Investigating cellular and molecular mechanisms of neurogenesis in Capitella teleta sheds light on the ancestor of Annelida.

BACKGROUND: Diverse architectures of nervous systems (NSs) such as a plexus in cnidarians or a more centralized nervous system (CNS) in insects and vertebrates are present across Metazoa, but it is unclear what selection pressures drove evolution and diversification of NSs. One underlying aspect of this diversity lies in the cellular and molecular mechanisms driving neurogenesis, i.e. generation of neurons from neural precursor cells (NPCs). In cnidarians, vertebrates, and arthropods, homologs of SoxB and bHLH proneural genes control different steps of neurogenesis, suggesting that some neurogenic mechanisms may be conserved. However, data are lacking for spiralian taxa. RESULTS: To that end, we characterized NPCs and their daughters at different stages of neurogenesis in the spiralian annelid Capitella teleta. We assessed cellular division patterns in the neuroectoderm using static and pulse-chase labeling with thymidine analogs (EdU and BrdU), which enabled identification of NPCs that underwent multiple rounds of division. Actively-dividing brain NPCs were found to be apically-localized, whereas actively-dividing NPCs for the ventral nerve cord (VNC) were found apically, basally, and closer to the ventral midline. We used lineage tracing to characterize the changing boundary of the trunk neuroectoderm. Finally, to start to generate a genetic hierarchy, we performed double-fluorescent in-situ hybridization (FISH) and single-FISH plus EdU labeling for neurogenic gene homologs. In the brain and VNC, Ct-soxB1 and Ct-neurogenin were expressed in a large proportion of apically-localized, EdU+ NPCs. In contrast, Ct-ash1 was expressed in a small subset of apically-localized, EdU+ NPCs and subsurface, EdU- cells, but not in Ct-neuroD+ or Ct-elav1+ cells, which also were subsurface. CONCLUSIONS: Our data suggest a putative genetic hierarchy with Ct-soxB1 and Ct-neurogenin at the top, followed by Ct-ash1, then Ct-neuroD, and finally Ct-elav1. Comparison of our data with that from Platynereis dumerilii revealed expression of neurogenin homologs in proliferating NPCs in annelids, which appears different than the expression of vertebrate neurogenin homologs in cells that are exiting the cell cycle. Furthermore, differences between neurogenesis in the head versus trunk of C. teleta suggest that these two tissues may be independent developmental modules, possibly with differing evolutionary trajectories.

Increased angiotensin II in ascites during severe ovarian hyperstimulation syndrome: role of early pregnancy and ovarian gonadotropin stimulation.

OBJECTIVE: To investigate the implications of the ovarian renin-angiotensin system (RAS) in the pathophysiology of the ovarian hyperstimulation syndrome (OHSS) in relation to gonadotropin stimulation and early pregnancy. DESIGN: A controlled clinical study comparing blood and simultaneously sampled peritoneal fluid (PF) from patients with severe OHSS and from controls without OHSS. SETTING: University Hospitals. PATIENT(S): Eleven patients with severe OHSS, 8 patients with ascites of other origin, 9 patients with a first-trimester pregnancy, and 15 patients stimulated with gonadotropins for IVF. MAIN OUTCOME MEASURE(S): Angiotensin II immunoreactivity was measured in blood and PF and analyzed by high-performance liquid chromatography (HPLC) in ascites from OHSS. RESULT(S): Angiotensin II immunoreactivity (pg/mL; mean +/- SE) was highest in the ascites from pregnant OHSS (1,669 +/- 418), reaching levels 5 times higher than in the plasma (331 +/- 61) and 100 times higher than in control ascites (17 +/- 6.7). Angiotensin II immunoreactivity was elevated in the PF during early pregnancy (211 +/- 68) and after gonadotropin stimulation (244 +/- 41) and was higher than in the plasma in both groups. Analysis by HPLC showed that the majority of Ang II immunoreactivity in the ascites of OHSS was because of true Ang II. CONCLUSION(S): Severe forms of OHSS, especially those associated with pregnancy, are consistently characterized by huge concentrations of Ang II immunoreactivity in the ascites, proved to be true Ang II by HPLC analysis. This may be due to the synergistic effects of exogenous and endogenous hCG on the ovarian RAS.

Features of the Renin-angiotensin system in ascites and pleural effusion during severe ovarian hyperstimulation syndrome.

PURPOSE: The purpose of this work was to investigate the ovarian renin-angiotensin system (RAS) during severe ovarian hyperstimulation syndrome (OHSS) with ascites and pleural effusion. METHODS: Two patients who developed severe OHSS after ovarian stimulation for in vitro fertilization were investigated. Both patients presented ascites and pleural effusion. Blood, ascites, and pleural fluid were simultaneously sampled during therapeutic paracentesis and thoracocentesis. Renin activity, active renin, prorenin, and angiotensin II immunoreactivity (Ang II-ir) were measured simultaneously in plasma, ascites, and pleural fluid. RESULTS: Prorenin, renin activity, active renin, and Ang II-ir levels were much higher than normal plasmatic laboratory norms in the three compartments. Prorenin and Ang II-ir levels were the highest in the ascites, while they were in the same range in the pleural fluid and in the plasma. CONCLUSIONS: The present findings provide additional evidence for the ovarian origin of the prorenin and Ang II-ir in the ascites of severe OHSS.

Prorenin and active renin concentrations in plasma and ascites during severe ovarian hyperstimulation syndrome.

The pathophysiology of ovarian hyperstimulation syndrome (OHSS) remains unclear. Several lines of evidence indicate that OHSS is associated with a stimulation of the renin-angiotensin system (RAS), but its functional significance as well as its role in the pathogenesis of the syndrome are not yet determined. OHSS is associated with high plasma and ascitic concentrations of total renin, renin activity (RA) and angiotensin II (Ang II). Their ovarian or renal origin is, however, still a matter of debate. To clarify these issues further, total renin, active renin, prorenin, RA and aldosterone were measured in plasma and ascites of nine patients who developed severe OHSS after in-vitro fertilization. Blood and ascites were sampled simultaneously during therapeutic paracentesis. Total renin and prorenin concentrations were significantly higher in the ascites (mean concentration +/- SE respectively of 5920 +/- 1430 mIU/l and 5250 +/- 1350 mIU/l) than in the plasma (respectively 3060 +/- 740 mIU/l and 2000 +/- 460 mIU/l) (P = 0.020 and 0.017 respectively). Conversely, active renin and RA concentrations tended to be lower, although not statistically significantly so in the ascites (respectively 670 +/- 190 mIU/l and 47 +/- 11 ng Ang I/ml/h) than in the plasma (respectively 1060 +/- 370 mIU/l and 75 +/- 21 ng Ang I/ml/h). Aldosterone concentrations were significantly higher in the serum (2609 +/- 374 pg/ml) than in the ascites (2025 +/- 347 pg/ml) (P = 0.015). The concentration gradient between plasma and ascites for total renin and prorenin supports the hypothesis of their ovarian origin in ascites and, to a large extent, in plasma, while it is likely that the high plasma active renin and RA concentrations reflect a peripheral activation of the RAS. In conclusion, the present findings are consistent with a marked stimulation of both ovarian and renal RAS during OHSS.

Periovulatory elevation of angiotensin II in the peritoneal fluid during the human menstrual cycle.

Local variations in the ovarian renin-angiotensin system were investigated in peritoneal fluid (PF) during normal menstrual cycles and after ovarian stimulation with gonadotropins. PF was collected either during laparoscopy or by transvaginal aspiration before ovocyte retrieval for in vitro fertilization. Renin activity (RA) and angiotensin II immunoreactivity (Ang II-ir) were assayed in PF and simultaneously collected blood. The level of Ang II-ir was higher in PF than in plasma throughout the cycle, where as RA was in the same range of magnitude in the two compartments. In PF, Ang II-ir reached levels 2-5 times higher in the periovulatory period (days 12-14 and 15-17 of the cycle) than those found during the other stages of the cycle, whereas plasmatic Ang II-ir remained stable. No significant change could be demonstrated in RA throughout the cycle in either PF or plasma. Ovarian stimulation induced a strong elevation of Ang II-ir in PF, but not in plasma. High performance liquid chromatography revealed that the majority of Ang II-ir in PF was true Ang II. In conclusion, these results show a periovulatory elevation of Ang II in PF during the cycle and a more pronounced rise after treatment with gonadotropins. These observations support the involvement of Ang II in the process of ovulation or fecundation.

Bioactive angiotensin (1-8) is the main component of angiotensin II immunoreactivity in human follicular fluid.

The ovarian renin-angiotensin system is involved in various aspects of human reproduction. As immunoreactive measurement of angiotensin II (ANG II) in follicular fluid (FF) relates to several angiotensin peptides with different biological activities, HPLC was used to characterize the molecular forms of the ANG II immunoreactivity in human FF. Samples of FF, obtained from gonadotropin-stimulated patients for in vitro fertilization, were collected at the time of oocyte retrieval. The C-terminal 2-8 heptapeptide was never detected. HPLC analysis revealed for the first time that the major component of the ANG II-IR in human FF was the biologically active octapeptide ANG II.

Angiotensin II immunoreactivity is elevated in ascites during severe ovarian hyperstimulation syndrome: implications for pathophysiology and clinical management.

OBJECTIVE: To investigate the ovarian renin-angiotensin system (RAS) during severe ovarian hyperstimulation syndrome (OHSS). DESIGN: Simultaneous sampling of blood and ascitic or peritoneal fluid (PF) during therapeutic paracentesis or laparoscopy. SETTING: University Hospital. PATIENTS: Twelve patients were investigated: three patients presenting severe OHSS, three patients with a spontaneous first trimester pregnancy, three normally cycling women during the early luteal phase, and three patients with ascites of nonovarian origin. MAIN OUTCOME MEASURE: Renin-like activity and angiotensin II (ANG II) immunoreactivity were measured simultaneously in the plasma and the ascites or PF. RESULTS: Angiotensin II immunoreactivity was much higher in the ascites or PF than in corresponding plasma during severe OHSS, first trimester pregnancy, and in the early luteal phase, while it was lower in ascites of nonovarian origin. Renin-like activity and ANG II immunoreactivity were the highest in the ascites of severe OHSS and in the PF from part of the patients with a spontaneous first trimester pregnancy. CONCLUSIONS: The present findings argue for the ovarian origin of the elevated renin-like activity and ANG II immunoreactivity in the ascites of severe OHSS and suggest a stimulatory role of hCG on the ovarian RAS whether during severe OHSS or first trimester spontaneous pregnancy. The vasoactive peptide ANG II may contribute to the maintenance of the ascites in severe OHSS but is probably not responsible for the formation of the ascites. The efficiency of paracentesis during severe OHSS could be explained at least partially by the removing of great amounts of ANG II from the peritoneal cavity.

Ethanol increases and vitamin D metabolites decrease the production of cyclic AMP by canine renal cortical membranes.

Ethanol 0.16% increased cyclic AMP production by canine renal cortical membranes in the basal state and when challenged with different parathyroid hormone or fluoride concentrations. 1,25-dihydroxycholecalciferol (1,25(OH)2D3) 40 pM completely inhibited this effect of ethanol and reversed cyclic AMP production to the level observed in buffer alone. The same inhibitory effect was observed with 25OHD3 and with 24,25-dihydroxycholecalciferol (24,25(OH)2D3). The inhibitory effect was related to the vitamin D metabolites' concentration and was maximal for 160 pM; it was independent of their biological activity. This suggests that the effect is mediated through an interaction with the membrane lipids. The effect of vitamin D metabolites on cyclic AMP production was also observed in the presence of serum proteins and should be taken into account if unextracted plasma is assayed in the renal cortical membrane system for PTH bioactivity.

Cationized serum albumin enhances basal and stimulated adenylate cyclase activity in canine renal membranes.

We examined the effects of cationized serum albumin on the canine renal membrane adenylate cyclase in the basal state and when stimulated with guanylyl-imidodiphosphate, PTH or NaF. Human albumin was cationized to an isoelectric point greater than 9.5 by the addition of hexamethylene diamine. Cationized albumin increased basal and stimulated cAMP production by the membranes and increased the sensitivity of the system to low doses of PTH (0.25 pmol/l), being usually inactive in buffer alone or in human serum albumin. These observations are comparable to those previously reported on thyroid membranes and cells from adrenal tumours and confirm that positively charged macromolecules can increase adenylate cyclase activity. A decrease in non-specific binding of PTH is only partly responsible for the increased sensitivity to the hormone. Though this increase in sensitivity is small, it could nevertheless be useful in the detection of biologically active PTH after extraction from the serum.

Cationized serum albumin enhances response of cultured fetal rat long bones to parathyroid hormone.

The effects of cationized serum albumin on the resorptive response to PTH and other agents were examined in organ cultures of fetal rat long bones. Human serum albumin (HSA) was cationized to an isoelectric point greater than 9.5 by addition of hexamethylene diamine. When cationized albumin (C-HSA) replaced HSA or BSA in the medium, resorption could be stimulated by 10- to 30-fold lower concentrations of synthetic (1-34) human or bovine PTH or intact 1-84 bovine PTH. Using C-HSA, significant resorption was obtained in some experiments with the concentration of PTH as low as 25 pM, but in most experiments 100 pM-400 pM concentrations were required. In contrast, 6.25 nM 1-34 PTH was required for a response in HSA. The sensitivity to stimulation of resorption by 1,25-dihydroxy-vitamin D and prostaglandin E2 was not increased. Hence, the increased sensitivity to PTH is most likely due to a selective protective effect of C-HSA, which might decrease nonspecific binding or degradation of the hormone.

Enhancement of parathyroid hormone-stimulated bone resorption by poly-L-lysine.

Poly-L-lysine (PL II; mol wt, 1000-4000) was added to fetal rat bones cultured in a chemically defined medium (BGJ) containing bovine serum albumin in the presence and absence of parathyroid hormone (PTH). Bone resorption was measured by the release of previously incorporated 45Ca. The addition of PL II at concentrations of 3-100 microgram/ml enhanced the stimulation of bone resorption by submaximal doses of PTH but had little effect on 45Ca release from control unstimulated cultures. Higher concentrations of PL II produced inhibition of 45Ca release. Dialysis of PL II did not alter enhancement or inhibition by PL II. PL II did not increase sensitivity to PTH in serum-supplemented cultures. Higher molecular weight PL II preparations were less effective. PL II did not enhance the resorptive response to 1,25-dihydroxyvitamin D, prostaglandin E2, osteoclast-activating factor, or bacterial endotoxin. The mechanism of the selective ability of PL II to enhance the response to low concentrations of PTH is unknown but may be due to the ability of this basic polypeptide to interfere with binding of PTH to sites other than the hormone receptor or to block degradation of PTH by bone.