Method of improving the development rate of porcine parthenogenetic embryos using exosome treatment

Abstract

The present invention relates to a method for improving the development rate of porcine parthenogenetic eggs through exosome treatment. By confirming that exosomes derived from follicle fluid not only mature the eggs during in vitro culture but also improve the quality of the eggs and thereby improve the blastocyst rate in subsequent parthenogenetic stages, the invention can be usefully utilized to improve the development rate of porcine embryos.

Description

Technology Field

[0001] The present invention relates to a method for improving the development rate of porcine parthenogenetic eggs through exosome treatment. Background Technology

[0003] In vitro embryo production is a technology that obtains embryos developed to a certain level by performing the series of processes—including egg maturation, fertilization, and embryonic development—outside the body, which typically occur in vivo. In the livestock industry, in vitro embryo production is a critical technology utilized in various fields, such as the mass production of animals for medical research and livestock. In particular, pigs are extensively studied in the fields of xenotransplantation and transgenic animals for the development of human disease models, as their organ properties are similar to those of humans.

[0004] Since production efficiency in in vitro culture varies depending on the degree to which the culture environment is similar to the in vivo environment, it is desirable to conduct in vitro production technology in an environment as similar as possible to the in vivo environment. However, the efficiency of in vitro production remains low due to in vitro environments unsuitable for culture, such as higher oxygen concentrations than in the body, exposure to light, and contamination of the culture medium; therefore, improvement of in vitro culture technology is necessary.

[0005] Therefore, research is actively underway to improve the efficiency of in vitro embryo production. External conditions such as temperature, atmosphere, and culture medium composition affect mitochondrial function, cytoskeletal development, and the function of other organelles in eggs and embryos, leading to significant differences in subsequent maturation and developmental stages. Numerous studies have reported that among these various factors, reactive oxygen species are involved in the development of cells, eggs, and embryos.

[0006] Currently, offspring are being produced in various animal species, including laboratory animals, through embryo transfer via in vitro maturation, fertilization, and culture of oocytes. However, it has been reported that in almost all animal species, the development rate of embryos after fertilization is lower when mature oocytes are cultured in vitro compared to when mature oocytes are fertilized and cultured in vivo.

[0007] Because the quality of in vitro matured oocytes is significantly lower than that of in vivo matured oocytes, many researchers are continuously conducting studies to improve the in vitro maturation conditions of porcine oocytes. Maturating immature oocytes in vitro has a major impact on the subsequent development of the oocyte and embryo. External conditions, such as temperature, atmosphere, and culture medium composition, affect mitochondrial function, cytoskeletal development, and the function of other organelles in oocytes and embryos, which can lead to significant differences in the maturation and developmental stages of the oocyte and embryo. For this reason, experiments are being conducted across various ranges, including cells, oocytes, and embryos, to elucidate the relationship between reactive oxygen species and development.

[0008] As prior art regarding a method for improving embryonic development efficiency in in vitro culture, Korean Published Patent No. 10-2020-0145804, "Composition for embryonic development containing melatonin and method for improving embryonic development efficiency using the same," discloses the effect of a composition containing melatonin on reducing reactive oxygen species and apoptosis in embryos, and Korean Published Patent No. 10-2018-0037967, "Culture medium," discloses the effect of reducing oxidative stress and increasing antioxidant capacity of an embryonic culture medium containing acetyl-carnitine and lipoic acid or a derivative thereof. Korean Registered Patent No. 10-1733818, titled "Composition of a medium for promoting maturation of mammalian oocytes and embryonic development and a method for promoting maturation of mammalian oocytes and embryonic development using the same," discloses that a medium composition for promoting maturation of mammalian oocytes and embryonic development containing tauroursodeoxycholate as an active ingredient inhibits apoptosis during in vitro embryonic culture, thereby increasing embryonic survival rate and promoting the maturation of oocytes and embryonic development. Additionally, Shen et al. disclose that treatment with recombinant porcine IL-6 during in vitro culture of porcine embryos increases the rate of development to the blastocyst stage and improves embryonic survival rate by reducing apoptosis.

[0009] Recently, the presence of exosomes responsible for signal transduction in porcine follicular fluid has been confirmed. Exosomes contain various signaling molecules and are known to regulate autocrine and paracrine functions by transmitting these signals to other cells.

[0010] Exosomes are endoplasmic reticulums ranging in size from tens to hundreds of nanometers, composed of a double phospholipid membrane identical in structure to a cell membrane, and contain proteins, nucleic acids (mRNA, miRNA, etc.), and other substances called exosome cargo. Exosome cargo contains a wide range of signaling factors, and these signaling factors are known to be specific to the cell type and regulated differently depending on the environment of the secreting cell.

[0011] Exosomes contain bioactive factors such as growth factors, similar to cells, and act as carriers that transport these bioactive factors between cells, that is, as a means of communication between cells. Exosomes are known to be released not only from animal cells such as stem cells, immune cells, fibroblasts, and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae.

[0012] In addition, exosomes are released from all animal cells, including mast cells, lymphocytes, astrocytes, platelets, neurons, endothelial cells, and epithelial cells, and are found in various body fluids such as blood, urine, mucus, saliva, bile, ascites, and cerebrospinal fluid. Exosomes can also pass through the blood-brain barrier (BBB) ​​and have high selective permeability, capable of penetrating the cell membranes of epidermal and endothelial cells, so they are also being utilized in the development of drug delivery systems (DDS) as nanocarriers for specific drugs.

[0013] Regulatory physiologically active molecules, such as proteins, lipids, RNA types (mRNA, miRNA, long non-coding RNA), and DNA fragments, encapsulated by the phospholipid bilayer membrane of exosomes, enable exosomes to mediate responses in various signaling pathways related to physiological and pathophysiological functions between adjacent and distant cells through the extracellular environment. The uptake of exosomes by embryos in the extracellular environment provides new insights into intracellular interactions between the oviduct epithelium and the developing embryo through exogenous biomolecule delivery tools.

[0014] To date, extensive research has been conducted on the function of signaling molecules through the analysis of mRNA or miRNA within exosomes derived from bovine or porcine follicular fluid, but no studies have yet been reported on the actual application of exosomes to oocyte maturation in vitro culture. While investigating the effects of exosomes isolated from porcine follicular fluid on the actual oocyte maturation rate and the development rate of parthenogenetic embryos when treated during the in vitro maturation culture process, the inventors completed the present invention by confirming that when follicular fluid from which exosomes had been removed was added to the in vitro maturation culture of porcine oocytes, the oocyte maturation rate and the blastocyst rate of parthenogenetic embryos decreased, and when exosomes were added to the general in vitro maturation culture medium of porcine oocytes, the oocyte maturation rate and the blastocyst rate of parthenogenetic embryos increased. The problem to be solved

[0017] The objective of the present invention is to provide a method for improving the development rate of porcine parthenogenetic eggs through exosome treatment. means of solving the problem

[0019] In order to achieve the above objective,

[0020] The present invention provides an exosome separated from follicular fluid.

[0021] In addition, the present invention provides a culture medium composition for promoting the development of a porcine embryo comprising the exosome as an active ingredient.

[0022] In addition, the present invention comprises 1) a step of collecting an in vitro matured oocyte; and

[0023] 2) a step of culturing the above-mentioned egg cell in vitro using the above-mentioned composition; thereby providing a culture method for promoting embryonic development.

[0024] In addition, the present invention provides an embryo produced by the above-described culture method.

[0025] In addition, the present invention provides a blastocyst produced by the above-described culture method. Effects of the invention

[0027] By using the medium composition for promoting the development of porcine embryos according to the present invention, which contains exosomes isolated from porcine follicular fluid as an active ingredient, the development rate of porcine parthenogenetic eggs can be improved, thereby enabling the production of porcine embryos with excellent quality and high efficiency. Brief explanation of the drawing

[0029] Figure 1 is a figure showing an experimental design to evaluate the effect on embryonic development rate when mature oocytes obtained by adding exosome-removed porcine follicle fluid to the in vitro maturation culture medium of porcine oocytes during in vitro culture. Figure 2 is a figure showing an experimental design to evaluate the effect on embryonic development rate when mature oocytes obtained by adding exosomes to an in vitro maturation culture medium of porcine oocytes containing general follicular fluid containing exosomes during in vitro culture. Specific details for implementing the invention

[0030] The present invention will be described in detail below.

[0032] The present invention provides an exosome separated from follicular fluid.

[0033] The above follicular fluid can be collected from mammals other than humans.

[0034] The above mammal may be a pig, but is not limited thereto.

[0035] The above embryo may have been formed through parthenogenetic activation (PA).

[0036] The term "parthenogenesis" in this invention refers to the growth and development of an embryo without fertilization, and is also referred to as parthenogenesis, asexual reproduction, or asexual development.

[0037] The term "exosome" in this invention refers to a small body composed of two layers of phospholipid membranes secreted by cells. These are known to perform intercellular signaling and to form disease-specific nucleic acids and proteins, which are then released into body fluids. They are also known to play an important role in intercellular communication through the intercellular transmission of a specific repertoire of nucleic acids, proteins, and lipids that are important for homeostasis. For example, they are involved in basic physiological processes such as neurotransmission, antigen presentation, immune response, organ development, and reproductive capacity, and are also involved in some pathological disorders including cancer progression, cardiovascular disease, inflammation, and prion propagation.

[0038] The exosomes mentioned above are secreted into the extracellular environment after a late endosome called a multivesicular body (MVB), containing intraluminal vesicles (ILVs), fuses with a cell membrane. The multivesicular body may fuse with the cell membrane to release ILVs, or it may fuse with a lysosome to degrade its contents. The substances present within the exosomes are not similar to the composition of the cytoplasm, and exosomes can selectively encapsulate RNA and proteins.

[0039] Although exosome generation is reported to occur either in a manner dependent on or independent of the endosomal sorting complex required for transport (ESCRT), the precise mechanism remains difficult to determine. Cells secrete proteins containing signal peptides via the endoplasmic reticulum-Golgi complex. Vesicles containing proteins with signal peptides move toward the cell membrane, fuse, and release the proteins outside the cell. However, proteins without signal peptides can be secreted via alternative non-classical secretory pathways. When proteins without signal peptides are secreted, they are released either without or with vesicles; while the exact mechanism of non-vesicular secretion pathways is unknown, some proteins are secreted via membrane pores and ATP-binding cassette transporters. Vesicular secretion occurs through extracellular vesicles containing exosomes, and secretion takes place via vesicles of various sizes.

[0040] The average diameter of the exosomes may be 50 to 300 nm, but is not limited thereto.

[0042] In addition, the present invention provides a culture medium composition for promoting the development of a porcine embryo comprising the exosome as an active ingredient.

[0043] The above embryos may be parthenogenetic embryos.

[0044] The term "embryonic development" in this invention refers to the process in which an embryo is formed after the fertilization of an animal's sperm and egg, and grows into a new individual through cell division and differentiation. Development is divided into the germinal stage, gastrulation, neural formation, and the development of organs. The germinal stage is the very first stage of development, referring to the period from the moment the sperm and egg are fertilized until the fertilized egg implants in the uterine wall. The fertilized egg undergoes cell division, resulting in cleavage, and forms a blastocyst through blastocyst formation.

[0045] In the present invention, "embryonic development" means that a parthenogenetic egg reaches the blastocyst stage through the 2-cell stage and 4-cell stage, and may be the germinal stage or pre-implantation embryo development.

[0046] An embryo is a term that defines the stage from when a fertilized egg begins its first cell division until it becomes a fetus. After formation, the embryo reaches the blastocyst stage, which develops through cell division into an inner cell mass (ICM) that will differentiate into a fetus and a trophectoderm (TE) that will differentiate into a placenta.

[0047] When a parthenogenetic egg in mammals is stimulated, free Ca enters the cytoplasm 2+ is flowing in, and Ca 2+ When the number increases, the environment becomes similar to that of a sperm invasion, causing the egg to be recognized as having been fertilized. At this point, the egg releases its second polar body, which is recognized as a nucleus and combines with the egg's nucleus to form an embryo. Through cell division, the embryo reaches the blastocyst stage.

[0048] The aforementioned "blastocyst" refers to a parthenogenetic egg that has developed to the point where, following the process of repeated cleavage and growth, it forms a blastocyst cavity and is distinguishable into an inner cell mass that will differentiate into a fetus and a trophoectomy that will differentiate into a placenta.

[0049] The above exosomes are 1 to 5 × 10 in the culture medium composition 6 It can be included in the amount of particles.

[0050] The above medium may be a micromanipulation culture medium prepared by adding 0.001 to 0.01% HEPES, 0.01 to 0.5% BSA, 0.001 to 0.005% penicillin G, and 0.001 to 0.006% streptomycin to TCM199.

[0051] The above composition can increase the blastocyst rate of unit eggs.

[0052] The above composition can increase the maturation rate of eggs.

[0054] In addition, the present invention comprises 1) a step of collecting an in vitro matured oocyte; and

[0055] 2) a step of culturing the above-mentioned egg cell in vitro using the above-mentioned composition; thereby providing a culture method for promoting embryonic development.

[0057] The above-mentioned eggs may be collected from mammals other than humans, and the said mammals may be pigs, but are not limited thereto.

[0058] The above oocyte may be an oocyte collected through in vitro maturation (IVM) of 40 to 50 hours.

[0059] After step 1) above, the method may include a step of removing cumulus cells by pipetting the collected in vitro matured oocytes in a PBS or TCM199 culture-based micromanipulation medium after hyaluronidase treatment.

[0060] In a specific embodiment, the hyaluronidase may be treated for 1 to 5 minutes, more specifically 1 to 3 minutes, and pipetting with the micromanipulation culture medium may be performed 20 to 60 times, more specifically 30 to 50 times, but is not limited thereto.

[0061] The above micromanipulation culture medium may be prepared by adding 0.001 to 0.01% HEPES, 0.01 to 0.5% BSA, 0.001 to 0.005% penicillin G, and 0.001 to 0.006% streptomycin to TCM199, but is not limited thereto.

[0062] In addition, it may include the step of transferring the egg in a culture medium mixed with micromanipulation medium and fusion medium and applying electrical stimulation.

[0063] The above electrical stimulation can be performed by placing an egg in a fusion solution (0.28 to 0.3 M mannitol, 0.1 mM MgSO4·7H2O, 0.05 mM CaCl2·2H2O, 0.01% BSA or 0.1% PVA) and under conditions where the voltage is 100 to 200 V, the electric length is 0.01 to 0.1 ms, and the pulse width is 30 to 70 μs; more specifically, under conditions where the voltage is 130 to 160 V, the electric length is 0.02 to 0.04 ms, and the pulse width is 40 to 60 μs, but is not limited thereto.

[0064] The above two electrical stimulations can be performed within the same range.

[0065] The above in vitro culture (IVC) can be carried out for 2 to 8 days, and preferably for 6 to 8 days.

[0066] The above egg may be an oocyte from which the cumulus cell has been removed.

[0067] The term "cumulus cell" in this invention refers to a granular cell surrounding an egg within a follicle. In the body, before ovulation, the egg initiates meiosis due to the action of hormones involved in egg maturation, and the cumulus cell swells along with the production of hyaluronic acid. The swollen cumulus cell can be described as the first gateway that allows sperm to enter the cytoplasm of the egg through chemical action by hyaluronidase secreted from the sperm head and the physical penetration power of the sperm. Research on the various functions performed by cumulus cells in the maturation, ovulation, and fertilization processes of mammalian eggs has been conducted in many animal species. It is known that cumulus cells influence post-fertilization pronucleus formation, sterile fertilization, and early development of the fertilized egg in sheep, rats, cattle, and pigs by participating in the maturation of the egg before ovulation. Recent studies on the in vitro fertilization of porcine oocytes have applied a method of co-culturing with sperm after completely or partially removing cumulus cells attached to the oocyte prior to fertilization, and there are reports that the removal of cumulus cells actually increased sperm penetration.

[0068] The above egg may be an oocyte with a polar body.

[0069] The term "oocyte" in this invention refers to a cell that ceases division and proliferation within the ovary during the egg formation period and enters the growth phase, serving as the source of the egg.

[0070] The above oocyte may include an oocyte in the metaphase II (MII) stage.

[0071] The term "polar body" in this invention refers to other cells that do not have an egg yolk.

[0073] In addition, the present invention provides an embryo produced by the above-described culture method.

[0074] In addition, the present invention provides a blastocyst produced by the above-described culture method.

[0076] In a specific embodiment, the inventors investigated how exosomes isolated from porcine follicle fluid affect the actual oocyte maturation rate and the development rate of parthenogenetic eggs when treated during an in vitro maturation culture process. In this case, the inventors checked the oocyte maturation rate and the blastocyst rate of parthenogenetic eggs when follicle fluid with exosomes removed was added to the in vitro maturation culture of porcine oocytes (Fig. 1) and when follicle fluid with exosomes added was added (Fig. 2).

[0077] As a result, it was confirmed that when exosomes were removed from the follicle fluid, the oocyte maturation rate and the blastocyst rate of parthenogenetic eggs decreased, and when exosomes were added to the normal in vitro maturation medium of porcine oocytes, the oocyte maturation rate and the blastocyst rate of parthenogenetic eggs increased.

[0079] The present invention will be explained in detail below through the following examples.

[0080] However, the following examples are merely illustrative of the present invention and do not limit the present invention.

[0082] <Example 1> Preparation of porcine follicular fluid and follicular fluid-derived exosomes

[0083] Exosome-free porcine follicle fluid (exosome-free pFF) was separated by ultracentrifugation.

[0084] Fresh follicular fluid or follicular fluid frozen at -80℃ was used, and in the case of frozen follicular fluid, it was thawed by placing it in room temperature water.

[0085] 50 ml of follicle fluid was filled tightly into a high-speed centrifugation tube, and after centrifugation at 12,000 × g at 4°C for 30 minutes, the supernatant was transferred to a 50 ml tube.

[0086] After filtering the sample using a 0.45 µm low-protein binding polyethersulfone (PES) syringe filter, the supernatant was transferred to an ultracentrifuge tube and centrifuged at 120,000 × g (or 29,900 rpm) at 4°C for 70 minutes.

[0087] The supernatant obtained thereafter was used as exosome-free pFF.

[0088] The pellet remaining in the tube was resuspensed in 500 µl of PBS and then transferred to a microcentrifuge tube.

[0089] Subsequently, exosomes were separated using size exclusion chromatography, and fractions with particle sizes mainly ranging from 50 to 150 nm in diameter were frozen for use in experiments.

[0091] <Example 2> Preparation of porcine oocytes

[0092] Pig ovaries collected from the slaughterhouse were washed three times with PBS (saline).

[0093] Follicles with a diameter of 3-6 mm were aspirated using a 10cc syringe (18 gauge needle) and collected in a 15ml tube.

[0094] After standing in a water bath for about 10 minutes, the supernatant was discarded, and a washing culture medium (PVA TL HEPES) was added and mixed. Then, the mixture was immersed (repeated 2-3 times), the supernatant was discarded, the pellet was transferred to a 100mm dish and mixed with the washing culture medium, and the eggs were found.

[0095] We prioritized finding and collecting cumulus-oocyte-complexes (COCs) in good condition that were covered with multiple layers of cumulus cells.

[0096] To induce in vitro maturation, selected COCs were placed in 4-well dishes containing primary maturation medium at a rate of about 50 to 100 per well and cultured in a 38.5°C incubator for 20 to 22 hours.

[0097] Afterward, mature porcine oocytes were collected through in vitro maturation for 42-44 hours. The cumulus cells of the collected oocytes were treated with hyaluronidase for 2 minutes, and then removed by pipetting 30 to 50 times with a pipette at an appropriate speed so as not to burst the oocytes in a micromanipulation culture medium.

[0098] After transferring oocytes from which cumulus cells had been removed to a micromanipulation culture medium, only oocytes with polar bodies were identified and selected, then transferred to a 4-well dish containing the micromanipulation culture medium and placed in an incubator.

[0099] To fuse the cytoplasm and polar bodies, a chamber was placed on a warm plate, 100 µl of fusion culture medium was added, the pedal was pressed 5 times and then removed, and about 60 µl was added.

[0100] The above fusion culture medium contains 0.28 to 0.3 M mannitol, 0.1 mM MgSO4·7H2O, 0.05 mM CaCl2·2H2O, 0.01% BSA or 0.1% PVA, and has a pH of 7.0-7.4.

[0101] Afterwards, two electrical stimuli were administered under the conditions of Table 1 below.

[0102] Oocytes were transferred from a micromanipulated 4-well dish to a 4-well dish containing a mixture of micromanipulated culture medium and fusion culture medium at ratios of 100:300, 200:200, 300:100, and 0:400. After transferring the oocytes sequentially starting from well 1, they were placed in the chamber. If the number of oocytes was large, the process was divided rather than performed all at once.

[0103] The number of eggs processed in the chamber was set to about 10 to 15.

[0104] After aligning the electrode body so that it is level with the chamber, electrical stimulation was applied.

[0105] condition unit voltage Voltage 147 V Battlefield Interval 0.03 ms pulse width Pulse width 50 ㎲

[0107] Oocytes activated by electrical stimulation were placed in an incubator for about 15 minutes to induce a fusion reaction.

[0108] After washing the fused oocytes three times in PZM-3 culture medium, they were sorted and transferred to 4-wells, then placed in an O2 incubator. Embryos were observed for 3 days to check and record cleavage, and the culture medium was replaced every 2 days.

[0110] <Experimental Example 1> Effects of Follicular Fluid-Derived Exosomes in In Vitro Culture Medium of Oocytes on Maturation of Immature Oocytes and Parthenogenesis of Mature Oocytes

[0112] 1-1. Confirmation of the effect on parthenogenesis embryonic development of mature oocytes when immature oocytes are matured by removing exosomes from the follicular fluid contained in the oocyte maturation culture medium (exosome-free).

[0113] To determine the effect on the maturation rate and quality of matured oocytes (parthenogenesis embryonic development rate) when immature oocytes are matured in an in vitro oocyte maturation culture medium prepared by adding exosome-free follicular fluid, an experiment was conducted under the following conditions (Fig. 1).

[0114] When preparing a porcine in vitro maturation culture medium based on TCM199 culture medium containing LH, FSH, EGF, D-glucose, Na₂Pyruvate, L-cysteine, penicillin G, and Streptomycin, 10% porcine follicle fluid is generally added. The porcine follicle fluid is filtered sequentially using filters with diameters of 0.8 μm, 0.45 μm, and 0.22 μm. Since exosomes range in size from 50 nm to 150 nm in diameter, they are contained in the filtered 10% porcine follicle fluid typically added. To prepare an exosome-free follicle fluid, a portion of the follicle fluid filtered sequentially above was ultracentrifuged at 120,000 xG for 70 minutes, and only the supernatant was prepared as the exosome-free follicle fluid. In the experiment, when preparing the culture medium, the culture medium was prepared by dividing it into three groups: a group without follicle fluid (instead, the volume was adjusted with DPBS), a group with exosome-free follicle fluid, and a group with normal follicle fluid.

[0115] (-)pFF: Culture medium supplemented with 10% DPBS

[0116] (+)pFF(exo-): Culture medium supplemented with 10% exosome-free pFF

[0117] (+)pFF: Culture medium supplemented with 10% pFF

[0118] Immature oocytes collected on the same day from the culture media of three experimental groups were cultured for 42-44 hours to mature. After treatment with hyaluronidase for 2 minutes, cumulus cells were removed by pipetting 30 to 50 times in micromanipulation medium at an appropriate speed that did not cause the oocytes to burst. The maturity of the oocytes from which cumulus cells had been removed was determined by checking under a microscope whether the first polar body was released.

[0119] In the parthenogenesis experiment, the mature oocytes (MII stage) obtained from each group were induced into parthenogenesis through a fusion process using electrical stimulation. The parthenogenetic oocytes were cultured in PZM-3 medium for about 7 to 10 days, and the medium was replaced every 2 days.

[0120] As a result, in the case of oocytes matured in a maturation culture medium containing exosomes removed, the maturation rate (Table 2) and parthenogenesis rate (Table 3) were lower than those matured in natural follicle fluid containing exosomes.

[0121] This inversely proves that exosomes have a positive effect on egg quality (parthenogenesis development rate) as well as egg maturation rate by confirming the negative effect of lowering egg maturation and development rates by removing exosomes from the follicular fluid added to the culture medium.

[0123] group Number of immature oocytes (GV stage) at the start of maturation culture Number of mature eggs (MII stage) Oocyte maturation rate (-)pFF 87 43 49.42 % (+)pFF (exo-) 87 58 66.66 % (+)pFF 87 67 77.01 %

[0125] group Number of unit eggs at the start of culture Number of blastocysts betrayal rate (-)pFF 43 1 2.32 % (+)pFF (exo-) 58 13 22.41 % (+)pFF 67 20 29.85 %

[0128] 1-2. Confirmation of the effects of additional exosomes added to a maturation culture medium containing 10% standard follicular fluid on the maturation rate of immature oocytes and parthenogenesis of embryonic development

[0129] To investigate the direct treatment effect of exosomes during in vitro culture of porcine oocytes, an experiment was conducted under the following conditions (Fig. 2).

[0130] (+)pFF: 500ml of culture solution with 10% pFF added

[0131] (+)pFF + exsome supplement: 500ml culture medium supplemented with 10% pFF + add 1μl of isolated exosome

[0133] 2.2 × 10⁶ in 500ml culture medium 6 It was confirmed that when particles (1 μl) were added, the maturation rate of immature oocytes (Table 4) and the blastocyst development rate during parthenogenesis (Table 5) increased compared to general in vitro maturation culture conditions.

[0134] This means that the addition of exosomes during in vitro maturation culture not only improves oocyte maturation but also enhances oocyte quality, thereby having a positive effect on the blastocyst rate during the subsequent parthenogenesis stage.

[0136] group Number of immature oocytes (GV stage) at the start of maturation culture Number of mature eggs (MII stage) Oocyte maturation rate (+)pFF 124 91 73.39 % (+)pFF (exo+) 124 103 83.06 %

[0138] group Number of unit eggs at the start of culture Number of blastocysts betrayal rate (+)pFF 89 18 20.22 % (+)pFF (exo+) 101 33 32.67 %

[0140] As described above, the inventors have confirmed that exosomes derived from follicular fluid not only improve the maturation of oocytes during in vitro culture but also enhance the quality of oocytes, thereby having a positive effect on the blastocyst rate in the subsequent parthenogenesis stage. Therefore, by using a culture medium composition for promoting porcine embryo development that includes exosomes isolated from porcine follicular fluid as an active ingredient according to the present invention, the development rate of porcine parthenogenesis eggs can be improved, allowing for the production of porcine embryos with excellent quality and high efficiency.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A culture medium composition for promoting the in vitro maturation of porcine oocytes and the development of porcine parthenogenetic eggs, comprising porcine follicular fluid and exosomes derived from porcine follicular fluid as active ingredients. Claim 6 delete Claim 7 In claim 5, the exosomes are 1 to 5 × 10 in the culture medium composition 6 A culture medium composition characterized by containing particles in an amount. Claim 8 In claim 5, the above composition is characterized by increasing the blastocyst rate of the unitarated egg. Claim 9 In claim 5, the composition is characterized by increasing the maturation rate of the egg. Claim 10 A culture method for promoting the development of a porcine parthenogenetic egg, comprising: 1) a step of collecting a porcine oocyte that has matured in vitro; and 2) a step of culturing the porcine oocyte in vitro using the composition of claim 5. Claim 11 delete Claim 12 delete Claim 13 A culture method according to claim 10, characterized in that the oocyte is an oocyte whose maturation is induced through 40 to 50 hours of in vitro maturation (IVM). Claim 14 A culture method according to claim 10, characterized in that the above-mentioned egg is an oocyte surrounded by cumulus cells. Claim 15 A culture method according to claim 10, characterized in that the above-mentioned egg is an oocyte having a polar body upon completion of maturation. Claim 16 A culture method according to claim 10, characterized in that the in vitro culture (IVC) is carried out for 2 to 8 days. Claim 17 Porcine parthenogenetic egg produced by the culture method that promotes the development of the parthenogenetic egg of claim 10. Claim 18 A porcine parthenogenetic blastocyst produced by the culture method that promotes the development of the parthenogenetic egg of claim 10.

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