Application of dbcAMP in the preparation of in vitro primordial follicle activator

Abstract

The present invention relates to the field of biotechnology, and in particular to the use of dbcAMP in the preparation of an in vitro primordial follicle activator. The present invention provides the use of a small molecule drug dbcAMP in the preparation of a preparation for primordial follicle activation. Experiments show that dbcAMP can increase the phosphorylation level of key proteins in the PI3K / mTOR signaling pathway when culturing mouse ovaries in vitro, thereby amplifying the effect of this signal cascade pathway and further promoting the activation of primordial follicles. At the same time, in situ injection of dbcAMP into the ovaries in vivo can also stimulate the activation of primordial follicles.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the application of dbcAMP in preparing an in vitro primordial follicle activator. Background Art

[0002] Assisted reproductive technology is the most effective means of assisting pregnancy at this stage. A prerequisite for traditional assisted reproductive technology is mature and healthy oocytes, which requires that the female patient's ovaries must have growing follicles that can respond to gonadotropin. However, patients with premature ovarian failure, polycystic ovary syndrome, and cancer after radiotherapy / chemotherapy do not have enough growing follicles in their ovaries that can respond to gonadotropin, making it difficult to obtain mature oocytes.

[0003] To solve this problem, traditional assisted reproductive technology must be improved. In fact, these patients still have a certain number of primordial follicles in their ovaries, so whether these primordial follicles can be used to help them give birth to offspring has attracted people's attention.

[0004] In recent years, with the help of transgenic mice and other models, many studies have confirmed the key role of two cells and two pathways in the activation of primordial follicles, namely the PTEN-PI3K / AKT signaling pathway in oocytes and the mTORC1 signaling pathway in pregranulosa cells. Based on the existing mechanism of primordial follicle activation, a new in vitro assisted reproductive technology has been born through clinical translation and application research, namely: in vitro activation of primordial follicles (abbreviated as in vitro activation, IVA).

[0005] IVA uses effective means to activate primordial follicles in vitro with drugs, and then performs traditional assisted reproduction when they grow to a stage where they can respond to hormones. This technology has successfully helped some infertile patients successfully give birth to the next generation. However, in the existing IVA clinical strategy, the safety and success rate of related drugs are still a major hidden danger. At the same time, patients need to undergo multiple laparoscopic surgeries, which take a long time and increase the pain of patients. Therefore, it is extremely important to develop a more efficient in vitro activation system for primordial follicles. Summary of the invention

[0006] In view of this, the present invention provides the use of dbcAMP in the preparation of an in vitro activator for primordial follicles. dbcAMP can increase the phosphorylation level of key proteins in the PI3K / mTOR signaling pathway, and further promote the activation of primordial follicles.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides the use of dbcAMP in preparing an mTOR agonist and / or a PI3K / AKT signaling pathway key protein agonist.

[0009] The present invention also provides the use of dbcAMP in preparing a preparation for improving the phosphorylation level of key proteins in mTOR and / or PI3K / AKT signaling pathways.

[0010] In some specific embodiments of the present invention, the key proteins of the mTOR and / or PI3K / AKT signaling pathways in the above applications are derived from ovaries.

[0011] The present invention also provides the use of dbcAMP in preparing medicine for primordial follicle activation in vitro.

[0012] The present invention also provides the use of dbcAMP in preparing medicine for promoting the transformation of primordial follicles into primary follicles.

[0013] The present invention also provides the use of dbcAMP in preparing drugs for assisted reproduction, wherein the assisted reproduction includes assisted pregnancy.

[0014] The present invention also provides a drug for activating primordial follicles in vitro, comprising dbcAMP and acceptable adjuvants and / or auxiliary agents.

[0015] In some specific embodiments of the present invention, the auxiliary materials and / or adjuvants in the above-mentioned medicine include Matrigel.

[0016] In some specific embodiments of the present invention, the content of dbcAMP in the above-mentioned drug includes 1 μmol / L to 10 μmol / L.

[0017] In some specific embodiments of the present invention, the content of dbcAMP in the above-mentioned drug is 10 μmol / L.

[0018] In some specific embodiments of the present invention, the excipients and / or auxiliary agents in the above-mentioned drugs include adhesives, fillers, disintegrants, lubricants, isotonicity regulators, solubilizers, solubilizers, preservatives, colorants, suspending agents, wetting agents, emulsifiers and / or surfactants.

[0019] The present invention also provides a method for activating primordial follicles, which comprises injecting the above-mentioned drug into the ovary.

[0020] In some specific embodiments of the present invention, the method for activating primordial follicles further comprises injecting the drug into the ovarian bursa.

[0021] The present invention also provides a method for activating primordial follicles in vitro, wherein ovarian tissue is cultured in an in vitro activation reagent;

[0022] The in vitro activating agent includes dbcAMP.

[0023] The present invention has the following effects:

[0024] 1. Adding dbcAMP to culture mouse ovaries in vitro can promote the activation of primordial follicles. The study found that the phosphorylation level of key proteins in the mTOR and PI3K / AKT signaling pathways in mouse ovarian tissue increased, but the total protein level remained unchanged; when adding dbcAMP to culture newborn mouse ovaries, the total number of follicles remained unchanged, but the number of activated follicles increased;

[0025] 2. dbcAMP can promote the activation of primordial follicles in vivo through in situ injection into the ovaries without affecting the total number of follicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0027] Figure 1 The embodiment of the present invention shows that 10 μmol / L dbcAMP was added to the ovaries of newborn 2-day-old mice, and samples were collected one day later to detect the expression of key proteins in the PI3K / AKT and mTOR signaling pathways; the left side shows the total protein of the key protein; the right side shows the phosphorylated key protein; "Control" is a control group without adding dbcAMP; the results show that after the ovaries of newborn mice were cultured with dbcAMP in vitro, it was found that dbcAMP did not affect the expression of the total protein of these molecules, but caused their active forms to be significantly upregulated, indicating that dbcAMP promotes the activation of primordial follicles through the activation of PI3K / AKT and mTOR pathways;

[0028] Figure 2 The present invention shows the effect of adding dbcAMP to culture follicles in newborn mouse ovaries; A is an ovarian tissue slice; B is the effect of 5-day dbcAMP culture on the total number of follicles and the number of activated growing follicles; C is the effect of dbcAMP on the total number of follicles on the 3rd, 5th and 7th days; D is the effect of dbcAMP on the proportion of follicle activation on the 3rd, 5th and 7th days; the study found that in the process of adding dbcAMP to culture newborn mouse ovaries, there was no significant difference in the total number of follicles, but the number of growing follicles increased, indicating that adding dbcAMP to culture mouse ovaries can promote the activation of primordial follicles; the results of 3-day, 5-day and 7-day in vitro culture of newborn mouse ovaries were statistically analyzed, and the results all showed that dbcAMP can significantly promote the activation of primordial follicles, but does not affect the total number of follicles in the ovaries; scale: 50μm;

[0029] Figure 3The figure shows that two weeks after the ovaries of 23-day-old mice were injected with dbcAMP in situ, the ovarian tissues were collected for tissue section staining and the number of follicles at each level was counted; wherein A is the ovarian tissue section; B is the effect of dbcAMP on the total number of follicles in vivo; C is the effect of dbcAMP on the number of growing follicles in vivo; the results show that dbcAMP can also significantly promote the activation of primordial follicles in vivo without affecting the total number of follicles; scale: 100 μm. DETAILED DESCRIPTION

[0030] The present invention discloses the use of dbcAMP in the preparation of an in vitro activator of primordial follicles. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the purpose. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0031] At present, the role of cAMP in the female reproductive system is mostly concerned with its relationship with the meiotic process of oocytes. The high level of cAMP in the cytoplasm of the oocytes in the growing follicles can maintain the first meiotic division of the oocytes in a long-term blocked state. Only when the synthesis of cAMP is downregulated or it is degraded, the oocytes can resume meiosis and mature. Studies have shown that the cAMP analog dbcAMP can inhibit the meiotic recovery of oocytes from antral follicles, that is, inhibit oocyte maturation. In addition, the rise of cAMP levels is required when early oocytes enter the first meiotic division, and changing the cAMP level with drugs in vitro can affect the formation of the primordial follicle bank. Because the physiological development process of follicles has distinct spatiotemporal specificity, the activation process of primordial follicles may have a completely different physiological regulation mechanism from the maturation of oocytes and the formation of primordial follicles. Before the present invention carried out related research, it was not clear whether cAMP played a role in the activation of mouse primordial follicles.

[0032] The technical problem to be solved by the present invention is to provide the use of dbcAMP in the preparation of an in vitro primordial follicle activator. Studies have shown that when dbcAMP is added during the culture of newborn mouse ovaries, the total number of follicles remains unchanged, while the number of primary follicles increases, indicating that some primordial follicles in the ovaries are activated and transformed into primary follicles.

[0033] The present invention provides the use of dbcAMP in preparing a preparation for activating mTOR.

[0034] mTOR (mammalian target of rapamycin) is a conserved tyrosine kinase that plays a key role in regulating cell proliferation, growth, survival, division, movement and angiogenesis. In the present invention, the mTOR is mTOR in the ovary. The present invention shows that dbcAMP treatment can increase the level of phosphorylated mTOR in mouse ovaries. That is, the present invention provides the use of dbcAMP in the preparation of a preparation that increases the level of phosphorylated mTOR.

[0035] The present invention also provides the use of dbcAMP in preparing a preparation for activating key proteins in the PI3K / AKT signaling pathway.

[0036] PI3K is a serine / threonine protein kinase that regulates various biological processes such as cell metabolism, cell proliferation, apoptosis, and cell cycle, mainly by activating the activity of AKT. In the present invention, the PI3K / AKT signaling pathway is the PI3K / AKT signaling pathway in the ovary. The present invention's research shows that dbcAMP treatment can increase the level of phosphorylated PI3K / AKT signaling pathway key proteins (AKT, FOXO3a, rpS6) in the mouse ovary. That is, the present invention provides the use of dbcAMP in the preparation of a preparation that increases the level of phosphorylated PI3K / AKT signaling pathway key proteins.

[0037] The invention also provides the use of dbcAMP in preparing an in vitro activator of primordial follicles.

[0038] The primordial follicles described in the present invention are primordial follicles of mice. The present invention's research shows that under in vitro culture conditions, dbcAMP has an agonistic effect on mTOR and AKT in the ovary, and has a promoting effect on the transformation of primordial follicles to primary follicles in the mouse ovary. In other words, dbcAMP can be used as an in vitro activator of primordial follicles.

[0039] The present invention provides an in vitro activation preparation for primordial follicles, which comprises dbcAMP.

[0040] In the preparation provided by the present invention, the concentration of dbcAMP is 1 μmol / L to 10 μmol / L.

[0041] In some specific embodiments, the concentration of dbcAMP is 10 μmol / L.

[0042] In the present invention, the preparation further comprises ovarian basal culture medium and phase-change biogel (Matrigel). Each 100 mL of the ovarian basal culture medium comprises water, 0.5 g DMEM, 0.53 g F12, 0.357 g HEPES, 0.2438 g NaHCO3, 0.006 g penicillin, and 0.005 g streptomycin.

[0043] The in vitro activation method of primordial follicles provided by the present invention is to culture ovarian tissue in the in vitro activation reagent. Specifically, the method comprises: preheating the in vitro activation reagent at 37°C, separating the ovaries of newborn mice in a sterile environment, transferring them to the preheated activation culture medium and culturing them for 1 to 5 days, and changing the medium every other day.

[0044] The in vivo activation method of primordial follicles provided by the present invention adopts the ovarian in situ injection method to inject the above preparation into the ovary.

[0045] The above-mentioned ovarian in situ injection method includes: after thoroughly mixing the drug and Matrigel on ice, anesthetizing the mouse, making a small incision at the location of the ovary on its back, finding the ovary, taking it out with tweezers and placing it on a latex pad, injecting Matrigel mixed with the drug into the ovarian sac, and after the injection is completed, moving the ovary back to the abdominal cavity and suturing the wound.

[0046] The present invention provides the use of a small molecule drug dbcAMP in the preparation of a preparation for primordial follicle activation. Experiments show that dbcAMP promotes the activation of primordial follicles by increasing the phosphorylation level of key proteins in the mTOR or PI3K / AKT signaling pathways, thereby increasing the number of primary follicles.

[0047] Unless otherwise specified, the raw materials, reagents, consumables and instruments used in the present invention are all common commercial products and can be purchased from the market.

[0048] The present invention will be further described below in conjunction with embodiments:

[0049] Example 1

[0050] 1. Experimental materials:

[0051] 2-day-old ICR female mice, 23-day-old ICR female mice.

[0052] 2. Experimental reagents:

[0053] Reagent A: Ovarian basal culture medium. The specific formula is shown in Table 1. The preparation process must be performed aseptically.

[0054] Table 1 Ovarian basal culture medium formula

[0055] DMEM 0.5g F12 0.53g HEPES 0.357g NaHCO3 0.2438g penicillin 0.006g Streptomycin 0.005g Ultrapure water Make up to 100mL

[0056] Reagent B: 10 mM dbcAMP (solvent is ovarian basal medium)

[0057] Reagent C: Sterile PBS

[0058] Reagent D: Matrigel

[0059] 3. Experimental process

[0060] (1) Add 3 mL of reagent A and 3 μL of reagent B to a 6-well plate, place a cell culture membrane in each well, and then preheat in a cell culture incubator at 37°C;

[0061] (2) After the ovaries of 2-day-old mice were separated in reagent C, they were transferred to the culture membrane in step 1 and cultured in a cell culture incubator to activate the primordial follicles in vitro. One day later, the phosphorylated forms of mTOR, AKT, FOXO3a, and rpS6 (p-mTOR, p-AKT, p-FOXO3a, and p-rpS6) and their total protein levels in the mouse ovarian tissue were collected and detected (the results are shown in Figure 1 shown);

[0062] (3) After culturing the ovaries of two-day-old mice in vitro for 3 to 7 days, the ovarian tissues were collected and sliced ​​to observe the development of follicles at all levels and to count the number of follicles (results are shown in Figure 2). Figure 2 , Table 2, Table 3, Table 4);

[0063] Table 2 Effect of dbcAMP culture concentration on the number of growing follicles and total follicles (unit: number)

[0064]

[0065] Table 3 Effect of dbcAMP on the total number of follicles (unit: number)

[0066]

[0067] Table 4 Effect of dbcAMP on the number of growing follicles (unit: piece)

[0068]

[0069] (4) The ovaries of 23-day-old mice were injected in situ. The left ovary was injected with Matrigel only as the control group, and the right ovary was injected with Matrigel mixed with dbcAMP (the content of dbcAMP in Matrigel was 10 μmol / L) as the treatment group. Two weeks after the operation, the ovaries were collected and sliced ​​to observe the development of follicles at various levels and to count the number of follicles (the results are shown in Figure 2). Figure 3 , as shown in Table 5).

[0070] Table 5 Effects of in situ injection of Matrigel or dbcAMP on follicle activation

[0071]

[0072] The results showed that: 1. Adding dbcAMP to culture mouse ovaries in vitro can promote the activation of primordial follicles. Further studies found that dbcAMP promoted the in vitro activation of mouse primordial follicles by activating the mTOR and PI3K / AKT signaling pathways; 2. dbcAMP can promote the activation of primordial follicles in vivo through in situ injection of ovaries. Therefore, dbcAMP can be used as a new safe and effective activation drug target for in vitro activation of primordial follicles in clinical practice, which will be helpful for further improving IVA technology in the future by simulating the activation of primordial follicles under physiological conditions.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of dbcAMP in the preparation of drugs for in vitro activation of primordial follicles.

2. The use according to claim 1, characterized in that The drugs include drugs for assisted reproduction, and the assisted reproduction includes assisted pregnancy.

3. The use according to claim 1, characterized in that The drug comprises dbcAMP and acceptable adjuvants and / or auxiliary agents.

4. The use according to claim 3, characterized in that The auxiliary materials and / or adjuvants include Matrigel.

5. The use according to claim 3 or 4, characterized in that The content of dbcAMP includes 1 μmol / L~10 μmol / L.

6. The use according to claim 1, characterized in that The in vitro activation comprises: taking ovarian tissue and culturing it in an in vitro activation reagent; The in vitro activating agent includes dbcAMP.

Images