Outer Basement Membrane Research Articles

Comparative analysis of testicular fusion in Spodoptera litura (cutworm) and Bombyx mori (silkworm): Histological and transcriptomic insights

Spodoptera litura commonly known as the cutworm, is among the most destructive lepidopteran pests affecting over 120 plants species. The powerful destructive nature of this lepidopteran is attributable to its high reproductive capacity. The testicular fusion that occurs during metamorphosis from larvae to pupa in S.litura positively influences the reproductive success of the offspring. In contrast, Bombyx mori, the silkworm, retains separate testes throughout its life and does not undergo this fusion process. Microscopic examination reveals that during testicular fusion in S.litura, the peritoneal sheath becomes thinner and more translucent, whereas in B.mori, the analogous region thickens. The outer basement membrane in S.litura exhibits fractures, discontinuity, and uneven thickness accompanied by a significant presence of cellular secretions, large cell size, increased vesicles, liquid droplets, and a proliferation of rough endoplasmic reticulum and mitochondria. In contrast, the testicular peritoneal sheath of B.mori at comparable developmental stage exhibits minimal change. Comparative transcriptomic analysis of the testicular peritoneal sheath reveals a substantial difference in gene expression between the two species. The disparity in differential expressed genes (DEGs) is linked to an enrichment of numerous transcription factors, intracellular signaling pathways involving Ca2+ and GTPase, as well as intracellular protein transport and signaling pathways. Meanwhile, structural proteins including actin, chitin-binding proteins, membrane protein fractions, cell adhesion, extracellular matrix proteins are predominantly identified. Moreover, the study highlights the enrichment of endopeptidases, serine proteases, proteolytic enzymes and matrix metalloproteins, which may play a role in the degradation of the outer membrane. Five transcription factors-Slforkhead, Slproline, Slcyclic, Slsilk, and SlD-ETS were identified, and their expression pattern were confirmed by qRT-PCR. they are candidates for participating in the regulation of testicular fusion. Our findings underscore significant morphological and trancriptomic variation in the testicular peritoneal sheath of S.litura compared to the silkworm, with substantial changes at the transcriptomic level coinciding with testicular fusion. The research provides valuable clues for understanding the complex mechanisms underlying this unique phenomenon in insects.

Collagen XIII Is the Key Molecule of Neurovascular Junctions in the Neuroendocrine System

Introduction: Axons of magnocellular neurosecretory cells project from the hypothalamus to the posterior lobe (PL) of the pituitary. In the PL, a wide perivascular space exists between the outer basement membrane (BM), where nerve axons terminate, and the inner BM lining the fenestrated capillaries. Hypothalamic axon terminals and outer BMs in the PL form neurovascular junctions. We previously had found that collagen XIII is strongly localized in the outer BMs. In this study, we investigated the role of collagen XIII in the PL of rat pituitaries. Methods: We first studied the expression of Col13a1, the gene encoding the α1 chains of collagen XIII, in rat pituitaries via quantitative real-time polymerase chain reaction and in situ hybridization. We observed the distribution of COL13A1 in the rat pituitary using immunohistochemistry and immunoelectron microscopy. We examined the expression of Col13a1 and the distribution of COL13A1 during the development of the pituitary. In addition, we examined the effects of water deprivation and arginine vasopressin (AVP) signaling on the expression of Col13a1 in the PL. Results: Col13a1 was expressed in NG2-positive pericytes, and COL13A1 signals were localized in the outer BM of the PL. The expression of Col13a1 was increased by water deprivation and was regulated via the AVP/AVPR1A/Gαq/11 cascade in pericytes of the PL. Conclusion: These results suggest that pericytes surrounding fenestrated capillaries in the PL secrete COL13A1 and are involved in the construction of neurovascular junctions. COL13A1 is localized in the outer BM surrounding capillaries in the PL and may be involved in the connection between capillaries and axon terminals.

Early Generation of New PrPSc on Blood Vessels after Brain Microinjection of Scrapie in Mice.

ABSTRACTAggregation of misfolded host proteins in the central nervous system is believed to be important in the pathogenic process in several neurodegenerative diseases of humans, including prion diseases, Alzheimer’s disease, and Parkinson’s disease. In these diseases, protein misfolding and aggregation appear to expand through a process of seeded polymerization. Prion diseases occur in both humans and animals and are experimentally transmissible orally or by injection, thus providing a controllable model of other neurodegenerative protein misfolding diseases. In rodents and ruminants, prion disease has a slow course, lasting months to years. Although prion infectivity has been detected in brain tissue at 3 to 4 weeks postinfection (p.i.), the details of early prion replication in the brain are not well understood. Here we studied the localization and quantitation of PrPSc generation in vivo starting at 30 min postmicroinjection of scrapie into the brain. In C57BL mice at 3 days p.i., generation of new PrPSc was detected by immunohistochemistry and immunoblot assays, and at 7 days p.i., new generation was confirmed by real-time quaking-induced conversion assay. The main site of new PrPSc generation was near the outer basement membrane of small and medium blood vessels. The finding and localization of replication at this site so early after injection have not been reported previously. This predominantly perivascular location suggested that structural components of the blood vessel basement membrane or perivascular astrocytes might act as cofactors in the initial generation of PrPSc. The location of PrPSc replication at the basement membrane also implies a role for the brain interstitial fluid drainage in the early infection process.

Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of adult mouse brain.

Fenestrated capillaries of the sensory circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis, the subfornical organ and the area postrema, lack completeness of the blood-brain barrier (BBB) to sense a variety of blood-derived molecules and to convey the information into other brain regions. We examine the vascular permeability of blood-derived molecules and the expression of tight-junction proteins in sensory CVOs. The present tracer assays revealed that blood-derived dextran 10k (Dex10k) having a molecular weight (MW) of 10,000 remained in the perivascular space between the inner and outer basement membranes, but fluorescein isothiocyanate (FITC; MW: 389) and Dex3k (MW: 3000) diffused into the parenchyma. The vascular permeability of FITC was higher at central subdivisions than at distal subdivisions. Neither FITC nor Dex3k diffused beyond the dense network of glial fibrillar acidic protein (GFAP)-positive astrocytes/tanycytes. The expression of tight-junction proteins such as occludin, claudin-5 and zonula occludens-1 (ZO-1) was undetectable at the central subdivisions of the sensory CVOs but some was expressed at the distal subdivisions. Electron microscopic observation showed that capillaries were surrounded with numerous layers of astrocyte processes and dendrites. The expression of occludin and ZO-1 was also observed as puncta on GFAP-positive astrocytes/tanycytes of the sensory CVOs. Our study thus demonstrates the heterogeneity of vascular permeability and expression of tight-junction proteins and indicates that the outer basement membrane and dense astrocyte/tanycyte connection are possible alternative mechanisms for a diffusion barrier of blood-derived molecules, instead of the BBB.

Accessibility of low‐molecular‐mass molecules to the median eminence and arcuate hypothalamic nucleus of adult mouse

Blood-derived molecules are able to access to the median eminence (ME) and arcuate hypothalamic nucleus (Arc) due to the lack of the blood-brain barrier. In the present study, we examined the accessibility of low-molecular-mass (LMM) molecules into parenchyma in the ME and Arc of adult mice by administration of Dextran 3000 (Dex3k), Dex10k, Evans blue (EB) and fluorescein isothiocyanate (FITC). In the external zone of the ME, the fluorescence of Dex3k, EB and FITC tracers generated an intensity gradient from fenestrated capillary, but that of Dex10k was detected only between the inner and outer basement membrane of pericapillary space. The fluorescence of FITC in the external zone of the ME was closely associated with axonal terminals and surrounded by cellular processes of tanycytes-like cells and astrocytes. In the ependymal/internal zone of the ME and Arc, the fluorescence of all LMM tracers was seen at tanycytes-like cells and neurons. The fluorescence of EB and FITC in these regions was not detected when brains were fixed during or before the administration of tracers. The inhomogeneity of accessibility for fluorescent tracers depended on routes for tracer administration. Thus, the present study indicates that the accessibility of LMM blood-derived molecules to parenchyma depends on fenestration of the capillary in the external zone of the ME and active transport of ependymal cells in the ependymal/internal zone of the ME and Arc.

Plasticity of the interface between oxytocin neurons and the vasculature in late pregnant rats: An ultrastructural morphometric study

Increased numbers of oxytocin-immunoreactive perivascular neurons have been shown to occur in the preoptic region and lateral hypothalamus of female rats around parturition. In the present study, electronmicroscopical immunocytochemistry and morphometry were used to examine such perivascular oxytocinergic neurons as well as those in the classical magnocellular nuclei in late pregnant and lactating rats. In 22 d pregnant animals and in rats killed after 2 d of lactation, numerous oxytocinergic neurons were found in direct apposition to the outer basement membrane of arterioles, venules, and capillaries. The distance between immunoreactive neurons and blood vessels was significantly lower in these animals than in 9 d lactating rats and in ovariectomized controls. It is likely that, around parturition, oxytocinergic perivascular neurons are uncovered by active retraction of glial elements. This plasticity is perhaps facilitated by changing hormonal conditions around parturition. The observed changes seem to be transitory and might reflect altered secretory properties of perivascular oxytocinergic neurons.

Monoamine innervation of the organum vasculosum laminae terminalis (OVLT): a high resolution radioautographic study in the rat.

The monoamine innervation of the organum vasculosum laminae terminalis (OVLT) was examined in the adult rat by light and electron microscope radioautography after intraventricular administration of tritiated serotonin [( 3H]5-HT) or dopamine [( 3H]DA). Radioautographic and biochemical controls after 5,7-dihydroxytryptamine or 6-hydroxydopamine lesioning established the respective serotonin (5-HT) and catecholamine (CA) identities of the axonal varicosities labeled under the conditions of the present experiments. For descriptive purposes, the OVLT was subdivided in three parts: two parenchymal zones, one juxtaventricular, the other juxtavascular, and the vascular core. Almost 10% of all axonal varicosities in the OVLT were found to be labeled with [3H]5-HT. This 5-HT innervation was most prominent in the rostrocaudal and ventrodorsal portions of the juxtaventricular zone and the dorsal aspect of the juxtavascular zone; there was none in the vascular core. [3H]DA-labeled varicosities were much less abundant and yet more numerous than earlier histofluorescent and immunohistochemical studies would have predicted. They predominated in the juxtavascular zone, where a majority presumably had a dopamine (DA) rather than a noradrenaline identity. Some were also found in the vascular core, where they most likely corresponded to peripheral autonomic noradrenaline endings. In the juxtaventricular zone of the OVLT, a significant proportion of the [3H]5-HT-labeled varicosity profiles could be observed to form axodendritic synapses, but in the juxtavascular zone no 5-HT or any [3H]DA-labeled ones were ever seen in synaptic junction. In the juxtavascular zone, the 5-HT and the presumed DA endings established close relationships with neurosecretory axons, and with astrocytic or tanycytic processes on which they occasionally formed "synaptoid contacts." A few endings of either type were also seen to about directly on the outer basement membrane of the perivascular space. It therefore appears probable that in OVLT monoamines influence neural and nonneural elements. At a proximal level of regulation (juxtaventricular zone), 5-HT could act both synaptically and nonsynaptically as an interneuronal transmitter or modulator. In contrast, distally (juxtavascular zone), both DA and 5-HT could be released as neurohormones in addition to modulating neurosecretion. 5-HT and DA varicosities in the OVLT could also behave as sensors for circulating factors that do not cross the blood-brain barrier.

Cystid Structure and Protrusion of the Polypide inCrisia(Bryozoa, Cyclostomata)

The ultrastructure of the cystid of Crisia eburnea has been studied. The cystid wall comprises an outer periostracum, a calcified layer and one inner cell layer, the ectoderm. The membranous sac, which consists of an outer basement membrane, a series of very thin annular muscle cells and an inner layer of epithelial cells, is interpreted as the detached mesoderm of the cystid wall. Accordingly, the atrial sphincter and the generally eight branched, longitudinal muscle cells connecting the terminal membrane and the membranous sac are interpreted as ectoderm. The membranous sac is attached to the cystid wall with two lateral ligaments and at four abfrontal areas: 1) a wide distal area, 2) an area at the origin of the retractor muscles, 3) a small area between 1 and 2, and 4) a small basal area at the origin of a pair of small muscle cells attached to the lowermost part of the caecum. We infer that the protrusion of the polypide is caused by a sequential contraction of the annular muscle cells of the membranous sac, starting basally and aided by the contraction of the longitudinal ectodermal muscle cells.

The hypothalamo-hypophysial system in acipenseridae. VI. The proximal neurosecretory contact region.

The proximal neurosecretory contact region (PCR) of Acipenseridae, a homologue of the tetrapod median eminence, has been studied by light, fluorescence and electron microscopy. It occupies the rostral and chiefly the ventral surfaces of the hypothalamic tuber cinereum. PAF-positive fibres occur in the zone of the preoptico-hypophysial tract but their terminal enlargements are concentrated mainly in the external zone. They make contact with the primary portal capillaries situated in the pia mater. Monoaminergic fibres and terminals with an intense green fluorescence are localized in the same regions. The fibres of some bipolar monoaminergic neurons of the PCR make contact both with the third ventricle and the primary portal capillaries. Three types of granule-containing neurosecretory fibres and terminals have been recognized in the PCR. Fibres of types A1 (d = 120-300 nm) and A2 (D = 100-170 nm) are peptidergic PAR-positive, although some fibres, including some of type A1, belong possibly to PAF-negative type. Monoaminergic type B fibres have granules 80-100 nm in diameter. Neurosecretory terminals and vascular "endfeet" of tanycytes make contact with the 70 nm thick outer basement membrane of the primary portal capillaries. Several laminae of thin horizontally oriented tanycyte processes form a boundary between the external zone and the preoptico-hypophysial tract. Few neuroglial cells with pale cytoplasm, numerous lysosomes and lipofuscin granules are seen in this region. It is hypothesized that, as in other vertebrates both peptide hypophysiotropic neurohormones and monoamines are discarged from the PCR into the portal circulation and affect the activity of the glandular cells of the pars distalis.

Influence of castration on incorporation of exogenous peroxidase into "synaptic" vesicles of the median eminence. II. The perivascular part of the palisade zone in male rats.

In the median eminence of male rats, nerve profiles in the immediate vicinity of portal capillaries have been divided into 4 categories on the basis of their vesicular content: profiles a with agranular "synaptic" vesicles of about 50 nm, b with similar agranular vesicles and also with granular vesicles of mainly 60-140 nm, c with granular vesicles alone and d without vesicles. Twenty-four hours after castration, the percentage of profiles of category a was significantly increased when compared with sham-operated animals, whereas the percentage of profiles of category b was significantly decreased. After intravenous injection of exogenous peroxidase, especially the nerve profiles located in direct contact with the outer basement membrane of the portal capillaries contained peroxidase positive "synaptic" vesicles. Injection off peroxidase after castration resulted in a significant increase in the percentage of nerve profiles containing both peroxidase positive and peroxidase negative "synaptic" vesicles (category a). It is suggested that, in certain nerve terminals, castration may lead to release off the content of granular vesicles, which may contain gonadotropin releasing factor. This release then may cause that nerve terminals with agranular vesicles and granular vesicles (category b) change in their vesicular content and are therefore classified as terminals with only agranular vesicles (category a). An increased turnover rate in the ultrastructurally affected terminals may be reflected in increased uptake of exogenous peroxidase. The observations imply that certain neurones projecting to portal capillaries in the median eminence are, directly or indirectly, sensitive to changes in the level of gonadal steroids.

The hypothalamo-hypophysial system of the frog Rana temporaria: II. Functional morphology of the external zone of the median eminence during metamorphosis

Changes in the external zone of the median eminence of Rana temporaria have been studied by light and electron microscopy, using a morphometric method. It has been found that nerve terminals complete their differentiation as judged by ultrastructural criteria only in metamorphosed frogs. In tadpoles before metamorphosis peptidergic A 2 terminals predominate in the external zone. During metamorphosis the percentage of the A 2 type terminals decreases and that of the B type terminals simultaneously increases. But even in metamorphosed frogs, this value as well as the total number of nerve terminals differ from those in adults. Thus, the development of the median eminence is not completed even in metamorphosed frogs. The capillaries of the primary portal plexus are superficial before metamorphosis. In metamorphosing frogs an intensive ingrowing of these capillaries occurs. The “intervascular channels” formed by the outer basement membrane are especially pronounced at the same time. Therefore, it is hypothesized that these channels provide routes for capillary ingrowth into the median eminence. The inner basement membrane is fragmented even in metamorphosed frogs. During metamorphic climax both a strong depletion of PAF-positive material and a decrease in the total concentration of granules in the A (A 2 and A 1) and B type terminals are revealed. The possibility of intensified discharge not only of peptide neurohormones (releasing factors), but also of monoamines from nerve terminals into the capillaries of the primary portal plexus is supposed.

Electron microscopic studies of the development of new neurohaemal contacts in the median eminence of the rat after hypophysectomy

In the normal rat the axons of the parvicellular neurosecretory system form terminals which are characterised by dense core vesicles of 80–160 nm diameter and which lie against the fenestrated portal capillaries in the upper neurohypophysis (median eminence). The magnocellular neurosecretory axons traverse the interanal zone of the median eminence and form terminals against the fenestrated capillaries of the distal neurohypophysis (neural lobe). In the neural lobe it is assumed that the more electron dense vesicles (of around 200 nm diameter) characterise the vasopressin containing terminals of neurones in the supraoptic nucleus and that the less numerous terminals with the more electron lucent vesicles (also of around 200 nm diameter) belong to the axons of the paraventricular neurones and contain oxytocin. For the first few days after hypophysectomy, the proximal stumps of the cut neurosecretory axons in the internal zone of the median eminence are distended with neurosecretory material while axonal sprouts which are rich in neurotubules and completely ensheathed in satellite cell cytoplasm gradually penetrate the external zone. Subsequently there is a massive hypertrophy of fenestrated capillaries and the newly formed axonal sprouts progressively lose their satellite cell cover and thus acquire neurohaemal contacts with the outer basement membrane of the perivascular space. The development of these contacts is associated with the appearance of increasing numbers of synaptic vesicles and prejunctional dense projections. This reconstitution of the morphological features of the normal neurohaemal contacts is associated with recovery of antidiuretic function. In the hypertrophic median eminence the neurosecretory axon terminals with the electron lucent type of vesicles far outnumber those with the electron dense vesicles. It is suggested that the greater degree of cell loss from the supraoptic as opposed to the paraventricular nuclei puts the paraventricular axons at an advantage in competing for space on the basement membrane around the perivascular space of the newly formed capillaries. It is suggested that an important factor in promoting the regeneration of the neurosecretory axons is the remarkable hypertrophy of the fenestrated capillaries against which they come to terminate.

Electron microscopy of neurohypophysis in normal, adrenaline-treated and pilocarpinetreated rabbits.

1. The fine structure of the neurohypophysis in normal, adrenaline-treated and pilocarpine-treated rabbits has been studied, using the electron microscope. The normal structure of the nerve terminal and pericapillary region is quite similar to that of the rat as described by Palay (1957) and Hartmann (1958). 2. Pituicytes are significantly different, however, and are classified into three types. The first one (Type A) is identical with the astrocyte of Farquhar and Hartmann (1957) and of Schultz, Maynard and Pease (1958). This type cell has a round or oval nucleus of low electron density and abundant watery cytoplasm with few formed elements. 3. The second type (Type B) is considered to be an atypical astrocyte. This type cell has a nucleus closely resembling that of Type A, but with abundant cytoplasm containing relatively numerous formed elements. The inverse relationship between relative numbers of Type A and Type B cells in a given gland suggests the possibility of one type being readily transformed into the other under normal conditions. The last type (Type C) has a dark, irregular nucleus and dense cytoplasm with many cell organelles. This cell is structurally identical with the microglia of Farquhar and Hartmann, and of Schultz, Maynard and Pease. A typical oligodendrocyte is not found. No secretory granules are present in pituicytes. The pituicytes are in contact with nerve fibers with a double membrane intervening, and sometimes enclose nerve fibers in invaginations of their cytoplasm. The cell bodies of the pituicytes are located in the parenchymatous region far from blood capillaries, and their processes, along with the nerve terminals, reach to the outer basement membrane of the pericapillary space. 4. During 1–2 hours after injection of adrenaline there was found to be a statistically significant increase in number and size of secretory granules, as well as structural changes of mitochondria in the nerve terminals, enlargement of the pericapillary space and endothelial alterations. These phenomena are considered to reflect a blocking of the release of secretory material after injection of adrenaline. 5. After injection of pilocarpine, a statistically significant decrease in number and size of neurosecretory granules, plus the appearance of many optically empty granules, structural changes of mitochondria and attenuation of the pericapillary space were observed. These phenomena are considered to be related to an acceleration of the release of secretory granules after injection of pilocarpine.