Expression profiles of core cell-cycle regulators and sperm cell specification markers pre- and post-mitotis of the germ cell. Schematic representation of tobacco germ cell-cycle progression during pollen-tube growth until near plasmogamy, depicted with reference to DNA content measurements of Nicotiana tabacum germ and sperm cells cycle . mRNA expression levels were determined by semi-quantitative RT-PCR with RNA isolated from mature bicellular pollen grains (MPG) and at four stages of pollen-tube growth: 4 h, 13 h, 24 h and 48 h, both pre- and post-PMII. Signal intensities were quantified using Image J software Java 1.6.0 (National Institutes of Health, USA) and normalized against the background signal to obtain relative intensities expressed as relative transcript levels. Genes were classified as repressors of G1/S transition; as transition, for those that counteract repressors; or as activators of G2/M transition and progression. NtDUO1-like is regarded as an integrator of germ cell cycle mitotic progression and sperm cell fate specification, with functional evidence from the Arabidopsis male gametophyte . Sperm cell fate markers are also reliably expressed pre and post germ cell mitosis to near karyogamy. Note: RBR1 levels are never minimal, accentuating the primary role of RBR1 in maintaining the vegetative cell nucleus at G0 .