Pulses Of Prostaglandin F2alpha Research Articles

Endometrial and luteal responses to a prostaglandin F2alpha pulse: a comparison between heifers and mares†.

In heifers and mares, multiple pulses of prostaglandin F2alpha (PGF) are generally associated with complete luteal regression. Although PGF pulses occur before and during luteolysis, little is known about the role of minor PGF pulses during preluteolysis on subsequent luteal and endometrial PGF production that may initiate luteolysis. Heifers (n = 7/group) and mares (n = 6/group) were treated with a single minor dose of PGF (3.0 and 0.5mg, respectively) during mid-luteal phase (12 and 10days postovulation respectively). After treatment, a transient decrease in progesterone (P4) concentrations occurred in heifers between Hours 0 and 2 but at Hour 4 P4 was not different from pretreatment. In mares, P4 was unaltered between Hours 0 and 4. Concentrations of P4 decreased in both species by Hour 24 and complete luteolysis occurred in mares by Hour 48. Luteal and endometrial gene expression were evaluated 4h posttreatment. In heifers, luteal mRNA abundance of PGF receptor and PGF dehydrogenase was decreased, while PTGS2, PGF transporter, and oxytocin receptor were increased. In the heifer endometrium, receptors for oxytocin, P4, and estradiol were upregulated. In mares, luteal expression of PGF receptor was decreased, while PGF transporter and oxytocin receptor were increased. The decrease in P4 between Hours 4 and 24 and changes in gene expression were consistent with upregulation of endogenous synthesis of PGF. The hypotheses were supported that a single minor PGF treatment upregulates endogenous machinery for PGF synthesis in heifers and mares stimulating endogenous PGF synthesis through distinct regulatory mechanisms in heifers and mares.

Effect of natural pre-luteolytic prostaglandin F2α pulses on the bovine luteal transcriptome during spontaneous luteal regression†.

The pulsatile pattern of prostaglandin F2alpha (PGF) secretion during spontaneous luteolysis is well documented, with multiple pulses of exogenous PGF necessary to induce regression using physiologic concentrations of PGF. However, during spontaneous regression, the earliest pulses of PGF are small and not associated with detectable changes in circulating progesterone (P4), bringing into question what, if any, role these early, subluteolytic PGF pulses have during physiologic regression. To investigate the effect of small PGF pulses, luteal biopsies were collected throughout natural luteolysis in conjunction with bihourly blood samples to determine circulating P4 and PGF metabolite to retrospectively assign biopsies to early and later regression. Whole transcriptome analysis was conducted on CL biopsies. Early PGF pulses altered the luteal transcriptome, inducing differential expression of 210 genes (Q < 0.05) during early regression, compared with 4615 differentially expressed genes during later regression. In early regression, few of these differentially expressed genes were directly associated with luteolysis, rather there were changes in local steroid and glutathione metabolism. Most (94%) differentially expressed genes from early regression were also differentially expressed during later regression, with 98% of these continuing to be altered in the same direction compared with CL at a similar stage of the cycle that had not yet been exposed to PGF. Thus, early, subluteolytic PGF pulses impact the luteal transcriptome, though not by altering steroidogenesis or causing direct inhibition of cellular function. Rather, small pulses alter pathways resulting in the removal of cellular support systems, which may sensitize the CL to later pulses of PGF.

Pregnancy-induced changes in the transcriptome of the bovine corpus luteum during and after embryonic interferon-tau secretion†.

Understanding luteal maintenance during early pregnancy is of substantial biological and practical importance. Characterizing effects of early pregnancy, however, has historically been confounded by use of controls with potential exposure to early Prostaglandin F2-alpha (PGF) pulses or differences in Corpus Luteum (CL) age. To avoid this, the present study utilized bihourly blood sampling to ensure control CL (n=6) were of a similar age to CL from pregnant animals (n=5), yet without exposure to PGF pulses. Additionally, CL from second month of pregnancy (n=4) were analyzed to track fate of altered genes after cessation of embryonic interferon tau (IFNT) secretion. The major alteration in gene expression in first month of pregnancy occurred in interferon-stimulated genes (ISGs), with immune/interferon signaling pathways enriched in three independent over-representation analyses. Most ISGs decreased during second month of pregnancy, though, surprisingly, some ISGs remained elevated in the second month even after cessation of IFNT secretion. Investigation of luteolytic genes found few altered transcripts, in contrast to previous reports, likely due to removal of controls exposed to PGF pulses. An exception to this trend was decreased expression of transcription factor NR4A1. Beyond luteolytic genes and ISGs, over representation analyses highlighted the prevalence of altered genes within the extracellular matrix and regulation of Insulin-like growth factor (IGF) availability, confirming results of other studies independent of luteolytic genes. These results support the idea that CL maintenance in early pregnancy is related to lack of PGF exposure, although potential roles for CL expression of diverse ISGs and other pathways activated during early pregnancy remain undefined.

Parthenogenetic bovine embryos secrete type I interferon capable of stimulating ISG15 in luteal cell culture.

Interferon tau (IFNT) is the pregnancy recognition signal in ruminants and is secreted by trophoblast cells. Paracrine action in the endometrium is well established by inhibiting luteolytic pulses of prostaglandin F2 alpha. Recently, endocrine action was documented in the corpus luteum, blood cell and liver. It was hypothesized that conditioned medium (CM) obtained from days 7, 9 and 12 parthenogenetic embryos alters luteal cell gene expression. The aim was to establish a bovine mixed luteal cell culture to evaluate cellular response associated to interferon stimulated genes, steroidogenesis and apoptosis. Conditioned medium was obtained from Days 7, 9 and 12 parthenogenetic (PA) embryos culture. Moreover, antiviral assay was performed on CM from Days 7, 9 and 12 to verify Type I interferon activity. Luteal cell culture was validated by steroidogenic and apoptotic genes (CYP11A1 , HSD3B1, BAX, BCL2, AKT and XIAP mRNA expression), and concentration of progesterone as endpoint. Luteal cell culture was treated with interferon alpha (IFNA) and CM from parthenogenetic embryos. Antiviral assay revealed Type I interferon activity on CM from embryos increasing on Days 9 and 12. ISG15 mRNA was greater in the mixed luteal cells culture treated with 1, 10 and 100ng/ml of interferon alpha (IFNA) and also on Days 7, 9 and 12 CM treatments. Concentration of progesterone was not altered in luteal cell culture regardless of treatments. Steroidogenic and apoptotic genes were similar among groups in luteal cell culture treated with different doses of IFNA or CM from PA embryos. In conclusion, parthenogenetic embryo-derived CM has antiviral activity, luteal cell culture respond to Type I interferon by expressing IGS15. These data indicate this model can be used for IFNT endocrine signaling studies.

Patterns of Gene Expression in the Bovine Corpus Luteum Following Repeated Intrauterine Infusions of Low Doses of Prostaglandin F2alpha1

Natural luteolysis involves multiple pulses of prostaglandin F2alpha (PGF) released by the nonpregnant uterus. This study investigated expression of 18 genes from five distinct pathways, following multiple low-dose pulses of PGF. Cows on Day 9 of the estrous cycle received four intrauterine infusions of 0.25 ml of phosphate-buffered saline (PBS) or PGF (0.5 mg of PGF in 0.25 ml of PBS) at 6-h intervals. A luteal biopsy sample was collected 30 min after each PBS or PGF infusion. There were four treatment groups: Control (n = 5; 4 PBS infusions), 4XPGF (4 PGF infusions; n = 5), 2XPGF-non-regressed (2 PGF infusions; n = 5; PGF-PBS-PGF-PBS; no regression after treatments), and 2XPGF-regressed (PGF-PBS-PGF-PBS; regression after treatments; n = 5). As expected, the first PGF pulse increased mRNA for the immediate early genes JUN, FOS, NR4A1, and EGR1 but unexpectedly also increased mRNA for steroidogenic (STAR) and angiogenic (VEGFA) pathways. The second PGF pulse induced immediate early genes and genes related to immune system activation (IL1B, FAS, FASLG, IL8). However, mRNA for VEGFA and STAR were decreased by the second PGF infusion. After the third and fourth PGF pulses, a distinctly luteolytic pattern of gene expression was evident, with inhibition of steroidogenic and angiogenic pathways, whereas, there was induction of pathways for immune system activation and production of PGF. The pattern of PGF-induced gene expression was similar in corpus luteum not destined for luteolysis (2X-non-regressed) after the first PGF pulse but was very distinct after the second PGF pulse. Thus, although the initial PGF pulse induced mRNA for many pathways, the second and later pulses of PGF appear to have set the distinct pattern of gene expression that result in luteolysis.

In Vivo Temporal Expression of Proteins Associated with Luteolysis Following Multiple Pulses of Prostaglandin F2alpha in Sheep.

In Vivo Temporal Expression of Proteins Associated with Luteolysis Following Multiple Pulses of Prostaglandin F2alpha in Sheep.

Necessity of Sequential Pulses of Prostaglandin F2alpha for Complete Physiologic Luteolysis in Cattle1

The luteolytic effects of exogenous prostaglandin F2alpha (PGF) that did and did not simulate natural 13,14-dihydro-15-keto-PGF (PGFM) pulses were studied during mid-diestrus in 42 Holstein heifers. Plasma concentrations of PGF were assessed by assay of PGFM. In experiment 1, a single intrauterine injection of 4.0 mg of PGF into the uterine horn ipsilateral to the corpus luteum resulted in a precipitous progesterone decline, whereas sequential injections of 0.25 or 1.0 mg every 12 h resulted in a stepwise decrease (P < 0.05) following each injection. A progesterone increase occurred during the first 5 min before the luteolytic decrease but only for the 4.0-mg dose. From the results of experiment 2, a 2-h intrauterine infusion of a total of 0.5 mg of PGF was judged to best simulate a natural PGFM pulse. In experiment 3, simulation of sequential pulses at 12-h intervals resulted in a continuous precipitous decrease in progesterone to <1 ng/ml by the beginning of the fourth simulated pulse. In contrast, a single simulated pulse resulted in a 6-h progesterone decrease to a constant concentration for 3 days after treatment, followed by a return to control concentrations. The mean +/- SEM interval between the pretreatment and posttreatment ovulations was shorter (P < 0.05) in the group with sequential simulated pulses (14 +/- 1 day) than in the group with a single pulse (21 +/- 1 day). Results indicated that excessive PGF doses may stimulate nonphysiologic progesterone responses and supported the hypothesis that sequential PGF pulses are required to stimulate natural luteolysis in cattle.

IN VIVO CHANGES IN PROTEIN EXPRESSION OF THE GELATINASES, MATRIX METALLOPROTEINASES -2 AND -9 (MMP-2 AND MMP-9), AND TISSUE INHIBITORS OF METALLOPROTEINASES -1 (TIMP-1) IN THE OVINE CORPUS LUTEUM (CL) AFTER MULTIPLE PULSES OF PROSTAGLANDIN F2ALPHA (PGF2A)

A model was established in sheep which mimics the frequency of prostaglandin F2alpha (PGF2a) pulses during the course of natural luteolysis. Using this model, we previously demonstrated that after one and two pulses of PGF2a, protein mediators of the extracellular matrix (ECM), tissue inhibitors of matrix metalloproteinases (TIMPs) -1 and -2, decreased dramatically in luteal tissue within one hour following each PGF2a pulse, which was accompanied several hours later by an increase in matrix metalloproteinase-2 (MMP-2) activity. The objective of the present study was to study the temporal protein expression patterns of the gelatinases, matrix metalloproteinases (MMP) -2 and MMP-9, and TIMP-1, in corpora lutea of sheep which received three systemic pulses of PGF2a (20ug/min/1hr) during the mid-luteal phase of the cycle. Intact cycling ewes were given 2 injections (7.5 mg) of Lutalyse and housed with a vasectomized marker ram to detect estrus (day 0). Sheep were then housed in metabolism cages, and infused with PGF2a via the right jugular vein to mimic the endogenous uterine pulses during luteolysis. Corpora lutea (day 10–12; n=4 sheep for each time point) were removed surgically one hr (-1hr) before the third pulse of PGF2a and at 1, 8, 16, and 24 hrs after the third infusion of PGF2a. Gelatin zymographic analysis identified the presence of three bands with relative molecular masses of approximately 85, 70, and 64 kDa. These bands co-migrated with MMP-9 (85kDa) and MMP-2 (70 and 64 kDa) gelatinase family members present in the positive control, which was conditioned medium of the fibrosarcoma cell line, HT-1080. An analysis of variance followed by Tukey's revealed no differences in the expression of the 64 kDa MMP-2 species, but the expression of the 70 kDa MMP-2 species between the 16 and 24 hr time points differed significantly (p<0.05). In our previous studies with one or two pulses of PGF2a, no differences were observed for MMP-9. In the present study, after three pulses of PGF2a, expression of the 85 kDa MMP-9 species tended to increase (p= 0.07) at 16 hrs relative to the pre- infusion (-1hr) time point. A band of approximately 30 kDa, which comigrated with a TIMP-1 recombinant protein, was identified by immunoblot analysis. Although expression of TIMP-1 fluctuated greatly, the variation among samples for each time point negated any differences (p>0.05) in the temporal expression of this protein. In summary, the increase of the 70 kDa MMP-2 species accompanied by an increasing trend of the 85 kDa MMP-9 species and varying expressions of TIMP-1 indicate changes in protein mediators of the CL in response to the third pulse of PGF2a. These results, taken together with our previous findings, indicate a continuum of changes in protein regulators of the ECM within the CL as luteolysis progresses. (Supported by USDA Grant #2004-35203- 14176) (poster)

Fetal-maternal interactions during the establishment of pregnancy in ruminants

This review integrates established information with new insights into molecular and physiological mechanisms responsible for events leading to pregnancy recognition, endometrial receptivity, and implantation with emphasis on sheep. After formation of the corpus luteum, progesterone acts on the endometrium and stimulates blastocyst growth and elongation to form a filamentous conceptus (embryo/fetus and associated extraembryonic membranes). Recurrent early pregnancy loss in the uterine gland knockout ewe model indicates that endometrial epithelial secretions are essential for peri-implantation blastocyst survival and growth. The elongating sheep conceptus secretes interferon tau (IFNT) that acts on the endometrium to inhibit development of the luteolytic mechanism by inhibiting transcription of the estrogen receptor alpha (ESR1) gene in the luminal (LE) and superficial ductal glandular (sGE) epithelia, which prevents estrogen-induction of oxytocin receptors (OXTR) and production of luteolytic prostaglandin F2-alpha pulses. Progesterone downregulates its receptors (PGR) in LE and then GE, correlating with a reduction of anti-adhesive MUC1 (mucin glycoprotein one) and induction of secreted LGALS15 (galectin 15) and SPP1 (secreted phosphoprotein one), that are proposed to regulate trophectoderm growth and adhesion. IFNT acts on the LE to induce WNT7A (wingless-type MMTV integration site family member 7A) and to stimulate LGALS15, CTSL (cathepsin L), and CST3 (cystatin C), which may regulate conceptus development and implantation. During the peri-implantation period, trophoblast giant binucleate cells (BNC) begin to differentiate from mononuclear trophectoderm cells, migrate and then fuse with the uterine LE as well as each other to form multinucleated syncytial plaques. Trophoblast giant BNC secrete chorionic somatomammotropin (CSH1 or placental lactogen) that acts on the endometrial glands to stimulate their morphogenesis and differentiated function. The interactive, coordinated and stage-specific effects of ovarian and placental hormones regulate endometrial events necessary for fetal-maternal interactions and successful establishment of pregnancy.

Temporal Associations among Pulses of 13,14-Dihydro-15-keto-PGF2alpha, Luteal Blood Flow, and Luteolysis in Cattle1

Luteal blood flow was studied in heifers by transrectal color-Doppler ultrasound. Data were normalized to the decrease in plasma progesterone to <1 ng/ml (Day 0 or Hour 0). Blood flow in the corpus luteum (CL) was estimated by the percentage of CL area with color flow signals. Systemic prostaglandin F2alpha (PGF) treatment (25 mg; n=4) resulted in a transient increase in CL blood flow during the initial portion of the induced decrease in progesterone. Intrauterine treatment (1 or 2 mg) was done to preclude hypothetical secondary effects of systemic treatment. Heifers were grouped into responders (luteolysis; n=3) and nonresponders (n=5). Blood flow increased transiently in both groups; induction of increased blood flow did not assure the occurrence of luteolysis. A transient increase in CL blood flow was not detected in association with spontaneous luteolysis when examinations were done every 12 h (n=6) or 24 h (n=10). The role of PGF pulses was studied by examinations every hour during a 12-h window each day during expected spontaneous luteolysis. At least one pulse of 13,14-dihydro-15-keto-PGF2alpha (PGFM) was identified in each of six heifers during the luteolytic period (Hours -48 to -1). Blood flow increased (P<0.02) during the 3-h ascending portion of the PGFM pulse, remained elevated for 2 h after the PGFM peak, and then decreased (P<0.03) to baseline. Results supported the hypothesis that CL blood flow increased and decreased with individual PGFM pulses during spontaneous luteolysis.

Biology of progesterone action during pregnancy recognition and maintenance of pregnancy.

Progesterone is the hormone of pregnancy and unequivocally required in all mammals for maternal support of conceptus (embryo/fetus and associated membranes) survival and development. The actions of progesterone are mediated by the progesterone receptor (PR). However, the endometrial lumenal (LE) and glandular epithelia (GE) of a number of species exhibit a loss of PR expression prior to the stages of uterine receptivity and implantation. In sheep, PR expression becomes undetectable in the endometrial LE after Day 11 and then in the GE after Day 13. Loss of PR in the GE appears to be required for onset of differentiated functions in terms of production of secretory proteins, such as uterine milk proteins (UTMP) and osteopontin (OPN). Therefore, the actions of progesterone on endometrial epithelia during most of gestation appear to be mediated by the endometrial stroma that remains PR-positive throughout pregnancy. Stromal cells produce several growth factors, such as hepatocyte growth factor (HGF) and fibroblast growth factors-7 and -10 (FGF-7, FGF-10), that have receptors expressed specifically in the endometrial epithelia. These factors may be progesterone-responsive and mediate epithelial-mesenchymal interactions that are crucial for support of pregnancy. Studies of the uterine gland knockout (UGKO) ewe indicate that uterine glands and, by default, their secretions are required for peri-implantation conceptus survival and growth. A complex servomechanism, involving hormones from the ovary and conceptus as well as endogenous betaretroviruses expressed in the endometrial LE and GE, is proposed to regulate endometrial gland differentiation and function during gestation. At estrus, estrogen increases PR expression in the endometrial epithelia. High levels of endogenous Jaagsiekte sheep retroviruses (enJSRVs) are expressed in the PR-positive endometrial LE and GE in response to increasing progesterone and are hypothesized to stimulate trophoblast proliferation and production of interferon (IFN) tau. IFN tau, the pregnancy recognition hormone produced by the trophoblast from Days 10 to 21, acts in a paracrine manner on the PR-negative endometrial LE and superficial GE to inhibit transcription of estrogen receptor alpha (ER) and oxytocin receptor (OTR) genes. These actions of IFN tau maintain progesterone production from the corpus luteum by abrogating release of luteolytic pulses of prostaglandin F2 alpha (PGF) from the endometrial epithelium. The antiluteolytic effects of IFN tau are dependent on progesterone. Progesterone stimulation over 8-10 days suppresses expression of the PR gene in the LE and then GE. Loss of the PR in the LE is concomitant with decreases in mucin glycoprotein one (MUC-1), an inhibitor of blastocyst implantation. As the conceptus begins implantation on Day 15, the binucleate trophectodermal cells then differentiate and produce placental lactogen (PL), a member of the prolactin (PRL) and growth hormone (GH) family. PL stimulates GE proliferation and production of secretory proteins, such as UTMP and OPN. Interestingly, the effects of PL on the GE appear to require the absence of PR and prior exposure to IFN tau. During mid-pregnancy, the mononuclear trophectodermal cells produce GH that can also act on a progestinized uterus to stimulate GE hypertrophy and secretory function. The actions of this servomechanism are proposed to stimulate GE hyperplasia from Days 20 to 50 and then GE hypertrophy and maximal differentiated function after Day 50 when the majority of fetal growth and development occurs during gestation.

Local action of trophoblast interferons in suppression of the development of oxytocin and oestradiol receptors in ovine endometrium.

Luteolysis in sheep is associated with uterine secretion of pulses of prostaglandin F2 alpha (PGF2 alpha) due to the action of luteal oxytocin on endometrial oxytocin receptors. For pregnancy to become established inhibition of oxytocin receptors is important as an antiluteolytic mechanism. The maternal recognition of pregnancy in cattle and sheep involves production, by the trophoblast, of a type 1 interferon (IFN-tau) that suppresses uterine development of oxytocin receptors and the generation of luteolytic episodes of PGF2 alpha. The action of IFN-tau in surgically prepared unilaterally pregnant ewes was investigated. Finn-Dorset ewes were anaesthetized on day 6 or 7 of the oestrous cycle and one uterine horn was surgically isolated at the uterine bifurcation from the body of the uterus. Ewes were mated at the subsequent oestrus either by a fertile or by a vasectomized ram and killed on day 13 or 16 after mating. On day 16, in the non-pregnant ewes, there was no measurable uterine IFN-tau but there were high concentrations of oxytocin receptors in both horns. In the pregnant ewes on day 16 after mating, the oxytocin receptor concentration was 45 +/- 11 fmol mg-1 protein in the pregnant horn and 585 +/- 131 fmol mg-1 in the non-pregnant horn. Antiviral activity was 5.8 x 10(7) +/- 5.2 x 10(7) U ml-1 in the pregnant horn and 2.9 x 10(3) +/- 1.2 x 10(3) U ml-1 in the non-pregnant horn. Thus, 16 days after mating, the pregnant horn exhibited high antiviral activity but oxytocin receptors were suppressed, while in the same endocrine environment (characteristic of pregnancy) there were low IFN-tau and high oxytocin receptor concentrations in the isolated horn equivalent to those expected at the onset of luteolysis. In situ hybridization to ovine mRNA encoding the oxytocin receptor and autoradiographic studies using the 125I-labelled oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin both showed that the large amount of oxytocin receptor message and binding sites in the endometrium of the isolated horn were localized in the luminal epithelium. Immunocytochemical studies showed that there was a suppression of oestradiol receptors in the pregnant horn but high concentrations equivalent to those at oestrus were present in the isolated horn. The content of progesterone receptors was low in the stromal tissue only in both horns, a pattern of localization similar to that seen in the late luteal phase and in early pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)