Neurotrophic regulation of fibroblast dedifferentiation during limb skeletal regeneration in the axolotl (Ambystoma mexicanum)

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a “critical-size defect” (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment.     

Orexin-B-like immunoreactivity localized in both luteinizing hormone- and thyroid-stimulating hormone-containing cells in the Nile tilapia (Oreochromis niloticus) pituitary

Immunohistochemical techniques were employed to examine orexin-like immunoreactivities in the pituitary of the Nile tilapia (Oreochromis niloticus). Rabbit anti-orexin-A serum and mouse anti-orexin-B monoclonal antibodies were used as primary antibodies. Orexin-B immunoreactive cells corresponded to luteinizing hormone (LH)- or thyroid-stimulating hormone (TSH)-containing cells, and all LH- and TSH-containing cells were immunoreactive for orexin-B. However, we found no orexin-A immunoreactive cells in the pituitary. In the Nile tilapia, an orexin-B-like substance may be secreted from LH- or TSH-containing cells and may regulate pituitary function, rather than the orexin-A-like substance in the pituitaries of Japanese seaperch and medaka.     

Ultrastructural localization of prolactin,growth hormone and luteinizing hormone by immunocytochemical techniques in the bovine pituitary

Summary The technique of ultrastructural immunocytochemistry involving the unlabeled antibody and the soluble peroxidase-antiperoxidase complex was used to identify and describe the prolactin (P) cells, somatotropic (STH) cells and luteinizing hormone (LH) cells in the bovine anterior pituitary gland. This method was used to localize the three hormones at the electron microscopic level. Staining of varying intensity was found on the secretory granules and on the small granules and vesicles within the Golgi complex. No stain was found in nuclei, on mitochondria or in the endoplasmic reticulum.     

Identification of the LH and TSH-secreting cells in the pituitary gland of the rhesus monkey

Summary Pituitary glands from juvenile (pre-pubertal) and adult male and female rhesus monkeys were examined following immunocytochemical staining with antisera to the beta subunits of ovine luteinizing hormone (LH) and of human thyroid stimulating hormone (TSH). The LH antiserum reacts with a cell that is PAS-positive, occurs singly and is randomly distributed throughout the pars distalis. The diameter of these cells is approximately 11.5 m. They do not seem to vary in number in either juveniles (pre-pubertals) or adults, or in males or females. There appears to be fewer LH cells in the pituitary glands of pregnant and lactating females. In addition to staining cells in the pars distalis, the antiserum also reacts with a population of cells located in the pars tuberalis.The cells that stain with the anti-TSH serum are confined primarily to the pars distalis. They are approximately 15.8 m in diameter and are generally found in groups or clusters located in the anterior and medial regions of the gland. The TSH cells vary in number from one animal to another; however, this variability is unrelated to the age or the sex of the animals. No demonstrable changes occur in the number of TSH cells during pregnancy or lactation.Supported by NIH General Research Support Grant RR05654The author wishes to express appreciation to the Hormone Distribution Program of NIAMDD for the preparations of ovine FSH, TSH and human TSH, and to Drs. H. Papkoff for the ovine LH and LH, L. Reichert for the human FSH, J. Vaitukaitis for the anti-human TSH, and L.A. Sternberger for the PAP complex     

Activity of lateral line efferents in the axolotl (Ambystoma mexicanum)

Summary Activity of efferent fibers was recorded from the ramus ophthalmicus superficialis of the head lateral line nerve and the ramus medialis of the trunk lateral line nerve of the axolotl Ambystoma mexicanum. Baseline activity and activity evoked by sensory stimuli were examined. Electrical stimulation of selected branches was used to determine the conduction velocity and the branching pattern of efferent fibers. The influence of lesions at different levels in the CNS on efferent activity was studied.Up to 5 units with baseline activity were found in a single ramus of the lateral line nerve. Discharge rates were variable and highly irregular; they differed between units of the same branch. Bursting activity occurred in 62% of the units. Movements of the animal were accompanied by activity in up to 8 efferent units in a single nerve.Efferent activity could be elicited or modified by stimulation of visual, labyrinthine, somatosensory, and lateral line systems. Stimulation of the electrosensory system had no effect. Individual efferent neurons innervated different fields in the lateral line periphery. Conduction velocities of efferent fibers ranged from 5 to 12 m/s.Efferent units received input from various sources at different brain levels up to the diencephalon. These in puts determined the baseline activity. The mechanosensory input was mediated at the medullary level.Abbreviations r.m. ramus medialis - r.o.s. ramus ophthalmicus superficialis - r.s. ramus superior     

Involvement of activin/BMP system in development of human pituitary gonadotropinomas and nonfunctioning adenomas

Roles of activin/bone morphogenetic protein (BMP) system in the pathogenesis of human pituitary adenoma remain unknown although these factors stimulate follicle-stimulating hormone (FSH) secretion in the normal pituitary. Here we demonstrated that type-I and -II subunit mRNAs of activin/BMP receptors are expressed in Pit-1-negative FSH-producing (FSH-oma) and nonfunctioning pituitary adenomas (NF-oma). Basal levels of serum FSH standardized by luteinizing hormone (LH) were markedly high in FSH-omas in contrast to NF-omas. However, gonadotropin-releasing hormone (GnRH)-induced increment of FSH standardized by that of LH was not changed in FSH-omas, suggesting that imbalanced FSH secretion by FSH-oma is not attributable to GnRH regardless of the expression of GnRH receptor. Although activin betaA subunit was detected in neither adenoma, the betaB subunit was expressed highly in FSH-omas and, to lesser extent, in NF-omas. As for BMPs, BMP-6 and -7 were detected in NF-omas while BMP-4 and -15 were not detected in either type of adenoma. In the presence of pituitary activin/BMP system, the levels of co-expressing follistatin mRNA in the tumors were reduced in FSH-oma compared with NF-oma, suggesting that endogenous follistatin is involved in FSH overproduction through inhibition of activin/BMP system independently of GnRH.     

Subcellular localization of thyrotropin-releasing hormone (TRH)- and Neuropeptide Y (NPY)-like immunoreactivity in the neurointermediate lobe of the frog pituitary

The presence of thyrotropin-releasing hormone (TRH) and neuropeptide Y (NPY) has been demonstrated in the neural lobe and in the intermediate lobe of the frog pituitary by immunocytochemistry on ultrathin sections of neurointermediate lobes obtained by cryoultramicrotomy. In the neural lobe, separate populations of TRH- or NPY-immunoreactive nerve fibers were observed. Both neuropeptides were contained in dense-core secretory vesicles about 200 nm in diameter. In intermediate lobe cells, TRH- and NPY-like immunoreactivities were observed in the cytoplasmic matrix and more sparsely in secretory granules. Occasionally, immunoreactive TRH could be visualized at the plasma membrane level. In the nucleus, both peptides were detected in the euchromatin, in the vicinity of the heterochromatin and in the nucleolus. Conversely, gonadotropin-releasing hormone-like immunoreactivity could not be detected. These results provide immunocytological evidence for the presence of endogenous TRH and NPY in frog melanotrophs indicating that these peptides may participate in the regulation of intermediate lobe secretion.     

Identification by immunofluorescence of prolactin-and somatotropin-producing cells in the pituitary gland of the Mexican axolotl (Ambystoma mexicanum,Shaw)

Summary The indirect immunofluorescence procedure was used to identify prolactin (LTH)-and somatotropin (STH)-producing cells in the pituitary of the Mexican axolotl. Histological staining techniques were employed to corroborate immunocytological results. The LTH cells are large, orange-staining cells (acidophils 1) distributed in the posterior two-thirds of the pars distalis. The STH cells are small, erythrosinophilic elements (acidophils 2) principally concentrated in the dorsal part of the pars distalis.     

Ultrastructural immunocytochemical localization of luteinizing hormone-releasing hormone (LH-RH) in the median eminence of the guinea pig

Summary LH-RH was localized at the ultrastructural level in axons and nerve terminals of the median eminence of the male guinea pig. LH-RH positive neuronal profiles were most concentrated in the medial-dorsal aspect of the infundibular stalk and in the post-infundibular median eminence at the level immediately following separation of the stalk from the base of the brain. LH-RH containing axon profiles were most abundant in the palisade zone; nerve terminals in contact with the hypophysial portal vasculature were relatively rare. The hormone was present within granules that measured 900–1,200 Å in axons of the palisade zone and 400–800 Å in nerve terminals abutting on the portal plexus. The differently sized granules represent heterogeneous populations.Supported in part by U.S. Public Health Service grant HD-09636 from the National Institutes of Health and RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center Publication No. 15-031The authors wish to thank Dr. Sandy Sorrentino, Jr. for the gift of antiserum to LH-RH and Dr. Ludwig Sternberger for the peroxidase.antiperoxidase complex     

Effects of luteinizing hormone releasing hormone on gonadotrophins in the hamster

The changes in serum gonadotrophins in male hamsters following one injection of 15 μg luteinizing hormone releasing hormone (LHRH) (Group A) were compared with those following the last injection of LHRH in animals receiving an injection approximately every 12 hr for 4 days (Group B) or 12 days (Group C). Peak follicle stimulating hormone (FSH) levels (ng/ml) were 1776±218 (Group A), 2904±346 (Group B), and 4336±449 (Group C). Peak luteinizing hormone (LH) values (ng/ml) were 1352±80 (Group A), 410±12 (Group B), and 498±53 (Group C). Serum FSH:LH ratios, calculated from the concentrations measured 16 hr after the last LHRH injections, were higher in Groups B and C than in Group A. Similar injections of LHRH (100 ng or 15 μg/injection) for 6 days elevated the serum FSH:LH ratio in intact males. Five such LHRH injections (100 ng/injection) blunted the rise in serum LH in orchidectomized hamsters. Direct effects of LHRH on gonadotrophin secretory dynamics or altered brain-pituitary-testicular interactions may alter the ratio of FSH to LH in the hamster.     

FMRF -amide-like immunoreactivity in brain and pituitary of the hagfish Eptatretus burgeri (Cyclostomata)

Summary Paraffin sections of brain and pituitary of the hagfish Eptatretus burgeri were immunostained with an antiserum to FMRF-amide. Immunoreactivity was visible in a large number of neurons in the posterior part of the ventromedial hypothalamus and in long neuronal processes extending cranially from the hypothalamus to the olfactory system and caudally to the medulla oblongata. FMRF-amide-like immunoreactivity was also found in cells of the adenohypophysis. These observations suggest that the hagfish possesses a brain FMRF-amide-like transmitter system and pituitary cells containing FMRF-amide-like material.Antisera to ACTH, -MSH and pancreatic polypeptide gave no immunoreaction in hagfish brain or pituitary. D aspartic acid - F phenylalanine - L leucine - M methionine - R arginine; - W tryptophan - Y tyrosine     

The ultrastructure of the cells of Leydig in the white-crowned sparrow (Zonotrichia leucophrys gambelii) in relation to plasma levels of luteinizing hormone and testosterone

In male White-crowned Sparrows subjected to 20 h daily photoperiods there is an approximately 3-fold increase in the plasma concentration of luteinizing hormone (LH) on the first long day after which a quasi-stable level is maintained for at least 42 days. This increase is followed by an increase in numbers of cells of Leydig and an enhancement of their steroidogenic features, a decrease in transitional interstitial cells, and an increase in plasma level of testosterone. With the decline in plasma LH, as photorefractoriness develops, the steroidogenic features of the cells of Leydig undergo disorganization. For as yet unexplainable reasons the plasma levels of testosterone decline before the decrease in plasma LH and before the degeneration of the steroidogenic features of the cells of Leydig.     

Postnatal appearance of luteinizing hormone-releasing hormone (LHRH)-like material in the pineal gland of the rat

Summary Immunoreactive luteinizing hormone-releasing hormone (LHRH)-like material has been demonstrated in the pineal gland of the adult rat. The objective of the present study was to examine the ontogenetic development of this LHRH-like substance in the rat pineal with the peroxidase-antiperoxidase (PAP) method of Sternberger. LHRH-like immunoreactive material was not observed in pineal glands of newborn rats. The amount of material increased progressively from the 6th–12th day of postnatal development. On day 12, the amount of LHRH-like immunoreactivity was consistent and comparable in all pineal glands of male and female animals examined.Supported by NIH Grant 1 R01 HD-12956     

Localization of melanin-concentrating hormone-like immunoreactivity in the brain and pituitary of the frog Rana ridibunda

The distribution of melanin-concentrating hormone (MCH) in the central nervous system of the frog Rana ridibunda was determined by the indirect immunofluorescence technique using antibodies against synthetic salmon MCH, generated in rabbits. The most prominent group of MCH-like containing perikarya was detected in the preoptic nucleus. Comparatively, a moderate number of cell bodies was observed in the dorsal infundibular nucleus and in the ventral thalamic area. Brightly immunofluorescent nerve bundles were found in the preoptic nucleus and in the ventral infundibular nucleus, coursing towards the internal zone of the median eminence and the pituitary stalk. An intense network of immunofluorescent fibers was localized in the neural lobe of the pituitary. The subcellular localization of MCH-like material was studied in the neurohypophysis using the immunogold technique. It was demonstrated that MCH-like material was contained in dense core vesicles (80–90 mm in diameter) within specific nerve terminals. The present findings indicate that, in amphibians, MCH-like peptide is located in specific hypothalamic neurons. Our data suggest that MCH may be released by neurohypophyseal nerve endings as a typical neurohormone.     

Ovarian estradiol modulates the stimulatory effect of ovulation-inducing factor (OIF) on pituitary LH secretion in llamas

This study was designed to: 1) characterize the effect of ovulation-inducing factor (OIF) on pituitary LH secretion in ovariectomized (OVX) llamas; and 2) determine the effect of OIF on LH secretion in OVX llamas pretreated with estradiol-17β (E-17β) or estradiol benzoate (EB). In Experiment 1, intact and OVX llamas (n = 5 or 6 per group) were assigned to a two by two factorial design: 1) Intact llamas treated with 1 mL of phosphate buffered saline (PBS); 2) Intact llamas treated with 1 mg of purified OIF; 3) OVX llamas treated with 1 mL of PBS; or 4) OVX llamas treated with 1 mg of purified OIF. In Experiment 2, intact and OVX llamas (n = 5 or 6 per group) were randomly assigned to the following groups: 1) Intact llamas treated with 1 mg of purified OIF; 2) OVX llamas treated with 1.0 mL of PBS; 3) OVX llamas treated with 1.0 mg of purified OIF; 4) OVX llamas primed with E-17β, followed by 1.0 mg of purified OIF. Experiment 3 was similar as described for Experiment 2, except that priming was done with EB. In Experiment 1, animal category by treatment and animal category by treatment by time interactions tended (P = 0.08) to affect LH concentration. The effect of OIF on LH released was partly restored (P < 0.05), to the values observed for the intact OIF-treated females, when OVX llamas were primed with E-17β or BE (Experiments 2 and 3). We concluded that peripheral estradiol concentrations in llamas partially modulates the effect of OIF on pituitary LH secretion; however, other ovarian factor(s) could also participate in this modulatory action.     

Pleiotropic effects of the Cardiac-lethal gene in the axolotl (Ambystoma mexicanum)

Embryos of the axolotl affected with the cardiac-lethal mutation form hearts that never begin to beat. A number of other traits characteristic of the mutant phenotype, including edema, underdeveloped gills, shorter stature, and aphagia (the inability to feed), were believed to be secondary effects of the absence of circulation. We have recently demonstrated that the pre-cardiac mesoderm is directly affected by the c gene, making it unresponsive to normal inductive signals. In this study, we replaced part or all of the mutant pre-cardiac mesoderm with wild-type tissue, to produce embryos with normally beating hearts and circulation. As expected, most of the other mutant characteristics were also corrected. However, otherwise normal individuals remained aphagic. All embryos with beating hearts containing mutant tissue also suffered from an unexpected circulatory arrest some time after the onset of circulation. This apparently indicates that there are at least two tissues other than the myocardium which appear to be directly affected by the c gene. These previously unsuspected pleiotropic effects of the mutation may involve poorly-characterized mesodermal-neural crest inductive interactions and may also lead to a greater understanding of the link between congenital heart defects and feeding difficulties in humans. © 1993Wiley-Liss, Inc.     

Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH)

Luteinizing hormone-releasing hormone (LHRH) analogues are used extensively for the treatment of various hormone-dependent diseases. However, none of the currently marketed derivatives can be administered orally. Modification of peptide sequences by attachment of carbohydrate moieties is a promising strategy that may increase the metabolic stability of the target peptide and enhance its transport across cell membranes, subsequently improving peptide bioavailability. In this study, either the N- or C-terminus of the LHRH peptide was altered by attachment of carbohydrate moieties. Caco-2 cells were chosen as an in vitro model to investigate both the permeability and stability of the new LHRH analogues. Our findings show that conjugating sugar moieties to the N-terminus of the LHRH peptide significantly increased both permeability and metabolic stability of most of the modified LHRH derivatives.     

Immunoreactive neuronal pathways of growth hormone-releasing hormone (GRH) in the brain and pituitary of the teleost Gadus morhua

Summary Using an antiserum directed against the C-terminus of hGRH(1–44)NH₂ and another recognizing the mid portion to C-terminal of hGRH(1–40)OH, we identify two immunocytochemically distinct GRH-immunoreactive systems in the brain of the codfish, Gadus morhua. The antiserum directed against GRF(1–44)NH₂ stains cell bodies exclusively in the rostral pars distalis. The other antiserum immunoreactive with GRF(1–40)OH reacts with a population of parvocellular and magnocellular neuronal cell bodies in the hypothalamus and with two major axonal pathways which project toward the median eminence and terminate primarily in the pars nervosa. These results indicate the presence of at least two forms of hGRH-like peptides in the teleost which may have different roles in the regulation of pituitary function.     

Amylin-like immunoreactivity in pancreatic X cells of the black-spotted frog Rana (Pelophylax) nigromaculata

In this study, we investigated the presence of ovoid or ellipsoidal amylin-immunoreactive cells of the pancreatic islets of the black-spotted frog Rana (Pelophylax) nigromaculata. Using double immunofluorescent staining, all amylin-immunoreactive cells were shown to be immuno-negative for insulin, glucagon, and somatostatin, and they were often observed in peripheral regions of clusters of insulin-immunoreactive cells. Under immunoelectron microscopy, amylin-immunoreactive signals were detected on the secretory granules in a specific type of endocrine cells. From our results, we conclude that the amylin-immunoreactive cells correspond to X cells among the 4 distinct types of endocrine cells (B, A/PP, D, and X) previously identified in the frog. Amylin secreted from X cells may regulate the hormone secretion from A/PP cells and/or B cells through a paracrine mechanism.     

Identification and morphometric evaluation of the synapses of optic nerve afferents in the optic tectum of the axolotl (Ambystoma mexicanum)

Summary The terminals of retinal afferents in the tectum of the axolotl have been identified ultrastructurally using techniques of horseradish peroxidase-filling and degeneration. The mitochondria in filled structures show a characteristic electron-lucent matrix. After both eyes have been removed, terminals with light mitochondria disappear from the area known to receive an optic input. In this area the presence of light mitochondria is almost always diagnostic of the retinal origin of a bouton. The synapses are similar to those assumed to be of retinal origin in other vertebrates. Detailed morphometric analysis has been carried out on identified optic synapses in the optic tectum of the axolotl.