Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.     

Changes in brain gonadotropin-releasing hormone, plasma estradiol 17-beta, and progesterone during the final reproductive cycle of the female sea lamprey, Petromyzon marinus.

Changes in ovarian morphology, brain gonadotropin-releasing hormone (GnRH), plasma estradiol, and progesterone were examined during the 1988 and 1989 spawning migrations of the adult female sea lamprey, Petromyzon marinus. There were significant increases through time in brain GnRH (1989) and plasma estradiol (1988 and 1989), with progesterone levels fluctuating (1988 and 1989) during the freshwater phase of the spawning migrations. In 1989, brain GnRH and plasma estradiol levels gradually increased through time until just prior to spawning when levels decreased. During 1988, there were no significant changes in GnRH, which may reflect lower temperatures in that year. These data provide new information on brain GnRH during the final maturational processes in the female sea lamprey.     

Distribution of lamprey gonadotropin-releasing hormone-III (GnRH-III) in brains of larval lampreys (Petromyzon marinus)

Two immunoreactive forms of gonadotropinreleasing hormone (GnRH), lamprey GnRH-I and lamprey GnRH-III, were found in neurons in larval sea lampreys (Petromyzon marinus). Using antisera preferentially directed against either lamprey GnRH-I or-III, dense reaction product was seen in cell bodies in the rostral hypothalamus and preoptic area. Reaction product was also dense in fibers to and within the neurohypophysis, in addition to numerous fibers which projected caudally, beyond the neurohypophysis through the mesencephalon. The majority of immunoreactive GnRH was lamprey GnRH-III, and when lamprey GnRH-I was seen, it was in cells that appeared to contain both forms of GnRH. A small number of cells found in the caudal hypothalamus contained only immunoreactive lamprey GnRH-III, and these may constitute a functional subgroup within the population of GnRH neurons. In animals undergoing metamorphosis there was a large increase in reaction product in all GnRH-containing cells and fibers. A striking change within the distribution of GnRH cells was localized to a distinct group of GnRH-immunoreactive cells (GnRH-I and-III) in the ventral anterior hypothalamic area. These cells were minimally detectable in larvae, but during metamorphosis became densely filled with immunoreactive product in perikarya and distal processes. The results are consistent with the hypothesis that lamprey GnRH-III is an important form of GnRH during the maturation of GnRH cells and fibers, and further indicates that these cells have attained their normal positions in the preoptic area and hypothalamus before metamorphosis.     

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.     

Nitrergic innervation and nitrergic cells in arteriovenous anastomoses

Nitrergic innervation and nitrergic epithelioid cells were studied in arteriovenous anastomoses of the tongue, ear, eye, and glomus organ of the finger in different species (rat, rabbit, dog, and man), by means of immunohistochemistry for nitric oxide synthase and enzyme histochemistry utilizing the catalytic activity of this enzyme (the NADPH-diaphorase reaction). Nitrergic perivascular fibers of the tongue were concentrated along the arterial tree and were maximal at the arteriovenous anastomoses in all species. Generally, fewer fibers were located around comparable segments of the episcleral eye vasculature. Only a few nitrergic fibers were found in the canine and rabbit ear, and in the glomus organ of the human finger; however, epithelioid cells in the tunica media of arteriovenous anastomoses of these organs were NADPH-diaphorase-positive and were moderately immunoreactive for nitric oxide synthase. In the epithelioid cells, the reaction product of the NADPH-diaphorase could also be demonstrated by transmission electron microscopy. The epithelioid cells were negative for the panneural and neuroendocrine marker PGP 9.5 confirming the myocytotic nature of these nitrergic cells. Thus, nitric oxide might play a role in mediating the vessel tone of arteriovenous anastomoses via nitrergic nerves or epithelioid cells.     

Sea lamprey Petromyzon marinus: an exception to the rule of homing in anadromous fishes

Anadromous fishes are believed to make regular circuits of migration in the sea before homing to their natal rivers. Sea lamprey Petromyzon marinus is an anadromous fish that is an exception to this life-history pattern. It also differs from other anadromous fishes in that its adult phase is parasitic, a feeding strategy that should make homing problematic for lamprey cohorts that become widely dispersed through transport by the diverse hosts they parasitize. We sequenced a portion of the mitochondrial DNA control region from sea lampreys collected from 11 North American east coast rivers to test for genetic evidence of homing. There were no significant differences (chi2=235.1, p=0.401) in haplotype frequencies among them, with almost 99 per cent of haplotypic diversity occurring within populations. These findings, together with concordant genetic results from other geographical regions and ancillary information on pheromonal communication, suggest that sea lamprey does not home but rather exhibits regional panmixia while using a novel 'suitable river' strategy to complete its life cycle.     

Changes in intestinal and hepatic thyroid hormone deiodination during spontaneous metamorphosis of the sea lamprey, Petromyzon marinus

We measured microsomal low-K(m) outer-ring deiodination (ORD) and inner-ring deiodination (IRD) activities for thyroxine (T(4)) and 3, 5,3'-triiodothyronine (T(3)) in intestine and liver in nonmetamorphosing (undersized) larvae, immediately premetamorphic larvae, animals in stages 1-7 of metamorphosis, and immediately postmetamorphic sea lampreys (Petromyzon marinus). For intestine: T(4)ORD activity was relatively low in nonmetamorphosing larvae, increased in premetamorphic individuals, was highest in stages 1 and 2 and was very low during stages 3-7; T(4)IRD activity was negligible until stage 3 but increased 4.7-fold through stages 3 to 7 such that T(4)IRD activity was 14 times T(4)ORD activity at stage 6; T(3)ORD activity was undetectable; T(3)IRD activity was not measured through stages 3-7 but correlated with T(4)IRD activity at other stages. For liver: deiodination was only measured up to stage 2 and in postmetamorphic animals; in contrast to intestine, T(4)ORD activity fell to low levels at stage 2 and was low during postmetamorphosis; T(4)IRD and T(3)IRD activities were very low and uninfluenced by developmental stage; T(3)ORD activity was undetectable. We conclude that (1) deiodination activity is usually much higher in intestine than in liver, (2) intestinal ORD and IRD activities change reciprocally so that ORD predominates in early metamorphosis but IRD predominates in mid and late metamorphosis, and (3) changes in intestinal deiodination may contribute to the characteristic depression of plasma T(4) and T(3) levels during spontaneous metamorphosis. J. Exp. Zool. 286:305-312, 2000.     

Distribution of gonadotropin-releasing hormone immunoreactive systems in the brain of the Senegalese sole, Solea senegalensis

The present paper reports the immunohistochemical distribution of the gonadotropin-releasing hormone (GnRH) structures in the brain of the Senegalese sole, Solea senegalensis. In this study, we have used two antibodies against the salmon GnRH and chicken GnRH-II forms and the streptavidin–biotin-peroxidase complex method. Immunoreactive cell bodies are observed at the junction between the olfactory bulbs and the telencephalon (terminal nerve ganglion cells), in the ventral telencephalon, in the preoptic parvocellular nucleus, and in the synencephalic nucleus of the medial longitudinal fasciculus. GnRH-immunoreactive fibres were found extensively throughout the brain, located in the telencephalon, preoptic area, hypothalamus, hypophysis, optic tectum, midbrain and rhombencephalon. The antisera used in this study against the two GnRH forms exhibited cross-reactivity on the same cell masses and did not allow cell populations expressing different GnRH forms to be discriminated clearly. However, anti-salmon GnRH immunostained the GnRH cells and fibres of the forebrain much more intensely, whereas the anti-chicken GnRH antiserum shows a higher immunoreactivity on synencephalic cells of the medial longitudinal fasciculus.     

Plasma cells in the ammocoete of Petromyzon marinus

Summary This study demonstrates for the first time the presence of mature and developing plasma cells in the spleen of non-immunized ammocoetes of Petromyzon marinus. Plasmocytes occur as electron-dense cells with much condensed chromatin and an extensively large developed and dilated rough endoplasmic reticulum. The importance of this finding is emphasized in relation to the evolution of the immune system.

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Résumé Ce travail démontre pour la première fois l'existence de plasmocytes murs et en développement dans la rate d'ammocètes de Petromyzon marinus non-immunisés. Les plasmocytes se présentent comme des cellules électroniquement denses avec de la chromatine condensée et un grand développement du réticulum endoplasmique granulaire. L'importance de cette découverte apparaît accentuée en relation avec l'évolution du système immunitaire.

     

Exposure to a putative alarm cue reduces downstream drift in larval sea lamprey Petromyzon marinus in the laboratory

An experimental mesocosm study suggested larval sea lamprey Petromyzon marinus detect and respond to an alarm cue released by dead adult conspecifics. Larvae exhibited a reduced tendency to move downstream when exposed to the cue and were less likely to move under continuous v. pulsed exposure. These findings support the hypothesis that short‐term exposure to the alarm cue would probably result in retraction into the burrow, consistent with the blind, cryptic lifestyle of the larval P. marinus.     

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.     

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.     

Developmental transformations in a normal series of embryos of the sea lamprey Petromyzon marinus (Linnaeus)

Lamprey development is of interest to evolutionary biologists because it can inform our understanding of primitive vertebrate developmental patterns. In this study, we describe and illustrate some of the principle landmarks of organogenesis in the embryonic sea lamprey Petromyzon marinus L. at different chronological ages. We examined 63 fixed embryos spanning Piavis developmental stages 11-18+ (5-70 days postfertilization) by gross observation and histology. This period begins at late neurulation stages and ends with the formation of the larva (ammocoete). A significant difference with some previous accounts is that the anus develops not from a persistent blastopore, but by secondary canalization and proctodeum formation at the former site of the blastopore. Further, we show that the ciliated bands of the pharyngeal roof originate in the esophagus, distinguishing it from the intestine. We clarify the epithelialization of the gut, showing that the secondary gut cavity is progressively epithelialized from each end. We identify possible germ cells in the coelomic and cloacal walls. Balfour's "subnotochordal rod" is lacking in our specimens; we suggest that he may have misinterpreted the corpus adiposum. Our study is of potential value to the growing number of biologists interested in lamprey development and provides a character set that will be used : 1) in a phylogenetic study of vertebrate development, and 2) to prepare a staging series for the lamprey based on parsimony analysis.     

Identification of squalamine in the plasma membrane of white blood cells in the sea lamprey, Petromyzon marinus

It is well established that innate mechanisms play an important role in the immunity of fish. Antimicrobial peptides have been isolated and characterized from several species of teleosts. Here, we report the isolation of an antimicrobial compound from the blood of bacterially challenged sea lamprey, Petromyzon marinus. An acetic acid extract from the blood cells of challenged fish was subjected to solid-phase extraction, cation-exchange chromatography, gel-filtration chromatography, and reverse-phase high-performance liquid chromatography, with the purified fractions assayed for antimicrobial activity. Surprisingly, antimicrobial activity in these fractions originated from squalamine, an aminosterol previously identified in the dogfish shark, Squalus acanthias. Further chromatographic and mass spectrometric analyses confirmed the identity of squalamine, an antimicrobial and antiangiogenic agent, in the active fraction from the sea lamprey blood cells. Immunocytochemical analysis localized squalamine to the plasma membrane of white blood cells. Therefore, we postulate that squalamine has an important role in the innate immunity that defends the lamprey against microbial invasion. The full biochemical and immunological roles of squalamine in the white blood cell membrane remain to be investigated.     

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.     

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.     

Neuronal responsiveness to gonadotropin-releasing hormone and its correlation with sexual receptivity in the rat

In the present study, an attempt was made to correlate the neuronal responsiveness of individual preoptic-septal (POA/S) units to iontophoretically applied GnRH with the onset of sexual receptivity. In both behavioral and electrophysiological studies, ovariectomized, estrogen-primed rats were used. In behaviorally tested rats, lordosis quotients (LQ) were determined at varying times following progesterone (P) injection. For electrophysiological studies, P was given 1 hr after the start of recording. GnRH was iontophoretically applied for 30 sec at 16 nA on spontaneously discharging cells. A unit was deemed excited or inhibited if a repeatable 30% change in discharge rate was observed. From 2-10 hours as the LQ increased from 17 to 90 the total number of GnRH sensitive cells did also. The majority of responsive cells were excited by the peptide. As receptivity displayed a sharp increase from 2 to 6 hours the mean responsiveness of cells excited by GnRH was significantly elevated over inhibitory responses. These findings confirm the E/P biasing effect on POA/S unit responses to GnRH. Moreover, they suggest that a dynamic relationship exists between GnRH responses at the cellular level and sexual behavior throughout the course of steroid-induced receptivity.