Effects Of Estrogen Receptor ?-specific Antagonist MPP On The Expressions Of G1/S Transition Associated Factors: Nanog, Cyclin D1 And Cyclin E In Mouse 2-cell Embryos

Objective1. To study the stage-specific subcellular localization of embryonic stem cell transcription factor Nanog in Kunming(KM) mouse preimplantation embryos;2. To study the effects of Estrogen Receptor ?(ER?)-specific antagonist MPP(20?M) on the subcellular localization of Nanog in mouse 2-cell embryos;3. To study the effects of MPP treatment on the subcellular localization of cell cycle refulatory proteins Cyclin D1 and Cyclin E;4. To study the effects of MPP treatment on the m RNA expression levels of Nanog, Cyclin D1 and Cyclin E;In order to elucidate the roles of ERa on the G1/S transition of mouse 2-cell embryos, and lay the theoritical foundations for further investigations regarding the functional mechanisms of ERa during mouse major zygotic genome activation. Methods1. Mouse GV oocytes, MII oocytes and the 1-cell embryos(at 21 h, 27 h post-h CG) and 2-cell embryos(at 33 h, 39 h, 45 h, 51 h post-h CG) were collected(note that the embryos collected at 33 h post-h CG contain 1-cells and those at 51 h contain 4-cells). And by using immunofluorescence techonology, the stage-specific subcellular localization of Nanog in mouse preimplantation embryos was detected.2. Mouse 1-cell embryos were collected at 27 h post-h CG, and cultured in vitro in KSOM medium(control group) and in the medium supplemented with 20?M MPP(experimental group); the 2-cell embryo samples in both groups were recovered at 45 h post-h CG, and the subcellular localization of Nanog protein was detected by immunofluorescence staining.3. Mouse 1-cell embryos were collected at 27 h post-h CG, and cultured in vitro in KSOM medium and in the medium supplemented with 20?M MPP; the 2-cell embryo samples in both groups were recovered at 45 h and 48 h post-h CG for the detection of subcellular localization of Cyclin D1 and Cyclin E respectively, by immunofluorescence staining.4. Mouse 1-cell embryos were collected at 27 h post-h CG, and cultured in vitro in KSOM medium and in the medium supplemented with 20?M MPP; 2-cell embryos(45h post-h CG) were recovered and by using Real Time PCR techonology, the m RNA expression levels of Nanog, Cyclin D1 and Cyclin E were detected. Results1. Immunofluorescence staining results showed that, Nanog was expressed neither in GV oocytes, MII oocytes, nor in 1-cells collected at 21 h, 27 h, 33 h post-h CG; however, in the 2-cells collected at 33 h, 39 h, 45 h and 51 h post-h CG, the expression of Nanog was detected; furthermore, the expression levels of Nanog at 39 h post-h CG was the highest, and declined at 45 h and 51h; besides, low expression levels of Nanog was detected in the 3-cells or 4-cells collected at 51 h post-h CG.2. After 27-45 h MPP(20?M) treatment, the total immunofluorescence intensity of Nanog was significantly lowered, comparing with the KSOM group(P<0.01), and the nuclear immunofluorescence intensity also declined significantly(P<0.01).3. After 27-45 h MPP(20?M) treatment, the total immunofluorescence intensity of Cyclin D1 was declined significantly, compared with the KSOM group(P<0.01),as well as the nuclear immunofluorescence intensity of Cyclin D1 was observed(P<0.05). After 27-48 h MPP(20?M) treatment, significant declination of the total immunofluorescence intensity(P<0.01), as well as nuclear immunofluorescence intensity(P<0.01) of Cyclin E was observed.4. Real Time-PCR results showed that, comparing with KSOM group, the m RNA expression levels of Nanog and Cyclin D1 in MPP(20?M) group did not changed significantly(P>0.05); while that of the Cyclin E declined significantly(P<0.01). ConclusionResults in the present study showed that, embryonic stem cell transcription factor Nanog expressed highest at 39 h post of h CG. The localization patterns indicate that Nanog might participate in regulating the G1/S transition of 2-cell embryos and major zygotic genome activation. After treatment with ER?-specific antagonist MPP(20?M), the expression levels of Nanog, Cyclin D1 and Cyclin E protein in the 2-cell embryos declined significantly. In addiation, Real Time-PCR detection showed that, after MPP treatment, the m RNA expression of Nanog and Cyclin D1 did not changed significantly, while that of Cyclin E declined significantly, which imply that the ER? regulates the translation of Nanog and Cyclin D1, while that on Cyclin E involves in transcription and translation level.