Endocrine secretion of prostaglandin F2alpha in cyclic gilts is decreased by intrauterine administration of exogenous oxytocin

When administered systemically, oxytocin (OT) stimulates secretion of uterine prostaglandin F2alpha (PGF2alpha) in swine, but the role of endometrially-derived OT in control of PGF2alpha release is not clear. This study determined the effect of exogenous OT, administered into the uterine lumen of intact cyclic gilts, on PGF2alpha secretion during late diestrus. Intrauterine infusion of 40USP units OT (in 30 ml 0.9% saline) was performed for 30 min (1 ml/min) into each uterine horn between 7:00 and 9:00 h on days 10, 12, 14 and 16 after estrus. Beginning 20 min before infusion, samples of jugular venous blood were drawn at 5-10-min intervals for 140 min for quantification of 13,14-dihydro-15-keto-PGF2alpha (PGFM), the major stable metabolite of PGF2alpha. Progesterone was analyzed in samples collected 0, 60 and 120 min after initiation of OT infusion. Treatment with OT did not alter plasma concentrations of PGFM on days 10 or 12 but decreased (P<0.001) PGFM concentrations for 40 min after onset of infusion on day 16. Concentrations of PGFM also were reduced in the pre-treatment samples on day 14 (P=0.05) and day 16 (P<0.001) in OT-infused gilts. Plasma progesterone declined (P<0.01) between days 10 and 16 in control-infused gilts but did not decline until after day 14 (P<0.001) in gilts infused with OT. These results indicate that when OT is administered into the uterine lumen of pigs during late diestrus, it has an anti-luteolytic effect to reduce endocrine secretion of PGF2alpha and delay the decline in progesterone that occurs during luteolysis.     

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.     

Effect of oxytocin on concentration of PGF2 alpha in the uterine lumen and subsequent endometrial responsiveness to oxytocin in pigs

The aim of this study was to determine the effect of oxytocin on PGF2 alpha secretion into the uterine lumen of pigs and subsequent endometrial responsiveness to oxytocin in vitro. Cyclic, pregnant and oestradiol-induced pseudopregnant gilts were injected i.v. with vehicle or 20 iu oxytocin 10 min before hysterectomy on day 16 after oestrus. Concentrations of PGF2 alpha and 13,14-dihydro-15-keto PGF2 alpha (PGFM) were significantly increased in uterine flushings collected at hysterectomy (P < 0.05) in pregnant oxytocin-injected gilts. Concentrations of PGF2 alpha and PGFM were greater (P < 0.001) in pregnant than in pseudopregnant and cyclic gilts, and greater (P < 0.01) in pseudopregnant than in cyclic gilts. The ratio of PGFM:PGF2 alpha tended to be greater in cyclic (P < 0.06) and pseudopregnant gilts (P < 0.1) than in pregnant gilts. At 85 +/- 5 min after oxytocin injection, endometrium from each gilt was incubated for 3 h for determination of phosphoinositide hydrolysis and PGF2 alpha secretion in response to treatment with 0 or 100 nmol oxytocin l-1. Endometrial phosphoinositide hydrolysis in response to 100 nmol oxytocin l-1 in vitro was greater (P < 0.05) in cyclic oxytocin-injected gilts than in cyclic vehicle-injected gilts. Treatment with oxytocin in vitro did not stimulate phosphoinositide hydrolysis significantly in vehicle- or oxytocin-injected pregnant gilts or pseudopregnant gilts. Endometrial PGF2 alpha secretion increased after treatment with 100 nmol oxytocin l-1 in vitro in cyclic vehicle-injected (P < 0.01), cyclic oxytocin-injected (P < 0.01), pregnant vehicle-injected (P = 0.06), pseudopregnant vehicle-injected (P < 0.05) and pseudopregnant oxytocin-injected (P < 0.05) gilts, but not in pregnant oxytocin-injected gilts. The increase in PGF2 alpha in pseudopregnant oxytocin-injected gilts was less (P < 0.05) than that in cyclic oxytocin-injected gilts. These results indicate that oxytocin increases the concentration of PGF2 alpha and PGFM in the uterine lumen during pregnancy and may upregulate endometrial responsiveness to oxytocin during late dioestrus in pigs, but does not have the latter effect during early pregnancy or oestradiol-induced pseudopregnancy.     

Secretion of PGF2alpha and oxytocin during hyperthermia in cyclic and pregnant heifers

The effects of acute heat stress (HS) and oxytocin (OT) injection on plasma concentrations of PGF2alpha and OT were examined in cyclic (C; n = 15) and pregnant (P; n = 11) dairy heifers. On Day 17 of synchronized estrous cycles, animals were randomly assigned to either thermoneutral (TN; 20 degrees C, 20% RH) or HS (42 degrees C, 60% RH) chambers. The jugular vein of each heifer was cannulated and blood samples collected hourly for 4 h, then every 15 min for an additional 3 h. Oxytocin (100 IU) was injected (IV) 5 h after the start of blood collection. Plasma samples were assayed subsequently for concentrations of 13,14-dihydro-15-keto PGF2alpha (PGFM) and OT. During the 7-h experiment, body temperature of HS heifers reached 41.2 degrees C as compared to 38.5 degrees C in control heifers. Plasma concentrations of PGFM increased (P<0.05) and peaked 30 min after OT injection in C (890 pg/ml) and P (540 pg/ml) heifers. In C heifers, heat stress failed to alter PGFM concentrations either before or after OT injection. In the P group, PGFM concentrations following OT injection tended to be higher in HS heifers were further TN heifers (peak values of 690 vs. 410 pg/ml). Pregnant TN and HS heifers were further classified as responders or non-responders to OT challenge according to a cutoff value for PGFM of 193 pg/ml (overall mean of C heifers minus 1 SD). Five of six HS and one of five TN pregnant heifers were classified as responders (P<0.06). Oxytocin concentrations in plasma prior to injection of exogenous OT were not affected by HS or pregnancy status. It is concluded that in C heifers, acute HS in vivo does not cause any further rise in PGF2alpha secretion. However, in P heifers, HS appears to antagonize suppressive effects of the embryo on uterine secretion of PGF2alpha, as indicated by the larger proportion of P heifers responding to OT challenge.     

Oxytocin (OT) action in uterine tissues of cyclic and early pregnant gilts: OT receptors concentration, prostaglandin F(2)alpha secretion, and phosphoinositide hydrolysis

Oxytocin (OT) is involved in the regulation of luteolysis in pigs. However, it is still not clear if OT is responsible for initiation of luteal regression in this species. The objectives of the study were: (1) to compare OT receptors (OTr) concentrations in endometrium and myometrium of cyclic and early pregnant pigs, (2) to examine the effect of OT on plasma PGF(2)alpha secretion during the progressive luteal regression, (3) to ascertain the effect of OT on inositol phosphates (IPs) accumulation in endometrial and myometrial cells of cyclic and early pregnant pigs. Concentrations of OTr on the endometrium and myometrium of cyclic (n = 33) (days 2-4; 11-13; 14-16; 18-20; day 21) and early pregnant (n = 4) (days 14-16) gilts were determined and they ranged from 7 +/- 3 (days 11-13) to 377 +/- 113 fmol/mg protein (day 21) in the endometrium and from 33 +/- 11 (days 2-4) to 167 +/- 28 fmol/mg protein (days 18-20) in the myometrium. In both tissues, concentrations of OTr were low during the luteal phase and increased (P < 0.01) during the follicular phase. In contrast to myometrial OTr, endometrial OTr during pregnancy were undetectable. In next experiment, mature gilts (n = 12) were injected with OT (20IU; i.v.) for three consecutive days starting on days 14 and 15 of the oestrous cycle and plasma PGF(2)alpha metabolite-13,14-dihydro-16-keto PGF(2)alpha (PGFM) concentration was determined. On days 15-16 and 16-17, OT increased plasma PGFM level. This effect was not observed on days 14-15 of the estrous cycle. A negative correlation was noticed between plasma concentrations of PGFM and progesterone (r = -0.3; P < 0.05). In last experiment, OT (100 nM) augmented (P < 0.01) an accumulation of inositol phosphates (IPs) in isolated myometrial cells on days 14-16 (n = 4) and 18-20 (n = 3) of the estrous cycle and on days 14-16 (n = 4) of pregnancy. Oxytocin-stimulated accumulation of IPs was not observed in endometrial cells. In summary: (1) concentrations of OTr on both the endometrium and myometrium were the highest during perioestrus-period in pigs, (2) myometrium of early pregnant sows possessed functional OTr, (3) oxytocin increased plasma PGFM concentration after initiation of luteolysis; and (4) OT-stimulated accumulation of IPs in myometrial, but not in endometrial cells. In conclusion, OT appears to not be involved in the initiation of luteal regression in sows and functional OTr are still present in the myometrium during early pregnancy (days 14-16).     

Effects of administration of oxytocin on embryonic survival in progestogen supplemented cattle.

Embryonic survival after administration of oxytocin (OT) was examined in 42 beef cows. All cows were bred (Day 0) and randomly assigned to receive either 25 mL saline (CON; n = 10), 100 IU OT + 20 mL saline (OT; n = 12), 100 IU OT + 1 g flunixin meglumine (OT + FM; inhibitor of prostaglandin endoperoxide synthase; n = 10), or 100 IU OT + lutectomy (OT + LUT; n = 10) administered (i.m.) at 8-h intervals on Days 5-8 after mating. Lutectomies were performed by transrectal digital pressure prior to initiation of treatments (0600, Day 5). All cows were fed 4 mg/head/day of melengesterol acetate (an orally administered exogenous progestogen) through Days 3-30 and were bled by jugular venipuncture at 0600 and 0700 h on Day 5 for determination of 13,14-dihydro-15-keto-PGF2a (PGFM). Pregnancy rates, as determined by transrectal ultrasonography at Day 30, were reduced in OT (33.3%) and OT + LUT (30%) groups compared to CON and OT + FM (80%; p < or = 0.03). Number of short cycles were increased in OT (n = 6/12) group compared to CON (n = 0/10; p < or = 0.009) and OT + FM (n = 1/10; p < or = 0.045). Mean change in PGFM from the 0600 to 0700 h bleed was different (p < or = 0.01) between the OT + LUT (31.6 +/- 11.0 pg/mL) group versus CON (-11.2 +/- 10.6 pg/mL) and OT + FM (-13.8 +/- 10.6 pg/mL) groups. Administration of oxytocin appears to decrease embryonic survival by stimulating uterine PGF2a. Thus, previous reports indicating that removal of the corpus luteum during progestogen supplementation and prior to PGF2a administration increases embryonic survival can be explained through interruption of the luteal oxytocin-uterine PGF2a feedback loop.     

Effect of pregnancy on oxytocin-induced release of prostaglandin F2 alpha in heifers

The effect of pregnancy on the release of prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin (OT) has been examined. Fourteen cyclic heifers received one intravenous injection of 1 IU OT (n = 6) or 100 IU OT (n = 8) 17, 18, or 19 days (Day 17-19) after the onset of estrus (Day 0). Five of these animals also received 100 IU OT at Days 6 and 13 to determine the effect of OT at different times of the cycle. Frequent blood samples were taken for 60 min before and for 90 min after OT injection for the measurement of 15-keto-13,14-dihydro-PGF2 alpha (PGFM) by radioimmunoassay. The experiment was then repeated using the same animals at Day 17-19 of pregnancy (confirmed by the recovery of an embryo the day after OT injection). Following the injection of 1 IU OT, plasma PGFM reached its peak within 30 min with the increase significantly lower (P less than 0.05) in pregnant (1.13 +/- 0.10-fold) than in nonpregnant animals (2.07 +/- 0.27-fold). However, because only 3 of the 6 cyclic animals showed a response to 1 IU OT, the dose was increased to 100 IU in subsequent experiments. The animals that received 100 IU at Days 6 and 13 had no significant increase in PGFM concentrations (1.18 +/- 0.05-fold and 1.01 +/- 0.04-fold, respectively). At Day 17-19 the increase in plasma PGFM reached its peak 5-15 min after 100 IU OT and the increase was significantly greater in nonpregnant (3.23 +/- 0.17-fold) than in pregnant (1.21 +/- 0.02-fold; P = 0.003) heifers. Six of 11 animals injected at Day 17-19 of the cycle showed a decrease in progesterone (P4) the day after OT administration. These data show that the release of PGF2 alpha in response to OT is suppressed in pregnant animals in vivo, suggesting an antiluteolytic role for the embryo in luteostasis.     

Effects of mating stimuli and oxytocin on plasma cortisol concentration in gilts

The involvement of oxytocin (OT) in the regulation of glucocorticoid secretion during stress reaction, parturition, and suckling has been documented in various species. In this study four in vivo experiments were conducted on gilts (1) to demonstrate the influence of mating stimuli on plasma cortisol concentration, (2) to test the effect of OT alone and (3) OT combined with OT-antagonist on cortisol secretion and (4) to clarify the role of progesterone and estradiol in cortisol response to exogenous OT. In experiment 1, plasma cortisol concentration in gilts (n=4) increased (p<0.05) from 16.1 +/- 5.3 ng ml(-)1 (control period: 30 min before mating) to 42.8 +/- 11.6 ng ml(-1) and 46.6 +/- 9.6 ng ml(-1) at the time of leaving the pen and during the first visual and olfactory contact with the boar, respectively. During coitus the elevation was maintained (48.8 +/- 9.8 ng ml(-1); p<0.05 vs. control). The plasma cortisol concentration returned to pre-mating levels within 30 min after mating. In experiment 2, gilts (n=7) were treated, according to Latin square design, with saline (2 ml; i.v.) and OT (10, 20, and 30 IU; i.v.). The magnitude of cortisol response (area under cortisol curve) was higher (p<0.01) only after treatments with 20 and 30 IU OT vs. control period (30 min before OT). Gilts (n=3) of experiment 3 were infused with OT-antagonist (Atosiban; 25 mg per gilt per 2 hours; i.v.) and then were injected with OT (20 IU; i.v.) 60 min after the beginning of Atosiban administration. Blockage of OT receptors by Atosiban reversed the stimulatory effect of OT on cortisol secretion. In experiment 4, ovariectomized gilts (n=25) primed (i.m.) with corn oil (n=7), progesterone (P4; n=7), estradiol benzoate (EB; n=4) or EB+P4 (n=7) were treated with OT (20 IU; i.v.). Plasma cortisol concentrations were increased following OT administration in all gilts of experiment 4. The highest cortisol response to OT was noted in gilts primed with EB+P4 (p<0.01 vs. other groups). In conclusion: (1) leaving the pens, visual and olfactory contact with the boar as well as coitus, increased plasma cortisol concentrations in gilts to similar levels; (2) exogenous OT (20 and 30 IU per gilt) increased cortisol plasma concentration, (3) this effect was abolished by OT-antagonist and (4) E2+P4 elevated cortisol response to OT. Oxytocin may be included to secretagogues of the hypothalamus-pituitary-adrenocortical axis in pigs.     

Involvement of gonadotropins in induction of luteolysis in pigs

In our previous study we have demonstrated that treatment of endometrial explants with LH increased 13,14-dihydro-15-ketoprostaglandin F(2alpha) (PGFM) accumulation in pigs. This was particularly visible on Days 14-16 of the estrous cycle. Action of gonadotropin in porcine endometrium appears to be mediated by LH/hCG receptors whose number is dependent on the day of the estrous cycle. In the current study i.v. infusion (1 hour) of hCG (200 IU) performed on Days 10 (n=4) and 12-14 (n=4) of the porcine estrous cycle did not affect plasma PGFM (ng/ml+/-SEM) concentrations. In contrast, administration of hCG on Days 15-17 produced, depending on plasma PGFM level before the infusion period, three different types of response: I. plasma PGFM surge of amplitude 0.62+/-0.15 was observed when the mean basal pre-infusion PGFM plasma level was 0.23+/-0.05 (n=6 gilts); II. the delayed PGFM surge of amplitude 0.62+/-0.15 was determined when basal pre-infusion PGFM level was 0.80+/-0.20 (n=6); and III. lack of PGFM response to hCG was found when basal pre-infusion PGFM level was 1.09+/-0.61 (n=6). Concentrations of plasma PGFM before and after saline infusion did not differ on Days 12-14 and 16 of the estrous cycle. In the next experiment blood samples were collected every 1 hour on Days 12-19 of the estrous cycle to determine concentrations of LH, PGFM and progesterone in four gilts. In particular gilts, plasma peaks of LH closely preceded surges of PGFM in 72.7, 84.6, 75.0 and 66.6 percent, respectively. The highest PGFM surges followed a decline in plasma progesterone concentration. We conclude that the increased PGF(2alpha) metabolite production after hCG infusion during the late luteal phase of the estrous cycle as well as the relationship between plasma LH and PGFM peaks suggest the LH involvement in the elevation of endometrial PGF(2alpha) secretion in pigs, and, in consequence, induction of luteolysis.     

Jugular plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha in prepubertal beef heifers treated with progestogen then challenged with oxytocin.

Prepubertal Angus crossbred heifers (n = 24) between 8 and 10 mo of age were used to determine if progestogen treatment would enhance jugular concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) after oxytocin (OT) injections. Heifers were stratified by age and weight and allotted to randomized treatments in a 2 x 2 factorial arrangement. Heifers were treated with either a norgestomet (NOR) implant (6 mg) for 9 d or no implant (0 mg; BLK). On d 8 of NOR treatment, jugular veins were catheterized and, on d 9, blood samples were collected every 15 min for 165 min. The first four samples were used to determine basal PGFM concentrations (an indirect measure of uterine PGF2 alpha release). After collection of the fourth sample, either OT (100 IU) or saline (0 IU; SAL) was injected via the jugular catheter. After the 165-min sample was collected, NOR implants were removed. Beginning 48 h after implant removal, a second 165- min blood sampling period was initiated. Average progesterone concentrations were less than 1 ng/ml during both bleeding periods. Within treatment, PGFM concentrations were similar between the first and second sampling periods; therefore, data within treatment were combined. Basal PGFM concentrations were higher (P < .01) in NOR-treated than in BLK heifers. Oxytocin did not increase PGFM concentrations in BLK-OT heifers; however, a marked increase in PGFM was detected in the NOR-OT heifers in response to oxytocin. Average PGFM concentration was greatest (P < .0001) in NOR-OT heifers, and PGFM profiles differed (P < .0001) between NOR-OT and each of the other treatment groups. Results from this study indicate that NOR increases basal PGFM and may "condition" the uterus to respond to OT in prepubertal heifers.     

Prolonged progesterone treatment of endometrial epithelial cells modifies the effect of estradiol on their sensitivity to oxytocin

Estradiol (E2), progesterone (P4), and oxytocin (OT) are important for the initiation of luteolysis in ruminants but the mechanisms involved are still poorly understood. The objective of this study was to determine if duration of exposure of bovine endometrial epithelial cells to P4 affected the response of the cells to E2. Endometrial epithelial cells, from cows at Days 1-3 of the estrous cycle, were cultured for 10, 17, and 21 days in the presence or absence of P4 (100 ng ml(-1)). After culture, each group of cells was incubated for a further 6, 12, 24 or 48 h with or without E2 (100 pg ml(-1)) and then incubated for 6 h with different doses of OT (2, 20, and 200 ng ml(-1)). E2 enhanced OT-stimulated PGF2 alpha secretion in cells cultured with P4 for 17 or 21 days, with a maximum effect after 24-h exposure, but not in cells cultured with P4 for 10 days. To determine the mechanism of action of E2, COX-1 and COX-2 were measured by Western blotting and OTR number was measured by saturation analysis. OT increased COX-2 (P<0.05), but there was no significant effect of E2 on the expression of either COX-1 or COX-2. E2 did, however, increase (P<0.001) the OTR number in cells cultured with P4 for 21 days, whereas it inhibited OTR in cells cultured for 10 days. These data show that E2 can stimulate PGF2 alpha secretion by increasing OTR expression in bovine endometrial cells in vitro, but only after exposure to P4.     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Neonatal oxytocin treatment modulates oxytocin receptor, atrial natriuretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression in rat heart

Oxytocin (OT) has been implicated in reproductive functions, induction of maternal behavior as well as endocrine and neuroendocrine regulation of the cardiovascular system. Here we demonstrate that neonatal manipulation of OT can modulate the mRNAs expression for OT receptor (OTR), atrial natriuretic peptide (ANP), endothelial nitric oxide synthase (eNOS) and estrogen receptor alpha (ERalpha) in the heart. On the first day of postnatal life, female and male rats were randomly assigned to receive one of the following treatments: (a) 50microl i.p. injection of 7microg OT; (b) 0.7microg of OT antagonist (OTA); or (c) isotonic saline (SAL). Hearts were collected either on postnatal day 1 or day 21 (D1 or D21) and the mRNAs expression of OTR, ANP, inducible NOS (iNOS), eNOS, ERalpha and estrogen receptor beta (ERbeta) were compared by age, treatment, and sex utilizing real time PCR. OT treatment significantly increased heart OTR, ANP and eNOS mRNAs expression on D1 in both males and females, ERalpha increased only in females. While there were significant changes in the relative expression of all types of mRNA between D1 and D21, there were no significant treatment effects observed in D21 animals. OTA treatment significantly decreased basal ANP and eNOS mRNAs expression on D1 in both sexes. The results indicate that during the early postnatal period OT can have an immediate effect on the expression OTR, ANP, eNOS, and ERalpha mRNAs and that these effects are mitigated by D21. Also with the exception of ERalpha mRNA, the effects are the same in both sexes.     

The influences of GnRH, oxytocin and vasoactive intestinal peptide on LH and PRL secretion by porcine pituitary cells in vitro.

The aim of the present study was to evaluate the possible direct effects of GnRH, oxytocin (OT) and vasoactive intestinal peptide (VIP) on the release of LH and PRL by dispersed porcine anterior pituitary cells. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) one month before slaughter. Gilts randomly assigned to one of the four groups were treated: in Group 1 (n = 8) with 1 ml/100 kg b.w. corn oil (placebo); in Group 2 (n = 8) and Group 3 (n = 8) with estradiol benzoate (EB) at the dose 2.5 mg/100 kg b.w., respectively, 30-36 h and 60-66 h before slaughter; and in Group 4 (n = 9) with progesterone (P4) at the dose 120 mg/ 100 kg b.w. for five consecutive days before slaughter. In gilts of Group 2 and Group 3 treatments with EB have induced the negative and positive feedback in LH secretion, respectively. Isolated anterior pituitary cells (10(6)/well) were cultured in McCoy's 5a medium with horse serum and fetal calf serum for 3 days at 37 degrees C under the atmosphere of 95% air and 5% CO2. Subsequently, the culture plates were rinsed with fresh McCoy's 5A medium and the cells were incubated for 3.5 h at 37 degrees C in the same medium containing one of the following agents: GnRH (100 ng/ml), OT (10-1000 nM) or VIP (1-100 nM). The addition of GnRH to cultured pituitary cells resulted in marked increases in LH release (p < 0.001) in all experimental groups. In the presence of OT and VIP we noted significant increases (p < 0.001) in LH secretion by pituitary cells derived from gilts representing the positive feedback phase (Group 3). In contrast, OT and VIP were without any effect on LH release in Group 1 (placebo) and Group 2 (the negative feedback). Pituitary cells obtained from OVX gilts primed with P4 produced significantly higher amounts (p < 0.001) of LH only after an addition of 100 nM OT. Neuropeptide GnRH did not affect PRL secretion by pituitary cells obtained from gilts of all experimental groups. Oxytocin also failed to alter PRL secretion in Group 1 and Group 2. However, pituitary cells from animals primed with EB 60-66 h before slaughter and P4 produced markedly increased amounts of PRL in the presence of OT. Neuropeptide VIP stimulated PRL release from pituitary cells of OVX gilts primed with EB (Groups 2 and 3) or P4. In contrast, in OVX gilts primed with placebo, VIP was without any effect on PRL secretion. In conclusion, the results of our in vitro studies confirmed the stimulatory effect of GnRH on LH secretion by porcine pituitary cells and also suggest a participation of OT and VIP in modulation of LH and PRL secretion at the pituitary level in a way dependent on hormonal status of animals.     

The influence of interleukin-1beta on oxytocin signalling in primary cells of human decidua

OBJECTIVE: Oxytocin (OT) and its corresponding receptor (OTR), synthesized within the pregnant uterus, play a key role in the process of (preterm) labor as part of a paracrine system that regulates symmetrical contractility. In the setting of intrauterine infection, a major cause of preterm labour and birth, decidua serves as a major source of cytokine production. The present study evaluates the time-dependent effect [0-24 h] of the inflammatory cytokine Interleukin-1beta (IL-1beta) treatment on OT signalling and OT stimulated prostaglandin release in primary cultures of human decidua. STUDY DESIGN: Primary cultures of human decidua (n=6) were treated with IL-1beta [5 ng/ml] for 0-24h and or indomethacin [100 microM]--an inhibitor of the prostaglandin synthesis--for 0-24 h or for 24 h. OT peptide expression, OTR binding, Inositol triphosphate production (IP(3)), and arachidonic acid (AA) as well as prostaglandin (PGE(2)) release were measured. RESULTS: IL-1beta transiently reduced cytoplasmic OT peptide at 4-6 h of IL-1beta incubation, while its secretion into the media was increased after 6 h of stimulation. The later was completely blocked by indomethacin. A decrease in OTR mRNA expression and a loss of OTR binding were detected after 8 h and 16 h of IL-1beta treatment, respectively. IL-1beta also decreased IP(3) production and AA release, but significantly enhanced OT mediated PGE(2) production. This effect was completely suppressed by the cyclooxygenase-2 (COX-2) inhibitor NS-398. CONCLUSION: Our data suggest, that IL-1beta indirectly increases OT secretion in primary cultures of human decidua in a time dependent fashion through the production of prostaglandins through COX-2 and that this increase in OT peptide may secondarily down-regulate the OTR and its signalling cascade. These findings might explain the poor effectiveness of oxytocin receptor antagonists as tocolytic agents in the setting of intrauterine infection.     

Inhibition of prostaglandin F2alpha synthesis and oxytocin receptor by progesterone antagonists in bovine endometrial cells in vitro

Oxytocin receptor (OTR) expression is suppressed by progesterone (P4) during the luteal phase of the estrous cycle and then it increases at the time of luteolysis, but its regulation is still not completely understood. In vitro studies to determine the mechanism of action are hindered because OTR spontaneously upregulates in vitro and it is impossible to alter expression with P4 or estradiol. During recent studies examining the effect of P4 and an antagonist (mifepristone) on PG secretion, we found that mifepristone attenuated OT-stimulated PG secretion from endometrial epithelial cells. The objective of the present study was to determine, whether this effect of mifepristone was due to changes in prostaglandin synthesis and/or OTR. A time-course showed that mifepristone (5 microM) had no significant effect after 24 h but by 72 h it decreased PGF(2alpha) secretion (P<0.01) and abolished the response of the cells to OT (P<0.01). The presence or absence of P4 did not affect the response to mifepristone. To determine the site of action of mifepristone, cells were cultured for 72 h with or without mifepristone and then COX-1 and COX-2 were measured by Western blotting and OTR was measured by saturation analysis. The results showed that mifepristone did not affect basal or PMA-stimulated expression of either COX-1 or COX-2 but did, however, decrease OTR number (P<0.05). These data demonstrate that OTR and the response to OT can be downregulated in endometrial epithelial cells in vitro via a mechanism involving the P4 receptor.     

Possible involvement of oxytocin and its receptor in the local regulation of prostaglandin secretion in the cat endometrium

Ovarian originated oxytocin (OT) is involved in several reproductive process, amongst them its role in the regulation/modulation of the estrous cycle in several species has been demonstrated. Although the systemic role of endometrial originated prostaglandins (PGs), especially prostaglandin F(2α) (PGF(2α)), is equivocal in cats, their possible involvement in the local regulation of uterine events during the estrous cycle is uncertain. We examined the spontaneous and LH-stimulated OT production in cultured luteal cells, the spatial and temporal arrangement of OT receptors (OTR) in a cat endometrium and, finally the effects of OT on PG secretion and prostaglandin-endoperoxide synthase (PTGS2) expression in the feline cultured endometrial cells. Uteri together with ovaries were collected from adult domestic cats (n=27) at different stages of the estrous cycle, after routine ovariohysterectomy procedures. The endometrial and luteal cells were separated enzymatically. Luteinizing hormone (LH) augmented OT secretion in cultured luteal cells 2-fold compared with control (P<0.05). Oxytocin receptor was abundantly expressed in different ovarian structure, as well as in uterine tissues collected at early/developing and mid-luteal phase. The secretion of PGF(2α) by endometrial epithelial cells was increased by OT at a dose 10(-7)M (P<0.001). Atosiban (specific OTR blocker) alone did not affect PG secretion but atosiban in combination with OT abolished the stimulating effect of OT on PGF(2α) secretion. Oxytocin augmented PGE(2) secretion at a dose 10(-7)M and 10(-6)M in the endometrial stromal cells (P<0.001). The treatment with atosiban did not abrogated positive effect of OT on PGE(2) production in the stromal cells. Effect of OT on PTGS2 mRNA expression, the rate-limiting enzyme in PG production, was examined by Real Time-PCR and PTGS2 mRNA expression was significantly affected by OT in both epithelial and stromal cell cultures (P<0.01). The present observations have shown that OT is locally produced by the early/developing corpora lutea and that corpora lutea delivered OT may regulate PG secretion in a cat endometrium especially at early- and mid-diestrus, by affecting PTGS2 mRNA expression.     

Effects of progesterone and estradiol on uterine secretion of prostaglandin f(2alpha)in response to oxytocin in ovariectomized sows

Thirty ovariectomized sows were used in an experiment designed to determine whether the ability of the porcine uterus to release prostaglandin (PG) F(2alpha) in response to oxytocin is regulated by progesterone (P(4)) and estradiol (E(2)). Sows were assigned to one of four treatment groups: 1) no steroids (ovariectomized controls; n = 8), 2) E(2) (n = 8), 3) P(4) (n = 7), or 4) E(2) + P(4) (n = 7). P(4) and E(2) were administered so as to mimic the normal temporal changes that occur in these hormones during the estrous cycle. A group of intact sows (n = 9) was included for comparison. All sows received an injection of oxytocin (30 IU, i.v.) on Days 12, 15, and 18 postestrus. Jugular venous blood samples were collected from 60 min before through 120 min after injection of oxytocin for quantification of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM). Preinjection baseline concentrations of PGFM, the magnitude of the PGFM response above baseline, and area under the PGFM response curve (AUC) were calculated for each sow on each day and compared among treatment groups by ANOVA. Among the ovariectomized sows receiving steroid replacement, baseline concentrations of PGFM were low on Day 12 postestrus in all four groups. On Days 15 and 18, baseline concentrations remained low in the two groups that did not receive P(4) but increased in those that did. Both the magnitude of the response to oxytocin and AUC were small on Day 12 postestrus in all 4 groups. By Day 15, the magnitude of the response and AUC increased in the group that received both P(4) and E(2) but remained low in the other three groups. By Day 18, responses to oxytocin were greater in both groups that received P(4) than in those that did not. Baseline concentrations were similar in intact sows and in those that received both P(4) and E(2) on all three days examined. The magnitude of the response and the AUC were greater in the ovariectomized sows receiving P(4) and E(2) replacement than in the intact control sows on Days 15 and 18 postestrus. From these results, we conclude that P(4) and E(2) interact to control the time when the uterus begins to secrete PGF(2alpha) in response to oxytocin and the amount of PGF(2alpha) secreted.     

Prostaglandin F2 alpha-induced release of oxytocin from bovine corpora lutea in vitro

Two experiments were conducted to study the in vitro effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2), and luteinizing hormone (LH) on oxytocin (OT) release from bovine luteal tissue. Luteal concentration of OT at different stages of the estrous cycle was also determined. In Experiment 1, sixteen beef heifers were assigned randomly in equal numbers (N = 4) to be killed on Days 4, 8, 12, and 16 of the estrous cycle (Day 0 = day of estrus). Corpora lutea were collected, an aliquot of each was removed for determination of initial OT concentration, and the remainder was sliced and incubated with vehicle (control) or with PGF2 alpha (10 ng/ml), PGE2 (10 ng/ml), or LH (5 ng/ml). Luteal tissue from heifers on Day 4 was sufficient only for determination of initial OT levels. Luteal OT concentrations (ng/g) increased from 414 +/- 84 on Day 4 to 2019 +/- 330 on Day 8 and then declined to 589 +/- 101 on Day 12 and 81 +/- 5 on Day 16. Prostaglandin F2 alpha induced a significant in vitro release of luteal OT (ng.g-1.2h-1) on Day 8 (2257 +/- 167 vs. control 1702 +/- 126) but not on Days 12 or 16 of the cycle. Prostaglandin E2 and LH did not affect OT release at any stage of the cycle studied. In Experiment 2, six heifers were used to investigate the in vitro dose-response relationship of 10, 20, and 40 ng PGF2 alpha/ml of medium on OT release from Day 8 luteal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E2 counteracts the effects of PGF2 alpha in indomethacin treated cycling gilts

Twenty crossbred gilts with at least 2 consecutive estrous cycles of 18 to 21 days in length were used to study the effects of prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) on luteal function in indomethacin (INDO) treated cycling gilts. Intrauterine and jugular vein catheters were surgically placed before day 7 of the treatment estrous cycle and gilts were randomly assigned to 1 of 5 treatment groups (4/group). With exception of the controls (Group I) all gilts received 3.3 mg/kg INDO every 8 h, Groups III, IV and V received 2.5 mg PGF2; 2.5 mg PGF2 alpha + 400 micrograms PGE2 every 4 hr, or 400 micrograms PGE2 every 4 h, respectively. All treatments were initiated on day 7 and continued until estrus or day 23. Jugular blood for progesterone analysis was collected twice daily from day 7 to 30. Estradiol-17 beta (E2-17 beta) concentrations were determined in samples collected twice daily, from 2 d before until 2 d following the day of estrus onset. When compared to pretreatment values, estrous cycle length was unaffected (P greater than 0.05) in Group I, prolonged (P less than 0.05) in Groups II, IV and V; and shortened (P less than 0.05) in Group III. The decline in plasma progesterone concentration that normally occurs around day 15 was unaffected (P greater than .05) in Group I; delayed (P less than 0.05) in Groups II, IV and V; and occurred early (P less than 0.05) in Group III. Mean E2-17 beta remained high (31.2 +/- 4.9 to 49.3 +/- 3.1 pg/ml) in Groups III and IV, while the mean concentrations in Groups III and V varied considerably (17.0 +/- 2.0 to 52.2 +/- 3.5 pg/ml). The results of this study have shown that PGE2 will counteract the effects of PGF2 alpha in INDO treated cycling gilts. The inclusion of PGF2 alpha appeared to either stimulate E2-17 beta secretion or maintain it at a higher level than other treatments.