Background
The corpus luteum (CL) is a transient endocrine gland whose primary secretory product is progesterone (P4). The life span of the CL and consequently 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, PGF
2α is recognized as an important if not the main luteolytic factor [
3‐
9]. During the ovarian cycle, the transition from early to mid-luteal phase is associated with changes in resistance/susceptibility to the luteolysin PGF
2α; in cows, the CL is resistant to exogenous PGF
2α prior to day 5 of the estrous cycle [
10‐
17]. The cellular basis controlling luteal function during these physiological transitions, although studied intensely, is incompletely understood.
In steroidogenic cells of the ruminant CL, PGF
2α activates its plasma membrane G-protein-coupled receptor, which in turn activates the membrane-bound phosphoinositide-specific phospholipase C (PLC), yielding inositol 1,4,5-trisphosphate (IP
3) and diacylglycerol [
18]. Indeed, in bovine luteal cells, PGF
2α stimulated phosphatidylinositol 4,5-biphosphate hydrolysis and mobilized intracellular Ca
2+ [
19]. Accordingly, calcium and protein kinase C (PKC) have been shown to be the intracellular mediators of PGF
2β actions in luteal cells [
20]. The regulatory effects of intracellular calcium concentration ([Ca
2+]i) on progesterone might be biphasic as there is also evidence for a calcium requirement to support P4 synthesis by bovine luteal cells and LH, a luteotrophic hormone, increases IP
3, and [Ca
2+]i in bovine luteal cells and in porcine granulosa cells [
21‐
23]. Therefore, there might exist thresholds of [Ca
2+]i that support or inhibit P4 synthesis.
Choudhary et al, [
17] tested the ability of increasing concentrations of PGF
2α to increase the [Ca
2+]i in large (LLC) and small (SLC) bovine luteal cells as function of development. Day-10 steroidogenic cells were more responsive to PGF
2α than Day-4 cell. Response amplitudes and number of responding cells were significantly affected by agonist concentration, luteal development and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations from Day-10 than from Day -4 cells. Furthermore, on Day-10, as concentrations of PGF
2α increased, larger percentages of SLC responded. Based on those results Choudhary et al proposed that the lower efficacy of PGF
2α in the early CL was likely related to signal transduction differences associated with the PGF
2α receptor at those two developmental stages [
17].
The array of PKC isozymes expressed in whole bovine CL includes α, βI, βII, ε and μ[
24‐
27]; and it has been demonstrated that the amount of PKCε expressed in the Day-10 CL is greater than in the Day-4 CL [
26]. The latter observation led Sen et al, to propose that differential expression of PKCε as a function of development could play a role in the observed transitional resistance/susceptibility to PGF
2α-induced luteal regression [
26,
27]. Sen et al, had further hypothesized that regulation of [Ca
2+]i was a cellular mechanism through which PKCε could mediate actions of PGF
2α on P4 secretion [
27]. Additionally, there is evidence indicating that when bovine follicular theca cells are isolated and their luteinization is induced under in vitro tissue culture conditions, they express PKCδ [
28]. As PKCδ has been reported to play an important role in other species such as in rabbits and rodents [
29,
30], this PKC isozyme might also be important for the physiology of the bovine ovary.
Endothelial cells of the bovine CL do not express PKCε, although they do express the other PKC isozymes described in the bovine CL [
31]. Data obtained with Western blot and immunohistological assays indicated that steroidogenic cells are the main source of PKCε in the bovine CL [
31]. Therefore, in experiment 1, in order to assess the potential physiological role of PKCε, we have used a siRNA strategy to down- regulate the expression of this PKC isozyme in luteal steroidogenic cells. In experiment 2, we used the PKCε down-regulated cells to test two hypotheses. Our first working hypothesis was that PKCε expression was necessary for PGF
2α to inhibit LH-stimulated P
4 secretion in vitro. The second working hypothesis was that PKCε was necessary for the expression of key genes of prostaglandin synthesis/metabolism that would favor PGF
2α synthesis; whereas in PKCε down regulated cells, the expression of key genes of prostaglandin synthesis/metabolism would be such that synthesis of PGE2 would be favored. Finally, in experiment 3, we tested the hypothesis that [Ca
2+]i is the cellular mechanism through which PGF
2α inhibits luteal progesterone. We reasoned that if a pharmacological treatment is used to increase [Ca
2+]i, this should inhibit luteal progesterone secretion with equally effectiveness, regardless of the developmental stage of the CL. Therefore, we used a pharmacological agent to increase [Ca
2+]i and examine its effects on LH-induced P
4 secretion in luteal cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL. Furthermore, this hypothesis was also tested by using a pharmacological agent to buffer any increase in [Ca
2+]i and examine, under conditions of low [Ca
2+]i, the anti-steroidogenic effect of PGF
2α on LH-induced P4 synthesis/secretion in cultures of luteal cells collected from mid-cycle (Day -10) CL.
Discussion
The roles of specific PKC isozymes in luteal physiology have received little attention to date. As discussed below, these studies were designed to test the effects of ablating PKCε expression in order to examine its hypothesized function. Previous studies had indicated that a potential function for PKCε might be to regulate quantitatively the intracellular calcium signal initiated by PGF
2α on one of its luteal targets, the steroidogenic cells. The present studies validate the effective and specific down-regulation of PKCε by siRNA technology and provide strong evidence about the function of this PKC isozyme in luteal physiology. The data support the overall hypothesis that down-regulating expression of PKCε reduces the effectiveness of PGF
2α in reducing progesterone synthesis/secretion. This observation extends the report that when PKCε was inhibited with PKCε-specific inhibitors, the PGF
2α – induced rise in [Ca
2+]i was decreased in LLC and SLC and that this in turn had consequences (at least in part) in the ability of PGF
2α to inhibit LH-stimulated P4 secretion at this developmental stage [
27]. As previously reported [
17], LH induced an increase in the amount of P4 secretion. Interestingly, in the group where PKCε expression was down -regulated, the inhibitory effect of PGF
2α on LH-stimulated P4 secretion was significantly mitigated (Fig.
3). This observation has an important physiological corollary: both PGF
2α-receptors and PKCε are expressed in the same luteal cell type. Therefore, the isozyme PKCε has an important compatible time (mid-luteal phase) and place (small and large luteal steroidogenic cells) of expression, for it to have a role in the luteal transition from resistance to sensitivity to luteolytic actions of PGF
2α. Furthermore, if PKCε expression is down -regulated (this study) or if its activation is inhibited [
27], the anti-steroidogenic effect of PGF
2α on LH-stimulated P4 secretion is impaired.
Experiment 2 also tested the hypothesis that down-regulating PKCε could influence the expression of key PG metabolizing enzymes that, in turn, could influence the balance of PG production from luteo-protective or luteotrophic to luteolytic. The mechanism for luteal resistance is not exactly known. However there is now evidence that regulation of key PG metabolizing enzymes observed during physiological states in which the life span of the CL is modified is likely to play an important role in this complex process [
41‐
49]. The selection of the examined genes was based on the available evidence that, because of their key positions in the PG biosynthetic pathway, these genes have been shown to determine the accumulation of luteolytic or luteotrophic classes of PG [
40‐
45]. For example, we examined the effects of down-regulating PKCε on the expression of PGE
2 and F synthases because of their more direct effect on determining whether PGH2 is metabolized to PGE
2 or PGF
2α. The results obtained were unexpected; the prediction was that because of low expression of PKCε, exogenous PGF
2α would not be able to induce high increases in the cytosolic concentration of calcium, and consequently, the expression of PGE
2 synthase/PGF
2α synthase ratio would favor PGE
2 synthesis. The above conditions would favor luteal function. However, it is worth pointing out the importance of looking beyond steady states of mRNA encoding these enzymes; sometimes regulation may be at the level of protein or even enzyme activity and additional work is necessary before rejecting the tested hypothesis.
The developmental significance of a regulatory role played by cytosolic calcium concentrations in mediating the inhibitory actions of PGF
2α is documented by results obtained in experiment 3. As reported in previous studies [
17], PGF
2α reduced LH-stimulated P4 secretion in Day-10 cells only. Basal P4 secretion was not affected by the PGF
2α-treatment at any of the two developmental stages tested. As the working hypothesis predicted, the pharmacological increase in [Ca
2+]i induced by A23187 effectively mimicked the inhibitory effect of PGF
2α in Day -10 steroidogenic cells. Furthermore, as predicted by the working hypothesis, the A23187 treatment also inhibited LH-stimulated P4 secretion in Day -4 steroidogenic cells. This inhibitory effect of A23187 is most likely due to its demonstrated effect in increasing the intracellular concentration of calcium ions [
27] in these cells and not due to other non-specific effects. This interpretation is also supported by the observation that treatment with A23187 had no negative effect on basal P4 secretion at any of the two developmental stages tested.
Further support for the significance of a regulatory role played by the increase in [Ca
2+]i in mediating the inhibitory actions of PGF
2α is documented by results obtained in experiment 3 where the cytoplasmic calcium buffering capacity of the cells was increased by Bapta-AM. At lower concentrations (0.1 and 1.0 μmol), the calcium buffering capacity of Bapta-AM was, most likely, at values that still allowed a PGF
2α-stimulated increase in [Ca
2+]i; which in turn, preserved the ability of PGF
2α to inhibit LH-stimulated P4 secretion (Fig.
6). However, as the calcium buffering capacity in the cytoplasm of the steroidogenic cells was increased by increasing the concentration of Bapta-AM (10 and 100 μmol), the calcium signaling feature of activating the PGF
2α receptors was most likely eliminated or at least reduced, and consequently, the ability of PGF
2α to inhibit LH-stimulated P
4 secretion was also significantly reduced (Fig.
6). Similar effects of Bapta-AM on basal and hormonal-stimulated steroidogenesis have been reported in MA-10 Leydig cells (34). Therefore, the results of experiment 3 stress the calcium requirement for PGF
2α to inhibit LH-stimulated P
4 secretion in the mid-phase CL and support the reported observation that the lower efficacy of PGF
2α to inhibit P
4 secretion in the early CL is related to the reduced ability of PGF
2α to increase the cytoplasmic concentration of calcium at this developmental stage [
17]. Taken together, the results obtained in the A23187 and Bapta-AM experiments, strongly support the proposed hypothesis that an attenuation of the luteolytic actions of PGF
2α is associated with a compromise in the ability of PGF
2α to induce a rise in [Ca
2+]i [
27]. Therefore these studies provide a strong linkage between the signal transduction utilized by the PGF
2α receptor at different developmental stages and quantitative aspects of the known intracellular mediator of PGF
2α actions in the CL, [Ca
2+]i. In this regard, species differences do exist, as in rat luteal cells the antigonadotropic action of PGF
2α is not mediated by elevated cytosolic calcium levels [
50]. It appears that the bovine CL therefore, has the following commonalities with human CL: 1) in both species, PGF
2α is luteolysin, 2) the luteolytic effect of PGF
2α appears only during mid- and late-luteal phase, and 3) in both, the humans and cows, changes in intracellular calcium appear to regulate luteal function ([
51] and this study).
In summary, the evidence presented here strongly supports the idea that PKCε, an isozyme highly expressed in steroidogenic luteal cells with acquired luteolytic response to PGF2α, has an important regulatory role in the ability of PGF2α to inhibit LH-stimulated P4 secretion in vitro at this developmental stage. The data presented strongly support the hypothesis that luteal resistance to the luteolytic actions of PGF2α is associated with a compromised ability of PGF2α to induce a rise in [Ca2+]i. If the PGF2α receptor and its associated signal transduction is bypassed with a pharmacological agent to increase the [Ca2+]i, the LH-stimulated P4 secretion in Day -4 steroidogenic cells is eliminated, an action that cannot be induced by PGF2α at this developmental stage. Conversely, if the increase in [Ca2+]i typically induced by PGF2α on Day-10 steroidogenic luteal cells is buffered by a pharmacological agent, then the ability of PGF2α to inhibit the LH-stimulated P4 secretion is abrogated.
Authors' contributions
MPG and AS made equal contributions to this study. MPG and AS were responsible for surgical procedures, all aspects of laboratory procedures, participated in the discussion, interpretation of results and in drafting the manuscript. EKI participated in the surgeries, design of the study, data analysis and drafting of the manuscript. JAF directed and participated in all aspects of the studies. All authors read and approved the final manuscript.