Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy

Increases inguanosine 3',5'-cyclic monophosphate (cGMP) induced bynitric oxide (NO), nitrovasodilators, and atrial peptides correlatewith relaxation of vascular smooth muscle. Relaxation of myometrialsmooth muscle by increases in cGMP, however, has required unusuallyhigh concentrations of the cyclic nucleotide. We tested the hypothesisthat the sensitivity of myometrium to relaxation by cGMP is increasedduring pregnancy. Aortic smooth muscle was more sensitive to relaxationby cGMP than myometrial tissues, and, contrary to our hypothesis,myometrium from pregnant rats was least sensitive. Although levels ofcGMP were elevated after treatment with the NO donor,S-nitroso-N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels ofcGMP-dependent protein kinase (PKG) were significantly decreased inmyometrium from pregnant rats compared with myometrium from nonpregnantcycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, andprogesterone antagonized this response. We conclude that1) myometrial tissues from pregnantrats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP isaccompanied by progesterone-mediated decreases in the level of PKGexpression.

     

Reduced expression of CRH receptor type 1 in upper segment human myometrium during labour

Background  

Corticotropin-releasing hormone (CRH) and CRH-related peptide are shown to modulate uterine contractility through two CRH receptor subtype, CRH-R1 and CRH-R2 during pregnancy. Through different signaling pathways, CRH-R1 maintains myometrial quiescence whereas CRH-R2 promotes smooth muscle contractility. We hypothesized that the expression of CRH receptors in myometrium might be changed during pregnancy and labour.     

Expression of alpha5 integrin (Itga5) is elevated in the rat myometrium during late pregnancy and labor: implications for development of a mechanical syncytium

The underlying mechanisms controlling uterine contractions during labor are still poorly understood. Integrins are heterodimeric, transmembrane receptors composed of alpha and beta subunits that can be found in focal adhesions. Because these structures play an important role in the regulation of smooth muscle contractility and cell adhesion, we hypothesized that alpha5 integrin mRNA (Itga5) and protein (ITGA5) expression would be induced in the rat myometrium during late pregnancy and labor. Itga5 mRNA expression was significantly increased (P < 0.05) from Day 17 to labor, noticeably decreasing 1 day postpartum (PP). Immunoblot analysis illustrated a continual increase in ITGA5 levels during pregnancy, labor, and PP, with levels reaching significance at labor (P < 0.05). Analysis of ITGA5 expression by immunocytochemistry demonstrated that it is primarily localized to myometrial cell membranes in the longitudinal muscle layer of the myometrium from before pregnancy to Day 6, and in both the longitudinal and circular muscle layers from Day 15 to PP. Treatment of late-pregnant rats with progesterone blocked labor and resulted in sustained expression of Itga5 mRNA expression to Day 24. In addition, immunocytochemistry experiments showed ITGA5 was detectable at higher levels in cell membranes of both myometrial layers in progesterone-treated animals on Days 23 and 24, compared with vehicle controls. We propose that ITGA5, with its sole known partner, ITGB1, may be important in promoting cellular cohesion during late pregnancy. This process may aid the development of a mechanical syncytium for efficient force transduction during the sustained, coordinated, and powerful contractions of labor.     

Regulation of myometrial beta 2-adrenergic receptors by progesterone and estradiol-17 beta in late pregnant rats

Rat myometrium exhibited a marked decrease in the concentration of beta 2-adrenergic receptors immediately before parturition, i.e., in the last 6 h of pregnancy. This phenomenon continued until the withdrawal of myometrial progesterone (-94% from Day 18 of pregnancy to term) and coincided with the sharp increase (+200%) of the myometrial concentration of estradiol. A linear positive correlation was found (r2 = 0.645) between the concentration of beta 2-adrenergic receptors and the log ratio of myometrial concentration of progesterone/myometrial concentration of estradiol (P/E2), suggesting a modulation of beta 2-adrenergic receptors by steroids. In rats with estrogen-dominated uteri (intact of ovariectomized late pregnant rats injected with estradiol), there was no change either in concentration or affinity of beta 2-adrenergic receptors relative to untreated control pregnant rats. In contrast, rats with progesterone-dominated uteri (intact or ovariectomized late pregnant rats treated with progesterone or ovariectomized rats) have an increased number of beta 2-adrenergic receptors, with a decreased affinity of these receptors compared to untreated control pregnant rats or to estrogen-treated rats. These results suggest that progesterone regulates the number of beta 2-adrenergic receptors in myometrium of late pregnant rats. The mechanisms by which progesterone exerts this regulation remains to be elucidated.     

Regulation of calcitonin gene-related peptide receptors in the rat uterus during pregnancy and labor and by progesterone.

Calcitonin gene-related peptide (CGRP) is a potent smooth muscle relaxant in a variety of tissues. We recently demonstrated that CGRP relaxes uterine tissue during pregnancy but not during labor. In the present study we examined whether uterine (125)I-CGRP binding and immunoreactive CGRP receptors are regulated by pregnancy and labor and by sex steroid hormones. We found that (125)I-CGRP binding to membrane preparations from uteri was elevated during pregnancy and decreased during labor and postpartum. Changes in immunoreactive CGRP receptors were similar to the changes in (125)I-CGRP binding in these tissues, suggesting pregnancy-dependent regulation of CGRP receptor protein. CGRP receptors were elevated by Day 7 of gestation, and a precipitous decrease in these receptors occurred on Day 22 of gestation prior to the onset of labor. Both (125)I-CGRP-binding and immunofluorescence studies indicated that CGRP receptors were localized to myometrial cells. Hormonal control of uterine CGRP receptors was assessed by the use of antiprogesterone RU-486, progesterone, and estradiol-17beta. RU-486 induced a decrease in uterine CGRP receptors during pregnancy (Day 19). On the other hand, progesterone prevented the fall in uterine CGRP receptors at term (Day 22). In addition, progesterone also increased uterine CGRP receptors in nonpregnant, ovariectomized rats, while estradiol had no effects. These hormone-induced changes in uterine CGRP receptors were demonstrated by (125)I-CGRP-binding, Western immunoblotting, and immunolocalization methods. These results indicate that CGRP receptors and CGRP binding in the rat uterus are increased with pregnancy and decreased at term. These receptors are localized to the myometrial cells, and progesterone is required for maintaining CGRP receptors in the rat uterus. Thus, the inhibitory effects of CGRP on uterine contractility are mediated through the changes in CGRP receptors and may play a role in uterine quiescence during pregnancy.     

Estradiol reduces F2alpha-isoprostane production in cultured human endothelial cells

Free radical-generated F(2alpha)-isoprostanes are a group of compounds with vasoconstrictor properties. To investigate whether estradiol exerts antioxidant actions modifying F(2alpha)-isoprostane production, cultured human umbilical vein endothelial cells were exposed to estradiol and other compounds and F(2alpha)-isoprostanes were measured in culture medium. Exposure to 1 and 10 nM estradiol for 24 h reduced F(2alpha)-isoprostane production by 36 and 49%, respectively (P < 0.001 vs. control). Exposure to antiestrogens alone (ICI-182780 or EM-652) slightly reduced F(2alpha)-isoprostanes (P < 0.05 vs. control), but much less than exposure to estradiol (P < 0.05). ICI-182780 reversed the estradiol-induced reduction of F(2alpha)-isoprostane concentration (P < 0.05). Along with time-course analysis, these results suggest that estradiol effects were mediated through estrogen receptor-dependent and -independent mechanisms. Progestogens alone (progesterone or medroxyprogesterone acetate) did not modify F(2alpha)-isoprostane production at any of the tested concentrations (1, 10, and 100 nM). Progesterone completely reversed estradiol-induced reduction of F(2alpha)-isoprostane production (P < 0.05 vs. control and estradiol), but medroxyprogesterone acetate did not (P < 0.05 vs. control).     

Structural and functional studies of myometrial gap junctions

Gap junctions are believed to be sites of metabolic and electrical coupling between cells. These contacts are present between myometrial cells immediately prior to and during parturition. We report the results of studies to investigate the control and the function of myometrial gap junctions. Injection of estradiol (500 micrograms/day) with or without progesterone into immature and ovariectomized mature rats demonstrated that estradiol stimulated whereas progesterone suppressed gap junction formation. Indomethacin treatment was also shown to potentiate the action of estradiol. Also, pregnant rats treated with oestradiol developed numerous myometrial gap junctions and aborted their fetuses. These results suggest that the steroid hormones and prostaglandins may control myometrial gap junction development. Diffusion studies of 3H-2-deoxyglucose in longitudinal myometrial strips revealed a significant increase in the diffusion coefficient in delivering versus ante-partum rat tissues. This indicates that there is increased metabolic transfer during parturition when gap junctions are present. The results of these studies show that steroid hormones and prostaglandins may regulate myometrial gap junctions and that metabolic, as well as electrical coupling, of uterine smooth muscle cells increase at parturition concomitant with the development of gap junctions.     

Immunohistochemical analysis of connexins 26, 32 and 43 in rat uterus during late pregnancy: lack of connexin 26 in the myometrium

The spatial and temporal patterns of expression of connexin 26, connexin 32 and connexin 43 were investigated in rat uterus at days 17, 19 and 22 of pregnancy and during delivery (23 days) by immunocytochemistry, Gap junctions, which are essential for the development of labour, are known to undergo rapid increase in the rat myometrium at the end of pregnancy. The expression of connexin 43, the major myometrial gap junction protein, was low throughout pregnancy but increased immediately before the onset of labour (day 22). It was found predominantly in the myometrium, although limited staining was also apparent in the stroma. Immunolabelling revealed the presence of connexins 26 and 32 in uterine luminal epithelial cells on days 17 and 19 of pregnancy, with a marked increase in connexin 26 expression at days 19, 22 and 23; however, marked expression of connexin 32 was apparent only at day 23. No immunoreactivity for either connexin 26 or 32 was found in myometrial cells at any stage of pregnancy. We conclude, contrary to other recent reports, that connexin 26 is not a gap junction protein of the rat myometrial smooth muscle cell. © 1998 Chapman & Hall     

Castration‐induced upregulation of muscle ERα supports estrogen sensitivity of motoneuron dendrites in a sexually dimorphic neuromuscular system

The spinal cord of rats contains the sexually dimorphic motoneurons of the spinal nucleus of the bulbocavernosus (SNB). In males, SNB dendrites fail to grow after castration, but androgen or estrogen treatment supports dendritic growth in castrated males. Estrogenic support of SNB dendrite growth is mediated by estrogen receptors (ER) in the target muscle. ERα expression in cells lacking a basal lamina (referred to as “extra‐muscle fiber cells”) of the SNB target musculature coincides with the period of estrogen‐dependent SNB dendrite growth. In the SNB target muscle, extra‐muscle fiber ERα expression declines with age and is typically absent after postnatal (P) day 21 (P21). Given that estradiol downregulates ERα in skeletal muscle, we tested the hypothesis that depleting gonadal hormones would prevent the postnatal decline in ERα expression in the SNB target musculature. We castrated male rats at P7 and assessed ERα immunolabeling at P21; ERα expression was significantly greater in castrated males compared with normal animals. Because ERα expression in SNB target muscles mediates estrogen‐dependent SNB dendrogenesis, we further hypothesized that the castration‐induced increase in muscle ERα would heighten the estrogen sensitivity of SNB dendrites. Male rats were castrated at P7 and treated with estradiol from P21 to P28; estradiol treatment in castrates resulted in dendritic hypertrophy in SNB motoneurons compared with normal males. We conclude that early castration results in an increase in ERα expression in the SNB target muscle, and this upregulation of ERα supports estrogen sensitivity of SNB dendrites, allowing for hypermasculinization of SNB dendritic arbors. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 921–935, 2013     

Progesterone receptors and ventilatory stimulation by progestin

Progestin is thought to be a ventilatory stimulant but its effectiveness in raising ventilation is variable in humans and other species. We hypothesized that the level of progesterone receptors was an important determinant of the ventilatory response to progestin. Since estradiol induces progesterone receptor formation, we compared the ventilatory effect of the synthetic progestin medroxyprogesterone acetate (MPA) given in combination with estradiol with the effects of estradiol alone, MPA alone, or vehicle (saline) in ovariectomized rats. Animals receiving MPA alone had low numbers of progesterone receptors (2.43 pmol/g uterine wt) and had no change in ventilation, arterial Pco2, or Po2. MPA administration raised ventilation 23 +/- 5%, lowered arterial Pco2 3.2 +/- 0.9 Torr (both P less than 0.01) and tended to raise arterial Po2 when given in combination with estradiol to animals with increased numbers of progesterone receptors (4.85 pmol/g uterine wt). Estradiol alone produced the highest number of progesterone receptors (12.3 pmol/g uterine wt) but had no effect on ventilation or arterial Pco2 and decreased arterial Po2. Combined estradiol plus MPA treatment produced a greater fall in arterial Pco2 than did treatment with MPA alone, estradiol, or saline (all P less than 0.05). These results suggest that both an elevation in progestin levels and progesterone receptor numbers are required to stimulate ventilation.     

Effects of 17 beta-estradiol on myometrial gap junctions and pregnancy in the rat

The effects of estradiol treatment on the development of myometrial gap junctions and premature labour were investigated using timed pregnant rats. In control animals myometrial gap junctions were infrequent between days 17 and 20 of pregnancy, but began to develop on day 21 and were at maximum frequency, size, and membrane area on day 22 during delivery. Gap junctions were completely absent from the myometrium 48 h after delivery. Animals treated with 500 micrograms 17 beta-estradiol/day starting on day 16 of pregnancy developed numerous myometrial gap junctions and delivered their pups prematurely on day 19. Similarly, treatment with 50 micrograms estradiol/day resulted in the development of myometrial gap junctions on day 20 of pregnancy and premature labour. However, treatment with various doses of estradiol up to and including 500 micrograms/day for 3 days beginning 1 day before delivery was not able to maintain the presence of myometrial gap junctions during the postpartum period. These results support the hypothesis that estradiol stimulates the development of myometrial gap junctions and that the presence of gap junctions in the myometrium is a requirement for the occurrence of term, as well as preterm labour. Furthermore, it is evident from this study that the postpartum regression of myometrial gap junctions is not dependent on the decrease in estradiol.     

Effects of tamoxifen citrate and cycloheximide on estradiol induction of rat myometrial gap junctions

Longitudinal muscle of myometrial tissues from immature rats were examined by quantitative thin section electron microscopy for the presence of gap junctions after treatment with estradiol with and without tamoxifen, and cycloheximide for 1-6 days. Gap junctions were present between myometrial cells on days 4, 5, and 6 after treatment with estradiol (500 micrograms/day). Tamoxifen administered concomitantly with estradiol over the 6-day period completely prevented induction of the junctions. Gap junctions were not detected in the myometrium after treatment with tamoxifen alone. Administration of cycloheximide together with estradiol on day 0 of the 6-day period had no effect on gap junction frequency but resulted in a reduction in gap junction size in the myometrium after continued treatment with the hormone. Treatment with cycloheximide on day 1, however, significantly suppressed the effect of further estradiol treatment on the induction of gap junctions in the myometrium. Junctions were not visible in the tissues from animals treated with cycloheximide alone or in the control groups treated with sesame oil. These results indicate that estradiol influences the presence of gap junctions in the myometrium by regulating the synthesis of gap junction proteins through the steroid receptor mechanism.     

Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores

IntracellularCa²⁺ release channels such asryanodine receptors play crucial roles in theCa²⁺-mediated signaling thattriggers excitation-contraction coupling in muscles. Although theexistence and the role of these channels are well characterized inskeletal and cardiac muscles, their existence in smooth muscles, andmore particularly in the myometrium, is very controversial. We have nowclearly demonstrated the expression of ryanodine receptorCa²⁺ release channels in ratmyometrial smooth muscle, and for the first time, intracellularCa²⁺ concentration experimentswith indo 1 on single myometrial cells have revealed the existence of afunctional ryanodine- and caffeine-sensitive Ca²⁺ release mechanism in 30% ofrat myometrial cells. RT-PCR and RNase protection assay on wholemyometrial smooth muscle demonstrate the existence of all threeryr mRNAs in the myometrium:ryr3 mRNA is the predominant subtype,with much lower levels of expression forryr1 andryr2 mRNAs, suggesting that theryanodine Ca²⁺ release mechanismin rat myometrium is largely encoded byryr3. Moreover, using intracellularCa²⁺ concentration measurementsand RNase protection assays, we have demonstrated that the expression,the percentage of cells responding to ryanodine, and the function ofthese channels are not modified during pregnancy.

     

Acute and chronic estrogen supplementation decreases uterine sympathetic innervation in ovariectomized adult virgin rats

Uterine innervation undergoes substantial reorganization associated with changes in reproductive status. Nerves innervating the uterus are decreased in pregnancy and puberty, and even the normal rodent estrous cycle is characterized by fluctuations in numbers of myometrial nerve fibers. During the follicular (proestrus/estrous) phase of the estrous cycle, intact nerves are rapidly depleted and then return over the next 2-3 days in the luteal (metestrus/diestrus) phase. We hypothesize that uterine nerve depletion is initiated by increased circulating estrogen in the follicular phase. However, studies have not shown whether estrogen can reduce uterine innervation and, if so, whether the time course is compatible with the rapid changes observed in the estrous cycle. These questions were addressed in the present study. Mature ovariectomized virgin rats received 17-beta-estradiol as a single injection (10 microg/kg s.c.) or chronically from timed-release pellets (0.1 microg/pellet for 3 weeks sustained release). Total (protein gene-product 9.5-immunoreactive) and sympathetic (dopamine beta-hydroxylase-immunoreactive) uterine innervation was assessed quantitatively. Both total and sympathetic innervation was abundant in uterine longitudinal smooth muscle of ovariectomized rats. However, following acute or chronic estrogen administration, total and sympathetic fiber numbers were markedly decreased. This was not due to altered uterine size, as reductions persisted after correcting for size differences. Our results indicate that sympathetic nerves are lost from uterine smooth muscle after estradiol treatment in a manner similar to that seen in the intact animal during estrus and pregnancy. This suggests that the rise in estradiol prior to estrus is sufficient to deplete uterine sympathetic innervation.     

The effect of estradiol on COX-2, EP2, and EP4 mRNA expression and the extracellular matrix in the cervix of the hypogonadotrophic, ovariectomized ewe

There is a degree of cervical relaxation in the ewe at estrus that is regulated by changes in prostaglandin synthesis, prostaglandin receptor expression, and changes in the cervical extracellular matrix. It is likely that these are regulated by changes in periovulatory hormones, particularly estradiol. This study determined the effect of estradiol benzoate on the mRNA expression of cyclooxygenase-2 (COX-2) and the prostaglandin E receptors EP₂ and EP₄, the concentration of cervical hyaluronan, and the proportion of smooth muscle and collagen in the cervix of the hypogonadotrophic ovariectomized ewe (Ovis aries). Ovariectomized hypogonadotrophic ewes were given 100 μg estradiol benzoate, and their cervices were collected 0, 24, and 48 h thereafter to determine the expression of cervical COX-2, EP₂, and EP₄ mRNA by in situ hybridization, the concentration of hyaluronan by ELISA, and the proportion of smooth muscle and collagen by Masson's trichrome staining. Estradiol benzoate increased the mRNA expression of COX-2 and EP₄ within 24 h after treatment (P < 0.05), whereas EP₂ mRNA, hyaluronan, and the ratio of smooth muscle to collagen did not change within 48 h after treatment. The COX-2, EP₂, and EP₄ mRNA expression were greatest in the smooth muscle layers (P < 0.05) and least in the luminal epithelium (P < 0.05). In conclusion, we inferred that estradiol regulates cervical COX-2 and EP₄ mRNA expression and may regulate cervical relaxation via the synthesis of prostaglandin E₂ and activation of the PGE₂ receptors EP₂ and EP₄.