The effect of interleukin-1β, interleukin-6, and tumor necrosis factor-α on estradiol-17β release in the myometrium: The in vitro study on the pig model

Estradiol-17β (E₂) is a potent regulator of early pregnancy and the estrous cycle in pigs. Production of E₂ occurs in the porcine myometrium, but the factors involved in its regulation are unknown. In this in vitro study, it was investigated whether interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α affect the release of E₂ from the porcine myometrium on Days 10 to 11, 12 to 13, and 15 to 16 of pregnancy and the estrous cycle. The expression of the cytochrome P450 family 19 (CYP19) gene and the presence of the aromatase cytochrome P450 protein in the myometrium confirmed the ability of the tissue to produce E₂. In gravid pigs, the expression of IL1RI mRNA and IL6R mRNA was markedly increased on Days 15 to 16 of gestation, whereas TNFRI mRNA was increased on Days 10 to 11 of gestation. In cyclic pigs, the expression of myometrial IL1RI mRNA did not differ among the studied days, although the expression of IL6R and TNFRI mRNAs was increased on Days 15 to 16. In gravid pigs, IL-1β, IL-6, and TNF-α increased myometrial E₂ secretion on Days 15 to 16 but did not affect E₂ release on Days 10 to 11 and 12 to 13 of pregnancy. In cyclic pigs, IL-1β, IL-6, and TNF-α did not increase myometrial E₂ release. In conclusion, IL-1β, IL-6, and TNF-α affected myometrial E₂ release in a manner that is dependent on the physiologic status of the female. The porcine myometrium expresses IL1RI, IL6R, and TNFRI genes and is the target tissue for IL-1β, IL-6, and TNF-α. In gravid pigs, IL-1β, IL-6, and TNF-α may increase myometrial release of E₂in vitro specifically on Days 15 to 16 of pregnancy. These findings may be of interest to researchers using pigs as an animal model for fetal programming.     

Denaturation and aggregation of lysozyme in water-ethanol solution

We have applied rheological methods for the analysis of ethanol-lysozyme interaction during the process of denaturation and aggregation of the protein. At low concentration of ethanol a destruction of the hydration shell of lysozyme is observed. With the increase in the ethanol concentration a structural transformation takes place. It leads to the formation of a protein aggregate with an elongated structure. The rheological characteristics of lysozyme-water-ethanol solution changes from Newtonian to pseudoplastic.     

The effect of oxytocin on cortisol and corticosterone secretion in cyclic gilts--in vivo and in vitro studies

Oxytocin (OT) may be implicated in the modulation of hypothalamo-pituitary-adrenal axis (HPA) at each level. In mature females the influence of OT on the HPA axis appeared to be dependent on ovarian steroid milieu and stress. In cyclic sows, the role of OT in the regulation of corticoid secretion is unknown. In the present study changes in plasma cortisol and corticosterone concentrations in response to exogenous OT (in vivo experiment) and a direct influence of OT on adrenocortical steroidogenesis (in vitro experiment) were determined in luteal- and follicular-phase gilts. In the luteal-phase gilts (n=5), OT injections increased both cortisol (p<0.01) and corticosterone (p<0.05) plasma concentrations, but in the follicular-phase gilts (n=5) only the concentration of cortisol (p<0.05) was elevated in response to the treatment. Areas under the cortisol and corticosterone curves calculated for 30 min period after the OT injection were statistically higher (p<0.05) during the luteal than the follicular phase. In the in vitro experiment, two doses of OT (10(-7) and 10(-6) M) increased (p<0.05) secretion of cortisol by porcine adrenocortical cells representing the luteal phase, but not the follicular phase. However, OT did not affect the release of corticosterone by the cells. Incubation of the cells with the OT-antagonist (10(-5) M) abolished the effects of OT on cortisol secretion. Thus, in the present study, stimulatory effects of OT on the hypothalamo-pituitary-adrenal axis were demonstrated in cyclic gilts. The changes in plasma corticoid concentrations in response to exogenous OT were more prominent during the luteal than the follicular phase of the estrous cycle. Moreover, the in vitro experiment revealed a possibility of direct action of OT on adrenocortical cells isolated from luteal phase gilts. These results suggest that OT may participate in the modulation of HPA axis activity in pigs.     

Oxytocin stimulates prostaglandin F2alpha secretion and prostaglandin F synthase protein expression in porcine myometrial tissue

The possibility of PGF(2)alpha production and presence of prostaglandin F synthase (PGFS; PGD(2) 11-ketoreductase) was studied in control and oxytocin (OT)-stimulated myometrial slices isolated from cyclic (Days 14-16) and early pregnant (Days 14-16) sows. Oxytocin (10(-7) M) stimulated (p<0.01) PGF(2)alpha production in both cycling and early pregnant myometrial slices. Prostaglandin F(2)alpha release was higher (p<0.01) in control as well as OT-treated myometrium of early pregnant sows in comparison to cycling myometrium. Prostaglandin F synthase expression at protein level was evident in myometrial slices of cyclic as well as early pregnant sows. The signals of PGFS was stronger (p<0.05) in cycling myometrium exposed to OT compared to that of control. There were no significant differences (p>0.05) in PGFS protein expression between control and OT-stimulated myometrial tissue of early-pregnant sows. The results of this study indicate the local PGF(2)alpha synthesis and the presence of PGFS in porcine cycling and early pregnant myometrial tissue. In addition, OT increased PGD(2) 11-ketoreductase protein expression in myometrium harvested during the porcine estrous cycle. However, the OT-stimulated PGF(2)alpha myometrial secretion was observed in both, cycling and pregnant gilts.     

In vitro effects of polychlorinated biphenyls (PCBs) on the contractility of bovine myometrium from the periovulatory stage of the estrous cycle

Strips of longitudinal myometrium from cows were obtained on days 19-21 and 1-5 of the estrous cycle and incubated (aerated atmosphere; 4 degrees C; 24, 48 or 72 h) with a mixture of PCBs Aroclor (Ar) 1248 or with one of three PCBs (77, 126 or 153), all at doses of 10 or 100 ng/ml. The force and frequency of spontaneous and oxytocin (OT; 10(-7)M)-stimulated contractions of each strip was registered by means of HSE Schuler Organbath. Contractions of myometrial strips in the presence and absence of PCBs were observed after 24, 48 and 72 h of incubation. All PCBs significantly affected myometrial contractions. A mixture of PCBs increased the spontaneous force of contractions after 24 h but decreased after 48 h. Individual congeners of PCB also amplified the force of contractions and in most cases this effect was dose-dependent. Response of myometrium to PCB-126 and PCB-153 or PCB-77 appeared after 24 h or 48 h of incubation. Incubation of myometrial strips with PCB congeners markedly amplified OT-stimulated contractions. This effect was less evident when tissue was pre-treated with a higher dose of PCBs. Pre-treatment with estrogen-like PCB-153 increased the spontaneous and OT-evoked frequency of myometrial contractions from days 19-21. The spontaneous force of myometrial strips' contractions as well as the effects evoked by PCBs and OT was higher before than after ovulation. In summary, PCBs affected both the force and frequency of uterine contractions. Thus, it can be concluded that PCBs may impair both ovum fertilization and blastocyst implantation in cows.     

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.     

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.     

The influence of estradiol and progesterone on the concentrations of uterine oxytocin receptors and plasma PGFM in response to oxytocin in ovariectomized gilts

Peripubertal gilts (n = 25) were treated with corn oil (CO) or ovarian steroids, one month following an ovariectomy. The first day of treatment was assigned as the first day of the experiment. The gilts received: Group (Gr) I (n = 4)--CO (2 mL x day(-1) from 1st to 12th day), Gr II (n = 4) and Gr III (n = 4)--progesterone (P4; 10 to 100 mg x day(-1) from 1st to 12th day), Gr IV (n = 5)--estradiol benzoate (EB; 400 microg x day(-1) from 1st to 3rd day), Gr V (n = 4) and Gr VI (n = 4)--EB + P4 (EB 400 microg x day(-1) from 1st to 3rd day, 20 microg x day(-1) at 6th and 9th day, 50 microg at 12th day plus P4 10 to 100 mg from 4th to 15th day). All gilts were injected with oxytocin (OT; 20 IU; i.v.) on the following days of the experiment: 13th (Gr I and Gr II), 15th (Gr III and Gr IV), 16th (Gr V) and 18th (Gr VI). Concentrations of the PGF2alpha metabolite--PGFM were determined in blood samples, collected from 30 min before to 120 min after OT injection. Baseline PGFM concentrations (30 min before OT) differed among treatment groups and were the highest in Gr V and Gr VI (P < 0.01 vs. other groups). The magnitude of the PGFM response to OT increased only in four of the five gilts of Gr IV and in three of the four gilts of Gr VI, and it was higher (P = 0.009) in Gr VI than in Gr IV. In the remaining groups, PGFM concentrations did not increase above the baseline in response to OT. The day after OT injection, oxytocin receptors (OTR) were found in the uterine tissues of all animals studied. The lowest OTR concentrations were in Gr I--75.5 +/- 11.2 fmol x mg protein(-1) and the highest in Gr IV--712.9 +/- 86.7 fmol x mg protein(-1); (P < 0.05 vs. other groups). The values of K of OTR differed among groups (P < 0.001) and ranged from 1.62 +/- 0.44 nM in Gr I to 12. 08 +/- 1.9 nM in Gr VI. A positive correlation (r = 0.54; P < 0.01) between plasma E2 and uterine OTR concentrations was observed. In conclusion, E2 and P4 are involved in both PGF2 synthesis/secretion and OTR formation, however, full PGF response to OT does not develop before puberty. Estrogens are evident stimulators of uterine OTR synthesis ingilts.     

Effect of continuous infusion of oxytocin on length of the oestrous cycle and luteolysis in cattle

In Exp. I oxytocin (60 micrograms/100 kg/day) was infused into the jugular vein of 3 heifers on Days 14-22, 15-18 and 16-19 of the oestrous cycle respectively. In Exp. II 5 heifers were infused with 12 micrograms oxytocin/100 kg/day from Day 15 of the oestrous cycle until clear signs of oestrus. Blood samples were taken from the contralateral jugular vein at 2-h intervals from the start of the infusion. The oestrous cycle before and after treatment served as the controls for each animal. Blood samples were taken less frequently during the control cycles. In Exp. III 3 heifers were infused with 12 micrograms oxytocin/100 kg/day for 50 h before expected oestrus and slaughtered 30-40 min after the end of infusion for determination of oxytocin receptor amounts in the endometrium. Three other heifers slaughtered at the same days of the cycle served as controls. Peripheral concentrations of oxytocin during infusion ranged between 155 and 641 pg/ml in Exp. I and 18 and 25 pg/ml in Exp. II. In 4 our of 8 heifers of Exps I and II, one high pulse of 15-keto-13,14-dihydro-prostaglandin F-2 alpha (PGFM) appeared soon after the start of oxytocin infusion followed by some irregular pulses. The first PGFM pulse was accompanied by a transient (10-14 h) decrease of blood progesterone concentration. High regular pulses of PGFM in all heifers examined were measured between Days 17 and 19 during spontaneous luteolysis. No change in length of the oestrous cycle or secretion patterns of progesterone, PGFM and LH was observed.(ABSTRACT TRUNCATED AT 250 WORDS)