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a Departments of Physiology,
b Internal Medicine, and
c Pathology, and
d Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female Sprague-Dawley rats were obtained from Charles River Breeding Labs. (Portage, MI) at 8 wk of age. Animals were housed under a controlled lighting schedule of 12L:12D (lights-on 0600–1800 h, experiment 1; lights-on 0600–1800 h or 0500–1700 h, experiment 2; lights-on 0500–1700 h, experiment 3). Examination of vaginal epithelium was carried out daily, and only those rats showing at least four consecutive 4-day cycles were used. Monitoring of vaginal epithelium began at 9–11 wk of age, and animals were killed between 11 and 17 wk of age. For experiment 3, rats were obtained from Charles River at 10 wk of age, after the animals had shown 2 consecutive 4-day estrous cycles. These rats were killed after a minimum of 2 consecutive 4-day cycles, at 12–14 wk of age. Animal procedures were approved by the University Committee on the Use and Care of Animals at the University of Michigan.
Experimental Design
In experiment 1, rats were killed either on the morning (0830–1100 h) of proestrus (n = 8) or estrus (n = 5), or on the afternoon (1700–1800 h) of estrus (n = 5). Rats were killed by decapitation following ether anesthesia, and the ovaries were immediately removed and placed on ice. One ovary was frozen in OCT compound (Miles Laboratories, Inc., Elkhart, IN) for immunohistochemistry for monocytes/macrophages (ED1-positive), differentiated macrophages (ED2-positive), and MCP-1 protein and for in situ detection of apoptosis in the corpora lutea of different ages. From the remaining ovary, specific populations or sets of corpora lutea were dissected: on proestrus, the corpora lutea of the current cycle; on estrus, the regressing corpora lutea from the preceding cycle. Once dissected, the corpora lutea from each rat were counted and weighed as a group.
In experiment 2, rats were killed on either the morning (0900–1000 h) of proestrus (n = 7) or the afternoon (1600–1800 h) of estrus (n = 7) as described above, and the ovaries were removed and placed on ice. Specific sets of corpora lutea were dissected from both ovaries: on proestrus, the corpora lutea of the current cycle; on estrus, the regressing corpora lutea from the preceding cycle. The dissected corpora lutea (n = 5–8) from one ovary were frozen as a group for sectioning; the sections were used for immunohistochemistry for monocytes/macrophages, differentiated macrophages, and MCP-1 protein and for in situ detection of apoptosis. The remaining dissected corpora lutea from each rat were counted and weighed as a group.
In experiment 3, rats were killed on either the morning or early afternoon (1000–1300 h) of proestrus (n = 10) or on the afternoon (1500–1700 h) of estrus (n = 10). Rats were killed as described for experiment 1 and the ovaries removed and placed on ice. Corpora lutea of the current cycle (proestrus) or regressing corpora lutea from the preceding cycle (estrus) were dissected from both ovaries, weighed, frozen in liquid nitrogen, and stored at -80°C for later analysis of total protein content.
Antibodies
A description of the polyclonal rabbit anti-rat MCP-1 antibody used for immunohistochemical staining has been published [12]. The monoclonal antibody against rat monocytes/macrophages (clone ED1) used for immunohistochemical staining was obtained from Chemicon International Inc. (Temecula, CA). The specificity of this antibody for rat monocytes/macrophages has been reported previously [13]. The monoclonal antibody against rat differentiated macrophages (clone ED2) was obtained from Serotec via Accurate Chemical & Scientific Corp. (Westbury, NY) and has been shown to specifically recognize differentiated, resident tissue macrophages [14]. Biotinylated secondary antibodies to mouse and rabbit immunoglobulin were obtained from Vector Laboratories (Burlingame, CA).
Immunohistochemistry
Frozen sections of ovaries or isolated corpora lutea were air-dried and fixed in 95% ethanol (10 min); they were then placed in 0.3% H2O2 in methanol (4°C) for 15 min (macrophages) or 30 min (MCP-1) to quench endogenous peroxidase activity. The tissue sections were rinsed three times (5 min each) in PBS containing 1% BSA (fraction V; PBS-1% BSA) and then incubated with 10% normal goat serum for 15 min (macrophages) or 30 min (MCP-1) at 37°C. After this blocking procedure, the sections were rinsed again in PBS-1% BSA and then incubated either with the rabbit anti-rat MCP-1 antibody or with a monoclonal antibody against either rat monocytes/macrophages or differentiated macrophages (1:200 dilution, 30 min at 37°C). After the incubation with primary antibody, the sections were rinsed in PBS-0.1% BSA and then exposed to either biotinylated goat anti-rabbit (MCP-1) or goat anti-mouse (monocytes/macrophages, differentiated macrophages) immunoglobulin (1:200 dilution; 30 min at 37°C). Detection of the antigen-antibody complex was achieved by using a Vector avidin-biotin-peroxidase kit and 3-amino-9 ethylcarbazole as the substrate. The tissue sections were counterstained with hematoxylin, rinsed in distilled water, and dipped in Scott's tap water (a mordant) before being mounted with aqueous mounting medium. MCP-1 staining was graded for intensity and for distribution by two independent observers using coded slides.
Nonspecific staining was assessed by replacement of primary or secondary antibodies with the appropriate serum and was undetectable in all instances.
In Situ Detection of Apoptosis
Nuclei exhibiting DNA fragmentation were detected by in situ hybridization using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Frozen sections were air-dried and fixed in 10% neutral buffered formalin for 10 min. They were then rinsed in two changes of PBS for 5 min each before being postfixed in ethanol:acetic acid (2:1) for 5 min at -20°C. After postfixing, sections were rinsed again in two changes of PBS and then quenched in 3% H2O2 in PBS for 5 min at room temperature. Sections were rinsed in PBS, and equilibration buffer from the kit was applied to cover each section; the buffer was blotted and replaced with a solution of terminal deoxynucleotide transferase (TdT). The sections were incubated with this enzyme for 1 h at 37°C to allow for tagging of 3' DNA ends with digoxigenin residues. The sections were next placed in stop buffer for 10 min, then rinsed with PBS and incubated with anti-digoxigenin antibody conjugated with peroxidase for 30 min at room temperature. After rinsing in PBS, sections were incubated for 6 min with diaminobenzidine substrate, washed in three changes of distilled water for 1 min each, and then washed in distilled water for 5 min. The sections were counterstained with methyl green for 10 min; this was followed by three washes in distilled water, three washes in 100% butanol, and three washes in xylene before mounting. Negative controls, in which the TdT enzyme solution was replaced with water, were run in every batch of slides stained; this replacement completely eliminated staining. Atretic follicles in whole ovarian sections served as a positive control for apoptotic nuclei. Whole ovarian sections containing these follicles were run in every batch of slides stained.
Assay of Total Luteal Protein
Specific sets of corpora lutea obtained in experiment 3 were homogenized in 500 µl modified RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA) containing detergents (1% Nonidet P-40 and 0.015% sodium deoxycholate) using a tissue homogenizer. The homogenate was diluted (1:30) for assay of total protein using the Bio-Rad Protein Microassay [15]. All standards and samples were assayed in triplicate.
Quantitation of Macrophages and Apoptotic Nuclei
Numbers of macrophages per high-power field were determined for coded slides by visual observation of immunodetectable cells. A light microscope with a x45 objective was used. For experiment 1, one section was examined to determine macrophage numbers, and as many fields as possible (1–4) were counted for each corpus luteum in the section (4–23 corpora lutea). New ovulations on estrus were easily distinguishable in whole ovarian sections and were not included as corpora lutea for this analysis. For experiment 2, two sections of the frozen group of corpora lutea (processed in different staining runs) were examined in the same manner as for experiment 1 (1–3 fields in 3–7 corpora lutea), and an average number of macrophages per high-power field was obtained for each rat.
For quantitation of apoptotic nuclei, two sections from each ovary or group of corpora lutea were examined as above, using coded slides, and an average number of apoptotic nuclei per high-power field was obtained. Again, as many fields as possible were counted for each corpus luteum in these sections, and new ovulations were excluded from analysis of whole ovarian sections.
Statistics
Comparisons among multiple groups were carried out using the Bonferroni multiple comparisons test. Comparisons between two groups were carried out using Student's t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monocytes/macrophages (ED1-positive) and differentiated macrophages (ED2-positive) were detected by immunohistochemistry in sections of whole ovaries from rats killed on the morning of proestrus (n = 6), the morning of estrus (n = 5), and the afternoon of estrus (n = 5). Cross sections of all corpora lutea, excluding the newly formed corpora lutea on estrus, were used for these analyses, without regard to age of the corpora lutea. Mean ± SEM numbers of luteal monocytes/macrophages (detected by the monoclonal antibody ED1) per high-power field were, respectively, 21.4 ± 2.1, 26.4 ± 4.0, and 27.9 ± 4.1 and were not significantly different among the three time points (p > 0.05; Fig. 1). Numbers of differentiated macrophages (detected by the monoclonal antibody ED2) per high-power field were, respectively, 6.4 ± 1.2, 10.9 ± 1.6, and 16.4 ± 2.5 (p < 0.05, proestrus vs. afternoon of estrus; Fig. 1).
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MCP-1 protein detected by immunohistochemistry was differentially distributed among time points. In ovarian sections from rats killed on proestrus (n = 6), MCP-1 was present in the center of the corpora lutea, with the remainder of the structure being essentially devoid of staining (Fig. 2, A and B). In ovarian sections from rats killed on estrus (n = 10), MCP-1 was present throughout the corpora lutea, as well as in the central area of the corpora lutea, as either a diffuse staining or as a number of localized foci of staining (Fig. 2, C–F). Coded slides were identified correctly as estrus or proestrus by two observers on the basis of quantity and distribution of luteal MCP-1 staining. In addition to staining in the corpora lutea, immunodetectable MCP-1 was seen in many follicles and vascular spaces throughout the ovary. This is not surprising, as MCP-1 is a secreted protein made by a number of cell types present in the ovary, including macrophages, epithelial cells, and fibroblasts [16, 17]. We have previously observed that MCP-1 is prevalent in atretic follicles, which also contain macrophages (unpublished results).
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Apoptotic nuclei were detected by in situ hybridization in sections of ovaries from rats killed on the morning of proestrus (n = 6), the morning of estrus (n = 5), and the afternoon of estrus (n = 5). Mean ± SEM numbers of apoptotic nuclei per high-power field were, respectively, 1.6 ± 0.2, 5.3 ± 1.3, and 4.5 ± 0.8 (p < 0.05, proestrus vs. morning of estrus; p < 0.05, proestrus vs. afternoon of estrus; Fig. 3). Cross sections of all corpora lutea, excluding the newly formed corpora lutea on estrus, were used in this analysis, without regard to age of the corpora lutea. Atretic follicles within the same sections exhibited striking numbers of apoptotic nuclei. Frequently, all granulosa cells visible in a follicular section would stain positively for DNA fragmentation (data not shown).
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Weights of individual corpora lutea were as follows: corpora lutea of the current cycle (proestrus) averaged 1.10 ± 0.09 mg (n = 8 rats), versus 0.87 ± 0.02 mg (n = 5 rats) for regressing corpora lutea from the previous cycle (estrus morning) and 0.75 ± 0.04 mg (n = 5 rats) for estrus afternoon (p < 0.05, proestrus vs. morning of estrus; p < 0.05, proestrus vs. afternoon of estrus).
Experiment 2
Monocytes/macrophages (ED1-positive) and differentiated macrophages (ED2-positive) were detected by immunohistochemistry in sections of isolated corpora lutea of the current cycle from rats killed on the morning of proestrus (n = 7) and in sections of isolated regressing corpora lutea from the previous cycle from rats killed on the afternoon of estrus (n = 7). Mean ± SEM numbers of luteal monocytes/macrophages per high-power field were, respectively, 1.4 ± 0.5 and 9.4 ± 0.9 (p < 0.001; Fig. 4). Numbers of differentiated macrophages per high-power field were, respectively, 1.1 ± 0.2 and 6.6 ± 1.6 (p < 0.05; Fig. 4).
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Mean ± SEM numbers of apoptotic nuclei per high-power field were respectively, 1.3 ± 0.2 and 4.7 ± 0.5 (p < 0.001; Fig. 5). Visually, the cells that appeared to be undergoing apoptosis were large round cells typical of the steroidogenic luteal cells. Total number of cell nuclei per high-power field was also determined as an indicator of whether cell shrinkage and tissue compression were taking place. This number was not significantly different between proestrus and the afternoon of estrus (260.3 ± 16 vs. 230.3 ± 16.5, respectively; p > 0.05).
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Weights of individual corpora lutea for experiment 2 were remarkably consistent with those observed in experiment 1. Corpora lutea of the current cycle on proestrus averaged 1.07 ± 0.03 mg (n = 7 rats), versus 0.76 ± 0.03 mg (n = 7 rats) for regressing corpora lutea from the previous cycle on the afternoon of estrus (p < 0.001).
MCP-1 protein detected by immunohistochemistry appeared differentially distributed among time points, as observed for whole ovarian sections in experiment 1. In sections of corpora lutea of the current cycle, MCP-1 was present almost exclusively in the center of the corpora lutea, with the remainder of the structure being essentially devoid of staining. In sections of regressing corpora lutea, MCP-1 was present throughout the corpora lutea. Again, coded slides were identified correctly as estrus or proestrus by two observers, with few misclassifications, based on intensity and distribution of MCP-1 staining.
Experiment 3
The average weight per corpus luteum of the current cycle on proestrus was 1.08 ± 0.02 mg (n = 10 rats); for regressing corpora lutea of the previous cycle on the afternoon of estrus, the value was 0.85 ± 0.03 mg (n = 10 rats; p < 0.001).
Protein per milligram luteal tissue was not different between the corpora lutea on proestrus and corpora lutea on estrus (108.4 ± 6.0 vs. 108.8 ± 5.9 µg/mg luteal tissue; n = 8 at each time point).
When expressed per corpus luteum, the values were 117.7 ± 6.5 vs. 92.9 ± 5.7 µg/corpus luteum, respectively; p < 0.05.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The recruitment of inflammatory/immune cells into the corpus luteum at the time of luteal regression has been observed in a number of species [18–24]; however, the underlying stimuli and mechanisms for this recruitment remain largely unknown. The chemokine, MCP-1, may have a prominent role. MCP-1 is expressed in the corpora lutea of several species [25–28]. It has multiple roles as a chemoattractant for monocytes, and as a factor that induces the production of reactive oxygen intermediates and lysosomal enzymes by monocytes [29–31]. The presence of MCP-1 staining at higher intensity and altered distribution within the corpora lutea on estrus compared to proestrus suggests that the presence of monocytes/macrophages in the corpus luteum at regression is not incidental and is associated with changes in expression of MCP-1. We have previously reported a similar association in regressing corpora lutea at the end of pregnancy [28], in regressing corpora lutea the day after mating [28], and in corpora lutea regressing due to the acute actions of prolactin injected in hypophysectomized rats [9].
Although intense staining for MCP-1 is observed in both the regressing corpora lutea of cycling rats and the corpora lutea of hypophysectomized rats treated with prolactin [9], we observe differential expression of cell markers for monocytes/macrophages in these animals. In hypophysectomized rats, although the number of monocytes/macrophages per high-power field is greatly increased by 72 h after onset of prolactin treatment [9], virtually none of these cells are positive for the ED2 antigen (based on personal communications). In contrast, in the current study we observed large numbers of ED2-positive cells, up to two thirds of the entire ED1-positive population. Expression of the ED2 antigen correlates with differentiated, resident macrophages, and adhesion is necessary to induce its expression [14]. The ED2 antigen is not expressed on monocytes, nor is it expressed on recently recruited inflammatory macrophages [14], which may explain the lack of staining in corpora lutea of rats treated with prolactin. According to van Rooijen et al. [32], it may take a week for ED1-positive cells to express the ED2 antigen. We observed larger numbers of both ED1- and ED2-positive cells per high-power field when regressing corpora lutea of all ages were addressed (experiment 1) than in corpora lutea entering regression on estrus (experiment 2). In addition, significant numbers of these immune cells were already present on proestrus when all regressing corpora lutea were used for analysis (experiment 1). This suggests that the infiltration of immune cells that occurs between proestrus and estrus is not transient and that it may continue as corpora lutea continue to regress, leading to larger numbers of monocytes/macrophages and resident, differentiated macrophages per high-power field in older corpora lutea.
We do observe increases in ED2-positive cells in corpora lutea entering regression on estrus, as compared to proestrus. It is possible that the time required for recruited macrophages to express the ED2 antigen is decreased in cycling rats as compared to hypophysectomized rats due to differences in the steroid milieu. The hypophysectomized rat, which lacks pituitary trophic hormones, has a low level of production of estradiol [8] and of glucocorticoids [33]. In addition, the absence of prolactin results in the conversion of progesterone to the inactive metabolite 20
-dihydroprogesterone by the corpora lutea [8, 34]. Thus the differential production of adrenal and ovarian steroids in the cycling rat as compared to the hypophysectomized rat may affect the differentiation of macrophages, at least some lineages of which express receptors for estrogens, androgens, and glucocorticoids [35–37]. The significant increase in numbers of ED2-positive macrophages in corpora lutea on estrus, as compared to proestrus, raises questions about the role of these cells in the tissue. The presence of ED2-positive cells may indicate increased activation of macrophages. Determining the level of expression of major histocompatibility complex (MHC) II molecules on these cells might provide further evidence as to whether ED2-positive cells have enhanced functional status. Brännström et al. [23] reported increased levels of MHC II-positive cells on Day 15 of pseudopregnancy and after Day 15 of pregnancy in the rat. However, the incidence of MHC II-positive cells did not correlate with changes in ED2-positive cells. We have observed recently that the administration of dexamethasone to prolactin-treated hypophysectomized rats is associated with an increase in the number of ED2-positive cells present in the corpora lutea (based on personal communications). Dexamethasone has previously been shown to increase the number of ED2-positive colonies arising during culture of bone marrow stromal cells of rats [38, 39], as has progesterone [39].
The physiological destruction of the corpora lutea in the rat is an orderly, choreographed process. Rat corpora lutea regress gradually over a period of several cycles [40] and are capable of a transient resurgence in growth in the case of a subsequent pregnancy [41]. In the hypophysectomized rat it is clear that the corpora lutea remain steroidogenically active, producing predominantly 20-dihydroprogesterone [8, 9]. While the inflammatory/immune events observed on estrus are associated with rapid physical destruction of the corpora lutea, measured by loss of luteal wet weight and total protein, the protein per milligram of tissue does not change over this interval (experiment 3), suggesting that the composition of the tissue remains stable, even as the tissue undergoes regression. One interpretation of the observations cited above is that the process of luteal regression in the rat involves the targeted deletion of individual cells, perhaps through apoptosis, leaving other cells in a relatively healthy steroidogenic condition.
An increase in apoptotic cell death in regressing corpora lutea on estrus suggests that apoptosis is one mechanism by which deletion of tissue occurs. Increases in luteal apoptosis after prolactin administration, or decreases after blockade of prolactin, have been previously reported [11]. It is also possible that macrophages play some role in deletion of luteal tissue, perhaps by phagocytosis of cells [24] or by the killing of cells through secreted products such as lysozyme and reactive oxygen intermediates [42]. Activated macrophages also produce cytokines such as tumor necrosis factor [43], which is present in corpora lutea at regression [20, 44, 45] and which has been shown to cause cell death in corpora lutea and luteal cells maintained in culture [46, 47]. Apoptosis, therefore, may occur first in response to a prolactin signal and then be perpetuated in the regressing tissue by the actions of macrophages that are also recruited in response to the prolactin signal. Evidence for continued tissue deletion after repeated exposures to prolactin is provided by the whole ovarian sections (experiment 1), in which corpora lutea of several different ages and regressive states were present. In these sections, despite this heterogeneity, a significant increase in apoptotic nuclei per high-power field was seen on the morning of estrus as compared to proestrus, and this increase was remarkably similar to that seen in corpora lutea first entering regression on estrus (experiment 2). This suggests that each prolactin surge might initiate a new round of apoptosis in all regressing corpora lutea. If this is so, then administration of prolactin every 12 h, as carried out in previous experiments with hypophysectomized rats [8, 9], may lead to a maximal rate of tissue deletion. While the non-ED2-positive macrophages recruited in response to prolactin are associated with rapid regression of the corpus luteum, when prolactin is delivered every 12 h [9], it is altogether possible that increased differentiation and activation of macrophages are required for similarly paced regression following one, solitary prolactin signal, as occurs on proestrus during the estrous cycle. At no time was the amount of apoptosis observed in the corpora lutea as dramatic and widespread as we observed in collapsing atretic follicles in the same sections, which is further evidence for a slow and controlled process of tissue deletion in the corpora lutea.
This study is the first to examine a single, most recent, generation of corpora lutea before (proestrus) and immediately after (estrus) the putative signal for regression as well as examining all populations of regressing corpora lutea. We have found that inflammatory/immune and regressive events are initiated (MCP-1 changes; monocyte/macrophage infiltration; apoptosis) and reinitiated (MCP-1 changes; apoptosis) at this time. The observed increases in monocyte/macrophage numbers and in apoptotic nuclei are consistent with the findings from previous studies [10, 11]. The timing of onset of these events is consistent with a role for the proestrous prolactin surge in initiating regression. However, other hormones, such as ovarian steroids and LH, are also elevated during this time period. It is possible that one or a combination of these factors participate in the initiation of inflammatory/immune events in corpora lutea observed on estrus. The recruitment of monocytes/macrophages to regressing corpora lutea entering regression between proestrus and estrus is consistent with the effects of prolactin treatment in the hypophysectomized rat [9]; however, the differentiation state of the macrophages present is altered, raising the question of what this differentiation means in terms of macrophage function in the tissue. The role of macrophages and other inflammatory cells in luteal regression remains an intriguing problem for future investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J.M. Bowen, Department of Physiology, University of Michigan, 7627 Medical Sciences II Building, Ann Arbor, MI 48109–0622. FAX: 734 936 8813; bowenjm{at}umich.edu
Accepted: October 27, 1998.
Received: July 1, 1998.
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J. M. Bowen and P. L. Keyes Repeated Exposure to Prolactin Is Required to Induce Luteal Regression in the Hypophysectomized Rat Biol Reprod, April 1, 2000; 63(4): 1179 - 1184. [Abstract] [Full Text]
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H. Inadera, T. Sekiya, T. Yoshimura, and K. Matsushima Molecular Analysis of the Inhibition of Monocyte Chemoattractant Protein-1 Gene Expression by Estrogens and Xenoestrogens in MCF-7 Cells Endocrinology, January 1, 2000; 141(1): 50 - 59. [Abstract] [Full Text] [PDF]
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J. M. Bowen and P. L. Keyes The Proestrous Prolactin Surge Is Not the Sole Initiator of Regressive Changes in Corpora Lutea of Normally Cycling Rats Biol Reprod, November 1, 1999; 61(5): 1208 - 1215. [Abstract] [Full Text]
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