Analysis Of Splicing Variants Of Luteinizing Hormone Receptor In Bovine Leydig Cells

The Luteinizing Hormone Receptor (LHR) is a member of the G-protein coupled receptor superfamily, which plays a key role in maintaining the amount, structure, and function of Leydig cells interaction with the Luteinizing Hormone (LH) and chorionic gonadotropin (CG). Various sizes of splice variants of the LHR mRNA have been found in several species, but their biological function still unknown. In this paper, we identified the LHR splicing variants in bovine testes and corpus luteum, and measured the secretion of testosterone as well as quantified the LHR mRNA expression in the bovine Leydig cells with the stimulation of LH.We detected 9 LHR splicing variants expressed in bovine testes and 6 in bovine corpus luteum, but most of their splicing patterns are diverse. The alternative splicing mechanisms that give rise to the receptor variants may be subject to metabolic regulation or physiological states, leading to modulation of tissue expression of the receptor variants and resulting in potential changes in LH action. Interestingly, we detected a special isoform (F form) that have not been found previously in other species. Isoform F has exon 10 and the first 428 bp of exon 11 missing, while some variants have a deletion of the first 266 bp of exon 11. If those splice variants are translated into proteins, the LHR protein is shorter than the full-length form, not only because an exon or several exons has been spliced out, but also because the splicing is out of frame, producing a premature stop codon and an alternative carboxyl-terminus.Leydig cells (LCs) were isolated from bovine testes by Percoll density gradient centrifugation. With stimulation of LH, the secretion of testosterone was increased along with the concentration of LH increased from 0 to 1×10-4 IU/ml, but declined thereafter to reach a nadir at the concentration of 1×10-3 IU/m as well as the expression of FL, A and B forms, which suggest that the isoforms FL, A and B are the main variants that play a key role in response to LH and the decrease of testosterone secretion may resulted from the decrease of the expression of those variants. The secretion of testosterone was progressively increased and a secondary peak in the concentration of 1×10-1 IU/ml, but the expression of splicing variants of FL, A and B are not increased, which suggest a small proportion of liganded receptors is sufficient to evoke full biological response.We describe the bovine LHR splicing pattern and gene expression in Leydig cells with LH stimulation. These results will pave the way for function study of those splicing variants.