Elsevier

Journal of Proteomics

Volume 204, 30 July 2019, 103401
Journal of Proteomics

iTRAQ-based quantitative proteomic analysis provides insights into strong broodiness in Muscovy duck (Cairina moschata) combined with metabolomics analysis

https://doi.org/10.1016/j.jprot.2019.103401Get rights and content

Highlights

  • APOV1, GAL, SAA, GNB5, VLDLR and CDK1 are interesting proteins related to reproductive performance.

  • The steroid biosynthesis and steroid hormone biosynthesis are important pathways associate with broodiness of birds.

  • 7-dehydrodesmosterol, 25-Hydroxyvitamin D3, 7-Dehydrocholesterol, Pregnanolone, Allopregnanolone and estrogen are important biomarkers.

  • Proteomics and metabolomics analysis found that upstream key enzymes effected the production of downstream metabolites.

Abstract

Much attention has been paid to the broodiness of the Muscovy duck, but the molecular mechanism of broodiness remains largely unknown. In this study, the ovary tissues of Muscovy ducks during the broody and laying periods were used to investigate differentially expressed proteins (DEPs) by the iTRAQ-based proteomics approach. A total of 335 DEPs were identified, including 139 up-regulated and 196 down-regulated proteins. Six proteins (APOV1, GAL, SAA, GNB5, VLDLR and CDK1) with higher changes in expression were selected, and these proteins are mainly involved in the pathways related to reproductive performance, such as Oocyte meiosis, and PI3K-Akt signaling pathway. Steroid biosynthesis was the most significantly enriched pathway by KEGG pathway enriched analysis. The qRT-PCR analysis was applied to verify the proteomic analysis. Meanwhile, metabolomics analysis found that several important differentially expressed metabolites (DEMs) (7-dehydrodesmosterol, 25-Hydroxyvitamin D3, 7-Dehydrocholesterol, Pregnanolone, Allopregnanolone and estrogen) that were also mainly involved in Steroid biosynthesis, Steroid hormone biosynthesis and Metabolic pathways. Crucially, the changes in the abundance of these metabolites are closely related to the changes in the protein abundance of proteins identified in the same pathway, and it is always the upstream key enzymes that influence the production of downstream metabolites.

Introduction

The Muscovy duck is a special kind of waterfowl that belongs to a different genus than domestic duck, and it has the characteristics of high heat resistance and high meat yield but strong broodiness and low egg-laying performance. It is quite challenging to reduce or eliminate the broodiness of the Muscovy duck. The broodiness of poultry has attracted great attention, and it is commonly regulated by the environment, nutrition, heredity and endocrinology of the ducks [[1], [2], [3], [4]]. There is a low heritability of broodiness, and it is difficult to eliminate broodiness by conventional breeding methods [5]. Many studies and our own previous studies have shown that the hypothalamic-pituitary-gonadal axis is a key regulatory axis for the regulation of reproductive performance in birds, in which ovaries play an important role. Ovaries secrete a variety of hormones, such as progesterone, estradiol, follicle-stimulating hormone, luteinizing hormone, androgen, and prolactin, which are considered to be the key hormones associated with broodiness, and most of these are steroid hormones [6,7]. These hormones affect the development and maturation of oocytes and follicles and ultimately promote or inhibit egg laying. Therefore, those genes, proteins, metabolites, growth factors and signaling pathways related to the synthesis, metabolism, and transport of steroid hormones have become the focus of current research on broodiness. Steroid biosynthesis, Steroid hormone biosynthesis, the GnRH signaling pathway, Calcium signaling pathway, PI3K-Akt signaling pathway, Progesterone-mediated oocyte maturation and Oxytocin signaling pathway are all key signaling pathways related to reproductive performance [[8], [9], [10]]. Studies have shown that the inhibition of steroid synthase in steroid biosynthesis causes ovarian function degeneration and triggers broody behavior [11]; the cholesterol side-chain cleavage enzyme gene in the steroid hormone biosynthesis pathway is associated with hormone release and follicular development [7,12]. In addition, the large number of follicular atresia and the degeneration of follicles during the broody period of birds is also related to the autophagy and apoptosis of granular cells [13,14]. However, how these factors affect the ovarian structure and function of Muscovy ducks to subsequently cause the broody behavior remains unclear.

In the recent years, Gel-free proteomic approaches based on mass spectrometry (MS) have been widely used in livestock and poultry breeding studies [15]. Some studies using proteomics techniques have achieved good results by iTRAQ methods, such as studies of the egg-laying performance of Huoyan geese [9], the molecular mechanisms for hyperprolificacy of Small Tail Han sheep [16], the diversity of wool fibers in sheep and goats [17], as well as studies in the plant and medical fields [[18], [19], [20]]. At the same time, an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based global metabolomics method was used to identify metabolites [21,22], combing univariate and multivariate statistical analyses to screen DEMs and to conduct pathway analysis [23]. Important metabolites selected as biomarkers have important research significance for the study of some physiological or pathological characteristics. Given our interest in obtaining a deeper understanding of the broodiness of Muscovy ducks, we undertook a combined proteomic and metabolomic investigation in ovary tissue of Muscovy ducks during the broody and laying periods. This study aims to reveal molecular mechanisms of Muscovy duck broodiness and to provide a new method for researching the broodiness of poultry.

Section snippets

Ethics statement

All animal experiments in this study were reviewed and approved by the Institutional Animal Care and Use Committee at the College of Animal Science, Fujian Agriculture and Forestry University. All of the following procedures were strictly performed according to the regulations and guidelines established by this committee.

Animal and tissue collection

In this study, small white feather female Muscovy ducks (MB lines, The MB lines were obtained by crossing two lines with divergent egg production rates by the Putian Guangdong

Identification of ovary tissue protein in Muscovy duck during the broody and laying periods

In the present study, a total of 297,472 spectra were generated from the iTRAQ experiment using the laying period as the control group and the broody period as the experimental group. Mascot search against the NCBI nr mallard sequence database and blast compared with several animal protein databases identified a total of 24,599 spectra, 22,044 unique spectra, 11,708 peptides, 11,217 unique peptides and 2759 proteins (Supplementary Table S2). Approximately 89% of these proteins had a coefficient

Interesting differentially expressed proteins between broody and laying periods

In this study, a total of 335 DEPs were identified by iTRAQ quantification between the broody and laying periods, and several (APOV1, GAL, SAA, GNB5, VLDLR and CDK1) important and significantly up-regulated or down-regulated proteins were selected that may be associated with the broodiness of Muscovy duck. APOV1 was the most up-regulated protein in the broody period. This small, hormonally regulated protein was found to play an important role in productive performance in quail [30]. It is a

Conclusion

The iTRAQ-based quantitative proteomics analysis and untargeted LC-MS/MS-based global metabolomics method utilized here identified many DEPs, DEMs and pathways, which have provided some insight into the molecular mechanisms of strong broodiness in Muscovy ducks. Among them, APOV1, GAL, SAA, GNB5, VLDLR and CDK1 are obviously up-regulated or down-regulated proteins, which are mainly involved in the pathways related to reproductive performance and may regulate the development and maturation of

Declaration of interest

All authors declare that they have no conflict of interest.

Acknowledgements

This project was supported by the National Natural Science Foundation of China (31001005), the Fujian Provincial Natural Science Foundation (2016J01092), the Agricultural Industrialization Project of Fujian Province (fjzycxny2017011), the Earmarked Fund for Modern Agro-industry Technology Research System of China (CaRS-43).

References (63)

  • J. Nimpf et al.

    Oocytes from the mutant restricted ovulator hen lack receptor for very low density lipoprotein

    J. Biol. Chem.

    (1989)
  • P.D. Dean et al.

    A soluble 2,3-oxidosqualene sterol cyclase

    J. Biol. Chem.

    (1967)
  • M.W. Huff et al.

    Lord of the rings – the mechanism for oxidosqualene:lanosterol cyclase becomes crystal clear

    Trends Pharmacol. Sci.

    (2005)
  • G. Jones et al.

    25-Hydroxyvitamin D-24-hydroxylase (CYP24A1): its important role in the degradation of vitamin D

    Arch. Biochem. Biophys.

    (2012)
  • P.J. Hansen

    Exploitation of genetic and physiological determinants of embryonic resistance to elevated temperature to improve embryonic survival in dairy cattle during heat stress

    Theriogenology

    (2007)
  • X. Wu et al.

    Cloning, expression, and polymorphism at the 5′-flanking region of the GnRH gene and their association with laying traits in Muscovy duck (Cairina moschata)

    Br. Poult. Sci.

    (2015)
  • X. Wu et al.

    Investigation of insulin-like growth factor-1 gene with egg-laying traits in the Muscovy duck (Cairina moschata)

    Can. J. Anim. Sci.

    (2016)
  • Z.Q. Xu et al.

    Polymorphisms in the 5′-flanking regions of the GH, PRL, and Pit-1 genes with Muscovy duck egg production

    J. Anim. Sci.

    (2015)
  • L.I. Ang et al.

    The change regularity of the concentrations of reproductive hormone during the broody period in Muscovy ducks

    Chin. J. Animal Vet. Sci.

    (2004)
  • P. Yan et al.

    Research progress on influencing factors and endocrine regulation mechanism of broodiness in poultry

    China Animal Husb. Vet. Med.

    (2018)
  • Q. Xu et al.

    Transcriptome profiling of the goose (Anser cygnoides) ovaries identify laying and broodiness phenotypes

    PLoS One

    (2013)
  • X. Luan et al.

    Comparative proteomic analysis of pituitary glands from Huoyan geese between pre-laying and laying periods using an iTRAQ-based approach

    PLoS One

    (2017)
  • Y.T. Wu et al.

    Activation of the PI3K-Akt-mTOR signaling pathway promotes necrotic cell death via suppression of autophagy

    Autophagy

    (2009)
  • X. Shen et al.

    Transcriptome sequencing reveals genetic mechanisms underlying the transition between the laying and brooding phases and gene expression changes associated with divergent reproductive phenotypes in chickens

    Mol. Biol. Rep.

    (2016)
  • Q. Xu et al.

    Identification and differential expression of microRNAs in ovaries of laying and broody geese (Anser cygnoides) by solexa sequencing

    PLoS One

    (2014)
  • J. Kovács et al.

    The fine structure of the follicular cells in growing and atretic ovarian follicles of the domestic goose

    Cell Tissue Res.

    (1992)
  • J. Yu et al.

    Transcriptome analysis of follicles reveals the importance of autophagy and hormones in regulating broodiness of Zhedong white goose

    Sci. Rep.

    (2016)
  • D.G. Lee et al.

    Proteomic analysis of reproduction proteins involved in litter size from porcine placenta

    Biosci. Biotechnol. Biochem.

    (2015)
  • X. Miao et al.

    Ovarian proteomic study reveals the possible molecular mechanism for hyperprolificacy of small tail Han sheep

    Sci. Rep.

    (2016)
  • A.C. Peterson et al.

    Development of a GC/Quadrupole-Orbitrap mass spectrometer, part I: design and characterization

    Anal. Chem.

    (2014)
  • A. Cano et al.

    Deciphering non-alcoholic fatty liver disease through metabolomics

    Biochem. Soc. Trans.

    (2014)
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    Biological significance: The Muscovy duck is a commercially and nutritionally important meat duck that is widely raised in the southeast coastal areas of China. However, it has the characteristics of strong broodiness, and poor egg-laying performance has restricted the rapid development of Muscovy duck breeding. Although previous studies have obtained some molecular mechanisms of broodiness by transcriptome and Long Non-Coding RNAs analysis, the global profiles of proteins and metabolites in tissue of Muscovy duck between broody and laying periods are still not available. Proteins and metabolites as functional implementers have potential research significance physiologically. The work herein also represents the first time that the combination analysis of proteomics and metabolomics was used to reveal the molecular mechanisms of broodiness in Muscovy ducks. The results of this study will provide more insights into the broodiness of Muscovy ducks at the level of proteins and metabolites.

    1

    These authors contributed equally to this work.

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