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.     

Developmental sensitivity of the bovine corpus luteum to prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1): is ET-1 a mediator of the luteolytic actions of PGF2alpha or a tonic inhibitor of progesterone secretion?

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly 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 in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.     

Effects of prostaglandin F2 alpha-induced luteolysis on the populations of cells in the ovine corpus luteum

Receptors for prostaglandin (PG) F2 alpha in the ovine corpus luteum are localized on large steroidogenic luteal cells. Therefore, it was hypothesized that during luteolysis, the first demonstrable effects of PGF2 alpha would occur in the population of large luteal cells. To test this hypothesis, the numbers and sizes of large and small luteal cells, fibroblasts, capillary endothelial cells, and pericytes were determined in corpora lutea collected 12, 24, or 36 h (6 animals/group) following administration of PGF2 alpha on Day 10 postestrus and from untreated ewes on Days 10 and 12 postestrus. The numbers and sizes of luteal cells were determined after enzymatic dissociation of the luteal tissue into single cell suspensions and by morphometric analysis of luteal slices. Serum levels of progesterone decreased (p less than 0.05) within 12 h of treatment, indicating that luteolysis was induced. Recovery of the two types of steroidogenic luteal cells following enzymatic dissociation was different (p less than 0.05). Recovery of both steroidogenic cell types decreased with time after PGF2 alpha treatment, suggesting that they had become more fragile. As determined by morphometry, the number of large luteal cells was not different at any time point examined; however, by 36 h after treatment, the average diameter of large luteal cells had decreased (p less than 0.05). In contrast, by 24 h after treatment, there was a decrease in the number of small luteal cells (p less than 0.05) but no change in their diameter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin f(2alpha) induces distinct physiological responses in porcine corpora lutea after acquisition of luteolytic capacity

This study examines differences in intracellular responses to cloprostenol, a prostaglandin (PG)F(2alpha) analog, in porcine corpora lutea (CL) before (Day 9 of estrous cycle) and after (Day 17 of pseudopregnancy) acquisition of luteolytic capacity. Pigs on Day 9 or Day 17 were treated with saline or 500 microgram cloprostenol, and CL were collected 10 h (experiment I) or 0.5 h (experiment III) after treatment. Some CL were cut into small pieces and cultured to measure progesterone and PGF(2alpha) secretion. In experiment I, progesterone remained high and PGF(2alpha) low in luteal incubations from either Day 9 or Day 17 saline-treated pigs. Cloprostenol increased PGF(2alpha) production 465% and decreased progesterone production 87% only from Day 17 luteal tissue. Cloprostenol induced prostaglandin G/H synthase (PGHS)-2 mRNA (0.5 h) and protein (10 h) in both groups. In cell culture, cloprostenol or phorbol 12, 13-didecanoate (PDD) (protein kinase C activator), induced PGHS-2 mRNA in luteal cells from both groups. However, acute cloprostenol treatment (10 min) decreased progesterone production and increased PGF(2alpha) production only from Day 17 luteal cells. Thus, PGF(2alpha) production is induced by cloprostenol in porcine CL with luteolytic capacity (Day 17) but not in CL without luteolytic capacity (Day 9). However, this change in PGF(2alpha) production is not explained by a difference in induction of PGHS-2 mRNA or protein.     

Immune cell-endothelial cell interactions in the bovine corpus luteum

Early embryonic mortality accounts for a substantial portionof reproductive failure in agriculturally important livestock,including the dairy cow. The maintenance of early pregnancyrequires a fully functional corpus luteum (CL) that is not susceptibleto regression following fertilization, yet the cellular mechanismsof luteal regression are not clearly understood. Immune-cellaccumulation within the CL at the time of regression is a well-documentedphenomenon in a variety of species. In the dairy cow, immune-cellaccumulation precedes luteal regression by several days andcoincides with an increase in expression of the chemokine monocytechemoattractant protein 1 (CCL2), suggesting that immune-mediatedevents promote tissue destruction. Recent studies indicate thatendothelial cells comprising the CL are a primary source ofCCL2 secretion. Moreover, although uterine-derived prostaglandinF₂ (PGF) initiates luteal regression in the cow, PGF does notdirectly provoke CCL2 secretion by luteal endothelial cells.Instead, PGF-induced luteal regression is thought to requirecooperative interaction among immune cells, endothelial cells,and steroidogenic cells of the CL to further promote CCL2 secretion,enhance immune-cell recruitment, and eliminate luteal tissue.This brief review focuses on putative interactions between immunecells and endothelial cells derived from the bovine CL thatresult in enhanced CCL2 expression and the elaboration of otherinflammatory mediators (for example, cytokines), which perpetuateluteal regression. Fundamental knowledge of immune-endocrineinteractions within the reproductive system of cows has relevanceto other CL-bearing mammals, including humans and endangeredanimals, particularly in the development of methods to controland/or improve fertility. Thus, it is a timely topic for thissymposium concerning ecological immunology and public health.     

Expression and activation of protein kinase C isozymes by prostaglandin F(2alpha) in the early- and mid-luteal phase bovine corpus luteum

Western blotting was used to identify the array of protein kinase C (PKC) isozymes expressed in the early (Day 4) and midcycle (Day 10) bovine corpus luteum (CL). PCKalpha, betaI, betaII, epsilon, and micro isozymes were detected in total protein samples prepared from both Day-4 and Day-10 corpora lutea. In contrast, specific antibodies for PKCgamma, eta, lambda, and theta isozymes failed to detect protein bands in the luteal samples. PKCbetaII and epsilon isozymes were expressed differentially at these two developmental stages of the bovine CL. In the Day-4 luteal samples, PKCepsilon was barely detectable; in contrast, in the Day-10 samples, the actin-corrected ratio for PKCepsilon was 1.16 +/- 0.13. This ratio was higher than the detected ratio for PKCbetaI and micro at this developmental phase of the CL (P < 0.01), but it was comparable with the ratio detected for the PCKalpha and betaII. The amount of PKCbetaII was, although not as dramatic, also greater in the Day-10 CL (actin-corrected ratio was 0.85 +/- 0.2) than in the Day-4 CL (0.35 +/- 0.09 [P < 0.01]). The actin-corrected ratios for all other PKC isozymes, alpha (Day 4 = 0.93 +/- 0.16, Day 10 = 0.97 +/- 0.09), betaI (Day 4 = 0.54 +/- 0.073, Day 10 = 0.48 +/- 0.74), and micro (Day 4 = 0.21 +/- 0.042, Day 10 = 0.21 +/- 0.38) were not different at these 2 days of the cycle. An experiment was designed to test whether activation of specific isozymes differed between CL that do or do not regress in response to PGF(2alpha). Bovine CL from Day 4 and Day 10 of the estrous cycle were collected and 1 mm CL fragments were treated in vitro for 0, 2.5, 5, 10 or 20 min with PGF(2alpha) (0.1, 1.0, and 10 nM) or minimal essential medium-Hepes vehicle. Translocation of PKC from cytoplasm to membrane fraction was used as indication of PKC activation by PGF(2alpha). Evidence for PKC activation was observed in both Day-4 and Day-10 luteal samples treated with 10 nM PGF(2alpha). Therefore, if PKC, an intracellular mediator associated with the luteal PGF(2alpha) receptor, contributes to the lesser sensitivity of the Day-4 CL, it is likely due to the differential expression of the epsilon and betaII isozymes of PKC at this stage and not due to an inability of the PGF(2alpha) receptor to activate the isozymes expressed in the early CL.     

PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells

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.     

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.     

Expression of cyclooxygenase 1 and 2 in the canine corpus luteum during diestrus

In the dog, unlike most other domestic animal species, corpus luteum (CL) life span is not affected by hysterectomy. Only in pregnant dogs, during the immediate prepartum decline of progesterone, does PGF2alpha clearly seem to act as an endogenous luteolytic agent. Whether endogenous PGF2alpha plays a role in the slow regression of the corpora lutea of the nonpregnant cycle is not known. To test for possible paracrine/autocrine effects of locally produced PGF2alpha, luteal expression of the key rate-limiting enzymes in prostaglandin biosynthesis, i.e. cyclooxygenase 1 and 2 (Cox1 and Cox2), was examined in dogs during diestrus, including the periods of CL formation, as well as early and late CL regression. Corpora lutea were collected by ovariohysterectomy from nonpregnant bitches 5, 15, 25, 35, 45 and 65 days after ovulation. On the mRNA-level, expression of Cox1 and Cox2 was tested by qualitative and quantitative, Real Time (Taq Man) RT-PCR; on the protein level, expression of Cox2 was studied by immunohistochemistry. The mRNA for Cox1 and Cox2 were detected at all stages of diestrus. Expression of Cox1 was lowest on Day 5 (ovulation = Day 0) and higher and nearly constant thereafter. Expression of Cox2-mRNA was distinctly cycle related and highest on Day 5; it decreased by Day 15 and remained constantly low until Day 65. Immunohistochemistry localized expression of Cox2 in the cytoplasm of luteal cells. Staining was restricted to Days 5 and 15, with stronger signals on Day 5. These data suggested that increased expression of Cox2 is associated with luteal growth and development and not luteal regression. Furthermore, the expression of Cox1 more likely reflected activity of a housekeeping gene.     

Effects of selective protein kinase c isozymes in prostaglandin2alpha-induced Ca2+ signaling and luteinizing hormone-induced progesterone accumulation in the mid-phase bovine corpus luteum

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.     

Equine luteal function regulation may depend on the interaction between cytokines and vascular endothelial growth factor: an in vitro study

We hypothesized that cytokines influence luteal angiogenesis in mares, while angiogenic factors themselves can also regulate luteal secretory capacity. Therefore, the purpose of this study was to evaluate the role of cytokines--tumor necrosis factor alpha (TNF), interferon gamma (IFNG) and Fas ligand (FASL)--on in vitro modulation of angiogenic activity and mRNA level of vascular endothelial growth factor A (VEGF), its receptor VEGFR2, thrombospondin 1 (TSP1), and its receptor CD36 in equine corpus luteum (CL) throughout the luteal phase. After treatment, VEGF protein expression was determined in midluteal phase (mid) CL cells. The role of VEGF on regulation of luteal secretory capacity was assessed by progesterone (P(4)) and prostaglandin E(2) (PGE(2)) production and by mRNA levels for steroidogenic enzymes 3-beta-hydroxysteroid dehydrogenase (3betaHSD) and PGE synthase (PGES). In early CL cells, TNF increased angiogenic activity (bovine aortic endothelial cell viability) and VEGF and VEGFR2 mRNA levels and decreased CD36 (real-time PCR relative quantification). In mid-CL cells, TNF increased VEGF mRNA and protein expression (Western blot analysis) and reduced CD36 mRNA levels, while FASL and TNF+IFNG+FASL decreased VEGF protein expression. In late CL cells, TNF and TNF+IFNG+FASL reduced VEGFR2 mRNA, but TNF+IFNG+FASL increased TSP1 and CD36 mRNA. VEGF treatment increased mRNA levels of 3betaHSD and PGES and secretion of P(4) and PGE(2). In conclusion, these findings suggest a novel auto/paracrine action of cytokines, specifically TNF, on the up-regulation of VEGF for angiogenesis stimulation in equine early CL, while at luteolysis, cytokines down-regulated angiogenesis. Additionally, VEGF stimulated P(4) and PGE(2) production, which may be crucial for CL establishment.     

Effects of prostaglandin F2 alpha on agonist-induced progesterone production in cultured bovine luteal cells

The present study examines the effects of prostaglandin F2 alpha (PGF2 alpha) on basal and agonist-stimulated progesterone (P4) production utilizing long-term, serum-free cultures of bovine luteal cells. During the first 24 h of culture, PGF2 alpha had no significant effect on P4 production, and was unable to inhibit either luteinizing hormone (LH)- or dibutyryl cAMP (dbcAMP)-stimulated increases in P4. Treatment with PGF2 alpha on Day 1 produced a moderate, nonsignificant (P greater than 0.05) inhibition of cholera toxin (CT)- and forskolin (FKN)-stimulated P4 synthesis. Beyond Day 1 of culture (Days 3-11), PGF2 alpha continued to have no significant effect on basal P4 production, but suppressed all stimulatory effects of LH, dbcAMP, CT and FKN. Treatment with indomethacin inhibited prostaglandin synthesis by the cultured cells and also elevated levels of P4 from Days 3 to 11 of culture. Concurrent treatment with PGF2 alpha suppressed the steroidogenic effect of indomethacin. From these studies it was concluded that in cultured bovine luteal cells, PGF2 alpha does not affect basal P4 production, but is able to inhibit agonist-stimulated P4 production at a site beyond the accumulation of cAMP. This inhibitory effect is not apparent during the first 24 h of culture, but appears after Day 1 and persists throughout the remaining 10 days of the culture period.     

Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2alpha-induced luteolysis of the sheep corpus luteum

Three experiments were designed to examine the mechanisms that govern prostaglandin (PGF2alpha)-induced regression of the sheep corpus luteum. Evidence is presented supporting the involvement of endothelin 1 (EDN1) in PGF2alpha-induced luteolysis. Experiment 1 measured effects of PGF2alpha when actions of EDN1 were blocked by sustained administration of a type-A endothelin (EDNRA) or type-B endothelin (EDNRB) antagonist in vivo. Experiment 2 examined antisteroidogenic actions of PGF2alpha and EDN1 in the presence of an EDNRA or EDNRB antagonist in Day-8 luteal minces. In experiment 3, luteal cellular expression of EDNRA and EDNRB was determined immunohistochemically. Relative gene expression of EDNRA and EDNRB receptors was examined by real-time RT-PCR in Day-8 sheep corpora lutea. EDNRA, but not EDNRB, participated in antisteroidogenic actions of EDN1. During the first 12 h after PGF2alpha-induced luteolysis, EDNRA antagonist did not prevent a decline in serum progesterone concentrations. Early actions of PGF2alpha are either direct or mediated by something other than EDN1. However, beyond 12 h after PGF2alpha, serum progesterone concentrations increased in EDNRA antagonist-treated animals until they were the same as saline-treated controls, whereas an EDNRB antagonist had no effect in vivo or in vitro. The EDNRA antagonist negated the antisteroidogenic actions of EDN1 but only partially abolished the actions of PGF2alpha in vitro. In contrast, the EDNRB antagonist was ineffective in abolishing antisteroidogenic actions of EDN1 and PGF2alpha. Whereas real-time RT-PCR demonstrated high expression of EDNRA and low expression of EDNRB, immunohistochemically, only EDNRA was located in small steroidogenic, endothelial, and smooth muscle cells. In summary, studies in ovine corpora lutea provided strong evidence that: 1) EDNRA, but not EDNRB, mediates antisteroidogenic actions of EDN1, 2) actions of PGF2alpha are both independent of and dependent upon mediation by EDN1, and 3) small steroidogenic cells are targets for antisteroidogenic effects of EDN1. Furthermore, the results from experiment 1 suggest that the intermediary role of EDN1 may be more important in later stages of luteal regression.