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a Department of Animal Sciences, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691-4096
b Department of Pharmacology, Okayama University Medical School, Okayama 700, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2) (n = 4 CL per time point). The greatest amount of MIF mRNA was observed in Day 5 CL compared with midcycle and Day 18 CL. Messenger RNA for MIF in CL collected 0.5 h post-PGF2 was greater than in midcycle and all other regressing CL. Immunohistochemical analysis (n = 4) revealed that MIF was present in the bovine CL throughout the estrous cycle and appeared to be localized to large luteal cells. It was concluded that MIF is produced within the bovine CL, mRNA expression is maximal in the early CL, and the protein is primarily localized to large luteal cells. The functional significance of MIF remains to be determined.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) and interferon-
(IFN-) [4]. In turn, MIF stimulates TNF- production by macrophages in an autocrine and paracrine fashion, thus functioning in a pro-inflammatory loop [4]. Together, these findings have prompted investigations leading to the discovery of MIF in a variety of tissues. MIF is also now known to be produced in differentiating and proliferating tissues such as early embryonic chicken lens [5], corneal epithelium [6], and basal layers of human skin epidermis [7]. Various reproductive organs such as the uterus, oviduct, and ovary of the pregnant mouse [8], as well as human granulosa cells [9] and rat Leydig cells, also produce and secrete MIF [10]. Perhaps most interesting is the discovery that MIF is secreted by the pituitary, and its function is to inhibit the anti-inflammatory and immunosuppressive effects of glucocorticoids, thus promoting immune responses [11].
Formation of the corpus luteum (CL) is a rapid event involving both proliferation and differentiation of follicular cells [12]. Immune cells and cytokines are thought to be involved in luteal formation and regression in a variety of species (reviewed in [13]). The bovine CL expresses the genes for the cytokines interleukin-1ß (IL-1ß), IFN-, and TNF- [14]. Additionally, it has been determined that these cytokines all have profound effects on luteal cells in vitro [15–18]. In light of its roles in cellular differentiation and immunoregulation, it seems logical to propose a role for MIF in the processes of formation and regression of the CL. Therefore, the objectives of this study were to determine whether the MIF gene is expressed in the bovine CL at various stages of luteal life span and regression and to elucidate the cellular localization of the MIF protein in the CL.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dairy cows exhibiting typical estrous cycles were used in accordance with protocols approved by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University. CL were removed via transvaginal incision [19] at the following time points during the estrous cycle: Day 5 (early), Days 9–12 (mid), Day 18 (late), and at 0.5, 1, 4, 12, 24, or 36 h (regressing) after a luteolytic injection of prostaglandin F2 (PGF2) administered on Days 9–12 of the cycle (n = 4 CL per time point). Each CL was immediately quartered upon collection. One quarter of the CL was fixed in Bouin's fixative (Sigma, St. Louis, MO), and the remainder of the tissue was immediately frozen in liquid nitrogen and stored until RNA or protein extraction. Blood samples were collected from the tail vein, and plasma progesterone was quantified by ELISA, as previously validated in this laboratory [20], to verify the stage of the estrous cycle and to confirm induction of luteal regression.
RNA Isolation
RNA was extracted from luteal tissue using TRIzol. Bovine peripheral blood lymphocytes were isolated to be used as positive controls for MIF mRNA. Blood (200 ml) was collected from the jugular vein into a sterile bottle containing sodium heparin at a final concentration of 140 IU/ml and centrifuged at 1000 x g for 10 min at room temperature. Buffy coats were aspirated and diluted 1:1 with sterile PBS, layered onto Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden), and centrifuged at 400 x g for 30 min at room temperature. The lymphocyte layers were aspirated, diluted 1:1 with PBS, and washed once at 400 x g and twice at 300 x g. Approximately 1.7 x 108 cells were seeded in RPMI containing 10% fetal calf serum, 2 mM glutamine, and 50 µg/ml gentamicin in a 75-cm2 flask. The lymphocytes were then treated with phorbol 12-myristate 13-acetate (1 ng/ml) together with ionomycin (5 ng/ml) to stimulate production of MIF [3]. For Northern analysis, the cells were harvested and the RNA extracted using TRIzol.
Generation of a Partial cDNA for Bovine MIF
Total RNA was extracted from CL removed 24 h after an i.m. injection of a luteolytic dose of PGF2 (25 ml Lutalyse; Upjohn, Kalamazoo, MI), using TRIzol (Gibco, Grand Island, NY) according to the manufacturer's instructions. Reverse transcription of the RNA was performed using murine Moloney leukemia virus reverse transcriptase and oligo(dT) primers. Polymerase chain reaction was carried out for 50 cycles (94°C denaturing for 30 sec, 53°C annealing for 30 sec, 72°C extension for 1 min) using a thermal cycler (Perkin-Elmer, Irvine, CA; Model 9700). The MIF primers were 5'-CTCTCCGAGCTCACCCAGCAG-3' (forward) and 5'-CGCGTTCATGTCGTAATAGTT-3' (reverse), corresponding to bases 58–78 and 292–312, respectively, of the human MIF cDNA sequence [6]. The resulting 255-base pair (bp) polymerase chain reaction product was ligated into the pGEM T-Easy vector (Promega, Madison, WI) and transformed into competent DH5a Escherichia coli (Gibco). The cloned plasmid was then purified and sequenced (ACGT, Inc., Northbrook, IL) to confirm its identity as MIF.
For manufacture of the cDNA probe, the insert was cut from the vector with the restriction enzyme EcoRI. The MIF insert served as a template to synthesize a [32P]dCTP (3000 Ci/mmol) (Amersham Pharmacia Biotech, Piscataway, NJ)-labeled cDNA probe using the random primer method.
Northern Analysis
Total RNA (20 µg) was separated by denaturing gel electrophoresis and transferred by capillary action to a Hybond-N nylon membrane (Amersham Pharmacia Biotech). The RNA was fixed to the membrane by baking between 2 sheets of Whatman (Clifton, NJ) filter paper in an 80°C oven for 2 h.
Prehybridization and hybridization were performed at 65°C in a Hybaid (Teddington, England) hybridization oven. The membrane was prehybridized in Rapid-Hyb hybridization buffer (Amersham Pharmacia Biotech) for 30 min. Approximately 1 x 106 cpm/ml of random primer-labeled [32P]cDNA (specific activity of the probe was approximately 1.9 x 109 cpm/µg) was denatured and added directly to the prehybridization buffer. After 1.5 h of hybridization, the membrane was washed with gentle agitation as follows: 20 min at room temperature in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), 0.1% (w:v) SDS; twice for 15 min at 65°C in single-strength SSC, 0.1% SDS; and once for 15 min at 65°C in 0.1-strength SSC, 0.1% SDS. The membranes were then exposed to Fuji (Tokyo, Japan) RX film at -70°C.
To normalize for loading differences among samples, the blots were stripped and labeled with an [32P]dCTP random primer-labeled human 18S cDNA probe (donated by Dr. Catherine Ernst, East Lansing, MI). Densitometric readings of MIF mRNA normalized for 18S were used for statistical analysis. The use of 18S as a standard for fully functional and regressing luteal tissue was validated by slot-blot analysis of increasing concentrations (5–35 µg) of luteal RNA. The blot was hybridized with the 18S probe as described above, and densitometric readings were used for linear regression analysis to determine the concentration at which 18S expression was maximal. There were no observable differences in 18S RNA in luteal tissue at the time points used in these experiments.
Western Analysis
For extraction of protein, the luteal tissue was homogenized in lysis buffer (1% igepal in PBS, 25 mM iodoacetamide, 1 mM PMSF) using a Polytron (Brinkmann Instruments, Westbury, NY) tissue homogenizer. The homogenate was then centrifuged at 40 000 x g for 1 h at 4°C, and the supernatant was used for protein analysis. Quantification of protein was performed by Bradford assay (Bio-Rad Labs., Richmond, CA). The protein (50 µg) was resolved by SDS-PAGE (4% stacking gel, 15% separating gel) and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA) for immunoblotting. After a 1-h incubation in blocking solution (20 mM Tris-buffered saline, pH 7.4, 0.1% Tween 20, 10% nonfat dry milk) at room temperature, the membrane was washed 3 times, 5 min each, in PBS and incubated with a 1:1500 dilution of the primary antibody (rabbit anti-bovine MIF polyclonal antiserum; Nishibori et al. [21]) in T-TBS (20 mM Tris-buffered saline, pH 7.4, 0.1% Tween 20, 1% BSA) at 4°C overnight. The membrane was then washed 3 times, 5 min each, in PBS and incubated with a 1:200 dilution of the secondary antibody (biotinylated goat anti-rabbit IgG; Vector, Burlingame, CA) in T-TBS for 30 min at room temperature. MIF protein (12 kDa) was detected using an avidin-biotin-peroxidase (ABC) kit (Vector), and diaminobenzidine/nickel chloride (Vector) was used as the substrate for the enzyme reaction.
Immunohistochemistry
The antibody to bovine MIF was used to localize MIF protein in paraffin sections of luteal tissue collected at the same times as those used for Northern analysis (n = 4 CL per time point). Cardiac muscle and pituitary tissue, used as positive and negative controls, respectively, were collected from necropsied cattle at the Ohio Agricultural Research and Development Center and fixed as described for luteal tissue. The tissue was processed, embedded in paraffin, and sectioned at a thickness of 6 µm. Four staining runs were performed with one run consisting of one slide from each time point to be analyzed.
For immunohistochemistry, the paraffin was cleared from the sections with xylene, and the tissues were rehydrated in a graded series of ethanol. The tissue sections were then quenched of endogenous peroxidase activity in 3% H2O2 in methanol for 5 min and blocked with 10% normal goat serum in 0.1 M PBS containing 1% BSA (PBS-1% BSA). Endogenous biotin was blocked with an avidin/biotin blocking kit (Vector) according to manufacturer's instructions. The sections were then washed 3 times, 5 min each, in PBS and incubated for 1 h at room temperature with a 1:2000 dilution (7 µg/ml) of rabbit polyclonal anti-MIF antiserum in PBS-1% BSA [21]. Negative controls consisted of elimination of the primary antibody or replacement of the primary antibody with nonimmune rabbit IgG (7 µg/ml). After washing 3 times, 5 min each, in PBS, the sections were incubated with a 1:200 dilution of biotinylated goat anti-rabbit IgG secondary antibody (Vector) for 30 min at room temperature. MIF was detected using an avidin-biotin-peroxidase (ABC) kit (Vector), and diaminobenzidine (Vector) was used as the substrate for the enzyme reaction. The sections were lightly counterstained with hematoxylin and dehydrated.
Statistical Analysis
The densitometric ratios of MIF:18S obtained from the Northern blots were examined by one-way ANOVA, and significant differences among treatments were subsequently established by the Student-Newman-Keuls method. The ratios from Days 5, 11, and 18 CL were log-transformed. Differences were considered significant at a level of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasma progesterone concentrations increased from Day 5 through Day 18 of the natural estrous cycle and decreased throughout PGF2-induced luteal regression. The progesterone value for individual animals was consistent with the stage at which the CL was collected (data not shown).
Cloning of Partial Sequence of MIF cDNA
The partial cDNA sequence obtained for bovine MIF was compared with that of the human and rat and was found to be 95% and 91% homologous, respectively. The sequence was submitted to GenBank and was assigned the accession number AF 119571.
Northern Analysis of MIF mRNA Expressionin the Bovine CL
A single band at approximately 0.6 kilobases (kb) was observed in all luteal RNA samples (Fig. 1A). This was the predicted size for the MIF mRNA. An intense signal was also observed at the same position in the stimulated lymphocyte positive control sample, further validating the identity of this band as MIF. A second unidentified band was also visible at 2.5–3 kb in some of the samples, most notably Day 5 and the positive lymphocyte control. Densitometric analysis of the 0.6-kb band, and normalization of these values to that of the respective 18S band, revealed that the greatest amount of MIF mRNA was in Day 5 CL compared with midcycle and Day 18 CL (Fig. 2A; P < 0.05). At 0.5 h post-PGF2, MIF mRNA was greater than in midcycle and all other regressing CL (Fig. 2B; P < 0.05).
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Western Analysis of MIF Protein
To confirm the specificity of the rabbit anti-bovine MIF primary antibody to be used for immunohistochemistry, Western analysis was performed. One CL from each time point of the natural estrous cycle and throughout regression was analyzed. The MIF antibody bound specifically to the 12-kDa MIF monomer and in some CL (Days 5, 11, 18, and 24 and 36 h post-PGF2) to the 24-kDa dimeric form of MIF (Fig. 3). The Western analysis was performed only to verify the specificity of the antibody and was not intended to be quantitative in any way.
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Immunohistochemical Localization of MIF in the Bovine CL
MIF protein appeared to be predominantly expressed in large luteal cells of the bovine CL throughout the estrous cycle and luteolysis (Figs. 4 and 5). Staining for MIF was readily observed during all three stages (Days 5, 11, and 18) of the estrous cycle (Fig. 4, A–C). In the Day 5 (early) CL, MIF appeared to be generally distributed throughout the tissue (Fig. 4A). Although it is difficult to distinguish different cell types with great certainty in the Day 5 CL, it appeared that some large cells exhibited more intense staining. In the midcycle CL, luteal cells are fully differentiated, and MIF was clearly observed in large luteal cells (Fig. 4B). At this time there also appeared to be a second group of large luteal cells that exhibited little to no staining for MIF. By Day 18 the overall intensity of the staining for MIF seemed to be slightly diminished; however, minor differences between the two large luteal cell populations could still be observed.
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Staining for MIF in sections from CL collected 0.5, 1, 4 (Fig. 4, D–F), and 12 h (Fig. 5A) post-PGF2 was generally similar to that for midcycle or Day 18 CL sections. As in the fully functional CL, MIF was found predominantly in the large luteal cells, with occasional lighter staining observed in smaller cells. By 24 and 36 h post-PGF2, however, several striking differences were observed (Fig. 5, B and C). In the 24-h post-PGF2 CL, marked deterioration of the vascular/interstitial tissue was readily observed surrounding large luteal cells that were intensely stained for MIF (Fig. 5B). At this time, vascular elements (small arrows) and probable immune cells (arrowhead), in addition to large luteal cells, stained positively for MIF. Finally, by 36 h post-PGF2 the overall intensity of staining for MIF seemed to be dramatically reduced; however, some vascular elements (small arrows) were still faintly stained for MIF (Fig. 5C). Rabbit IgG and negative controls (no primary antibody) were absent of any staining for MIF in all tissues examined (Fig. 5D).
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The pituitary, a positive control tissue, contained cells with strong cytoplasmic and occasional nuclear staining for MIF (Fig. 5E). Cardiac muscle was used as a negative control tissue. There was no staining for MIF in the myocardial cells; however, some staining of blood vessels was apparent (Fig. 5F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Functionally, MIF may serve multiple roles depending on the stage of the estrous cycle. There is evidence that immune cells and cytokines are involved in all stages of luteal life span, and that many immune cells and cytokines have opposing functions during formation and regression of the CL (reviewed in [13]). Therefore, it is not surprising that MIF is present during these stages of the luteal life span. As described by Brännström and Norman [13], luteinization is similar to tumor formation; and accordingly, macrophages are found in large numbers in the newly formed CL. Luteinization is also similar to wound healing. Macrophages within wounds ingest dead and senescent cells and extracellular debris, as well as producing factors that promote collagen synthesis, fibroplasia, and angiogenesis. These factors include fibroblast growth factor, insulin-like growth factor-1, transforming growth factor-ß, prostacyclin, interleukin-1, and TNF- [22, 23]. Therefore, one of the functions of MIF in the early developing CL may be to activate and retain macrophages within the tissue to promote tissue remodeling and angiogenesis during luteinization. The presence of greater quantities of MIF mRNA during luteinization is also consistent with previous reports of MIF in other differentiating and proliferating tissues [5–7]. One of the many putative functions of MIF is to regulate cellular differentiation. It was recently reported that granulosa cells secrete greater concentrations of MIF when stimulated by hCG [24]. Perhaps MIF is stimulated in the luteinizing follicle by the LH surge.
By Day 11, the cells of the CL are fully differentiated and functional. Examination of a Day 11 CL stained for MIF reveals that the large luteal cells stain positively for MIF. Closer scrutiny reveals two distinct populations of large luteal cells, one population that stains intensely for MIF and another that contains little to no positive staining for MIF. In human follicles, MIF mRNA and protein are present in the granulosa cells but not in the theca cells [9]. Large luteal cells of cattle are both granulosa and theca derived [25]; it is possible that since the granulosa cells express the MIF gene, it is only the granulosa-derived large luteal cells that continue to express the gene once the CL is fully differentiated. Therefore, the large luteal cells that do not stain positively for MIF may be theca-derived large cells.
During PGF2-induced luteal regression, a significant increase in MIF mRNA was observed in CL collected at 0.5 h. By 1 h post-PGF2, the steady-state quantities of MIF mRNA had returned to midcycle levels and remained low through 36 h. Although the MIF protein was not quantitatively assessed in this study, it was clear from the immunohistochemical analysis that MIF was present throughout luteal regression, at least up to 24 h post-PGF2. By 24 h post-PGF2 the CL was visibly deteriorated. The interstitial tissue of the CL was almost completely degenerated, but the remaining large luteal cells and vascular elements were stained intensely for MIF. Light staining of vascular elements could also be observed in CL collected 36 h post-PGF2. Synthesis of MIF in the vascular elements at the end of luteolysis may be at least partly stimulated by the milieu of cytokines, prostaglandins, and other molecules, such as endothelin-1, present at this time.
During luteal regression, it can be hypothesized that the primary role of MIF is to regulate immune cell function. Monocyte chemoattractant protein-1 (MCP-1) is produced by the bovine and ovine CL and is up-regulated during the time of luteolysis [26, 27]. In the ovine CL, MCP-1 was found to be maximally up-regulated between 4 and 8 h after PGF2 administration; however, in situ hybridization revealed that the MCP-1 message was not colocalized with large luteal cells, which contain the PGF2 receptors [27]. Therefore, it is possible that PGF2 may indirectly stimulate MCP-1 in the bovine CL via activation of an intermediate signal, perhaps MIF. Since MIF mRNA is elevated 0.5 h after PGF2 administration, several hours before MCP-1 message is increased, MIF from the large luteal cells may activate resident macrophages and/or T cells, which in turn could produce MCP-1 to recruit monocytes into the regressing CL [28, 29]. Continued production of MIF throughout regression may function to retain and stimulate macrophages to phagocytose cellular debris [30, 31] during the final phases of luteolysis. Messenger RNA for other cytokines such as TNF- and IFN- have been found in the regressing CL [14]. Both TNF- and IFN- increase luteal prostaglandin production and inhibit progesterone synthesis in bovine luteal cells [16, 17]. These cytokines are also known to stimulate MIF production, and MIF in turn is known to stimulate TNF- production in a pro-inflammatory loop [4]. Additionally, MIF may stimulate major histocompatibility complex (MHC) class II molecules on luteal cells and therefore play a more direct role in luteal regression. The ability of MIF to induce HLA-DR (human MHC type II) gene expression in human monocytes was demonstrated by Weiser et al. [32]. Both class I and class II MHC molecules are present on bovine luteal cells [33], and class II MHC molecules increase during luteolysis [34].
In conclusion, MIF mRNA is most abundant during the early phase of the estrous cycle and is rapidly elevated following PGF2 administration. Not only is the MIF gene expressed in the bovine CL, but the MIF protein is also present throughout the estrous cycle and appears to be localized to large luteal cells. During luteinization, MIF may be present as a differentiation/proliferation factor of luteal cells and/or to activate resident macrophages present at this time. During luteolysis, PGF2 may stimulate MIF to assist with the facilitation of luteal regression, and MIF may in turn serve to activate macrophages and T cells. Additionally, MIF may stimulate other cytokines, increase MHC class II molecule expression, and stimulate macrophage phagocytosis of dead luteal cells [30–32]. At this time, the functional significance of MIF within the CL remains to be elucidated, and further studies are currently in progress.
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FOOTNOTES |
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1 This research was supported by USDA Grant No. 96-35203-3355 to J.L.P. Salaries and research support provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. 
2 Correspondence: J.L. Pate, Department of Animal Sciences, Ohio Agricultural Research and Development Center, 1680 Madison Avenue, Wooster, OH 44691-4096. FAX: 330 263 3949; pate.1{at}osu.edu
3 Current address: Center for Reproductive Sciences, Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160.
Accepted: November 10, 1999.
Received: July 12, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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alters bovine luteal cell synthetic capacity and viability. Endocrinology 1992; 130:854–860.. Biol Reprod 1998; 58:169–174. induction of major histocompatibility complex antigens on cultured bovine luteal cells. Biol Reprod 1989; 40:453–457.[Abstract]This article has been cited by other articles:
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  E. Nilsson, N. Rogers, and M. K Skinner Actions of anti-Mullerian hormone on the ovarian transcriptome to inhibit primordial to primary follicle transition Reproduction, August 1, 2007; 134(2): 209 - 221. [Abstract] [Full Text] [PDF]
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 M. J. Cannon and J. L. Pate Indoleamine 2,3-Dioxygenase Participates in the Interferon-gamma-Induced Cell Death Process in Cultured Bovine Luteal Cells Biol Reprod, March 1, 2006; 74(3): 552 - 559. [Abstract] [Full Text] [PDF]
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M. J. Cannon and J. L. Pate Expression and Regulation of Interferon {gamma}-Inducible Proteasomal Subunits LMP7 and LMP10 in the Bovine Corpus Luteum Biol Reprod, April 1, 2003; 68(4): 1447 - 1454. [Abstract] [Full Text] [PDF]
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