Neurogenesis in the vomeronasal epithelium of adult garter snakes: 3. Use of H3-thymidine autoradiography to trace the genesis and migration of bipolar neurons

Use of H3-thymidine autoradiography and unilateral vomeronasal (VN) axotomy has permitted us to demonstrate directly the existence of VN stem cells in the adult garter snake and to trace continuous bipolar neuron development and migration in the normal VN and deafferentated VN epithelium in the same animal. The vomeronasal epithelium and olfactory epithelium of adult garter snakes are both capable of incorporating H3-thymidine. In the sensory epithelium of the vomeronasal organ, H3-thymidine-labeled cells were initially restricted to the base of the undifferentiated cell layer in animals surviving 1 day following H3-thymidine injection. With increasing survival time, labeled cells progressively migrated vertically within the receptor cell column toward the apex of the bipolar neuron layer. In both the normal and denervated VN epithelium, labeled cells were observed through the 56 days of postoperative survival. In the normal epithelium, labeled cells were always located within the matrix of the intact receptor cell columns. However, labeled cells of the denervated epithelium were always located at the apical front of the newly formed cell mass following depletion of the original neuronal cell population. In addition, at postoperative days 28 and 56, labeled cells of the denervated VN epithelium achieved neuronal differentiation and maturation by migrating much farther away from the base of the receptor cell column than the labeled cells on the normal, unoperated contralateral side. This study directly demonstrates that basal cells initially incorporating H3-thymidine are indeed stem cells of the VN epithelium in adult garter snakes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal transduction in the vomeronasal organ of garter snakes: ligand-receptor binding-mediated protein phosphorylation.

The vomeronasal (VN) system of garter snakes plays an important role in several species-typical behaviors, such as prey recognition and responding to courtship pheromones. We (X.C. Jiang et al., J. Biol. Chem. 265 (1990) 8736-8744 and Y. Luo et al., J. Biol. Chem. 269 (1994) 16867-16877) have demonstrated previously that in the snake VN sensory epithelium, the chemoattractant ES20, a 20-kDa glycoprotein derived from electric shock-induced earthworm secretion, binds to its receptor which is coupled to PTX-sensitive G-proteins. Such binding results in elevated levels of IP3. We now report that ES20-receptor binding regulates the phosphorylation of two membrane-bound proteins with molecular masses of 42- and 44-kDa (p42/44) in both intact and cell-free preparations of the VN sensory epithelium. ES20 and DAG regulate the phosphorylation of p42/44 in a similar manner. ES20-receptor binding-mediated phosphorylation of p42/44 is rapid and transient, reaching a peak value within 40 seconds and decaying thereafter. Phosphorylation of p42/44 appears to be regulated by the countervailing actions of a specific membrane-bound protein kinase and a protein phosphatase. The phosphorylation of these membrane-bound proteins significantly reduces the activity of G-proteins as evidenced by a decrease in GTPase activity, but has little effect on ligand-receptor binding. These findings suggest that p42/44 play a role in modulating the signal transduction induced by ES20 in the vomeronasal system.     

Developmental changes in cytochrome oxidase histochemistry in the main and accessory olfactory bulbs of embryonic and neonatal garter snakes (Thamnophis sirtalis spp.)

Developmental studies examining the changes in oxidative metabolic activity are useful for understanding how and if the vomeronasal and olfactory systems respond to stimulation during embryogenesis. Garter snakes are good candidates for examining the potential functionality of the vomeronasal system in utero. In adult garter snakes, the vomeronasal system mediates many behaviors. Neonatal garter snakes exhibit these same behaviors, and the vomeronasal system has been shown to mediate feeding behavior in neonates. Using cytochrome oxidase histochemistry, we examined changes in the oxidative metabolic activity of main and accessory olfactory bulbs of embryonic and neonatal garter snakes (Thamnophis sirtalis sirtalis and T. s. parietalis). Cytochrome oxidase staining is greater in the accessory olfactory bulb than in the main olfactory bulb of embryonic garter snakes. However, neonates show no differences in the staining of the accessory and main olfactory bulbs, suggesting a change in the stimulation of the main olfactory bulb after birth. This is the first report of cytochrome oxidase histochemistry in reptiles and in the vomeronasal system of embryonic vertebrates. © 1993 Wiley-Liss, Inc.     

Species specificity of methyl ketone profiles in the skin lipids of female garter snakes,genus Thamnophis

One of the relatively few vertebrate pheromones to be chemically identified, the female sex pheromone of the red-sided garter snake (Thamnophis sirtalis parietalis) is a series of saturated and monounsaturated methyl ketones contained within female skin lipids. During the breeding season, this pheromone is responsible for eliciting male courtship behaviors and males are able to utilize pheromonal variation to discriminate among females. While the pheromone system of the red-sided garter snake has been the subject of many studies, relatively little is known about the pheromone systems of other garter snakes. Through chemical analyses, we demonstrate that female skin lipids of the red-spotted garter snake (Thamnophis sirtalis concinnus), northwestern garter snake (Thamnophis ordinoides), and plains garter snake (Thamnophis radix) contain similar methyl ketones. The methyl ketone profiles of these snakes differ qualitatively from one another and from the methyl ketone profiles of red-sided garter snakes with differences particularly pronounced between sympatric species. Our results provide evidence that the use of methyl ketones in sexual signaling may be ubiquitous for Thamnophis species and suggest that these compounds could play a role in reproductive isolation between species in this genus.     

Light and electron microscopic observations on the normal structure of the vomeronasal organ of garter snakes

The vomeronasal epithelium of adult garter snakes (Thamnophis sirtalis and T. radix) was studied by light and electron microscopy. The sensory epithelium is extraordinarily thick, consisting of a supporting cell layer, a bipolar cell layer, and an undifferentiated cell layer. The supporting cell layer is situated along the luminal surface and includes supporting cells and the peripheral processes (dendrites) of bipolar neurons. The luminal surfaces of both supporting cells and bipolar neurons are covered with microvilli. Specializations of membrane junctions are always observed between adjacent cells in the subluminal region. Below the supporting cell layer, the epithelium is characterized by a columnar organization. Each column contains a population of bipolar neurons and undifferentiated cells. These cells are isolated from the underlying vascular and pigmented connective tissue by the presence of a thin sheath of satellite cells and a basal lamina. Heterogeneity of cell morphology occurs within each cell column. Generative and undifferentiated cells occupy the basal regions and mature neurons occupy the apical regions. Transitional changes in cell morphology occur within the depth of each cell column. These observations suggest that the vomeronasal cell column is the structural unit of the organ and may represent the dynamic unit for cell replacement as well. A sequential process of cell proliferation, neuronal differentiation, and maturation appears to occur in the epithelium despite the adult state of the animal.     

Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate

The cell-free supernatant of sterile inflammatory peritoneal exudates contains a phospholipase A2 that participates in the digestion of Escherichia coli killed by polymorphonuclear leukocytes or by the purified bactericidal/permeability increasing protein (BPI) of these cells. This phospholipase A2 has been purified, and the sequence of the NH2-terminal 39 amino acids has been determined and compared with sequences of both BPI-responsive and BPI-nonresponsive phospholipases A2 from snake venoms and mammalian pancreas. The high concentration and location of basic residues in the NH2-terminal region is a common feature of BPI-responsive phospholipases A2 and may characterize those phospholipases A2 participating in inflammatory events.     

Female snake sex pheromone induces membrane responses in vomeronasal sensory neurons of male snakes

The vomeronasal organ (VNO) is important for activating accessory olfactory pathways that are involved in sexually dimorphic mating behavior. The VNO of male garter snakes is critically important for detection of, and response to, female sex pheromones. In the present study, under voltage-clamp conditions, male snake VNO neurons were stimulated with female sexual attractiveness pheromone. Thirty-nine of 139 neurons exhibited inward current responses (reversal potential: -10.6 +/- 2.8 mV). The amplitude of the inward current was dose dependent, and the relationship could be fitted by the Hill equation. Under current-clamp conditions, application of pheromone produced membrane depolarizing responses and increases in firing frequency. These results suggest that the female pheromone directly affects male snake VNO neurons and results in opening of ion channels, thereby converting the pheromone signal to an electrical signal. The response to female pheromone is sexually dimorphic, that is, the pheromone does not evoke responses in VNO neurons of female snakes. An associated finding of the present study is that the female sex pheromone, which is insoluble in aqueous solutions, became soluble in the presence of Harderian gland homogenate.     

Biophysical costs associated with tetrodotoxin resistance in the sodium channel pore of the garter snake, <Emphasis Type="Italic">Thamnophis sirtalis</Emphasis>

Tetrodotoxin (TTX) is a potent toxin that specifically binds to voltage-gated sodium channels (NaV). TTX binding physically blocks the flow of sodium ions through NaV, thereby preventing action potential generation and propagation. TTX has different binding affinities for different NaV isoforms. These differences are imparted by amino acid substitutions in positions within, or proximal to, the TTX-binding site in the channel pore. These substitutions confer TTX-resistance to a variety of species. The garter snake Thamnophis sirtalis has evolved TTX-resistance over the course of an arms race, allowing some populations of snakes to feed on tetrodotoxic newts, including Taricha granulosa. Different populations of the garter snake have different degrees of TTX-resistance, which is closely related to the number of amino acid substitutions. We tested the biophysical properties and ion selectivity of NaV of three garter snake populations from Bear Lake, Idaho; Warrenton, Oregon; and Willow Creek, California. We observed changes in gating properties of TTX-resistant (TTXr) NaV. In addition, ion selectivity of TTXr NaV was significantly different from that of TTX-sensitive NaV. These results suggest TTX-resistance comes at a cost to performance caused by changes in the biophysical properties and ion selectivity of TTXr NaV.     

Structure of LEP100, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA

LEP100, a membrane glycoprotein that has the unique property of shuttling from lysosomes to endosomes to plasma membrane and back, was purified from chicken brain. Its NH2-terminal amino acid sequence was determined, and an oligonucleotide encoding part of this sequence was used to clone the encoding cDNA. The deduced amino acid sequence consists of 414 residues of which the NH2-terminal 18 constitute a signal peptide. The sequence includes 17 sites for N-glycosylation in the NH2-terminal 75% of the polypeptide chain followed by a region lacking N-linked oligosaccharides, a single possible membrane-spanning segment, and a cytoplasmic domain of 11 residues, including three potential phosphorylation sites. Eight cysteine residues are spaced in a regular pattern through the lumenal (extracellular) domain, while a 32-residue sequence rich in proline, serine, and threonine occurs at its midpoint. Expression of the cDNA in mouse L cells resulted in targeting of LEP100 primarily to the mouse lysosomes.     

Cryptic forcible insemination: male snakes exploit female physiology, anatomy, and behavior to obtain coercive matings

Whether males can inseminate uncooperative females is a central determinant of mating system evolution that profoundly affects the interpretation of phenomena such as multiple mating by females, mate choice, reproductive seasonality, and courtship tactics. Forcible insemination is usually inferred from direct physical battles between the sexes and has been dismissed on intuitive grounds for many kinds of animals. For example, snakes have elongate flexible bodies (making it difficult for a male to restrain a female physically), males are typically smaller than females, and copulation requires female cloacal gaping to enable intromission. Male garter snakes (Thamnophis sirtalis) do not display any overt aggression during courtship and simply lie over the female and exhibit rhythmic pulsating caudocephalic waves of muscular contraction; previous studies have interpreted this behavior as a mechanism for eliciting female receptivity. In contrast, we show that male garter snakes forcibly inseminate females. They do so by taking advantage of specific features of snake physiology, respiratory anatomy, and antipredator behavior. The snake lung extends along most of the body, with the large posterior section (the saccular lung) lacking any respiratory exchange surface. Rhythmic caudocephalic waves by courting male garter snakes push anoxic air from the saccular lung forward and across the respiratory surfaces such that females cannot obtain oxygen. Their stress response involves cloacal gaping, which functions in other contexts to repel predators by extruding feces and musk but in this situation permits male intromission. Thus, superficially benign courtship behaviors may involve cryptic coercion even in species for which intuition dismisses any possibility of forcible insemination.     

Discordant patterns of population structure for two co‐distributed snake species across a fragmented Ontario landscape

Aim The goal of our study was to investigate the effects of a fragmented landscape on the genetic population structure of two sympatric snake species that differ in habitat preference. The eastern garter snake (Thamnophis sirtalis sirtalis) is a common, habitat generalist, whereas the endangered eastern foxsnake (Mintonius [Elaphe] gloydi) is rarer, geographically restricted, and a marsh‐specialist. We were most interested in comparing the genetic population structure of both species and identifying any natural and human‐created features of the landscape that overlap with genetic disjunctions. Location Southwestern Ontario, Canada, surveying over half of the remaining range of the eastern foxsnake. Methods We utilized DNA microsatellite markers to examine genetic population structure of both species. The number of genetically distinct clusters for each species was determined using both Bayesian spatial assignment and spatial principal component analyses (sPCA). Genetic clusters were overlaid onto a habitat map to deduce possible physiognomic barriers to gene flow. Results Spatial assignment revealed three genetic clusters for garter snakes and five for foxsnakes. Each individual garter snake had a near equal probability of membership to two or more clusters with no cluster mapping onto a discrete geographic region, indicating that garter snakes comprise a single genetic population. The identified foxsnake clusters correspond to geographically circumscribed locations on the landscape, roughly coincident with isolated patches of suitable habitat. sPCAs revealed significant global allelic structure for foxsnakes, but not for garter snakes. No significant local structure was found for either species. Main Conclusions Our results imply that foxsnakes and garter snakes are differentially impacted by the same landscape or have dramatically different effective population sizes. Unsuitable intervening habitat such as agricultural tracts and roads between existing populations of foxsnakes appears to act as barriers to gene flow, while garter snake movement appears unrestricted by these features. Our findings have important implications for the management of eastern foxsnakes.     

Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

Purification and complete sequence of a small proteolipid associated with the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

The purified plasma membrane H(+)-ATPase of Schizosaccharomyces pombe and Saccharomyces cerevisiae display, in addition to the catalytic subunit of 100 kDa, a highly mobile component, soluble in chloroform/methanol. Chloroform/methanol extraction of S. cerevisiae plasma membranes led to isolation of a low molecular weight proteolipid identical to that present in purified H(+)-ATPase. NH2-terminal amino acid sequencing revealed a 38-residue polypeptide with a calculated molecular mass of 4250 Da. The polypeptide lacks the first two NH2-terminal amino acids as compared with the deduced sequence of the PMP1 gene (for plasma membrane proteolipid) isolated by hybridization with an oligonucleotide probe corresponding to an internal amino acid sequence of the proteolipid. The polypeptide is predicted to contain an NH2-terminal transmembrane segment followed by a very basic hydrophilic domain.     

Isolation from earthworms of a proteinaceous chemoattractant to garter snakes

We have isolated a potent proteinaceous chemoattractant from aqueous washes of earthworm (Lumbricus terrestris) for garter snakes (Thamnophis sirtalis) by means of a covalent chromatography. It contained free sulfhydryl groups and showed an apparent mass of 20 kDa. The chemoattractive activity of this protein could be destroyed by heating as well as by proteolysis. Its activity could also be reversibly blocked by mixed disulfide formation with dithiodipyridine, suggesting that the free sulfhydryl(s) was essential for its function as a chemoattractant. This bioactive material had a tendency to form intermolecular crosslinked aggregates during isolation, if reducing agents were not included. Some of the high-molecular-weight aggregates cochromatographed with earthworm cuticle collagen on Ultragel AcA 34 or 44 columns. In contrast to an earlier report by D. M. Kirschenbaum, N. Schulman, and M. Halpern [1986) Proc. Natl. Acad. Sci. USA 83, 1213-1216) the purified earthworm collagen showed no chemoattractive activity to garter snakes.     

Chemosensitive conductance and inositol 1,4,5-trisphosphate-induced conductance in snake vomeronasal receptor neurons

Snake vomeronasal receptor neurons in slice preparations were studied using the patch-clamp technique in the conventional and nystatin-perforated whole-cell configurations. The mean resting potential was approximately -70 mV; the average input resistance was 3 GOmega. Neurons required current injection of only 1-10 pA to display a variety of spiking patterns. Intracellular dialysis of 100 microM inositol 1,4,5-trisphosphate (IP(3)) evoked an inward current in 38% of neurons, with an average peak amplitude of 16.4 +/- 2.8 pA at a holding potential of -70mV. Application of 100 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate (F-IP(3)), a derivative of IP(3), also evoked an inward current in 4/8 (50%) neurons (32.6 +/- 58 pA at -70 mV, n = 4). The reversal potentials of the induced components were estimated to be -14 +/- 5 mV for IP(3) and -17 +/- 3 mV for F-IP(3). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP(3)-evoked inward current to 1.6 +/- 1.1 pA at -70 mV (n = 6). With Cs(+)-containing internal solution, neither the Ca(2+)-ATPase inhibitor thapsigargin (1-50 microM) nor the Ca(2+)-ionophore ionomycin (10 microM) evoked a significant current response, suggesting that IP(3) can elicit current response in the neurons without mediation by intracellular Ca(2+) stores. Intracellular application of 1 mM cAMP evoked no detectable current response. Extracellular application of chemoattractant for snakes evoked a very large inward current. The reversal potential of the chemoattractant-induced current was similar to that of the IP(3)-induced current. The present results suggest that IP(3) may act as a second messenger in the transduction of chemoattractants in the garter snake vomeronasal organ.     

Chemo-attractant for the garter snake: characterization of vomeronasally-mediated response-eliciting components of earthworm wash--III

Heating a 0.1 N HCl solution of lyophilized garter snake, Thamnophis sirtalis, chemoattractant obtained from earthworm, Lumbricus terrestris, surface washings, in a boiling water bath, and separation by gel chromatography, gave two fractions, F2, of high molecular weight and containing all the chemoattractant activity, and F4, of low molecular weight. The activity is lost between 15 and 30 min of heating. A similarly treated 0.1 N NaOH solution of lyophilized washings gave no F2 fraction and an F4 fraction in increasing amount. All the chemoattractant activity is lost between 0 and 15 min. Separations were monitored by assays for protein and carbohydrate.     

Environmental and seasonal adaptations of the adrenocortical and gonadal responses to capture stress in two populations of the male garter snake, Thamnophis sirtalis

Stress and reproduction are generally thought to work in opposition to one another. This is often manifested as reciprocal relationships between glucocorticoid stress hormones and sex steroid hormones. However, seasonal differences in how animals respond to stressors have been described in extreme environments. We tested the hypothesis that garter snakes, Thamnophis sirtalis, with limited reproductive opportunities will suppress their hormonal stress response during the breeding season relative to conspecifics with an extended breeding season. The red-sided garter snake, T.s. parietalis, of Manitoba, Canada, has a brief breeding season during which males displayed no change in either plasma levels of testosterone or corticosterone, which were both elevated above basal levels, in response to capture stress. During the summer, capture stress resulted in increased plasma corticosterone and decreased testosterone. During the fall, when mating can also occur, males exhibited a significant decrease in testosterone but no increase in corticosterone in response to capture stress. The red-spotted garter snake, T.s. concinnus, of western Oregon, has an extended breeding season during which males displayed a stress response of increased plasma corticosterone and decreased testosterone levels. The corticosterone response to capture stress was similar during the spring, summer, and fall. In contrast, the testosterone response was suppressed during the summer and fall when gametogenesis was occurring. These data suggest that male garter snakes, in both populations, seasonally adapt their stress response but for different reasons and by potentially different mechanisms. J. Exp. Zool. 289:99-108, 2001.