| [Background]Nearly30years, assisted reproductive technology (ART)developed rapidly. It isestimated that1%of children in the western world are born with the help of assistedreproductive techniques. At present,commonly used methods of clinical ART include invitro fertilisation and embryo transfer (IVF-ET), intracytoplasmic single spermmicroscopic injection (ICSI), preimplantation genetic diagnosos(PGD), donor eggsIVF-ET and gametes and embryos frozen technology. Oocytes of ART are mostgained by controlling the ovulation, in a certain extent, which improved the pregnancyrate of ART. But there are some disadvantages and complications during the controllingsuper ovulation, such as ovarian hyperstimulation syndrome(OHSS), the the potentialrisk of breast cancer and ovarian cancer, the high cost of medical treatment and longmedical time, and so on. Therefore, it is hightime to looking for a new method ofgaining mature oocytes.In vitro maturation (IVM) of immature oocytes is that make the immatureoocytes collecting from the ovary, in culture conditions in vitro, finally mature and havefertilization ability by simulating oocytes mature environment, which prove to be theeffective ways to solve the problems. IVM technology of Immature oocytes generates aLarge number of mature oocytes that used in vitro fertilization,supportingpretransplantation embryo development,which play an important role in ART. However,IVM technology still exists some disadvantages because it has not been set up the idealculturing system. At present, medium used in the in vitro maturation system is adding serum, albumin, all kinds of cytokines and hormone to basal medium, in a certain extent,which improved the efficiency of in vitro maturation. But there are somedeficiency,including low oocyte maturation rate, nonsynchronous maturation ofnucleus and cytoplasm and poor quality of oocytes, so that it must optimize culturesystem in vitro.Bone marrow mesenchymal stem cells(MSCs) is a kind of adult stem cells,which ispresent in the marrow cavity of animals.It not only possesses update and multiplexdifferentiation function but also can secrete many kinds of cytokines,such as epidermalgrowth factor(EGF), insulinlike growth factors(IGF), leukemia inhibitory factor(LIF),transforming growth factor(TGF),fibroblast growth factor(FGF),bone morphogeneticprotein(BMP), interleukin-6(IL-6),and so on. Granulosa cells, follicular theca cells,oocytes and stromal cells can also secrete those a series of cytokines during the growthand development of follicular, which promote and regulate oocytes differentiation,development and maturation.Our early study have demonstrated conditioned medium(CM) of mesenchymal stem cells can promote mice oocytes in ovarian tissuedevelopment, maturation and the development of embryos. Therefore, MSCsmicroenvironment may be a kind of ideal IVM system, which can simulate the in vivomicroenvironment of oocytes maturation, thus make oocytes have good developmentpotential, enhance IVF rate and embryonic development ability. This study will furtherevaluate conditioned medium of MSCs promoting mice immature oocyte maturation,embryonic development, and analysis the bioactive factor of it, so that provides thebasis for improving IVM culturing system.[Objective]To investigate the effects of conditioned medium (CM) of mesenchymal stem cells(MSCs) on the in vitro maturation of mouse immaturate oocytes, subsequentdevelopment of embryo and its regulation mechanism, so that optimize culture systemin vitro, provid basis.[Methods] 1) Mouse MSCs were isolated and cultured to harvest CM.2) Three categories of immature germinal vesicle (GV) stage oocytes werecollected and cultured in the basal medium (DMEM, Stempro) and CM (DCM,SCM) respectively. The optimal time and maturation rate of the in vitromaturation oocyte were surveyed.3) Oocytes were stained with Fluorescein diacetate(FDA), Hoechst33342andProdium iodide(PI) to evaluate the cell viability.4) The behaviors of cortical granules (CG) and spindle complexes were examinedwith a fluorescence microscope to evaluate cytoplasmic maturation of IVMoocyte.5) IVM and in vivo oocytes were in vitro fertilization and embryo cultured tocount fertility rate,4-cell rate and blastocyst rate respectively. Development situation ofcultured embryo in vitro were observated by morphology.6) Blastocysts were stained with hoechst33342to evaluate the quality byobservation formation of inner cell mass and trophoblast cell, and counting thenumber of total cell of Blastocysts.7) Proteins of conditioned medium(CM) of MSCs were collected andconcentrated to process electrophoresis in order to identify the factors in CMby the application of chromatography-mass spectrometry analyses.[Results]1) Three categories of GV oocytes cultured in CM were all achieved highermaturation rate than those cultured in basal medium. The optimal maturationtime for category A and B GV oocytes were16h while24h for category C.2) Oocytes keep good viability in CM culture condition during IVM. Thedistribution of CG and movement of spindle complexes in IVM oocytes, whichwere well consistent with in vivo matured oocytes, showed good markers ofcytoplasmic maturation. 3) The embryos of IVM oocytes after IVF showed developmental well. Thefertility rate,4-cell formation rate,blastocyst formation rate and the number oftotal cells of IVM group were not statistically different compared the controlgroup(p>0.05). The fluorescence pictures of total cells of IVM group blastocystswere consistent with the in vivo group.4) Over50kinds of proteins were checked out by the application ofchromatography-mass spectrometry analyses, but the bioactive factors whichplay a role in the IVM oocyte still needs to be further identified.[Conclusion]Conditioned medium of MSCs is an efficient IVM system, which benefit tonuclear and cytoplasm maturation of mouse IVM oocytes synchronously and improvethe embryonic potential development; Conditioned medium of mesenchymal stemcells contain a variety of bioactive factors,but the factors which play a role in the IVMoocyte still needs to be further identified. |
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