A review on inflammation and angiogenesis as key mechanisms involved in the pathogenesis of bovine cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of reproductive failure in dairy cattle. The main aim of this review is to discuss some aspects related to inflammation and angiogenesis that seem to be involved in the development of follicular cysts in domestic animals, with special emphasis on the bovine species, in an attempt to elucidate the relationship between these two processes in the early stages of persistence and in the development of bovine COD. We describe the changes in the expression of cytokines and angiogenic factors that seem to generate disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. Results show that pro-inflammatory and anti-inflammatory cytokines behave as regulators of angiogenesis through direct and indirect effects, like overexpression of pro-angiogenic factors, particularly in bovine ovarian cells from follicular cysts and persistent follicles. We conclude that, in dairy cattle, an imbalance in the expression of cytokines and pro-angiogenic growth factors related to ovulation and the processes associated with it would contribute to follicular persistence and to the recurrent appearance of COD.

Contribution of the VEGF system to the follicular persistence associated with bovine cystic ovaries.

Cystic ovaries (CO) characterize a disorder frequently found in dairy cattle. However, despite the contributions by several researchers, the mechanism that leads to ovulatory failure has not yet been completely elucidated. Thus, the aim of this study was to examine the mRNA expression of bovine vascular endothelial growth factor (VEGFA)-164, VEGFA-164b and VEGF receptors (VEGFR1 and VEGFR2) by real-time PCR and protein expression by immunohistochemistry, immunofluorescence and Western blot in follicular fluid from dairy cows with spontaneous CO and in an experimental model of follicular persistence induced by prolonged treatment with progesterone. Results showed that both VEGFA isoforms and receptors were coexpressed in granulosa and theca interna cells and in follicular fluid of ovaries from all the groups evaluated. VEGFA-164, VEGFA-164b and VEGFR2 protein expression was higher in theca cells of persistent follicles from group P0 (expected time of ovulation) than in those from dominant follicles (as reference structure) from the control group (p < 0.05). Also, VEGFA-164 expression was higher in theca cells of cysts than in those of dominant follicles of the control group (p < 0.05). In follicular fluid, VEGFA-164 expression was higher in persistent follicles from group P5 (5 days of follicular persistence) than in the control, P0 and P15 groups, and higher in cysts than in dominant follicles from the control group (p < 0.05). This study provides evidence of an altered expression of VEGFA-164, VEGFA-164b and VEGFR2 during the formation of persistent follicles and cysts in cows. Together, these results evidence that early development of CO in cows is concurrent with an altered expression of these growth factors and that these alterations may contribute to the follicular persistence, angiogenic dysregulation and ovulatory failure found in cows with follicular cysts.

New experimental methodology, setup and LabView program for accurate absolute thermoelectric power and electrical resistivity measurements between 25 and 1600 K: application to pure copper, platinum, tungsten, and nickel at very high temperatures.

In this paper we describe an experimental setup designed to measure simultaneously and very accurately the resistivity and the absolute thermoelectric power, also called absolute thermopower or absolute Seebeck coefficient, of solid and liquid conductors/semiconductors over a wide range of temperatures (room temperature to 1600 K in present work). A careful analysis of the existing experimental data allowed us to extend the absolute thermoelectric power scale of platinum to the range 0-1800 K with two new polynomial expressions. The experimental device is controlled by a LabView program. A detailed description of the accurate dynamic measurement methodology is given in this paper. We measure the absolute thermoelectric power and the electrical resistivity and deduce with a good accuracy the thermal conductivity using the relations between the three electronic transport coefficients, going beyond the classical Wiedemann-Franz law. We use this experimental setup and methodology to give new very accurate results for pure copper, platinum, and nickel especially at very high temperatures. But resistivity and absolute thermopower measurement can be more than an objective in itself. Resistivity characterizes the bulk of a material while absolute thermoelectric power characterizes the material at the point where the electrical contact is established with a couple of metallic elements (forming a thermocouple). In a forthcoming paper we will show that the measurement of resistivity and absolute thermoelectric power characterizes advantageously the (change of) phase, probably as well as DSC (if not better), since the change of phases can be easily followed during several hours/days at constant temperature.

Design and performance of a versatile curved-crystal spectrometer for high-resolution spectroscopy in the tender x-ray range.

A complete in-vacuum curved-crystal x-ray emission spectrometer in Johansson geometry has been constructed for a 2-6 keV energy range with sub natural line-width energy resolution. The spectrometer is designed to measure x-ray emission induced by photon and charged particle impact on solid and gaseous targets. It works with a relatively large x-ray source placed inside the Rowland circle and employs position sensitive detection of diffracted x-rays. Its compact modular design enables fast and easy installation at a synchrotron or particle accelerator beamline. The paper presents main characteristics of the spectrometer and illustrates its capabilities by showing few selected experimental examples.

Glargine blood biotransformation: in vitro appraisal with human insulin immunoassay.

AIM: Glargine, a long-acting insulin analogue, is metabolized in the bloodstream and in subcutaneous tissue. Glargine metabolism and its implications for diabetes therapy remain poorly understood. The aim of our study was to assess in vitro the glargine blood biotransformation and its inter-individual variability. METHODS: Formation of M1 glargine metabolite in vitro was studied with Elecsys Insulin immunoassay in pools of sera and sera from patients spiked with glargine. Elecsys Insulin assay is specific of human insulin, does not recognize glargine and its M2 metabolite but does recognize its M1 metabolite. RESULTS: Glargine incubation with serum resulted in M1 metabolite formation which was detected and characterized as an enzymatic process: metabolite kinetics were dependant on temperature, substrate concentration and serum proportion. Carboxypeptidase inhibitors and chelating agents partially inhibited the activity of the enzyme(s). Glargine biotransformation was decreased when blood was collected on EDTA tubes. After 30 min incubation of glargine (100 mU/l) in 69 sera at 37 degrees C, percentage of glargine converted into M1 ranged from 46% to 98% (mean 72%; S.D. 11%). CONCLUSION: Glargine blood biotransformation is an enzymatic process probably involving serum carboxypeptidase(s). Metabolite formation is rapid and non negligible. Inter-individual variability of glargine biotransformation is noteworthy and should be confronted to M1 metabolite bioactivity which has not been fully documented yet.

[Proposed reference values for nine methods for measurement of free thyroxin]. / Proposition de valeurs de référence pour neuf techniques de dosage de la thyroxine libre dans le sérum.

In connection with a comparative study of nine kits for the measurement of free thyroxin, we determined reference values in a adult control group of 81 women and 73 men. The correlations observed between the kits are associated with very large differences in the results obtained. The reference ranges are more or less broad according to the kits, but narrower than those offered by the manufacturers.

[Macroprolactin detection: a new approach]. / Détection de la macroprolactine: une nouvelle approche.

Macroprolactin is a complex of prolactin with immunoglobulins (IgG) that has limited or no biological activity in vivo. Immunoassays for prolactin have variable reactivity with macroprolactin. Therefore the presence of macroprolactin should be considered in the differential diagnosis of hyperprolactinemia. We compared a valid screening test for macroprolactin, polyethyleneglycol (PEG) precipitation, with the determination of the ratio of the results of two prolactin assays: Elecsys with high cross-reactivity with macroprolactin and Centaur with low cross-reactivity. In 59 negative samples subjected to the PEG test (precipitation < 50%), the Elecsys/Centaur ratio ranged between 1.11 and 1.45. Among 35 positive samples (precipitation > 60%), 33 had, as expected, an increased ratio (over 1.45), 1 a normal ratio and 1 a decreased ratio (1.07). This decreased ratio could be due to a particular form of macroprolactin poorly recognised by the Elecsys assay. Among 5 samples in the grey zone (precipitation between 50 and 60%), the ratio was increased in 2, normal in 1 and decreased in 2. Apart from one false negative case (normal ratio with positive PEG test), the results of the Elecsys/Centaur ratio method were in good agreement with those of the PEG test. The ratio method could be helpful for samples with PEG test results in the grey zone, before undertaking a complete analysis of circulating molecular forms by gel filtration chromatography. Out of the 5 five samples in the grey zone, the ratio was 4 times out of the reference range: 2 increased, 2 decreased. Our results also underline the necessity of reevaluating the Centaur prolactin reference range from samples without macroprolactin.

Concordance of eight kits for antithyroid peroxidase autoantibodies determination.

Numerous methods are proposed to quantify antithyroid peroxidase autoantibodies. No standardization exists but most assays use the standard MRC 66/387 with a calibration factor. Costs of the tests vary between the different kits. We evaluated the concordance of eight peroxidase autoantibodies assay kits in two centres, using a panel of sera from 269 subjects: controls (n=100), patients with autoimmune thyroid disease (n=77; Graves' disease, Hashimoto's thyroiditis), patients with non-autoimmune thyroid disease (n=69; nodular goiter, differentiated thyroid carcinoma) and individual sera with thyroglobulin antibodies only (n=23). The concordance between the eight methods was high, ranging from 88.3% to 98.8% with the total panel of sera. The majority of assays demonstrated high diagnostic performance. We encountered some false-positive results at borderline positive levels, and the nonrecognition of some sera by competitive assays.

[Two-center evaluation of eight kits for antithyroid peroxidase autoantibodies determinations]. / Evaluation bicentrique de huit kits utilisés pour la recherche des anticorps antithyroperoxydase.

We compared eight antithyroid peroxidase antibody assay kits in two centres, by use of panel sera from 269 patients: controls (n = 100), patients with autoimmune thyroid diseases (n = 77 Graves' disease, Hashimoto's thyroiditis), with non autoimmune thyroid diseases (n = 69 nodular goiter, differentiated thyroid carcinoma), and with autoimmune disease without thyroid pathology (n = 23 diabetic subjects, rheumatoid polyarthritis). On the controls sera we observed different distributions of values. The cut-off values of each kit was, in most cases, similar to the value noted in the manufacturer's instructions. In the clinical study, we observed few differences. The majority of assays demonstrated high diagnostic performance. Some false positive results and the non assessment of some sera by competitive immunoassay were observed.

Characterization of FSH-regulated genes isolated by mRNA differential display from pig ovarian granulosa cells.

The present authors have isolated FSH-regulated genes from primary granulosa cell cultures with or without Follicle Stimulating Hormone (FSH) treatment using mRNA differential display. mRNA differential display consists of amplification of partial sequences of cDNAs (150-400 bp) corresponding to 3' ends of cellular messenger RNAs, and thus, generates 3' expressed sequence tags (3' ESTs). Five thousand cDNA bands were examined, among which the present authors have isolated and sequenced 16 different FSH-regulated products. These sequences were compared with those available in databases. Three of the sequences showed similarity to identified genes from other species (bovine NADH dehydrogenase subunit 4, Xenopus chromosome sequence-associated polypeptide E and transformation-sensitive protein IEF SSP) and four others with human ESTs. Regulation of the corresponding genes has been checked by RT-PCR since most of these are expressed at a low level. FSH-regulation was confirmed for 12 mRNAs (four down- and eight up-regulated). The present authors have also mapped 12 of these ESTs on porcine chromosomes regions using a somatic cell hybrid panel.

Evaluation of Elecsys free triiodothyronine assay: relevance of age-related reference ranges.

OBJECTIVE: This study aimed at evaluating Elecsys free triiodothyronine (FT3) assay performed on an Elecsys 2010 system, while paying special attention to age relationship in euthyroid subjects. DESIGN AND METHODS: FT3 levels were measured in 149 euthyroid control subjects aged between 2 and 92 years old, 33 hyperthyroid and particular euthyroid patients: female in the last 3 months of pregnancy (n = 30), nonthyroidal ill hospitalized in medical (NTlm, n = 31), or intensive care units (NTlc, n = 31) and amiodarone-treated (n = 27). RESULTS: FT3 was inversely related to age in controls (r = -0.67). Three reference ranges were used: below 20 years 4.5-9.0 pmol/L, between 20 and 60 years 3.9-7.2, and over 60 years 2.4-6.5. Compared to age-matched controls, FT3 decreased in pregnancy, NTlm, NTlc, and amiodarone groups. Use of age-related reference ranges improved the specificity markedly in amiodarone patients and to a lesser extent in NTlm and TClc patients. CONCLUSIONS: The reliability of the Elecsys FT3 assay was found to be satisfactory for clinical use, when the age of patients was taken into account.

Improved specificity of a new direct assay for urinary cortisol: application in corticoid treated patients.

Twenty four hours urinary free cortisol (UFC) excretion has been determined in 35 eucortisolic control patients, in seven of them before and after tetracosactide (Synacthen) stimulation and in 18 patients treated by anti-inflammatory steroids. Results of the new direct Immunotech RIA (DIm) were compared to those of the INCSTAR RIA kit with (ECA) or without methylene chloride extraction (DCA). In controls DIm UFC (106.2+/-45.8 nmol/24h) was significantly lower than DCA UFC (397+/-119 nmol/24h) and than ECA UFC (127+/-49 nmol/24h). After tetracosactide stimulation, median of DCA/DIm ratio decreased from 3.61 to 1.88 whereas ECA/DIm ratio did not change significantly (1.31 to 1.06). In treated patients most DCA and ECA results were over the upper limit of controls but only 5 DIm results were increased. DIm assay showed good specificity and practicability and may be used with benefit in the evaluation of the adrenal gland function.

Two centre evaluation of seven thyrotropin kits using luminescent detection.

We compared seven thyrotropin luminescent immunometric assay kits in two centres, by use of panel sera from 438 patients: controls (n = 203) and different groups of subjects: hyperthyroidism (n = 42), hypothyroidism (n = 46), non-thyroidal illness (n = 102), geriatrics (n = 24) and selected patients previously treated for thyroid cancer and maintained on suppressive doses of L-thyroxine (n = 17), anti-thyrotropin antibody (n = 4). We did not observe any significant differences in analytical tests among the seven methods on the Probioqual control sera, Anemia control serum and human serum pools. The linearity of serial dilutions was found with all kits. Some variations were noticed at extreme dilutions. The within-assay precision was acceptable in all cases. The functional sensitivity limits were estimated from 20% compound precision profile: they ranged from 0.011 to 0.030 mU/l. In the clinical study, the seven assay demonstrated high diagnostic performance. Some interference by heterophilic antibodies were observed.

A first catalog of genes involved in pig ovarian follicular differentiation.

As a first step toward the characterization of genetic expression in pig ovaries, we have selected 238 clones by differential hybridization from a pig granulosa cell cDNA library, using probes prepared from RNA extracted from either untreated or FSH-treated cells and, in order to generate expressed sequence tags (ESTs), we have performed 3' and 5' single-pass sequencing of these clones. Sequences of the 3' end of the 167 clones that produced informative sequence data were first compared with each other, revealing a redundancy level of 21%. Sequences from the 136 unique clones were analyzed for similarities with sequence data included in Genbank and EMBL databases. Among these unique clones, 54 (40%) matched significantly with sequences from either Genbank of EMBL: 4 with known genes in pig, 35 matched with previously reported human genes, and 15 with other mammalian genes. Eighty-two clones (60%) showed no significant match with any gene or DNA sequence in the Genbank and EMBL databases and thus may represent new pig transcripts.

Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles.

In the sheep as in many mammalian species, growth and atresia of antral follicles are characterized, respectively, by a decrease and a high increase in the intrafollicular levels of insulin-like growth factor binding proteins of less than 40 kDa (IGFBPs < 40 kDa), mainly IGFBP-2, -4, and -5. The objective of this study was to investigate whether such changes are associated with changes in follicular expression of the corresponding mRNA. For this purpose, ovaries were recovered from ewes slaughtered at the end of follicular phase (i.e., 30 h after progestagen sponge removal; control ewes) or at 24 h, 36 h or 72 h after hypophysectomy (hypox) performed 30 h after sponge removal. The expression of mRNA of IGFBPs of less than 40 kDa (IGFBPs < 40 kDa mRNA) was studied in ovine antral follicles from control and hypox ewes by in situ hybridization using [35S]-labeled human IGFBP-2, -4, and -5 cRNA as probes. In control ewes, IGFBP-2 mRNA was mainly expressed in granulosa as a gradient in healthy follicles, the expression being higher in granulosa cells close to the basal membrane than in granulosa cells bordering the antrum and within the cumulus. The level of IGFBP-2 mRNA was lower both in granulosa cells close to the basal membrane and in those bordering the antrum from small follicles than in the corresponding compartments of granulosa cells from large healthy follicles (p < 0.05). In healthy follicles, IGFBP-4 and -5 mRNA were mainly expressed in thecal cells. No change in level of IGFBP-4 mRNA was observed between small and large follicles, whereas the level of IGFBP-5 mRNA tended to be lower in thecal cells from large compared to small follicles (p = 0.055). In atretic follicles, expression of IGFBPs < 40 kDa mRNA strongly increased in granulosa (IGFBP-2 and -5, p < 0.01) and in thecal cells (IGFBP-2 and -4, p < 0.01). In hypox ewes, the chronology of changes in expression of follicular IGFBPs < 40 kDa mRNA and in intrafollicular levels of the corresponding proteins was studied during atresia of large antral follicles. Early atresia of large follicles was associated with a strong decrease in intrafollicular estradiol levels (p < 0.001); an increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001) an increase in both IGFBP-2 (p < 0.001) and -5 (p < 0.01) mRNA expression in granulosa and thecal cells; but no changed in IGFBP-4 mRNA expression. Late atresia of large follicles was associated with a further decrease in intrafollicular estradiol levels (p < 0.01); a further increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001); an increase in IGFBP-4 (p < 0.01) and -5 (p < 0.05) mRNA expression in theca and granulosa, respectively; a decrease in IGFBP-5 mRNA expression in theca (p < 0.05); but no further increase in IGFBP-2 mRNA expression. Overall, these data suggest that the decrease and the increase in expression of mRNA of follicular IGFBPs < 40 kDa during follicular growth and atresia, respectively, are involved in the decrease and the increase in intrafollicular levels of the corresponding proteins. Moreover, the increases in expression of follicular IGFBPs < 40 kDa during atresia of large follicles in hypophysectomized ewes followed a specific time course, the increase in IGFBP-2 and -5 mRNA expression being early than the increase in IGFBP-4 mRNA expression.