siRNAs that enhance the developmental capacity of cryopreserved oocytes and their applications

Abstract

This invention discloses siRNA for enhancing the developmental capacity of cryopreserved oocytes and its applications, belonging to the field of oocyte cryopreservation technology. The provided siRNA molecule for enhancing the developmental capacity of cryopreserved oocytes has the nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2. The invention also describes the application of the provided siRNA molecule in the preparation of formulations that enhance the developmental capacity of cryopreserved oocytes. Specifically, it provides siRNA that targets and inhibits Atf5 gene expression. When microinjected into the cytoplasm of oocytes, it can specifically silence the target gene mRNA expression, significantly reducing Atf5 gene expression, maintaining mitochondrial function under cryopreservation stress, and improving the developmental capacity of cryopreserved oocytes.

Description

Technical Field

[0001] This invention belongs to the field of cryopreservation technology for oocytes, and particularly relates to siRNA and its application in improving the developmental capacity of cryopreserved oocytes. Background Technology

[0002] Oocyte cryopreservation is a fundamental method of fertility preservation and plays a crucial role in assisted reproductive technology. Despite significant advancements in oocyte cryopreservation technology, the developmental capacity of frozen oocytes decreases after thawing, and low freezing efficiency has become a major limiting factor for their application. Mitochondrial function is essential for normal oocyte development, and mitochondria are extremely sensitive to temperature and osmotic pressure changes during cryopreservation and thawing. Studies have shown that abnormal mitochondrial morphology and spatial distribution in frozen oocytes can lead to mitochondrial dysfunction during freezing. Given the critical role of mitochondria in oocyte development, maintaining mitochondrial function can improve the developmental capacity of frozen oocytes.

[0003] The mitochondrial unfolded protein response (mtUPR) is the main response mechanism of mitochondria to external environmental stimuli. Mitochondrial molecular chaperone heat shock protein 60 (Hsp60) is a biomarker of mtUPR and plays a crucial role in maintaining mitochondrial function. Activated transcription factor 5 (Atf5) is a key regulator of mtUPR in mammals, and studies have shown that Atf5 is essential for organelle recovery after mitochondrial stress in mammalian cells. Currently, the effects of Atf5 on mitochondrial function and developmental capacity in frozen oocytes are unknown.

[0004] RNA interference (RNAi) is an effective method for regulating target gene expression, efficiently and specifically inhibiting the expression of target mRNAs. Exogenous small interfering RNAs (siRNAs) are the main RNAi molecules, inducing the formation of silencing complexes to achieve posttranscriptional gene regulation. RNAi has proven to be a highly adaptable and powerful therapeutic strategy, with several siRNA drugs already approved and several RNAi-based therapies advancing into clinical trials. However, the use of Atf5-targeting siRNAs to improve the developmental capacity of frozen oocytes has not yet been reported. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes siRNAs that enhance the developmental capacity of cryopreserved oocytes and their applications. A siRNA molecule that inhibits Atf5 gene expression was discovered, and its impact on the developmental capacity of cryopreserved oocytes was evaluated, with the aim of maintaining mitochondrial function and improving the developmental capacity of cryopreserved oocytes under cryopreservation stress.

[0006] To achieve the above objectives, the present invention provides an siRNA molecule that enhances the developmental capacity of cryopreserved oocytes, the nucleotide sequence of which is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0007] The present invention also provides the application of the siRNA molecule in the preparation of formulations that enhance the developmental capacity of cryopreserved oocytes.

[0008] Preferably, the cryopreservation is performed at -196°C.

[0009] Preferably, the siRNA molecule maintains mitochondrial function and improves the developmental capacity of cryopreserved oocytes by inhibiting the expression of the Atf5 gene.

[0010] More preferably, the nucleotide sequence of the Atf5 gene is shown in SEQ ID NO.3.

[0011] The present invention also provides a formulation for improving the developmental capacity of cryopreserved oocytes, comprising the siRNA molecule.

[0012] Preferably, the formulation further includes pharmaceutically acceptable excipients.

[0013] Preferably, the preparation is an injectable formulation.

[0014] The present invention also provides a nucleic acid construct for improving the developmental capacity of cryopreserved oocytes, the nucleic acid construct comprising the siRNA molecule.

[0015] The present invention also provides a method for improving the developmental capacity of cryopreserved oocytes, comprising the following steps: after obtaining oocytes, injecting the siRNA molecule, and then cryopreserving them.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] This invention uses mouse oocytes as a model to silence the Atf5 gene in oocytes via RNA interference, thereby maintaining mitochondrial function under cryopreservation stress, enhancing oocyte cryopreservation tolerance, and improving the developmental capacity of frozen oocytes. A siRNA targeting and inhibiting Atf5 gene expression is provided, with nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2. In oocyte cryopreservation, the siRNA is microinjected into the oocyte cytoplasm. The siRNA specifically silences the target gene mRNA expression. Results show that Atf5 gene expression in oocytes is significantly reduced after siRNA injection, mitochondrial function is maintained under cryopreservation stress, and the developmental capacity of frozen oocytes is improved. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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 these drawings without creative effort.

[0019] Figure 1 To determine the effect of freezing on oocyte developmental capacity, A shows representative images of GVBD and PBE in fresh and frozen oocytes, with a scale bar of 100 μm; B shows a graph analyzing the proportion of GVBD and PBE in fresh and frozen oocytes.

[0020] Figure 2 To determine the effect of freezing on mitochondrial function in oocytes, the following measurements were taken: A) TMRM fluorescence staining to detect mitochondrial membrane potential in fresh and frozen oocytes (scale bar: 100 μm); B) TMRM fluorescence intensity analysis in fresh and frozen oocytes; C) 2'7'-DCFHDH fluorescence staining to detect ROS levels in fresh and frozen oocytes (scale bar: 100 μm); D) 2'7'-DCFHDH fluorescence intensity analysis in fresh and frozen oocytes; and E) ATP content detection in fresh and frozen oocytes.

[0021] Figure 3 To determine the effect of freezing on the expression of ATF5 and HSP60 in oocytes, A shows the immunofluorescence staining images of ATF5 in fresh and frozen oocytes (scale bar: 100 μm), B shows the immunofluorescence staining images of HSP60 in fresh and frozen oocytes (scale bar: 50 μm), C shows the fluorescence intensity analysis of ATF5 in fresh and frozen oocytes, and D shows the fluorescence intensity analysis of HSP60 in fresh and frozen oocytes.

[0022] Figure 4 To determine the effect of low Atf5 expression on the developmental capacity of frozen oocytes, A shows that injection of siRNA can reduce Atf5 expression in oocytes; B shows representative images of GVBD and PBE in frozen and siAtf5-frozen oocytes (scale bar: 100 μm); and C shows the proportion of GVBD and PBE in frozen and siAtf5-frozen oocytes.

[0023] Figure 5 To determine the effect of low Atf5 expression on mitochondrial function in frozen oocytes, A shows the detection of mitochondrial membrane potential in frozen and siAtf5-frozen oocytes by TMRM fluorescence staining (scale bar: 100 μm), B shows the analysis of TMRM fluorescence intensity in frozen and siAtf5-frozen oocytes, and C shows the expression analysis of genes related to mitochondrial dynamics in frozen and siAtf5-frozen oocytes.

[0024] Figure 6 To determine the effect of low Atf5 expression on mitochondrial function in fresh oocytes, the following measurements were taken: A) TMRM fluorescence staining to detect mitochondrial membrane potential in fresh and siAtf5 oocytes (scale bar: 100 μm); B) TMRM fluorescence intensity analysis in fresh and siAtf5 oocytes; C) ATP content analysis in fresh and siAtf5 oocytes; and D) expression analysis of genes related to mitochondrial dynamics in fresh and siAtf5 oocytes. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1

[0031] Atf5 gene expression analysis in frozen oocytes

[0032] 1. Oocyte Acquisition

[0033] Six-week-old female ICR strain mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were kept under a 12-hour dark / 12-hour light cycle and allowed free access to food and water. After a one-week acclimatization period, they were used in experiments. Mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG). Forty-eight hours later, the mice were euthanized by cervical dislocation. The ovaries were removed and placed in pre-equilibrated HX-M199 solution (4 hours prior), and gently rinsed to remove excess impurities. The ovaries were then transferred to clean culture dishes, and the ovaries were thoroughly minced using a sterile blade to release the follicular contents. Pre-equilibrated HX-M199 solution was then added again, and cumulus-oocyte complexes (COCs) were collected using a pipette into fresh pre-equilibrated HX-M199 solution. COCs were aspirated using a pipette slightly larger than the oocyte diameter to completely detach the granulosa cells.

[0034] 2. Oocyte freezing and thawing

[0035] Freezing method: First, equilibrate the oocytes in a pretreatment solution (10% EG (v / v) + 10% DMSO (v / v)) for 30 seconds, then transfer them to a vitrification solution (Dulbecco's phosphate-buffered saline (DPBS) containing 0.5 M sucrose, 30% Ficoll (w / v), 15% EG (v / v) and 15% DMSO (v / v), hold for 25 seconds, and aspirate the tip of the OPS. Then, rapidly immerse the OPS containing the oocytes in liquid nitrogen for storage (-196°C).

[0036] Thawing method: Remove the OPS capillary tube from liquid nitrogen and quickly immerse it in thawing medium (DPBS containing 0.5M sucrose) for 5 minutes.

[0037] 3. In vitro maturation of oocytes

[0038] Oocytes were washed three times in IVM medium (Cat. M7292, Sigma) and then cultured in IVM. After 2 h or 12 h of IVM culture, the GVBD rate and PBE rate were recorded, respectively.

[0039] 4. Mitochondrial membrane potential detection

[0040] Mitochondrial membrane potential was measured using the TMRM fluorescent probe (Cat. I34361, Invitrogen). Oocytes were stained with 100 nM TMRM working solution in M2 medium containing milrinone for 30 min at 37°C. After washing three times with M2 medium containing milrinone, images were captured using an Olympus IX71 inverted fluorescence microscope. Fluorescence intensity was measured using EZ-C1 FreeViewer software, and the average fluorescence intensity per unit area within the ROI was determined to quantify the fluorescence of individual oocyte images.

[0041] 5. Detection of reactive oxygen species (ROS) levels

[0042] ROS levels were measured using the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFHDA; Cat.D399, Invitrogen). Oocytes were stained with 1 mM 2',7'DCFHDA working solution diluted with M2 medium containing milrinone for 30 min at 37°C. After washing three times with M2 medium containing milrinone, images were captured using an Olympus IX71 inverted fluorescence microscope. Fluorescence intensity was measured using EZ-C1 FreeViewer software, and the average fluorescence intensity per unit area within the ROI was determined to quantify the fluorescence of individual oocyte images.

[0043] 6. ATP level detection

[0044] ATP levels were measured using an enhanced ATP assay kit (Cat.S0027, Beyotime). Oocytes (10 oocytes per sample) were transferred to 50 μL of lysis buffer and vortexed to lyse the cells. The ATP assay working solution (50 μL / well) was added to a new 96-well plate and incubated at room temperature for 5 min. Five standard wells were set up, with standard samples (0 μL, 2 μL, 4 μL, 8 μL, and 16 μL) added to the 96-well plate. The volume was then adjusted to 50 μL with lysis buffer. For the sample wells, 50 μL of sample lysis buffer was added. ATP levels were measured using an Infinite F200 microplate reader.

[0045] 7. Immunofluorescence staining detection

[0046] GV stage oocytes were fixed for 30 min at room temperature in immunofluorescence staining fixation solution (Cat. P0098, Beyotime), permeabilized for 1 h at room temperature with PBS solution containing 0.1% Triton X-100 and 0.1% PVA, and then blocked for 1 h at room temperature in blocking solution (PBS solution containing 3% BSA, 0.1% Triton X-100, and 0.1% PVA). The oocytes were then incubated overnight at 4°C with anti-Atf5 (1:200, Cat. AF2563, Beyotime) and anti-HSP60 (1:200, Cat. bs-0191R, Bioss) antibodies. Images were captured and analyzed using a Nikon A1 confocal microscope.

[0047] 8. Data Analysis

[0048] All statistical analyses were performed using GraphPadPrism8 software. Independent t-tests and chi-square tests were used to assess differences between the two groups. All experiments were performed at least three times, and results are expressed as mean ± SEM. A p-value < 0.05 was considered statistically significant.

[0049] result

[0050] 1. Freezing can lead to a decrease in the developmental capacity of oocytes.

[0051] Representative images of GVBD and PBE during in vitro maturation of fresh and frozen oocytes are shown below. Figure 1 As shown in Figure A, statistical analysis indicated that freezing significantly reduced the proportions of GVBD (80.64±2.15 vs 71.80±2.21, P<0.05) and PBE (90.74±2.32 vs 68.86±7.93, P<0.05) oocytes (e.g., Figure 1 (B)

[0052] 2. Freezing can cause abnormal mitochondrial function in oocytes.

[0053] Detection of mitochondrial membrane potential in fresh and frozen oocytes, such as Figure 2 As shown in Figure A. Compared with the fresh group, the mitochondrial membrane potential of frozen oocytes was significantly decreased (9838.00±205.90 vs 8350.00±162.50, P<0.01, as shown in Figure A). Figure 2 As shown in Figure B). Figure 2 As shown in Figure C, 2'7'-DCFHDH fluorescent staining was used to detect ROS levels in oocytes. ROS levels in frozen oocytes were significantly higher than those in fresh oocytes (9690.00±111.90 vs 10567.00±245.20, P<0.01). Figure 2 (As shown in D). Furthermore, ATP levels in frozen oocytes were significantly decreased (1.20±0.02 vs 1.01±0.02, P<0.01, as shown in Figure D). Figure 2 (As shown in E).

[0054] 3. Freezing can cause abnormally high levels of ATF5.

[0055] Immunofluorescence staining to detect ATF5 protein expression in oocytes (e.g.) Figure 3 As shown in Figure A), the results showed that ATF5 expression in frozen oocytes was significantly increased (152.40±10.34 vs 416.40±53.08, P<0.01). Figure 3 (As shown in Figure B). Further detection of the expression of the mtUPR biomarker HSP60 (e.g., Figure 3Immunofluorescence staining results showed that HSP60 expression in the frozen group was significantly higher than that in the fresh group (543.90±22.54 vs 669.10±27.24, P<0.01). Figure 3 (As shown in D).

[0056] Example 2

[0057] Low expression of Atf5 can improve the developmental capacity of frozen oocytes.

[0058] The methods for oocyte acquisition, freezing and thawing, in vitro maturation, and mitochondrial membrane potential detection are the same as in Example 1. The difference from Example 1 is that after oocyte acquisition, siRNA that inhibits Atf5 expression is microinjected to construct oocytes with low Atf5 expression. The changes in mitochondrial functional parameters such as mitochondrial dynamics after freezing are evaluated. The specific protocol is as follows:

[0059] 1. siRNA sequence

[0060]

[0061] Three pairs of siRNAs were designed based on the Atf5 nucleotide sequence, and a negative control siRNA was used as a control. All siRNA sequences are shown in Table 1 below:

[0062] Table 1 siRNA sequences

[0063] Gene Sequence (5'-3') Serial Number NC-siRNA-F UUCUCCGAACGUGUCACGUTT SEQ ID NO.4 NC-siRNA-R ACGUGACACGUUCGGAGAATT SEQ ID NO.5 Atf5-siRNA#722-F GCCCUUGCCCACCUUUGACTT SEQ ID NO.1 Atf5-siRNA#722-R GUCAAAGGUGGGCAAGGGCTT SEQ ID NO.2 Atf5-siRNA#820-F GCUUGUCAACCCUGCCUGUTT SEQ ID NO.6 Atf5-siRNA#820-R ACAGGCAGGGUUGACAAGCTT SEQ ID NO.7 Atf5-siRNA#964-F UCAGGUACCGCCAGAGGAATT SEQ ID NO.8 Atf5-siRNA#964-R UUCCUCUGGCGGUACCUGATT SEQ ID NO.9

[0064] 2. siRNA microinjection

[0065] The siRNA was diluted to 20 μM, and approximately 10 pL of siRNA was injected intracytoplasm into GV-stage oocytes using a FemtoJet4i microinjection apparatus (Eppendorf). The injected oocytes were then cultured for 24 h in M2 medium containing 2.5 μM milrinone at 37°C and 5% CO2. The V_NC group and V_si group represent oocytes injected with negative siRNA and Atf5 siRNA sequences, respectively.

[0066] 3. Detection of mitochondrial dynamics gene expression

[0067] RNA was extracted using the TRIzol chloroform extraction method. PrimeScript TM RT Master Mix (Perfect RealTime; Cat.RR036A, Takara) was used for reverse transcription to cDNA. Primer efficiency was tested to ensure specificity. Prepare the reaction system using Tip Green qPCR SuperMix (+Dye II) (Cat. AQ142-22, Transgen). Perform qRT-PCR using an ABI 7500 qRT-PCR system (Applied Biosystems). 2 -△△Ct The method was used to calculate the relative expression levels of the target genes, with Ppia and Rpl7 used as internal reference genes. Table 2 lists all primer sequences.

[0068] Table 2 Primer sequences

[0069]

[0070]

[0071] result

[0072] 1. Low expression of Atf5 can improve the developmental capacity of frozen oocytes.

[0073] like Figure 4As shown in Figure A, microinjection of Atf5 siRNA significantly reduced Atf5 expression in oocytes, with the Atf5-siRNA#722-F (SEQ ID NO.1) and Atf5-siRNA#722-R (SEQ ID NO.2) groups showing the most significant reduction in Atf5 expression. The development of frozen oocytes in the negative control group (V_NC) and the selected Atf5-siRNA#722-F (SEQ ID NO.1) and Atf5-siRNA#722-R (SEQ ID NO.2) injection groups (V_si) is shown below. Figure 4 As shown in Figure B, the proportion of GVBD in frozen oocytes in the V_si group was significantly increased (60.02±1.73 vs 68.52±1.91, P<0.05). Figure 4 (As shown in C), and the PBE ratio has also improved to some extent.

[0074] 2. Low expression of Atf5 can improve mitochondrial function in frozen oocytes.

[0075] Detection of mitochondrial membrane potential in two groups of frozen oocytes (e.g.) Figure 5 The results showed that the mitochondrial membrane potential of frozen oocytes injected with siRNA molecules Atf5-siRNA#722-F (SEQ ID NO.1) and Atf5-siRNA#722-R (SEQ ID NO.2) was significantly increased (6655.00±204.40 vs 7463.00±268.20, P<0.05). Figure 5 (As shown in B). Further analysis revealed that, compared with the V_NC group, the expression of mitochondrial dynamics-related genes (Drp1, Opa1) was significantly increased in the V_si group (e.g., ...). Figure 5 (As shown in C).

[0076] Example 3

[0077] Low expression of Atf5 is closely related to mitochondrial function in fresh oocytes.

[0078] The methods for oocyte acquisition, in vitro maturation, mitochondrial membrane potential, and ATP content detection were the same as in Example 1. The siRNA molecules Atf5-siRNA#722-F (SEQ ID NO.1) and Atf5-siRNA#722-R (SEQ ID NO.2) were selected. Oocytes with low Atf5 expression were constructed by microinjection. The methods for mitochondrial dynamics-related gene expression were the same as in Example 2. The effect of low Atf5 expression on mitochondrial function of fresh oocytes was evaluated.

[0079] result

[0080] TMRM staining was used to detect the mitochondrial membrane potential (MMT) of oocytes in the fresh negative control group (F_NC) and the groups injected with siRNA molecules Atf5-siRNA#722-F (SEQ ID NO.1) and Atf5-siRNA#722-R (SEQ ID NO.2) (F_si). Figure 6 As shown in Figure A), compared with F_NC, the mitochondrial membrane potential of the F_si group was significantly decreased (7410.00±217.20 vs 6664.00±230.50, P<0.05). Figure 6 As shown in B), and as Figure 6 As shown in Figure C, the ATP level in the F_si group was significantly decreased (0.98±0.01 vs 0.85±0.04, P<0.01). Further analysis of mitochondrial dynamics-related gene expression in oocytes from both groups revealed that the expression of Drp1, Fis-1, Opa1, Mfn1, and Mfn2 in the F_si group oocytes was significantly decreased (e.g., ...). Figure 6 (D).

[0081] In summary, the above results indicate that elevated Atf5 levels in frozen oocytes are associated with mitochondrial dysfunction and decreased oocyte developmental capacity. Using the siRNA method, the siRNA molecules Atf5-siRNA#722-F (SEQ ID NO.1) and Atf5-siRNA#722-R (SEQ ID NO.2) were prepared, which can effectively inhibit Atf5 expression, thereby specifically maintaining mitochondrial function in frozen oocytes and improving their developmental capacity.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of siRNA molecules, characterized in that, The siRNA molecule is used to improve the developmental capacity of cryopreserved oocytes, and its nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The use of the siRNA molecule as described in claim 1 in the preparation of a formulation to enhance the developmental capacity of cryopreserved oocytes.

3. The application according to claim 2, characterized in that, The cryopreservation is performed at -196°C.

4. The application of an siRNA molecular formulation, characterized in that, The siRNA molecule is used to improve the developmental ability of cryopreserved oocytes, and is the siRNA molecule described in claim 1.

5. The application according to claim 4, characterized in that, The formulation also includes pharmaceutically acceptable excipients.

6. The application according to claim 4, characterized in that, The preparation is an injectable formulation.

7. A nucleic acid construct used to improve the developmental capacity of cryopreserved oocytes, characterized in that, The nucleic acid construct includes the siRNA molecule of claim 1.

8. A method for improving the developmental capacity of cryopreserved oocytes, characterized in that, Includes the following steps: After obtaining oocytes, the siRNA molecule of claim 1 is injected and then cryopreserved.

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