Concentrations of steroids and expression of messenger RNA for steroidogenic enzymes and gonadotropin receptors in bovine ovarian follicles of first and second waves and changes in second wave follicles after pulsatile LH infusion

Abstract

The objectives were to compare expression of mRNA for cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase Delta(4), Delta(5) isomerase (3beta-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or 4 days using a computer-controlled syringe pump (n=5-6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25 microg/h; W2LH). During infusion, blood samples were collected at 12min intervals for 8h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or 4 after emergence of follicular waves. Follicles were frozen and stored at -80 degrees C. Follicular fluid (FF, 50 microl) was collected for determination of progesterone (P4), oestradiol-17beta (E2) and androstenedione (A4) concentrations. Frozen sections (14 microm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P450scc, P450c17, P450arom, 3beta-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E2 and A4 in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E2 and A4 concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P450c17 and 3beta-HSD. In conclusion, follicular fluid concentrations of E2 and A4 were lower in W2S than in W1S and E2 and A4 concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3beta-HSD.