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

  • Emna Ouni
    Affiliations
    From the ‡Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium;
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  • Didier Vertommen
    Affiliations
    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium;
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  • Maria Costanza Chiti
    Affiliations
    From the ‡Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium;
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  • Marie-Madeleine Dolmans
    Affiliations
    From the ‡Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium;

    Gynecology Department, Cliniques Universitaires Saint-Luc, Brussels, Belgium
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  • Christiani A. Amorim
    Correspondence
    To whom correspondence should be addressed:Pôle de Recherche en Gynécologie, Université Catholique de Louvain, 52 Avenue Mounier, Bte. B1.52.02, 1200 Brussels, Belgium. Tel.:+322-764-5237;
    Affiliations
    From the ‡Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium;
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Open AccessPublished:February 23, 2018DOI:https://doi.org/10.1074/mcp.RA117.000469
      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 (
      • World report on disability
      ). 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 (
      • Vogel C.
      • Marcotte E.M.
      Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.
      ). Moreover, even abundant mRNA transcripts may be translated inefficiently or degrade rapidly, resulting in lower than expected levels of protein expression (
      • 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.
      ).
      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 (
      • 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.
      ,
      • 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.
      ), emphasizing the functional selectivity of the basement membrane toward plasma proteins (
      • 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.
      ). 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 (
      • 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.
      ). To our knowledge, the only proteomic analysis of human ovaries performed to date was conducted by Wang et al., (2005) (
      • 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.
      ). The study identified 138 proteins by two-dimensional (2D) electrophoresis and MALDI-TOF mass spectrometry, but without differentiation of ovarian extracellular matrix (ECM)
      The 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 epithelium-derived factor; PI3K-Akt, phosphoinositide 3-kinase serine/threonine kinase Akt; PSM, peptide spectrum match; RAS, renin-angiotensin system; SLRP, small leucine-rich proteoglycan; TBS, tris-buffered saline; VEGF, vascular endothelial growth factor; αSMA, alpha-smooth muscle actin.
      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 epithelium-derived factor; PI3K-Akt, phosphoinositide 3-kinase serine/threonine kinase Akt; PSM, peptide spectrum match; RAS, renin-angiotensin system; SLRP, small leucine-rich proteoglycan; TBS, tris-buffered saline; VEGF, vascular endothelial growth factor; αSMA, alpha-smooth muscle actin.
      proteins, despite the ECM's important role in defining the follicular environment and orchestrating cellular organization and function. Indeed, the only aspect of the ovarian ECM to have been investigated so far is the basement follicular membrane, and this was done through immunohistochemical tests, which only offer a limited characterization restricted by the type and number of antibodies used (
      • Heeren A.M.
      • van Iperen L.
      • Klootwijk D.B.
      • de Melo Bernardo A.
      • Roost M.S.
      • Gomes Fernandes M.M.
      • Louwe L.A.
      • Hilders C.G.
      • Helmerhorst F.M.
      • van der Westerlaken L.A.
      • Chuva de Sousa Lopes S.M.
      Development of the follicular basement membrane during human gametogenesis and early folliculogenesis.
      ,
      • Rodgers R.J.
      • Irving-Rodgers H.F.
      • Russell D.L.
      Extracellular matrix of the developing ovarian follicle.
      ).
      The goal of this study was therefore to provide a draft map of functional proteins identified in human ovarian cortex and complement available ovarian ECM data. By using 2D liquid chromatography-mass spectrometry (2D-LC/MS) and ensuring adequate sample preparation, we aimed to shed light on potential key intra- and extracellular proteins in reproduction, to form a basis for comparative studies between normal and pathological ovaries and open the door to improved bioengineering techniques for creation of better biomimetic scaffolds.

      DISCUSSION

      The present study confidently identified 1508 proteins in fresh and frozen human ovarian cortex by 2D-LC/MS. Using bioinformatic tools and data mining analysis, we gained a deeper understanding of the function, cellular distribution and signaling role of these proteins. Although we do not yet fully discern the implication of all of them in the ovarian function, we report the most complete proteomic characterization of human ovarian cortex made to date, including a detailed description of the human ovarian ECM composition, which will lead to a better understanding of the follicle environment.
      The technique described can be broadly applied to different tissues of unknown composition to provide a basic understanding of their most abundant proteins and ECM characteristics in one fraction analysis without ECM enrichment. Tissue digestion with Liberase before sample preparation for MS enabled solubilization of major fibrous proteins and thus facilitated analysis of attached molecules and characterization of the ECM. An enzymatic tissue digestion method was chosen because of the high insolubility of ECM proteins, even in strong detergents, which might hinder their detection by MS. Moreover, enzymatic digestion led to rupture of some cellular membranes, which yielded further information on intracellular proteins.
      A large panel of proteins was identified within our data set, some of which had only been demonstrated in vitro, or in animal models, but never in humans. We therefore turned to gene ontology and pathway analysis to better understand their biological role in ovarian tissue, as well as their interactions.
      Fundamental cellular biological processes and several oocyte-related events were detected, namely cell communication pathways involving ECM-receptor interaction, focal adhesion and gap junctions, emphasizing the primordial functional importance of ovarian cell interaction and communication. Focal adhesion proteins made up the second largest group. Proteins included in this category were considered important for ovarian function, because focal adhesion is a key means by which cells sense and respond to the extracellular environment. Conformation of these proteins can vary in response to physical forces, and hence their function (
      • Wu C.
      Focal adhesion: a focal point in current cell biology and molecular medicine.
      ). This fact can be correlated with the importance of biomechanical regulation mechanisms within the ovary and the impact of the mechanical properties of scaffolds used in 3D culture on ovarian cell fate in vitro, (
      • Wood C.D.
      • Vijayvergia M.
      • Miller F.H.
      • Carroll T.
      • Fasanati C.
      • Shea L.D.
      • Brinson L.C.
      • Woodruff T.K.
      Multi-modal magnetic resonance elastography for noninvasive assessment of ovarian tissue rigidity in vivo.
      ,
      • West E.R.
      • Xu M.
      • Woodruff T.K.
      • Shea L.D.
      Physical properties of alginate hydrogels and their effects on in vitro follicle development.
      ). Thus, greater awareness of ovarian focal adhesion proteins might provide more insights into ovarian cell communication and implication in reproduction. Gap junction channels, another detected protein category, allow direct cell-cell communication and diffusion of fundamental nutrients and chemical cues essential for follicular development (
      • McGinnis L.K.
      • Kinsey W.H.
      Role of focal adhesion kinase in oocyte-follicle communication.
      ). Their assembly is promoted by the WNT/β-catenin pathway of gap junction proteins, wherein β-catenin, a protein documented in our MS data and immunohistochemical results, plays a key role in reproduction by influencing estradiol synthesis and adversely affecting follicular development (
      • DaganWells Z.H.a.
      Molecular aspects of follicular development.
      ,
      • Parakh T.N.
      • Hernandez J.A.
      • Grammer J.C.
      • Weck J.
      • Hunzicker-Dunn M.
      • Zeleznik A.J.
      • Nilson J.H.
      Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin.
      ) (Fig. 7D,).
      Other pathways of interest were also detected, such as cyclic GMP-protein kinase G (cGMP/PKG) (supplemental Data S1B,) implicated in oocyte meiotic arrest (
      • Vaccari S.
      • Weeks J.L.
      2nd; Hsieh, M., Menniti, F. S., Conti, M., Cyclic GMP signaling is involved in the luteinizing hormone-dependent meiotic maturation of mouse oocytes.
      ), and the renin-angiotensin system (RAS) that is presumed to regulate oocyte maturation and quality. Up to day, RAS involvement in hormonal regulation remains unclear because of significant differences between species (
      • Herr D.
      • Bekes I.
      • Wulff C.
      Local Renin-Angiotensin system in the reproductive system.
      ). Hence, by means of our data set acquired by MS, we hope to contribute to the elucidation of unexplored proteins in the RAS pathway in the human ovary (supplemental Data S1C,).
      Numerous coagulation- and angiogenesis-related proteins were identified and further elucidated by KEGG pathway analysis, such as coagulation factors (e.g., fibrinogen, prothrombin and plasminogen), as well as those linked to the coagulation and platelet-associated regulatory system (e.g., antithrombin, von Willebrand factor, platelet-activating factor acetylhydrolase IB and serpins), revealing their potential function in reproduction. In 2014, Bódis et al., suggested the role of the platelet-associated regulatory system (PARS) in regulating activity of the hypothalamo-hypophyseal ovarian system and its function in inducing and stimulating follicular and oocyte maturation and steroid hormone secretion in the ovary (
      • Bodis J.
      • Papp S.
      • Vermes I.
      • Sulyok E.
      • Tamas P.
      • Farkas B.
      • Zambo K.
      • Hatzipetros I.
      • Kovacs G.L.
      “Platelet-associated regulatory system (PARS)” with particular reference to female reproduction.
      ). In the light of recent discoveries, coagulation proteins in the ovary appear to occupy new roles beyond plugging blood leakage that go as far as stem cell awakening in the ovary and ovarian rejuvenation (
      • Bukovsky A.
      Novel methods of treating ovarian infertility in older and POF women, testicular infertility, and other human functional diseases.
      ,
      • Hirota Y.
      • Osuga Y.
      • Yoshino O.
      • Koga K.
      • Yano T.
      • Hirata T.
      • Nose E.
      • Ayabe T.
      • Namba A.
      • Tsutsumi O.
      • Taketani Y.
      Possible roles of thrombin-induced activation of protease-activated receptor 1 in human luteinized granulosa cells.
      ), clearly requiring further investigation.
      Although pro-angiogenic factors have been widely explored in the ovary, mainly vascular endothelial growth factor (VEGF), anti-angiogenic factors are still under-investigated. Among identified growth factors, we report detection of pigment epithelium-derived factor (PEDF), a glycoprotein known to have potent physiologic anti-angiogenic activity that negates VEGF action (
      • Chuderland D.
      • Ben-Ami I.
      • Bar-Joseph H.
      • Shalgi R.
      Role of pigment epithelium-derived factor in the reproductive system.
      ), and thus plays a potential anti-tumoral role (
      • Cheung L.W.
      • Au S.C.
      • Cheung A.N.
      • Ngan H.Y.
      • Tombran-Tink J.
      • Auersperg N.
      • Wong A.S.
      Pigment epithelium-derived factor is estrogen sensitive and inhibits the growth of human ovarian cancer and ovarian surface epithelial cells.
      ). PEDF may also function as a gonadal protectant thanks to its anti-inflammatory and anti-oxidative abilities (
      • Yamagishi S.
      • Nakamura K.
      • Ueda S.
      • Kato S.
      • Imaizumi T.
      Pigment epithelium-derived factor (PEDF) blocks angiotensin II signaling in endothelial cells via suppression of NADPH oxidase: a novel anti-oxidative mechanism of PEDF.
      ,
      • Wang J.J.
      • Zhang S.X.
      • Mott R.
      • Chen Y.
      • Knapp R.R.
      • Cao W.
      • Ma J.X.
      Anti-inflammatory effects of pigment epithelium-derived factor in diabetic nephropathy.
      ), which are two important functions in reproduction, considering that oxidative stress and inflammation have been correlated with infertility in women (
      • Chuderland D.
      • Ben-Ami I.
      • Bar-Joseph H.
      • Shalgi R.
      Role of pigment epithelium-derived factor in the reproductive system.
      ). Moreover, high levels of PEDF secreted before ovulation may induce apoptosis in ovarian surface epithelium cells surrounding the follicle to facilitate release of oocyte (
      • Auersperg N.
      • Wong A.S.
      • Choi K.C.
      • Kang S.K.
      • Leung P.C.
      Ovarian surface epithelium: biology, endocrinology, and pathology.
      ).
      Other rarely explored proteins in human ovaries were documented in our data set, namely 14-3-3 protein isoforms delta and epsilon, which are among the top 50 most abundant proteins. 14-3-3 proteins are known to be central mediators modifying cell-signaling processes, including cell cycle regulation and apoptosis (
      • Pozuelo Rubio M.
      • Geraghty K.M.
      • Wong B.H.
      • Wood N.T.
      • Campbell D.G.
      • Morrice N.
      • Mackintosh C.
      14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.
      ,
      • Aitken A.
      14-3-3 proteins: a historic overview.
      ,
      • Morrison D.K.
      The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development.
      ,
      • Freeman A.K.
      • Morrison D.K.
      14-3-3 Proteins: diverse functions in cell proliferation and cancer progression.
      ). It has been suggested that these proteins are involved, at least in the Xenopus, genus, in maintaining prophase I arrest in germinal vesicle-intact oocytes by sequestering the key phosphatase, in meiosis resumption M-phase inducer phosphatase 2 (CDC25B), in an inactive state (
      • Duckworth B.C.
      • Weaver J.S.
      • Ruderman J.V.
      G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A.
      ).
      In view of the lack of knowledge of the follicular environment, particularly ECM proteins, this study provides the most comprehensive description available of healthy human ovarian ECM. ECM protein recognition in raw MS data was achieved by comparison of confidently detected proteins with the Matrisome Project data set (
      • Naba A.
      • Clauser K.R.
      • Ding H.
      • Whittaker C.A.
      • Carr S.A.
      • Hynes R.O.
      The extracellular matrix: Tools and insights for the “omics” era.
      ), an in silico, identified ECM protein set, founded on the characteristic domain-based organization of ECM proteins (
      • Naba A.
      • Clauser K.R.
      • Hoersch S.
      • Liu H.
      • Carr S.A.
      • Hynes R.O.
      The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices.
      ). Hence, we were able to define not only the ovarian ECM, but also the matrisome: an extended definition of the ECM and associated proteins. The dominant collagen type in the ovaries was revealed to be collagen VI, which we showed to be ubiquitously expressed throughout the ECM by immunohistochemistry. It is a basement membrane-anchoring molecule that interacts with collagen IV and may have additional cytoprotective and regulatory functions (
      • Cescon M.
      • Gattazzo F.
      • Chen P.
      • Bonaldo P.
      • Collagen V.I.
      at a glance.
      ). In addition, it has been suggested that type VI collagen microfibrils are resistant to MMPs but are susceptible to degradation by serine proteases, which are enzymes secreted by granulocytes and neutrophils and known to be present in the ovary during the inflammatory phase preceding ovulation (
      • Iwahashi M.
      • Muragaki Y.
      • Ooshima A.
      • Nakano R.
      • Type V.I.
      collagen expression during growth of human ovarian follicles.
      ).
      Within the category of glycoproteins, we identified laminin, fibrillin and thrombospondin, fundamental proteins of the basement membrane, of key importance for cellular attachment, cell proliferation and ECM organization. Most importantly, however, they share EGF-like intrinsic domains, which might bind to EGF receptors and modulate its signaling following their release by ECM proteolysis.
      Within the category of proteoglycans, perlecan (HSPG2) was of interest. Like many proteoglycans, HSPG2 is able to bind growth factors and cytokines and sequester them in the ECM and may be crucially important in the action of basic fibroblast growth factor (bFGF), a key growth factor with pro-angiogenic and anti-apoptotic effects in the ovary. Localized in the basement membrane, HSPG2 provides a barrier, which is both size- and charge-selective, and promotes cell adhesion, endothelial cell growth and regeneration. In the ovary, it has been identified as a major estrogen-binding protein in follicular fluid (
      • Bentov Y.
      • Jurisicova A.
      • Kenigsberg S.
      • Casper R.F.
      What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles?.
      ).
      Another proteoglycan that was unexpectedly detected is OGN, also known as osteoglycin. Its down-regulation in vascular smooth muscle cells results in an increased cell proliferation (
      • Deckx S.
      • Heggermont W.
      • Carai P.
      • Rienks M.
      • Dresselaers T.
      • Himmelreich U.
      • van Leeuwen R.
      • Lommen W.
      • van der Velden J.
      • Gonzalez A.
      • Diez J.
      • Papageorgiou A.P.
      • Heymans S.
      Osteoglycin prevents the development of age-related diastolic dysfunction during pressure overload by reducing cardiac fibrosis and inflammation.
      ), which can provide insights into possible vascular stimulation of the ovarian tissue grafting site by controlling OGN expression.
      In osteoblasts, bone morphogenetic protein 2 (BMP-2) increases OGN expression, whereas in the ovary, the same protein is implicated in primordial follicle assembly during fetal life (
      • Chakraborty P.
      • Roy S.K.
      Bone morphogenetic protein 2 promotes primordial follicle formation in the ovary.
      ), which might suggest the possible involvement of OGN in ovarian fetal remodeling and follicular assembly under the action of BMP-2. Further research is nevertheless needed to elucidate its function in the ovary, especially in adult life.
      In the ovary, enzymatic ECM degradation is required to allow activated follicle expansion. However, to protect surrounding cells from proteolysis, different protease inhibitors appear to be secreted in the ovary, such as protein AMBP, alpha-2-macroglobulin and serpins. The latter is the most widely detected ECM regulator protein family and its expression is closely related to ovarian function (
      • Ny T.
      • Wahlberg P.
      • Brandstrom I.J.
      Matrix remodeling in the ovary: regulation and functional role of the plasminogen activator and matrix metalloproteinase systems.
      ). Indeed, folliculogenesis stage-specific expression of serpins seems to participate in the growth and atresia of follicles (
      • Hayashi K.G.
      • Ushizawa K.
      • Hosoe M.
      • Takahashi T.
      Differential gene expression of serine protease inhibitors in bovine ovarian follicle: possible involvement in follicular growth and atresia.
      ), as it may control ECM remodeling (
      • Law R.H.
      • Zhang Q.
      • McGowan S.
      • Buckle A.M.
      • Silverman G.A.
      • Wong W.
      • Rosado C.J.
      • Langendorf C.G.
      • Pike R.N.
      • Bird P.I.
      • Whisstock J.C.
      An overview of the serpin superfamily.
      ).
      Most of ECM-affiliated proteins have seldom been studied, especially in the human ovary, where we detected galectin-1 and galectin-3. Galectins are a family of β-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. In 2004, Walzel H et al., demonstrated the inhibitory effect of galectin-1 on the steroidogenic activity of granulosa cells, interfering with hormone-receptor interaction and resulting in decreased responses to FSH stimulation in pigs (
      • Walzel H.
      • Brock J.
      • Pohland R.
      • Vanselow J.
      • Tomek W.
      • Schneider F.
      • Tiemann U.
      Effects of galectin-1 on regulation of progesterone production in granulosa cells from pig ovaries in vitro.
      ). Although galectin-3 has similar functions to galectin-1, it has been associated with loss of progesterone synthesis in the mouse ovary, showing increased presence in atretic preantral follicles and the later stages of luteolysis (
      • Nio J.
      • Iwanaga T.
      Galectins in the mouse ovary: concomitant expression of galectin-3 and progesterone degradation enzyme (20alpha-HSD) in the corpus luteum.
      ). This protein been described in a variety of tissues, but not explicitly in healthy human ovarian cortex. It plays a role in diverse biological events, such as embryogenesis, angiogenesis, adhesion, cellular proliferation, apoptosis and modulation of immunity and inflammatory processes (
      • Devouassoux-Shisheboran M.
      • Deschildre C.
      • Mauduit C.
      • Berger G.
      • Mejean-Lebreton F.
      • Bouvier R.
      • Droz J.P.
      • Fenichel P.
      • Benahmed M.
      Expression of galectin-3 in gonads and gonadal sex cord stromal and germ cell tumors.
      ). Overexpression of both galectins is related to ovarian carcinoma; therefore, unraveling the regulatory mechanisms might provide therapeutic solutions.
      The third category of matrisome-associated proteins includes ECM-secreted factors. Proteins in this group were the least abundant, as expected, but several S100 proteins emerged as the most abundant protein type in this category. In fact, binding of calcium to the S100 protein causes structural rearrangement, exposing a target-binding surface. Target-binding results in a range of responses, from inflammation (S100A13) and cytoskeletal reorganization (S100A10), to cell growth control (S100A9) and tumor suppression (
      • Donato R.
      S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles.
      ,
      • Santamaria-Kisiel L.
      • Rintala-Dempsey A.C.
      • Shaw G.S.
      Calcium-dependent and -independent interactions of the S100 protein family.
      ). However, little is known about the action of these calcium-binding proteins in the ovary.
      To our knowledge, this is the first time that fresh human ovarian cortex has been analyzed by MS and compared with frozen tissue cryopreserved using the protocol that has so far generated 13 live births after transplantation in our hospital. This comparison demonstrates the suitability of cryopreserved tissue to accurately represent the proteomic composition of fresh tissue, proving that it can be used to conduct more extended MS analyses.
      In conclusion, our study provides an accurate first draft map of human ovarian cortex, with identification of its ECM proteins. It represents the first step in human ovary characterization, essential for development of a biomimetic artificial ovary and greater understanding of fertility in women.

      DATA AVAILABILITY

      Raw MS proteomic data were deposited in the ProteomeXchange Consortium database via the PRIDE partner repository with the data set identifier PXD008183 (https://www.ebi.ac.uk/pride/archive/projects/PXD008183).

      Acknowledgments

      We thank Olivier Van Kerk and Gaëtan Herinckx for their technical assistance, and Lieven Desmet for his advice on biostatistical evaluation. We also thank Mira Hryniuk, BA, for reviewing the English language of the manuscript.

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