Second messenger systems in the action of LH and oxytocin on porcine endometrial cells in vitro

LH/hCG as well as oxytocin receptors are present in the porcine endometrium. Oxytocin increases phosphatidylinositol hydrolysis in this tissue, but its action on adenylate cyclase activity is disputed. The second messenger system responding to LH/hCG in endometrial cells has not been established. In this study, we investigated the involvement of protein kinase A and C signaling mechanisms in the action of LH on porcine endometrial cells in vitro. The possibility of cAMP accumulation after treatment of endometrial cells with oxytocin was also investigated. Endometrial tissue was obtained from gilts during Days 12-15 of the estrous cycle. To study the adenylate cyclase system, endometrial cells were cultured for 48 h and then incubated with different doses of LH or oxytocin for 15, 30, 60, and 180 min. To study the phospholipase C system, dispersed cells were first labeled with myo-[3H]inositol and then treated with increasing doses of LH or 100 nM of oxytocin for 30 min. Time- and dose-dependent effect of LH and oxytocin on cAMP concentration was observed. After 30 min of incubation only the highest dose of LH (100 ng/ml) was able to increase cAMP concentration in medium (P < 0.05). Longer periods (1 and 3 h) caused increased cAMP accumulation after treatment with 10 and 100 ng/ml of LH (P < 0.001). Oxytocin-stimulated cAMP concentration was observed after 1 h when only the highest dose (1000 nM) of hormone was used (P < 0.01) and after 3 h of incubation with doses of 10-1000 nM (P < 0.01). LH (10 and 100 ng/ml) increased inositol phosphates (IPs) accumulation in endometrial cells after 30 min of incubation (P < 0.01). Oxytocin involvement in IPs synthesis was more apparent than was LH (P < 0.001 versus P < 0.01). This is the first demonstration that LH receptor signaling leads to increased cAMP generation as well as IPs turnover in porcine endometrium. Oxytocin-dependent cAMP production in endometrial cells of swine was found after longer periods (3 h) of incubation. Our observations lead to the conclusion that both protein kinase A and C second messenger systems are involved in LH action and that oxytocin is able to stimulate adenylate cyclase activity in porcine endometrial cells.     

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).     

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.     

Control of endometrial oxytocin receptor and uterine response to oxytocin by progesterone and oestradiol in the ewe

The effects of administration of progesterone and oestradiol on ovine endometrial oxytocin receptor concentrations and plasma concentrations of 13,14-dihydro-15-keto prostaglandin F-2 alpha (PGFM) after oxytocin treatment were determined in ovariectomized ewes. Ewes received progestagen pre-treatment, progesterone and/or oestradiol in 11 different treatment schedules. Progestagen pre-treatment decreased oxytocin receptor concentrations in endometrium from ewes treated subsequently with either progesterone for 5 days or progesterone for 5 days plus oestradiol on Days 4 and 5 of progesterone treatment. Oestradiol increased endometrial oxytocin receptor concentrations when administered on Days 4 and 5 of 5 days progesterone treatment. Progestagen pre-treatment followed by progesterone treatment for 12 days caused a large increase in oxytocin receptors and no further increase occurred when ewes were given oestradiol on Days 11 and 12, or when progesterone was withdrawn on Days 11 and 12, or these two treatments were combined. Oxytocin administration caused an increase in plasma PGFM concentrations in ewes which did not receive progestagen pre-treatment, and subsequently received progesterone treatment for 5 days and oestradiol treatment on Days 4 and 5 of progesterone treatment. Similarly treated ewes which received progestagen pre-treatment did not respond to oxytocin. Oxytocin administration also increased plasma PGFM concentrations in ewes which received progestagen pre-treatment followed by progesterone treatment for 12 days, progesterone treatment for 12 days plus oestradiol on Day 11 and 12 of progesterone treatment, progesterone withdrawal on Day 11 and 12, or progesterone withdrawal and oestradiol treatment combined. The results indicate that (1) progesterone pre-treatment affects oxytocin receptor concentrations in the endometrium and uterine responsiveness to oxytocin and (2) progesterone treatment alone for 12 days after a treatment which mimics a previous luteal phase and oestrus is sufficient to induce oxytocin receptors and increase oxytocin-induced PGF release. These results emphasize the importance of progesterone and provide information which can be used to form an hypothesis for control of luteolysis and oestrous cycle length in the ewe.     

Differential effects of progesterone treatment on the oxytocin-induced prostaglandin F2 alpha response and the levels of endometrial oxytocin receptors in ovariectomized ewes.

The oxytocin-induced uterine prostaglandin (PG) F2 alpha response and the levels of endometrial oxytocin receptors were measured in ovariectomized ewes after they had been given steroid pretreatment (SP) with progesterone and estrogen to induce estrus (day of expected estrus = Day 0) and had subsequently been treated with progesterone over Days 1-12 and/or PGF2 alpha over Days 10-12 postestrus. The uterine PGF2 alpha response was measured after an i.v. injection of 10 IU oxytocin on Days 13 and 14, using the PGF2 alpha metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), as an indicator for PGF2 alpha release. The levels of oxytocin receptors in the endometrium were measured on Day 14. During the treatment with progesterone, the peripheral progesterone concentrations were elevated and remained above 1.8 ng/ml until the morning of Day 14. The PGFM responses to oxytocin in untreated controls and SP controls were low on both Days 13 and 14 whereas the levels of endometrial oxytocin receptors in the same ewes were high. Treatment with progesterone either alone or in combination with PGF2 alpha significantly (p less than 0.04) increased the PGFM response on Day 14 and reduced the levels of endometrial oxytocin receptors; treatment with PGF2 alpha alone had no effect. It is concluded that progesterone promotes the PGFM response to oxytocin while simultaneously suppressing the levels of endometrial oxytocin receptors. PGF2 alpha treatment had no effect on either the uterine secretory response to oxytocin or the levels of oxytocin receptors in the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Endocrine profiles of dairy cows following experimentally induced clinical mastitis during early lactation

Concentrations of LH, cortisol, estradiol-17beta (E(2)), prolactin and 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM) were determined in cows with experimentally induced clinical mastitis during early lactation. Cows free of intramammary infection (IMI) and in the luteal phase of the estrous cycle were balanced by lactation number and days in milk and assigned to either control (n=5) or treatment (n=5) groups. Treated cows were infected experimentally (day 0), in two mammary quarters, with Streptococcus uberis and developed clinical mastitis within 60 h after inoculation as evidenced by increased mastitis scores, elevated rectal temperatures, mammary swelling and isolation of S. uberis pathogen. Four days following bacterial challenge, blood samples were collected every 20 min for 8 h for determination of PGFM and LH following administration of oxytocin and GnRH, respectively. Blood samples were also collected on days 0, 4 and 7 of the experiment to determine concentrations of E(2), prolactin and cortisol. Four days after bacterial challenge, concentrations of cortisol were higher (P=0.04) in experimentally infected cows than controls. Experimentally challenged cows had increased (P=0.02) concentrations of cortisol on days 4 and 7 compared with day 0. Control cows had no significant increase in blood cortisol during the experimental period. Baseline concentrations of PGFM did not differ between groups; however, peak concentrations of PGFM following oxytocin challenge were elevated (P=0.006) in cows with clinical mastitis compared with control animals. Prolactin, E(2) and LH did not differ between cows with clinical mastitis or controls. Experimentally induced mastitis during early lactation elevated concentrations of cortisol during the luteal phase of the estrous cycle. Furthermore, mastitic cows demonstrated an increased PGFM response following oxytocin administration. Altered reproductive efficiency in cows with clinical mastitis caused by Gram-positive pathogens may be the result of increased uterine sensitivity to prostaglandin F(2alpha) (PGF(2alpha)).     

Control of luteolysis in the one-humped camel (Camelus dromedarius)

Blood plasma concentrations of 13,14-dihydro-15-keto PGF2 alpha (PGFM) were measured in groups of mature non-pregnant and pregnant camels to study PGF2 alpha release patterns around the time of luteolysis and the timing of the signal for pregnancy recognition. Injection of each of four camels with 10 and 50 mg of PGF2 alpha showed clearly that five times the dose of exogenous hormone produced five times the amount of PGFM in peripheral plasma, thereby indicating that, as in other animal species, PGFM is the principal metabolite of PGF2 alpha in the camel. Serial sampling of three non-pregnant camels on each of days 8, 10 and 12, and three pregnant camels on day 10, after ovulation for 8 h showed a significant (P < 0.05) rise in mean plasma PGFM concentrations only on day 10 in the non-pregnant, but not the pregnant, animals. A single intravenous injection of 20, 50 or 100 iu oxytocin given to three groups of three non-pregnant camels on day 10 after ovulation did not increase their basal serum PGFM concentrations. However, daily treatment of six non-pregnant camels between days 6 and 15 (n = 3) or 20 (n = 3) after ovulation with 1-2 g of the prostaglandin synthetase inhibitor, meclofenamic acid, inhibited PGF2 alpha release and thereby resulted in continued progesterone secretion throughout the period of meclofenamic acid administration. These results showed that, as in other large domestic animal species, release of PGF2 alpha from, presumably, the endometrium controls luteolysis in the dromedary camel. Furthermore, reduction in the amount of PGF2 alpha released is associated with luteal maintenance and the embryonic signal for maternal recognition of pregnancy must be transmitted before day 10 after ovulation if luteostasis is to be achieved. However, the results also indicate that, in contrast to ruminants, the release of endometrial PGF2 alpha in the non-pregnant camel may not be controlled by the release of oxytocin.     

Role of phospholipase C in mediating oxytocin-induced release of prostaglandin F2 alpha from ovine endometrial tissue

Two experiments were conducted to determine if the ability of oxytocin to stimulate release of prostaglandin (PG)F2 alpha from ovine uterine tissue involved activation of phospholipase C (PLC). In the first experiment, 9 ewes were injected with progesterone for 11 d (12 mg/d, im). On days 11 and 12, ewes received an injection of estradiol (100 micrograms, im). Caruncular endometrial tissue was collected on d 13 and incubated in the presence or absence of oxytocin (10(-6) M). Concentrations of PGF2 alpha and its metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), in culture media were determined by radioimmunoassay. PLC activity was determined by measuring the intracellular accumulation of 3H-inositol phosphates after preincubation with 3H-inositol. Concentrations of PGF2 alpha and total PGF (PGF2 alpha + PGFM) in culture media were greater for explants treated with oxytocin than for controls (p. less than .02, p less than .06, respectively). A similar effect of oxytocin on intracellular concentrations of 3H-inositol phosphates was observed (p less than .01). A second experiment was conducted to determine if agonists of second messengers, produced by activation of PLC, could stimulate release of PGF2 alpha from ovine endometrial tissue. Seven ewes were treated with progesterone and estradiol as in experiment 1. Explants of caruncular tissue from each ewe were incubated with 1) control medium, 2) A23187 (10(-5) M), 3) oxytocin (10(-6) M), 4) phorbol 12-myristate 13-acetate (PMA, 10(-7) M), 5) PMA + A23187 and 6) PMA + oxytocin. Significant stimulatory effects of oxytocin, PMA and A23187 on concentrations of PGF2 alpha and total PGF in culture media were observed (p. less than .05, p less than .1, p less than .1, respectively). In conclusion, oxytocin stimulated release of PGF2 alpha and activity of PLC in explants of ovine endometrial tissue in vitro. Second messengers associated with activation of PLC enhanced release of PGF2 alpha from ovine endometrial tissue.     

Ovine trophoblast protein-one inhibits development of endometrial responsiveness to oxytocin in ewes

In experiment (Exp) 1, 12 cyclic ewes had catheters placed into each uterine horn on Day 7 (estrus = Day 0). On Days 11-15, 6 ewes received twice-daily intrauterine infusions of 1.5 mg serum protein (SP) into each uterine horn and 6 ewes received infusions of 1.08 mg SP + 0.42 mg ovine conceptus secretory proteins (oCSP) containing 25 micrograms ovine trophoblast protein-one (oTP-1) as determined by radioimmunoassay (25-35% bioactive by antiviral assay). SP-infused and oCSP-infused ewes had similar plasma 13,14-dihydro-15-keto prostaglandin F2 alpha (PGF2 alpha) profiles in response to oxytocin on Day 11, but SP ewes became more responsive (p less than 0.01) to oxytocin on Days 13 and 15 than oCSP ewes. SP ewes also had greater incorporation of [3H]inositol into inositol trisphosphate (IP3) (+3449%, p less than 0.01) and total inositol phosphate (IP) (+760%, p less than 0.08), in response to oxytocin, than did oCSP ewes (+553 and +168% for IP3 and total IP, respectively) in endometrium collected at ovariectomy/hysterectomy on Day 16. Mean CL weights on Day 16 and mean concentrations of progesterone in plasma collected at 12-h intervals on Days 6-16 were not different for SP and oCSP ewes, but concentrations of progesterone were lower (p less than 0.05) in SP ewes on Days 15-16 than for oCSP ewes. These results indicate that oTP-1 may prevent luteolysis by inhibiting development of endometrial responsiveness to oxytocin and, therefore, reduce oxytocin-induced synthesis of IP3 and PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increases in the oxytocin-induced prostaglandin F2 alpha response and reduction in the concentrations of endometrial oxytocin receptors in ewes in response to progesterone.

Ovariectomized ewes were given progesterone and oestrogen priming as steroid pretreatment and subsequently treated with progesterone, prostaglandin F2 alpha (PGF2 alpha), or both. In Expt 1, plasma concentrations of the metabolite 13,14-dihydro-15-keto-PGF2 alpha (PGFM) were measured after an i.v. injection of oxytocin. There was little PGFM response in the untreated control ewes or in the pretreated ewes. Treatment with PGF2 alpha alone had no effect (P greater than 0.05), whereas treatment with progesterone either alone or with PGF2 alpha significantly (P less than 0.05) increased the uterine PGFM response to oxytocin. In Expt 2, chronically ovariectomized ewes had high concentrations of endometrial oxytocin receptors. Treatment with PGF2 alpha alone did not alter the concentrations of the receptors. Treatment with progesterone either alone or with PGF2 alpha significantly (P less than 0.05) reduced the concentrations of the receptors. It is concluded that progesterone promotes the PGFM response to oxytocin, but simultaneously suppresses the concentrations of endometrial oxytocin receptors.     

Fetal-maternal secretion of prostaglandins in the cow

A study was conducted to measure the blood plasma concentrations of prostaglandin F2 alpha (PGF2 alpha), 13,14-dihydro-15-keto-prostaglandin F (PGFM), 6-keto-prostaglandin F1 alpha (6-keto), prostaglandin uterine artery, uterine vein, umbilical artery and umbilical vein in 24 cows from days 80 to 260 of pregnancy. Blood was collected during surgery and all prostaglandins were measured using specific radioimmunoassay procedures. Results indicate that PGF2 alpha blood levels are higher in the umbilical vessels and uterine vein than in the ovarian vein and uterine artery. PGFM and PGE2 showed a trend towards higher values in the umbilical than in the maternal vessels, but the levels of 6-keto and TBX2 were not different among the vessels studied. No differences across time could be observed in any of the prostaglandins measured, partly due to the great variability in blood levels among animals during the same stage of pregnancy.     

Evidence that platelet-activating factor suppresses uterine oxytocin-induced 13,14-dihydro-15-keto-prostaglandin F2 alpha release and phosphatidylinositol hydrolysis in the ewe.

This study was conducted to determine whether platelet-activating factor (PAF) (1) attenuated oxytocin-induced secretion of the prostaglandin (PG) F2 alpha metabolite, PGFM, by the ovine uterus in situ and (2) inhibited the generation of the inositol phosphate secondary messengers by endometrial tissue in response to oxytocin challenge in vitro. Ovariectomized ewes received steroid replacement to mimic the luteal phase. Six ewes received intrauterine injections of 200 micrograms PAF/uterine horn/day on Days 11-15, and 6 ewes were treated with vehicle. All ewes received 1 microgram oxytocin i.v. on Days 13-16. Pretreatment of ewes with PAF significantly suppressed PGFM release in response to oxytocin on Days 14 and 15 (p less than 0.005) compared to vehicle-treated ewes. PAF was not administered on Day 16, and the PGFM response to oxytocin was not different between groups. In a second experiment, ewes were given intrauterine injections of 200 micrograms PAF/uterine horn/day (n = 8) or vehicle (n = 7) on Days 11-15, and all ewes received 1 microgram oxytocin i.v. on Days 13 and 14. On Day 15 the uterus was removed, and the incorporation of 3H-inositol into inositol phosphates was determined in caruncular endometrium. Treatment of ewes with PAF in vivo reduced inositol monophosphate (IP1) generated by oxytocin (10(-6) M) by 56.4%, compared to that in endometrium from vehicle-treated controls, and also inhibited the incorporation of 3H-inositol into glycerophosphoinositol (GPI). If PAF was added to the endometrium during the incubation in vitro, the attenuation of inositol phosphate generation did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Uterine blood supply as a main factor involved in the regulation of the estrous cycle--a new theory

The paper presents a new theory on the physiological mechanism of initiation of luteolysis, function of endometrial cells and protection of corpus luteum. This theory is based on previous studies published by the authors and their coworkers on the retrograde transfer of PGF2alpha in the uterine broad ligament vasculature during the estrous cycle, early pregnancy and pseudopregnancy. The studies were focused on cyclic changes in uterine blood supply and the apoptosis of endometrial cells. Moreover, the results of many other authors are cited. The statements of the theory are as follows: 1. The initiation of luteolysis is a consequence of regressive changes in the endometrium which are due to the reduction of the uterine blood supply below the level necessary to provide for the extended needs of active endometrium. 2. During the luteal phase, both a considerable increase in uterine weight and a decrease in blood flow through the uterine artery, resulting from increasing progesterone concentration, reduce the uterine blood supply. In comparison to the volume of blood flowing to the porcine uterus during the estrus period, only 30-40% of the blood volume is determined on day 12 of the estrous cycle. The uterine weight at that time is 40-60% larger than that in the early luteal phase. Thus, due to the considerable constriction of uterine blood vessels, there is a discrepancy between the requirement for oxygen and other factors transported by blood and the possibility of supplying the uterus with these substances. After reaching the threshold of uterine blood supply level, which in pigs takes place around day 12 of the estrous cycle, regressive changes and PGF2alpha release from endometrial cells occurs. 3. Estrogens and progesterone are the major factors affecting blood flow in vessels supplying the uterus. The factors that modulate, complement and support vasodilation and vasoconstriction are: PGE2, LH, oxytocin, cytokines, neurotransmitters and other local blood flow regulators. In some animal species these modulators, especially those of embryonic origin, may be crucial for the status of uterine vasculature. 4. During early pregnancy, the action of embryo signals (estrogens, cytokines), endometrial PGE2 as well as LH results in the relaxation of the uterine artery (pigs: day 12) and, consequently, in an increase in uterine blood supply. This reaction of the maternal recognition of pregnancy effectively prevents regressive changes in well developed endometrial cells to occur. 5. Local uptake and retrograde transfer of PGF2alpha into the uterine lumen during early pregnancy protects corpus luteum from PGF2alpha luteolytic action. 6. During the period of regressive changes resulting from the limited uterine blood supply, endometrial cells restrain PGF2alpha synthesis. They are, however, still capable of releasing prostaglandin when uterine blood supply is improved after the embryo appears in the uterus. This potential capability for PGF2alpha synthesis was demonstrated in in vitro studies when endometrial cells collected during its regressive phase were incubated in medium and stimulated by LH and oxytocin. 7. Prostaglandin F2alpha pulses in venous blood flowing from the uterus do not confirm pulsatile secretion of PGF2alpha. The pulses may result from the pulsatile excretion of PGF2alpha with venous blood according to the rhythmic uterine contractions associated with oxytocin secretion. 8. The results supporting this concept are presented and discussed in due course. The critique of Bazer and Thatcher's theory on exocrine versus endocrine secretion of prostaglandin F2alpha during the estrous cycle is also depicted.     

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.     

Luteinising hormone receptor kinetic and LH-induced prostaglandin production throughout the oestrous cycle in porcine endometrium

The present studies were performed to determine the LH/hCG receptor concentration and to evaluate the LH effect on prostaglandin production in porcine endometrium throughout the oestrous cycle. LH/hCG receptors in cell membrane preparations of the endometrium were found from days 12-14 and 15-16 of the oestrous cycle but not in preparations from days 6-7 and 18-20 using the ligand radioreceptor assay. Western blot analysis revealed, however, that the endometrium from all stages of the oestrous cycle contains a 75-kDa immunoreactive LH receptor protein similar to corpora lutea. The incubation of endometrial explants with LH (0, 1, 10 and 100 ng x mL(-1)) resulted in an increase of 13,14-dihydro-15-keto-PGF2alpha accumulation in a dose-dependent manner on days 5, 10, 14 and 16 of the oestrous cycle. The most effective dose was 10 ng LH x mL(-1) on days 5-16, but the strongest effect was found on days 14 and 16 (7.40 +/- 0.14 versus 12.75 +/- 1.40 and 5.67 +/- 0.35 versus 9.4 +/- 1.25 ng x 100 mg(-1) tissue/6 h, respectively; P < 0.01). It was also observed that 10 and 100 ng x mL(-1) of LH significantly increased cyclo-oxygenase expression to 135.2 and 123.5% respectively, above the control value (P < 0.01) on day 16 of the oestrous cycle. Our data suggest that LH receptors are of physiological significance in the porcine endometrium, since LH induces cyclooxygenase synthesis and increases prostaglandin production.     

Oxytocin-induced release of prostaglandin F2 alpha in postpartum beef cows: comparison of short versus normal luteal phases

The first postpartum ovulation after early weaning of calves (30 35 days of age) from cows is normally followed by a short luteal phase (6 10 days) unless the animals are pretreated with a progestogen (e.g. norgestomet). Reduced luteal lifespan in cattle is reportedly due to the premature release of a luteolysin (presumably prostaglandin F2 alpha [PGF2 alpha]). Therefore, the objective was to determine if oxytocin-induced release of PGF2 alpha (measured by the stable PGF2 alpha metabolite, 15-keto-13,14-dihydro PGF2 alpha [PGFM]) was greater for cows having a short compared to a normal luteal phase on Day 5 following the first postpartum estrus (Day 0). Thirty postpartum beef cows were randomly assigned into three groups (n = 10 per group) expected to have short (Short d 5) or normal (Norgestomet d 5 and Norgestomet d 16) luteal phases. Cows in Norgestomet d 5 and d 16 groups received Norgestomet (progestogen) implants for 9 days beginning 21 23 days postpartum. On Day 5 (Short d 5 and Norgestomet d 5) or Day 16 (Norgestomet d 16) following first postpartum estrus, each animal was injected (i.v.) with 100 IU oxytocin. In addition, cows in the Short d 5 group were subdivided into two groups following second estrus (normal luteal phase, n = 5 per group) to receive 100 IU oxytocin on Day 5 (Normal d 5) or 16 (Normal d 16), respectively. Estrous cycle length (means +/- SE) for cows in the Short d 5 group (8.7 +/- 0.4 days) was shorter (p less than 0.01) than for cows in all other groups (21.1 +/- 0.3 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in concentration of uterine oxytocin receptors and decrease in response to 13,14-dihydro-15-keto prostaglandin F2 alpha in ewes after withdrawal of exogenous progesterone.

Ovariectomized ewes were treated with progesterone and oestradiol to induce oestrus (day of expected oestrus = day 0) and with progesterone on days 1 to 12. The concentrations of endometrial oxytocin receptors and the 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) response induced by oxytocin were measured on days 12, 14, 16 and 18 after the cessation of progesterone treatment on day 12, by a receptor binding assay and direct radioimmunoassay, respectively. During the period of treatment, the concentrations of plasma progesterone were high and remained above 2 ng ml-1 until day 13 when they dropped rapidly to less than 0.5 ng ml-1 by day 14. The concentrations of oxytocin receptors in endometrium of control ewes were high (820.7 +/- 91.7 (SEM) fmol mg-1 protein). Treatment with progesterone significantly (P < 0.01) reduced the concentrations of the receptors on days 12 and 14 (144.1 +/- 65.0 and 200.4 +/- 45.4 fmol mg-1 protein, respectively). The receptor concentrations then increased to relatively high values on day 16 (1021.4 +/- 216.6 fmol mg-1 protein) and remained high until day 18 (677.7 +/- 103.4 fmol mg-1 protein).(ABSTRACT TRUNCATED AT 250 WORDS)