171 EXPRESSION OF ANDROGEN-PRODUCING ENZYMES IN NELORE AND ANGUS HEIFERS WITH LOW AND HIGH NUMBERS OF FOLLICLES IN THE OVARY

Abstract

During follicle development, androgens are synthesised by thecal cells under LH influence through the enzymatic conversion of the androgen precursors. In cattle, androgen concentration is positively correlated with the number of antral follicles. This experiment was designed to assess mRNA levels of androgen-producing enzymes (CYP11A1, CYP17A1, HSD3B, HSD17B) in Nelore and Angus heifers with high (HFC) and low follicle counts (LFC). Eighteen Nelore and 22 Angus heifers (≈24 months old) were kept on Brachiaria brizantha grass supplemented with a mix of grains. To determine the number of follicles, heifers were scanned with an ultrasound device (Mindray Vet DPS 2200, São Paulo, Brazil) with a 7.5-MHz probe 1 day after ovulation for 3 consecutive oestrous cycles. The first and third (before slaughter) oestrous cycles were synchronized with 2 doses of PGF_2α 11 days apart. Animals were slaughtered ≈24 h after ovulation of the third cycle; 3 follicles from 2 to 4 mm in diameter were dissected from the ovary contralateral to the CL. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Gene expression was evaluated using oligo-dT reverse transcription, Sybr Green Master Mix, and Step One Plus (AB, Foster City, CA, USA) Real-Time PCR System. Samples were analysed in duplicates, and CT values were normalized to the housekeeping gene PPIA using the ΔCT method. The ΔCT values were compared using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with follicle as the repeated measure and cow as the subject. We tested the effects of breed, follicle number, and millimeters on gene expression. Individual differences between groups (HFC or LFC) within breed were analysed using the Diff function (SAS 9.2). Results were significant when P < 0.05. Follicle count was higher (P < 0.04) in Nelore 32 ± 3.1 (LFC = 16; HFC = 52) when compared with Angus 20 ± 2.6 (LFC = 10; HFC = 27) heifers. Follicle diameter did not differ between breeds or groups. Expression of CYP11A1 mRNA was higher in Angus heifers (P < 0.01). However, there was no difference in CYP11A1 mRNA expression within groups in Angus heifers, whereas HFC Nelore heifers expressed higher levels of CYP11A1 mRNA than LFC Nelore heifers (P < 0.04). Expression of CYP17A1 was not different between breeds or groups within each breed. Expression of HSD3B mRNA was higher (P < 0.04) in Nelore heifers (P < 0.004) when compared with follicles derived from Angus heifers but not different between HFC and LFC groups within each breed. Expression of HSD17B was higher in Nelore heifers (P < 0.001) but not different between HFC and LFC groups within each breed. In conclusion, Nelore heifers had a higher expression of hydroxysteroid dehydrogenases (HSD3B and HSD17B). As androgens have been demonstrated to stimulate early follicle development (Yang and Fortune 2006 Biol. Reprod. 75, 924–932), these data are indicative that androgens may be involved in the mechanisms determining different follicle counts across cattle subspecies but not between groups within each breed.

Funding and scholarship for Loureiro, Ereno, Favoureto, and Pupulim was from FAPESP.