Oxygen deficiency caused by a decrease in the blood supply is known to induce various responses of cells. Because luteal blood flow has been shown to decrease during luteolysis, a low-oxygen condition seems to be an integral part of the environment during luteolysis. To determine whether a low-oxygen condition is associated with functional luteolysis, we examined the influence of reduced oxygen tension on the luteal progesterone (P4) generating system in cultured bovine midluteal cells. Luteal cells obtained from midcycle corpus luteum (d 8-12) were incubated under different O2 concentrations (20, 10, 5, 3% O2) with or without LH for 24 h. P4 production decreased with decreasing O2 concentration but was significantly stimulated by LH regardless of O2 concentration. After 8 h of culture, both basal and LH-stimulated P4 production was significantly lower under 3% O2 than under 20% O2. Low-oxygen condition also inhibited pregnenolone production. Cytochrome P450 side-chain cleavage enzyme (P450scc) mRNA expression, measured by quantitative PCR, decreased under low-oxygen condition in both non-LH-treated and LH-treated cells. Low-oxygen condition did not affect the expressions of steroidogenic acute regulatory protein mRNA or protein, whereas steroidogenic acute regulatory protein mRNA expression was stimulated by LH during 4 h of culture. Low-oxygen condition also did not affect 3 beta-hydroxysteroid dehydrogenase/Delta 5-Delta 4 isomerase mRNA expression or the activity of the enzyme in the cells, regardless of the incubation period. The overall results indicate that a low-oxygen condition decreases P4 synthesis by attenuating P450scc production and P450scc activity in bovine luteal cells and suggest that oxygen deficiency is an essential condition for the progression of luteolysis in cattle.

Prolactin (PRL) is known to be synthesized not only in the anterior pituitary, but also in other organs including the ovary. Among its various functions, PRL is regarded as the most important constituent of the luteotropic complex in rodents and pigs. The purpose of the present study was to determine whether PRL is produced locally in bovine corpus luteum (CL) and to determine its possible roles in CL. In the present study, we examined changes during the luteal phase in (1) the expressions of PRL and PRL receptors (long form: l-PRLR, short form: s-PRLR) in CL and (2) the localization of PRL in CL. We also measured the levels of PRL mRNA in cultured luteal cells and luteal endothelial cells. Furthermore, the effect of PRL on progesterone (P4) and prostaglandin (PG) F2alpha production by cultured bovine luteal cells was examined. Semiquantitative RT-PCR analysis revealed that the mRNAs for PRL and its two receptors, l- and s-PRLR, were expressed in all luteal stages examined. PRL mRNA expression was less in the regressed stage (days 19-21 after ovulation) than in the other stages. Both l-PRLR and s-PRLR mRNA expressions were higher in the late luteal stage (days 15-17) than in the other stages, while the ratio of l-PRLR to s-PRLR was less in the regressed stage than in the other stages. PRL mRNA was also detected in cultured luteal cells and luteal endothelial cells. PRL protein was immunohistochemically detected only in CL of the mid- and regressed stages. It was detected in smooth muscle cells of the intraluteal arterioles and endothelial cells but not in luteal cells and other cell types of CL. Exposure of cultured luteal cells obtained from mid-stage CL (days 8-12) to bovine PRL (100, 200 ng/ml) for 24 hr did not affect P4 and PGF2alpha production by the cells. The present study demonstrates for the first time the expressions of PRL and PRLR mRNA in bovine CL throughout the luteal phase. The overall results strongly suggest that the bovine CL is an extrapituitary site of PRL production.

Abstract. Structural luteolysis occurs by apoptosis of luteal cells. The present study examined the effects of activators of well-characterized second messengers on Fas and caspase-3 mRNA expression and on P4 production in luteal cells in order to trace the pro-and anti-apoptotic factors in the bovine corpus luteum (CL). Cultured bovine mid luteal cells were treated for 24 h with a cyclic AMP analogue (8-bromo cyclic AMP; 8br-cAMP; 2.5 mM), a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate; PMA; 10 µM), or calcium ionophore (A23187; 10 µM). Fas and caspase-3 mRNA expression was inhibited by 8br-cAMP and PMA but was increased by A23187 (P<0.05). In addition, P4 production by bovine luteal cells was stimulated by 8br-cAMP and PMA, whereas it was inhibited by A23187, compared with untreated controls (P<0.05). The overall results suggest that cAMP and PKC suppress apoptosis in bovine luteal cells through inhibition of Fas and caspase-3 mRNA expression and through stimulation of P4 production. Therefore, substances that activate cAMP or PKC may act as survival factors in the bovine CL. Furthermore, substances that mobilize Ca 2+ may act as apoptotic factors by stimulating Fas and caspase-3 expression in the bovine luteal cells.