Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.     

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.     

Involvement of perivascular neuropeptide Y nerve fibres in uterine arterial vasoconstriction in conjunction with pregnancy

The perivascular neuropeptide Y (NPY) innervation and its relation to adrenergic nerves of uterine arteries from non-pregnant and pregnant guinea pigs was analyzed immunocytochemically. The NPY content of the uterine artery was, in addition, measured radioimmunologically (RIA). Vasomotor effects of NPY per se and in combination with other vasoconstrictors were examined using a sensitive in vitro method. Pregnancy did not visibly affect density and distribution of NPY-immunoreactive fibres. The NPY fibres contained in addition immunoreactivity to dopamine-beta-hydroxylase (marker for noradrenergic neurons). RIA revealed a slight decrease of NPY content during pregnancy, probably due to the increased smooth muscle volume of uterine arteries. The contractile effect of NPY on uterine arteries was weak, while vasoconstriction induced by various agonists was potentiated by NPY, particularly during pregnancy. It is concluded that perivascular NPY-containing nerve fibres may be involved in the dramatic blood flow alterations that occur in the uterine circulation in connection with pregnancy and partus.     

Organization of the guinea-pig uterine innervation. Distribution of immunoreactivities for different neuronal markers. Effects of chemical- and pregnancy-induced sympathectomy

Summary The structural organization of the guinea-pig uterine innervation was investigated by an immunofluorescence method using neurofibrillary protein (NF) and neuron-specific enolase (NSE) as general neuronal markers. NF- and NSE-immunoreactive nerve trunks and non-varicose nerves formed continuous networks similar to nerves with analogue morphology and with immunoreactivities for tyrosine hydroxylase (TH; adrenergic nerves) and neuropeptide Y (NPY). NF- and NSE-immunoreactive non-varicose nerves occurred in the myometrium and along vessels, where TH- and NPY-immunoreactive varicose nerves were also comparatively frequent. After chemical sympathectomy all TH- and NPY-immunoreactive varicose nerves and most NF- and NSE-immunoreactive non-varicose nerves disappeared, suggesting colocalization of TH, NPY, NF and NSE immunoreactivities. During pregnancy all NF-, NSE-, TH- and NPY-immunoreactive nerve structures disappeared in the foetus-bearing uterine horns whereas in the cervix and non-foetus-bearing uterine horns only the myometrial TH- and NPY-immunoreactive varicose nerves disappeared. After parturition there was a complete structural restoration of all types of immunoreactive nerves in previously non-foetus-related tissue. The reinnervation of this tissue followed a similar time-course to that after chemical sympathectomy. In contrast, the reinnervation of previously foetus-related tissue was much slower and incomplete.In conclusion, the whole autonomic uterine innervation undergoes overt structural changes during pregnancy and these changes are related to the foetus-bearing regions.     

An immunohistochemical study on the innervation of SP-IR nerves in the cerebral arteries of the bent-winged bat

Summary The overall distribution of substance P (SP) immunoreactive (IR) nerves surrounding the cerebral arteries of the bent-winged bat were investigated immunohistochemically. In this microchiropteran species, the walls from the vertebral artery to the caudal part of the basilar artery have considerably well-developed plexuses of SP-IR nerves, whereas no demonstrable SP-IR fibers were found in the crostral part of the basilar artery, and in more rostrally located arteries the nerve supply was very sparse or occasionally lacking. This innervation pattern has not yet been established for the cerebral arterial systems of other mammals that have been studied under normal conditions, but it is very similar to the pattern of SP-IR innervation observed in the guinea pig and cat of which the trigeminal ganglia have been destroyed. From the combination of this and other immunohistochemical findings, it is suggested that SP-IR nerves innervating the vertebral and basilar arteries of the bent-winged bat originate from the upper cervical dorsal root ganglia (DRG) and enter the cranial cavity along the vertebral artery and through the meninges.Abbreviations BA basilar artery - CSN cervical spinal nerves - ICS internal carotid system - SCG superior cervical ganglion - SNB sympathetic nerve bundle - VA vertebral artery - VBS vertebro-basilar system     

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.     

Calcitonin gene-related peptide in the rat uterus: presence in nerves and effects on uterine contraction

The influence of calcitonin gene-related peptide (CGRP) on rat uterine activity was examined in concert with the anatomical distribution of CGRP-like immunoreactivity in the uterus. CGRP-like immunoreactivity was localized in nerve fibers; these peptide-containing nerves were abundant throughout the mesometrium of the uterine horn and appeared to innervate mesometrial smooth muscle and vascular smooth muscle. In the uterine wall, CGRP-like immunoreactive fibers were prevalent in the myometrium, endometrium and the endocervix. Fibers in the endometrium and endocervix appeared to form a plexus subjacent to the epithelium and some fibers penetrated the epithelium as an intraepithelial plexus. The action of CGRP (10(-9) to 10(-6) M) on acetylcholine (10(-6) or 10(-5) M)-stimulated uterine activity was examined in vitro. Exogenously applied CGRP induced a dose-dependent relaxation of acetylcholine-stimulated uterine contractions. CGRP had no effect on basal uterine tension. The localization of CGRP-like immunoreactivity in nerves and the relaxing effect of CGRP suggests a role for CGRP-containing nerve fibers in the regulation of uterine activity.     

Galanin and calcitonin gene-related peptide immunoreactivity in nerves of the rat uterus: localization, colocalization, and effects on uterine contractility.

Immunoreactivity to the neuropeptides galanin (GAL) and calcitonin gene-related peptide (CGRP) was examined in nerves in the rat uterus as a prelude to studying their effects on uterine contractility. With immunocytochemical techniques, GAL immunoreactivity (GAL-I) and CGRP-I were localized in myometrial nerves throughout the uterine horns and cervix, with nerves immunoreactive for CGRP being more numerous. Immunocytochemical double-labeling studies revealed GAL coexists with CGRP in a subpopulation of CGRP-I nerve fibers, i.e., GAL-I was not present in all CGRP-I nerves. Effects of these neuropeptides on uterine contractility were examined on in vitro preparations of uterine horns from diethylstilbestrol-treated rats. GAL (10(-5) to 10(-8) M) stimulated uterine contraction in a dose-related manner. CGRP had no effect on basal uterine tension, but CGRP (10(-7) M) reduced GAL-stimulated (10(-7) M) uterine contraction by 92.5%. These results demonstrate that GAL- and CGRP-I are present in, and coexist in, some uterine nerves, presumably afferent nerves. GAL and CGRP could be released from afferent fibers in an "efferent fashion" and influence uterine contractility, GAL having a contractile effect and CGRP having a relaxing effect.     

Quantitative analysis of the density and pattern of adrenergic innervation of blood vessels

Summary Automated quantitative image analysis (QIAF) was used to measure and compare the adrenergic nerve plexuses of 4 blood vessels from the guinea pig, demonstrated by glyoxylic acid fluorescence (GAF). The results showed considerable quantitative variation of plexus density, size of bundles, and numbers of varicosities. A range of alternative procedural and anatomical sources of variability were investigated and assessed. The carotid artery was found to have a dense plexus with more nerves than that of the mesenteric artery; the mesenteric vein and abdominal aorta had sparse plexuses. The carotid artery plexus, despite the density of its nerves, possessed only half the number of varicosities of the mesenteric artery plexus. This sparse varicosity population was shown to have a similar density to the varicosities demonstrated by QIAF in the scattered nerves of the mesenteric vein and abdominal aorta. QIAF confirmed visual estimates of adrenergic plexus density, and was able to demonstrate less obvious differences of nerve density and size, and varicosity populations, between the different plexuses studied. The method is applicable to stretch preparations and transverse sections of many adrenergically innervated tissues.     

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.     

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.     

Distribution, origin and sensitivity to capsaicin of primary afferent substance P-immunoreactive nerves in the heart

This report is intended as an overview of the distribution, origin and sensitivity to capsaicin of substance P-immunoreactive (SP-I) primary afferent cardiac nerves. Immunohistochemical and physiological methods were employed to compare the presence and density of these nerve fibers in the guinea pig and rat hearts. SP-I fibers are numerous in the guinea pig heart including the parietal pericardium, atria, ventricles, valves, coronary arteries and around intrinsic cardiac ganglion cells. The rat heart contains few SP-I fibers. Vagotomy does not influence the number of intensity of immunoreactive fibers in the guinea pig heart. By stimulating the atrium or ventricle and recording from the second or third thoracic dorsal roots Ad1, Ad2 and C fibers were demonstrated in the atria, but only Ad fibers in the guinea pig ventricle; in addition, only Ad fibers were recorded from the vagus nerves. Only Ad1 fibers were demonstrated in the rat heart. Treatment with capsaicin depletes the SP-I and decreases the conduction velocity of C-fibers and some Ad2 fibers in the guinea pig heart. We suggest that SP-I primary afferent nerve fibers are unmyelinated (C-type) or small myelinated (Ad2-type) nerves in the guinea pig heart and that their cell bodies of origin are predominantly in dorsal root ganglia.     

An immunohistochemical study on the innervation of SP-IR nerves in the cerebral arteries of the bent-winged bat

The overall distribution of substance P (SP) immunoreactive (IR) nerves surrounding the cerebral arteries of the bent-winged bat were investigated immunohistochemically. In this microchiropteran species, the walls from the vertebral artery to the caudal part of the basilar artery have considerably well-developed plexuses of SP-IR nerves, whereas no demonstrable SP-IR fibers were found in the rostral part of the basilar artery, and in more rostrally located arteries the nerve supply was very sparse or occasionally lacking. This innervation pattern has not yet been established for the cerebral arterial systems of other mammals that have been studied under normal conditions, but it is very similar to the pattern of SP-IR innervation observed in the guinea pig and cat of which the trigeminal ganglia have been destroyed. From the combination of this and other immunohistochemical findings, it is suggested that SP-IR nerves innervating the vertebral and basilar arteries of the bent-winged bat originate from the upper cervical dorsal root ganglia (DRG) and enter the cranial cavity along the vertebral artery and through the meninges.     

Co-existence and origin of peptidergic and adrenergic nerves in the guinea pig uterus

Summary The occurrence and distribution of peptidergic nerves in the guinea pig uterus was studied by means of immunocytochemistry using numerous neuropeptide antisera. Neuropeptide Y (NPY)-immunoreactive (IR) nerves were the most abundant, whereas substance P (SP)-, calcitonine gene-related peptide (CGRP)-, and neurokinin A (NKA)-IR nerves were less frequent, and peptide histidine isoleucine (PHI)-IR nerves were the most sparse. Chemical sympathectomy by means of 6-hydroxydopamine, and capsaicin treatment revealed the division of the peptidergic nerves into three separate populations: (1) NPY-IR nerves, which co-existed with adrenergic nerves, (2) SP-, CGRP-and NKA-IR nerves, which mutually co-existed, and (3) PHI-IR nerves. Parallel-running adrenergic/NPY-IR and SP-IR nerves could be found with very similar although not completely identical morphological appearance. Paracervical ganglia contained neurotensin-and dynorphin A-IR cell bodies in addition to cell bodies with immunoreactivities similar to those in prevertebral ganglia. Combined retrograde tracing with True blue and immunocytochemistry showed that the adrenergic and NPY-IR uterine nerves originate in paracervical and prevertebral ganglia. In the prevertebral ganglia the cellular origin was the same for adrenergic and NPY-IR nerves. In contrast, SP-, CGRP-,and NKA-IR nerves originated in dorsal root ganglia. At full-term pregnancy all the neuropeptide immunoreactivities had vanished, probably reflecting a fetus-induced general nerve degeneration.     

Plasmodium falciparum parasitaemia in the first half of pregnancy, uterine and umbilical artery blood flow, and foetal growth: a longitudinal Doppler ultrasound study

ABSTRACT: BACKGROUND: During early pregnancy, the placenta develops to meet the metabolic demands of the foetus. The objective of this analysis was to examine the effect of malaria parasitaemia prior to 20 weeks' gestation on subsequent changes in uterine and umbilical artery blood flow and intrauterine growth restriction. METHODS: Data were analysed from 548 antenatal visits after 20 weeks' gestation of 128 women, which included foetal biometric measures and interrogation of uterine and umbilical artery blood flow. Linear mixed effect models estimated the effect of early pregnancy malaria parasitaemia on uterine and umbilical artery resistance indices. Log-binomial models with generalized estimating equations estimated the effect of early pregnancy malaria parasitaemia on the risk of intrauterine growth restriction. RESULTS: There were differential effects of early pregnancy malaria parasitaemia on uterine artery resistance by nutritional status, with decreased uterine artery resistance among nourished women with early pregnancy malaria and increased uterine artery resistance among undernourished women with early pregnancy malaria. Among primigravidae, early pregnancy malaria parasitaemia decreased umbilical artery resistance in the late third trimester, likely reflecting adaptive villous angiogenesis. In fully adjusted models, primigravidae with early pregnancy malaria parasitaemia had 3.6 times the risk of subsequent intrauterine growth restriction (95% CI: 2.1, 6.2) compared to the referent group of multigravidae with no early pregnancy malaria parasitaemia. CONCLUSIONS: Early pregnancy malaria parasitaemia affects uterine and umbilical artery blood flow, possibly due to alterations in placentation and angiogenesis, respectively. Among primigravidae, early pregnancy malaria parasitaemia increases the risk of intrauterine growth restriction. The findings support the initiation of malaria parasitaemia prevention and control efforts earlier in pregnancy.     

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.     

Changes in cholinergic and noradrenergic nerves in the pregnant and postpartum uterus of the albino rat and guinea pig

The present study was undertaken to investigate the structural changes in both cholinesterase (ChE)-positive nerve fibers and adrenergic nerves with formaldehyde-induced fluorescence in pregnant and postpartum uteri of both the albino rat and guinea pig. Particular attention was directed to the relationship between these changes and the local factors associated with the growing fetus. ChE reaction was absent in the control and pregnant uterus of the guinea pig. In the albino rat, there were signs of degeneration in pregnancy. These were evidenced by vacuolation of large nerve trunks and the presence of focal segments with very faint reaction along the course of the nerve bundles. Myometrial segments from fetus-containing horns showed some fragmented nerve fibers, but at the same time some other normal ones. Most of the fine nerve bundles gave a weak reaction. Three weeks after delivery, multiple ChE fibers were found in the uterus of the albino rat. The normal appearance was, however, not regained and some nerve fibers were still fragmented. Noradrenergic (NA) nerve fibers were disintegrated and markedly reduced in number in the myometrium of the pregnant uterus of both the guinea pig and albino rat, particularly in the uterine horns that were distended by fetuses. The number of NA fibers was not significantly reduced in the tubal ends of the albino rat uterus. Three weeks after delivery, normal NA fibers were seen in the myometrium of both the albino rat and guinea pig uterus. Nerves with reduced fluorescence reaction were observed less frequently.     

Pregnancy-stimulated growth of vascular smooth muscle cells: Importance of protein kinase C-dependent synergy between estrogen and platelet-derived growth factor

Dramatic smooth muscle cell (SMC) growth occurs in the uterine artery during pregnancy. The potential for pregnancy-associated growth may also exist at other vascular sites. We tested the hypothesis that increased growth of uterine artery SMC isolated from pregnant (vs. nonpregnant) guinea pigs would be detectable in culture, that pregnancy-associated phenotypic changes would also be found in nonuterine vascular cells (aortic SMC), and that the enhanced growth would be dependent on estrogen, peptide growth factors like platelet-derived growth factor (PDGF), and protein kinase C (PKC). Growth responses were measured by [³H]-thymidine incorporation and cell counts. Uterine artery SMC from pregnant guinea pigs grew to a higher plateau density with serum stimulation, had increased spontaneous DNA synthesis and persistent growth following serum withdrawal, and were more responsive to 3–30 ng/ml PDGF-BB than nonpregnant cells. Aortic SMC from pregnant animals also grew to a higher plateau density and had enhanced responsiveness to PDGF-BB. This increased response to PDGF-BB by pregnant uterine artery and aortic SMC (40–233% increase over nonpregnant PDGF result) was reproduced in nonpregnant cells by pretreatment for 1–24 h with 17-beta(β)-estradiol (30–100 nM). Neither the pregnancy-induced difference nor the estradiol pretreatment was associated with increased PDGF-BB binding activity. The synergistic effect of 17β-estradiol was partially (62%) reproduced with 17-alpha(α)-estradiol, an isomer which does not bind the estrogen receptor. This suggested that 17β-estradiol modulates the PDGF-BB response by both estrogen-receptor- and nonreceptor-mediated mechanisms. To test if the estrogen effects were dependent on PKC, two different antagonist strategies (3 μM dihydrosphingosine and phorbol-ester-induced downregulation) were applied prior to 17α- or β-estradiol and blocked the enhanced responses to PDGF. The synergistic effect of 17β-estradiol on PDGF was then reproduced by 1 h pretreatment with the cell-permeable PKC activator, 10 nM PMA. We conclude that pregnancy stimulates increased growth of uterine and aortic SMC in vitro which is dependent on estrogen, PDGF, and PKC and may be important in vascular remodeling during pregnancy. © 1996 Wiley-Liss, Inc.     

Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

Ontogenetic development of the guinea pig uterine innervation. An immunohistochemical study of different neuronal markers, neuropeptides and S-100 protein

The ontogenetic development of the guinea pig uterine autonomic innervation was studied immunohistochemically using neurofibrillary protein (NF) and neuron specific enolase (NSE) as general neuronal markers, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) as specific markers for adrenergic innervation and S-100 protein as marker for Schwann cell structure and/or function. In addition, comparisons were made of the development of the different populations of peptide-containing nerves. The structure and time of appearance were similar for nerves with NF-, NSE-, TH- and DBH-immunoreactivities, which were first present in the organ periphery as coarse nerve trunks, then extending centrally and branching into non-varicose nerves. From these, varicose nerves developed first in relation to vessels and then in association with the myometrial smooth musculature. Development was completed earlier in the cervix than in the uterine horns suggesting differences in local environment. In comparison, S-100 nerve-immunoreactivity appeared later but attained complete development more rapidly than axonal structures. Neuropeptide Y-immunoreactive nerves showed a similar developmental pattern to presumed adrenergic nerves, further verifying the assumption of intraneuronal localization of NPY in uterine adrenergic nerves. Other peptide-containing nerves were developed later probably reflecting differences in neuronal growth properties.