The Expression Pattern Of α-Tubulin During Early Embryonic Cell Division And Follicular Development

The reason for the infertility of the majority is that the molecular biology and developmental molecular mechanisms of female infertility or abnormal pregnancy are still unclear.Oosperm is the beginning of life,and early embryonic lethality is also closely related to infertility.The ovum is successfully discharged and combined with sperm,undergoes cell division and differentiation into individuals,In the middle of the pre-cell division,the spindle consisting of the centrosome and its binding proteins is assembled to form a mitotic device in order to align the chromosomes neatly with the equatorial plate until the chromosomes are evenly distributed into the two daughter cells.Microtubules are assembled from tubulin subunits and are one of the major components of the cytoskeleton.Tubulin is a dimer composed of α/β-tubulin(α/β-tubulin),and a-Tubulin is one of the major structures of intracellular free tubulin.Oogenesis occurs through primordial follicles,primary follicles,secondary follicles,antral follicles,ovum maturation,and corpus luteum formation.Studies have shown that the focus of oocyte maturation and subsequent embryo maturation is in the follicular cavity,and the proliferation of granulosa cells in the follicular cavity is the key to ensuring oocyte maturation.The follicular cavity formed,and parts of the granulosa cells around the oocyte proliferate and differentiate into cumulus cells.The cumulus-oocyte complex gradually formed,and the other part proliferates and proliferates.The exchange of information between cells and cells is also particularly important in the process of ovum production.When the follicular cavity does not formed,the preantral follicle does not affected by gonadotropins(Gn).At this time,the follicular development is regulated by the exchange between the oocyte and the surrounding somatic cells,and after the follicular cavity is formed.The development of oocytes affected by Gn.At this time,gonadotropin(PMSG)promotes oocytes by promoting the interaction of follicle stimulating hormone(FSH)with follicle stimulating hormone receptor(FSHR)to go further mature.In this study,we found embryonic lethality in transgenic mice based on the study of the role of tubulin a-Tubulin in the cell cycle.FN-T transgenic mice were used as experimental group,129 wild-type mice.For the control group,the ovaries of the reproductive system were used to study the effects of tubulin a-Tubulin on the ovum formation process of transgenic mice under breeding conditions and under exogenous gonadotropin conditions,and the possible causes of embryonic lethality.In this experiment,immunofluorescence staining,conventional paraffin section staining,immunohistochemistry and RT-PCR were used to investigate the expression pattern of cytoskeletal protein a-Tubulin in early embryonic cell division and follicular development.The analysis results are as follows:1.In mouse 1-cell embryos,a-Tubulin pulls chromosomes in a parasitic spindle structure and arranges them neatly on the equatorial plate to participate in cell mitosis.In mouse embryonic 2-cell phase,a-Tubulin connects adjacent cell bodies with a fusiform linker until the division is completed;in the mouse 4-8 cell stage,part of α-Tubulin forms a centrosome concatenation between adjacent cell bodies in a polymeric state to ensure normal cell division.RNA interference knocked down a-Tubulin,and the embryonic phenotype showed multinuclear multipolar phenomenon,indicating that tubulin a-Tubulin has an irreplaceable role in cell division.In the process of obtaining embryos,we found that embryos lethal in the transgenic mice under the same conditions and follicular congestion in the ovary,as an extension to carry out further experiments.2.Histopathological analysis showed that morphologically,the ovary of FN-T transgenic mice under ovulation-promoting conditions had significant hyperemia compared with wild-type mice and FN-T transgenic mice under normal physiological conditions,and the volume has increased as well.The appearance of the follicular cavity is similar to an elliptical shape.The alternation of volume and size expressed as the lengths of the horizontal and vertical axes of the four groups of follicles.The results showed that under normal conditions,the length of the ovary of the FN-T transgenic mice was significantly different from that of the wild type mice and the ovary of FN-T transgenic mice under normal physiological conditions(P<0.001).this result is consistent with the above morphological ovarian volume increase results.The a-Tubulin immunofluorescence staining of mouse ovary showed that a-Tubulin was abundantly expressed in granulosa cells in the follicular cavity during ovum development,and the pairing at high magnification was closely related to granulosa cell proliferation.There is an important connection between ovum production.3.TUNEL kit test results:under ovulation-promoting conditions,there are a large number of apoptosis primary,secondary,antral follicles,physalides and luteal phaseas of follicular development in FN-T transgenic mice,especially the granulosa cells.The number of apoptosis in the wild-type mice under the same conditions and the transgenic and wild-type mice under normal physiological conditions are significantly increased,and there are differences.Especially in the late stage of follicular development,after the formation of luminal follicles,the damaged granule cell layer appears,and the granulosa cells appear nuclear disintegration and pile up apoptosis.This indicates that due to exogenous gonadotropin,the abnormal polymerization or depolymerization of a-Tubulin in the ovary of FN-T transgenic mice leads to the granulosa cells unable to undergo normal proliferation and differentiation and a large number of apoptosis,resulting in the ovum cannot be successfully discharged,even embryos are lethaled.4.RT-PCR results showed that the expression levels of apoptosis-related genes Caspase-3,Bax and Bcl-2 were up-regulated in FN-O transgenic mice compared with wild-type mice,and there were significant differences.(P<0.01);for FN-T transgenic mice,the genes Caspase-3,Bax and Bcl-2 were up-regulated at the mRNA level compared with wild-type mice,and there was a difference between them(P<0.05).Conclusion:For accomplishment of cell division,a-Tubulin plays a critical role in cell division especial in mouse early embryonic development.In the 1-cell stage of mouse embryo,the cell division involves in the polymerization of microtubule spindle;in the 2-8 cell stage,the adjacent cell body is pulled by the polymerization state of the shuttle-type linker to help the cell accurately.In the ovarian tissue of FN-T transgenic mice,granulosa cells around the follicle play an important role in the maturation of the mother cells and the formation of the ovum.The function of the cytoskeletal protein a-Tubulin in the granulosa cells around the follicle is affected,causing the ovarian granulosa cells to directional apoptosis,resulting in the oocyte not being able to develop normally,and the ovum cannot be discharged normally.This research,it can provide a strong scientific basis for female infertility,and can be used as reference data for the treatment of early embryo death.