However, the modulators of intraluteal PG synthesis are still not well understood. The first step of PG synthesis is the release of arachidonic acid from the phospholipid of cell membranes by phospholipase A2 [12, 13]. Thereafter, arachidonic acid is metabolized to PGH2 by cyclooxygenase Vorinostat solubility (COX)-1 and COX-2 [14, 15]. PGH2 is converted to PGF by PGF synthase (PGFS) or to PGE2 by PGE2 synthase (PGES) [16], and PGE2 is converted to PGF by carbonyl reductase (CBR1), which has the same activity as 9-ketoprostaglandin reductase [17]. LH has been shown to stimulate PGF production by cultured bovine luteal cells [18]. Moreover, human chorionic gonadotropin, whose molecular structure is similar to that of LH, stimulates COX-2 expression in bovine luteal cells [19].

Thus, we hypothesized that LH modulates PG production in the bovine CL by stimulating the expressions of COX-2 and/or other PG synthases and that these actions help to maintain CL function. In the present study, to determine whether LH promotes cell viability by regulating P4 and intra-luteal PG production, we examined the effects of LH on 1) the mRNA and protein expressions of COX-1, COX-2, PGFS, PGES and CBR1; 2) the production of PGF, PGE2 and P4; and 3) cell viability using a luteal cell culture system. Materials and Methods Collection of bovine CLs Ovaries with CLs from Holstein cows were collected at a local abattoir within 10�C20 min after exsanguination. Luteal stages were classified as early, developing, mid, late or regressed by macroscopic observation of the ovary and uterus [20].

For cell culture experiments, the ovaries with mid-CLs (days 8�C12 of the estrous cycle) were submerged in ice-cold physiological saline and transported to the laboratory. Cell isolation Luteal cells were obtained as described previously [21]. Briefly, mid-CL tissue from five cows was enzymatically dissociated, and the resulting cell suspensions were centrifuged (5 min at 50 �� g) three times to separate the luteal cells (pellet) from endothelial cells and other types of luteal nonsteroidogenic cells (supernatant). The dissociated luteal cells were suspended in a culture medium, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (D/F, 1:1 [v/v]; no. D8900; Sigma-Aldrich, St. Louis, MO, USA) containing 5% calf serum (no. 16170�C078; Life Technologies, Grand Island, NY, USA) and 20 ��g/ml gentamicin (no.

15750�C060; Life Technologies), under 5% CO2 in air. Cell viability was greater than 80%, as assessed by trypan blue exclusion. The cells in the cell suspension after centrifugation consisted of about 70% small luteal cells, 20% large luteal cells, 10% endothelial cells Brefeldin_A or fibrocytes and no erythrocytes. Cell culture The dispersed luteal cells were seeded at 2.0 �� 105 viable cells in 1 ml in 24-well cluster dishes (no. 662160; Greiner Bio-One, Frickenhausen, Germany) for evaluating mRNA expression and PG and P4 production, at 1.