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Childs (Moriarity), Gwen V.; Ellison, Dayle G.; Garner, Laura L.
doi: 10.1002/aja.1001580403pmid: 6257101
This study was designed to elucidate hormone storage patterns in gonadotropes with the use of ultrastructural immunocytochemistry on serial ultrathin sections. Sets of six serial sections were stained for beta chains of LH, FSH, or the C‐Terminal sequence of ACTH, and 430 cells cut in triple or double serial section were collected from a group of seven normal adult male rats. Approximately 50‐88% of the cells contained both LH and FSH, and most of these were Type I cells which are distinguished by their round shape and heterogeneous populations of secretion granules. Cells containing only FSH or LH constituted, on average, 19% of the population. These were a mixed group, morphologically, and included Type II cells distinguished by their angular shape and population of secretion granules, 250 nm in average diameter. Also among the FSH cells (and a few LH cells in two of the rats) were Type III cells, which resemble the corticotrope. On average, 10% of the serially sectioned cells contained only ACTH. Our findings show the presence of subpopulations of gonadotropes containing only one of the hormones, in numbers large enough to support the hypothesis that they may be partly responsible for the nonparallel release of gonadotropins. Also, the FSH‐LH cells seemed to vary in their staining intensity for the two hormones, suggesting that the gondaotropes are a fluid, heterogeneous population of cells capable of storing both or only one of the hormones.
Ferrand, Raymond; Fremont, Patrick H.; Dubois, Maurice P.
doi: 10.1002/aja.1001580404pmid: 6257102
Epithelial rudiments of adenohypohysis were removed from chick and quail embryos between days 3 and 5 of development. Chick rudiments were grafted for 11–13 days onto the chorioallantoic membrane of decapitated chick embryo hosts. Quail rudiments were cultivated in vitro for 6 days. Both grafted and cultivated Rathke's pouches differentiated into adenohypophyseal tissue. The adenohypophyseal tissue cultured on chorio‐allantoic membrane exhibited cells reacting with the following immune sera: anti‐β‐(1–24)ACTH, anti‐α‐(17–39)‐ACTH, anti‐α‐endorphin, anti‐β‐endorphin and anti‐β‐LPH, which also gave a positive reaction when applied to adenohypophysis of corresponding age which had differentiated in situ. In situ, corticotrophs were located exclusively in the cephalic lobe of adenohypophysis. Therefore, the differentiation of corticotrophs in the whole graft, i.e., from both cephalic and caudal lobes of Rathke's pouch, showed that the cells of the caudal lobe, or at least some of them, were uncommitted when the rudiment was removed. In vitro, tissue derived from Rathke's pouch contained cells reacting with antibodies to β‐(1–24)‐ACTH, α‐(17–39)‐ACTH, and β‐LPH, as did adenohypophysis from quail embryos of corresponding age (9–10 days), differentiated in situ. The differentiation of quail Rathke's pouch in vitro corroborates that differentiation can occur without influence from hypothalamus and, moreover, shows that at least some kinds of cells can differentiate without influence exerted by any other encephalic factors, and in the absence of mesenchyme. The question arises whether fibroblastic cells derived from Rathke's pouch cells act as feeder‐cells and/or secrete some factors promoting differentiation.
Shiino, Masataka; Fujihara, Noboru; Rennels, Edward G.
doi: 10.1002/aja.1001580405pmid: 6779620
Female Sprague‐Dawley rats were hypophysectomized and the anterior pituitary gland was immediately placed under the kidney capsule. For 1 week after surgery, groups of pituitary autograft‐bearing animals were treated with twice‐daily injections of estradiol 17β (E), progesterone (P), estradiol 17β and progesterone (EP), or luteinizing hormone‐releasing hormone (LRH). Within 2–4 hours following the last injection, the pituitary grafts were removed and placed into organ culture. They were maintained in culture with or without added LRH (10−7 M) for 1 hour at 37°C. The culture media were then frozen for later radioimmunoassay of FSH and LH. The tissues were kept in culture for an additional 24 hours, at which time they were fixed and prepared for immunocytochemistry or electron microscopy.
Watkins, Wayne B.; Moore, Robert Y.
doi: 10.1002/aja.1001580406pmid: 7006372
The distribution of those cells in the anterior pituitary gland of the rat which stain immunohistochemically with rabbit anti‐human calcitonin serum has been examined. Immunoreactive cells were confined primarily to the ventral surface of the gland and possessed both a distribution and morphology distinct from the corticotrophs. Staining of serial thin sections with rabbit anti‐rat TSH‐subunit serum resulted in an immunoreaction in those cells that stained for calcitonin. However, not all the thyrotrophs gave a positive immunoreaction for calcitonin. It is concluded from this study that it is inappropriate to attribute calcitonin as being part of the 31K‐dalton precursor for adrenocorticotropin, a hypothesis that was proposed earlier. In the immunohistochemical reaction with anti‐calcitonin serum, it was found that relatively high concentrations of antigen (500 μg/ml) were required in absorption experiments in order to inhibit staining. Furthermore, the staining of thyrotrophs with the anti‐calcitonin serum was inhibited after preadsorption of the primary antiserum with excess rat β‐TSH (1000 μg/ml). Because of these immunochemical characteristics, it is questionable whether the calcitonin‐like material observed in the rat pituitary gland is chemically identical to that of thyroidal calcitonin.
Baskin, Denis G.; Erlandsen, Stanley L.; Parsons, Jonathan A.
doi: 10.1002/aja.1001580407pmid: 7006373
The aim of this study was to identify the neoplastic endocrine cells which contain growth hormone (GH) and prolactin (PRL), in the MtTW15 mammosomatotropic tumor, with ultrastructural immunocytochemistry. We used tumors recovered after 5 to 11 weeks of tumor development, from normal (untreated) rats and from rats treated with the progestin medroxyprogesterone acetate (MPA)—a stimulator of GH secretion in these tumors. Immunocytochemical staining was done with the peroxidase‐antiperoxidase technique on ultrathin sections of tumor that had been fixed in glutaraldehyde and postfixed in osmium tetroxide. Immunospecific staining for PRL was found over small (150 nm) secretion granules, whereas staining for GH was deposited on the larger secretion granules (250 nm). Tumors from MPA‐treated rats contained profuse numbers of neoplastic cells with large, GH‐positive granules. Immunocytochemical staining for GH and PRL was also found in crinophagic, lysosome‐like inclusions, particularly in cells that contained many secretion granules. The results support the hypothesis that GH and PRL are produced by separate neoplastic endocrine cell types in the MtTW15 mammosomatotropic tumor, and demonstrate the value of ultrastructural immunocytochemical analysis for functional classification of cell types in chromophobic pituitary adenomas.
doi: 10.1002/aja.1001580408pmid: 7006374
Among 92 surgically removed pituitary adenomas immunostained for prolactin and growth hormone, 70 showed positive staining for prolactin. The majority of these (54) was associated with hyperprolactinemia leading to amenorrhea (and often galactorrhea) in women of reproductive age. Similar tumors, asymptomatic or conducive to disturbances of sexual function, were found in six hyperprolactinemic men. Among nine acromegalics, seven had immunostained lactotrophs associated with the somatotrophic adenomas cells, but only two of these had hyperprolactinemia. In all of the remaining tumors that had at least some immunoreactive lactotrophs, mild hyperprolactinemia had been present. This indicates that immunostaining of pituitary tumors for prolactin correlated well with elevated plasma prolactin levels, except in the case of mixed somatolactotrophic adenomas. The patterns of distribution of immunoreactive prolactin in adenoma cells are illustrated. Since only some of the prolactin‐producing adenomas stained with carmoisine–a dye that has been suggested as a marker for prolactin cells–immunocytochemistry is the method of choice for the identification of prolactin‐secreting adenomas.
Tougard, C.; Picart, R.; Tixier‐Vidal, A.
doi: 10.1002/aja.1001580409pmid: 7006375
Three aspects of the secretory process in male rat prolactin (PRL) cells grown in primary cultures for 7–14 days have been investigated by cytochemical methods. The subcellular localization of prolactin has been studied using preembedding or postembedding immunocytochemical methods after various fixatives. With postembedding method, PRL is localized essentially in secretory granules. The maximum intensity of staining is obtained with PAF fixative. With the preembedding method, subcellular localization of the staining varies depending on the fixative. After PAF‐fixation, positive staining is observed on secretory granules, ground cytoplasm, the outer face of some RER cisternae and, in a few cells, on the innermost Golgi cisternae, as well as on masses of condensing secretory material. After Ohtsuki's hypotonic fixative followed by saponin permeabilization, PRL is visualized within the totality of RER cisternae, including the perinuclear cisternae and the peripheral saccules on the cis‐Golgi face. Secretory granules are unstained. Membrane traffic was investigated using the Con A‐HRP indirect method as a tracer of surface saccharides. Plasma membrane, coated with Con A‐HRP at 4°C, is slowly internalized at 37°C. This involves both randomly distributed invaginations and capping. The final step of endocytosis (1–2 hours) is located in the Golgi zone, where very few smooth membranes are stained. In contrast, a conspicuous deposit is found around the dense content of secretory granules. This suggests a recycling of internalized membrane and a transfer of Con A‐HRP from the inner face of smooth cisternae to the secretory material. The internalization of Con A‐HRP‐coated membrane leads to an inhibition of PRL release starting after 30 minutes. This is accompanied by a marked increase of acid phosphatase activity, mostly around forming and mature secretory granules.
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