mRNA Expression Of Related Genes Research Articles

Effects of Daidzein on mRNA Expression of Gonadotropin Receptors and P450 Aromatase in Ovarian Follicles of White Silky Fowls

Effects of daidzein on expression of mRNAs of gonadotropin receptors (FSHR, LHR) and P450 aromatase (P450arom) were evaluated in ovarian follicles of white silky fowls. The hens were 13 months old in the post-peak period of egg laying and were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at 10 mg/kg for 7 consecutive weeks. The mRNA expression of related genes was measured by semi-quantitative RT-PCR in the granulosa layers of the preovulatory follicle (PRF: Fl, F2 ...) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF) and atretic follicle (ATF). Results showed that daidzein supplementation significantly increased the number of SYF and LWF (p<0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F3 to Fl, but LHR mRNA displayed opposite developmental changes. P450arom mRNA was highest in the SYF, but was very low in the granulosa layers after follicles finished selection. Treatment with daidzein resulted in increased mRNA expression of FSHR in F3 granulosa layer, LHR in granulosa layers of F3 to Fl and P450arom in LWF (p<0.05). These results indicated that dietary supplementation of daidzein up-regulated mRNA expression of gonadotropin receptors and P450arom to improve the development of preovulatory follicles in white silky fowls after the peak-laying period.

Chronic metabolic acidosis stimulated transcellular and solvent drag-induced calcium transport in the duodenum of female rats

Chronic metabolic acidosis results in a negative calcium balance as a result of bone resorption and renal calcium loss. However, reports on the changes in intestinal calcium transport have been controversial. The present investigation therefore aimed to study the effects of chronic metabolic acidosis induced by 1.5% NH(4)Cl administration on the three components of duodenal calcium transport, namely, solvent drag-induced, transcellular active, and passive paracellular components, in rats using an in vitro Ussing chamber technique. The relative mRNA expression of genes related to duodenal calcium transport was also determined. We found that 21-day chronic metabolic acidosis stimulated solvent drag-induced and transcellular active duodenal calcium transport but not passive paracellular calcium transport. Our results further demonstrated that an acute direct exposure to serosal acidic pH, in contrast, decreased solvent drag-induced calcium transport in a pH-dependent fashion but had no effect on transcellular active calcium transport. Neither the transepithelial resistance nor duodenal permeability to Na(+), Cl(-), and Ca(2+) via the passive paracellular pathway were altered by chronic metabolic acidosis, suggesting that widening of the tight junction and changes in the charge-selective property of the tight junction did not occur. Thus the enhanced duodenal calcium transport observed in chronic metabolic acidosis could have resulted from a long-term adaptation, possibly at the molecular level. RT-PCR study revealed that chronic metabolic acidosis significantly increased the relative mRNA expression of duodenal genes associated with solvent drag-induced transport, i.e., the beta(1)-subunit of Na(+)-K(+)-ATPase, zonula occludens-1, occludin, and claudin-3, and with transcellular active transport, i.e., transient receptor potential vanilloid family Ca(2+) channels 5 and 6 and plasma membrane Ca(2+)-ATPase isoform 1b. Total plasma calcium and free ionized calcium and magnesium concentrations were also increased, whereas serum parathyroid hormone and 1alpha,25-dihydroxyvitamin D(3) levels were not changed. The results indicated that 21-day chronic metabolic acidosis affected the calcium metabolism in rats partly through enhancing the mRNA expression of crucial duodenal genes involved in calcium absorption, thereby stimulating solvent drag-induced and transcellular active calcium transport in the duodenum.

Retinoid-dependent mRNA expression and poly-(A) contents in bovine oocytes meiotically arrested and/or matured in vitro.

The presence of retinoic acid (RA) during in vitro maturation (IVM) improves bovine oocyte quality and developmental potential. In this work, we investigated the underlying molecular mechanisms. Cumulus-oocyte complexes were meiotically arrested by roscovitine and/or matured in defined medium containing RA, 1% ethanol (vehicle), or no additives. Cumulus-free oocytes were analyzed for poly-(A) mRNA contents and relative mRNA expression of genes involved in cell cycle regulation (cyclin B1 and H1) and antioxidative defence (Mn-superoxide dismutase and glucose-6-phosphate dehydrogenase). Poly-(A) mRNA increased after meiotic inhibition and decreased with IVM completion, both in meiotically arrested and permissively matured oocytes, i.e., matured without previous meiotic arrest. RA dramatically increased poly-(A) mRNA in meiotically arrested oocytes, but more than half of the poly-(A) mRNA disappeared during maturation. Irrespective of oocyte origin, transcripts were detected for all the genes analyzed. IVM, with or without previous meiotic inhibition, increased expression of cyclin B1 and glucose-6-phosphate dehydrogenase, and decreased cyclin H1 and Mn-superoxide dismutase. Except for a decreasing of Mn-superoxide dismutase in meiotically arrested and matured oocytes, RA did not affect mRNA expression. Ethanol led to an abnormal poly-(A) mRNA profile and expression of all the genes analyzed. RA does not modify expression of cyclin B1 and HI genes in the bovine oocyte, and probably does not generate oxidative stress. In addition, RA enhanced mRNA amount as measured by poly-(A) mRNA contents.

235RETINOID-DEPENDENT MRNA EXPRESSION IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

As a transcription factor, retinoic acid (RA) can activate or silence a wide number of genes, thus inducing differentiation in cell systems and playing a role in cell cycle regulation. However, little is known of RA-dependent gene expression in the oocyte. Bovine oocytes and cumulus cells express most RA receptors, and the presence of 9-cis-RA during in vitro maturation (IVM) is beneficial to oocyte development (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). The present work analyzes the relative abundance of various developmentally important gene transcripts in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were manipulated in defined medium with polyvinyl-alcohol (DM-PVA). Those COCs undergoing prematuration were cultured for 24h in DM-PVA with 25μM roscovitine. For IVM, some prematured COCs were cultured for 24h in DM-PVA containing pFSH, LH and E2. Incubations were made at 39°C in an atmosphere of 5% CO2 in air and high humidity. Within experiments, COCs were cultured with nM 9-cis-RA 5, in 1% ethanol (both as vehicle and inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Using Real Time PCR (10 oocytes per group) (Rizos et al., 2003 Biol. Reprod. 68, 236) we examined the relative mRNA expression of genes involved in protection against free oxygen radicals (Mn-superoxide dismutase, MnSOD), glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH) and cell cycle events (Cyclin B1 and H1). Data (of 4 replicates) were analyzed by ANOVA and Duncan test (P&amp;lt;0.05). Regarding immature oocytes, prematuration in 1% ethanol increased cyclin B1 expression and decreased cyclin H1, while 9-cis-RA increased G6PDH. Maturation without additives increased cyclin B1 and G6PDH, but decreased cyclin H1 and MnSOD expression;; opposite trends were observed under increasing ethanol dosages (3% and 5%). Maturation with 1% ethanol or 9-cis-RA enhanced cyclin B1 and G6PDH, while reducing cyclin H1 and MnSOD expressions. The presence of 9-cis-RA during both prematuration and maturation processes tended to show more prominent effects than the ones observed when it was present only during prematuration or maturation alone. In our study, in presence of 9-cis-RA during both prematuration and maturation processes, the expression of cyclin B1 and G6PDH tended to increase, while cyclin H1 and MnSOD tended to decrease. However, the differences with the control group without additives were not significant. Our study during both prematuration and maturation processes show that beneficial effects of RA on oocyte developmental competence may not be related to the alteration of mRNA expression of the four genes analyzed. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175; 2003-05783).