Ultrastructural properties and immunolocalization of relaxin in the cytoplasmic electron-dense granules of large luteal cells during pregnancy in the cow

• Published in: Journal of Reproduction and Development 2001, Vol.47(4), pp.217-225
• Main Authors: Kohsaka, T. (Shizuoka Univ. (Japan). Faculty of Agriculture), Sasada, H, Sato, E, Bamba, K, Hashizume, K
• Format: Article
• Language: English
• Subjects: Bovine; CORPUS LUTEUM; COWS; Immunocytochemistry; RELAXIN; ULTRASTRUCTURE
• ISSN: 0916-8818; 1348-4400
• DOI: 10.1262/jrd.47.217

In cattle, there is no direct evidence for subcellular localization of relaxin. This study investigated the ultrastructural features of the cytoplasmic electron-dense granules of bovine corpora lutea from the 4th month of gestation to near term, and the immunolocalization of relaxin in these granules. Corpora lutea collected from 28 Holstein cows were fixed with Bouin's solution for light microscopy or with 4% paraformaldehyde containing 0.3% glutaraldehyde followed by 1% osmium tetroxide for electron microscopy. Immunocytochemical detection of relaxin was done using polyclonal rabbit antiserum raised against purified porcine relaxin, and was performed by the peroxidase anti-peroxidase method or the protein A-gold technique for light and electron microscopy, respectively. Light microscopic immunocytochemistry showed immunostaining for relaxin in large luteal cells but not in small luteal cells. In the 4th month of pregnancy, weakly positive immunostaining of a few large cells was note. Subsequently, the number of relaxin-positive cells and the intensity of immunostaining both increased gradually from the 7th month of pregnancy to near term. Ultrastructural examination revealed that the large luteal cells contained two types of cytoplasmic electron-dense granules, one being small granules of 100-300nm in diameter and the other being large granules of 500-1800nm. The small granules were abundant by the 7th month of pregnancy, decreased in number by the 9th month, and were absent near term. These granules were observed in the process of being released by exocytosis from the cell surface of the large luteal cells, especially in 4th to 6th months of pregnancy. In contrast, large granules were rare in the 4th month and then increased gradually to reach a maximum near term, and no evidence of their release from the luteal cells was obtained. After incubation with relaxin antiserum and protein A-gold, gold particles indicating relaxin immunoreactivity labelled the dense cores of most of the large granules but did not label small granules. These results indicate that the large luteal cells are the source of relaxin in the bovine corpus luteum during pregnancy and that electron-dense large granules are the subcellular site of relaxin storage within these cells.