69 results about "Cumulus Cell" patented technology

The present invention relates generally to the fields of reproductive medicine. More specifically, the present invention relates to a method determining the developmental stage of human cumulus cells issues from MII oocyte.

A genetic means of determining whether a female subject produces "pregnancy competent" oocytes is provided. The means comprises detecting the level of expression of one or more genes that are expressed at characteristic levels (upregulated or downregulated) in cumulus cells derived from pregnancy competent oocytes. This characteristic gene expression level, or pattern referred to herein as the "pregnancy signature", also can be used to identify subjects with underlying conditions that impair or prevent the development of a viable pregnancy, e.g., pre-menopausal condition, other hormonal dysfunction, ovarian dysfunction, ovarian cyst, cancer or other cell proliferation disorder, autoimmune disease and the like. Microarrays containing "pregnancy signature" genes or corresponding polypeptides provide another preferred aspect of the invention. Still further, the subject invention can be used to derive animal models, e.g., non-human primate animal models, for the evaluation of the efficacy of putative female fertility treatments.

A genetic means of determining whether a female subject produces "pregnancy competent" oocytes is provided. The means comprises detecting the level of expression of one or more genes that are expressed at characteristic levels (upregulated or downregulated) in cumulus cells derived from pregnancy competent oocytes. This characteristic gene expression level, or pattern referred to herein as the "pregnancy signature", also can be used to identify subjects with underlying conditions that impair or prevent the development of a viable pregnancy, e.g., pre-menopausal condition, other hormonal dysfunction, ovarian dysfunction, ovarian cyst, cancer or other cell proliferation disorder, autoimmune disease and the like. Microarrays containing "pregnancy signature" genes or corresponding polypeptides provide another preferred aspect of the invention. Still further, the subject invention can be used to derive animal models, e.g., non-human primate animal models, for the evaluation of the efficacy of putative female fertility treatments. Additionally, an improved RNA amplification protocol is provided herein referred to as the CRL amplification protocol which is suitable for reproducibly amplifying all the RNAs expressed by a cell sample, even when only a few cells are available.

A method for evaluating the quality of mammalian oocytes comprises determining the expression level of one or more of the genes ACPP, AQP11, CCDC126, CLU, CYP11 A1, CYP19A1, EGR3, FN1, FOSL2, GMNN, HRAS, HSD3B2, HS-D17B1, HSD11B2, HSDL1, IGF1, IGFBP4, IGFBP5, IRS1, KCNK3, KLF6, NEK6, SMAD7 or STC1 in a test sample derived from a cumulus cell or granulosa cell associated with the oocyte, and comparing the expression level of said at least one marker gene expression in the sample with the expression level in a control. Differential expression of the gene between the sample and the control is indicative of the quality of the oocyte.

The invention relates to a method for in-vitro fertilization and embryo culture by collecting bovine oocytes in vivo. Specifically, on one hand, the bovine in-vitro fertilization embryo culture methodcomprises the following steps of: taking follicular fluid derived from ovum pick-up of bovine living body, picking out a cumulus-oocyte complex wrapped with at least containing three layers of cumulus cells under a stereoscopic microscope, putting the cumulus-oocyte complex into a transport culture solution, and transporting the cumulus-oocyte complex back to a laboratory within 24 hours; washingCOCs once in an oocyte maturation culture solution and then transferring into a new maturation culture solution for culturing for 22-24 h under the culture conditions that the temperature is 38.8 DEGC, the concentration of CO2 is 5.5-6.5% and the humidity is saturated; performing in vitro fertilization; and carrying out embryo in vitro culture and preservation. The invention also relates to a transport culture solution for oocytes. The method and the transportation culture solution disclosed by the invention show excellent technical effects as shown in the specification.

Methods are provided for evaluating an oocyte for fertilization and implantation. For example, methods are provided for determining whether an oocyte expresses, or does not express, one or more of a group of markers identified as differently expressed between chromosomally normal and chromosomally abnormal oocytes. Also provided, for example, are methods for determining whether a cumulus cell expresses, or does not express, one or more of a group of markers identified as differently expressed between cumulus cells associated with chromosomally normal oocytes and cumulus cells associated with chromosomally abnormal oocytes. Methods are provided for the detection of marker expression of differentially expressed genes at the RNA level, as well as at the protein level.

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 provides a screening culture method for sheep oocytes in vitro, which comprises the following steps: step 1: ovaries which were killed less than half an hour ago are put into saline water at the temperature of 30DEG C to 40DEG C and containing penicillin and streptomycin, washed for 3 to 4 times within 3 hours, and oocytes are picked out; 25 to 30mu mol / L brilliant cresyl blue is put into 35DEG C to 39DEG C water bath to be dyed for 85 to 92min, the cytoplasm is blue and is washed for 3 to 4 times by in vitro maturation fluid, is put into 55 to 78mul / drip in vitro maturation culture fluid by 25 to 30m / drip, and is cultured in 5 percent CO2 by 95 percent; step 2: cumulus cells are removed from oocytes which are maturated in vitro for 25 to 28 hours; IVF washing liquid is used to wash for 2 to 4 times, and is dripped into 50 to 70mu l fertilization fluid by 25 to 30m / drop; semen is unfrozen, the fertilization fluid is moved in, supernatant fluid is centrifugurated for 4 to 5min and removed, the precipitated sperms are added into fertilization fluid drips by the density of 2 to 4*106 / ml and incubated; eggs which are fertilized for 12 to 18 hours are treated in the above step and then moved into a four-hole culture plate to be cultured; and step 3: reagent egg absorption liquid, brilliant cresyl blue maturation liquid, dyeing liquor SOF, the fertilization fluid, the culture fluid and sheep serum are prepared.

The present invention relates to the competence of oocytes to fertilization, uterine implantation and development into a living being. The invention describes ovarian markers whose expression is predicative of oocyte competency that are detected and / or measured in follicular fluid, cumulus cells and / or follicular cells of a mammal. Also described are methods for evaluating competence of mammalian oocytes, methods for selecting a mammalian oocyte for assisted reproduction (AR), and screening methods for identifying stimulatory or inhibitory compounds to mammalian oocyte competence.

The invention discloses an optimized mice somatic cell nuclear transplantation method. According to the mice somatic cell nuclear transplantation method, three kinds of cells, including cumulus cell, fetal fibroblasts, and embryonic stem cell, are adopted to construct reconstructed embryos; the activation efficiencies of three reconstructed embryos in different activation mediums are compared, development efficiencies in different solutions are compared, and pregnancy efficiencies of different embryo transplantation methods are compared; it is concluded that calcium-free KSOM activation medium is a universal high efficiency activation medium, and the activation efficiency is about 93.5%; KSOM-AA culture medium is suitable for in vitro culture of the reconstructed embryos of cumulus cells and embryonic stem cells, and the best culture medium for fetal fibroblasts is alpha MEM culture medium; when the reconstructed embryos are at 2-cell period, nuclear transplanted animals can be obtained via transplantation of embryos through bilateral fallopian tubes, wherein for each fallopian tube, transplantation of 15 embryos is carried out. The optimized mice somatic cell nuclear transplantation method is capable of increasing reconstructed embryo cleavage rate, blastula development rate, cloning animal birth rate, and survival rate, so that the success rate of obtaining embryonic stem cells from the reconstructed embryos is increased.

Cumulus cell (CC) gene expression is explored as an additional method to morphological scoring to choose the embryo with the highest chance to pregnancy. The present invention relates to a novel method of identifying biomarker genes for evaluating the competence of a mammalian oocyte in giving rise to a viable pregnancy after fertilization, based on the use of live birth and embryonic development as endpoint criteria for the oocytes to be used in an exon level analysis of potential biomarker genes. The invention further provides CC-expressed biomarker genes thus identified, as well as prognostic models based on the biomarker genes identified using the methods of the present invention.

The invention discloses a pig oocyte in-vitro maturation culture solution and a preparation method and application thereof, and belongs to the technical field of oocyte in-vitro maturation culture. The problems of low in-vitro maturation rate and development rate of porcine oocytes currently are solved. The pig oocyte in-vitro maturation culture solution is prepared from a basal culture solution TCM-199, penicillin, streptomycin, NaHCO3, 4-hydroxyethylpiperazine ethane sulfonic acid, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, epidermal growth factor, porcine follicular fluid, pregnant mare serum gonadotropin, human chorionic gonadotropin and tannic acid. According to the pig oocyte in vitro maturation culture solution, the oocyte in-vitro maturation quality, the cumulus cell diffusion index, the parthenogenetic embryo blastocyst rate and blastocyst total cell number, and the in-vitro fertilization embryo cleavage rate, blastocyst rate and blastocyst total cell number can beimproved.

A genetic means of determining whether a female subject produces “pregnancy competent” oocytes is provided. The means comprises detecting the level of expression of one or more genes that are expressed at characteristic levels (upregulated or downregulated) in cumulus cells derived from pregnancy competent oocytes. This characteristic gene expression level, or pattern referred to herein as the “pregnancy signature”, also can be used to identify subjects with underlying conditions that impair or prevent the development of a viable pregnancy, e.g., pre-menopausal condition, other hormonal dysfunction, ovarian dysfunction, ovarian cyst, cancer or other cell proliferation disorder, autoimmune disease and the like. Microarrays containing “pregnancy signature” genes or corresponding polypeptides provide another preferred aspect of the invention. Still further, the subject invention can be used to derive animal models, e.g., non-human primate animal models, for the evaluation of the efficacy of putative female fertility treatments.

The invention relates to a mouse denuded oocyte (DO) in vitro maturation technology, belonging to the technical field of biology. The invention is characterized in that a cumulus cell uses cystine and cysteamine to improve GSH synthesis and developmental capacities of the DO and, through cumulus cell co-culture and addition of cysteamine and cystine, an in vitro maturation system is established, which completely recovers the developmental capacity of the DO. The DO is co-cultured with mouse or goat cumulus cells in TCM-199 mature culture fluid added with 100-200 mumol / l of cysteamine and 250-300 mumol / l of cystine, the blastocyst rate reaches the level of mature COC cultured by adding the same mercapto-compound. After transplanting 2-cell embryos from the mature DO through co-culture and from COC respectively, the differences in pregnancy rate and number born are not obvious. The invention, for the first time in the world, establishes an in vitro mature system for completely recovering the developmental capacity of mouse denuded oocyte, which is of great importance in the research and application of both animal embryo engineering and human-assisted reproductive technology.

The invention relates to an egg cells automatic recognition and sorting apparatus, which comprises a negative pressure generator, an egg cell image acquisition device, light source equipment, a follicular fluid collection container, a sorting switch and a central controller which is connected to the negative pressure generator, the egg cell image acquisition device and the sorting switch. The negative pressure generator and the follicular fluid collection container are communicated to a conduit used for sucking and conveying a follicular stock solution with egg cells, and the negative pressure generator is used for forming negative pressure in the tube to complete puncture and suck the follicular stock solution with egg cells into the conduit. The egg cell image acquisition device and the light source equipment are arranged at two sides of a conveying path of the conduit for cooperating to obtain the egg cell image information, the obtained egg cell image information is sent to the central controller, the central controller controls the sorting switch arranged on the conduit to be switched on / off according to the egg cell image information, so that separation of egg cell (a cumulus cell compound) and follicular fluid in the follicular stock solution with egg cells can be realized, and then the required egg cells can be obtained.

The present invention relates to a method for selecting a competent oocyte or a competent embryo by determining the expression level of specific microRNA species in a body fluid or in cumulus cells.

The invention discloses a method for improving the developmental capacity of lamb in-vitro embryos and provides a method for accelerating in-vitro maturation of lamb ova. The method comprises the following steps: carrying out in-vitro maturation culture of in-vitro lamb cumulus-oocytes complexes in an ovum maturation accelerating culture solution added with adult lamb follicular fluid, and achieving the in-vitro maturation of the lamb ova. Through the method, the ovum maturation accelerating culture solution added with adult lamb follicular fluid is used for culturing the lamb oocytes; after the ova are mature, the ova can produce in-vitro embryos through in-vitro fertilization; the developmental capacity of the lamb embryos can be obviously improved; the method is simple and feasible and is convenient in material sources.

The present invention relates to field of fertility. The invention identifies biological ovarian markers from follicular cells, from follicular fluid, from cumulus cells and from oocytes which are indicative of follicular maturity in mammals. Described are methods for improving ovarian stimulation, methods for assessing maturity of a mammalian ovarian follicle, methods for optimizing in vitro maturation (IVM) and methods for classifying an embryo, these methods being based on assessment of expression level of ovarian markers indicative of maturity. Also described are methods for screening compounds stimulatory of or inhibitory to mammalian follicular maturation, kits for evaluating follicular maturity.

The invention provides a cumulus cell recognition and sorting device. The cumulus cell recognition and sorting device comprises a liquid road system, an imaging system and a control system; and the liquid road system comprises an ovum taking needle, a main input tube, a cell sorting device, a cell buffering device, a waste liquid buffering device, a main output tube, a peristaltic pump and a wasteliquid box. An inlet of the cell sorting device is connected with the ovum taking needle through the main input tube, a first outlet of the cell sorting device is connected with the cell buffering device through a cumulus cell inlet tube, and a second outlet of the cell sorting device is connected with the waste liquid buffering device through a waste inlet tube. The main output tube is connectedwith the cell buffering device through a first waste liquid outlet tube and is connected with the waste liquid buffering device through a second waste liquid outlet tube, a three-way valve is arranged between first waste liquid outlet tube, the second waste liquid outlet tube and a end opening adjacent to the main output tube, and the main output tube is sequentially connected with the peristaltic pump and the waste liquid box. By using the cumulus cell recognition and sorting device, cumulus cells can be automatically recognized and sorted, and the accuracy of recognizing and sorting the cumulus cells is improved.

The invention provides a human oocyte in vitro maturation culture solution containing Endothelin-1. The human oocyte in vitro maturation culture solution comprises liquid A and liquid B, wherein the liquid A comprises ET-1, r-FSH and 10% SSS, the liquid B comprises ET-1, r-FSH, hCG and 10% SSS, and solvent is conventional basic culture solutions. Researches show that the culture solution and culture method are capable of promoting human oocyte in vitro maturation especially cytoplasmic maturation and can increase embryo development potential after human oocyte in vitro fertilization. The culture solution aiming at the current situation that development imbalance of cytoplast and cell nucleus during immature human oocyte in vitro culture affects embryo in vitro development potential is developed by utilizing the biological function and effect of ET-1 in cumulus cells. The culture solution has the advantages that the culture solution is applicable to human oocyte in vitro culture, an IVM process can be optimized favorably, and the culture solution is worthy of popularization and application.

The present invention relates to a biomarker for diagnosing or predicting reactivity of an ovary to FSH and a use thereof. According to a composition for diagnosing or predicting reactivity of an ovary to FSH, a kit, and a method using same in accordance with one aspect, the degree of reactivity of the ovary can be easily diagnosed using miRNA present in blood, follicular fluid, granule cells, and cumulus cells, and thus, it is easy to predict or diagnose symptoms or diseases caused by abnormal reactivity of an ovary to FSH.

The invention discloses a porcine oocyte in-vitro maturation culture solution as well as a preparation method and application thereof and belongs to the technical field of oocyte in-vitro maturation culture. In order to solve the problems of low porcine oocyte in-vitro maturation rate and development rate at present, the invention provides the porcine oocyte in-vitro maturation culture solution aswell as the preparation method and application thereof. The culture solution comprises a base culture solution TCM-199, penicillin, streptomycin, NaHCO3, 4-hydroxyethylpiperazine ethane sulfonic acid, polyvinyl alcohol, sodium pyruvate, insulin, cysteine, an epidermal growth factor, a porcine follicular fluid, pregnant mare serum gonadotropin, human chorionic gonadotropin and ramelteon. The culture solution can increase the oocyte in-vitro maturation quality, the cumulus cell diffusion index, the glutathione level, the parthenogenetic embryo blastocyst rate and blastocyst total cell number, the in-vitro fertilization embryo cleavage rate, the blastocyst rate and blastocyst total cell number and decrease the ROS level of oocytes.

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 method for separating pig cumulus stem cells, which comprises the following steps of: extracting a cumulus-oocyte complex from a young sow, performing in-vitro maturation culture on oocyte cells, digesting and separating cumulus cells, collecting digested cumulus cells for culture, performing digestive passage inoculation onto a feeder layer when a cell colony appears and a larger cell colony is formed, and culturing in a cell factor-containing culture solution, wherein the separately cultured cumulus stem cells have bird nest cell growthform, round shapes and largernuclei, express unique markers Oct4, Nanog and SSEA1, and are subjected to in-vitro differentiation to form nerve cells, muscle cells and liver cells derived from three germ layers; and results show that the cumulus stem cells are separated from pig cumulus cells.

Methods are provided for evaluating an oocyte for fertilization and implantation. For example, methods are provided for determining whether an oocyte expresses, or does not express, one or more of a group of markers identified as differently expressed between chromosomally normal and chromosomally abnormal oocytes. Also provided, for example, are methods for determining whether a cumulus cell expresses, or does not express, one or more of a group of markers identified as differently expressed between cumulus cells associated with chromosomally normal oocytes and cumulus cells associated with chromosomally abnormal oocytes. Methods are provided for the detection of marker expression of differentially expressed genes at the RNA level, as well as at the protein level.

A genetic means of identifying “pregnancy competent” oocytes is provided. The means comprises detecting the level of expression of one or more genes that are expressed at characteristic levels (upregulated or downregulated) in cumulus cells derived from pregnancy competent oocytes. This characteristic gene expression level, or pattern referred to herein as the “pregnancy signature”, also can be used to identify subjects with under-lying conditions that impair or prevent the development of a viable pregnancy, e.g., pre-menopausal condition, other hormonal dysfunction, ovarian dysfunction, ovarian cyst, cancer or other cell proliferation disorder, autoimmune disease and the like. In preferred embodiments the pregnancy signature will comprise one or more of AB-CA6, NCAM1, OLFML3, PTPRA, SDF4, GPR137B, DDIT4, DUSP1, GPR137B, IDUA, KCTD5, NDNL2, SLC26A3, and TERF2IP.

The present invention provides a buffalo oocyte enucleation method which comprises the following steps: 1) a cumulus oophorus oocyte cell complex is taken form ovary of a slaughter house for in vitro maturation culture for 22-24h under the conditions of 38.5 DEG C, 5% CO2 and saturated humidity; 2) the maturation-cultured COCs are gently and slowly blew and beaten by 100muL pipette tip for removal of cumulus oophorus cells to left oocytes; and 3) selecting MII oocytes containing first polarbody for enucleation; 4) transferring the buffalo oocytes into a culture solution containing 7.5 mug / mL cytochalasin B (CB) for culture for 30min under the conditions of 38.5 DEG C, 5% CO2 and saturated humidity; 5) transferring the treated buffalo oocytes into 30-40 muL of an enucleation operating liquid covered with paraffin oil; and 6) enucleating in an inverted microscope provided with a spindle body imaging system. By use of the cytochalasin B (CB) for culture of the oocytes and use of the spindle body imaging system for auxiliary enucleation, the efficiency of enucleation can be effectively improved.

The invention provides a method for performing high-throughput screening on high-quality oocytes. The method comprises the following steps: enabling oocytes in an in-vitro culture environment to be mature through mature liquid I and II, removing cumulus cells with a cumulus removing solution, performing initial selection on the integrity of the cytomembrane structure of the oocytes, enabling perivitelline space not to occur in the oocytes with an incomplete semipermeable membrane, eliminating one part of inferior oocytes in the process, and performing re-selection in an isosmotic hormone-freesolution, wherein if the cytomembranes of the initially selected oocytes cannot recover to the state before treatment with the cumulus removing solution, the oocytes must be eliminated, so that high-quality oocytes can be screened rapidly and with high throughput and are applied to development of productive cloning work.

The invention discloses a granular cell stripping solution and a preparation method thereof. The granular cell stripping solutionis prepared fromabase solutioncontaining an antibacterial agent, a phenol red indicator, 20 to 22mmol / Lof hyaluronidase, andaprotein additive, namely human serum albumin, 33 to 36mmol / L ofHEPES, 10.5 to 12.0 mmol / L ofTES, 10.5 to 12.0 mmol / L of DIPSO , and 3.5 to 5.0 mmol / L of arginine. The compositions can provide effective functions for stripping of cumulus cells of oocytes and surrounding granular cell clusters, provide sufficient energy substances during in vitrostripping operation, and provide a more stable buffer system, and increase the fertilization rate, special nutrients needed during the periodoflater stage of fertilizationare also provided forthe oocytes at the later stage of fertilization, an ideal environment is provided for in vitro treatmentand pre-cultivation of ova, and further development of a reproduction technology and increasing of thesuccess rate are assisted advantageously.