Abstract. The objective of the present study was to elucidate whether luteolytic prostaglandin F2α (PGF) plays roles in regulating the nitric oxide (NO) generating system in luteal endothelial cells (LECs). Reverse transcriptase PCR, immunoblotting and immunostaining revealed the presence of PGF receptor mRNA (521 bp) and protein (64 kDa) in cultured LECs obtained from the mid-stage corpus luteum. When cultured LECs were exposed to 0.1 μM-10 μM PGF, NO production was significantly stimulated by PGF at 24 h. When LECs were exposed to 1 μM PGF for 2, 6 and 24 h, PGF did not affect the expressions of endothelial NO synthase (eNOS) mRNA and protein. On the other hand, PGF stimulated the expression of inducible NOS (iNOS) mRNA (P<0.05) and protein (P<0.05) at 2 h, but not at 6 and 24 h. By observing the conversion of [ 3 C]L-arginine to [ 3 C]L-citrulline, we found that PGF stimulated NOS activity in cultured LECs at 2 h (P<0.05). The overall findings indicate that bovine LECs are a target for PGF and that PGF stimulates iNOS expression and NOS activity in bovine LECs. Stimulation of the NO generating system and NOS activity by PGF may result in increasing local NO production followed by luteolysis. Key words: eNOS, iNOS, Luteal endothelial cell, Nitric oxide, Prostaglandin F2α (PGF2α) (J. Reprod. Dev. 55: [418][419][420][421][422][423][424] 2009) he corpus luteum (CL) is a transient endocrine gland essential for regulation of ovarian cycles as well as for establishment and maintenance of pregnancy. If pregnancy does not occur, the bovine corpus luteum starts to regress between days 17-19 after ovulation [1]. Functional and structural regression of the CL in mammals is induced by the pulsatile release of prostaglandin F2α(PGF) from the uterus and the ovary [2][3][4]. Although regarded as a physiological luteolysin in the cow, the local mechanism involved in the luteolytic action of PGF remains unclear.The actions of PGF are mediated by the PGF receptor (FPr) located in the plasma membrane. Bovine luteal cell membranes have the ability to bind PGF throughout the estrous cycle [5,6]. However, there is no consensus on the presence of FPr in the bovine luteal vasculature. Cavicchio et al. reported the absence of FPr mRNA in luteal endothelial cells (LECs) isolated from early pregnant cows [7]. More recent studies using endothelial cells (ECs) derived from cyclic bovine CLs have demonstrated that freshly isolated and cultured LECs express FPr mRNA [8,9]. Furthermore, FPr has been immunohistologically demonstrated to be expressed in luteal tissue including steroidogenic cells and large blood vessels in the peripheral region of the mature CL [10].Directly treating pure populations of luteal steroidogenic cells with PGF does not inhibit basal progesterone (P4) production [11,12], although intramuscular injections of PGF analogues induce a rapid decrease in P4 production [13]. Similarly, a decrease in P4 release has been observed when bovine luteal cells (LCs) were exposed to PGF in the presence of endothelin-1 (EDN1) [14,...

Abstract. To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) α-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2α in unfrozen and frozen-thawed LECs until passage 10. LECs were obtained from developing corpora lutea (CL; days 5-7 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. The LECs were frozen at -80 C or further cultured and/ or passaged until passage 10 in DMEM/Ham's F-12 supplemented with 10% calf serum. The hormonal productions of unfrozen and frozen/thawed LECs were compared through passages 2-10. When both the unfrozen and frozen/thawed cells reached confluence, the culture medium was replaced with fresh medium containing 0.1% bovine serum albumin (BSA), and the cells were incubated with TNFα (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2α by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2α by LECs was not altered by passage and storage at -80 C, whereas the basal production of ET-1 decreased from passage 2 and 3 to passage 4 in the unfrozen LECs and from passage 2 to passage 3 in the frozen/thawed LECs. However, production of ET-1 by the unfrozen and frozen/thawed LECs was similar between passages 4-10 and passages 3-10, respectively. Exposure of LECs to TNFα increased (P<0.05) ET-1 and PGF2α production by the unfrozen and frozen-thawed LECs in all passages examined. Thus, LECs obtained from developing CLs and stored until passage 10 can be used for study of the physiology of LECs in vitro. Key words: Bovine, Corpus luteum, Cytokines, Endothelin-1, Endothelium, Prostaglandins (J. Reprod. Dev. 53: [473][474][475][476][477][478][479][480] 2007) he corpus luteum (CL) is a complex organ that mainly consists of endothelial cells (ECs), steroidogenic cells, fibroblasts, and immune cells [1,2]. After ovulation, thecal microvessels invade the granulosal cell layer, and ECs rapidly proliferate in the early CL constituting more than 50% of the total cells in the mid-cycle CL [3][4][5]. The dense vascular network established at mid-cycle is essential for adequate CL function. It also enables an intricate cross-talk between steroidogenic and luteal endothelial cells (LECs) [6]. The products of LECs are directly associated with the capacity of

Abstract. The corpus luteum (CL) is mainly composed of luteal steroidogenic cells (LSCs) and luteal endothelial cells (LECs). Cell death of LSCs and LECs is essential for structural luteolysis. Therefore, it is important to understand the mechanisms regulating cell death in both types of luteal cells. We previously reported that a treatment combining tumor necrosis factor α (TNF) and interferon γ (IFNG) induced cell death in LSCs and that LECs express TNF receptor type I (TNFRI). To investigate the mechanism of cell death in LECs, in the present study we determined the effects of the same cytokines on cell viability and TNFRI mRNA expression in cultured LECs. To induce cell death in LECs, LECs were treated with TNF or IFNG (0, 0.05, 0.5, 1.0, 2.5 nM) and a combination of TNF (0.5 nM) and IFNG (0.5 nM) for 24 h. The viability of LECs was reduced by a single treatment with TNF or IFNG dose-dependently (P<0.05). Cell viability was further decreased by treatment with a combination of TNF and IFNG (P<0.05). Cells with DNA fragmentation (TUNEL-positive cells) were observed after the treatment with TNF and IFNG. Semi-quantitative RT-PCR analysis revealed that treatment with IFNG alone or in combination with TNF increased the expression of TNFRI mRNA compared with the control (P<0.05). In summary, TNF and IFNG increased cell death in cultured bovine LECs. The upregulation of TNFRI mRNA expression by IFNG suggests that TNF and IFNG synergistically affect the death of LECs resulting in acute luteolysis. Key words: Bovine, Cell death, Corpus luteum, Luteal endothelial cell, Luteolysis (J. Reprod. Dev. 56: [515][516][517][518][519] 2010) he corpus luteum (CL) is a transient organ that forms from the wall of a Graafian follicle following ovulation and secretes progesterone (P4) [1]. It reaches structural and functional maturity by the mid luteal phase and then begins to regress after Day 17 postovulation of a non-fertile cycle. In cows, luteal regression is characterized by a reduction in P4 production (functional luteolysis) and tissue degeneration by apoptosis (structural luteolysis) [2,3].Development of the bovine CL is associated with intensive angiogenesis [4], so that the mature CL becomes one of the most highly vascularized organs in the body [5][6][7], and vascular endothelial cells account for up to 50% of the total cells of the mid CL [8,9]. Recently, capillaries without smooth muscle and a few blood vessels with smooth muscle were found in the center of the mid bovine CL [10], and we reported that the capillaries disappeared, but not the large blood vessels during luteolysis [11]. Luteal endothelial cells (LECs) are the first cells to undergo programmed cell death (apoptosis) during luteal regression, resulting in the loss of capillaries [12,13].The number of leukocytes in the bovine CL (e.g., T lymphocytes, macrophages) increases at the time of luteolysis [14], and leukocytes are known to produce a variety of cytokines, including tumor necrosis factor α (TNF) and interferon γ (IFNG). A combination of IFNG and ...