γ-Amino Butyric Acid Control of Arginine Vasopressin Release from the Ewe Hypothalamus In Vitro: Sensitivity to Oestradiol

The present study aims to ascertain the influence of gamma-amino butyric acid (GABA)(A or B) receptors on arginine vasopressin (AVP) release in vitro and determine whether E(2) modulates GABA-AVP interaction. Within 10 min of ewe killing, saggital midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus along with the median eminence, 2-mm thick, two per ewe) were dissected, placed in oxygenated minimum essential media (MEM)-alpha at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without E(2) (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to a specific GABA(A or B) receptor agonist or antagonist at various doses (0.1-10 mm). GABA(A) (muscimol; no E(2), n = 7 perifusion chambers, with E(2), n = 11) or GABA(B) (baclofen; no E(2), n = 8, with E(2), n = 15) agonists (10 mm) did not influence AVP concentrations. However, AVP release increased (p < 0.05) 20-30 min after exposure to 10 mm GABA(A or B) antagonists (bicuculline, no E(2), n = 7: from 4.6 +/- 0.7 to 33.0 +/- 0.4, with E(2), n = 17: from 11.9 +/- 1.4 to 32.8 +/- 6.0; CGP52432, with E(2), n = 14: from 14.0 +/- 2.6 to 28.8 +/- 3.9 pg/ml). At the end of the collection period, hypothalamic slices responded to KCl (100 mm) with AVP efflux (p < 0.05). GABA(B) but not GABA(A) antagonist-stimulated AVP release was enhanced in the presence of E(2). In summary, AVP release is under the inhibitory influence of GABA input with further potentiation by E(2) through GABA(B) receptors in vitro.     

Immunolocalization of Aquaporins 1 and 9 in the Ram Efferent Ducts and Epididymis

Aquaporins (AQPs) are essential membrane protein channels for the transport of water across membranes. Fluid movement in the epididymis is important for modulation of the luminal environment, in which sperm mature and reside. This study was designed to understand the morphology and localization of AQPs in ram efferent ducts (ED) and epididymis. For this purpose, the epididymis of seven animals were removed for histologic and immunohistochemical analyses. AQP1 immunoreactivity was observed in the apex of the ED, and AQP9 was found adjacent to the nuclei of the epithelial cells of the ED. The epithelial lining of ram epididymis is pseudostratified columnar and presents principal, basal, apical and narrow cells. In the initial segment (IS), a moderate reaction for AQP1 was observed in the apical cytoplasm of epithelial cells. An intense reactivity for AQP1 was noted over the microvilli of principal cells and in spermatozoa in the caput. In the corpus and cauda, AQP1 was noted only over the endothelial cells of vascular channels located in intertubular spaces. A weak‐to‐moderate reaction for AQP9 was observed in the nuclei of epithelial cells in the IS, caput and corpus of the epididymis. In the cauda, an intense reaction to AQP9 was observed in the epithelial border. In the IS, caput and corpus, the reactivity for AQP9 differed from those observed in domestic animals. The cauda showed a pattern similar to that previously described. These results indicate that AQPs 1 and 9 have reversed locations and roles in rams, suggesting activity variations related with fluid and solute absorption throughout the epididymis.     

Kisspeptin,c‐Fos and CRFR Type 2 Co‐expression in the Hypothalamus after Insulin‐Induced Hypoglycaemia

Normal reproductive function is dependent upon availability of glucose and insulin‐induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c‐Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin‐treated ewes had an increased expression of c‐Fos. Furthermore, the percentage of kisspeptin cells co‐expressing c‐Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre‐optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.     

A geographical approach to identify phosphorus-efficient genotypes among landraces and wild ancestors of common bean

Cultivars of common bean (Phaseolus vulgaris L.) capable of yielding well at low levels of native or added phosphorus (P) are highly desirable in many tropical production systems. The objective of the present study was to identify geographical regions which might be sources of such genotypes. A total of 364 landraces, cultivars and wild genotypes, drawn from a broad geographic range, were divided on the basis of growth habit into four field trials, each comprising two levels of P, stressed and unstressed, on an infertile Andosol in Popayan, Colombia. The regression relationship between grain yield per plant in the presence and in the absence of stress was determined, and each genotype's deviation from this relationship was used as a measure of P-efficiency. There was highly significant variation in efficiency among genotypes in all growth habits, and in climbing beans there were consistent regional differences, superior genotypes being identified with greater frequency among those from Bolivia, West Mexico and South Mexico-West Guatemala. The latter region was promising for prostrate bush genotypes also. Wild beans in general performed relatively poorly; it appears that P-efficiency traits in P. vulgaris have been acquired during or after domestication. These results confirm that genetic differences in P-efficiency exist among common bean genotypes and suggest that these are related to geographic origin. Furthermore, the use of a representative sample of germplasm can help to identify segments of the gene bank that are especially promising as sources of desirable traits. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variability in cultivated common bean beyond the two major gene pools

It is generally accepted that two major gene pools exist in cultivatedcommon bean (Phaseolus vulgaris L.), a MiddleAmerican and an Andean one. Some evidence, based on unique phaseolin morphotypesand AFLP analysis, suggests that at least one more gene pool exists incultivated common bean. To investigate this hypothesis, 1072 accessions from acommon bean core collection from the primary centres of origin, held at CIAT,were investigated. Various agronomic and morphological attributes (14categorical and 11 quantitative) were measured. Multivariate analyses,consisting of homogeneity analysis and clustering for categorical data,clustering and ordination techniques for quantitative data and nonlinearprincipal component analysis for mixed data, were undertaken. The results ofmost analyses supported the existence of the two major gene pools. However, theanalysis of categorical data of protein types showed an additional minor genepool. The minor gene pool is designated North Andean and includes phaseolintypes CH, S and T; lectin types 312, Pr, B and K; and mostly A5, A6 and A4 types-amylase inhibitor. Analysis of the combined categorical data ofprotein types and some plant categorical data also suggested that some othergermplasm with C type phaseolin are distinguished from the major gene pools.     

Homogeneity of meats prepared for analysis with a commercial food processor: collaborative study

The food processor was evaluated as an alternative to the food chopper and bowl cutter for preparing meat samples for analysis in AOAC procedure 24.001. Samples of 6 meat types--cooked sausage, pork sausage, canned ham, hamburger, water-added ham, and smoked ham--were distributed to 9 laboratories for preparation using a food processor. The resulting 54 samples were sent to a USDA-accredited laboratory for analysis in triplicate for moisture, protein, and fat. Standard deviations and their 95% confidence intervals calculated for the analytical results were compared with USDA Performance Standards. With few exceptions, the upper limits were lower than the Performance Standards and for the exceptions, the intervals included the Performance Standards. By these criteria, the food processor is as effective in preparing homogeneous samples as the preparation procedures used to set the Performance Standards. Collaborators found the processor faster to use and easier to clean than the food chopper. Use of the food processor has received interim approval as an alternative to the food chopper or bowl cutter in AOAC procedure 24.001 for preparing meat samples for analysis.