Method for improving in-vitro fertilization efficiency of low-concentration sperms

Abstract

The invention relates to the technical field of animal breeding, in particular to a method for improving the in-vitro fertilization efficiency of low-concentration sperms. Comprising the following steps: treating sperms with functional proteins; the functional protein is MFG-E8; the concentration of the functional protein ranges from 250 ng / mL to 750 ng / mL. The protein MFG-E8 capable of improving the sperm motility and the in-vitro fertilization efficiency is obtained through research. In a fertilization environment, by adding the protein MFG-E8, the fertilization rate of sperms can be effectively increased, and the in-vitro fertilization efficiency under the condition of low-concentration sperms is remarkably improved. The method provided by the invention can be applied to the in-vitro fertilization technology of various mammals, and provides a new optimization strategy for the assisted reproduction technology.

Description

Technical Field

[0001] This invention relates to the field of animal reproductive technology, and in particular to a method for improving the efficiency of in vitro fertilization with low concentrations of sperm. Background Technology

[0002] In natural fertilization in mammals, sperm travel through the vagina, cervix, and uterus, eventually reaching the fallopian tubes to await fertilization. During this process, sperm undergo rigorous selection within the body; therefore, sperm count and motility determine the success of fertilization. In recent years, the incidence of male infertility has been steadily increasing, with oligospermia and asthenospermia being significant contributing factors. In vitro fertilization and embryo transfer (IVF-ET), a common clinical application within the assisted reproductive technology (ART) system, is a key method for treating infertility. Beyond infertility treatment, the development of animal husbandry also largely relies on IVF technology, which utilizes high-quality sperm. IVF and other assisted reproductive technologies have significantly improved the breeding efficiency of superior livestock, providing crucial support for genetic improvement and performance optimization.

[0003] In vitro fertilization-electrosurgical (IVF-ET) is a routine procedure in ART (Assisted Reproductive Technology). During in vitro fertilization, it simulates the process of sperm swimming towards oocytes in vivo, allowing for sperm selection. However, the decreased fertilization rate due to insufficient sperm count and poor sperm motility remains a technical bottleneck limiting the success rate of assisted reproduction. While intracytoplasmic sperm injection (ICSI) can significantly improve in vitro fertilization rates, it lacks sperm selection, is complex, costly, and may cause mechanical damage to oocytes during microinjection. Therefore, developing new methods to improve the efficiency of in vitro fertilization with low sperm concentrations is of great significance for improving reproductive outcomes in patients with oligospermia and optimizing reproductive efficiency in livestock farming. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for improving the efficiency of in vitro fertilization with low concentrations of sperm.

[0005] In a first aspect, the present invention provides the use of a functional protein, or a composition comprising the functional protein, in improving sperm motility; said functional protein is MFG-E8.

[0006] This invention has found that in vitro fertilization fluid containing milk fat globule epidermal growth factor 8 (MFG-E8) has higher in vitro fertilization efficiency (stronger fertilization capacity) without affecting the developmental capacity of the embryo.

[0007] In a second aspect, the present invention provides the use of a functional protein, or a composition comprising the functional protein, in the preparation of a reagent or medicament for improving sperm motility; said functional protein is MFG-E8.

[0008] Thirdly, the present invention provides the use of a functional protein, or a composition containing the functional protein, in improving the fertilization capacity of sperm; said functional protein is MFG-E8.

[0009] Fourthly, the present invention provides the use of a functional protein, or a composition comprising the functional protein, in the preparation of a reagent or medicament for improving the fertilization capacity of sperm; said functional protein is MFG-E8.

[0010] Furthermore, the MFG-E8 comprises the following amino acid sequence: i) The amino acid sequence as shown in SEQ ID NO.1; ii) An amino acid sequence with the same function obtained by adding, substituting, or deleting one or more amino acid sequences from the amino acid sequence shown in SEQ ID NO.1.

[0011] The amino acid sequence shown in SEQ ID NO.1: MQVSRVLAALCGMLLCASGLFAASGDFCDSSLCLNGGTCLTGQDNDIYCLCPEGFTGLVCNETERGPCSPNPCYNDAKCLVTLDTQRGDIFTEYICQCPVGYSGIHCETETNYYNL DGEYMFTTAVPNTAVPTPAPTPDLSNNLASRCSTQLGMEGGAIADSQISASSVYMGFMGLQRWGPELARLYRTGIVNAWTASNYDSKPWIQVNLLRKMRVSGVMTQGASRAGRAEY LKTFKVAYSLDGRKFEFIQDESGGDKEFLGNLDNNSLKVNMFNPTLEAQYIKLYPVSCHRGCTLRFELLGCELHGCSEPLGLKNNTIPDSQMSASSSYKTWNLRAFGWYPHLGRLD NQGKINAWTAQSNSAKEWLQVDLGTQRQVTGIITQGARDFGHIQYVASYKVAHSDDGVQWTVYEEQGSSKVFQGNLDNNSHKKNIFEKPFMARYVRVLPVSWHNRITLRLELLGC.

[0012] Furthermore, the encoding gene of MFG-E8 includes the following nucleotide sequence: i) The nucleotide sequence shown in SEQ ID NO.2; ii) A nucleotide sequence encoding a protein with the same function obtained by adding, substituting, or deleting one or more nucleotide sequences from the nucleotide sequence shown in SEQ ID NO.2.

[0013] The nucleotide sequence shown in SEQ ID NO.2:

[0014] Furthermore, the sperm is mammalian sperm, preferably sperm from cattle, sheep, pigs, dogs, horses, cats, or mice.

[0015] Fifthly, the present invention provides a sperm motility enhancer, comprising: a functional protein, wherein the functional protein comprises: MFG-E8.

[0016] In a sixth aspect, the present invention provides a method for improving sperm motility or fertilization capacity, comprising: treating sperm with a functional protein; wherein the functional protein is MFG-E8; and the concentration of the functional protein is 250~750 ng / mL.

[0017] Furthermore, the concentration of the sperm is (1~2)×10⁻⁶. 4 per mL.

[0018] In existing in-vitro fertilization (IVF) procedures, a sperm count of 15-20 million sperm per mL is generally required (i.e., the WHO standard for normal sperm concentration), and the proportion of motile sperm (progressive motility) typically needs to reach 40% or more. In cases of low sperm concentration, the risk of fertilization failure increases significantly. However, this invention adds the aforementioned functional protein to the fertilization fluid, enabling it to meet the requirements of a low sperm concentration environment, which is of significant value in the current field of IVF.

[0019] Further, the treatment includes: adding the functional protein to the fertilization fluid during in vitro fertilization and incubating it under the conditions of pH=7~7.6, temperature 35~40℃, humidity above 95% and CO2 concentration of 3~10%.

[0020] The fertilization medium described in this invention is a fertilization culture medium that provides a fertilization environment for sperm and oocytes during in vitro fertilization. For example, T6 fertilization medium (also known as T6 culture medium), HTF culture medium, and commercially available multi-stage culture medium can be used.

[0021] Furthermore, the T6 fertilized fluid comprises: Glucose, KCl, MgCl2·6H2O, NaCl, NaHCO3, NaH2PO4, H2O, CaCl2·2H2O, Phenol red, Penicillin G / Streptomycin sulfate (GIBCO 1308300) and Bovine serum albumin (BSA).

[0022] Further, the T6 fertilization solution comprises: glucose 0.5~2g / L, KCl 0.1~0.5g / L, MgCl2•6H2O 0.05~0.2g / L, NaCl 5~10g / L, NaHCO3 1~5g / L, NaH2PO4 0.02~0.1g / L, CaCl2•2H2O 0.1~0.5g / L, Phenol red 1~5mL / L, Penicillin G / Streptomycin sulfate (GIBCO 1308300) 5~20mL / L and Bovine serum albumin (BSA) 10~30g / L, with the balance being H2O.

[0023] Furthermore, the fertilization fluid also includes: T6 culture medium and 10-50 mg / mL bovine serum albumin.

[0024] Furthermore, the incubation is for more than 5 hours.

[0025] Further, after the incubation, early embryos (early mouse embryos from the fertilized egg stage to the blastocyst stage) are obtained, and then cultured in CZB culture medium at pH=7~7.6, temperature 35~40℃, humidity above 95% and CO2 concentration of 3~10%; when reaching the 4-cell stage, they are transferred to CZB culture medium containing glucose and cultured until the blastocyst stage.

[0026] The present invention has the following beneficial effects: This invention has discovered the protein MFG-E8, which can improve sperm motility and in vitro fertilization (IVF) efficiency. Adding MFG-E8 to the fertilization fluid during IVF can increase the IVF rate from approximately 69% to approximately 84%, an increase of about 15%. The method provided by this invention is simple to operate and, while improving the fertilization rate, does not affect embryo development rate or embryo quality, which has significant application value in the field of assisted reproduction. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this 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 some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1This is a statistical chart of the in vitro fertilization efficiency of low-concentration sperm using T6 fertilization fluid with different concentrations of MFG-E8, as provided in Example 1 of the present invention. The experiment was performed in three biological replicates. The vertical axis represents the percentage of in vitro fertilization rate, and the horizontal axis represents the different concentrations of MFG-E8 added to the T6 fertilization fluid.

[0029] Figure 2 This is a statistical chart of sperm motility after incubation in conventional T6 fertilization fluid (0 ng / mL group) and T6 fertilization fluid with 500 ng / mL MFG-E8 added (500 ng / mL group), provided in Example 2 of the present invention; the vertical axis is the percentage of sperm motility, and the horizontal axis is the group.

[0030] Figure 3 This is a statistical graph of the linear velocity of sperm after incubation in conventional T6 fertilization fluid (0 ng / mL group) and T6 fertilization fluid with 500 ng / mL MFG-E8 added (500 ng / mL group), as provided in Example 2 of the present invention; the vertical axis represents the linear velocity of sperm, and the horizontal axis represents the group.

[0031] Figure 4 This is a statistical graph of sperm velocity curves after incubation in conventional T6 fertilization fluid (0 ng / mL group) and T6 fertilization fluid with 500 ng / mL MFG-E8 added (500 ng / mL group), as provided in Example 2 of the present invention; the vertical axis represents sperm velocity curves, and the horizontal axis represents the group.

[0032] Figure 5 This is a statistical chart of the average path velocity of sperm after incubation in conventional T6 fertilization fluid (0 ng / mL group) and T6 fertilization fluid with 500 ng / mL MFG-E8 added (500 ng / mL group), as provided in Example 2 of the present invention; the vertical axis represents the average path velocity of sperm, and the horizontal axis represents the group.

[0033] Figure 6 This is a statistical chart of sperm ATP levels after incubation in conventional T6 fertilization medium (0 ng / mL group) and T6 fertilization medium supplemented with 500 ng / mL MFG-E8 (500 ng / mL group), as provided in Example 2 of this invention; the vertical axis is 10. 4 The total ATP content of each sperm cell is shown on the x-axis, which represents the grouping.

[0034] Figure 7 This is a statistical chart of the number of sperm bound to a single oocyte after incubation in conventional T6 fertilization fluid (0 ng / mL group) and T6 fertilization fluid with 500 ng / mL MFG-E8 added (500 ng / mL group), provided in Example 3 of the present invention; the vertical axis represents the number of sperm bound to a single oocyte, and the horizontal axis represents the group. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0037] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0038] Example 1: Application of MFG-E8 in improving the efficiency of in vitro fertilization with low concentrations of sperm This embodiment includes the following process: The ICR strain mice used in this invention were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. The mice were housed in the SPF-grade experimental animal breeding room of Inner Mongolia University, under controlled conditions of temperature (22±2℃), humidity (40%~60%), light cycle (12 h / 12 ​​h), and free access to food and water. The experimental procedures were conducted in accordance with regulations and ethical requirements.

[0039] 1. Collection and in vitro capacitation of mature sperm from the epididymal tail of mice Male mice aged 8 weeks and older were euthanized by cervical dislocation. The epididymal tail was cut off and held in place with absorbent paper. A small incision was made in the epididymal tail with ophthalmic scissors, and sperm was forcefully squeezed out. The sperm was then transferred to in vitro capacitation droplets and incubated for 1.5 h under the following conditions: pH 7.2-7.4, temperature 37℃, humidity 100%, and carbon dioxide concentration 5%. The in vitro capacitation droplets were prepared using the commercially available in vitro capacitation solution C-TYH (72021, Nanjing Nanjin Biotechnology Co., Ltd., China): 200 μL of C-TYH sperm capacitation droplets were prepared in a culture dish and covered with paraffin oil. Before use, the droplets needed to be equilibrated in an incubator at pH 7.2-7.4, temperature 37℃, humidity 100%, and carbon dioxide concentration 5% for at least 4 h.

[0040] 2. In vitro fertilization of mouse oocytes Superovulation in female mice: Six-week-old ICR female mice were selected and injected subcutaneously with 5 IU PMSG, followed by an intraperitoneal injection of 5 IU hCG 48 h later. Mature oocytes were collected between 14 and 16 h after hCG injection.

[0041] In vitro fertilization: After euthanizing the female mouse by cervical dislocation, the dilated portion of the fallopian tube was excised, and the oocytes were extracted under a microscope. The oocytes were then placed in four different solutions: standard T6 fertilization droplets (0 ng / mL), T6 fertilization droplets supplemented with 100 ng / mL MFG-E8 (100 ng / mL), T6 fertilization droplets supplemented with 500 ng / mL MFG-E8 (500 ng / mL), and T6 fertilization droplets supplemented with 1000 ng / mL MFG-E8 (1000 ng / mL). Pre-capacitated sperm (sperm concentration 1×10⁻⁶) was then added to the other side of each fertilization droplet. 4 (Number of embryos per mL). Incubate for 6 hours at pH 7.2-7.4, 37°C, 100% humidity, and 5% carbon dioxide. After incubation, transfer the fertilized embryos to CZB medium and culture at pH 7.2-7.4, 37°C, 100% humidity, and 5% carbon dioxide. When the embryos reach the 4-cell stage, transfer them to CZB medium containing glucose (5.5 mM) and continue culturing until the blastocyst stage.

[0042] Fertilization rate = number of Zygote embryos / number of MII stage oocytes in in vitro fertilization × 100%.

[0043] 2-cells rate = (Number of 2-cells embryos / Number of Zygote embryos) × 100%.

[0044] 4-cell rate = number of 4-cell embryos / number of Zygote embryos × 100%.

[0045] Morula rate = (Number of Morula embryos / Number of Zygote embryos) × 100%.

[0046] Blastocyst rate = (Number of Blastocyst embryos / Number of Zygote embryos) × 100%.

[0047] Table 1. Composition of T6 fertilized fluid

[0048] Table 2 Mouse embryonic development fluid CZB

[0049] Statistical analysis was performed on the in vitro fertilization rate and subsequent embryo development rate: such as Figure 1As shown, the in vitro fertilization rate of the 500 ng / mL group was significantly higher than that of the 0 ng / mL, 100 ng / mL, and 1000 ng / mL groups (P<0.05), while the in vitro fertilization rates of the 100 ng / mL and 1000 ng / mL groups were not significantly different from those of the 0 ng / mL group (P>0.05). This indicates that 500 ng / mL is the optimal working concentration for improving in vitro fertilization efficiency. Therefore, this invention focuses on comparing whether there is a significant difference in embryo development rate between the 500 ng / mL group and the 0 ng / mL group. As shown in Table 3 (data are presented as mean+SD, and there is a significant difference between the two groups marked with different superscripts a and b, i.e., P<0.05), adding 500 ng / mL MFG-E8 to T6 fertilization fluid does not affect subsequent embryo development. The above results indicate that adding 500 ng / mL MFG-E8 to T6 fertilization fluid can improve the efficiency of in vitro fertilization without negatively impacting subsequent embryonic development.

[0050] Table 3. Statistics on embryo development rates after in-vitro fertilization with T6 fertilization fluid containing different working concentrations of MFG-E8.

[0051] Therefore, the present invention determines that the optimal working concentration of MFG-E8 added to T6 fertilization fluid is 500 ng / mL, and this concentration is used for subsequent operations.

[0052] Example 2: Application of MFG-E8 in enhancing sperm motility This embodiment includes the following process: 1. Collection and in vitro capacitation of mature sperm from the epididymal tail of mice See Example 1.

[0053] 2. Computer-aided sperm analysis Captured, mature epididymal sperm were placed in conventional T6 fertilization medium (0 ng / mL group) and T6 fertilization medium supplemented with 500 ng / mL MFG-E8 (500 ng / mL group) and incubated for 1 h at pH 7.2-7.4, 37℃, 100% humidity, and 5% carbon dioxide concentration. 2.5 μL of the sperm mixture was then placed on a dedicated sperm detection plate. The kinematic parameters of the sperm, including sperm motility, straight-line velocity (VSL), curvilinear velocity (VCL), and average path velocity (VAP), were analyzed using a computer-assisted sperm analysis (CASA) system.

[0054] 3. Sperm ATP level detection Captured, mature epididymal sperm were placed in standard T6 fertilization medium and T6 fertilization medium supplemented with 500 ng / mL MFG-E8, respectively, and incubated for 1 hour at pH 7.2-7.4, 37°C, 100% humidity, and 5% carbon dioxide. After incubation, the sperm concentration was adjusted to 1×10⁻⁶. 4 Sperm were collected at 600 g / mL and centrifuged for 4 min to remove the fertilization fluid, retaining the sperm precipitate. The sperm were then resuspended in 500 μL of 1×PBS, centrifuged again at 600 g for 5 min, and washed twice to remove residual fertilization fluid and impurities. Detection was performed according to the instructions of the Adenosine 5′-triphosphate (ATP) bioluminescent somatic cell assay kit (FLASC-1KT, Thermo, USA).

[0055] Statistical analysis was performed on the data obtained from the above experiments: Figures 2-5 As shown, the sperm motility rate in the 500 ng / mL group ( Figure 2 VSL ( Figure 3 VCL ( Figure 4 VAP ( Figure 5 All were significantly higher than the 0 ng / mL group ( P <0.05). By Figure 6 As shown, the ATP level of sperm in the 500 ng / mL group was significantly higher than that in the 0 ng / mL group ( P<0.05). The above results indicate that adding 500 ng / mL MFG-E8 to the fertilization fluid can improve sperm motility by increasing sperm APT levels, thereby improving the efficiency of in vitro fertilization with low concentrations of sperm.

[0056] Example 3: Application of MFG-E8 in enhancing sperm fertilization capacity This embodiment includes the following process: 1. Sperm fertilization capacity test The same number of oocytes were placed in both standard T6 fertilization medium (0 ng / mL group) and T6 fertilization medium supplemented with 500 ng / mL MFG-E8 (500 ng / mL group), with a concentration of 1×10⁻⁶. 4 Sperm counts / mL were incubated for 1 h at pH 7.2-7.4, 37°C, 100% humidity, and 5% carbon dioxide. After incubation, oocytes were gently washed twice in in vitro manipulation solution M2, then transferred to 4% paraformaldehyde fixative (8% paraformaldehyde and 1×PBS mixed 1:1) for 30 min. The oocytes were then washed three times (5 min each) with in vitro manipulation solution to remove residual fixative. The fixed oocytes were then incubated in Hoechst 33342 staining solution in the dark for 5 min to stain the sperm nuclei. The oocytes were then washed three times (5 min each) with in vitro manipulation solution.

[0057] Table 4 Mouse oocyte in vitro manipulation solution M2

[0058] This invention uses a laser confocal microscope to observe and photograph samples, and counts the number of sperm fertilizing each oocyte in two groups to assess sperm fertilization capacity. Figure 7 As shown, the average number of sperm fertilizations per oocyte in the 500 ng / mL group was significantly higher than that in the 0 ng / mL group ( P <0.05). The above results indicate that adding 500 ng / mL MFG-E8 to the fertilization fluid can enhance the fertilization capacity by improving the binding ability of sperm to oocytes, thereby achieving the goal of improving the efficiency of in vitro fertilization with low concentrations of sperm.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a functional protein, or a composition containing the functional protein, in improving sperm motility; said functional protein is MFG-E8.

2. The use of a functional protein, or a composition comprising the functional protein, in the preparation of a reagent or medicament for improving sperm motility; said functional protein being MFG-E8.

3. The use of a functional protein, or a composition containing the functional protein, in enhancing the fertilization capacity of sperm; said functional protein is MFG-E8.

4. The use of a functional protein, or a composition comprising the functional protein, in the preparation of a reagent or pharmaceutical for improving the fertilization capacity of sperm; said functional protein being MFG-E8.

5. The application according to any one of claims 1-4, characterized in that, The sperm is from mammals, preferably from cattle, sheep, pigs, dogs, horses, cats, or mice.

6. A sperm motility enhancer, characterized in that, Includes: a functional protein, wherein the functional protein is MFG-E8.

7. A method for improving sperm motility or in vitro fertilization efficiency, characterized in that, include: Sperm were treated with functional proteins; The functional protein is MFG-E8; The concentration of the functional protein is 250~750 ng / mL.

8. The method according to claim 7, characterized in that, The concentration of the sperm was (1~2)×10. 4 per mL.

9. The method according to claim 7 or 8, characterized in that, The process includes: adding the functional protein to the fertilization fluid during in vitro fertilization and incubating it under the conditions of pH=7~7.6, temperature 35~40℃, humidity above 95% and CO2 concentration of 3~10%.

10. The method according to claim 9, characterized in that, The fertilization fluid also includes: T6 culture medium and 10-50 mg / mL bovine serum albumin.

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