Degradation of a developmentally regulated mRNA in Xenopus embryos is controlled by the 3' region and requires the translation of another maternal mRNA.

By injecting the appropriately constructed plasmids into one-cell Xenopus embryos, we determined that the 3' region of the maternal Xenopus Eg2 mRNA confers instability on the chimeric mRNA transcribed from these plasmids. This instability, like that of the maternal Eg2 transcript, was abolished by treatment of the embryos with cycloheximide. Analysis of the polysome distribution of the maternal Eg2 mRNA in cycloheximide-treated and untreated embryos showed that Eg2 mRNA was released from polysomes after fertilization and that the stabilization caused by cycloheximide treatment was not due to a reloading of ribosomes onto the mRNA. Insertion of a stable hairpin loop (delta G = -50 kcal/mol) 5' to the reporter gene in the injected plasmid caused a 10- to 20-fold decrease in translation from the transcribed mRNAs. This decrease in translation did not abolish the instability conferred by the 3' Eg2 region. Therefore, the degradation of these chimeric mRNAs in Xenopus embryos requires the translation of another maternal mRNA coding for a trans-acting factor involved in mRNA degradation. Further restriction of the 3' Eg2 region, placed 3' to the reporter gene, showed that a cis-acting instability-conferring sequence is contained in a 497-nucleotide fragment.

Elevated beta-human chorionic gonadotropin and testosterone in cord serum of male infants of diabetic mothers.

Leydig cell hyperplasia is a common histological finding in male infants of diabetic mothers. The functional correlates of this histological finding were investigated by measuring beta hCG, testosterone, androstenedione, dihydrotestosterone, and progesterone in mixed cord serum of male and female infants of diabetic mothers (n = 40) and normal mothers (n = 40) at term. Male and female infants of diabetic mothers had significantly higher cord serum beta hCG levels than male and female controls. Male infants of diabetic mothers had significantly higher cord serum testosterone concentrations than male controls, female controls, and female infants of diabetic mothers. Cord serum testosterone concentrations were similar in female infants of diabetic mothers and female controls. In the male infants of diabetic mothers, there was a significant positive correlation between beta hCG and testosterone (r = 0.64; P less than 0.01). There was no significant correlation between beta hCG and testosterone in the male controls (r = -0.15; P = NS). There was no significant difference in cord serum dihydrotestosterone in any group tested. Cord serum progesterone was significantly higher in the males than in the females. Cord serum androstenedione was lower in the infants of diabetic mothers than in the controls. This study suggests that the Leydig cell hyperplasia found in male infants of diabetic mothers is due, in part, to elevated concentrations of hCG and is accompanied by elevated testosterone concentrations in the fetal compartment.

Potassium chloride effects on the hormonal signal transduction mechanisms underlying phasic myometrial contractions.

These studies sought to test the hypothesis that potassium-stimulated phasic myometrial contractions utilize cytosolic calcium oscillation-like mechanisms comparable to those activated in response to oxytocin. Uterine tissue was obtained from pro-oestrus/oestrus Sprague-Dawley rats. In vitro isometric contraction studies were performed using longitudinal myometrial strips; computer digitalized contraction data were analyzed for contraction area, and normalized for tissue cross-section area. Dose-response studies were performed using potassium chloride with and without inhibitors of cytosolic calcium oscillation mechanisms. Qualitative inositol-phosphate production studies were performed after preloading uterine tissue with [3H]inositol; subsequently, the individual inositol-phosphates produced in response to stimulation were isolated by anion exchange chromatography. Potassium chloride over a concentration of 10 to 30 mM produced a dose-related increase in phasic contractile activity. The potassium-stimulated phasic contractions were significantly suppressed in response to inhibition of phospholipase C, stimulation of protein kinase C, inhibition of calcium-induced calcium release, and prevention of extracellular calcium influx. The qualitative inositol-phosphate production studies confirmed activation of phospholipase C in response to 20 mM potassium. These studies have provided support for the hypothesis that potassium-stimulated phasic myometrial contractions activate intracellular signal transduction mechanisms comparable to those activated in response to hormonal uterotonic agonists.

Recurrent inversion of the puerperal uterus managed with 15(s)-15-methyl prostaglandin F2 alpha and uterine packing.

The present report describes an unusual case of recurrent puerperal uterine inversion causing major postpartum hemorrhage. Blood replacement, oxytocin, and ergot therapy along with manual reduction failed to prevent immediate recurrence, but treatment with 15(S)-15-methyl prostaglandin F2 alpha (Prostin 15M) and uterine packing were successful. It is recommended that 15(S)-15-methyl prostaglandin F2 alpha be available in all obstetric suites for the management of similar emergencies.

Pregnancy complicated by the Kasabach-Merritt syndrome.

The Kasabach-Merritt syndrome is the association of cavernous hemangiomas and consumption coagulopathy marked by anemia, thrombocytopenia, and hypofibronigenemia. Exacerbation of the consumption coagulopathy has been described in the 2 previous reports of this syndrome when associated with pregnancy. The authors report a third patient whose delivery and postopartum course were marked by increased coagulation abnormalities and subsequent hemorrhage. This patient's 32-day hospital course and need for multiple blood transfusions, clotting factors, platelets, heparin, and finally epsilon-aminocaproic acid underscore the need for patients with this syndrome to deliver in a referral center hospital where replacement therapy and hematologic consultation are readily available.

Pregnancy complicated by homozygous hypercholesterolemia.

The course of pregnancy in a woman with homozygous type IIa hypercholesterolemia is described. Despite prepartum cholesterol levels as high as 700 mg/dL, her cholesterol level increased further during gestation. Pathological examination of the placenta did not reveal insufficiency or vasculopathy.

Delivery of the second twin.

A retrospective chart review of all twin deliveries between April 1, 1977, and March 20, 1980, at the Boston City Hospital and the Brigham and Women's Hospital revealed corrected neonatal mortality rates of 0 for 74 twins in nonvertex presentation delivered by cesarean section and 0 for 76 second-born twins extracted vaginally. Breech extraction of 76 second-born twins weighing more than 1499 g at birth was not associated with a statistically significant increase in the incidence of 5-minute Apgar scores of 7 or less when compared to 74 similarly asymptomatic twins in nonvertex presentation delivered by cesarean section. Vaginal delivery may be considered when the second twin, weighing more than 1500 to 2000 g, is in breech presentation.

Continuous subcutaneous insulin therapy during early pregnancy.

Intensive metabolic control of diabetes is probably important during formation of the embryo early in pregnancy. The purpose of this study was to determine the efficacy and complications of continuous subcutaneous insulin infusion therapy during the fifth to the tenth week of gestation. Twenty-four insulin-dependent subjects were trained to use blood glucose self-monitoring and the Auto Syringe portable insulin infusion pump (AS6C). Regular insulin was administered as a basal infusion of 18 +/- 8 U/24 hours (+/- SD) (12.2 +/- 3.9 mU . kg-1 . h-1) and as bolus injections of 6 +/- 3 U before meals and 1.2 +/- 1 U before snacks. Reasonable control of fasting (119 +/- 30 mg/dL) and postprandial (133 +/- 34 mg/dL) hyperglycemia was achieved, accompanied by an average of 2.2 +/- 1.5 symptomatic hypoglycemic episodes per week. The frequency of complications with this new therapy declined as the authors gained experience in teaching the system. The persistence of good diabetic control in many of the subjects after they returned to conventional insulin therapy points to the need for a controlled trial of continuous subcutaneous insulin infusion therapy versus intensive conventional therapy in pregnancy.

The relationship between oxytocin, phosphoinositide-specific phospholipase C, and phasic myometrial contractions.

OBJECTIVES: Oxytocin-stimulated uterine contractions are associated with repetitive cycles of elevated cytosolic calcium, i.e., cytosolic calcium oscillations. The studies in this report were performed to test the hypothesis that phosphoinositide-specific phospholipase C (PI-PLC) is an important component of the myometrial intracellular oscillator. METHODS: In vitro isometric contraction studies were performed using longitudinal strips of myometrium from nonpregnant, adult Sprague-Dawley rats. Cumulative dose-response studies were performed using oxytocin and aluminum fluoride with and without 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate (NCDC), an inhibitor of PI-PLC. RESULTS: Stimulation of G-proteins coupled to PI-PLC with aluminum fluoride resulted in a significant increase in phasic myometrial contractions comparable to those produced by oxytocin. Inhibition of PI-PLC with NCDC resulted in significant suppression of oxytocin- and aluminum fluoride-stimulated myometrial contractions. In contrast, doses that suppressed agonist-stimulated contractions had only a minimal effect on KCl-stimulated tonic myometrial contractions. CONCLUSIONS: These studies provide significant support for the novel hypothesis that PI-PLC is an important component of the agonist-stimulated cytosolic calcium oscillator that generates phasic myometrial contractions.

Effects of sodium and calcium channel blockade on cytosolic calcium oscillations and phasic contractions of myometrial tissue.

OBJECTIVE: These studies sought to test the hypothesis that agonist-stimulated cytosolic calcium oscillations and phasic myometrial contractions are dependent on calcium influx through dihydropyridine-sensitive calcium channels, but not sodium influx through tetrodotoxin-sensitive sodium channels. METHODS: Cytosolic calcium imaging studies and in vitro isometric contraction studies were performed using uterine tissue from proestrus/estrus Sprague-Dawley rats. The calcium imaging studies were performed after loading partial thickness strips of myometrium with Fura-2. For the in vitro isometric contraction studies, the contraction data were computer digitalized, analyzed for contraction area, and normalized for cross-section area. The effects of nifedipine (1.0-5 mumol/L), a calcium channel blocker, were compared to tetrodotoxin (0.01-1 mumol/L), a sodium channel blocker. RESULTS: Oxytocin-stimulated simultaneous cytosolic calcium oscillations and phasic contractions were completely inhibited by 1 mumol/L nifedipine; in contrast, 1 mumol/L tetrodotoxin had no effect on the oxytocin-stimulated calcium oscillations and contractions. Oxytocin, aluminum fluoride, potassium chloride, and ionomycin stimulated in vitro phasic myometrial contractions. Tetrodotoxin had no effect on these agonist-stimulated phasic contractions, whereas nifedipine produced a significant, dose-related inhibition of the phasic contractile activity. CONCLUSIONS: The studies described in this report support the hypothesis that the influx of extracellular calcium is an important component of the cellular mechanisms responsible for the cytosolic calcium oscillations occurring during phasic myometrial contractions. In contrast, sodium influx through tetrodotoxin-sensitive sodium channels does not appear to play a comparably important role.

Thrombin-stimulated uterine contractions in the pregnant and nonpregnant rat.

OBJECTIVE: Thrombin generated during the active clotting of blood appears to be a potent uterotonic agonist; however, the mechanism underlying this effect on uterine smooth muscle is not well understood. We performed studies to confirm the uterotonic effects of thrombin and to determine whether prostaglandin production plays a role during the uterotonic effects of thrombin or clotting blood. METHODS: Uterine contraction studies were performed using adult nonpregnant and near-term pregnant rats. The in vitro isometric contraction studies used uterine strips pretreated with indomethacin or vehicle (ethanol), which were then stimulated with thrombin. For the in vivo contraction studies, rats were pretreated with intraperitoneal injections of indomethacin or vehicle (ethanol) then stimulated by intraluminal injection of fresh rat blood or thrombin into the uterus. The contraction data were acquired using isometric force transducers, were computer digitized, normalized for spontaneous activity, and statistically analyzed. Prostaglandin (PG) F2alpha was measured using an enzyme-linked immunoassay. RESULTS: The in vitro contraction studies demonstrated that both thrombin and actively clotting blood produce a significant increase in the frequency and intensity of uterine contractions. Thrombin stimulation was associated with a 54% increase in PGF2alpha concentration in vitro; indomethacin (1 microM) pretreatment completely inhibited that increase in PGF2alpha production. Despite the suppression of PGF2alpha production, pretreatment with indomethacin had no inhibitory effect on thrombin-stimulated contractile activity. In vivo contraction studies further confirmed that indomethacin (2 mg/kg) pretreatment had no effect on blood- or thrombin-stimulated contractile activity. CONCLUSIONS: We confirmed that thrombin and thrombin produced by actively clotting blood had a robust uterotonic effect in the rat and that prostaglandin production did not play a significant role in thrombin-stimulated contractions.

Intracellular signaling and phasic myometrial contractions.

This article reviews recently reported observations regarding the intracellular signal transduction mechanisms involved in the generation of phasic contractions occurring in myometrial tissue. The presence of cell surface receptors for classic uterotonic agonists (including oxytocin, norepinephrine, vasopressin, acetylcholine, and prostaglandins [PGs]) has been well described; all are seven-membrane-spanning, G protein-coupled receptors. Occupancy of these receptors, coupled through members of the Gq and/or Gi families of heterotrimeric G proteins, results in stimulation of the phospholipase C-beta (PLC-beta) isoforms. Nonclassic uterotonic agonists, such as growth factors and cytokines, also activate the phosphatidylinositol (PI)-signaling pathway, in this case through tyrosine kinase receptor-mediated activation of the phospholipase C-gamma (PLC-gamma) isoforms. Several recent reports have demonstrated that activation of the PI-signaling pathway in uterine myocytes results in the development of cytosolic calcium oscillation-like phenomena. These cytosolic calcium oscillations appear to arise from repetitive cycles of emptying and refill of the endoplasmic reticulum calcium stores along with the influx of extracellular calcium. Calcium release from the endoplasmic reticulum calcium stores appears to be mediated by the inositol trisphosphate-sensitive and the ryanodine-sensitive receptor/channels; isoforms for both the these receptor/channels have been shown to be expressed in myometrial tissue. In summary, receptor-mediated activation of the PI-signaling pathway and the generation of cytosolic calcium oscillations appear to produce intermittent calcium transients that result in the development and maintenance of phasic myometrial contractions.

The mechanisms underlying Bay K 8644-stimulated phasic myometrial contractions.

OBJECTIVE: Phasic myometrial contractions appear to be produced by calcium transients resulting from the activation of the phosphatidylinositol-signaling pathway. Bay K 8644, an L-type calcium channel activator, produces an increase in frequency and intensity of phasic myometrial contractions. These studies were performed to test the hypothesis that Bay K 8644-stimulated contractions were mediated through mechanisms involving phosphoinositide-specific phospholipase C activation and cytosolic calcium oscillation-like mechanisms. METHODS: In vitro contraction studies and intracellular calcium imaging were performed on longitudinal strips of uterine tissue obtained from mature virgin Sprague-Dawley rats. Isometric contraction data were computer digitized, analyzed for contraction area, and normalized for cross-sectional area. Dose-response studies were performed using previously reported inhibitors of cytosolic calcium oscillation mechanisms. In addition, qualitative inositol-phosphate production studies were performed after prelabeling uterine tissue in vitro with 3H-inositol. Subsequently, the labeled inositol phosphates were separated and recovered using anion exchange chromatography. RESULTS: Bay K 8644 produced periodic calcium transients or oscillations along with a dose-related increase in contractile activity and a significant increase in inositol-phosphate production. In contrast, neomycin (an inhibitor of phospholipase C), adenine (an inhibitor of calcium-induced calcium release), nifedipine (an L-type calcium channel blocker), and EGTA (a calcium chelator) significantly inhibited Bay K 8644-stimulated contractile activity. CONCLUSIONS: These results are consistent with the hypothesis that Bay K 8644, through its facilitation of increased intracellular calcium, results in the activation of the phosphatidylinsitol-signaling pathway and cytosolic calcium oscillation-like phenomena, thereby resulting in the generation of phasic myometrial contractions.

Constitutive androstane receptor expression in the rat cervix during gestation.

OBJECTIVE: To investigate expression of the constitutive androstane receptor (CAR) in the pregnant rat cervix. METHODS: Rat uterine tissue was obtained on gestational days 12, 16, 20, 21, and 22 (the day of parturition), and postpartum day 1. In addition, liver, lung, kidney, heart, and skeletal muscle tissue were obtained. Expression of the two known CAR isoforms was evaluated using reverse transcriptase-polymerase chain reaction. RESULTS: These studies confirmed CAR expression in the liver; however, CAR was not demonstrated in the myometrium or cervical tissue. CONCLUSIONS: The currently described CAR1 and CAR2 isoforms are not expressed in rat uterine tissue; therefore, they do not appear to participate in parturition in the pregnant rat.

Tyrosine kinase-mediated activation of cytosolic calcium oscillations and phasic myometrial contractions.

These studies sought to test the hypothesis that tyrosine kinase-stimulated phasic myometrial contractions are mediated by activation of the phosphatidylinositol (PI)-signaling pathway and the generation of cytosolic calcium oscillations. For these studies, uterine tissue was obtained from adult female Sprague-Dawley white rats during the proestrus/estrus phase of the cycle. In vitro contraction studies were performed using pervanadate (a tyrosine phosphatase inhibitor) with and without inhibitors of the PI-signaling pathway, including 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (a phospholipase C inhibitor), thimerosal (an inositol-trisphosphate receptor/channel inhibitor), and Ruthenium red (a ryanodine receptor inhibitor), and with oxytocin or prostaglandin F2 alpha (two classic uterotonic agonists). Cytosolic calcium studies were performed using Fura-2-loaded myometrial strips. During these studies, pervanadate was observed to produce cytosolic calcium oscillations and phasic contractions in myometrial tissue comparable to those produced in response to oxytocin and prostaglandin F2 alpha. The pervanadate-stimulated phasic contractions were significantly suppressed in response to inhibition of phospholipase C, the inositol-trisphosphate receptor, and the ryanodine receptor, thereby confirming the importance of the PI-signaling pathway during tyrosine kinase-associated myometrial activity. Further confirming the important and shared role for the PI-signaling pathway during pervanadate-stimulated myometrial contractions, no significant additive effects were observed when classic uterotonic agonists such as oxytocin or prostaglandin F2 alpha were combined with pervanadate.

Gq-protein alpha subunit expression and distribution in pregnant rat myometrial tissues.

OBJECTIVE: Multiple G-protein isoforms play an integral role in signal transduction; the Gq subtype of G-protein alpha subunits is involved in the activation of the phosphatidylinositol signaling pathway. The studies described herein evaluate the expression of Gq, along with Gs and Gi, in pregnant and nonpregnant rat myometrial tissues. METHODS: Myometrium and other tissues were obtained from nonpregnant and timed-pregnant Sprague-Dawley rats. Western blot studies were performed using polyclonal G-protein isoform-specific antibodies. Immunohistochemical studies were performed using the same antibodies with specimens of myometrium, intestine, and skeletal muscle. RESULTS: The Western blot studies confirmed differential expression of all types of G-protein alpha subunit subtypes in rat myometrial tissues. In pregnant rat myometrium, the expression of Gq and Gs was sustained through day 22, whereas, Gi expression decreased on day 20 and remained low through the remainder of gestation. The immunohistochemical studies revealed significant staining for Gq and Gs in the myometrial layers of the pregnant and nonpregnant rat uterus; in contrast, immunostaining for Gi was minimal in nonpregnant myometrium, and even lower in myometrium from pregnant uteri. CONCLUSIONS: These studies have confirmed expression of the Gq, Gi, and Gs alpha subunits in rat myometrial tissue. Immunohistochemistry confirmed that Gq was expressed at high levels in the myometrial layer of the pregnant and nonpregnant uterus. These observations support the hypothesis that Gq expression is critically important for the transduction of hormone signals, such as those responsible for the generation of phasic myometrial contractions.

Tissue-specific protein kinase C isoform expression in rat uterine tissue.

OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a key role during the generation of agonist-stimulated phasic myometrial contractions. Protein kinase C (PKC), a component of this signaling pathway, has been previously shown to produce feedback inhibition of agonist-stimulated myometrial contractions. The studies described in this report were performed to survey the tissue-specific expression of several PKC isoforms in the rat uterus. METHODS: Uterine tissue was obtained from timed pregnant and normally cycling adult female Sprague-Dawley rats. Immunohistochemical studies were performed using the Vectastain ABC immunostaining technique and PKC isoform-specific polyclonal antibodies. Western blot studies were performed using myometrial tissue separated into cytosol and membrane fractions by differential centrifugation. RESULTS: These studies confirmed significant expression of the PKC-alpha, -beta 2, -delta, -eta, and -zeta isoforms in myometrium from pregnant and estrus rats, whereas only trace or no expression of the PKC-beta 1, -gamma, -epsilon, and -theta isoforms was observed. Expression of the PKC-alpha, -beta 2, and -eta isoforms decreased modestly during the latter days of gestation; in contrast, PKC-delta and -zeta remained stable during this period. The immunohistochemical studies confirmed expression of the PKC-alpha, -beta 2, -delta, -eta, and -zeta isoforms in both circular and longitudinal smooth-muscle layers of the near-term pregnant rat uterus. CONCLUSION: In summary, these studies have confirmed significant levels of expression of several isoforms of PKC in estrus and near-term pregnant rat uterine tissue, which was most prominent in the smooth-muscle cells of the myometrium.

Absence of alpha-2 adrenergic effects on cAMP production in a genital tract smooth muscle cell line.

This study sought to evaluate alpha-2 and beta adrenergic modulation of cAMP production in the DDT1 MF-2 transformed smooth muscle myocyte. After stimulation with forskolin or adrenergic agonists with or without subtype specific antagonists, cAMP production was determined. These experiments confirmed an increase of cAMP in response to forskolin, isoproterenol, epinephrine, and norepinephrine; the adrenergic stimulation was inhibited by propranolol. On the other hand, the alpha-2 agonist clonidine did not inhibit cAMP production. Likewise, alpha-2 receptor blockade did not increase cAMP production in response to epinephrine. These studies, therefore, suggest that the DDT1 MF-2 myocyte does not contain a significant population of functional alpha-2 adrenergic receptors.

Gestational modulation of myometrial proteins in the timed-pregnant Sprague-Dawley rat.

These studies sought to test the hypothesis that the expression of myometrial proteins is modulated as the onset of parturition approaches. Myometrial proteins from timed-pregnant rats were analyzed utilizing sodium dodecylsulfate polyacrylamide gel and 2-dimensional electrophoresis, and Western blot techniques. SDS-PAGE gels demonstrated increased expression of at least 10 protein bands from 17 to 200+ KD. 2-dimensional gels confirmed the presence of at least five groups of gestationally modulated proteins. Western blots for phosphoinositide-specific phospholipase C demonstrated significant modulation of the expression of three isozymes. These studies have confirmed differential expression of myometrial proteins near term in the timed-pregnant rat; some of which play an important role in intracellular signal transduction in response to hormones and pharmacologic agents.

Adrenergic stimulation of diacylglycerol production in genital tract smooth muscle myocytes.

Diacylglycerol (DAG) production has not been reported in previous studies that have characterized inositol phosphate production during alpha-1 adrenergic receptor signal transduction in the DDT1 MF-2 genital tract myocytes. The current study sought to measure norepinephrine (NE)-stimulated DAG production in these transformed myocytes utilizing thin layer chromatography. DAG production was characterized as an alpha-1 adrenergic mediated event utilizing subtype specific adrenergic agonist and antagonists. DAG production occurred in response to physiologic concentration of NE, was apparent by 30 s and was significantly increased by 2 min. Maximal DAG production was unaffected by pretreatment of the myocytes for 96 h with testosterone, which has previously been shown to induce a doubling of alpha-1 adrenergic receptors in these cells. In contrast, testosterone pretreatment did result in a shift of the dose-response curve resulting in a significantly lower EC50 for NE in the treated cells compared to control myocytes. In conclusion, these studies have confirmed that DAG production occurs as a component of alpha-1 adrenergic signal transduction in the DDT1 MF-2 myocytes; transduction events that were modulated by testosterone resulting in increased agonist sensitivity.