Embryo culture method and embryo culture solution for improving quality of pig somatic cell nuclear transfer embryo

Abstract

The invention discloses an embryo culture method and an embryo culture solution for improving the quality of a pig somatic cell nuclear transfer embryo, and a small molecule substance X1 capable of specifically binding to a RepA structural domain of Xist is added into a pig early embryo culture solution, so that the development quality of a pig SCNT embryo is improved.

Description

Technical Field

[0001] The invention relates to pig in vitro embryo culture, in particular to an early embryo culture solution added with a small molecule substance X1 and an early embryo culture method for improving the quality of pig somatic cell nuclear transplantation embryos. Background Art

[0002] Somatic cell nuclear transfer (SCNT) technology can enable highly differentiated somatic cells to acquire totipotency through reprogramming, thereby developing into new animal individuals. However, the cloning efficiency of SCNT technology has been extremely low for a long time, mainly manifested in the high rate of early embryonic development, death of cloned animals during pregnancy or perinatal period, and developmental abnormalities of cloned animals after birth, which seriously hinders the promotion and application of this technology. It is widely believed that abnormal histone modification, failure of donor cells to be fully reprogrammed, and zygotic genome activation disorders are the main reasons for the low cloning efficiency of SCNT technology.

[0003] X-chromosome inactivation (XCI) refers to the inactivation of one X chromosome in female mammals to achieve the same gene dosage expression as male mammals. XCI is mainly caused by Xist (X-inactive specific transcript) expression regulation, Xist High expression leads to downregulation of X-linked gene expression throughout the X chromosome, resulting in abnormal histone modification and XCI abnormalities. For example, some blastomeres of SCNT female embryos have two Xist All loci are highly expressed and both X chromosomes are inactivated. The only X chromosome in some blastomeres of male cloned embryos is also highly expressed. Xist , and inactivation occurs. In practice, many cloned animals fail due to reprogramming errors. Xist Abnormal expression and XCI abnormality. Abnormal expression of X-linked genes was detected in the stillbirths of cloned cattle and cloned pigs. However, XCI abnormality is not the only reason for abnormal embryo development and reduced number of blastocysts, which affects the quality and efficiency of cloned embryos. For example, in the early embryo culture of pig cloned embryos (pig oocyte parthenogenetic embryos), there is a problem of poor blastocyst quality caused by delayed X chromosome inactivation (see: CN118813526A).

[0004] Studies have shown (see: Targeting Xist with compounds that disrupt RNAstructure and X inactivation) that the small molecule X1 can specifically bind to the RepA domain of Xist in vitro and in vivo. The binding of the small molecule X1 reduces the conformational space of RepA, replaces homologous interacting protein factors (PRC2 and SPEN), inhibits histone H3K27me3, and inhibits the initiation of X chromosome inactivation. However, the small molecule X1 in this study was used to treat embryonic stem cells, and it was found that it would affect the embryoid growth of female embryonic stem cells after treatment. At the same time, only the X chromosome inactivation itself was focused on, and it was not related to the blastocyst rate (and the quality of early embryo development). Summary of the invention

[0005] The purpose of the present invention is to provide an embryo culture method and embryo culture medium for improving the quality of pig somatic cell nuclear transfer embryos, and to improve the development quality of pig somatic cell nuclear transfer (SCNT) embryos by adding small molecule substance X1 to the in vitro early embryo culture medium.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: In one aspect, a method for culturing an embryo is provided, comprising the following steps: The porcine SCNT embryos were constructed and transferred into porcine early embryo culture medium and supplemented with 4-15 μmol·L -1 The pig SCNT embryo is then cultured to the blastocyst stage, and the pig early embryo culture medium contains 1-2 mmol·L -1 (i.e. 1-2 mM) glutamine and 5-8 mmol·L -1 (i.e. 5-8 mM) hypotaurine.

[0007] Preferably, the small molecule substance X1 is added to the pig early embryo culture medium to a final concentration of 5 to 10 μmol·L -1 The small molecule X1 inhibited histone H3K27me3 in pig SCNT embryos, reduced the occurrence of abnormal X chromosome inactivation in pig SCNT embryos, and improved the development quality of pig SCNT embryos. Preferably, the small molecule substance X1 is added to the pig early embryo culture medium to a final concentration of 5 to 8 μmol·L -1 The addition of small molecule substance X1 can increase the number of blastocyst cells and the blastocyst rate.

[0008] Preferably, the porcine early embryo culture medium is supplemented with 1-2 mmol·L -1 Glutamine and 5-8 mmol·L-1 Hypotaurine PZM-3.

[0009] Preferably, the culture conditions of the porcine SCNT embryos also include: 38.0-38.5°C, 4.8%-5.2% CO 2 , 94.8%~95.2% N 2 , and saturated humidity; or, 38.0-38.5°C, 4.5%-5.5% CO 2 , 94.5%~95.5% N 2 , and saturated humidity.

[0010] Preferably, the construction of porcine SCNT embryos specifically comprises the following steps: culturing porcine oocytes in an in vitro maturation culture medium for 44 to 46 hours, then forming reconstructed embryos through nuclear transplantation with porcine somatic cells, and fusing the reconstructed embryos to obtain porcine SCNT embryos to be cultured (for example, the reconstructed embryos can be transferred into porcine early embryo culture medium after being electrofused and recovered).

[0011] Preferably, the porcine somatic cells are selected from porcine fibroblasts that have been starved in advance (generally, the culture medium of porcine fibroblasts is replaced with a serum-free cell culture medium 22 to 24 hours before somatic cell nuclear transplantation, thereby performing starvation treatment).

[0012] In another aspect, a porcine SCNT embryo culture medium is provided, wherein the embryo culture medium comprises a basal medium and a 4-15 μmol·L -1 Small molecule substance X1.

[0013] Preferably, the embryo culture medium comprises a basal culture medium and a final concentration of 5 to 10 μmol·L -1 Small molecule substance X1.

[0014] Preferably, the embryo culture medium comprises a basal culture medium and a 5- to 8-μmol·L -1 Small molecule substance X1.

[0015] Preferably, the basic culture medium comprises the following components: 107-109 mmol·L -1 NaCl, 9-11 mmol·L -1 KCl, 0.33~0.36 mmol·L -1 KH 2 PO 4 , 0.35~0.45 mmol·L -1 MgSO 4 , 25~26 mmol·L -1 NaHCO 3 , 0.18~0.22 mmol·L -1 Sodium pyruvate, 1.8-2.2 mmol·L -1 Calcium lactate pentahydrate, 1.8% to 2.1% (v / v) essential amino acid solution BME, 0.9% to 1.1% (v / v) non-essential amino acid solution MEM, 2.8 to 3.2 g·L -1 BSA and 9.5-10.5 μg mL -1 Gentamicin.

[0016] Preferably, the basal culture medium is selected from the following porcine early embryo culture medium: PZM-3, or improved PZM-3.

[0017] Preferably, the improved PZM-3 is added with 1-2 mmol·L -1 Glutamine and 5-8 mmol·L -1 Hypotaurine PZM-3.

[0018] The beneficial effects of the present invention are embodied in: The present invention effectively prevents X chromosome inactivation abnormality of pig SCNT embryos and improves the development quality of pig SCNT embryos by adding a small molecule X1 (the small molecule X1 is known to have the activity of specifically binding to the RepA domain of Xist) within a certain concentration range to pig early embryo culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the structural formula of small molecule substance X1.

[0020] Figure 2A The results of immunofluorescence staining of the cell number in the control group porcine somatic cell nuclear transplanted blastocyst (one embryo was randomly selected on the 7th day).

[0021] Figure 2B 5 μmol·L -1 The results of immunofluorescence staining observation of cell number in porcine somatic cell nuclear transplanted blastocysts (one embryo was selected on the 7th day) in group X1.

[0022] Figure 2C 10 μmol·L -1 The results of immunofluorescence staining observation of cell number in porcine somatic cell nuclear transplanted blastocysts (one embryo was selected on the 7th day) in group X1. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention, but not to limit the scope of protection of the present invention.

[0024] 1. Source and preparation of reagents (1) PBS for embryos Formula: 0.203 mg·mL -1 KCl, 8.05 mg mL -1 NaCl, 0.121 mg mL -1 MgCl 2 , 0.036 mg·mL -1 Sodium pyruvate, 1 mg mL -1 D-glucose, 1.153 mg·mL -1 Na 2 HPO 4 , 0.2 mg·mL -1 KH 2 PO 4 , 0.132mg·mL -1 CaCl 2 ·2H 2 O, 0.121 mg mL -1 MgCl 2 6H 2 O and 3% (v / v) FBS.

[0025] (2) Oocyte maturation medium OM Formula: 3.05 mmol·L -1 Glucose, 0.91 mmol·L -1 Sodium pyruvate, 0.57 mmol·L -1 L-cysteine, 1% (w / v) ITS-G, 0.1% (w / v) PVA, 10 IU·mL -1 eCG, 10 IU·mL -1 hCG, 2.5 IU·mL -1 FSH, 40 ng·mL -1 FGF2, 20 ng mL -1 IGF1, 20 ng·mL -1 LIF, 10 ng mL -1 EGF and 10 μg mL -1 Gentamicin.

[0026] (3) 0.1% hyaluronidase Dissolve the hyaluronidase powder in embryo PBS at a concentration of 0.1% (w / v), filter and aliquot, and store at 4 °C.

[0027] (4) Pig early embryo culture medium After the solution is prepared according to the formula of PZM-3, 1 mM glutamine and 5 mM hypotaurine are added (to the final concentration), which is the improved PZM-3. The glutamine in the improved PZM-3 improves the ability of the embryo to develop to the blastocyst stage, and the hypotaurine has an antioxidant effect, which can overcome the oxidative effect of the embryo's metabolism during development, thereby overcoming the developmental block phenomenon.

[0028] Improved PZM-3 formula: 108 mmol·L -1 NaCl, 10 mmol·L -1 KCl, 0.35 mmol·L -1 KH 2 PO 4 , 0.4 mmol·L -1 MgSO 4 、25.07 mmol·L -1 NaHCO 3 , 0.2 mmol·L -1 Sodium pyruvate, 2 mmol·L -1 Calcium lactate pentahydrate, 2% (v / v) essential amino acid solution BME, 1% (v / v) non-essential amino acid solution MEM, 3 g·L -1 BSA, 10 μg mL -1 Gentamycin, 1 mmol·L -1 Glutamine and 5 mmol·L -1 Hypotaurine.

[0029] The non-essential amino acid solution MEM was purchased from Sigma, product number M7145 (w / v), and the formula is as follows: 0.89 g·L -1 L-alanine, 1.5 g L -1 L-asparagine, 1.33 g·L -1 L-aspartic acid, 1.47 g·L -1 L-glutamic acid, 0.75 g·L -1 Glycine, 1.15 g·L -1 L-proline and 1.05 g·L -1 L-serine.

[0030] The essential amino acid solution BME was purchased from Sigma, product number B6766 (w / v), and the formula is as follows: 1.05 g·L -1 L-arginine, 0.6 g·L -1 L-cysteine, 0.4 g·L -1 L-histidine, 1.3 g L -1 L-isoleucine, 1.3 g·L-1 L-Leucine, 1.849 g·L -1 L-lysine, 0.375 g·L -1 L-methionine, 0.825 g·L -1 L-phenylalanine, 1.2 g·L -1 L-threonine, 0.2 g·L -1 L-Tryptophan, 0.9 g·L -1 L-tyrosine and 1.175 g·L -1 L-Valine.

[0031] (5) Small molecule substance X1 (SPEN-IN-1) The structural formula of small molecule X1 is as follows Figure 1 The product was purchased from Taoshu Biological Technology Co., Ltd. with the serial number T60044. The product was diluted with DMSO and the storage concentration was 1 mmol·L -1 . The 1 mmol·L -1 The small molecule substance X1 stock solution was diluted to 5 μmol·L -1 and 10 μmol·L -1 , i.e., to prepare a solution containing 5 μmol·L -1 and 10 μmol·L -1 The culture medium of small molecule substance X1 was used as the subsequent 5 μmol·L -1 X1 group and 10 μmol·L -1 The porcine SCNT embryo culture experiment in group X1 (specifically, the modified PZM-3 was added with a final concentration of 5 μmol·L -1 and 10 μmol·L -1 Small molecule substances X1).

[0032] (6) Cytochalasin B (CB), PBS-PVA, and BTX solution were purchased from Sigma.

[0033] 2. Collection and culture of porcine oocytes Pig ovaries were obtained from a local slaughterhouse in Yangling (April to August 2023), placed in 30-35°C saline with penicillin-streptomycin, and sent to the laboratory within 2 hours. 75% alcohol and saline were preheated to 38.5°C in advance, and the blood stains on the ovaries were washed with 75% alcohol first, and then the ovaries were washed 3-4 times with saline, and finally the ovaries were placed in a 38.5°C water bath to keep warm. A negative pressure pump was connected to a blood collection tube equipped with a No. 9 syringe needle to extract follicular fluid from ovarian follicles with a diameter of 3 to 8 mm, collected in a 15 mL centrifuge tube, centrifuged at 50 g for 1 min, and the supernatant was discarded until 2 to 3 mL of precipitate and follicular fluid remained in the tube. 2 mL of resuspended follicular fluid was added to a 60 mm culture dish, and cumulus-oocyte complexes with three or more layers of granulosa cells and uniformly black cytoplasm were selected under a stereomicroscope. The complexes were placed in PBS for embryos, washed clean, and washed once in OM, and cultured in OM that had been equilibrated for more than 2 h and covered with paraffin oil. The incubator conditions were 38.5°C, 5% CO 2 , and saturated humidity.

[0034] 3. Somatic Cell Nuclear Transfer After the oocyte (specifically the cumulus oocyte complex) is cultured for 46 hours, it can be taken out and placed under a stereo microscope with a hot stage. At this time, take a suitable micropipette, insert the tip, sterilize the tip with a flame and burn it blunt, then blow it. When most of the granulosa cells around the oocyte are blown naked, use an egg picking needle to absorb the oocyte, move it into 0.1% hyaluronidase (Hy), use a micropipette to blow it repeatedly for no more than 5 minutes, and then pick it out with an egg picking needle. Put it in the embryo PBS that has been balanced in advance, adjust the refractive angle under the stereo microscope (to make it more three-dimensional so that the first polar body can be seen), and gently blow the oocyte with an egg picking needle. The dorsal polar body can be seen, and the oocytes with polar bodies are collected and placed in the pre-balanced OM for operation. Balance 1 droplet of cytochalasin B (CB) operating solution and 1 droplet of PBS-PVA operating solution in a 35mm culture dish, and then cover it with paraffin oil. The oocyte was placed in a microdroplet of CB operating solution, and then the oocyte was sucked onto the fixed needle. The polar body was exposed to the right side of the field of vision by moving the nucleus removal needle. At this time, the zona pellucida was inserted to start the aspiration of the cytoplasm and polar body. Then the pre-starved porcine fibroblasts (female and male, separated from the edge of the pig ear; collected in Rongchang District, Chongqing in August 2023) were added to PBS-PVA and resuspended. Then, the porcine fibroblasts were transferred into the microdroplet of PBS-PVA operating solution using a micropipette, and then the porcine fibroblasts of appropriate size were aspirated with the nucleus removal needle and injected into the cytoplasm and zona pellucida of the oocyte. After the nuclear transplantation was completed, the reconstructed embryo was placed in the pre-balanced OM for about 30 minutes. The pre-balanced BTX solution was taken out, made into a microdroplet and covered with oil, and the reconstructed embryo was moved into the BTX microdroplet. The fusion process was performed using the microelectrode method, in which the field strength of the electric fusion instrument was 2.0KV / cm, the frequency was single, and the time was 30μs. After the electric fusion, it was placed in the pre-balanced OM to recover for 1h.

[0035] 4. Early embryo culture After the reconstructed embryos recovered for 1 hour, embryos with fused somatic cells (i.e., pig SCNT embryos) were selected under a microscope and transferred into a culture medium supplemented with small molecule substance X1 for treatment (i.e., in vitro embryo culture test). One embryo was cultured with 10 μL of culture medium, and 50 embryos were cultured in a culture dish with 500 μL of culture medium. A control group was also set up. The experiment was divided into three groups: the control group (cultured with modified PZM-3), 5 μmol·L -1 Group X1 (using modified PZM-3 supplemented with 5 μmol·L -1 Small molecule substances x1 and cultured) and 10 μmol·L -1 X1 group (using modified PZM-3 supplemented with 10 μmol·L -1 Small molecule x1 and culture). Incubator conditions were 38.5 °C, 5% CO 2, 95% N 2 , and saturated humidity.

[0036] (V) Immunofluorescence staining of blastocyst H3K27me3 After 7 days of culture, the pig SCNT embryo (blastocyst) was fixed with immunofluorescence staining fixative at 4°C overnight, washed with PBS-PVA for 5 minutes, repeated 3 times, washed off the fixative, and permeabilized in 0.1% TritonX-100 at room temperature for 30 minutes. Repeat the washing steps, wash off the permeabilization solution, and place in immunofluorescence staining blocking solution for 2 to 3 hours at room temperature. Wash off the blocking solution and incubate in the primary antibody at 4°C overnight. Wash off the primary antibody, incubate with the secondary antibody at room temperature for 2 hours, and wash off the secondary antibody. Finally, stain with DAPI for 8 minutes. Draw a box on the slide with vaseline, add 2μL of anti-fluorescence quencher, place the cleaned embryo in the drop of anti-fluorescence quencher, cover with a coverslip and seal with transparent nail polish, and observe the results under a fluorescence microscope.

[0037] 6. Immunofluorescence staining of blastocyst cell number After 7 days of culture, the porcine SCNT embryos (blastocysts) were fixed with immunofluorescence staining fixative at 4°C overnight, washed with PBS-PVA for 5 minutes, and repeated 3 times. After washing away the fixative, the embryos were placed in DAPI nucleus staining for 8 minutes. A square was drawn on the slide with vaseline, and 2 μL of anti-fluorescence quencher was added. The cleaned embryos were placed in the drop of anti-fluorescence quencher, covered with a coverslip and sealed with transparent nail polish, and the results were observed under a fluorescence microscope.

[0038] (VII) Test results and analysis 1. Cleavage rate and blastocyst rate of porcine somatic cell nuclear transfer embryos After one day of in vitro culture, the cleavage rate was counted; on the seventh day, the blastocyst development rate (i.e., blastocyst rate) was counted.

[0039] Table 1. Effects of adding small molecule X1 on the development of nuclear transplanted embryos

[0040] Note: Table 1 * The difference was significant (P<0.05) compared with the control group.

[0041] As shown in Table 1, there was no significant difference in the cleavage rate between the treatment groups and the control group (P>0.05). -1 The blastocyst rate was significantly different between the X1 group and the control group (P<0.05). -1 The blastocyst rate of the X1 group was not significantly different from that of the control group (P>0.05). The results showed that the small molecule X1 had no effect on the cleavage of early pig embryos, and the addition of 5 μmol·L -1The small molecule substance X1 can increase the blastocyst rate of nuclear transfer embryos.

[0042] 2. Cell Number of Porcine Somatic Cell Nuclear Transfer Blastocysts Table 2. Cell number analysis of porcine somatic cell nuclear transfer blastocysts

[0043] Note: Table 2 * The difference was significant (P<0.05) compared with the control group.

[0044] The results showed (see Table 2) that 5 μmol·L -1 X1 group (i.e., 5 μmol·L -1 Small molecule X1) can significantly increase the number of cells in porcine somatic cell nuclear transplantation blastocysts (see also Figure 2A , Figure 2B , Figure 2C ).

[0045] 3. H3K27me3 and XCI in porcine somatic cell nuclear transfer blastocysts H3K27me3 staining was performed on female and male somatic cell nuclear transfer embryos (blastocysts) cultured to day 7, and the X chromosome inactivation of somatic cell nuclear transfer embryos was determined by the staining results. For female embryos, one H3K27me3 staining point on the nucleus indicates normal XCI, and two H3K27me3 staining points indicate abnormal XCI; for male embryos, one H3K27me3 staining point indicates abnormal XCI.

[0046] Table 3. H3K27me3 staining analysis of porcine female somatic cell nuclear transfer blastocysts

[0047] Table 4. H3K27me3 staining analysis of porcine male somatic cell nuclear transfer blastocysts

[0048] Note: Table 3 * Represents a significant difference (P < 0.05). Compared with the control group, except for the percentage of 1 H3K27me3 and 2 H3K27me3, the remaining percentage not shown is 0 H3K27me3; Table 4 * Represents a significant difference (P < 0.05). Compared with the control group, except for the percentage of 1 H3K27me3, the remaining percentage not shown is 0 H3K27me3.

[0049] From Table 3 and Table 4, we can see that 5 μmol·L -1 X1 group and 10 μmol·L -1X1 group could significantly reduce the abnormal X chromosome inactivation in both female and male pig somatic cell nuclear transfer embryos.

[0050] In summary, the present invention, through the in vitro culture test of pig SCNT embryos with the addition of small molecule substance X1 in early embryo culture medium and combined with immunofluorescence staining, proves that small molecule substance X1 increases the blastocyst rate and the total number of blastocyst cells of pig somatic cell nuclear transfer embryos and reduces XCI abnormalities of pig somatic cell nuclear transfer embryos. Therefore, adding small molecule substance X1 to pig early embryo culture medium plays an important role in improving the development quality of pig somatic cell nuclear transfer embryos and improving XCI abnormalities.

Claims

1. An embryo culture method, characterized in that: The following steps are involved: The porcine SCNT embryos were transferred into porcine early embryo culture medium and supplemented with 4-15 μmol·L -1 The small molecule substance X1, the pig early embryo culture medium contains 1-2 mmol·L -1 Glutamine and 5-8 mmol·L -1 Hypotaurine, and then culturing the porcine SCNT embryos to the blastocyst stage.

2. The embryo culture method according to claim 1, characterized in that: The small molecule substance X1 is added to the pig early embryo culture medium to a final concentration of 5 to 10 μmol·L -1 .

3. The embryo culture method according to claim 1, characterized in that: The small molecule substance X1 is added to the pig early embryo culture medium to a final concentration of 5 to 8 μmol·L -1 .

4. The embryo culture method according to claim 1, characterized in that: The porcine early embryo culture medium is supplemented with 1-2 mmol·L -1 Glutamine and 5-8 mmol·L -1 Hypotaurine PZM-3.

5. The embryo culture method according to claim 1, characterized in that: The construction of the porcine SCNT embryo specifically includes the following steps: culturing porcine oocytes in an in vitro maturation culture medium for 44 to 46 hours, then forming a reconstructed embryo through nuclear transplantation with porcine somatic cells, and fusing the reconstructed embryo to obtain a porcine SCNT embryo to be cultured.

6. A porcine SCNT embryo culture medium, characterized in that: The embryo culture solution comprises a basic culture medium and a final concentration of 4 to 15 μmol·L -1 Small molecule substance X1.

7. The porcine SCNT embryo culture medium according to claim 6, characterized in that: The embryo culture solution includes a basic culture medium and a final concentration of 5 to 10 μmol·L -1 Small molecule substance X1.

8. The porcine SCNT embryo culture medium according to claim 6, characterized in that: The embryo culture solution includes a basic culture medium and a final concentration of 5 to 8 μmol·L -1 Small molecule substance X1.

9. The porcine SCNT embryo culture medium according to claim 6, characterized in that: The basic culture medium includes the following components: 107-109 mmol·L -1 of NaCl, 9-11 mmol·L -1 KCl, 0.33~0.36 mmol·L -1 KH2PO4, 0.35~0.45 mmol·L -1 MgSO4, 25~26 mmol·L -1 NaHCO3, 0.18~0.22 mmol·L -1 Sodium pyruvate, 1.8-2.2 mmol·L -1 Calcium lactate pentahydrate, 1.8% to 2.1% essential amino acid solution BME, 0.9% to 1.1% non-essential amino acid solution MEM, 2.8 to 3.2 g·L -1 BSA and 9.5-10.5 μg mL -1 of gentamicin.

10. The porcine SCNT embryo culture medium according to claim 6, characterized in that: The basic culture medium is selected from PZM-3 or improved PZM-3, wherein the improved PZM-3 is supplemented with 1 to 2 mmol·L -1 Glutamine and 5-8 mmol·L -1 Hypotaurine PZM-3.

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