Category: Ovary - Part 2

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.

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MATERIALS AND METHODS(2)

RNA Preparation

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

32P-Labeled Probes

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).

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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

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Rat Ovary

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.

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