237 EXPRESSION OF THE FULL-LENGTH AND ALTERNATIVELY SPLICED BOVINE LH RECEPTOR mRNAs IN GRANULOSA CELLS FROM FOLLICLES ≥ 7 mm IN DIAMETER

Abstract

The objective of this study was to characterize the pattern of gene expression of LH receptor (LHR) transcripts from bovine antral follicles from 5 to 14 mm in diameter (theca and granulosa cells, TC and GC, respectively) in crossbreed cattle and from cultured GC. From ovaries collected in abattoir (Bos indicus × B. taurus cattle), antral follicles were dissected, and samples of TC and GC were obtained for total RNA extraction (Trizol protocol). Steroid concentrations in the follicular fluid were determined by RIA. Samples of GC cultured for 6 days (obtained from B. taurus follicles), were treated with FSH (0 (control), 1, or 10 ng) and processed for total RNA extraction. Total RNA (1 µg) was utilized in the RT reaction (SuperScript III; Invitrogen, Brasil Ltd., São Paulo, Brazil). The PCR conditions were 29 cycles of 95°C, 60°C, and 70°C (1 min each) for denaturation, annealing, and extension (PTC200 thermocycler; MJ Research, Biozym, Landgraaf, The Netherlands). Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify the fragment from exon 9 to exon 11 (LHRBC; full-length amplicon with 1240 bp). To investigate a putative site of alternative splicing on exon 3 specific primers were utilized to amplify a fragment containing the exons 2 to 9 (LHRA). As an internal control of the PCR, GAPDH expression was used. After sequencing, four isoforms were detected from the LHRBC fragment two with deletion of exon 10 (M2 and M4) and two with deletion of part of exon 11 (M3 and M4) as well as the fragment without deletions (M1). There was no correlation (P > 0.05; Spearman correlation) between LHRBC isoform expression and steroid hormones or follicular diameter (TC). However, estradiol and progesterone concentrations (r > 0.51 and P < 0.01) and follicular diameter (r > 0.82 and P < 0.01) were correlated with expression of the four LHRBC isoforms (GC). Expression of isoforms from fragment LHRBC was observed in GC from follicles with a diameter of 7 mm. In six follicles with a diameter of 7 mm, only one (16.7%) expressed LHR in GC, whereas most of the follicles e 8 mm expressed LHR (87.5%, 21/24). No LHR isoforms were detected in GC from follicles (n = 7) d 6 mm in diameter. From LHRA fragment amplification, an alternative transcript with a deletion of 75 bp, homologous to the rat exon 3, was detected by sequencing. In cultured GC (without FSH treatment), only a weak LHR expression (in vitro control) was observed when compared with the in vivo control (TC sample). The treatment with FSH (1 or 10 ng) was effective to induce LHR expression in cultured GC, however, with a different pattern of expression (lower M1/M2 ratio) when compared to in vivo GC samples (0.8 ± 0.14 vs. 3.5 ± 0.66; mean ± SEM, P < 0.01, unpaired t-test). It is concluded that, in GC from follicles with a diameter e 7 mm, the gene expression of the LHR was positively correlated with follicular diameter and with estradiol and progesterone concentration in the follicular fluid. Treatment with FSH was effective in inducing LHR expression in cultured GC, however, with a different pattern than in the in vivo control. Additionally, sites of alternative splicing were detected in exons 3, 10, and 11 from the bovine LHR gene.

This work was supported by a Fellowship from FAPESP.