Expression Of RNA-binding Protein LIN28in Primordial Germ Cell Develop Of Male Embryo

Human infertility has increasingly become a serious issue for human health.Besides acquired damages and pollution effects, inherited defects play an importantrole. Although their congenital damages are still unknown, some evidences show thatthey are related with gonad development and gene defects. Germ cells are importantfor gonad development because their formation and expansion affect gonads function.Reproductive cells is an important part of the gonads, their formation and expansionin gonad development plays a decisive role, which affect reproductive function.Human primordial germ cells, PGCs, including egg and sperm, is the commonorigin of cells (ancestral cell) of all kinds of germ cells. PGCs is closely related toreproductive development, playing an important role in the process of human survivaland development. Human PGCs were first detected in the allantoic endoderm andmesenchyme at about22days after gestation. At5-6weeks after gestation, PGCsmigration to the original genital ridge. Within a series of amplification and initialdifferentiation, migrated PGCs form5-7million oogoniums or spermatogoniums.These primitive cells surrounded by support cells and somatic cells, form the earlysex gland. The mature process of PGCs appeared to be developed periodically andprecisely controlled. This experiment is mainly about migrated PGCs study, in whichis a decision period of cell’s proliferation and gender differentiation. The period iscontrolled by inside and outside factors of cells. Through the induction of embryonic stem cells in vitro for reproductive stem cells, the scientists found that the mainexternal factors Bone Morphogenetic Protein (BMP). While complex internal factorsincluding four types: Transcription factors regulation, RNA-binding proteins (RBP)regulation, pluripotency gene regulation and epigenetic regulation.LIN28is a highly conserved RNA-binding protein that is indispensable inprocess of germ cell proliferation and differentiation factors. It’s also reported that isclosely related with the progression of human testicular germ cell tumors. Throughmice study, it’s found LIN28highly expressed in embryonic stem cells and earlyembryonic development process. By Induced in vitro experiment, LIN28is foundhighly expressed in human embryonic stem cells and improve the induced pluripotentstem cells to the reproductive efficiency of stem cells. While, OCT4, a well-knownearly germ cell marker, plays a key role in maintaining the pluripotency andself-renewal of embryonic stem cells. Because of its high expression during the earlygestation, OCT4is usually used in common reproduction studies. So far, however,there are no other stable index to judge the different development stages of the fetusgerm cells. To this end, we choose another pluripotent marker LIN28compared withOCT4. Looking for suitable marker for different stage in germ cell developmentand so as to shed light on the occurrence and development of human germ cell,maturation and fertilization of regulating mechanism is the key of diagnosis andtreatment for infertility and improve the basis and key of eugenic and superior nurture.It will also provide important theoretical basis for assisted reproductive technologywhich is recently the main measure for infertility and help to create new birth control.ObjectiveThis topic explores function and mechanism of LIN28and OCT4in PGCs andearly embryonic development, to analysis expression development trends of OCT4and LIN28protein in fetal develop. Experimental purpose is to provide theoreticaland experimental basis for further study formation and proliferation of PGCs. And atthe same time, it is discussed main cause of infertility, fetal development and geneticdefect, which will provide theoretical basis for infertility, embryo transplant failure,abortion, and diagnosis and prevention of genetic disease. Materials and MethodsSpecimen collection is about human fetal testicular tissue from multiplepregnancy reduction specimens, abortion and induced labor specimens of6to20weeks of pregnancy which a total of cases is20. Firstly rapid frozen section andimmunohistochemical fluorescence staining is needed. Then Using laser confocalmicroscope photographed and statistics LIN28and OCT4positive cells. Thespecimens are divided by gestational weeks (7wks,8wks,9wks,10wks,11-12wks,13-14wks) into6groups,(6weeks which is not found two factor expression and20weeks which is only LIN28single factor expression of20weeks induced labororganization is not included), each group of n=3, statistics of different period twofactor expression changes and trends. The experimental data is showed in the form ofx s. Quantitative data which conforms to normal distribution and homogeneity ofvariance is analyzed by one-way ANOVA. Application SPSS17.0statistical softwareis put into use for statistical processing. Inspection level for a=0.05.Results1．High expression of LIN28in fetal testes during7-14weeksIn human fetal testes, high expression of OCT4and LIN28was consistentlydetected during7-14weeks. Our study found these OCT4-positive cells have a lot oftypical characteristics of reproductive mother cells in morphology, circular, rules, thenucleus, the higher nuclear mass ratio. These identified as PGCs positive cells.2．Relative changes of expression level of LIN28and OCT4LIN28expression gradually increased during the key stage of testesdevelopment and, while at11weeks, the relative positive cell of OCT4continues todecline. After13weeks, OCT4-positive cells were rarely observed. There are onlyLIN28expression visible at testes samples of20weeks.3．Subcellular localization change analysis of LIN28It is well known that OCT4staining is confined in the cell nuclei during theearly fetal development. From week7, LIN28was also detected in the nucleoli ofgerm cells. At week8–9, the localization occurs both in the nuclei and cytoplasm, while at week10, some germ cells had LIN28only in the cytoplasm and by week11–13, almost all of them displayed LIN28exclusively in the cytoplasm.4．Colocalization pattern of LIN28and OCT4When the PGCs express two protein at the same time, namely under thefluorescent red (LIN28) and green (OCT4) double staining, we think thatColocalization is existed in the specific cells of the period. The co-staining of LIN28and OCT4showed that the two proteins localize similarly in large, round germ cells.First, about60%of positive cells co-expressed both proteins at the nuclear level.Then, the percentage gradually increased and both proteins were expressed in thenuclei and cytoplasm because of the differential expression of LIN28. At week11, thenumber of cells in which the two proteins colocalized reached approximately100%.Discussions1. LIN28is highly expressed during7-20w PGCs of fetal testes and its expressionincrease with embryo develop.By its high specific expression of LIN28duringthis period, it is confirmed the feasibility and stability as a germ cell markupfactors.2. It is observed that there were special expression pattern change of LIN28fromnuclear localization to cytoplasmic localization in early PGCs.3. Study found LIN28with totipotency OCT4gene has colocalization patternpositioning deeper interaction relationship, to control the primordial germ cellformation and common development.InnovationsThis experiment systematically describes the expression pattern and subcellularlocalization change of RNA-binding protein LIN28in primordial germ cells of earlyfetal developmental testis.