123 EVALUATION OF LUTEINIZING HORMONE RECEPTOR (LHR) OVER THE ESTROUS CYCLE IN CANINE OVARIES

Abstract

Luteinizing hormone (LH) plays a major role in reproductive processes. This hormone exerts its effects through binding to its receptor LHR, which is one of the 7 transmembrane domain G-protein-coupled receptor superfamilies. Compared with other mammals, an early acquisition of LH receptors on granulosa cells has been reported in dogs around the period of LH surge, during the proestrous-oestrous transition. However, there are no available data with respect to the follicular stage at which LHR starts to become expressed. Hence, the aim of this study was to determine the LHR pattern in the canine ovarian follicles at different stages of development. The ovaries were obtained from 1- to 7-year-old bitches at proestrus/oestrus, anestrus, and diestrus stages following ovariohysterectomy. Follicular cells were mechanically recovered from follicles (n = 2,043) distributed into 4 types (preantral, small antral, medium antral, and large antral). Intrafollicular amounts of LHR were assessed by Western blot method (with the goat polyclonal anti-LHR antibody SC-26341 at 1:100 dilution) and results were evaluated by a 2-way ANOVA (follicle type, stage of oestrous cycle). The LHR was detected in dog follicles in all reproductive phases with patterns varying with stage of follicular development over the reproductive cycle. The antibody against human LHR, which had previously been proven to cross-react with canine LHR, revealed 2 bands at ~90 and ~65 kDa, probably representing the matured protein and its precursor, respectively. Both bands LHR appeared already at preantral follicles increasing (P < 0.05) with growth. Densitometric analysis of each immunoreactive band showed that LHR level changed among the different development stages and phases of reproductive cycle. These bands appeared to be specific for LH, as the secondary antibody alone did not produce cross-reactivity. During proestrus/oestrus, follicular cells expressed mostly the precursor form, increasing significantly with an increase in follicular diameter. The mature form showed a lower (P < 0.05) intensity band, which increased from the preantral to preovulatory stage. At diestrus, the relative abundance of these bands was different between the precursors and mature forms, with a higher expression of precursor form in all follicle stages and rising (P < 0.05) immunoreactivity from the preantral to medium antral stage. The mature form also exhibited LHR variation among follicle sizes (P < 0.05). At anestrous, both bands were expressed with increasing (P < 0.05) levels from preantral to antral follicle stages. A high proportion of LHR was presented as immature forms in all follicles stages during different phases of the oestrous cycle. In conclusion, the LHR is differentially expressed in dogs over the oestrous cycle, increasing during development, and the precursor protein is the most predominant LHR form present in canine follicles.

This study was funded by Grant FONDECYT 1140658.