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Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits

In some induced-ovulating species, beta nerve growth factor (β-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant β-NGF from rabbit tissue (rrβ-NGF) to address the following objectives: 1) to compare rabbit...

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Autores principales: Sanchez-Rodriguez, Ana, Abad, Paloma, Arias-Alvarez, María, Rebollar, Pilar G., Bautista, José M., Lorenzo, Pedro L., García-García, Rosa M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639004/
https://www.ncbi.nlm.nih.gov/pubmed/31318941
http://dx.doi.org/10.1371/journal.pone.0219780
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author Sanchez-Rodriguez, Ana
Abad, Paloma
Arias-Alvarez, María
Rebollar, Pilar G.
Bautista, José M.
Lorenzo, Pedro L.
García-García, Rosa M.
author_facet Sanchez-Rodriguez, Ana
Abad, Paloma
Arias-Alvarez, María
Rebollar, Pilar G.
Bautista, José M.
Lorenzo, Pedro L.
García-García, Rosa M.
author_sort Sanchez-Rodriguez, Ana
collection PubMed
description In some induced-ovulating species, beta nerve growth factor (β-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant β-NGF from rabbit tissue (rrβ-NGF) to address the following objectives: 1) to compare rabbit β-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrβ-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrβ-NGF added to the seminal dose in rabbit does. The NGF gene in rabbit prostate tissue was sequenced by Rapid Amplification of cDNA Ends and annotated in GenBank (KX528686). Recombinant rβ-NGF was produced in CHO cells and purified by affinity chromatography. Once confirmed by Western blotting and mass spectrometry (MALDI-TOF) that the amino acid sequence of the recombinant protein corresponded to β-NGF, its functionality was validated in PC12 cells in a successful dose-response study over 8 days. The amino acid sequence of prostate rabbit NGF differed to that of other species mainly in its receptor binding sites. In all the spontaneous ovulating species examined, compared with rabbit, alanine and proline residues, which interact with the high-affinity receptor, were replaced by a serine. In rabbits, asparagine and methionine were substituted by lysine at the low-affinity receptor binding site. In time- and dose-response experiments, the in vitro addition of rrβ-NGF to the ejaculate did not affect sperm viability whereas sperm motility parameters were enhanced by the addition of 1 μg/mL of the neuropeptide. Addition of this same concentration of rrβ-NGF to the seminal dose administered via the intravaginal route in does induced ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery. Our findings suggest that rrβ-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed.
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spelling pubmed-66390042019-07-25 Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits Sanchez-Rodriguez, Ana Abad, Paloma Arias-Alvarez, María Rebollar, Pilar G. Bautista, José M. Lorenzo, Pedro L. García-García, Rosa M. PLoS One Research Article In some induced-ovulating species, beta nerve growth factor (β-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant β-NGF from rabbit tissue (rrβ-NGF) to address the following objectives: 1) to compare rabbit β-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrβ-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrβ-NGF added to the seminal dose in rabbit does. The NGF gene in rabbit prostate tissue was sequenced by Rapid Amplification of cDNA Ends and annotated in GenBank (KX528686). Recombinant rβ-NGF was produced in CHO cells and purified by affinity chromatography. Once confirmed by Western blotting and mass spectrometry (MALDI-TOF) that the amino acid sequence of the recombinant protein corresponded to β-NGF, its functionality was validated in PC12 cells in a successful dose-response study over 8 days. The amino acid sequence of prostate rabbit NGF differed to that of other species mainly in its receptor binding sites. In all the spontaneous ovulating species examined, compared with rabbit, alanine and proline residues, which interact with the high-affinity receptor, were replaced by a serine. In rabbits, asparagine and methionine were substituted by lysine at the low-affinity receptor binding site. In time- and dose-response experiments, the in vitro addition of rrβ-NGF to the ejaculate did not affect sperm viability whereas sperm motility parameters were enhanced by the addition of 1 μg/mL of the neuropeptide. Addition of this same concentration of rrβ-NGF to the seminal dose administered via the intravaginal route in does induced ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery. Our findings suggest that rrβ-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed. Public Library of Science 2019-07-18 /pmc/articles/PMC6639004/ /pubmed/31318941 http://dx.doi.org/10.1371/journal.pone.0219780 Text en © 2019 Sanchez-Rodriguez et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Sanchez-Rodriguez, Ana
Abad, Paloma
Arias-Alvarez, María
Rebollar, Pilar G.
Bautista, José M.
Lorenzo, Pedro L.
García-García, Rosa M.
Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits
title Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits
title_full Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits
title_fullStr Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits
title_full_unstemmed Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits
title_short Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits
title_sort recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639004/
https://www.ncbi.nlm.nih.gov/pubmed/31318941
http://dx.doi.org/10.1371/journal.pone.0219780
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