Absence of penile erections during paradoxical sleep. Peculiar penile events during wakefulness and slow wave sleep in the armadillo.

The electroencephalogram (EEG) together with electromyogram (EMG) of the ischiocavernosus, bulbocavernosus and levator penis muscles were chronically monitored across behavioral states of the armadillo Chaetophractus villosus. This animal has a very long penis, which exhibits remarkable phenomena during wakefulness (W), slow wave sleep (SWS) and paradoxical sleep (PS). During W it remains retracted within a skin receptacle. During SWS penile protrusion can be observed together with very complex movements. Protrusion is a non erectile event during which the penis remains out of its receptacle but without rigidity. Penile erections are observed only during SWS. Contrasting with other mammals, no erections occur during PS. During this phase the penile muscles share the atonia of the body musculature characteristic of that phase. Some reflections on mechanisms of those penile events are presented.

Acyl-CoA-binding protein in the armadillo Harderian gland: its primary structure and possible role in lipid secretion.

Similar to those of other species, the Harderian glands of armadillo produce an abundant lipid secretion, most of which is composed of 1-alkyl-2,3-diacylglycerol. Biosynthesis of this component is apparently performed with the participation of one cytosolic pool of acyl-CoA and another of free fatty acids. The acyl-CoA-binding protein (ACBP) is present at a concentration at least 7-fold that of the heart-type fatty acid-binding protein (H-FABP), though lower than that in other armadillo organs such as liver and brain. The ACBP complete amino acid sequence was determined by Edman degradation of peptides generated by cleavage of the protein with cyanogen bromide, endopeptidase Glu-C, and trypsin. ACBP consists of 86 residues and has a calculated molecular mass of 9783 Da, taking into account that an acetyl group is blocking the N-terminus. Identity percentages between armadillo Harderian gland ACBP and other known ACBPs show that the protein belongs to the liver-specific ACBP isoform (L-ACBP). The fact that the ACBP concentration is higher than that of FABP suggests that the Harderian gland is able to store acyl-CoA amounts in ACBP larger than those of fatty acids in H-FABP for 1-alkyl-2,3-diacylglycerol synthesis.

Gonadotropin-releasing hormone (GnRH) variants in a lizard brain: is mammalian GnRH being expressed?

In reptiles as in other vertebrates, multiple forms of gonadotropin-releasing hormone (GnRH) within a single brain have been identified. In this group the following GnRH molecular variants have been characterized either by direct or indirect methods: chicken GnRH I (cGnRH-I), chicken GnRH II (cGnRH-II), salmon GnRH (sGnRH) and several unidentified GnRH-like forms. In the present study GnRH variants were investigated in brain extracts of the lizard Tupinambis teguixin (= T. merinae) by combining high-performance liquid chromatography (RP-HPLC) followed by radioimmunoassays (RIA). Two peaks showing GnRH immunoreactivity with the elution position of synthetic mammalian GnRH (mGnRH) and cGnRH-II were detected. Both peaks were further analyzed with different radioimmunoassay systems specific for mGnRH, cGnRH-I, and cGnRH-II. Pooled fractions corresponding to the first eluting peak showed no crossreactivity when analyzed with a cGnRH-I specific assay and logit-log displacement curves were not significantly different from those of synthetic mGnRH with homologous RIA systems. The second peak showed immunological characteristics of cGnRH-II when analyzed with a specific antiserum. The first ir-GnRH peak was selected for further RP-HPLC purification showing similar chromatographic behavior as mGnRH synthetic standard. We demonstrated the absence of cGnRH-I in this lizard using well-characterized antisera.

Characterisation of glycoconjugate sugar residues in the vomeronasal organ of the armadillo Chaetophractus villosus (Mammalia, Xenarthra).

Conventional carbohydrate histochemistry and the binding patterns of 21 lectins were analysed to characterise the glycoconjugate content in the components of the vomeronasal organ of the armadillo Chaetophractus villosus. The mucomicrovillous complex of the sensory epithelium bound most of the lectins studied. No reaction was observed with Con A, PSA, S-Con A and SBA, and the sustentacular cells were-stained with UEA-I, DSL, LEL, STL and Con A. The vomeronasal receptor neurons were labelled with S-WGA, WGA, PNA, UEA-I, STL, Con A, S-Con A, ECL and RCA120. The basal cell layer reacted with S-WGA, WGA, LCA, UEA-I, DSL, LEL, STL, Con A, JAC and VVA. The nonsensory epithelium exhibited a differential staining in relation to the different components. The mucociliary complex stained with ECL, DBA, JAC, RCA120, STL, LCA, PHA-E, PHA-L, LEL, BSL-I and VVA. However, SJA and UEA-I stained the mucus complex lining a subpopulation of columnar cells. The cytoplasm and cell membranes of columnar cells was labelled with DBA, DSL and LCA. The apical region of these cells exhibited moderate reactivity with LEL and SJA. None of the lectins bound specifically to secretory granules of the nonsecretory cells. Basal cells of the nonsensory epithelium were labelled with DSL, LEL, LCA, BSL-I and STL. The vomeronasal glands showed a positive reaction with WGA, DSL, LEL, LCA, DBA, PNA, RCA120 and SBA. Subpopulations of acinar cells were observed with ECL, S-WGA, Con A, S-Con A and DBA. PNA and RCA120 stained the cells lining the glandular ducts. In comparison with previous results obtained in the olfactory mucosa of the same group of armadillos, the carbohydrate composition of the vomeronasal organ sensory epithelium differed from the olfactory sensory epithelium. This is probably related to the different nature of molecules involved in the perireceptor processes.

Immunoidentification of CRF-like material in the intermaxillary glands during Bufo arenarum development.

The presence of corticotropin-releasing factor-like material in the intermaxillary glands was studied by immunocytochemical techniques during the metamorphosis of Bufo arenarum. The intermaxillary glands appeared at stage XV (midprometamorphosis) with CRF-like material slightly immunoreactive. These glands are located posterior to the premaxillae and between the nasal capsules in the roof of the mouth and are formed of alveoli or tubules. During metamorphic climax, corticotropin-releasing factor-like material was identified strongly immunostained at the apices of the secretory cells. It was observed that collecting ducts of the gland open to the anterior palatal surface suggesting that the secretion could be ingested by tadpoles. Our results clearly showed that ir-CRF-like material present in the intermaxillary glands is ingested by tadpoles during metamorphosis and could play an important role during amphibian development.

Ultrastructural characterisation of the olfactory mucosa of the armadillo Dasypus hybridus (Dasypodidae, Xenarthra).

The ultrastructure of the olfactory mucosa of the armadillo Dasypus hybridus was studied. A comparison with the olfactory mucosa of another armadillo (Chaetophractus villosus) was made. The olfactory mucosa of D. hybridus shows many features which are similar to those of other mammals. Interestingly, it differs from the olfactory mucosa of the armadillo C. villosus. A suggestion is made that these differences may be due to differences in the digging habits of these species. In Dasypus, the supporting cells (SCs) showed dense vacuoles, multivesicular bodies and lysosome-like bodies probably related with the endocytotic system. The SCs show a dense network of SER presumably associated with xenobiotic mechanisms. The olfactory receptor neurons exhibit lysosome-like bodies and multivesicular bodies in their perikarya. These organelles suggest the presence of an endocytotic system. Duct cells of Bowman's glands exhibit secretory activities. Bowman's glands are compound-branched tubulo-acinar mixed glands with merocrine secretory mechanisms.

Thyrotropin-releasing hormone immunoreactivity in the brain and the pituitary during Bufo arenarum development.

The ontogeny of the thyrotropin releasing hormone (TRH) neuronal system was evaluated by immunocytochemistry in Bufo arenarum. The first appearance of TRH immunoreactive fibers was at early premetamorphosis. These fibers were found in small numbers and weakly stained in the median eminence and pars nervosa. With the advance of larval development, TRH-like material stained intensely and tended to aggregate in the median eminence, pars nervosa and pars intermedia. At climax stages immunoreactive fibers and perikarya (weakly stained) were also identified in the preoptic area. In adult specimens TRH perikarya and neuronal fibers were found in the preoptic and infundibular nuclei of the hypothalamus and in the amygdala, septum and diagonal band of Broca of the telencephalon. In addition, TRH neuronal fibers and endings were found in the preoptic-hypophyseal tract, the external zone of the median eminence, the pars nervosa and pars intermedia. Fibers were absent in the pars distalis. This study represents the first immunocytochemical demonstration of TRH in Bufo species, and serves as a basis for clarification of the neuroendocrine regulation of metamorphosis.

Corticotropin-releasing factor accelerates metamorphosis in Bufo arenarum: effect on pituitary ACTH and TSH cells.

The actions of several neuropeptides as hypothalamic mediators in the regulation of Bufo arenarum metamorphosis were investigated. Prometamorphic larvae were injected with 1.5 microg thyrotropin-releasing hormone (TRH), 2 microg ovine corticotropin-releasing factor (oCRF), 2 microg mammalian gonadotropin-releasing hormone (mGnRH), 2 microg human growth hormone-releasing hormone (hGHRH), or Holtfreter solution (control group). Larvae received two injections with the same dose: one at the beginning of the experiment and the other 7 days later. Several morphologic parameters (total length, tail length, wet weight, hind limb length, and metamorphic stages) were measured as indicators of growth and metamorphic development. These measurements were taken in 20 larvae per treatment or control group at the beginning of the experiment, at day 7 and at day 14 when the experiment ended. We observed that only the administration of exogenous CRF stimulated resorption of the tail and accelerated the rate of metamorphosis. In the pituitary of CRF-treated larvae we observed that thyrotropin (TSH) and adrenocorticotropic hormone (ACTH) producing cells showed a weaker immunoreactivity, a decrease in cell number and a reduction of volume density when compared with normal larvae. In conclusion, the results obtained indicate a possible role for CRF in Bufo arenarum metamorphosis. CRF may regulate interrenal and thyroid activity by acting directly upon TSH and ACTH cells. On the other hand, TRH, GnRH and GHRH were inactive in stimulating growth or metamorphosis of Bufo arenarum. J. Exp. Zool. 286:473-480, 2000.

Identification and localisation of glycoconjugates in the olfactory mucosa of the armadillo Chaetophractus villosus.

Conventional histochemistry and the binding patterns of 22 biotinylated lectins were examined for characterisation of glycoconjugates in the components of the olfactory mucosa of the armadillo Chaetophractus villosus. The mucous lining the olfactory epithelium showed binding sites for DSL, WGA, STL, LEL, PHA-E and JAC. Only the basilar processes of the supporting cells stained for Con-A and S-Con A. The olfactory receptor neurons stained with LEL, LCA, Con A, S-Con A, JAC and PNA. The layer of basal cells did not react with any of the lectins studied. Bowman's glands in the lamina propria showed subpopulations of acinar cells reacting with SBA, S-WGA, WGA, STL, Con A, PSA, PNA, SJA, VVA, JAC and S-Con A, but in our optical studies with lectins we were unable to differentiate between mucous and serous cells in the way that is possible on electron microscopy. The ducts of Bowman's glands were labelled with S-WGA, STL, LEL, PHA-E, BSL-I and JAC. This histochemical study on the glycoconjugates of the olfactory mucosa in the order Xenarthra provides a basis for further experimental investigations.

Differential distribution of gonadotropin-releasing hormone variants in the brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia).

1. In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic-hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested. 2. The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents. 3. In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve-septopreoptic and the midbrain systems. 4. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. 5. To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.

The vomeronasal organ of the South American armadillo Chaetophractus villosus (Xenarthra, Mammalia): anatomy, histology and ultrastructure.

The vomeronasal organ (VNO) is a chemoreceptive structure that has not been extensively studied in the Xenarthran order. Tissue samples from the VNO of the armadillo Chaetophractus villosus were prepared for light and electron microscopy. The VNO is located in the anterior part of the base of the nasal septum. It is tubular in shape, approximately 18 mm in length and opens in the rostral region of the nasal cavity and with a blind caudal end. Its lumen is lined by sensory (SE) and nonsensory (NSE) epithelium. The SE shows sensory, supporting and basal cells whereas the NSE contains ciliated and nonciliated secretory cells and basal cells. At the ultrastructural level, the sensory cells appear as bipolar neurons with conspicuous microvilli on their free surface. The supporting cells of the SE contain numerous membrane-bound vesicles in their apical regions. A peculiar feature not found in other mammals, is the presence of concentric whorls of RER cisterns frequently observed in their basal expansions. Infiltrating plasma cells can be detected in the SE basal region close to the dorsal junctional area. This region also exhibits an unusual type of basal cell, probably responsible for the generation of new vomeronasal receptor neurons. The ciliated NSE cells exhibit numerous ovoids or irregularly shaped membranous protrusions projecting from the plasma membrane of the cilia. As far as we know, this is the first study reporting the presence of this feature in ciliated NSE cells. The nonciliated cells are characterised by scarce large secretory granules and apical microvilli. The vomeronasal glands are compound-branched tubuloacinar glands with serous acinar cells. Four types of secretory granules are present. The ducts of these glands reach the lumen in the dorsolateral region between the NSE and SE. Hypolemmal nerve terminals were observed contacting secretory cells. Fenestrated and nonfenestrated capillaries constitute the vascular supply to these glands. Plasma cells, intimately associated with acinar cells, were frequently observed.

A simple method for taking photographs of histological sections without using neither photographic camera nor microscope

A simple photographic method for histological sections is described. This is a contact method which does not require the use of photographic camera. It is very similar to the contact print method used routinely in photographic laboratories. It is performed by placing the slides with the histological section, coverslip downwards, over the emulsion of the negative film. Then, they are illuminated with a common photographic enlarger during a brief period of time. Thus, a negative contact print of the section is obtained. This technique yields photographs of high quality focus, sharply contrasted with great detail. These results may also be obtained with low-contrast stained sections. This method is most suitable for photography of large or medium-sized histological sections (macros). This is often the case of neuroanatomical and immunohistochemical studies. Our method, being simple and quick, is recommended for routine use in laboratories

A simple method for taking photographs of histological sections without using neither photographic camera nor microscope

A simple photographic method for histological sections is described. This is a contact method which does not require the use of photographic camera. It is very similar to the contact print method used routinely in photographic laboratories. It is performed by placing the slides with the histological section, coverslip downwards, over the emulsion of the negative film. Then, they are illuminated with a common photographic enlarger during a brief period of time. Thus, a negative contact print of the section is obtained. This technique yields photographs of high quality focus, sharply contrasted with great detail. These results may also be obtained with low-contrast stained sections. This method is most suitable for photography of large or medium-sized histological sections (macros). This is often the case of neuroanatomical and immunohistochemical studies. Our method, being simple and quick, is recommended for routine use in laboratories

Olfactory mucosa of the South American armadillo Chaetophractus villosus: an ultrastructural study.

The sense of olfaction in armadillos plays an important role, suggested by the great development of the nasal structures, olfactory bulbs, and related brain regions. The mammalian olfactory mucosa is a privileged site of neuronal death and regeneration during the whole life span. A detailed knowledge of its ultrastructure is convenient for gaining insight into the factors controlling those phenomena. We performed this work in species not previously studied in order to provide a firm basis for further research on those factors. No information is available on the histology and ultrastructure of the olfactory mucosa in the order Xenarthra to which armadillos belong. Samples from the endoturbinals of the armadillo Chaetophractus villosus were prepared for light and electron microscopic examination by the usual conventional means. The olfactory epithelium of Chaetophractus villosus shows the classical three types of cells: supporting cells, olfactory receptor neurons, and basal cells. The olfactory neurons and the basal cells were similar to that described in other species. Two different types of supporting cells are described. An outstanding characteristic of the supporting cells is the normal presence of abundant phagosomes, apical secretory granules, apocrine-like protrusions, and highly developed smooth endoplasmic reticulum. Apoptotic bodies are frequently found in the infranuclear cytoplasm of supporting cells. The ductular epithelium of Bowman's glands reveals secretory activity. The lamina propria shows mixed Bowman's glands. Great development of smooth endoplasmic reticulum is observed in the mucous acinar cells. Evidence for merocrine and apocrine mechanisms in the Bowman's glands is presented. The presence of apoptotic bodies and phagosomes in supporting cells suggests a participation in the cellular events induced by cell death and proliferation of the olfactory epithelium. The variety of characteristics exhibited by the supporting cells of the olfactory mucosa may contribute to a deeper understanding of their scarcely known functions.

Identification and neuroanatomical distribution of immunoreactivity for mammalian gonadotropin-releasing hormone (mGnRH) in the brain and neural hypophyseal lobe of the toad Bufo arenarum.

The presence and distribution of gonadotropin-releasing hormone (GnRH) in sexually mature specimens of Bufo arenarum was studied by reverse phase/high performance liquid chromatography (RP-HPLC) combined with radioimmunoassay and immunocytochemistry. The analysis of brain extracts with RP-HPLC followed by radioimmunoassay with PBL#45 antiserum showed the presence of only one peak with immunoreactivity for GnRH (ir-GnRH) having the chromatographic and immunological characteristics of mammalian GnRH (mGnRH). This peak was further analyzed with two mGnRH-specific antisera, EL-15 and m1076, yielding serial dilution displacement curves parallel to those obtained with the mGnRH synthetic standard. Immunocytochemical results with the monoclonal antibody LRH13 showed the presence of a terminal nerve-septo-preoptic system with neurons and fibers distributed from the olfactory bulb, septal area, and anterior preoptic area toward the hypothalamus and hypophyseal neural lobe. The main group of ir-GnRH fibers and neurons was identified in the anterior preoptic area. These neurons appear to be the origin of fibers that, after surrounding the preoptic recess, border the dorsal surface of the optic chiasma, extend through the infundibulum, traverse the external layer of the median eminence, and end in the pars nervosa.

The brain of the armadillo Dasypus hybridus. A general view of its most salient features

The most salient neuroanatomical features of the brain of the seven banded armadillo Dasypus hybridus are described. The microscopic characteristics were studied by serial transverse and sagittal paraffin sections, stained with Nissl and Klüver-Barrera technique. This analysis comprises the telencephalon, diencephalon and mesencephalon. The most outstanding features of this brain are: 1) Great development of rhinencephalic structures (olfactory bulbs, olfactory tubercles, anterior commissure and pyriform cortex). 2) The relative size of the induseum griseum strongly suggests that this animal would be useful for a variety of studies on this structure. 3) The high position of the rhinal fissure on the lateral hemispheric wall determines the smallness of neocortex. Therefore, this armadillo is also useful for decortication studies. 4) Absence of a distinct pineal organ. 5) Conspicuous subfornical and subcommissural organs. 6) Absence of a distinct intermediate lobe in the hypophysis

The brain of the armadillo Dasypus hybridus. A general view of its most salient features

The most salient neuroanatomical features of the brain of the seven banded armadillo Dasypus hybridus are described. The microscopic characteristics were studied by serial transverse and sagittal paraffin sections, stained with Nissl and Kl³ver-Barrera technique. This analysis comprises the telencephalon, diencephalon and mesencephalon. The most outstanding features of this brain are: 1) Great development of rhinencephalic structures (olfactory bulbs, olfactory tubercles, anterior commissure and pyriform cortex). 2) The relative size of the induseum griseum strongly suggests that this animal would be useful for a variety of studies on this structure. 3) The high position of the rhinal fissure on the lateral hemispheric wall determines the smallness of neocortex. Therefore, this armadillo is also useful for decortication studies. 4) Absence of a distinct pineal organ. 5) Conspicuous subfornical and subcommissural organs. 6) Absence of a distinct intermediate lobe in the hypophysis

Presence of a fatty acid-binding protein in the armadillo Harderian gland.

A fatty acid-binding protein from the cytosolic fraction of the armadillo Chaetophractus villosus Harderian gland was purified to homogeneity by a procedure based on gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein has an apparent molecular mass of 14 kDa. N-terminal sequence analysis showed that the protein has a blocked N-terminus. For internal amino acid sequencing, the protein was digested in-gel and the resulting peptides were fractionated by reverse-phase high performance liquid chromatography and subjected to automated Edman degradation. Partial amino acid sequencing suggests that it belongs to the heart type. Moreover, it cross-reacted with anti-serum to rat heart fatty acid-binding protein but not with rat intestinal and liver anti-sera. A very slow cross-reaction was also found with anti-serum to rat ALBP. This is the first time that a fatty acid-binding protein has been reported in a Harderian gland.

Chromatographic and immunological identification of GnRH (gonadotropin-releasing hormone) variants. Occurrence of mammalian and a salmon-like GnRH in the forebrain of an eutherian mammal: Hydrochaeris hydrochaeris (Mammalia, Rodentia).

The molecular variants of Gonadotropin releasing hormone (GnRH) in brain extracts of the eutherian mammal Hydrochaeris hydrochaeris (Mammalia, Rodentia) were characterized. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) with different antisera was used. Two different forebrain regions (olfactory bulbs and preoptic-hypothalamic region) were analyzed. Characterization of RP-HPLC fractions from preoptic-hypothalamic extracts with three different RIA systems revealed two immunoreactive GnRH (ir-GnRH) peaks coeluting with mammalian GnRH (mGnRH) and salmon GnRH (sGnRH) synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. Similar results were obtained from olfactory bulb extracts with the same methodology. However, a third ir-GnRH peak in a similar position to that of chicken GnRH II (cIIGnRH) synthetic standard was revealed. As far as we know, this is the first report showing chromatographic and immunological evidences for the presence of a second GnRH variant in the forebrain of an eutherian mammal.

A simple method for taking photographs of histological sections without using neither photographic camera nor microscope.

A simple photographic method for histological sections is described. This is a contact method which does not require the use of photographic camera. It is very similar to the contact print method used routinely in photographic laboratories. It is performed by placing the slides with the histological section, coverslip downwards, over the emulsion of the negative film. Then, they are illuminated with a common photographic enlarger during a brief period of time. Thus, a negative contact print of the section is obtained. This technique yields photographs of high quality focus, sharply contrasted with great detail. These results may also be obtained with low-contrast stained sections. This method is most suitable for photography of large or medium-sized histological sections (macros). This is often the case of neuroanatomical and immunohistochemical studies. Our method, being simple and quick, is recommended for routine use in laboratories.