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

The objectives were to compare expression of mRNA for cytochrome P450 chole sterol side-chain cleavage (P450scc), cytochrome P450 17 alpha -hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3 beta -hydroxysteroid de hydrogenase Delta (4), Delta (5) isomerase (3 beta -HSD), FSH receptor (FSH r) and LH receptor (LHr) in bovine ovarian follicles of the first and secon d waves of the bovine oestrous cycle and to determine if LH infusion change s growth, steroidogenesis and gene expression in second wave follicles. Tra nsrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n = 31). Saline or puri fied bovine LH was infused intravenously into cows at emergence of follicul ar 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 mug/h; W2LH). During infusion, blood samples were collected at 1 2 min intervals for 8 h via i.v. catheters for measurement of serum LH conc entrations. Ovaries were removed from cows on days 2 or 4 after emergence o f follicular waves. Follicles were frozen and stored at -80 degreesC. Folli cular fluid (FF, 50 mul) was collected for determination of progesterone (P -4) oestradiol-17 beta (E-2) and androstenedione (A(4)) concentrations. Fro zen sections ( 14 mum) were used for in situ hybridization to measure expre ssion of mRNA (% pixel intensity) for P450scc, P450c17, P450arom. 3 beta -H SD. FSHr. and LHr. LH infusion resulted in a serum LH pattern (high frequen cy) similar to the early luteal phase. There were no significant difference s in size of follicles among the three treatment groups. Follicular fluid c oncentrations of E-2 and A(4) in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase incr eased follicular fluid E-2 and A(4) concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increas e in follicular fluid E-2 on day 2 in wave 2 follicles after LH infusion oc curred possibly through an increase in mRNA expression of P450c17 and 3 bet a -HSD. In conclusion, follicular fluid concentrations of E-2 and A4 were l ower in W2S than in W1S and E-2 and A(4) concentrations were restored by in fusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3 b eta -HSD. (C) 2001 Elsevier Science B.V. All rights reserved.