Oestradiol Potentiates Hormone Secretion and Neuronal Activation in Response to Hypertonic Extracellular Volume Expansion in Ovariectomised Rats

Secretion of vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) is an essential mechanism for the maintenance of hydromineral homeostasis. Secretion of these hormones is modulated by several circulating factors, including oestradiol. However, it remains unclear how oestradiol exerts this modulation. In the present study we investigated the participation of oestradiol in the secretion of VP, OT and ANP and in activation of vasopressinergic and oxytocinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to extracellular volume expansion (EVE). For this purpose, ovariectomised (OVX) rats treated for 7 days with vehicle (corn oil, 0.1 ml/rat, OVX+O group) or oestradiol (oestradiol cypionate, 10 mu g/kg, OVX+E group) were subjected to either isotonic (0.15 m NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 m NaCl, 2 ml/100 g b.w., i.v.) EVE. Blood samples were collected for plasma VP, OT and ANP determination. Another group of rats was subjected to cerebral perfusion, and brain sections were processed for c-Fos-VP and c-Fos-OT double-labelling immunohistochemistry. In OVX+O rats, we observed that both isotonic and hypertonic EVE increased plasma OT and ANP concentrations, although no changes were observed in VP secretion. Oestradiol replacement did not alter hormonal secretion in response to isotonic EVE, but it increased VP secretion and potentiated plasma OT and ANP concentrations in response to hypertonic EVE. Immunohistochemical data showed that, in the OVX+O group, hypertonic EVE increased the number of c-Fos-OT and c-Fos-VP double-labelled neurones in the PVN and SON. Oestradiol replacement did not alter neuronal activation in response to isotonic EVE, but it potentiated vasopressinergic and oxytocinergic neuronal activation in the medial magnocellular PVN (PaMM) and SON. Taken together, these results suggest that oestradiol increases the responsiveness of vasopressinergic and oxytocinergic magnocellular neurones in the PVN and SON in response to osmotic stimulation.

CB(1) modulation of hormone secretion, neuronal activation and mRNA expression following extracellular volume expansion

The endocannabinoid system includes important signaling molecules that are involved in several homeostatic and neuroendocrine functions. In the present study, we evaluated the effects of the type 1 cannabinoid (CB(1)) receptor antagonist, rimonabant (10 mg/kg, p.o.), on hormone secretion, neuronal activation and mRNA expression in the hypothalamus following isotonic (I-) or hypertonic (H-) extracellular volume expansion (EVE). The total nitrate content in the PVN and SON was also assessed under the same experimental conditions. Our results showed that OT and AVP plasma concentrations were increased in response to H-EVE, while decreased AVP levels were found following I-EVE. Accordingly, both I- and H-EVE stimulated oxytocinergic neuronal activation, as evidenced by the increased number of c-Fos/OT double labeled neurons in the hypothalamus. The vasopressinergic cells of the PVN and SON, however, were only activated in response to H-EVE. Furthermore, increased amounts of both AVP and OT mRNAs were found in the hypothalamus following EVE. Pretreatment with rimonabant significantly potentiated hormone secretion and also vasopressinergic and oxytocinergic neuronal activation induced by EVE, although decreased AVP and OT mRNA expression was found in the hypothalami of rimonabant pretreated groups. In addition, the nitrate content in the PVN and SON was not altered in response to EVE or rimonabant pretreatment. Taken together, these results suggest that the CB(1) receptor may modulate several events that contribute to the development of appropriate responses to increased fluid volume and osmolality. (C) 2010 Elsevier Inc. All rights reserved.

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Neuronal maturation in an experimental model of brain tissue heterotopia in the lung

Neural maturation involves diverse interaction and signaling mechanisms that are essential to the development of the nervous system. However, little is known about the development of neurons in heterotopic brain tissue in the lung, a rare abnormality observed in malformed babies and fetuses. The aim of this study was to identify the neurons and to investigate their maturation in experimental brain tissue heterotopia during fetal and neonatal periods. The fetuses from 24 pregnant female Swiss mice were used to induce brain tissue heterotopia on the 15th gestational day. Briefly, the brain of one fetus of each dam was extracted, disaggregated, and injected into the right hemithorax of siblings. Six of these fetuses with pulmonary brain tissue implantation were collected on the 18th gestational day (group E18), and six others were collected on the 8th postnatal day (group P8). The brain of each fetus from dams not submitted to any experimental procedure was collected on the 18th gestational day (group CE18) and on the 8th postnatal day (group CP8) to serve as a control for neuronal quantitation and maturation. Immunohistochemical staining of NeuN was used to assess neuron quantity and maturation. The NeuN labeling index was greater in the postnatal period than in the fetal period for the experimental and control groups (138 > E18 and CP8 > CE18), although there were fewer neurons in experimental than in control groups (P8 < CP8 and El 8 < CE1 8) (P < 0.005). These results indicate that fetal neuroblasts/neurons not only survive a dramatic event such as mechanical disaggregation, in the same way as it happens in human cases, but also they retain their development in heterotopia, irrespective of local tissue influences.

Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. (C) 2011 Elsevier B.V. All rights reserved.

Correlation between mastitis occurrence and the count of microorganisms in bulk raw milk of bovine dairy herds in four selective culture media

Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of oil(? or more healthy animals. Mastitis is all inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk ill four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were Used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman`s correlation coefficient was calculated in order to compare the Occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective Culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacteria I-growth mastitis rates and log(10) of KF Streptoccocus Agar plate Count and there were two positive correlations between coagulase-positive staphylococci and log(10) of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 mu g/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX-blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

The neuroprotective effect of dental pulp cells in models of Alzheimer`s and Parkinson`s disease

Aim of the present study was to investigate the neuroprotective effect of dental pulp cells (DPCs) in in vitro models of Alzheimer and Parkinson disease. Primary cultures of hippocampal and ventral mesencephalic neurons were treated for 24 h with amyloid beta (A beta(1-42)) peptide 1-42 and 6-OHDA, respectively. DPCs isolated from adult rat incisors were previously cultured in tissue culture inserts and added to the neuron cultures 2 days prior to neurotoxin treatment. Cell viability was assessed by the MTT assay. The co-culture with DPCs significantly attenuated 6-OHDA and A beta(1-42)-induced toxicity in primary cultures of mesencephalic and hippocampal neurons, and lead to an increase in neuronal viability in untreated cultures, suggesting a neurotrophic effect in both models. Furthermore, human dental pulp cells expressed a neuronal phenotype and produced the neurotrophic factors NGF, GDNF, BDNF, and BMP2 shown by microarray screening and antibody staining for the representative proteins. DPCs protected primary neurons in in vitro models of Alzheimer`s and Parkinson`s disease and can be viewed as possible candidates for studies on cell-based therapy.