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Regular Article |
a Graduate School of Natural Science and Technology
b Graduate School of Medical Science, Kanazawa University, Takara-machi, Kanazawa, Ishikawa 920-0934, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum, cytokines, female reproductive tract, gene regulation, ovulatory cycle
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The corpus luteum, a transient endocrine tissue formed from the ovarian follicle after ovulation [4], is responsible for the maintenance of early pregnancy but regresses in the absence of mating [5]. Regression of the corpus luteum, or luteolysis, is an essential event for the ovulatory cycle, and this process is believed to be regulated by various hormones and cytokines, probably in a manner that depends on the animal species [5, 6]. Previous studies have shown that immune cells, including monocytes/macrophages and lymphocytes, accumulate, that apoptotic luteal cells increase in number, and that the expression of some cytokines is activated during regression of the corpus luteum [5, 6]. In particular, augmented expression of monocyte chemoattractant protein-1 (MCP-1), a C-C chemokine that provokes migration of monocytes/macrophages toward the place of inflammation, in regressive corpora lutea at the mRNA [7–11] and protein [7, 11–14] levels has been reported by several investigators. However, the possibility that monocytes/macrophages accumulate in corpora lutea because of the action of MCP-1 and play a role in luteolysis has been only speculative.
Cells undergoing apoptosis are rapidly and selectively eliminated from the organism by phagocytosis, and this phenomenon is considered to contribute to the maintenance of tissue homeostasis [15–17]. Because luteal cells are induced to undergo apoptosis, phagocytic clearance of those cells is likely to cause a decrease in luteal weight and size. If this is so, then the presence of phagocytes in regressive corpora lutea should be essential for elimination of dying luteal cells. We hypothesized that MCP-1, synthesized in response to luteal cell apoptosis, induces migration of phagocytic monocytes/macrophages to corpora lutea where luteal cells are apoptosing and that the accumulated phagocytic cells engulf dying luteal cells. As the first step toward verifying this possibility, we simultaneously determined the expression of MCP-1 mRNA, the occurrence of apoptosis in luteal cells, and the accumulation of monocytes/macrophages in corpora lutea of normally cycling rats, and we compared the localization, timing, and levels of these events during the estrous cycle. The results generally, though not completely, supported our hypothesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experimental procedures were conducted with the approval of the animal ethics committee of our university. Female Donryu rats (7–8 wk old) (Japan SLC, Shizuoka, Japan) were housed under controlled photoperiod of 12L:12D (lights-on, 0800–2000 h). Vaginal cells were collected at noon and cytologically examined to determine their phase in the estrous cycle, which consists of proestrus, estrus, metestrus, and diestrus. Animals that showed a regular 4-day estrous cycle for more than two consecutive weeks were considered to be normally cycling and were used in the experiments. The animals were anesthetized with ether, and their ovaries were removed at various estrous phases.
Section Preparation
Isolated ovaries were immersed in PBS containing 4% (w/v) paraformaldehyde at 4°C for 12 h and then rinsed with PBS. They were further immersed successively in 10% and 20% (w/v) sucrose-containing PBS at 4°C for 12 h and then embedded with OCT compound (Sakura Finetechnical, Tokyo, Japan) on dry ice/ethanol. The ovaries were then frozen-sectioned at 10-µm thickness on glass slides coated with Vectabond Reagent (Vector, Burlingame, CA) and used for histochemical analyses.
In Situ Hybridization
In situ detection of MCP-1 mRNA in ovarian sections was carried out using RNA probes as previously described [18]. In brief, the 380-base pair DNA fragment corresponding to part of the protein-coding sequence of the rat MCP-1 cDNA [19] was inserted into pBluescript KS+ vector (Stratagene, La Jolla, CA) and used as a template for RNA probe synthesis. Antisense and sense RNA probes were synthesized in vitro in the presence of digoxigenin-labeled UTP (Dig RNA Labeling Kit; Roche Diagnostics, Mannheim, Germany) and dissolved in a hybridization buffer consisting of 20 mM Tris-HCl (pH 8), 2.5 mM EDTA, 0.3 M NaCl, 10% (w/v) dextran sulfate, 1x Denhardt solution, 1 mg/ml of yeast RNA, and 50% (v/v) formamide. The sections were treated successively with Triton X-100 (0.3% ;obv/v;cb), proteinase K (1 µg/ml), and paraformaldehyde (4% ;obw/v;cb). They were supplemented with the hybridization buffer containing either the sense or the antisense probe, incubated at 60°C for 16 h, and treated with RNase A to remove unreacted probes. An alkaline phosphatase-conjugated antidigoxigenin antibody was then added to the samples, and hybridization signals were visualized by adding a coloring solution containing nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt. The samples were counterstained with methyl green, dehydrated, covered with Entellan New (Merck, Darmstadt, Germany), and examined by microscopy.
Antibodies
To generate an anti-rat class B scavenger receptor type I (SR-BI) antiserum, the synthetic peptide corresponding to amino acid residues 110–132 of SR-BI of the rat [20] with an extra Cys residue at the carboxyl terminus was coupled to keyhole limpet hemocyanin, emulsified with Freund adjuvant, and injected into rabbits. Antibody titers in rabbit sera were monitored by an enzyme-linked immunosorbent assay. The antiserum obtained gave a discrete signal corresponding to SR-BI in Western blots of lysates of rat liver and testes (the data will be made public elsewhere). An anti-mouse Fas antiserum (named P4), which has been successfully used to detect Fas-expressing spermatogenic cells in sections of mouse testes [21], was used as a second antibody to localize luteal cells. Anti-rat CD68 monoclonal antibody clone ED1, which binds to monocytes and macrophages [22], and a polyclonal antibody recognizing activated caspase-3 were purchased from Chemicon (Temecula, CA) and Promega (Madison, WI), respectively.
Immunohistochemistry
Ovarian sections were treated with 0.2% (v/v) Triton X-100 for permeabilization of the plasma membrane and with 0.3% (w/v) hydrogen peroxide for inactivation of endogenous peroxidase. The sections were then blocked with 5% (v/v) swine serum and incubated with anti-SR-BI antiserum, anti-Fas antiserum, anti-CD68 antibody, or antiactivated caspase-3 antibody. The samples were subsequently reacted with either biotinylated anti-rabbit immunoglobulin (Ig) G antibody (Vector) for reaction with the anti-SR-BI or the antiactivated caspase-3 antibody or with biotinylated anti-mouse IgG antibody (Zymed, San Francisco, CA) for reaction with the anti-CD68 antibody, followed by a treatment with horseradish peroxidase-conjugated streptavidin (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Signals were visualized by adding a coloring solution consisting of 50 mM Tris-HCl (pH 7.5), 0.002% (w/v) hydrogen peroxide, and 0.1 mg/ml of 3,3-diaminobenzidine tetrahydrochloride. The samples were treated and examined by microscopy as for in situ hybridization. For simultaneously detecting the hybridization and immunohistochemical signals, ovarian sections were first analyzed by in situ hybridization, the results recorded, and the immunohistochemistry subsequently conducted with no additional permeabilization step. In the analyses with antiactivated caspase-3 or anti-Fas antibody, treatment of ovarian sections with 0.3% (w/v) SDS was substituted for the treatment with Triton X-100 and proteinase K before the hybridization reaction. After the antibody reactions, the samples were treated and examined by microscopy as described above.
TUNEL Assay
Ovarian sections were fixed with 1% (w/v) paraformaldehyde, washed, and refixed with ethanol/acetic acid. The sections were treated with 3% (w/v) hydrogen peroxide and subjected to the TUNEL assay in which synthesized DNA was labeled with digoxigenin (ApopTag; Intergen, Purchase, NY). The reactions were then supplemented with a horseradish peroxidase-conjugated antidigoxigenin antibody, and signals were visualized and examined by microscopy as described for immunohistochemistry. Nuclei with fragmented DNA were stained brown, whereas normal nuclei were stained green. For simultaneous detection of the TUNEL and hybridization signals, the sections were first subjected to in situ hybridization and subsequently analyzed by the TUNEL assay as reported previously [23].
Numerical Analysis of Histochemical Data
The embedded ovaries were first roughly divided into three blocks, and 12 serial sections were prepared from each block. Two to three sections were used for each of four types of histochemical examination: in situ hybridization of MCP-1 mRNA, TUNEL, immunohistochemistry for detection of activated caspase-3 or monocytes/macrophages. Corpora lutea that extended through the sections were chosen and analyzed for the number of positive cells in each assay; most detectable corpora lutea (n = 5–8) were examined at proestrus (2000 and 2300 h) and estrus (all time points), whereas randomly chosen corpora lutea (n = 4) were examined at all other time points (the number of apoptotic cells was small). The number of positive cells present in the entire area of the luteal cross-section was determined either manually (hybridization signals) or digitally (the others), and the average number (per 0.16 mm2 of the luteal section) was given to each corpus luteum as the score for each histochemical assay. No significant difference was found in the results of any assays among the three blocks.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We first examined whether the gene coding for MCP-1 is expressed in luteal cells or other cell types present in the corpus luteum. For this purpose, sections of ovaries from normally cycling rats were analyzed simultaneously by in situ hybridization to detect MCP-1 mRNA and by immunohistochemistry to locate cell type markers. In situ hybridization was carried out using RNA probes that were synthesized in vitro and possessed either the antisense or the sense sequence of rat MCP-1 mRNA. The antisense probe clearly hybridized to the cytoplasm of cells present in corpora lutea, whereas the sense probe did not give signals (Fig. 1A), indicating that cells containing MCP-1 mRNA were present in the corpus luteum. We found that only a small fraction of cells in the tissue express the Mcp-1 gene. We then conducted immunohistochemistry to identify cell type(s) of the MCP-1 mRNA-expressing cells. Localization of CD68-positive cells was first determined to locate monocytes/macrophages in the sections. Many cells positive for anti-CD68-antibody staining were seen, but none of the positive cells contained the MCP-1 mRNA hybridization signal (Fig. 1B). An antiserum recognizing SR-BI was then used to identify steroidogenic luteal cells [24]. The antiserum bound to cells that were abundantly distributed throughout the ovary, including in corpora lutea and the periphery of follicles (i.e., theca cells), and cells containing hybridization signals for MCP-1 mRNA were also positive for SR-BI (Fig. 1C). We then immunohistochemically localized luteal cells using another antibody. When luteal sections were analyzed with an antiserum recognizing Fas, the expression of which increases in regressive corpora lutea [25, 26], cells containing MCP-1 mRNA were positively stained with the antibody (Fig. 1D). These results indicate that the Mcp-1 gene is expressed in luteal cells, but not in monocytes/macrophages, of corpora lutea in normally cycling rats.
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Examination of MCP-1 mRNA Expression During the Estrous Cycle
We next determined the level of Mcp-1 gene expression during the estrous cycle. Ovarian sections were prepared from rats at various estrous phases, which were assessed by histological examination of cell populations collected from vaginas. The level of MCP-1 mRNA expression showed a sharp peak at the beginning of the estrous phase (Fig. 2A, top). We then examined the same samples to detect apoptotic luteal cells using two methods: Immunohistochemistry was employed for identifying cells with activated caspase-3, and the TUNEL assay was used for locating cells with fragmented DNA. Apoptotic cells were detectable in corpora lutea by immunohistochemistry with the antiactivated caspase-3 antibody, which bound to the cytoplasm of luteal cells (Fig. 2B, left). The latter method similarly gave fewer (but clear) signals in corpora lutea as well as in follicles undergoing atresia, probably because of detection of apoptotic granulosa cells (Fig. 2B, right). Quantification of these results revealed that the number of luteal cells undergoing apoptosis, as assessed by either method, began to increase at the end of proestrus and reached a maximum at estrus (Fig. 2A, middle two panels). These results agree with those reported by Matsuyama et al. [27]. However, no conclusion could be made in the present study regarding change in the number of monocytes/macrophages, because variation was high at all time points (Fig. 2A, bottom).
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Relation of MCP-1 mRNA Expression to Luteal Cell Apoptosis and Monocyte/Macrophage Accumulation
The concomitance of MCP-1 mRNA expression and apoptosis during the estrous cycle raised the possibility that apoptotic luteal cells express the Mcp-1 gene. This possibility was tested by analyzing luteal sections simultaneously for the occurrence of MCP-1 mRNA expression and apoptosis (Fig. 3). The results showed that cells bound by the antiactivated caspase-3 antibody were distinct from those cells positive for MCP-1 mRNA (Fig. 3, left). The TUNEL-positive cells were rarely found in the area where many MCP-1 mRNA-expressing cells were detectable (Fig. 3, right). These results clearly indicate that the cells expressing MCP-1 mRNA are nonapoptotic luteal cells.
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At least four generations of regressing corpora lutea coexist in the ovary of cycling rats at any phase of the estrous cycle. We therefore analyzed individual corpora lutea at estrus to examine possible correlations among the levels of MCP-1 mRNA expression, apoptosis, and monocyte/macrophage accumulation (Fig. 4). Because most corpora lutea differed in size, we compared the number of positive cells in a given area of the corpus luteum. We found that corpora lutea with more MCP-1 mRNA-positive cells contained fewer apoptotic cells at all time points, and vice versa. This suggests that MCP-1 mRNA expression and apoptosis are induced in different corpora lutea, although these events seem to be synchronized during the estrous cycle, as indicated by the overall number of corpora lutea containing cells positive for these phenomena (Fig. 2A). In contrast, the correlation between the levels of MCP-1 mRNA expression and monocyte/macrophage accumulation was ambiguous, again because of high variation in the number of monocytes/macrophages from one experiment to another.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-administered sheep. The reasons for this discrepancy are not certain at present, but they could include 1) the fact that small luteal cells in the sheep express MCP-1, 2) a difference in the conditions of the animals (normal rats vs. prostaglandin F2-treated sheep), or 3) a difference in the antibodies used to identify steroidogenic cells. Penny et al. [10] has raised the possibility that MCP-1 is expressed in CD5-positive T lymphocytes in corpora lutea of the cow; however, their experimental procedure was not accurate enough to identify cell types expressing MCP-1. Penny et al. compared two serial luteal sections, one analyzed by in situ hybridization for MCP-1 mRNA and the other by immunohistochemistry with an anti-CD5 antibody. The level of MCP-1 mRNA expression increased slightly later than that of luteal cell apoptosis during the estrous cycle (Fig. 2A). Accumulation of mRNA after transcription induction is, in general, a rapid event compared with the activation of caspase-3, which takes hours to occur irrespective of the type of apoptotic stimuli. It can thus be concluded that Mcp-1 gene expression is preceded by the induction of apoptosis. However, the signal that initiates the apoptotic process is unlikely to simultaneously trigger expression of the Mcp-1 gene: MCP-1 mRNA-expressing luteal cells were not apoptotic, and moreover, corpora lutea with higher levels of MCP-1 expression contained fewer apoptotic cells. Further studies are needed to clarify a possible causal connection between apoptosis and MCP-1 expression in luteal cells. In the present study, high variation in the number of monocytes/macrophages in individual corpora lutea retarded examination of possible correlation between the levels of MCP-1 expression and phagocyte accumulation. This is probably because recent and older generations of corpora lutea were not separately analyzed. Bowen et al. [13, 28] showed that the number of macrophages increases in corpora lutea entering estrus. Thus, expression of the Mcp-1 gene in luteal cells, induction of luteal cell apoptosis, and accumulation of monocytes/macrophages in corpora lutea likely are all induced at the entrance of the estrous phase. Another approach is necessary to verify the possibility that monocytes/macrophages invade corpora lutea by the action of MCP-1.
The pituitary hormone prolactin is the most likely candidate for initiating the involution of corpora lutea [29, 30]. Other factors, however, also seem to be involved in the phenomenon, including the pituitary hormones FSH and LH; the steroid hormone progesterone, which is secreted from the corpus luteum; glucocorticoids; and cytokines, including prostaglandin F2, tumor necrosis factor , and inhibin/activin. These hormones and cytokines might directly or indirectly regulate apoptosis in luteal cells. Kuranaga et al. [31–33] have proposed that prolactin stimulates the expression of Fas ligand in lymphocytes present in corpora lutea and that, in turn, this induces apoptosis in Fas-expressing luteal cells. In fact, the levels of mRNA and protein expression of Fas and Fas ligand increase in the rat corpus luteum during pregnancy and postpartum [25], and the amount of Fas mRNA increases in corpora lutea of the cow at regressive phases [26]. This is an intriguing idea, because the amount of MCP-1 mRNA increases in human glioma cells after treatment with an apoptosis-inducing anti-Fas antibody [34]. Our results indicate that apoptosing luteal cells themselves do not contain detectable amounts of MCP-1 mRNA. Thus, cells undergoing Fas-mediated apoptosis may somehow activate expression of the Mcp-1 gene in bystander nonapoptotic luteal cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Yoshinobu Nakanishi, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan. FAX: 81 76 234 4480; nakanaka{at}kenroku.kanazawa-u.ac.jp
Received: 28 February 2002.
First decision: 26 March 2002.
Accepted: 11 June 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Biol Reprod 1998 58:169-174This article has been cited by other articles:
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M. J Cannon, J. S Davis, and J. L Pate The class II major histocompatibility complex molecule BoLA-DR is expressed by endothelial cells of the bovine corpus luteum Reproduction, May 1, 2007; 133(5): 991 - 1003. [Abstract] [Full Text] [PDF]
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