79 results about "Mature oocyte" patented technology

An IVF system for successfully utilizing spermatozoa separated into X-chromosome bearing and into Y-chromosome bearing population for insemination. The IVF system includes fertilization medium that can shorten the time from insemination to cleavage and a portable incubator for the transportation of maturing oocytes and inseminated oocytes comrprising a straw (19) and an incubation element (20) that can be sealed with a cap (22).

An IVF system for successfully utilizing spermatozoa separated into X-chromosome bearing and into Y-chromosome bearing population for insemination. The IVF system includes fertilization medium that can shorten the time from insemination to cleavage and a portable incubator for the transportation of maturing oocytes and inseminated oocytes comprising a straw (19) and an incubation element (20) that can be sealed with a cap (22).

The invention provides a cultivation method of cross-bred wagy. The method comprises the steps of culturing cross-bred wagy embryo in vitro and transplanting the cultured embryo into uterus of receptor cow to breed, wherein the embryo in-vitro culture method comprises the following steps: (1) taking an ox with 99.9% of Japanese wagy hereditary characters as a paternal line, and Holstein cow as a maternal line, respectively extracting semen of the paternal line and ovocyte of the maternal line; (2) culturing the ovocyte in ovocyte follicle fluid; (3) performing in-vitro fertilization on mature ovocyte in an in-vitro fertilization culture solution; (4) culturing the fertilized ovum in the embryo culture solution; and (5) vitrifying the in-vitro embryo. The in-vitro cultured embryo is transplanted into the uterus of receptor cow by a bilateral uterine lining embryo transplantation method. At last, the cross-bred first filial generation wagy new strain with characters of Japanese wagy and Holstein cow is cultivated, and has high table purpose value and good production performance.

The extracorporeal culture and mature method of immature oocyte from ovary tissue block includes selecting marrow mesenchyme stem cell MSC of female mouse as the feeder cell, compounding MSC culture liquid and agar and covering the culture rack, adding HCG in 3 IU / ml, and adhering the ovary tissue block on the rack to culture. In the co-culture system, estradiol, progestin and luteotopin contents are increased obviously, and after culturing, there is mature oocyte discharged. The method is significant in surviving rear endangered species of animals, breeding fine variety of animal, cell biological research of embryo stem cell, etc.

The invention discloses mature optimized human oocyte cytoplasm fluid. The invention develops the mature optimized human oocyte cytoplasm fluid by adding some important hormones, energy substances and efficient antioxidant melatonin on the basis of low-sugar TCM199 tissue culture solution and patient autoserum. The invention develops a culture solution which is suitable for mature optimization of the oocyte cytoplasm in IVF-ET period and also is suitable for reutilization after in vitro maturity of the immature oocyte in the period. The mature optimized human oocyte cytoplasm fluid is capable of improving the maturity degree of the cytoplasm of the matured oocyte. A clinical result proves that the high-quality embryo rate of the oocyte is greatly increased, the development potential of the immature oocyte is increased and some immature oocytes in the IVF-ET period have a reutilization value.

The invention discloses a simple in-vitro maturation culture method for oocytes, which comprises the following steps: A1, collecting oocytes; A2, preparing a closed air bag and performing in-vitro maturation culture of oocytes; and A3, performing parthenogenetic activation and in-vivo culture of oocytes, namely after 22 to 24 hours of in-vitro maturation culture, selecting mature oocytes discharging first polar bodies, activating by a chemical process in the 28th hour, culturing for 3 days in merogenesis culture solution, culturing for 4 days in blastaea culture solution, and observing and counting a blastaea rate after 7 to 9 days. When a self-prepared closed air bag system is used, the in-vitro culture of the oocytes and embryos can be accomplished by using a common biochemical culture tank or water bath pot, so the dependency on expensive instruments is reduced and a research doorsill is reduced. When the simple closed air bag is used for in-vitro culture of the oocytes, blastaea can be grown, and the in-vitro maturation rate, merogenesis rate and blastaea rate are higher than those of oocytes cultured in a normal CO2 culture tank.

The invention discloses a nutrient solution for maturation of oocyte in vitro and a method for culturing oocyte using the nutrient solution, and relates to a nutrient solution for nutrient solution for oocyte and a method for culturing the oocyte with the nutrient solution. The nutrient solution and the culturing method using the nutrient solution solve the problem that the viability of oocyte culturing in vitro is low. The nutrient solution for maturation of oocyte in vitro is composed of normal nutrient solution of oocyte, vitamin B12, fetal calf serum, FSH, LH, 17 betas estradiol, sodium pyruvate, EGF, penicillin and streptomycin sulphate. The oocyte is placed into the utrient solution for maturation of oocyte in vitro, the maturation culture on the oocyte lasts for 20-24 hours in the environment in which the volume concentration of CO2 is 5%, and a mature oocyte is acquired. The nutrient solution for maturation of oocyte in vitro can decrease stress injury caused by the external environment, the cytoplasm maturing of the oocyte is promoted, and the parthenogenetic activation of the matured oocyte matured in vitro and the embryos developmental potential after being fertilized are improved.

The method of obtaining immature oocyte from ovary tissue through separating or extracting for extracorporeal culture includes selecting marrow mesenchyme stem cell MSC of female mouse as the feeder cell or culturing the immature oocyte in the MSC culture liquid. After culturing, the co-culture system has obviously increased estradiol, progestin and luteotopin contents, and over 90 % of the immature oocyte become mature. The method is significant in surviving rear endangered species of animals, breeding fine variety of animal, cell biological research of embryo stem cell, etc.

A method of producing a cloned or genetically modified non-human mammalian embryo comprising: (a) providing a cell culture comprising a plurality of in vitro matured oocytes; (b) preferentially selecting from the cell culture a rapidly matured oocyte or developmentally competent oocyte; (c) transferring DNA from a donor cell derived from non-human mammalian tissue to the matured oocyte to form a nuclear transfer unit; and (d) culturing said nuclear transfer unit to form an embryo. At the initiation of maturation, oocytes are preferably beyond the GV-II stage of prophase I. Porcine oocytes most preferably mature in about 20-28 hours.

Methods and kits are provided for assessing the ovarian reserve and predicting the ovarian response to fertility treatments in a female subject. The serum levels of MIS are shown to be positively correlated with the production and retrieval of mature oocytes and serve as prognostic indicators for the female response to fertility treatment. The MIS levels can be monitored prior to and during fertility treatment and are useful to adjust the timing and dosage of treatments in order to produce optimal outcome in individual patients, to avoid ovarian hyperstimulation, or to indicate cancellation of an unsuccessful treatment. MIS can also be administered to women to stimulate follicle development and to prevent depletion of ovarian reserve.

The invention provides a method for fertilizing oocytes in vitro of young stock twice, which comprises the following steps of: performing FSH super-ovulation processing on 45 to 100-day young female animals which are receptors and taking more than 100 oocytes from each young female animal for later use; mixing the oocytes in vitro matured for 25 to 26 hours and sperms to finish in-vitro fertilization of the first time; and after 5 to 6 hours, performing the in-vitro fertilization of the second time, transferring the fertilized oocytes into a culture plate for culture after 13 to 14 hours, and counting a clearage rate after 48 hours. The matured oocytes need treating with 0.1 percent hyaluronidase for 30 to 60 seconds, and then are added into a fertilization medium for incubation after cumulus cells are partially removed from the oocytes in a blowing and sucking way, wherein the concentration of the fertilization medium is 50 to 70 mu l per drop and is well balanced for 2 to 5 hours; semen is unfrozen with a water bath and is placed into a CO2 box, and after 20 to 25 minutes, supernatant fluid is extracted and centrifugated for 2 to 5 hours, and then the precipitated sperms are added into the fertilization medium for incubation according to the density of 2-4*106 sperms per ml; ova fertilized for 5 to 6 hours are taken out and are placed into the fertilization medium; the sperms are unfrozen by the similar method, are added into the fertilization medium, and after 13 to 14 hours, are washed and transferred into a well-balanced four-hole culture plate for culture, wherein each hole of the four-hole culture plate contains 500 mu l of the washed solution. The method has the advantages of simplicity, practicability and the capacity of making the fertility rate of young cows and ewes reach 80 to 92 percent while blastocyst rate reach 40 to 50 percent.

The invention relates to an immature oocyte in vitro maturation promoting culture medium and application thereof. The immature oocyte in vitro maturation promoting culture medium is screened out through research on effects of different additives on immature oocytes and prepared by adding 20-100 mu M of BAPTA-AM in normal oocyte in vitro maturation culture solution. The immature oocyte in vitro maturation promoting culture medium is safe and free to side and toxic effects, enables meiosis of the immature oocytes, particularly those in level 3 cumulus-oocyte complexes, improves the in vitro development of the oocytes, promotes maturation, improves the cleavage rate and the blastula development rate after the mature oocytes are parthenogenetically-activated and further improves embryo quality. Application of the immature oocyte in vitro maturation promoting culture medium provides favorable support for the technology of in vitro propagation of animal embryos.

The present invention addresses the problem of providing a method for differentiating primordial germ cells into functionally mature GV stage oocytes by in vitro culture. The present invention pertains to a method for differentiating primordial germ cells into functional GV stage oocytes in vitro including (a) a step for forming secondary follicles by culturing primordial germ cells and feeder cells adjacent to the primordial germ cells under conditions that eliminate the effects of estrogen or factors having a similar function to estrogen, (b) a step for partially cleaving the bonds between the granulosa cell layer and the thecal cell layer among the oocyte, granulosa cell layer, and thecal cell layer that constitute the formed secondary follicles, and (c) a step for differentiating the oocytes into functional GV stage oocytes by culturing the oocytes and granulosa cell layer that constitute the secondary follicles and the thecal cell layer in medium including a polymer compound.

The present invention provides a novel oocyte maturation medium or / and embryo culture medium with a chemically defined supplement to produce matured oocytes at high efficiency. The inventive medium or supplement comprises three growth factors, namely, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF-1) in a synergistic combination. Methods for oocyte and embryo culture are also provided.

The invention discloses a preparation method of heterogeneous in-vitro fertilization embryos of cows and cattle. The preparation method includes that cow ovaries abandoned in a slaughter house are adopted and subjected to gradient washing through normal saline in a temperature increasing type, oocytes are extracted for in-vitro maturation, and high-quality in-vitro matured oocytes are obtained; high-yield cow sperms are unfreezed and placed into a fertilization medium to be gently patted through a pipette, the cow sperms float in an incubator after overturning up and down for several times, in-vitro fertilization is performed, and the in-vitro fertilization embryos of the cows and the cattle are prepared. The oocytes are obtained through the ovaries abandoned in the slaughter house, so that utilization rate of cow resources is increased; through improvement of technical details, good effect is achieved; utilization of good male herbs can be greatly increased, the good male herbs can obtain a great deal of offspring, and function of the preparation method in genetic modification of herbs is expanded.

The invention belongs to the field of assisted reproduction and particularly relates to vitrification refrigerating liquid. According to the vitrification refrigerating liquid provided by the invention, taxol is added to serve as a cryoprotectant and the vitrification refrigerating liquid obtained with reference to the taxol adding mode is treated, so that the development rate of in vitro fertilized blastocyst after the oocyte is thawed can be increased. The taxol serving as an additive in the refrigerating liquid can achieve the effects of reducing freezing injury of the mature oocytes and improving the development capability of the embryo after fertilization. The optimal concentration of the adding mode is obtained by researching the adding concentration of the taxol. The vitrification refrigerating liquid provided by the invention can greatly increase recovery rate after the frozen mature oocytes are thawed and the development rate of the blastula and achieves the protective effecton the cell ultrastructure.

Using electroporation, it is possible to activate the natural reprogramming potential of living Xenopus laevis oocytes and pass it on to donor cells placed with eggs in one electroporation chamber. We demonstrated that co-electroporation at 150 v / cm / 25 μF of mature oocytes with ˜105 cells / ml of suspension of various normal and cancerous human cell lines, such as bone marrow stromal cells, foreskin fibroblasts, pre-adipocytes, CD4+ T-lymphocytes, cheek cells, cervical carcinoma (HeLa) cells and breast adenocarcinoma (MCF-7) cells, reprograms donor cells into iPSc-like cells, which form colonies on irradiated MEF feeders. The iPSc-like cells generated by this study resemble human embryonic stem cells in colony morphology and expression of stem cell-associated transcription factors, including Oct3 / 4, Nanog, SOX-2, Rex-1, TRA-1-60 and SSEA-1. New method obviates the use of retroviral or lentiviral gene delivery vectors and other “non-parental” reprogramming approaches.

The invention relates to a method for improving the developmental capacity of sheep mature oocytes after vitrification refrigeration, belonging to the technical field of biology. The method comprises the steps of micro-injection fertilization of sheep mature oocytes and activation of fertilized eggs, wherein the activation of the fertilized eggs adopts a direct current pulse-chemical activation-direct current pulse mode, and experiments prove that the embryonic developmental capacity of the vitrified sheep mature oocytes subject to is greatly improved after the vitrified sheep mature oocytes is subject to intracytoplasmic sperm injection and is activated by the activation method of the invention. Compared with the conventional external fertilization method, the cleavage rate and the blastocyst rate are respectively increased to 13.7% and 6.8%. At the same time, by comparing the chromosome ploidy of embryos subject to different treatments, the result indicates that the proportion of normal-ploidy embryos (71.5%) obtained by the ECE method is apparently higher than that (45.4%) obtained by the common chemical activation method and no obvious difference exists compared with the external fertilization groups.

The invention discloses a culture solution for promoting in-vitro maturation of porcine oocyte, the culture solution takes an M199 culture solution without HEPES as a basic culture solution, and porcine follicular fluid, L-cysteine, sodium pyruvate, epidermal growth factor, insulin, gonadotropin, chorionic gonadotropin and WNT5A cytokine are also added. When the culture solution is used for carrying out in-vitro culture on the porcine oocytes, the in-vitro maturation efficiency and quality of the oocytes can be effectively improved, the obtained mature oocytes are used for carrying out porcinein-vitro fertilization and somatic cell nuclear transfer embryo production, and the blastocyst development rate and quality of the mature oocytes are also remarkably improved.

The invention relates to a buffalo oocyte in-vitro maturation (IVM) culture method. The method is characterized by comprising the following steps: oocyte preparation, IVM culture droplet preparation and in-vitro maturation culture. The IVM culture fluid is composed of TCM199, FBS, oFSH, 17bet-E2 and GSH. The buffalo oocyte IVM culture method can obviously promote the maturation of oocyte cytoplasms, and solves the problem of low embryo ectogenesis rate after the buffalo in-vitro mature oocytes are fertilized. The maturation culture fluid can promote the maturation of the buffalo oocytes, and has favorable effects on maintaining the morphology of the oocytes and early embryos.

The invention discloses a method for improving the parthenogenetic development potential of vitrified mature oocytes, wherein oocytes are frozen in a melatonin-containing freezing liquid, and then arethawed with a melatonin-containing sucrose solution, the thawed oocytes are cultured, the cultured oocytes are subjected to parthenogenetic activation, and finally the parthenogenetically activated embryo is cultured in vitro. According to the present invention, the method has advantages of simple process and short time consumption, and can effectively improve the parthenogenetic activation development potential of vitrified mature oocytes.

The present invention provides compositions and methods for regulating fertility in mammals. In general, the invention relates to a novel protein produced by oocytes named JY-1, and nucleic acids encoding the JY-1 protein, for controlling folliculogenesis and early embryonic development, particularly in monoovulatory species. In particular, the present invention provides nucleic acid and amino acid sequences encoding JY-1, vectors for the expression of JY-1, host cells expressing JY-1, RNAi probes for reducing levels of JY-1 message, and antibodies to JY-1. Specifically, developing and mature oocytes express JY-1 in vivo, while granulosa cells treated in vitro with recombinant JY-1 (rJY-1) protein reduced cell proliferation while increasing progesterone synthesis and estradiol production. Further, reducing JY-1 protein in developing embryos in vitro using inhibitory siRNA constructs corresponded with arrested blastocyte maturation.

The invention provides an extraction method of a mouse ovarian tissue fluid. The method comprises the following steps: placing mouse ovaries in a basal medium; mashing and dissloving the ovarian tissue fluid in the basal medium to form a pre-culture solution. The invention also provides an in-vitro maturation culture method of mouse oocyte, and the method comprises: transferring an immature mouse oocyte into a mature culture solution for in-vitro culture, wherein the mature culture solution is formed by adding the pre-culture solution in the basal medium of oocytes. In addition, the invention also provides a method for obtaining a test-tube mouse through embryo transplantation, and the method comprises: carrying out in-vitro fertilization and embryo transplantation on the oocyte matured by the in-vitro maturation culture method. By using the ovarian tissue fluid provided by the invention in the in-vitro maturation of the mouse oocyte, the cleavage rate of the in-vitro fertilized mature egg can be effectively increased, and is nearly the same as the that of the in-vivo mature egg; and finally, the test-tube mouse can be obtained through embryo transplantation.

The invention discloses a method for evaluating the influences of smoking on the quality of oocytes. Specifically, oocytes are cultured to be mature in culture solutions containing different concentrations of cotinine. Finally, the influences of smoking on the quality of oocytes are evaluated by detecting some indicators, such as the maturation rate of oocytes, chromosomal aneuploidy, actin distribution, mitochondrial membrane potential, intracellular reactive oxygen species level, spindle morphology, chromosome arrangement of mature oocytes, sperm binding number after in-vitro fertilization,pronucleus formation rate after parthenogenetic activation and the like. Through a large number of objective tests, the influences of smoking on the quality of oocytes are evaluated, thus providing areference of theoretical data for the maintenance of reproductive capacity of female smokers.

The use of IL-17 for the in vitro maturation of mammalian oocytes is described. The in vitro matured oocytes may be used for in vitro fertilization protocols.

The present invention provides a method for producing human cloned embryos by employing inter-species nuclear transplantation technique. The method for producing human cloned embryos of the invention comprises the steps of: preparing donor somatic cell lines collected from human; maturing oocytes collected from ovary of cow in vitro; removing the cumulus cells surrounding the oocytes; cutting a portion of zona pellucida of the matured oocytes to make a slit, and squeezing out a portion of cytoplasm including the first polar body through the slit to give enucleated recipient oocytes; transferring a nucleus to the recipient oocyte by injection of the donor cells to the enucleated recipient oocytes, followed by the subsequent electrofusion and activation of the electrofused cells to give embryos; and, postactivating and culturing the embryos in vitro. The human cloned embryos of the invention can be employed to obtain the human embryonic stem cells, which may be widely applied in biological and medical fields.

The invention discloses a porcine oocyte in-vitro maturation culture solution added with a chemotactic factor ligand 14 (CXCL14). The culture solution can improve the quality of in-vitro maturation of oocytes. The quality of the oocytes is closely related to a parthenogenetic activation technology, an in-vitro fertilization technology and a somatic cell cloning technology, and improvement of the quality of the in-vitro mature oocytes plays an extremely important role in the fields of livestock production, medical reproduction, life science research and the like.

The invention belongs to the field of animal breeding and particularly relates to an artificial reproduction-hybridization joint breeding method for a meat goat. The method comprises the steps of selecting one goat variety with good meat performance as a female parent, selecting another goat variety with a large body size as a male parent; culturing oocytes of the female parent in vitro into mature oocytes containing a first polar body, and soaking semen of the male parent with a semen motility enhancement liquid and then preparing a semen suspension; carrying out in vitro fertilization on themature oocytes and the semen suspension, culturing zygotes into a hybrid blastocyst form, transplanting hybrid blastocysts into a body of the female parent for developing to natural delivery in the body of the female parent and obtaining hybrid F1; and backcrossing the hybrid F1 and the female parent, backcrossing backcross descendants and the female parent for multiple times until a breeding objective is met, and carrying out crossbred fixing to obtain a new variety / line with good characters. According to the method, an artificial assistant reproduction technology is combined with a traditional hybridization technology, so that the number of animal embryos can be increased, the survival rate of the animal embryos can be improved and the method has a good application prospect in the fieldof animal breeding.

The invention discloses a method for establishing a source of heredofamilial premature ovarian failure and inducing differentiation of totipotential stem cells to original genital ridge cells. The method is characterized in that adult cells of patients with heredofamilial premature ovarian failure are applied and reprogrammed into induced totipotential stem cells in vitro, and then are induced and differentiated into precursor cells of the oocytes, namely the original genital ridge cells. The method is a method for obtaining original genital ridge cells from patients with heredofamilial premature ovarian failure, lays foundation for further obtaining manure oocytes, and is expected to be applied in the field of reproductive medicine and translational medicine.

A microchip includes a first chamber and second chamber formed to a predetermined depth in a member made of polydimethylsiloxane (PDMS); and a channel (microchannel) connecting the first chamber to the second chamber. These components mimic in vivo organs which produce by selecting capacitated motile sperm from semen and causing the selected sperm to meet mature oocyte, and these components are capable of effectively producing motile capacitated good-quality sperms from sperms ejaculated outside the body.