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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0608—Germ cells
  • C12N5/0609—Oocytes, oogonia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/30—Hormones
  • C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/30—Hormones
  • C12N2501/38—Hormones with nuclear receptors
  • C12N2501/39—Steroid hormones
  • C12N2501/392—Sexual steroids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2517/00—Cells related to new breeds of animals
  • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

• the present invention relates to a new system for in vitro oocyte maturation, hereinafter named as "follicular system”.
• the proposed follicular system comprises a two-step culture method combined with a strategy to obtain oocytes specifically suitable to the culture protocol, aiming to improve oocyte maturation.
• the proposed follicular system aims to provide to the oocyte a more physiological environment for its development in order to fully acquire meiotic and developmental competence, which are both related to the oocyte's capability to complete meiotic division, to be fertilized and to develop into a viable embryo.
• the invention herein proposed further relates to a kit comprising the basic medium and the components of the two-step culture method, as well as the protocol for obtaining the oocytes at the perfect developmental stage for the culture system.
• IVP in vitro embryo production
• IVP has limited efficiency. To illustrate, only about 30-35% of the female gametes (oocytes) retrieved from cows and submitted to IVP are able to generate viable embryos.
• oocytes that are isolated from the ovary are very heterogeneous and come from follicles at different stages of development. Also, as with the exception of the ovulatory follicle, no other follicle reaches the ovulatory stage of development in the same ovarian cycle (in mono-ovu!atory species as bovine and humans, estrous or menstrual cycle, respectively); the vast majority of oocytes remaining in the ovary is destined to regress and die during the process named follicular atresia.
• US20090137478 relate to methods that use hormones (LH and FSH, for example) to promote in vitro and in vivo oocyte maturation, respectively.
• hormones LH and FSH, for example
• CN103923878 disclose the combination of hormone, cytokines and growth factors for the same purpose. Although reproductive biologists have not discovered yet the "magic formula" for IVM, several culture systems including a pre-matu ration phase utilizing pharmacological agents capable of delaying nuclear maturation (meiotic resumption) have been tested without consistent success. This is mainly due to the lack of gradually in the stimulation during the pre- maturation time.
• the present invention relates to a follicular system for oocyte in vitro maturation, which aims to improve the efficiency of IVP by providing to the oocyte a more physiological and suitable environment for the acquisition of competence to complete meiosis, to be fertilized and to produce an embryo with high developmental potential.
• the proposed follicular system comprises a two-step culture method combined with a strategy to obtain oocytes specifically suitable to the culture protocol, aiming to improve oocyte maturation.
• the culture contains agents present in the ovarian follicle and known to be involved in the regulation of the cumulus-oocyte complex maturation. Also, with a view to obtain oocytes in a maturation stage appropriate to the composition of the follicular system, the consideration of follicular growth stage in the female donor is also important. Therefore, the follicular system includes a strategy to synchronize and stimulate the growth of ovarian follicles in the donor aiming to obtain oocytes specifically suitable to the culture protocol.
• the invention herein proposed further relates to a kit comprising the basic medium and the components of the two-step culture method, as well as the protocol for obtaining the oocytes at the perfect developmental stage for the culture system.
• Another novel aspect of the system proposed herein is the synchronization of ovarian follicular activity in the donor to obtain a homogeneous population of oocytes at a developmental stage specifically compatible with the culture system.
• IVM oocyte maturation
• the present invention relates to a follicular system for in vitro oocyte maturation using a two-step culture method combined with a strategy to obtain a homogeneous population of oocytes specifically suitable to the culture protocol, aiming to improve oocyte maturation.
• the system aims to provide to the oocyte a more physiological environment for its development in order to fully acquire meiotic and developmental competence.
• the invention further relates to a kit comprising the basic medium and the components of the two-step culture method, as well as the protocol for obtaining the oocytes at the perfect developmental stage for the culture system.
• Figure 1 is a graphic that shows the effect of different culture media on the percentage of oocytes in germinal vesicle (GV), germinal vesicle breakdown (GVBD) and metaphase I (Ml) after 6 hours of culture.
• GV germinal vesicle
• GVBD germinal vesicle breakdown
• Ml metaphase I
• Figure 2 is a graphic that shows the effect of different culture media on the percentage of cumulus-oocyte complexes with opened gap-junction mediated communications.
• the invention disclosed herein relates to a follicular system for in vitro oocyte maturation, which combines a two-step culture method and a strategy to obtain a homogeneous population of oocytes specifically suitable to the culture protocol, aiming to improve oocyte maturation.
• the invention aims to improve the efficiency of IVP by providing to the oocyte a more physiological and suitable environment for its development in order to fully acquire meiotic and developmental competence to be fertilized and to produce an embryo with high
• a follicular system which comprises biological agents in a two-step culture method, namely:
• the two-step culture method is combined with a synchronization strategy to obtain oocytes specifically suitable to the culture protocol, since the pattern of follicular growth in the female donor is an important factor to obtain oocytes in a maturation stage appropriate to the composition of the follicular system.
• the two-step culture method of the follicular system herein disclosed comprises a base medium, which is supplemented with peptides and hormones that are naturally present in the follicular fluid, at concentrations close to their physiological concentrations for each step.
• the follicular system is used to improve oocyte maturation in cattle.
• the commonly used base medium [TCM 199 with Earle's salts, glutamine, NaHC03, amikacin (75pg/mL), piruvate (22 g/mL) and bovine serum albumin (4mg/mL)] is supplemented with the following constituents (dose ranges for each constituent at each step of the culture system are shown in the table below):
• NPPC neuropeptide type C
• IGF1 insulin-like growth factor 1
• AREG amphiregulin
• FGFs fibroblast growth factors
• BMP bone morphogenetic proteins
• FGFs 2, 10 and 17 Fibroblast Growth Factors
• Bone Morphogenetic Proteins (BMP 5 and GDF9) 50- 50 ng/mL
• fibroblast growth factors The presence of fibroblast growth factors, bone morphogenetic proteins and cumulin is not mandatory.
• the proposed follicular system comprises physiological agents that prevent meiotic resumption and progression, while supporting the metabolism of cumulus cells and their communication with the oocyte.
• the oocytes be obtained at a specific stage of development that can benefit from the culture system. Due to this fact, the follicular system is combined with a synchronization strategy to obtain oocytes specifically suitable to the culture protocol.
• the protocol for ovarian synchronization and stimulation of follicle growth combines ultrasound guided follicle aspiration (aiming to eliminate old follicles from previous cycles larger than 5 mm and to induce the emergence of a synchronized cohort of follicles) with stimulation of follicle growth by the administration of rBST (recombinant bovine somatrotopin, 250-500 mg, subcutaneously) 7 days before follicle aspiration and/or FSH (100-300 mg, intramuscularly) at the same day of the synchronizing follicle aspiration. Synchronized oocytes will be collected by ultrasound guided follicle aspiration 2-4 days later.
• the synchronization protocol shall be adapted for use in other species and women, using species-specific doses and hormonal protocols.
• the follicular system for improving oocyte maturation of the invention comprises the two-step culture system combined with the follicular synchronization in the oocyte donor, as detailed above.
• the purpose of the pre-maturation step is to expose the cumulus- oocyte complex to physiological agents that stimulate the functions of the cumulus cells and their communication with the oocyte and the overall cross-talk between the two compartments.
• Figure 1 demonstrate that the pre-maturation phase of the follicular system herein proposed effectively arrested nuclear maturation for 6 hours, as indicated by the maintenance of the germinal vesicle stage (GV).
• GV germinal vesicle stage
• basic corresponds to basic medium (without follicular factors);
• FS corresponds to basic medium plus estradiol, progesterone, androstenedione, FSH and IGF1 close to physiological concentrations and
• FS + NPPC corresponds to basic medium plus estradiol, progesterone, androstenedione, FSH, IGF1 and NPPC close to physiological concentrations.
• the oocyte is arrested at prophase of the first meiotic division since early stages of follicular development.
• the breakdown of the germinal vesicle corresponds to the disappearance of the nuclear envelope and marks the resumption of meiosis.
• the invention further relates to a kit comprising the basic medium and the components of the two-step culture method as well as the protocol for obtaining the oocytes at the perfect developmental stage for the culture system.
• the commonly used base medium [TCM 199 with Earle's salts, glutamine, NaHC0 3 , amikacin (75pg/ml_), piruvate (22pg/mL) and bovine serum albumin (4mg/ml_)3 is supplemented with hormones and peptides, which are provided in the following concentration ranges.
• the protocol further proposes the utilization of the culture system described above, in combination with ultrasound guided follicle aspiration and stimulation of follicle growth by the administration of hormones, in order to obtain a homogeneous population of oocytes at a developmental stage appropriate to the culture system.

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• 2016
  • 2016-02-24 BR BR112018068474-0A patent/BR112018068474B1/pt not_active IP Right Cessation
  • 2016-02-24 WO PCT/BR2016/000019 patent/WO2017143412A1/en active Application Filing

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