Method for producing mammalian fertilized egg

Abstract

[Problem] To provide a means for improving the growing ability of a fertilized egg. [Solution] The present invention provides a method for producing a fertilized egg of a mammal, the method comprising culturing a mammalian fertilized egg in vitro in the presence of a TLR2 activator. The present invention also provides: a method for providing reproductive assistance to a mammal, the method further comprising transplanting, into the uterus of a mammal, a fertilized egg after being cultured; and an agent that is for improving the growing ability of a mammalian fertilized egg, and that contains a TLR2 activator. According to the present invention, the growing ability of a mammalian fertilized egg can be improved.

Description

Method for producing mammalian fertilized eggs

[0001] The present invention relates to a method for producing a mammalian fertilized egg, a method for assisted reproduction in a mammal, and the use of a Toll-like receptor (TLR) 2 activator in these methods.

[0002] Assisted reproductive technologies such as in vitro fertilization and embryo transfer have become widespread with the aim of improving the reproductive capacity of mammals. However, current assisted reproductive technologies have not been able to achieve high conception rates; for example, the conception rate of dairy cows is below 50%. Low conception rates in livestock animals impose a serious economic burden on livestock producers, resulting in increased production costs due to an increased number of embryo transfers, open periods, and extended calving intervals.

[0003] Mammalian pregnancy progresses through a complex process involving sperm migration within the female reproductive tract, physiological changes in sperm, sperm-egg interactions, early embryonic development, and induction of maternal immune tolerance. Because various factors can interfere with the progression of fertilization and implantation, thereby affecting fertility and conception rates, elucidating the mechanisms regulating these processes is essential for improving reproductive performance.

[0004] Toll-like receptors (TLRs) are receptors responsible for recognizing pathogens and activating innate immunity. TLR2, a member of the TLR family, is thought to play an important role in bovine reproduction. For example, Non-Patent Document 1 discloses that TLR2, present in both bovine sperm and uterine epithelium, activates the innate immune response of the uterus against sperm, thereby eliminating excess sperm, dead sperm, and abnormal sperm from the uterus and promoting embryo implantation, and that TLR2 present in the oviduct is also involved in the innate immune response against sperm.

[0005] Furthermore, Non-Patent Document 2 reports that activating sperm TLR2 with a TLR2 agonist in an in vitro fertilization system improved the fertilization rate and development rate.

[0006] Thus, progress has been made in elucidating the role of TLR2 in the interactions between sperm, the uterus, and the fallopian tubes at the fertilization stage, but it remains unclear how TLR2 functions in embryonic development after fertilization.

[0007] Akthar I. et al., Reproduction, Fertility and Development: 139-148. (2022)Ma D. et al., Frontiers in Cell and Developmental Biology 10: 810961. (2022)

[0008] The present invention provides a means for improving the developmental competence of fertilized eggs.

[0009] The present inventors have found that activating TLR2 in fertilized eggs improves the developmental ability of the fertilized eggs.

[0010] The present disclosure provides the following: Item 1. A method for producing a mammalian fertilized egg, comprising culturing a mammalian fertilized egg ex vivo in the presence of a TLR2 activator. Item 2. The method of Item 1, wherein a 1-cell to 16-cell fertilized egg is cultured from the morula stage to the blastocyst stage in the presence of a TLR2 activator. Item 3. The method of Item 1, wherein a 2-cell to 8-cell fertilized egg is cultured from the morula stage to the blastocyst stage in the presence of a TLR2 activator. Item 4. The method of any one of Items 1 to 3, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist. Item 5. A method for assisted reproduction in a mammal, comprising culturing a mammalian fertilized egg ex vivo in the presence of a TLR2 activator, and implanting the cultured fertilized egg into the uterus of a mammal. Item 6. Item 5. The method of Item 5, wherein fertilized eggs from the 1-cell stage to the 16-cell stage are cultured from the morula stage to the blastocyst stage in the presence of a TLR2 activator. Item 7. The method of Item 5, wherein fertilized eggs from the 2-cell stage to the 8-cell stage are cultured from the morula stage to the blastocyst stage in the presence of a TLR2 activator. Item 8. The method of any one of Items 5 to 7, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist. Item 9. An agent for improving the developmental competence of a mammalian fertilized egg, comprising a TLR2 activator. Item 10. The agent of Item 9, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist. Item 11. A culture medium for producing a mammalian fertilized egg, comprising a TLR2 activator. Item 12. Item 12. The medium according to Item 11, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist. Item 13. A kit for improving the developmental competence of a mammalian fertilized egg, comprising a TLR2 activator. Item 14. The kit according to Item 13, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist.

[0011] According to the present invention, the developmental ability of mammalian fertilized eggs can be improved.

[0012] 3A shows images of bovine early embryos immunostained for TLR2. Live imaging images from 36 to 156 hours after fertilization of bovine embryos cultured in the presence (lower row, TLR2 ago) or absence (upper row, control) of a TLR1 / TLR2 agonist. Images stained with Hoechst 33342, TUNEL, and PI ( FIG. 3A ) of bovine embryos cultured in the presence (TLR2 ago) or absence (control) of a TLR1 / TLR2 agonist up to day 7 after fertilization are also shown. Also shown are graphs of the apoptotic index, number of TUNEL-positive cells, and total cell number ( FIG. 3B ). 4A shows Magic Red-stained images of bovine embryos cultured until day 7 after fertilization in the presence (TLR2 ago) or absence (Control) of a TLR1 / TLR2 agonist, and a graph showing relative cathepsin B activity (FIG. 4B). 4B shows graphs (leftmost in the figure) showing the proportions of blastocysts (BL), expanded blastocysts (ExB), and escaped blastocysts (HB) in bovine embryos cultured until day 7 after fertilization in the presence (TLR2 ago) or absence (Control or Con) of a TLR1 / TLR2 agonist, and graphs (rightmost in the figure) showing the expression of five types of genes in blastocysts and expanded blastocysts.

[0013] The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. The upper and lower limit values ​​of each numerical range shown in this disclosure can be arbitrarily combined. In this disclosure, numerical ranges expressed using "to" or "-" mean ranges that include the numerical values ​​at both ends as upper and lower limits, unless otherwise specified.

[0014] TLR2 is a transmembrane protein composed of an extracellular domain with an LRR (leucine rich repeat) structure, a transmembrane domain, and a TIR (Toll / Interleukin-1 receptor) domain that initiates intracellular signal transduction, and as a membrane surface receptor it recognizes a variety of PAMPs (pathogen-associated molecular patterns) derived from foreign organisms such as bacteria, fungi, viruses, and protozoa. TLR2 is known to form a heterodimer with TLR1 or TLR6 on the cell surface and induce intracellular signal transduction.

[0015] In the present disclosure, a TLR2 activator refers to a substance capable of inducing intracellular signaling via TLR2 in a target mammal. An example of a TLR2 activator is a PAMP recognized by TLR2. Examples of such PAMPs include lipopolysaccharide (LPS), lipoprotein, lipoteichoic acid, lipomannan, glycosylphosphatidylinositol (GPI), zymosan, and hemagglutinin.

[0016] Examples of TLR2 activators are substances that can bind to a TLR1 / TLR2 heterodimer or a TLR2 / TLR6 heterodimer and induce intracellular signaling via TLR2. TLR2 activators that bind to a TLR1 / TLR2 heterodimer are called TLR1 / TLR2 agonists, and TLR2 activators that bind to a TLR2 / TLR6 heterodimer are called TLR2 / TLR6 agonists. The above-mentioned PAMPs may include either or both of a TLR1 / TLR2 agonist and a TLR2 / TLR6 agonist.

[0017] A TLR1 / TLR2 agonist may bind to either or both of TLR1 and TLR2 constituting a TLR1 / TLR2 heterodimer, while a TLR2 / TLR6 agonist may bind to either or both of TLR2 and TLR6 constituting a TLR2 / TLR6 heterodimer.

[0018] Examples of TLR1 / TLR2 agonists include triacyl lipopeptides such as Pam3 lipopeptides, for example, Pam3CSK4 (S-[2,3-bis(palmitoyloxy)propyl]-Cys-Ser-Lys-Lys-Lys-Lys) and salts thereof, and CU-T12-9 and salts thereof.

[0019] Examples of TLR2 / TLR6 agonists include diacyl lipopeptides such as Pam2 lipopeptides, for example, Pam2CSK4 (N-palmitoyl-S-(2,3-bis(palmitoyloxy)propyl)-Cys-Ser-Lys-Lys-Lys-Lys) and salts thereof, MALP-2, and Fibroblast-Stimulating Lipopeptide-1.

[0020] The chemical formula for an exemplary TLR2 activator is shown below.

[0021] In the present disclosure, the mammalian fertilized egg may be a fertilized egg of a non-human mammal or a human fertilized egg. Non-human mammals may be, for example, primates other than humans, ruminants such as cattle, yaks, water buffaloes, sheep, goats, reindeer, red deer, dromedaries, llamas, and alpacas, livestock animals such as horses and pigs, and pet animals such as dogs and cats, with ruminants, particularly cattle, being preferred. The fertilized egg is preferably an in vitro fertilized egg. The fertilized egg may be one from which the zona pellucida has been removed, but preferably one that still has the zona pellucida.

[0022] Generally, in vitro fertilized eggs are prepared through a series of steps, including collection of eggs and sperm from a living organism, maturation culture of the eggs, sperm processing, and subsequent fertilization (insemination) of the eggs and sperm. After fertilization, the fertilized eggs are washed and transferred to a medium for culture (also called embryo culture or developmental culture).

[0023] In the culture of fertilized eggs, in the case of cattle, an initial developmental test is usually conducted 1.5 to 2 days after the start of culture to evaluate the developmental stage. Fertilized eggs that are determined to be degenerated eggs (abnormal eggs) or one-cell stage embryos (presumed to be unfertilized eggs) in the initial developmental test are excluded from culture as necessary, and fertilized eggs at the two-cell stage or higher are further cultured. In this disclosure, the term "embryo" is used interchangeably with "fertilized egg."

[0024] The present disclosure provides a method for producing a mammalian fertilized egg, which comprises culturing a mammalian fertilized egg in vitro in the presence of a TLR2 activator.

[0025] Culturing of fertilized eggs in the presence of a TLR2 activator can be carried out by culturing the fertilized eggs in a medium containing the TLR2 activator. The medium can be prepared by adding the TLR2 activator to a known embryo culture medium, such as BO-IVC medium or CR1aa medium.

[0026] The TLR2 activator may be present in the medium from the start of culturing the fertilized eggs or from the middle of the culturing. The TLR2 activator may be present in the medium from the time when culturing of the fertilized eggs is resumed after the initial developmental test, preferably for fertilized eggs determined by the initial developmental test to be at the two-cell stage or higher, more preferably for fertilized eggs determined by the initial developmental test to be at the four-cell stage or higher.

[0027] The fertilized eggs to be cultured in the presence of a TLR2 activator are fertilized eggs at a developmental stage from the 1-cell stage to the blastocyst stage, for example, from the 1-cell stage to the 16-cell stage, preferably from the 2-cell stage to the 8-cell stage, and more preferably from the 4-cell stage to the 8-cell stage. Without being bound by theory, it is believed that the genome of the fertilized egg becomes activated and its energy requirement increases when the egg enters the 4-cell stage, and it is thought that it would be preferable to apply a TLR2 activator to fertilized eggs at this stage.

[0028] In general, oocytes are matured and cultured with cumulus cells attached to the periphery, and then fertilized. If a TLR2 activator is present during fertilization, it may strongly activate the TLR2 possessed by the cumulus cells surrounding the mature oocyte, causing an inflammatory reaction in the more sensitive cumulus cells and potentially reducing the fertilization rate. Therefore, it is preferable to have the TLR2 activator present in the medium for culturing the fertilized oocytes, rather than in the fertilization medium.

[0029] The concentration of the TLR2 activator can be set appropriately taking into consideration factors such as the type of mammal, the type of TLR2 activator, the state of the fertilized egg, etc. For example, when culturing bovine fertilized eggs using Pam3CSK1 or Pam2CSK4 as the TLR2 activator, the concentration of the TLR2 activator in the medium can be 10 ng / ml to 500 ng / ml, for example, 20 ng / ml to 400 ng / ml, 30 ng / ml to 300 ng / ml, 40 ng / ml to 200 ng / ml, or 50 ng / ml to 100 ng / ml.

[0030] The TLR2 activator can be present in the culture medium, for example, while a 1-cell to 16-cell fertilized egg develops from the morula stage to the blastocyst stage, preferably while a 2-cell to 8-cell fertilized egg develops from the morula stage to the blastocyst stage, and more preferably while a 4-cell to 8-cell fertilized egg develops from the morula stage to the blastocyst stage.

[0031] The fertilized eggs are cultured until they have developed to a level where they can be expected to implant in the uterus after transplantation, preferably from the morula stage to the blastocyst stage, more preferably until they have developed to the blastocyst stage. After culturing, the fertilized eggs are evaluated for their developmental stage and quality (size, color, aggregation, shape, etc. of individual cells), and those that have been determined to have developed well and are of high quality are subjected to transplantation into the uterus.

[0032] Morula-stage fertilized eggs include early morula and morula, and blastocyst-stage fertilized eggs include early blastocyst, blastocyst, expanded blastocyst, erupted (hatched) blastocyst, and expanded (hatched) blastocyst. In the present disclosure, any of these fertilized eggs can be used for transplantation, but in the case of cattle, it is recommended to transplant embryos with a complete zona pellucida in consideration of the washing procedure during transplantation, so it is preferable to use fertilized eggs that have not yet reached the expanded blastocyst stage for transplantation.

[0033] As described above, by culturing mammalian fertilized eggs in the presence of a TLR2 activator, the developmental competence of the fertilized eggs can be improved. Improvement in developmental competence means at least one of an improvement in the blastocyst development rate (blastocyst rate), an improvement in the embryo expansion rate, a shortened embryo growth time (embryonic growth promotion), a decrease in the number of apoptotic cells, and an increase in the total number of blastocyst cells. By implanting fertilized eggs with improved developmental competence into the uterus, it is expected that the conception rate will increase. Furthermore, by improving the developmental competence of fertilized eggs, it is possible to secure more fertilized eggs available for implantation into the uterus in a shorter time.

[0034] Furthermore, as described above, it was confirmed that culturing bovine fertilized eggs in the presence of a TLR2 activator enhances the expression of interferon tau (IFN-τ) in the fertilized eggs. IFN-τ is known to play an important role in the maternal recognition of pregnancy and the maintenance of corpus luteum function in ruminants such as cattle. Therefore, it is expected that the fertilized eggs obtained by culturing bovine fertilized eggs in the presence of a TLR2 activator will have increased fertility.

[0035] Thus, the present disclosure provides a method for producing a mammalian fertilized egg for embryo transfer, a method for producing a mammalian fertilized egg with improved developmental potential, and a method for producing a mammalian fertilized egg with an increased conception rate, which comprises culturing the mammalian fertilized egg in vitro in the presence of a TLR2 activator.

[0036] The present disclosure also provides a method for assisted reproduction in a mammal, comprising in vitro culturing of mammalian fertilized eggs in the presence of a TLR2 activator, and transplanting the cultured fertilized eggs into the uterus of a mammal. The method for assisted reproduction can also be described as a method for performing in vitro fertilized egg-embryo transfer. The mammalian fertilized eggs obtained by in vitro culture in the presence of a TLR2 activator may be used for uterine transplantation as is, or may be cryopreserved by a known method before being used for uterine transplantation. The fertilized eggs are preferably transplanted into the uterus of a mammal of the same species. Unless otherwise specified, the preparation of in vitro fertilized eggs, the culture and evaluation of the fertilized eggs, and the cryopreservation and transplantation of the cultured fertilized eggs into the uterus can be performed using known methods.

[0037] The present disclosure also provides an agent for improving the developmental competence of a mammalian fertilized egg, comprising a TLR2 activator. The agent may be a composition containing a pharmaceutically acceptable ingredient other than the TLR2 activator. The pharmaceutically acceptable ingredient is one that does not interfere with the effect of the TLR2 activator or the development of the fertilized egg, and examples of such ingredients include a solvent capable of dissolving the TLR2 activator, an excipient, a buffer, an isotonicity agent, and the like. The pharmaceutically acceptable ingredient may also be an ingredient that is preferably used in embryo culture, such as a growth factor (EGF, IGF-I, etc.), L-carnitine, reduced glutathione, linoleic acid albumin, and the like. The agent and composition may be used in the method for producing the mammalian fertilized egg described above.

[0038] The present disclosure also provides a culture medium for improving the developmental competence of mammalian fertilized eggs, comprising a TLR2 activator. The medium may be a known embryo culture medium containing a TLR2 activator, and may further contain pharmaceutically acceptable components such as growth factors (EGF, IGF-I, etc.), L-carnitine, reduced glutathione, linoleic acid albumin, etc. The medium can be used in the above-mentioned method for producing mammalian fertilized eggs.

[0039] The present disclosure also provides a kit for improving the developmental competence of a mammalian fertilized egg, comprising a TLR2 activator. The kit comprises the TLR2 activator, and may further comprise pharmaceutically acceptable ingredients other than the TLR2 activator, a medium, a container, an instruction manual, etc. The kit can be used in the method for producing a mammalian fertilized egg described above.

[0040] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0041] Materials and Methods (1) Collection and In Vitro Maturation (IVM) of Cumulus Cell Complexes (COCs). Bovine ovaries collected at a slaughterhouse in Obihiro, Hokkaido, were stored in 1% penicillin-streptomycin-containing saline (Gibco) at approximately 35-36°C and transported to the laboratory within 1-2 hours. COCs were aspirated from individual, visible antral follicles measuring 2-6 mm in diameter using a 10-ml syringe equipped with an 18G needle. Oocytes with homogeneous cytoplasm and surrounded by at least three layers of densely packed cumulus cells were then washed three times with oocyte collection medium (OCM, Functional Peptide Institute, Inc.) supplemented with 10% fetal bovine serum (FBS, Biowest). Poor quality oocytes (fragmented oocytes, degenerated oocytes) were discarded, and only good quality oocytes were cultured in a high performance modified 199 medium (HP-M199; Functional Peptide Institute, Inc.) supplemented with 10 ng / ml epidermal growth factor (EGF, Sigma) and 10% FBS under mineral oil at 5% CO ２ The mixture was matured in vitro at 38.5°C in humidified air for 22-24 hours.

[0042] (2) Preparation of sperm and in vitro fertilization (IVF) Three types of semen straws collected from different Holstein bulls were thawed at 37°C for 30 seconds and washed with SP-TALP (Sperm-Tyrode's Albumin Lactate Pyruvate) by centrifugation at 1500 rpm for 5 minutes at room temperature, repeated 2-3 times. After washing, sperm were extracted at 2 × 10 in fertilization medium IVF100 (Functional Peptide Institute, Inc.). ６ Finally, 10-15 COCs and sperm were incubated in 5% CO２ The cells were co-cultured in a humidified atmosphere at 38.5°C for 6 hours.

[0043] (3) In vitro culture (IVC) After 6 hours of fertilization, cumulus cells were removed by repeated pipetting using 300 μg / ml hyaluronidase. In each experiment, presumed fertilized eggs (n=40-45) were cultured in 5% O2 medium. ２ , 5% CO ２ , 90%N ２ The cells were transferred to 400 μl of BO-IVC medium (IVF-bioscience) in a 4-well dish at 38.5° C. in a humidified atmosphere, and then overlaid with mineral oil.

[0044] (4) Immunostaining Embryos at each stage (2-cell, 4-cell, 8-16-cell, morula, and blastocyst) were washed three times with PBS containing 1 mg / ml polyvinyl alcohol (PVA-PBS) and fixed in 4% paraformaldehyde (PFA) at room temperature for 30 minutes. After washing, the embryos were transferred to a drop of PBS-PVA containing 0.1% Tween-20 (Sigma) and permeabilized at room temperature for 30 minutes. After careful washing, the embryos were incubated in 5% BSA / PBS at room temperature for 2 hours. They were then incubated overnight at 4°C with a primary antibody (rabbit polyclonal anti-TLR2 antibody, dilution 1:200). A negative control was prepared without the primary antibody. After washing, the embryos were transferred to a drop of secondary antibody (Alexa Flour 546-labeled goat anti-rabbit IgG, Invitrogen, dilution 1:200) and incubated at room temperature for 2 hours, after which they were imaged using a fluorescence microscope.

[0045] (5) Treatment with TLR2 activators. The TLR1 / TLR2 agonist Pam3CSK4 (abcam) and the TLR2 / TLR6 agonist Pam2CSK4 (InvivoGen) were each dissolved in ethanol and added to BO-IVC medium at concentrations of 1 to 1000 ng / ml. For comparison, the TLR1 / TLR2 antagonist CU-CPT22 (Calbiochem) was dissolved in DMSO and added to a separate BO-IVC medium at concentrations of 2 to 40 μM. Embryos at the 4-cell stage or higher on day 1.5 after fertilization were cultured using these media until day 7 after fertilization, and the numbers of blastocysts, expanded blastocysts, and hatched blastocysts were then counted.

[0046] (6) Differential Staining of Apoptotic Nuclei by TUNEL Assay Blastocysts cultured up to day 7 after fertilization were permeabilized for 20 seconds using 30 μg / ml propidium iodide (PI) and 0.2% Triton X-100. They were then immediately transferred to a 200 μl drop of 4% PFA containing 30 μg / ml bisbenzimide (Hoechst 33342) and fixed at room temperature for 20 minutes. Next, the blastocysts were carefully washed and transferred to a 50 μl drop of In Situ Cell Death Detection Kit (Roche) and incubated at 5% CO according to the manufacturer's instructions. ２ The blastocysts were then left to stand in the dark at 38.5° C. for 45 minutes. Thereafter, the blastocysts were washed for 5 minutes each, mounted on slide glasses using Mounting Medium (H-1000, Vectashield), and observed under a fluorescent microscope to count the number of cells.

[0047] (7) Detection of Intracellular Cathepsin B Activity Intracellular cathepsin B activity was detected for blastocysts cultured up to day 7 after fertilization using the Magic Red (registered trademark) CTSB Detection Kit (MR-RR) 2. Briefly, blastocysts were cultured in 500 μl of PBS containing 2 μl of reaction mix at 5% CO ２The blastocysts were incubated under PBS at 38.5°C for 20-30 minutes. After three washes with PBS-PVA, the blastocysts were mounted on slides and immediately photographed under a fluorescent microscope. All images to detect cathepsin B activity were acquired with the same exposure time and analyzed using Image J software.

[0048] (8) Gene Expression Analysis by Quantitative Real-Time PCR RNA was extracted from blastocysts cultured up to day 7 after fertilization using the Arcturus PicoPure RNA Isolation Kit (Arcturus) according to the manufacturer's protocol and stored at -80°C until use. Genomic DNA was removed from the extracted RNA using the QuantiTect® Reverse Transcription Kit (QIAGEN), and cDNA was prepared. Briefly, 14 μl of mRNA sample was placed in an Eppendorf tube (200 μl) containing 1 μl of Quantiscript Reverse Transcriptase, 4 μl of 5× Quantiscript RT Buffer, and 1 μl of RT Primer Mix, heated at 42°C for 15 minutes, 95°C for 3 minutes, and then kept at 4°C.

[0049] Quantitative real-time PCR was performed using the primer set shown in Table 1, QuantiTect SYBR Green PCR Master Mix (QIAGEN), and iCycler iQ (Bio-Rad Laboratories). Samples were heated at 95°C for 15 minutes, followed by 40 cycles of 95°C for 15 seconds, 51.5-58.9°C for 15 seconds, and 72°C for 30 seconds. Ct values ​​were normalized using Histone H2A as an internal standard, and relative gene expression was analyzed using the ΔΔCt method.

[0050] (9) Statistical Analysis Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software) and SPSS software version 17.0. One-way analysis of variance (ANOVA) was used to compare mean differences between groups. Data are expressed as mean ± standard error.

[0051] [Test Example: Analysis of TLR2 Expression] The expression of TLR2 in early bovine embryos was analyzed by immunostaining. Strong expression of TLR2 was confirmed in all embryos from the 2-cell stage to the blastocyst stage (Figure 1).

[0052] Example 1 Promotion of Bovine Embryo Development by TLR2 Activation (1-1) Embryos at the 4-cell stage or higher on day 1.5 after fertilization were cultured until day 7 after fertilization using a medium supplemented with the TLR1 / TLR2 agonist Pam3CSK4 at a concentration of 1 to 1000 ng / ml, or, for comparison, a medium supplemented with the TLR1 / TLR2 antagonist CU-CPT22 at a concentration of 2 to 40 μM. The number of blastocysts was counted, and the blastocyst rate was calculated from the number of blastocysts relative to the total number of embryos cultured.

[0053] The TLR1 / TLR2 agonist increased the number and rate of blastocysts (Table 2), whereas the TLR1 / TLR2 antagonist decreased both the number and rate of blastocysts (Table 3). Because high concentrations of the TLR1 / TLR2 agonist inhibited blastocyst formation, subsequent tests were performed at a concentration of 100 ng / ml. Furthermore, subsequent tests were performed at 5 μM of the TLR1 / TLR2 antagonist, the lowest concentration at which a significant difference in blastocyst formation was observed.

[0054] (1-2) Embryos at the 4-cell stage or higher on day 1.5 after fertilization were cultured until day 7 after fertilization using medium supplemented with a TLR1 / TLR2 agonist at 100 ng / ml or medium supplemented with a TLR1 / TLR2 antagonist at 5 μM. The numbers of blastocysts, expanded blastocysts, and hatched blastocysts were counted, and the incidence of each relative to the total number of embryos cultured was calculated. The TLR1 / TLR2 agonist promoted the formation of not only blastocysts but also expanded and hatched blastocysts, while the TLR1 / TLR2 antagonist showed the opposite tendency (Table 4).

[0055] Live cell imaging images of embryos cultured in a medium supplemented with a TLR1 / TLR2 agonist are shown in Figure 2. In Figure 2, the arrow indicates a blastocyst. At the start of culture, 1.5 days after fertilization, the embryos were generally at the four-cell stage, and in the control group, multiple blastocysts were observed by 6.5 days after fertilization. In contrast, in the presence of a TLR1 / TLR2 agonist, multiple blastocysts were observed by 5.5 days after fertilization, and the number of blastocysts further increased by 6.5 days after fertilization.

[0056] (1-3) Embryos at the 4-cell stage or higher on day 1.5 after fertilization were cultured until day 7 after fertilization using medium supplemented with 100 ng / ml of the TLR2 / TLR6 agonist Pam2CSK4. The number of blastocysts was counted, and the blastocyst rate was calculated from the number of blastocysts relative to the total number of embryos cultured. The TLR2 / TLR6 agonist increased the number and rate of blastocysts, similar to the TLR1 / TLR2 agonist (Table 5).

[0057] From the above, it was confirmed that activation of TLR2 promotes embryonic development and improves the blastocyst rate, expansion rate, and growth rate.

[0058] Example 2. Suppression of apoptosis in bovine embryos by TLR2 activation (2-1) Embryos 1.5 days after fertilization were cultured until day 7 after fertilization using a medium supplemented with a TLR1 / TLR2 agonist at 100 ng / ml. Nuclear DNA fragmentation was detected by a TUNEL assay to analyze apoptosis in the embryos. The numbers of Hoechst-positive cells, TUNEL-positive cells, and PI-positive cells were counted to determine the total cell number, the number of apoptotic cells, and the number of trophoblast cells, respectively.

[0059] Representative staining images are shown in Figure 3A, and graphs of the apoptotic index (the percentage of apoptotic cells among all cells), the number of TUNEL-positive cells, and the total number of cells are shown in Figure 3B. In the presence of TLR1 / TLR2 agonists, the percentage of apoptotic cells decreased and the total number of cells increased.

[0060] Furthermore, embryo apoptosis was analyzed by detecting cathepsin B activity in the cultured embryos. A representative staining image is shown in Figure 4A, and a graph of Magic Red fluorescence intensity, which indicates cathepsin B activity, is shown in Figure 4B. Similar to the results of the TUNEL assay, a decrease in the proportion of apoptotic cells was confirmed in the presence of TLR1 / TLR2 agonists.

[0061] Example 3. Changes in gene expression in bovine embryos due to TLR2 activation Embryos on day 1.5 after fertilization were cultured until day 7 after fertilization using medium supplemented with 100 ng / ml of TLR1 / TLR2 agonist. The numbers of blastocysts, expanded blastocysts, and eclosed blastocysts were counted, and the development rate of each relative to the total number of embryos cultured was calculated. Next, mRNA was extracted from each blastocyst and expanded blastocyst, and the gene expression of IFN-τ, the pro-inflammatory cytokine TNF-α, caspase-3, an indicator of apoptosis, Oct-4, an indicator of inner cell mass, and Cdx-2, an indicator of trophoblast cells, was analyzed by quantitative real-time PCR.

[0062] In the presence of TLR1 / TLR2 agonists, IFN-τ gene expression was increased in both blastocysts and expanded blastocysts, and caspase-3 gene expression was decreased in expanded blastocysts (Figure 5).

[0063] Examples 2 and 3 confirmed that activation of TLR2 suppresses embryonic apoptosis and increases IFN-τ gene expression.

Claims

1. A method for producing a mammalian fertilized egg, which comprises culturing a mammalian fertilized egg in vitro in the presence of a TLR2 activator.

2. The method according to claim 1, wherein fertilized eggs from the 1-cell stage to the 16-cell stage are cultured from the morula stage to the blastocyst stage in the presence of a TLR2 activator.

3. The method according to claim 1, wherein fertilized eggs from the 2-cell stage to the 8-cell stage are cultured from the morula stage to the blastocyst stage in the presence of a TLR2 activator.

4. The method according to any one of claims 1 to 3, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist.

5. A method for assisted reproduction of a non-human mammal, comprising culturing in vitro a fertilized egg of the non-human mammal in the presence of a TLR2 activator, and implanting the cultured fertilized egg into the uterus of the mammal.

6. The method according to claim 5, wherein fertilized eggs from the 1-cell stage to the 16-cell stage are cultured from the morula stage to the blastocyst stage in the presence of a TLR2 activator.

7. The method according to claim 5, wherein fertilized eggs from the 2-cell stage to the 8-cell stage are cultured from the morula stage to the blastocyst stage in the presence of a TLR2 activator.

8. The method according to any one of claims 5 to 7, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist.

9. An agent for improving the developmental competence of a mammalian fertilized egg, comprising a TLR2 activator.

10. The agent according to claim 9, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist.

11. A medium for producing mammalian fertilized eggs, comprising a TLR2 activator.

12. The medium according to claim 11, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist.

13. A kit for improving the developmental competence of a mammalian fertilized egg, comprising a TLR2 activator.

14. The kit of claim 13, wherein the TLR2 activator is a TLR1 / TLR2 agonist or a TLR2 / TLR6 agonist.

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