Growth and cellular proliferation of ovine corpora lutea throughout the estrous cycle

Abstract

This study was conducted to determine the rates of growth and cellular proliferation of ovine corpora lutea (CL) throughout the estrous cycle. To determine the cellular labeling index (LI), ewes received an iv injection of bromodeoxyuridine (BrdU) 1 h before death on days 2, 4, 8, 12, or 15 (day 0 = estrus; n = 6-12 ewes/day). At death, CL were weighed, and samples of each were fixed in Carnoy's solution or frozen until analyzed for DNA, protein, and progesterone contents. Nuclear incorporation of BrdU was determined in paraffin-embedded tissue sections by using a primary antibody against BrdU and a fluorescent (fluorescein isothiocyanate-labeled) secondary antibody, and sections were counterstained with propidium iodide (a nuclear stain). The labeling index (BrdU-labeled nuclei as a proportion of propidium iodide-labeled nuclei) of each CL was determined by using dual channel interactive laser cytometry and image analysis. Moreover, BrdU and 3 beta-hydroxysteroid dehydrogenase (a marker for steroidogenic cells) or BrdU and factor VIII (a marker for endothelial cells) were immunolocalized in tissue sections by using double immunohistochemical or dual immunofluorescent staining, respectively. Results demonstrated that cellular proliferation was greatest (LI, 34.1 +/- 2.1%) on day 2 and decreased (P < 0.01) through day 15 (LI, 0.7 +/- 0.1%) of the estrous cycle. The results of the immunohistochemical studies provide evidence that both parenchymal (steroidogenic) and nonparenchymal (e.g. endothelial, fibroblastic) luteal cells proliferated throughout the ovine estrous cycle. Conversely, from days 2-12 of the estrous cycle, fresh weight and DNA content of CL increased linearly (P < 0.01; 8- and 10-fold, respectively), then decreased (P < 0.02) from days 12-15. Ratios of protein/DNA on days 2, 4, and 8 were similar and were greater (P < 0.02) than those on days 12 and 15, which also were similar. These data demonstrate that growth of the ovine CL is extremely rapid, linear from days 2-12, and primarily due to hyperplasia. In addition, the high rate of cellular proliferation is associated primarily with nonsteroidogenic cells, a large proportion of which appear to be endothelial cells. Data such as these will enable us to determine the factors that are important in regulating luteal growth and development in normal and pathological conditions.