The enzymes in cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism in human corpora lutea: dependence on luteal phase, cellular and subcellular distribution

Eicosanoids synthesized within corpus luteum are presumed to regulate luteal function in women. However, the potential cellular source(s) of the eicosanoids, whether small and large luteal cells differ in eicosanoid synthesis and whether eicosanoids other than prostaglandin (PG)E2, PGF2 alpha and 6-keto-PGI1 alpha can be synthesized, have not been investigated. The present immunocytochemical studies were undertaken to answer these questions using mono and polyclonal antibodies to several enzymes in arachidonic acid metabolism by cyclooxygenase and lipoxygenase pathways. Human corpora lutea from early (n = 5), mid (n = 6) and late (n = 3) luteal phases were specifically immunostained for all the enzymes. All the enzymes were present in small and large luteal cells as well as in non luteal cells. However, small luteal cells contained more immunoreactive 5-lipoxygenase, PGD2 and PGF2 alpha synthases; large luteal cells contained more TXA2 synthase and 12-lipoxygenase; small and large luteal cells contained similar amounts of cyclooxygenase and PGI2 synthase. In all the cells, immunoreactive PGD2, PGI2 and TXA2 synthases increased from early to mid luteal phase and then declined in late luteal phase. Cyclooxygenase, 5- and 12-lipoxygenases and PGF2 alpha synthase, on the other hand, increased from early to mid and mid to late luteal phases. Immunoreactive cyclooxygenase and 5- and 12-lipoxygenases were present primarily in rough endoplasmic reticulum (ER) and/or smooth ER and cytoplasm. Quite unexpectedly, all three enzymes were also found in nuclear membranes, condensed chromatin and especially at the perimeter of condensed chromatin. Dispersed chromatin contained very little or no immunoreactive enzyme. These results indicate that regulation of human luteal function by eicosanoids synthesized within the corpus luteum is complex involving perhaps a) small and large luteal as well as non luteal cells, b) eicosanoids which have not been previously considered to play a role in luteal function and c) coordinate regulation of more than one enzyme in the pathways of arachidonic acid metabolism.     

Role of the DLL4-NOTCH system in PGF2alpha-induced luteolysis in the pregnant rat

We investigated the expression and cell localization of NOTCH1, NOTCH4, and the delta-like ligand DLL4 in corpus luteum (CL) from pregnant rats during prostaglandin F2alpha (PGF2alpha)-induced luteolysis. We also examined serum progesterone (P(4)) and CL proteins related to apoptosis after local administration of the notch inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT). Specific staining for NOTCH1 and NOTCH4 receptors was detected predominantly in large and small luteal cells. Furthermore, in line with the fact that the notch intracellular domain is translocated to the nucleus, where it regulates gene expression, staining was evident in the nuclei of luteal cells. In addition, we detected diffuse cytoplasmic immunostaining for DLL4 in small and large luteal cells, in accordance with the fact that DLL4 undergoes proteolytic degradation after receptor binding. The mRNA expression of Notch1, Notch4, and Dll4 in CL isolated on Day 19 of pregnancy decreased significantly after administration of PGF2alpha. Consistent with the mRNA results, administration of PGF2alpha to pregnant rats on Day 19 of pregnancy decreased the protein fragment corresponding to the cleaved forms of NOTCH1/4 CL receptors. In contrast, no significant changes were detected in protein levels for the ligand DLL4. The local intrabursal administration of DAPT decreased serum P(4) levels and increased luteal levels of active caspase 3 and the BAX:BCL2 ratio 24 h after the treatment. These results support a luteotropic role for notch signaling to promote luteal cell viability and steroidogenesis, and they suggest that the luteolytic hormone PGF2alpha may act in part by reducing the expression of some notch system members.     

AKT-independent phosphorylation of TSC2 and activation of mTOR and ribosomal protein S6 kinase signaling by prostaglandin F2alpha

Prostaglandin F2alpha (PGF2alpha) is an important mediator of corpus luteum (CL) regression, although the cellular signaling events that mediate this process have not been clearly identified. It is established that PGF2alpha binds to a G-proteincoupled receptor (GPCR) to stimulate protein kinase C (PKC) and Raf-MEK-Erk signaling in luteal cells. The present experiments were performed to determine whether PGF2alpha stimulates the mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase 1 (S6K1) signaling pathway in steroidogenic luteal cells. We demonstrate that PGF2alpha treatment results in a timeand concentration-dependent stimulation of the phosphorylation and activation of S6K1. The stimulation of S6K1 in response to PGF2alpha treatment was abolished by the mTOR inhibitor rapamycin. Treatment with PGF2alpha did not increase AKT phosphorylation but increased the phosphorylation of Erk and the tumor suppressor protein tuberous sclerosis complex 2 (TSC2), an upstream regulator of mTOR. The effects of PGF2alpha were mimicked by the PKC activator PMA and inhibited by U0126, a MEK1 inhibitor. The activation of mTOR/S6K1 and putative down stream processes involving the translational apparatus (i.e. 4EBP1 phosphorylation, release of 4EBP1 binding in m(7)G cap binding assays, and the phosphorylation and synthesis of S6) were completely sensitive to treatment with rapamycin, implicating mTOR in the actions of PGF2alpha. Taken together, our data suggest that GPCR activation in response to PGF2alpha stimulates the mTOR pathway which increases the translational machinery in luteal cells. The translation of proteins under the control of mTOR may have implications for luteal development and regression and offer new strategies for therapeutic intervention in PGF2alpha-target tissues.     

Prostaglandin F2alpha-mediated activation of apoptotic signaling cascades in the corpus luteum during apoptosis: involvement of caspase-activated DNase

Prostaglandin F(2alpha) (PGF(2alpha)) acting via a G protein-coupled receptor has been shown to induce apoptosis in the corpus luteum of many species. Studies were carried out to characterize changes in the apoptotic signaling cascade(s) culminating in luteal tissue apoptosis during PGF(2alpha)-induced luteolysis in the bovine species in which initiation of apoptosis was demonstrable at 18 h after exogenous PGF(2alpha) treatment. An analysis of intrinsic arm of apoptotic signaling cascade elements revealed that PGF(2alpha) injection triggered increased ratio of Bax to Bcl-2 in the luteal tissue as early as 4 h posttreatment that remained elevated until 18 h. This increase was associated with the elevation in the active caspase-9 and -3 protein levels and activity (p < 0.05) at 4-12 h, but a spurt in the activity was seen only at 18 h posttreatment that could not be accounted for by the changes in the Bax/Bcl-2 ratio or changes in translocation of Bax to mitochondria. Examination of luteal tissue for FasL/Fas death receptor cascade revealed increased expression of FasL and Fas at 18 h accompanied by a significant (p < 0.05) induction in the caspase-8 activity and truncated Bid levels. Furthermore, intrabursal administration of specific caspase inhibitors, downstream to the extrinsic and intrinsic apoptotic signaling cascades, in a pseudopregnant rat model revealed a greater importance of extrinsic apoptotic signaling cascade in mediating luteal tissue apoptosis during PGF(2alpha) treatment. The DNase responsible for PGF(2alpha)-induced apoptotic DNA fragmentation was found to be Ca(2+)/Mg(2+)-dependent, temperature-sensitive DNase, and optimally active at neutral pH conditions. This putative DNase was inhibited by the recombinant inhibitor of caspase-activated DNase, and immunodepletion of caspase-activated DNase from luteal lysates abolished the observed DNA fragmentation activity. Together, these data demonstrate for the first time temporal and spatial changes in the apoptotic signaling cascades during PGF(2alpha)-in-duced apoptosis in the corpus luteum.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro*

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-1 (IGF-1) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-I (IGF-I) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.     

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2alpha induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2alpha on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares (n=8/group) were randomized into three groups: control (no treatment or biopsy), saline+biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2alpha+biopsy (5mg PGF2alpha at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline+biopsy group. At Hour 24 (12h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower (P<0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2alpha increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P<0.006), but decreased mRNA for LH receptor to 44% (P<0.05), steroidogenic acute regulatory protein to 22% (P<0.001), and aromatase to 43% (P<0.1) of controls. There was no difference in mRNA levels for PGF2alpha receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2alpha-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2alpha induced the synthesis of intraluteal PGF2alpha. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2alpha in mares.     

Administration of prostaglandin f(2 alpha) during the early bovine luteal phase does not alter the expression of ET-1 and of its type A receptor: a possible cause for corpus luteum refractoriness

Luteal regression is initiated by prostaglandin F(2 alpha) (PGF(2 alpha)). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF(2 alpha). Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF(2 alpha)-induced luteolysis. Therefore, in this study, we compared the effects of PGF(2 alpha) administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF(2 alpha) receptors (PGF(2 alpha)-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF(2 alpha) analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF(2 alpha) analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF(2 alpha) injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450(scc)) was only affected when PGF(2 alpha) was administered during midcycle. Nevertheless, PGF(2 alpha) elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF(2 alpha)-R was transiently affected. Such effects probably result from PGF(2 alpha) acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF(2 alpha), possibly the endothelium, could yet be nonresponsive during the early luteal phase.     

Actions of prostaglandin F2alpha and prolactin on intercellular adhesion molecule-1 expression and monocyte/macrophage accumulation in the rat corpus luteum

Expression of intercellular adhesion molecule-1 (ICAM-1) and the accumulation of monocytes/macrophages are inflammatory events that occur during PRL (PRL)-induced regression of the rat corpus luteum. Here we have compared the ability of prostaglandin F2alpha (PGF) and PRL to induce, in rat corpora lutea, inflammatory events thought to perpetuate luteal regression. Immature rats were ovulated with eCG-hCG and then hypophysectomized (Day 0), which resulted in a single cohort of persistent, functional corpora lutea. On Days 9-11, the rats received twice daily injections of saline, PGF (Lutalyse, 250 microg/injection), or PRL (312 microg/injection) to induce luteal regression. Surprisingly, luteal weight and plasma progestin concentrations (progesterone and 20alpha-dihydroprogesterone) did not differ between PGF-treated rats and controls; whereas both luteal weight and plasma progestins declined significantly in PRL-treated rats. Furthermore, corpora lutea of PGF-treated rats and controls contained relatively minimal ICAM-1 staining and few monocytes/macrophages. In contrast, but as expected, corpora lutea of PRL-treated rats stained intensely for ICAM-1 and contained numerous monocytes/macrophages. In an additional experiment, there was no indication that luteal prostaglandin F2alpha receptor mRNA diminished as a result of hypophysectomy. These findings suggest that prolactin, not PGF, induces the inflammatory events that accompany regression of the rat corpus luteum.     

Prostaglandin E1 and F2alpha specific binding in bovine corpora lutea: comparison with luteolytic effects.

Preliminary characterization indicated the presence of separate prostaglandin (PG)E1 and (PG)F2alpha binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 x 10(-9)M and 1.1 x 10(-8)M for PGE1 and PGF2alpha, respectively. Competition of several natural prostaglandins for the PGE1 and PGF2alpha bovine luteal specific binding sites indicates specificity for the 9-keto or 9alpha-hydroxyl moiety, respectively. Differences in relative ability to inhibit 3H-PG binding were found due to sensitivity to the absence or presence of the 5, 6-cis-double bond as well. Bovine luteal function was affected following treatment of heifers with 25 mg PGF2alpha as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contract, treatment with 25 mg PGE1 resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE1 treatment compared to pretreatment values. In so far as data obtained in vitro on PGF2alpha relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF2alpha induced luteolysis in the bovine, PGF2alpha relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE1 relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.     

Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha)

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).     

Changes in corpus luteum content of prostaglandin F2 alpha and E in the adult pseudopregnant rat

Conflicting reports exist regarding the source of luteolytic PGF2 alpha in the rat ovary. To assess the quantities of different PGs, measurements of PGF2 alpha, PGE and PGB were performed by radioimmunoassay in the adult pseudopregnant rat ovary throughout the luteal lifespan. Ovaries of 84 rats were separated by dissection into two compartments, corpora lutea of pseudopregnancy and remainder of ovary. Tissue samples were homogenized and prostaglandins extracted and determined by radioimmunoassay. During the mid-luteal and late-luteal phases, levels of PGs were significantly higher in the corpora lutea of pseudopregnancy than in the remainder of ovary. An increase of PGF2 alpha-content in the corpus luteum was registered with peak-levels of 53.9 +/- 8.5 (mean +/- SEM, N = 18) ng/g tissue wet weight at day 13 of pseudopregnancy. PGE-levels reached peak-values at day 11 of pseudopregnancy (271.6 +/- 28.4 ng/g w w, mean +/- SEM, N = 12). PGB-levels were below detection limits in all compartments for all ages studied. The present study demonstrates increased availability of PGF2 alpha in the corpus luteum during the luteolytic period, and points toward either increased luteal synthesis or luteal binding of PGF2 alpha during the luteolytic period.     

Changes in expression of 11beta-hydroxysteroid dehydrogenase type-1, type-2 and glucocorticoid receptor mRNAs in porcine corpus luteum during the estrous cycle

The objective of the present study was to determine whether glucocorticoid (GC) and its receptor (GC-R) are expressed in the porcine corpus luteum (CL), and whether GC influences porcine luteal hormone production. The gene expressions of 11beta-hydroxysteroid dehydrogenase type 1 (11-HSD1), type 2 (11-HSD2), GC-R, and the concentrations of GC were determined in the CL of Chinese Meishan pigs during the estrous cycle. Moreover, the effects of GC on progesterone (P(4)), estradiol-17beta (E(2)), and prostaglandin (PG) F2alpha secretion by cultured luteal cells were investigated. Messenger RNAs of the 11-HSD1, 11-HSD2, and GC-R were clearly expressed in the CL throughout the estrous cycle. The 11-HSD1 mRNA level in the CL was higher at the regressed stage than at the other stages (P < 0.05), whereas 11-HSD2 mRNA was lower at the regressed stage than at the other stages (P < 0.05). GC-R mRNA level was higher at the regressed stages than at the other stages (P < 0.01). Concentrations of GC were lower in the regressed CL than in the other stages (P < 0.01). When the cultured luteal cells obtained from mid-stage CL (Days 8-11) were exposed to GC (50-5,000 ng/ml), P(4) and PGF2alpha secretion by the cells were reduced (P < 0.05), whereas GC had no effect on E(2) secretion by the cells. The overall results suggest that GC is regulated locally by 11-HSD1 and 11-HSD2 in the porcine CL. GC inhibits P(4) and PGF2alpha production from luteal cells via their specific receptors, implying GC plays some roles in regulating porcine CL function throughout the estrous cycle.     

Mechanisms of gonadotropin-releasing hormone and prostaglandin action on luteal cells

Both gonadotropin-releasing hormone (GnRH) and prostaglandin F2 alpha (PGF2 alpha) can inhibit cAMP and progesterone production in the corpus luteum; however, their mechanism of action is not known. GnRH or PGF2 alpha causes a rapid and marked increase of labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The incorporation of radioactivity is increased as early as 2 and 5 min into PA and PI, respectively. The labelling of the other phospholipids is not affected. GnRH and PGF2 alpha exert their stimulatory effects on PA-PI turnover at a mean effective dose value of ca. 15 and 100 nM, respectively. Their effects appeared to be additive when both agents were present in the same incubations. Interestingly, addition of the calcium ionophore A23187 also causes a dramatic increase of PA-PI turnover in luteal cells. By contrast, human chorionic gonadotropin and isoproterenol, agents that stimulate cAMP and progesterone production in luteal cells, as well as PGE2 (1 microM), all fail to alter phospholipid labelling; dibutyryl or 8-bromo-cAMP (2-5 mM) actually attentuates the GnRH or PGF2 alpha effect on PI and PA. A very similar PA-PI response to GnRH and PGF2 alpha has also been observed using rat granulosa cells in culture. It seems that following their binding to membrane receptors, GnRH and PGF2 alpha may share a common mechanism in the ovarian cell, possibly involving the stimulation of PA-PI metabolism.     

Hormonal regulation of free intracellular calcium concentrations in small and large ovine luteal cells

The second messengers mediating hormonal regulation of the corpus luteum are incompletely defined, particularly for the primary luteolytic hormone prostaglandin F2 alpha (PGF2 alpha). In this study, hormonally induced changes in free intracellular calcium concentrations were measured in individual small and large ovine luteal cells by using computer-assisted microscopic imaging of fura-2 fluorescence. This technique could readily detect transient increases in free calcium concentrations within both small and large luteal cells after treatment with 1 microM of the calcium ionophore, A23187. Treatment with PGF2 alpha (1 microM) caused a dramatic increase in free calcium concentrations in large (before = 73 +/- 2 nM; 2 min after PGF2 alpha = 370 +/- 21 nM; n = 33 cells) but not in small (before = 66 +/- 4 nM; 2 min after PGF2 alpha = 69 +/- 8 nM; n = 12 cells) luteal cells. The magnitude and timing of the calcium response was dose- and time-dependent. The PGF2 alpha-induced increase in free intracellular calcium is probably due to influx of extracellular calcium, since inclusion of inorganic calcium channel blockers (100 microM manganese or cobalt) attenuated the response to PGF2 alpha and removal of extracellular calcium eliminated the response. In contrast to PGF2 alpha, luteinizing hormone (LH) (100 ng/ml) caused no change in intracellular levels of free calcium in small or large luteal cells, even though this dose of LH stimulated (p less than 0.01) progesterone production by small luteal cells. Therefore, alterations in free calcium concentrations could be the intracellular second message mediating the luteolytic action of PGF2 alpha in the large ovine luteal cell.