Use of gpld1 protein in preparation of reagent for inhibiting polyspermy of pig oocytes

Abstract

The application discloses application of GPLD1 protein in preparation of a reagent for inhibiting polyspermy of pig oocytes, and belongs to the technical field of animal husbandry. The application discloses application of GPLD1 protein in preparation of a reagent for inhibiting polyspermy of pig oocytes, 1000 ng / mL and 2000 ng / mL GPLD1 can significantly reduce the polyspermy incidence of pig oocytes in vitro, and improve the development capacity of in vitro fertilization embryos.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry technology, and more specifically to the application of GPLD1 protein in the preparation of reagents that inhibit polyspermy in porcine oocytes. Background Technology

[0002] In vitro fertilization (IVF) of porcine oocytes provides crucial research materials and methods for fundamental theoretical research and practical application of gametogenesis, fertilization, and early embryonic development mechanisms in pigs. This technology not only improves the utilization rate of superior germplasm resources and shortens the reproductive interval of core breeding stock, accelerating the genetic improvement process, but also provides technical support for biomedical research such as the construction of disease model pigs and the preparation of xenogeneic organ donors. Polyspermia is an abnormal fertilization process characterized by multiple sperm entering the oocyte, resulting in the production of polyploid zygotes and causing abnormal embryonic development. The incidence of polyspermia in porcine oocytes is as high as 50% to 90% in vitro, and close to 30-40% in vivo, far exceeding that of other species such as mice and humans. This directly restricts the establishment of an efficient in vitro production system for porcine embryos. Therefore, effectively inhibiting the incidence of polyspermia and ensuring the developmental quality of subsequent porcine embryos is one of the important directions of current IVF technology research.

[0003] Therefore, providing the application of GPLD1 protein in the preparation of reagents that inhibit polyspermy in porcine oocytes is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides the application of GPLD1 protein in the preparation of reagents that inhibit polyspermy in porcine oocytes, effectively inhibiting the occurrence of polyspermy and ensuring the quality of embryonic development in the later stages.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of GPLD1 protein in the preparation of reagents to inhibit polyspermy in porcine oocytes.

[0007] Furthermore, the application of GPLD1 in the preparation of drugs for regulating or intervening in polyspermy of porcine oocytes.

[0008] Furthermore, a porcine oocyte culture medium that inhibits polysperm fertilization is prepared by adding the following components to a HEPES-free M199 basal culture medium: 1 mg / mL cysteine, 0.44 mg / mL sodium pyruvate, 10 ng / mL epidermal growth factor, 10% porcine follicular fluid, 50 ng / mL insulin, 5 IU / mL gonadotropin, 5 IU / mL human chorionic gonadotropin, 11.3 ng / mL kanamycin, and 1000-2000 ng / mL GPLD1 protein.

[0009] Furthermore, the porcine oocyte culture medium is used in the preparation of a reagent to inhibit polysperm fertilization.

[0010] Furthermore, the porcine oocyte culture medium is used in the preparation of drugs that enhance the developmental capacity of in vitro fertilized embryos.

[0011] As can be seen from the above technical solution, compared with the prior art, this invention discloses the application of GPLD1 protein in the preparation of reagents to inhibit polyspermy in porcine oocytes. In vitro fertilization results of oocytes showed that the percentage of double pronuclei produced in the oocyte maturation culture groups with 1000 and 2000 ng / mL GPLD1 was significantly higher than that in the group without GPLD1. These results indicate that 1000 and 2000 ng / mL GPLD1 significantly reduced the incidence of polyspermy in in vitro porcine oocytes and improved the developmental capacity of in vitro fertilized embryos. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0013] Figure 1 For the present invention, immunofluorescence staining was performed on the MII group of the GV-control group and the group with exogenous GPLD1 (1000ng / mL); the first column red is GPLD1 staining, the second column is cortical granule CGs staining, the third column is bright field image, the fourth column is stained DNA, and the fifth column is a combination of all images in the first four columns.

[0014] Figure 2 This invention relates to different scenarios of oocyte fertilization;

[0015] Figure 3As the control group of this invention, the oocytes of the exogenously added GPLD1 group were used to statistically analyze the MII oocyte maturity rate (MII%), polyspermy rate (more than 2 pronuclear PN), 2C cleavage rate (2C%), and blastocyst rate (Blast%). The numbers show the p-values ​​of the significance between the two groups (p<0.05 is defined as significant). The MII oocyte maturity rate was calculated based on whether the first polar body (PB1) was expelled. The 2C cleavage rate was calculated based on whether the first cleavage occurred and whether the fertilized egg divided into two cells. The blastocyst rate was calculated based on whether the embryo had a blastocoel and blastocyst expansion. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] PVA-TLHEPES solution: Dissolve 6.6622 g of sodium chloride, 0.2311 g of potassium chloride, 0.1680 g of sodium bicarbonate, 0.0410 g of sodium dihydrogen phosphate, 1.443 mL of sodium lactate, 0.1017 g of magnesium chloride hexahydrate, 0.2940 g of calcium chloride dihydrate, 2.3830 g of HEPES, 0.0275 g of sodium pyruvate, 0.0750 g of kanamycin, 0.5000 g of polyvinyl alcohol, and 0.0100 g of phenol red in 1 L of ultrapure water, and adjust the osmotic pressure to 297 mOsm. All other experimental reagents were commercially available.

[0018] Example 1

[0019] 1) Porcine oocyte culture medium:

[0020] Composition of porcine oocyte culture medium: The following components were added to HEPES-free M199 basal culture medium (ThermoFisher-11150059): 1 mg / mL cysteine, 0.44 mg / mL sodium pyruvate, 10 ng / mL epidermal growth factor, 10% porcine follicular fluid, 50 ng / mL insulin, 5 IU / mL gonadotropin, 5 IU / mL human chorionic gonadotropin, 11.3 ng / mL kanamycin, and 1000 ng / mL GPLD1 protein (AntibodySystem, YHF70501).

[0021] 2) Preparation method of porcine oocyte culture medium:

[0022] (1) Obtaining porcine follicular fluid: Fresh porcine ovarian tissue was obtained from COFCO Meat Products Co., Ltd. in Wuhan, Hubei Province, and transported to the laboratory within 1 hour in a 38℃ physiological saline thermos. Subsequently, follicular fluid of 2-8 mm from the surface of the ovary was extracted using a syringe with a 12G needle and transferred to a 50 mL centrifuge tube. The centrifuge tube was placed in a 38℃ incubator for 30 minutes to allow the cumulus granulosa cell-oocyte complex (COCs) to settle at the bottom of the centrifuge tube. Then, the supernatant was separated by centrifugation at 3000 rpm for 30 minutes, and the supernatant was filtered through a 0.22 μm filter to obtain sterile porcine follicular fluid.

[0023] (2) Add cysteine, sodium pyruvate, epidermal growth factor, insulin, gonadotropin, human chorionic gonadotropin, kanamycin and GPLD1 to the M199 basic culture medium without HEPES to obtain the culture medium.

[0024] (3) Mix the culture medium obtained in step (2) with the sterile porcine follicle fluid obtained in step (1) to obtain porcine oocyte culture medium.

[0025] 3) Methods for preparing oocytes

[0026] Porcine ovaries were obtained and placed in a 38°C saline incubator, then transported to the laboratory within one hour. Follicular fluid from follicles 2–8 mm in diameter (GV stage) on the ovarian surface was then extracted using a syringe equipped with a 12G needle and injected into 50 mL centrifuge tubes. The centrifuge tubes were placed in a 38°C incubator and incubated for 30 minutes. During this time, cumulus-oocyte complexes (COCs) would deposit at the bottom of the centrifuge tube. After discarding the supernatant follicular fluid, an equal volume of PVA-TLHEPES solution was added, and the tubes were gently inverted to resuspend the COCs. Finally, the liquid was poured into 100 mm culture dishes, and well-morphologically sound COCs were selected under a stereomicroscope using a 500 μm glass needle. These COCs were then cultured in porcine oocyte culture medium (IVM) for 42 hours, after which mature oocytes were collected (one group of MII oocytes were processed).

[0027] Example 2

[0028] 1) Porcine oocyte culture medium:

[0029] Composition of porcine oocyte culture medium: The following components were added to HEPES-free M199 basal culture medium (ThermoFisher-11150059): 1 mg / mL cysteine, 0.44 mg / mL sodium pyruvate, 10 ng / mL epidermal growth factor, 10% porcine follicular fluid, 50 ng / mL insulin, 5 IU / mL gonadotropin, 5 IU / mL human chorionic gonadotropin, 11.3 ng / mL kanamycin, and 2000 ng / mL GPLD1 protein.

[0030] 2) Preparation method of porcine oocyte culture medium:

[0031] (1) Obtaining porcine follicular fluid: Fresh porcine ovarian tissue was obtained from COFCO Meat Products Co., Ltd. in Wuhan, Hubei Province, and placed in a 38℃ physiological saline thermos. It was transported to the laboratory within 1 hour. Subsequently, follicular fluid of 2-8 mm from the surface of the ovary was extracted using a syringe with a 12G needle and transferred to a 50 mL centrifuge tube. The centrifuge tube was placed in a 38℃ incubator for 30 minutes to allow the cumulus granulosa cell-oocyte complex (COCs) to settle at the bottom of the centrifuge tube. Then, the supernatant was separated by centrifugation at 3000 rpm for 30 minutes, and the supernatant was filtered through a 0.22 μm filter to obtain sterile porcine follicular fluid.

[0032] (2) Add cysteine, sodium pyruvate, epidermal growth factor, insulin, gonadotropin, human chorionic gonadotropin, kanamycin and GPLD1 to the M199 basic culture medium without HEPES to obtain the culture medium.

[0033] (3) Mix the culture medium obtained in step (2) with the sterile porcine follicle fluid obtained in step (1) to obtain porcine oocyte culture medium.

[0034] 3) Methods for preparing oocytes

[0035] Porcine ovaries were obtained and placed in a 38°C saline incubator, then transported to the laboratory within one hour. Follicular fluid from follicles (GV stage oocytes) at a depth of 2–8 mm on the ovarian surface was then extracted using a syringe equipped with a 12G needle and injected into 50 mL centrifuge tubes. The centrifuge tubes were placed in a 38°C incubator and incubated for 30 minutes. During this time, cumulus-oocyte complexes (COCs) would deposit at the bottom of the centrifuge tube. After discarding the supernatant follicular fluid, an equal volume of PVA-TLHEPES solution was added, and the tubes were gently inverted to resuspend the COCs. Finally, the liquid was poured into a 100 mm culture dish, and well-morphologically sound COCs were selected under a stereomicroscope using a 500 μm glass needle (at this point, some GV stage oocytes were collected for subsequent experiments). These were then cultured in porcine oocyte culture medium (IVM) for 42 hours, after which mature oocytes were collected (two groups of MII oocytes were treated).

[0036] Comparative Example 1

[0037] 1) Porcine oocyte culture medium:

[0038] Composition of porcine oocyte culture medium: The following components were added to M199 basal culture medium (ThermoFisher-11150059) without HEPES: 1 mg / mL cysteine, 0.44 mg / mL sodium pyruvate, 10 ng / mL epidermal growth factor, 10% porcine follicular fluid, 50 ng / mL insulin, 5 IU / mL gonadotropin, 5 IU / mL human chorionic gonadotropin, and 11.3 ng / mL kanamycin.

[0039] 2) The preparation method of porcine oocyte culture medium is the same as in Example 1, but without the addition of GPLD1 protein.

[0040] 3) Methods for preparing oocytes

[0041] Porcine ovaries were obtained and placed in a 38°C saline incubator, then transported to the laboratory within one hour. Follicular fluid from follicles 2–8 mm in diameter (GV stage) on the ovarian surface was then extracted using a syringe equipped with a 12G needle and injected into 50 mL centrifuge tubes. The centrifuge tubes were placed in a 38°C incubator and incubated for 30 minutes. During this time, cumulus-oocyte complexes (COCs) would deposit at the bottom of the centrifuge tube. After discarding the supernatant follicular fluid, an equal volume of PVA-TLHEPES solution was added, and the tubes were gently inverted to resuspend the COCs. Finally, the liquid was poured into 100 mm culture dishes, and well-morphologically sound COCs were selected under a stereomicroscope using a 500 μm glass needle. These COCs were then cultured in porcine oocyte culture medium (IVM) for 42 hours, after which mature oocytes (control group MII oocytes) were collected.

[0042] Experimental Example 1

[0043] Oocytes at the GV and MII stages were collected, and immunofluorescence staining of cortical granules, GPLD1, and nuclear DNA was performed. The specific procedures are as follows:

[0044] Cortical granule activity in porcine oocytes was detected using peanut lectin (FITC conjugated peanutlectin, 1:200 dilution, L7381, Sigma). GV, control (Comparative Example 1), treatment group 1 (Example 1), and treatment group 2 (Example 2) MII oocytes were placed in HEPES-free M199 basal culture medium (ThermoFisher-11150059) containing peanut lectin and incubated for 30 min in the dark at 37°C and 5% CO2. The oocytes were then washed three times in HEPES-free M199 basal culture medium. The oocytes were then incubated for 30 min at room temperature in fixation solution (0.4 g PFA (Sigma) dissolved in 10 ml PBS), followed by three washes with washing buffer (0.05 g PVA (Sigma) dissolved in 100 ml PBS). After washing three more times, the oocytes were transferred to permeabilization buffer (20 μl Triton-X100 (Sigma) dissolved in 10 ml PBS) and incubated for 30 min at room temperature. After three more washes, the oocytes were transferred to blocking buffer (Beyotime P0023B) and incubated for 2 h at room temperature. Then, they were directly transferred to antibody dilution buffer (Beyotime) containing 0.5% (v / v) GPLD1 antibody (SantaCruz, sc-365096) and incubated at 4°C for 24 h. After three more washes, the oocytes were transferred to antibody dilution buffer containing 0.1% (v / v) fluorescent secondary antibody (Beyotime Alexa Fluor 488) and incubated for 1 h at room temperature. Finally, the oocytes were placed on a glass slide containing DAPI (Beyotime nuclear staining agent) and pressed into a slide. The distribution of GPLD1 protein, cortical granules and nuclear DNA were observed and photographed under a laser confocal microscope (Zeiss).

[0045] The expression localization of GPLD1 in porcine oocytes was determined, and the results are shown in [Figure number missing]. Figure 1 .

[0046] Immunofluorescence staining results showed a significant increase in GPLD1 protein levels during the GV and MII stages, with GPLD1 protein highly enriched on the oocyte plasma membrane, suggesting it may play a role in influencing sperm-egg binding. Immunofluorescence staining of the MII stage with exogenous GPLD1 protein supplementation in the culture medium showed higher GPLD1 protein levels compared to the control group, indicating the feasibility of exogenous GPLD1 supplementation. Furthermore, immunofluorescence staining of the cortical granules of oocytes revealed co-localization of GPLD1 protein expression within the cortical granules.

[0047] Experimental Example 2

[0048] (1) In vitro fertilization of porcine oocytes:

[0049] Frozen boar semen (from Baijunda Technology Development (Beijing) Co., Ltd.) was thawed in a 50°C water bath for 16 seconds, then washed for 5 minutes with 10 mL of preheated modified Tris buffer (mTBM) medium. After centrifugation at 1900 g for 5 minutes and resuspending, 50 μL of the sperm suspension (5 × 10⁻⁶) was collected. 5 (Sperm / mL) was added to a 50 μL droplet of mTBM medium containing 30 mature oocytes. After fertilization at 38.5°C and 5% CO2 for 4 hours, excess sperm on the zona pellucida surface was removed by gentle pipetting using mTBM medium. The fertilized eggs were then transferred to PZM3 medium for 14 hours of culture.

[0050] Preparation of modified Tris buffer medium (mTBM): Dissolve 0.6610 g of sodium chloride, 0.2240 g of potassium chloride, 0.1100 g of calcium chloride dihydrate, 0.2420 g of triaminomethane, 0.1980 g of glucose, and 0.0550 g of sodium pyruvate in 100 mL of ultrapure water.

[0051] Preparation of PZM3 medium: Dissolve 0.6311 g sodium chloride, 0.2106 g sodium bicarbonate, 0.0746 g potassium chloride, 0.0048 g potassium dihydrogen phosphate, 0.0099 g magnesium sulfate heptahydrate, 0.0616 g calcium lactate, 0.0055 g sodium pyruvate, 0.0146 g L-glutamine, 0.0546 g taurine, 1 mL BME amino acid solution, 1 mL MEM non-essential amino acid solution, 0.0050 g gentamicin, 0.0066 g penicillin, 0.0050 g streptomycin, and 0.3000 g bovine serum albumin in 100 mL embryonic water, and adjust the osmotic pressure to 297 mOsm.

[0052] (2) Assessment of polyspermia:

[0053] Polyspermia assessment was performed 18 hours post-fertilization using pronuclear counting. The procedure was as follows: Fertilized eggs were washed with 0.2% polyvinyl alcohol (w / v, dissolved in PBS) and fixed at room temperature for 30 minutes in 4% paraformaldehyde (w / v, dissolved in PBS). After three rinses, the samples were stained with 15 μg / mL 4',6-diamidinyl-2-phenylindole (DAPI, BioBio, Shanghai) at room temperature in the dark for 3 minutes. Finally, antifluorescence quenching mounting medium (BioBio) was added to the samples and placed on glass slides. Images were acquired using a confocal microscope system (Zeiss LSM 800, Oberkochen, Germany) under a standard objective lens, with all samples using the same scanning parameters.

[0054] (3) Development rate statistics:

[0055] The MII maturation rate of porcine oocytes in the control group, treatment group 1, and treatment group 2 was statistically analyzed. Subsequently, in vitro fertilization was performed. After 24 hours of culture in PZM3 medium, the 2C cleavage rate was observed and counted under a stereomicroscope. After 144 hours, the number of embryos that reached the blastocyst stage was observed and the blastocyst development rate was counted.

[0056] Experimental results:

[0057] (1) Fluorescence images of zygotes with different pronuclear numbers (DAPI):

[0058] DNA-DAPI staining was performed on fertilized eggs 18 hours after in vitro fertilization. The results are shown in [Figure 1]. Figure 2 .

[0059] 0PN: Fertilization failed, and the embryo did not form a pronucleus;

[0060] 2PN: Two pronuclei, which is a normal embryo fertilized by a single sperm.

[0061] 3-4PN: Polyspermized embryos, with more than two depolymerized pronuclei in the zygote.

[0062] Figure 2 The results show different cases of oocyte fertilization. 2PN is normal monosperm fertilization, and embryos larger than 2PN are uniformly identified as polysperm fertilization.

[0063] (2) The effects of GPLD1 on oocyte maturation, polyspermy, and blastocyst development rate are shown in [reference needed]. Figure 3 Tables 1-4.

[0064] Table 1. Statistics on polysperm fertilization rate

[0065]

[0066] As shown in Table 1, compared with the control group, the polysperm fertilization rate decreased by about 18% after adding 1000 ng / mL GPLD1 protein to the oocyte maturation culture medium in treatment group 1, and by about 15% after adding 2000 ng / mL GPLD1 protein to the oocyte maturation culture medium in treatment group 2.

[0067] Table 2. Statistics on MII egg maturity rate

[0068]

[0069] Table 32C Cleavage Rate Statistics

[0070]

[0071] Table 4. Blastocyst Rate Statistics

[0072]

[0073] Note: Each experiment was conducted at least 3 times; maturity rate is presented as mean ± standard deviation; different superscript letters in the same column indicate significant differences (p<0.05, Duncan's test for one-way ANOVA).

[0074] As shown in Table 2-4, there were no significant differences in the MII maturation rate, 2C cleavage rate, and subsequent blastocyst development rate between treatment group 1 and treatment group 2 compared with the control group.

[0075] Figure 3 The results in Tables 1-4 indicate that the addition of GPLD1 protein at concentrations of 1000 and 2000 ng / mL can significantly reduce the incidence of polyspermia in pigs in vitro, but has no significant effect on oocyte maturation, cleavage rate, and blastocyst development rate.

[0076] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of GPLD1 protein in the preparation of reagents to inhibit polyspermy in porcine oocytes.

2. A porcine oocyte culture medium with an inhibitory effect on polysperm fertilization, characterized in that, The following components were added to the HEPES-free M199 basal culture medium: 1 mg / mL cysteine, 0.44 mg / mL sodium pyruvate, 10 ng / mL epidermal growth factor, 10% porcine follicular fluid, 50 ng / mL insulin, 5 IU / mL gonadotropin, 5 IU / mL human chorionic gonadotropin, 11.3 ng / mL kanamycin, and 1000-2000 ng / mL GPLD1 protein.

3. The use of the porcine oocyte culture medium according to claim 2 in the preparation of a reagent to inhibit polysperm fertilization of porcine oocytes.

4. The use of the porcine oocyte culture medium according to claim 2 in the preparation of a drug for improving the developmental ability of porcine in vitro fertilized embryos.

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