Abstract
The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.
Highlights
During early embryogenesis, the first wave of the embryo development process is the maternal-to-zygotic transition (MZT) in which embryos are under maternal signaling control until zygotic gene activation (ZGA) [1,2,3]
To determine the molecular mechanism by which granzyme g (Gzmg) is upregulated at the mouse two-cell stage, we investigated whether two-cell stage-specific enhancers are in the Gzmg promoter sequence
We found a novel isoform in the N-terminal domain (NTD)-deleted signal transducer and activator of transcription 3 (STAT3) protein that exhibited abundant signals in the pronuclei of the zygotes when the anti-C-20 antibody was used for the whole-mount in situ immunofluorescence analysis (Figure 6B)
Summary
The first wave of the embryo development process is the maternal-to-zygotic transition (MZT) in which embryos are under maternal signaling control until zygotic gene activation (ZGA) [1,2,3]. MZT processing involves maternal clearance and activates a cascade of early zygotic genes. Maternal clearance involves the storage and degradation of maternal transcripts and proteins that are necessary for oocyte maturation and fertilization and constitutes the first wave of ZGA [4,5,6]. ZGA, maternal transcripts and proteins trigger zygotic gene activation. MZT dramatically reprograms terminally differentiated germ cells into totipotent embryos and initiates the embryo development process [1,2]. A minor wave of ZGA begins at the zygote stage, and the major ZGA wave is detected at the two-cell stage [7]
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