Proteomes of the Female Genital Tract During the Oestrous Cycle
2016; Elsevier BV; Volume: 15; Issue: 1 Linguagem: Inglês
10.1074/mcp.m115.052332
ISSN1535-9484
AutoresClément Soleilhavoup, Cindy Riou, Guillaume Tsikis, Valérie Labas, Grégoire Harichaux, Philippa Kohnke, Karine Reynaud, G. de, Nadine Gérard, Xavier Druart,
Tópico(s)Reproductive Physiology in Livestock
ResumoThe female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed. The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed. The success of fertilisation in mammals is linked to the correct migration of spermatozoa in the different compartments of the female genital tract and the adequate timing of their interaction with the female gamete. In many mammalian species including human, the deposition of semen in the vagina is followed by the sperm migration through the cervix, the uterus and then the oviduct before reaching the site of fertilisation. This sperm transit within the female tract includes mechanical and biochemical interactions with the luminal fluids leading to selection of spermatozoa able to fertilize the oocyte. The first physiological barrier the spermatozoa will have to go through is the cervix whose vaginal side is covered by a highly viscous mucus, the cervical vaginal fluid (CVF) 1The abbreviations used are:CVFcervical vaginal fluidHSPheat shock protein.. The CVF proteome was analyzed in humans in various physiological conditions (1.Zegels G. Van Raemdonck G.A. Tjalma W.A. Van Ostade X.W. 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Proteomics. 2007; 6: 708-716Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The amount of CVF increases at the time of ovulation concomitantly with a higher state of hydration and a reduced viscosity, to facilitate sperm migration (12.Katz D.F. Slade D.A. Nakajima S.T. Analysis of pre-ovulatory changes in cervical mucus hydration and sperm penetrability.Adv. Contracept. 1997; 13: 143-151Crossref PubMed Scopus (73) Google Scholar). The mechanical properties of the mucus are essential to select spermatozoa with the highest fertilizing ability, i.e. with normal morphology and efficient mobility. The main structural components of the cervical mucus are mucins, highly glycosylated, high-molecular-weight proteins assembled into a filamentous and viscous mesh (13.Gipson I.K. Mucins of the human endocervix.Front. Biosci. 2001; 6: 1245-1255Crossref PubMed Google Scholar). 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Therefore, the quantification of proteins such as mucins in the cervical mucus along the cycle is required to better understand the relation between the proteome and the mechanical properties of the mucus. cervical vaginal fluid heat shock protein. Attached to the cervix, the uterus is layered by an endometrial tissue which is capable of physiological remodeling in response to the oestrous cycle and the presence of the embryo (15.Chae J.I. Kim J. Lee S.G. Jeon Y.J. Kim D.W. Soh Y. Seo K.S. Lee H.K. Choi N.J. Ryu J. Kang S. Cho S.K. Lee D.S. Chung H.M. Koo A.D. Proteomic analysis of pregnancy-related proteins from pig uterus endometrium during pregnancy.Proteome Sci. 2011; 9: 41Crossref PubMed Scopus (24) Google Scholar). Therefore many studies focused on the activation of the uterus genome during pregnancy (16.Forde N. Lonergan P. Transcriptomic analysis of the bovine endometrium: What is required to establish uterine receptivity to implantation in cattle?.J. Reprod. 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During this phase, the uterus and the embryo have a biochemical dialog leading to modifications of the uterus transcriptome aiming to support embryo development. But few studies investigated the uterus activity along the cycle, including at oestrous when the uterus is putatively interacting with spermatozoa. The expression of the transcriptome of the endometrium along the oestrous cycle was investigated in bovine (20.Mitko K. Ulbrich S.E. Wenigerkind H. Sinowatz F. Blum H. Wolf E. Bauersachs S. Dynamic changes in messenger RNA profiles of bovine endometrium during the oestrous cycle.Reproduction. 2008; 135: 225-240Crossref PubMed Scopus (99) Google Scholar), equine (21.Gebhardt S. Merkl M. Herbach N. Wanke R. Handler J. Bauersachs S. Exploration of Global Gene Expression Changes During the Estrous Cycle in Equine Endometrium.Biol. Reprod. 2012; 87: 1-13Crossref Scopus (39) Google Scholar) and mouse (22.Yip K.S. Suvorov A. Connerney J. Lodato N.J. Waxman D.J. Changes in mouse uterine transcriptome in estrus and prestrous.Biol. Reprod. 2013; 89: 1-12Crossref Scopus (26) Google Scholar). In human, the proteome of the uterus along the menstrual cycle was studied either on endometrium epithelium (23.Li J. Tan Z. Li M. Xia T. Liu P. Yu W. Proteomic analysis of endometrium in fertile women during the prereceptive and receptive phases after luteinizing hormone surge.Fertility Sterility. 2011; 95: 1161-1163Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 24.Rai P. Kota V. Sundaram C.S. Deendayal M. Shivaji S. Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium.Proteomics Clin. Appl. 2010; 4: 48-59Crossref PubMed Scopus (35) Google Scholar, 25.Chen Q. Zhang A. Yu F. Gao J. Liu Y. Yu C. Zhou H. Xu C. Label-free proteomics uncovers energy metabolism and focal adhesion regulations responsive for endometrium receptivity.J. 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Ametzazurra A. Alkorta N. García-Velasco J.A. Matorras R. Prieto B. González S. Nagore D. Simón L. Elortza F. Comprehensive proteomic analysis of human endometrial fluid aspirate.J. Proteome Res. 2009; 8: 4622-4632Crossref PubMed Scopus (108) Google Scholar). However, the variation of the proteome of the uterine fluid during the oestrous cycle is not known. The oviduct is connected to the uterus through the utero-tubal junction. After having migrated through the uterus, the spermatozoa transit through this utero-tubal junction to fix in the caudal isthmus of the oviduct, the site of the sperm reservoir (30.Coy P. García-Vázquez F.A. Visconti P.E. Avilés M. Roles of the oviduct in mammalian fertilization.Reproduction. 2012; 144: 649-660Crossref PubMed Scopus (195) Google Scholar). The quantification of expression of oviduct proteins in the presence of spermatozoa has revealed the dialog between the gametes and the oviduct (31.Georgiou A.S. Sostaric E. Wong C.H. Snijders A.P. Wright P.C. Moore H.D. Fazeli A. Gametes alter the oviductal secretory proteome.Mol. Cell. Proteomics. 2005; 4: 1785-1796Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 32.Georgiou A.S. Snijders A.P. Sostaric E. Aflatoonian R. Vazquez J.L. Vazquez J.M. Roca J. Martinez E.A. Wright P.C. Fazeli A. Modulation of the oviductal environment by gametes.J. Proteome Res. 2007; 6: 4656-4666Crossref PubMed Scopus (129) Google Scholar). The regulation of expression of genes in the oviduct along the oestrous cycle was shown using oviduct cells from females in oestrus or luteal phase in porcine (33.Seytanoglu A. Georgiou A.S. Sostaric E. Watson P.F. Holt W.V. Fazeli A. Oviductal cell proteome alterations during the reproductive cycle in pigs.J. Proteome Res. 2008; 7: 2825-2833Crossref PubMed Scopus (54) Google Scholar) and bovine species (34.Bauersachs S. Rehfeld S. Ulbrich S. Mallok S. Prelle K. Wenigerkind H. Einspanier R. Blum H. Wolf E. Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle.J. Mol. Endocrinol. 2004; 32: 449-466Crossref PubMed Scopus (117) Google Scholar). Again, an extensive description of the proteome of the oviduct fluid along the oestrous cycle is lacking. Therefore, the aims of this study are 1) to provide for the first time an integrated analysis of the luminal proteomes of the female genital tract by performing a proteomic study of inner cervical mucus, uterine fluid and oviduct fluid in the same biological model, and 2) to quantify the abundance of these luminal proteins throughout the oestrous cycle. The animal model chosen for this study was the sheep because, in this species, the migration of the spermatozoa through the female genital tract, especially in the cervical lumen and the utero-tubal junction, is subjected to a high rate of selection. Therefore, the protein components identified in the lumen of these regions of the tract are candidates for an interaction with spermatozoa during their transit. The proteomes were also assessed after an exogenous hormonal induction of estrus to investigate the endocrine control of the proteomes of each luminal fluid. Otherwise indicated, chemicals were purchased by Sigma-Aldrich (Saint Quentin Fallavier, France). The proteome from the secretions of three segments of the female genital tract (cervix, uterus, and oviduct) was analyzed according to the stage of the oestrous cycle (oestrus versus luteal phase) and the type of cycle (spontaneous versus synchronized). Females used in this study were adult fertile Ile-de-France ewes housed at the INRA Experimental Farm. The experiment took place during natural season of reproduction in France (December). A group of 8 ewes in spontaneous oestrus was identified within the INRA flock by detection of oestrus with a teaser ram. Among these animals, 4 ewes in estrus were slaughtered on the first day of detection of oestrus whereas the other 4 animals were slaughtered 10 days later, during the luteal phase. Another group of 8 females were synchronized using a hormonal treatment. Females received a vaginal sponge impregnated with fluorogestone acetate (20 mg) for 14 days followed by an injection of 400 IU PMSG (Pregnant Mare Serum Gonadotropin) at the time of sponge removal. Oestrus occurred 48 h after sponge removal. Four ewes in synchronized oestrus were slaughtered on the day of estrus whereas the 4 remaining ewes were slaughtered 10 days after oestrus during the luteal phase. As such, four groups of ewes were available: spontaneous estrus, spontaneous luteal phase, synchronized estrus and synchronized luteal phase. Immediately after slaughter of the females, the genital tracts were collected and dissected. The cervix was longitudinally opened with surgical scissors and the inner cervical mucus was aspirated using a positive displacement pipette suited for viscous media (Gilson MicroMan). Each uterine horn was separated from the cervix and the oviduct, and was flushed from the utero-tubal junction to the bottom of the horn with 1 ml of PBS (phosphate buffer saline). Each oviduct was flushed from the isthmus to the ampulla with 200 μl of PBS. The fluids from each uterine horn and each oviduct were kept separate and provided biological variability within each animal. The uterine and oviduct fluids were centrifuged at 10,000 × g to remove the cellular debris and stored at −20 °C until use. The stage of each ewe (estrus or luteal phase) was confirmed first by the presence/absence of corpora lutea or preovulatory follicles on the ovaries during fluids collection then by blood progesterone assay. The fluids from each region of the genital tract (cervix, uterus, and oviduct) were then pooled by group of ewes according to their physiological state (spontaneous estrus, synchronized estrus, spontaneous luteal phase, and synchronized luteal phase). Protein concentration was determined in pool samples using Uptima BC Assay kit (Interchim, Montluc̦on, France.) according to manufacturer's instructions and using bovine serum albumin as a standard. Each sample was migrated separately in triplicate (20 μg per lane) on a 10% SDS-PAGE (50V, 30 min). Gels were stained with Coomassie (G-250) and each lane was cut horizontally in 3 bands for quantitative proteomic analysis. After SDS-PAGE and cutting of the bands, each band was in-gel digested with bovine trypsin (Roche Diagnostics GmbH, Mannheim, Germany) as previously described (35.Labas V. Grasseau I. Cahier K. Gargaros A. Harichaux G. Teixeira-Gomes A.P. Alves S. Bourin M. Gerard N. Blesbois E. Qualitative and quantitative peptidomic and proteomic approaches to phenotyping chicken semen.J. Proteomics. 2015; 112: 313-335Crossref PubMed Scopus (75) Google Scholar). Each band was washed in water/acetonitrile (1:1) for 5 min followed by a second wash in acetonitrile for 10 min. Cysteine reduction and alkylation were performed by successive incubations in solutions of 10 mm dithiothreitol in 50 mm NH4HCO3 for 30 min at 56 °C and 55 mm iodoacetamide in 50 mm NH4HCO3 for 20 min at room temperature in the dark, respectively. Gel slices were washed by an incubation in 50 mm NH4HCO3: acetonitrile (1:1) for 10 min followed by an incubation in acetonitrile for 15 min. Proteins were digested overnight in 25 mm NH4HCO3 with 12.5 ng/μl trypsin (Sequencing Grade, Roche, Paris, France). The resulting peptides were extracted from the gel using an incubation in 0.1% formic acid, acetonitrile (1:1) for 10 min followed by an incubation for 5 min, in acetonitrile. The two collected extractions were pooled with the initial digestion supernatant, dried in a SpeedVac, reconstituted with 30 μl of 0.1% formic acid, 2% acetonitrile, and sonicated for 10 min. To complete the data provided by the in-gel digestion, the in-solution digestion of the cervical mucus, uterus fluid, and oviduct fluid was performed. Each sample (7 μg of total proteins) was diluted in 1% Rapigest (Waters, Milford, MA) in TEAB 100 mm. Cysteine reduction and alkylation were performed by successive incubations in solutions of 10 mm TCEP for 1 h at 37 °C and 50 mm iodoacetamide for 30 min at room temperature in the dark. Proteins were digested overnight with 0.1 μg/μl trypsin (Sequencing Grade, Roche, Paris, France). The resulting peptides were incubated in 1% formic acid to precipitate the Rapigest and centrifuged during 5 min at 10,000 × g. The supernatant was collected and dried in a SpeedVac. TFA (trifluoroacetic acid) was then added at the final concentration of 1% and the peptides were desalted using Zip Tips U-C18 (Millipore, Billerica, MA). Peptides were eluted in a small volume of a solution containing organic solvent (50:50 acetonitrile: 0.1% TFA in water), dried in a SpeedVac, reconstituted with 30 μl of 0.1% formic acid, 2% acetonitrile, and sonicated for 10 min. All experiments were performed on a dual linear ion trap Fourier transform mass spectrometer (FT-MS) LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) coupled to an Ultimate® 3000 RSLC Ultra High Pressure Liquid Chromatographer (Dionex, Amsterdam, The Netherlands). Five microliters of each sample was loaded on trap column for desalting and separated using nano-column as previously described (35.Labas V. Grasseau I. Cahier K. Gargaros A. Harichaux G. Teixeira-Gomes A.P. Alves S. Bourin M. Gerard N. Blesbois E. Qualitative and quantitative peptidomic and proteomic approaches to phenotyping chicken semen.J. Proteomics. 2015; 112: 313-335Crossref PubMed Scopus (75) Google Scholar). The gradient consisted of 4–55% B for 90 min at 300 nl/min flow rate. The eluate was ionized using a Thermo Finnigan Nanospray Ion Source 1 with a SilicaTip emitter of 15 μm inner diameter (New Objective, Woburn, MA). Standard mass spectrometric conditions for all experiments were spray voltage 1.2 kV, no sheath and auxiliary gas flow; heated capillary temperature, 275 °C; predictive automatic gain control enabled, and an S-lens RF level of 60%. Data were acquired using Xcalibur software (version 2.1; Thermo Fisher Scientific, San Jose, CA). The instrument was operated in positive data-dependent mode. Resolution in the Orbitrap was set to r = 60,000. In the scan range of m/z 300–1800, the 20 most intense peptide ions with charge states ≥2 were sequentially isolated (isolation width, 2 m/z; 1 microscan) and fragmented using collision induced dissociation. The ion selection threshold was 500 counts for MS/MS, and the maximum allowed ion accumulation times were 200 ms for full scans and 50 ms for collision induced dissociation-MS/MS in the LTQ. Target ion quantity for FT full MS was 1e6 and for MS/MS it was 1e4. The resulting fragment ions were scanned at the "normal scan rate" with q = 0.25 activation and activation time of 10 ms. Dynamic exclusion was active during 30 s with a repeat count of 1. The lock mass was enabled for accurate mass measurements. Polydimethylcyclosiloxane (m/z, 445.1200025, (Si(CH3)2O)6) ions were used for internal recalibration of the mass spectra. Raw data files were converted to MGF using Proteome Discoverer software (version 1.2; Thermo Fischer Scientific, San Jose, USA). Precursor mass range of 350–5000 Da and signal to noise ratio of 1.5 were the criteria used for generation of peak lists. In order to identify the proteins, the peptide and fragment masses obtained were matched automatically against the Swissprot_2013.01 database (66153 entries). MS/MS ion searches were performed using MASCOT Daemon and search engine (version 2.3; Matrix Science, London, UK). The parameters used for database searches include trypsin as a protease with allowed two missed cleavage, carbamidomethylcysteine (+57 Da), oxidation of methionine (+16) and N-terminal protein acetylation (+42) as variable modifications. The tolerance of the ions was set to 5 ppm for parent and 0.8 Da for fragment ion matches. Mascot results from the target and decoy databases were incorporated to Scaffold 3 software (version 3.6, Proteome Software, Portland, OR). Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (36.Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3896) Google Scholar). Peptides were considered distinct if they differed in sequence. Protein identifications were accepted if they could be established at greater than 95.0% probability as specified by the Protein Prophet algorithm and contained at least two identified peptides (FDR < 1%). Scaffold 3 Q+ software was employed (version 3.6, Proteome Software, Portland, USA) using spectral count quantitative module. All proteins with greater than two peptides identified in SwissProt database with high confidence were considered for protein quantification. To eliminate quantitative ambiguity into protein groups, we ignored all the spectra matching any peptide which is shared between proteins. Thereby, quantification from normalized spectral counts was carried out on distinct proteins. Student's t test was done to characterize changes between two samples. Statistically significant differences were considered for p < 0.05. The Uniprot accession numbers were submitted to the UniProt Knowledgebase (UniProtKB) and the Gene Ontology data were retrieved for all the 940 identified proteins. The subcellular location data were used to generate a pie graph. A panel of 16 main biological functions was used to categorize the proteins and generate a pie graph. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository (37.Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Perez-Riverol Y. Reisinger F. Rios D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: 29Google Scholar) with the data set identifier PXD000299. Primary antibodies directed against the following proteins were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA): zinc α glycoprotein (dilution 1/500, sc-11358), lactoferrin (1/500, sc-52694), α-enolase (1/500, sc-15343), CD109 (1/500, sc-98793), myosin 9 (1/500, sc-98978), ceruloplasmin (1/500, sc-21240), Hsp105 (1/500, sc-1805). Primary antibodies directed against the following proteins were purchased from Abcam (Cambridge, England): gelsolin (1/1000, ab11081), valosin containing protein/VCP (1/2000, ab11433), heat shock protein (HSP) 90 β (1/1000, ab82522), heat shock protein 70 (HSPA2, 1/200, ab1428), and HSPA8 (1/2500, ab1427). Primary antibodies against angiotensin converting enzyme and acrosin were produced from our laboratory after immunization of rabbits with purified angiotensin converting enzyme and acrosin. The antibody directed against bovine oviductin (OVGP or Oviduct specific glycoprotein) was a generous gift from Dr O'Day-Bowman (Laboratory of Dr. Harold Verhage, University of Illinois) The second antibody was goat anti rabbit HRP (1/5000, A6154) for rabbit primary antibodies and goat anti mouse HRP (1/5000, A4416) for mouse primary antibodies. The chemiluminescent HRP substrate was SuperSignal West Pico and West Femto Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). Each sample was migrated separately in triplicate (20 μg per lane) on a 8–16% gradient SDS-PAGE (180V, 60 min). Liquid transfer of proteins was performed over 75 min at 100V at 4 °C. The western blots were blocked with TBS-Tween 20 (0.5%, w/v), supplemented with lyophilized low-fat milk (5% w/v). Membranes were incubated with primary antibodies under mild ag
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