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.     

Functional and histochemical characterization of a uterine adrenergic denervation process in pregnant rats

The time course of pregnancy-induced changes in the contractile responses of isolated uterine rings and sympathetic innervation pattern were studied using electric field stimulation and histofluorescence techniques, respectively, in intact and 6-hydroxydopamine-treated rats. Neurally mediated contractions elicited by field stimulation (0.6 msec, 1-70 Hz, 40 V) were measured in uterine preparations obtained from nonpregnant, 6-hydroxydopamine-treated and 5-, 10-, 15-, 18-, and 22-day (term) pregnant rats. At all frequencies, the amplitudes of contractions were highest in nonpregnant uteri. Stimulation at 1-2.5 Hz evoked contractions in 10-day pregnant uteri but failed to cause contractions on Day 5 and from Day 15 onward. In uterine preparations obtained from term and from 6-hydroxydopamine-treated rats, contractions could not be evoked by stimulation at 1-20 Hz. Fluorescence histochemistry of uterine adrenergic nerves revealed rich perivascular and myometrial innervation in nonpregnant and in pregnant rats through Day 10. Degeneration and loss of adrenergic nerve fibers was apparent by Day 15, and fluorescent myometrial and perivascular nerves were practically absent by Day 22. These findings demonstrate a progressive, frequency-related reduction of nerve-mediated uterine contractions beginning in midterm pregnancy, in parallel with a gradual loss of adrenergic nerve fibers. Pregnancy-induced nerve degeneration may promote the development of nonsynaptic alpha-adrenergic uterine contractile activity towards term. The reduced responsiveness of uterine smooth muscle to electric field stimulation in early pregnancy appears to be unrelated to alterations in uterine innervation but may be related to changes associated with implantation.     

Uterine trauma and limb defects

The temporary clamping of the uterine blood vessels on one side of the uterus during late pregnancy in the rat (days 14-16) results in hemorrhage and tissue necrosis in the extremities of the fetuses from the experimental side and occasionally from the control side. A further series of experiments showed that similar fetal hemorrhage followed the temporary clamping (45 minutes) of the uterine wall or uterine fat, excluding major uterine vessels; handling the uterus for 5 minutes; and stretching of the uterine blood vessels. A low incidence of fetal hemorrhage was also associated with laparotomy alone, but the fetuses were unaffected by extensive handling of the uterus through the abdominal wall or by intraperitoneal anesthesia. Fetal hemorrhage was also induced by a short episode of severe maternal hyperthermia but not by a high dose of ethanol given by gavage. These results suggest that a range of uterine trauma may result in fetal hemorrhage, perhaps through a common mechanism.     

Uterine autonomic nerve innervation plays a crucial role in regulating rat uterine mast cell functions during embryo implantation

To explore the potential mechanism of how uterine innervations would affect the uterine mast cell (MC) population and functions during the periimplantation. We herein first examined the consequence of uterine neurectomy on embryo implantation events. We observed that amputation of autonomic nerves innervating the uterus led to on-time implantation failure in rats. Exploiting MC culture and ELISA approaches, we then further analyzed the effect of neurectomy on cellular histamine levels and its release from uterine MCs, to elucidate the relation of the autonomic nerves and local cellular immunity in the uterine during early pregnancy. We observed that disconnection of autonomic nerve innervation significantly increased the population of uterine MCs. Most interestingly, these increased number of uterine MCs in neuroectomized rats contained a much reduced cellular level of histamine. Our subsequent challenge experiments revealed that uterine MCs in nerve amputated rats exhibited enhanced histamine releasing rate in response to substance P and antiIgE, suggesting loss of nerve innervation in the uterus not only increases the population of uterine MCs, but also facilitates the release of histamine from MCs, thus subsequently interfere with the normal implantation process. Collectively, our findings provide a new line of evidence supporting the concept that immune–neuro-endocrine network plays important role during pregnancy establishment and maintenance.     

Congenital facial palsy and emotion processing: The case of Moebius syndrome

According to the Darwinian perspective, facial expressions of emotions evolved to quickly communicate emotional states and would serve adaptive functions that promote social interactions. Embodied cognition theories suggest that we understand others' emotions by reproducing the perceived expression in our own facial musculature (facial mimicry) and the mere observation of a facial expression can evoke the corresponding emotion in the perceivers. Consequently, the inability to form facial expressions would affect the experience of emotional understanding. In this review, we aimed at providing account on the link between the lack of emotion production and the mechanisms of emotion processing. We address this issue by taking into account Moebius syndrome, a rare neurological disorder that primarily affects the muscles controlling facial expressions. Individuals with Moebius syndrome are born with facial paralysis and inability to form facial expressions. This makes them the ideal population to study whether facial mimicry is necessary for emotion understanding. Here, we discuss behavioral ambiguous/mixed results on emotion recognition deficits in Moebius syndrome suggesting the need to investigate further aspects of emotional processing such as the physiological responses associated with the emotional experience during developmental age.     

Uterine adrenergic and cholinesterase-positive nerves and myometrial catecholamine concentrations during pregnancy in sheep

Uterine adrenergic and cholinesterase (AChE)-positive innervation of the sheep uterus during anestrus and at 4 stages of pregnancy were examined by histochemical methods. In addition, uterine and cervical myometrium concentrations of norepinephrine (NE) and dopamine (DA) were determined using high-performance liquid chromatography. During anestrus, adrenergic and AChE-positive nerve fibers in the uterine myometrium and endometrium were primarily associated with the vasculature. Innervation of myometrial smooth muscle was almost exclusively by adrenergic fibers. In the endometrium, fibers of both types were observed closely associated with endometrial glands, and adrenergic fibers were observed in the connective tissue beneath the luminal epithelium. Density of uterine innervation decreased by day 65 of pregnancy with an additional decrease by day 105. Myometrial NE concentrations were higher in the cervix than the uterus. Uterine NE concentrations generally were not affected by pregnancy. Although cervical NE per gram of tissue decreased during pregnancy, this effect of pregnancy was not detected when NE was expressed per microgram of DNA. Myometrial DA concentrations were higher in uterine segments than in the cervix. DA concentrations decreased during pregnancy in all tissues except the posterior uterine segment. The DA to NE ratio in the uterus was greater than that for the cervix and was not generally affected by the stage of pregnancy. These results demonstrate that cholinergic and adrenergic nerves supply the sheep uterus. Decreasing fiber density during pregnancy suggests that a majority of the innervation to the sheep uterus is supplied by 'short' nerve fibers whose activity is regulated by steroids of pregnancy. The possible role of DA as a neurotransmitter in the sheep uterus is discussed.     

Development of the graft versus host reaction and its influence on pregnancy in mice following heparin administration]

The influence of heparin on the graft-versus-host reaction (GVHR) and the peculiarities attending the development of pregnancy in female animals which survived after the GVHR were studied. Preliminary administration of heparin to the recipients prevented their death or increased their life span. An intensification of the GVHR was noted after the administration of heparin to donors or its addition to the transplanted cells. In mice which survived after the GVHR as a result of heparin administration the intrauterine fetuses death and abortions were noted in 60-100% of cases during subsequent pregnancy (3 to 6 months after the cell transplantation). In the case of repeated pregnancies of these female animal pathology of pregnancy was less frequent; however, some of the offspring displayed the rant syndrome. No such disturbances of pregnancy were observed in mice given heparin alone or in those which survived after the transplantation of lymphoid cells only. The sustained pregnancy promoted the intensification of the GVHR induced in the female animals earlier after the heparin administration.     

Death receptors mediate the adverse effects of febrile-range hyperthermia on the outcome of lipopolysaccharide-induced lung injury

We have shown that febrile-range hyperthermia enhances lung injury and mortality in mice exposed to inhaled LPS and is associated with increased TNF-α receptor activity, suppression of NF-κB activity in vitro, and increased apoptosis of alveolar epithelial cells in vivo. We hypothesized that hyperthermia enhances lung injury and mortality in vivo by a mechanism dependent on TNF receptor signaling. To test this, we exposed mice lacking the TNF-receptor family members TNFR1/R2 or Fas (TNFR1/R2(-/-) and lpr) to inhaled LPS with or without febrile-range hyperthermia. For comparison, we studied mice lacking IL-1 receptor activity (IL-1R(-/-)) to determine the role of inflammation on the effect of hyperthermia in vivo. TNFR1/R2(-/-) and lpr mice were protected from augmented alveolar permeability and mortality associated with hyperthermia, whereas IL-1R(-/-) mice were susceptible to augmented alveolar permeability but protected from mortality associated with hyperthermia. Hyperthermia decreased pulmonary concentrations of TNF-α and keratinocyte-derived chemokine after LPS in C57BL/6 mice and did not affect pulmonary inflammation but enhanced circulating markers of oxidative injury and nitric oxide metabolites. The data suggest that hyperthermia enhances lung injury by a mechanism that requires death receptor activity and is not directly associated with changes in inflammation mediated by hyperthermia. In addition, hyperthermia appears to enhance mortality by generating a systemic inflammatory response and not by a mechanism directly associated with respiratory failure. Finally, we observed that exposure to febrile-range hyperthermia converts a modest, survivable model of lung injury into a fatal syndrome associated with oxidative and nitrosative stress, similar to the systemic inflammatory response syndrome.     

Birth weight corrected for gestational age is related to the incidence of Down's syndrome pregnancies.

Three recent studies reported that early depletion of the primordial follicle pool is likely to be an independent risk factor for Down's syndrome pregnancies. The size of the primordial follicle pool at birth is determined by oogenesis and by the rate of follicle atresia during the intra uterine period. Since intra uterine growth retardation was reported to be associated with a significantly reduced primordial follicle pool at birth, we investigated the possibility of a relation between low birth weight for gestational age and the risk of a Down's syndrome pregnancy. In a case control study, 95 women with a history of a Down's syndrome pregnancy and 85 controls provided information on their own birth weight and length of gestation. Birth weight standard deviation scores, indicating the difference in birth weight from a reference group, were significantly lower in Down's syndrome mothers than in controls. These findings illustrate that the risk of a Down's syndrome pregnancy is related to a low birth weight corrected for gestational age, possibly by a causal relation between intra uterine growth retardation and the size of the primordial follicle pool.     

Biochemical changes in the developing rat central nervous system due to hyperthermia

Rat embryos at 10 days of gestation were exposed to 43 degrees C for 8 minutes by submerging the exteriorized right uterine horn in heated saline solution and then reinserting the uterine horn into the abdominal cavity. At 15 days, the fetuses were removed, and cells from the cerebral hemispheres were dissociated and grown as primary cultures. Embryos from the left uterine horn served as controls. No morphological changes were observed between the cultures of cells from control and heat-exposed embryos at different days in culture. However, exposure of embryos to hyperthermia at 10 days significantly affected the developmental pattern of activities of acetylcholine esterase associated with cholinergic neurons and of 2',3'-cyclic nucleotide phosphohydrolase associated with oligodendrocytes and myelin membrane formation. These results suggest that hyperthermia at 10 days of gestation in the rat may lead to an impairment in the development of neurons and oligodendrocytes in the central nervous system.     

Glucocorticoid regulation of human and ovine parturition: the relationship between fetal hypothalamic-pituitary-adrenal axis activation and intrauterine prostaglandin production

Birth in many animal species and in humans is associated with activation of hypothalamic-pituitary-adrenal function in the fetus and the increased influence of glucocorticoids on trophoblast cells of the placenta and fetal membranes. We suggest that in ovine pregnancy glucocorticoids directly increase fetal placental prostaglandin production, and indirectly increase prostaglandin production by maternal uterine tissues through the stimulation of placental estradiol synthesis. The events of ovine parturition are compared with those of human parturition. In the latter, we suggest similar direct effects of glucocorticoids on prostaglandin synthesis and metabolism in fetal membranes and similar indirect effects mediated by glucocorticoid-stimulated increases in intrauterine corticotropin-releasing hormone expression.     

Surgical correction of genital prolapse in three rhesus monkeys

Three adult female rhesus monkeys (Macaca mulatta) in a breeding colony of approximately 75 adult females developed a clinical condition characterized by protrusion of the cervix through the vulva during pregnancy and/or following parturition. The Gilliam round-ligament uterine ventro-suspension procedure (hereafter called the Gilliam uterine suspension or uterine suspension procedure) was used to return the cervix to a normal anatomical position. Following the procedure, one female delivered a normal live infant, but reprolapsed. After a second suspension procedure, she again became pregnant and delivered a normal live infant without a reoccurrence of the prolapse. A second animal never became pregnant despite repeated breedings to different males for two years. The third animal became pregnant twice following the procedure. The first pregnancy terminated in abortion at two months of gestation, while the second pregnancy ended in an apparent dystocia, necessitating a cesarean section and delivery of a dead fetus. The animal died post-operatively. This surgical procedure successfully salvaged one of these animals which otherwise had no reproductive future.     

Maternal fever and neural tube defects

It has been proposed that hyperthermia in the pregnant woman is associated with neural tube defects in her offspring. We analyzed retrospective interview data for a maternal history of probable febrile illness during the first trimester of pregnancy among mothers of infants with anencephaly or spina bifida. There were two control groups--mothers of infants with Down syndrome and mothers of infants with cleft lip or palate. With the Down syndrome group serving as controls, the incidence of febrile illness among mothers of all infants with neural tube defects was significantly elevated. With the cleft group as controls, the fever incidence was not significantly increased in the neural tube defect groups. When the combined cleft and Down syndrome controls were used, only mothers of the spina bifida group had an elevated fever incidence. Epidemiology data suggest an association of maternal fever during pregnancy with neural tube defects in the offspring.     

The placental problem: Linking abnormal cytotrophoblast differentiation to the maternal symptoms of preeclampsia

The placenta is a remarkable organ. In normal pregnancy its specialized cells (termed cytotrophoblasts) differentiate into various specialized subpopulations that play pivotal roles in governing fetal growth and development. One cytotrophoblast subset acquires tumor-like properties that allow the cells to invade the decidua and myometrium, a process that attaches the placenta to the uterus. The same subset also adopts a vascular phenotype that allows these fetal cells to breach and subsequently line uterine blood vessels, a process that channels maternal blood to the rest of the placenta. In the pregnancy complication preeclampsia, which is characterized by the sudden onset of maternal hypertension, proteinuria and edema, cytotrophoblast invasion is shallow and vascular transformation incomplete. These findings, together with very recent evidence from animal models, suggest that preeclampsia is associated with abnormal placental production of vasculogenic/angiogenic substances that reach the maternal circulation with the potential to produce at least a subset of the clinical signs of this syndrome. The current challenge is to build on this knowledge to design clinically useful tests for predicting, diagnosing and treating this dangerous disorder.     

Cow attributes,herd management,and reproductive history events associated with abortion in cow-calf herds from Western Canada

The primary objective of this study was to identify herd management and cow characteristics that are associated with abortion in cow-calf herds in Western Canada. Reproductive events were closely monitored in 29,713 cows in 203 herds from the beginning of the breeding season in 2001 through the calving season in 2002. Herd management and cow-level risk factors such as age, body condition score, and previous reproductive history were measured through a series of herd visits by project personnel and detailed individual animal records maintained by the herd owner. Pregnancy status was assessed in fall of 2001 by the herd veterinarian. Cows most likely to abort were replacement heifers, cows that were more than 10 years of age, cows with a body condition score of less than or equal to or 5 of 9 at pregnancy testing, or with twin pregnancies. Cows vaccinated for bovine viral diarrhea virus and infectious bovine rhinotracheitis and bred on community pastures were less likely to abort than cows from community pastures that were not vaccinated. Cows bred on community pastures that were not vaccinated were also more likely to abort than cows that were not on community pastures regardless of vaccination status. Adverse calving-associated events such as severe dystocia, problems such as uterine prolapse or retained placentas, abortion or calf death within 1 hour of birth were also associated with an increased risk of abortion the subsequent calving season after accounting for all other factors.     

Reproductive biology in the era of genomics biology

Current and emerging technologies in reproductive biology, including assisted reproductive technologies and animal cloning, are discussed in the context of the impact of genomics era biology. The discussion focuses on the endocrinology associated with establishment and maintenance of pregnancy, fetal-placental development, lactation, and neonatal survival. Various aspects of uterine biology, including development during the neonatal period and function in adult females, are discussed with respect to reproductive efficiency. It is clear that combining strategies for use of conventional animal models for studying the reproductive system with new genomics technologies will provide exceptional opportunities in discovery research involving data integration and application of functional genomics to benefit animal agriculture and the biomedical community. New and emerging biotechnologies and comparative genomics approaches will greatly advance our understanding of genes that are critical to development of the reproductive system and to key events at each stage of the reproductive cycle of females and males.     

Expression of scaffolding, signalling and contractile-filament proteins in human myometria: effects of pregnancy and labour

Successful parturition requires the co-ordination of numerous myometrial signalling events to allow for timely and efficient uterine contractions. Late pregnancy and labour onset in humans may be associated with changes in the expression of myometrial proteins implicated in such uterine contractile signal integration. Accordingly, in myometria from non-pregnant women and pregnant women, not in labour or in labour, we examined the content of putative plasmalemmal scaffolding proteins (caveolin-1 and -2) and compared these to the proportions of signal transducing rho-associated kinases (ROKalpha and beta) and contractile filament-associated proteins alpha-actin, myosin regulatory light chain (MLC(20)) and h-caldesmon. There was no effect of pregnancy or labour on the proportion of caveolin, ROK betaor alpha-actin. However, pregnancy was associated with a decrease in ROKalpha and MLC(20) such that ROK alpha: alpha-actin and MLC(20): alpha-actin ratios were reduced compared to myometria of non-pregnant women. In contrast, h-caldesmon was up-regulated in pregnancy resulting in an elevated h-caldesmon: alpha-actin ratio. There were, however, no further significant changes in ROK alpha, MLC(20) or h-caldesmon expression with spontaneous or oxytocin-induced labour. These data suggest that the mechanism(s) integrating myometrial signalling events with the onset of human labour does not involve differential alterations of the cellular expressions of caveolins, ROK, alpha-actin, MLC(20) or h-caldesmon.     

Pregnancy reduces noradrenaline but not neuropeptide levels in the uterine artery of the guinea-pig

Summary Using histochemical, immunohistochemical and biochemical techniques, noradrenaline-, neuropeptide Y-, vasoactive intestinal polypeptide-, substance P- and calcitonin gene-related peptide-containing nerve fibres were studied in the uterine artery of virgin, progesterone-treated and pregnant guinea-pigs. Morphological changes following hormone treatment or in pregnancy were also evaluated in a quantitative study on semithin sections of the uterine artery. In late pregnancy, the number of noradrenalinecontaining nerve fibres, which formed the densest plexus in virgin animals, was significantly decreased, a finding supported by a significant reduction in noradrenaline levels. This reduction was not mimicked by systemic progesterone treatment. In contrast, the innervation of the uterine artery by neuropeptide Y-containing nerve fibres was increased in pregnancy, while the other peptidergic nerves and peptide levels were unchanged after progesterone treatment and in pregnancy. These changes led to a predominance of innervation by neuropeptide Y- rather than noradrenaline-containing nerve fibres in late pregnancy. No morphological changes were detected following progesterone treament, but pregnancy led to a marked increase in the cross-sectional area of the vessel accompanied by an increase in the thickness of the media.     

Teratogen update: methotrexate

Methotrexate and aminopterin are folic acid antagonists that inhibit dihydrofolate reductase, resulting in a block in the synthesis of thymidine and inhibition of DNA synthesis. Methotrexate has been used for the treatment of malignancy, rheumatic disorders, and psoriasis and termination of intrauterine pregnancy. Recently, methotrexate has become a standard treatment for ectopic pregnancy. The misdiagnosis of an intrauterine pregnancy as an ectopic pregnancy can result in exposure of a continuing pregnancy to dose levels of methotrexate of 50 mg/m(2) (maternal body surface area). Experimental animal studies have associated methotrexate therapy with embryo death in mice, rats, rabbits, and monkeys. Structural malformations have been most consistently produced in rabbits at a maternal dose level of 19.2 mg/kg. Abnormalities in rabbits include hydrocephalus, microphthalmia, cleft lip and palate, micrognathia, dysplastic sacral and caudal vertebrate, phocomelia, hemimelia, syndactyly, and ectrodactyly. Based on human case reports of methotrexate exposure during pregnancy, a methotrexate embryopathy has been described that includes growth deficiency, microcephaly, hypoplasia of skull bones, wide fontanels, coronal or lambdoidal craniosynostosis, upswept frontal scalp hair, broad nasal bridge, shallow supraorbital ridges, prominent eyes, low-set ears, maxillary hypoplasia, epicanthal folds, short limbs, talipes, hypodactyly, and syndactyly. This syndrome may be associated with exposures between 6 and 8 weeks after conception and dose levels of 10 mg/week or greater. More recent case reports of methotrexate exposure for the misdiagnosis of ectopic pregnancy involve treatment before 6 weeks after conception and have raised the suggestion of a distinct syndrome due to such early exposures. Tetralogy of Fallot and perhaps other neural crest cell-related abnormalities may be features of this early syndrome. A disproportionality analysis of methotrexate and aminopterin case reports and series provides support for pulmonary atresia, craniosynostosis, and limb deficiencies as reported more often than expected in methotrexate-exposed children. Denominator-based data will be welcome to better define elements of a methotrexate embryopathy and possibly to distinguish an early exposure syndrome from anomalies traditionally associated with methotrexate exposure.