KR20140055651A - Medium compositions for oocytes maturation and embryo development of mammals and methods for oocytes maturation and embryo development of mammals using the same - Google Patents
Medium compositions for oocytes maturation and embryo development of mammals and methods for oocytes maturation and embryo development of mammals using the same Download PDFInfo
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Abstract
Description
The present invention relates to a medium composition for promoting oocyte maturation and embryo development in a mammal, and a method for promoting oocyte maturation and embryo development using the same, and more particularly, to a medium composition for mammalian oocyte maturation and embryo development including a mammalian animal including tauroursodeoxycholate And to a method for promoting mammalian oocyte maturation and embryo development using the medium for promoting oocyte maturation and embryo development.
Unfolded or misfolded proteins accumulate in the endoplasmic reticulum (ER) and activate the Unfolded Protein Response (UPR), allowing cells to respond to stress. ER is the main cell organs of cell membrane proteins and lipid synthesis and is involved in the maintenance of intracellular calcium homeostasis. ER homeostasis fluctuates, or an overload in the regulatory mechanism leads to apoptosis of cells due to sustained stress. These mechanisms of ER stress induced apoptosis in mammals are not well known, but it is understood that EIF2AK3 (PERK), ERN1 (IRE1a) and ATF6 activate UPR in ER stress processes.
In mouse and human somatic cells, X-box-binding protein 1 (XBP1) is known to be the main activator controlling the expression of the UPR gene. In the stress state, and the transfer of the XBP1 gene is derived, transcripts generates a XBP -1 (XBP1 -s) as a result of the non-splicing is typically 26 ribonucleotides is removed by splicing endoribonuclease. Only the spliced XBP- 1 migrates to the nucleus of the cell and activates the UPR gene. Thus, XBP1 -s are in vivo and in lt; RTI ID = 0.0 > ER < / RTI > stress in vitro .
On the other hand, there are various attempts to mature mammalian embryos in order to provide a more effective method of in vitro fertilization. For example, Korean Patent Registration No. 10-1107329 entitled " Medium Composition Containing Tauroluxodeoxycholic Acid and Method for Mass Production of a Mammalian Embryo Using the Invention "discloses that an in vitro fertilized egg or a cloned embryo is added with taurolosodeoxycholic acid A method of culturing in a medium to enable mass production of embryos is disclosed.
The object of the present invention is to provide a medium composition for promoting oocyte maturation and embryo development of a mammal and to provide a method of using the medium composition to mature mammalian oocytes and to promote embryonic development .
In order to solve the above problems, the present invention provides a medium composition for promoting oocyte maturation and embryo development in a mammal containing an apoptosis inhibitor by ER stress as an active ingredient .
Preferably, the ER stress-induced apoptosis inhibitor is tauroursodeoxycholate.
The present invention also provides a method for promoting oocyte maturation and embryo development in a mammal which inhibits apoptosis caused by ER stress.
The method is preferably carried out by culturing the mammalian oocyte in a medium containing tauroursodeoxycholate.
It is preferable that the above tauroursodeoxycholate is added to the medium at a concentration of 10-100 μM.
The culture medium composition of the present invention and mammalian oocyte maturation and method for promoting embryonic development using the same can promote efficient oocyte maturation and embryo development in mammalian oocytes.
FIGS. 1A and 1B show the results of detection of resistant XBP-1 in mouse oocyte and preimplantation embryos,
FIGS. 2A-2C show the results of detection of XBP- 1 splicing in mouse preimplantation embryos,
FIGS. 3A and 3B are the results of measuring the effect of a stress inducer in mouse embryo development,
FIGS. 4A to 4D show the results of measuring the expression of active XBP-1 in a 1-cell step embryo,
5A and 5B are the results of confirming the concentration dependency of the distribution of XBP-1 in the stress inducer,
6A and 6B show the results of confirming the effect of TUDCA on apoptosis in mouse blastocyst,
7A to 7D show the results of detecting the ER stress signal in the pig system,
8 is a photograph showing the expression pattern of XBP- 1 mRNA in porcine oocyte and preimplantation embryos,
Figures 9a-9c show the effect of TUDCA on the maturation of porcine oocytes,
Figures 10a-10d show the effect of TUDCA on the quality of porcine blastocyst.
Hereinafter, the present invention will be described in detail.
The inventors of the present invention have conducted extensive research to develop a method capable of promoting embryonic development by matured oocytes in large quantities in vitro using mammalian oocytes, and as a result, it has been found that an apoptosis- To inhibit apoptosis caused by ER stress in the cells, thereby inducing the maturation of oocytes and promotion of embryonic development, thereby completing the present invention.
Accordingly, the present invention provides a medium composition for promoting oocyte maturation and embryo development in a mammal containing an apoptosis inhibitor by ER stress as an active ingredient.
The ER stress is caused by intracellular UPR and plays an important role in a series of processes leading to apoptosis.
The ER stress-induced apoptosis inhibitor is tauroursodeoxycholate (TUDCA) represented by the following formula (1).
≪ Formula 1 >
Taurosoldoxylcholate is a type of bile acid which is an analogue of urushodeoxycholic acid and is mainly used for the treatment of cirrhosis. The taurosoldoxylcholate contained in the medium component for promoting oocyte maturation and embryo development of the mammal of the present invention is a compound of formula (1), which is equivalent to the effect of promoting oocyte maturation and embryo development in a mammal, Taurosoldoxylcholate as well as derivatives thereof can also be included.
The taurosoldoxylcholate contained in the medium composition for mammalian oocyte maturation and embryo development promotion of the present invention is preferably added at a concentration of 10 to 100 μM.
The mammalian oocyte maturation and embryo development promoting medium may be added to the basic medium used for culturing germ cells of a conventional mammal by adding the tauroursodeoxycholate of the present invention, , Carbon sources, amino acids, bovine serum albumin, other co-factors, and the like, can be used as general media known to those skilled in the art. Commercially available media include, for example, NCSU-23 or PZM-3.
The term "mammal" as used herein includes, but is not limited to, pigs, cows, goats, sheep and mice except humans.
The term " oocytes "of the present invention refers to cells that have stopped growing proliferation and entered the growth phase in the ovary during the process of oocyte formation in mammals.
The term " maturation "of the present invention means that the oocyte has grown to such an extent that it can be fertilized with the sperm.
The term "embryos" of the present invention refers to mature oocytes and embryos developed and modified by sperm.
The term " development "of the present invention refers to the process in which an embryo is matured as an individual.
The present invention also provides a method for promoting oocyte maturation and embryo development in a mammal which inhibits apoptosis caused by ER stress.
The method is preferably carried out by culturing the mammalian oocyte in a medium containing tauroursodeoxycholate.
It is preferable that the above tauroursodeoxycholate is added to the medium at a concentration of 10-100 μM.
Hereinafter, the present invention will be described in more detail with reference to examples.
[ Example ]
All chemicals used in this example were purchased from Sigma (St. Louis, MO, USA) unless otherwise noted. In addition, all animal experiments used in this example were carried out in accordance with the regulations of the Animal Experiment Ethics Committee of Chungnam National University. All data used in this example were analyzed using Fisher's protected least significant difference (LSD), which included one-way ANOVA and general linear models of the Statistical Analysis System (SAS, Cary, NC). The deviation is < 0.05.
Example One
1. Materials and Methods
Preparation of mouse oocytes and embryos
Oocytes of the vesicle stage were isolated as cumulus-oocyte complexes (COCs) from ICR female mice (Charles River). Five to seven week old female mice were injected with 5 IU PMSG to induce superovulation and sacrificed after 48 hours. Ovaries were collected in FHM medium (Millipore). COCs were mechanically removed and oocytes were washed by pipetting in FHM medium containing 0.1% (w / v) hyaluronidase. ovary cells containing germinal vesicles were collected. Mature MII oocytes were injected with PMSG and injected with 5
Embryonic in vitro culture Wow TUDCA treatment
To confirm the effects of TM (tunicamycin), sorbitol and TUDCA on embryonic development, the two-cell stage embryos were harvested at 44h phCG and cultured in the presence or absence of TM, sorbitol or TUDCA. The required drug concentration was diluted with stock solution to M16. Twenty-five to thirty embryos were placed in culture medium of 40 μL droplets, covered with mineral oil, and cultured at 37 ° C under 5% (v / v) CO 2 .
RT - PCR
To analyze gene expression, various stages of mature oocytes and early embryonic stages were collected. Total RNA was isolated from RNeasy Mini Kits (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. For the reverse transcription reaction, the final 20 μL volume of total RNA (0.5 mg oligo-dT, 1 × RT buffer, 10 mM dithiothreitol, 10 mM dNTPs) was subjected to a reverse transcription reaction at 37 ° C. for 50 minutes and 70 ° C. for 15 minutes, Lt; / RTI > Each RT-PCT reaction was 4 μL cDNA, 10 pm / μL forward and reverse primers (Table 1). 3 times repeatedly. The mRNA concentration of each sample was measured by β-actin mRNA.
(° C)
(bp)
Immunofluorescence Analysis
Several stages of mouse embryos were fixed in 4% (v / v) paraformaldehyde for 30 min at room temperature and infiltrated with 0.1% (v / v) Triton-100 for 30 min. Bovine embryos and embryos were blocked by overnight incubation in PBS containing 3% (w / v) BSA at 4 ° C and incubated with rabbit polyclonal anti-XBP-1 antibody (Santa Cruz Biochemicals, Santa Cruz, CA, USA) for 1 hour at < RTI ID = 0.0 > 37 C. < / RTI > After washing with PBS containing 0.5% (v / v) Tween-20, the sample was reacted with anti-rabbit FITC-conjugated secondary antibody in a blocking solution for 60 minutes at 37 ° C. Next, samples of various developmental stages were analyzed using VECTASHIELD Mounting Medium. Images were acquired using a Zeiss scanning laser confocal microscope and analyzed using the LSM Image Browser. At least 20 oocytes or embryos were measured at each step.
Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling ( TUNEL ) Assay
Blastocyst was washed three times in PBS (pH 7.4) containing polyvinylpyrrolidone (PVP; 1 mg / mL) and fixed with PBS containing 4% (v / v) paraformaldehyde for 1 hour at room temperature. After fixation, parthenotes were washed with PVA-PBS and incubated with 0.3% (v / v) Triton-X100 for 1 hour at room temperature. Twice washing the embryos in PVA-PBS and, fluorescin-conjugated dUTP and terminal deoxynucleotidyl transferase; were incubated for 1 hour at room temperature in a dark room condition (in situ Cell Death Detection Kit Roche , Mannheim, Germany). To mark the entire nucleus, 40 μg / mL propidium iodide (PI) and 50 μg / mL RNaseA were counted for 1 hour at 37 ° C., and the embryo was mounted on a thin cover slip and observed with a confocal microscope.
Western Blotting
Various stages of mouse embryos (n = 100 per sample) were washed three times with PVA-PBS and resuspended in extraction buffer (PRO-PREP; Intron Biotechnology, Seong, Korea). The extracted proteins were separated on a 10% (w / v) SDS-PAGE using a Bio-Rad apparatus (Bio-Rad, Hercules, Calif.) And transferred to the membrane using a Bio-Rad Mini Trans-Blot Cell. The membranes were blocked with Tris-buffered saline containing 5% (w / v) skimmed milk and 0.5% (v / v) Tween-20, The antibody-antibody complexes were incubated for 15 min with primary antibodies against XBP-1 and β-actin (Santa Cruz Biochemicals) dissolved in Tris buffered saline containing 10% v / v Tween-20, -rabbit IgG peroxidase conjugates using an ECL detection kit (Amersham Bioscience, Piscataway, NJ, USA). All experiments were repeated three times.
2. result
Mouse oocytes and preimplantation embryos In XBP -1 expression position
It was confirmed that most of the mouse XBP-1 in the GV, two-cell, four-cell, eight-cell, morula and blastocyst stages is present in the nucleus and partially in the cytoplasm (FIG. Conversely, it was confirmed that mouse XBP-1 in the one-cell stage was mainly present in the cytoplasm (FIG. 1A). In the meiosis stage, XBP-1 was located in the spindle microtubules in metaphase I, but the signal intensity in the spindle microtubules was weaker as it progressed to pro-metaphase II and metaphase II (Fig. 1a).
In early-stage embryos, XBP -One mRNA Splicing Characteristic
RT-PCR analysis, two-cell, four-cell, morula and blastocyst, but are not Singh ㅅseu spliced XBP -1s and a splicer in step XBP -1u mRNA is detected therefrom, the one-cell stage, only XBP -1u mRNA (Fig. 2A). Westrn Bolt analysis showed the same results (Fig. 2b). These results suggest that XBP-1 plays an important role in the development of mouse preimplantaion embryos.
Effect of culture stress on mouse embryo development
When one-cell embryos were treated with 2 μg / mL TM or 25 mM sorbitol, blastocyst development rate was significantly reduced (FIG. 3). However, in the absence of TM or sorbitol, approximately 80% of the two-cell stage embryos developed into blastocyst after 60 hours. However, embryos treated with 5 μg / mL or 50 mM sorbitol or more stopped development at the two-cell stage and did not progress to blastocyst. These results suggest that sustained culture stress may arrest embryos at the two-cell stage.
stress To inducing agent by one - cell In the step-embryo active XBP -1 induction
To confirm the relationship between culture stress and ER stress, one-cell stage mouse embryos were cultured in the presence or absence of TM or sorbitol. RT-PCR results in normal one-cell embryo splicer but washing the XBP -1 is not verified, in the case of 1μg / mL TM or a one-cell embryo treated with 25mM sorbitol was confirmed that the splicer Singh XBP -1 (Fig. 4a). These results were also the same in western blot analysis (Fig. 4c). In addition, immunostaining analysis showed that XBP-1 protein was detected only in the cytoplasm in the case of the normal one-cell embryo not treated with TM or sorbitol. However, in the one-cell embryo treated with 1 μg / mL TM or 25 mM sorbitol, XBP-1 protein was identified (Figure 4b). These results suggest that ER stress induction in early stage embryos is optimal at a concentration of 1 μg / mL TM or 25 mM sorbitol.
Stress-induced Two - cell In the stage embryo XBP -1 protein
In the absence of stress inducers such as TM and sorbitol, two-cell step embryos did not show XBP-1 in the nucleus (Figure 5a), except that some signal was detected in the cytoplasm. In addition, XBP-1 in two-cell stage embryos was rearranged into nuclei when treated with a high concentration of stress inducer and TUDCA. These results were also the same in western blot analysis (Fig. 5B). When we synthesize these results, we can predict that only XBP-1 present in the nucleus is functional.
In mouse embryo development TUDCA Effect of
To determine whether TUDCA could inhibit ER stress-induced apoptosis, one-cell embryo was divided into a control group treated with stress inducer and an experimental group treated with stress inducer and TUDCA to observe development of blastocyst, The results are shown in Table 2 below. As shown in Table 2, TUDCA showed a marked increase in the number of developed individuals as well as the rate of development into blastocyst.
blastocyst Stress in To inducing agent by Apoptosis Induction effect
Apoptosis was not observed in blastocyst treated with 1 μg / mL TM or 25 mM sorbitol. However, in the case of blastocyst treated with 5 μg / mL TM or 50 mM sorbitol, DNA fragmentation was markedly increased, and when TUDCA was treated together, blastocyst apoptosis was significantly reduced (FIG. 6). These results suggest that TUDCA attenuates apoptosis induced by ER stress in mouse embryo development.
Example 2
1. Materials and Methods
Cell culture and ER Stress condition
Porcine embryo fibroblast (PEF) cells derived from porcine embryos (25-26 days of gestation) were incubated with 10% (v / v) heat-inactivated fetal bovine serum, 1% (w / v) nonessential amino acids % (w / v) gentamicin in Dulbecco's modified Eagle medium. PFE cells were placed in wet conditions at 5% (v / v) CO 2 and 37 ° C. To induce ER stress, seeded PEF cells in a six well dish were treated with TM (2 μg / mL in dimethyl sulfoxide) and harvested 6 hours later.
Of porcine oocyte in vitro maturation
100 to 130 kg of ovarian sows from slaughterhouses were collected and placed in 0.9% (w / v) saline containing 75 μg / ml potassium penicillin G and 50 mg / ml streptomycin sulfatefmf. I moved to the lab. The cumulus-oocyte complex was aspirated from the follicle through an 18-gauge needle and placed in a 10 ml syringe. The cells were washed three times with TL-HEPES medium (HEPES-buffered Tyrode lactate; Sigma), and the oocytes were matured in IVM medium under conditions of 5% (v / v) CO 2 and 38.5 ° C using 4-well multi-dish. The media used for maturation of oocytes were 10% v / v follicular fluid, 0.57 mM cysteine, 10 ng / ml β-mercaptoethanol, 10 ng / ml epidermal growth factor, 10 IU / ml equine chorionic gonadotropin (eCG) and NCSU-23 containing human chorionic gonadotropin (hCG). After 22 hours of incubation, the oocytes were immediately washed three times with the medium and further incubated with the medium for 22 hours in the absence of the hormone.
Parthenogenic activation
in After in vitro maturation, cumulus cells were removed by repeated pipetting in TL-HEPES containing 0.1% (w / v) polyvinyl alcohol (PVA) and 0.3% (w / v) hyaluronidase. The oocytes were applied to an activation solution containing 0.3 M D-mannitol, 0.1 mM MgSO 4 , 0.05 mM CaCl 2 , and 0.01% (w / v) PVA. The oocytes were stimulated with a DC pulse of 15 kV / cm for 30 μs using an ECM 2001 Electro Cell Manipulator (BTX). After activation, the oocytes were immediately washed three times with the culture medium, transferred to 50 μL of culture medium, and then covered with mineral oil in a 60 cm diameter dish. The cultivation was carried out for 7 days under conditions of 38.5 ° C and 5% (v / v) CO 2 . Porcine Zygote Medium-3 (PZM-3) was used for in vitro culture (IVC) medium.
RT - PCR
To analyze genes associated with apoptosis, various stages of mature oocytes and early stage embryos were screened. RT-PCR was performed in the same manner as in Example 1, except that the primer set of Table 3 below was used.
gene
primer
seq.
No.
Tm
(° C)
you
(bp)
Immunofluorescence
Germinal Vesicle (GV), GV breakdown (GVBD), metaphase Ⅰ (M Ⅰ) and metaphase Ⅱ (M Ⅱ) oocytes were collected at 0, 6, 22 and 44 hours respectively and transferred to maturation medium. In addition, 1-cell, 2-cell, 4-cell, 8-cell, morula and blastocyst embryos were obtained at 10, 36, 72, 96 and 144 hours after activation of mature oocytes. The following steps were carried out in the same manner as in Example 1 above.
Dual Differential staining
The differential staining of ICM and trophectoderm in blastocyst (day 6) was performed by Machaty Z. et. al., Development of early porcine embryos in vitro and in vivo. Biol Reprod 1998; 59; 451 to 455. < tb >< TABLE >
TUNEL Assay
The procedure of Example 1 was repeated.
activation Oxygen species Measure
Mature oocytes were incubated for 20 min at 39 ° C in the presence of 10 mM 2 ', 7'-dichlorodihydrofluorescein diacetate (DCHFDA), immediately washed three times with TCM-199 medium and then analyzed by epifluorescence microscopy using a wavelength of 450-490 nm (Olympus) to detect intracellular fluorescence from DCHFDA and to monitor luminescence at 515-565 nm. Images were acquired with a digital camera (Nikon) attached to the microscope and the mean gray value of the fluorescent oocytes was measured with ImageJ software (National Institutes of Health). The experiment was repeated three times using 20-25 oocytes.
Western blot analysis
Metaphase I (MI), metaphase II (MII) oocytes and blastocyst (n = 50 per sample) were washed three times with PVA-PBS and resuspended in extraction buffer (PRO-PREP; Intron Biotechnology, Seong, Korea) . The extracted proteins were separated on a 10% (w / v) SDS-PAGE using a Bio-Rad apparatus (Bio-Rad, Hercules, Calif.) And transferred to the membrane using a Bio-Rad Mini Trans-Blot Cell. The membranes were blocked with Tris-buffered saline containing 5% (w / v) skimmed milk and 0.5% (v / v) Tween-20, (ERK1 / 2), mouse monoclonal anti-phospho-MAPK3 / 1 (ERK1 / 2) or mouse monoclonal anti-caspase-1 (ERK1 / 2) dissolved in Tris buffered saline containing anti- 3 antibody (Santa Cruz Biochemicals) primary antibody and washed with Tris-buffered saline containing 0.5% (v / v) Tween-20 for 15 minutes. Antigen-antibody complexes were incubated with anti-mouse IgG or anti-rabbit IgG peroxidase conjugates were detected using an ECL detection kit (Amersham Bioscience, Piscataway, NJ, USA). All experiments were repeated three times. Β-actin was used as a control.
2. result
Porcine oocytes and preimplantation embryos in XBP -1 distribution
When PEF cells were treated with TM for 6 hours, XBP-1 was distributed both in nucleus and cytoplasm (Fig. 7A). According to western blot analysis, treatment of PEF cells with TM for 6 hours resulted in a significant increase in XBP-1s and no change in XBP-1u, consistent with the immunostaining assay (FIG. 7b). When PEF cells were simultaneously treated with TM and TUDCA, the degree of expression of XBP-1 protein increased by TM was decreased.
The XBP-1-specific antibody was used to determine the intracellular distribution of XBP-1 in mature oocytes and early-stage embryos. The result was confirmed to be present in nuclei in GV, 4-cell, morula and blastocyst, MII), metaphase II (MII), and 1-cell, 2-cell, and 8-cell stages (FIG. In addition, in 4-cell, the expression of XBP-1 in the nucleus was remarkable by TM (Fig. 7D).
Pig from the oocyte and early stage embryo XBP -One mRNA Splicing Characteristic
PEF cells were treated with TM for 6 hours, and the expression levels of XBP-1s and XBP-1u were confirmed by RT-PCR (Fig. 8A). As a result, both XBP-1s and XBP-1u were confirmed in GV, 4-cell, morula and blastocyst stages but only XBP-1u was observed in MI, MII, 1-cell and 2-cell stages ).
in vitro In pig embryo development TUDCA Effect of
The concentration of TUDCA in PZM-3 medium was confirmed to promote the development of oocytes. As shown in Table 4, it was confirmed that the addition of TUDCA increased cleavage of oocytes. In addition, the development of embryos to the blastocyst stage in the presence of TUDCA is significantly increased compared to the control.
group
oocytes (n)
ectoderm
MAPK pathway Of oocytes through On maturity TUDCA Effect of
Maturation of oocytes cultured in TCM-199 medium supplemented with 50 μM TUDCA to GVBD, MI, and MII stages was significantly increased compared to the control (Table 5).
Concentrations of reactive oxygen species (ROS) and MAPK pathway activity affected the maturation of oocytes. ROS concentration in oocytes cultured in the presence of 50 μM TUDCA was significantly reduced compared to the control (FIG. 9A). As a result of western blot analysis, the expression of pMAPK3 / 1 (ERK1 / 2) protein in MII oocytes cultured in the presence of TUDCA was significantly increased compared to the control. However, there was no significant change in the expression of pMAPK3 / 1 (ERK1 / 2) protein in MII oocytes cultured in the presence of TUDCA (Fig. 9b and Fig. 9c).
in vitro in TUDCA Pigs by blastocyst of quality evaluation
The quality of porcine blastocyst cultured in the presence of TUDCA was compared with the control cultures supplemented with TM (Table 6).
group
oocytes (n)
ectoderm
As shown in Table 6, it can be seen that when TUDCA was treated, the cleavage rate of oocytes, the development rate to blastocyst, and the number of total nuclei were significantly increased compared to the TM treatment group. From these results, it can be predicted that the level of apoptosis reflects the quality of blastocyst. The TUNEL assay shows that DNA fragmentation is significantly reduced in the TUDCA treated group compared to the control group (FIGS. 10A and 10B).
TUDCA Pig blastocyst of Apoptosis effect
The expression level of proapoptotic gene of blastocyst cultured in the presence or absence of TUDCA was confirmed by wuantitative PCR. Expression of BCL2L1 mRNA was increased compared to the control group, but BAX and TP53 decreased (FIG. 10c).
Western blot analysis showed that expression of caspase-3 in blastocyst treated with TUDCA was significantly lower than that of the control (Fig. 10d).
The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
Claims (5)
Wherein the ER stress-induced apoptosis inhibitor is tauroursodeoxycholate.
Wherein the mammalian oocyte is cultured in a medium containing tauroursodeoxycholate.
Wherein the taurosoldecoxycholate is added in a concentration of 10 to 100 [mu] M in the medium.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019117454A1 (en) * | 2017-12-12 | 2019-06-20 | 순천향대학교 산학협력단 | Medium additive for highly efficient cell transformation using cell organelle stress regulation factor |
| KR20220080758A (en) * | 2020-12-07 | 2022-06-15 | 을지대학교 산학협력단 | Composition for cultivating fertilized eggs containing adiponectin as an active ingredient |
| KR20220125889A (en) * | 2021-03-05 | 2022-09-15 | 을지대학교 산학협력단 | Composition for cultivating fertilized eggs containing anti-miR-210-oligonucleotide as an active ingredient |
| CN115287253A (en) * | 2022-07-26 | 2022-11-04 | 五邑大学 | Application of oroxin A or analog thereof in preparation of product for improving quality of in-vitro embryo |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019117454A1 (en) * | 2017-12-12 | 2019-06-20 | 순천향대학교 산학협력단 | Medium additive for highly efficient cell transformation using cell organelle stress regulation factor |
| KR20220080758A (en) * | 2020-12-07 | 2022-06-15 | 을지대학교 산학협력단 | Composition for cultivating fertilized eggs containing adiponectin as an active ingredient |
| KR20220125889A (en) * | 2021-03-05 | 2022-09-15 | 을지대학교 산학협력단 | Composition for cultivating fertilized eggs containing anti-miR-210-oligonucleotide as an active ingredient |
| CN115287253A (en) * | 2022-07-26 | 2022-11-04 | 五邑大学 | Application of oroxin A or analog thereof in preparation of product for improving quality of in-vitro embryo |
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