Morphological variation between non-native lake- and stream-dwelling pumpkinseed Lepomis gibbosusin the Iberian Peninsula.

The objective of this study was to test if morphological differences in pumpkinseed Lepomis gibbosus found in their native range (eastern North America) that are linked to feeding regime, competition with other species, hydrodynamic forces and habitat were also found among stream- and lake- or reservoir-dwelling fish in Iberian systems. The species has been introduced into these systems, expanding its range, and is presumably well adapted to freshwater Iberian Peninsula ecosystems. The results show a consistent pattern for size of lateral fins, with L. gibbosus that inhabit streams in the Iberian Peninsula having longer lateral fins than those inhabiting reservoirs or lakes. Differences in fin placement, body depth and caudal peduncle dimensions do not differentiate populations of L. gibbosus from lentic and lotic water bodies and, therefore, are not consistent with functional expectations. Lepomis gibbosus from lotic and lentic habitats also do not show a consistent pattern of internal morphological differentiation, probably due to the lack of lotic-lentic differences in prey type. Overall, the univariate and multivariate analyses show that most of the external and internal morphological characters that vary among populations do not differentiate lotic from lentic Iberian populations. The lack of expected differences may be a consequence of the high seasonal flow variation in Mediterranean streams, and the resultant low- or no-flow conditions during periods of summer drought.

Gonad plasticity and gametogenesis in the endangered Spanish toothcarp Aphanius iberus (Teleostei: Cyprinodontidae).

The Spanish toothcarp Aphanius iberus is an endangered species which inhabits small rivers, creeks, salt marshes and marine salt pans in the Mediterranean coast of Spain. No differences in weights were observed among females or males taken from different environments. Analyses of the morphology of the gonads and the gametogenesis were performed in fish taken from different environments by comparing gamete development in females and in males and gonadal cell proliferation in the testis. A high degree of plasticity was observed in the gonad morphology of A. iberus. Females possess two ovaries which show non-restricted oogenesis with all germ cell stages within the same ovigerous lamellae, while males possess gonads without any clear division with the typical restricted pattern observed in cyprinodontid fish. Some females and males showed asymetrically developed gonads. Proliferation of germ cells in testis is located only in the periphery of the gonads corresponding with primary and secondary spermatogonia. Salinity did not influence gonad plasticity or the appearance of mature germ cells in either females or males. This is the first study to provide a microscopic description of oogenesis and spermatogenesis in A. iberus at extreme different environmental conditions.

Differential expression and processing of chromogranin A and secretogranin II in relation to the secretory status of endocrine cells.

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

Melanotrope cells as a model to understand the (patho)physiological regulation of hormone secretion.

Regulation of hormone secretion is a complex process that comprises the sequential participation of numerous subcellular mechanisms. Hormone secretion is dictated by extracellular stimuli that are transduced intracellularly into activation/deactivation of different mechanisms, such as hormone expression, processing and exocytosis, which will ultimately determine the precise availability of hormone to be secreted. Malfunction in any of these steps may result in deficient or excessive hormone release and the subsequent appearance of endocrine disorders. Given the complexity of this system, it is difficult to find appropriate cellular models wherein to investigate the multiple components of the secretory process in a physiologically relevant, experimentally manipulable setting. In this review, we present recent evidence on the use of the intermediate lobe (IL) of the pituitary as a powerful tool to understand different aspects of the regulated secretory pathway. IL is composed of a single endocrine cell type, alpha-melanocyte stimulating hormone (alpha-MSH)-producing melanotropes, a fact that greatly facilitates its study. Furthermore, melanotropes can be separated using classic cell separation techniques into two cell subtypes showing opposite morphophysiological phenotypes of hypo- and hypersecretory cells. Comparison of their gene expression fingerprints has unveiled the existence of certain genes preferentially expressed in each melanotrope subtype. Because of their direct participation in the secretory pathway, we postulate that characterization of these gene products in an endocrine cell type may represent novel and useful markers for reliably determining the general secretory status in an endocrine gland, as well as a valuable new tool to further investigate this complex process.

Secretory and morphological heterogeneity of porcine somatotropes during postnatal development.

Previous studies from our laboratory have demonstrated that porcine somatotropes can be separated into two subpopulations of low (LD) and high density (HD) by centrifugation in a Percoll gradient. The two subsets are present throughout porcine postnatal growth, although their relative proportions vary with age. In prepubertal animals, HD cells exhibit higher secretory granule content and release more GH than LD cells under basal culture conditions. In the present study, we analysed the ultrastructure of separated LD and HD cells from neonate and mature female pigs, and quantified cell size as well as the relative abundance of several subcellular organelles on immunoidentified somatotropes. Subsequently, GH release under basal conditions was assessed for cultures of unseparated cells and also for LD and HD somatotropes obtained at different stages of postnatal development. Results from the morphometric study demonstrated that LD somatotropes were significantly smaller in size, contained less secretory granules and displayed a more developed endoplasmic reticulum than their HD counterparts, regardless of the age of the pituitary donors. In terms of secretory ability, a significant age-associated decrease in GH release was observed in monolayer cultures of unseparated cells from prepubertal and mature pigs compared to neonates. A similar decline in GH-releasing ability was detected for cultures of HD cells. For LD cells, GH secretion only decreased significantly in mature animals. In spite of the divergent pattern followed by both subpopulations during growth, HD somatotropes released significantly more GH than LD somatotropes at the three ages studied. Taken together, our findings demonstrate that the population of porcine somatotropes is mainly composed of two subtypes, LD and HD, which differ in density, morphology and basal secretory activity. These differences are essentially maintained during porcine postnatal development. The progressive reduction in the secretory capacity of HD and LD somatotropes, coupled to the decrease in the relative abundance reported for the HD subpopulation, provides the cytological basis for a better understanding of the decline in GH release associated with age in pigs.

Heterogeneity of growth hormone (GH)-producing cells in aging male rats: in vitro GH releasing activity of somatotrope subpopulations.

Studies on the age-related decline of growth hormone (GH) release have ignored that the population of GH-producing cells (somatotropes) is heterogeneous. In aging male rats, centrifugation of dispersed pituitary cells in a density gradient yields two somatotrope subpopulations, i.e. low- (LD) and high-density (HD) cells. A previous analysis of ultrastructure and GH mRNA levels has shown that storage and biosynthetic features were inversely related in both subsets. Furthermore, ultrastructural and molecular differences between LD- and HD-cells were retained throughout the rat lifespan, suggesting that the heterogeneity of somatotropes may have a biological meaning. Accordingly, the main objective of the present study was to analyze the functional heterogeneity of the somatotrope population during the aging process in male rats. For this purpose, the response of LD- and HD-somatotropes from 5-, 19-, and 26-month-old male rats was analyzed with an optimized cell immunoblot assay both under basal conditions, and after GH-releasing factor (GRF) and/or somatostatin (SS) treatments. Simultaneous measurements of hormonal release, intracellular GH content, and cell size were performed at the single-somatotrope level. Average values for those parameters were significantly higher in HD- than in corresponding LD-cells, such differences being irrespective of age or treatment. Releasing activity and GH content were significantly reduced with age in both subpopulations. GRF stimulated GH release from LD- and HD-somatotropes, and the GRF responsiveness was similar in both subpopulations and in all ages. On the other hand, SS prevented GRF-stimulated GH release in most cases. At the level of single cells, both releasing activity and cell size showed a significant, linear dependence on intracellular GH content, correlations being irrespective of age, subpopulation, or treatment. Taken together, our results demonstrate that LD- and HD-somatotrope subpopulations display quantitative differences in releasing activity that are essentially retained through aging. This functional heterogeneity is more dependent on the basal GH release of these somatotrope subsets than in their responsiveness to GRF and SS. The present findings suggest that the reduction in secretory activity at the single somatotrope level observed in both subpopulations underlies the age-related decline of pituitary GH release. Finally, a theoretical model of secretory cycle is proposed which might contribute to the understanding of the biological meaning of the somatotrope subpopulations in aging male rats.

Heterogeneity of growth hormone (GH)-producing cells in aging male rats: ultrastructure and GH gene expression in somatotrope subpopulations.

Mammalian aging is characterized by a decline in the content and release of pituitary growth hormone (GH). However, few studies on the age-related changes in the population of GH-producing cells (somatotropes) have been carried out. We have investigated whether changes in number, ultrastructure and GH gene expression in subpopulations of somatotropes could explain the reduced GH release in aged rats. Three representative ages were studied: adult (5-month-old), old (19-month-old), and senescent (26-month-old) male rats. The total number of immunoreactive-GH cells per pituitary gland remained invariable to age. The separation of dispersed pituitary cells on a density gradient yielded two somatotrope subpopulations, of low density (LD) and high density (HD). Both subpopulations were equally represented in adults, whereas in old and senescent rats a predominance of LD-somatotropes was observed. Morphometric analysis showed that subpopulations exhibited storage and biosynthetic features inversely related. In LD-somatotropes, rough endoplasmic reticulum (RER) was more prominent but secretory granules (SG) were less abundant than in HD somatotropes. Concurrently, in situ hybridization for GH mRNA showed that GH gene expression was higher in LD-cells. Differences between subpopulations were essentially retained through the animals' lifespan, but small-sized SG, reduced RER, and low GH mRNA levels were inherent to aging both in LD- and in HD-somatotropes. The present findings demonstrate that the reduced content of pituitary GH in aged male rats is not due to a diminished number of GH-producing cells, but to the numerical predominance of scarcely granulated LD-somatotropes, combined with the decline in GH biosynthetic capacity observed in both subpopulations. In addition, age-related changes in ultrastructure and GH gene expression suggest a chronic inhibition of GH release and/or a weak stimulation of GH biosynthesis affecting both subpopulations.

Somatostatin increases growth hormone (GH) secretion in a subpopulation of porcine somatotropes: evidence for functional and morphological heterogeneity among porcine GH-producing cells.

Previous results demonstrate that porcine somatotropes can be separated by density gradient centrifugation into low density (LD) and high density (HD) subpopulations. In rat, two analog somatotrope subpopulations differ morphologically and functionally. In an attempt to determine whether morphological differences were also present within LD and HD porcine somatotropes, we undertook a quantitative electron microscope study of the subcellular organelles of immunoidentified LD and HD somatotropes. In addition, to test for the existence of functional differences, cultures of separated HD and LD subpopulations were treated for 4 h with or without 10 microM GRF-(1-29) and/or 100 microM somatostatin (SRIF), and porcine GH release and intracellular content were evaluated using a homologous enzyme immunoassay. Morphometric results demonstrate that LD somatotropes are smaller in size (P < 0.05) and contain fewer secretory granules (P < 0.05) and more rough endoplasmic reticulum (P < 0.05) than HD somatotropes. In terms of secretion, LD somatotropes showed a classical response; GRF increased GH release 1.7-fold (n = 6; P < 0.05) over the control value, whereas treatment with SRIF alone did not affect basal GH release in this subpopulation, but partially blocked GRF-induced GH release. HD somatotropes responded to GRF with a similar 1.7-fold increase in GH release. However, SRIF administered alone or in combination with GRF exerted a paradoxical stimulatory effect on HD somatotropes (2.15- and 2.12-fold over control value, respectively; n = 6; P < 0.05). These results demonstrate that the porcine somatotrope population is composed of two major subpopulations that display a distinctive pattern of ultrastructural organization and a markedly divergent secretory response to in vitro SRIF treatment.

Presence of mammosomatotropic (MS) cells in mink (Mustela vison) adenohypophysis.

This study was undertaken to investigate the presence of mammosomatotrophs (MS) cells in the suckling mink. Using the double immunolabeling procedure, with colloidal gold as label, we demonstrated the existence of MS cells in these animals. Only one type of MS cells has been observed. These cells showed a great morphological similarity to classic prolactin (PRL) cells. MS cells of suckling mink were pleomorphic in appearance with many processes, their nuclei were irregular and their Golgi apparatus and endoplasmic reticulum were poorly developed. Their secretory granules were small (about 144 nm in mean diameter) and round. Two types of secretory granules have been found: monohormonal including PRL (the more frequent) and growth hormone (GH) (very scanty) granules, and bihormonal granules distributed between the former. We propose that MS cells of the mink, like other species, could represent an intermediate cell type in the transformation process of GH cells into PRL cells.

Morphological and functional heterogeneity of frog melanotrope cells.

Previous reports have described the heterogeneity of different pituitary cell types on the basis of morphological and physiological criteria. In the present study, we investigated the possible existence of distinct subpopulations of melanotrope cells in the intermediate lobe of the pituitary of the frog, Rana ridibunda. Separation of dispersed pars intermedia cells in a Percoll density gradient made it possible to isolate two fractions of melanotrope cells whose morphological and functional properties were further characterized. Analysis of the relative volume and number of various cellular organelles showed that high-density cells had a larger number of secretory granules than low-density cells. Concurrently, radioimmunoassay quantification revealed that the concentration of alpha-melanocyte-stimulating hormone (alpha-MSH) was 2 times higher in the heavy cell population. The rate of secretion of alpha-MSH from cultured melanotrophs was significantly higher in low-density than in high-density cells. Thyrotropin-releasing hormone (TRH) was more potent in stimulating alpha-MSH release from the low-density than from the high-density cell subset. In contrast, the response to TRH persisted for a longer time in the high-density cell subpopulation. Taken together, these data demonstrate the existence of two subpopulations of melanotrope cells, and indicate that the low-density cells have a secretory rate substantially greater than high-density cells.

Hormonal storage patterns and morphological heterogeneity of porcine gonadotrope cells during postnatal development.

Previous reports indicate that gonadotrope cells of the porcine pituitary gland can be separated into three subpopulations of low- (1.049 g/cm3), middle- (1.062 g/cm3) and high- (1.087 g/cm3) density in a continuous Percoll density gradient. The aim of this work was to study the hormonal storage patterns and morphological features of these subpopulations at three representative ages of the postnatal development: neonatals (30-day-old animals), prepubertals (5-6-month-old animals) and matures (16-18-month-old animals). The low-density subpopulation, present at the three ages studied, was mainly composed of bihormonal LH/FSH cells in neonatal and monohormonal LH cells in prepubertal and mature animals. On the other hand, middle- (only present in prepubertal and mature animals) and high-density subpopulations (only present in neonatal and prepubertal animals) were mainly composed of bihormonal LH/FSH gonadotropes. In ultrastructural terms, these subpopulations exhibit a correlation between density and morphology irrespective of the animal's age. The low-density subpopulation was composed of poorly granulated cells with highly developed biosynthetic machinery (rough endoplasmic reticulum and Golgi complex), while high-density cells were of opposite morphology, with a highly granulated cytoplasm and poorly developed rough endoplasmic reticulum and Golgi complex. The middle-density subpopulation was composed of poorly granulated cells with scarcely developed biosynthetic machinery. In conclusion, these results indicate that porcine gonadotrope cells during postnatal development are composed of three subpopulations of different hormonal storage patterns and morphology. The presence of these subpopulations at the different stages of postnatal development strongly suggests that their proportions may play a major role in the endocrine control process.

Application of a Percoll density gradient to separate and enrich porcine pituitary cell types.

The application of a Percoll density gradient cell separation procedure to the pituitary gland of neonatal, prepubertal and mature pigs is described. After enzymatic dispersion, cell viability was 90% to 98% as determined by uptake of Trypan Blue. Recovery of dissociated cells after application of the gradient ranged from 80% to 95%. The dissociated cells were separated in several fractions that were characterized immunocytochemically using different antisera. We obtained highly enriched fractions of gonadotrope (gonadotropins), somatotrope (growth hormone) and lactotrope (prolactin) cells for each age. Concentration of the cell types (purity) was higher than 60% in the following fractions: 1) gonadotropin cells, fraction 15 (1.033 g/cc of density) from mature animals; 2) growth hormone cells, fraction 3 (1.121 g/cc of density) from neonatal animals and fraction 9 (1.087 g/cc) from prepubertal animals; and 3) prolactin cells, fraction 7 (1.094 g/cc) and 10 (1.082 g/cc) from neonatal animals and fraction 14 (1.051 g/cc) from mature pigs. In thyrotrope (thyroid-stimulating hormone) and corticotrope (adrenocorticotropin) cells, enriched fractions were also obtained, although the values of purity were lower (20% to 58%). In conclusion, the proposed cell separation and enrichment technique is suitable for the isolation, purification and examination of porcine pituitary cell types and subpopulations, and offers major advantages such as simplicity, rapidity, efficiency and reproducible results.

Different exocytotic morphology in amphibian prolactin and growth hormone cells stimulated in vitro with TRH.

Exocytotic process in growth hormone (GH) and prolactin cells (PRL) of the frog anterior pituitary have been examined using an experimental design that has been previously demonstrated to increase the release of hormone from both cell types. Hemipituitaries of the same animals were superfused either with medium alone or containing 100 ng/ml of thyrotropin-releasing hormone (TRH) for 24 hr. PRL and GH cells were identified by the colloidal gold method using anti-human prolactin and anti-ovine growth hormone as primary antisera. In hemipituitaries cultured with medium alone, PRL and GH cells showed few exocytotic figures with different morphology in both cells types. In TRH treated hemipituitaries, PRL cells showed numerous exocytotic vacuoles containing immunoreactive granulated material that was preferentially located near basal lamina. On the other hand, GH cells showed higher amount of exocytotic vacuoles containing heterogeneous immunoreactive material, located along the cell membrane. In PRL cells single secretory granules are secreted, whereas GH cells showed multigranular exocytosis. These results indicate that in PRL and GH amphibian cells exocytotic process has a different polarity and morphology and that this process increases with TRH stimulation.

Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.

The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.

Effects of ovine CRF on amphibian pituitary ACTH and TSH cells in vivo: a quantitative ultrastructural study.

The subcellular responses of amphibian adrenocorticotropic (ACTH) and thyrotropic (TSH) pituitary cells to the in vivo administration of ovine corticotropin-releasing factor was investigated. For this purpose, groups of six Rana perezi adult frog (three males and three females) were given daily injections of ovine CRF and sacrificed at 6 hr, 24 hr, and 4 days after the first injection. Immunogold staining, applied to ultrathin sections using antisera to human ACTH (1-39) and human beta-TSH identified ACTH and TSH cells, respectively. Morphometry was used to evaluate the volume density (Vv) changes of the rough endoplasmic reticulum, Golgi complex, and secretory granules and the numerical density of the latter. CRF significantly reduced the Vv of the secretory granules in both cell types, taken as indicative of short-term enhanced hormonal release. The peptide also stimulated the development of the cellular biosynthetic machinery, although this effect was detected at an earlier stage in ACTH cells than in TSH cells. These results show for the first time the occurrence of cellular response of amphibian adrenocorticotropes and thyrotropes to CRF and suggesting that this peptide regulates ACTH and TSH production. Moreover, each type of cell differed in its sensitivity to the peptide. After long-term treatment the cytological response of ACTH cells to CRF seemed to decrease, while TSH cells remained active.

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: effects on prolactin and thyrotropic cells.

The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human-beta-thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thyrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.

Annulate lamellae in frog adenohypophysis under normal and experimental conditions.

Cytoplasmic annulate lamellae have been observed in frog (Rana ridibunda) adenohypophysis pars distalis from normal spring animals and from others which were submitted to experimental conditions inducing selective activation of different cell types. Cell activation, because of either the normal active period in the frog cycle or the experimental treatments, seems to be correlated with the occurrence annulate lamellae. These annulate lamellae consist of a succession of two relatively parallel membranes interrupted periodically by discontinuities similar to nuclear pores. Sometimes they have been observed connected to endoplasmic reticulum.