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c Departments of Cell Biology,
d Pathology,
e and Physiology, Faculty of Medicine, University of Córdoba, 14071 Córdoba, Spain
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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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The dynamics of luteal cell populations during the cycle is not fully understood. Studies in domestic ruminants indicate that small (theca-derived) luteal cells proliferate, whereas large (granulosa-derived) luteal cells do not [2, 3, 19]. In the human ovary, segregation of theca-lutein and granulosa-lutein cells is retained throughout the cycle [20]. Proliferation of endothelial luteal cells has been studied in the human [16] and nonhuman primate [17] ovary, although the existence or absence of proliferative activity in luteal steroidogenic cells in the human CL is not clear [21, 22]. However, changes in the ratio of theca-lutein to granulosa-lutein cells during the cycle have been reported [3].
We have studied the presence, numbers, and characteristics of macrophages in the human cyclic CL, covering comprehensively the changes that occur both throughout the cycle and at the different CL compartments. These changes have been related to the proliferative activity of luteal cells (as a marker of luteogenesis) and to the presence of apoptotic cells (as a marker of luteolysis).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue samples were obtained from the archives of the Department of Pathology from hysterectomized-ovariectomized menstruating women showing normal CL and no clinical history of endocrine pathology. The stage of the menstrual cycle was determined by evaluation of the endometrium [23] and CL [20]. Forty-five tissue samples (with concordant endometrial and luteal dating) were selected. The following phases were considered: early (Days 15–19 of the standard cycle, corresponding to young CL, 1–5 days of age), mid (Days 20–24, mature CL, 6–10 days of age), and late (Days 25–27, old CL, 11–13 days of age). Day 14 (day of ovulation) was considered as CL Day 0. When menstruation was present (Day 28), it was considered Day 1 of the following cycle. For quantitative data, three CL per day of the cycle were studied. Six tissue samples, corresponding to Day 14, were not analyzed quantitatively because they showed large variation, probably due to the time elapsed from ovulation.
Immunohistochemistry
Immunohistochemistry was performed on routinely neutral-buffered formaldehyde-fixed, paraffin-embedded tissues. Monoclonal antibodies against CD68 antigen (Dako Diagnostica, Sevilla, Spain) and against Ki67 antigen (Concepta, Barcelona, Spain) for the identification of proliferating cells were used. Sections (5-µm-thick) were placed on poly-L-lysine-coated slides and, after dewaxing and rehydration in a graded ethanol series, were incubated in 2% hydrogen peroxide in methanol for 30 min to inhibit endogenous peroxidase and then were washed in PBS. Sections destined to be immunostained for Ki67 antigen were predigested in 0.1% (w:v) trypsin (Difco, Detroit, MI) in PBS containing 0.1% (w:v) sodium chloride for 20 min at 37°C. After being washed in distilled water, these sections, as well as those destined to be stained for CD68 antigen, were immersed in 10 mM citrate buffer and submitted to antigen retrieval in a microwave oven (2 x 5 min at 700 W). Afterwards, the sections were allowed to cool at room temperature and were washed in PBS, blocked with normal rabbit serum, and incubated overnight with the primary antibodies (anti-Ki67 1:1 and anti-CD68 1:200). The sections were then processed according to the avidin-biotin-peroxidase complex (ABC) technique following previously described methods [24]. Sections were counterstained with hematoxylin. Positive controls for Ki67 antigen immunostaining were provided by healthy growing follicles present in the sections, which showed abundant Ki67-immunostained (Ki67+) granulosa cells. Negative controls for both Ki67 and CD68 antigens were run by omitting the first antibody. Sections were counterstained with hematoxylin. Adjacent sections were stained with hematoxylin and eosin.
Tissue Compartments
The structure of the human CL [7, 20] is depicted in Figure 1. The major compartment is the granulosa-lutein layer (GLL), composed of GLC and stromal cells (SC) corresponding to vascular endothelial and perivascular cells, fibroblasts, and macrophages. Theca-lutein areas (TLA) line the outer aspect of the CL as well as the septae and are composed of theca-lutein cells (TLC) and SC. Finally, the inner boundary tissue (IBT) lines the inner aspect of the CL and is composed of SC. IBT was considered to extend for 75 µm beyond the inner edge of the GLL. Differences between GLL and TLA were not clear in some areas on Day 15 because of the disruption of the basement membrane and on Day 16 because of the lack of complete morphological luteinization of the GLC. Therefore, differential counts in these areas were performed from Day 17 onward. Similarly, the IBT was not evident before Day 18 (CL Day 4), and counts were performed from this point onward. The central cavity was not considered because it showed extreme variation among CL and ranged from a small gap in some CL to a large blood-filled cavity in others.
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Cell Counting
The total number of cells per CL is rather difficult to obtain because of difficulty in determining the volume of the CL. Therefore, in this study, cell counts were expressed as the number of cell profiles per area unit of tissue section, which is proportional to the total number of cells. These counts have previously been used to estimate the number of different cell types within the CL [3, 4, 6]. Counts were performed with a test point reticle attached to the microscope. Cell counts were performed in three nonadjacent sections per CL and were carried out by two independent observers. Differences between them were lower than 10%, and the mean values were considered. For macrophages, the number of CD68+ cells were counted. Since both the shape and the cytoplasmic volume of macrophages changed considerably through the cycle, only cells showing the nucleus in the section were counted. This prevented overestimation of macrophage numbers when these cells showed large dendritic cytoplasm (Fig. 2). Counts were performed systematically throughout the CL section. The test area was located at the outer aspect of the CL section; successive microscopic fields were observed by moving down at 150-µm intervals. When the IBT was reached, the field of view was moved laterally, then upwards at 150-µm intervals to reach the outer aspect. This procedure was systematically repeated throughout the whole CL section. Since the volume of the GLL changed considerably during the cycle (because of the increase in the cytoplasm of GLC from early to midluteal phase and to the decrease in GLC cytoplasm from mid to late luteal phase), the density of macrophages could appear to be modified. To minimize this effect, steroidogenic cells (recognizable from Day 17 onward by their large round nuclei with prominent nucleolus and large cytoplasm) were also counted, and macrophages in the GLL were also expressed relative to the number of GLC (number of macrophages/100 GLC). In the thecal luteal area and IBT, the area of the section (when it did not correspond to the whole test area) was determined by point counting.
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To assure that Ki67+ cells in these tissues corresponded to cells undergoing proliferation, the number of Ki67+ cells per area unit was related to the number of mitotic cells per area unit. For this, the numbers of both Ki67+ cells and mitotic cells were counted systematically (as for macrophage counting) in the whole CL. From Day 17 onward, the number of Ki67+ cells and the number of GLC in the GLL, and the number of Ki67+ cells, as well as the number of TLC (recognizable by their round nuclei and vacuolated cytoplasm) in TLA and the number of Ki67+ cells in the IBT, were counted.
Results were expressed as the number of Ki67+ cells per area unit, and the labeling indices (percentage of Ki67+ cells) for GLC and TLC were obtained by dividing the number of Ki67+ GLC or TLC per area unit by the total number (immunostained or not) of GLC or TLC per area unit, respectively.
Apoptotic cells were recognized by their morphological features [25]. They showed shrunken eosinophilic cytoplasm and condensed chromatin. Apoptotic cells were counted in hematoxylin and eosin-stained sections, according to the same schedule.
Statistical analysis was carried out by ANOVA and Tukey's test for multiple comparison among means. Because differences among consecutive days were gradual, and in order to simplify the results, comparisons were established among means in the different luteal phases. Mean ± SD in each luteal phase corresponded to the pooled data of the different days of the cycle corresponding to that phase. Differences at the 0.05 level were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On Day 14 (day of ovulation), the ruptured follicles showed variable features. In some cases, macrophages were exclusively found among the theca cells, whereas in others, some macrophages had invaded the granulosa cell layer. On Days 15 and 16 (CL Days 1 and 2) macrophages were already present among GLC. On Day 16, macrophages were already present in the inner aspect of the GLL and showed large round or elongated cytoplasm (Fig. 2A). On Days 17–19, macrophages were abundant and uniformly distributed in both TLA and GLL (Fig. 3A), as well as in IBT, which was present from Day 18 onward. Most macrophages showed round or elongated cytoplasm, although some dendritic macrophages were observed, mainly on Day 19. On Days 20–24, there were prominent changes in both the distribution and the shape of macrophages. Macrophages accumulated at the boundary between TLA and GLL (Fig. 2D), as well as at the boundary between GLL and IBT (Fig. 2E). Macrophages located in the GLL showed dendritic features, with large cytoplasmic processes that surrounded blood vessels and GLC (Figs. 2B and 3B), whereas macrophages showing round cytoplasm were scarce. On Days 25–27, regressive changes were evident in the CL. Isolated GLC showed extreme vacuolization of the cytoplasm and expression of CD68 immunoreactivity (Fig. 2L). These CD68+ GLC were clearly distinguished from macrophages because they showed large round nuclei, vacuolated cytoplasm, and weaker immunostaining. Macrophages were scarce in the GLL (Fig. 3C) and showed small round or elongated cytoplasm (Fig. 2, C and K), but dendritic macrophages were almost absent. These changes were more evident on Days 26–27. However, macrophages continued to be abundant in the TLA and IBT. Quantitative data on macrophages are presented in Figure 4 and Table 1. Considering the whole CL, macrophages reached their highest density by the end of the early luteal phase (Days 18–19), and their number was maintained during most of the midluteal phase (Days 20–23). A significant decrease in the number of macrophages was found in the late luteal phase (Days 25–27). In the TLA, the number of macrophages was significantly higher at the midluteal phase, and significantly decreased in the late luteal phase. In the GLL, the number of macrophages was significantly higher at the end of the early luteal phase, showed a slight but significant decrease at the midluteal phase, and showed a new decrease at the late luteal phase. Similar changes were observed when the number of macrophages was expressed with respect to the number of GLC (to avoid the appearance of changes in the density of macrophages due to the changes in the volume occupied by the cytoplasm of GLC throughout the cycle). In the IBT, the number of macrophages significantly increased from the early to the midluteal phase and remained unchanged during the late luteal phase.
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Proliferating Cells
On Days 15–16, abundant Ki67+ cells were observed (Fig. 5A). Although many of these cells seemed to correspond to steroidogenic cells, the distinction between steroidogenic and stromal cells was unclear at this time. On Days 17–19, proliferating cells were less abundant. Ki67+ steroidogenic (either theca-lutein or granulosa-lutein) cells were observed frequently (Fig. 2, F and G, and Fig. 5B). Most Ki67+ cells corresponded, however, to stromal, mainly vascular, cells. The number of proliferating cells decreased from the early to the midluteal phase. However, Ki67+ TLC were abundant during the midluteal phase, and isolated Ki67+ GLC were observed during the midluteal (Figs. 2H and 5C) and even at the late luteal phase. At the midluteal phase, most Ki67+ GLC were observed in the inner zone, near the IBT (Fig. 5C).
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Quantitative data are presented in Figure 6 and Table 1. The densities of Ki67+ and mitotic cells were parallel. Densities were very high during the early luteal phase and showed significant decreases at midluteal and late luteal phases. In the TLA and GLL, the proliferative activity of stromal cells was higher than that of steroidogenic cells. In the TLA, a relatively high number of proliferating steroidogenic cells were found during the early and midluteal phases, whereas the number of proliferating cells was significantly decreased in the late luteal phase. In the GLL, the number of proliferating cells significantly decreased from Day 19 onward. In relation to the labeling indices, about 15% of the TLC proliferated during the early and midluteal phases, whereas about 5% of GLC proliferated on Days 17–19. Significant decreases were found at the late luteal phase. In the IBT, a high number of proliferating cells were found during the early and midluteal phases, and a significant decrease was found in the late luteal phase.
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Apoptotic Cells
Apoptotic cells were found in very low numbers throughout the cycle in the GLL. On Day 15, these cells were relatively frequent near the inner aspect of the CL. In the TLA, apoptotic cells were practically absent during early and midluteal phases. Relatively abundant apoptotic cells were found in both TLA and GLL in the late luteal phase. Most of these cells seemed to correspond to vascular endothelial cells (Fig. 2, J and K). Quantitative data are shown in Figure 6 and Table 1. A significant increase in the number of apoptotic cells was found on Days 25–27 in both TLA and GLL.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to the changes in the number of macrophages, striking differences in the morphology of macrophages among the different luteal phases were observed. Whereas at early and late luteal phases macrophages were round or elongated, in the mature CL, macrophages showed dendritic features. Dendritic macrophages showed increased surface contacts with GLC and microvasculature. Previous studies have shown that physical contact of macrophages with granulosa cells stimulated progesterone synthesis [29]. Furthermore, the recruitment of macrophages and the existence of morphological changes, as an expression of maturational changes in these cells, indicate the existence of a modulation of macrophage morphology (and probably function) by GLC-derived factors. The invasion of the newly formed CL by macrophages is most likely due to the release of chemotactic factors by GLC. In the rat, the expression of a chemotactic factor, monocyte chemoattractant protein-1 (MCP-1), has recently been reported [30, 31]. In the human CL, granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA is expressed during early and midluteal phases [32], which are coincident with the recruitment and maturation of macrophages. The release of chemotactic factors for macrophages from GLC could also explain the accumulation of these cells at the edges of the GLL, observed during the midluteal phase. MCP-1 and GM-CSF also induce differentiation and activation of macrophages [33, 34], and could act as local regulators of macrophages. Petrovská et al. [4], reported that MHC class II positive cells were abundant in the GLL at the midluteal phase. Expression of MHC class II molecules is a classical marker of macrophage activation [14]. Additionally, several data indicate that ovarian macrophages can be hormonally regulated. Progesterone modifies interleukin-1 [35] and superoxide anion [36] production by macrophages, and LH receptor immunoreactivity has been demonstrated in ovarian macrophages in the rat [37]. Thus, macrophages in the CL are probably regulated by a combination of endocrine and paracrine signals.
Otherwise, several lines of evidence suggest that macrophages are also involved in the luteolytic process in many species [3, 6, 7]. Although this study does not preclude the existence of a possible role for macrophages during structural luteolysis in subsequent cycles, these data clearly indicate that during the functional cycle of the human CL, macrophages seem to be related to luteogenesis and progesterone production rather than to CL decay. In this study, identification of macrophages was based on CD68 immunoreactivity. The decrease in the number of macrophages during the late luteal phase seems to be due to a decrease in the number of macrophages and not to a loss of antigen expression, since other signs of decay of the macrophage population such as a decrease in cell size and loss of dendritic features were also observed. Furthermore, previous authors [4] using a different macrophage marker (CD14) also reported a decrease in the number of macrophages in the GLL during the late luteal phase.
Ki67 antigen is a marker of cell proliferation [38] and has been used previously for the study of proliferative activity in the primate CL [17]. In this study, the number of Ki67+ cells was well correlated with the number of mitotic cells, which indicates that Ki67 antigen is an adequate marker for proliferating cells in this tissue. Studies on the proliferative activity in the human CL have been devoted to the proliferation of endothelial cells during CL vascularization [16]. In the present study, stromal (mainly vascular) cells comprise the majority of proliferating cells in the CL, which agrees with previous studies in human and nonhuman primates [16, 17]. However, this study demonstrated for the first time that about 5% of GLC (and about 15% of TLC) were proliferating at the end of the early luteal phase and that a smaller proportion of these cells remained cycling during midluteal phase. In a previous study in the primate ovary, no cells staining for both 3ß-hydroxysteroid dehydrogenase and Ki67 antigen were observed [17], which suggests that steroidogenic cells do not proliferate. In this study, enzymatic activity was not detected, because frozen sections were not available and because it was beyond the objective of the study, and therefore, steroidogenic cells were recognized by morphological criteria. The characteristic morphological features of luteal steroidogenic cells allow clear distinction from stromal cell types. However, the steroidogenic status of these cells cannot be ascertained, although no morphological differences between Ki67 positive and negative GLC or TLC were observed. The functional significance of the existence of a subpopulation of proliferating luteal steroidogenic cells is not clear. Although the majority of GLC arise by differentiation of the granulosa cells already present in the preovulatory follicle, a part of the final GLC population is derived from GLC proliferation. No clear relationship between proliferative activity and macrophages was found. Although the data of this study do not lead us to discard a possible role for macrophages in the proliferative activity of luteal cells at the end of the early luteal phase and during the midluteal phase, most of the proliferative activity occurs on Days 15–17, when macrophages were still scarce. Vascularization (which is responsible for most of this early proliferative activity) seemed to be dependent on stimulatory factors derived from GLC. It has been reported that vascular endothelial growth factor is extensively expressed in the luteal cells forming the CL [39].
Increased apoptosis and decreased proliferative activity appeared to be involved in the cyclic CL regression. The majority of apoptotic cells observed in this study in the late luteal phase corresponded to vascular cells, which agrees with previous data indicating that endothelial injury is an early marker of CL regression [7, 40], and with studies demonstrating DNA cleavage in the CL [41]. However, the presence of apoptotic cells did not seem to be related to macrophages, since apoptosis was coincident with a decrease in the number and size of macrophages at the late luteal phase.
In summary, the observed numerical and maturational changes in macrophages in the human cyclic CL provide the morphological basis for a functional interaction with steroidogenic luteal cells and support the hypothesis that macrophages act as local regulators in the CL.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: F. Gaytán, Dept. of Cell Biology, School of Medicine, 14071 Córdoba, Spain. FAX: 34-57-218288; fi1begac{at}lucano.uco.es
Accepted: April 2, 1998.
Received: December 30, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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