RESUMO
OBJECTIVES: The aim of the present study was to find a possible polymorphism in the promoter region of the osteopontin (OPN) gene, as a potential mutation region, connected with the transcription factor-binding sites or regulatory sequences and to estimate the expression of this gene in ovaries and oviduct of sows. MATERIAL AND METHODS: Sixty wbp x pbz sows after the first mating were slaughtered and tissues samples of the ovaries and oviduct were taken. Primer pairs for PCR analysis were designed on the basis of swine osteopontin 5' end sequence driven from GenBank. This study's amplified DNA fragment, spanning 274 bp of the promoter region, was chosen because of its contents of tree specific sites: type II collagen silence sequence (CACCTCC) at -682 (from the transcription initiation site), glucocortIcoid response site (TGTCCT) at -658 and CAAT box -592. This DNA fragment was subjected to a MSSCP analysis. RESULTS: A different MSSCP pattern was shown which indicates that mutation is located in this region. The samples of different conformers were sequenced and the A --> G transitions was identified in two positions -617 and -608. Restriction analysis of the DNA was performed, but unfortunately none of the known restriction enzymes recognized the novel SNPs, which is why the specific primer pairs characteristic for nucleotide A or for nucleotide G were chosen. In the second stage of the presented study the total RNA was extracted from the tissues of the ovaries and oviduct and the complementary DNA (cDNA) was synthesized. CONCLUSION: The real-time PCR analysis to determined the expression dynamics of the OPN gene in pig tissues was performed.
Assuntos
Osteopontina/genética , Polimorfismo de Fragmento de Restrição , Reprodução/genética , Sus scrofa/genética , Animais , Sequência de Bases , Feminino , Dados de Sequência Molecular , Ovário/fisiologia , Oviductos/fisiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The present studies were undertaken to examine the effect of copper and nickel salts and their complexes with GnRH on LH release from the pig anterior pituitary cells in vitro. The potency of Cu-GnRH and Ni-GnRH binding to GnRH receptors with iodinated GnRH as a radioactive tracer was also verified. The incubation of pig pituitary cells with Cu and Ni acetate salts showed no effect of the studied ions on LH release at any concentration used. However, nickel salt at a lower dose (10(-10) and 10(-9) M) tended to decrease LH output. By contrast, the native GnRH as well as its metal complexes significantly stimulated LH release after three hours of treatment and Cu-GnRH was found to be the most effective. The results showed that Cu and Ni complexes with GnRH but not their acetate salts are effective in LH release from pig pituitary cells collected from adult female pigs.
Assuntos
Cobre/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Luteinizante/metabolismo , Níquel/farmacologia , Adeno-Hipófise/metabolismo , Acetatos/farmacologia , Fatores Etários , Animais , Ligação Competitiva , Cátions/metabolismo , Cátions/farmacologia , Células Cultivadas , Cobre/metabolismo , Relação Dose-Resposta a Droga , Feminino , Técnicas In Vitro , Níquel/metabolismo , Adeno-Hipófise/citologia , Sus scrofaRESUMO
Copper (Cu) is one of the essential trace metals which are necessary in maintaining the functioning of living organisms. The current knowledge on the role of copper in animal reproduction is presented in the article. Our studies have shown that complexes of copper (Cu²+) with gonadotropin-releasing hormone (GnRH) are even more effective in the release of LH than native GnRH. Moreover, Cu-GnRH is more potent in inducing in vivo release of FSH than LH. Copper complexes with GnRH interact with GnRH receptors (GnRHR) and modulate intracellular signaling in the gonadotrope cells of the anterior pituitary. Copper plays also a significant role in maintaining normal fetus development in mammals.