The Study Of Retinoic Acid On Regulation Of Germ Cell Proliferation And Meiosis In Embryonic Chickens

For the development pattern of chicken embryos is unique, we could further understand of the development process and influential factors by study of chicken primordial germ cell (PGC) in vitro. We can acquire many study models by genetic manipulation in PGC of chicken, which can be used as an ideal model for reproductive biology and developmental biology to reveal intrinsic and external molecular mechanisms underlying regulation in proliferation and differentiation of germ cells, gonad development and gametogenesis. In this study, PGC were isolated from Hailan chicken embryo, and then subcultured on feed layer with somatic cells after initial primary culture. In addition, the effect of RA on proliferation of cultured PGC and meiosis initiation of germ cells in embryonic chicken ovary were evaluated, together with underlining mechanisms. Meanwhile, we also investigate the production of transgenic chicken by LV-siRNA and get a model of Raldh2knock down. These studies will provide theoretic guidance for further investigation of early development and preparation of transgenic poultry.1. Isolation, culture and identification of chicken PGCPGC were collected from stage14chicken embryonic bloods, stage19genital ridges or stage28gonads with a fine glass needle under a microsurgery scope. For primary culture, cell suspension containing both PGC and somatic cells was seeded onto gelatin-treated6-well culture plates at a density of1×106/well in KO-DMEM supplemented with7.5%fetal calf serum (FCS),10ng/ml leukemia inhibitory factor (LIF),10ng/ml human basic fibroblast growth factor (bFGF),0.1mM MEM nonessential amino acids.0.1mM2-mercaptoethanol,2mM L-glutamine (Glu),100U/ml penicillin and100μg/ml streptomycin, PGC were cultured at38.5℃in an air atmosphere containing5%CO2with60%-70%relative humidity. To trace the origin of the colonies, the primary formed colonies were picked up with a fine glass needle and then subjected to RT-PCR analysis for expression of PGC-specific markers. For further subculture, colonies that were positive for PGC markers were picked up and digested to single cell suspension and reseeded onto6-well dish. The third passage PGC were identified by staining of Alkaline phosphatase (AKP), periodic acid-Schiff regent (PAS), c-kit, stage-specific embryonic antigens (SSEA-1,3.4) and EMA-1immunocytochemistry, and the expression of the pluripotency-associated genes PouV, Nanog and Sox2was analyzed by RT-PCR. The results all confirmed the characteristics of cultured PGC which indicated that the primary and subculture models of PGC could be used for studies about regulation of PGC proliferation and differenciation.2. Effects of RA on adhesion and proliferation of chicken PGCIn the present study, the proliferating effect of RA on PGC was investigated along with the intracellular PI3K/Akt-mediated NF-κB signaling cascade and PKC/(3-catenin signaling. Results showed that RA significantly promoted PGC proliferation in a dose-and time-dependent manner, which was confirmed by BrdU incorporation. RA induced PGC aggregation by increasing expression of E-cadherin and α/β-catenins. However, this promoting effect was attenuated obviously by sequential inhibitors of LY294002for PI3K, KP372-1, for Akt SN50for NF-κB and PKC inhibitor H7, respectively. Furthermore, Western blot analysis manifested increased Akt phosphorylation (Ser ") of PGC after stimulation with RA, but this effect was abolished by LY294002or KP372-1. Meanwhile, treatment with RA increased expression of NF-κB and decreased IκBα expression that were inhibited by SN50. Moreover, blockade of PI3K or Akt activity resulted in inhibiting NF-κB translocation from the cytoplasm to the nucleus. E-cadherin and β-catenin protein expression levels also were increased by RA addition. Finally, the up-regulated mRNA expression of cell cycle regulating genes (cyclin D1and E, cyclin-dependent kinases6and2) was observed in the RA-treated cells. Flow cytometry analysis confirmed that RA-treated cultured PGC populations displayed a significant increase in the proportion of S and G2phase cells. Again, this stimulation was remarkably retarded by combined treatment with LY294002, KP372-1, SN50and H7respectively. Therefore, these results suggest that RA promote proliferation of the cultured chicken PGC via the PI3K/Akt mediated NF-κB signaling cascade and PKC/β-catenin signaling pathway.3. Effect of RA on meiosis initiation in the chicken embryoIn the present study, the effect of RA on meiosis initiation in embryonic germ cells was investigated by organ culture in vitro and RNA interference. The results showed the profile of meiosis in chicken embryo ovary-meiotic germ cells were increased gradually since day15.5(first detectable) to day18.5(widespread) by immunofluorescence of SCP3and yH2AX. And the expression of Stra8is specifically up-regulated at day12.5, Scp3and Dmcl became elevated at day15.5. In addition, we observed increased mRNA levels for Raldh2but deceased gradually for Cyp26bl during meiosis that evidence for a requirement of RA accumulation to sustain meiosis. The expression of RARβ also presented slightly increase after day15.5. But in males, the expression of these genes keeps low level throughout development. We also found that the culture ovaries from day10.5and12.5were able to initiate meiosis, and meiosis is stimulated by RA compared with control (P<0.05). Finally, Raldh2shRNA remarkably abolished RA-dependent functions, indicating that RA synthesis and signaling is crucial for meiosis initiation. Taken together, these studies indicate that RA and signal are important in regulating the onset of meiosis in the chicken embryonic ovary. We reported here for the first time that Raldh2knock-down could significantly reduced germ cells to enter meiosis.4. Production of transgenic chicken via LV-siRNA systemWe designed three pairs of shRNA specific for Raldh2by RNA interference to knock down its function and constructed expressing vector of shRNA The day15.5ovarian cells were transfected with either shRNA specific for Raldh2. or a non-targeting shRNA as a negative control. The efficiency of transfection is about80%-90%. Among of these shRNAs, shRNA2produced the most significant inhibitory effect on Raldh2mRNA expression. RNA interference of Raldh2prevented the appearance of meiotic cells by immunofluorescence of SCP3. The Lentivirus vector specific for Raldh2shRNA was constructed and packaged. Then the virus was injected into X stage chicken embryos for production of transgenic chicken. The chicken model of Raldh2knock down was successfully obtained. The results showed that there were no obviously difference between transgenic chicken and wild chicken, as the morphology of ovary. The GFP was detected by PCR, which confirmed the extra gene was injected to genome successfully. The expression of Raldh2was decreased significantly by qRT-PCR. This research provide theoretical basis for understanding the molecular mechanisms of regulation of oogenesis and follicular development.The above results indicated that the co-culture model of somatic cells and PGC from chicken embryo could be used to study on proliferation and differentiation. PGC were characterized by staining of AKP, PAS, c-kit, SSEA-1,3,4and EMA-1immunocytochemistry. The pluripotency of PGC was also detected by the expression of the pluripotency-associated genes (PouV,Nanog and Sox2). We investigated the molecular mechanisms of RA induced PGC proliferation by this model. Furthermore, we described the morphology and genetic changes during meiosis initiation in chicken embryonic ovary, then studied the regulating effect of RA on meiosis initiation by organ culture in vitro and RNA interference. These findings provide theoretic guidance and experimental platform for studying the development of germ cells and preparation of chimeras and transgenic poultry.