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in the Porcine Corpus Luteum1
a Departments of Obstetrics and Gynecology and
b Laboratory Medicine, University of South Dakota School of Medicine, Sioux Falls, South Dakota 57105-1570
c Center for Reproductive Sciences, Departments of Anatomy and Cell Biology and
d Molecular and Integrative Physiology, University of Kansas School of Medicine, Kansas City, Kansas 66160
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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within the porcine corpus luteum (CL). 1) Sections of frozen or paraffin-embedded CL from various stages of the estrous cycle were incubated with the following primary antibodies: anti-human recombinant TNF, anti-porcine macrophage-specific antigen, or anti--actin (marker of pericyte and smooth muscle cells). Dolichos biflorus lectin-peroxidase was used as an endothelial cell label. Positive immunostaining for TNF was apparent in porcine CL throughout the estrous cycle. TNF immunoreactivity was primarily localized in cells along septal/vascular tracts, and exhibited spatial and temporal distribution similar to that of cells labeled with anti-macrophage antibodies. Large luteal cells exhibited weak staining for TNF in paraffin sections, whereas microvascular endothelial cells were consistently negative in both frozen and paraffin sections. 2) Enriched subpopulations of macrophages, endothelial cells, and large and small luteal cells were isolated by density gradient and immunomagnetic bead separation techniques. TNF secretion by each subpopulation was determined by measuring bioactive TNF in incubation media using a specific in vitro bioassay. Macrophage subpopulations secreted up to 100-fold greater quantities of bioactive TNF (up to 400 pg/106 cells) than did other subpopulations. In contrast, endothelial cell and small luteal cell subpopulations released very small amounts (< 8 pg/106 cells) of bioactive TNF. Large luteal cells secreted slightly greater amounts of TNF (10–15 pg/106 cells). Local macrophages appear to be the primary source of TNF in the porcine CL.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), interleukins, and interferons in ovarian follicle development, ovulation, and steroidogenesis [1–3]. Studies on several animal species and humans suggest a role of cytokines in luteal development, function, and regression of the corpus luteum (CL). For example, TNF and interferon 
induced apoptosis of cultured mouse luteal cells [4], and plasma interleukin (IL)-1 concentrations were elevated during the luteal phase of the human menstrual cycle [5]. TNF injection caused regression of blood vessels and decreased progesterone concentration in the rabbit CL [6]. Cytokines, particularly TNF, had inhibitory effects on luteal cell progesterone production [7]. TNF administration by microdialysis within the CL induced functional luteal regression in pigs [8]. It has been suggested that an increase in the number of TNF receptors or a sudden release of TNF may inhibit the luteotropic action of LH and induce luteal regression by inhibition of adenyl cyclase [3]. Conversely, TNF may have a luteotrophic effect in the early stages of luteal development [3].
Despite the interest in ovarian cytokines, intraluteal sources of cytokines including TNF have not been clearly resolved. TNF and IL-1 are among the major cytokines produced by macrophages [9]. Macrophages have been identified in the CL of most species, and increased numbers of macrophages during luteal regression have been demonstrated in the rabbit [10], rat [11], pig [12], and human [13, 14]. However, cytokines can be secreted by other cell types having paracrine/autocrine function [2]. It has been reported that endothelial cells (EC) produce TNF in the porcine CL [15], but this conclusion has not been confirmed. The aim of the present study was to elucidate the cellular origin of TNF in the porcine CL. The present results indicate that macrophages, not EC, are the major source of TNF secretion in the porcine CL.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries from 100–110-kg gilts were collected at a local abattoir and transported on ice to the laboratory within 20 min after slaughter. Ovaries were classified as early (Days 4–6), mid (Days 8–12), and late (Days 14–18) estrous cycle on the basis of gross morphology [16]. CL were immediately excised and dissected in ice-cold Ham's F-10 containing 1% BSA. Luteal tissue was either processed for immunocytochemistry or enzymatically dissociated for in vitro experiments.
Immunocytochemistry
A slice (2- to 3-mm thick) of a prominent CL from each ovary (n = 3 ovaries at each stage of the estrous cycle, i.e., 9 total) was fixed in neutral-buffered formalin and embedded in paraffin. Luteal slices from six different ovaries (n = 2 at each stage) were rapidly frozen in liquid nitrogen-cooled isopentane and stored at -70°C. Paraffin-embedded tissue was sectioned at 4-µm thickness, mounted on Superfrost Plus slides (Fisons, Houston, TX), and stored at room temperature for subsequent immunocytochemical staining. Frozen tissue was sectioned (6 µm) on a cryostat at -20°C, thaw-mounted on 0.5% gelatin-coated slides, air-dried for 3 h at room temperature, and stored at -70°C.
The immunocytochemical staining procedure was performed by a horseradish peroxidase two-step staining technique with DAKO Envison System kits (DAKO Corporation, Carpinteria, CA). Paraffin-embedded tissue sections were dehydrated in a 60°C oven for 30 min, deparaffinized in xylene (2 x 3 min), and rehydrated in decreasing concentrations of ethanol. Sections were incubated with target retrieval reagent (DAKO) by placement in a water bath prewarmed to 60°C and then allowed to cool to 30°C. Total incubation time was about 20 min. Frozen sections were brought to room temperature, fixed in acetone for 10 min, and air-dried.
With 5-min rinses between steps, both paraffin-embedded and frozen tissue sections were incubated sequentially in 0.1 M PBS buffer for 5 min, peroxidase block reagent (0.3% hydrogen peroxide; DAKO) for 5 min, and one of the following primary antibodies for 10 min at room temperature: polyclonal rabbit anti-human recombinant TNF (1:2000); polyclonal rabbit anti-human recombinant IL-1 (1:200; Sigma Chemical Co., St. Louis, MO); monoclonal (mAb) mouse anti-porcine macrophage-specific antigen (Po-M1; 1:1000; VMRD, Inc., Pullman, WA); or -actin mAb (1:50; Boehringer-Mannheim, Indianapolis, IN). Immunostaining was completed by successive 10-min incubations in DAKO peroxidase-labeled polymer conjugated with secondary antibody (goat anti-rabbit and goat anti-mouse lgA/G/M), and AEC substrate-chromogen (3-amino-9-ethylcarbazole). Staining with Dolichos biflorus (DBA) lectin-peroxidase (1:500 dilution; Sigma) followed the same protocol, except that incubation with polymer (second antibody) was omitted. DBA lectin specifically binds to porcine endothelium via N-acetyl-D-glucosamine residues on the cell surface [17]. Slides were counterstained with hematoxylin and coverslipped. Evaluation of slides included serial staining of adjacent sections and observations by at least three individuals. Multiple slides from multiple runs were examined for each CL. Morphometric analyses were performed to estimate densities of macrophage- and TNF-positive cells. A 150 x 150-µm square was superimposed on freeze-framed images of luteal sections using an Optimas 4.02 image analysis system (BioScan, Inc., Edmonds, WA). Stained cells within the defined area were manually counted. Ten random fields on each of two slides from different ovaries at each stage of the cycle were analyzed.
The TNF antiserum neutralized TNF bioactivity produced by lipopolysaccharide (LPS)-stimulated white blood cells (16.3 x 106) isolated from fresh porcine blood as described previously for rat TNF in a similar bioassay [18], verifying its reactivity with porcine TNF. The IL-1 antiserum was used to immunoblot a whole luteal extract from multiple mid/late-cycle CL, resulting in visualization of a single major band at the appropriate molecular weight. Procedural controls for immunocytochemistry included 1) omission of primary antibody, second antibody, or detection reagent and 2) substitution of primary antibody with nonimmune mouse/rabbit sera. No specific staining was detected in any of the control sections.
Isolation of Luteal Cell Subpopulations
CL from individual ovaries were pooled and dissociated with collagenase (0.16% w:v; type IV; Worthington Biochemicals, Freehold, NJ) and deoxyribonuclease I (0.02% w:v; Sigma) to yield a suspension of freely dispersed cells (15–30 x 106 cells/ovary). Highly enriched subpopulations of macrophages, small luteal cells (SLC), large luteal cells (LLC), and EC were sequentially isolated from the dispersed cell suspensions by immunomagnetic bead separation coupled with density gradient centrifugation as described below.
Dispersed luteal cell suspensions were incubated with 50 µg anti-macrophage mAb (the same as used for immunocytochemistry) for 30 min at 4°C with constant gentle mixing. Cells were centrifuged at 200 x g for 8–10 min and then washed twice in cold PBS + 0.1% BSA to remove unbound mAb. After the second wash, cells were resuspended in PBS/BSA containing a suspension of prewashed superparamagnetic polystyrene microspheres (30 x 106 beads/ml; Dynabeads M-450; Dynal, Lake Success, NY) precoated with rat anti-mouse IgM. The cell-bead suspension was incubated for an additional 30 min at 4°C with gentle rocking in a glass 13 x 100-mm test tube, and then placed in a magnetic particle concentrator (MPC; Dynal). Beads (with bound macrophages) were held against the sides of the test tube by a magnetic field. After 2–3 min, the supernatant was carefully aspirated with a Pasteur pipette, transferred to a separate test tube, and held at 4°C. The beads were then washed and re-placed in the MPC, and the supernatant was aspirated. The process was repeated a total of three times. After the final wash, the macrophages (with beads still bound) were resuspended in PBS/BSA and used for in vitro experiments (described below).
A Percoll (Sigma) gradient of 50%, 30%, and 10% in PBS was prepared in a 15-ml conical centrifuge tube. The macrophage-free cell suspension (from above) was overlaid on the Percoll gradient and centrifuged at 400 x g for 45 sec as previously described [19]. The SLC fraction was recovered from the 10%/30% interphase, whereas the LLC fraction was recovered from the 50% phase [19]. SLC and LLC fractions were washed and resuspended in PBS/BSA.
EC were isolated from SLC and LLC fractions as follows. Dynabeads M-450 (tosylactivated; 4 x 108 beads/ml; Dynal) were coated by overnight incubation at room temperature with 0.3 mg DBA-lectin (Sigma) in 2.0 ml 0.5 M borate solution, pH 9.5. After thorough washing, DBA-coated beads (6–10 x 107 beads/ml) were incubated at 4°C for 10 min with SLC or LLC fractions. Cell-bead suspensions were then placed in the MPC for 3–5 min until all beads adhered to the side of the test tube. The supernatants were gently aspirated as described above and reserved as the final SLC and LLC subpopulations. Beads (with bound EC) were washed 2–3 times and resuspended in 3 ml of 0.2 M N-acetyl-D-galactosamine (Sigma) solution. EC were competitively displaced from the beads by the N-acetyl-D-galactosamine. The cell-bead suspension was again placed in the MPC, and the supernatant, which now contained a highly purified suspension of unbound EC, was gently aspirated. Post-sorting cell viability on the basis of trypan blue dye exclusion was 
90% in all subpopulations.
Cell Incubations and TNF Bioassay
Immediately after sorting, all cell subpopulations were incubated for 2 h at 37°C in a shaking water bath in PBS/BSA ± LPS (from Escherichia coli; 1.25 µg/ml; Sigma). LPS (endotoxin) stimulates TNF secretion from macrophages [20]. At the end of the incubation, the spent medium was collected and stored at -20°C until bioassayed for TNF.
TNF concentrations were measured in duplicate 100-µl volumes of incubation media using a sensitive and specific bioassay based on the cytolytic activity of TNF on mouse cell line L929 [18]. TNF bioactivity was referenced against a murine TNF standard curve, and TNF secretion by each subpopulation was expressed as pg/106 cells. Anti-human TNF neutralized the bioactivity of TNF released from porcine cells in these experiments. Details of this assay have been previously published [18].
Statistics
TNF production by isolated cell subpopulations was analyzed by factorial ANOVA. Data were transformed to achieve homogeneity of variance. One-way ANOVA with repeated measures was performed across subpopulations within each stage of the cycle. When significance was found by ANOVA, means were compared by paired t-test. Morphometric data were analyzed by chi-square. Significance was assumed at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunolocalization of TNF in porcine CL is shown in Figure 1. Positive immunostaining was evident in all CL examined. Moderate to intense immunostaining was present in numerous cells located near the periphery of the CL and along major vascular tracts in sections from mid-stage CL (Fig. 1, A and B). Although TNF immunoreactivity was evident in many cells surrounding blood vessels (Fig. 1B), it did not localize in EC labeled with DBA lectin-peroxidase (Fig. 1C). As shown in Figure 1B (insert), EC lining the small vessels were invariably devoid of immunostaining. Some TNF staining was similar to that of vascular smooth muscle cells and pericytes labeled with -actin (Fig. 1D), but -actin immunoreactivity was much more limited in distribution and was restricted to larger vessels. Moreover, many LLC exhibited weak immunostaining for TNF (Fig. 1, A and B).
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A similar distribution of TNF immunoreactivity was apparent in early-cycle CL, in which stained cells were evident in peripheral regions (Fig. 1E). Some LLC in early CL also displayed weak immunoreactivity. Moderate and intense immunostaining was evident throughout the parenchyma in sections from late CL (Fig. 1F).
Several anti-macrophage antibodies that were screened (e.g., Po-M1, VMRD, Inc.; HAM56 and RAM11; DAKO) failed to show reactivity in sections of paraffin-embedded tissue. Therefore, a specific mAb directed against porcine macrophages (Po-M1) was used on sections of frozen tissue to compare macrophage and TNF immunoreactivity. HAM56 antibody showed a similar distribution of staining but appeared to be less specific.
A few macrophages were localized around blood vessels in early- and mid-cycle CL (Fig. 2A). In contrast, numerous macrophages were present along vascular tracts and throughout the parenchyma in late CL (Fig. 2B). Macrophage and TNF immunostaining in frozen sections revealed similar distributions in all sections examined irrespective of the stage of the estrous cycle (Fig. 2, A–D). Moreover, the overall densities of macrophage- and TNF-positive cells were similar at all stages (Table 1).
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To help confirm that macrophages were a primary site of cytokine expression in the porcine CL, another cytokine, IL-1, was immunolocalized. IL-1 immunoreactivity was weak or absent from early- and mid-cycle CL (Fig. 2E) but was evident in numerous cells in late CL (Fig. 2F). Although fewer cells were stained, the distribution of IL-1 immunoreactivity in late tissue was similar to that of TNF and macrophages.
TNF Bioactivity in Isolated Cell Subpopulation
Immunomagnetic bead and density gradient sorting of cells yielded highly enriched subpopulations of macrophages, SLC, LLC, and EC. Macrophage and EC subpopulations were consistently 95% pure on the basis of postsorting microscopic examination. As previously described, LLC subpopulations were 85% pure for cells 25 µm in diameter that stained histochemically for 3ß-hydroxysteroid dehydrogenase (HSD) [19]. The major contaminating cells in the LLC subpopulation were large clumps of EC, which were less efficiently removed by the immunomagnetic bead technique than well-dispersed EC. SLC subpopulations were > 60% pure for 3ß-HSD-positive cells 
21 µm in diameter [19]. Contaminating cells appeared to include EC, leukocytes, and fibrocytes. More complete removal of EC from the SLC and LLC fractions could be accomplished by increasing the number and concentration of beads. However, this decreased the purity of the EC preparations and significantly increased the cost. The number of cells recovered in each subpopulation at each stage of the estrous cycle is shown in Table 2.
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Secretion of bioactive TNF by luteal cell subpopulations is shown in Figure 3. Under unstimulated conditions (without LPS), the macrophage subpopulation secreted 60 to > 100-fold more TNF than did EC or SLC subpopulations at early- and mid-estrous cycle and 10-fold more in late-cycle subpopulations. LLC generally produced somewhat greater amounts of TNF than EC and SLC, but always much less than macrophages. In the presence of LPS, macrophage TNF secretion was stimulated 2-fold (p < 0.05, paired t-test) at each stage of the estrous cycle. Conversely, TNF secretion by all other subpopulations was not significantly stimulated by LPS, although that by EC and SLC tended (p < 0.10) to be modestly increased (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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immuno- and bioactivity is predominantly associated with local macrophages within the porcine CL. Localization of TNF in macrophages is consistent with previous results in the human ovary [21] but does not agree with an earlier report [15] that TNF localizes within the endothelium in porcine CL. The detection of TNF immuno- and bioactivity in LLC indicates that these steroidogenic cells may also be local sites of expression of TNF, as previously suggested by Wuttke and coworkers, who observed TNF mRNA in these cells [22].
The localization of TNF immunoreactivity in macrophages and its absence in microvascular EC is in direct contrast to previous results reported by Hehnke-Vagnoni et al. [15]. Those investigators concluded that TNF was localized in and secreted by EC and that macrophages were not significant sources of TNF in porcine CL. The contradictory results may be partly due to the use of different antibodies and/or techniques. The reactivity of the antibody used in the present study with porcine TNF was validated by immunoneutralization of TNF bioactivity in preparations of LPS (endotoxin)-stimulated porcine white blood cells. In contrast to using only frozen sections, we compared TNF immunoactivity in paraffin-embedded tissue sections as well. When compared with frozen sections, paraffin sections provided better histology, permitting more precise cellular localization. In fact, the temporal and spatial distribution of TNF activity was comparable in both frozen and paraffin sections. Although vascular pericytes and smooth muscle cells cannot be ruled out as possible sources of TNF, comparison of TNF immunostaining with that of EC (DBA-lectin) clearly revealed dissimilar localization. Specific high-affinity binding sites for TNF have been identified on porcine small cell populations containing predominantly SLC and EC [23]. Because EC are primary targets of TNF action in other tissues, it is probable that luteal microvascular EC express TNF receptors. Therefore, confusion may arise in distinguishing receptor-bound versus secretory TNF activity. Furthermore, the distribution of IL-1 in porcine CL, like that of TNF, is consistent with that of macrophages. IL-1 is an established immune mediator produced by resident macrophages, and these cells have been presumed to be the main source of this cytokine in the ovary [24].
The profound difference in the in vitro secretion of bioactive TNF between the macrophage subpopulation and other subpopulations corroborates our immunocytochemical results. Because endotoxin (LPS) is a potent macrophage activator, the LPS-induced stimulation of TNF secretion by the macrophage subpopulations further verifies the macrophage origin of the TNF activity. Moreover, the tendency of LPS to increase TNF secretion in the EC and SLC subpopulations suggests that the activity in these cell subpopulations may arise largely from contaminating macrophages. Conversely, the presence of significant TNF bioactivity in media from LLC subpopulations suggests that the LLC themselves may be synthesizing and secreting modest amounts of TNF. This is consistent with the detection of TNF mRNA in porcine LLC [22].
Macrophages are present in high numbers within the human CL, particularly within the theca-derived regions [25]. Macrophage density increases at the time of CL regression [14], and macrophages are the primary immune cells present during luteolysis [26]. In rabbit CL, the number of macrophages increases before the onset of luteolysis [10, 27], and macrophages invade the regressing CL in the mouse [28] and guinea pig [29]. In the present study, the density of macrophages and TNF-positive cells increased in regressing CL, supporting the view that TNF is involved in luteolysis in the pig. A significant rise in TNF following the decline in progesterone levels has been demonstrated by continuous-flow microdialysis in cows undergoing spontaneous or induced luteolysis [30], and TNF is a powerful inhibitor of steroidogenesis in the porcine CL [22]. Consistent with the presence of specific TNF receptors on luteal EC [23], TNF injection into the parenchyma of rabbit ovaries induced regression of blood vessels and a decline in serum progesterone concentration [6]. Moreover, proliferation of EC from the rabbit CL was inhibited by TNF [31]. Therefore, it appears that a paracrine interaction may exist in the CL, as in other tissues, whereby microvascular EC are targets of TNF secreted by neighboring macrophages. The physiologic details of this interaction during the luteal lifespan remain to be elucidated. However, it seems likely from our current understanding that the increase in macrophage density during luteal regression may contribute to vascular demise via a TNF-mediated mechanism.
In summary, the immunocytochemical localization of TNF paralleled that of macrophages but not that of EC. In addition, high levels of bioactive TNF were secreted by isolated subpopulations of luteal macrophages, whereas minimal amounts of TNF were secreted by EC and SLC subpopulations. These results provide compelling evidence that local macrophages are the principal source of TNF in the porcine CL. The temporal and spatial distribution of TNF is consistent with a physiologic role in luteal development, function, and regression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: John Brannian, Dept. of Ob/Gyn, University of South Dakota, Health Sciences Center, 1400 W. 22nd St., Sioux Falls, SD 57105–1570. FAX: 605 357 1528; jbrannia{at}sundance.usd.edu
Accepted: August 11, 1998.
Received: March 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in follicular development, ovulation and the life span of the corpus luteum. Domest Animal Endocrinol 1997; 14:1–15.[CrossRef][Medline] in the corpus luteum of nonpregnant and pregnant pigs. Biol Reprod 1995; 53:1339–1344.[Abstract] (TNF-) production and localization of macrophages and T lymphocytes in the rabbit corpus luteum. Endocrinology 1988; 122:1185–1187.This article has been cited by other articles:
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  R. Wu, K. H. Van der Hoek, N. K. Ryan, R. J. Norman, and R. L. Robker Macrophage contributions to ovarian function Hum. Reprod. Update, March 1, 2004; 10(2): 119 - 133. [Abstract] [Full Text] [PDF]
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 M. Abdo, S. Hisheh, and A. Dharmarajan Role of Tumor Necrosis Factor-Alpha and the Modulating Effect of the Caspases in Rat Corpus Luteum Apoptosis Biol Reprod, April 1, 2003; 68(4): 1241 - 1248. [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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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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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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