The role of sensory neurons in cervical ripening: effects of estrogen and neuropeptides.

Central nervous system nuclei and circuits, such as the medial preoptic, ventromedial and paraventricular nuclei of the hypothalamus, play important roles in reproduction and parturition, and are influenced by estrogen. Peripheral autonomic and sensory neurons also play important roles in pregnancy and parturition. Moreover, the steroid hormone estrogen acts directly, not only on the reproductive tract organs (uterus and cervix), but also on the central and peripheral nerves by regulating expression of various neuronal genes. The peripheral primary afferent neurons innervating the uterine cervix relay mechanical and biochemical sensory information induced by local cervical events and by passage of fetuses, to the spinal cord and supraspinal centers. Consequently, the birth process in mammals is influenced by the combined action of neurons and hormones. Peripheral sensory stimuli, induced physiologically by fetal expulsion or mechanically by vaginocervical stimulation, alter behavior, as well as autonomic and neuroendocrine systems. Recent evidence indicates that primary afferent neurons innervating the cervix, in addition to their sensory effects, likely exert local "efferent" actions on the ripening cervix near term. These efferent effects may involve estrogen-regulated production of such neuropeptides as substance P and calcitonin gene-related peptide in lumbosacral dorsal root ganglia, and their release in the cervix. Collectively, these findings suggest an interrelationship among estrogen, cervix-related sensory neurons, and local cervical events near term.     

Galanin-immunoreactive nerves in the female rat paracervical ganglion and uterine cervix: distribution and reaction to capsaicin

Summary The presence and distribution of galanin-immunoreactivity was examined in the uterine cervix and paracervical autonomic ganglia of the female rat. Some animals were treated with capsaicin to determine if galanin-immunoreactivity was present in small-diameter primary afferent nerves. Other animals were treated with the noradrenergic neurotoxin 6-hydroxydopamine to ascertain if galanin-immunoreactivity was present in sympathetic noradrenergic nerves. Galanin-immunoreactive nerve fibers were sparse in the cervical myometrium and vasculature, but numerous in the paracervical ganglion where they appeared to innervate principal neurons. Immunoreactivity was also present in dorsal root ganglia, dorsal horn of spinal cord, and inferior mesenteric ganglia. Capsaicin treatment resulted in a marked reduction of galanin-immunoreactivity in the spinal cord dorsal horn, but not in the dorsal root ganglia, paracervical ganglia, or cervix (although there was a substantial reduction of substance P-, neurokinin A-, and calcitonin gene-related peptide-immunoreactivity in the dorsal horn, dorsal root ganglia, and uterine cervix). 6-Hydroxydopamine treatment did not cause any appreciable change in the galanin-immunoreactivity in any tissues. We conclude that galanin-like immunoreactivity is expressed in nerve fibers innervating the paracervical ganglia and uterine cervix of the female rat. This immunoreactivity is probably present in afferent nerves and could play a role in neuroendocrine reflexes and in reproductive function.     

Guinea-pig interpubic joint (symphysis pubica) relaxation at parturition: Underlying cellular processes that resemble an inflammatory response

Background  

At term, cervical ripening in coordination with uterine contractions becomes a prerequisite for a normal vaginal delivery. Currently, cervical ripening is considered to occur independently from uterine contractions. Many evidences suggest that cervical ripening resembles an inflammatory process. Comparatively little attention has been paid to the increased flexibility of the pelvic symphysis that occurs in many species to enable safe delivery. The aim of this study was to investigate whether the guinea-pig interpubic joint relaxation process observed during late pregnancy and parturition resembles an inflammatory process.     

Effects of capsaicin on reproductive function in the female rat: Role of peptide-containing primary afferent nerves innervating the uterine cervix in the neuroendocrine copulatory response

Summary Nerves immunoreactive for the peptides substance P, neurokinin A, calcitonin gene-related peptide or cholecystokinin-octapeptide innervate the uterine cervix in the rat. Nerve terminals are associated with the myometrial and vascular smooth muscle and are distributed throughout the endocervix. These nerves degenerate following neonatal capsaicin treatment indicating that they are small-diameter, unmyelinated, C-type primary afferent nerves. Adult female rats, treated with capsaicin as neonates, exhibit decreased fertility following mating and diminished sensitivity to the induction of pseudopregnancy following copulomimetic electrical stimulation of the cervix. The results also demonstrate that hypothalamo-adenohypophyseal-ovarian interactions, corpus luteum progesterone secretion and uterine sensitivity to progesterone are normal in capsaicin-treated rats. Taken together, these data suggest that the reproductive dysfunction observed in capsaicin-treated rats is due to destruction of the afferent limb of the neuroendocrine copulatory response that facilitates the luteal progesterone secretion necessary to support pregnancy or pseudopregnancy. Thus, it is concluded that the afferent limb of this neuroendocrine response in the rat consists primarily of unmyelinated, peptide-containing, C-type primary afferent nerves.A preliminary report of this study was presented at the Substance P and Neurokinins Symposium in Montreal, 1986, and published in the symposium proceedings (Traurig et al. 1987)     

Expression of pituitary adenylate cyclase activating peptide in the uterine cervix, lumbosacral dorsal root ganglia and spinal cord of rats during pregnancy

The uterine cervix is highly innervated by the sensory nerves containing neuropeptides which change during pregnancy and are regulated, in part, by estrogen. These neuropeptides act as transmitters both in the spinal cord and cervix. The present study was undertaken to determine the expression pattern of the neuropeptide pituitary adenylate cyclase activating peptide (PACAP) in the cervix and its nerves during pregnancy and the influence of estrogen on this expression using immunohistochemistry, radioimmunoassay and RT-PCR. PACAP immunoreactivity was detected in nerves in the cervix, lumbosacral (L6-S1) dorsal root ganglia (DRG) and spinal cord. PACAP immunoreactivity was highest at day 15 of pregnancy in the cervix and dorsal spinal cord, but then decreased over the last trimester of pregnancy. However, levels of PACAP mRNA increased in the L6-S1 DRG at late pregnancy relative to early pregnancy. DRG of ovariectomized rats treated with estrogen showed increased PACAP mRNA synthesis in a dose-related manner, an effect partially blocked by the estrogen receptor (ER) antagonist ICI 182,780. We postulate that synthesis of PACAP in L6-S1 DRG and utilization in the cervix and spinal cord increase over pregnancy and this synthesis is the under influence of the estrogen-ER system. Since PACAP is expressed by sensory nerves and may have roles in nociception and vascular function, collectively, these data are consistent with the hypothesis that sensory nerve-derived neuronal factors innervate the cervix and play a role in cervical ripening.     

Timing of neutrophil activation and expression of proinflammatory markers do not support a role for neutrophils in cervical ripening in the mouse

The mechanisms that facilitate remodeling of the cervix in preparation for and during parturition remain poorly understood. In the current study, we have evaluated the timing of inflammatory cell migration in cervix through comparisons between wild-type mice and steroid 5alpha-reductase type 1 null mice (Srd5a1-/-), which fail to undergo cervical ripening due to insufficient local progesterone metabolism. The timing of migration and distribution of macrophages, monocytes, and neutrophils were examined using cervices from wild-type and Srd5a1-/- mice before Day 15 (d15) and during cervical ripening (late d18), and postpartum (d19). Neutrophil numbers were quantitated by cell counts and activity was estimated by measurement of myeloperoxidase activity. The mRNA and/or protein expression of neutrophil chemoattractants, CXCL2 and CXCL1, and other proinflammatory and adhesion molecules, including IL1A, IL1B, TNF, CCL11, CCL5, CCL3, ITGAM, and ICAM1, were measured in cervices collected before, during, and after birth. The effect of neutrophil depletion on parturition was tested. Tissue macrophages, myeloperoxidase activity, and expression of proinflammatory molecules are not increased within the cervix until after birth. Neutrophil numbers do not change after birth and neutrophil depletion before term has no effect on timing or success of parturition. These results suggest that cervical ripening does not require neutrophils. Moreover, neutrophil activation and a general inflammatory response are not initiated within the cervix until shortly after parturition. The timing of inflammatory cell migration and activation in pregnant cervix suggest a role for these cells in postpartum remodeling of the cervix rather than in the initiation of cervical ripening at parturition.     

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.     

Effects of RU486 on the interstitial collagenase in the process of cervical ripening in the pregnant rat.

Changes in interstitial collagenase activity in the rat uterine cervix during ripening were clarified in a time-dependent manner. Premature delivery was induced by an antiprogesterone agent, RU486, for rats in late pregnancy. The presence of interstitial collagenase in the extract from the rat cervical tissue was demonstrated, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using the natural and unaffected collagen as a substrate. The collagenase activity was determined as the release of digested peptides from the radio-labeled collagen. Our experiments with RU486 were performed in rats on the 18th day of pregnancy. A single administration of RU486 (15 mg/kg) resulted in the premature delivery of all treated rats within 30 h after the injection (average time was 23.9 h). The marked increase in cervical wet weight was observed up to the time to premature delivery along with a significant acceleration from 18 h after the administration of RU486. In this state, the cervical collagenase activity was enhanced, the highest levels being recorded at 21 h after the administration. The interstitial collagenase in the uterine cervix appears to play a significant role in the regulation mechanisms of cervical ripening in late pregnant rats.     

The parturition defect in steroid 5alpha-reductase type 1 knockout mice is due to impaired cervical ripening.

Successful delivery of the fetus (parturition) depends on coordinate interactions between the uterus and cervix. A majority (70%) of mice deficient in the type 1 isozyme of steroid 5alpha-reductase fail to deliver their young at term and thus manifest a parturition defect. Using in vitro and in vivo measurements we show here that rhythmic contractions of the uterus occur normally in these mutant mice at the end of gestation. In contrast, the cervix of the mutant animal fails to ripen at term as judged by biomechanical, histological, and endocrinological assays. Impaired metabolism of progesterone in the cervix of the mutant mice in late gestation leads to an accumulation of this steroid in the tissue. We conclude that a failure of cervical ripening underlies the parturition defect in mice lacking steroid 5alpha-reductase type 1 and that this enzyme normally plays an essential role in cervical progesterone catabolism at the end of pregnancy.     

Vagal afferents from the uterus and cervix provide direct connections to the brainstem

Previous anatomical studies demonstrated vagal innervation to the ovary and distal colon and suggested the vagus nerve has uterine inputs. Recent behavioral and physiological evidence indicated that the vagus nerves conduct sensory information from the uterus to the brainstem. The present study was undertaken to identify vagal sensory connections to the uterus. Retrograde tracers, Fluorogold and pseudorabies virus were injected into the uterus and cervix. DiI, an anterograde tracer, was injected into the nodose ganglia. Neurectomies involving the pelvic, hypogastric, ovarian and abdominal vagus nerves were performed, and then uterine whole-mounts examined for sensory nerves containing calcitonin gene-related peptide. Nodose ganglia and caudal brainstem sections were examined for the presence of estrogen receptor-containing neurons in ”vagal locales." Labeling of uterine-related neurons in the nodose ganglia (Fluorogold and pseudorabies virus) and in the brainstem nuclei (pseudorabies virus) was obtained. DiI-labeled nerve fibers occurred near uterine horn and uterine cervical blood vessels, in the myometrium, and in paracervical ganglia. Rats with vagal, pelvic, hypogastric and ovarian neurectomies exhibited a marked decrease in calcitonin gene-related peptide-immunoreactive nerves in the uterus relative to rats with pelvic, hypogastric, and ovarian neurectomies with intact vagus nerves. Neurons in the nodose ganglia and nucleus tractus solitarius were immunoreactive for estrogen receptors. These results demonstrated: (1) the vagus nerves serve as connections between the uterus and CNS, (2) the nodose ganglia contain uterine-related vagal afferent neuron cell bodies, and (3) neurons in vagal locales contain estrogen receptors.     

Calcitonin gene-related peptide-receptor component protein expression in the uterine cervix, lumbosacral spinal cord, and dorsal root ganglia

The neuropeptide calcitonin gene-related peptide (CGRP) may play a role in neurogenic inflammation, tissue remodeling of the uterine cervix, promoting vasodilation, parturition, and processing of sensory information in the spinal cord. CGRP-immunoreactive nerves of the cervix and spinal cord have been studied but cellular identification of the CGRP receptor has received little attention. CGRP-receptor component protein (CGRP-RCP) is a small protein associated with the CGRP receptor; thus, immunostaining for the CGRP-RCP can be used to identify sites of the CGRP receptor. We determined sites of CGRP-RCP immunoreactivity relative to the presence of CGRP-ir nerve fibers in the female rat uterine cervix, spinal cord, and dorsal root ganglia. CGRP-RCP immunoreactivity was expressed in the dorsal horn of the spinal cord, venules of the uterine cervix, and perikarya of sensory neurons in dorsal root ganglia. CGRP-immunoreactive fibers were adjacent to CGRP-RCP-immunoreactive vessels in the cervix and among CGRP-RCP-immunoreactive structures in the dorsal horn of the spinal cord. This suggests CGRP-RCP is associated with structures innervated by CGRP nerves and these interactions may be changed in tissues in response to an appropriate stimulus.     

Characterization of vascular endothelial growth factor (VEGF) in the uterine cervix over pregnancy: effects of denervation and implications for cervical ripening.

Bilateral neurectomy of the pelvic nerve (BLPN) that carries uterine cervix-related sensory nerves induces dystocia, and administration of its vasoactive neuropeptides induces changes in the cervical microvasculature, resembling those that occur in the ripening cervix. This study was designed to test the hypothesis that (a) the cervix of pregnant rats expresses vascular endothelial growth factor (VEGF) and components of the angiogenic signaling pathway [VEGF receptors (Flt-1, KDR), activity of protein kinase B, Akt (phosphorylated Akt), and endothelial nitric oxide synthase (eNOS)] and von Willebrand Factor (vWF) and that these molecules undergo changes with pregnancy, and (b) bilateral pelvic neurectomy (BLPN) alters levels of VEGF concentration in the cervix. Using RT-PCR and sequencing, two VEGF isoforms, 120 and 164, were identified in the rat cervix. VEGF, VEGF receptor-1 (Flt-1), eNOS, and vWF immunoreactivities (ir) were localized in the microvasculature of cervical stroma. Their protein levels increased during pregnancy but decreased to control levels by 2 days postpartum. VEGF receptor-2 (KDR)-ir was confined to the epithelium of the endocervix. BLPN downregulated levels of VEGF by a third. Therefore, the components of the angiogenic signaling pathway are expressed in the cervix and change over pregnancy. Furthermore, angiogenic and sensory neuronal factors may be important in regulating the dynamic microvasculature in the ripening cervix and may subsequently play a role in cervical ripening and the birth process.     

P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy

ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X₃-receptor subtype showed P2X₃- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X₃-receptor-immunoreactive DRG neurons to the cervix. Some P2X₃-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X₃-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition.     

Macrophage trafficking in the uterus and cervix precedes parturition in the mouse.

Immune activation is implicated in the etiology of preterm labor, but little is known about macrophage number or distribution in the uterus or cervix at term. This study tested the hypothesis that macrophages migrate into the reproductive tract before the onset of parturition. Paraffin-embedded sections from the mid-uterine horn and cervix of C3/HeN mice on Days 15 and 18 of pregnancy, the day of birth (Day 19), and 1 day postpartum were stained with a pan-macrophage marker to analyze cell numbers and distribution. During pregnancy, uterine macrophages were dispersed in endometrium, usually associated with vasculature and subluminal epithelium. In myometrium, macrophages were clustered in stromal connective tissue; near term and postpartum, cells appeared to surround the muscle bundles. Total macrophage numbers were increased on Day 15 relative to those in nonpregnant controls, declined before birth, and increased postpartum. In the cervix, macrophages congregated in subepithelium, often perivascular or near ganglia. Macrophage numbers in the cervix peaked on Day 18, then declined to nonpregnant levels by the day after birth. Thus, macrophage numbers in the uterus were inversely related to those in the cervix. These findings raise the possibility that macrophages and their products may be involved in uterine contractility and cervical remodeling during the processes of parturition.     

The peptidergic innervation of the guinea pig uterine artery in pregnancy

The influence of pregnancy on the density and pattern of the peptidergic innervation of the guinea pig uterine artery was studied. Whole mount stretch preparations of the uterine artery from estrus and late pregnant guinea pigs were processed for the immunohistochemical demonstration of neuropeptide Y (NPY)-, vasoactive intestinal polypeptide (VIP)-, calcitonin gene-related peptide (CGRP)- and substance P (SP)- immunoreactive nerve fibres. In late pregnancy the density of NPY- and CGRP- containing nerve fibres was remarkably decreased, while that of VIP- and SP- immunoreactive nerves showed a moderate reduction. The meaning and the possible physiological relevance of the decreased density of peptide-immunoreactive nerves in the uterine artery in late pregnancy are discussed.     

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in pelvic nerves supplying the posterior large intestine of the toad,Bufo marinus

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in the posterior portion of the large intestine of the toad was studied using single- and dual-label immunohistochemistry. Neurons containing colocalized galanin/somatostatin or vasoactive intestinal peptide alone were observed along intramural pelvic nerves. Some of the galanin/somatostatin neurons also contained 5-hydroxytryptamine. Synaptic boutons containing colocalized calcitonin gene-related peptide/vasoactive intestinal peptide were associated with the galanin/somatostatin neurons. The muscle of the large intestine was also innervated by axons containing galamin/somatostatin, vasoactive intestinal peptide/calcitonin gene-related peptide or vasoactive intestinal peptide alone. Nerve fibres containing calcitonin gene-related peptide/substance P, probably representing primary afferent nerves, were also associated with muscle bundles. Submucosal blood vessels carried dense plexuses of fibres containing vasoactive intestinal peptide alone or and calcitonin gene-related peptide/substance P. Adrenergic perivascular nerves also contained galanin and neuropeptide Y.     

Regulation of hyaluronan synthases in mouse uterine cervix

We examined the expression pattern of hyaluronan synthase (HAS) mRNAs in the uterine cervix of pregnant mice. The expression levels of HAS-1 and -2 mRNAs peaked at delivery, whereas that of HAS-3 mRNA peaked on the 15th day of pregnancy. The regulation of HAS mRNA expression was examined in pregnant mouse uterine cervical fibroblasts. The expression of HAS-1, -2, and -3 mRNAs was significantly augmented by interleukin-1beta (IL-1beta). Progesterone significantly interfered with expression of HAS-1 and -2 mRNAs, but significantly increased the expression of HAS-3 mRNA. Low-molecular-weight hyaluronan significantly enhanced only the expression of HAS-1 mRNA. These results indicate that HAS in the uterine cervix is regulated in a complex manner by IL-1beta, progesterone, and low-molecular-weight hyaluronan, of which changes in the cervical tissue and serum closely participate in uterine cervical ripening and/or inflammation.