Oviductal Cell Proteome Alterations during the Reproductive Cycle in Pigs Adil Seytanoglu,† A. Stephen Georgiou,† Edita Sostaric,†,# Paul F. Watson,§ William V. Holt,‡ and Alireza Fazeli*,† Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Level 4, The Jessop Wing, Tree Root Walk, Sheffield S10 2SF, United Kingdom, Royal Veterinary College, London, NW1 0TU, United Kingdom, and Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, United Kingdom Received January 8, 2008
The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. During the reproductive cycle, the reproductive system undergoes various changes, including alterations in the number of different cell types in the oviductal epithelium and changes in the height of oviductal cells. Maintaining the unique oviductal environment required for the fertilization and early embryonic development comes with an energy cost to the organism. Therefore, it is hypothesized that structural and functional changes to the oviduct during the reproductive cycle represent vital preparations for the development of suitable environments for conception and embryo support. Here, we aimed to identify the changes in protein expression profile that occur during the follicular and luteal stages of the reproductive cycle in oviductal epithelial cells. The porcine oviductal epithelial cell proteomes from the follicular and luteal stages of the reproductive cycle were contrasted after separation by 2-D gel electrophoresis. Several oviductal epithelial cell proteins were up- or down-regulated during the reproductive cycle. We checked the quantitative changes of two of these molecules during different stages of the reproductive cycle using Western blot analysis. Finally, a number of these proteins were identified using tandem mass spectrometry. The results demonstrated distinctive differences in the proteomic profiles of the oviduct between follicular and luteal phases of the reproductive cycle. Keywords: Oviduct • Epithelial Cells • Reproductive Cycle • Quantitative Proteomics
Introduction The mammalian oviduct plays a crucial role in events leading to the establishment of pregnancy. These events include the final maturation of gametes, fertilization, early embryonic development and transport of the embryo to the uterus.1,2 Understanding oviductal physiology will lead to the identification of mechanisms involved in the fertilization process and early embryonic development, and may facilitate the development of new treatments for human infertility, as well as for the breeding of farm animals. In mammals, the oviduct undergoes several structural and biological changes during the reproductive cycle.3 The reproductive cycle in mammals is divided into two main phases, the follicular and luteal, which correlate with sex hormone fluctuations (mainly progesterone and estrogen) before (follicular) and after (luteal) ovulation. If fertilization does not happen after ovulation, the luteal phase, characterized by high circulating * To whom correspondence should be addressed. Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, The Jessop Wing, Level 4, Tree root walk, S10 2SF Sheffield, U.K. Tel, 01142268195; fax, 0114|2268538; e-mail,
[email protected]. † University of Sheffield. # Current address: Utrecht University, Department of Equine Sciences, Yalelaan 12, 3584 CM Utrecht, The Netherlands. § Royal Veterinary College. ‡ Zoological Society of London. 10.1021/pr8000095 CCC: $40.75
2008 American Chemical Society
progesterone levels, is followed by another follicular phase, when estrogen levels increase as the preovulatory ovarian follicle expands and matures. The detailed pattern of the reproductive cycle, for example, the overall duration of a cycle and length of the follicular phase, is a species-specific characteristic. Additionally, some species such as, horses, sheep and goats exhibit seasonal breeding, while humans, cows and pigs do not have a clearly defined breeding season. Moreover, some species ovulate spontaneously, while others, especially felids, only ovulate in response to mating. The domesticated pig, studied here, can maintain estrous cyclicity throughout the year, unless mated, with the cycle recurring about every 21 to 22 days.4,5 The oviduct itself is a tubular structure connecting the uterus and the ovary. The oviducts are relaxed and flaccid during luteal phase of the cycle. However, they gradually change during the follicular phase and by the beginning of estrus display a characteristic spatial organization.1 The oviduct undergoes various other changes throughout the estrous cycle,6 including alteration in the number of different cell types in the oviductal epithelium2 and changes in the height of oviductal cells.7 The surface epithelium lining the oviductal lumen consists mainly of columnar epithelial cells. These oviductal epithelial cells (OEC) are of great interest in fertility research as they are the main site of contact and interaction between the female Journal of Proteome Research 2008, 7, 2825–2833 2825 Published on Web 06/10/2008
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Seytanoglu et al. 8
reproductive tract and gametes/embryos. As the follicular and luteal phases of the reproductive cycle are correlated with functionally different metabolic, secretory and interactory requirements, we hypothesize that these will be reflected by variations in qualitative and quantitative differences in the proteome. These differences influence gamete function, gamete interaction with the OECs, fertilization and early embryonic development within the oviduct. For example, upon ejaculation, mammalian spermatozoa are briefly exposed to seminal plasma but then enter the uterine environment prior to reaching the oviduct. Once within the oviductal isthmus during the follicular phase of the reproductive cycle, significant modification of motility occurs as this region is involved in storing spermatozoa for periods of varying duration prior to fertilization.9 Observations of spermatozoa taken from the isthmic reservoir of several species have consistently shown that their motility is suppressed within the oviduct, for example.10–13 Furthermore motility modulation has also been observed in spermatozoa exposed to oviductal fluid14 or proteomic components of OEC,15 emphasizing the role of oviductal proteins in mediating gamete function. In the current investigation, we aimed to identify proteins altered at different stages of the reproductive cycle in porcine OEC. Proteins associated with the follicular phase are likely to be involved in modulation of gamete function and sperm storage within oviduct, while those found during the luteal phase are more likely to be involved in providing embryo support. The OEC proteomes from the follicular and luteal stages of the reproductive cycle were separated by 2-D gel electrophoresis and contrasted to detect significant differences. Several OEC proteins were indeed altered during the reproductive cycle and some were identified using tandem mass spectrometry. Finally, we verified the quantitative changes of some of these molecules during different stages of the reproductive cycle using Western blot analysis. The results supported the hypothesis that there are distinctive differences between the proteomic profiles of the oviduct at follicular and luteal phases of the reproductive cycle.
Materials and Methods Isolation and Culture of Oviductal Epithelial Cells. Pig reproductive tracts were obtained from a local slaughterhouse (G Wood and sons, Mansfield, U.K.) and transferred to the laboratory at ambient temperature. The ovaries were inspected for signs of follicular growth, recent ovulation or corpora lutea and divided into follicular and luteal groups. Ovaries with corpora lutea were designated as luteal ovaries. Those with large dominant follicles were designated as follicular phase ovaries. Oviducts attached to the ovaries that did not fall into these categories were discarded. Oviducts were cut away from the ovary and the uterine horn, and excess tissue was trimmed away. Oviducts were rinsed with PBS (Gibco, Invitrogen, Paisley, U.K.) and each oviduct was flushed with PBS. The oviducts were tied with a piece of cotton tread at one end and filled with 0.25% (w/v) collagenase (Sigma-Aldrich, Poole, U.K.) in Hank’s balanced salt solution, HBSS (Gibco). Then, the other end of each oviduct was sealed. The oviducts containing collagenase were pooled accordingly and were incubated in HBSS at 39 °C, 5% CO2 for 2 h. They were then removed from HBSS and opened at the ampullar end. The oviductal fluid was squeezed out and the extracted liquid from each group was collected separately. Two milliliters of red blood cell lysing buffer (Sigma, Poole, U.K.) was added to the oviductal extracts and mixed. 2826
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Following a brief centrifugation at 300g, the fluid was removed and the pellets were resuspended in warm (37 °C) HBSS (Gibco). The tubes were recentrifuged at 300g for 5 min. These steps were repeated until the pellets were cleared of the majority of red blood cells and other debris. Cell pellets were resuspended in medium 199 (M199; Gibco), containing 10% fetal calf serum (FCS; Gibco), 100 units/mL penicillin (Sigma), 100 units/mL streptomycin (Sigma) and 100 units/mL amphotericin (Sigma). The cells were cultured in 75 cm2 tissue flasks (Nunc, VWR International Ltd., Leichs, U.K.) at 39 °C, 5% CO2 for 18 h. At the end of this period, the flasks were viewed under a light microscope (Eclipse TS100, Nikon, Japan) to confirm the attachment of viable OECs to the culture flask. The media in the flasks were discarded without disturbing the OECs to remove dead/unattached cells as well as any other debris. The contents of the flasks were washed with PBS (37 °C) (Gibco). OEC Proteomic Sample Preparation. Cells were harvested by scraping and collected in tubes containing 2 µL of protease inhibitor cocktail (Sigma) and PBS (+Mg, +Ca) (Gibco). Luteal and follicular OECs were homogenized by sonication for 1 h using a sonicator (Sonomatic 575H, Jencons Scientific Ltd., Leighton Buzzard, U.K.). After sonication, the cells were centrifuged at 1000g for 5 min at 4 °C to remove any insoluble material. The supernatant, containing the soluble OEC extract, was used in further experiments. Clean-Up and Isolelectric Focusing of the OEC Proteins. OEC extracts were concentrated using Vivaspin concentrators (3 kDa molecular weight cutoff) (Sigma-Aldrich). A Plus-one 2D clean up kit (Amersham Biosciences, Buckinghamshire, U.K.) 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 (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)). The Bicinchoninic acid (BCA; Sigma) assay was performed 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 1.3 µg of protein/ µL in buffer A, after which 0.5% (v/v) immobilized pH gradient (IPG) buffer pH 4-7 (Amersham Biosciences) and 0.002% (w/ v) bromophenol blue were added. Dithiothrietol (DTT) (Amersham Biosciences) and thiourea were then added to give a final concentration of 40 mM DTT and 2 M thiourea. A total of 450 µg of protein was used to rehydrate Immobiline Drystips (pH 3-10, 18 cm-linear) (Amersham Biosciences) using the manufacturer’s guidelines. The strips were rehydrated overnight. Proteins were resolved by isoelectric focusing using Ettan IPGphor Isoelectric Focusing system (Amersham Biosciences). Ettan IPGphor 3 Control Software Version 1.1 build 27 was used to monitor the actual voltage passing through the strips. The protocol used for pH 3-10 strips was 500 V (0.5 kVh), 1000 V (1 kVh), 8000 V (24 kVh) and 8000 V (12 kVh). 2-D Gel Electrophoresis and the Image Analysis of the 2-D Gels. The focused strips were washed in 15 mL of SDS equilibration buffer (50 mM Tris-HCl (1.5 M, pH 8.8), 6 M urea, 30% (v/v) 87% glycerol, 2% SDS (w/v), and trace bromophenol blue in distilled-deionized water) containing 1% (w/v) of DTT (Fluka) while gently rocking on a rocker (Stuart See-Saw Rocker SSL4) for 15 min. Another wash was carried out with the same amount of buffer containing 2.5% (w/v) iodoacetamide (Sigma).
Modulation of Oviductal Cell Proteome during Reproductive Cycle Strips were then affixed onto homogeneous 12.5% polyacrylamide SDS-PAGE slab gels (2550 × 2100 × 1 mm). The second dimension was performed in the EttanDalt vertical system (Amersham Biosciences) at 25 °C. The gels were run for half an hour at 5 W (W) per gel and 17 W/gel for 4 h. The gels were placed in 7% glacial acetic acid in 40% (v/v) methanol to fix the proteins. The gels were stained with Brilliant Blue GColloidal (Sigma) and 20% (v/v) methanol. They were stained for 2 h and destained with 10% acetic acid in 25% (v/v) methanol for 1 min. The gels were rinsed with 25% (v/v) methanol and further destained in 25% (v/v) methanol for up to 24 h. Gels were scanned using an Image Scanner II flatbed scanner (Amersham Biosciences) and LabScan software (Amersham Biosciences). The gel spots were analyzed using software ImageMaster 2D Platinum v6.0 (Amersham Biosciences). They were detected automatically by the software, and further modified and verified manually. Spot volumes in each gel were normalized using Image Master 2D Platinum software. To normalize, the staining intensity of each spot was adjusted against the sum total of intensities of the detectable spots in the gels. This normalization corrected for minor differences in protein loading among the same group of gels. Only gel spots that showed an increase/decrease of 2-fold or more in each group were selected for identification using liquid chromatography electrospray ionization tandem mass spectrometry (LCESI-MS/MS). Protein spots were excised from the gel and destained in 200 mM ammonium bicarbonate (BDH Laboratories Supplies, Poole, U.K.) with 40% acetonitrile (ACN) (BDH) at 37 °C for 30 min. Once the spots destained completely, the solution was removed and spots were dried in a vacuum centrifuge (miVac DNA concentrator, Barnstead Genevac, Ipswich, U.K.) at 37 °C for 15 min. Proteins were digested with 20 ng/µL of sequencing grade modified trypsin (Promega, Southampton, U.K.) in 50 mM ammonium bicarbonate at 37 °C for 12 h. The supernatant from 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 000g and the supernatants were subjected to LC-ESI-MS/MS. Identification of Protein Spots Using LC-ESI-MS/MS. For the liquid chromatographic (LC) separations of the tryptic digests of the protein samples, a reverse phase CapLC system (Waters, Manchester, U.K.) was used. After desalting peptides using a PepMap18 microguard column (300 µm internal diameter × 1 mm) (LC-Dionex, Leeds, U.K.), the peptides were eluted in a 60 min gradient. The compositions of the hydrophilic and hydrophobic solvents were 5% (v/v) ACN, 0.1% (v/ v) formic acid and 95% (v/v) ACN, 0.1% (v/v) formic acid, respectively. The column eluent was sprayed directly into the nanoESI source of a Q-TOF micro (Waters). Following an initial MS scan, ions were selected for collision induced dissociation (CID) automatically by Mass Lynx software (Waters). The CID selection criteria for the samples were set for 2+ and 3+ ions within the range of 400-2000 m/z above 10 ion counts. Spectra
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were searched against the Swiss-Prot database (Swiss-Prot 50.4) in a sequence query search using MASCOT 2.0 software (www.matrixscience.com). The results were filtered to meet only mammalian matches and trypsin was used as the cleavage enzyme with 2 missed cleavage sites permitted. The peptide tolerance was set as 0.5 Da, while the MS/MS tolerance was set as 0.3 Da. Carbamidomethyl modification of cysteine and oxidized methionine were set as variable modifications. The match results were considered valid only if ion scores were above the threshold of statistical significance values that were automatically generated by MASCOT. Western Blotting. Following the identification of the proteins, to further verify the results of the gel-based comparison made, we selected heat shock protein 70 and cytokeratin proteins showing opposite levels of expression in different phases. To carry out Western blotting, 10 µg of protein was separated by SDS-PAGE using precast gels (4-20% Precise Protein Gels, Pierce). Gels were run at 25 mA for approximately 2 h using a Mini-Protean II Gel Electrophoresis System (BioRad Laboratories Ltd.). Resolved proteins were transferred to a Polyvinylidene fluoride (PVDF) Immobilon PSQ transfer membrane (0.2 µm pore size) (Millipore) using a Bio-Rad Mini Trans-blot electrophoretic transfer cell (Bio-Rad Laboratories Ltd.). 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. Following this, the membranes were washed in TTBS and incubated with the primary antibodies of either HSP70 (mouse Anti-HSP70 monoclonal antibody, Stressgen, Victoria, Canada) or cytokeratin (monoclonal Anti-Pan cytokeratin, Sigma) at dilutions of 1:4500 in 2% milk powder in TTBS for 2 h rolling at room temperature. Following three washes of membranes with TTBS, the membranes were incubated with secondary antibodies, anti-mouse IgG (Sigma) for HSP70 and for cytokeratin at dilutions of 1:100 000 in 2% milk powder in TTBS, rolling for 90 min. At the end of this process, the PVDF membranes were washed. The fluorescence signal produced after detection of the blotted proteins was detected using SuperSignal West Dura Extended Duration Substrate (Pierce) in the dark and exposed to X-ray films (Sigma) laid on top of the membranes. Experimental Design. Experiments were designed to compare and identify the alterations in OEC proteomic profiles during the reproductive cycle using 2-D gel analysis, mass spectrometry and Western blotting. To compare the OEC proteins from follicular and luteal phases of the cycle, we used 2-D gel analysis. For this purpose, we first identified the optimum amount of OEC proteins to be used in 2-D gels. We used three different protein amounts of follicular OEC proteins, 300, 450, and 600 µg, to investigate the optimum amount of proteins for 2-D gel analysis. After the focusing of proteins for the first dimension, gels were run for the second dimension. Gels were stained using Coomassie stain and were compared for optimum focusing and visibility of protein spots. After the optimization of the gel load, 2-D gels of follicular and luteal OEC proteins were run using 450 µg of protein and were stained with Coomassie. Three sets of gels were prepared from a pool of protein samples from each of the follicular and luteal OEC protein groups. The pool of follicular and luteal protein samples were collected by seven separate visits to slaughterhouse in different days. This was done to gather a homogeneous sample pool, avoiding variation that may exist between different individuals. The gels were analyzed as described using gel analysis software. The gel spots identified as increasing or Journal of Proteome Research • Vol. 7, No. 7, 2008 2827
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Figure 1. Representative image of 2-D of follicular OEC polyacrylamide gel. Each gel was loaded with 450 µg of protein and stained with Coomassie. The Coomassie-stained gel shows the separated spots of follicular OEC sample. Marked points and match identity numbers show some of the spots detected that have their quantity more than g2.0-fold increased in the follicular samples in compare to the lutal oviductal epithelial cells. The identities of these spots are described in Table 1.
decreasing more than 2-fold in either phase were selected for LC-ESI-MS/MS identification. To verify the changes in the gelbased results, we used Western blot analysis to verify the alteration of two of identified proteins cytokeratin and HSP70 at the different stages of the reproductive cycle.
Results Protein samples of 300, 450, and 600 µg from follicular OEC were used to produce 2-D gels. The sample load of 450 µg produced the optimum isoelectric focusing with the highest number of protein spots detected for further analysis (data not shown). Therefore, 450 µg of protein load was used for the comparison between luteal and follicular OEC protein samples. Representative images of follicular and luteal OEC gels are depicted in Figures 1 and 2, respectively. The consistency of three replicate gels produced from follicular OEC protein samples was investigated by comparing the percentage volumes of identical spots in the three follicular OEC protein sample gels against each other. The follicular gels were found to be more than 96% homologous. Similarly, the homology of the luteal gels was above 96%. A total of 330 protein spots were 2828
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detected in the follicular OEC protein samples and 510 in luteal OEC protein samples. Protein spots with 2-fold or more change between follicular and luteal OEC protein samples are listed in Supplementary Tables 1 and 2, respectively. Comparing the gels of follicular and luteal OEC protein samples revealed 51 protein spots that were upregulated more than 2-fold in the follicular group. The protein spot with the highest alteration in the follicular group showed an increase of more than 10-fold. Additionally, there were 27 protein spots that were upregulated more than 2-fold in the luteal phase, with the highest increase of more than 7.27fold. Only protein spots that were altered 2-fold or more between follicular and luteal phase samples were excised from the gels and used for mass spectrometry analysis. Not all the excised spots submitted for mass spectrometry analysis returned reliable data for determination of the identity of excised protein spot. The summary information of identified protein spots is depicted in Table 1. Information regarding the identified peptides for each spot, potential identified protein with Uniprot primary accession code (www.ebi.uniprot.org/index.shtml) and
Modulation of Oviductal Cell Proteome during Reproductive Cycle
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Figure 2. Representative image of 2-D of follicular OEC polyacrylamide gel. Each gel was loaded with 450 µg of protein and stained with Coomassie. The Coomassie-stained gel shows the separated spots of luteal OEC sample. Marked points and match identity numbers show some of the spots detected that have their quantity more than g2.0-fold increased in the luteal samples in compare to the follicular oviductal epithelial cells. The identities of these spots are described in the Table 1.
the potential reproductive function of the respected protein are included in Table 1. Two of the OEC identified proteins (Table 1), showing more than 2-fold alteration between follicular and luteal phases of the reproductive cycle, heat shock protein 70 (HSP70) and cytoskeletal keratin 19, were selected for further comparison using Western blot analysis. The Western blot analysis confirmed the changes observed using 2-D gel electrophoresis (Figure 3).
Discussion Previous studies investigating the changes in the oviductal epithelium during the reproductive cycle have demonstrated alterations in the morphology,6 gene expression16 and biosynthetic activities17 of the oviductal cells. In the current investigation, we produced high resolution 2-D gels of soluble cytoplasmic proteins from isolated OECs derived from follicular and luteal stages of the reproductive cycle. The results clearly demonstrated that the cytoplasmic proteomic profile of the OECs is altered during different phases of the reproductive cycle. Overall, we found 51 and 27 protein spots showing an
increase and decrease during the follicular phase compared to that of the luteal phase, respectively. The identities of eight of the upregulated and six of downregulated protein spots were characterized using mass spectrometry. We took several precautions to ensure that the findings from the 2-D gel analysis were not due to artifacts and were specifically due to the differences in the oviductal proteomic profiles in follicular and luteal phases of the reproductive cycle. To minimize potential dedifferentiation of the OECs due to the in vitro culture, cells obtained from both groups were not cultured for more than 18 h, minimizing in vitro cell dedifferentiation. This short in vitro culture period allowed us to separate the OECs from other contaminating cells such as red and white blood cells. Furthermore, oviducts were obtained from pigs originating from the same breed, within the same age group that had the same diet. Western blot analysis using anti-HSP70 antibody and a monoclonal anti-pan cytokeratin clone C-II verified the quantitative results obtained using 2-D gel electrophoresis analysis. In agreement with 2-D gel image analysis results, the Western Journal of Proteome Research • Vol. 7, No. 7, 2008 2829
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(202)
P08728
Keratin, type I cytoskeletal 19
primary protein name
Creatine kinase B-type
Actin, cytoplasmic 1
8
18
12
potential function
-
Plays a role in channel-mediated K+ and Ca2+ transport in human syncytiotrophoblast43 -
694
731
619
159
100
272
MOWSE score (Mascot)
43858
53463
47532
47923
69225
84723
theoretical protein MW (Da)
17
20
21
4
3
9
peptides identified
sample peptide sequence(s)
K.IIAPPERK.Y, K.AGFAGDDAPR.A, R.GYSFTTTAER.E K.IIAPPERK.Y, K.DSYVGDEAQSK.R, R.VAPEEHPVLLTEAPLNPK.A K.LLIEMEQR.L, K.GGNMKEVFTR.F, R.LEQGQAIDDLVPAQK.-
R.VATVSLPR.S, K.LSSPATLNSR.V
K.FLPFKVVEK.K, K.NQLTSNPENTVFDAKR.L
K.IALLEEAR.R, K.QRIDEFEAM, R.LQDYEEKTR.K
R.SGYLLPDTK.A, K.GVVDSDDLPLNVSR.E
K.SILFVPTSAPR.G
K.LLEGEESR.L, R.EYQELMNVK.L, R.ASLEAAIADAEQR.G K.FASFIDK.V, R.QLETLAQEK.L, R.ASLEAAIADAEQR.G
K.AEFVEVTK.L, K.YLYEIAR.R, K.YICDNQDTISSK.L R.ADNLIPGTR.A, R.AQQLEQIR.K, K.YVPYVGDSKR.A R.IILLAEGR.L, K.QAQYLGMSR.E, K.LTEKQAQYLGMSR.E
K.TASDFISK.M, R.LKATQVSK.G, K.AGALNSNDAFVLK.T
sample peptide qasequence(s)
(B) OEC Protein Spots Increased 2-Fold or More in Luteal Phase Compared to the Follicular Phase
42674
41579
41579
2
23
3
2
1
peptides identified
2.26 ( 0.041
3.44 ( 0.046
2.50 ( 0.014
2.21 ( 0.013
2.67 ( 0.027
2.02 ( 0.011
average fold change ( SEM (Increase in luteal phase)
2.00 ( 0.044
2.39 ( 0.087
2.42 ( 0.235
3.18 ( 0.07
2.57 ( 0.157
2.76 ( 0.565
2.96 ( 1.169
2.80 ( 0.089
average fold change ( SEM (Increase in follicular phase)
(A) OEC protein spots increased 2-fold or more in follicular phase compared to the luteal phase, (B) OEC protein spots increased 2-fold or more in luteal phase compared to the follicular phase.
(199)
P05786
a
(147)
Q710C4
Serum albumin precursor Myo-inositol 1-phosphate synthase A1 Adenosylhomocysteinase May play a key role in the control of methylations via regulation of the intracellular concentration of adenosylhomocysteine (Uniprot) Keratin, type II cytoskeletal 8
(50)
(30)
P20305
(65)
match ID
primary accession (Uniprot)
sP02769
(166)
P05124
Q6AYK3
Gelsolin precursor
(160)
P60712
Actin, cytoplasmic 1
47966
62
254
(138)
P60712
Calreticulin precursor
72301
836
-
(90)
P18418
Heat shock 70 kDa protein 5
68586
91
549
(41)
Q3TI47
Ezrin
70871
82
-
(35)
P31976
Tumor rejection antigen gp96
92369
theoretical protein MW (Da)
44
371
(22)
Q3TUD6
potential function
MOWSE score (Mascot)
Potential target for phosphorylation on the surface of spermatozoa, may have a postspermatogenic role37 May play a role in the formation of a functional zona receptor complex on the surface of mammalian spermatozoa38 May control the ionic pump activity39and cavitation of the blastocyst40 Hsp70 might be present at very low amounts but is sufficient for the receptor to function41 May be involved in signal transduction events during or after sperm-egg interactions at fertilization42 -
(15)
Q95M18
Endoplasmin precursor
match ID
primary accession (Uniprot)
primary protein name
(A) OEC Protein Spots Increased 2-Fold or More in Follicular Phase Compared to the Luteal Phase
Table 1. Oviductal Epithelial Cell (OEC) Protein Spots Altered More than 2-Fold during Reproductive Cyclea
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Modulation of Oviductal Cell Proteome during Reproductive Cycle
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Figure 3. Western blot analysis of HSP 70 and cytoskeletal keratin. Antibodies against HSP 70 and cytoskeletal keratin were used to semiquantitatively compare the protein levels in follicular and luteal OEC samples. HSP 70 was increased in follicular oviductal samples compared to luteal samples and cytoskeletal keratin decreased in follicular samples compared to the luteal OEC samples. The Western blot analysis confirmed the changes observed using 2-D gel electrophoresis.
blot analysis also showed HSP70 upregulation in the follicular phase compared to the luteal phase. It is well-known that oviductal physiology undergoes cyclical changes under the influence of steroid hormones (reviewed in ref 18). These cyclical changes in the oviduct are associated with the production of an optimal microenvironment for fertilization and early embryonic development. To understand the maternal interaction with gametes and embryo, it is important to decipher and characterize different components of this environment. So far, comparison of the secretory oviductal proteins produced by the oviduct at different phases of the reproductive cycle has been the usual strategy for identifying the steroid regulated proteins. Porcine oviductal epithelial cells secrete at least 14 major proteins into the culture medium,19 including oviduct-specific glycoprotein, tissue inhibitor of metalloproteinase and plasminogen activator inhibitor-1.18 The expression of oviduct-specific glycoprotein is steroid-regulated20 and it has been shown that this protein is involved in modulation of sperm-zona binding and improving the efficiency of porcine fertilization in vitro.21 Three other reported secretory oviductal proteins with temporal and spatial regulation during the reproductive cycle are complement component C3b, the carboxy-terminal propeptide of alpha 1 (III) procollagen (PIICP), and the heavy chain variable region of IgA.22 The temporal and spatial differences in expression of these proteins in the oviduct at estrus and during early pregnancy suggest that they may protect the luminal environment and participate in extracellular matrix remodeling and gamete interactions.22 Other factors such as plasminogen activator inhibitor (PAI)-1 have been partially purified from the porcine oviduct during the estrous cycle and early pregnancy.23 Immunogold electron microscopy localized PAI-1 to the secretory granules and cilia of the isthmus.24 The expression of PAI-1 is steroid-regulated.18 It has been proposed that the isthmic PAI may play a role in protecting the preimplantation embryo from proteolytic degradation as well as in the regulation of extracellular matrix turnover and remodelling.24 All identified proteins in the current investigation that were altered during the reproductive cycle were further searched in the database of National Center for Biotechnology Information, NCBI (http://www.ncbi.nlm.nih.gov/)25 for more detailed information. Three of the nine proteins identified as being altered in oviductal OEC during the follicular phase belong to the heat shock protein (HSP)
family, namely, endoplasmin, tumor rejection antigen GP96 and heat shock protein 70-kDa protein 5. The presence of HSPs in the oviduct has been reported before. HSP60, for example, is reported to be abundant in the apical plasma membrane of the OECs.26 While HSPs are classically considered to be expressed mainly during cell stress, this is not necessarily the case for their expression in the oviduct. Previously, the presence of HSPs in healthy oviductal epithelial cells has been reported.26 However, the exact functions of the HSPs in the oviduct are yet not known. It is probable that HSPs play a major role in the modulation of the maternal communication with gametes and embryos. Georgiou and colleagues identified an up-regulation in the release of the HSPs by the oviduct in response to sperm arrival in the oviduct.27 They suggested that HSPs may have a beneficial role in the final maturational stages of gametes within the female reproductive tract and maintaining their viability. In line with that hypothesis, we found that HSP70 is upregulated in the oviduct during the follicular phase of the cycle, indicating that the oviduct increases HSP70 production at estrus to prepare its environment for the arrival of gametes. Another protein found to be increased in oviductal cells during the follicular phase compared to the luteal phase of the cycle was calreticulin. The increase in calreticulin is potentially correlated with HSPs, as the function of calreticulin is to act as a calcium binding chaperone, promoting correct folding of proteins. During the follicular phase of the cycle, the oviduct has a higher biosynthetic activity and produces higher amounts of proteins compared to the luteal phase of the cycle.17,28 It can therefore be envisaged that an increase in the production of calreticulin is essential for the increased protein production in the oviduct during the follicular phase. In addition, maintaining a high level of biosynthetic activity in the oviduct during the follicular phase would obviously result in increased energy demands at this site during the follicular phase compared to that of the luteal phase. The human homologue of creatine kinase B-type protein plays an important role modulating the energy demand in tissues that have large, fluctuating energy demands (including skeletal muscle, heart, brain and spermatozoa) by reversibly catalyzing the transfer of phosphate between ATP and phosphogens.25 The increase in production of creatine kinase in the oviduct during the follicular phase is probably essential for maintaining the oviductal higher metabolic activity during the follicular phase. Journal of Proteome Research • Vol. 7, No. 7, 2008 2831
research articles Another identified protein, ezrin, which was one of the proteins found to be upregulated during the follicular phase of the reproductive cycle compared to the luteal phase, belongs to the ERM (ezrin-radisin-moesin) family. It has a key role in cell surface adhesion by serving as an intermediate between the plasma membrane and the Actin cytoskeleton.29 It was reported that ezrin also plays a vital role in determining the survival of cells through the activation of phosphatidylinositol3-kinase/Akt pathway30 and that ezrin is present in mouse embryos through preimplantation development.31 In the light of these facts, the identification of ezrin and its upregulated presence in the oviduct during the follicular phase may suggest its contribution to oviductal preparation to support the potential zygote. When examining the upregulated oviductal proteins during the luteal phase compared to the follicular phase of the cycle, we found that gelsolin, which is both a cytoplasmic protein and secreted Ca2+-dependent Actin filament severing protein,32 is reduced in the follicular phase relative to those of the luteal phase. It is known that low levels of gelsolin protein are mainly present in undifferentiated and proliferative cell phenotypes, whereas high gelsolin expression is a property of differentiated and nonproliferative cells.33,34 Importantly, the gelsolin related cytoskeletal regulator Fliih (Flightless I homologue) is essential in the mammalian development, showing the potential importance of the identified protein in successful embryogenesis.35 Although homozygous mice knockouts of gelsolin are viable and fertile, they are reported to have defects in fibroblast motility.36 We suggest that the downregulation of the gelsolin protein in the follicular phase reflects the increased proliferation of the OECs observed during the follicular phase of the reproductive cycle. As gelsolin plays an important role in the development of the embryo in the oviduct before implantation, it is remarkable that gelsolin is synthesized by the OECs. It remains to be investigated if Gelsolin produced by OEC is also secreted into the lumen of the oviduct to interact with the potential zygote. Cytokeratins are intermediate filament keratins found in the intracytoplasmic cytoskeleton of epithelial tissue. There are two types of cytokeratins: the low weight, acidic type I cytokeratins and the high weight, basic or neutral type II cytokeratins. Cytokeratins are usually found in pairs comprising a type I cytokeratin and a type II cytokeratin. The 2-D gel image analysis and mass spectrometry identified upregulation of both type I and II cytokeratins in the luteal phase OEC samples compared to the follicular phase. With the use of a pan cytokeratin antibody that recognizes several different types of type I and II cytokeratins (cytokeratin 4, 5, 6, 8, 10, 13 and 18), two different cytokeratin molecules were identified in Western blot analysis. Both of these molecules showed upregulation in the luteal OEC samples compared to the follicular samples confirming the 2-D gel image analysis and mass spectrometry data. It must be noted that the observed protein alterations occurring during the reproductive cycle are likely to represent only a small fraction of the actual proteomic changes occurring in OECs. Even in the current investigation, we were unable to determine the identity of all protein spots detected to be differentially expressed in gels between follicular and luteal samples. Future experiments focusing on different compartments of oviductal cells may help in understanding further the nature of proteomic changes during the reproductive cycle in the female reproductive tract. Potentially, focusing on the surface proteome of the OECs may reveal a better understand2832
Journal of Proteome Research • Vol. 7, No. 7, 2008
Seytanoglu et al. ing about the interactions of the oviduct, gametes and the embryo. Furthermore, it is imperative to determine whether our findings are also echoed with other species such as the cow and sheep. Intraspecies proteomic differences may provide important insights into evolutionary variations in the reproductive mechanisms of different species.
Acknowledgment. We thank Mrs. Christine Bruce and Mrs. Sarah Elliott for their technical assistance and Department of Envoirnment Food and Rural Affairs, U.K., for financial support of this project. Supporting Information Available: Tables of oviductal epithelial cell protein spots with 2-fold or more increase in follicular phase compared to the luteal phase and in luteal phase compared to the follicular phase. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Yaniz, J. L.; Lopez-Gatius, F.; Hunter, R. H. Scanning electron microscopic study of the functional anatomy of the porcine oviductal mucosa. Anat. Histol. Embryol. 2006, 35 (1), 28–34. (2) Reischl, J.; Prelle, K.; Schol, H.; Neumuller, C.; Einspanier, R.; Sinowatz, F.; Wolf, E. Factors affecting proliferation and dedifferentiation of primary bovine oviduct epithelial cells in vitro. Cell Tissue Res. 1999, 296 (2), 371–383. (3) Donnez, J.; Casanas-Roux, F.; Caprasse, J.; Ferin, J.; Thomas, K. Cyclic changes in ciliation, cell height, and mitotic activity in human tubal epithelium during reproductive life. Fertil. Steril. 1985, 43 (4), 554–559. (4) Hafez, B.; Hafez, E. S. E.; Reproduction in Farm Animals, 7th ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2000; p 509. (5) Hammond, J.; Hammond, J.; Bowman, J. C.; Robinson, T. J.; Hammond’s Farm Animals, 5th ed.; Hammond, J., Jr.; Bowman, J. C,.; Robinson, T, J,., eds.; Edward Arnold: London, 1983. (6) Crow J, A. N. N.; Lewin, J.; Shaw, R. W. Morphology and ultrastructure of Fallopian tube epithelium at different stages of the menstrual cycle and menopause. Hum. Reprod. 1994, 9 (12), 2224– 2233. (7) Abe, H.; Oikawa, T. Observations by scanning electron microscopy of oviductal epithelial cells from cows at follicular and luteal phases. Anat. Rec. 1993, 235 (3), 399–410. (8) Fazeli, 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 (4), 509–517. (9) Birkhead, T. R.; Moller, A. P. Sexual selection and the temporal seperation of the reproductive events: sperm storage data from reptiles, birds and mammals. Biol. J. Linnean Soc. 1993, 50, 295– 311. (10) Bedford, J. M.; Breed, W. G. Regulated storage and subsequent transformation of spermatozoa in the fallopian tubes of an Australian marsupial, Sminthopsis crassicaudata. Biol. Reprod. 1994, 50 (4), 845–854. (11) Burkman, L. J.; Overstreet, J. W.; Katz, D. F. A possible role for potassium and pyruvate in the modulation of sperm motility in the rabbit oviducal isthmus. J. Reprod. Fertil. 1984, 71 (2), 367– 376. (12) Overstreet, J. W.; Cooper, G. W. Reduced sperm motility in the isthmus of the rabbit oviduct. Nature 1975, 258 (5537), 718–719. (13) Overstreet, J. W.; Katz, D. F.; Johnson, L. L. Motility of rabbit spermatozoa in the secretions of the oviduct. Biol. Reprod. 1980, 22 (5), 1083–1088. (14) McNutt, T. L.; Olds-Clarke, P.; Way, A. L.; Suarez, S. S.; Killian, G. J. Effect of follicular or oviductal fluids on movement characteristics of bovine sperm during capacitation in vitro. J. Androl. 1994, 15 (4), 328–336. (15) Satake, N.; Elliott, R. M.; Watson, P. F.; Holt, W. V. Sperm selection and competition in pigs may be mediated by the differential motility activation and suppression of sperm subpopulations within the oviduct. J. Exp. Biol. 2006, 209 (Pt 8), 1560–1572. (16) Bauersachs, S.; Rehfeld, S.; Ulbrich, S. E.; 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 (2), 449–466.
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