Gametes Alter the Oviductal Secretory Proteome
2005; Elsevier BV; Volume: 4; Issue: 11 Linguagem: Inglês
10.1074/mcp.m500119-mcp200
ISSN1535-9484
AutoresA. Stephen Georgiou, Edita Šoštarić, Chi Huey Wong, Ambrosius P. Snijders, Phillip C. Wright, H. D. M. Moore, Alireza Fazeli,
Tópico(s)Proteins in Food Systems
ResumoThe mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes. The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes. The mammalian oviduct is the venue of important events leading to the establishment of pregnancy. These events include final maturation and transport of the female and male gametes, fertilization, cleavage-stage embryonic development, and transport of the embryo to the uterus. In mammals, the physiological interaction between gametes, embryos, and oviductal epithelia involves intimate and specific contact between the two cell types (1Fazeli A. Duncan A.E. Watson P.F. Holt W.V. Sperm-oviduct interaction: induction of capacitation and preferential binding of uncapacitated spermatozoa to oviductal epithelial cells in porcine species.Biol. Reprod. 1999; 60: 879-886Google Scholar, 2Fazeli A. Elliott R.M. Duncan A.E. Moore A. Watson P.F. Holt W.V. In vitro maintenance of boar sperm viability by a soluble fraction obtained from oviductal apical plasma membrane preparations.Reproduction. 2003; 125: 509-517Google Scholar, 3Hunter R.H. Nichol R. Transport of spermatozoa in the sheep oviduct: preovulatory sequestering of cells in the caudal isthmus.J. Exp. Zool. 1983; 228: 121-128Google Scholar, 4Green C.E. Bredl J. Holt W.V. Watson P.F. Fazeli A. Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro.Reproduction. 2001; 122: 305-315Google Scholar, 5Talbot P. Shur B.D. Myles D.G. Cell adhesion and fertilization: steps in oocyte transport, sperm-zona pellucida interactions, and sperm-egg fusion.Biol. Reprod. 2003; 68: 1-9Google Scholar, 6Talbot P. Geiske C. Knoll M. Oocyte pickup by the mammalian oviduct.Mol. Biol. Cell. 1999; 10: 5-8Google Scholar). During the estrous or menstrual cycle, the mammalian oviduct undergoes significant endocrine-induced morphological, biochemical, and physiological changes. These changes establish an essential microenvironment within the oviduct. Oviductal fluid is a crucial part of this milieu and consists of transudate from serum together with specific compounds synthesized by the luminal epithelium. The current dogma states that the oviductal environment and the composition of oviductal fluid are solely under the influence of the hormonal changes in the oviduct (7Leese H.J. The formation and function of oviduct fluid.J. Reprod. Fertil. 1988; 82: 843-856Google Scholar, 8Buhi W.C. Alvarez I.M. Kouba A.J. Secreted proteins of the oviduct.Cells Tissues Organs. 2000; 166: 165-179Google Scholar, 9Buhi W.C. Characterization and biological roles of oviduct-specific, oestrogen-dependent glycoprotein.Reproduction. 2002; 123: 355-362Google Scholar). However, in recent years, several investigations from our laboratory and others have challenged this view by providing evidence of transcriptional changes in the oviduct in response to gametes irrespective of oviductal hormonal status (10Fazeli A. Affara N.A. Hubank M. Holt W.V. Sperm-induced modification of the oviductal gene expression profile after natural insemination in mice.Biol. Reprod. 2004; 71: 60-65Google Scholar, 11Bauersachs S. Blum H. Mallok S. Wenigerkind H. Rief S. Prelle K. Wolf E. Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach.Biol. Reprod. 2003; 68: 1170-1177Google Scholar, 12Lee K.F. Yao Y.Q. Kwok K.L. Xu J.S. Yeung W.S. Early developing embryos affect the gene expression patterns in the mouse oviduct.Biochem. Biophys. Res. Commun. 2002; 292: 564-570Google Scholar). %Although these data provide strong evidence in relation to the modulation of the oviductal environment by gametes, they lack information regarding changes to the oviductal proteomic profile, for example the secretory profile. In mammals, not all the changes in the transcriptome are translated into proteomic alterations due to post-translational modifications. Ellington et al. (13Ellington J.E. Ignotz G.G. Ball B.A. Meyers-Wallen V.N. Currie W.B. De novo protein synthesis by bovine uterine tube (oviduct) epithelial cells changes during co-culture with bull spermatozoa.Biol. Reprod. 1993; 48: 851-856Google Scholar) and Thomas et al. (14Thomas P.G. Ignotz G.G. Ball B.A. Brinsko S.P. Currie W.B. Effect of coculture with stallion spermatozoa on de novo protein synthesis and secretion by equine oviduct epithelial cells.Am. J. Vet. Res. 1995; 56: 1657-1662Google Scholar) provide the only evidence that at least spermatozoa can influence the (secretory) proteomic profile of oviductal epithelial cells. These investigations have reported de novo protein synthesis in oviductal epithelial cell monolayers in response to spermatozoa in vitro. However, they failed to obtain the identity of the de novo synthesized proteins. In the present investigation, we report alterations in the secretory oviductal proteome in response to oocytes and spermatozoa using quantitative 2D1gel electrophoresis and mass spectrometry. Secretory oviductal proteomic changes specific to each gamete were defined. Furthermore the results obtained by 2D gel electrophoresis were verified in two candidate proteins using Western blot analysis. Bioinformatic analysis indicated that the majority of these proteins may have a role in maintenance and protection of gametes in the oviduct. This is the first report to demonstrate an oviduct-specific proteomic response to both of the gametes. Boar semen obtained from JSR Healthbred Limited (Yorkshire, UK) was collected, then diluted, and stored for 24 h in Beltsville thawing solution (15Pursel V.G. Johnson L.A. Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure.J. Anim. Sci. 1975; 40: 99-102Google Scholar). Immediately prior to use, diluted boar semen was washed three times with PBS by centrifugation and resuspension at 500 × g for 10 min. Sperm concentration was measured using a hemocytometer, and the proportion of motile sperm was determined. The washed semen sample concentration was adjusted to 1 × 106 motile sperm/ml in PBS. Gilt reproductive tracts were obtained from a local abattoir on the day of slaughter. These were transported to the laboratory at room temperature in PBS supplemented with 100 units/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), and 0.25 μg/ml amphotericin B (Invitrogen). The ovaries did not show any signs of cyclicity such as follicular growth, ovulation, or corpora lutea. Oviducts were cut away from the ovary and the uterine horn and were used in experiments as described elsewhere. Cumulus-oocyte complexes (COCs) were aspirated from medium sized ovarian follicles (3–6-mm diameter) using a 19-gauge needle. Oocytes were washed three times in M199 (Invitrogen) supplemented with 10% (v/v) fetal calf serum (FCS) (Invitrogen), 100 units/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), and 0.25 μg/ml amphotericin B (Invitrogen). COCs were cultured in groups of 50/well in 4-well plates (Nalge Nunc International, Hereford, UK) for up to 48 h at 37 °C with 5% CO2 in air. COCs with an expanded cumulus were collected for inclusion in further experiments and washed three times in PBS. The nuclear status of oocytes after culture in this in vitro maturation (IVM) protocol was determined previously by 4,6-diamino-2-phenylindole staining described by Mori et al. (16Mori C. Hashimoto H. Hoshino K. Fluorescence microscopy of nuclear DNA in oocytes and zygotes during in vitro fertilization and development of early embryos in mice.Biol. Reprod. 1988; 39: 737-742Google Scholar). COCs were denuded by vortexing for 3 min and then fixed for 15 min in 2.5% (w/v) glutaraldehyde, washed with PBS, stained with 2.5% (w/v) 4,6-diamino-2-phenylindol (Sigma, Dorset, UK), and mounted on slides. The nuclear state of the stained oocytes was assessed under a fluorescence microscope. Our IVM protocol previously resulted in 76% of oocytes completing at least meiosis I stage of nuclear maturation after 48 h. The oviducts were washed three times with PBS supplemented with 100 units/ml penicillin (Invitrogen) and 100 μg/ml streptomycin (Invitrogen). Oviducts were sealed at one end using a piece of cotton thread and filled with ∼1 ml of PBS (Invitrogen) using a syringe. The other oviduct end was then closed, and the sealed PBS-filled oviducts were placed in glass beakers and covered with PBS. Beakers were incubated at 37 °C and 5% CO2 in air for 2 h. The cotton thread was cut at both ends of the oviduct, and oviducts were flushed with ∼2 ml of PBS, which was discarded. Washed oviduct pairs were utilized in subsequent co-incubation experiments with gametes. Each oviduct was sealed at one end using a piece of cotton thread. One of each oviduct pair (originating from the same animal) was filled with 1 ml of PBS before sealing the oviduct (control). The other oviduct from each pair was filled with 1 ml of 1 × 106 motile sperm/ml in PBS before sealing the oviduct (sperm, test) or PBS containing 10 IVM oocytes (oocyte, test). Sealed oviducts were placed in glass beakers and covered with PBS. Beakers were incubated at 37 °C and 5% CO2 in air for 18 h. The 18-h incubation time was chosen to allow enough time for exposure of gametes to the oviduct while making the experimental design practical. An aliquot of washed semen sample diluted in PBS (1 × 106 motile sperm/ml) was incubated at 37 °C and 5% CO2 in air for 18 h to serve as the control sperm sample. Oocytes in PBS (10 IVM oocytes/ml) were incubated at 37 °C and 5% CO2 in air for 18 h to serve as a control oocyte sample. After sperm-oviduct and oocyte-oviduct co-incubations, the cotton thread was cut at both ends of the oviduct, and oviducts were flushed with ∼2 ml of PBS. All fluid was collected. Ten milliliters of the control sperm sample and 10 ml of the control oocyte sample were then added to the conditioned medium collected from PBS-filled oviducts whose oviduct counterparts had been incubated with sperm and oocyte samples, respectively. A protease inhibitor solution (PMSF, Sigma) was added to the collected media to a final concentration of 1 mℳ. All samples were immediately centrifuged at 2,000 × g for 10 min to remove spermatozoa, detached oviductal cells, or any other debris. The supernatant was collected and ultracentrifuged at 100,000 × g for 30 min at 4 °C. The clarified medium (conditioned medium) was collected and stored at −70 °C. The lactate dehydrogenase (LDH) activity of all the collected oviductal fluid samples was examined using a colorimetric assay for cytotoxicity (Oxford Biomedical Research) according to the manufacturer's instructions. Briefly 100 μl of conditioned medium was added to 100 μl of the kit substrate mixture and incubated at room temperature for 30 min in the dark. Fifty microliters of 1 ℳ hydrochloric acid was added to terminate the reaction, and absorbance was read at 490 nm using a Benchmark 96-well plate reader (Bio-Rad). Conditioned medium was concentrated using 3-kDa molecular mass cut off Centricon microconcentrators (Millipore). Control sperm- and oocyte-conditioned media supernatants (no oviduct) that had been incubated alongside the oviduct-gamete co-culture supernatants were concentrated 10 times further than the oviduct-gamete co-culture supernatants. A Plus-one 2D clean up kit (Amersham Biosciences) was used according to the manufacturer's instructions to purify, desalt, and remove all impurities from the protein samples. The resulting protein pellet was dissolved in buffer A (8 ℳ urea, 2% (w/v) CHAPS). The bicinchoninic acid (BCA) assay was performed as described previously by Smith et al. (17Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Measurement of protein using bicinchoninic acid.Anal. Biochem. 1985; 150: 76-85Google Scholar) to determine the protein concentration of all samples. Briefly 10 μl of each protein sample was added to 200 μl of 2% (v/v) copper sulfate solution in BCA and incubated at 37 °C in the dark for 30 min. Absorbance was read at 570 nm using a Benchmark 96-well plate reader (Bio-Rad). Samples were diluted to a concentration of 1.7 μg of protein/μl in buffer A after which 0.5% (v/v) IPG buffer pH 4–7 (Amersham Biosciences) and 0.002% (w/v) bromphenol blue were added. DTT (Amersham Biosciences) was then added to give a final concentration of 40 mℳ DTT. Two to eight hundred micrograms of protein were used to rehydrate 18-cm, pH 4–7 IPG strips (Amersham Biosciences). Proteins were resolved in the first dimension by IEF for a total of 33,500 V-h using the IPGphor isoelectric focusing system (Amersham Biosciences). The IEF program started with 500 V for 500 V-h followed by a step-and-hold increase to 1000 V for 1000 V-h and finally to a step-and-hold increase to 8000 V for 32,000 V-h. After focusing, IPG strips were equilibrated to reduce protein disulfide bonds in 10 ml of equilibrating solution per strip (6 ℳ urea, 30% (v/v) glycerol, 2% (w/v) SDS, 50 mℳ Tris-HCl, pH 8.8, 0.25% (w/v) bromphenol blue, and 1% (w/v) DTT) with gentle rocking for 15 min. The free cysteine residues of proteins were then alkylated to prevent reformation of disulfide bonds by rocking each strip for 15 min in 10 ml of solution containing 6 ℳ urea, 30% (v/v) glycerol, 2% (w/v) SDS, 50 mℳ Tris-HCl, pH 8.8, 0.25% (w/v) bromphenol blue, and 2.5% (w/v) iodoacetamide. These strips were then affixed onto homogeneous 12.5% SDS-polyacrylamide slab gels (2550 × 2100 × 1 mm). The second dimension was performed in the EttanDalt vertical system (Amersham Biosciences) at 25 °C. The 2D gels were fixed overnight in 7% (v/v) acetic acid in 40% (v/v) methanol at room temperature. Gels were then rinsed with water and stained with colloidal Coomassie Brilliant Blue G250 (Sigma) for 2 h at room temperature. Gels were destained in 10% acetic acid in 25% (v/v) methanol for 1 min, washed, and stored in 25% (v/v) methanol at room temperature. Images were captured by scanning using an Image Scanner II flat bed scanner (Amersham Biosciences) and LabScan software (Amersham Biosciences). Gel images were calibrated and normalized using ImageMaster 2D Platinum image analysis software (Amersham Biosciences) to allow for quantitative comparison between gels. To assess the reproducibility of 2D PAGE, gels were prepared in triplicate. The staining intensity of each spot was normalized against the sum total of intensities of all detectable spots in the 2D gel; this normalization performed by ImageMaster 2D Platinum software corrects for any minor differences in protein loading among replicate gels. The triplicate normalized gels were then subjected to triplicate curation in which the gel with the most spots (detected using 2D Platinum software) was chosen as the reference gel (designated gel I), and the two other gels were called gel II and gel III, respectively. First, gel I was curated against the reference gel, and then gel III was curated against it. Next gel II was curated against gel III. Gels were then analyzed for differential protein expression by using 2D Platinum software to calculate and compare the volume of each spot on every gel. Spot volume was calculated as the volume above the spot border situated at 75% of the spot height (measured from the peak of the spot). Finally gels were checked manually to check that protein spots expressed differentially in the reference gel were also different in the other two gels within a triplicate. Only protein spots that changed more than 2-fold in magnitude in at least one experimental day (up or down) and were observed to be altered in the same direction in all three experimental days were considered to be altered. Differentially expressed proteins were excised from the gel, washed in 25% (v/v) methanol, and then incubated for 1 h at 37 °C in Coomassie destain solution consisting of 40% (v/v) acetonitrile (VWR International Ltd., Leicester, UK) in 200 mℳ ammonium bicarbonate. The destain solution was removed, and spots were incubated with ACN for 15 min at room temperature. ACN was subsequently removed, and the spots were dried in a vacuum centrifuge. Dried spots were stored at 4 °C until required. Proteins were digested with 20 ng/μl sequencing grade modified trypsin (Promega, Southampton, UK) in 50 mℳ ammonium bicarbonate at 37 °C for 12 h. The supernatant from the trypsin digest was transferred to a siliconized microcentrifuge tube. Peptides were sequentially extracted three times by incubation with peptide extraction solution consisting of 25 mℳ ammonium bicarbonate (10 min at room temperature), 5% formic acid (15 min at 37 °C), and ACN (15 min at 37 °C). Each extraction was followed by centrifugation and removal of supernatants. The original supernatant and the supernatants from the three sequential extractions were combined and dried in a vacuum centrifuge for 4–6 h. The dried peptides were dissolved in 7 μl of 0.1% (v/v) formic acid in 3% (v/v) ACN in water. Samples were centrifuged for 5 min at 12,000 × g, and the supernatants were subjected to LC-ESI-MS/MS. Liquid chromatographic separations of the tryptic digests were performed using a reverse phase CapLC™ system (Waters, Manchester, UK). Peptides were desalted by a PepMap C18 microguard column (300-μm internal diameter × 1 mm) (LC-Dionex, Leeds, UK) and were then transferred to the analytical column (PepMap C18; 75-μm internal diameter × 15-cm column (LC-Dionex). The peptides were eluted in a 60-min gradient. The compositions of the hydrophilic and hydrophobic solvents were 5% ACN, 0.1% formic acid and 95% ACN, 0.1% formic acid. The column eluent was sprayed directly into the nano-ESI source of a Q-TOF microcolumn (Waters). An initial MS scan was performed, and selection of ions for CID was automated by Mass Lynx software (Waters). CID selection criteria were set for 2+ and 3+ ions within the range of 400–2000 m/z above 10 ion counts. Alternatively LC separations of the tryptic digests were performed on a PepMap C18 reverse phase capillary column (LC-Dionex) and eluted in a 30-min gradient via an LC Packings Ultimate nano-LC directly into the mass spectrometer. The compositions of the hydrophilic and hydrophobic solvents were 5% ACN, 0.1% formic acid and 95% ACN, 0.1% formic acid. An Applied Biosystems QStarXL electrospray ionization quadrupole time-of-flight tandem mass spectrometer (ESI-qQ-TOF) was used for mass spectrometric analysis. Analyst Qs software (Applied Biosystems) was used for data acquisition and data analysis. The data acquisition on the mass spectrometer was performed using information-dependent acquisition. After each TOF-MS scan, two peaks with charge states 2 or 3 were selected for tandem mass spectrometry. Spectra were searched against the Mass Spectrometry Data Base (MSDB) in a sequence query search using MASCOT 2.0 software (www.matrixscience.com). The taxonomy was limited to filter for only mammalian matches, and trypsin was used as the cleavage enzyme with one missed cleavage site allowed. The peptide tolerance was set to 1.0 Da, and the MS/MS tolerance was set to 0.3 Da. Carbamidomethyl modification of cysteine and oxidized methionine were set as variable modifications. Matches were considered valid if MS/MS data for multiple unique peptides per protein were identified, each with ions scores above the threshold of statistical significance (values generated by MASCOT). Manual examination of MS/MS data was performed for single peptide matches. In these cases, a continuous stretch of peptide sequence covered by either the y- or b-ion series was required for the protein match to be considered valid. Conditioned medium proteins (30 μg of protein) were separated by one-dimensional gel electrophoresis using self-cast homogeneous 12.5% SDS-polyacrylamide gels. Gels were run at 25 mA for ∼2 h using a Mini-Protean II™ gel electrophoresis system (Bio-Rad). Resolved proteins were transferred to a PVDF Immobilon PSQ transfer membrane (0.2-μm pore size) (Millipore) using a Bio-Rad Mini Trans-blot electrophoretic transfer cell. Following transfer, membranes were blocked with 5% (w/v) nonfat milk powder in Tris-buffered saline containing 0.1% v/v Tween 20 (TBST) overnight at 4 °C. Blocked membranes were then incubated with either of the following antibodies: mouse anti-heat shock 70-kDa protein (HSP70) monoclonal antibody (Stressgen Biotechnologies Corp., Victoria, Canada) or rabbit anti-peroxiredoxin II polyclonal antibody (LabFrontier, Seoul, Korea). Antibodies were diluted 1:1000 and 1:2000, respectively, in 5% (w/v) milk powder in TBST and incubated for 1 h at room temperature. After three washes with TBST, membranes were incubated in horseradish peroxidase-conjugated secondary antibodies for 1 h. After three more washes with TBST, immunoreactive proteins on the membranes were detected using SuperSignal West Dura chemiluminescent reagents (Perbio Science UK Ltd., Northumberland, UK) and exposure to Hyperfilm ECL high performance chemiluminescent film (Amersham Biosciences). Experiments were designed to compare the oviductal secretory protein profile of oviduct pairs in response to gametes. Three experimental groups were established. In the first experimental group, one oviduct was incubated with spermatozoa, and its counterpart was incubated with PBS. In the second experimental group, one oviduct was incubated with oocytes, and its counterpart was incubated with PBS. Finally in the third experimental group, one oviduct was incubated with spermatozoa, and its counterpart was incubated with oocytes. A total of 10 oviductal pairs were used in each experimental group. All three experiments were undertaken on three separate occasions. The reproducibility of the 2D electrophoresis technique was assessed by creating three 2D electrophoresis gels using oviductal proteins obtained from the first experimental group. An altered oviductal secretory protein profile was examined across the three experimental repetitions (three experimental days). Any proteins showing greater than 2-fold alteration in expression level in at least one repetition and the rest of repetitions showing changes in agreement with that were considered as differentially expressed/produced in response to gametes. These proteins were selected for further examination using LC-ESI-MS/MS. Proteins present in oviductal fluid after 18 h of oviductal co-culture with PBS or sperm were loaded onto 2D gels at 200, 600, and 800 μg of total protein. The images of the resulting Coomassie-stained gels were analyzed using 2D Platinum software to assess the effect of protein loading on the ability to detect regulatory differences between PBS- and sperm-incubated oviducts. Both the total number of protein spots and detectable regulatory differences increased when the gels were loaded with 600 μg compared with 200 μg of total protein (data not shown). Loading the gels with 800 μg resulted in poor quality isoelectric focusing and, as a consequence, resulted in the detection of fewer total proteins and fewer regulatory differences. Therefore, 600 μg of total protein loading was considered optimal for this particular experimental program. Each gel was found to comprise more than 500 protein spots. Representative 2D gels for PBS- and sperm- or oocyte-incubated oviducts are shown in Figs. 1and2. The gels were highly reproducible among each triplicate, showing 94–97% homology (Table I).Fig. 2Representative 2D gels of oviductal fluid proteins from PBS-filled, control oviducts (A) and oocyte-filled, test oviducts (B). Protein abbreviations and UniProt accession numbers are as follows: SOD, Cu,Zn-SOD, P04178; Ddah2, dimethylarginine dimethylaminohydrolase 2, Q6MG60; Apolipoprotein, apolipoprotein A-I precursor, P18648; Haptoglobin, haptoglobin precursor, Q8SPS7 (see Table III for the accession numbers of all other proteins).View Large Image Figure ViewerDownload (PPT)Table IPercent homology between triplicate gelsReference gelGel IIGel IIISperm1009696PBS1009497 Open table in a new tab Triplicate independent oviductal fluid proteins prepared on three independent experimental days were also examined for reproducibility. Homology was found to be 73–79% (Table II), indicating a biological variation in the precise composition of oviductal fluid dependent upon the biological specimen examined. Therefore, gels from each experimental day were compared with each other to account for this biological variation. Any proteins showing greater than 2-fold alteration in expression level in at least one biological batch repetition and the rest of the biological batch repetitions showing changes in agreement with that were considered as differentially expressed in response to gametes.Table IIPercent homology between gels obtained on three different experimental daysReference gelExperiment IIExperiment IIISperm1007473PBS1007678Oocyte1007473PBS1007678Oocyte1007772Sperm1007073 Open table in a new tab Identification of the oviductal secretory proteome changes in response to the presence of gametes in the oviduct was based on the comparison of oviductal fluid collected from multiple independent biological repetitions. The comparison of oviductal fluid proteins from oviducts incubated with PBS versus sperm identified 20 spots that were reproducibly differentially regulated across all three biological batches of which nine were up- and 11 were down-regulated (shown in Table III). Five spots were reproducibly differentially regulated by the presence of oocytes in the oviduct; three were up- and two were down-regulated. Only one of these protein spots, superoxide dismutase (SOD), was commonly regulated by both the sperm and oocyte presence in the oviduct. The direct comparison of oviductal fluid proteins from oviducts incubated with sperm versus oocytes identified nine spots that were reproducibly differentially regulated of which six were up- and three were down-regulated. Three of these protein spots, heat shock 70-kDa protein 1A, triose-phosphate isomerase, and ribonuclease UK114, confirm the up-regulatory changes identified in the comparison of oviductal fluid proteins from oviducts incubated with PBS versus sperm. There was no detectable protein presence in any of controls taken during experiments as determined by the BCA assay.Table IIIOviductal proteins regulated by the presence of sperm and oocytesPredicted proteinFunctional categoryUniProt accession no.Molecular masspIPeptidesCoverageMowse scoreAverage -fold change ± S.E.kDa%Oviductal proteins regulated by presence of spermatozoa in the oviduct (n = 6)Nucleophosmin (h)MiscQ6V96216.14.4919472Unique to sperm (3)CCT8 protein (h)PMRQ7Z75930.15.248211+3.1 ± 0.1 (3)Cytoskeleton-associated protein 1aR.LIEEEAQASAIPVGSR.C; precursor mass, 1668.79 (expt), 1668.87 (calc); Δ, −0.08 Da; charge, 2+. (p)MiscQ8HXL428.65.11699+2.5 ± 0.9 (1)Triose-phosphate isomerase (r)MetP4850023.96.8426206+2.2 ± 0.3 (2)Heat shock 70-kDa protein 1A (p)PMRP3493030.94.735154+1.8 ± 0.0 (1)Elongation factor 1-βbK.SPAGLQVLNDYLADK.S; precursor mass, 1602.89 (expt), 1602.83 (calc); Δ, 0.07 Da; charge, 2+. (p)MiscP2941226.44.51877+1.7 ± 0.3 (2)Heat shock 70-kDa protein 1A (p)PMRP3493031.54.655263+1.6 ± 0.5 (1)Protein CutA precursor (h)MiscQ9NYQ914.04.9320148+1.4 ± 0.4 (1)Thioredoxin (p)AntP8246011.85.1331167+1.4 ± 0.5 (2)λ chain C (p)MiscP0184623.96.7951453−1.5 ± 0.4 (1)Osmotic stress protein 94 (h)PMRO9575729.85.033139−1.6 ± 0.4 (1)Phosphoglyc
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