivate the follicle, and this culture plate not only can make the follicle be three-dimensional growth mode at whole developmental process, makes the granulosa cell around the oocyte obtain more abundant nutrient substance and space moreover, can comparatively be favorable to the development of follicle before the chamber. Meanwhile, after inhibitor Y27632 is added, the expression levels of Gdf9 and Bmp15 are increased, and the follicle proliferation and development are promoted.

Description

Application of ROCK inhibitor in promotion of up-regulation of oocyte gene Bmp15 and Gdf9 expression level
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a ROCK inhibitor in promoting expression levels of oocyte genes Bmp15 and Gdf9 and preventing spindle assembly abnormality and a kit thereof.
Background
The ovary is an important reproductive endocrine organ in female mammals, and the follicle plays an important role in the whole ovarian development process as the basic structure and functional unit of the ovary. In recent years, the trend towards the younger patients with tumors has been increasing, and although the survival of these patients is being prolonged by the constantly updated medical techniques, the fertility of women is seriously impaired by the chemotherapy and radiotherapy of the mainstream technology. Therefore, the ovarian tissue or immature ovarian follicles are preserved before radiotherapy and chemotherapy, and the ovarian tissue is moved back after tumor recovery or ovarian follicles are matured and cultured in vitro, so that the method can be used as an effective means for maintaining the fertility or endocrine function of women.
Rho kinase (ROCK) is an effector of GTPase Rho and coordinates multiple cellular behaviors such as cell proliferation, apoptosis, division, migration, differentiation, etc. Y27632 is an inhibitor of the ROCK pathway and plays an important role in cell proliferation and apoptosis. However, no record is found that ROCK inhibitor Y27632 can improve the expression level of oocyte-specific genes Bmp15 and Gdf 9.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides application of a ROCK inhibitor in promoting the expression level of oocyte genes Bmp15 and Gdf9 and preventing spindle assembly abnormality and a kit thereof, and aims to discuss the influence of Y27632 on the maturation of oocytes, the proliferation of granulosa cells and the like, particularly the influence on the expression level of oocyte specific genes Bmp15 and Gdf9 in the process of in vitro development of mouse follicles from pre-luminal follicles to mature follicles.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
the application of ROCK inhibitor in promoting the up-regulation of the expression level of oocyte specific genes Bmp15 and Gdf 9.
Further, the ROCK inhibitor is ROCK inhibitor Y27632.
A medicine for promoting the up-regulation of the expression levels of oocyte-specific genes Bmp15 and Gdf9 comprises the ROCK inhibitor.
An agent for improving the quality of an oocyte and promoting the development of the oocyte, which can improve the expression level of genes Bmp15 and Gdf 9.
Further, the agent comprises the ROCK inhibitor described above.
A kit for promoting the expression of oocyte specific genes Bmp15 and Gdf9 comprises the ROCK inhibitor.
A medicament for preventing abnormal assembly of oocyte spindles, comprising the ROCK inhibitor of claim 1 or 2.
An oocyte cryoprotectant comprising the ROCK inhibitor.
An in vitro culture reagent for oocyte, comprising the ROCK inhibitor.
The invention has the beneficial effects that:
this application has adopted the serum-free culture system, simultaneously, in order to simulate the follicle in internal growing environment, this research has adopted the ultralow adsorption culture plate to cultivate the follicle, and this culture plate not only can make the follicle be three-dimensional growth mode at whole developmental process, makes the granulosa cell around the oocyte obtain more abundant nutrient substance and space moreover, can comparatively be favorable to the development of follicle before the chamber. Meanwhile, after inhibitor Y27632 is added, the expression levels of Gdf9 and Bmp15 are increased, and the follicle proliferation and development are promoted.
Drawings
FIG. 1 shows follicles after 0, 2 and 4 days of in vitro culture;
FIG. 2 shows the distribution of oocyte spindle microtubule tubulin detected by immunofluorescence staining; scale 10 μm;
FIG. 3 shows the effect of ROCK inhibitor Y27632 on the expression level of oocyte-specific genes Bmp15 and Gdf 9; p < 0.01, P < 0.001 compared to control;
FIG. 4 shows the effect of ROCK inhibitor Y27632 on the proliferation and development of follicular granulosa cells; p < 0.001.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
1. Laboratory animal
The pre-luminal follicles were obtained from female mice of ICR strain at 12.5d clean after birth, and oocytes were obtained from female mice at 21d after birth, all purchased from the laboratory animals center of Ningxia medical university. The mice are raised in a controlled environment at room temperature (20 +/-2) DEG C and humidity (50 +/-10)%, and are free to eat and eat in 12h light and 12h dark cycle. The experiment is approved by the ethical committee of medical research of Ningxia medical university, and the experimental process conforms to the regulations of animal experimental research management.
2. Primary reagents and consumables
L-15, alpha-MEM, insulin-transferrin-selenium additive (ITS), Fetal Bovine Serum (FBS), fluorescent secondary antibody and Live/dead cell activity detection kit are purchased from Thermo Fisher company; sodium pyruvate (PNa) and Bovine Serum Albumin (BSA) were purchased from Sigma company; follitropin (FSH), chorionic gonadotropin (HCG), Pregnant Mare Serum Gonadotropin (PMSG) were purchased from Ningbo second hormone plant; epidermal Growth Factor (EGF) was purchased from PEPROTECH; the trace RNA extraction kit is purchased from Omega company; reverse transcription kit was purchased from Abm; the fluorescent quantitative PCR kit is purchased from TAKARA company; the Tubulin antibody was purchased from abcam; ultra-low adsorption 96-well plates were purchased from Corning.
3. Main instrument equipment
Stereoscopic microscope, inverted fluorescence microscope, confocal microscope were purchased from Nikon corporation; incubators were purchased from Thermo corporation; a micro-spectrophotometer was purchased from Thermo corporation; fluorescent quantitative PCR instrument purchased from ABI corporation; the centrifuge was purchased from Eppendorf Inc.
Example 1
1. Isolation of Pre-luminal follicles in mice
After the mice were sacrificed by cervical dislocation, bilateral mouse ovaries were obtained under a scope of a stereoscope and quickly placed in L-15 operating fluid containing 10% FBS. Under a dissecting microscope, the oviduct part connected to the ovary and redundant ovary mesentery and fat tissue are removed, and a single follicle is mechanically separated by a Gunafen injection needle, so that the in-vitro separation time of each ovary is not more than 20 min. The screened follicle basement membrane is complete and has 2-3 layers of granular cells, and the center has the pre-luminal follicle of the round oocyte with a clear structure.
2. In vitro culture of Pre-luminal ovarian follicles
After 2-4 hours in advance, 100 mu L/hole of follicle growth culture solution (alpha-MEM culture medium + 3% BSA + 1% ITS + 1% cyan/streptomycin +0.1IU/mL FSH +0.33mM PNA +50 mu g/mL Vc) is placed in a 96-well ultra-low adsorption plate, and 5% CO is carried out at 37 DEG C2Culture ofAnd pre-balancing the breeding box. The isolated individual pre-luminal follicles were washed with a follicle growth medium using a thin glass pipette and then placed in wells for culture. And (3) changing the culture solution half every other day, changing the culture solution to oocyte maturation solution (follicle growth culture solution +1.5IU/mL HCG +10ng/mL EGF) in the afternoon of the 8 th day, selecting mature follicles or Cumulus Oocyte Complexes (COCs) on the next day, treating the oocytes with hyaluronidase, picking out the oocytes, and observing the cytoskeleton staining.
3. Collection of oocytes
Injecting PMSG (10 IU/mouse) into the abdominal cavity of an ICR female mouse 21d after birth for 48h, injecting HCG (10 IU/mouse) for 16h, taking an oviduct, piercing the oviduct expansion part under a microscope to obtain COCs, and treating with hyaluronidase to obtain oocytes at each period.
Example 2 extraction of follicular RNA, reverse transcription of cDNA and fluorescent quantitative PCR
After collecting follicles cultured in vitro for 8d, washing the follicles with PBS for 3 times, the procedure in the instruction in the RNA extraction kit is strictly followed, the RNA concentration and purity are measured with a microspectrophotometer, and then the detection of the relevant genes is carried out according to the requirements of the reverse transcription kit and the fluorescent quantitative PCR kit (the primer sequences are shown in Table 1). And analyzing and charting the obtained result Graphpad prism software.
TABLE 1 fluorescent quantitative PCR primer sequences
Figure BDA0002969973240000051
Example 3 follicular activity assay
To assess and confirm the survival status of the follicles, 8d follicles were stained with fluorescent reagents Calcein AM and Eethidium homomodimer (EthD-1) in the dark and observed for follicular activity under a confocal laser microscope after being left in an incubator at 37 ℃ for 30 min. In which intracellular esterases, which are present extensively in living cells, are converted enzymatically by cell-permeable Calcein AM into intense, homogeneous green fluorescence. EthD-1 enters cells with damaged cell membranes and emits bright red fluorescence after binding to nucleic acids. The survival status of the follicles was judged according to the ratio of green light and red light emitted from granulosa cells and oocytes, and the results are shown in FIG. 1.
As shown in FIG. 1, the number of follicular granulosa cells increases with the number of days of culture, and after 4 days, a follicular chamber begins to form, and after the development reaches 8 days, a follicular maturation culture is performed, so that cumulus oocyte complexes can be eliminated. From the morphology of the follicles, there was no significant difference between the control group and the treated group, but the granulosa cells in the treated group were more closely arranged. The results showed no statistical difference between the two groups (P > 0.05) by measuring the change in diameter of the follicles (Table 2) and the survival rate, cavitation rate and oocyte exclusion rate of the follicles (Table 3) on different days.
TABLE 2 diameter of in vitro cultured follicles: (
Figure BDA0002969973240000061
μm)
Figure BDA0002969973240000062
TABLE 3 growth rate of in vitro cultured follicles: (
Figure BDA0002969973240000063
％)
Figure BDA0002969973240000064
EXAMPLE 4 immunofluorescence staining of oocytes
The oocytes after the degranulation treatment with hyaluronidase were fixed in a fixing solution of 2% paraformaldehyde + 0.02% TritonX-100 for 30min, and then washed in PBS for 3 times, 5min each time. The oocytes were then transferred to Tubulin antibody containing 0.5% FBS overnight at 4 ℃. The next day, PBS was washed 3 times for 5min each time, then transferred to fluorescein isothiocyanate labeled goat anti-rabbit IgG, and incubated at 37 ℃ for 1 h. PBS washing 3 times, each time 5min, 10ng/mL DAPI light-shielding incubation 15min, final PBS washing 3 times to transfer the oocyte to the slide glass, (fluorescence quencher resistant) nail polish mounting, fluorescence microscope observation, its results are shown in figure 2 and figure 3.
After 9d of follicle culture, spindle microtubule Tubulin staining was performed on the expulsed oocytes, and it was found that abnormal spindle assembly was observed in the oocytes of the control group (indicated by arrows in FIG. 2), but not in the treatment group (FIG. 2), and the upregulation of the expression of the oocyte-specific genes Bmp15 and Gdf9 in the treatment group was quantitatively detected in real time by fluorescence (FIG. 3).
Example 5 Effect of Y27632 on growth and development of follicular granulosa cells
Real-time fluorescent quantitative detection shows that the expression of the gene Pcna reflecting the proliferation state of the cells and the expression of the granular cell specific gene Fshr in the treatment group are obviously up-regulated (figure 3), and the difference has statistical significance (P is less than 0.05). Indicating that the proliferation of the granular cells is more obvious after the inhibitor is added.
This application has adopted the serum-free culture system, simultaneously, in order to simulate the follicle in internal growing environment, this research has adopted the ultralow adsorption culture plate to cultivate the follicle, and this culture plate not only can make the follicle be three-dimensional growth mode at whole developmental process, makes the granulosa cell around the oocyte obtain more abundant nutrient substance and space moreover, can comparatively be favorable to the development of follicle before the chamber.
Gdf9 and Bmp15 belong to transforming growth factor beta superfamily, are specifically expressed in oocytes, play an important role in regulating and controlling early follicular development and granulosa cell and membrane cell functions, and are important paracrine factors in ovaries. The detection result shows that after the inhibitor Y27632 is added, the expression levels of Gdf9 and Bmp15 are increased, and the follicle proliferation and development are promoted.

Claims (9)

  1. Use of a ROCK inhibitor for promoting the up-regulation of the expression levels of oocyte-specific genes Bmp15 and Gdf 9.
  2. 2. Use according to claim 1, wherein said ROCK inhibitor is ROCK inhibitor Y27632.
  3. 3. A drug for promoting the up-regulation of the expression levels of oocyte-specific genes Bmp15 and Gdf9, comprising the ROCK inhibitor according to claim 1 or 2.
  4. 4. An agent for improving the quality of an oocyte and promoting the development of the oocyte, wherein the agent can improve the expression level of genes Bmp15 and Gdf 9.
  5. 5. An agent according to claim 4, wherein said agent comprises a ROCK inhibitor according to claim 1 or 2.
  6. 6. A kit for promoting the expression of oocyte specific genes Bmp15 and Gdf9, the kit comprising the ROCK inhibitor of claim 1 or 2.
  7. 7. A medicament for preventing abnormal assembly of oocyte spindles, comprising the ROCK inhibitor according to claim 1 or 2.
  8. 8. An oocyte cryoprotectant comprising the ROCK inhibitor of claim 1 or 2.
  9. 9. An in vitro culture reagent for oocyte, comprising the ROCK inhibitor according to claim 1 or 2.
CN202110253455.4A 2021-03-10 2021-03-10 Application of ROCK inhibitor in promotion of up-regulation of oocyte gene Bmp15 and Gdf9 expression level Pending CN113005075A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102363766A (en) * 2011-11-02 2012-02-29 青岛农业大学 Method for promoting in vitro maturation of immature oocytes by using activin A (ActA)
CN110129402A (en) * 2019-04-15 2019-08-16 宁夏医科大学 A kind of research method that Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102363766A (en) * 2011-11-02 2012-02-29 青岛农业大学 Method for promoting in vitro maturation of immature oocytes by using activin A (ActA)
CN110129402A (en) * 2019-04-15 2019-08-16 宁夏医科大学 A kind of research method that Rho-ROCK signal pathway inhibitor Y27632 influences mouse eggs bubble ectogenesis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
YOON YOUNG KIM等: "The Expression Profile of Angiotensin System on Thawed Murine Ovaries", 《TISSUE ENGINEERING AND REGENERATIVE MEDICINE》, vol. 13, 17 December 2016 (2016-12-17), pages 725 *
俞晓丽等: "Rho/ROCK抑制剂Y27632对小鼠腔前卵泡体外培养的影响", 《宁夏医科大学学报》 *
俞晓丽等: "Rho/ROCK抑制剂Y27632对小鼠腔前卵泡体外培养的影响", 《宁夏医科大学学报》, vol. 41, no. 8, 31 August 2019 (2019-08-31), pages 770 - 772 *
崔静等: "动物重要经济性状基因的分离与应用", 中国农业大学出版社, pages: 364 *

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