New molecular markers involved in immune system homeostasis and hemopoietic organ development are differentially regulated during oocytes in vitro maturation

The growth and maturation of the oocyte is a dynamic process which requires a variable supply of hormones, growth factors and energy. These needs are met partially by the surrounding somatic cells and the cumulus-oocyte complex, which communicate bi-directionally via gap junctions. Identifying and analyzing protein expression in the oocyte can provide insight in its development and growth. Further, like bone marrow stem cells, if relevant marker genes are found in oocytes, there is a potential for the oocyte to be manipulated into becoming hemopoietic stem cells. In this study, porcine oocytes were isolated and subjected to microarray analysis to compare the oocyte gene expression in vivo and in vitro maturation (IVM). Genes identified belonged to both ‘hemopoietic or lymphoid organ development’(GO:0048534) and ‘immune system development’ (GO:0002520), and the markers can be used to identify several activities such as cell migration, neurogenesis and proliferation. The following are the identified genes and all were downregulated after IVM to varying degrees: ID2, VEGFA, TGFBR3, INHBA, CDK6, BCL11A, MYO1E, ITGB1, EGR1, NOTCH2, SPTA1, KIT and TPD52. Our results should provide new markers to further investigate oocyte development and growth regulation.Running title: Markers of hemopoietic organ development

EMBRYONIC HAEMOPOIESIS IN EARLY ONTOGENY OF AFRICAN CATFISH (CLARIAS GARIEPIUS)

The study of the growth of blood cells and hemopoietic organs of claravia catfish ( Clarias gariepius ) grown in the closed loop water systems on the basis of "RANTOP AGRO-5" LLC in the Krasnodar region. Test materials (prolarvae and larvae aged 5, 10, 15, 20 and 25 days of active feeding) were selected in the spring-summer period of 2013-2014. Prolarvae in mesenchyma of forming mesonephros which begins to develop after hatching had primordial precursor cell and blast blood cells between forming vesicles. There took place differentiation of erythropoietic cells: erythroblasts, pronormoblasts and basophilic normoblasts. Accumulation of hemoglobin in erythrocytes indicates that since the first day of hatching, the blood starts to perform transport function - transportation of oxygen. The rudiment of thymus was observed in larvae aged 10 days. This organ generated lymphocytepoietic cells. The central hemopoietic organ - spleen - was originally registered as a mesenchymal rudiment at the age of 10 days. At the age of 25 days, development of the organ stroma is not finished in clarid catfish larvae. Reticular tissues develop actively. Separate lymphoid clumps in the spleen structure have not been found. Melano-macrofagic centres are also unformed. Qualitative analysis of haemopoiesis showed that in spleen there take place development of all types of blood cells: erythropoiesis, granulopoiesis and agranulopoiesis.

Stem Cell Emergence and Hemopoietic Activity Are Incompatible in Mouse Intraembryonic Sites

In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) region. Here, we address the following question: can the P-Sp/AGM environment support hemopoietic differentiation as well as generate stem cells, and, conversely, are other sites where hemopoietic differentiation occurs capable of generating stem cells? Although P-Sp/AGM generates de novo hemopoietic stem cells between 9.5 and 12.5 days post coitus (dpc), we show here that it does not support hemopoietic differentiation. Among mesoderm-derived sites, spleen and omentum were shown to be colonized by exogenous cells in the same fashion as the fetal liver. Cells colonizing the spleen were multipotent and pursued their evolution to committed progenitors in this organ. In contrast, the omentum, which was colonized by lymphoid-committed progenitors that did not expand, cannot be considered as a hemopoietic organ. From these data, stem cell generation appears incompatible with hemopoietic activity. At the peak of hemopoietic progenitor production in the P-Sp/AGM, between 10.5 and 11.5 dpc, multipotent cells were found at the exceptional frequency of 1 out of 12 total cells and 1 out of 4 AA4.1+ cells. Thus, progenitors within this region constitute a pool of undifferentiated hemopoietic cells readily accessible for characterization.

BLOOD CELL FORMATION AND DISTRIBUTION IN RELATION TO THE MECHANISM OF THYROID-ACCELERATED METAMORPHOSIS IN THE LARVAL FROG

1. Thyroid-accelerated metamorphosis in the larval frog is accompanied by changes in the hemopoietic centers and in the blood cell distribution in the various regions of the body. These changes are interpreted as results of the fundamental change in basal metabolic rate induced by the thyroid treatment. 2. There is initiation of the shift of hemopoietic locus from the kidney, the larval hemopoietic organ, to the spleen, the adult hemopoietic organ. The spleen, being chiefly an erythrocyte producer, becomes of greater importance with the transition from the lower metabolic rate to the higher, since greater erythropoiesis becomes necessary to supply the physical basis for the maintenance of the higher metabolic rate. 3. It is suggested that the appearance of red bone marrow in the later history of the frog is correlated with a still higher metabolic rate. Phylogenetically, in the vertebrate series, red bone marrow is also associated with higher metabolic rate. 4. The new metabolic rate initiated in tadpoles by thyroid administration sets up a demand for (a) erythrocytes, (b) granulocytes and lymphoid phagocytes for distribution to regions of regressive change, (c) lymphocytes, (1) as progenitors of erythrocytes, granulocytes and phagocytes, (2) for promoting growth of cells in regions of progressive change. 5. Upon the hemopoietic reserve, which in the last analysis is the lymphocyte (and its mesenchymal precursor), depends the extent to which metamorphosis will proceed. Inability on the part of the hemopoietic centers, chiefly the spleen, to keep pace with the demand for blood cells during metamorphosis results in metamorphic stasis, a condition of anemia which is usually followed by death. 6. The growth-promoting function of leucocytes, as demonstrated by Carrel, is probably to be ascribed to the lymphocyte component of leucocytes. 7. The granulocytes have probably also a glandular function, and may exert a lytic effect upon adjacent tissues in regions of regressive change.