Each PCR cycle consisted of 45-sec denaturing at 94°C, 45-sec annealing at 60°C, and 2-min extension at 72°C with the final extension for 10 min. Resultant PCR products were cloned using pCR-Script Amp SK(+) Cloning Kit (Stratagene Cloning Systems) and sequenced to verify the authenticity of the products. The 18S rRNA was synthesized from cDNA containing the 80-bp fragment of a highly conserved region of human 18S rRNA (pT7 RNA 18S; Ambion, Austin, TX). RT-PCR-generated cDNAs were linearized with either £coRI or Nod. The RNA probes were labeled with [32P]UTP (400 Ci/mmol; Amersham International, Aylesbury, UK) using either T3 or T7 RNA polymerase and reagents supplied by Promega (Madison, WI). Resultant RNA probes protect 620-, 412-, 772-, and 254-nt mRNAs for 11(3HSD1, 11(3HSD2, GR, and MR, respectively. The specific activities of these probes were 6.7 X 108 cpm/|xg for 110HSD1, 110HSD2, GR, and MR and 4.4 X 103 cpm/^g for 18S rRNA.
Category: Ovary - Part 2
Ovaries were homogenized in ice-cold 4 M guanidium thiocyanate solution containing 25 mM sodium citrate, 0.5% (w:v) sarcosyl, and 0.1 M (3-mercaptoethanol (all from Sigma). Total RNA was extracted with phenol-chlo-roform. buy yasmin online
The 11(3HSD1, 11(3HSD2, GR, and MR RNA probes were synthesized from DNAs generated by reverse tran-scription-polymerase chain reaction (RT-PCR). Oligonucleotide primer pairs of 23-26 nucleotides were obtained from Cruachem Ltd. (Glasgow, UK). The lengths of resultant DNAs were as follows: 11pHSD1, 620 base pairs (bp) (nucleotides [nt] 109-728, GenBank accession no. J05107); 11(3HSD2, 412 bp (nt 534-945, U22424); GR, 772 bp (nt 1383-2154, M14053); MR, 331 bp (nt 3206-3536, M36074).
Animals and Treatments
Immature female Wistar rats (21-25 days old; Charles River UK, Margate, UK) were kept in a temperature-controlled room on a 12L:12D cycle and fed rat chow and water ad libitum. Animals were assigned to three groups and treated (subcutaneous injection in PBS vehicle) as follows before being killed: 1) eCG (Sigma Chemical Co.. Poole, UK; 10 IU for 48 h: group P); 2) eCG (10 IU for 48 h) and hCG (Sigma; 10 IU for 12 h after eCG treatment: group H); 3) controls (no treatment: group C). To examine expression of 11pHSD mRNAs during luteinization, animals were treated in the same manner as group H and killed at 0, 3, 6, 9, 12, 24, and 120 h after the hCG injection. Ovaries were removed and kept on ice in PBS. They were cleaned under a dissection microscope then deep frozen in liquid nitrogen. All animal handling and treatments were performed according to the guidance issued by the Home Office, UK. buy ventolin inhalers
In glucocorticoid (G) and mineralocorticoid (M) target organs, access of these steroids to their receptors (Gr and MR) is regulated by two 11p-hydroxysteroid dehydrogenase (11pHSD) isoforms: NADP+-dependent bidirectional 11pHSD type 1 (11pHSD1) with predominant reductase activity and low binding affinity for cortisol or corticosterone, and NAD+-dependent dehydrogenase type 2 (11pHSD2) with high binding affinity for cortisol or corticosterone. In G target organs such as the liver, 11pHSD1 converts cortisone to cortisol (human) and 11-dehydrocorticosterone to corticosterone (rodents), and ensures that GR is appropriately stimulated by active G. In M target organs such as the kidney and colon, 11 (3HSD2 catalyzes active G to inactive forms, thereby protecting nonselective MR from inappropriate stimulation by active G.