MSH Cells Research Articles

Establishment and characterization of a permanent heart cell line from largemouth bass Micropterus salmoides and its application to fish virology and immunology

Largemouth bass (Micropterus salmoides) is among the most important cultured fish species in China and various viral diseases have greatly affected the largemouth bass industry. We established a permanent cell line from the heart of M. salmoides (MSH) that has been subcultured over 70 times and showed optimal growth at 27 °C using Medium 199 supplemented with 10% fetal bovine serum. MSH cells consisted predominantly of fibroblast-like cells and their karyotypes revealed diploid numbers for most cells at passage 60 with 2n = 64. Sequencing of cytochrome oxidase I and 16S rRNA genes confirmed that MSH cells were derived from M. salmoides. The cell line was tested for Mycoplasma contamination and found to be negative. MSH cells were successfully transfected with a GFP reporter gene indicating that these cells can be utilized for gene expression studies. The MSH cells showed susceptibility to M. salmoides rhabdovirus (MSRV), Largemouth bass ranavirus (LMBV), Infectious spleen and kidney necrosis virus(ISKNV), Spring viremia of carp virus (SVCV), Tilapia lake virus (TiLV) and Grass carp reovirus genotype I (GCRV-I) but were resistant to nervous necrosis virus (NNV). PCR assay and electron microscopy further confirmed that all the tested viruses except NNV can replicate in MSH cells. The replication efficiency of MSRV, LMBV, ISKNV, SVCV, TiLV and GCRV-I ranged from 107.24 to 108.82 TCID50/mL. In addition, type I interferon genes were induced with only NNV infection. In conclusion, we successfully generated an immortal cell line from the heart tissue of M. salmoides and the newly established MHS cell line was highly susceptible to many fish viruses that can be useful for future genetic, virological and immunological studies.

Protective effect of forsythiaside A on OVA-induced asthma in mice

Forsythiaside A (FSA), an active constituent isolated from the Chinese medicinal herb Forsythia suspensa, has been known to have anti-inflammatory effect. However, the effect of FSA on allergic airway inflammation remains unclear. The aim of this study was to investigate the effect of FSA on OVA-induced asthma in mice. Mice model of asthma was induced by OVA. OVA-induced airway hyperresponsiveness (AHR) and inflammatory cells in BALF were detected. The production of IgE, IL-4, IL-5, IFN-γ, and IL-13 were detected by ELISA. The effects of FSA on Nrf2 and NF-κB signaling pathways were detected by western blot analysis. The results showed that treatment of FSA significantly attenuated OVA-induced lung histopathological changes. FSA inhibited OVA-induced AHR and inflammatory cells in BALF. OVA-induced IgE, IL-4, IL-5, and IL-13 production were also inhibited by FSA. Western blot analysis showed that treatment of FSA inhibited OVA-induced NF-κB activation. Treatment of FSA dose-dependently up-regulated the expression of Nrf2 and HO-1. In addition, we found that FSA up-regulated the expression of Nrf2 and HO-1 both in A549 cells and MS-H cells. Taken together, FSA suppressed inflammatory responses in OVA-induced asthma through activating Nrf2/HO-1 signaling pathway. FSA may be a promising potential preventive agent for asthma treatment.

Immunocytochemical and ultrastructural identification of adenohypophyseal cells in Ctenopharyngodon idella (Cypriniformes: Cyprinidae) during gonadal differentiation

The adenohypophysis was studied by immunocytochemical and ultrastructural methods in juvenile grass carp (Ctenopharyngodon idella) from natural reproduction in Northern Italian rivers. The adenohypophysis included the rostral pars distalis (RPD), the proximal pars distalis (PPD) and the pars intermedia (PI), all deeply penetrated by branches of the neurohypophysis (Nh). The prolactin (PRL), adrenocorticotropic (ACTH), somatotropic (GH), thyrotropic (TSH), gonadotropic type I (GtH I) and type II (GtH II), somatolactin (SL), melanotropic (MSH) and endorphin (END) cells were identified with antisera raised against piscine and human pituitary hormones. In juveniles of 51-69mm of total body length (TL) with undifferentiated gonads, the PRL cells, arranged in thick strands, occupied most of the RPD. The ACTH and GH cells organized in cords bordering Nh were, respectively, confined to RPD and PPD. The TSH cells were scattered among ACTH cells in RPD and among GH cells in PPD. Cells simultaneously immunoreactive to anti-follicle stimulating hormone and to anti-croaker gonadotropin were intermingled among GH and TSH cells, which were mostly in the dorsal PPD. The SL cells were detected in PI layers bordering the Nh. The MSH and END cells were intermingled in PI and, unlike what observed in other teleosts, their respective antisera did not cross-react. In individuals of 78-112mm TL with gonads at the beginning of differentiation, the GtH II cells were detected in PPD; all other cell types increased in number. These results, supported by ultrastructural investigations, suggest that SL and GtH II cells are directly involved in gonadal differentiation in C. idella.

Sexual Maturation Modulates Expression of Nuclear Receptor Types in Laser-Captured Single Cells of the Cichlid (Oreochromis niloticus) Pituitary

The role of steroid/thyroid hormones in the regulation of endocrine cells at the level of the pituitary has remained unclear. Therefore, using single-cell quantitative real-time PCR, we examined absolute amounts of transcripts for nuclear receptors [estrogen receptors (ERs) alpha, beta, and gamma; androgen receptors (ARs) a and b; glucocorticoid receptors (GRs) 1, 2a, and 2b; and thyroid hormone receptors (TRs) alpha1, alpha2, and beta] in pituitary cells of immature (IM) and mature (M) male tilapia, Oreochromis niloticus. In the two reproductive stages, ACTH cells expressed only ERbeta, whereas all other pituitary cell types expressed ERalpha + beta, and a subpopulation coexpressed ARa, ARb, GR1, GR2b, and TRbeta but lacked ERgamma, GR2a, TRalpha1, and TRalpha2. IM males had high percentages of LH cells (IM 46.0% vs. M 10.0%), GH cells (IM 23.3% vs. M 7.9%), and prolactin cells (IM 68.8% vs. M 6.0%) with ERbeta, and TSH cells (IM 19.2% vs. M 0.0%) and MSH cells (IM 25.6% vs. M 0.0%) with ERalpha + TRbeta. A high percentage of FSH cells in IM males expressed ERbeta (IM 46.9% vs. M 18.8%), and FSH cells in M males showed significantly high GR1 transcripts (IM 76.0 +/- 5.0 vs. M 195.0 +/- 10.7 copies per cell; P < 0.05), suggesting that FSH cells are regulated differently in the two reproductive stages. Coexpression of ERalpha + beta in high percentages of cells of the GH family (GH, IM 43.8% vs. M 14.3%; prolactin, IM 8.3% vs. M 59.7%; somatolactin, IM 22.2% vs. M 42.2%) suggests that the expression of both ERs is important for functionality. Thus, differential coexpression of genes for nuclear receptors in subpopulations of pituitary cell types suggests multiple steroid/thyroid hormone regulatory pathways at the level of the pituitary during the two reproductive stages.

Proportions of melanocyte stimulating hormone‐immunoreactive cells in the adenohypophysis of Silky fowl and hyperpigmentation‐free cockerels

ABSTRACTAlpha‐melanocyte stimulating hormone (α‐MSH) is one of the main regulators for melanocytes, and the adenohypophysis is one of the major tissues for the synthesis of this hormone. The Silky fowl is a characteristic breed of chicken with hyperpigmentation throughout the body. The involvement of the adenohypophysis in the hyperpigmentation of this breed is not known. In the present study, the proportion of melanocyte stimulating hormone‐immunopositive cells (MSH cells) in the adenohypophysis was immunocytochemically compared between Silky, Red Cornish × New Hampshire (RN) crossbred and Japanese bantam cockerels at 15 weeks of age. After the body and gland were weighed, the adenohypophyseal cells were enzymatically dispersed and immunostained for α‐MSH, and the immunopositive MSH cells were counted. The weights of the body and adenohypophysis were heaviest in the RN crossbred, followed by the Silky and lightest in the bantam cockerels. In contrast, the ratio between adenohypophysis and bodyweight was much larger in the Silky and the bantam than in the RN crossbreed (P &lt; 0.05). The population of MSH cells in the adenohypophysis was larger in the Silky (14.3%) than in the RN crossbreed (8.0%) and the bantam (8.1%) cockerels (P &lt; 0.05). From these results, it was concluded that prepubertal Silky cockerel have numerous MSH cells in the adenohypophysis suggesting a relationship to hyperpigmentation.

Three GnRH receptor types in laser-captured single cells of the cichlid pituitary display cellular and functional heterogeneity

The role of multiple gonadotropin-releasing hormone receptor (GnRH-R) types in the regulation of gonadotropic and nongonadotropic cells remains speculative. To address this issue, we developed a technology integrating laser-captured microdissection of single digoxigenin-labeled pituitary cells coupled with real-time quantitative PCR to examine the expression profiles of three endogenous GnRH-R types (R1, R2, and R3) in immature and mature males of tilapia Oreochromis niloticus. Here, in addition to gonadotropes (luteinizing and folicle-stimulating hormone, FSH), we show GnRH-Rs are also present in lactotropes, somatotropes, thyrotropes, melanotropes (melanocyte-stimulating hormone, MSH), corticotropes and somatolactin cells. Subpopulations of pituitary cells express single (42.9%), multiple (32.4%) or lack (24.7%) GnRH-Rs. In immature males, the percentage of FSH cells containing combinations of GnRH-Rs was significantly higher (R1+R2: 24%, P <0.05; R1+R2+R3: 25%, P <0.01) than in mature males, whereas the percentage showing only R1 and R1 and R3 transcripts (P <0.05) was higher in mature males. Significantly greater copies of R1 and R3 transcripts were found in MSH cells of immature and mature males, respectively (P <0.05). GnRH-R transcripts in other pituitary cells (lactotropes, R1 and R2; somatolactin cells/thyrotropes/corticotropes, R1, R2, and R3) were significantly higher in mature males (P <0.05) but were unaltered in somatotropes and luteinizing hormone cells. Thus, FSH and MSH cells are required for both reproductive states, whereas other pituitary cells are recruited only during testicular maturation. The differential expression of GnRH-Rs in gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity of mechanisms controlling normal sexual development.

Glycoconjugate profiles of adrenocorticotropic and melanotropic cells in the pituitary of adult sea lampreys (Petromyzon marinus): a lectin histochemical study.

In the lamprey, adrenocorticotropin (ACTH) and melanotropins (MSHs) are produced from two distinct precursors, proopiocortin (POC) and proopiomelanotropin (POM). Both POC and POM have been suggested to be glycoproteins. The present study aimed to demonstrate glycoconjugates in ACTH and MSH cells in the pituitary of adult sea lampreys (Petromyzon marinus) by means of a lectin histochemistry. A total of 19 kinds of lectins were tested. ACTH cells were distributed in both the rostral pars distalis and the proximal pars distalis, and were stained positively with N-acetylglucosamine binding lectins (i.e., succinylated wheat germ agglutinin), N-acetylgalactosamine binding lectins (i.e., soybean agglutinin), D-mannose binding lectins (i.e., Lens culinaris agglutinin), and D-galactose binding lectins (i.e., Erythrina cristagall lectin). MSH cells were distributed in the pars intermedia, and were stained with N-acetylgalactosamine binding lectins (i.e., Dolichos biflorus agglutinin), D-mannose binding lectin (Pisum sativum agglutinin) and D-galactose binding lectins (i.e., peanut agglutinin). These results suggested that ACTH and MSH cells produce different types of glycoconjugates which may be attributed to the difference in glycoconjugate moieties between the precursor proteins, POC and POM.

Immunocytochemical Demonstration of Melanotropic and Adrenocorticotropic Cells from the Gilthead Sea Bream (Sparus aurata L., Teleostei) by Light and Electron Microscopy: An Ontogenic Study

In the pituitary of gilthead sea bream, Sparus aurata, melanotropic (MSH) and adrenocorticotropic (ACTH) cells were identified at the light and electron microscopic levels using rabbit anti-synthetic αMSH (MSH) and anti-human ACTH (1–24) (ACTH) sera. The distribution of these cell types was followed from hatching to 48 months. The techniques used included the peroxidase anti-peroxidase (PAP) method, conventional electron microscopy, and an immunogold technique. Using PAP, MSH (immunoreactive to both anti-MSH and anti-ACTH) and ACTH (immunoreactive to anti-ACTH) cells were detected from hatching onward. These cells were distinguished ultrastructurally in 1-day-old larvae. Immunogold labeling was first detected in MSH cells in 5-day-old larvae, while ACTH cells were only immunogold labeled in adults. In newly hatched larvae, MSH cells were located from the middle to the posterior region of the adenohypophysis, while ACTH cells were found in the dorsoanterior region, next to the hypothalamus. At this age, both cell types were scarce. As the fish developed, these cell types progressively increased in number: MSH cells made up a layer surrounding the neurohypophysis (NH) in the pars intermedia (pi), whereas ACTH cells bordered the developing NH in the rostral pars distalis (rpd). From 82 days onward, a few MSH cells were observed in the proximal pars distalis (ppd) next to the pi and some ACTH cells were seen in the ppd next to the rpd. In adult specimens, both MSH and ACTH cells were adjacent to the stellate cells and showed processes and synaptic-like structures. MSH cells exhibited numerous round secretory granules with a granular content and of varying electron density and compactness. These granules were immunogold labeled with anti-MSH serum. Electron-dense secretory granules near the Golgi complex immunoreacted with anti-MSH, anti-ACTH, or with both antisera. ACTH cells exhibited round secretory granules with a homogeneous, high electron-dense core and a narrow, clear halo. These granules immunoreacted with anti-ACTH serum. The main ultrastructural features that characterize the MSH and ACTH cells of adults appeared early during ontogeny. Involutive MSH and ACTH cells were only observed in adult specimens.

Stress response to waterborne Cu during early life stages of carp, Cyprinus carpio

Following in vitro fertilization, eggs/embryos and larvae of the common carp ( Cyprinus carpio) were exposed to 0 (control), 0.3 or 0.8 μmol·l −1 Cu in artificially prepared fresh water for 168 h. The total amounts of Cu, Na, Ca, adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone ( α-MSH) and cortisol were measured in homogenates of eggs (up to 60 h post fertilization, hpf), isolated embryos (between 60 hpf and hatching) and free-swimming larvae. Only in embryos of eggs exposed to 0.8 μmol·l −1 Cu a significant accumulation of Cu was observed as well as a concurrent increase in the incidence of spinal cord deformation and larval mortality. Further, when exposed to 0.8 μmol·l −1 Cu, the whole-body Ca and Na contents were lower at 48 and 72 hpf compared to the controls and those exposed to 0.3 μmol·l −1 Cu. Conversely, in larvae (>72 hpf) exposed to 0.3 μmol·l −1 Cu, the Ca content was elevated from 96 hpf onwards. At 48 hpf and onwards, the whole-body ACTH and cortisol contents of the embryos exposed to 0.8 μmol·l −1 Cu were higher than those in either controls or those exposed to 0.3 μmol·l −1 Cu. By 96 hpf, ACTH and cortisol contents of the group exposed to 0.3 μmol·l −1 Cu also surpassed those in controls. The α-MSH content in both Cu exposed groups was lower than in controls from 48 hpf onwards. It thus appears that ACTH cells and MSH cells in early life stages of carp exposed to waterborn Cu respond differently; we conclude that in prehatch carp pituitary corticotropes and interrenal cortisol producing cells respond to the chemical stressor Cu and that the resulting hormonal changes provide a sensitive diagnosis for stress as well as toxicity tests.

Ontogeny of ACTH and MSH cells in Japanese flounder ( Paralichthys olivaceus) in relation to albinism

Japanese flounder larvae were reared for 43 days in glass aquariums with and without sand in the bottom to study the influence of the substrate on the pigmentation pattern, pituitary growth and ACTH and MSH production. Similar results were obtained in terms of survival, growth and ocular side pigmentation in both groups. However, a clear difference was observed in blind side pigmentation and only 28% of the fish cultured with sand developed this abnormality, contrasting with 58% of those reared without sand. A clear increase in the pituitary volume was observed during larval development, being maximal at post metamorphosis. No differences in the pituitary volume could be found among pigmented, albino and ambicoloured juveniles, although those reared with sand had a larger pituitary than those reared without sand. ACTH and MSH producing cells were detected by immunohistochemistry. The proportion of the pituitary occupied by ACTH or MSH cells (ratio) was highest at early larval stages, and decreased with development to a minimum value at the climax of metamorphosis. No significant difference in the ACTH ratio was found between the two groups, although juveniles reared without sand showed a slightly higher ratio, probably reflecting the stress experienced by these fish. The MSH ratio was significantly higher in fish reared with sand. No significant differences were found in the ACTH and MSH ratios among pigmented, albino and ambicoloured fish. Fish produced with a sandy bottom showed a lower degree of hyperpigmentation and possessed larger pituitaries that had higher MSH ratios than those reared without sand.

Immunocytohistochemical characterization of pituitary cells of the bluefin tuna, Thunnus thynnus L.

In this paper we report the first complete mapping of the pituitary in a tuna species. The various different adenohypophysis cell types of the bluefin tuna, Thunnus thynnus L. have been identified and located using different antisera against mammalian and piscine hormones and various histochemical techniques: PAS, Alcian Blue pH 2.5 and lectins -ConA and WGA(Neutral and Acidic Glycoproteins); Bromophenol Blue (Proteins) and Tioglycollate-Ferric-Ferricianide-FeIII (-S-S- groups). Prolactin (PRL) and adrenocorticotrophic (ACTH) cells were located in the rostral pars distalis (RPD) of the pituitary, while the proximal pars distalis (PPD) displayed gonadotrophic (GTH), thyrotrophic (TSH), somatotrophic (GH) and also a few PRL cells. Moreover, somatolactin (SL) and melanotrophic (MSH) cells were identified inside the pars intermedia (PI). Interestingly, some SL-immunoreactive fibers were also detected in the neurohypophysis. Some GTH cells were also located on the exterior surface of the PI. Glycoproteins containing mannose (Man) and/or glucose (Glc); N-acetyl-glucosamine (GlcNAc) and/or sialic acid sugar residues, as well as -S-S- groups, were observed in GTH, TSH and SL cells. The Bromophenol Blue technique stained amphiphilic SL, acidophilic GH cells and weakly ACTH cells. GH and ACTH cells were unreactive to PAS, Alcian Blue, Tioglycollate-Ferric-Ferricianide-FeIII and lectin (Con A and WGA) techniques. Finally, PAS reaction was positive in amphiphilic SL cells, which were PbH unreactive, while MSH and ACTH cells were stained with PbH technique.

Pituitary Proopiomelanocortin-Derived Peptides and Hypothalamus–Pituitary–Interrenal Axis Activity in Gilthead Sea Bream (Sparus aurata) during Prolonged Crowding Stress: Differential Regulation of Adrenocorticotropin Hormone and α-Melanocyte-Stimulating Hormone Release by Corticotropin-Releasing Hormone and Thyrotropin-Releasing Hormone

Plasma levels of cortisol, growth hormone (GH), adrenocorticotropin hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), N-acetyl-β-endorphin, in vitro ACTH-stimulated cortisol secretion, and in vitro corticotropin-releasing hormone (CRH)- and thyrotropin-releasing hormone (TRH)-stimulated ACTH and α-MSH secretion were investigated in gilthead sea bream exposed to high stocking density (30 kg m−3) for 23 days. Within 3 days after the onset of crowding, plasma levels of cortisol, ACTH, α-MSH, and N-acetyl-β-endorphin were above control values. After 7 days, plasma parameters had returned to control levels, but at 23 days, cortisol, α-MSH, and N-acetyl-β-endorphin levels were again elevated over controls, indicating a long-term activation of the melanotrope cells. In contrast, crowding stress elicited a prolonged reduction in plasma GH levels concomitant with the increased hypothalamus–pituitary–interrenal axis (HPI) activation. Crowding stress enhanced cortisol secretory activity of the unstimulated interrenal cells. However, interrenal tissue from crowded fish in vitro displayed an attenuated response to ACTH stimulation compared with tissue from control fish, indicating a desensitization of these cells to ACTH during crowding. The involvement of pituitary proopiomelanocortin-derived peptides in the HPI axis of sea bream is indicated by the observed modulation of the CRH and TRH responsiveness of the corticotropes and melanotropes in crowded fish. At day 1, when there were crowding-induced plasma increases in ACTH and α-MSH, there was an attenuated CRH-stimulated but not TRH-stimulated, ACTH release. However, at that time, CRH- and TRH-induced responses of α-MSH secretion, and the unstimulated secretory activity of the MSH cells, were enhanced in crowded sea bream. These data provide evidence for stimulatory roles of multiple hypothalamic (CRH and TRH) and pituitary (ACTH and α-MSH) peptides in the activation of the hypothalamus–pituitary–interrenal axis under crowding conditions in sea bream.

Differentiating Ability of the Pars Intermedia in Hypothalectomized Frog Tadpoles

It is generally accepted that hypothalectomy of frog tadpoles at the open neurula stage results in failure of the pars intermedia to develop. A pale body color is assumed to be evidence that the hypothalamus was completely removed. The present study, however, shows that hypothalectomized Rana japonica can develop into either albino, as already reported, or darkly pigmented tadpoles. In order to determine the extent to which the intermediate lobe can develop in these hypothalectomized tadpoles, their adenohypophyses were examined immunohistochemically by using anti-αMSH (melanocyte-stimulating hormone). In all the dark-colored larvae a pars intermedia had formed, though its size was very small. In the pale-colored tadpoles, on the other hand, the pars intermedia frequently failed to differentiate, but it was observed in 4 of 13 hypothalectomized larvae. In view of other investigators' data showing the complete absence of ACTH (adrenocorticotropin) cells in hypothalectomized tadpoles, hypophyses were also stained with anti-ACTH. Immunoreactive ACTH cells were detected in hypothalectomized tadpoles irrespective of the body pigmentation, although their incidence was lower than in normal controls. These data indicate that contact between the infundibulum and adenohypophysis is not absolutely essential for differentiation of MSH and ACTH cells in the frog.

Immunocytochemical and histological studies on the hypophyseal-gonadal system in the freshwater nile tilapia, oreochromis niloticus (L.), during sexual maturation and spawning in different habitats

The activities of the hypophyseal-gonadal system of O. niloticus in different habitats were investigated using immunocytochemical and histological techniques. The observed physico-chemical results indicated that Lake Nasser water characteristics are within the allowed and desired safety baseline levels. In contrast, Lake Manzalah exhibited high levels of Ca(2+), Mg(2+), SO(4)(2-) and heavy metals (Zn, Pb, and Cd). These water conditions affected the activity of the hypophyseal-gonadal axis of tilapia. The secretory and the synthetic activities of GTH and SL cells in the pituitary gland, in general, underwent obvious normal changes during gonad maturation and spawning of O. niloticus in Lake Nasser. In Lake Manzalah, the secretory activity of GTH and SL cells declined. This may be due to the effect of the high levels of heavy metals which interfere with the release of hormones and disturb the feedback mechanisms. In addition, the activity of both PRL and GH cells in Lake Nasser was higher than that of Lake Manzalah, but the synthetic activity of the ACTH and MSH cells was higher in Lake Manzalah. This may be related to the increased stress on fishes and the dark polluted water in Lake Manzalah. Both the environmental and the endocrine impacts in Lake Manzalah caused a decline in the gonadal activity as reflected by the decrease of sperm amount in the ripe testis, the appearance of oocytes in the testis and the degeneration of ripe oocytes (atresia) during the spawning season. J. Exp. Zool. 284:343-354, 1999. Copyright 1999 Wiley-Liss, Inc.

Immunocytochemical and histological studies on the hypophyseal‐gonadal system in the freshwater Nile Tilapia, Oreochromis niloticus (L.), during sexual maturation and spawning in different habitats

The activities of the hypophyseal-gonadal system of O. niloticus in different habitats were investigated using immunocytochemical and histological techniques. The observed physico-chemical results indicated that Lake Nasser water characteristics are within the allowed and desired safety baseline levels. In contrast, Lake Manzalah exhibited high levels of Ca2+, Mg2+, SO42– and heavy metals (Zn, Pb, and Cd). These water conditions affected the activity of the hypophyseal-gonadal axis of tilapia. The secretory and the synthetic activities of GTH and SL cells in the pituitary gland, in general, underwent obvious normal changes during gonad maturation and spawning of O. niloticus in Lake Nasser. In Lake Manzalah, the secretory activity of GTH and SL cells declined. This may be due to the effect of the high levels of heavy metals which interfere with the release of hormones and disturb the feedback mechanisms. In addition, the activity of both PRL and GH cells in Lake Nasser was higher than that of Lake Manzalah, but the synthetic activity of the ACTH and MSH cells was higher in Lake Manzalah. This may be related to the increased stress on fishes and the dark polluted water in Lake Manzalah. Both the environmental and the endocrine impacts in Lake Manzalah caused a decline in the gonadal activity as reflected by the decrease of sperm amount in the ripe testis, the appearance of oocytes in the testis and the degeneration of ripe oocytes (atresia) during the spawning season. J. Exp. Zool. 284:343–354, 1999. © 1999 Wiley-Liss, Inc.

Human pigmentation genetics: the difference is only skin deep.

There is no doubt that visual impressions of body form and color are important in the interactions within and between human communities. Remarkably, it is the levels of just one chemically inert and stable visual pigment known as melanin that is responsible for producing all shades of humankind. Major human genes involved in its formation have been identified largely using a comparative genomics approach and through the molecular analysis of the pigmentary process that occurs within the melanocyte. Three classes of genes have been examined for their contribution to normal human color variation through the production of hypopigmented phenotypes or by genetic association with skin type and hair color. The MSH cell surface receptor and the melanosomal P-protein are the two most obvious candidate genes influencing variation in pigmentation phenotype, and may do so by regulating the levels and activities of the melanogenic enzymes tyrosinase, TRP-1 and TRP-2.

Immunocytochemical and ultrastructural characterization of melanotropin and adrenocorticotropin cells from the Mediterranean yellowtail (Seriola dumerilii, Risso 1810).

Melanotropin (MSH) and adrenocorticotropin (ACTH) are pituitary hormones derived from a common precursor: the proopiomelanocortin (POMC), which is processed differently in the melanotropic and corticotropic cells of several vertebrates. While ACTH is a major final product in corticotropes, it is further processed into alpha-MSH and corticotropin-like intermediate lobe peptide (CLIP) in melanotropes. Cells which are immunoreactive to ACTH (ACTH cells) and to both alpha-MSH and ACTH (MSH cells) have been described in a number of teleosts, including the Mediterranean yellowtail, by light microscopic immunocytochemistry. However, these cells have been ultrastructurally characterized only in a few species. In this paper, we use electron microscopy to identify and characterize the cells producing MSH and ACTH in M. yellowtail (Seriola dumerilii). Pituitaries from adult specimens were dissected and processed for conventional and immunocytochemical electron microscopy. An immunogold technique was performed using anti-synthetic alpha-MSH and anti-human (h) ACTH (1-24) sera. MSH cells had round secretory granules with a granular content of varying electron density and compactness, which were immunogold-labeled with anti-alpha-MSH. Homogeneous and electron-dense secretory granules found in the Golgi area of these cells reacted with both anti-alpha-MSH and anti-hACTH (1-24). ACTH cells had round secretory granules with a homogeneous and medium or high electron-dense core and narrow clear halo, which were grouped in the cell area near the neurohypophysis (NH). Some granules showed an osmiophilic semicore in the medium electron-dense content, which has not been described in other teleost pituitary cells. Immunogold-labeling over the secretory granules only was obtained with all the antisera used. Some ACTH cells showed involutive features. MSH and ACTH are respective final products of the POMC in two ultrastructurally different cells of the pituitary of M. yellowtail, MSH and ACTH cells. The immature granules in the Golgi area of MSH cells seem to be the site of proteolitic cleavage of ACTH into alpha-MSH and CLIP.

Immunocytochemical and ultrastructural characterization of melanotropin and adrenocorticotropin cells from the mediterranean yellowtail (Seriola dumerilii, Risso 1810)

Background Melanotropin (MSH) and adrenocorticotropin (ACTH) are pituitary hormones derived from a common precursor: the proopiomelanocortin (POMC), which is processed differently in the melanotropic and corticotropic cells of several vertebrates. While ACTH is a major final product in corticotropes, it is further processed into α-MSH and corticotropin-like intermediate lobe peptide (CLIP) in melanotropes. Cells which are immunoreactive to ACTH (ACTH cells) and to both α-MSH and ACTH (MSH cells) have been described in a number of teleosts, including the Mediterranean yellowtail, by light microscopic immunocytochemistry. However, these cells have been ultrastructurally characterized only in a few species. In this paper, we use electron microscopy to identify and characterize the cells producing MSH and ACTH in M. yellowtail (Seriola dumerilii). Methods Pituitaries from adult specimens were dissected and processed for conventional and immunocytochemical electron microscopy. An immunogold technique was performed using anti-synthetic α-MSH and anti-human (h) ACTH (1-24) sera. Results MSH cells had round secretory granules with a granular content of varying electron density and compactness, which were immunogold-labeled with anti-α-MSH. Homogeneous and electron-dense secretory granules found in the Golgi area of these cells reacted with both anti-α-MSH and anti-hACTH (1-24). ACTH cells had round secretory granules with a homogeneous and medium or high electron-dense core and narrow clear halo, which were grouped in the cell area near the neurohypophysis (NH). Some granules showed an osmiophilic semicore in the medium electron-dense content, which has not been described in other teleost pituitary cells. Immunogold-labeling over the secretory granules only was obtained with all the antisera used. Some ACTH cells showed involutive features. Conclusions MSH and ACTH are respective final products of the POMC in two ultrastructurally different cells of the pituitary of M. yellowtail, MSH and ACTH cells. The immature granules in the Golgi area of MSH cells seem to be the site of proteolitic cleavage of ACTH into α-MSH and CLIP. Anat. Rec. 249:74–80, 1997. © 1997 Wiley-Liss, Inc.

D2-like dopamine receptor mediates dopaminergic or gamma-aminobutyric acidergic inhibition of melanotropin-releasing hormone release from the pars intermedia in frogs (Rana nigromaculata).

Frogs can adapt to their background by making their skin color lighter or darker as necessary, and this adaptation is regulated by MSH. We investigated the mechanism inhibiting MSH release from the pars intermedia (PI) of the pituitary gland in frogs (Rana nigromaculata) by ultrastructural immunohistochemistry and bioassay using the melanophore index. The PI contained fibers immunoreactive for tyrosine hydroxylase, gamma-aminobutyric acid (GABA), and neuropeptide Y, which made synaptic contacts with MSH cells. The synapses had an asymmetric profile with small round and large-cored synaptic vesicles. The skin of frogs adapted to a white background became darker after administration of 6-hydroxydopamine or autografting of the PI into the anterior chamber of the eye. The skin of autografted frogs became lighter after the administration of dopamine or GABA into the anterior chamber. Lightening of skin color with dopamine was inhibited by a D2 receptor antagonist (sulpiride), and the effect of GABA was blocked by both sulpiride and a GABAA receptor antagonist (bicuculline). These results indicate that MSH release from the PI in frogs may be inhibited by dopaminergic nerves via the D2-like receptor and by GABAergic nerves via the D2-like and GABAA receptors.

Immunohistochemical studies on the development of the hypothalamo-hypophysial system in Xenopus laevis.

Few attempts have been made to clarify the relational development of the hypothalamo-adenohypophysial and -neurohypophysial systems in species higher than amphibians. The appearance and topographical distribution of endocrine and neuroendocrine cells and fibers in these systems were immunohistochemically examined in the larvae of Xenopus laevis from immediately before hatching (stage 32, Nieuwkoop and Faber's classification) to the end of metamorphosis (stage 66). (1) Each endocrine cell differentiated until the middle premetamorphic period. MSH cells initially appeared in the posterior half of the pituitary anlage at stage 35/36, followed by the differentiation of GH cells at stage 39 in the middle part, PRL cells at stage 46 in the anterior half of the pituitary anlage, and LH cells at stage 50 in the posterior two thirds of the pars distalis. With the progression of development, the cells which differentiated at early stages shifted from their initial positions; MSH cells, to the pars intermedia; and GH cells, to the posterior half of the pars distalis. 2) Oxytocin and vasopressin fibers were observed at stage 47/48 in the median eminence, and converged to the pars nervosa at later stages. 3) Neuroendocrine fibers innervated the median eminence during the middle premetamorphic to prometamorphic period: SOM fibers, at stage 45; CRH, 47/48; GRH, 48; dopamine, 58; and LHRH, 60. The cells containing these hormones were observed in the (presumptive) preoptic and/or infundibular nuclei. These results suggest the following three chronological steps in the development of hypothalamo-hypophysial systems and their target organs: independent development of target organs at early developmental stages; appearance of hypophysial hormones to control the development of target organs at middle developmental stages; appearance of hypothalamic hormones to control the function or maturation of the hypophysis at late developmental stages.