Studies on Early Development of Primordial Germ Cells and Their Repopulation Capacity in Chicken

Abstract

Germ cells are the only population to transfer genetic and epigenetic information across generations. Studying their origin and characteristics is one of important topics in developmental and evolutionary biology. Among various species, the origin and specification of germ cells in birds have not been clearly defined. Besides, information about cleavage stages, the period that the developmental processes start and germ cells may arise, is insufficient, so that comprehensive studies are needed. Also, the research on the restorable proliferation of primordial germ cells (PGCs) in the gonads that can contribute to understand regenerative medicine is not studied, so far. In the present study, we analyzed the developmental dynamics of intrauterine embryo development in chicken and studied the origin of germ cells by tracing the expression of germ cell-specific genes. Also, we investigated the compensatory proliferation of PGCs in the gonads after busulfan treatment. The first study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from hens and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions. Based on the information of intrauterine chick embryos, we investigated origin of germ cells in chicken. We found that cDAZL mRNA was expressed specifically in germ cells throughout all stages of development in company with our previous study. During oocyte-to-zygote transition, cDAZL mRNA was localized in the perinuclear region of oocyte and the central region of zygote as a granule. In the EM study of zygote, electron-dense granules with numerous mitochondria were observed During intrauterine embryo development, cDAZL gene showed kinetics of expression pattern

localization in cleavage furrows during initial cleavages (EGK.I-III), subcellular localization in presumptive primordial germ cells (pPGCs) during cleavage progression (EGK.IV-VI) and finally diffused in the cytoplasm of PGCs during late intrauterine stages (EGK.VI-X) that indicated primordial germ cells (PGCs) may first arise at least from EGK.VI. cDAZL and CVH protein was also co-localized in cleavage furrows and PGCs. In addition, PGCs which were found at EGK.VI-X were located not only in the most upper layer (future epiblast) but also in the lower layers (future hypoblast) that indicated chicken PGCs were already present not only in the epiblast but also in the hypoblast in pre-streak stage embryos. Phosphorylation of RNA polymerase II in PGCs was synchronized with other somatic cells that indicated global transcriptional repression in PGCs during specification is not a common mechanism in chicken. Taken together, these results suggest that the germ plasm in chicken are consistently located in the center of embryo during specification, and PGCs are distinguished from somatic cells by germline-specific transcription at EGK.VI-VII. Little is known about the cellular responses of PGCs after treatment with toxic chemicals such as busulfan during embryo development. Thus, we investigated the elimination, restorative ability, and cell cycle status of endogenous chicken PGCs after busulfan treatment. Busulfan was emulsified in sesame oil by a dispersion-emulsifying system and injected into the chick blastoderm (embryonic stage X). Subsequently, we conducted flow cytometry analysis to evaluate changes in the PGC population and cell cycle status, and immunohistochemistry to examine the germ cell proliferation. Results of flow cytometry and immunohistochemistry analyses after busulfan treatment showed that the proportion of male PGCs at embryonic day 9 and female PGCs at embryonic day 7 were increased by approximately 60% when compared to embryonic day 5.5, indicating the existence of a compensatory mechanism in PGCs in response to the cytotoxic effects of busulfan. Results of cell cycling analysis showed that the germ cells in G0/G1 phase were significantly decreased, while S/G2/M-phase germ cells were significantly increased in the treatment group compared to the untreated control group in both 9-day-old male and female embryos. In addition, in the proliferation analysis with 5-ethynyl-2′-deoxyuridine (EdU) incorporation, we found that the proportion of EdU positive cells among VASA (VASA homolog) positive cells in the 9-day embryonic gonads of busulfan treated group was significantly higher than control group. We conclude that PGCs enter a restoration pathway by promoting their cell cycle after experiencing a cytotoxic effect. Through our results, intrauterine embryo development and cleavage progression of chicken are different from those of mammals. To know the mechanisms of cleavage progression, lineage specification and polarity formation, gene-expression patterns and functional studies should be done in the future. Also, studying of the biological function for polyspermy will contribute to understanding of the unique developmental characteristics in chicken. Germ cells seem to be specified by the predetermined mode in chicken. However, whether signaling pathways and zygotic genome activation (ZGA) are related to specification or not should be identified through further studies for clarify this. Also, in the future, it should be identified what mechanisms regulate the reconstitution of PGC population after busufan treatment. Taken together, our results in the present study will contribute to understanding for developmental dynamics of early chick embryo and germ cells, and potency of primordial germ cells.