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Expression of reproductive genes during fetal
development in cattle
J. H. Hernandez-Medranoa, K. Dereckaa, A. Petersa, B. K. Campbellb, aSchool of Biosciences, The University of Nottingham, Loughborough, LE12 5RD, UK; bDivision of Human Development, The University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH, UK In humans and rodents, a-gonadotrophin (a-SU) is the earliest subunit expressed in the fetal pituitary gland1, while in sheep fetuses, pituitary mRNA for oestradiol receptors (ERa and ERb) was reported at 80 d of gestation2. Conversely, in cattle information regarding expression of reproductive genes is scarce. The aim of the present study was to describe gene expression of gonadotrophin subunits (a-SU, LHb and FSHb), GnRH receptor (GnRHR), ER-a, ER-b and inhibin/activin subunits (a, bA and bB) in the fetal pituitary gland at different stages of development in cattle using a multiplexed PCR system, which allowed evaluation of up to 30 genes in a single PCR reaction.
Bovine fetal pituitary glands were dissected, frozen in liquid nitrogen and gestational age (GA) estimated using crown-rump length measurements. Fetuses were grouped into first (1st, <90 d; n=4); early (91 to 150 d; n=6) and late (151 to 180 d; n=3) second (2nd); and third (3rd; >210 d; n=3) trimesters of gestation. Pituitary gland RNA was extracted and DNase treated before multigene expression assays were carried out (GenomeLab GeXP Genetic Analysis System; Beckman-Coulter Inc.). Gene specific primers (F and R) attached to common universal tag sequences were designed and individually evaluated using a positive RNA control (adult pituitary RNA), before fetal samples were analysed. Controls included reactions in which template RNA and reverse transcriptase were not added and an internal quality control of the system (Kanamycin resistance RNA). Individual gene expression was normalised using three housekeeping genes (Actin B, GAPDH and Histone 2A) and resulting values were compared using one-way ANOVA.
Expression of a-SU and FSHb subunits was observed at 65 d and LHb at 75 d of GA. Expres- sion of all gonadotrophin subunits increased from 1st to 2nd trimester (P<0.05). Despite an increase in LHb and a-SU gene expression during 3rd trimester (P<0.05), FSHb expression did not change (P>0.05). At 65 d of GA, expression of GnRHR and ER-b was observed, while ER-a expression occurred during the early 2nd trimester (GA=91 to 150 d). GnRHR expression tended (P=0.063) to increase with age, whereas expression of ERs did not change (P>0.05) throughout gestation. Inhibin/activin subunit gene expression was detected during the 1st trimester (GA<90d), and did not change during pregnancy (P>0.05).
In conclusion, the reproductive genes studied were expressed in fetal pituitary glands between 65 and 150 d of gestation. A differential pattern of gene expression according to age was observed, with some genes showing higher expression with increasing GA (i.e., a-gonadotrophin and LHb), while others did not change throughout pregnancy. Whether this differential pattern of expression is linked to developmental changes observed during fetal life requires further research. Study funded by The University of Nottingham/Pfizer and CONACYT-Mexico. 1Simmons et al., 1990. Genes and Development 4:695-711.
2Shaub et al., 2008. Gene Expression Patterns 8:457-463.
Characteristics of bovine follicular growth and follicular
function under low plasma progesterone concentrations

induced by using the “corpus luteum absent” model
M. Matsuia, K. Yamanea, H. Leea, S. Hanedaa, A. Miyamotob, and Y-I. MiyakeaaDepartment of Applied Veterinary Medicine; bGraduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan Failure of a positive feedback response to estradiol (E ) may cause follicular cysts in lactating dairy cows1. Progesterone (P ) exposure of the hypothalamus may prevent cyst formation by maintaining this postive E feedback mechanism2. We recently demonstrated that repeated follicular aspirations could prevent CL formation, maintain low plasma P concentrations and induce a persistent follicle (CL absent model). Follicular growth may be altered under low plasma P concentrations. The ef- fects of continuous low plasma P concentrations on the growth and function of follicles destined to become persistent were examined using the CL absent model in the present study. Eleven cows received PGF at 10 d after spontaneous ovulation. At 36 h after PGF injection, the follicular fluid (FF) of all follicles with a diameter > 6 mm was aspirated using an ultrasound-guided technique. Follicular aspirations were repeated at 3, 6 and 9 d after first aspiration. Five cows showed no CL formation and maintained low plasma P concentrations continuously from the time of first follicle aspiration. Measurement of diameter and collection of FF from the largest follicle were performed at 5 (d5NCL), 7 (d7NCL) or 9 d (d9NCL) after the last aspiration (CL absent model). The day of FF collection was set up as d 0 again and then the measurement of follicle diameter and collection of FF were repeated. In the control group, the dominant follicle at 6 d after ovulation was aspirated to induce new follicular recruitment. The diameter of the largest follicle was examined and its FF was collected at 5 (d5C), 7 (d7C) or 9 d (d9C) after the start of follicle recruitment in the control group. Concentration of E , total insulin-like growth factor-I (IGF-I) and free IGF-I in FF were analyzed. Blood samples for hormonal analysis (E and total IGF-I) were also collected at the same The diameter of d9NCL was greater than that of d9C. Concentration of E in plasma and FF were greater in d5NCL, d7NCL and d9NCL compared with d5C, d7C and d9C, respectively. Total IGF-I concentrations in FF of CL absent model were greater than that of control. In the CL absent model, free IGF-I concentrations in FF tended to be greater compared with the control group. Free IGF-I in FF of d9NCL was greater than that of d9C. Follicular growth and E synthesis within the dominant follicle were stimulated during follicular growth in the CL absent model. Increased LH pulse frequency under continuous low plasma P levels induced by the CL absent model may alter follicular growth and function and then induce a persistent follicle.
1Dobson and Alam, 1987. J Endocrinol 113:167-171.
2Gümen and Wiltbank, 2002. Biol Reprod 66:1689-1695.



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