Peptide-activator of cyclic nucleotide phosphodiesterases in the rat and human uterus

The presence of the cyclic nucleotide phosphodiesterase peptide-activator previously isolated from the calf myometrium was studied in uterine tissue of rats at different functional states (in puberty, estrus and diestrus stages, pregnancy) and in human myometrium and myoma. The peptide isolated from calf myometrium, immature rat myometrium and uterine tissue in myoma caused a 3-4-fold stimulation of the phosphodiesterase activity.     

Expression of estrogen receptor alpha and beta in myometrium of premenopausal and postmenopausal women

Although a clear role for estrogen receptor (ER) alpha has been established, the contribution of ERbeta in estrogen-dependent development, growth and functions of the myometrium is not understood. As a first step towards understanding the role of ERbeta, we have examined the expression of ERalpha and ERbeta in the human myometrium. With competitive RT-PCR assays, the level of ERbeta mRNA was 10-200 times lower than that of ERalpha mRNA in both premenopausal and postmenopausal myometrium. In premenopausal myometrium, the expression pattern of ERbeta mRNA during the menstrual cycle was similar to that of ERalpha mRNA, with highest levels in peri-ovulatory phase. In postmenopausal myometrium, ERbeta mRNA was significantly higher than it was in premenopausal myometrium, while the level of ERalpha mRNA was lower. The net result was a change in the ratio of ERbeta to ERalpha mRNA expression. The ratio changed from 0.6-1.5 in premenopausal to 2.5-7.6 in postmenopausal myometrium. In premenopausal women, the gonadotropin releasing hormone analogue, leuprorelin acetate, elicited a decrease in ERalpha and an increase in ERbeta mRNA expression to cause a postmenopausal receptor phenotype. Estradiol, on the other hand, reversed ERalpha and ERbeta mRNA expression and their ratio in postmenopausal myometrium to those of premenopausal myometrium. Immunohistochemical staining and Western blot analysis of ERalpha and ERbeta with semiquantitative analysis showed good agreement between mRNA and protein levels. The data indicate that coordinated expression of ERalpha and ERbeta might be necessary for normal estrogen action in myometrium. Furthermore, estrogen appears a dominant regulator of both receptors in the myometrium.     

Electrical coupling in rat myometrium during pregnancy.

Gap junctions were found in rat myometrium only during parturition of abortion. A comparison was made between the values of the length constants obtained from longitudinally cut strips of rat myometrium at the midterm stage of pregnancy and during parturition. No significant difference was found between these values. No significant difference was found between length constants measured in myometrium from rats near term (22 days) and those during parturition, or between midterm myometrium and myometrium taken from animals aborting at midterm following ovariectomy. It was concluded that the appearance of gap junctions in parturient myometrium does not alter the spread of passive current in the longitudinal axis of the cells in these tissues. It is suggested that gap junctions are not required for spread of passive current, and that other structures may provide an alternate path.     

Prostaglandin E2 and F2 alpha in mid-pregnant rat uterus and at parturition

Prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) produced by 15 days pregnant rat myometrium, by parturient rat myometrium and myometrium plus endometrium were measured in vitro. The results showed that the PGs produced by parturient myometrium were higher than these obtained during mid-pregnancy. Myometrium with endometrium released more PGs than myometrium alone, and the addition of arachidonic acid (AA) at 10 DM did not show any significant effect. Exogenous progesterone or estradiol-17b at a concentration of 1 Dmol had no effect on parturient uterine PG secretions.     

Adenylate cyclase of myometrium and its regulation by cAMP-dependent protein kinase

The purpose of this study was to examine activity of adenylate cyclase (AC) and its cAMP-dependent phosphorylation by cAMP-dependent protein kinase (PKA) in the membrane of rabbit myometrium. Isoproterenol (IP) significantly increased AC activity of nonpregnant rabbits myometrium plasma membranes. However, during pregnancy AC of myometrium plasma membranes did not respond to IP. Phosphorylation of the myometrium membrane by PKA was followed by significantly decreased response of AC to IP.     

Differential production of prostaglandins within the human uterus

The ability of broken cell preparations of human endometrium, myometrium and a mixture of endometrium and myometrium to convert 14C arachidonic acid to prostaglandins (PG's) was compared. Endometrium metabolished arachidonic acid predominantly to a mixture of PGF2 alpha and PGE2. A similar weight of myometrium showed relatively little activity, the major product identified was 6 oxo PGF1 alpha. However, a combination of endometrium and myometrium showed an enhanced conversion of arachidonic acid to 6 oxo PGF1 alpha associated with a decreased production of PGF2 alpha and PGE2. This suggests that human endometrium and myometrium differ in their ability to metabolize arachidonic acid and in their ability to convert the endoperoxides formed, to PG's.     

Increased MMPs expression and decreased contraction in the rat myometrium during pregnancy and in response to prolonged stretch and sex hormones

Normal pregnancy is associated with uterine relaxation to accommodate the stretch imposed by the growing fetus; however, the mechanisms underlying the relationship between pregnancy-associated uterine stretch and uterine relaxation are unclear. We hypothesized that increased uterine stretch during pregnancy is associated with upregulation of matrix metalloproteinases (MMPs), which in turn cause inhibition of myometrium contraction and promote uterine relaxation. Uteri from virgin, midpregnant (day 12), and late-pregnant rats (day 19) were isolated, and myometrium strips were prepared for measurement of isometric contraction and MMP expression and activity using RT-PCR, Western blot analysis, and gelatin zymography. Oxytocin caused concentration-dependent contraction of myometrium strips that was reduced in mid- and late-pregnant rats compared with virgin rats. Pretreatment with the MMP inhibitors SB-3CT (MMP-2/MMP-9 Inhibitor IV), BB-94 (batimastat), or Ro-28-2653 (cipemastat) enhanced contraction in myometrium of pregnant rats. RT-PCR, Western blot analysis, and gelatin zymography demonstrated increased mRNA expression, protein amount, and activity of MMP-2 and MMP-9 in myometrium of late-pregnant>midpregnant>virgin rats. Prolonged stretch of myometrium strips of virgin rats under 8 g basal tension for 18 h was associated with reduced contraction and enhanced expression and activity of MMP-2 and MMP-9, which were reversed by MMP inhibitors. Concomitant treatment of stretched myometrium of virgin rats with 17β-estradiol (E2), progesterone (P4), or E2+P4 was associated with further reduction in contraction and increased MMP expression and activity. MMP-2 and MMP-9 caused significant reduction of oxytocin-induced contraction of myometrium of virgin rat. Thus, normal pregnancy is associated with reduced myometrium contraction and increased MMPs expression and activity. The results are consistent with the possibility that myometrium stretch and concomitant increase in sex hormones during pregnancy are associated with increased expression/activity of specific MMPs, which in turn inhibit uterine contraction and promote uterine relaxation.     

Expression of ATP-sensitive potassium channels in human pregnant myometrium

Background  

Potassium channels play critical roles in the regulation of cell membrane potential, which is central to the excitability of myometrium. The ATP-sensitive potassium (KATP) channel is one of the most abundant potassium channels in myometrium. The objectives of this study were to investigate the protein expression of KATP channel in human myometrium and determine the levels of KATP channel in lower and upper segmental myometrium before and after onset of labour.     

EMG activity of the muscular and stromal layer of the cervix in relation to EMG activity of the myometrium and cervical dilatation in PGF2alpha induced parturition in the cow

The goal of this study was to quantify and characterize the electromyographic (EMG) activities in the cervical outer muscular layer and in the cervical stromal layer, and to characterize their relationship with myometrial EMG activity and cervical dilatation during PGF2alpha-induced parturition in term pregnant cows. We continuously measured the EMG activity of the uterine myometrium and cervical outer muscular layer as well as the cervical stromal layer in five cows using bipolar electrodes while at the same time measuring changes in the cervical diameter with ultrasound cervimetry. This we did from the moment a prostaglandin analogue was injected until the expulsion of the calf. In contrast to the cervical stromal layer, the cervical outer muscular layer showed distinct EMG activity, which began to increase at about the same time as the EMG activity of the myometrium, i.e. some 12 h before the start of cervical dilatation. However, the rate of this increase was lower than in the myometrium and it was not characterized, like in the myometrium, by an increase in maximum EMG amplitude. Although the cervical outer muscular layer showed contracture and contraction like EMG activity in unison with in the myometrium, it was also characterized by a more irregular EMG activity, which occurred independently from the myometrium. These data suggest that while the outer muscular layer of the cervix may be considered to be a caudal continuation of the myometrium, it also displays activity independently from the myometrium. The physiological relevance of this activity remains to be explored.     

A polyclonal antiserum against the rabbit progesterone receptor recognizes the human receptor: immunohistochemical localization in rabbit and human uterus

A polyclonal antiserum, raised in guinea pigs immunized with the 116,000 Mr rabbit uterine progesterone receptor (PR), was used to demonstrate immunoreactive PR in frozen fixed sections of rabbit and human uterus. In both species, PR localization was exclusively nuclear. For the rabbit uterus, staining intensity was greatest in the myometrium, followed by endometrial stroma, glands, and luminal epithelium. In premenopausal human endometrium and myometrium there was intense staining of nuclei from proliferative phase glands and myometrium. In the secretory phase the glands failed to stain, yet immunostaining persisted in the myometrium.     

Gestational alterations in phospholipase C coupled muscarinic response

In the pregnant rat, carbachol-induced phosphoinositol hydrolysis by myometrium at the placental attachment region progressively decreased toward term, whereas hydrolysis was relatively stable in the myometrium of the non-attachment region. Tritium-quinuclidinyl benzilate binding increased in the myometrium of non-attachment regions as pregnancy progressed. At placental attachment sites binding remained relatively stable until parturition when it increased. Apparently the myometrium associated with the placental attachment site is less sensitive to cholinergic influence during pregnancy compared with the non-attachment site when evaluated by muscarinic activation of phospholipase C or ligand binding.     

Immunolocalization and expression of small‐conductance calcium‐activated potassium channels in human myometrium

Small‐conductance calcium‐activated potassium (SK3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro. The aims of this study were to identify the precise localization of SK3 channels and to quantify SK3 mRNA expression in myometrium from pregnant and non‐pregnant women. Myometrial biopsies were obtained from pregnant (n = 11) and non‐pregnant (n = 11) women. The expression of SK3 channels was assessed using immunohistochemistry and SK3 mRNA was determined by qRT‐PCR. In non‐pregnant myometrium SK3 immunoreactivity was observed in CD34 positive (CD34⁺) interstitial Cajal‐like cells (ICLC), now called telocytes. Although CD34⁺ cells were also present in pregnant myometrium, they lacked SK3 immunoreactivity. Furthermore, the immunohistochemical results showed that SK3 expression in vascular endothelium was similar between the two groups. CD117 immunoreactivity was only detected in small round cells that resemble mast cells. Compared to non‐pregnant myometrium we found significantly less SK3 mRNA in pregnant myometrium. We demonstrate that SK3 channels are localized solely in CD34⁺ cells and not in smooth muscle cells, and that the molecular expression of SK3 channels is higher in non‐pregnant compared to pregnant myometrium. On the basis of our previous study and the present findings, we propose that SK3 activators reduce contractility in human myometrium by modulating telocyte function. This is the first report to provide evidence for a possible role of SK3 channels in human uterine telocytes.     

Hormone-dependent characteristics of the myocyte cell surface in the human myoma and myometrium cultured in diffusion chambers

The normal and the pathologically changed human myometrium was cultured in the diffusion chamber implanted in the subcutaneous cellular tissue of the rat. In the absence of hormonal influence the growth of myometrium culture is only insignificant, while no myoma cell growth was found at all. Estradiol stimulates the growth and development of the myometrium culture and the myoma. A combined action of estradiol and progesterone stimulates the collagen formation.     

Calcium homeostatic pathways change with gestation in human myometrium

A rise in intracellular calcium is the primary trigger for contractile activity in pregnant human myometrium. It is hypothesized that key proteins involved in myometrial calcium homeostasis are gestationally regulated and play an important role in the preparation for labor. The aims of the study were to investigate the role of sarcoplasmic reticulum Ca ATPases (SERCAs) in regulating spontaneous contractile activity in myometrium, and to determine the expression of SERCA isoforms 2a and 2b, and the plasma membrane Ca ATPase (PMCA), at term and during labor. Western blot analysis demonstrated that the expression of SERCA 2a and 2b significantly increased in myometrium of women in labor compared with those not in labor. The augmentation of contractile activity in laboring myometrium in the presence of a SERCA 2 inhibitor, cyclopiazonic acid (CPA), demonstrated the functional significance of this observation. It is interesting that the application of CPA in the presence of a calcium-activated potassium channel inhibitor to term nonlabor myometrium mimicked the response of myometrium from women in active labor to CPA alone. We conclude that the activity of SERCA isoforms becomes increasingly important in the maintenance of regular contractile activity during labor and may compensate for the functional loss of other calcium control pathways at term.     

足月妊娠PGE2及其受体表达与引产成功率的关系

目的研究足月妊娠子宫平滑肌与蜕膜组织中前列腺素E2（Prostaglandin E2,PGE2）的浓度、子宫平滑肌中PGE2受体-2（PGE2 recepor-2,PGER2）蛋白的表达与缩宫素引产成功率的关系。方法选择缩宫素引产成功与缩宫素引产失败的孕妇,于剖宫产术中取子宫平滑肌及子宫蜕膜组织。分别行ELISA法检测组织匀浆中PTGE2的浓度,Western blot检测子宫平滑肌中PTGER2蛋白的表达。结果缩宫素引产成功组比缩宫素引产失败组子宫平滑肌、子宫蜕膜组织中PTGE2浓度显著增高（P〈0．01）;缩宫索引产成功组比缩宫素引产失败组子宫平滑肌组织中PTGER2蛋白的表达也明显增高（P〈0．01）。结论子宫平滑肌与蜕膜组织中PTGE2浓度、子宫平滑肌组织中PTGER2蛋白的表达量与缩宫素引产成功率关系密切。     

Nitric oxide synthase activity in human trophoblast, term placenta and pregnant myometrium

To investigate the possible role of nitric oxide (NO) produced locally or intramurally in the quiescence of the pregnant myometrium, nitric oxide synthase (NOS) activity was measured in samples from first trimester (villous, and non villous-trophoblast), term placenta and pregnant myometrium. Trophoblast tissue was obtained from psychosocial termination of pregnancy (9 – 12 weeks' gestation) whereas placenta and myometrium, from the same patient, at deliveries by Caesarean section. NOS activity was measured in both cytosolic and particulate fractions by the formation of ¹⁴C-citrulline from ¹⁴C-arginine. Western immunoblotting was used to identify the endothelial NOS (eNOS) and neuronal (nNOS) isoforms. The activity of NOS in particulate fractions from all preparations was considerably higher than the cytosolic fractions. Activity in all fractions except the myometrium was highly Ca-dependent. More than 50% of particulate NOS from the myometrium was Ca-independent. NOS activity was highest in the villous trophoblast and there was a significant difference between the villous and non-villous trophoblast. In placenta and myometrium, NOS was 2–4 fold and 20–28-fold lower than the villous trophoblast, respectively. Western blot analysis showed clearly eNOS in the particulate fraction and a weak eNOS band in the cytosolic fractions, whereas nNOS was not detectable in any of the fractions. In view of the marginal activity of NOS in the myometrium, NO produced by the trophoblast and placenta could play a significant role in maintaining uterine quiescence by paracrine effect.     

Increase in concentrations of prostaglandin endoperoxide synthase and prostacyclin synthase in human myometrium in late pregnancy

Concentrations of prostaglandin endoperoxide synthase (i.e. cyclooxygenase; PGH synthase) and prostacyclin synthase (PGI synthase) were quantified with specific radioimmunometric assays in human myometrium during the last trimester of pregnancy (n = 23) and in non-pregnant controls (n = 8). Pregnant myometrium contained 3 times more PGH synthase per mg microsomal protein than non-pregnant myometrium (p less than 0.01) but there was no increase with increasing gestational age in the third trimester nor with the onset of labor. In pregnancy, as compared to the non-pregnant state, there was no significant change in the PGI synthase content of myometrial microsomes, but significantly more PGI synthase was recovered in other subcellular fractions (p less than 0.01). This suggests that pregnancy affects preferential changes in the subcellular distribution of PGI synthase in myometrial cells. Relative to its PGI synthase content pregnant myometrium contained twice as much PGH synthase as non-pregnant myometrium (p less than 0.01). This may offer further evidence that PGH synthase rather than PGI synthase itself is the rate limiting factor in myometrial PGI2 production. On the other hand, the much larger increase in PGH synthase than in PGI synthase in pregnant as compared to non-pregnant myometrium, may serve to promote preferential synthesis of prostaglandins that are potent myometrial stimulants and of critical importance in human parturition.     

超声造影对子宫内膜病变良恶性鉴别诊断价值的研究

目的：探讨超声造影检查子宫内膜病变灌注特征及其在子宫内膜良恶性疾病中的鉴别诊断价值。方法：选择50例经阴式或腹式超声检查发现子宫内膜病变的术前患者。造影剂选用SonoVue,剂量为2．4mL,以团注方式由肘部浅静脉注入进行实时超声造影检查,观察宫腔内病变和子宫肌层、子宫内膜造影剂的显影特征及增强方式等超声造影表现,绘制时间-强度曲线（TIC）,与病理结果对照并分析各病灶的造影剂灌注特征,比较良、恶性病变的曲线参数的差别。结果：50例子宫内膜病变患者,29例良性病变和21例恶性病变。19例子宫内膜良性病变造影剂多同时或晚于子宫肌层增强,增强均匀,廓清晚于子宫肌层或同步于肌层。16例子宫内膜癌病变增强迅速,呈整体强化,病变区造影剂廓清,出现早于肌层,表现为”快进快出”。通过对时间．强度曲线的分析得出,良性病变与恶性病变平均绝对峰值强度、上升时间和斜率差别有统计学意义。结论：子宫内膜病变不同超声造影灌注模式特征和时间一强度曲线（TIC）不同,并对其良恶性鉴别诊断有重要意义。