In vitro and in vivo effects of GABA, muscimol, and bicuculline on lamprey GnRH concentration in the brain of the sea lamprey (Petromyzon marinus)

gamma-Aminobutyric acid (GABA) is a neurotransmitter with a demonstrated neuroregulatory role in reproduction in most representative species of vertebrate classes via the hypothalamus. The role of GABA on the hypothalamus-pituitary axis in lampreys has not been fully elucidated. Recent immunocytochemical and in situ hybridization studies suggest that there may be a neuroregulatory role of GABA on the gonadotropin-releasing hormone (GnRH) system in lampreys. To assess possible GABA-GnRH interactions, the effects of GABA and its analogs on lamprey GnRH in vitro and in vivo were studied in adult female sea lampreys (Petromyzon marinus). In vitro perfusion of GABA and its analogs at increasing concentrations (0.1-100 microM) was performed over a 3-h time course. There was a substantial increase of GnRH-I and GnRH-III following treatment of muscimol at 100 microM. In in vivo studies, GABA or muscimol injected at 200 microg/kg significantly increased lamprey GnRH concentration in the brain 0.5 h after treatment compared to controls in female sea lampreys. No significant change in lamprey GnRH-I or GnRH-III was observed following treatment with bicuculline. These data provide novel physiological data supporting the hypothesis that GABA may influence GnRH in the brain of sea lamprey.     

Immunocytochemical study of fibronectin in the sea lamprey,Petromyzon marinus,and the Atlantic hagfish,Myxine glutinosa

Summary Immunoreactive fibronectin-like material was localized within tissues of agnathans (hagfishes and lampreys) by an immunoperoxidase technique. Fibronectin was detected in basement membranes and in loose and dense connective tissues throughout the agnathan body. A fibronectin-like component was also identified in the plasma of both lampreys and hagfishes. The results indicate that fibronectin or a fibronectin-like material is a major component of agnathan connective tissues. Although there were some variations in the localization of fibronectin both between the lamprey and the hagfish and between agnathan and other vertebrate tissues, the generalized pattern of distribution of fibronectin in the agnathans supports the view that this protein, like that in higher vertebrates, plays a role in cellmatrix adhesion and tissue organization.     

Ultrastructural study of the retinal pigment epithelium during metamorphosis in the sea lamprey (Petromyzon marinus L.)

Summary The sequence of morphological changes in the retinal pigment epithelium during the metamorphic period of the sea lamprey Petromyzon marinus L. has been investigated using electron microscopy. At early metamorphic stages (stages I and II), photoreceptors are present in a small zone of the retina. During these stages, the lateral surface of the epithelial cells shows zonulae occludentes and adhaerentes. The degree of cell differentiation varies throughout the retinal pigment epithelium. Cells covering the differentiated photoreceptors in the central retina have phagosomes, whereas pigment granules appear only in the retinal pigment epithelium dorsal to the optic nerve head. Most epithelial cells have myeloid bodies; their morphology is more complex around the optic nerve head. At stage III, when photoreceptors develop over the whole retina, the distribution of cytoplasmic organelles is almost homogeneous in the retinal pigment epithelium. Subsequently, the basal plasma membrane of the epithelial cells becomes progressively folded and their apical processes enlarged. In addition, extensive gap junctions develop between retinal pigment cells. In late metamorphic stages, noticeable growth of myeloid bodies occurs and consequently the retinal pigment epithelium resembles that of the adult. This study also describes, for the first time, the presence of wandering phagocytes in the retinal pigment epithelium of lampreys; their role in melanosome degradation is discussed.     

Isolation and identification of petromyzestrosterol, a polyhydroxysteroid from sexually mature male sea lamprey (Petromyzon marinus L.)

Petromyzestrosterol (1), a novel polyhydroxylated steroid, was identified from water conditioned with sexually mature male sea lamprey (Petromyzon marinus), a jawless vertebrate animal. Along with this novel steroid, two known steroids, 7α,12α,24-trihydroxy-5α-cholan-3-one-24-sulfate (3k PZS) and 7α,12α,24-trihydroxy-5α-cholan-24-sulfate (PZS), were isolated. Structures of these compounds were unequivocally established by spectroscopic analyses and by comparison with spectra of known compounds. Electro-olfactogram recordings (EOG) showed that 1 at nanomolar concentrations was stimulatory for the olfactory epithelium of adult females. 3k PZS, known to function as a male sex pheromone, was more stimulatory than 1 for the female olfactory epithelia. The concentration-response curve of 3k PZS was exponential in shape with steep slopes between 10(-10) and 10(-6) mol L(-1). The concentration-response curve for 1 was shallower than that for 3k PZS.     

Morphology of the gills of larval and parasitic adult sea lamprey,Petromyzon marinus L.

The general morphology of the gills is similar in larval (ammocoetes) and parasitic adult sea lampreys, Petromyzon marinus, despite different methods of ventilation necessitated by their feeding habits. The gill lamellae are supported by randomly-distributed pillar cells which enclose blood spaces and collagen columns. The distribution of these cells in lampreys is different from that of higher fishes and it may be inefficient for respiratory exchange. The presence of cytoplasmic microfilaments suggests that these cells have the ability to reduce the lamellar blood spaces through contraction. Marginal channels at the tips of the lamellae are lined only by endothelial cells. The thickness of the water-blood pathway in lampreys falls within the range described for higher fishes, with the most efficient gas exchange likely occurring at the lamellar tips where only a single layer of epithelial cells is present. The abrupt increase in height of the epithelium near the lamellar bases in adults, compared to the gradual transition in height along the lamellae in ammocoetes, is perhaps reflective of higher oxygen requirements during the parasitic stage. The consistent appearance of wide, lateral intercellular spaces within the respiratory epithelium of lampreys indicates possible involvement of these spaces in transport. Mucous secretion appears to be an important function of the superficial platelet cells in ammocoetes. “Mitochondria-rich” and “mitochondria-poor” superficial cells are observed in both ammocoetes and adults, with the mitochondria-rich cells more prevalent toward the lamellar bases. The possibility that at least some of these cells may be involved in absorption is discussed. Mitochondria-rich cells in the interlamellar region are morphologically different in ammocoetes and adults but all possess an abundance of smooth endoplasmic reticulum and hence resemble “chloride cells” of higher fishes. The similarity of these cells in the parasitic adult lamprey to chloride cells of marine fishes may reflect the potential of the adult lamprey to osmoregulate in salt water. A scarcity of these cells in ammocoetes and their resemblance to chloride cells in freshwater fishes may reflect the restriction of larval lampreys to a freshwater habitat.     

Immunohistochemical observations of the endocrine pancreas during metamorphosis of the sea lamprey,Petromyzon marinus L.

Summary Light-microscopic immunohistochemistry was used to localize insulin- and somatostatin-immunoreactive cells within developing endocrine pancreatic tissue of metamorphosing lampreys, Petromyzon marinus. The extrahepatic common bile duct and a portion of the intrahepatic bile duct develop into the caudal portion of the endocrine pancreas. The cranial pancreas is composed of follicles originating in the intestinal and diverticular epithelia, thus following the method of formation of pancreatic follicles from gut epithelium in larvae. In both the cranial and caudal portions, and in an intermediate cord of isolated follicles which connect these two major masses, insulin-immunoreactive cells appear first and are followed by cells showing somatostatin-immunoreactivity. In all stages of metamorphosis individual endocrine cells demonstrate immunoreactivity to a single hormone. Biliary atresia in lamprey may have some adaptive significance in providing cells that produce a caudal endocrine pancreas.Supported by NSERC of Canada grant No. A5945 and MRC of Canada grant No. MA8629 to JHY     

Immunocytochemical demonstration of growth hormone,prolactin and somatostatin-like immunoreactivities in the brain of larval,young adult and upstream migrant adult sea lamprey,Petromyzon marinus

Summary Growth hormone, prolactin and somatostatinlike immunoreactivities were demonstrated in the brains of larval, young adult (parasitic) and upstream migrant adult sea lampreys, Petromyzon marinus, by means of immunoperoxidase techniques. Growth hormone (GH) and prolactin (PRL) were observed within separate perikarya in the nucleus praeopticus, within fibers in the commissura praeinfundibularis, and in nerve endings within the neurohypophysis of larval and adult-stage lampreys. Cell bodies demonstrating immunoreactive growth hormone were more numerous than those reactive for prolactin. Unlike in the upstream migrant adult lamprey, no GH or PRL was demonstrated in the adenohypophysis of larval or parasitic lamprey.Somatostatin (SRIF)-like immunoreactive neurons were demonstrated in the nucleus commissurae praeinfundibularis, anterior and posterior pars ventralis hypothalami, pars dorsalis thalami, and the tegmentum motorium rhombencephali of larval, parasitic and upstream migrant adult lampreys. Many of the SRIF containing neurons within the hypothalamus were cerebrospinal fluid (CSF)-contacting cells. SRIF fibers were found throughout most of the brain predominating within the nucleus praeopticus, pars ventralis hypothalami, and the nucleus interpeduncularis. No SRIF immunoreactivity was found within the neurophyophysis. The possible functions of these peptides within the brain of the lamprey are discussed.     

Migration and spawning energetics of the anadromous sea lamprey,Petromyzon marinus

Synopsis Energy expended in migration and reproduction was determined from measurements of caloric concentration and body and gonodal weight for nontrophic sea lampreys collected from different sites along the St. John River, New Brunswick. The estimated cost of locomotion in swimming the 140 km which separates the estuary from the spawning redds was 300 and 260 kcal for males and females respectively. Acutal distance which lampreys swam as well as mean swimming speed were estimated from a linear regression equation relating energy expenditure for locomotion and body weight. Energy expenditure for breeding was considerably greater than that catabolized throughout the upstream migration.     

Growth and intermediary metabolism of larval and metamorphosing stages of the landlocked sea lamprey,Petromyzon marinus L

Synopsis Seasonal changes in blood, liver and muscle substrate (glucose, glycogen and lipid) concentrations and enzyme (pyruvate kinase (PyK), fructose diphosphatase (FDPase), NADP-isocitrate dehydrogenase (ICDH), malic enzyme (ME) and the hexose monophosphate shunt dehydrogenases (HMSD)) activities were assessed in ammocoete and metamorphosing stages of a stream stock of the landlocked sea lamprey, Petromyzon marinus L. In all developmental stages studied, muscle rather than liver tissue served as the main site of carbohydrate and fat storage. Blood glucose and muscle lipid exhibited a positive relationship while liver HMSD and muscle ME activity, a negative relationship, with ammocoete weight. These responses were attributed to a proliferation of red fibers and adipocytes in the ammocoete muscle as the time of metamorphosis approched. Muscle lipid stores of ammocoetes in their last year of larval life increased dramatically during the fall and winter preceding metamorphosis. Changes in tissue enzyme activity of ammocoetes in their last year of larval life indicated that the liver was the site of amino acid incorporation into fat while muscle was the site of lipogenesis from glucose. During the non-trophic period of metamorphosis, stored material was catabolized to provide energy for protein synthesis.     

A critical review of the discovery and application of a migratory pheromone in an invasive fish, the sea lamprey Petromyzon marinus L.

The sea lamprey Petromyzon marinus , an ancient and parasitic agnathan fish native to the North Atlantic, invaded the Laurentian Great Lakes approximately a century ago triggering its fisheries to collapse. Presently, this species is held in check through a toxicant-based control programme directed by a bi-national treaty organization that seeks to develop a more broadly based integrated pest management (IPM) strategy. After a long and difficult search, the most active components of a migratory pheromone for the sea lamprey were identified and synthesized. This pheromone has remarkable potency and considerable potential to enhance various control strategies that fall within an IPM scheme. The pheromone is comprised of a mixture of at least three sulphated steroids: petromyzonamine disulphate (PADS), petromyzosterol disulphate (PSDS) and petromyzonol sulphate (PS). A steroidal sex pheromone appears to have similar promise. These discoveries, which are prompting development of the first pheromonally guided pest management programme for an invasive fish, have revealed significant scientific and policy challenges. Among the former are the needs to acquire long-term and significant support for the science, to synthesize sufficient quantities of cues, and to deploy and measure pheromonal components in natural waters. Among the latter are the needs to regulate this new type of 'pesticide', protect these technologies from exploitation and promote future research on this and other invasive fish species. Lessons from these experiences may be applicable to the study of other invasive fishes.     

Metamorphosis of the olfactory organ of the sea lamprey (Petromyzon marinus L.): Morphological changes and morphometric analysis

In larval sea lampreys (Petromyzon marinus), a small, relatively inconspicuous olfactory organ sac contains small, densely packed olfactory receptor neurons and sustentacular cells. During metamorphosis, the larval organ transforms into a prominent lamellar structure with large distinct olfactory epithelial cells that is characteristic of the adult lamprey. In the present study, scanning electron microscopy and light microscopy are used to examine changes during the seven stages (1–7) of metamorphosis. The magnitude of growth over the course of metamorphosis is evident from the doubling of the relative weight of the nasal sac. During early metamorphosis (stages 1 and 2), the larval olfactory organ enlarges, and by stage 3 specific adult structures begin to form, namely a nasal valve between the nasal tube and the organ, lamellar folds, and diverticuli of the accessory olfactory organ. Subsequent development involves widening of the cells lining the lamellar folds to the form characteristic of postmetamorphic lampreys. Although the cells in the troughs initially retain numerical density values that are significantly higher than those on the lamellar surfaces, by stage 7 values decline both in troughs and along lamellar surfaces to those observed in adults. These results show that although expansion of the olfactory organ is ongoing throughout metamorphosis, remodeling occurs early (by stage 3). This timing provides space for extensive olfactory receptor neuron neurogenesis and differentiation and correlates with the transformation of some organs that were previously examined. This is the first report in any species of olfactory receptor neuron zonation based on morphometric characteristics. J. Morphol. 231:41–52, 1997. © 1997 Wiley-Liss, Inc.     

Morphological changes in the liver of the sea lamprey, Petromyzon marinus L., during metamorphosis: I. Atresia of the bile ducts

The bile ducts in the liver of larval sea lamprey, Petromyzon marinus, undergo programmed degeneration during metamorphosis. The degenerative process is most dramatic in the middle metamorphic stages (3-5), and is asynchronous, occurring more rapidly in small peripheral biliary components than in larger, medial ducts. All classes of bile ducts within the biliary tree exhibit similar histological changes during regression. The initial evidence of degeneration in the epithelium is a folding of the basal lamina, and this is accompanied by cell shrinkage and disruption of cell order. "Shedding" of microvilli and cytoplasmic constituents then takes place at the apical surface resulting in the accumulation of periodic acid-Schiff positive membranous debris in the Lumen. The appearance of "hyalin bodies" in the lumen coincides with the depletion of intermediate-sized filaments from the cytoplasmic matrix. Numerous, large dense bodies, myelin figures, and autophagic vacuoles are consistently observed in necrotic cells. Following cytolysis, bile duct remnants become ensheathed within regions of fibrosis. Ultimately, these fibrous regions are replaced with cords of hepatocytes. By stage 7, all bile ducts have disappeared. The events of biliary atresia in lampreys are comparable to tissue regression which is associated with normal development and pathological conditions in other vertebrates but are particularly reminiscent of those in human biliary atresia. The unique ability of the adult lamprey to service without bile ducts enhances the value of this organism as an experimental model for studying human biliary atresia.     

Distribution of immunoreactive thyroxine in the endostyle and thyroid tissue of the sea lamprey Petromyzon marinus L., 1758

Anti-thyroxine (T₄) immunostaining in the endostyle and thyroid tissue during metamorphosis of sea lamprey Petromyzon marinus suggested that T₄ synthesis occurs on the luminal surface of the apical membrane of selected cells of the endostyle, and that a 'classical' endocrine thyroid function was only evident in post-transformed animals.     

Ultrastructure of chloride cells in young adults of the anadromous sea lamprey, Petromyzon marinus L., in fresh water and during adaptation to sea water.

The chloride cells in the interlamellar areas of the gills of young adult, anadromous sea lampreys, Petromyzon marinus L., captured in fresh water undergo structural modification during the adaptation of these animals to sea water. In fresh water the chloride cells are partially overlapped by mucus-secreting superficial cells and contain an extensive reticulum of cytoplasmic tubules, which are confluent with both lateral and basal plasma membranes, numerous mitochondria, a Golgi complex of moderate size, and numerous apical vesicles. Adaptation to sea water results in a retraction of the superficial cells, exposing the entire apical surface of the chloride cells, and a proliferation of both cytoplasmic tubules and mitochondria. Extensive enlargement of the Golgi complex in the chloride cells of these animals suggests the involvement of this organelle in the proliferation of cytoplasmic tubules. The extracellular tracer, ruthenium red, enters the tubules from the lateral or basal intercellular spaces in both freshwater- and seawater-adapted animals but never enters either tubules or vesicles from the apical surfaces, indicating that these are not confluent. The presence of dividing basal cells and newly-forming chloride cells, combined with evidence of degeneration of chloride cells, suggests that there is a turnover of this cell type. Both superficial and basal cells are phagocytic and involved in heterophagy of degenerating chloride cells. This phenomenon occurs in both fresh water and sea water indicating that the chloride cells may be functional in both environments.     

Morphological changes in the liver of the sea lamprey, Petromyzon marinus L., during metamorphosis. II. Canalicular degeneration and transformation of the hepatocytes

Degeneration of all bile canaliculi takes place in the liver of the sea lamprey, Petromyzon marinus, during metamorphosis. Disintegration of microvilli is observed during earlier stages, and membranous debris ultimately accumulates within the canalicular lumina. Complete occlusion of the lumina and disorganization of junctional complexes is followed by a complete loss of the exocrine biliary pole of hepatocytes and a reorganization of these cells into solid cords. An increase in the size and number of acid phosphatase-containing cytoplasmic bodies coincides with the events of canalicular degeneration. These secondary lysosomes apparently participate in some manner in the isolation and disposal of iron and other bile constituents which no longer can be excreted in bile canaliculi. The loss of the exocrine biliary pole of hepatocytes is concomitant with vascular disturbances in the form of disordered arrangements of sinusoidal endothelial cells and an increase in the population of activated Kupffer cells involved in erythrophagocytosis. The significance of the shift in functional organization of the liver in adult lampreys is discussed in relation to physiological changes in this organism and to human hepatic cholestasis, for which this organism is a potentially valuable experimental model.