低氧对胚胎干细胞增殖的影响

目的:观察间歇性低氧和持续性低氧对体外培养的胚胎干细胞(ES细胞)增殖的影响.方法:利用细胞记数法和BrdU (5-溴脱氧尿苷)掺入的流式细胞分析检测细胞增殖,并用RT -PCR的方法检测低氧诱导因子(HIF-1a)的表达变化.结果:①将ES细胞分别放在低氧(3%～10% O2)和常氧(20% O2)的环境中培养24 h后,在低氧环境中培养的ES细胞数较常氧组明显减少;②将ES细胞分别给予间歇性低氧刺激(3%～10% O2),每天10 min,连续4 d后,发现3%低氧组较常氧对照组的细胞增殖明显升高.③用RT-PCR方法观察HIF-1a的表达与细胞增殖的关系,发现在常氧环境中培养的ES即有HIF-1a的表达,ES细胞在持续低氧24 h或间歇性低氧(3%～10% O2)刺激4 d后对HIF-1a的表达均无明显影响.结论:间歇性低氧(3% O2)可明显促进体外培养的ES细胞增殖,而持续性低氧抑制ES细胞增殖,间歇性低氧(3% O2)刺激促进ES细胞增殖的机制尚有待于进一步的研究.     

Effects of hypoxia on embryonic development in two Ambystoma and two Rana species

Oxygen available to amphibian embryos fluctuates widely and is often very low. We investigated the effects of oxygen partial pressure (1. 3-16.9 kPa) on embryonic development and hatching of two salamander (Ambystoma) and two frog (Rana) species. In Ambystoma, chronic hypoxia resulted in slowed development, delayed hatching, and embryos that were less developed at the time of hatching. Although hypoxia was not lethal to embryos, temporary developmental abnormalities were observed in Ambystoma at oxygen partial pressures of 3.8 kPa and below. Posthatching survival decreased below 3.3 kPa. In Rana, hypoxia did not affect developmental rate, presumably because hatching occurs at a very early stage of development relative to Ambystoma. However, Rana embryos hatched sooner in hypoxia than in normoxia, resulting in less developed embryos at the time of hatching. The results suggest that embryonic hypoxia may negatively affect survival and fitness in these species.     

Effects of chronic hypoxia on microcirculation of the central nervous system during embryonic development

In the attempt to know the factors influencing the vasculogenesis and to verify whether the vessel formation and growth are influenced, during different ontogenetic periods, by oxygen deficiency, the intraneural vascular network has been morphometrically analyzed in chicken embryo optic tectum under conditions of aerogenic hypoxia. Chicken eggs, incubated under routine conditions, have been half-painted with melted wax at the 2nd incubation day (i.d.). Fragments of optic tectum, isolated from living embryos at the 8th, 14th and 17th i.d., have been fixed and embedded according to the usual E/M procedures. A parallel series of normally developed embryos of 8, 14 and 17 incubation days has been likewise prepared. On semithin sections from the hypoxic specimens and normal control embryos the area occupied by vessels (Av), the number of vessels (Nv) and the diameter of the radially directed ones (Dv) have been evaluated. The preliminary results indicate that hypoxia evokes, from the 8th to the 17th i.d., an increment of the Av parameter, due first to microvessel neoformation and enlargement from the 8th to the 14th i.d., then from the 14th to the 17th i.d. only to a growth of new capillary branches. The response of the vascular network to the hypoxic condition is more marked from the 8th to the 14th i.d., t.i. when, in relation to the differentiation and the stratification of the neurons, the metabolic requirements of the developing tectum are presumably increased. O2 deficiency causes severe developmental disorders of the cyto- and mieloarchitecture of the tectum: the vascular response can apparently prevent actual damages only when the hypoxic condition lasts for a relatively short time.     

The effect of graded hypoxia on the embryonic chick heart

Embryonic ventricular function in the chick was measured in response to graded levels of hypoxia. Myocardial contractility, as measured by cinephotoanalysis and expressed as shortening fraction, was significantly depressed after 1 hour of moderate hypoxia (6% O2) and after 5 hours of milder (16% O2 and 11% O2) levels of hypoxia (P less than .05). Microscopy confirmed associated myocyte damage with cell death noted after 5 hours of moderate hypoxic stress. Heart rate change was not related to the severity of hypoxia. The greatest level of tachycardia was noted with conditions of mildest hypoxia (16% O2). The data confirm that cardiac contractility, as measured by shortening fraction, is depressed on exposure to hypoxia, with impairment of function related to the severity of the hypoxic conditions.     

Effects of colchicine on the formation and looping of the tubular heart of the embryonic chick

The role of microtubules in the early development of the chick embryo heart was studied. The microtubules were disrupted by treatment of the embryos with colchicine. The embryos were divided for study into three groups: (I) before the fusion of the paired cardiac primordia; (II) before the starting of the cardiac loop, and (III) during the formation of the looping process. Colchicine did not impair the fusion of the paired heart primordia nor the acquisition of an asymmetric C-shaped form. However, the normal counterclockwise movement of the heart loop was prevented. From these results we conclude that the formation of the tubular heart and its looping are independent of the integrity of the microtubular system. Under the effects of colchicine the developing myocytes rounded up bulging into the pericardial cavity. The cell contours became scarcely discernible and the individual cell surfaces gave rise to blebs and ruffles of different sizes. In older embryos, clefts of different sizes appeared between the myocardial cells. The effects of colchicine on the pulsatile activity of the heart were recorded. These effects, as well as those on the cell surface characteristics, were found to be age dependent. The more mature the hearts were, the more resistant to colchicine they became. The developmental significance of the results reported here is discussed.     

Effects of FGF9 on embryonic Sertoli cell proliferation and testicular cord formation in the mouse

Proliferation and cord formation by embryonic Sertoli cells are pivotal events involved in testis morphogenesis. A number of growth factors have been implicated in mediating these events. However, the exact level of involvement and importance of each as yet remains elusive. We have adopted an in vitro approach to assess developing mouse Sertoli cells, whereby they are cultured in the presence or absence of fibroblast growth factor (FGF9) and/or extracellular matrix (ECM) gel, since previous studies have shown that ECM gel aids Sertoli cell differentiation. The present findings corroborate this effect, but in addition demonstrate that in the presence of FGF9 (10 ng/ml), cells undergo greater proliferation than those cultured on gel alone. They also display a differentiated epithelial phenotype, with appositional contact of cell membranes in cord-like aggregations. In addition we have shown that cultured Sertoli cells generally express a smaller truncated, nuclear form of the FGFr3, although in the presence of FGF9 and absence of gel, the larger, cytoplasmic form of the receptor is also expressed. Immunolocalisation of FGFr3 in Sertoli cells of whole testes revealed a temporal expression pattern profile, with high levels being abundant in the embryonic testicular cords and at puberty, but an absence in adult Sertoli cells. Our findings suggest that FGF9 plays an important role in proliferation and organisation of embryonic Sertoli cells during testis morphogenesis.     

Effects of hypoxia on pulmonary microvascular volume

To determine the effects of alveolar hypoxia on pulmonary microvascular volume, X-ray microfocal angiographic images of isolated perfused dog lung lobes were obtained during passage of a bolus of radiopaque contrast medium during both normoxic (alveolar gas, 15% O(2), 6% CO(2), and 79% N(2)) and hypoxic (3% O(2), 6% CO(2), and 91% N(2)) conditions. Regions of interest (ROIs) over the lobar artery and vein at low magnification and a feeding artery ( approximately 500 microm diameter) and the nearby microvasculature (vessels smaller than approximately 50 microm) at high magnification were identified, and X-ray absorbance vs. time curves were acquired under both conditions from the same ROIs. The total pulmonary vascular volume was calculated from the flow and the mean transit time for the contrast medium passage from the lobar artery to lobar vein. The fractional changes in microvascular volume were determined from the areas under the high-magnification X-ray absorbance curves. Hypoxia decreased lobar volume by 13 +/- 3% (SE) and regional microvascular volume by 26 +/- 4% (SE). Given the morphometry of the lung vasculature, these results suggest that capillary volume was decreased by hypoxia.     

Effects of hypoxia on vertebrate blood vessels

Hypoxia contracts mammalian respiratory vessels and increases vascular resistance in respiratory tissues of many vertebrates. In systemic vessels these responses vary, hypoxia relaxes mammalian vessels and contracts systemic arteries from cyclostomes. It has been proposed that hypoxic vasoconstriction in cyclostome systemic arteries is the antecedent to mammalian hypoxic pulmonary vasoconstriction, however, phylogenetic characterization of hypoxic responses is lacking. In this study, we characterized the hypoxic response of isolated systemic and respiratory vessels from a variety of vertebrates using standard myography. Pre-gill/respiratory (ventral aorta, afferent branchial artery, pulmonary artery) and post-gill/systemic (dorsal and thoracic aortas, efferent branchial artery) from lamprey (Petromyzon marinus), sandbar shark (Carcharhinus plumbeus), yellowfin tuna (Thunnus albacares), American bullfrog (Rana catesbeiana), American alligator (Alligator mississippiensis), Pekin duck (Anas platyrhynchos domesticus), chicken (Gallus domesticus) and rat (Rattus norvegicus) were exposed to hypoxia at rest or during pre-stimulation (elevated extracellular potassium, epinephrine or norepinephrine). Hypoxia produced a relaxation or transient contraction followed by relaxation in all pre-gill vessels, except for contraction in lamprey, and vasoconstriction or tri-phasic constriction-dilation-constriction in all pulmonary vessels. Hypoxia contracted systemic vessels from all animals except shark and rat and in pre-contracted rat aortas it produced a transient contraction followed by relaxation. These results show that while the classic "systemic hypoxic vasodilation and pulmonary hypoxic vasoconstriction" may occur in the microcirculation, the hypoxic response of the vertebrate macrocirculation is quite variable. These findings also suggest that hypoxic vasoconstriction is a phylogenetically ancient response.     

Effects of chronic hypoxia on kidney function

Renal disfunctions which appear in the chronic respiratory insufficient patient are analysed, as well as the participation of the arterial blood hypoxemia in their genesis. Renal clearances of Na, K, Cl, Ca, Mg and Pi, and those of urea and creatinine, were lower in 36 patients having chronic hypoxemia than in 15 normosemic controls, showing significant statistical differences for Na, K, Cl, Ca and urea. The correlations between the clearances of these substances and the pO2 arterial blood levels had a greater statistical significance than can be established with pCO2 or [H+] levels. Thus, the existence of a causal dependency between renal disfunction and hypoxemia may be deduced.     

Effects of dithiothreitol and boar on pronuclear formation and embryonic development following intracytoplasmic sperm injection in pigs

The objective of this study was to improve normal fertilization, male pronuclear formation and embryonic development following intracytoplasmic injection of dithiothreitol (DTT)-treated boar spermatozoa. To determine the effect of DTT treatment, frozen-thawed boar spermatozoa were treated with DTT for 0, 10, 30, and 60 min, and injected into porcine oocytes. The effects of DTT and male difference on normal fertilization and embryonic development were investigated. The mean normal fertilization rate in the groups treated with DTT for 30 min (73.8%) and 60 min (74.9%) was higher (P < 0.05) than that in the control group (49.3%). The mean blastocyst formation rate in the group treated with DTT for 30 min (23.2%) was higher (P < 0.05) than that in the other groups (8.7-10.9%). Among boars there was no difference in normal fertilization, but there was a significant difference between the non-treated and the DTT-treated groups. The mean rate of blastocyst formation was different (P < 0.05) among boars, and between the non-treated and DTT-treated groups. The mean number of cells in blastocysts was similar among the boars and between the non-treated and the DTT-treated groups. In conclusion, DTT treatment for 30 min increased the rate of normal fertilization and embryonic development to the blastocyst stage. Furthermore, the rate of blastocyst formation of oocytes injected with spermatozoa differed among boars.     

Extracellular proteases and embryonic pattern formation

At least three genes that play crucial roles in dorsal-ventral patterning of the Drosophila embryo appear to encode extracellular proteases. These proteases are involved in the generation of localized extracellular ligands for membrane receptors. Because the sequences of these gene products closely resemble those of mammalian enzymes that have been studied in detail biochemically, it is possible to draw on the wealth of information on the biochemical mechanisms that regulate protease activity to make inferences about how proteases can be used to generate spatial asymmetries within fields of cells.