Characterization of the canine 3β-hydroxysteroid dehydrogenase and its expression in the corpus luteum during diestrus

3 beta-Hydroxysteroid dehydrogenase (3 beta HSD) is a key enzyme in the synthesis of bioactive steroid hormones. Objectives of the present study were to clone canine 3 beta HSD and to investigate its expression in dog corpora lutea (CL) covering the periods of their formation, early and late regression (days 5, 15, 25, 35, 45, 65 after ovulation). Complete complementary DNA sequence was amplified by RACE PCR. Subsequent cloning revealed that the canine ovarian 3 beta HSD transcript was composed of a 5'-untranslated region (5'-UTR) of 126 nucleotides, an open reading frame (ORF) of 1122 nucleotides and a 3'-UTR of 441 nucleotides. The putative ORF encoded a 374 amino acid protein which remains highly conserved (79-85% identity) between species. The transient expression of the cloned canine 3 beta HSD in a mammalian heterologous cell expression system (HEK293T cells) identified the 3 beta HSD activity as the only activity of this canine enzyme (absence of any detectable 17-hydroxysteroid dehydrogenase activity).

Qualitative RT-PCR revealed expression of 3 beta HSD on all days investigated and the signals were strongest on days 5 and 15, with day 25 intensity tending to decrease. However, variability between individual animals was high. The significant decrease in the expression of 3 beta HSD towards the end of diestrus as indicated by Real Time PCR (p <0.01) and immunhistochemistry may indicate that the provision of progesterone is controlled by availability of the enzyme rather than the substrate. (c) 2006 Elsevier Ltd. All rights reserved.