Quantitative Changes in Macrophage Distribution in Normal Mouse Ovary Over the Course of the Estrous Cycle Examined With an Image Analysis System

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Abstract
PROBLEM: The current study considered the distribution of macrophages within the major ovarian structures throughout the estrous cycle. METHODS: Immunohistochemical analyses were carried out using an avidin-biotin-peroxidase staining method and the rat anti-mouse macrophage monoclonal antibody anti-Mac-1 was applied to stain macrophages. A computer-assisted image analysis system was used to quantify and compare the distribution of macrophages within individual ovarian structures during the estrous cycle. The following morphological structures were analyzed: primordial, preantral, antral, pre-Graafian, and atretic follicles; first-, second-, and third-generation corpora lutea; and the interstitium. The analysis included follicular and corpus luteum substructures: theca, granulosa cells, and interstitium. The system allows the estimation of macrophage distribution as a macrophage density per μm2 of the defined area. RESULTS: Primordial and preantral follicles did not contain macrophages during all stages of the estrous cycle. In antral, pre-graafian, and graafian follicles, macrophages were located and quantified only in the theca and were not detected in the granulosa cell layer. In contrast, atretic follicles showed macrophage localization in both thecal and granulosa cell layers. Macrophages were present in small numbers in the granulosa luteal cell layer and in high numbers in the thecal layer of newly developing corpora lutea. In the second generation of corpus luteum, macrophages followed the same pattern of distribution, while old corpora lutea contained significantly higher numbers of macrophages in both thecal and luteal cell layers. Surprisingly, significant quantitative changes in the macrophages distribution were detected over the course of the estrous cycle. Macrophage density was significantly higher in proestrus and metestrus when compared with the density in diestrus and estrus in most of the studied substructures with the exception of atretic follicles. Atretic follicles showed high macrophage density throughout the cycle with a two-fold higher density at metestrus. CONCLUSION: Macrophages were present in the mouse ovary over the course of the estrous cycle. The greatest numbers of macrophages appearing in corpora lutea and in atretic follicles suggest a role for macrophages in corpus luteum differentiation and follicular atresia. Their patterns of distribution at proestrus and metestrus within microenvironmental compartments suggests a functional correlation with the events of ovarian development.

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