Stage-specific changes in protein phosphorylation during preimplantation development in the mouse

To gain insight into the role of protein phosphorylation during early mammalian development, seven mouse preimplantation stages were metabolically labeled with radioactive orthophosphate and the radiolabeled proteins identified using gel electrophoresis and autoradiography. The results obtained indicate that there are marked differences in protein phosphorylation patterns between the zygote and two-cell stage and between the morula and blastocyst stage. In addition, there is a compaction-specific change in the phosphorylation profile of three components of Mr 37,000. This compaction-specific change takes place during compaction in the eight-cell embryo; thus, it is the first biochemical change specifically correlated to this important event of early development.     

Myelination in rat brain: changes in myelin composition during brain maturation

Abstract— Myelin was isolated from rat brains during development by a procedure giving fractions of constant purity at all ages. The lipid composition of these fractions and of whole brains of littermates was determined. The amount of myelin recovered per brain was a nearly linear function of the logarithm of age from the youngest (15 days) to the oldest (425 days) animals studied. With the exception of the earliest age point, the isolated myelin accounted for approximately 40 per cent of total brain galactolipid, evidence that a constant fraction (calculated to be 60 per cent) of myelin was recovered at all ages. Although the lipid-protein ratio of the myelin was constant with age, marked changes were seen in the amounts of cerebroside, sulphatide, phosphatidylcholine and desmosterol. The total galactolipid increased from 21 per cent of the total lipid at age 15 days to about 31 per cent at maturity. Phosphatidylcholine decreased from 17 to 11 per cent during the same period. Desmosterol decreased from 2.5 per cent of the total sterol to 0.2-0.3 per cent. All of these changes were complete between 2 and 5 months of age; no other ‘lower phase’ lipids showed significant changes with age. Although qualitatively similar to those reported by others, the changes differed in magnitude, with more stability in the levels of cholesterol and phosphatidalethanolamine with development. A sensitive indicator of the maturation of myelin was the mole ratio galactolipid/phosphatidylcholine, which varied from 1.2 at age 15 days to 2.8 at maturity. The maximum rate of myelination occurred at 20 days of postnatal age when myelin was deposited at the rate of 3.5 mg day^?1 brain^?1. However, at this age the rat brain had only 15 per cent of its eventual complement of myelin. The rate of accumulation of cerebroside in the whole brain paralleled that of myelin, and was the only lipid to show this relationship. Myelin deposition appeared to be almost solely responsible for the continued increase in brain weight after about 100 days of age.     

Effect of a high fat intake on protein utilization during ontogenetic development

Male rats (Wistar strain, Velaz, Prague) aged 30, 90 and 150 days were fed 14 days ad libitum on a high fat diet (containing 40% margarine) and their growth (PER, NPR) and utilization (NPU, LPU) parameters of protein (casein) biological value were compared with those of animals given a standard gel containing 10% margarine (the diets were isoenergetic). The course of gluconeogenesis in their liver was also determined by measuring phosphoenolpyruvate carboxykinase (PEPCK) activity. A high fat intake had a negative effect on the growth parameters of protein biological value, PER and NPR, in all three age groups and on the utilization parameters NPU and LPU in 30- and 90-day-old rats. The nonsignificant increase in NPU and LPU in the oldest animals was evidently related to the equal protein intake compared with the control, indicating that proteins need to be utilized more economically in the presence of a raised fat intake; owing to their far lower fat intake compared with 90-day-old animals on a high fat diet, the fat had a less negative effect on their protein utilization than in the younger age group. The negative effect of a high fat intake was confirmed by high activation of gluconeogenesis in 30- and 90-day-old animals and by raised phosphoenolpyruvate carboxykinase activity in 150-day-old rats, in which the supposition that gluconeogenesis would be highly activated if the diet were administered for a period other than 14 days cannot be ignored. The biological and biochemical methods employed in this study can be used with a wider perceptual range of dietary nutrients to determine optimum nutrient values under different conditions.     

Changing iron content of the mouse brain during development

Iron is crucial to many processes in the brain yet the percentages of the major iron-containing species contained therein, and how these percentages change during development, have not been reliably determined. To do this, C57BL/6 mice were enriched in (57)Fe and their brains were examined by M?ssbauer, EPR, and electronic absorption spectroscopy; Fe concentrations were evaluated using ICP-MS. Excluding the contribution of residual blood hemoglobin, the three major categories of brain Fe included ferritin (an iron storage protein), mitochondrial iron (consisting primarily of Fe/S clusters and hemes), and mononuclear nonheme high-spin (NHHS) Fe(II) and Fe(III) species. Brains from prenatal and one-week old mice were dominated by ferritin and were deficient in mitochondrial Fe. During the next few weeks of life, the brain grew and experienced a burst of mitochondriogenesis. Overall brain Fe concentration and the concentration of ferritin declined during this burst phase, suggesting that the rate of Fe incorporation was insufficient to accommodate these changes. The slow rate of Fe import and export to/from the brain, relative to other organs, was verified by an isotopic labeling study. Iron levels and ferritin stores replenished in young adult mice. NHHS Fe(II) species were observed in substantial levels in brains of several ages. A stable free-radical species that increased with age was observed by EPR spectroscopy. Brains from mice raised on an Fe-deficient diet showed depleted ferritin iron but normal mitochondrial iron levels.     

Proteome profile changes during mouse testis development

Spermatogenesis is a process of terminal differentiation that results in the formation of mature sperm. In the first wave of this differentiation in the mouse testis, different cell types appear in the seminiferous epithelium at specific times. These cytological changes must be accompanied by changes in protein expression patterns. The aim of the present study was the comparative analysis of proteomic profiles of the soluble proteins expressed at different stages of mouse testis development (8, 18 and 45 postnatal days). Conspicuous variations in their accumulation (representing up or downregulation) were detected over the course of development. Using mass spectrometry (MALDI-TOF), 44 proteins or variant forms were identified. Proteins with redox or antioxidant activity were identified in high proportions; others involved in lipid and carbohydrate metabolic pathways, as well as a number of proteins or isoforms not previously characterized in testis were also detected. These results contribute to identify changes in soluble protein associated to the complex process of male germ cell differentiation.     

Regenerative capability in the hindlimb of Xenopus laevis during ontogenetic development

The regenerative capacity of hindlimb of Xenopus laevis was investigated by amputating the limbs at four levels in various developmental stages including younger postmetamorphosed froglets. Amputations of limbs were performed at the base of limb in stages 50, 51, 52, 53, 54, 55, 58, and 60 (Nieuwkoop and Faber's table), at the middle of limb bud in stages 50, 51, 52 and 54, and at mid-thigh and mid-shank in stages 58 and 60, and the froglets in 2 and 3 cm in snout-vent length. In the present experiments the regenerative capacity of limbs was expressed by the rate of regeneration and morphogenesis. Tadpoles in the stages after 55 failed to regenerate when the limbs were amputated at base level, but individuals in all the other experimental series exhibited regeneration in various rates irrespective of the level of amputation and the stage. The regenerative capacity increased distally along the proximo-distal axis of the limb when amputated at the same stage, while regeneration was better in younger stages than that in older stages when amputations were made at the same levels. The regenerates obtained by amputation of limbs in stages between 50 and 54, were mainly digitated in that they had 5 toes with 3 claws which is the same pattern with the normal limb, 4 toes with 2 claws, 3 toes with 2 claws or one, and 2 toes with one claw etc. Tadpoles at stage 50 could regenerate toes and claws without defect, but in the later the regenerative capacity gradually declined by reducing the number of toes and claws and accompanied by malformation of skeleton as the stage proceeded. The tadpoles in stages after 58, and the froglets of 2 and 3 cm, produced various types of heteromorphic regenerates of shapes such as cone, spike or rod of which the centra were occupied with cartilage rods. However these regenerates showed no morphological differences according to the developmental stages. These heteromorphic regenerates continued their growth even after one year without any sign of development of digitated feet.     

Pattern of expression of the podocalyxin gene in the mouse brain during development

We studied the expression pattern of the major renal protein Podocalyxin during the development of mouse brain using in situ hybridization. Podocalyxin mRNA was widely expressed at least from E14, the first age we studied, and expression remained high until adulthood. The highest levels of expression were postnatal. Podocalyxin expression was particularly elevated in the cortical plate, the hippocampus and cerebellum, and in several basal forebrain nuclei.     

PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in cholesterol homeostasis through enhanced degradation of the LDL receptor (LDLR) in liver. As novel inhibitors/silencers of PCSK9 are now being tested in clinical trials to treat hypercholesterolemia, it is crucial to define the physiological consequences of the lack of PCSK9 in various organs. LDLR regulation by PCSK9 has not been extensively described during mouse brain development and injury. Herein, we show that PCSK9 and LDLR are co-expressed in mouse brain during development and at adulthood. Although the protein levels of LDLR and apolipoprotein E (apoE) in the adult brain of Pcsk9(-/-) mice are similar to those of wild-type (WT) mice, LDLR levels increased and were accompanied by a reduction of apoE levels during development. This suggests that the upregulation of LDLR protein levels in Pcsk9(-/-) mice enhances apoE degradation. Upon ischemic stroke, PCSK9 was expressed in the dentate gyrus between 24 h and 72 h following brain reperfusion. Although mouse behavior and lesion volume were similar, LDLR protein levels dropped ～2-fold less in the Pcsk9(-/-)-lesioned hippocampus, without affecting apoE levels and neurogenesis. Thus, PCSK9 downregulates LDLR levels during brain development and following transient ischemic stroke in adult mice.     

Monoclonal antibody to chick embryo hyaluronan-binding protein: changes in distribution of binding protein during early brain development.

A monoclonal antibody, MAb IVd4, that recognizes hyaluronan-binding protein (HABP) from chick embryo brain has been produced and characterized. By immunoblotting, MAb IVd4 was shown to recognize three proteins in chick embryo brain of molecular weight 93, 90, and 69 kDa; this interaction was inhibited by addition of hyaluronan hexasaccharides. Overlay of transblots with [3H]hyaluronan showed binding to proteins of similar molecular weight. MAb IVd4 blocked binding of [3H]hyaluronan to brain HABP and to simian virus-transformed 3T3 cells, indicating a possible relationship with the 85-kDa hyaluronan receptor of these cells. The distribution of HABP during early brain development was analyzed by immunohistochemistry. Immunoreactivity was uniform in newly formed neuroectoderm but became more concentrated in the roof of the brain during the second day of embryonic development. As the neuroectoderm becomes layered, the HABP was increasingly restricted to the forming plexiform layer, an area enriched in neural cell processes. Immunoreactivity was greatly enhanced by pretreatment of tissue with hyaluronidase, presumably due to removal of hyaluronan bound to the HABP, and was abolished on treatment with hyaluronan hexasaccharide, presumably due to inhibition of HABP-antibody interaction. These results suggest that a hyaluronan receptor is involved in early cellular events in brain development.