Shinya Aramaki

Molecular cloning and expression of dead end homologue in chicken primordial germ cells

Chicken primordial germ cells (PGCs) dynamically migrate towards the prospective gonadal area through the germinal crescent region and bloodstream at early embryonic stages. To date, chicken PGCs have been mainly identified by histochemical and immunohistochemical methods or by their morphological characteristics. However, their origin, migration and differentiation are not fully understood because of the lack of specific PGC molecular markers. Here, we have isolated the chicken dead end homologue (CDH) in order to clone its full-length cDNA with an open reading frame of 329 amino acids. The RNA-binding motif present in CDH at amino acids 54–133 was highly homologous to those in the dead end proteins of human, mouse and Xenopus. The temporal and spatial distribution of PGCs was also investigated by in situ hybridization (ISH) on whole-mount embryos with CDH cRNA as a probe. Chicken embryos from stage X to stage 20 were subjected to ISH. The hybridized samples were then sectioned to analyse the translocation of PGCs. CDH-positive cells could be counted from stage X to stage 4, with minimally 30 cells at the blastderm and approximately 260 cells at the germinal crescent. Thus, specific expression of CDH mRNA has been established in chicken PGCs located at the blastderm, germinal crescent and prospective gonadal area by ISH and reverse transcription/polymerase chain reaction. We conclude that isolated CDH is specifically expressed in chicken PGCs during embryogenesis.

Expression of rat uterine serine proteinases homologous to mouse implantation serine proteinase 2.

Implantation serine protease (ISP) was first identified in the uteri of pregnant mice. It is thought that ISP may have an important role in the initiation of implantation. However, the expression status and detailed functions of ISP remain unclear. In this study, the expression of ISP was investigated in the rat uterus. The analysis of two rat genes registered in GenBank, accession nos. XM_220240 and XM_577076, exhibited high identities to the mouse ISP2 genes, respectively at an mRNA level. We labeled the former as rISP2a and the latter as rISP2b. Using RT-PCR, we found that both genes were expressed in the uterus. Specifically, rISP2a mRNA was detected in the uterus throughout pregnancy, whereas rISP2b mRNA was only expressed in the uterus from day 5 of pregnancy until the end of gestation. Expression of both genes was observed specifically within the endometrial gland epithelium. Furthermore, rISP2a was also observed to be expressed in the fetus and placenta, whereas rISP2b expression was observed in the fetus but not in the placenta. An expressional signal of the rISP2a gene was observed in the spongiotrophoblasts, giant cells and decidual endometrium in the placenta. In the embryo, the ventral specific region was positive in rISP2a and rISP2b gene expression. These findings indicate the possibility that the presently examined genes with high identity to mouse ISP2 may play some role not only during the implantation phase, but also in the development of the placenta and embryo.