PPAR ligand association with prostaglandin F2α and E2 synthesis in the pig corpus luteum-An in vitro study.

The present study evaluated the involvement of PPARs in prostaglandin (PG) E2 and F2α production in the corpus luteum (CL) of pigs on days 10-12 and 14-16 of the estrous cycle or pregnancy. The tissue explants were incubated for 6h in the presence of PPARα, PPARß, PPARγ ligands. The concentration of PGs in the incubation media was determined by radioimmunoassay, while mRNA abundance of PG synthetases (PGES and PGFS) was analyzed by quantitative real-time PCR. It was found that L-165,045 and rosiglitazone stimulated PGES synthesis on days 10-12 of the estrous cycle, whereas all factors that were assessed did not affect PGE2 release. The PGFS mRNA abundance in the CL did not change in the presence of PPAR ligands during the assessment periods. However, PPARß agonist inhibited PGF2α secretion on days 10-12 of the estrous cycle and on days 14-16 of pregnancy. Interestingly, PPAR antagonists, MK 886, GW 9662 or T0070907 decreased PGF2α release by the slices on days 10-12 of the estrous cycle. It is concluded that the CL has a different susceptibility (greatest during mid-luteal phase of the estrous cycle) to the PPAR ligands, which is related to the physiological status of animal. The inhibition of PGF2α release and augmentation of PGES mRNA concentration during mid-luteal phase of the estrous cycle might suggest luteotropic properties of PPAR ligands.

Zygote arrest 1, nucleoplasmin 2, and developmentally associated protein 3 mRNA profiles throughout porcine embryo development in vitro.

Maternal effect genes (MEGs) are expressed in oocytes and embryos and play an important role in activation of the embryonic genome. An abnormality in the expression of these genes may lead to arrest of embryonic cleavage or to altered transcription of factors responsible for further embryonic development. In vitro-produced porcine embryos have a lower developmental potential than embryos produced in vivo. We hypothesized that in vitro embryo culture conditions have an effect on the expression of MEGs at various developmental stages, which may affect their developmental potential. Here, using real-time polymerase chain reaction, we examined mRNA profiles of the MEGs, zygote arrest 1 (ZAR-1), nucleoplasmin 2 (NPM2), and developmentally associated pluripotency protein 3 (DPPA3), in porcine oocytes and embryos produced in vitro and in vivo. Further, we evaluated the effect of the combined addition of EGF, interleukin 1ß, and leukemia inhibitory factor to the porcine in vitro embryo production system on mRNA profiles of selected MEGs. Finally, we studied localization of the MEG protein products in in vitro-obtained oocytes and embryos using confocal microscopy. We found that the ZAR-1 mRNA profile differed throughout in vitro and in vivo embryo development. In the embryos produced in vitro, the decrease in ZAR-1 mRNA levels was observed at the 2-cell stage, whereas in in vivo embryos, ZAR-1 mRNA levels declined significantly starting at the 4-cell stage (P < 0.05). In vitro culture conditions affected transiently also DPPA3 mRNA levels at the 4-cell stage (P < 0.05). There was no difference in the NPM2 mRNA profile during in vitro and in vivo embryo development. The ZAR-1 and DPPA3 proteins were localized in the cytoplasm of the oocytes and embryos, whereas the NPM2 protein was found both in the cytoplasm and in the nucleus. All proteins were expressed until blastocyst stage. The addition of EGF and cytokines to the culture medium decreased DPPA3 mRNA levels in 8-cell embryos (P < 0.05). This study indicated that IVC conditions affect ZAR-1 mRNA levels before the 4-cell stage, which may disturb the activation of the embryonic genome in pigs. The expression of the proteins after the 4-cell to 8-cell transition indicates that these factors play a role beyond activation of the embryonic genome. Supplementation of the culture media with EGF and cytokines affects DPPA3 mRNA levels after maternal to embryonic transition.

Peroxisome proliferator activated receptor ligands affect progesterone and 17ß-estradiol secretion by porcine corpus luteum during early pregnancy.

In the present study we investigated the effect of peroxisome proliferator activated receptor (PPAR) ligands on progesterone (P4) and 17ß-estradiol (E2) secretion and 3b-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3ß-HSD) mRNA abundance in porcine corpora lutea (CL) collected on days 10-12 and 14-16 of the estrous cycle or pregnancy. The PPAR agonists reduced P4 secretion by the CL during pregnancy whereas they were ineffective during the estrous cycle. An inhibitory effect of WY-14643 (PPARα agonist) on P4 release was noted on days 14-16 of pregnancy. The treatment of the CL with L-165,045 (PPARß agonist) diminished P4 release by the tissue during both stages of pregnancy. A natural PPARγ agonist, PGJ2, reduced P4 release on days 14-16 or days 10-12 of pregnancy, respectively. Rosiglitazone (PPARγ agonist) inhibited P4 secretion by the CL on days 10-12 of pregnancy. In turn, PPARα ligands effect on E2 release was differential. While PPARγ activator diminished E2 secretion by the CL explants during all tested stages of the estrous cycle and pregnancy, PPARß ligands did not induce any change in E2 level. In turn, PPARß agonist reduced E2 release by the tissue during both stages of pregnancy but did not affect the secretion during the estrous cycle. In the present study there was a lack of PPAR ligands effect on 3ß-HSD mRNA abundance. In summary, the results suggest that PPARs are involved in the regulation of progesterone and 17ß-estradiol release by porcine CL. Porcine CL indicates a different receptivity to PPAR ligands depending on the reproductive status of animals.

The involvement of peroxisome proliferator activated receptors (PPARs) in prostaglandin F2α production by porcine endometrium.

In the present study, we investigated the in vitro effects of peroxisome proliferator activated receptor (PPAR) ligands on PGF2α secretion and mRNA expression of prostaglandin F synthase (PGFS) in porcine endometrial explants collected on days 10-12 and 14-16 of the estrous cycle or pregnancy. The explants were incubated for 6h with: PPARα ligands - WY-14643 (agonist) and MK 886 (antagonist); PPARß ligands - l-165,041 (agonist) and GW 9662 (antagonist); PPARγ ligands - 15d-prostaglandin J2 (PGJ2, agonist), rosiglitazone (agonist) and T0070907 (antagonist). The expression of PGFS mRNA in the endometrium and the concentration of PGF2α in culture media were determined by real time RT-PCR and radioimmunoassay, respectively. During the estrous cycle (days 10-12 and 14-16), the agonists - WY-14643 (PPARα), l-165,041 (PPARß), PGJ2 and rosiglitazone (PPARγ) - increased PGF2α secretion but did not affect PGFS mRNA abundance. During pregnancy (days 10-12 and 14-16), PPARα and PPARγ ligands did not change PGF2α release, whereas PPARß agonist augmented PGF2α release on days 14-16 of pregnancy. In addition, WY-14643 and l-165,041 increased PGFS mRNA level in both examined periods of pregnancy. PPARγ agonist (PGJ2) and antagonist (T0070907) enhanced PGFS mRNA abundance in the endometrium on days 10-12 and 14-16 of pregnancy, respectively. The results indicate that PPARs are involved in the production of PGF2α by porcine endometrium, and that the sensitivity of the endometrium to PPAR ligands depends on reproductive status of animals.

In vitro effect of peroxisome proliferator activated receptor (PPAR) ligands on prostaglandin E2 synthesis and secretion by porcine endometrium during the estrous cycle and early pregnancy.

Peroxisome proliferator activated receptors (PPARs) are ligand-dependent transcriptional factors which are expressed in distinct tissues of the female reproductive system, including the ovary, uterus and placenta. An important role of PPARs in the regulation of reproductive processes has been previously highlighted in rodents. In the present study we investigated the in vitro effect of PPAR ligands on prostaglandin E2 (PGE2) release and prostaglandin E synthase (PGES) gene expression in the endometrial explants collected from cyclic (days 10-12 and 14-16 of the estrous cycle) or pregnant (days 10-12 and 14-16) pigs. A stimulatory (p<0.05) effect of rosiglitazone (PPARγ agonist) on PGE2 accumulation was noted during both stages of the estrous cycle and both stages of pregnancy, whereas a higher (p<0.05) PGES mRNA level was observed on days 10-12 of the estrous cycle and on days 14-16 of gestation when compared to the controls. The activation of PPARß by L-165,041 augmented (p<0.05) PGE2 release by the endometrium on days 14-16 of the estrous cycle and on days 14-16 of pregnancy, but the increase (p<0.05) in PGES mRNA abundance was noted on days 10-12 of the estrous cycle and during both stages of pregnancy. A stimulatory (p<0.05) effect of WY-14643 (agonist) and MK 886 (antagonist) on PGE2 release was noted on days 10-12 of the estrous cycle, and days 14-16 of pregnancy, respectively. There was a lack of change in PGES mRNA abundance in the endometrium exposed to PPARα ligands. We conclude that PPARs are mediators of prostaglandin E2 synthesis/accumulation in porcine endometrium during the luteal phase of the estrous cycle and the time of periimplantation.

Participation of analogues of lysophosphatidic acid (LPA): oleoyl-sn-glycero-3-phosphate (L-alpha-LPA) and 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT) in uterine smooth muscle contractility of the pregnant pigs.

Recent studies show that a representative of phospholipids, namely lysophosphatidic acid (LPA) and its receptors (LPA1.3) play a significant role in the reproductive processes, i. a, in the modulation of the uterine contractility. The participation of LPA3 in the reproductive processes has been revealed in mice and has not been studied in gilts. Therefore, in the present study we investigated the role/action of LPA and its receptors LPA1, LPA2 and LPA3 on the contraction activity in the porcine uterus. The study was conducted on an experimental model in which the pig uterus consisted of the one whole uterine horn and a part of the second horn, both connected with the uterine corpus. Uterine strips consisting of the endometrium with the myometrium (ENDO/MYO) and myometrium (MYO) alone were collected on days 12-14 of the estrous cycle (control group; n = 5) or pregnancy (experimental group; n = 5). Two analogues of LPA at increasing doses were used: oleoyl-sn-glycero-3-phosphate (L-alpha-LPA, a selective agonist of LPA1 and LPA2 receptors; 10(-7) M; 10(-6) M and 10(-5) M) and 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT, a selective agonist of LPA3 receptor; 68 nM; 136 nM and 680 nM). L-alpha-LPA caused an increase in the contraction tension, amplitude and frequency of ENDO/MYO from the uterine horn with the developing embryos. This effect was not observed in MYO in both groups examined. In the ENDO/MYO strips of the uterine horn with developing embryos, OMPT significantly increased the contraction tension at the highest dose (680 nM) and amplitude at all doses examined, while frequency of contractions was decreased at doses of 136 nM and 680 nM. In the MYO strips of the uterine horn with embryos a significant increase in the contraction tension and amplitude after the highest dose of OMPT was observed. The results obtained imply the important role of receptors LPA1, LPA2 and LPA3 in the contraction activity of the porcine uterus during early pregnancy.

The quantitative expression of peroxisome proliferator activated receptor (PPAR) genes in porcine endometrium through the estrous cycle and early pregnancy.

Peroxisome proliferator activated receptors (PPAR) are a family of the nuclear receptors which play an important role as transcriptional factors. The aim of the present study was to determine the expression of PPARs (α, ß, γ) mRNA in the porcine endometrium during the estrous cycle and periimplantation period. Gilts were divided into two groups (cyclic and pregnant), synchronized and superovulated. The animals from the first group were slaughtered throughout the estrous cycle: 2-4, 5-8, 9-10, 11-12, 13-15, 16-17 and 18-21. Gilts from the second group were inseminated and slaughtered at different days of pregnancy to create subgroups: 5-8, 9-10, 11-12, 13-15, 16-17, 18-21, 21-30. PPAR mRNA expression in the endometrium was analyzed by real-time PCRs. During the estrous cycle the expression of PPARγ1 mRNA was significantly higher on days 13-15 than at the remaining stages. The expression of PPARα and ß transcripts showed a similar pattern and the lowest levels were observed on days 2-4, 16-17 and 18-21 in comparison with the remaining stages (days 5-8, 9-10, 11-12). During pregnancy a significant increase in the expression of PPARγ1 mRNA was noted on days 16-17, 18-21 and 22-30 compared to earlier stages. The transcript level of PPARß was significantly lower on days 11-12 and 22-30 than on days 5-8, 9-10, 13-15. mRNA expression of PPARα was high on days beginning from 5-8 until 18-21 and significantly dropped on days 22-30. The results indicate that the endometrial expression of PPARs genes fluctuates during the estrous cycle and pregnancy. PPARα and PPARß transcript levels show similar profiles during the estrous cycle. The decrease of both transcripts concentration on days 10-12 and 22-30 days in pregnant gilts implicates their role in maternal recognition of pregnancy and the end of implantation, respectively.

Conceptus signals for establishment and maintenance of pregnancy in pigs - lipid signaling system.

Establishment of pregnancy in pigs requires estrogen secretion by the conceptus. The developmental changes of embryo before implantation and embryo-uterine cross talk are dependent on various biological molecules secreted by the endometrium and conceptus. An integral part of maternal recognition of pregnancy seems to be also the lipid signaling system consisting of prostaglandin (PG) F2 alpha and E2 and/or lysophosphatic acid (LPA). The downstream enzymes in PG synthesis pathway are: microsomal PGE synthase-1 (mPGES-1), PGF synthase (PGFS) and prostaglandin 9-ketoreductase/carbonyl reductase (CBR1) which catalyzes conversion of PGE 2 into PGF2 alpha. In contrast to mPGES-1, endometrial PGFS is highly increased on days 13-15 similarly as CBR1 on days 16-17 of the estrous cycle. Potential mechanism by which a conceptus inhibits luteolysis is changing the PGE2/PGF2 alpha ratio in favor of PGE2. It may be result of high expression of mPGES-1 in trophoblast and endometrium on days 10-13 of pregnancy and simultaneously the down-regulation of PGFS and CBR1 in conceptuses during this period. The conceptus can alter expression of endometrial CBR1 to modulate the PGE2/PGF2 alpha ratio in the uterus during the maternal recognition of pregnancy. High expression of conceptus and endometrial terminal PG synthases and CBR1 after initiation of blastocyst attachment suggest their involvement in early placentation. The higher LPA3 receptor mRNA expression during the early pregnancy compared to corresponding period of estrous cycle could indicate an important role of LPA and its receptor during the peri-implantation stage of pregnancy in pigs. Above results suggest that the lipid signaling system is an integral part of establishment and maintenance of pregnancy in the pig.

Expression of prostaglandin synthesis pathway enzymes in the porcine corpus luteum during the oestrous cycle and early pregnancy.

Prostaglandins (PGs) of luteal origin may have paracrine and/or autocrine actions on the functions of the corpus luteum (CL). Previously, we have shown that enzymes of PG synthesis pathway such as prostaglandin E synthase (mPGES-1), prostaglandin F synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1) are important in regulation of PG production in the conceptuses and endometrium of cyclic and pregnant pigs. Therefore, localization and expression patterns of these enzymes were determinated in porcine CL. The PGFS protein content was lower in metestrus and higher around luteolysis, and then decreased in late regressing CL. PGFS protein levels were lower on days 5-8 of pregnancy and did not differ between days 10 and 25. Elevated expression of mPGES-1 mRNA was found in early luteal phase. The mPGES-1 protein content, similarly to PGFS, was higher during luteolysis. mPGES-1 mRNA and protein levels were constant between days 5 and 25 of pregnancy. PGFS and mPGES-1 expression was down-regulated on days 16-17 of the oestrous cycle when compared to the corresponding days of pregnancy. Enhanced mPGES-1/PGFS ratio occurred during early luteal phase and days 5-8 of pregnancy. Expression of CBR1 mRNA and protein was constant during the cycle and pregnancy. Our studies revealed higher mPGES-1/PGFS ratios in the CL during early luteal phase and corresponding days of pregnancy that could favor PGE(2) synthesis and may be important in the control of luteal development. However, PG synthesis in the endometrium/conceptus rather than in the CL could be involved in luteolysis and maternal recognition of pregnancy in pigs.

The influence of embryo presence on prostaglandins synthesis and prostaglandin E2 and F2alpha content in corpora lutea during periimplantation period in the pig.

We determined the expression of PGE2 synthase (mPGES-1), PGF synthase (PGFS), carbonyl reductase/prostaglandin 9-ketoreductase (CBR1) genes and the content of PGE2, PGF2alpha in porcine corpora lutea on Days 12-14 of pregnancy and Days 12-14 of the estrous cycle. For this study we used a surgically-generated model in which one of the uterine horns was cut transversely and a part of this horn was detached from the uterine corpus. The expression of mPGES-1, PGFS, and CBR1 genes and mPGES-1/PGFS ratio were significantly higher in corpora lutea of the pregnant gilts compared to the corpora lutea from the parallel ovaries of the cyclic gilts. There was no difference in mPGES-1, PGFS, CBR1 genes expression and mPGES-1/PGFS ratio between corpora lutea ipsi-(CL1) and contralateral (CL2) to the uterine horn with the developing embryos. The highest content of PGE2 was found in CL1 of the pregnant gilts. The PGE2/PGF2alpha ratio was significantly higher in CL1 of the pregnant gilts compared to corpora lutea from parallel ovary of the cyclic gilts. We suggest that the activity of the investigated genes is induced by compounds of embryonic origin which are not distributed only to the ipsilateral ovary but are transported within the mesometrium to both ovaries in a more systemic manner.

Inhibition of luteolysis and embryo-uterine interactions during the peri-implantation period in pigs.

Inhibition of luteolysis and establishment of pregnancy in pigs results from oestrogen secretion by the conceptuses and requires progesterone produced by the corpus luteum (CL). An integral part of maternal recognition of pregnancy in the pig is the redirection of prostaglandin (PG) F2alpha secretion from endocrine (blood) to exocrine (uterus) direction and an increase of PGE2 synthesis in both the endometrium and conceptus. Uterine and conceptus PGE2 synthases play an integrated role in establishing the PGE2:PGF2alpha ratio necessary for luteal maintenance. The luteolytic or luteotrophic changes in the CL are synchronised with the release of maternal pituitary and ovarian hormones. The presence of uterine oxytocin (OT) and luteinising hormone (LH) receptors are important for the luteolytic effect of PGF2alpha. Conceptus oestrogen secretion coincides with autocrine and paracrine dialogue between the multiple conceptuses and uterine biological compounds and their receptors in trophoblast and endometrium.

Progesterone production in bovine luteal cells treated with drugs that modulate nitric oxide production.

The aim of this study was to investigate the influence of nitric oxide (NO) donors (S-nitroso-L-acetyl penicillamine, spermine-NO complex and sodium nitroprusside) and NO synthase inhibitors (N(omega)-nitro-L-arginine methyl ester, N(omega)-nitro-l-arginine, and (+/-)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine) on progesterone production by dispersed bovine luteal cells cultured for 24 h. All NO donors inhibited progesterone production and increased nitrite or nitrate concentration in the medium in a dose-dependent manner. Secretion of progesterone was reduced to 75% (P < 0.01), 56% (P < 0.001) and 37% (P < 0.001) by S-nitroso-L-acetyl penicillamine; to 65% (P < 0.001), 45% (P < 0.001) and 33% (P < 0.001) by spermine-NO complex and to 77% (P < 0.05), 74% (P < 0.01) and 54% (P < 0.001) by sodium nitroprusside treatments at concentrations of 10(-5), 10(-4) and 10(-3) mol l(-1), respectively, compared with the concentration of this hormone measured in cells cultured in medium alone. NO synthase inhibitors decreased significantly (P < 0.05) nitrite or nitrate concentration and increased progesterone secretion with different potency at different doses. Significant increases in progesterone production were observed after N(omega)-nitro-L-arginine methyl ester treatment at a concentration of 10(-5) mol l(-1) and 10(-4) mol l(-1), and after N(omega)-nitro-l-arginine administration at a concentration of 10(-6) mol l(-1) (P < 0.01) and 10(-5) mol l(-1) (P < 0.05), compared with the concentration of this hormone measured in control cells. The results indicate that both NO donors and NO synthase inhibitors regulate steroidogenesis in cultured bovine luteal cells from days 10 to 14 of the oestrous cycle; however, the degree of progesterone inhibition by NO donors and stimulation by NO synthase inhibitors was dependent on the drug used.

Neural regulation of the bovine corpus luteum.

The ovarian noradrenergic stimulation or noradrenaline (NA) administration directly to the ovary in cow increases ovarian oxytocin (OT) release and post-translational processing of OT synthesis within a few minutes has been established in both in vivo and in vitro studies. Furthermore, NA affects progesterone secretion and its synthesis by an increase of cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase activity. This effect is mediated via luteal cell beta(1)- and beta(2)-receptors. Their total amount correlates with peripheral progesterone concentrations during the luteal phase and this reflects the ability of the ovary to react to beta-stimulation. On the other hand, ovarian denervation causes a decrease of steroidogenic activity in the CL, an increase of beta-receptors on luteal cells, a delay in follicular development and the disruption of cyclicity. Moreover, decrease of progesterone secretion by 20-30% was seen after brief pharmacological blockade of ovarian beta-receptors in the mid-cycle of cattle. We assume that tonic beta-stimulation of the CL ensures the basal secretion of progesterone, whereas acute noradrenergic activation supports the CL during stressful situations which could impair its function. Conversely, long-lasting increase in blood catecholamine concentrations markedly decreases the number of beta-receptors in CL, presumably due to their down-regulation. Concentrations of dopamine (DA) within the CL are highly correlated with those of NA during the estrous cycle, and are higher in the newly-formed than in the developed corpus luteum, the regressed corpus luteum or the corpus luteum of pregnant females. Bovine CL can synthesise de novo NA from DA as a precursor. Concluding, presented data indicate that noradrenergic stimulation can be an important part of mechanism supporting secretory function of CL.

Direct inhibitory effect of progesterone on oxytocin-induced secretion of prostaglandin F(2alpha) from bovine endometrial tissue.

The effect of progesterone on oxytocin-induced secretion of prostaglandin (PG) F(2alpha) from bovine endometrial tissue explants was examined. Endometrial tissue from the late luteal phase were preincubated for 20 h in control medium. Explants were then treated for 6 h with control medium, oxytocin (10(-7) M), progesterone (10(-5) M), or both hormones. Oxytocin increased the medium concentration of 13,14-dihydro-15-keto-PGF(2alpha), whereas progesterone completely suppressed the stimulatory effect of oxytocin. In experiment 2, isolated endometrial epithelial cells were incubated with progesterone (10(-5) M), oxytocin (10(-7) M), and combinations of these hormones with or without actinomycin D (1 ng/ml). Only oxytocin stimulated secretion of PGF(2alpha), and this response was suppressed by progesterone. Oxytocin induced a rapid increase in intracellular concentrations of Ca(2+) detected within 1 min of exposure of epithelial cells from the same cows. Progesterone pretreatment diminished this response. In experiment 3, direct effects of progesterone (2 nM-20 microM) on binding of (3)H-oxytocin to the membrane preparation from epithelial cells were determined by saturation analysis. Oxytocin binding was suppressed by progesterone at every dosage tested. Progesterone is capable of suppressing the ability of oxytocin to induce endometrial secretion of PGF(2alpha). This effect appears to be mediated through a direct interference in the interaction of oxytocin with its own receptor.

Concentrations of catecholamines, ascorbic acid, progesterone and oxytocin in the corpora lutea of cyclic and pregnant cattle.

To determine if there are inter-relationships between progesterone, oxytocin (OT), dopamine (DA), noradrenaline (NA) and ascorbic acid, these compounds were measured in the corpus luteum (CL) from cattle at different stages of the oestrous cycle (n = 42) and from 1-5 months of pregnancy (n = 27). They were measured by radioimmunoassay (RIA), high performance liquid chromatography (HPLC) and colorimetric methods. Corpora lutea were collected from heifers and cows within 30 min of slaughter on days 1-5, 6-10, 11-16 and 17-21 of the oestrous cycle. The stage of pregnancy was determined on the basis of foetal size and development. Each CL was divided into four parts and stored in liquid nitrogen. For hormone estimation, the tissue was homogenised/powdered and suspended in phosphate buffer (for OT and progesterone), 0.1 M trichloracetic acid (TCA; for catecholamines) or in ice-cold metaphosphoric acid (for ascorbic acid). There were no significant differences in the measured parameters between cows and heifers, and so the data were combined. The concentration of DA was correlated with NA (r = 0.66; P < 0.001) during the oestrous cycle and was highest in newly formed CL (P < 0.01) as compared with early CL, regressed CL and CL of pregnant females. NA was negatively correlated (P < 0.01) with progesterone (r = -0.53) and OT (r = -0.41). In contrast, progesterone and OT were positively correlated with each other (r = 0.81; P < 0.01) during all stages of the oestrous cycle, but not during pregnancy. The lowest concentrations of ascorbic acid were observed in regressed CL. Ascorbic acid concentrations were correlated (P < 0.01) with those of progesterone (r = 0.68), OT (r = 0.42) and DA (r = -0.37). Luteal concentrations of ascorbic acid, progesterone and OT followed a pattern consistent with the development and regression of the CL. Luteal concentrations of catecholamines were not consistent with this pattern.

Physiological importance of dopamine as a noradrenaline precursor in the corpus luteum.

1. Both in vivo and in vitro studies have demonstrated that the adrenergic innervation of the ovary affects corpus luteum (CL) secretory function in many species. 2. In cattle, ovarian noradrenergic stimulation or the administration of noradrenaline (NA) to the ovary increases ovarian oxytocin (OT) secretion and post-translational processing of OT synthesis within a few minutes. Furthermore, NA affects both progesterone release and its synthesis by increasing cytochrome P450sec and 3 beta-hydroxysteroid dehydrogenase activity. This effect is mediated via luteal cell beta 1- and beta 2-adrenoceptors. 3. The total number of luteal beta-adrenoceptors correlates with peripheral progesterone concentrations during the luteal phase. Conversely, ovarian denervation causes a decrease of steroidogenic activity in the corpus luteum, an increase in beta-adrenoceptors on luteal cells, a delay in follicular development and the disruption of cyclicity, as well as other effects. Moreover, brief pharmacological blockade of ovarian beta-adrenoceptors in the mid-cycle of cattle decreases progesterone secretion by 20-40%. 4. We conclude that tonic beta-adrenoceptor stimulation of the CL ensures the basal secretion of progesterone, whereas acute noradrenergic activation supports the CL during stressful situations that could impair its function. Dopamine concentrations within the CL are highly correlated with those of NA during the oestrous cycle and are higher in the newly formed compared with the developed CL, the regressed CL or the CL of pregnant females.

Involvement of ovarian steroids in basal and oxytocin-stimulated prostaglandin (PG) F2 alpha secretion by the bovine endometrium in vitro.

It is assumed that exposure of endometrium to spontaneously secreted luteal hormones stimulates PGF2 alpha secretion and modifies oxytocin (OT) influence on the bovine uterus. At first, the time-dependent effect of endogenous luteal products on endometrial PGF2 alpha secretion was examined. Endometrial strips (100 mg) from slaughtered heifers (Days 11 to 17 of the cycle) were incubated alone or with luteal cells (1 x 10(5) cells/mL). The highest PGF2 alpha secretion by the endometrium under influence of hormones secreted from luteal cells was observed after 12 h of incubation compared with the control (P < 0.001). Then, endometrium (Days 11 to 17) was incubated with luteal cells and concomitantly with antagonists of P4 and OT. The P4 antagonist prevented the stimulatory effect of endogenous luteal hormones on PGF2 alpha secretion (P < 0.05), but the OT antagonist did not. Further, direct effects of exogenous P4, OT and estradiol (E2) on endometrial PGF2 alpha secretion (Days 11 to 17) were examined. Both OT and P4 increased PGF2 alpha secretion (P < 0.05); E2 alone had no effect on PGF2 alpha secretion, but it amplified the P4 effect (P < 0.05). Finally, we studied the effect of endogenous luteal products on OT-stimulated PGF2 alpha secretion from endometrium. When endometrium (Days 11 to 17) was incubated without luteal cells, OT stimulated PGF2 alpha secretion (P < 0.001), whereas incubation of endometrium with luteal cells abolished the stimulatory effect of OT on PGF2 alpha secretion (P < 0.001). These treatments did not affect PGF2 alpha secretion from the endometrium collected on Days 1 to 4. In conclusion, P4 stimulates PGF2 alpha secretion by the endometrium and E2 amplifies this effect. As long as the endometrium is under the influence of P4, ovarian OT does not affect PGF2 alpha secretion.

Influence of noradrenaline on progesterone synthesis and post-translational processing of oxytocin synthesis in the bovine corpus luteum.

Noradrenaline (NA) influences secretory function of the bovine corpus luteum (CL), stimulating secretion of progesterone and ovarian oxytocin (OT). To study whether NA is able to stimulate progesterone synthesis and to affect post-translational OT processing, different doses of NA alone or in combination with different doses of OT were added to bovine CL slices from 8 to 13 d of the estrous cycle. To determine which receptors NA affects, and if dopamine (DA) also affects CL function, we used NA or DA combined with a beta-antagonist (propranolol). The results indicated that NA stimulates both luteal progesterone and OT content; furthermore, it increased the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA), terminal enzymes in synthesis of these 2 hormones. The stimulating effect of NA was inhibited by propranolol and by pre-treatment of CL slices with high OT doses. Post-translational processing of OT synthesis by PGA activation was also stimulated by DA, but this effect was inhibited by beta-receptor blocker. Thus DA acts in CL as a NA precursor. In conclusion, it can be assumed that the noradrenergic system affects CL secretory function on different levels of regulation. Furthermore, a high concentration of OT in CL prevents NA from activating PGA and thus decreases post-translational OT synthesis.

Uterine secretion of prostaglandin F2alpha stimulated by different doses of oxytocin and released spontaneously during luteolysis in cattle.

The objectives were to determine the involvement of oxytocin (OT) in the stimulation of prostaglandin F2alpha (PGF) secretion during luteolysis in cattle. On days 16-17 of the oestrous cycle, catheters were inserted into the aorta abdominalis of heifers for OT or saline infusion and into the jugular vein for blood sample collection. The following day, heifers were assigned to one of three experimental groups (Gr): Gr I - 10 IU OT (n=4); Gr II - 20 IU OT (n=4); Gr III - 50 IU OT (n=4). Blood samples were collected every 10 min during a 1-h control period before treatment and every 5-10 min for 2 h after OT treatment. In Gr IV (n=5), a catheter was inserted into the jugular vein on day 15 of the cycle and blood samples were collected every 15 min for 12 h on days 16-19. Plasma concentrations of progesterone, PGF metabolite, 13, 14-dihydro-15-keto-prostaglandin F2alpha (PGFM) and OT were determined. Within 5 min of infusion of 10 or 20 IU OT, peripheral concentrations of OT (7-12 pg/mL) increased by about 200 and 350-500 pg/mL, respectively. These doses did not affect plasma concentrations of PGFM or progesterone within 1.5 h. Fifty IU of OT increased its maximal peripheral concentration to 1500 pg/mL, which is over 20 times greater than that observed physiologically. Concentrations of plasma PGFM in Gr III increased from basal concentrations (5065 pg/mL) to 150-250 pg/mL (P < 0.01) within 10-30 min. During luteolysis, PGFM pulses ranged between 250 and 600 pg/mL on days 16-19 of the cycle (Gr IV), whereas coincident pulses of OT, and those appearing between spikes of PGFM, were never above 75 pg/mL. Only 50% of OT pulses coincided with pulses of PGFM, and 54 % of PGFM pulses coincided with a pulse of OT. Results indicate that luteolytic PGF secretion in cattle is not directly dependent upon ovarian OT.

Changes in ovarian oxytocin secretion as an indicator of corpus luteum response to prostaglandin F(2alpha) treatment in cattle.

Exogenous prostaglandin F(2alpha) (PGF(2alpha)) rapidly increases ovarian oxytocin (OT) release and decreases progesterone (P4) secretion in cattle. Hence, the measurement of OT secretion (the area under the curve and the height of the peak) after different doses of Oestrophan - PGF(2alpha) analogue (aPGF(2alpha)) on Days 12 and 18 of the estrous cycle (estrus = day 0), could be a suitable indicator of corpus luteum (CL) sensitivity to PGF(2alpha) treatment. Mature heifers (n = 36) were used in this study. Blood samples were collected from the jugular vein for the estimation of OT, P4 and 13, 14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM). In Experiment 1, different doses of aPGF(2alpha) (400, 300, 200 and 100 microg) given on Day 12 of the estrous cycle (n = 8) shortened (P < 0.05) the cycle duration (15.2 +/- 0.6 d) compared with that of the control (21.7 +/- 0.4 d). Successive heifers were also treated on Day 12 with 200 (n = 2), 100 (n = 2), 75 (n = 2) or 50 microg aPGF(2alpha) (n = 2). Only the 50 microg aPGF(2alpha) dose did not cause CL regression, although it increased OT concentrations to levels comparable to those observed during spontaneous luteolysis (50 to 70 pg/ml). In Experiment 2, on Day 18 of the cycle heifers (n = 8) were treated with 50, 40, 30 and 20 microg aPGF(2alpha). There was a dose-dependent effect of aPGF(2alpha) on OT secretion on Day 18 of the estrous cycle (r = 0.77; P < 0.05). In Experiment 3, an injection of 500 microg aPGF(2alpha) on Day 12 (n = 4) and 50 microg aPGF(2alpha) on Day 18 (n = 4) caused a similar (P > 0.05) increase in the OT concentration (288.5 +/- 23.0 and 261.5 +/- 34.7 pg/ml, respectively). Thus the effect of the same dose of aPGF(2alpha) (50 microg) on OT secretion was different on Days 12 and 18 of the cycle. To evoke similar OT secretion on Days 12 and 18 the dose of aPGF(2alpha) on Day 18 could be reduced 10-fold, confirming that CL sensitivity to PGF(2alpha) appears to increase in the late luteal phase.