The mare is a seasonal breeder and undergoes a period of ovarian transition in spring between winter anoestrus and cyclicity. During spring transition LH concentrations are low and many mares have successive large anovulatory follicular waves which reach the size of preovulatory follicles. Follicular angiogenesis is essential for growth and health of preovulatory follicles. The aim of the present study was to investigate the morphology and vascularity of transitional anovulatory follicles. On gross inspection, the wall of transitional follicles was visibly less well vascularized than that of preovulatory follicles. Histologically, it could be seen that the theca was only poorly developed in transitional follicles. Immunostaining for factor VIII showed that there were significantly (P < 0.05) fewer blood vessels in the theca of transitional follicles. There was substantially less (P < 0.001) proliferative activity, measured by immunostaining for Ki67, in the endothelial cells and granulosa cells of transitional follicles compared with preovulatory follicles. Preovulatory follicles had a heavy band of immunostaining in the theca for vascular endothelial growth factor (VEGF), whereas staining was sparse in the transitional follicles. It was concluded that the poor vascularity and development of the theca layer in transitional follicles could be related to low circulating LH, and possibly other trophic hormones, and are likely to be the key factors in explaining the steroidogenic incompetence of transitional anovulatory follicles.

Precise pharmacological control of the corpus luteum is important in the manipulation of the oestrous cycle in mares. Angiogenesis plays a key role in the growth and regression of the corpus luteum; therefore, influencing the vasculature of the corpus luteum may offer a novel method for controlling its lifespan. In the present study, changes in angiogenesis and vascular expression of endothelial growth factor (VEGF) were evaluated throughout the luteal phase and after PGF 2␣ -induced luteolysis. Corpora lutea were collected from mares in the early luteal phase (days 3-4), mid-luteal phase (day 10), early regression (day 14), late regression (day 17), and at 12 and 36 h after administration of PGF 2␣ on day 10 of the oestrous cycle. Immunohistochemistry was used to localize Von Willebrand factor and Ki67 in endothelial and proliferating cells, respectively. VEGF mRNA and protein were localized by in situ hybridization and immunohistochemistry. The proliferation index of endothelial cells was intense in the early luteal phase. The early and mid-luteal phases were characterized by a dense network of capillaries. The microvasculature started to regress by day 14. After administration of PGF 2␣ , vasodilation was observed after 12 h, but after 36 h, luteal degeneration was accompanied by a significant decrease in vascularity. VEGF mRNA and protein were expressed mainly in the luteal cells during the early and mid-luteal phases and expression declined at early regression (day 14). However, immunostaining for VEGF protein was high in late luteal regression (day 17) and 36 h after PGF 2␣ administration. These findings indicate a close temporal association between VEGF expression and angiogenesis in the equine corpus luteum during its functional lifespan.

In mares, little information is available on the type of cell death that occurs during natural and induced luteal regression. Corpora lutea were collected from mares in the early luteal phase, days 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) after PGF2alpha administration on day 10. Histological and ultrastructural sections were examined and TUNEL was used to detect DNA fragmentation. In early luteal regression, there were more pyknotic luteal cells and extracellular round dense bodies compared with the mid-luteal phase. By late regression, there was a significant decline (P < 0.01) in the number of round dense body clusters and a marked accumulation of lipid. Twelve and 36 h after PGF2alpha administration, changes were similar to those seen in natural regression, but there was also a marked infiltration of neutrophils. Accumulation of lipid was not apparent until 36 h after PGF2alpha administration. Ultrastructural examination revealed rarefaction and distortion of the mitochondrial cristae in most of the luteal cells by the mid-luteal phase. Luteal cells showed shrinkage, accumulation of lipid with foamy appearance, and disruption in both smooth endoplasmic reticulum and mitochondria during natural and induced regression. Some luteal cells showed fragmented or pyknotic chromatin characteristic of apoptosis. Other luteal cells showed crenation of the nuclear membrane and shrinkage of the nucleus, features not characteristic of apoptotic cell death. In late regression, capillaries were obstructed by swollen endothelial cells and round dense bodies. These results show that structural regression may be initiated as early as the mid-luteal phase, and is clearly visible by day 14 in natural regression and 12 h after induced regression. Apoptosis did appear to be involved in luteolysis in the equine corpus luteum, but non-apoptotic changes were also observed in some luteal cells during regression. Accumulation of lipid was a late feature of luteal regression.

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