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.     

Alterations to the subepithelial layers of the larval alimentary tract during metamorphosis in the sea lamprey Petromyzon marinus,with emphasis on tissue interactions

Light-microscopic histochemistry and conventional electron microscopy were used to study the changes to the subepithelial layers in the larval esophagus of the sea lamprey Petromyzon marinus during metamorphosis. During early stages of metamorphosis, smooth muscle cells of the muscularis mucosae and tunica muscularis dedifferentiate into myofibroblast-like cells, which make contact with the basal lamina of the overlying mucosal epithelium. During later stages, these myofibroblast-like cells redifferentiate into smooth muscle cells, reforming the muscularis mucosae and tunica muscularis. Alterations to the extracellular matrix occur concomitantly.     

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 identification of photoreceptor proteins in hypothalamic cerebrospinal fluid-contacting neurons of the larval lamprey (Petromyzon marinus)

Antibodies directed against different visual pigment opsins, and an antibody raised against the C terminal of the -subunit of retinal G protein (transducin) labelled cerebrospinal fluid-contacting cells located within the hypothalamus (postoptic commissural nucleus and ventral hypothalamic nucleus) of ammocoete lampreys (Petromyzon marinus). These antibodies also labelled photoreceptor cells within the retina and the pineal and parapineal organs, but no other areas of the brain. Despite considerable behavioural and physiological evidence for the existence of deep brain photoreceptors, numerous studies have failed to identify photoreceptor proteins within the basal brain. The results presented in this paper support our recent results in the lizard Anolis carolinensis, suggesting that a group of cerebrospinal fluid-contacting neurons within the vertebrate brain have a photosensory capacity. We speculate that these cells mediate extraocular and extrapineal photoreception in nonmammalian vertebrates.     

Neuroendocrine and behavioral responses to weak electric fields in adult sea lampreys (Petromyzon marinus)

We characterized the behavioral and neuroendocrine responses of adult sea lampreys (Petromyzon marinus) to weak electric fields. Adult sea lampreys, captured during upstream spawning migration, exhibited limited active behaviors during exposure to weak electric fields and spent the most time attached to the wall of the testing arena near the cathode (−). For adult male sea lampreys, exposure to weak electric fields resulted in increased lamprey (l) GnRH-I mRNA expression but decreased lGnRH-I immunoreactivities in the forebrain, and decreased Jun (a neuronal activation marker) mRNA levels in the brain stem. Similar effects were not observed in the brains of female sea lampreys after weak electric field stimulation. The influence of electroreception on forebrain lGnRH suggests that electroreception may modulate the reproductive systems in adult male sea lampreys. The changes in Jun expression may be associated with swimming inhibition during weak electric field stimulation. The results for adult sea lampreys are the opposite of those obtained using parasitic-stage sea lampreys, which displayed increased activity during and after cathodal stimulation. Our results demonstrate that adult sea lampreys are sensitive to weak electric fields, which may play a role in reproduction. They also suggest that electrical stimuli mediate different behaviors in feeding-stage and spawning-stage sea lampreys.     

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.     

Behavioral thermoregulation by Ammocoete larvae of the sea lamprey (Petromyzon marinus) in an electronic shuttlebox

Ammocoete larvae of the sea lamprey Petromyzon marinus, a member of the primitive vertebrate class Agnatha, were tested for thermoregulatory behavior in an electronic shuttlebox (ichthyotron). The final preferendum derived from pooled data for 24 individually tested ammocoetes was characterized by a mean of 13.6 ± 0.17 (s.e.m.)°C, a mode and median of 140°C, and a range of voluntarily occupied temperatures from 10–19°C over a 3-day period.     

Light and electron microscopic immunocytochemical localization of thyroglobulin in the thyroid gland of the anadromous sea lamprey,Petromyzon marinus L., during its upstream migration

Summary Antibodies made against thyroglobulin (TG) were used in an immunocytochemical study for the light and electron microscopic localization of TG in the thyroid gland of the anadromous sea lamprey, Petromyzon marinus, during its upstream migration. TG was found in the follicular lumen and in some colloid droplets within the follicular cells. Except for an immunoreactive product observed in a small portion of the interstitial connective tissue, the location of TG in the lamprey was similar to that in the thyroid of the rat.Supported by National Research Council of Canada Grant no. A5945 to J.H.Y. We thank Dr. F.W.W. Beamish and Mr. R. Robinson who helped in the capture of the lamprey     

Immunocytochemical localization of insulin and somatostatin in the endocrine pancreas of the sea lamprey Petromyzon marinus L., at various stages of its life cycle

Summary Light-microscopic immunocytochemistry and routine staining techniques were used to localize insulin and somatostatin-immunoreactive cells within the endocrine pancreatic tissue of the lamprey, Petromyzon marinus, during various stages of the life cycle. The endocrine pancreas of larvae consists solely of follicles of insulin-immunoreactive cells surrounding the junction of oesophagus, intestine and bile duct. Somatostatin-immunoreactive cells are restricted to the intestinal epithelium. In both parasitic and upstream-migrating adults the endocrine pancreas consists of cranial and caudal portions, both containing separate populations of insulin and somatostatin-immunoreactive cells.Supported by NSERC of Canada grant no. A5945 to JHY     

The pronephros of the early ammocoete larva of lampreys (Cyclostomata,Petromyzontes): Fine structure of the renal tubules

Summary The renal tubules of the paired pronephros in early larvae (ammocoetes) of two lamprey species, Lampetra fluviatilis and Petromyzon marinus, were studied by use of light-, scanning- and transmission electron microscopy. They consist of (1) a variable number of pronephric tubules (3 to 6), and (2) an excretory duct. By fine-structural criteria, the renal tubules can be divided into 6 segments. Each pronephric tubule is divided into (1) the nephrostome and (2) the proximal tubule, the excretory duct consisting of (3) a common proximal tubule followed by (4) a short intermediate segment, and then by a pronephric duct composed of (5) a cranial and (6) a caudal section. The epithelium of the nephrostome displays bundles of cilia. The cells of the proximal tubule possess a brush border, many endocytotic organelles and a system of canaliculi (tubular invaginations of the basolateral plasmalemma). The same characteristics are encountered in the epithelium of the common proximal tubule; however, the number of these specific organelles decreases along the course of this segment in a posterior direction. In the intermediate segment, the epithelium appears structurally nonspecialized. The cells of the cranial pronephric duct lack a brush border; they have an extensive system of canaliculi and numerous mitochondria. The caudal pronephric duct is lined by an epithelium composed of light and dark cells; the latter are filled with mitochondria and the former contain mucus granules beneath the luminal plasmalemma. The tubular segments found in the pronephros are the same in structure and sequence as in the lamprey opisthonephroi. However, only the nephrostomes and proximal tubules occur serially in the pronephros, while the common proximal tubule, the intermediate segment and the cranial pronephric duct form portions of a single excretory duct.This paper is dedicated to the memory of Professor W. Bargmann, long-time editor of Cell and Tissue Research, the author of a splendid review on the structure of the vertebrate kidney and a master of German scientific writing.     

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called “CLARITY” was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.     

Effects of ammonia,chloroquine, and monensin on the vacuolar apparatus of an absorptive epithelium

Summary The distribution of two major immunoreactive forms of somatostatin, somatostatin-14 and somatostatin-34, within the brain, pancreas and intestine of adult lampreys, Petromyzon marinus, was identified using antisera raised against these peptides. Immunostaining of the brain is similar in juveniles and upstream migrants, and somatostatin-14 is the major somatostatin form demonstrated. A few somatostatin-34-containing cells are localized within the olfactory bulbs, thalamus and hypothalamus, but cells immunoreactive to anti-somatostatin-34 in the hypothalamus and thalamus do not co-localize somatostatin-14. Immunostaining of pinealocytes within the pineal pellucida with anti-somatostatin-14 may infer a novel function for this structure. Somatostatin-14 and somatostatin-34 are co-localized within D-cells of the cranial pancreas and caudal pancreas of juveniles and upstream migrants. Numerous somatostatin-34-immunoreactive cells are distributed within the epithelial mucosa of the anterior intestine but not all of these cells cross-react with anti-somatostatin-14. It appears that somatostatin-34 is the major somatostatin in the pancreo-gastrointestinal system of adult lampreys.     

Distribution of FMRFamide-like immunoreactivity in the brain and neurohypophysis of the lamprey,Lampetra japonica

Summary Distribution of molluscan cardio-excitatory tetrapeptide Phe—Met—Arg—Phe—NH₂ (FMRFamide) was determined by means of immunohistochemistry in the brain and neurohypophysis of the lamprey, Lampetra japonica. Many FMRFamide-like immunoreactive neurons were found in the periventricular nuclear region and in a region near the mammillary recess. Neurons situated in the former region were larger. The immunoreactive cell groups were shown to be located at sites differing from those of the AF-positive cell groups. The fibers of immunoreactive neurons extended in all directions within the brain and towards the spinal cord, some reaching the third ventricle and capillaries. Thus, FMRFamide-like immunoreactive peptides appear to function as neurotransmitters or neuromodulators and possibly also as neurohormones. FMRFamide-like immunoreactive material was rarely observed in the posterior neurohypophysis (neural lobe), but was noted to be present to a limited extent in the caudal part of the anterior neurohypophysis (median eminence). It would thus follow that FMRFamide-like immunoreactive neurons may not necessarily be related to the hypothalamo-neural lobe system, but may possibly be associated with the hypothalamoadenohypophysial system. The pineal body showed no FMRFamide-like immunoreactivity.     

Movement of radio-tagged anadromous sea lamprey during the spawning migration in the River Mondego (Portugal)

The available spawning habitat for the anadromous sea lamprey, Petromyzon marinus L., population that enters the River Mondego has been drastically reduced in the last 20 years. The installation of a fish passage in the first impassable dam, the Açude-Ponte, would enable sea lamprey to recolonise the 34.6-km river stretch between the Açude-Ponte and Raiva dams. In order to assess the suitability of the upstream river stretches for this species, 10 radio-tagged sea lamprey were released upstream of the Açude-Ponte dam and tracked continuously throughout the entire migratory path. Lamprey were unable to pass over intact weirs that had been built for recreational purposes. Sea lamprey movements were more frequent during dusk and night than other periods. Increased river discharge at night, resulting from the operation of the Raiva power station, stimulated lamprey movements but reduced ground speeds recorded. After reaching a certain location, some of the animals maintained their position for several weeks, before undergoing a new movement. Two of the main tributaries of this river stretch were used by some sea lamprey, indicating that the animals were able to find these historical spawning grounds.     

Morphological analysis of geographic variation of sea lamprey ammocoetes in Portuguese river basins

The main objective of this study was to compare the morphological variability of sea lamprey (Petromyzon marinus L.) larvae from the main Portuguese river basins. Samples were collected in rivers Minho, Lima, Cávado, Vouga, Mondego, Tejo and Guadiana. Specimens were analysed in terms of morphometric (linear body measures) and meristic (number of myomeres) characters to investigate the hypothesis of population fragmentation between river basins caused by some degree of homing behaviour. The discriminant analysis showed a morphological segregation of the studied populations based on the characters head, tail and branchial length. The discriminatory power of the meristic characters was comparatively weaker, with the number of trunk myomeres, and to some extent the head myomeres, being responsible for the reduced separation between groups. Both analyses were consistent in identifying the cephalic region as the most important morphological feature to discriminate populations of sea lamprey larvae in the Portuguese territory. The largest cephalic region of the ammocoetes sampled in the northern river basins may be responsible for a better feeding efficiency and, consequently, higher values of condition factor. Guest editors: S. Dufour, E. Prévost, E. Rochard & P. Williot Fish and diadromy in Europe (ecology, management, conservation)     

Structure of the cumulus oophorus at the time of fertilization

Summary The paired external glomus of the fully developed pronephros has been studied in early larvae (ammocoetes) of 2 lamprey species, Lampetra fluviatilis and Petromyzon marinus, several weeks after hatching and newly hatched, by use of light-, scanning (SEM) and transmission (TEM) electron microscopy. Three weeks after hatching the glomus is a complex of capillary loops supplied by a single arteriole branching from the aorta. The glomus consists of 3 cell types: podocytes, fenestrated endothelium, and mesangial cells. A basement membrane, which has a close contact to the podocytes, is the only continuous barrier between blood and the coelomic cavity. The glomus exhibits all fine-structural elements known to be essential for function in the glomeruli of other vertebrates. We therefore assume the pronephric glomus of lampreys to be functional in ultrafiltration, with the ultrafiltrate released into the coelomic cavity. In newly hatched larvae, the structure of the glomus is not fully developed. In this earlier stage several afferent arterioles supply each glomus. The endothelial cells in the glomar capillaries still lack regular epithelial organization and resemble mesenchymal cells. However, the presence of typical podocytes stretching over a continuous basement membrane suggests that the tissue is already capable of ultrafiltration.This paper is dedicated to the memory of Professor W. Bargmann, long-time editor of Cell and Tissue Research, the author of a splendid review on the structure of the vertebrate kidney and a master of German scientific writing     

FMRFamide-like immunoreactivity in the brain of the Pacific hagfish,Eptatretus stouti (Myxinoidea)

Summary The distribution of FMRFamide-like immunoreactivity was investigated in the brain of a myxinoid, the Pacific hagfish,Eptatretus stouti, by means of immunocytochemistry. In the forebrain, labelled cell bodies occurred in the infundibular nucleus of the hypothalamus and some closely adjacent nuclei. Labelled fibers formed a diffuse network in the forebrain, but there was no evidence for the presence of intracerebral ganglionic cells of the terminal nerve or a central projection of the terminal nerve. In the hindbrain, a group of labelled cells was found in the trigeminal sensory nucleus. A distinet terminal arborization occurred in the ventrally adjacent nucleus A of Kusunoki and around the nuclei of the branchial motor column. These findings suggest that FMRFamide may play a role in the central control of branchiomotor activity.     

Communicating (gap) junctions between chloride cells in the gill epithelium of the lamprey,Geotria australis

Summary Freeze-fracture replicas show that communicating (gap) junctions are present between chloride cells in the gill epithelium of young adults of the Southern Hemisphere lamprey, Geotria australis, acclimated to full-strength sea water. The junctions, which were already present when these lampreys were migrating downstream, may help coordinate the secretory activities of the chloride cells during the marine phase of the lamprey life cycle.     

Distribution of aromatase-immunoreactive cells in the mouse forebrain

Summary The distribution of aromatase-immunoreactive cells was studied by immunocytochemistry in the mouse forebrain using a purified polyclonal antibody raised against human placental aromatase. Labeled perikarya were found in the dorso-lateral parts of the medial and tuberal hypothalamus. Positive cells filled an area extending between the subincertal nucleus in the dorsal part, the ventromedial hypothalamic nucleus in the ventral part, and the internal capsule and the magnocellular nucleus of the lateral hypothalamus in the lateral part. The same distribution was seen in the two strains of mice that were studied (Jackson and Swiss), and the number of immunoreactive perikarya did not seem to be affected by castration or testosterone treatment. No immunoreactivity could be detected in the medial regions of the preoptic area and hypothalamus; these were expected to contain the enzyme based on assays of aromatase activity performed in rats and on indirect autoradiographic evidence in mice. Our data raise questions concerning the distribution of aromatase in the brain and the mode of action of the centrally produced estrogens.