Department of Physiology, Faculty of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain.
Department of Biochemistry and Molecular Biology, Faculty of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain.
PLoS One. 2019 Jul 18;14(7):e0219780. doi: 10.1371/journal.pone.0219780. eCollection 2019.
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.
在一些诱导排卵的物种中，β 神经生长因子（β-NGF）在排卵中具有重要作用，尽管兔子的数据仍不确定。在这项研究中，我们首先从兔子组织中合成了功能性重组 β-NGF（rrβ-NGF），以达到以下目的：1）比较兔子β-NGF 氨基酸序列与其他诱导或自发排卵物种的序列；2）评估 rrβ-NGF 对兔子精子活力和运动的影响，以及 3）研究 rrβ-NGF 添加到兔精液剂量中对体内排卵的诱导作用。通过快速扩增 cDNA 末端和在 GenBank（KX528686）中注释，对兔子前列腺组织中的 NGF 基因进行测序。通过亲和层析从 CHO 细胞中生产重组 rβ-NGF 并进行纯化。通过 Western blot 和质谱（MALDI-TOF）证实重组蛋白的氨基酸序列与β-NGF 相对应后，在 PC12 细胞中进行了为期 8 天的成功剂量反应研究，验证了其功能。与其他物种相比，前列腺兔子 NGF 的氨基酸序列主要在其受体结合位点发生了变化。在所检查的所有自发排卵物种中，与兔子相比，与高亲和力受体相互作用的丙氨酸和脯氨酸残基被丝氨酸取代。在兔子中，低亲和力受体结合位点的天冬酰胺和蛋氨酸被赖氨酸取代。在时间和剂量反应实验中，体外向精液中添加 rrβ-NGF 不会影响精子活力，而添加 1μg/ml 的神经肽可增强精子运动参数。通过阴道途径向精液中添加相同浓度的 rrβ-NGF 可诱导排卵，导致 LH 峰延迟，从而增加血浆孕酮水平、妊娠和分娩。我们的研究结果表明，rrβ-NGF 可能是兔生物技术和辅助生殖技术的有用选择，但需要进一步研究。
