Differential expression of LHCGR and its isoforms is associated to the variability in superovulation responses of Gir...

The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3 ± 1.2 ova/embryos per flush, n = 5) or poor responders (1.1 ± 0.3 ova/embryos per flush, n = 5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8 mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater^®. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P < 0.01) as well as isoforms S10, S11 and S10 + 11, compared to good responders. In both phenotypes, superovulation down-regulated total LHCGR expression (-0.5 ± 0.2 and -0.9 ± 0.0 for good and poor responders, respectively; P < 0.05). However, in poor responders the exogenous FSH treatment up-regulated the S10 (2.4 ± 2.0; P < 0.05), S10 + 11 (3.8 ± 3.2; P < 0.01), and S1 isoforms (1.8 ± 1.3; P < 0.05), compared to good responders We conclude that down-regulation of total LHCGR, associated to up-regulation of their inactive isoforms, may have compromised follicle development and thus contributed to the low efficiency of superovulation in heifers with a poor responder phenotype.