Amplification of R-spondin1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary.

R-spondin1 is a secreted regulator of WNT signaling, involved in both embryonic development and homeostasis of adult organs. It can have a dual role, acting either as a mitogen or as a tumor suppressor. During ovarian development, Rspo1 is a key factor required for sex determination and differentiation of the follicular cell progenitors, but is downregulated after birth. In human, increased RSPO1 expression is associated with ovarian carcinomas, but it is not clear whether it is a cause or a consequence of the tumorigenic process. To address the role of Rspo1 expression in adult ovaries, we generated an Rspo1 gain-of-function mouse model. Females were hypofertile and exhibited various ovarian defects, ranging from cysts to ovarian tumors. Detailed phenotypical characterization showed anomalies in the ovulation process. Although follicles responded to initial follicle-stimulating hormone stimulation and developed normally until the pre-ovulatory stage, they did not progress any further. Although non-ovulated oocytes degenerated, the surrounding follicular cells did not begin atresia. RSPO1-induced expression not only promotes canonical WNT signaling but also alters granulosa cell fate decisions by maintaining epithelial-like traits in these cells. This prevents follicle cells from undergoing apoptosis, leading to the accumulation of granulosa cell tumors that reactivates the epithelial program from their progenitors. Taken together, our data demonstrate that activation of RSPO1 is sufficient in promoting ovarian tumors and thus supports a direct involvement of this gene in the commencement of ovarian cancers.

Peroxisome-proliferator-activated receptor delta mediates the effects of long-chain fatty acids on post-confluent cell proliferation.

Nutritional long-chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes. It is now established that peroxisome-proliferator-activated receptors (PPARs) play crucial functions in the control of gene expression and the level of cell differentiation. PPARgamma, which is activated by specific prostanoids, is a key factor in activating terminal differentiation and adipogenesis. We have recently demonstrated that PPARdelta, once activated by fatty acids, drives the expression of a limited set of genes, including that encoding PPARgamma, thereby inducing adipose differentiation. Thus far, the mechanism of action of fatty acids in the control of preadipocyte proliferation has remained unknown. We show here that PPARdelta is directly implicated in fatty acid-induced cell proliferation. Ectopic expression of PPARdelta renders 3T3C2 cells capable of responding to treatment with long-chain fatty acids by a resumption of mitosis, and this effect is limited to a few days after confluence. This response is restricted to PPARdelta activators and, for fatty acids, takes place within the range of concentrations found to trigger differentiation of preadipocytes both in vitro and in vivo. Furthermore, the use of a mutated inactive PPARdelta demonstrated that transcriptional activity of the nuclear receptor is required to mediate fatty acid-induced proliferation. These data demonstrate that PPARdelta, as a transcription factor, is directly implicated in fatty acid-induced proliferation, and this could explain the hyperplastic development of adipose tissue that occurs in high-fat-fed animals.

Phagocytosis reveals a reversible differentiated state early in the development of the mouse embryo.

Mural trophectoderm cells of the mouse embryo possess a phagocytic potential as early as 3.5 days post coitum (d.p.c.). This first differentiated function shows a graded variation along the embryonic-abembryonic axis, from a maximal activity in the non-dividing cells of the abembryonic pole to a complete lack of activity in the replicating polar trophectoderm overlying the inner cell mass (ICM). This pattern can be explained by a negative control exerted by the ICM. Addition of FGF4, a factor secreted by ICM cells, strongly inhibited phagocytosis while inducing resumption of DNA synthesis in mural trophectoderm cells, revealing a reversible, FGF4-dependent differentiation state. Under conditions in which a small cluster of mural trophectoderm cells (<10) had internalized large particles, these otherwise morphologically normal embryos could not implant in the uterus, indicating that cells at the abembryonic pole have a critical role in initiating the implantation process. At post-implantation stages (6.5-8.5 d.p.c.), the ectoplacental cone and secondary giant cells derived from the polar trophectoderm also contained active phagocytes, but at that stage, differentiation was not reversed by FGF4.

Ca increase in secretory granules of stimulated mast cells.

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray micro-analysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components. In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines: It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue. Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.

Calcium signals in and around the nucleus in sea urchin eggs.

A transient rise in cytoplasmic Ca2+ activity in the sea urchin egg occurs during fertilization due to calcium release from an intracellular store. Using a combination of the Ca2+ sensitive dye Calcium Green dextran and the Ca2+ insensitive dye tetramethylrhodamine dextran we have obtained confocal ratio images of free cytoplasmic calcium distribution during the fertilization calcium wave. We can also trigger calcium release using calcium-releasing agonists such as InsP3, ryanodine and cADP-ribose. Calcium levels are in all cases similar within nucleus and in the cytoplasm. A striking result from confocal calcium imaging is that the fertilization calcium wave is not the only spatio-temporal calcium signal observed after fertilization. In fact, a second calcium wave propagates through the egg as pronuclear migration begins; this wave also originates at the point of sperm entry. A global calcium increase is also recorded during the fusion of the male and female pronuclei. We conclude that calcium concentrations in the nucleus are similar to those in the cytoplasm during these calcium transients, that a remnant at the point of sperm entry can originate a second propagating calcium wave and that a global calcium transient occurs at the time of pronuclear fusion.

Redundant mechanisms of calcium-induced calcium release underlying calcium waves during fertilization of sea urchin eggs.

Propagating Ca2+ waves are a characteristic feature of Ca(2+)-linked signal transduction pathways. Intracellular Ca2+ waves are formed by regenerative stimulation of Ca2+ release from intracellular stores by Ca2+ itself. Mechanisms that rely on either inositol trisphosphate or ryanodine receptor channels have been proposed to account for Ca2+ waves in various cell types. Both channel types contributed to the Ca2+ wave during fertilization of sea urchin eggs. Alternative mechanisms of Ca2+ release imply redundancy but may also allow for modulation and diversity in the generation of Ca2+ waves.

Thimerosal reveals calcium-induced calcium release in unfertilised sea urchin eggs.

The fertilisation calcium wave in sea urchin eggs triggers the onset of development. The wave is an explosive increase in intracellular free calcium concentration ([Ca2+i]) that begins at the point of sperm entry and crosses the egg in about 20 s. Thimerosal is a sulphydryl reagent that sensitises calcium release from intracellular stores in a variety of cell types. Treatment of unfertilised eggs with thimerosal causes a slow increase [Ca2+i] that results eventually in a large, spontaneous calcium transient and egg activation. At shorter times after thimerosal treatment, egg activation and the calcium transient can be triggered by calcium influx through voltage-gated calcium channels, a form of calcium-induced/calcium release (CICR). Thimerosal treatment also reduces the latency of the fertilisation calcium response and increases the velocity of the fertilisation wave. These results indicate that thimerosal can unmask CICR in sea urchin eggs and suggest that the ryanodine receptor channel based CICR may contribute to explosive calcium release during the fertilisation wave.

Net calcium and acid release at fertilization in eggs of sea urchins and ascidians.

Sea urchin eggs lose about 10-30% of their total calcium content upon fertilization. We have investigated the mechanism of this calcium-loss with an ion-selective vibrating probe system. Upon fertilization of Arbacia punctulata and Lytechinus pictus eggs we could measure a calcium efflux signal with an average duration of 204 +/- 26 s and 146 +/- 46 s, respectively. Measurements of hydrogen ion signals in normal and in low sodium media showed that the release of cortical vesicle material from these eggs lasts for about 30 and 50 s, respectively. The data indicate that most of the calcium that is lost from sea urchin eggs originates from the cytosol in which it is released during fertilization and then pumped out through the plasma membrane. Calcium loss due to cortical granule release accounts for less than 14% of the total loss measured. We also measured a substantial post-fertilization calcium efflux in eggs of Phallusia mammilata, with an average duration of 265 +/- 18 s followed by smaller periodic effluxes that corresponded to oscillations in the [Ca2+]i during contractile waves in these eggs. These data, together with the lack of cortical granules in ascidian eggs, indicate that Phallusia eggs also pump out a substantial amount of calcium through the plasma membrane after fertilization.

Ryanodine Activates Sea Urchin Eggs: (sea urchin egg/activation/ryanodine/inositol phosphate/heparin).

We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP3 , produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner 45 Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP3 stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg++ , ruthenium red and heparin. Our results suggest that ryanodine-sensitive intracellular calcium channels exist in the sea urchin egg.

The calcium content of cortical granules and the loss of calcium from sea urchin eggs at fertilization.

In many species, fertilization triggers a wave of cortical granule exocytosis in the egg that is the consequence of an increase in intracellular free calcium concentration. We have measured the total calcium content of cortical granules from two species of sea urchins by quantitative X-ray microanalysis and spectrometric measurements. Our results show that cortical granules: (1) contain a high concentration of total calcium (around 30 and 95 mM for Paracentrotus lividus and Arbacia lixula, respectively), (2) represent a major cortical storage site of calcium in the egg (5 and 11% of total egg calcium for P. lividus and A. lixula, respectively), and (3) exchange part of their accumulated calcium by an ATP dependent mechanism. In addition we have confirmed that at fertilization, sea urchin eggs lose a sizeable amount of their calcium (7% for P. lividus and 15% for A. lixula). The kinetics and magnitude of the loss suggest that some of this calcium could be provided by cortical granules during exocytosis.

Calcium pools in sea urchin eggs: roles of endoplasmic reticulum and mitochondria in relation to fertilization.

A preparation of sea urchin eggs permeabilized with digitonin (40 microM for 2.5 min) was used to study the kinetic characteristics of the two cellular compartments suspected to play a key role in cellular calcium transfer during fertilization: an ATP-dependent Ca2+ pool (Km = 0.47 microM; Vm = 0.48 nmol/min.mg protein) probably located in the endoplasmic reticulum and a mitochondrial Ca2+ pool (Km = 1.50 microM; Vm = 0.12 nmol/min.mg protein). Fertilization triggered a decrease in the rate of ATP dependent uptake by the non-mitochondrial pool (Km = 0.59 microM; Vm = 0.15 nmol/min.mg protein) while it transiently increased the Ca2+ uptake into mitochondria (2 min post-fertilization: Km = 2.20 microM; Vm = 0.40 nmol/min.mg protein). Microanalysis studies performed on quickly frozen, freeze substituted and embedded eggs showed a transient Ca2+ enrichment of mitochondria soon after fertilization thus suggesting that mitochondria behave as a Ca2+ sink at fertilization. Results are discussed in relation to the role of endoplasmic reticulum and mitochondria in handling free calcium during the early period following sea urchin egg fertilization.

Calcium in sea urchin egg during fertilization.

Calcium plays a strikingly important role in two of the major events in developmental biology: cell activation and differentiation. In this review we begin with the location and quantity of intracellular calcium in sea urchin oocytes, and then discuss the changes that occur during fertilization and egg activation, placing special emphasis on the mobilization and redistribution of intracellular calcium. We also discuss the propagation of the calcium wave and the role of the burst of calcium on the process of reorganizing the egg cortex at fertilization.

Renal effects of caffeine in preterm infants.

Renal functions were assessed during two 12-hour periods, before and after the administration of a single oral dose of caffeine (15 mg/kg) in 13 preterm infants. Each infant acted as his own control. Heart rate, mean blood pressure, hematocrit, blood pH, arterial PO2 and PCO2 remained stable throughout the two successive periods. Mean protein serum level slightly increased from period I to period II. Urine flow rate (+63 +/- 57%), water output/input ratio (+49 +/- 54%) and creatinine clearance (+79 +/- 102%) increased significantly after the administration of caffeine.

X-ray microanalysis of calcium containing organelles in resin embedded tissue.

The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites: calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation. Resin embedding is less demanding than cryomicrotomy and gives better images: it can be used after cryosubstitution in the presence of oxalic acid. This technique was tested, and applied to several cell types.

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution. Validity of the method.

Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2 +/- 0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3 +/- 1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of 45Ca from prelabelled eggs. Exchangeable 45Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.

Smectite reduces gastroesophageal reflux in newborn infants.

We assessed the efficacy of a natural clay (smectite) on the frequency and the duration of acid (pH less than 4) and very acid (pH less than 3) gastroesophageal reflux (GER) measured by 24-hour continuous pH recording (CPR). Twenty newborn infants were enrolled in this double-blind controlled study owing to pathological CPR in supine position. After inclusion, all the patients were maintained in the 30-degree elevated prone position and received either smectite (3 g/day; n = 10) or placebo (n = 10) for 7 days. On the 8th day, a second 24-hour CPR was performed in supine position. The postural therapy alone (placebo group) was followed by a significant decrease in the numbers of acid GER (p less than 0.05) during the second CPR. The combination of postural therapy and smectite treatment was followed by a decrease in the number of acid (p less than 0.05) and very acid GER (p = 0.01), the percentages of time spent at pH below 4 (p less than 0.05) and 3 (p less than 0.01) and the maximal duration of acid GER (p less than 0.05).

[Esophageal pH-monitoring in asymptomatic and symptomatic newborn infants]. / Etude de la pH-métrie oesophagienne chez les nouveau-nés asymptomatiques et symptomatiques.

The supine distal esophageal pH was recorded over 24 hours, in low birth weight infants. Forty-seven of them were asymptomatic (group T) and 93 presented with digestive symptoms (regurgitations and/or vomiting) associated (group DA; n = 49) or not (group D; n = 44) with a history of apneas, bradycardias, cyanosis or pallor fits. On the recording day, post-natal ages (weeks), conceptional ages (weeks) and weights (g) were not different among the 3 groups (T: 4.8 +/- 2.6; 40.1 +/- 2; 2,590 +/- 210. DA: 4.7 +/- 3.3; 40 +/- 3.3; 2,980 +/- 640. D: 4.7 +/- 2.8; 41.1 +/- 3.3; 2,710 +/- 620). In the 3 groups all the features of the acid and highly acid gastroesophageal refluxes (GER) were significantly more marked in late post-prandial stages (LPPS) than in early post-prandial stages (EPPS) (p less than 0.001). Slightly acid GER were more frequent in LPPS than in EPPS for groups T and DA (p less than 0.01). No significant difference could be found when comparing group T with groups D, DA and with a sample of 21 infants presenting with vomiting.