A Draft Map of the Human Ovarian Proteome for Tissue Engineering and Clinical Applications

Abstract

Fertility preservation research in women today is increasingly taking advantage of bioengineering techniques to develop new biomimetic materials and solutions to safeguard ovarian cell function and microenvironment in vitro, and in vivo,. However, available data on the human ovary are limited and fundamental differences between animal models and humans are hampering researchers in their quest for more extensive knowledge of human ovarian physiology and key reproductive proteins that need to be preserved. We therefore turned to multi-dimensional label-free mass spectrometry to analyze human ovarian cortex, as it is a high-throughput and conclusive technique providing information on the proteomic composition of complex tissues like the ovary. In-depth proteomic profiling through two-dimensional liquid chromatography-mass spectrometry, Western blotting, histological and immunohistochemical analyses, and data mining helped us to confidently identify 1508 proteins. Moreover, our method allowed us to chart the most complete representation so far of the ovarian matrisome, defined as the ensemble of extracellular matrix proteins and associated factors, including more than 80 proteins. In conclusion, this study will provide a better understanding of ovarian proteomics, with a detailed characterization of the ovarian follicle microenvironment, in order to enable bioengineers to create biomimetic scaffolds for transplantation and three-dimensional in vitro, culture. By publishing our proteomic data, we also hope to contribute to accelerating biomedical research into ovarian health and disease in general. Fertility preservation research in women today is increasingly taking advantage of bioengineering techniques to develop new biomimetic materials and solutions to safeguard ovarian cell function and microenvironment in vitro, and in vivo,. However, available data on the human ovary are limited and fundamental differences between animal models and humans are hampering researchers in their quest for more extensive knowledge of human ovarian physiology and key reproductive proteins that need to be preserved. We therefore turned to multi-dimensional label-free mass spectrometry to analyze human ovarian cortex, as it is a high-throughput and conclusive technique providing information on the proteomic composition of complex tissues like the ovary. In-depth proteomic profiling through two-dimensional liquid chromatography-mass spectrometry, Western blotting, histological and immunohistochemical analyses, and data mining helped us to confidently identify 1508 proteins. Moreover, our method allowed us to chart the most complete representation so far of the ovarian matrisome, defined as the ensemble of extracellular matrix proteins and associated factors, including more than 80 proteins. In conclusion, this study will provide a better understanding of ovarian proteomics, with a detailed characterization of the ovarian follicle microenvironment, in order to enable bioengineers to create biomimetic scaffolds for transplantation and three-dimensional in vitro, culture. By publishing our proteomic data, we also hope to contribute to accelerating biomedical research into ovarian health and disease in general. The World Health Organization has ranked infertility in women as the fifth highest serious global disability (1.World report on disability Lancet. 1977; 377Google Scholar). Based on this critical assessment, several strategies have been developed to preserve and even restore fertility in women. Although the human ovary is relatively well understood in terms of secretory patterns of ovarian hormones and the pathogenesis of ovarian diseases, little is known about the molecular composition and regulation of the microenvironment that directs the development and function of ovarian follicles, the functional units that mainly reside in the ovarian cortex and play an important role in oogenesis and gonadal hormone secretion. A few studies have focused on mRNA expression to provide a complete characterization of gene expression in the ovary. However, study of gene expression at the mRNA level yields no information about post-transcriptional modifications (2.Vogel C. Marcotte E.M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.Nat. Rev. Genet. 2012; 13: 227-232Crossref PubMed Scopus (2504) Google Scholar). Moreover, even abundant mRNA transcripts may be translated inefficiently or degrade rapidly, resulting in lower than expected levels of protein expression (3.Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Global analysis of protein expression in yeast.Nature. 2003; 425: 737-741Crossref PubMed Scopus (2995) Google Scholar). On the other hand, by focusing on final gene products, a proteomic approach has the advantage of investigating complex biological events and diseases, providing more conclusive information. For several years now, ovarian proteomic studies have concentrated on characterizing follicular fluid composition in human preovulatory follicles (4.Jarkovska K. Martinkova J. Liskova L. Halada P. Moos J. Rezabek K. Gadher S.J. Kovarova H. Proteome mining of human follicular fluid reveals a crucial role of complement cascade and key biological pathways in women undergoing in vitro fertilization.J. Proteome Res. 2010; 9: 1289-1301Crossref PubMed Scopus (75) Google Scholar, 5.Ambekar A.S. Kelkar D.S. Pinto S.M. Sharma R. Hinduja I. Zaveri K. Pandey A. Prasad T.S. Gowda H. Mukherjee S. Proteomics of follicular fluid from women with polycystic ovary syndrome suggests molecular defects in follicular development.J. Clin. Endocrinol. Metab. 2015; 100: 744-753Crossref PubMed Scopus (94) Google Scholar), emphasizing the functional selectivity of the basement membrane toward plasma proteins (6.Angelucci S. Ciavardelli D. Di Giuseppe F. Eleuterio E. Sulpizio M. Tiboni G.M. Giampietro F. Palumbo P. Di Ilio C. Proteome analysis of human follicular fluid.Biochim. Biophys. Acta. 2006; 1764: 1775-1785Crossref PubMed Scopus (125) Google Scholar). Efforts have also been made to identify ovary-related transcription in different species to explore the complex functions of the ovary in an integrated manner. This is necessary because of obvious difficulties in obtaining human ovaries, as well as the feasibility of conducting experimental research in humans because of ethical and logistical constraints. He et al., (2015). described ovarian proteomics in rhesus monkeys through identification of 4325 proteins to provide a basis for future studies of human reproductive disorders using this animal as a model (7.He H. Teng H. Zhou T. Guo Y. Wang G. Lin M. Sun Y. Si W. Zhou Z. Guo X. Huo R. Unravelling the proteome of adult rhesus monkey ovaries.Mol. Biosyst. 2014; 10: 653-662Crossref PubMed Scopus (9) Google Scholar). To our knowledge, the only proteomic analysis of human ovaries performed to date was conducted by Wang et al., (2005) (8.Wang L. Zhu Y.F. Guo X.J. Huo R. Ma X. Lin M. Zhou Z.M. Sha J.H. A two-dimensional electrophoresis reference map of human ovary.J. Mol. Med. 2005; 83: 812-821Crossref PubMed Scopus (41) Google Scholar). The study identified 138 proteins by two-dimensional (2D) electrophoresis and MALDI-TOF mass spectrometry, but without differentiation of ovarian extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; 2D-LC/MS, two-dimensional liquid chromatography mass spectrometry; bFGF, basic fibroblast growth factor; BMP-2, bone morphogenetic protein 2; CGMP-PKG, cyclic GMP-protein kinase G; DAB, 3,3′-diaminobenzidine; EGF, epithelial growth factor; FDR, false discovery rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HILIC, hydrophilic interaction liquid chromatography; HSA, human serum albumin; HSPG2, perlecan; IGF, insulin-like growth factor; IGFALS, insulin-like growth factor-binding protein acid-labile subunit; MEM, minimal essential medium; MMP, matrix metalloproteinase; OGN, mimecan; ORA, overrepresentation enrichment analysis; PARS, platelet-associated regulatory system; PCNA, proliferating cell nuclear antigen; PEDF, pigment factor; kinase system; growth factor; abbreviations used are: ECM, extracellular matrix; 2D-LC/MS, two-dimensional liquid chromatography mass spectrometry; bFGF, basic fibroblast growth factor; BMP-2, bone morphogenetic protein 2; CGMP-PKG, cyclic GMP-protein kinase G; DAB, 3,3′-diaminobenzidine; EGF, epithelial growth factor; FDR, false discovery rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HILIC, hydrophilic interaction liquid chromatography; HSA, human serum albumin; HSPG2, perlecan; IGF, insulin-like growth factor; IGFALS, insulin-like growth factor-binding protein acid-labile subunit; MEM, minimal essential medium; MMP, matrix metalloproteinase; OGN, mimecan; ORA, overrepresentation enrichment analysis; PARS, platelet-associated regulatory system; PCNA, proliferating cell nuclear antigen; PEDF, pigment factor; kinase system; growth factor; the important role in the follicular and and the only of the ovarian to have been so far is the basement follicular and this was through immunohistochemical only a limited characterization by the and of used L. 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