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Regular Article |
a Department of Cell Biology, Physiology and Immunology and
b Department of Pathology, School of Medicine, University of Cordoba, 14004 Cordoba, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, corpus luteum, corticosterone, glucocorticoid receptor, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Glucocorticoids (GCs) disrupt the ovarian cycle [6–8]. Pathological conditions such as Cushing's syndrome and physical or psychological stress adversely affect fertility [9]. Moreover, GCs inhibit progesterone secretion [10–12], although stimulatory effects have been reported in hypophysectomized rats [13]. Glucocorticoid receptors are expressed in ovarian cells [13, 14], which implies the potential for direct actions of GCs in the ovary. Glucocorticoids have been found to interfere with luteolysis [11, 15–17], although the mechanisms of these actions are not fully understood. Aside from their possible central effects on hypothalamic and pituitary hormones [18–20], GCs may interfere with structural luteolysis by multiple local mechanisms. Glucocorticoids inhibit prostaglandin synthesis [17, 21], macrophage recruitment and activation [22, 23], and cytokine release [24, 25].
The objective of this study was to analyze the effect of the glucocorticoid dexamethasone (DEX) on structural luteolysis in the cyclic rat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female cycling rats of the Wistar strain (mean weight 300 g) were used. The animals were maintained under controlled light (14L:10D; lights-on at 0500 h) and temperature (21°C) and had free access to rat chow and tap water. The stage of the cycle was checked daily by examining vaginal smears. Only rats showing at least 2 consecutive 4-day cycles were used. All experiments were carried out in accordance to the Guide for the Care and Use of Laboratory Animals and were approved by the Ethical Committee of the University of Cordoba.
Animals were given daily s.c. injections of 0.025, 0.1, 0.4, or 1 mg/rat DEX in 200 µl of olive oil or vehicle for 3 days (from estrus to diestrus), and 5 animals/group were killed on the morning (1000 h) and the evening (2100 h) of proestrus and on the morning (1000 h) of estrus. DEX doses in this range have been previously found to interfere with luteolysis [17].
Tissue Processing
Right ovaries were dissected and fixed in Bouin-Hollande fluid for 24 h and processed for paraffin embedment. Five-micrometer-thick sections were cut, stained with hematoxylin and eosin, and used for morphological evaluation. Left ovaries were fixed in 4% paraformaldehyde (PFA) in Sorensen buffer (pH 7.3) for 24 h and processed for paraffin embedment. Four-micrometer-thick sections were cut, placed on poly-L-lysine-coated slides and used for immunohistochemistry.
In Situ TUNEL
Apoptotic cell detection was based in the method described by Gavrieli et al. [26], with an in situ cell death detection kit (POD; Boehringer Mannheim, Mannheim, Germany) according to the instructions of the supplier but with slight modifications. After dewaxing and hydration, endogenous peroxidase was blocked by incubation for 30 min in 2% H2O2 in methanol. After washing with PBS, sections were submitted to protease treatment with Proteinase K (5 mg/ml in 20 mM Tris, 2 mM CaCl2, pH 7.2, from 5 to 25 min at 37°C; DAKO Diagnostica). Sections were washed again and treated with TUNEL reaction mixture (containing terminal deoxynucleotidyl transferase and label solution) for 60 min at 37°C. After washing in PBS, sections were incubated with the anti-label antibody conjugated with horseradish peroxidase (POD). Negative controls were incubated with label solution lacking terminal deoxynucleotidyl transferase. The 3' end-bound POD was visualized by incubation in 0.03% diaminobenzidine tetrahydrochloride (Type IV; Sigma, St. Louis, MO), 0.01% H2O2 in 0.1 M Tris buffer (pH 7.6) for 1 min. Sections were darkened in 1% copper sulfate for 5 min and counterstained with hematoxylin, 0.04% (w:v) 3,3'-diaminobenzidine in 0.05 M Tris-HCl (Sigma), pH 8, and 3% H2O2. Positive controls in the same sections were apoptotic cells in atretic follicles.
Immunohistochemical Detection of GC Receptors
Glucocorticoid receptors (GCRs) were detected with the polyclonal antibody GR (M20; Santa Cruz Biotechnology, Santa Cruz, CA). PFA-fixed sections (4 µm thick) were dewaxed and rehydrated. Endogenous peroxidase was inhibited with 2% H2O2 in methanol for 30 min. After being washed in distilled water, these sections were immersed in 10 mM citrate buffer and incubated in a microwave oven (2 x 5 min at 700 W) for antigen retrieval. Sections were allowed to cool at room temperature, washed in PBS, blocked with 10% normal rabbit serum for 2 h, and incubated overnight with the primary antibody (diluted 1:100). Negative controls were incubated with preimmune serum or PBS instead of the primary antibody. Sections were then processed according to the avidin-biotin complex (ABC) method, as previously described [4]. PFA-fixed control tissues (heart, spleen, and lung) were fixed, processed, embedded, and prepared for immunohistochemistry in the same way.
Immunohistochemical Detection of Macrophages
Macrophages were detected with the monoclonal antibody ED1, following previously described methods [4]. PFA-fixed sections (4 µm thick) were placed on poly-L-lysine-coated slides. After dewaxing and inhibition of peroxidase with 2% H2O2 in methanol for 30 min, sections were rinsed in PBS, blocked with 10% preimmune serum for 2 h, and incubated overnight with mouse monoclonal ED1 antibody (diluted 1:400). Negative controls were incubated with preimmune serum or PBS instead of the primary antibody. Sections were then processed according to the ABC method, as previously described [4].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CLs of DEX-treated rats showed a clearly different pattern of cell death. DEX-induced changes were particularly evident with the higher doses (0.4 and 1 mg). The following description corresponds to these doses; differences with lower doses (0.025 and 0.1 mg) are reported thereafter. On the morning of proestrus, morphological changes were already evident and affected exclusively the last CL generation (corresponding to the CL of the current cycle), whereas regressing CLs of previous cycles did not show morphological changes (Fig. 2A). In CLs of the current cycle, 3 different concentric zones could be distinguished (Fig. 2A): a peripheral zone showing steroidogenic cells with normal features, a midzone in which steroidogenic cells showed nuclear and cytoplasmic swelling (Fig. 2, A and B), and an inner zone in which steroidogenic cells showed slight pyknosis, a punctate chromatin pattern, and some degree of cytoplasmic condensation (Fig. 2, A and C). The most relevant feature at this time was the presence of high numbers of apoptotic cells that showed the characteristic morphological features of this type of cell death [27], i.e., shrunken eosinophilic cytoplasm, chromatin margination, and fragmentation into apoptotic bodies at advanced stages (Fig. 3A), and were specifically stained with the TUNEL assay (Fig. 3, B and C). Apoptotic cells were observed throughout the CL section and lined the blood vessels, although at advanced stages of apoptosis they were detached from the vessel wall and found in the vessel lumen (Fig. 3A). These cells were assumed to correspond to endothelial cells because of their location lining the blood vessel lumen and by the extreme scarcity of healthy endothelial cells in the last CL generation (Figs. 2B and 3, A–C)
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On the evening of proestrus, only 2 concentric zones could be distinguished in the last CL generation (Fig. 4A): a peripheral zone of apparently normal tissue (except for the scarcity of endothelial cells lining blood vessels) and a central zone with clear morphological signs of initial necrosis (Fig. 4, A and C). Tissue architecture was disrupted, and steroidogenic cells showed pyknotic nuclei or karyolysis and cytoplasmic condensation (Fig. 4C). Some apoptotic cells were still found in blood vessel remnants. Both nuclei and cytoplasm were nonspecifically stained with the TUNEL method in central necrotic areas (Figs. 3D and 5A). On the morning of estrus, as on the previous day, alterations were found exclusively on the last generation of regressing CL. These CL showed a narrow peripheral zone of apparently healthy steroidogenic cells and a central zone of advanced necrosis, in which nuclei were indistinct and only ghost outlines of luteal cells and blood vessels were present (Fig. 4, B and D). In this zone, macrophages immunostained for ED1 antigen and polymorphonuclear leukocytes were found in low numbers. Newly formed CLs derived from the ovulatory event of the early estrus were present (Fig. 5B). In the previous generations of regressing CLs, apoptotic steroidogenic cells (as observed in vehicle-injected rats from the evening of proestrus) and necrotic areas were not found (Fig. 5B).
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The alterations described above were dose related. A direct dose-related relationship was found for the presence of necrotic areas. With the lower DEX doses (0.025 and 0.1 mg), CL alterations were found in only a percentage of the animals (33% and 60% of rats, respectively). In affected animals, only small areas of the CL section were affected (Fig. 6A). These necrotic zones had a higher cellularity because of the abundance of infiltrating macrophages (Fig. 6B) and polymorphonuclear leukocytes (Fig. 6C). An inverse dose-related relationship was found for the presence of the physiological apoptotic response of steroidogenic cells during the transition from proestrus to estrus. With the lower doses, apoptotic cells were abundant in nonnecrotic areas, similar to the situation in vehicle-treated rats.
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Immunolocalization of GCR was performed in intact cycling rats. Clear immunostaining was found exclusively in the endothelial cells of some ovarian compartments. In growing and preovulatory follicles, endothelial cells in the blood vessels of the theca layer showed intense cytoplasmic immunostaining (Fig. 7A) at all stages of the cycle. Blood vessels in the ovarian stroma were negative (Fig. 7A). In the CL, immunostaining showed variable intensity depending on the day of the estrous cycle. At estrus, endothelial cells invading the newly formed CL showed cytoplasmic staining. At metestrus, intense cytoplasmic immunostaining was found in endothelial cells throughout the CL section (Fig. 7B). A progressive decrease in the intensity of the immunostaining was found from metestrus to proestrus (Fig. 7C), and immunostaining was absent on the morning of estrus in the regressing CL of the previous cycle (Fig. 7D). Consequently, regressing CLs from previous cycles were not immunostained. Although slight nuclear immunostaining was observed in different luteal cell types along the cycle, it was difficult to differentiate from background, and these cells were considered negative. In contrast, intense nuclear staining was present in routine control tissues (heart, spleen, and lung). In these tissues, however, endothelial cells of the blood vessels were not immunostained (Fig. 7E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In DEX-treated rats on the morning (1000 h) of proestrus, apoptosis (as detected by both morphological features and staining with the TUNEL assay) affected exclusively the endothelial cells. This apoptosis was coincident with degenerative changes in luteal cells compatible with the initial stages of ischemic necrosis [27]. The subsequent evolution of the luteal tissue, showing clear morphological signs of necrosis on the evening (2100 h) of proestrus and advanced necrosis on the morning (1000 h) of estrus, was in agreement with the well-documented morphological changes characteristic of ischemic necrosis, i.e., progressive changes from cell swelling to piknosis and cytoplasmic eosinophilia leading to disruption of tissue architecture and disappearance of cell nuclei, leaving ghost outlines of cells [27]. Selective apoptosis of endothelial cells seemed to be the cornerstone of the major alterations induced by DEX in the luteal tissue. GCs induce apoptosis in several cell types, including lymphocytes [28], osteoblasts/osteocytes [29], and placental cells [30]. Selective apoptosis of endothelial cells after GC treatment has not been previously reported. However, some data suggest the induction of apoptosis of endothelial cells by GC in other tissues. In in vitro model systems of angiogenesis [31, 32], growing capillaries were destroyed after 4 days of DEX treatment. The endothelial cells "rounded up," which is suggestive of the apoptotic mode of cell death. In contrast, it has been reported that GC can suppress apoptosis in glomerular endothelial cells [33] and testicular germ cells [34], which suggests that GCs differentially regulate apoptosis in different tissues. Apoptosis of endothelial cells has been reported during physiological luteolysis in several species, such as guinea pig [35], cow [36], and human [37]. However, this process is not massive, does not induce necrosis, and does not severely compromise blood supply to the regressing CL, which continues to be a well-vascularized organ throughout regression [38]. These findings are in accordance with the presence of intact blood vessels in the CLs of vehicle-treated rats after the apoptotic burst during the transition from proestrus to estrus. Moreover, the apoptosis of endothelial cells in DEX-treated rats was already present on the morning of proestrus, before the preovulatory PRL surge triggering apoptosis in intact cycling rats, and seemed to be independent of hormonal preovulatory surges.
Necrosis of the luteal tissue affected only the last generation of regressing CLs. The previous generations of CLs, which were also functionally inactive, did not show necrotic areas. This finding is in keeping with the immunohistochemical localization of GCR in the endothelial cells of the last CL generation. In the ovary, intense GCR immunostaining was found in those tissue compartments in which active angiogenesis was occurring, i.e., in the theca layer of growing follicles and in newly formed CLs. In older CLs and in the ovarian stroma, blood vessels that have a low turnover, similar to that in vessels in other anatomical locations [39], did not show GCR immunostaining. Whether newly formed blood vessels in other locations outside the ovary also show GCR immunostaining is not known.
In this study, GCRs were found in the cytoplasm of endothelial cells. In contrast to other intracellular steroid receptors such as those of estrogens and androgens, which seem to be located exclusively in the nucleus, GCR has been reported to be located in both the nucleus and the cytoplasm [40–42] in different tissues. In this study, the nuclear location was predominant in extraovarian tissues. Whether the induction of apoptosis of endothelial luteal cells was mediated by classical intracellular GCR is not known. Although most known effects of GC are mediated by the intracellular GCR inducing changes in gene expression (i.e., genomic responses [43]), recent evidence indicates that GCs may also induce nongenomic responses through interaction with specific GCRs in the cell membrane [44]. In this sense, DEX induces lymphoid cell apoptosis though activation of membrane receptors [45].
The effects of GC reported here occurred at pharmacological doses. Glucocorticoid therapy is useful for a number of chronic conditions, including rheumatoid arthritis, chronic obstructive lung disease, chronic active hepatitis, and inflammatory bowel disease, and is widely used at very high short-term or long-term doses. Some of the side effects of GC treatment involve blood vessel injury. Up to 25% of patients receiving corticosteroid therapy underwent ischemic or avascular necrosis of bone [46]. In this sense, endothelial cell apoptosis deserves attention as a possible explanation for the deleterious effects of GCs, particularly in those cases involving vascular injury.
It is generally assumed that the main deleterious actions of GC in disrupting the ovarian cycle are the result of the inhibition of hypothalamic GnRH output, thus preventing the release of LH and FSH and creating a state of hypogonadotropic hypogonadism [18, 19, 47]. However, in the present study, ovulation, indicated by the presence of newly formed CLs on the morning of estrus, was not blocked even with the higher DEX doses, whereas direct effects on the CL were observed even with the lowest DEX doses. In previous studies, GCs have inhibited progesterone secretion in other experimental models in the rat [10, 12] or in other species [11]. Different mechanisms, including direct effects on the CL [12], have been proposed. The data of this study raise the possibility that vascular damage could be underlying inhibitory effects on the CL and indicate that peripheral actions of GCs should be considered when studying the effects of these drugs on the reproductive system.
The inhibition of the normal luteolytic process in rats treated with the higher doses of DEX was consistent with results of previous studies reporting inhibition of luteolysis in rats treated with immunosuppressive doses of GC [17]. In this study, inhibition of apoptosis of steroidogenic cells was observed only in the nonnecrotic areas of the last CL generation or in CLs of previous generations. The inhibition of apoptosis may be due to multiple mechanisms, such as inhibition of the PRL surge [20], blocking of macrophage infiltration [16], inhibition of macrophage activation or cytokine release [24, 25] or inhibition of prostaglandin synthesis [17, 21]. Dose-related differences were also found for the number of infiltrating macrophages and polymorphonuclear leukocytes. These cells were abundant with the lower DEX doses but were scarce with the higher doses. This scarcity was likely related to the anti-inflammatory actions of GC [23, 48] and has been previously reported after GC treatment in the CLs of different species [16, 17].
The presence of GCR immunoreactivity and the specific induction of apoptosis in the endothelial cells by DEX treatment suggest that luteal blood vessels are a target for GCs in the rat ovary.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work has been subsidized by grants 1FD97-1065-CO3-03 and PM98-0167 from the Dirección General de Enseñanza Superior e Investigación Científica, Spain. 
2 Correspondence. FAX: 57 34 218288; fi1sacrj{at}uco.es
Accepted: August 29, 2001.
Received: May 21, 2001.
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REFERENCES |
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and its type I receptor in luteal regression: induction of programmed cell death in bovine corpus luteum-derived endothelial cells. Biol Reprod 2000; 63:1905-1912This article has been cited by other articles:
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  M. Myers, M. C. Lamont, S. van den Driesche, N. Mary, K. J. Thong, S. G. Hillier, and W. C. Duncan Role of Luteal Glucocorticoid Metabolism during Maternal Recognition of Pregnancy in Women Endocrinology, December 1, 2007; 148(12): 5769 - 5779. [Abstract] [Full Text] [PDF]
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 C. Stocco, C. Telleria, and G. Gibori The Molecular Control of Corpus Luteum Formation, Function, and Regression Endocr. Rev., February 1, 2007; 28(1): 117 - 149. [Abstract] [Full Text] [PDF]
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 J Aguilar, H M Fraser, H Wilson, E Clutton, D J Shaw, and E D Watson Temporal relationship between proliferating and apoptotic hormone-producing and endothelial cells in the equine corpus luteum Reproduction, July 1, 2006; 132(1): 111 - 118. [Abstract] [Full Text] [PDF]
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 C. J. Hemmer, H. A. Lehr, K. Westphal, M. Unverricht, M. Kratzius, and E. C. Reisinger Plasmodium falciparum Malaria: Reduction of Endothelial Cell Apoptosis In Vitro Infect. Immun., March 1, 2005; 73(3): 1764 - 1770. [Abstract] [Full Text] [PDF]
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 R. Sasson and A. Amsterdam Stimulation of Apoptosis in Human Granulosa Cells from in Vitro Fertilization Patients and Its Prevention by Dexamethasone: Involvement of Cell Contact and Bcl-2 Expression J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3441 - 3451. [Abstract] [Full Text] [PDF]
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