Two types of mammosomatotropes in mouse adenohypophysis

Summary Two types of mammosomatotropes (MS), the small-granule and vesicle-granule MS, were detected in mouse adenohypophysis by electron microscopy and immunohistochemistry. Both cell-types were immunoreactive to prolactin (PRL) and growth hormone (GH) antisera. The small-granule MS contained small, round, solid secretory granules about 100 nm in diameter, and were smaller than the classical GH and PRL cell-types. The vesicle-granule MS contained secretory granules like cored vesicles, and were larger than classical GH and PRL cells. Small-granule MS were immunoreactive to both PRL and GH antisera in the same region of the cell cytoplasm; the vesicle-granule MS, however, were immunoreactive to only PRL antiserum in most cytoplasmic areas, and a positive response to both PRL and GH antisera was confined to only certain small areas.     

Multiple hormone storage by cells of the human pituitary

While immunostaining serial semi-thin sections of acrylic resin-embedded normal human pituitary using antisera to human pituitary hormones, it became clear that several cells were stained by more than one antiserum. The tissue had been surgically excised from a patient with a prolactinoma. The tumor, which was immunoreactive only with antiprolactin antiserum, was distinctly different from the pieces of tissue under study which had normal pituitary architecture and demonstrated immunoreactivity with antisera against all six of the common pituitary hormones. A major immunoelectron microscopic investigation, using immunocolloidal gold and immunoperoxidase methods, revealed cells in which follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) were co-localized to the same electron-dense granules. Some similar cells also possessed electron-lucent granules immunoreactive only for anti-PRL antiserum. Adrenocorticotrophic hormone (ACTH) and PRL were also found in the same cell but were very largely localized to separate, morphologically different populations of electron-dense and -lucent storage granules. By employing double immunolabeling, a few granules in the ACTH/PRL cells were shown to be immunoreactive to both anti-ACTH and anti-PRL antisera. The possibility that the multipotential stem cells is discussed.     

Subcellular distribution of secretogranins I and II in GH3 rat tumoral prolactin (PRL) cells as revealed by electron microscopic immunocytochemistry

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.     

Regulation of chromogranin B/secretogranin I and secretogranin II storage in GH4C1 cells

GH4C1 cells are a rat pituitary tumor cell strain in which the level of cellular prolactin (PRL) and PRL-containing secretory granules can be regulated by hormone treatment. The chromogranins/secretogranins (Sg) are a family of secretory proteins which are widely distributed in the secretory granules of endocrine and neuronal cells. In the present study, we investigated in GH4C1 cell cultures the regulation of the cell content of the Sg by immunoblotting and the relationship between the storage of Sg I and Sg II and PRL by double immunocytochemistry. GH4C1 cells grown in the presence of gelded horse serum, a condition in which these cells contain a low level of secretory granules, contained low levels of PRL, Sg I, and Sg II. Treatment of GH4C1 cells with a combination of 17 beta-estradiol, insulin, and epidermal growth factor for 3 days, known to induce a marked increase in the number of secretory granules, increased the cell contents of PRL, Sg I, and Sg II. To determine whether the induction of PRL was morphologically associated with that of the Sg, the distribution of PRL and the Sg was determined by double immunofluorescence microscopy. After hormone treatment, 54% of cells showed positive PRL immunoreactivity, fluorescence being extranuclear and consistent with staining of the Golgi zone and secretory granules. Forty-six percent of PRL-positive cells stained coincidently for Sg I, while 72% of the PRL cells were also reactive with anti-Sg II. To determine whether PRL storage was associated with storage of at least one of the Sg, cells were stained with anti-PRL and anti-Sg I and anti-Sg II together. Eighty-six percent of PRL cells stained for one or the other of the Sg. Therefore, PRL storage in GH4C1 cell cultures is closely but not completely associated with the storage of Sg I and/or II.     

The ultrastructure of prolactin cells in the annual cyprinodont Cynolebias whitei during its life cycle

Summary Prolactin (PRL) cells were studied electron-microscopically and morphometrically in the annual cyprinodont fish, Cynolebias whitei during its life cycle. In prehatching larvae, PRL cells possessed small secretory granules, giant mitochondria and a well-developed Golgi apparatus. During hatching, no changes were observed in the volume density of the secretory granules, indicating that no increased release of PRL occurs at hatching. A significant change in the composition of PRL cells, i.e., the volume densities per cytoplasm volume of the different organelles, occurred between one day and one week of age. Thereafter, only minor differences were observed between age groups, indicating that no major changes occur in PRL cell activity during the lifespan of C. whitei. However, the volume density per cell volume of the nucleus decreased steadily with age during the lifespan. A comparison of the PRL cells in young and adult fish reared in fresh water (FW) with siblings reared from hatching in diluted sea water (1/3 SW) did not reveal any differences with respect to the volume densities of the organelles, including the secretory granules. However, significant differences were observed with respect to the diameter, electron-dense content and affinity to anti- PRL serum of the secretory granules. These differences indicate that, despite the similar volumetric composition of the PRL cells, their secretory granules contain a substantially higher concentration of PRL in FW-reared fish than in 1/3 SW-reared fish.     

Immunocytochemical identification of growth hormone cells in the adenohypophysis of the golden hamster

Somatotrophs or growth hormone (GH) cells in the adenohypophysis of golden hamsters were identified by immunocytochemical staining with polyclonal rabbit anti-human GH. They were oval or columnar in shape, and had secretory granules of two size ranges, 90-150 nm and 280-320 nm, which were present in the same cells; no subtypes of GH cells were observed. Secretory granules were located in the peripheral portion of the cytoplasm or concentrated at the vascular pole of the cell. Flattened cisternae of the rough endoplasmic reticulum in parallel array and a moderately developed Golgi apparatus were often found in the cytoplasm. No sex difference was noticed in the population ratio of GH cells. Immunocytochemical staining with anti-GH or anti-prolactin (PRL) antibodies on separate adjacent sections revealed that the GH and PRL were stored in two different cell types.     

Effect of decreased environmental salinity on growth hormone cells in the gilthead sea bream (Sparus aurata)

The effect of decreased environmental salinity on growth hormone producing cells (GH cells) of the adenohypophysial proximal pars distalis has been studied in the gilthead sea bream ( Sparus aurata L.) adapted to sea water (SW, 980 mosmol kg ⁻¹) and brackish water (BW, 200 mosmol kg ⁻¹). A combined immunocytochemical, morphometric and electron microscopic study was carried out. GH cells offish adapted to BW occupied a greater hypophysial volume (about 21% of the total hypophysial volume in BW, 17% in SW) and had a larger nuclear area (mean 16 μm² in BW, 13 μm² in SW) than GH cells of SW-adapted fish. The immunoreactivity against a salmon GH-antiserum was lower in BW (mean optical density 142 in BW, 159 in SW). Ultrastructural characteristics of GH cells of BW-adapted fish were distended rough endo-plasmatic reticulum and large secretory granules (about 216 nm in diameter for BW, 209 nm in SW). Volumetric, densitometric and ultrastructural evidence suggested that the synthesis and release of GH were activated in S. aurata adapted to hypo-osmotic environment.     

Immunocytochemical studies on prolactin cells in the adenohypophysis of the golden hamster

Mammotrophs or prolactin (PRL) cells were identified in the adenohypophysis of adult golden hamsters by immunocytochemical techniques with a polyclonal anti-PRL, that was proved to be specific to PRL by the dot immunoblotting test. Postembedding immunostaining was performed on Araldite thin sections by immunoperoxidase and immunogold methods. PRL cells were classified into three types according to the different size of the secretory granules. The Type A cells were usually small and angular or oval in shape, and had secretory granules ranging in diameter from 100-230 nm, and showed poorly developed organelles. The Type B and C cells were larger and round or ovoid in shape, contained larger granules, 230-280 nm and 280-570 nm, respectively, and displayed well developed organelles. Immunoreactive PRL cells in the male pituitaries were far less numerous than in the nonpregnant female glands, and were mostly of the Type A and B, whereas in the female the Type C and B cells predominated. In pregnant females, Type C cells became activated and increased in number, while the other two types decreased in proportion. In lactating females, Type A and B cells significantly increased in number at the expense of the Type C cells; meanwhile, the exocytosis of secretory granules was frequently found in all types of PRL cells. The present findings suggest that Type C and B PRL cells, especially the former, are potent in producing and releasing PRL and highly responsive to various physiological stimuli, while Type A cells are probably relatively inert in synthetic activity.     

Immunohistochemical and ultrastructural study of anterior pituitary cells in the female Afghan pika,Ochotona rufescens rufescens

An immunohistochemical study of the anterior pituitary gland of the female Afghan pika was carried out to distinguish the ultrastructural features of GH, PRL, ACTH, TSH and LH cells. The histochemically identified GH cells resembled ultrastructurally oval or round GH cells of the rat laden with large, dense secretory granules. PRL cells were divided into three subtypes based on differences in the diameter of their spherical secretory granules. They lacked polymorphic or irregularly shaped secretory granules. ACTH cells resembled ultrastructurally, in some respects, Siperstein's "corticotrophs" of the rat with peripheral arrangement of secretory granules. However, they were not always stellate, but elongate or angular in shape. The dense secretory granules were concentrated in the peripheral area of cytoplasm. TSH cells were non-stellate, but usually oval in shape, containing the smallest spherical secretory granules (100-200 nm in diameter). Almost all LH cells reacted also with FSH antiserum. They were irregular in shape, sometimes in contact with or surrounded the GH cells. They contained an abundance of medium-sized secretory granules (140-260 nm in diameter) which were larger than those in the LH cells of the female rat throughout the estrous cycle. Large secretory granules in the LH cells of the female pika seemed to be related to the endocrine state of persistent estrus.     

Effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) release and cell morphology in human pituitary adenoma cell cultures

Six GH adenomas and three prolactinomas were investigated by light- and electron-microscopic morphological and immunocytochemical methods and the effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) secretion was tested in vitro. The tumour cells of the acromegalic patients revealed both GH and PRL immunoreactivity while prolactinomas showed only PRL activity. All the adenomas stained immunocytochemically also for VIP. By electron microscopy, the tumours included two densely and two sparsely granulated GH, two mixed GH/PRL, and three sparsely granulated PRL adenomas. The dissociated cells were explanted, and cultured in vitro. The cultures in micro test plates were treated with VIP at different concentrations between 10(-5)-10(-12) M. GH and PRL contents in the culture media were measured by radioimmunoassay. GH release was significantly stimulated by VIP in a dose-dependent manner over the whole concentration range, while VIP was effective on the PRL release only at 10(-6)-10(-7) M concentration. The cells of a mixed adenoma were grown in Petri dishes and used for ultrastructural and immunocytochemical studies. The cytoplasmic structure of the cells treated with VIP corresponded to that of active hormone-secreting cells with large ergastoplasmic fields and Golgi zones containing secretory granules. Massive exocytotic events were encountered mainly in the GH-type cells. GH and PRL double immunocytochemistry showed the predominance of GH cells, many of them containing low amounts of PRL as well. Cells predominantly containing PRL were spread among them, they also might contain GH as well. Some of the cells contained only a single immunoreactive hormone. The intensity of gold labelling of the secretory granules appeared higher in the VIP-treated cells than in the untreated control ones which showed a cytoplasmic structure characteristic of glandular cells with low secretory activity. As all the adenoma cells both contained and reacted to VIP, our results are in agreement with an autocrine or paracrine effect of this peptide. The fine structure of the cells in the cultures treated with VIP supply an additional argument to the assumption that VIP may serve as a growth factor for these cell types.     

Quantitative aspects of growth hormone cell maturation in the normal and little mutant mouse

Growth hormone (GH) cells were analyzed by means of ultrastructural morphometry in the pars distalis of pituitary glands from male adult and immature normal (C57BL) and homozygous little (lit/lit) mutant mice. Thin sections were exposed to anti-GH serum and processed immunocytochemically with the colloidal-gold technique. In the pars distalis of adult lit/lit mice, the mean volume density of GH cells/total tissue was 24% of the normal value, granules/GH cells was 58% of normal, and granules/total tissue was only 12% of normal. Deficits in all of these parameters likewise occurred in immature glands, though to a lesser extent than in the adults. The results indicate that the GH deficiency in this mutant reflects quantitative deficits in both the secretory granule content of GH cells, as well as the GH cell content of the gland, with the latter being the more severely affected.     

CXCL14-like immunoreactivity in growth hormone-containing cells of urodele pituitaries

Immunohistochemical techniques were employed to investigate the distribution of a chemokine, namely, CXCL14-like immunoreactivity in the axolotl (Ambystoma mexicanum) and Japanese black salamander (Hynobius nigrescens) pituitaries. CXCL14-immunoreactive cells concentrated at an area of the pars distalis adjacent to the pars intermedia. We found that these cells correspond to the cells immunoreactive to an antibody against rat growth hormone (GH). Immunoelectron microscopy indicated that the CXCL14-like substance and GH coexisted on the secretory granules in the axolotl pituitary. Western blot analysis of axolotl pituitary extracts revealed the anti-human CXCL14 antibody labeled an approximately 16.6-kDa band that was not labeled by the anti-GH antibody. The CXCL14-like substance in the pars distalis may participate in GH functions in these species.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.     

Postnatal development of growth hormone and prolactin cells in male and female rat pituitary. An immunocytochemical light and electron microscopic study

Localization and ultrastructural maturation of prolactin (PRL) and growth hormone (GH) cells were studied in pituitaries from neonatal, immature (4-6 weeks old), and adult rats (2-3 months old) by light and electron microscopic immunocytochemistry. The distribution pattern of these cells did not change with age. Both cell types were concentrated laterodorsally, with PRL cells adjacent to the intermediate lobe and GH cells nearer the center of the pars distalis. Labeling density of the immunogold reaction was highest for both hormones in immature rats. In neonatal and immature rats, one PRL cell type with granules 200 nm in diameter was present. In adult rats, two types of PRL cells were present: one containing polymorphous granules measuring about 500 nm (prevalent in female rats), the other with spherical granules about 200 nm (prevalent in male rats). No changes were detected in GH cells during maturation.     

斜带石斑鱼生长激素/催乳素家族基因cDNAs的分子克隆及其进化意义

从斜带石斑鱼垂体提取总。RNA,再取其50ng合成SMART cDNA。从所构建的垂体SMART cDNA质粒文库中筛选到生长激素／催乳素基因家族的2个成员的全长cDNA片段：生长激素(GH)基因全长为938bp,编码204个氨基酸;催乳素基因(PRI．)全长为1429bp,编码212个氨基酸。采用计算机软件Mega 2和CLUSTAL W1．64b对9种鱼的生长激素／催乳素基因家族的3个成员(GH、PRL和生长催乳素SL)的氨基酸序列进行系统分析,构建NJ分支系统树,对于序列中的插入／缺失位点则采用Pairaise Deletion,1000次自展(Bootstrap)分析计算各节点支持率。根据3个基因的氨基酸序列构建的系统树表明,石斑鱼与金头鲷、金鲈和牙鲆聚成一类,虹鳟与大马哈鱼聚成一类,鲫鱼与鲶鱼聚成一类,鳗鲡成另外一类。根据石斑鱼全长cDNA推断的氨基酸序列比较表明,SL相对GH和PRL有较高的保守性。石斑鱼的GH、PRL和SL的氨基酸同源性在24％～31％,但其C-端的氨基酸同源性较高,尤其是C-端的3个Cys是严格保守的。其中SL与GH的同源性(30．8％)高于与PRL的同源性(25．6％),GH和PRL的同源性最低(24．1％)。     

Immunocytochemical study of the GH cells in the anterior pituitary gland of human fetus II. Anencephalic fetus

In order to elucidate the effects of hypothalamic regulation on the morphology of GH cells, light and electron microscopic immunocytochemical examinations were carried out comparing GH cells in the anterior pituitary gland of anencephalic fetus with those of normal fetuses. Three types of GH cells were identified in the anterior pituitary gland of anencephalic fetus as well as in the normal fetus. Type-I is a small, round cell containing a few small secretory granules. Type-III is a large, polygonal cell with numerous large secretory granules. Type-II is a polygonal cell with medium-sized secretory granules. The Type-II GH cell was predominant in both anencephalic and normal fetuses. The most striking difference between anencephalic and normal fetuses was the presence of atypical forms of the Type II cell. These were polygonal cells containing secretory granules, which were either immunopositive or immunonegative to anti-human GH (anti-hGH) serum. Furthermore, two other types of GH cells were identified. The somatomammotroph (SM cell) contained GH and PRL in different granules within the same cell. Also, a different type of the GH cell was noted containing two varieties of secretory granules; one was immunolabeled only with anti-hGH and the other was not immunolabeled to either anti-hGH or anti-human PRL (anti-hPRL). From these results, we suggest that an absence of hypothalamic regulation in the anencehpalic does not seriously modify GH cell morphology but induces an altered GH storage pattern in some of the cells.     

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.     

Immunocytochemical identification of prolactin cells in the pituitary gland of tilapia larvae (Oreochromis mossambicus: Teleostei)

Summary Using an antiserum to highly purified chum salmon prolactin, prolactin cells were identified in the putative rostral pars distalis of newly hatched tilapia larvae (Oreochromis mossambicus) by the immunogold method for the electron microscope. In the putative rostral pars distalis, some cells had another kind of secretory granule which was much less numerous, much smaller in size, and without immunoreactivity to salmon prolactin antiserum. Controls incubated with salmon prolactin-preabsorbed antiserum or normal serum showed no immunoreactive cells, confirming the specificity of the antiserum. The possible role of prolactin in the osmoregulation of tilapia larvae is discussed.