Innervation of amphibian reproductive system. Histological and ultrastructural studies.

In the present study we describe for the first time in anuran amphibians the histological and ultrastructural characteristics of innervation in the female reproductive organs. The observations in Rhinella arenarum revealed the presence of nerve fibers located predominantly in the ovarian hilium and in the oviduct wall. In both organs the nerves fibers are placed near blood vessels and smooth muscles fibers. In the present study the histological observations were confirmed using antibodies against peripherin and neurofilament 200 proteins. Ultrastructural analyses demonstrated that the innervation of the reproductive organs is constituted by unmyelinated nerve fibers surrounded by Schwann cells. Axon terminals contain a population of small, clear, translucent vesicles that coexist with a few dense cored vesicles. The ultrastructural characteristics together with the immunopositive reaction to tyrosine hydroxylase of the nerve fibers and the type of synaptic vesicles present in the axon terminal would indicate that the reproductive organs of R. arenarum females are innervated by the sympathetic division of the autonomic nervous system.

Mating regulates neuromodulator ensembles at nerve termini innervating the Drosophila reproductive tract.

Upon mating, regions of the female reproductive tract mature and alter their function [1-3], for example to facilitate storage of sperm or control the release of eggs [4-6]. The female's nervous system and neuromodulators play important roles in her responses to mating [7-13]. However, it is difficult to reconcile the reproductive tract's many changing but coordinated events with the small set of neuromodulators present [14-18]. We hypothesized that each part of the reproductive tract contains a characteristic combination of neuromodulators that confer unique identities on each region and that postmating changes in these combinations coordinate subsequent actions. We examined the presence, locations, and levels of neuromodulators and related molecules ("signaling molecules") in the reproductive tract of Drosophila melanogaster females before and after mating: the biogenic amine octopamine, which regulates ovulation rate in Drosophila and locusts [7, 14-20]; serotonin, which regulates muscle contraction in locust oviducts [21]; and the FMRF amide dromyosuppressin, which regulates contraction of Drosophila heart muscle [22] and may regulate muscle contractions in the reproductive tract, if it is expressed there. We find that separate aspects of mating (sperm, seminal proteins, and physical effects) independently modulate the release of signaling molecules. Each reproductive tract subregion displays a characteristic combination of signaling molecule release, resulting in a unique functional identity. These patterns, and thus functions, change reproducibly after mating. Thus, one event (mating) promotes new combinations of signaling molecules that endow different parts of the reproductive tract with unique temporal and spatial identities that facilitate many aspects of fertilization.

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling.

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg's release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin's increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.

Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct.

BACKGROUND: In both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Drosophila is a model system for tubular organ development, few studies have addressed the development of the fly's oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules (e.g. Muscle LIM protein 84B, Coracle, Neuroglian) have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise. RESULTS: Using electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings. CONCLUSION: We uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ.

The sympathetic postganglionic and sensory innervation of oviducal magnum in hen: a choleratoxin subunit B-conjugated horseradish peroxidase study.

The anatomy of the extrinsic innervation of the avian magnum has not been accurately demonstrated previously. In the present study, choleratoxin subunit B-conjugated horseradish peroxidase (CB-HRP) was used as a retrograde tracer to determine the sympathetic postganglionic and sensory innervation of the magnum of hens. With regard to the sympathetic postganglionic innervation, following CB-HRP injections under the serosa of the magnum, CB-HRP-positive neurons were found bilaterally in the C12-LS13 ganglia of the sympathetic chain, splanchnic ganglia and adrenal ganglia. The number of labelled neurons in the left ganglia of the sympathetic chain and splanchnic ganglia was approximately 2.1 times that in the right ganglia. This suggests that the unilateral magnum is bilaterally innervated with sympathetic postganglionic nerves, the left nerves being predominant. With regard to the sensory innervation, following tracer injections, CB-HRP-positive neurons were found bilaterally in the spinal ganglia C13-LS12, jugular ganglia and nodose ganglia. The number of positive cells in the left ganglia was about 2.2 times that in the right ganglia. In the spinal ganglia, 85.6% of the labelled neurons were in the T5-LS2 and LS8-LS11 ganglia. These results suggest that the sensory nerve fibres of the magnum reach the central nervous system principally via two groups of spinal ganglia and vagus nerves, and that the innervation is bilateral although the left-hand route predominates. Moreover, 45.7% of all the CB-HRP-labelled neurons were found in the rectal region of the intestinal nerve of Remak (INR), which suggests that the INR plays a very important role in the functional regulation of the magnum.

Immunoreactivities to protein gene product 9.5, neurofilament protein and neuron specific enolase in nerves in the oviduct of the sexually immature ostrich, Struthio camelus.

The present study investigated the distribution of nerves in the oviduct of the sexually immature ostrich. The presence of protein gene product 9.5, neurofilament protein and neuron specific enolase nerve fibres were demonstrated in the infundibulum, magnum, isthmus, shell gland and vagina. Nerve fibres containing protein gene product 9.5, neuron specific enolase and neurofilament protein were particularly numerous in the tunica muscularis and intermuscular connective tissue areas of the shell gland and vagina. The presence of a large number of nerves in these oviductal regions is probably important in the coordination of muscle contraction. An interesting finding of the study was the presence of protein gene product 9.5 and neuron specific enolase immunopositive nerve fibres in the walls of blood vessels. It is likely that these nerves are autonomicin nature and play a role in the regulation of blood flow. This study has shown the presence of an extensive neural network in the oviduct of the ostrich. In addition, the results of the investigation have shown that the neuronal markers protein gene product 9.5, neurofilament protein and neuron specific enolase can be used to demonstate nerve fibres in the ostrich.

Oviduct contraction in Drosophila is modulated by a neural network that is both, octopaminergic and glutamatergic.

Fertility is a highly complex and regulated phenomenon essential for the survival of any species. To identify Drosophila fertility-specific neural networks, we used a GAL4/UAS enhancer trap genetic screen that selectively inactivates groups of neurons. We identified a GAL4 line (bwktqs) that has a female sterile phenotype only when it expresses the tetanus toxin light chain (TeTxLC). These flies lack oviduct contraction, lay almost no eggs, sperm accumulate in the oviducts, and fewer than normal are seen in the storage organs. In insects, two neuroactive substances are important for oviduct contraction: octopamine (OA), a monoamine that inhibits oviduct contraction, and glutamate (Glu), a neurotransmitter that induces contraction. It is known that octopaminergic neurons of the thoracic abdominal ganglion (TAG) modulate oviduct contraction, however, the glutamatergic neurons that innervate the oviduct have not been identified yet and the interaction between these two neuroactive substances is not well understood. Immunostaining experiments revealed that the bwktqs line trapped an octopaminergic neural network that innervates the genital tract. We show that wt like oviduct contraction in TeTxLC-inactivated flies can only be rescued by simultaneous application of Glu and OA suggesting that the abdominal bwktqs neurons are both octopaminergic and glutamatergic, the use of an agonist and an antagonist for Glu receptors as well as their direct visualization confirmed its participation in this phenomenon. Our work provides the first evidence that adult abdominal type II visceral innervations co-express Glu and OA and allows us to re-evaluate the previous model of neuronal network controlling insect oviduct contraction.

Evidence for a possible neurotransmitter/neuromodulator role of tyramine on the locust oviducts.

Visualization of the tyraminergic innervation of the oviducts was demonstrated by immunohistochemistry, and the presence of tyramine was confirmed using high-performance liquid chromatography coupled to electrochemical detection. Oviducts incubated in high-potassium saline released tyramine in a calcium-dependent manner. Stimulation of the oviducal nerves also resulted in tyramine release, suggesting that tyramine might function as a neurotransmitter/neuromodulator at the locust oviducts. Tyramine decreased the basal tension, and also attenuated proctolin-induced contractions in a dose-dependent manner over a range of doses between 10(-7) and 10(-4) M. Low concentrations of tyramine attenuated forskolin-stimulated cyclic AMP levels in a dose-dependent manner. This effect was not blocked by yohimbine. High concentrations of tyramine increased basal cyclic AMP levels of locust oviducts in a dose-dependent manner; however, the increases in cyclic AMP were only evident at the highest concentrations tested, 5 x 10(-5) and 10(-4) M tyramine. The tyramine-induced increase in cyclic AMP shared a similar pharmacological profile with the octopamine-induced increase in cyclic AMP. Tyramine increased the amplitude of excitatory junction potentials at low concentrations while hyperpolarizing the membrane potential by 2-5 mV. A further increase in the amplitude of the excitatory junction potentials and the occurrence of an active response was seen upon washing tyramine from the preparation. These results suggest that tyramine can activate at least three different endogenous receptors on the locust oviducts a putative tyramine receptor at low concentrations, a different tyramine receptor to inhibit muscle contraction, and an octopamine receptor at high concentrations.

Allatostatin in ovaries, oviducts, and young embryos in the cockroach Diploptera punctata.

The quantity and localization of -Phe-Gly-Leu-amide allatostatins (-F-G-L-amide AST) was determined by ELISA and immunohistochemistry in ovaries and oviducts and in pre-dorsal closure embryos. AST in the cytoplasm of basal oocytes gradually increased from 4 to 35 fmol/ovary pair from the start (day 2) to the completion of vitellogenesis (day 6), then rapidly increased to 121 fmol/ovary pair during choriogenesis. In oviducts, AST-immunoreactivity was found in nerves to the muscle layer and in epithelial cells. AST-immunoreactivity in oviduct epithelial cells increased during vitellogenesis. A marked increase in quantity of AST in oviduct tissue between completion of chorion formation and immediately after ovulation appears to result from AST released from oocytes as they travel down the oviducts because AST content of newly ovulated eggs was 40% lower than late stage chorionated oocytes, and these oocytes released AST when incubated in saline. AST in embryos, localized in yolk cells, decreased as embryos approached dorsal closure. That this material in ovaries and embryos is AST was confirmed by its ability to inhibit JH synthesis in vitro and identification by MALDI-TOF mass spectrometry of a peptide with a mass corresponding to that of a Diploptera punctata AST. These findings indicate likely novel functions for ASTs: facilitation of ovulation and utilization of yolk.

NADPH-d positive neurons of the lizard Podarcis s. sicula oviduct and their relationship to 17beta-estradiol hormone.

The distribution of nicotinamide adenine dinucleotide phosphate reduced diaphorase (NADPH-d) containing neurons was examined in the oviduct of the lizard Podarcis s. sicula and the relationship between these neurons and 17beta-estradiol hormone was studied. NADPH-d-histochemistry and indirect immunofluorescence method were applied to cryostat sections. NADPH-d-nerve structures were found throughout the oviduct. Positive neurons were primarily located in the reproductive oviduct, and were more numerous in the intermuscular and circular muscle layers than in the mucosa. The vagina revealed a reactive nerve population denser than elsewhere. The NADPH-d-positive neurons densities and the 17beta-estradiol plasma levels coincided throughout the lizard sexual cycle. In addition, after 17beta-estradiol treatments, non-reproductive lizards showed an increase of NADPH-d neurons. We suppose that nitric oxide (NO) neurons play an estrogen-dependent role in the oviduct muscle motility.

Developmental changes in galanin in lumbosacral sympathetic ganglionic neurons innervating the avian uterine oviduct and galanin induction by sex steroids.

We recently found lumbosacral sympathetic ganglionic galanin neurons innervating the quail uterine oviduct. Galaninergic innervation of the uterine muscle may be essential for avian oviposition, as galanin evoked oviposition through a mechanism of induction of vigorous uterine contraction. The questions arising from these findings are: what changes occur in galanin expression in the sympathetic ganglionic galanin neuron during development, and what is the hormonal factor(s) that induces galanin expression in this neuron? Therefore, the present study examined the developmental changes in galanin of the quail sympathetic ganglionic neuron and uterus, and the effect of administration of ovarian sex steroids on galanin induction. Immature birds reared under long-day photoperiods from 4 weeks of age demonstrated progressive increases in galanin levels both per unit ganglionic protein (concentration) and per ganglia (content) concurrent with ganglionic development during weeks 4--13. The uterine galanin content and uterine weight also increased progressively during the same period, but the galanin concentration in the uterus at 4 weeks was high due to the much smaller tissue mass. Immunocytochemical analysis with anti-galanin serum showed that immunoreactive ganglionic cells were few and small at 4 weeks and increased progressively thereafter. Administration of oestradiol-17 beta to immature birds at 3 weeks of age for 1 week increased both the galanin concentration and content in the ganglia without ganglionic growth. A marked increase in galanin-immunoreactive ganglionic cells was detected following oestradiol treatment. In contrast, progesterone increased ganglionic galanin levels, but the effects were low. Expression of the mRNAs encoding oestrogen receptor-alpha and -beta (ER alpha and ER beta) in the ganglionic tissue was verified by RT-PCR/Southern blot analysis. Immunocytochemical staining with anti-ER serum further revealed an intense immunoreaction restricted to the nucleus of ganglionic neurons. These results suggest that ovarian sex steroids, in particular oestradiol-17 beta, contribute as hormonal factors to galanin induction, which takes place in the lumbosacral sympathetic ganglionic neurons innervating avian uterine oviduct during development. Oestradiol may act directly on this ganglionic neuron through intra-nuclear receptor-mediated mechanisms to induce galanin.

Evidence for the innervation of sperm storage tubules in the oviduct of the turkey (Meleagris gallopavo).

The presence of neural tissue and smooth muscle elements in the vicinity of the oviductal sperm storage tubules at the uterovaginal junction was assessed by several modes of light microscopy. Isolated neurones and small ganglia were identified in the uterovaginal junction of the turkey oviduct. The nerve cell bodies were observed in the tunica mucosa by bright field microscopy. Immunoreactivity against neurofilament antibody and recombinant fragment C of the tetanus toxin reacted with nerve fibres and the nuclei of neurones. Fluorescence microscopy and confocal laser scanning microscopy revealed that nerve fibres continued from the base of the tunica mucosa into the plicae. Axons appeared to terminate on, or run immediately adjacent to, individual sperm storage tubules. Neither phalloidin reacting with F-actin nor the monoclonal antibody against alpha-smooth muscle actin detected smooth muscle fibres in the tissue encapsulating individual sperm storage tubules. In contrast, F-actin was strongly localized in the apical region of the epithelial cells of the sperm storage tubule and in smooth muscle elements in the tunica mucosa and tunica muscularis. These observations present the first evidence for the innervation of the sperm storage tubules. It is suggested that a previously unrecognized neural factor may function in oviductal sperm storage in, and release of spermatozoa from, the sperm storage tubules of hens.

Distribution of efferent neurones innervating the oviduct in the pig.

This study was aimed, by means of the retrograde tracing technique, at disclosing the distribution of efferent neurones innervating the porcine oviduct. The fluorescent retrograde tracer Fast Blue was injected into the wall of the right oviduct in six juvenile pigs during laparotomy performed under anaesthesia. After a recovery period of 3 weeks the animals were reanaesthetised, perfused with 4% buffered paraformaldehyde (pH 7.4) and different ganglia, thought to be potent sources of the efferent innervation, were collected. The occurrence and distribution of Fast Blue-positive neurones were studied in the sympathetic chain and prevertebral ganglia, including the coeliac-superior mesenteric ganglion complex, adrenal ganglion, aorticorenal ganglion, ovarian ganglion and inferior mesenteric ganglion. The labelled neurones were found only in the right, ipsilateral ganglia. The largest number of Fast Blue-positive neurones was found in the inferior mesenteric ganglion, ovarian ganglion and in the coeliac-superior mesenteric ganglion complex. In the inferior mesenteric ganglion, the Fast Blue-positive neurones showed a tendency to gather in the dorso-cranial and the dorso-caudal region of the ganglion, forming two discrete "oviductal centres". The aortico-renal and adrenal ganglion contained a smaller population of Fast Blue-positive nerve cell bodies. The smallest number of Fast Blue-positive neurones was found in the sympathetic chain ganglia (T14-L5). The localisation of Fast Blue-positive neurones in the sympathetic chain ganglia and prevertebral ganglia suggests that these nerve structures play a fundamental role in the efferent innervation of the porcine oviduct.

Innervation of the pigeon oviduct: correlation of NADPH diaphorase with acetylcholinesterase, tyrosine hydroxylase, and neuropeptides.

The motility of the avian oviduct is controlled by hormones and neurons, but little is microscopically known about a neural network in the oviduct. The present study was investigated to determine the distribution of nitric oxide-synthesizing neurons in the oviduct of the pigeon by histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). The NADPH-d reaction was seen in the neurons and fibers. NADPH-d neurons were mainly distributed around the arterioles of the intermuscular tissue in the upper oviduct (infundibulum, magnum, and isthmus); in addition, NADPH-d neurons were also seen in the smooth muscle layers and lamina propria in the lower oviduct (uterus and vagina). NADPH-d neurons were found singly or in small groups of two-eight cell bodies. The number of NADPH-d neurons was smallest in the infundibulum, gradually increased toward the vagina. NADPH-d was also shown to be strongly positive in many neurons in the ganglia of the vaginal adventitia. Bundles of NADPH-d fibers ran in the smooth muscle layer, surrounded blood vessels, or connected with small groups of NADPH-d neurons by forming strands. Thin fibers branched from these bundles and constituted a finer network in the smooth muscle layer and lamina propria. Acetylcholinesterase staining in neurons and fibers showed a similar pattern of NADPH-d distribution in the oviduct. By double staining, 70 approximately 77% of neurons showed colocalization of NADPH-d and acetylcholinesterase in the uterus and vagina. Tyrosine hydroxylase immunoreactivity stained only nerve fibers and were distributed largely around blood vessels in the oviduct. Nerve fibers immunoreactive for calcitonin-gene related peptide, galanin, methionine-enkephalin, substance P, or vasoactive intestinal peptide were found sparsely in the oviduct. These results demonstrate that nitrergic neurons make up a large subpopulation of intrinsic neurons that are closely associated with a cholinergic system in the pigeon oviduct, thus suggesting that nitric oxide and acetylcholine could be used to modify the relaxation of the avian oviduct.

Existence of galanin in lumbosacral sympathetic ganglionic neurons that project to the quail uterine oviduct.

Oviposition in birds is conducted by vigorous contractions of the uterine oviduct. We recently isolated an oviposition-inducing peptide that was identified as avian galanin from mature quail oviducts. This peptide was localized in neuronal fibers terminating in muscle layers in the uterine oviduct and evoked vigorous uterine contractions through binding to receptors located in the uterus. However, no cell bodies that express avian galanin were detected in the uterus or other oviduct regions. To understand the control mechanism of avian oviposition by galanin, we identified the neurons that synthesize galanin and project to the uterus with the combination of retrograde labeling with neurobiotin and immunocytochemistry for galanin in mature Japanese quails. Retrograde labeling with neurobiotin from the uterus revealed that lumbosacral sympathetic ganglionic neurons located in the uterine side projected their axons to the uterine muscle layer. Abundant elementary granules were observed in somata of the retrogradely labeled sympathetic ganglionic neurons, suggesting that labeled neurons may function as a neurosecretory cell. Immunocytochemical analysis with the antiserum against avian galanin showed an intense immunoreaction restricted to somata of the retrograde-labeled ganglionic neurons. Preabsorbing the antiserum with avian galanin resulted in a complete absence of the immunoreaction. Competitive enzyme-linked immunosorbent assay using antigalanin serum confirmed that avian galanin existed in the sympathetic ganglionic neurons. Expression of the avian galanin messenger RNA in the neurons was further verified by Northern blot analysis. In addition, both avian galanin and its messenger RNA in the neurons were highly expressed in mature birds, unlike in immature birds. These results suggest that lumbosacral sympathetic ganglionic neurons innervating the uterine muscle produce avian galanin in mature birds. Because this peptide acts directly on the uterus to evoke oviposition through a mechanism of the induction of vigorous uterine contraction, galaninergic innervation of the uterine oviduct may be essential for avian oviposition.

Galanin in the lizard oviduct: its distribution and relationships with estrogen, VIP and oviposition.

The distribution of neurons containing galanin immunoreactivity (Gal/IR) has been detected in the oviduct of the lizard Podarcis s. sicula during the main phases of its sexual cycle and after 17beta-estradiol treatment. Indirect immunofluorescence technique was applied both to cryostatic sections and whole mount preparations, and Western blot analysis, with an antibody directed against mammalian galanin (Gal), was performed with lizard oviduct extracts. Colocalization of Gal with vasoactive intestinal polypeptide (VIP) was also studied as well as Gal effects on egg deposition. In the quiescent oviduct of non-reproductive females, scanty Gal/IR fibres were found in the uterine-vaginal segment. During the reproductive period a gradual increase of positive nerve fibres and cell bodies were found distally in the lizard oviduct and the vagina revealed a reactive nerve population denser than elsewhere. Gal-IR nerve structures were present either in the musculature or mucosa and in the intermuscular layer they were organized in a nerve network. In the oviduct of non-reproductive females, 17beta-estradiol administration induced a significant increase of neurons containing Gal/IR. This hormone could be involved in the egg laying by means of galanin action and this hypothesis is supported by the induction of premature oviposition in pre-ovulatory females after Gal administration. Western blot analysis validates this peptide as true Gal, recognising one protein band with a molecular weight (3.2 kDa), similar to that of porcine Gal. Double labelling studies showed the co-presence of Gal and VIP in some neurons.

Proctolin's role in neurally evoked contractions of the locust oviducts.

The effects of proctolin (RYLPT) on neurally evoked contractions of locust oviduct muscle were studied to examine the role of proctolin as a cotransmitter. Increasing the number of stimuli in a burst (from one to 30 stimuli) resulted in an increase in amplitude of contraction of locust oviduct muscle. Proctolin was capable of increasing the amplitude of neurally evoked contractions at lower-stimulus regimes (one- and two-stimulus bursts) but did not do so at higher-stimulus regimes (five- and 10-stimulus bursts). The effects of proctolin were dose dependent within the one- and two-stimulus regimes, with thresholds at 10(-9) M and maxima at 2.5 x 10(-8) M. Addition of proctolin increased the basal tonus and size of a postcontraction relaxation of the oviduct muscle in a dose-dependent manner during all stimulus regimes. However, the effect of proctolin on basal tonus and the postcontraction relaxation was much less at the higher stimulus regimes. Previously, several proctolin analogues have been tested for their ability to antagonize proctolin-induced contractions of the oviduct muscle. Since proctolin is proposed to be a cotransmitter at this neuromuscular junction, one of these analogues, cycloproctolin, was used to antagonize proctolin's effects on neurally evoked contractions. In the presence of the antagonist, the maximum amplitude induced by application of proctolin was decreased by 22.7%, while the proctolin-induced increase in basal tonus was decreased by 45.8%. Finally, the maximum increase in the size of the postcontraction relaxation caused by proctolin was lowered by 32.0%. The results of the present study show that exogenously applied proctolin is an excitant of the oviduct muscle at lower, rather than higher, stimulus regimes, and this latter inaction may be due to the corelease of endogenous proctolin during increased neural stimulation.

NADPH-diaphorase-, nitric oxide synthase- and VIP-containing nerve structures in the hen oviduct: a histochemical and immunohistochemical study.

The enzyme NADPH-diaphorase (NADPH-d; a marker of NO producing or nitrergic neurons) and the neuropeptide VIP have been detected in the nerve structures of the hen oviduct by histochemical and immunohistochemical techniques performed on cryostatic sections and whole mount preparations. In the upper four segments of the oviduct, i.e. the infundibulum, magnum, isthmus and shell gland, nitrergic and VIP-positive nerve structures were particularly numerous in the intermuscular and mucosal layers, and were represented by fibres and cell bodies showing mainly a perivascular distribution. Functionally, such perivascular structures were related to the blood flow regulation. Different types of nitrergic pyrenophora were recognized in the walls of the shell gland on the basis of their peculiar morphology. In the distal zone of the oviduct, the vagina, nitrergic and VIP-positive nerve fibres were widely diffused in the circular muscle, which was particularly thick in this segment. The source of at least part of such fibres was probably represented by large nerve cell bodies scattered in the layer and containing NO and VIP colocalized. Functionally these cells retained inhibitory motor neurons causing relaxation of the vaginal smooth muscle.

Structural characterization of peripheral nerve cells and nerve-muscle junctions of the oviduct of stable fly (Diptera:Muscidae).

Fine structure of both peripheral nerve cells and neuromuscular junctions associated with the oviduct of stable fly. Stomoxys calcitrans (L.), was described. Twelve or more multipolar peripheral neurons were found along major branch nerves that enter the ovipositor. Several were suspended in the haemacoel and others were in close proximity to the surface of the oviduct. Some peripheral neurons contained an abundance of neurosecretory granules that ranged in size from 32 to 180 nm in diameter. No glial elements enveloped the perikarya of such cells. Neurosecretory axons were usually found in the boundary region of large nerves just beneath the stroma. Peripheral nerve cells in close apposition to oviduct muscles were generally non-neurosecretory and were ensheathed in a glial perineurium. Peripheral neurons were surrounded occasionally by an extensive network of extracellular spaces in the glial perineurium. Other smaller neurons were found within large nerve trunks. Nerve-muscle junctions contained large clusters of synaptic vesicles and occasionally included small groups of neuro-secretory granules. Many nerve terminals on the surface of the oviduct contained a complex postsynaptic folding of sarcolemma. Active transmission sites were indicated by increased densities along the neurolemma. Some neurohemal release sites were also evident.

Sexual differentiation in the CNS of the moth, Manduca sexta. I. Sex and segment-specificity in production, differentiation, and survival of the imaginal midline neurons.

We analyzed the development of several sets of postembryonic sex-specific motoneurons in Manduca sexta which belong to a group of homologous lineages of neurons called the imaginal midline neurons (IMNs). Adult female oviduct motoneurons and male sperm duct motoneurons are IMNs that show similar anatomical features and differentiate during metamorphosis, despite appearing in different segments: A7 for oviduct neurons, A9 for sperm duct neurons. These cells are born at the same time and, initially, similar sets are found in A7 and A9 ganglia of larvae of both sexes. The dimorphic adult pattern is generated by sex-specific production and cell death. A7 IMNs differentiate in both sexes through early pupal stages, whereupon they disappear in the male and become the oviduct motoneurons in the female. A9 IMNs are overproduced in the male, and subsequent cell death reduces male cell number and eliminates the small complement of female cells; the surviving male cells develop into the sperm duct motoneurons. Similar IMN arrays are generated in nongenital ganglia, but show non-sex-specific fates. This suggests that both the sex of these cells and their segment of residence play major roles in their subsequent differentiation.