Prostaglandin F(2alpha) receptor in the corpus luteum: recent information on the gene, messenger ribonucleic acid, and protein

The prostaglandin (PG) F(2alpha) receptor (FPr) in the corpus luteum is essential for maintaining normal reproductive cyclicity in many species. Activation of this seven-transmembrane spanning receptor at the end of the cycle leads to a decrease in progesterone and the demise of the corpus luteum (luteolysis). Recently, the gene structure of the FPr in three mammalian species has been elucidated; however, promoter regulation of the gene is still poorly understood. The FPr mRNA is extremely low in steroidogenic follicular cells (theca or granulosa) but is expressed at high levels in the corpus luteum, particularly in the large luteal cells. Treatment with PGF(2alpha) decreased FPr mRNA expression in luteal cells in most species that have been studied. Key amino acids have been suggested to be critical for binding of FPr to PGF(2alpha) based on three-dimensional modeling and comparisons with other G-protein-coupled receptors. Moieties of the PGF(2alpha) molecule that are essential for binding or specificity of binding to the FPr have been identified by radioreceptor binding studies. In this article, recent information is reviewed on the structure of the FPr gene, regulation of luteal FPr mRNA, and receptor/ligand interaction requirements for the FPr protein.     

The expression of tumor necrosis factor-alpha,its receptors and steroidogenic acute regulatory protein during corpus luteum regression

Background  

Corpus luteum (CL) regression is known to occur as two parts; functional regression when steroidogenesis declines and structural regression when apoptosis is induced. Previous studies suggest this process occurs by the production of luteolytic factors, such as tumour necrosis factor-alpha (TNF-alpha).     

Treatment with prostaglandin F2alpha increases expression of prostaglandin synthase-2 in the rat corpus luteum

Recent studies indicate that the corpus luteum (CL) may be a source of prostaglandin F2alpha (PGF2alpha) for regression. We investigated expression of mRNA and protein for prostaglandin G/H synthase (PGHS) in the CL of immature superovulated rats following administration of PGF2alpha. We observed an increase in mRNA for PGHS-2, the induced isoform, at 1 h and protein at 8 and 24 h after treatment. One hour after PGF2alpha, there was also a progressive decrease in plasma progesterone concentration. There were no changes, however, in expression of PGHS-1, the constitutive isoform, over the 24 h sampling period. These results indicate that PGHS-2 increases following PGF2alpha treatment and that expression of this enzyme in the rat CL may contribute to the luteolytic mechanism.     

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).     

Prostaglandin F2-alpha receptor (FPr) expression on porcine corpus luteum microvascular endothelial cells (pCL-MVECs)

Background  

The corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested.     

Expression of steroidogenic enzyme messenger ribonucleic acid and cortisol production in adrenocortical cells isolated from halothane-sensitive and halothane-resistant pigs

Stress susceptibility in pigs is inherited by a single recessive gene (Hal(n)), and homozygous individuals can be identified by exposure to halothane anesthesia. Previous studies have shown that in stress-susceptible pigs, exposure to a high ambient temperature resulted in a twofold increase in corticotropin (ACTH) and lower plasma cortisol. To determine whether there is a fundamental difference in adrenocortical function between halothane-sensitive (HAL-S) and halothane-resistant (HAL-R) pigs, independent of other factors influencing the hypothalamic-pituitary-adrenal (HPA) axis, we compared cortisol responses to ACTH and 8-bromo-cyclic AMP (8-Br-cAMP) in HAL-S and HAL-R pig adrenocortical cells in vitro. We also determined directly the accumulation of four different mRNAs encoding cholesterol side-chain cleavage cytochrome P450 (P450(scc)), 17alpha-hydroxylase cytochrome P450 (P450(17alpha)), 21-hydroxylase cytochrome P450 (P450(c21)) and 11beta-hydroxylase cytochrome P450 (P450(11beta)) in HAL-S pig adrenal cells and compared them to HAL-R pigs. A time- and dose-dependent increase in medium content of cortisol and cAMP was observed after ACTH treatment. 8-Br-cAMP also caused a time- and dose-dependent increase in cortisol production in the medium. Addition of ACTH or 8-Br-cAMP to HAL-S and HAL-R male Lanyu small-ear miniature pig adrenocortical cells increased cortisol production in a dose- and time-related manner. However, cells isolated from HAL-S pigs had a lower cortisol production in response to ACTH or 8-Br-cAMP compared to those from HAL-R pigs. Treatment of cultured cells with 8-Br-cAMP (0.5 mM) for 18 h resulted in a significant increase in P450(scc), P450(17alpha), P450(c21), and P450(11beta) mRNA levels. In the absence of 8-Br-cAMP, the four genes were expressed constitutively in both HAL-S and HAL-R pig adrenal cells. Densitometric scanning of the autoradiograph indicated that the relative amounts of P450(scc) and P450(17(alpha)) mRNAs in HAL-S pig adrenal cells were between 48% and 53% of those detected in HAL-R pig adrenal cells (P < 0.05). No difference in the amounts of P450(c21) and P450(11beta) was seen in HAL-S and HAL-R pig adrenal cells. Addition of 8-Br-cAMP (0.5 mM) resulted in a uniform increase in the levels of all four P450 mRNAs in both HAL-S and HAL-R pig adrenal cells. However, the amounts of P450(scc) mRNA in HAL-S pig adrenal cells were 67% (P < 0.05) of those measured in HAL-R pig adrenal cells, whereas the amounts of P450(17alpha ), P450(c21), and P450(11beta) mRNAs were similar in these cells. Our data suggest an HPA axis defect in HAL-S pigs at the adrenal level. This defect appears to be at the level of P450scc gene expression, which could be partially related to reduced cortisol production by ACTH stimulation.     

Prostaglandin E and F2alpha receptors in bovine corpus luteum plasma membranes are two different macromolecular entities.

Greater numbers of prostaglandin (PG) F_2α as compared to PGE receptors were solubilized from bovine corpus luteum plasma membranes by sodium deoxycholate (SDC) concentrations from 0.01% to 0.1%. However, at 0.5% SDC concentration, virtually 100% of both PGs receptors were solubilized. When these solubilized PGs receptors were chromatographed on a calibrated Sepharose 6B column, they exhibited a small but reproducible difference in their distribution coefficients. The above results suggest that PGE and PGF_2α receptors represent two different macromolecular entities with a relatively small size difference between them.     

Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor “processing” within 4 h of administration followed by recovery or “replenishment” of ER levels to the initial level by 20 h. The term “processing” has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor “processing” constitutes disappearance of receptor protein and the later “replenishment” phase represents new ER protein rather than recycling of “processed” receptor. Progesterone-action, on the other hand, influenced only the “replenishment” phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was “processed” and “replenishment” already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.     

Canine prostaglandin F2alpha receptor (FP) and prostaglandin F2alpha synthase (PGFS): molecular cloning and expression in the corpus luteum

In the dog luteolysis is not affected by hysterectomy. This observation led to the hypothesis that paracrine/autocrine rather than endocrine mechanisms of PGF2alpha are responsible for luteal regression in the dioestric bitch. The present experiments tested for the capacity of canine CL to produce and respond to PGF2alpha by qualitatively and quantitatively determining the expressions of PGFS, the enzyme converting PGH2 into PGF2alpha, and the PGF2alpha-receptor (FP) in CL of non-pregnant dogs during dioestrus. Canine PGFS and FP were isolated and cloned; both genes show a high homology (82-94%) when compared to those of other species. Relatively weak FP mRNA expression was detected on day 5 of dioestrus. It had increased by day 25 and remained constant thereafter. In situ hybridization (ISH) localized FP solely to the cytoplasm of the luteal cells, suggesting that these cells are the only luteal targets of PGF2alpha in this species. Only negative results were obtained for the expression of PGFS in canine CL by routine qualitative RT-PCR. When Real Time (TaqMan) PCR was applied, repetitively more negative than positive results were obtained at all timepoints. Any positive measurements observed at any point were neither repeatable nor related to the stage of dioestrus. This led us to conclude that expression of PGFS is either absent or present at very low level only. These data suggest that luteal regression in non-pregnant bitches is not modulated by PGF2alpha. However, the FP seems to be constitutionally expressed, explaining the receptivity of canine CL to exogenous PGF2alpha.     

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.     

Role of intraluteal prostaglandin F(2alpha), progesterone and oxytocin in basal and pulsatile progesterone release from developing bovine corpus luteum

The present study examined the role of intra-luteal prostaglandin (PG) F(2alpha), progesterone (P4) and oxytocin (OT) on the corpus luteum function by using specific hormone antagonists. Luteal cells from the developing CL (days 5-7 of the estrous cycle) were exposed to P4 antagonist (onapristone, OP, 10(-4)M), OT antagonist (atosiban, AT; 10(-6)M) or indomethacin (INDO; 10(-4)M), for 12h and then stimulated with PGF(2alpha) (10(-8)M) for 4h. Pre-treatment of the cells with OP, AT or INDO resulted in an increase in P4 secretion in response to PGF(2alpha). To examine the temporal effects of P4, OT and PGs on P4 secretion, dispersed luteal cells were pre-exposed to OP, AT or INDO for 1, 2, 4, 6 or 12h. Prostaglandin F(2alpha) stimulated P4 secretion (P<0.05) after 2h of pre-exposition. In the microdyalisis study, the spontaneous release of P4 from developing CL tissue was of pulsatile nature with irregular peaks at 1-2h intervals. Treatment with OP increased the number of P4 peaks (P<0.05), whereas AT and INDO significantly reduced the number of P4 peaks detected (P<0.05). Interestingly, INDO completely blocked the pulsatile nature in the release of P4, but it secretion remained stable throughout the experimental period. These results demonstrate that luteal PGF(2alpha), OT, and P4 are components of an autocrine/paracrine intra-ovarian regulatory system responsible for the episodic (pulsatile) release of P4 from the bovine CL during the early luteal phase.     

Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.