Abstract
The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.PKCepsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKCepsilon expression was ablated by 75%, the inhibitory effect of PGF2alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKCepsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKCepsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 μmol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKCepsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2alpha.
Highlights
The corpus luteum (CL) is a transient endocrine gland whose primary secretory product is progesterone (P4)
Experiment 1 Culturing steroidogenic cells collected from the Day -6 CL spontaneously induced the expression of PKCε
Expression of PKCε was induced gradually by the tissue culture conditions, and as Day-6 luteal cells were cultured up to 6 days, PKCε expression had been spontaneously increased to values comparable to those seen in Day-10 CL
Summary
The corpus luteum (CL) is a transient endocrine gland whose primary secretory product is progesterone (P4). The life span of the CL and the amount of P4 it secretes is regulated according to reproductive physiological status. Substances reducing P4 secretion and shortening the luteal life span are said to be luteolytic [1,2]. In most species, including human beings, PGF2α is recognized as an important if not the main luteolytic factor [39]. The transition from early to mid-luteal phase is associated with changes in resistance/ susceptibility to the luteolysin PGF2α; in cows, the CL is resistant to exogenous PGF2α prior to day 5 of the estrous cycle [10,11,12,13,14,15,16,17]. The cellular basis controlling luteal function during these physiological transitions, studied intensely, is incompletely understood
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