Recombinant production of rabbit β-Nerve Growth Factor and its biological effect on rabbit sperm

Abstract

The neurotrophin {beta}-Nerve Growth Factor ({beta}-NGF) is flourishing as a protein with important roles in the ovulation induction process in induced-ovulation species but data in rabbits are still inconclusive, probably due to the species-specificity effect of the neurotrophin to trigger the ovulation. Moreover, {beta}-NGF seems to have a role in sperm function. To clarify these functionalities we aimed, in the present research: 1) to newly synthesize a functional recombinant {beta}-NGF from rabbit (rr{beta}-NGF), 2) to reveal differences in the amino acid sequence of rabbit {beta}-NGF compared to other sequences of induced and spontaneous ovulator species, and 3) to assess the effects of rr{beta}-NGF on sperm viability and motility. The nucleotide sequence of NGF from rabbit prostate was sequenced by Rapid Amplification of cDNA Ends (RACE) and annotated in GenBank (KX528686). Then, rr{beta}-NGF was produced in CHO cells and purified by affinity chromatography. Western blot and MALDI-TOF analyses confirmed the correct identity of the recombinant protein. rr{beta}-NGF functionality was validated in PC12 cells through a successful dose-response effect along 8 days. The comparison of the amino acid sequences of NGF between rabbit and other species suggested some relevant substitutions at its binding site to both the high-(TrkA) and the low-(p75) affinity receptors. The addition of rr{beta}-NGF in rabbit sperm, in a time- and dose-response study, did not affect its viability but slightly changed some of its motility parameters at the highest concentration used (100 ng/ml). Thus, it can be considered that this new recombinant protein may be used for biotechnological and reproduction assisted techniques in ovulation-induced species.