Granzyme Gis expressed in the two-cell stage mouse embryo and is required for the maternal-zygotic transition

Abstract

BackgroundDetailed knowledge of the molecular and cellular mechanisms that direct spatial and temporal gene expression in pre-implantation embryos is critical for understanding the control of the maternal-zygotic transition and cell differentiation in early embryonic development. In this study, twenty-three clones, expressed at different stages of early mouse development, were identified using differential display reverse transcription polymerase chain reaction (DDRT-PCR). One of these clones, which is expressed in 2-cell stage embryos at 48 hr post-hCG injection, shows a perfect sequence homology to the gene encoding the granzyme G protein. The granzyme family members are serine proteases that are present in the secretory granules of cytolytic T lymphocytes. However, the pattern of granzyme G expression and its function in early mouse embryos are entirely unknown.ResultsUpon the introduction of an antisense morpholino (2 mM) against granzyme G to knock-down endogenous gene function, all embryos were arrested at the 2- to 4-cell stages of egg cleavage, and the de novo synthesis of zygotic RNAs was decreased. The embryonic survival rate was dramatically decreased at the late 2-cell stage when serine protease-specific inhibitors, 0.1 mM 3,4-dichloroisocoumarin (3,4-DCI), and 2 mM phenyl methanesulphonyl fluoride (PMSF), were added to the in vitro embryonic culture medium. Survival was not affected by the addition of 0.5 mM EDTA, a metalloproteinase inhibitor.ConclusionWe characterized for the first time the expression and function of granzyme G during early stage embryogenesis. Our data suggest that granzyme G is an important factor in early mouse embryonic development and may play a novel role in the elimination of maternal proteins and the triggering of zygotic gene expression during the maternal-zygotic transition.

Are all granzymes cytotoxicin vivo?

“…Orphan granzymes are also found in cell types other than cytotoxic immune cells. For example: gzmN is expressed in spermatocytes and spermatids of adult mice testes (Takano et al, 2004); GzmD is present in activated mast cells (Ronnberg et al, 2013); and GzmG is expressed in mouse 2-cell stage embryos (Tsai et al, 2010). The physiological role/s of these granzymes is still enigmatic, especially as in the majority of cases they are produced without perforin, therefore one can only speculate about possible internal or extracellular functions.…”

Imprinted Genes and Satellite Loci Are Differentially Methylated in Bovine Somatic Cell Nuclear Transfer Clones

“…The DNase reaction was terminated with heat inactivation at 65°C. Firststrand cDNA synthesis was performed using 2 lg of RNA with oligo(dT) primers and Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega) in a total volume of 25 lL (Tsai et al, 2010). Quantitative RT-PCR was conducted using specific sets of primers (Table S1) for each analyzed gene.…”

A Chinese herbal medicine, Gexia-Zhuyu Tang (GZT), prevents dimethylnitrosamine-induced liver fibrosis through inhibition of hepatic stellate cells proliferation

“…The total cellular RNA was isolated from liver tissues using a TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. The RNA was subsequently treated with deoxyribonuclease I (MBI Fermentas Inc., Lithuania, Germany) to remove any genomic DNA contamination (Tsai et al, 2010). Approximately 900 ng of total RNA was reverse-transcribed with MuLV Reverse Transcriptase using the GeneAmp RNA PCR Kit (Applied Biosystems, Foster, CA) and oligo d(T)16 primers.…”