Histological study of angiogenesis and cell death in the equine corpus luteum

Abstract

The corpus luteum (CL) is formed from the preovulatory follicle after ovulation and its main function is the secretion of progesterone, which is essential for the establishment of pregnancy. The CL is associated with massive vascularisation followed by rapid regression during luteolysis. In the nonfertile cycle, progesterone production falls and luteal structural regression takes place. If conception occurs, the CL continues to produce progesterone.

The CL provides a unique example of tightly controlled angiogenesis and cell death. Little is known about these processes in the equine CL.

Elucidation of angiogenesis and luteolysis during natural and PGF2a-induced regression will provide new insights into the control of the corpus luteum, increase our understanding of the physiology of the corpus luteum and may help in advancing and developing other methods for induction of luteolysis.

Corpora lutea were collected from mares in the early luteal phase: Day 3-4 (n=4), mid-luteal phase: Day 10 (n=5), early regression: Day 14 (n=4), late regression: Day 17 (n=4), and 12 and 36 h (n=3 per group) following PGF2(X administration on day 10 Luteal angiogenesis was assessed by measuring cell proliferation, endothelial cell content and vascular endothelial growth factor (VEGF) mRNA and protein expression in luteal sections by quantitative immunocytochemistry. For cell death, sections were examined at both histological and ultrastructural levels and TdT-mediated X-dUtp nick end labelling (TUNEL) was used to detect DNA fragmentation. The combination ofTUNEL immunostaining and ultrastructural examination provided definitive identification of the types of cell death involved in the CL. It was demonstrated that apoptotic and non-apoptotic mechanisms were involved in the demise of the CL.

The presence of fragmented chromatin, pyknotic cells, round dense bodies and phagocytosis were considered as apoptotic features. Other changes (crenation of the nuclear membrane with shrinkage of the nucleus) seen in some luteal cells indicated there is an additional nonapoptotic form of cell death at luteolysis. Angiogenesis is a dynamic process of endothelial cell proliferation and microvessel formation regulated by the production of angiogenic factors, especially VEGF. It was established that the early luteal phase was associated with intense endothelial cell proliferation, expression of VEGF and the growth of the microvascular network.

Angiogenesis continued during the mid-luteal phase at a reduced level.

However, VEGF remained high and the full microvasculature had been established for optimal progesterone secretion. During regression, the marked progesterone decline was associated with a decline in endothelial cell proliferation, VEGF expression and reduction in vascularity. Tumour necrosis factor a (TNFa), Nitric oxide (NO) and basic fibroblast growth factor (bFGF) immunostaining in the luteal cells during the luteal phase may indicate a paracrine and/or autocrine role for these factors in the CL and may play a role in luteal development and regression. This thesis has described for the first time the cellular changes in angiogenesis and regression post PGF2a-induced regression. The findings revealed massive neutrophils and vasodilation as well as a similar pattern of other cellular changes to those undergone during natural luteolysis. This study increases our understanding of equine CL control. It demonstrates that luteal angiogenesis is important for luteal function and it is likely that VEGF is essential for luteal angiogenesis. Luteolysis and cell death play a crucial role in ovarian cyclicity and the demise of luteal tissue represents both apoptotic and non-apoptotic pathways.