Masami Shibaya

Progesterone Is a Suppressor of Apoptosis in Bovine Luteal Cells

Abstract Progesterone is suggested to be a suppressor of apoptosis in bovine luteal cells. Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells. Furthermore, apoptosis is known to be controlled by the bcl-2 gene/protein family and caspases. This study was undertaken to determine whether intraluteal progesterone (P4) is involved in Fas L–mediated luteal cell death in the bovine corpus luteum (CL) in vitro. Moreover, we studied whether an antagonist of P4 influences gene expression of the bcl-2 family and caspase-3 and the activity of caspase-3 in the bovine CL. Luteal cells obtained from the cows in the midluteal phase of the estrous cycle (Days 8–12 of the cycle) were exposed to a specific P4 antagonist (onapristone [OP], 10−4 M) with or without 100 ng/ml Fas L. Although Fas L alone did not show a cytotoxic effect, treatment of the cells with OP alone or in combination with Fas L resulted in killing of 30% and 45% of the cells, respectively (P < 0.05). DNA fragmentation was observed in the cells treated with Fas L in the presence of OP. The inhibition of P4 action by OP increased the expression of Fas mRNA (P < 0.01); however, it did not affect bax or bcl-2 mRNA expression (P > 0.05). Moreover, OP stimulated expression of caspase-3 mRNA (P < 0.01). The overall results indirectly show that intraluteal P4 suppresses apoptosis in bovine luteal cells through the inhibition of Fas and caspase-3 mRNA expression and inhibition of caspase-3 activation.

Roles of Tumor Necrosis Factor-α of the Estrous Cycle in Cattle: An In Vivo Study

Abstract We have suggested in a previous in vitro study that tumor necrosis factor-α (TNFα) plays a role in the initiation of luteolysis in cattle. The aim of the present study was to examine the influence of different doses of TNFα on the estrous cycle in cattle by observing the standing behavior and measuring peripheral concentrations of progesterone (P4) during the estrous cycle. Moreover, we evaluated the secretion of P4, oxytocin (OT), nitric oxide (NO), and luteolytic (prostaglandin F2α [PGF2α] and leukotriene C4 [LTC4]) and luteotropic (PGE2) metabolites of arachidonic acid in peripheral blood plasma as parameters of TNFα actions. Mature Holstein/Polish black and white heifers (n = 36) were treated on Day 14 of the estrous cycle (Day 0 = estrus) by infusion into the aorta abdominalis of saline (n = 8), an analogue of PGF2α (cloprostenol, 100 μg; n = 3) or saline with TNFα at doses of 0.1 (n = 3), 1 (n = 8), 10 (n = 8), 25 (n = 3), or 50 μg (n = 3) per animal. Peripheral blood samples were collected frequently before, during, and up to 4 h after TNFα treatment. After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. Lower doses of TNFα (0.1 and 1 μg) decreased the P4 level during the estrous cycle and consequently resulted in shortening of the estrous cycle (18.8 ± 0.9 and 18.0 ± 0.7 days, respectively) compared with the control (22.3 ± 0.3 days, P < 0.05). One microgram of TNFα increased the PGF2α (P < 0.001) and NO (P < 0.001) concentrations and decreased OT secretion (P < 0.01). Higher doses of TNFα (10, 25, 50 μg) stimulated synthesis of P4 (P < 0.001) and PGE2 (P < 0.001), inhibited LTC4 secreton (P < 0.05), and consequently resulted in prolongation of the estrous cycle (throughout 30 days, P < 0.05). Altogether, the results suggest that low concentrations of TNFα cause luteolysis, whereas high concentrations of TNFα activate corpus luteum function and prolong the estrous cycle in cattle.

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms.

Prostaglandins (PGs) are known to modulate the proper cycllcity of bovine reproductive organs. The main luteolytlc agent in ruminants Is PGF2α, whereas PGE2 has luteotropic actions. Estradiol 17ß (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2α and PGE2synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2α synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2α than stromal cells, increasing the PGF2α, to PGE2 ratio. The epithelial and stromal cells were prelncubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a transiation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2- Although the action of E2 on PGF2α synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2action, phytoestrogens did not cause Intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2α and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2α synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2α to PGE2 ratio.

Tumor Necrosis Factor-α (TNF α) Inhibits Progesterone and Estradiol-17β Production from Cultured Granulosa Cells: Presence of TNFα Receptors in Bovine Granulosa and Theca Cells

The aim of this study was to investigate whether functional tumor necrosis factor-α (TNFα) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P4) and estradiol-17β (E2) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 ± 0.15 to 6.9 ± 1.40 nM). Moreover, TNFα receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFα (rhTNFα; 0.06-6 nM) inhibited E2 secretion in a dose-dependent fashion (P<0.01), but did not affect P4 secretion. In addition, rhTNFα inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P4 and E2 secretion by the cells (P<0.01). These results indicate the presence of functional TNFα receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFα plays a role in regulating their secretory function.

Involvement of the Cytoskeleton in Oxytocin Secretion by Cultured Bovine Luteal Cells

Abstract A number of substances have been implicated in the regulation of oxytocin (OT) secretion from bovine corpus luteum in vivo. However, isolated bovine luteal cells cultured in a monolayer lose the ability to secrete OT in response to stimulatory substances. The present study investigated how cell-to-cell contact and the cytoskeleton affect OT secretion by isolated bovine luteal cells. In experiment 1, bovine midluteal cells (Days 8–12 of the estrous cycle) were stimulated with prostaglandin F2α (PGF2α; 1 μM), noradrenaline (NA; 10 μM), or growth hormone (GH; 5 nM) in two culture systems: In one system, cell monolayers were incubated in 24-well culture plates, and in the other system, aggregates of cells were incubated in glass tubes in a shaking water bath. The cells cultured in a monolayer underwent considerable spreading and showed a variety of shapes, whereas the cells cultured in glass tubes remained fully rounded during the experimental period and soon formed aggregates of cells. Although PGF2α, NA, and GH did not stimulate OT secretion by the monolayer cells, all tested substances stimulated OT secretion by the aggregated cells (P < 0.01). In experiment 2, the monolayer cells were pre-exposed for 1 h to an antimicrofilament agent (cytochalasin B; 1 μM) or two antimicrotubule agents (colchicine or vinblastine; 1 μM) before stimulation with PGF2α, NA, or GH. Although PGF2α, NA, and GH did not stimulate OT secretion by the monolayer cells in the presence of colchicine or vinblastine, they all stimulated OT secretion in the presence of cytochalasin B (P < 0.001). The overall results show that OT secretion by bovine luteal cells depends on microfilament function and cell shape. Moreover, the aggregate culture system that allows three-dimensional, cell-to-cell contact seems to be a good model for studying OT secretion by isolated bovine luteal cells.