Immunohistochemical analysis of the development of olfactory organs in two species of turtles Pelodiscus sinensis and Mauremys reevesii.

The nasal cavity of turtles is composed of the upper and lower chambers, lined by the upper and lower chamber epithelia, respectively. In many turtles including the Reeve's turtle Mauremys reevesii, the upper chamber epithelium contains ciliated olfactory receptor neurons (ORNs) and the lower chamber epithelium contains microvillous ORNs. However, in the olfactory organ of the Chinese soft-shelled turtle Pelodiscus sinensis, both the upper and lower chamber epithelia contain ciliated ORNs. In the present study, we immunohistochemically examined the developmental process of olfactory organs in soft-shelled turtle and the Reeve's turtle to clarify the developmental origins of the lower chamber epithelium in these turtles. Obtained data indicate that olfactory organs of these turtles have identical origin and follow similar process of development, suggesting that, in the lower chamber epithelium of the nasal cavity, ciliated ORNs differentiate in soft-shelled turtle whereas microvillous ORNs differentiate in the Reeve's turtle.

Conserved and non-conserved characteristics of porcine glial cell line-derived neurotrophic factor expressed in the testis.

Glial cell line-derived neurotrophic factor (GDNF) is essential for the self-renewal and proliferation of spermatogonial stem cells (SSCs) in mice, rats, and rabbits. Although the key extrinsic factors essential for spermatogonial proliferation in other mammals have not been determined, GDNF is one of the potential candidates. In this study, we isolated porcine GDNF (pGDNF) cDNAs from neonatal testis and generated recombinant pGDNF to investigate its biological activity on gonocytes/undifferentiated spermatogonia, including SSCs. In porcine testis, long and short forms of GDNF transcripts, the counterparts of pre-(α)pro and pre-(ß)pro GDNF identified in humans and rodents, were expressed. The two transcripts encode identical mature proteins. Recombinant pGDNF supported proliferation of murine SSCs in culture, and their stem cell activity was confirmed by a transplantation assay. Subsequently, porcine gonocytes/undifferentiated spermatogonia were cultured with pGDNF; however, pGDNF did not affect their proliferation. Furthermore, GDNF expression was localised to the vascular smooth muscle cells, and its cognate receptor GFRA1 expression was negligible during spermatogonial proliferation in the testes. These results indicate that although pGDNF retains structural similarity with those of other mammals and conserves the biological activity on the self-renewal of murine SSCs, porcine SSCs likely require extrinsic factors other than GDNF for their proliferation.

Histochemical and ultrastructural analyses of the lubrication systems in the olfactory organs of soft-shelled turtle.

In general, the nasal cavity of turtles is divided into two chambers: the upper chamber, lined with the olfactory epithelium containing ciliated olfactory receptor cells, and the lower chamber, lined with the vomeronasal epithelium containing microvillous receptor cells. In the nasal cavity of soft-shelled turtles, however, differences between the upper and lower chamber epithelia are unclear due to the presence of ciliated receptor cells in both epithelia. In the olfactory organ of vertebrates, the surface of sensory epithelium is covered with secretory products of associated glands and supporting cells, playing important roles in the olfaction by dissolving odorants and transporting them to the olfactory receptors. Here, the associated glands and supporting cells in the olfactory organ of soft-shelled turtles were analyzed histochemically and ultrastructurally. The upper chamber epithelium possessed associated glands, constituted by cells containing serous secretory granules; whereas, the lower chamber epithelium did not. In the upper chamber epithelium, secretory granules filled the supranuclear region of supporting cells, while most of the granules were distributed near the free border of supporting cells in the lower chamber epithelium. The secretory granules in the supporting cells of both epithelia were seromucous, but alcian blue stained them differently from each other. In addition, distinct expression of carbohydrates was suggested by the differences in lectin binding. These data indicate the quantitative and qualitative differences in the secretory properties between the upper and lower chamber epithelia, suggesting their distinct roles in the olfaction.

Immunohistochemical analysis for G protein in the olfactory organs of soft-shelled turtle, Pelodiscus sinensis.

In turtles, the epithelia lining the upper and lower chambers of the nasal cavity project axons to the ventral and dorsal parts of the olfactory bulbs, respectively. In a semi-aquatic soft-shelled turtle, Pelodiscus sinensis, more than 1,000 odorant receptor genes have been found, but it is not known where they are expressed. In this study, we aimed to clarify the distribution of cells expressing these genes in the olfactory organs of soft-shelled turtles. Immunoreactions for the Gαolf, the α subunit of G protein coupled to the odorant receptors, were detected on the surface of epithelia lining both the upper and lower chambers of the nasal cavity. The receptor cells in the epithelium of both chambers possessed cilia on the tip of their dendrites, whereas microvillous, non-ciliated, receptor cells were not found. These data suggest that the odorant receptor genes are expressed by the ciliated receptor cells in the upper and lower chamber epithelia. Precise location of the vomeronasal epithelium is not known at present.

Immunohistochemical and lectin histochemical studies on the developing olfactory organs of fetal camel.

Little is known about the development of the olfactory organs of camel. In this study, prenatal development and neuronal differentiation of the vomeronasal organ (VNO) and the olfactory epithelium (OE) of the one-humped camel were studied by immunohistochemistry and lectin histochemistry. A neuronal marker, protein gene product (PGP) 9.5, but not a marker of fully differentiated olfactory receptor cells, olfactory marker protein, intensely labeled the olfactory receptor cells of the VNO and OE at 395 mm, 510 mm, and 530 mm fetal ages, indicating that the olfactory receptor cells are differentiated, but not fully matured both in the VNO and the OE. In 187 mm and 190 mm fetuses, PGP 9.5 yielded faint immunoreactive signals in the VNO, but not in the OE, although the presence of olfactory receptor cells were demonstrated in both tissues by intense WGA and LEL stainings. We conclude that the camel VNO and OE bear differentiated, but still immature receptor cells; in addition, the onset of neuronal differentiation seems to be somewhat earlier in the VNO than in the OE till half of the prenatal life.

Immunohistochemical studies for the neuronal elements in the vomeronasal organ of the one-humped camel.

The neuronal elements of the vomeronasal organ (VNO) of camel were investigated immunohistochemically. PGP 9.5 labeled the receptor cells in the vomeronasal sensory epithelium, but not the supporting or basal cells. OMP stained some receptor cells, but no immunoreactive signals for OMP were detected in the non-sensory epithelium. PLCß2 labeled scattered cells in the sensory epithelium and a larger number of cells in the non-sensory epithelium. Double labeling immunohistochemistry revealed that the PLCß2-positive cells were surrounded by substance P-positive nerve fibers. Collectively, these data suggest that the camel VNO bears, in addition to the mature vomeronasal receptor cells, trigeminally-innervated solitary chemosensory cells which are expected to play a substantial role in the control of stimulus access to the VNO.

Cell-surface DEAD-box polypeptide 4-immunoreactive cells and gonocytes are two distinct populations in postnatal porcine testes.

DEAD-box polypeptide 4 (DDX4) is an evolutionally conserved ATP-dependent RNA helicase that is exclusively expressed in germ cell lineage. Although DDX4 is believed to reside and function in the cytoplasm, recent studies in mice and humans suggest that its epitope is expressed on the cell surface of a small subpopulation in the ovary, putative oogonial stem cells. No study has examined whether such cell-surface DDX4(+) cells exist in the testes of any species. In this study, we explored cell-surface DDX4(+) cells in postnatal porcine testes before the onset of spermatogenesis, where gonocytes, which are the precursors of spermatogonial stem cells, are the only germ cell population. Transfection experiments demonstrated that recombinant porcine DDX4 can be expressed on the cell surface, and cell-surface DDX4-immunoreactive cells were identified in the testis by flow cytometry. Although the DDX4-expressing cells identified in the testis were indeed gonocytes, the cell-surface DDX4-immunoreactive cells expressed negligible DDX4 mRNA and protein levels. Furthermore, they did not express other germ cell markers, such as ZBTB16, NANOS2, and DAZL, but prominently expressed early primordial germ cell markers, such as PRDM1, IFITM3, and EPCAM. Nonetheless, the cell-surface DDX4-immunoreactive cells generated neither germ cell colonies nor teratomas following transplantation into immunocompromised mouse testes. Taken together, these results demonstrate that testicular cell-surface DDX4-immunoreactive cells are not germ cells and constitute a distinct subpopulation that is different from gonocytes. Moreover, the subpopulation in porcine testes might be species specific because no DDX4-immunoreactive cells were found in postnatal mouse testes.

Phylogenic studies on the olfactory system in vertebrates.

The olfactory receptor organs and their primary centers are classified into several types. The receptor organs are divided into fish-type olfactory epithelium (OE), mammal-type OE, middle chamber epithelium (MCE), lower chamber epithelium (LCE), recess epithelium, septal olfactory organ of Masera (SO), mammal-type vomeronasal organ (VNO) and snake-type VNO. The fish-type OE is observed in flatfish and lungfish, while the mammal-type OE is observed in amphibians, reptiles, birds and mammals. The MCE and LCE are unique to Xenopus and turtles, respectively. The recess epithelium is unique to lungfish. The SO is observed only in mammals. The mammal-type VNO is widely observed in amphibians, lizards and mammals, while the snake-type VNO is unique to snakes. The VNO itself is absent in turtles and birds. The mammal-type OE, MCE, LCE and recess epithelium seem to be descendants of the fish-type OE that is derived from the putative primitive OE. The VNO may be derived from the recess epithelium or fish-type OE and differentiate into the mammal-type VNO and snake-type VNO. The primary olfactory centers are divided into mammal-type main olfactory bulbs (MOB), fish-type MOB and mammal-type accessory olfactory bulbs (AOB). The mammal-type MOB first appears in amphibians and succeeds to reptiles, birds and mammals. The fish-type MOB, which is unique to fish, may be the ancestor of the mammal-type MOB. The mammal-type AOB is observed in amphibians, lizards, snakes and mammals and may be the remnant of the fish-type MOB.

Time-dependent changes in cardiovascular function during copulatory behavior induced by the hand method in the male dog.

PURPOSE: Ejaculation in the male dog consists of three fractions. Observation of behavior and measurement of heart rate (HR), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were researched sequentially, and a fundamental examination of the features of sympathetic nerve activity during copulatory behavior induced by the hand method in the male dog was undertaken. METHODS: We investigated the breeding capability of male dogs. HR, plasma NA level and plasma Ad levels were measured during ejaculation induced by the hand method. RESULTS: HR was 125.8 ± 6.0 beats/min at rest, and peaked during mounting at 195.2 ± 8.2 beats/min. Moreover, HR at 3 min after the first fraction decreased to values similar to those at rest. Plasma NA and Ad concentrations during copulatory behavior induced by the hand method did not differ significantly from those at rest. However, although there was no significant difference, plasma NA concentration during ejaculation of the third fraction peaked at about 1.8 times the baseline value. CONCLUSIONS: In the male dog, excitation of sympathetic nerves of long duration during erection of the penis and ejaculation is questionable. However, inhibition of sympathetic nerves and activation of parasympathetic nerves is thought to occur during erection of the penis and ejaculation.

Histological and lectin histochemical studies on the olfactory and respiratory mucosae of the sheep.

The olfactory and respiratory mucosae of the Corriedale sheep were examined using lectin histochemistry in order to clarify the histochemical and glycohistochemical differences between these two tissues. The olfactory epithelium was stained with 13 lectins out of 21 lectins examined, while the respiratory epithelium was positive to 16 lectins. The free border of both of the olfactory and respiratory epithelia was stained with 12 lectins: Wheat germ agglutinin (WGA), succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL), Datura stramonium lectin (DSL), Soybean agglutinin (SBA), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-120), Erythrina cristagalli lectin (ECL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L). The associated glands of the olfactory mucosa, Bowman's glands, were stained with 13 lectins. While both the goblet cells and mucous nasal glands were stained with 8 lectins; five of them (WGA, s-WGA, STL, Vicia villosa agglutinin (VVA) and ECL) were mutually positive among the Bowman's glands, mucous nasal glands and the goblet cells. These findings indicate that the glycohistochemical characteristics of the free borders of both olfactory and respiratory epithelia are similar to each other, suggesting that secretions from the Bowman's glands and those of the goblet cells and mucous nasal glands are partially exchanged between the surface of two epithelia to contribute the functions of the respiratory epithelium and the olfactory receptor cells, respectively.

A morphological study of the vomeronasal organ and the accessory olfactory bulb in the Korean roe deer, Capreolus pygargus.

The vomeronasal organ (VNO) and accessory olfactory bulb (AOB) of the Korean roe deer (Capreolus pygargus) were studied histologically to evaluate their morphological characteristics. Grossly, the VNO, encased by cartilage, has a paired tubular structure with a caudal blind end and a rostral connection through incisive ducts on the hard palate. In the VNO, the vomeronasal sensory epithelium (VSE) consists of galectin-3-positive supporting cells, protein gene product (PGP) 9.5-positive receptor cells, and basal cells. The vomeronasal respiratory epithelium (VRE) consists of a pseudostratified epithelium. The AOB strata included a vomeronasal nerve layer (VNL), a glomerular layer (GL), a mitral/tufted cell layer, and a granular cell layer. All lectins used in this study, including Bandeiraea simplicifolia agglutinin isolectin B4 (BSI-B4), soybean agglutinin (SBA), Ulex europaeus agglutinin I (UEA-I), and Triticum vulgaris wheat germ agglutinin (WGA), labeled the VSE with varying intensity. In the AOB, both the VNL and the GL reacted with BSI-B4, SBA, and WGA with varying intensity, but not with UEA-I. This is the first morphological study of the VNO and AOB of the Korean roe deer, which are similar to those of goats.

Lectin histochemical studies on the vomeronasal organ of the sheep.

The vomeronasal organ of sheep was examined using lectin histochemistry in order to compare the types and amounts of the glycoconjugates among various components of the vomeronasal sensory and non-sensory epithelia. In the vomeronasal sensory epithelium, Dolichos biflorus agglutinin (DBA) stained particular cells, located at the same level as the vomeronasal receptor cells, while the distribution, shape and number of the stained cells did not correspond to those of the vomeronasal receptor cells. Datura stramonium lectin (DSL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L) labeled the basal cells of both vomeronasal sensory and non-sensory epithelia. While, Wheat germ agglutinin (WGA), Succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL) and Ricinus communis agglutinin-I (RCA-120) labeled the basal cells of the sensory epithelium, and Bandeiraea simplicifolia lectin-I (BSL-I) stained the basal cells of the non-sensory epithelium, respectively. Seventeen lectins labeled the free border of both vomeronasal sensory and non-sensory epithelia, while Sophora japonica agglutinin (SJA), Jacalin and Pisum sativum agglutinin (PSA) labeled neither free border of the sensory nor that of non-sensory epithelia. The expression pattern of glycoconjugate was similar, but not identical, in the free border between the sensory and non-sensory epithelia. These results indicate that there are dissimilar features in the type and amount of glycoconjugates between the vomeronasal sensory and non-sensory epithelia, and at the same time, among the various cell types either in the vomeronasal sensory or non-sensory epithelium.

Localization of the primordial vomeronasal organ and its relationship to the associated gland in lungfish.

The lungfish, the closest fish to tetrapods, has two types of sensory epithelia in the olfactory organ: the lamellar olfactory epithelium and the recess epithelium. The former resembles the olfactory epithelium of ordinary teleosts and the latter resembles the vomeronasal organ of tetrapods with respect to the G-protein expressions and the morphological properties of olfactory receptor cells. In contrast to the lamellar olfactory epithelium covering the surface of olfactory lamella, the recess epithelium, together with the glandular epithelium, lines the recesses at the base of olfactory lamellae and is separated from the surrounding tissues by nonsensory epithelium. In the present study, we examined the distribution of these recesses and the relationship between the recess epithelium and the associated gland in the nasal sac of lungfish. We found that the posterior part of the nasal sac contained more recesses than the anterior one, and the medial one contained more recesses than the lateral one. In addition, virtually all recesses consisted of both the recess epithelium and the glandular epithelium. Furthermore, the glandular epithelium was invariably situated proximal to the midline raphe of the nasal sac, and the recess epithelium distal to it. Possible roles of the recess epithelium and the glandular epithelium are discussed.

Analysis of glycoproteins produced by the associated gland in the olfactory organ of lungfish.

The olfactory organ of African lungfish, Protopterus annectens, contains two distinct sensory epithelia: the lamellar olfactory epithelium and the recess epithelium. These epithelia correspond to the olfactory epithelium and the vomeronasal organ of tetrapods, respectively. In contrast to the lamellar olfactory epithelium, which has no associated gland, the recess epithelium is equipped with associated glands. Although the glandular cells and/or the supporting cells are generally presumed to secrete proteins involved in the function of olfactory sensory epithelia, the properties of these proteins in lungfish have not been evaluated to date. In this study, we investigated the associated glands in the olfactory organ of lungfish by transmission electron microscopy and found that the glandular cells contain numerous secretory granules and secrete them from the apical membrane. In addition, we analyzed the olfactory organ by lectin histochemistry using 16 biotinylated lectins. All lectins labeled the secretory granules in the glandular cells with different staining patterns from those of the supporting cells in the lamellar olfactory epithelium or in the recess epithelium. Furthermore, lectin blotting analysis showed that multiple bands were detected by the lectins which specifically labeled the glandular epithelium of the olfactory organ. These results indicate that the secretory products of the associated glands in the recess epithelium have different properties from those of the supporting cells in the olfactory sensory epithelia and contain multiple glycoproteins with different carbohydrate moieties.

Histological and ultrastructural characteristics of the primordial vomeronasal organ in lungfish.

Many vertebrates have two anatomically distinct olfactory organs--the olfactory epithelium and the vomeronasal organ--to detect chemicals such as general odorants and pheromones in their environment. The vomeronasal organ is not present in fish but is present in vertebrates of a higher order than amphibians. Among all extant fishes, the lungfish is considered to be genetically and phylogenetically closest to tetrapods. In this study, we examined the olfactory organs of African lungfish, Protopterus annectens, by lectin histochemistry, immunohistochemistry, and transmission electron microscopy. Two types of sensory epithelia were identified in the olfactory organ--the olfactory epithelium covering the surface of lamellae and the sensory epithelium lining the recesses both at the base of lamellae and in the wall of the nasal sac--and designated here as the lamellar olfactory epithelium and the recess epithelium, respectively. Based on analysis of G-protein expression and ultrastructure, the lamellar olfactory epithelium resembled the olfactory epithelium of ordinary teleosts and the recess epithelium resembled the vomeronasal organ of tetrapods. Furthermore, lectin histochemistry demonstrated that the axons from the recess epithelium converge and project to the ventrolateral part of the olfactory bulb, suggesting that lungfish possess a region homologous to the accessory olfactory bulb of tetrapods. Based on these results, it seems appropriate to refer to the recess epithelium as "a primordium of the vomeronasal organ." This study may provide important clues to elucidate how the vomeronasal organ emerged during the evolution of vertebrates.

Galectin-3 immunohistochemistry in the vomeronasal organ of the domestic pig, Sus scrofa.

The immunohistochemical localization of galectin-3, a ß-galactoside-binding protein, was studied in the vomeronasal organ (VNO) of fetal, 1-day-old, and 6-month-old pigs. In all age groups, the porcine VNO consisted of vomeronasal sensory epithelium (VSE) located medially and non-sensory vomeronasal respiratory epithelium (VRE) located laterally. In the pig, the VNO epithelium increased in height with postnatal development from fetus to adult. In the VSE of all stages examined, galectin-3 immunostaining was seen in the supporting cells and free border, but not in receptor or basal cells. Galectin-3 immunostaining was seen in all layers of the VRE, and the intensity increased with postnatal development. In the lamina propria, galectin-3 was detected in some ductal epithelial cells and the vomeronasal nerve sheath, but not in the acini of the Jacobson glands in all age groups. In view of these observations, we postulate that galectin-3 plays a role in cell survival and cell adhesion in both the VSE and VRE of porcine VNO in all age groups.

Developmental changes affecting lectin binding in the vomeronasal organ of domestic pigs, Sus scrofa.

This study investigated the developmental changes of glycoconjugate patterns in the porcine vomeronasal organs (VNOs) and associated glands (Jacobson's glands) from prenatal (9 weeks of gestation) and postnatal (2 days after birth) to the sexually mature stage (6 months old). The VNO of pigs (Sus scrofa) was examined using the following: Dolichos biflorus agglutinin (DBA), Bandeiraea simplicifolia agglutinin isolectin B4 (BSI-B4), Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), and soybean agglutinin (SBA). At the fetal stage, all lectins examined were detected mainly in the free border of the vomeronasal epithelium, but few (WGA and UEA-I) and or absent in the VNO cell bodies. At the postnatal and sexually mature stages, the reactivity of some lectins, including WGA, UEA-I, DBA and SBA, were shown to increase in the VNO sensory epithelium as well as the free border. The increased reactivity of lectins as development progressed was also observed in Jacobson's gland acini. These findings suggest that binding sites of lectins, including those of WGA, UEA-I, DBA, and SBA, increase during development from fetal to postnatal growth, possibly contributing to the increased ability of chemoreception in the pig.

Phylogenic outline of the olfactory system in vertebrates.

Phylogenic outline of the vertebrate olfactory system is summarized in the present review. In the fish and the birds, the olfactory system consists only of the olfactory epithelium (OE) and the olfactory bulb (B). In the amphibians, reptiles and mammals, the olfactory system is subdivided into the main olfactory and the vomeronasal olfactory systems, and the former consists of the OE and the main olfactory bulb (MOB), while the latter the vomeronasal organ (VNO) and the accessory olfactory bulb (AOB). The subdivision of the olfactory system into the main and the vomeronasal olfactory systems may partly be induced by the difference between paraphyletic groups and monophyletic groups in the phylogeny of vertebrates.

Morphological changes in the endocrine and exocrine pancreas of rats after experimental obstructive jaundice.

Obstructive jaundice causes multiple malfunctions in various organs including the pancreas. To establish how such malfunctions occur, we experimentally induced obstructive jaundice through bile duct ligation (BDL) using rats, measured serum bilirubin, amylase and insulin levels, and examined histological, immunohistochemical and cytological changes in the pancreas at 3 days, 1 week, and 4 weeks after the BDL. Morphometrical analysis was also conducted. Serum amylase levels steeply increased at 3 days, and then decreased at 1 and 4 weeks after the BDL to lower than the control level. In contrast, the number of zymogen granules decreased at 3 days after the BDL, then increased and eventually surpassed the control group at 4 weeks after the BDL. On the other hand, serum insulin levels dramatically decreased at 3 days after the BDL but recovered to a level close to that of the control group at 1 week after the BDL. At 4 weeks after the BDL, however, the serum insulin levels again showed a marked decline. Slight decrease in insulin immunoreactivity and number of insulin granules were observed at 4 weeks after the BDL. Cholecystokinin receptors (CCK-R) were expressed in both acinar and islet cells; their immunoreactivity significantly decreased in the acinar cells at 4 weeks after the BDL. Our results suggest that CCK may play a role in regulating changes in the pancreas after obstructive jaundice.

Fine structure of the vomeronasal organ in the grass lizard, Takydromus tachydromoides.

The squamates are composed of many taxa, among which there is morphological variation in the vomeronasal organ (VNO). To elucidate the evolution of chemoreception in squamate reptiles, morphological data from the VNO from a variety of squamate species is required. In this study, the morphology of the VNO of the grass lizard Takydromus tachydromoides was examined using light and electron microscopy. The VNO consists of a pair of dome-shaped structures, which communicate with the oral cavity. There are no associated glandular structures. Microvilli are present on the apical surfaces of receptor cells in its sensory epithelium, as well as on supporting cells, and there are centrioles and ciliary precursor bodies on the dendrites. In addition to ciliated cells and basal cells in the non-sensory epithelium, there is a novel type of non-ciliated cell in T. tachydromoides. They have constricted apical cytoplasm and microvilli instead of cilia, and are sparsely distributed in the epithelium. Based on these results, the variation in the morphology of the VNO in scincomorpha, a representative squamate taxon, is discussed.