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a Department of Vet Clinical Studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, Scotland, United Kingdom
b Division of Development and Reproduction, Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, Scotland, United Kingdom
c Department of Obstetrics and Gynaecology, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden
d Department Obstetrics and Gynaecology, University of Edinburgh, Centre for Reproductive Biology, Edinburgh, EH3 9EW, Scotland, United Kingdom
e University of Nottingham, School of Biological Sciences, Division of Agriculture and Horticulture, Sutton-Bonington Campus, Loughborough, Leicestershire, LE12 5RD, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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metabolite was high at the time of tissue collection. T lymphocyte populations, identified by immunohistochemistry, had a distribution similar to that of cells expressing MCP-1 mRNA within the CL, but other cell types were also involved. These results demonstrate an increase in MCP-1 mRNA after functional luteolysis in the cow, which may be related to the influx of macrophages that occurs at this time.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interleukin-1 stimulate MCP-1 mRNA expression and secretion of the active protein [6, 7]. In recent years it has been suggested that cytokines, in particular the cytokine products of immune cells, may be involved in controlling ovarian function [8, 9]. The properties of specific monocyte attraction and production by cell types that are abundant in luteal tissue first led workers to consider a role for MCP-1 in initiating the influx of macrophages that is observed in the late-stage corpus luteum (CL). Macrophages are involved in the rapid destruction of luteal tissue that occurs after functional luteolysis [10, 11]. MCP-1 has been described in porcine luteal cells [12], and increased expression is seen in the CL of the rat prior to the influx of monocytes/macrophages in late-stage luteal tissue and following prolactin-induced regression [13, 14], findings that support the hypothesis of MCP-1 involvement in luteolysis.
As in other species, macrophages are involved in destruction of bovine luteal tissue after luteolysis [15], and a role for MCP-1 in controlling macrophage influx in the bovine CL has been suggested [16]. In addition, we have shown previously that there is a significant increase in the number of T lymphocytes (specifically CD8+, cytotoxic/suppressor T cells) within the bovine CL from Day 16 of the estrous cycle onward, prior to luteolysis [17]. The reason for this influx of T lymphocytes is unclear, but T lymphocytes are a potential source of MCP-1 [5]. In this study our primary aim was to investigate the expression of MCP-1 mRNA within the bovine CL during luteolysis using both the reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. In addition, in order to assess whether T lymphocytes might be involved in MCP-1 mRNA expression in the bovine CL, we used immunohistochemistry to analyze the distribution of these cells within sections of luteal tissue and correlated this with the distribution of MCP-1 mRNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The estrous cycles of 18 Holstein-Friesian heifers were synchronized using progesterone-releasing intravaginal devices (PRID; Sanofi Animal Health, Watford, UK) for 12 days and an injection of 500 µg cloprostenol, a synthetic prostaglandin F2 (PGF2; Estrumate; Schering Plough, Hertfordshire, UK) 2 days before PRID removal. The CL were collected at slaughter on Days 15–20 of the estrous cycle (estrus = Day 0). Luteal tissue was snap frozen in dry ice/isopentane and then stored at -70°C. Blood samples (7 ml) were collected once daily for 1–2 days prior to slaughter, and at slaughter, into lithium heparin-treated (143 U/tube) blood sample tubes (Vacutainer; Becton Dickinson, Oxford, UK). The plasma was collected after centrifugation at 1000 x g for 30 min and stored at -20°C prior to analysis of progesterone and 15-keto-13,14-dihydro-PGF2 (PGFM, the stable metabolite of PGF2) content.
Progesterone Assay and PGFM Assay
Plasma progesterone concentrations were measured using an RIA described by Corrie et al. [18], as modified by Law et al. [19]. All samples were measured in a single assay with an intraassay coefficient of variation of 13.9%. The assay sensitivity was 0.1 ng/ml, and values of less than 1 ng/ml were considered to indicate that functional luteolysis had taken place.
In addition to progesterone concentrations, PGFM was also measured in all plasma samples collected during the experiment. The samples were assayed by Professor Hans Kindahl, Swedish University of Agricultural Sciences, according to the method described by Granström and Kindahl [20]. The limit of detection was 8–10 pg/ml for 0.5 ml of plasma.
Extraction of mRNA and RT-PCR
RNA was isolated from CL using the guanidium thiocyanate method [21]. Concentrations were estimated by absorbance at 260 nm, and the samples were stored in diethyl pyrocarbonate-treated H2O at -80°C (1-µg aliquots) until required. The A260/A280 ratio for all RNA samples was > 1.6. First strand cDNA synthesis was carried out using a modification of the method described previously [22]. Briefly, total RNA (1 µg) was reverse transcribed in RTase buffer (Tris-HCl [250 mM, pH 8.3], KCl [375 mM], MgCl2 [15 mM]; Gibco BRL, Life Technologies, Paisley, Scotland, UK), dNTP mix (0.5 µM; Pharmacia Biotech, UK), random hexamers (50 µM; Pharmacia Biotech), RNasin (4 U; Promega UK Ltd., Southampton, UK), and Superscript II reverse transcriptase (13.5 U; Gibco BRL) at 37°C for 60 min. The reverse transcriptase reaction medium was diluted to 100 µl after the addition of 8 µl 10-strength PCR buffer and stored at -20°C. The PCR reaction was carried out using 6 µl of the diluted reverse transcriptase reaction (equivalent to 0.06 µg of the original total RNA) in 12 µl dH2O, 1 µl 10-strength PCR buffer, 100–200 pmol each of 5' and 3' primers, and 1 U Taq DNA polymerase (Gibco BRL) in a total volume of 20 µl.
The oligomers used for the PCRs were 5'-AACAGCTTCCCGCTGAAAC-3' and 5'-TCTGCACATAACTCCTTGCC-3' and corresponded to positions 22–41 (exon 1) and 291–272 (exon 3) of the bovine MCP-1 cDNA [23], respectively. Samples were heated to 94°C for 5 min and amplified for 30 cycles (93°C for 30 sec; 60°C for 30 sec; 72°C for 30 sec) using a Biometra Personal Cycler (Biometra, Maidstone, Kent, UK). The final 72°C incubation was continued for a further 5 min. Reverse transcriptase blanks (no RNA) and PCR blanks (no cDNA products) were included in each analysis. Products were visualized by ethidium bromide staining after electrophoresis on 4% agarose gels (NuSieve GTG Agarose; FMC Bioproducts, Rockland, ME). In all experiments, reverse transcriptase and PCR blanks were negative. Identities of the PCR product were confirmed by restriction endonuclease digestion and by DNA sequencing of representative samples. Quantification of the PCR products was performed using an NIH-Image analyzer (Bethesda, MD). RT-PCR for ATPase was used as an interassay control. The ATPase primers were 5'-ACGAACACCACT CCTGGATGAGC-3' and 5'-CACGGACGTCTCCAGGCTGTGTA-3', corresponding to positions 21–43 and 191–213 of a bovine plasma membrane calcium-pumping ATPase cDNA [24]. Under the conditions described here, the amount of amplified MCP-1 and ATPase cDNA produced was proportional to the number of thermocycles of the PCR reaction and the mass of RNA added to the reverse transcriptase reaction.
MCP-1 Riboprobes
The MCP-1 cDNA amplified by the PCR reaction described in the previous section was cloned into pGEM-T (Promega) using the manufacturer's procedure. The insert was sequenced to determine its identity and orientation within the plasmid. The construct was linearized with NcoI and used as a template to generate 35S-labeled antisense RNA using SP6 DNA-dependent RNA polymerase. A sense RNA probe was prepared using T7 DNA-dependent RNA polymerase after linearization with NsiI.
In Situ Hybridization
In situ hybridization was performed on luteal tissue from a limited number of animals. CL were selected from the group of cows that had not undergone luteolysis (progesterone > 1 ng/ml at slaughter, n = 3) and from the group of cows that had undergone luteolysis (progesterone < 1 ng/ml at slaughter, n = 3). Four serial sections were cut (14 µm) from each CL. Sections for in situ hybridization were dehydrated, fixed, and probed with 35S-labeled MCP-1 riboprobes [25]. After the final high-stringency wash, the sections were dipped in autoradiographic K2 photographic emulsion (Ilford Ltd., Mobberley, Cheshire, UK) and exposed for 2 wk at 4°C. Sections were then developed (Kodak D-19; Eastman Kodak, Rochester, NY) and fixed (Ilford Hypam fixer; Ilford Ltd.) before staining in hematoxylin and eosin. The sections were finally mounted in DPX mountant before microscopic examination using both light- and darkfield illumination on a Nikon Microphot-SA microscope (Nikon Instruments, Garden City, NY).
Image Analysis
The intensity of the in situ hybridization signal was analyzed using an NIH-Image analysis system (NIH, Bethesda, MD). The number of graphic pixels occupied by silver grains (identified by a set gray threshold) within a defined area of the tissue section was counted and presented as a percentage of the total pixel number within the defined area. The hybridization intensity is therefore presented as the percentage of occupied pixels within a defined area of the tissue. Three serial sections were analyzed for each CL. Two were probed with the antisense RNA probe, and the remaining section was probed with the sense probe. Background hybridization intensity, measured with the sense RNA probes, was subtracted from the measurements obtained with the antisense probes to give the final hybridization signal. Within each CL, three separate fields were analyzed for each probe.
Immunohistochemistry
Immunohistochemistry was performed on sections of frozen luteal tissue serial to those used for in situ hybridization. T lymphocytes were identified in sections of luteal tissue using an avidin-biotin complex (ABC) method [26], modified as described by Cobb and Watson [27]. The monoclonal antibody used was a specific marker for CD5+ T lymphocytes, CC17 (Institute for Animal Health, Compton, UK). This monoclonal antibody identifies mature T lymphocytes including CD8+ cytotoxic/suppressor T lymphocytes and CD4+ helper/inducer T lymphocytes [28]. Immunohistochemistry was performed using a Vectastain ABC kit (Vector Laboratories, Peterborough, UK). The peroxidase substrate used to visualize the product was 3-amino-9-ethyl carbazole (Vector Laboratories). Sections of bovine lymph node were processed with each batch of CL as positive control samples, and negative controls were included in which the monoclonal antibody was replaced with normal mouse serum.
Statistics
MCP-1 mRNA expression, measured by in situ hybridization, was analyzed by ANOVA using a mixed model. The data were fitted to the model Y = µ + Xi + Aj(i) + Bk(ij) + Cl(ijk) + ijklm, where Xi (fixed effect) represents CL collected before (i = 1) and after (i = 2) the onset of natural luteolysis, Aj represents cow number within treatments (random effect), Bk represents slide number within cows (random effect), and Cl represents replicate within slides (random effect). A similar procedure was performed for the reverse transcriptase data. In this case Bk represents the experiment number and Cl represents the replicate within experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thirteen animals had systemic progesterone concentrations consistent with a functional CL (systemic progesterone concentration > 1 ng/ml) at the time of slaughter. Plasma progesterone concentration (mean ± SEM) was 3.6 ± 1.0 ng/ml (range 1.9–6.9 ng/ml). Five animals had undergone luteolysis at the time of slaughter (systemic progesterone concentration < 1 ng/ml). The mean plasma progesterone concentration for these animals was 0.6 ± 0.13 ng/ml (range 0.2–0.9 ng/ml).
PGFM Analysis
PGFM is an accurate indicator of PGF2 concentrations in plasma [29], but analysis of single daily blood samples for PGFM concentrations is of limited use because of the pulsatile nature of PGF2 release [30]. However, previous studies using the same assay technique have demonstrated that systemic concentrations of more than 300 pM (105 pg/ml) are only likely to occur during luteolysis [30]. Therefore PGFM concentrations were used to identify animals that experienced luteolytic concentrations of PGF2 prior to, or at the time of, slaughter.
The 5 animals that had undergone luteolysis all showed PGFM concentrations higher than 105 pg/ml in at least 1 sample collected either 1 or 2 days before or at the time of slaughter. Nine of the twelve animals with a functional CL (progesterone > 1 ng/ml) at the time of slaughter had PGFM concentrations higher than 105 pg/ml on the day of slaughter (range 108–243 pg/ml), but all samples collected from these animals in the days preceding slaughter contained less than 105 pg/ml PGFM (range 45–78 pg/ml). These results, in combination with the progesterone results, indicate that these cows were undergoing luteolysis at the time of the trial as would be expected at this stage of the estrous cycle. During natural luteolysis, progesterone concentrations do not fall to below 1 ng/ml (taken to indicate completion of functional luteolysis) until 36–48 h after the first large pulses of PGF2 are released [30].
RT-PCR
A 270-base pair (bp) cDNA product was amplified by RT-PCR. The PCR products from 8 animals (3 preluteolytic and 5 postluteolytic) are shown in Figure 1a. The identity of the band was confirmed by DNA sequencing and was 100% homologous with the expected cDNA sequence described by Wempe et al. [23]. The intensity of the amplified cDNA product, measured after ethidium bromide staining of agarose gels, was significantly greater (p < 0.05) in luteal tissue from animals that had already undergone functional luteolysis (n = 5) compared to the other animals in the group (n = 9) that still had a functional CL (Fig. 2). There was no difference in the intensity of the amplified band obtained with the ATPase oligomers in CL collected before and after the onset of structural luteolysis (Fig. 1b).
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In Situ Hybridization
In situ hybridization was performed on luteal tissue from a subset of animals before (n = 3) and after (n = 3) the onset of functional luteolysis. Representative in situ results for both groups of animals are presented in Figure 3. The intensity of the signal (the number of graphic pixels containing a silver grain) was significantly greater (p < 0.05) in CL from cows after functional luteolysis compared to CL collected before luteolysis (Table 1). In the preluteolytic animals the most intense signal was seen in a CL collected from a cow (number 959) that, out of the three preluteolytic animals studied, had the highest plasma PGFM concentration and lowest progesterone concentration at the time of tissue collection (Table 1).
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Immunohistochemistry
Immunohistochemistry was performed on serial sections from the same CL used for in situ hybridization (Fig. 4). Cells that stained positively with the CD5+ T lymphocyte marker were present in all sections of CL studied. They were distributed throughout the luteal tissue as individual cells and in small clumps. Positive staining was absent from negative control samples (Fig. 4c).
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When compared to serial sections from the in situ hybridization studies, areas of luteal tissue were seen in which the distribution of cells expressing mRNA for MCP-1 was similar to the distribution of T lymphocytes (Fig. 4, a and b); however, there were also other regions where this was not the case. In addition, significant numbers of T lymphocytes were also present in CL where MCP-1 mRNA expression was minimal (preluteolysis).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression of mRNA encoding MCP-1 in the CL is not unexpected because of the large number of cell types present within luteal tissue that are potential sources of MCP-1. These include endothelial cells, fibroblasts, lymphocytes, and macrophages [2]. These cell types are present throughout the estrous cycle, although their proportions relative to large and small luteal cells vary [17, 31, 32]. Detailed studies will be required to identify all of the different cell types involved in expression of MCP-1 mRNA in the bovine CL.
We have shown previously that there is an increase in the numbers of CD8+ T lymphocytes in bovine luteal tissue from Day 15 of the estrous cycle onward [17]. The role of these cells at this stage of the estrous cycle is not clear, although recently there has been increasing interest in the potential local effects of immune cells and their cytokine products, particularly around key events such as ovulation and luteolysis [33, 34]. Uterine PGF2 is known to be the luteolytic factor in the cow [35]. However, events occurring at the level of the CL around the time of luteolysis are much less clearly defined.
T lymphocytes are a potent source of MCP-1 [5, 36]. They may also act indirectly, through their cytokine products, including tumor necrosis factor and interleukin-1, to promote expression of MCP-1 mRNA by other cell types [6, 7]. In the present study, T lymphocytes were distributed in a pattern similar to that for those cells expressing mRNA for MCP-1 in some areas of luteal tissue. It is possible that one of the roles of the increased numbers of T lymphocytes in the CL around natural luteolysis [17] is the production of MCP-1 to recruit macrophages that are then involved in structural luteolysis. However, it is likely that other, as yet uncharacterized, cell types within the CL are also significant in the production of MCP-1 around the time of luteolysis. Further studies will be required to more clearly identify the cell types involved at different stages of luteolysis.
There must also be a signal that causes increased expression of mRNA encoding MCP-1 within luteal tissue. The expression of mRNA encoding MCP-1 was significantly increased in CL from animals after functional luteolysis and also from one animal prior to the completion of functional luteolysis (progesterone > 1 ng/ml, cow 959, Table 1). The potentially significant finding in this individual cow was the high systemic concentration of PGFM in plasma at the time of CL collection. MCP-1 mRNA expression as demonstrated by RT-PCR and in situ hybridization was higher in this animal compared to the other preluteolytic cows. This suggests that PGF2 released during luteolysis may be involved in activating the expression of mRNA encoding MCP-1 in the CL. Future studies will address this hypothesis.
Other factors are also likely to be involved in increased expression of MCP-1 mRNA. For example, increased amounts of tumor necrosis factor have been measured in the bovine CL after functional luteolysis [37]. As discussed previously, MCP-1 expression may be enhanced by this cytokine as well as other cytokines that have not been investigated in detail within the bovine CL [6, 7]. Declining progesterone concentrations during luteolysis may also be the stimulus for enhanced MCP-1 mRNA expression in the cow, as has been suggested in the rat [14]. However, this appears less likely when the single cow discussed above is considered (cow 959, Table 1). The increase in MCP-1 expression occurred while progesterone concentrations were still high.
It is not clear from the results of the present study whether MCP-1 is also produced by luteal cells themselves. Hosang et al. [12] described expression of MCP-1 by luteal cells from the pig, but this study used dispersed luteal tissue that would contain large numbers of endothelial cells and fibroblasts that could themselves act as the source of MCP-1.
In conclusion, the results of this study provide further evidence supporting a role for MCP-1 in the attraction of monocytes/macrophages into luteal tissue after functional luteolysis. Further studies are required to determine the factors involved in regulating MCP-1 expression and to establish the cell types involved.
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FOOTNOTES |
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2 Correspondence. FAX: 0131 650 6588; lesleya{at}lab0.vet.ed.uk
Accepted: July 13, 1998.
Received: November 5, 1997.
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  T. Kisliouk, A. Friedman, E. Klipper, Q.-Y. Zhou, D. Schams, N. Alfaidy, and R. Meidan Expression Pattern of Prokineticin 1 and Its Receptors in Bovine Ovaries During the Estrous Cycle: Involvement in Corpus Luteum Regression and Follicular Atresia Biol Reprod, May 1, 2007; 76(5): 749 - 758. [Abstract] [Full Text] [PDF]
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 D. H. Townson Immune cell-endothelial cell interactions in the bovine corpus luteum Integr. Comp. Biol., December 1, 2006; 46(6): 1055 - 1059. [Abstract] [Full Text] [PDF]
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A. R. Liptak, B. T. Sullivan, L. E. Henkes, M. P.B. Wijayagunawardane, A. Miyamoto, J. S. Davis, B. R. Rueda, and D. H. Townson Cooperative Expression of Monocyte Chemoattractant Protein 1 Within the Bovine Corpus Luteum: Evidence of Immune Cell-Endothelial Cell Interactions in a Coculture System Biol Reprod, May 1, 2005; 72(5): 1169 - 1176. [Abstract] [Full Text] [PDF]
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K. Nagaosa, A. Shiratsuchi, and Y. Nakanishi Determination of Cell Type Specificity and Estrous Cycle Dependency of Monocyte Chemoattractant Protein-1 Expression in Corpora Lutea of Normally Cycling Rats in Relation to Apoptosis and Monocyte/Macrophage Accumulation Biol Reprod, November 1, 2002; 67(5): 1502 - 1508. [Abstract] [Full Text] [PDF]
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 V. A. Cavicchio, J. K. Pru, B. S. Davis, J. S. Davis, B. R. Rueda, and D. H. Townson Secretion of Monocyte Chemoattractant Protein-1 by Endothelial Cells of the Bovine Corpus Luteum: Regulation by Cytokines But Not Prostaglandin F2{alpha} Endocrinology, September 1, 2002; 143(9): 3582 - 3589. [Abstract] [Full Text] [PDF]
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K. H. H. Wong, H. Negishi, and E. Y. Adashi Expression, Hormonal Regulation, and Cyclic Variation of Chemokines in the Rat Ovary: Key Determinants of the Intraovarian Residence of Representatives of the White Blood Cell Series Endocrinology, March 1, 2002; 143(3): 784 - 791. [Abstract] [Full Text] [PDF]
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D. H. Townson, C. L. O'Connor, and J. K. Pru Expression of Monocyte Chemoattractant Protein-1 and Distribution of Immune Cell Populations in the Bovine Corpus Luteum Throughout the Estrous Cycle Biol Reprod, February 1, 2002; 66(2): 361 - 366. [Abstract] [Full Text] [PDF]
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K. K. Olson, L. E. Anderson, M. C. Wiltbank, and D. H. Townson Actions of Prostaglandin F2{{alpha}} and Prolactin on Intercellular Adhesion Molecule-1 Expression and Monocyte/Macrophage Accumulation in the Rat Corpus Luteum Biol Reprod, March 1, 2001; 64(3): 890 - 897. [Abstract] [Full Text]
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E. Asselin, G. A. Johnson, T. E. Spencer, and F. W. Bazer Monocyte Chemotactic Protein-1 and -2 Messenger Ribonucleic Acids in the Ovine Uterus: Regulation by Pregnancy, Progesterone, and Interferon-{{tau}} Biol Reprod, March 1, 2001; 64(3): 992 - 1000. [Abstract] [Full Text]
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A. Friedman, S. Weiss, N. Levy, and R. Meidan Role of Tumor Necrosis Factor {alpha} and Its Type I Receptor in Luteal Regression: Induction of Programmed Cell Death in Bovine Corpus Luteum-Derived Endothelial Cells Biol Reprod, December 1, 2000; 63(6): 1905 - 1912. [Abstract] [Full Text]
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K. K. Olson and D. H. Townson Prolactin-Induced Expression of Intercellular Adhesion Molecule-1 and the Accumulation of Monocytes/Macrophages During Regression of the Rat Corpus Luteum Biol Reprod, June 1, 2000; 62(6): 1571 - 1578. [Abstract] [Full Text]
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S. E. Bove, M. G. Petroff, M. Nishibori, and J. L. Pate Macrophage Migration Inhibitory Factor in the Bovine Corpus Luteum: Characterization of Steady-State Messenger Ribonucleic Acid and Immunohistochemical Localization Biol Reprod, April 1, 2000; 62(4): 879 - 885. [Abstract] [Full Text]
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