Immunohistochemical, electron microscopic and morphometric studies of estrogen-induced rat prolactinomas after bromocriptine treatment

To clarify the effects of bromocriptine on prolactinoma cells in vivo, immunohistochemical, ultrastructural and morphometrical analyses were applied to estrogen-induced rat prolactinoma cells 1 h and 6 h after injection of bromocriptine (3 mg/kg of body weight). One h after treatment, serum prolactin levels decreased markedly. Electron microscopy disclosed many secretory granules, slightly distorted rough endoplasmic reticulum, and partially dilated Golgi cisternae in the prolactinoma cells. Morphometric analysis revealed that the volume density of secretory granules increased, while the volume density of cytoplasmic microtubules decreased. These findings suggest that lowered serum prolactin levels in the early phase of bromocriptine treatment may result from an impaired secretion of prolactin due to decreasing numbers of cytoplasmic microtubules. At 6 h after injection, serum prolactin levels were still considerably lower than in controls. The prolactinoma cells at this time were well granulated, with vesiculated rough endoplasmic reticulum and markedly dilated Golgi cisternae. Electron microscopical immunohistochemistry revealed positive reaction products noted on the secretory granules, Golgi cisternae, and endoplasmic reticulum of the untreated rat prolactinoma cells. However, only secretory granules showed the positive reaction products for prolactin 6 h after bromocriptine treatment of the adenoma cells. An increase in the volume density of secretory granules and a decrease in the volume densities of rough endoplasmic reticulum and microtubules was determined by morphometric analysis, suggesting that bromocriptine inhibits protein synthesis as well as bringing about a disturbance of the prolactin secretion.     

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.     

Radioautographic analysis of the secretory pathway for glycoproteins in principal cells of the mouse epididymis exposed to [3H] fucose

The secretory process for glycoproteins in principal cells of the mouse caput epididymis was studied by electron microscope radioautography at intervals after exposure to [3H] fucose in vitro. The large Golgi apparatus showed very heavy labeling at the initial interval, followed by a steady decline in percent of grains and relative grain concentrations. Conversely, the epididymal lumen and the apical cell surface began low and increased in radioactivity at the 30-min interval. The extensive sparsely granulated endoplasmic reticulum showed modest increases in percent of grains and relative grain concentrations 30 min after administration of the percursor. Subdivision of the sparsely granulated reticulum into "intermediate" profiles (some ribosomes attached to the membranes) and "smooth" profiles (lacking ribosomes) showed that this increase was due to silver grains assigned to the smooth portions. After the initial interval, high relative grain concentrations were calculated for vesicles. The results indicate that glycosylation of epididymal secretory glycoproteins occurs in the Golgi apparatus, which is, therefore, not bypassed as its morphological features had suggested. The kinetics of the secretory process in the principal cells includes 15 to 30 min for synthesis of the polypeptide parts of secretory products and addition of sugars in the Golgi apparatus, and a similar time for subsequent release from the Golgi apparatus, transport to the apical end of the cell and discharge to the lumen. Ribosome-studded (intermediate) portions of the sparsely granulated endoplasmic reticulum are probably involved in synthesis of polypeptide parts of secretory products, while vesicles or smooth portions of the sparsely granulated reticulum may play a role in intracellular transport of glycoproteins.     

Effect of chronic suppression of growth hormone secretion, by SMS 201-995 on lactation in mice

The effect of chronic suppression of growth hormone (GH) secretion by SMS 201-995 on lactation was studied in primiparous C3H/He mice. Mammary gland DNA content on day 12 of lactation was significantly lower in SMS 201-995 treated mice than in the control. There were little differences between groups in mammary gland RNA content and litter growth on day 12 of lactation. That was associated with a slightly higher RNA/DNA ratio and a significant increase in food intake during lactation. These results indicate that inhibited mammary gland growth by GH suppression has little effect on lactation. The smaller mammary gland can compensate by increasing its secretory activity.     

SMS 201-995 and thyroid function in acromegaly: acute, intermediate and long-term effects.

The acute (TRH-stimulation test), intermediate (0-6 days administration), and long-term (0-30 months administration) effects of SMS 201-995 (octreotide) treatment on thyroid function were studied. Subcutaneous injection of 100 micrograms SMS 201-995 one hour before 200 micrograms TRH intravenously reduced serum TSH response area by more than 50% in 8 healthy volunteers. After 3 days of continuous subcutaneous infusion (CSI) of SMS 201-995 in 9 acromegalic patients (100 micrograms/24 h) a slight but significant decrease in serum total triiodothyronine (TT3) and a concomitant increase in serum TSH were demonstrated, indicating an initial inhibitory effect on peripheral deiodination of thyroxine. After a further 3 days treatment serum T3 and TSH had returned to prevalues. Six of the nine acromegalics were treated with SMS 201-995 (100-1500 micrograms/24 h) and admitted for diurnal hormone profiles on 13 occasions over 30 months. Apart from a barely significant increase in serum TSH, no changes in thyroid function were noted. The study was especially designed to detect minute changes over time in thyroid hormones. The only long-term effect of SMS 201-995 was the barely significant clinically irrelevant increase in serum TSH, possibly caused by a slight inhibition of peripheral deiodination of thyroxine.     

Unexpected growth-stimulatory effect of somatostatin analogue on cultured human pancreatic carcinoid cells.

Therapeutic efficacy of a synthetic somatostatin analogue for the treatment of carcinoid tumors is still controversial. In vivo studies performed in our laboratory showed that a somatostatin analogue, SMS 201-995, significantly inhibited growth of human pancreatic carcinoid (BON) tumors xenotransplanted into athymic nude mice. In the present study, however, SMS 201-995 did not inhibit in vitro growth of BON cells, but rather SMS 201-995 stimulated growth in a dose-dependent fashion. The growth-stimulatory effect was likely mediated through the reduction of cyclic AMP production. Unsuccessful treatment of certain types of carcinoid tumor with SMS 201-995 may be partly due to the direct growth-stimulatory effect of SMS 201-995 on carcinoid cells.     

Morphogenesis and significance of fibrous bodies in human pituitary adenomas

In the course of light and electron microscopic studies of 142 surgically-removed human pituitary adenomas, 28 tumors were found containing fibrous bodies composed of type II microfilaments with an average width of 115A. These spherical structures, measuring up to 4-5 micrometer occur exclusively in sparsely granulated growth hormone cells and acidophil stem cells, but as revealed by the immunoperoxidase technique, contain no growth hormone. Fibrous bodies are located in the Golgi region and are consistently associated with Golgi membranes and smooth-surfaced endoplasmic reticulum. Their association with centrioles is thought to be anatomical rather than functional. Several adenoma cells possess spherical formations composed entirely of smooth-walled membranes or transitional forms between smooth tubules and type II microfilaments, suggesting that smooth membranes may play a key role in the production of fibrillar substance. Fibrous bodies appear to be reliable morphologic markers and are valuable in the differential diagnosis of pituitary adenomas.     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

Improvement of diabetes after treatment with somatostatin analogue SMS 201-995 in an acromegalic patient

The beneficial effects of long acting somatostatin analogue SMS 201-995 in an acromegalic patient affected by severe diabetes mellitus are reported. Neither human insulin alone nor human insulin plus bromocriptine allowed satisfactory metabolic control though, with the latter treatment, virtually normal plasma GH levels were reached. Conversely, addition of SMS 201-995 to insulin treatment led to normalization of blood glucose. This result was obtained with a dose of SMS 201-995 of 400 micrograms/day and only after 3 weeks of therapy.     

Somatostatin analogue (SMS 201-995) decreases plasma levels of corticotropin (ACTH) and corticotropin-releasing hormone in a patient with ectopic ACTH-producing tumors

Effects of long-acting somatostain analogue (SMS 201-995) on plasma corticotropin (ACTH) and corticotropin-releasing hormone (CRH) levels were studied in a patient (63-year-old woman) with ectopic ACTH-producing tumors associated with type I multiple endocrine neoplasia (MEN-I). The patient had undergone bilateral adrenalectomy. Plasma CRH, as well as plasma ACTH, beta-endorphin and alpha-MSH, increased. The hormone levels were dramatically decreased by acute administration of SMS 201-995. Moderately higher doses of dexamethasone (0.05 or 0.1 mg/kg a day) did not decrease plasma CRH or ACTH. An extremely high dose of dexamethasone (0.2 mg/kg a day), however, decreased plasma ACTH, but failed to decrease plasma CRH. Acute administration of SMS 201-995 further lowered the level of plasma ACTH even in this condition. In addition to the decrease in ACTH, SMS 201-995 decreased plasma CRH. Chronic administration of SMS 201-995 continuously decreased plasma CRH, ACTH and beta-endorphin. The decrease in these hormone concentrations accompanied the disappearance of hyperpigmentation. These results suggested that SMS 201-995 inhibits hypersecretion not only of ACTH but also of CRH, and that the agent is therapeutically useful in normalizing the hypersecretion of these hormones.     

Cell density and growth-dependent down-regulation of both intracellular calcium responses to agonist stimuli and expression of smooth-surfaced endoplasmic reticulum in MC3T3-E1 osteoblast-like cells

A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.     

Somatostatin analog treatment of acromegaly: new aspects

Ten acromegalics received daily doses of 200-300 micrograms of a long-acting somatostatin analog, SMS 201-995 (Sandostatin, SMS), for an average of 64 weeks. Basal mean GH values of 44 +/- (SE) 7.8 ng/ml had fallen into the normal range at the end of the observation period (mean 64 weeks). This effect was accompanied by a substantial drop in somatomedin-C values. Reduction of pituitary tumor size could be documented in 3 of 6 patients. Whereas SMS did not affect high plasma PRL in 4 microprolactinoma patients, lactotrophs turned sensitive to this agent in mixed GH/PRL tumors. In a comparative study between SMS and bromocriptine, the former normalized circulating GH in 10 of 17 acromegalics in an acute trial, whereas bromocriptine was effective in only 5. A combination of both substances was effective in 2 of 3 patients who were insensitive to single drug administration. Cultures of GH-secreting tumor cells showed a statistically significant hormone decrease in the medium when exposed to SMS. However, in some instances, a diminution of the GH contents of the tumor cells was also observed, presumably as the basis for intracellular breakdown and clinical tumor shrinkage.     

Annulate lamellae in adenomas of human pituitary glands.

Twelve nontumorous adenohypophyses and 36 various pituitary adenomas, removed by surgery, have been investigated by electron microscopy in order to shed some light on annulate lamellae, primarily on their ultrastructural features, incidence, origin, fate and functional significance. No annulate lamellae were found in the nontumorous adenohypophyses and in 33 pituitary adenomas. They were, however, detected in two adenomas consisting of undifferentiated cells and one adenoma composed of sparsely granulated prolactin cells indicating that these unique membrane configurations cannot be regarded as an exceedingly rare finding and, furthermore, that they may be disclosed not only in undifferentiated but occasionally in highly differentiated cells. Annulate lamellae may arise from endoplasmic reticulum and/or nuclear envelope and consist of arrays of smooth walled double membrane sheets exhibiting regularly spaced interruptions as well as continuities with the endoplasmic reticulum. No relationship was established between annulate lamellae and adenohypophysial secretory activity. Our findings seem to be consistent with the view that annulate lamellae are present in those cells which have the tendency to proliferate.     

Growth hormone and prolactin immunoreactivity in the pituitary gland of postnatal little (lit) mice

Homozygous little (lit/lit) mutant mice exhibit a growth lag which is manifested at approximately two weeks postnatally. Functional aspects of the development of pituitary growth hormone (GH) cells and prolactin (PRL) cells were thus analyzed by means of colloidal gold immunocytochemistry at the ultrastructural level in lit/lit mice and their normal counterparts ranging in age from 5 days postnatally to adulthood. In the adult normal and lit/lit pituitaries, secretory granules in GH cells and PRL cells showed a positive immunoreaction to their respective antisera, as did granules in both cell-types at 5 days postnatally. By 14 days some GH cells in lit/lit pituitaries appeared to be less densely populated with granules than GH cells in normal pituitaries, but a positive immunoreaction continued to occur even in sparsely granulated GH cells. PRL cells showed ultrastructural features in lit/lit pituitaries which were similar to those in normal mice, and immunoreactivity was present at all stages examined. The results indicate that since differences in granule reactivity were not evident between lit/lit and normal GH cells, despite ultrastructural morphologic differences which were present by 14 days postnatally, manifestations of the defect in lit/lit may be primarily quantitative in terms of numbers of granules and/or numbers of GH cells. With respect to PRL cells, neither morphologic nor functional aberrations could be observed; thus, a deficit in PRL hormone production might be the result of a more subtle defect than that in GH cells.     

An ultrastructural study of the clitellar epithelium of the earthworm Metaphire posthuma (vail.)

The ultrastructure of clitellar epithelium of Metuphire posthuma revealed mainly three types of secretory cells. Most prominent among these are the large slender granular cells which contain a large number of secretory granules filling in the entire columncr region of the cell. The secretory granules are 2-4mu in diameter with a limiting membrane and containing numerous tiny vesicles in a matrix of varying electron density. Basolateral rough endoplasmic reticulum and extensive Golgi cisternae were seen interspersed with the secretory granules. The Golgi cisternae in these cells were quite prominent extending all around the secretory granules. The secretory granules of type 2 cells are spheroid bodies with motley appearance due to varying electron density of the matrix. The immature granules contain fibrillar material. Type 3 cells contained electron lucent membrane-bound mucous like secretory granules which are reticulated with filamentous materials. All the three cell types open to the exterior at the cuticular region which is characterised by the presence of numerous microvilli.     

Characterization of human pituitary adenomas in cell cultures by light and electron microscopic morphology and immunolabeling

The morphology and hormone production of pituitary adenoma cell cultures were compared in order to highlight their characteristic in vitro features. Cell suspensions were prepared from 494 surgical specimens. The 319 viable monolayer cultures were analyzed in detail by light microscopy and immunocytochemistry within two weeks of cultivation. Some cultures were further characterized by scanning, transmission and immunogold electron microscopy. The viability and detailed in vitro morphology of adenoma cells were found to be characteristic for the various types of pituitary tumors. The sparsely granulated growth hormone, the corticotroph and the acidophil stem cell adenomas provided the highest ratio of viable cultures. Occasionally, prolonged maintenance of cells resulted in long-term cultures. Furthermore, a variety of particular distributions of different hormone-containing granules were found in several cases. Both light microscopic and ultrastructural analyses proved that the primary cultures of adenoma cells retain their physiological features during in vitro cultivations. Our in vitro findings correlated with the routine histopathological examination. These results prove that monolayer cultures of pituitary adenoma cells can contribute to the correct diagnosis and are valid model systems for various oncological and neuroendocrinological studies.     

Mutant vasopressin precursor in the endoplasmic reticulum of the Brattleboro rat. Ultrastructural evidence from individual “vasopressin” cells localized with the light microscope by use of a new gold/silver method for immunostain enhancement

Summary This ultrastructural study demonstrates that the vasopressin immunoreactivity found in the occasional, densely stained cells in the hypothalamus of the homozygous Brattleboro rat is localized in the rough endoplasmic reticulum. 50-m Vibratome sections were stained with anti-vasopressin serum by use of a peroxidase method with 3,3-diaminobenzidine as chromogen. The diaminobenzidine end-product has a specific capability to bind gold particles from a chloroauric acid solution and the bound gold was used to precipitate silver grains from a silver developer. The stained sections were flat embedded in resin and ultrathin sections were cut of areas containing the immuno-identified occasional cells. In these densely stained, vasopressin-immunoreactive cells of homozygous Brattleboro rats the rough endoplasmic reticulum was dilated. The lumen of the reticulum contained both end-products of diaminobenzidine and gold/silver grains, but some parts of the reticulum appeared unstained. No other cell organelles were immunostained and no secretory granules were found. In control rats, gold/silver deposits were found throughout the cytoplasm of vasopressin-immunoreactive cells. In these immunostained cells secretory granules were seen.     

Plasma alpha-subunit levels during the treatment of pituitary adenomas with the somatostatin analog (SMS 201-995)

The effect os SMS 201-995 (Sandostatin), a long-acting somatostatin analog, on different types of pituitary adenomas including alpha-subunit elevation is illustrated in this report. Treatment induced a fall in hCG levels in a woman with a pituitary adenoma producing only alpha-subunit. In 3 acromegalic patients, there was only a partial drop in GH and alpha-hCG. The same effect was observed in a woman with menopausal FSH and LH levels. SMS reduced plasma TSH and alpha-hCG in a case of thyrotropic adenoma. Two patients exhibiting FSH- and alpha-hCG-secreting adenomas did not respond to acute administration of SMS 201-995. More patients have to be treated before a definitive statement can be made on the usefulness of somatostatin analogs in the management of different types of pituitary adenomas.     

Effects of thyroidectomy, propylthiouracil, and thyroxine on pituitary content and immunocytochemical staining of thyrotropin (TSH) and thyrotropin releasing hormone (TRH)

Previous studies have demonstrated immunocytochemical staining for beta chains of thyroid stimulating hormone (TSH-beta) in rough endoplasmic reticulum of pituitary cells hypertrophied after thyroidectomy ("thyroidectomy cells") (Moriarty CG(1976): J Histochem Cytochem (24:846; Moriarty GC, Tobin RB (1976): J Histochem Cytochem 24:1140). Here we report the localization of thyrotropin releasing hormone (TRH) in serial sections of the same pituitaries to determine if it could be found at similar sites. No staining for TRH was found in hypertrophied TSH cells formed 42 days after the surgery, or after 14, 34, and 70 days of propylthiouracil (PTU) treatment. The loss in immunostaining in the PTU-treated rats was correlated with radioimmunoassay (RIA) measurements that showed a 65% reduction in anterior pituitary TRH content after 34, 70, and 98 days of PTU treatment (from 22.9--7.8 pg/mg wet wt) and a 50% reduction in TSH content after 34 days of treatment. When thyroxine was administered to hypothyroid rats for 3 days before death, our previous studies had demonstrated intense staining for TSH in granules inside the rough endoplasmic reticulum. In this study, the radioimmunoassay showed that TSH content rose dramatically in the hypothyroid animals treated with PTU for 77 days and thyroxine for 2 days before death (from 8.5--64.1 mU/mg wet wt); however, the rise in TRH content was minimal (5.8--9.8 pg/mg wet wt). The immunocytochemical stain for TRH correlated well with the RIA showing a weak reaction mainly on small granules in the cytoplasm. No reaction for TRH was found in rough endoplasmic reticulum. These results suggest that TRH and TSH storage sites are dissimilar in the hypothyroid rat. The presence of stain for TRH in granules in the cytoplasm suggests that it might play a role in the storage or packaging of TSH. Its absence in profiles of rough endoplasmic reticulum staining intensely for TSH suggests that it is not synthesized at this site. No definite conclusions about its origin can be drawn at this time.     

Stereological study of gonadotropes in the frog,Rana pipiens,after GnRH stimulation in vitro

Summary Previous physiological results have indicated the existence of two releasable pools of gonadotropins in amphibian pituitaries: an acute releasable pool that appears independent of protein synthesis, and a storage pool involved in chronic release that depends on protein synthesis. To elucidate the ultrastructural localization of these pools and the morphological changes induced in gonadotrope cells after treatment with gonadotropin-releasing hormone, we carried out a morphometric study of immuno-identified gonadotrope cells using an in vitro superfusion system. Treatment with gonadotropin-releasing hormone induced a degranulation of small (110–255 nm) and medium (236–360 nm) secretory granules as well as hypertrophy of the endoplasmic reticulum and Golgi complex. Simultaneous incubation with gonadotropin-releasing hormone and cycloheximide inhibited the release of secretory granules although the endoplasmic reticulum and Golgi complex were hypertrophied. These morphological results strongly suggest: (1) that gonadotropin-releasing hormone induces degranulation and hypertrophy of the biosynthetic machinery in gonadotrope cells; and (2) that the activation of the endoplasmic reticulum and Golgi complex by stimulation with gonadotropin-releasing hormone is independent of protein synthesis, while the release of secretory granules is protein synthesis-dependent. In addition, the second or storage pool of gonadotropin is associated mainly with the small and medium secretory granules.