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Infusion of oxytocin induces successful delivery in prostanoid FP-receptor-deficient mice
Molecular and Cellular Endocrinology 283 (2008) 32–37
Infusion of oxytocin induces successful delivery in prostanoid FP-receptor-deficient mice Masaki Kawamata a,1 , Masahide Yoshida a , Yukihiko Sugimoto b , Tadashi Kimura c , Yutaka Tonomura a , Yuki Takayanagi d , Teruyuki Yanagisawa e , Katsuhiko Nishimori a,∗ a Department of Molecular Biology, Graduate School of Agriculture, Tohoku University, Sendai, Miyagi 981-8555, Japan Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan c Division of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan d Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan e Department of Molecular Pharmacology, Graduate School of Medicine, COE “CRESCENDO”, Tohoku University, Sendai, Miyagi 980-8575, Japan b
Received 30 January 2007; received in revised form 30 October 2007; accepted 30 October 2007
Abstract The dramatic increase of oxytocin (OT) receptor (OTR) in the myometrium as well as circulating progesterone withdrawal has been thought to be the most important factor in the induction and accomplishment of parturition since delivery fails in prostaglandin F2␣ receptor (FP) knockout (FP KO) mice. The expression levels of OTR mRNA/protein were not dramatically increased in the near-term uteri of FP KO mice. However, OT-induced myometrial contractions and the concentration–response curves in FP KO in vitro were almost similar to those in wild-type (WT) mice. OT-infusion (0.3 U/day) enabled FP KO mice to experience successful delivery, and furthermore the duration until the onset was hastened by a higher dose of OT (3 U/day). The plasma progesterone levels of FP KO females were maintained at high levels, but decreased during labor by OT-infusion (3 U/day). These results suggest that OT has potentials to induce strong myometrial contractions in uterus with low expression levels of OTR and luteolysis in ovary, which enabled FP KO females to undergo successful delivery. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Oxytocin; Oxytocin receptor; FP; Parturition; Knockout mice; Uterus
1. Introduction Oxytocin (OT) is a well-known neurohypophysial hormone, which stimulates uterine contractions to facilitate parturition (Gimpl and Fahrenholz, 2001). At or near term, the sensitivity of human uterine response to OT is much greater than that between the twentieth and thirtieth week of gestation (Caldeyro-Barcia and Theobald, 1968). The near-term myometrium is extremely sensitive to OT and the increased responsiveness to OT occurs in parallel with an increase in the number of uterine OT binding sites in rats (Fuchs et al., 1983; Soloff et al., 1979), humans (Fuchs et al., 1984), rabbits (Maggi et al., 1988, 1991), and ∗
Corresponding author. Tel.: +81 22 717 8770; fax: +81 22 717 8883. E-mail addresses: [email protected] (M. Kawamata), [email protected] (K. Nishimori). 1 Present address: Section for Studies on Metastasis, National Cancer Center Research Institute 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Tel.: +81 3 3542 2511; fax: +81 3 3541 2685. 0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2007.10.012
cows (Fuchs et al., 1992). Corresponding increases in uterine OT receptor (OTR) mRNA in late pregnancy and at parturition has been reported in cows (Ivell et al., 1995), rats (Larcher et al., 1995; Clerget et al., 1997), humans (Kimura et al., 1996), and sheep (Wathes et al., 1996; Wu et al., 1996). Prostaglandin F2␣ receptor (FP) knockout mice (FP KO) fail to undergo parturition due to the absence of luteolysis and/or of OTR elevation in uterus. In such mice, ovariectomy at 19.0 days of gestation (19.0 G) restored the induction of the luteolysis in ovary followed by the OTR elevation in uterus, which enabled successful delivery (Sugimoto et al., 1997). On the other hand, OTR KO (Takayanagi et al., 2005) as well as OT KO (Nishimori et al., 1996; Young et al., 1996) mice revealed that OT/OTR signal was not essential for the onset of labor since they could undergo normal parturition at term. Moreover, we recently reported that myometrial contractility in vitro was mainly controlled by the contribution of L-type Ca2+ channels, store-operated Ca2+ channels (SOCs), and Rho signal pathway rather than simply by OTR quantity (Kawamata et al., 2007). These reports sug-
M. Kawamata et al. / Molecular and Cellular Endocrinology 283 (2008) 32–37
gest that up-regulation of OTR is not essential for myometrial contractions to induce successful delivery, and the failure of parturition in FP KO mice may be due to the inhibitory mechanisms supported by high levels of progesterone. On the other hand, previous studies contradictorily reported that OT had a luteolytic (Shirasuna et al., 2007) or a luteotrophic (Gross et al., 1998; Imamura et al., 2000) effect on ovary. Therefore, the role of OT/OTR at term is not well established. In this study, we examined the effects of exogenous OT on the FP KO pregnant mice to clarify the potentials for contractile activity of uterus with low expression levels of OTR, and the regulation of the circulating progesterone levels. 2. Materials and methods 2.1. Tissues and preparations C57BL/6, FP KO (Sugimoto et al., 1997), and OTR KO (Takayanagi et al., 2005) mice (8–13 weeks) were used. Mice were housed on a 12-light:12dark cycle. All experiments complied with the Guidelines for the Care and Use of Laboratory Animals in Tohoku University. To obtain pregnant mouse uteri, a female mouse was mated with a male overnight. The next morning, if a copulation plug was detected, was designated as 0.5 days of gestation (0.5 G). The uteri of pregnant mice were obtained at 19.0 G. The animals were euthanized by cervical dislocation and the uteri were isolated. Plasma progesterone levels were measured by radioimmunoassay (Diagnostic Products, Los Angeles, CA).
2.2. Binding assay The proteins of the membrane fraction from mouse whole uteri were prepared by homogenization and sonication in a 20-fold volume of buffer 1 (10 mM Tris–HCl, pH 7.4, containing 1.5 mM EDTA, 0.5 mM dithiothreitol, 1.0 g/ml antipain, 1.0 g/ml leupeptin, 1.0 g/ml pepstain A, 1.0 g/ml chymostain, 1.0 g/ml aprotinin, 0.4 M phenylmethylanesulfonyl fluoride). The supernatant, after centrifugation at 1000 × g for 10 min at 4 ◦ C, was subjected to centrifugation at 160,000 × g for 30 min at 4 ◦ C. The subsequent pellet containing membrane proteins was resuspended in 5 ml of buffer 2 (50 mM Tris–maleate, pH 7.6, containing 10 mM MgSO4 , 1.0 g/ml antipain, 1.0 g/ml leupeptin, 1.0 g/ml pepstain A, 1.0 g/ml chymostain, 1.0 g/ml aprotinin, 0.4 M phenylmethylanesulfonyl fluoride). The resulting suspension was subjected to a second centrifugation at 160,000 × g for 30 min at 4 ◦ C. The final pellet was dispersed in buffer 2 to obtain a protein concentration of 4.5–19.0 mg/ml. The membrane preparations were divided into aliquots that contained 75 ng protein (final concentration, 0.75 mg/ml) and then incubated with 0.6 nM [3 H] OT in a final volume of 100 l for 1 h at 22 ◦ C. The incubation was terminated by filtering the suspension, rinsing with 50 mM Tris–HCl (pH 7.4) and drying with 70% ethanol. Scatchard analyses were performed with increasing concentrations of nonradioactive OT. Nonspecific binding was measured by the addition of 0.2 M nonradioactive OT.
2.3. Northern blot analysis We analyzed the expression levels of otr mRNA in longitudinal smooth muscle of uteri at 19.0 G. After uteri were fixed by using insect pins in silicon-dish filled with PBS, longitudinal smooth muscle was carefully separated from circular muscle under a dissecting microscope. Total RNA was extracted from longitudinal smooth muscle of uteri according to Chomczynski and Sacchi (1987), and 5 g of total RNAs were loaded on formaldehydeagarose gels. These RNA samples were subjected to electrophoresis and transferred onto Byodyne B nylon membrane (Pall BioSupport, USA), and blocked before hybridization. The probes were labeled with Mega-prime DNA labeling system (Amersham Biosciences, Japan) with [32 P]-dCTP. After the detection of otr expression, the membrane was stripped and reprobed
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for glyceraldehydes-3-phosphate dehydrogenase (gapd) to ensure equal loading.
2.4. Measurements of tension in vitro The myometrium was prepared by cutting in the longitudinal direction without including the endometrium under a dissecting microscope. Contractions were recorded isometrically using a micro-easy-magnus system (UC-5A, UFER, Kyoto, Japan) as described in our previous studies (Kawamata et al., 2003, 2004). In brief, the myometrial strip was equilibrated under a resting tension of 2 mN in normal physiological salt solution containing 140.0 mM NaCl, 5.0 mM KCl, 1.2 mM MgCl2 , 1.8 mM CaCl2 , 23.8 mM NaHCO3 , and 11.1 mM glucose. The 35 mM KCl solution was made by substituting NaCl with equimolar KCl. These solutions were saturated with a 95% O2 and 5% CO2 mixture at 37 ◦ C and pH 7.4. Each strip was repeatedly exposed to 35 mM KCl solution for 15 min until the contractile responses became stable. OT was cumulatively added to the strips every 5 min under normal physiological salt solution. All results are expressed as percentage of the maximum response to the 35 mM KCl solution. This concentration of KCl was sufficient to evoke the maximum contraction in mice (Osa, 1973). The pD2 values, maximum responses and slope values of those concentration–response curves for OT (Sigma–Aldrich, St. Louis, MO) were computer-fitted to the equation as follows (Yanagisawa et al., 1989): [OT]n E = ; Emax ([OT]n + ECn50 )
pD2 = − log[EC50 ]
2.5. Continuous infusion of OT On day 19.0 or 17.0 of gestation, each gravid female mouse underwent the subcutaneous implantation of a micro-osmotic pump (alzet, Cupertino, CA, USA) under diethylether anesthesia. Pumps were loaded with OT (Sigma–Aldrich, St. Louis, MO, USA) dissolved in distilled and filtrated water at the concentrations necessary to provide 0, 0.3, or 3 U/day. 1 U is equal to 61.0 g or 60.6 nmol. The time until the onset of labor was defined as delivery of the first pup by observation of gravid females every 6 h following pump implantation.
2.6. Statistics The results of the experiments are expressed as the mean ± S.E.M. ANOVA and student’s t-test were used for statistical analysis of the results and a P value less than 0.05 was accepted as being significantly different.
3. Results 3.1. Expression levels of OTR The ligand binding assay showed that OT binding sites in 19.0 G of whole uteri were markedly lower in FP KO (5.60 ± 0.83 fmol/g protein, n = 3, P < 0.01) than WT mice (14.2 ± 1.3 fmol/g protein, n = 4) (Fig. 1A). The affinity of OTR for OT (KD value) was equivalent in each sample (WT, 49.5 ± 15.7 nM; FP KO, 38.5 ± 3.7 nM). We analyzed the expression levels of otr mRNA at 19.0 G in longitudinal smooth muscles since the contractile responses to OT were measured in the longitudinal direction. As a small amount of the longitudinal smooth muscle layers could be obtained from mouse uteri, it was very difficult to perform the ligand binding assay. Thus, we performed Northern blot analysis by using only 5 g of total RNA samples. The expression levels of otr at 19.0 G in longitudinal smooth muscles of FP KO mice were lower than those of WT mice
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Fig. 1. Expression levels of OTR. (A) The binding capacity of OT was analyzed by a ligand binding assay in samples from the whole uteri of 19.0 G WT (n = 4, open column) and 19.0 G FP KO mice (n = 3, closed column). Each column represents the mean ± S.E.M. and significant differences were examined by ANOVA and t-test (** P < 0.01). (B) Northern blot analysis for otr mRNA was performed in samples from longitudinal smooth muscles of 19.0 G WT and 19.0 G FP KO mice. Five micrograms of total RNA were loaded. gapd is shown as a control. The blot was sequentially hybridized with otr and gapd cDNA probes.
(Fig. 1B), which corresponded to the result of the ligand binding assay. 3.2. Contractile responses to OT We examined the contractile responses to OT in myometrium of FP KO (n = 7) and WT (n = 7) mice at 19.0 G in vitro. Representative recordings were shown in Fig. 2A and B. OT was cumulatively added to the strips in concentrations from 0.01 to 100 nM every 5 min. The amplitude in each concentration in FP KO was similar to that in WT (Fig. 2C). Although the concentration–response curve in FP KO tended to shift to the right, the contractile features derived from the analysis of the curves (Fig. 2C) were also similar between WT (n = 7) and FP KO (n = 7) in regard to the maximum responses (232.0 ± 13.6%; 207.9 ± 7.37%), pD2 (9.06 ± 0.16; 8.73 ± 0.06), slope (3.60 ± 0.61; 1.79 ± 0.28) or the lowest concentration of OT to initiate contraction (134.8 ± 1.38; 59.7 ± 1.28 pM), respectively. The summarized data were shown in Table 1. 3.3. Effect of OT-infusion in FP KO mice We verified whether such contractile responses also occur and influence the gestation in FP KO mice in vivo. FP KO Table 1 Characterization of OT-induced contractions in the pregnant mouse myometrium Max (%) WT (n = 7) FP KO (n = 7)
pD2
232.0 ± 13.6 9.06 ± 0.16 207.9 ± 7.37 8.73 ± 0.06
Slope (nH )
Initiation (pM)
3.60 ± 0.61 1.79 ± 0.28
134.8 ± 1.38 59.7 ± 1.28
The data are presented as means ± S.E.M. The initiation values (the lowest concentration of OT to initiate contraction) were calculated on the basis of the logarithm. ANOVA was used for statistical analysis and there was no significant difference.
females could not commence parturition by the implantation of the vehicle pumps (0 U/day), and the pups died in uteri and were delivered 82 h after the implantation at 19.0 G (n = 3). On the other hand, the OT-pump (0.3 U/day) actively induced labor 16.5 h after implantation and the pups were born healthily (n = 4). Moreover, the higher rate (3 U/day) hastened the timing of labor, 6 h after implantation, compared to the infusion of 0.3 U/day (Fig. 3, n = 4, P < 0.01). 3.4. Effect of OT-infusion in OTR KO mice We examined the effect of OT-infusion in OTR KO mice to remove a possibility for a cross reactivity of OT to other receptors which induced successful delivery by OT-infusion in FP KO mice. Takayanagi et al. (2005) reported that OTR KO mice could undergo normal parturition in spite of no contractile response to OT in myometrium of OTR KO mice at 19.0 G in vitro. The OT-infusion rate of 3 U/day was chosen to induce imminent preterm labor since that of 2.5 U/day delivered pups about 24 h after the implantation of OT-pumps in gravid WT mice at 15.5 G (Imamura et al., 2000). In the present study, the OTpumps (3 U/day) were implanted into WT or OTR KO females at 17.0 G. The OT-pump (3 U/day) induced imminent preterm labor 24 ± 3.5 h after the implantation in WT mice (n = 3), and the time until labor was significantly shorter than the case of the vehicle pump in WT mice (54 ± 3.5 h, n = 3, P < 0.01; Fig. 4). OTR KO females delivered pups 53 ± 2.9 h after the implantation (n = 4), which was similar to the cases of the vehicle pump in WT mice and the previous results of 19.3 ± 0.1 G in OTR KO mice (n = 13; Takayanagi et al., 2005). 3.5. Measurement of plasma progesterone levels We examined plasma progesterone levels in FP KO mice 6 h after implantation of OT (3 U/day) at 19.0 G.
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Fig. 3. Effects of continuous OT-infusion on the time until the onset of labor. Micro-osmotic pumps were implanted into FP KO females at 19.0 G, and the duration until the onset of labor was determined. Labor was actively induced by the OT-infusion (0.3 U/day, n = 4, gray column or 3 U/day, n = 4, closed column) but not by the vehicle administration (0 U/day, n = 3, open column). Time until the onset of labor was defined as delivery of the first pup by observation of gravid females every 6 h following pump implantation. The data show the mean ± S.E.M. and significant differences were examined by ANOVA and t-test. * P < 0.05 vs. 0 U/day; ## P < 0.01 vs. 0.3 U/day; a The pups died in uteri and were delivered 82 h after the implantation at 19.0 G.
Fig. 2. Contractile responses to OT in pregnant mouse myometrium. (A and B) The typical trace of 19.0 G WT (A) or FP KO (B) was shown. (C) The concentration–response curves for OT-induced contractions in WT (n = 7, open circle) and FP KO (n = 7, closed circle) are shown. Contractions were induced in all tissues by 35 mM KCl solution, and the maximum responses were 2.33 ± 0.49 or 1.78 ± 0.21 mN in WT or FP KO, respectively. OT was cumulatively added to the strips every 5 min under normal physiological salt solution. One hundred percent represents the maximum response of the contractions induced by 35 mM KCl. The data show the mean ± S.E.M. and significant differences were examined by ANOVA.
The plasma progesterone levels of FP KO mice with OTinfusion (9.04 ± 2.45 ng/ml, n = 4) were 5-fold lower that those of FP KO mice with vehicle-infusion (46.8 ± 3.9 ng/ml, n = 5, P < 0.001) and 8-fold higher than those of WT mice without infusion (1.16 ± 0.17 ng/ml, n = 5, P < 0.05; Fig. 5).
Fig. 4. Effects of continuous OT-infusion on the time until the onset of labor. Micro-osmotic pumps (0 U/day, n = 3; 3 U/day, n = 3) were implanted into WT (open column) or OTR KO (3 U/day, n = 4, closed column) females at 17.0 G. The duration until the onset of labor was determined. Time until the onset of labor was defined as delivery of the first pup by observation of gravid females every 6 h following pump implantation. The data show the mean ± S.E.M. and significant differences were examined by ANOVA and t-test. ** P < 0.01 vs. WT (0 U/day); ## P < 0.01 vs. OTR KO (3 U/day).
4. Discussion In the present study, we found that FP KO females could undergo successful delivery by infusion of OT (0.3 or 3 U/day, Fig. 3) at 19.0 G although the failure of parturition in FP KO females has been thought to be due to weak contractile responses to OT because of the low expression levels of OTR in myometrium. There are two possible mechanisms by which OT-infusion could induce delivery in FP KO females. One is an induction of strong uterine contractions, and the other is plasma progesterone withdrawal. The in vitro study clearly showed that FP KO myometrium with the low expression levels of
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OTR had a potential to induce almost the same contractile responses to OT as the WT myometrium at 19.0 G (Figs. 1 and 2 and Table 1). The results imply that such uterine contractions would also occur in FP KO females by the OT-infusion in vivo. Sugimoto et al. (1997) reported that the uterus of FP KO mice had little contractile responses to OT compared to that of WT mice in vivo. The discrepancy may be resulted from the differences in the methods of contractile measurement, the doses and roots of administration of OT, and the period of pregnancy, term or preterm. In the previous study (Sugimoto et al., 1997), uterine contractions at term were measured as an increase in intrauterine pressure in vivo by use of a small balloon catheter inserted into the uterine horn, and the pressure curve of OT was represented based on spontaneous contractions and the transient increases in pressure were measured after bolus intravenous administration of OT. In contrast, in the present study, preterm myometrial contractions were measured in vitro and the cumulative concentration–response curve of OT was represented based on the contractions induced by 35 mM KCl, which shows the intense and tonic contractions induced by pharmacological high concentrations of OT (Fig. 2). The whole uteri are exposed to many contractile regulatory factors especially in progesterone, resulting in suppression of uterine contractions in FP KO mice in vivo differently from in vitro experiment using a small stripe of myometrium. We could consider that the concentration–response curves of our result (Fig. 2C) are not much different from those of the previous study (Sugimoto et al., 1997) since the curve of FP KO mice of the present study tended to shift to the right (Fig. 2C). As reported in our previous study (Kawamata et al., 2007), the slight rightward shift of the curve was due to the sparseness of phasic contractions at low concentration of OT in FP KO mice compared to those in WT mice (Fig. 2A and B). FP KO uterus might not induce enough contractions to accomplish delivery like WT uterus when the physiological concentration of OT was released from the posterior lobe of hypophysis in a pulsatile fashion in vivo. In the present study, however, we used higher doses of OT with the continuous infusion compared to the previous study (Sugimoto et al., 1997), which resulted in the delivery of pups even in FP KO females. Although OTR is well known to be desensitized by OT stimulation in human myometrium (Plested and Bernal, 2001), the smaller amount of OTR in response to the continuous infusion of OT in the pregnant myometrium of FP KO mice in vivo seemed to induce delivery with myometrial contractions which were shown in vitro study almost the same as those from wild mice. As OTR is expressed in corpus luteum of pregnant mouse ovary (Imamura et al., 2000), OT might progress luteolysis. It is reported that intraluteal OT locally modulate secretion of endothelin 1 as well as PGF2␣ within the corpus luteum, which induce luteolysis (Shirasuna et al., 2007). In contrast, the previous works using the OT-pump, which were similar to our study, showed a luteotrophic effect of OT (Imamura et al., 2000). The report showed a significant delay in the onset of labor (20.1 G) in WT mice by the implantation of OT infused at 1 U/day at 15.5 G, and the serum progesterone levels at 19.0 G were higher than the case of vehicle implantation. However,
preterm labor was observed one day after the implantation of OT infused at 2.5 U/day (Imamura et al., 2000). Although it is unknown whether the uterine contractions or the progesterone withdrawal induced the preterm labor, these results imply that both the doses of OT and the timing of application critically relate to the onset of labor. In the present study, since we implanted OT-pump in FP KO females at 19.0 G, the gestational age may cause the different results from the previous study. We also implanted OT-pump infused at 3 U/day in WT females at 17.0 G. In these females, it took longer time (16.5 h) for the onset of labor compared to the FP KO females (6 h) implanted at 19.0 G. This might be due to the difference of contractile activity in gestational ages rather than the velocity of progesterone withdrawal. In most smooth muscle organs, stretching leads to contraction (Renfree, 2000). Fetus growth induces myometrial stretching followed by contributions to parturition (Smith, 2007). The plasma progesterone levels of FP KO females during delivery by the OT-infusion (3 U/day) were 5-fold lower than those of FP KO females with the vehicle-infusion, but were 8-fold higher than those of WT females that underwent spontaneous delivery (Fig. 5). The results indicate that the decreased levels of plasma progesterone might be insufficient to compel FP KO females to accomplish delivery, and that the combination with the strong uterine contractions is necessary. We also demonstrated that both effects of OT on the uterine contractions and the progesterone withdrawal were initiated to mediate only via OTR because OTR KO females could not undergo preterm labor by the infusion of OT at 17.0 G (Fig. 4). Therefore, in this study, we have clarified two aspects of OT functions during parturition to induce strong uterine contraction even with low expression levels of OTR in myometrium and to produce progesterone withdrawal probably due to luteolysis, which enabled FP KO females to undergo successful delivery by OT-infusion.
Fig. 5. The plasma progesterone levels in mice. Plasma progesterone levels were measured in WT females without infusion (n = 5, open column) or FP KO females (closed column) 6 h after the implantation of OT-pump (0 U/day, n = 5; 3 U/day, n = 4) at 19.0 G. The time represents 19.25 G when FP KO females start delivery by OT-infusion (3 U/day). The data show the mean ± S.E.M. and significant differences were examined by ANOVA and t-test. * P < 0.05, *** P < 0.001 vs. WT; ### P < 0.001 vs. FP KO (0 U/day).
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Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research (No. 14360046) from the Ministry of Education, Science and Culture of Japan and by Sankyo Foundation of Life Science. References Caldeyro-Barcia, R., Theobald, G.W., 1968. Sensitivity of the pregnant human myometrium to oxytocin. Am. J. Obstet. Gynecol. 102, 1181. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Clerget, M.S., Elalouf, J.M., Germain, G., 1997. Quantitative reverse transcription and polymerase chain reaction analysis of oxytocin and vasopressin receptor mRNAs in the rat uterus near parturition. Mol. Cell. Endocrinol. 136, 79–89. Fuchs, A.R., Periyasamy, S., Alexandrroa, M., Soloff, M.S., 1983. Correlation between oxytocin receptor concentration and responsiveness to oxytocin in pregnant rat myometrium. Endocrinology 113, 742–749. Fuchs, A.R., Fuchs, F., Husslein, P., Soloff, M.S., 1984. Oxytocin receptors in the human uterus during pregnancy and parturition. Am. J. Obstet. Gynecol. 150, 734–741. Fuchs, A.R., Helmer, H., Behrens, O., Liu, H.C., Antonian, L., Chang, S.M., Fields, M.J., 1992. Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors precedes onset of labor. Biol. Reprod. 47, 937–944. Gimpl, G., Fahrenholz, F., 2001. The oxytocin receptor system: structure, function, and regulation. Physiol. Rev. 81, 629–683. Gross, G.A., Imamura, T., Luedke, C., Vogt, S.K., Olson, L.M., Nelson, D.M., Sadovsky, Y., Muglia, L.J., 1998. Opposing actions of prostaglandins and oxytocin determine the onset of murine labor. Proc. Natl. Acad. Sci. U.S.A. 95, 11875–11879. Imamura, T., Luedke, C.E., Vogt, S.K., Muglia, L.J., 2000. Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, R1061–R1067. Ivell, R., Rust, W., Einspanier, A., Hartung, S., Fields, M., Fuchs, A.R., 1995. Oxytocin and oxytocin receptor gene expression in the reproductive tract of the pregnant cow: rescue of luteal oxytocin production at term. Biol. Reprod. 53, 553–560. Kawamata, M., Mitsui-Saito, M., Kimura, T., Takayanagi, Y., Yanagisawa, T., Nishimori, K., 2003. Vasopressin-induced contraction of uterus is mediated solely by the oxytocin receptor in mice, but not in humans. Eur. J. Pharmacol. 472, 229–234. Kawamata, M., Tonomura, Y., Kimura, T., Yanagisawa, T., Nishimori, K., 2004. The differential coupling of oxytocin receptors to uterine contractions in murine estrous cycle. Biochem. Biophys. Res. Commun. 321, 695–699. Kawamata, M., Tonomura, Y., Kimura, T., Sugimoto, Y., Yanagisawa, T., Nishimori, K., 2007. Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor. Am. J. Physiol. Endocrinol. Metab. 292, E992–E999. Kimura, T., Takemura, M., Nomura, S., Nobunaga, T., Kubota, Y., Inoue, T., Hashimoto, K., Kumazawa, I., Ito, Y., Ohashi, K., Koyama, M., Azuma,
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C., Kitamura, Y., Saji, F., 1996. Expression of oxytocin receptor in human pregnant myometrium. Endocrinology 137, 780–785. Larcher, A., Neculcea, J., Breton, C., Arslan, A., Rozen, F., Russo, C., Zingg, H.H., 1995. Oxytocin receptor gene expression in the rat uterus during pregnancy and the estrous cycle and in response to gonadal steroid treatment. Endocrinology 136, 5350–5356. Maggi, M., Genazzani, A.D., Giannini, S., Torrisi, C., Baldi, E., di Tomaso, M., Munson, P.J., Rodbard, D., Serio, M., 1988. Vasopressin and oxytocin receptors in vagina, myometrium, and oviduct of rabbits. Endocrinology 122, 2970–2980. Maggi, M., Peri, A., Giannini, S., Fantoni, G., Guardabasso, V., Serio, M., 1991. Oxytocin and V1 vasopressin receptors in rabbit endometrium during pregnancy. J. Reprod. Fertil. 91, 575–581. Nishimori, K., Young, L.J., Guo, Q., Wang, Z., Insel, T.R., Matzuk, M.M., 1996. Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc. Natl. Acad. Sci. U.S.A. 93, 11699–11704. Osa, T., 1973. Some factors affecting the contractile responses of pregnant mouse myometrium. Jpn. J. Physiol. 23, 401–417. Plested, C.P., Bernal, A.L., 2001. Desensitisation of the oxytocin receptor and other G-protein coupled receptors in the human myometrium. Exp. Physiol. 86, 303–312. Renfree, M.B., 2000. Maternal recognition of pregnancy in marsupials. Rev. Reprod. 5, 6–11. Shirasuna, K., Shimizu, T., Hayashi, K.G., Nagai, K., Matsui, M., Miyamoto, A., 2007. Positive association, in local release, of luteal oxytocin with endothelin 1 and prostaglandin F2alpha during spontaneous luteolysis in the cow: a possible intermediatory role for luteolytic cascade within the corpus luteum. Biol. Reprod. 76, 965–970. Smith, R., 2007. Parturition. N. Engl. J. Med. 356, 271–283. Soloff, M.S., Alexandrova, M., Fernstrom, M.J., 1979. Oxytocin receptors: triggers for parturition and lactation? Science 204, 1313–1315. Sugimoto, Y., Yamasaki, A., Segi, E., Tsuboi, K., Aze, Y., Nishimura, T., Oida, H., Yoshida, N., Tanaka, T., Katsuyama, M., Hasumoto, K., Murata, T., Hirata, M., Ushikubi, F., Negishi, M., Ichikawa, A., Narumiya, S., 1997. Failure of parturition in mice lacking the prostaglandin F receptor. Science 277, 681–683. Takayanagi, Y., Yoshida, M., Bielsky, I.F., Ross, H.E., Kawamata, M., Onaka, T., Yanagisawa, T., Kimura, T., Matzuk, M.M., Young, L.J., Nishimori, K., 2005. Pervasive social deficits, but normal parturition, in oxytocin receptordeficient mice. Proc. Natl. Acad. Sci. U.S.A. 102, 16096–16101. Wathes, D.C., Smith, H.F., Leung, S.T., Stevenson, K.R., Meier, S., Jenkin, G., 1996. Oxytocin receptor development in ovine uterus and cervix throughout pregnancy and at parturition as determined by in situ hybridization analysis. J. Reprod. Fertil. 106, 23–31. Wu, W.X., Verbalis, J.G., Hoffman, G.E., Derks, J.B., Nathanielsz, P.W., 1996. Characterization of oxytocin receptor expression and distribution in the pregnant sheep uterus. Endocrinology 137, 722–728. Yanagisawa, T., Ishii, K., Hashimoto, H., Taira, N., 1989. Differential coupling to positive inotropic responses of cyclic AMP produced by stimulation of beta1- and beta2-adrenergic receptors. J. Cardiovasc. Pharmacol. 13, 64–75. Young 3rd, W.S., Shepard, E., Amico, J., Hennighausen, L., Wagner, K.U., LaMarca, M.E., McKinney, C., Ginns, E.I., 1996. Deficiency in mouse oxytocin prevents milk ejection, but not fertility or parturition. J. Neuroendocrinol. 8, 847–853.
Infusion of oxytocin induces successful delivery in prostanoid FP-receptor-deficient mice Masaki Kawamata a,1 , Masahide Yoshida a , Yukihiko Sugimoto b , Tadashi Kimura c , Yutaka Tonomura a , Yuki Takayanagi d , Teruyuki Yanagisawa e , Katsuhiko Nishimori a,∗ a Department of Molecular Biology, Graduate School of Agriculture, Tohoku University, Sendai, Miyagi 981-8555, Japan Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan c Division of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan d Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan e Department of Molecular Pharmacology, Graduate School of Medicine, COE “CRESCENDO”, Tohoku University, Sendai, Miyagi 980-8575, Japan b
Received 30 January 2007; received in revised form 30 October 2007; accepted 30 October 2007
Abstract The dramatic increase of oxytocin (OT) receptor (OTR) in the myometrium as well as circulating progesterone withdrawal has been thought to be the most important factor in the induction and accomplishment of parturition since delivery fails in prostaglandin F2␣ receptor (FP) knockout (FP KO) mice. The expression levels of OTR mRNA/protein were not dramatically increased in the near-term uteri of FP KO mice. However, OT-induced myometrial contractions and the concentration–response curves in FP KO in vitro were almost similar to those in wild-type (WT) mice. OT-infusion (0.3 U/day) enabled FP KO mice to experience successful delivery, and furthermore the duration until the onset was hastened by a higher dose of OT (3 U/day). The plasma progesterone levels of FP KO females were maintained at high levels, but decreased during labor by OT-infusion (3 U/day). These results suggest that OT has potentials to induce strong myometrial contractions in uterus with low expression levels of OTR and luteolysis in ovary, which enabled FP KO females to undergo successful delivery. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Oxytocin; Oxytocin receptor; FP; Parturition; Knockout mice; Uterus
1. Introduction Oxytocin (OT) is a well-known neurohypophysial hormone, which stimulates uterine contractions to facilitate parturition (Gimpl and Fahrenholz, 2001). At or near term, the sensitivity of human uterine response to OT is much greater than that between the twentieth and thirtieth week of gestation (Caldeyro-Barcia and Theobald, 1968). The near-term myometrium is extremely sensitive to OT and the increased responsiveness to OT occurs in parallel with an increase in the number of uterine OT binding sites in rats (Fuchs et al., 1983; Soloff et al., 1979), humans (Fuchs et al., 1984), rabbits (Maggi et al., 1988, 1991), and ∗
Corresponding author. Tel.: +81 22 717 8770; fax: +81 22 717 8883. E-mail addresses: [email protected] (M. Kawamata), [email protected] (K. Nishimori). 1 Present address: Section for Studies on Metastasis, National Cancer Center Research Institute 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Tel.: +81 3 3542 2511; fax: +81 3 3541 2685. 0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2007.10.012
cows (Fuchs et al., 1992). Corresponding increases in uterine OT receptor (OTR) mRNA in late pregnancy and at parturition has been reported in cows (Ivell et al., 1995), rats (Larcher et al., 1995; Clerget et al., 1997), humans (Kimura et al., 1996), and sheep (Wathes et al., 1996; Wu et al., 1996). Prostaglandin F2␣ receptor (FP) knockout mice (FP KO) fail to undergo parturition due to the absence of luteolysis and/or of OTR elevation in uterus. In such mice, ovariectomy at 19.0 days of gestation (19.0 G) restored the induction of the luteolysis in ovary followed by the OTR elevation in uterus, which enabled successful delivery (Sugimoto et al., 1997). On the other hand, OTR KO (Takayanagi et al., 2005) as well as OT KO (Nishimori et al., 1996; Young et al., 1996) mice revealed that OT/OTR signal was not essential for the onset of labor since they could undergo normal parturition at term. Moreover, we recently reported that myometrial contractility in vitro was mainly controlled by the contribution of L-type Ca2+ channels, store-operated Ca2+ channels (SOCs), and Rho signal pathway rather than simply by OTR quantity (Kawamata et al., 2007). These reports sug-
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gest that up-regulation of OTR is not essential for myometrial contractions to induce successful delivery, and the failure of parturition in FP KO mice may be due to the inhibitory mechanisms supported by high levels of progesterone. On the other hand, previous studies contradictorily reported that OT had a luteolytic (Shirasuna et al., 2007) or a luteotrophic (Gross et al., 1998; Imamura et al., 2000) effect on ovary. Therefore, the role of OT/OTR at term is not well established. In this study, we examined the effects of exogenous OT on the FP KO pregnant mice to clarify the potentials for contractile activity of uterus with low expression levels of OTR, and the regulation of the circulating progesterone levels. 2. Materials and methods 2.1. Tissues and preparations C57BL/6, FP KO (Sugimoto et al., 1997), and OTR KO (Takayanagi et al., 2005) mice (8–13 weeks) were used. Mice were housed on a 12-light:12dark cycle. All experiments complied with the Guidelines for the Care and Use of Laboratory Animals in Tohoku University. To obtain pregnant mouse uteri, a female mouse was mated with a male overnight. The next morning, if a copulation plug was detected, was designated as 0.5 days of gestation (0.5 G). The uteri of pregnant mice were obtained at 19.0 G. The animals were euthanized by cervical dislocation and the uteri were isolated. Plasma progesterone levels were measured by radioimmunoassay (Diagnostic Products, Los Angeles, CA).
2.2. Binding assay The proteins of the membrane fraction from mouse whole uteri were prepared by homogenization and sonication in a 20-fold volume of buffer 1 (10 mM Tris–HCl, pH 7.4, containing 1.5 mM EDTA, 0.5 mM dithiothreitol, 1.0 g/ml antipain, 1.0 g/ml leupeptin, 1.0 g/ml pepstain A, 1.0 g/ml chymostain, 1.0 g/ml aprotinin, 0.4 M phenylmethylanesulfonyl fluoride). The supernatant, after centrifugation at 1000 × g for 10 min at 4 ◦ C, was subjected to centrifugation at 160,000 × g for 30 min at 4 ◦ C. The subsequent pellet containing membrane proteins was resuspended in 5 ml of buffer 2 (50 mM Tris–maleate, pH 7.6, containing 10 mM MgSO4 , 1.0 g/ml antipain, 1.0 g/ml leupeptin, 1.0 g/ml pepstain A, 1.0 g/ml chymostain, 1.0 g/ml aprotinin, 0.4 M phenylmethylanesulfonyl fluoride). The resulting suspension was subjected to a second centrifugation at 160,000 × g for 30 min at 4 ◦ C. The final pellet was dispersed in buffer 2 to obtain a protein concentration of 4.5–19.0 mg/ml. The membrane preparations were divided into aliquots that contained 75 ng protein (final concentration, 0.75 mg/ml) and then incubated with 0.6 nM [3 H] OT in a final volume of 100 l for 1 h at 22 ◦ C. The incubation was terminated by filtering the suspension, rinsing with 50 mM Tris–HCl (pH 7.4) and drying with 70% ethanol. Scatchard analyses were performed with increasing concentrations of nonradioactive OT. Nonspecific binding was measured by the addition of 0.2 M nonradioactive OT.
2.3. Northern blot analysis We analyzed the expression levels of otr mRNA in longitudinal smooth muscle of uteri at 19.0 G. After uteri were fixed by using insect pins in silicon-dish filled with PBS, longitudinal smooth muscle was carefully separated from circular muscle under a dissecting microscope. Total RNA was extracted from longitudinal smooth muscle of uteri according to Chomczynski and Sacchi (1987), and 5 g of total RNAs were loaded on formaldehydeagarose gels. These RNA samples were subjected to electrophoresis and transferred onto Byodyne B nylon membrane (Pall BioSupport, USA), and blocked before hybridization. The probes were labeled with Mega-prime DNA labeling system (Amersham Biosciences, Japan) with [32 P]-dCTP. After the detection of otr expression, the membrane was stripped and reprobed
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for glyceraldehydes-3-phosphate dehydrogenase (gapd) to ensure equal loading.
2.4. Measurements of tension in vitro The myometrium was prepared by cutting in the longitudinal direction without including the endometrium under a dissecting microscope. Contractions were recorded isometrically using a micro-easy-magnus system (UC-5A, UFER, Kyoto, Japan) as described in our previous studies (Kawamata et al., 2003, 2004). In brief, the myometrial strip was equilibrated under a resting tension of 2 mN in normal physiological salt solution containing 140.0 mM NaCl, 5.0 mM KCl, 1.2 mM MgCl2 , 1.8 mM CaCl2 , 23.8 mM NaHCO3 , and 11.1 mM glucose. The 35 mM KCl solution was made by substituting NaCl with equimolar KCl. These solutions were saturated with a 95% O2 and 5% CO2 mixture at 37 ◦ C and pH 7.4. Each strip was repeatedly exposed to 35 mM KCl solution for 15 min until the contractile responses became stable. OT was cumulatively added to the strips every 5 min under normal physiological salt solution. All results are expressed as percentage of the maximum response to the 35 mM KCl solution. This concentration of KCl was sufficient to evoke the maximum contraction in mice (Osa, 1973). The pD2 values, maximum responses and slope values of those concentration–response curves for OT (Sigma–Aldrich, St. Louis, MO) were computer-fitted to the equation as follows (Yanagisawa et al., 1989): [OT]n E = ; Emax ([OT]n + ECn50 )
pD2 = − log[EC50 ]
2.5. Continuous infusion of OT On day 19.0 or 17.0 of gestation, each gravid female mouse underwent the subcutaneous implantation of a micro-osmotic pump (alzet, Cupertino, CA, USA) under diethylether anesthesia. Pumps were loaded with OT (Sigma–Aldrich, St. Louis, MO, USA) dissolved in distilled and filtrated water at the concentrations necessary to provide 0, 0.3, or 3 U/day. 1 U is equal to 61.0 g or 60.6 nmol. The time until the onset of labor was defined as delivery of the first pup by observation of gravid females every 6 h following pump implantation.
2.6. Statistics The results of the experiments are expressed as the mean ± S.E.M. ANOVA and student’s t-test were used for statistical analysis of the results and a P value less than 0.05 was accepted as being significantly different.
3. Results 3.1. Expression levels of OTR The ligand binding assay showed that OT binding sites in 19.0 G of whole uteri were markedly lower in FP KO (5.60 ± 0.83 fmol/g protein, n = 3, P < 0.01) than WT mice (14.2 ± 1.3 fmol/g protein, n = 4) (Fig. 1A). The affinity of OTR for OT (KD value) was equivalent in each sample (WT, 49.5 ± 15.7 nM; FP KO, 38.5 ± 3.7 nM). We analyzed the expression levels of otr mRNA at 19.0 G in longitudinal smooth muscles since the contractile responses to OT were measured in the longitudinal direction. As a small amount of the longitudinal smooth muscle layers could be obtained from mouse uteri, it was very difficult to perform the ligand binding assay. Thus, we performed Northern blot analysis by using only 5 g of total RNA samples. The expression levels of otr at 19.0 G in longitudinal smooth muscles of FP KO mice were lower than those of WT mice
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Fig. 1. Expression levels of OTR. (A) The binding capacity of OT was analyzed by a ligand binding assay in samples from the whole uteri of 19.0 G WT (n = 4, open column) and 19.0 G FP KO mice (n = 3, closed column). Each column represents the mean ± S.E.M. and significant differences were examined by ANOVA and t-test (** P < 0.01). (B) Northern blot analysis for otr mRNA was performed in samples from longitudinal smooth muscles of 19.0 G WT and 19.0 G FP KO mice. Five micrograms of total RNA were loaded. gapd is shown as a control. The blot was sequentially hybridized with otr and gapd cDNA probes.
(Fig. 1B), which corresponded to the result of the ligand binding assay. 3.2. Contractile responses to OT We examined the contractile responses to OT in myometrium of FP KO (n = 7) and WT (n = 7) mice at 19.0 G in vitro. Representative recordings were shown in Fig. 2A and B. OT was cumulatively added to the strips in concentrations from 0.01 to 100 nM every 5 min. The amplitude in each concentration in FP KO was similar to that in WT (Fig. 2C). Although the concentration–response curve in FP KO tended to shift to the right, the contractile features derived from the analysis of the curves (Fig. 2C) were also similar between WT (n = 7) and FP KO (n = 7) in regard to the maximum responses (232.0 ± 13.6%; 207.9 ± 7.37%), pD2 (9.06 ± 0.16; 8.73 ± 0.06), slope (3.60 ± 0.61; 1.79 ± 0.28) or the lowest concentration of OT to initiate contraction (134.8 ± 1.38; 59.7 ± 1.28 pM), respectively. The summarized data were shown in Table 1. 3.3. Effect of OT-infusion in FP KO mice We verified whether such contractile responses also occur and influence the gestation in FP KO mice in vivo. FP KO Table 1 Characterization of OT-induced contractions in the pregnant mouse myometrium Max (%) WT (n = 7) FP KO (n = 7)
pD2
232.0 ± 13.6 9.06 ± 0.16 207.9 ± 7.37 8.73 ± 0.06
Slope (nH )
Initiation (pM)
3.60 ± 0.61 1.79 ± 0.28
134.8 ± 1.38 59.7 ± 1.28
The data are presented as means ± S.E.M. The initiation values (the lowest concentration of OT to initiate contraction) were calculated on the basis of the logarithm. ANOVA was used for statistical analysis and there was no significant difference.
females could not commence parturition by the implantation of the vehicle pumps (0 U/day), and the pups died in uteri and were delivered 82 h after the implantation at 19.0 G (n = 3). On the other hand, the OT-pump (0.3 U/day) actively induced labor 16.5 h after implantation and the pups were born healthily (n = 4). Moreover, the higher rate (3 U/day) hastened the timing of labor, 6 h after implantation, compared to the infusion of 0.3 U/day (Fig. 3, n = 4, P < 0.01). 3.4. Effect of OT-infusion in OTR KO mice We examined the effect of OT-infusion in OTR KO mice to remove a possibility for a cross reactivity of OT to other receptors which induced successful delivery by OT-infusion in FP KO mice. Takayanagi et al. (2005) reported that OTR KO mice could undergo normal parturition in spite of no contractile response to OT in myometrium of OTR KO mice at 19.0 G in vitro. The OT-infusion rate of 3 U/day was chosen to induce imminent preterm labor since that of 2.5 U/day delivered pups about 24 h after the implantation of OT-pumps in gravid WT mice at 15.5 G (Imamura et al., 2000). In the present study, the OTpumps (3 U/day) were implanted into WT or OTR KO females at 17.0 G. The OT-pump (3 U/day) induced imminent preterm labor 24 ± 3.5 h after the implantation in WT mice (n = 3), and the time until labor was significantly shorter than the case of the vehicle pump in WT mice (54 ± 3.5 h, n = 3, P < 0.01; Fig. 4). OTR KO females delivered pups 53 ± 2.9 h after the implantation (n = 4), which was similar to the cases of the vehicle pump in WT mice and the previous results of 19.3 ± 0.1 G in OTR KO mice (n = 13; Takayanagi et al., 2005). 3.5. Measurement of plasma progesterone levels We examined plasma progesterone levels in FP KO mice 6 h after implantation of OT (3 U/day) at 19.0 G.
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Fig. 3. Effects of continuous OT-infusion on the time until the onset of labor. Micro-osmotic pumps were implanted into FP KO females at 19.0 G, and the duration until the onset of labor was determined. Labor was actively induced by the OT-infusion (0.3 U/day, n = 4, gray column or 3 U/day, n = 4, closed column) but not by the vehicle administration (0 U/day, n = 3, open column). Time until the onset of labor was defined as delivery of the first pup by observation of gravid females every 6 h following pump implantation. The data show the mean ± S.E.M. and significant differences were examined by ANOVA and t-test. * P < 0.05 vs. 0 U/day; ## P < 0.01 vs. 0.3 U/day; a The pups died in uteri and were delivered 82 h after the implantation at 19.0 G.
Fig. 2. Contractile responses to OT in pregnant mouse myometrium. (A and B) The typical trace of 19.0 G WT (A) or FP KO (B) was shown. (C) The concentration–response curves for OT-induced contractions in WT (n = 7, open circle) and FP KO (n = 7, closed circle) are shown. Contractions were induced in all tissues by 35 mM KCl solution, and the maximum responses were 2.33 ± 0.49 or 1.78 ± 0.21 mN in WT or FP KO, respectively. OT was cumulatively added to the strips every 5 min under normal physiological salt solution. One hundred percent represents the maximum response of the contractions induced by 35 mM KCl. The data show the mean ± S.E.M. and significant differences were examined by ANOVA.
The plasma progesterone levels of FP KO mice with OTinfusion (9.04 ± 2.45 ng/ml, n = 4) were 5-fold lower that those of FP KO mice with vehicle-infusion (46.8 ± 3.9 ng/ml, n = 5, P < 0.001) and 8-fold higher than those of WT mice without infusion (1.16 ± 0.17 ng/ml, n = 5, P < 0.05; Fig. 5).
Fig. 4. Effects of continuous OT-infusion on the time until the onset of labor. Micro-osmotic pumps (0 U/day, n = 3; 3 U/day, n = 3) were implanted into WT (open column) or OTR KO (3 U/day, n = 4, closed column) females at 17.0 G. The duration until the onset of labor was determined. Time until the onset of labor was defined as delivery of the first pup by observation of gravid females every 6 h following pump implantation. The data show the mean ± S.E.M. and significant differences were examined by ANOVA and t-test. ** P < 0.01 vs. WT (0 U/day); ## P < 0.01 vs. OTR KO (3 U/day).
4. Discussion In the present study, we found that FP KO females could undergo successful delivery by infusion of OT (0.3 or 3 U/day, Fig. 3) at 19.0 G although the failure of parturition in FP KO females has been thought to be due to weak contractile responses to OT because of the low expression levels of OTR in myometrium. There are two possible mechanisms by which OT-infusion could induce delivery in FP KO females. One is an induction of strong uterine contractions, and the other is plasma progesterone withdrawal. The in vitro study clearly showed that FP KO myometrium with the low expression levels of
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OTR had a potential to induce almost the same contractile responses to OT as the WT myometrium at 19.0 G (Figs. 1 and 2 and Table 1). The results imply that such uterine contractions would also occur in FP KO females by the OT-infusion in vivo. Sugimoto et al. (1997) reported that the uterus of FP KO mice had little contractile responses to OT compared to that of WT mice in vivo. The discrepancy may be resulted from the differences in the methods of contractile measurement, the doses and roots of administration of OT, and the period of pregnancy, term or preterm. In the previous study (Sugimoto et al., 1997), uterine contractions at term were measured as an increase in intrauterine pressure in vivo by use of a small balloon catheter inserted into the uterine horn, and the pressure curve of OT was represented based on spontaneous contractions and the transient increases in pressure were measured after bolus intravenous administration of OT. In contrast, in the present study, preterm myometrial contractions were measured in vitro and the cumulative concentration–response curve of OT was represented based on the contractions induced by 35 mM KCl, which shows the intense and tonic contractions induced by pharmacological high concentrations of OT (Fig. 2). The whole uteri are exposed to many contractile regulatory factors especially in progesterone, resulting in suppression of uterine contractions in FP KO mice in vivo differently from in vitro experiment using a small stripe of myometrium. We could consider that the concentration–response curves of our result (Fig. 2C) are not much different from those of the previous study (Sugimoto et al., 1997) since the curve of FP KO mice of the present study tended to shift to the right (Fig. 2C). As reported in our previous study (Kawamata et al., 2007), the slight rightward shift of the curve was due to the sparseness of phasic contractions at low concentration of OT in FP KO mice compared to those in WT mice (Fig. 2A and B). FP KO uterus might not induce enough contractions to accomplish delivery like WT uterus when the physiological concentration of OT was released from the posterior lobe of hypophysis in a pulsatile fashion in vivo. In the present study, however, we used higher doses of OT with the continuous infusion compared to the previous study (Sugimoto et al., 1997), which resulted in the delivery of pups even in FP KO females. Although OTR is well known to be desensitized by OT stimulation in human myometrium (Plested and Bernal, 2001), the smaller amount of OTR in response to the continuous infusion of OT in the pregnant myometrium of FP KO mice in vivo seemed to induce delivery with myometrial contractions which were shown in vitro study almost the same as those from wild mice. As OTR is expressed in corpus luteum of pregnant mouse ovary (Imamura et al., 2000), OT might progress luteolysis. It is reported that intraluteal OT locally modulate secretion of endothelin 1 as well as PGF2␣ within the corpus luteum, which induce luteolysis (Shirasuna et al., 2007). In contrast, the previous works using the OT-pump, which were similar to our study, showed a luteotrophic effect of OT (Imamura et al., 2000). The report showed a significant delay in the onset of labor (20.1 G) in WT mice by the implantation of OT infused at 1 U/day at 15.5 G, and the serum progesterone levels at 19.0 G were higher than the case of vehicle implantation. However,
preterm labor was observed one day after the implantation of OT infused at 2.5 U/day (Imamura et al., 2000). Although it is unknown whether the uterine contractions or the progesterone withdrawal induced the preterm labor, these results imply that both the doses of OT and the timing of application critically relate to the onset of labor. In the present study, since we implanted OT-pump in FP KO females at 19.0 G, the gestational age may cause the different results from the previous study. We also implanted OT-pump infused at 3 U/day in WT females at 17.0 G. In these females, it took longer time (16.5 h) for the onset of labor compared to the FP KO females (6 h) implanted at 19.0 G. This might be due to the difference of contractile activity in gestational ages rather than the velocity of progesterone withdrawal. In most smooth muscle organs, stretching leads to contraction (Renfree, 2000). Fetus growth induces myometrial stretching followed by contributions to parturition (Smith, 2007). The plasma progesterone levels of FP KO females during delivery by the OT-infusion (3 U/day) were 5-fold lower than those of FP KO females with the vehicle-infusion, but were 8-fold higher than those of WT females that underwent spontaneous delivery (Fig. 5). The results indicate that the decreased levels of plasma progesterone might be insufficient to compel FP KO females to accomplish delivery, and that the combination with the strong uterine contractions is necessary. We also demonstrated that both effects of OT on the uterine contractions and the progesterone withdrawal were initiated to mediate only via OTR because OTR KO females could not undergo preterm labor by the infusion of OT at 17.0 G (Fig. 4). Therefore, in this study, we have clarified two aspects of OT functions during parturition to induce strong uterine contraction even with low expression levels of OTR in myometrium and to produce progesterone withdrawal probably due to luteolysis, which enabled FP KO females to undergo successful delivery by OT-infusion.
Fig. 5. The plasma progesterone levels in mice. Plasma progesterone levels were measured in WT females without infusion (n = 5, open column) or FP KO females (closed column) 6 h after the implantation of OT-pump (0 U/day, n = 5; 3 U/day, n = 4) at 19.0 G. The time represents 19.25 G when FP KO females start delivery by OT-infusion (3 U/day). The data show the mean ± S.E.M. and significant differences were examined by ANOVA and t-test. * P < 0.05, *** P < 0.001 vs. WT; ### P < 0.001 vs. FP KO (0 U/day).
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Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research (No. 14360046) from the Ministry of Education, Science and Culture of Japan and by Sankyo Foundation of Life Science. References Caldeyro-Barcia, R., Theobald, G.W., 1968. Sensitivity of the pregnant human myometrium to oxytocin. Am. J. Obstet. Gynecol. 102, 1181. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Clerget, M.S., Elalouf, J.M., Germain, G., 1997. Quantitative reverse transcription and polymerase chain reaction analysis of oxytocin and vasopressin receptor mRNAs in the rat uterus near parturition. Mol. Cell. Endocrinol. 136, 79–89. Fuchs, A.R., Periyasamy, S., Alexandrroa, M., Soloff, M.S., 1983. Correlation between oxytocin receptor concentration and responsiveness to oxytocin in pregnant rat myometrium. Endocrinology 113, 742–749. Fuchs, A.R., Fuchs, F., Husslein, P., Soloff, M.S., 1984. Oxytocin receptors in the human uterus during pregnancy and parturition. Am. J. Obstet. Gynecol. 150, 734–741. Fuchs, A.R., Helmer, H., Behrens, O., Liu, H.C., Antonian, L., Chang, S.M., Fields, M.J., 1992. Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors precedes onset of labor. Biol. Reprod. 47, 937–944. Gimpl, G., Fahrenholz, F., 2001. The oxytocin receptor system: structure, function, and regulation. Physiol. Rev. 81, 629–683. Gross, G.A., Imamura, T., Luedke, C., Vogt, S.K., Olson, L.M., Nelson, D.M., Sadovsky, Y., Muglia, L.J., 1998. Opposing actions of prostaglandins and oxytocin determine the onset of murine labor. Proc. Natl. Acad. Sci. U.S.A. 95, 11875–11879. Imamura, T., Luedke, C.E., Vogt, S.K., Muglia, L.J., 2000. Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, R1061–R1067. Ivell, R., Rust, W., Einspanier, A., Hartung, S., Fields, M., Fuchs, A.R., 1995. Oxytocin and oxytocin receptor gene expression in the reproductive tract of the pregnant cow: rescue of luteal oxytocin production at term. Biol. Reprod. 53, 553–560. Kawamata, M., Mitsui-Saito, M., Kimura, T., Takayanagi, Y., Yanagisawa, T., Nishimori, K., 2003. Vasopressin-induced contraction of uterus is mediated solely by the oxytocin receptor in mice, but not in humans. Eur. J. Pharmacol. 472, 229–234. Kawamata, M., Tonomura, Y., Kimura, T., Yanagisawa, T., Nishimori, K., 2004. The differential coupling of oxytocin receptors to uterine contractions in murine estrous cycle. Biochem. Biophys. Res. Commun. 321, 695–699. Kawamata, M., Tonomura, Y., Kimura, T., Sugimoto, Y., Yanagisawa, T., Nishimori, K., 2007. Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor. Am. J. Physiol. Endocrinol. Metab. 292, E992–E999. Kimura, T., Takemura, M., Nomura, S., Nobunaga, T., Kubota, Y., Inoue, T., Hashimoto, K., Kumazawa, I., Ito, Y., Ohashi, K., Koyama, M., Azuma,
37
C., Kitamura, Y., Saji, F., 1996. Expression of oxytocin receptor in human pregnant myometrium. Endocrinology 137, 780–785. Larcher, A., Neculcea, J., Breton, C., Arslan, A., Rozen, F., Russo, C., Zingg, H.H., 1995. Oxytocin receptor gene expression in the rat uterus during pregnancy and the estrous cycle and in response to gonadal steroid treatment. Endocrinology 136, 5350–5356. Maggi, M., Genazzani, A.D., Giannini, S., Torrisi, C., Baldi, E., di Tomaso, M., Munson, P.J., Rodbard, D., Serio, M., 1988. Vasopressin and oxytocin receptors in vagina, myometrium, and oviduct of rabbits. Endocrinology 122, 2970–2980. Maggi, M., Peri, A., Giannini, S., Fantoni, G., Guardabasso, V., Serio, M., 1991. Oxytocin and V1 vasopressin receptors in rabbit endometrium during pregnancy. J. Reprod. Fertil. 91, 575–581. Nishimori, K., Young, L.J., Guo, Q., Wang, Z., Insel, T.R., Matzuk, M.M., 1996. Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc. Natl. Acad. Sci. U.S.A. 93, 11699–11704. Osa, T., 1973. Some factors affecting the contractile responses of pregnant mouse myometrium. Jpn. J. Physiol. 23, 401–417. Plested, C.P., Bernal, A.L., 2001. Desensitisation of the oxytocin receptor and other G-protein coupled receptors in the human myometrium. Exp. Physiol. 86, 303–312. Renfree, M.B., 2000. Maternal recognition of pregnancy in marsupials. Rev. Reprod. 5, 6–11. Shirasuna, K., Shimizu, T., Hayashi, K.G., Nagai, K., Matsui, M., Miyamoto, A., 2007. Positive association, in local release, of luteal oxytocin with endothelin 1 and prostaglandin F2alpha during spontaneous luteolysis in the cow: a possible intermediatory role for luteolytic cascade within the corpus luteum. Biol. Reprod. 76, 965–970. Smith, R., 2007. Parturition. N. Engl. J. Med. 356, 271–283. Soloff, M.S., Alexandrova, M., Fernstrom, M.J., 1979. Oxytocin receptors: triggers for parturition and lactation? Science 204, 1313–1315. Sugimoto, Y., Yamasaki, A., Segi, E., Tsuboi, K., Aze, Y., Nishimura, T., Oida, H., Yoshida, N., Tanaka, T., Katsuyama, M., Hasumoto, K., Murata, T., Hirata, M., Ushikubi, F., Negishi, M., Ichikawa, A., Narumiya, S., 1997. Failure of parturition in mice lacking the prostaglandin F receptor. Science 277, 681–683. Takayanagi, Y., Yoshida, M., Bielsky, I.F., Ross, H.E., Kawamata, M., Onaka, T., Yanagisawa, T., Kimura, T., Matzuk, M.M., Young, L.J., Nishimori, K., 2005. Pervasive social deficits, but normal parturition, in oxytocin receptordeficient mice. Proc. Natl. Acad. Sci. U.S.A. 102, 16096–16101. Wathes, D.C., Smith, H.F., Leung, S.T., Stevenson, K.R., Meier, S., Jenkin, G., 1996. Oxytocin receptor development in ovine uterus and cervix throughout pregnancy and at parturition as determined by in situ hybridization analysis. J. Reprod. Fertil. 106, 23–31. Wu, W.X., Verbalis, J.G., Hoffman, G.E., Derks, J.B., Nathanielsz, P.W., 1996. Characterization of oxytocin receptor expression and distribution in the pregnant sheep uterus. Endocrinology 137, 722–728. Yanagisawa, T., Ishii, K., Hashimoto, H., Taira, N., 1989. Differential coupling to positive inotropic responses of cyclic AMP produced by stimulation of beta1- and beta2-adrenergic receptors. J. Cardiovasc. Pharmacol. 13, 64–75. Young 3rd, W.S., Shepard, E., Amico, J., Hennighausen, L., Wagner, K.U., LaMarca, M.E., McKinney, C., Ginns, E.I., 1996. Deficiency in mouse oxytocin prevents milk ejection, but not fertility or parturition. J. Neuroendocrinol. 8, 847–853.