Subcellular localization of gonadotrophic hormones LH and FSH in frog adenohypophysis using double-staining immunocytochemistry

We applied double post-embedding immunocytochemical methods using specific antibodies against bullfrog (Rana catesbeiana) luteinizing hormone (LH) and follicle-stimulating hormone (FSH) with immunogold staining (5- and 20-nm particles) to determine the subcellular localization of both gonadotropins and to observe their immunostaining patterns in anterior pituitary of the frog Rana pipiens. Results showed that individual gonadotrophs may store either one or both gonadotropins in a given secretory granule and in large globules (lysosomes?). Most gonadotrophs (50-88%) contain both hormones; 12-50% contain only FSH, and only a few (0-7%) contain LH alone. Individual secretory granules, even in cells that contain both hormones, may contain only one or both gonadotropin molecules. Evaluation of the percentage of monohormonal and multihormonal secretory granules revealed that multihormonal secretory granules were the most numerous and that LH monohormonal secretory granules were the least numerous. These results indicate that cellular storage of gonadotropin in amphibian pituitary is similar to that described for mammals, where a single cell type containing both gonadotropins predominates. Variability in hormone content both of cells and of granules in all individuals is consistent with the hypothesis that frog pituitary possesses a single multipotential gonadotroph.     

Ultrastructural immunocytochemical localization of LH and FSH in the pituitary of the untreated male rat

Summary Rapid freeze-substitution fixation was employed in immunocytochemical studies on the localization of LH and FSH in the typical gonadotrophs of the anterior pituitary in the untreated male rat; a modification of a recently described ferritin antibody method (Inoue et al. 1982) was used in these studies. It was shown that rapid freeze-substitution fixation provides good preservation not only of the ultrastructure but also of the antigenicity. Both LH and FSH were clearly demonstrated in the same gonadotrophic cells, but the subcellular localization of these gonadotrophins differed: (i) LH was mainly located in small secretory granules, 250–300 nm in diameter; (ii) FSH was mainly present in large secretory granules, up to 500 nm in diameter. In the pituitary gland of the adult male rat, all gonadotrophs that react to antibodies against gonadotrophins are characterized by small and large secretory granules. Other types of cells of the anterior pituitary containing either small secretory granules or resembling corticotrophs with secretory granules assembled at cell periphery did not react to either anti-LH beta or anti-FSH beta serum.For light microscopy, the peroxidase antibody method was used. All of the gonadotrophin-positive cells contain both LH and FSH. None of the pituitary cells reacted to antibody against only one gonadotrophin. However, some cells are LH-rich while other cells are FSH-rich.     

Discrimination of LH,FSH, TSH and ACTH in dissociated porcine anterior pituitary cells by light and electron microscope immunocytochemistry

Summary Using the PAP unlabelled antibody method, LH, FSH, TSH and ACTH were localized at light microscope level in cultured cells dissociated from the porcine adenohypophysis. Antisera were shown to be specific for the subunits of the porcine glycoprotein hormones by radioimmunoassay and absorption studies. Using these antisera, it was found that LH and FSH were contained within the same cell, with TSH in a separate cell. When absorbed with LH, anti-porcine ACTH stained a separate distinct population of ACTH cells.Adjacent ultra-thin sections stained with anti-pLH and anti-pFSH, and examined at electron microscope level, showed that the ovoid, 150–400 nm secretory granules of the LH/FSH gonadotrophs contained both LH and FSH.The authors gratefully acknowledge the technical assistance of Carole Smith and Adrian Walsh     

Ultrastructural localization of gonadotrophic hormones in the porcine pituitary using the immunoperoxidase technique

Summary Using specific antibodies against subunits of porcine LH and FSH, the pituitary cells which produce these two gonadotrophins were identified in the porcine pituitary at the ultrastructural level using the immunoperoxidase technique. It was clearly shown that most of the gonadotrophic cells are responsible for the production of both FSH and LH. However, some cells, only positive for FSH or LH, indicate that the concentrations of FSH and LH vary from cell to cell. At the ultrastructural level, the FSH/LH cells contain one class of secretory granules strongly positive for both FSH and LH as well as large negatively stained dense bodies. These findings indicate that the one cell-one hormone concept may not apply to gonadotrophic hormones; a FSH/LH cell cannot be distinguished from a LH or a FSH cell without immunocytochemical identification of its hormonal content.Abbreviations p-LH porcine luteinizing hormone - p-LH porcine LH subunit - p-LH porcine LH subunit - p-FSH porcine follicule stimulating hormone - p-FSH porcine FSH subunit - b-TSH bovine thyrotropic hormone     

Ultrastructural localization of thyrotropin (TSH) in the porcine anterior pituitary

Summary Two different immunocytochemical techniques based on specific antibodies against -subunits of porcine, rat and bovine TSH were applied at the ultrastructural level to identify the TSH cells in the porcine anterior pituitary and to compare the subcellular localization of the hormone.The post-embedding method on serial ultrathin sections revealed the localization of TSH in the granules of a specific cell type, negative for the other hormones. TSH was found in polyhedral cells characterized (i) by their content of granules that were the smallest of all the cell types examined, and (ii) by their flattened or slightly dilated RER cisternae. The pre-embedding method applied to isolated cells permitted a good penetration of antisera and the maintenance of antigenicity in sites inaccessible to the post-embedding method. Thus, immunoreactivity of TSH was detected in the secretory granules, the cytoplasmic matrix and in portions of the rough endoplasmic reticulum, in association with some membranes and inside some saccular structures.     

Estrogen target cells in the pituitary of platyfish,Xiphophorus maculatus

Summary The distribution of estradiol-concentrating cells in the pituitary of the platyfish, Xiphophorus maculatus, is studied after the injection of ³H estradiol-17 by thaw-mount autoradiography. Autoradiograms prepared 2–8 h after the injection show nuclear concentration of radioactivity in certain cells of the proximal pars distalis, while no nuclear labeling is found in cells of the rostral pars distalis, pars intermedia and pars nervosa. Radioactively labeled cells are identified as gonadotropes by a combined technique of thaw-mount autoradiography and immunoperoxidase staining with antiserum to ovine LH. Approximately 80% of the immunoreactive LH cells show a concentration of radioactivity in their nuclei. These observations indicate that in teleosts, as in mammals, estradiol has a direct effect on pituitary gonadotropes.Supported by PHS grant NS09914. The authors are grateful to Dr. D.G. Humm for providing the platyfish     

Targeted ablation of pituitary gonadotropes in transgenic mice.

LH, FSH, and TSH are heterodimeric glycoprotein hormones composed of a common alpha-subunit and unique beta-subunits. The alpha-subunit is produced in two distinct specialized cell types of the pituitary gland: gonadotropes, which synthesize LH and FSH, and thyrotropes, which synthesize TSH. We have demonstrated that 313 base pairs of the bovine-alpha subunit promoter direct expression of diphtheria toxin A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotrope ablation. Lack of gonadotrope activity was verified by RIA and immunohistochemical staining for LH. The phenotype of these transgenic mice is nearly identical to mice homozygous for the spontaneous mutation, hpg, which is due to a deletion in the gene encoding GnRH. Thyrotrope function was judged normal based on overall growth of the animals, appearance of their thyroids, T4 levels measured by RIA, and immunohistochemical staining for TSH. The ablation of gonadotropes but not thyrotropes suggests that separate cis-acting elements are necessary for expression of the alpha-subunit gene in these two cell types. Pituitary content of ACTH and GH was apparently normal, while PRL synthesis and storage were reduced. Thus, in a pituitary almost completely devoid of gonadotropes, most other pituitary functions were normal. This suggests that most pituitary cells are able to differentiate independently of terminal gonadotrope differentiation and can function in the absence of paracrine signaling provided by gonadotropes.     

Evidence for FSH-like material in ACTH granules of certain corticotropic cells in the pituitary of the pig

Summary The ultrastructural immunocytochemical peroxidase-antiperoxidase technique used on serial sections reveals that certain corticotropic cells contain both ACTH and FSH, but not LH (ACTH/FSH cells). To determine the specificity of the anti-FSH staining in these cells, immunocytochemical absorption experiments were performed. The results indicate that (1) anti-FSH and -ACTH antisera are bound to different antigens in the corticotropic cell, and (2) anti-FSH staining is specific for a FSH-like antigen. In the ACTH/FSH cells both hormones are stored in the same secretory granules, distributed among other granules that contain only ACTH.     

Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

Immunohistochemical detection of adenohypophyseal cells containing hormones in Rana ridibunda

Summary The pars distalis of the pituitary of Rana ridibunda captured throughout the spring and summer was examined with immunofluorescence techniques using antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, four different cell types are recognized: 1) cells immunoreactive to anti-bovine LTH, 2) cells immunoreactive to anti-bovine STH, 3) cells immunoreactive to anti-ovine LH, and 4) cells immunoreactive to anti-synthetic ACTH (1–24). Their distribution and morphology as well as their staining characteristics (classical histological techniques) are reported in this study.     

Immunocytochemistry of the pituitary glycoprotein hormones.

The storage sites of the pituitary glycoprotein hormones were identified with the use of electron microscopic immunocytochemical techniques and antisera to the beta (beta) chains of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH). The TSH cells in normal rats is ovoid or angular and contains small granules 60-160 nm in diameter. In TSH cells hypertrophied 45 days after thyroidectomy, staining is in globular patches in granules or diffusely distributed in the expanded profiles of dilated rough endoplasmic reticulum. The gonadotrophs (FSH and LH cells) exhibited three different morphologies. Type I cells are ovoid with a population of large granules and a population of small granules. Staining for FSHbeta or LHbeta was intense and specific only in the large granules (diameter of 400 nm or greater). Type II cells are angular or stellate and contain numerous secretory granules averaging 200-220 nm in diameter. They predominate during stages in the estrous cycle when FSH or LH secretion is high. Type III cells look like adrenocorticotropin (ACTH) cells in that they are stellate with peripherally arranged granules. They generally stain only with anti-FSHbeta and their staining can not be abolished by the addition of 100 ng ACTH. In preliminary quantitative studies of cycling females, we found that on serial sections FSH cells and LH cells show similar shifts to a more angular population of cells during stages of active secretion. However, the shifts are not in phase with one another. Furthermore, there are at least 1.5 times more FSH cells than LH cells at all stages of the cycle. Our collection of serial cells shows that some cells (usually type I or II) stain for both gonadotropic hormones, whereas others (usually type II or III) contain only one.     

Ultrastructural localization of gonadotropin-releasing hormone in the porcine gonadotropic cells

Summary The comparative ultrastructural localization of LH, FSH and GnRH clearly shows that the granules of the FSH/LH cells contain all three hormones. The separate storage of LH and FSH in a significant number of cells, which in the same granules also display GnRH, may suggest that LH-RH is also FSH-RH and may help to explain the non-parallel release of LH and FSH under some functional conditions.     

Effects of estradiol and calcium on gonadotrophic cells in middle-aged female rats

The effects of multiple treatment with estradiol dipropionate (EDP) or calcium glucoheptonate (Ca) or a combination of the two on gonadotrophic cells in the pituitary pars distalis of middle-aged female rats were examined. The animals were treated daily for two weeks with EDP (0.625mg i.p./kg body weight) or Ca (11.4mg/kg body weight) or EDP+Ca. Luteinising (LH) and follicle stimulating hormone (FSH)-producing cells were examined by immunohistochemistry using antisera to the specific (beta) -subunits of LH and FSH and a peroxidase–anti-peroxidase immunohistochemical procedure. Plasma levels of FSH and LH were measured by radio-immune assay. A stereological method for determining morphometric parameters in immunopositive FSH and LH cells was used. The number of gonadotrophs per unit area (mm²), their cellular volume and relative volume densities, as well as plasma levels of FSH and LH, were decreased in all treated females in comparison with the controls. The most significant decrease of these parameters was observed in EDP-treated animals. Such changes were also expressed in Ca-treated animals, but the alterations were less distinct. These results demonstrate that multiple EDP or Ca application to middle-aged female rats is able to inhibit, directly or indirectly, the morphofunctional state of gonadotrophic cells in the pituitary pars distalis.     

A whole range of fine structural criteria for immunohistochemically identified LH cells in rats

Summary Pituitaries from normal, young and adult male rats were fixed either in sublimate-formalin or in glutaraldehyde-osmium. In adjacent Paraplast sections, almost all the gonadotrophs were immunostained with both LH and FSH antisera. The rat LH and FSH antisera used were shown to be highly specific by the absorption test and by double antibody radioimmunoassay. Thin and thick adjacent Epon sections were prepared for EM and immunohistochemical examination. Cells stained with the rat LH antiserum were identified by LM, and then observed in detail by EM. On the basis of these observations we suggest that the LH cells are arranged in a sequence of basophils, i.e., Types II/III, III, III/IV and IV: Type II/III basophils are elongate with a cytoplasmic process and less vesiculated. They have morphological features of Type II (classical thyrotrophs) and also of Type III basophils. Type III basophils are oval in shape and moderately vesiculated. Both Types II/III and III basophils can be divided into two classes of cell characterized mainly by the existence of only small secretory granules (150–220 nm in diameter) (Type A) or by the coexistence of small and large (350–500 nm) (Type B). Type III/IV basophils are cells intermediate between types III and IV basophils, and moderately vesiculated with an abundance of secretory granules (150–300 nm in diameter). Type IV basophils are large, spherical or oval cells whose RER cisternae are conspicuously dilated; they contain less numerous secretory granules (150–300 nm in diameter). It is concluded that LH cells are not a single cell type, but include a wide range of subtypes.     

RCA I-binding patterns of the Golgi apparatus

The distribution in the Golgi apparatus of binding sites for the galactose-specific Ricinus communis I lectin (RCA I) was studied in differently specialized cells, including goblet cells and absorptive enterocytes of the rat small intestine as well as acinar cells of the rat embryonic pancreas and submandibular gland. For the purpose of localizing the binding reactions, a pre-embedment method using horseradish peroxidase for electron microscopic visualization, and a post-embedding technique making use of the colloidal gold system were employed. The reactions obtained, localizing cell constituents which contain saccharides with terminal or internal beta-D-galactosyl residues, labeled diverse Golgi subcompartments. The goblet cells showed intense RCA I staining of the cisternae of the trans side of the Golgi stacks. The reaction was weak in the medial cisternae and the cis side of the stacks mostly was devoid of label. In the absorptive cells, in addition to the RCA I reaction of trans Golgi elements, binding sites for this lectin were concentrated in the stacks' medial section. In the embryonic acinar cells, accessible galactosyl residues were either confined to the trans and/or medial cisternae, or distributed across elements of all the stacked saccules. In the latter stacks, the reactions mostly were weak in the cis cisternae and increased in intensity towards the trans side. As regards the respective labeling patterns, similar percentages were calculated for the early and late stages of development: they were approximately 62% for the pattern which showed RCA I label limited to trans/medial cisternae and approximately 38% for the "cis-to-trans"-distributed RCA I reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-existence of gonadotrophins (FSH,LH) and thyrotrophin (TSH) in single anterior pituitary cells of the musk shrew,Suncus murinus

According to recent immunocytochemical studies of anterior pituitary cells, it is obvious that the one cell-one hormone theory must be modified. Many pituitary morphologists have demonstrated that there are some cells that contain two hormones. In this study, we demonstrate by means of immuno-electronmicroscopy the co-existence of gonadotrophins (FSH and LH) and thyrotrophin (TSH) in the same anterior pituitary cells of the musk shrew. These cells were remarkably altered in their ultrastructural features by either gonadectomy or thyroidectomy. Double labeling for gonadotrophins and thyrotrophin was present not only in the same cells but also in the same secretory granules. Our ability to demonstrate co-existence of gonadotrophins and thyrotrophin in the same cell may be due to our selection of fixative and embedding media for electron-microscopic immunocytochemistry. Our conclusion that gonadotrophins and thyrotrophin are produced in a single cell type of the anterior pituitary gland in the musk shrew, i.e., thyrogonadotrophs, suggests the need to consider a modification of the classic scheme for classification of anterior pituitary cells.     

Morphology of the mammotrophs and gonadotrophs in the anterior pituitary gland of rats with protein-calorie malnutrition

The structure of the anterior pituitary gland of rats with protein-calorie malnutrition was studied at the light and electron microscopic levels. Male Sprague-Dawley rats were fed a low-protein diet from 20 to 50 days of age. The hypophyses from these animals, along with those from age-matched controls, were then removed and fixed for light microscopic immunocytochemical analyses, using anti-rat PRL or anti-ovine LH beta serum, or for routine ultrastructural study. The overall number of mammotrophs and gonadotrophs appeared to be unaffected by the deficiency in dietary protein. However, the nuclei, in both cell types, were significantly smaller in the malnourished rats. Cytoplasmic volume was also apparently less than in control rats, leading to the "crowding" of adjacent cell types. Additionally, in the malnourished rats, the mammotrophs assumed a less polygonal profile, while the gonadotrophs, which were more irregularly shaped in the central zone, rarely displayed the negative image of a Golgi complex. Ultrastructurally, the mammotrophs in the experimental rats had a less extensively developed granular endoplasmic reticulum and Golgi complex. The number of secretary granules was not different; however, their size was markedly less and their shape was round to ovoid, rather than pleomorphic. Two types of gonadotrophs were distinguished, based on the classification of Kurosumi et al. ('76). The type-I cell (former "FSH" cell) had a smaller Golgi complex, less vesicular endoplasmic reticulum and smaller secretory granules. The major alteration in the type-II gonadotroph (former "LH" cell) was the smaller size of the secretory granules. The morphological changes found in this study support our previous physiological observations (Herbert, '80), which demonstrate that prolactin and gonadotrophin secretion are severely impaired in rats suffering from protein-calorie malnutrition.     

Localization of gonadotrophic hormones in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH¹, selective immunochemical staining was localized mostly in the same cell type in the pars distalis and pars tuberalis of the dog pituitary gland. However, some cells were consistently shown to react solely with antisera to either LH or FSH. The cells stained for FSH were at least 1.5 times less numerous than those shown to contain LH. In the pars distalis of adult male dogs the immunoreactive gonadotrophs varied greatly in their relative proportion and were mostly shown to be much less numerous than in bitches in the anestrus phase of the sexual cycle. These cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. The performic acid-alcian blue (pH 0.2)-PAS-orange G procedure stained the FSH/LH cells blue or turquoise, demonstrating TSH cells (blue-purple), ACTH/MSH cells (red-purple) and PRL cells (orange-red). The FSH/LH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not apply to gonadotrophic hormones, although some cells seem to be the source of either FSH or LH.Abbreviations for Pituitary Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Dr. H. Wiemann for the statistical evaluation and to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistanceRecipient of a Research Scholarship from the Arabic Republic of Egypt     

Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures

Intact anterior pituitary tissue and primary anterior pituitary cultures were stained with 1:30,000 anti-TRH and 1:10,000 anti-GnRH using the peroxidase antiperoxidase immunocytochemical technique. Stains applied to serial ultrathin sections of intact pituitaries showed that TRH immunoreactivity could be localized in secretory granules of thyrotropes, gonadotropes and corticotropes whereas GnRH immunoreactivity was found only in gonadotropes and corticotropes. Long-term primary pituitary cultures were studied to remove the anterior pituitary cells from hypothalamic influences. In these cell populations both TRH and GnRH immunoreactivity persisted. In addition, quantification of the stained cells at the light microscopic level demonstrated that the volume fraction of TRH and GnRH immunoreactive cells remained constant up to 3 weeks of culture. Studies of serial ultrathin sections through cells from these cultures showed TRH or GnRH localized in secretory granules of cells that contained LH and ACTH, but not TSH. Both liquid and solid phase immunoabsorption specificity controls were used to validate the immunocytochemical stains. These studies suggest that the pituitary TRH and GnRH immunoreactivities may not be completely of hypothalamic origin, but may also be endogenous to a subpopulation of unique multihormonal pituitary cells.     

Collagen formation — observations on its intracellular packaging and transport

Summary Odontoblasts, osteoblasts and fibroblasts of young rats were examined in the electron microscope after staining thin sections either with lead citrate alone or with uranyl acetate prior to lead citrate.With lead citrate alone, collagen fibrils in the extracellular matrix stand out as lucent structures against a moderately electron dense background. Within the cells, lucency is restricted to certain dilated portions of the Golgi saccules as well as to the secretory granules located nearby and in the secretory pole of the cells. The lucency present in these compartments may be attributed to fibrils that are similar to the lucent collagen fibrils in the extracellular matrix. Other cellular compartments, e.g. the rough ER, do not display lucency.When preparations are stained with uranyl acetate prior to lead citrate, lucency is observed neither in the matrix nor in the cells. In the matrix, collagen fibrils are easily identifiable by their cross banded pattern. In the odontoblasts, dilated portions of Golgi saccules between the outer and inner face contain filaments aligned in parallel that are approximately 3 000 Å in length. In saccules on the inner face filament aggregates are present, some of them exhibiting a cross banding pattern. In secretory granules, however, the contents appear rather homogeneous.It is suggested that filament aggregates of collagen can assemble in the Golgi apparatus from filamentous units. These are transported through the cell by way of secretion granules and are discharged to the extracellular matrix by exocytosis.This investigation was supported by grants of the Medical Research Council of Canada. The author wishes to express appreciation to Dr. C. P. Leblond for his guidance in the course of this work.