Phosphorylation of Rat Spermatidal Protein TP2 by Sperm-specific Protein Kinase A and Modulation of Its Transport into the Haploid Nucleus

Transition protein 2 (TP2), which is expressed during stages 12–15 of mammalian spermiogenesis, has been shown to undergo phosphorylation immediately after its synthesis. We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA). We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA). Reverse transcription-PCR analysis indicated that Cs-PKA was present in the haploid spermatids and absent from premeiotic germ cells. The rat Cs-PKA transcript was amplified and sequenced using the isoform-specific primers. The sequence of rat Cs- PKA at the N terminus differs from mouse and human by one amino acid. Western blot analysis using specific anti- C \alpha 1 antibodies revealed that C \alpha 1-PKA is absent in haploid spermatid cytosol. We have also established an in vitro nuclear transport assay for the haploid round spermatids. Using this assay, we have found that the cytoplasmic factors and ATP are absolutely essential for translocation of TP2 into the nucleus. Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus

Phosphorylation of Rat Spermatidal Protein TP2 by Sperm-specific Protein Kinase A and Modulation of Its Transport into the Haploid Nucleus

Transition protein 2 (TP2), which is expressed during stages 12–15 of mammalian spermiogenesis, has been shown to undergo phosphorylation immediately after its synthesis. We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA). We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA). Reverse transcription-PCR analysis indicated that Cs-PKA was present in the haploid spermatids and absent from premeiotic germ cells. The rat Cs-PKA transcript was amplified and sequenced using the isoform-specific primers. The sequence of rat Cs- PKA at the N terminus differs from mouse and human by one amino acid. Western blot analysis using specific anti- C \alpha 1 antibodies revealed that C \alpha 1-PKA is absent in haploid spermatid cytosol. We have also established an in vitro nuclear transport assay for the haploid round spermatids. Using this assay, we have found that the cytoplasmic factors and ATP are absolutely essential for translocation of TP2 into the nucleus. Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus

Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

The impact of embryonic enhanced green fluorescent protein (EGFP)-expression on development is not clear. In this study, we comprehensively assessed EGFP-expression pattern and its effect on early mouse development, following pronuclear-microinjection of the EGFP-transgene, containing chicken-$\beta$-actin promoter and cytomegalovirus enhancer. Preimplantation embryos exhibited differential EGFP-expression patterns. While blastocyst development of non-expressing embryos was 77.3$\pm$1.8%, that of expressing embryos was only 43.9$\pm$1.6% (P < 0.0001). Developmental competence of embryos negatively correlated (r = -0.99) with the levels of EGFP-expression. Faint-, moderate-, and intense-expressing embryos developed to 83.1$\pm$5.3%, 50$\pm$5%, and 9.5$\pm$3.9% blastocysts, respectively (P < 0.002). Interestingly, blastocysts expressing faint–moderate levels of EGFP were developmentally competent through the post-implantation period and delivered viable transgenic ‘green’ mice, following embryo transfer. These results indicate that hyper-expression of EGFP affects preimplantation development and faint–moderate level of its expression is compatible with normal embryogenesis in the mouse

Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

The impact of embryonic enhanced green fluorescent protein (EGFP)-expression on development is not clear. In this study, we comprehensively assessed EGFP-expression pattern and its effect on early mouse development, following pronuclear-microinjection of the EGFP-transgene, containing chicken-$\beta$-actin promoter and cytomegalovirus enhancer. Preimplantation embryos exhibited differential EGFP-expression patterns. While blastocyst development of non-expressing embryos was 77.3$\pm$1.8%, that of expressing embryos was only 43.9$\pm$1.6% (P < 0.0001). Developmental competence of embryos negatively correlated (r = -0.99) with the levels of EGFP-expression. Faint-, moderate-, and intense-expressing embryos developed to 83.1$\pm$5.3%, 50$\pm$5%, and 9.5$\pm$3.9% blastocysts, respectively (P < 0.002). Interestingly, blastocysts expressing faint–moderate levels of EGFP were developmentally competent through the post-implantation period and delivered viable transgenic ‘green’ mice, following embryo transfer. These results indicate that hyper-expression of EGFP affects preimplantation development and faint–moderate level of its expression is compatible with normal embryogenesis in the mouse

Identification of Two Novel Zinc Finger Modules and Nuclear Localization Signal in Rat Spermatidal Protein TP2 by Site-directed Mutagenesis

Spermatidal protein TP2, which appears transiently during stages 12-16 of mammalian spermiogenesis, is a DNA condensing zinc metalloprotein with a preference to GC-rich DNA. We have carried out a detailed site-directed mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc. Two zinc fingers modules have been identified involving 4 histidine and 4 cysteine residues, respectively. The modular structure of the two zinc fingers identified in TP2 define a new class of zinc finger proteins that do not fall into any of the known classes of zinc fingers. Transfection experiments with COS-7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus. The nuclear localization signal in TP2 was identified to be $^{87}GKVSKRKAV^{95}$ present in the C-terminal third of TP2 as a part of an extended NoLS sequence

Embryo culture-based generation of enhanced green fluorescent protein-transgenic mice

One of the limitations of transgenesis is low efficiency. In this study, we generated transgenic mice harboring the enhanced green fluorescent protein (EGFP) gene, under the control of chicken-β-actin promoter and cytomegalovirus enhancer, using two approaches and compared their efficiencies. One involved culture of EGFP-injected embryos developing through EGFP-expressing “green” blastocysts, followed by their transfer to uterus. The second was oviductal-transfer of EGFP-injected-eggs. Embryo culture-based-transgenesis (ECBT) produced 100% transgenic mice, unlike the second approach. Moreover, ECBT required reduced number of recipients and markedly increased pregnancy rates. Of the nine founders, seven exhibited ubiquitous EGFP-expression, one (GU1) was a mosaic and the other (G18) was non-expressing. The molecular basis for this was attributed to repeat-induced gene silencing, since the G18 had a high copy number ( 99/genome) of the non-mutated and non-rearranged EGFP-transgene integrated at a single site. Our results show the superiority of ECBT over the conventional oviductal approach for generating transgenic “green” mice