LOCALIZATION AND CHANGES OF ENOS IN THE CORPUS LUTEUM AFTER ADMINISTRATION OF PGF2ALPHA DURING EARLY AND MID LUTEAL PHASE IN THE COW

Abstract

We have indicated that luteal blood flow (BF) temporarily but drastically increases in the peripheral vasculature of mature corpus luteum (CL) at 0.5–2 hours after prostaglandin F2alpha (PGF) administration to induce luteolysis in the cow. However, this phenomenon has not been seen in early CL. We hypothesized that this acute increase of luteal BF occurs due to vasodilation induced by the local action of nitric oxide (NO) on the resistance vessels that possess smooth muscle layer, and might be one of the first signs to start the cascade of luteolysis. Firstly, we aimed to determine the site-dependent expression of endothelial nitric oxide synthase (eNOS) protein together with distribution of resistance vessels and capillaries. The CLs were obtained from local abattoir (early; Days2–5 n=5, mid; Days8–12 n=5), and were compared in immunohistochemical localization of eNOS with distribution of resistance vessels and capillaries. The number of the resistance vessels and capillaries were determined by immunohistochemistry for smooth muscle actin (SMA), and expressed as the number of vessels per 40,000 square micro meter. Also, the expression of eNOS protein was confirmed as the percentage of eNOS positive areas per 40,000 square micro meter. To examine the sitedependent effect, the area in the CL was divided into center area (without connective tissue) and periphery (1mm from the border of the luteal tissue and ovarian parenchyma) of the CL. The number of the resistance vessels was high only in periphery of the mid CL (early; 1.7±0.5 in periphery vs. N.D. in center, mid; 6.7±1.5 in periphery vs. 0.3±0.3 in center). The number of capillaries was at the same level between periphery and center in the early and mid CL respectively (early; 11.3±1.7 in periphery vs. 14.3±0.9 in center, mid; 7.0±1.2 in periphery vs. 7.7±1.5 in center). However, the number of capillaries decreased from early to mid stage. The eNOS protein in early CL was localized evenly in periphery and center. (periphery; 1.4±0.2, center; 1.3±0.2). In the mid CL, eNOS protein was localized mainly in periphery of the CL (periphery;1.4±0.1, center; 0.1±0.01). Secondly, to define the effect of PGF on the eNOS expression, PGF or saline was treated to cows (n=4–5, each treated group) during early (Day4) or mid luteal phase (Day10–12). At 30 min after treatment, we observed the luteal BF using the color Doppler ultrasound, and confirmed the luteal BF increase in the mid CL of the PGF treated group only. Cows were then ovariectomized, the CL was stained immunohistochemically for eNOS, and its mRNA expression was determined with real-time PCR. PGF increased both eNOS protein and mRNA expression only in the periphery of the mid CL (P<0.01), but not in the center of the mid CL and both area of the early CL. These results indicate that capillaries and eNOS in the early CL are distributed homogeneously between periphery and center, but in the mid CL, the resistance vessels and eNOS are evolved only in periphery. In conclusion, PGF stimulates eNOS expression together with luteal BF increase in the resistance vessels in the periphery of the matured CL. (poster)