Tissue culture studies on human pituitary tumours: radioimmunoassayable anterior pituitary hormones in the culture medium.
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Adenoma, Chromophobe↗
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Anterior pituitary function before and after trans-sphenoidal microsurgical resection of pituitary tumors.
Anterior pituitary lobe function was measured pre- and postoperatively in 40 patients with pituitary tumors who were managed surgically by the trans-sphenoidal approach. Of 23 patients with normal anterior lobe function preoperatively, 21 (91%) were normal postoperatively. Of 17 preoperatively impaired patients, 7 (40%) regained normal function, 3 (18%) improved, 4 (24%) remained the same, and 3 (18%) were further impaired by the surgery. In this small series, the likelihood of recovery of pituitary function varied inversely with the degree of preoperative impairment and the size of the tumor, suggesting that more aggressive surgical management of small, minimally symptomatic pituitary tumors is justified.
Adenoma↗
Tissue culture studies on human pituitary tumours: long term release of anterior pituitary hormones into the culture medium.
A study was undertaken to determine the length of time that human pituitary tumours are capable of releasing anterior pituitary polypeptide hormones in vitro under basal conditions and to study the spectrum of hormone release by functioning and "non-functioning" pituitary neoplasms. Fragments from the pituitary tumours of 10 patients in the following categories: 1 Cushing's disease, 2 with amenorrhoea-galactorrhoea, 3 with acromegaly, and 4 with "non-functioning" pituitary tumours and from 2 normal human anterior pituitary glands were placed in primary culture immediately after surgery. The in vitro release of human growth hormone (hGH), prolactin (Prl), thyrotrophin (TSH), adrenocorticotrophin (ACTH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) was measured by specific radioimmunoassays at the end of each week in culture. Hormone release was surveyed from 6 weeks to 6 months depending upon the survival of the culture. Hormone release patterns were compared with clinical and pathological data. In the initial week of the study, all 6 anterior pituitary polypeptides were detected in the media from the 2 control pituitaries and from 4 of the tumours (1 amenorrhoea-galactorrhoea and 3 acromegaly) in concentrations up to 100 ng/ml of medium while 5 of the 6 hormones were readily detectable in the media from 2 additional tumour samples (Cushing's disease and 1 "non-functioning" pituitary tumour). The media of the remaining 4 tumours contained at least 3 of the 6 hormones (1 amenorrhoea-galactorrhoea and 3 "non-functioning" pituitary tumours). After 6 months in culture, the 6 hormones were readily detectable in at least 1 of the 5 surviving cultures and hGH (up to 800 ng/ml) and LH were each detectable in the media from 2 cultures. Although most of the hormone concentrations in the media decreased with length of time in culture, there were 2 exceptions. First in the media from 5 of the 12 cultures from both controls and tumours, Prl concentrations increased after 50 to 80 days culture. This increase usually lasted for several weeks before Prl levels again began to decline. The second unusual finding occurred in a tumour from a patient with acromegaly in the media of which hGH levels rose from 60 ng/ml to 800 ng/ml between days 125 and 174. These findings of prolonged hormone release in vitro give promise of future usefulness of tissue culture methods for study of polypeptide hormone releasing mechanisms and long-term production of human anterior pituitary hormones for use in research and possible therapy.
Acromegaly↗
[Specific role of sex steroids on the pituitary level. Sex steroids and anterior pituitary secretion].
Although sex steroids were known to play a role in the control of LH, FSH, TSH and prolactin secretion, in vivo experiments could not discriminate between hypothalamic and pituitary sites of action. In this study, the specific action of sex steroids at the anterior pituitary level could be achieved using rat adenohypophyseal cells in primary culture. While estrogens stimulated the sensitivity of the LH and FSH responses to LHRH, androgens had differential effects on the secretion of the two gonadotropins: marked inhibition of LH and stimulation of FSH secretion. Progesterone, on the other hand, while having no effect in the absence of estrogens, could reverse the stimulatory effect of estrogens on LH release while it led to a stimulation of FSH secretion. Estrogens and thyroid hormone exert respective stimulatory and inhibitory effects on TSH secretion by a direct action at the pituitary level. These effects appear to be mediated changes of the level of adenohy-pophyseal TRH receptors. A close correlation was observed between the specificity of binding of the dopamine agonist (3H)dihydroergocryptine and the control of prolactin release in cells in culture, thus supporting the physiological importance of the dopamine receptor in the control of prolactin release. The high degree precision of this system permits assessment of activity of not only dopamine agonists and antagonists, but also of compounds having mixed agonist-antagonistic activity. Preincubation of anterior pituitary cells with 17beta-estradiol not only stimulated basal and TRH-induced prolactin release but, more unexpectedly, led to an almost complete reversal of the inhibitory effect of dopamine agonists on prolactin secretion. Besides its own interest, the adenohypophyseal cell culture system could well be used as a model system for study of the interaction between estrogens and dopaminergic action.
Androgens↗
Glycoproteins in membranes of secretory granules of the anterior pituitary gland.
Rat anterior pituitary glands were examined by electron microscopy after staining with five different histochemical stains. Histochemical reactions were observed in the cell coat, cell membrane and the membrane surrounding the secretory granules in all anterior pituitary cells following staining with phosphotungstic acid (PTA), chromic acid and PTA, the periodic acid-thiosemicarbazide-silver protein method (PA-TSC-SP) of Thiéry, ruthenium red and concanavalin A. The staining was abolished when the sections were preincubated with pronase, neuraminidase or trypsin and subsequently exposed to PTA, chromic acid and PTA or PA-TSC-SP. The possible functional role of the glycoproteins present in the membrane surrounding the secretory granules is considered.
Animals↗
GNRH interaction with anterior pituitary. I. Determination of the affinity and number of receptors for GNRH in ovine anterior pituitary.
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Anestrus↗
Molecular weight forms of adrenocorticotropic hormone secreted by primary cultures of mouse anterior pituitary.
Extracts of mouse anterior pituitary cells in monolayer culture were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate the molecular weight forms of ACTH. The gels were sliced and each segment was eluted. The eluates were assayed for ACTH immunoactivity. Approximately 10% of the immunoactivity in the extracts was found to be present as 20,000--32,000 mol wt ACTH. The remainder of the immunoactivity was equally distributed between two forms of ACTH with apparent molecular weights of 11,800 and 4,500. This distribution is very similar to that found in extracts of mouse anterior pituitary. Mouse anterior pituitary cultures were incubated for 2 h in serum-free tissue culture medium. ACTH was concentrated from the medium and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The medium was found to contain predominantly the 4,500 and 11,800 forms of ACTH. When vasopressin was added to these cultures (100 ng/ml), the rate of secretion of ACTH was more than doubled in serum free medium. Analysis of the medium from vasopressin-stimulated cultures showed that the 4,500 and 11,800 mol wt forms of ACTH were again the predominant forms present.
Adrenocorticotropic Hormone↗
Characterization of the mannosyl and fucosyl transferases in the ovine anterior pituitary glands.
Ovine anterior pituitary glands contain mannosyl- and fucosyl-transferases localized in the microsomes and able to incorporate mannose or fucose as such from GDP-mannose or GDP-fucose into endogenous glycoproteins. The requirements and conditions necessary for maximum activity were investigated. The value of the Km is very similar for the two enzyme systems, 3 X 10(-7) M in the case of mannosyl-transferases and 5 X 10(-7) M in the case of fucosyl-transferases.
Animals↗
Relationship between the follicular cells and marginal layer cells of the anterior pituitary.
In the anterior pituitaries of adult rats of both sexes, follicular cells resemble marginal layer cells in fine structure. Numerous occurrences of cilia were electronmicroscopically confirmed on the free surface of both cells. The absence of secretory granules, the poor development of endoplasmic reticula and abundant free ribosomes and polysomes are the other coincidental cytological properties between both cells showing the immaturity. Thus, the two cells seem to be intimately related. The follicular cells may play an important role in the mitotic activity of the gland. Cilia and microvilli on the free surface of follicular cells are not easily changed by some experimental conditions.
Animals↗
[Inhibition of LH-RH induced release of gonadotropins by substance P in cultured rat anterior pituitary cells].
Using anterior pituitary cells cultured for 7 days and then incubated for 4 hrs, substance P, an undecapeptide, inhibited the stimulatory effect of LH-RH on the release of LH-RH on the release of LH and FSH. This inhibitory effect, which was similar for both gonadotropins was only observed when the adenopituitary cells were put in culture at DI and Proestrus stages of the oestrous cycle. Furthermore substance P partly inhibited the basal release of FSH at DI and DII stages but did never affected that of LH.
Animals↗
A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary gland in vitro.
Rat anterior pituitary explants were incubated with PGI2, PGH2 and PGE2 in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE2 was found to be about 20 times more potent than PGI2 while PGH2 was slightly more effective than PGI2. The results suggest that PGI2 does not play a AMP was measured. PGE was found to be about 20 times more potent than physiological role in cyclic AMP mediated events in the rat anterior pituitary.
Animals↗
Functional heterogeneity in somatotrophs isolated from the rat anterior pituitary.
Somatotrophs in suspensions of anterior pituitary cells from adult male rats can be separated into 2 fractions by density gradient centrifugation. In addition to their different densities, somatotrophs in these 2 fractions can be distinguished morphologically by their staining characteristics and ultrastructure. Somatotrophs of lesser density (type I; approximately 1.068 g/cm3) have fewer secretory granules and a more extensive Golgi apparatus than the somatotrophs of greater density (type II; approximately 1.073 g/cm3). Responsiveness of type I and type II cells to secretory agents (i.e., dibutyryl cyclic adenosine monophosphate, somatostatin, thyroxine, and hydrocortisone) was evaluated by GH radioimmunoassay. Type I cells were consistently more responsive (% GH release) than type II cells. During 7 days in culture, type I cells produced more (approximately 200%) GH than they initially contained, whereas type II cells did not show evidence of increased GH production. Hydrocortisone significantly stimulated GH production in type I, but not type II cells. These results support the hypothesis that at least 2 functionally distinct populations of somatotrophs are present in the anterior pituitary gland of the adult male rat.
Animals↗
Long-term organ culture of rat anterior pituitary glands.
An organ culture system for neonatal rat anterior pituitary glands has been developed in which cellular integrity and responsiveness to LH releasing hormone (LHRH) are maintained for at least 4 days. Anterior pituitary glands from Sprague-Dawley rats were cultured individually at 37 C in an atmosphere of 95% O2-5% CO2 in BGJb culture medium, Fitton-Jackson modification. Histological examination of 5-day-old rat pituitary glands cultured for 96 h in control medium revealed no evidence of tissue necrosis. By contrast, 40-day-old-rat anterior pituitary glands were centrally necrotic after only 24 h of incubation under the same conditions. The neonatal glands were treated for 24 h with synthetic LHRH, and medium and pituitary LH FSH concentrations were determined by double antibody radioimmunoassay. On the first day of culture, LHRH caused a dose-related release of LH with the minimal effective dose between 10(-10) and 10(-9)M. Near-maximal release appeared to be attained with 10(-6)M LHRH, which induced a 12-fold elevation in medium LH over controls. Release was evident within 3 h following stimulation with 10(-9)M LHRH. Pituitary glands were responsive to this dose at 0,24 and 72 h of culture with progressively increasing ratios between LHRH-stimulated and control LH levels of 3,13, and 24, respectively. Although FSH release by LHRH was not observed on the first day of culture, on the second day LHRH caused a dose-related secretion of this hormone. Specificity of the pituitary response was demonstrated for LH and TSH release following addition of LHRH and thyrotropin releasing hormone (TRH). The simplicity, sensitivity, reproducibility, and long-term viability of this neonatal rat anterior pituitary organ culture system make it a valuable technique for the study of gonadotropin secretion.
Age Factors↗
The effects of LH-RH on the concentration of 3',5'-cyclic AMP and RNA synthesis in rat anterior pituitary.
In an experiment performed to investigate the effect of LH-RH on the anterior pituitary of rats, we studied the changes in the concentration of 3',5'-cyclic AMP in the anterior pituitary and the uridine uptake by the anterior pituitary following the intravenous injection of 100 ng LH-RH. The results obtained are summarized as follows: 1) The tissue level of 3',5'-cyclic AMP in the anterior pituitary reached the highest peak 15 min after the injection of LH-RH. 2) The uridine uptake of the anterior pituitary began to increase 15 min after the LH-RH injection and attained a maximum level (5 to 6 times the normal level) in 30 min. 3) The fact that the uridine uptake was inhibited to a considerable extent by the addition of actinomycin D to the incubation medium suggested that uridine was incorporated into the RNA fraction.
Animals↗
Influence of cholinergic receptor blockade and stimulation on the anterior pituitary mitotic activity.
The effects of pilocarpine and atropine on the mitotic incidence in the anterior pituitary in male and ovariectomized estradiol-pretreated female rats was investigated. It was shown that pilocarpine stimulated, while atropine inhibited, the anterior pituitary mitotic activity. It was also shown that carbachol enhanced the mitotic incidence in the organ-cultured anterior pituitary and its effect was blocked by scopolamine. The existence of the muscarinic cholinergic receptor within the anterior pituitary, as well as its involvement in the control of the anterior pituitary mitotic activity, is suggested.
Animals↗
Granules and Golgi vesicles with differential reactivity to ACTH antiserum in the corticotroph of the rat anterior pituitary.
Slices of unembedded rat anterior pituitaries, fixed with a periodate-lysine-paraformaldehyde (PLP) fixative, were incubated with guinea pig antiserum to ACTH and stained with a peroxidase-conjugated IgG fraction of anti-guinea pig gamma-globulin serum from rabbits. The fine structure of the stained cells was identical to that of the ACTH-secreting cell, as described by Siperstein and coworkers. Immunoreactive granules were mainly located at the periphery of the cell. Numerous granules of the inner cytoplasm and also the Golgi complex were nonreactive to the antiserum. The differential labeling for granules and Golgi apparatus peptide.
Acid Phosphatase↗
Specific subclones derived from a multipotential clone of rat anterior pituitary cells.
Several functional subclones of rat anterior pituitary cells were established from our 2A8 clone which apparently contains a heterogenous population of committed and uncommitted cells. On the basis of the hormones secreted into the culture media, as measured by radioimmunoassay, these subclones were divided into four categories, i.e., subclones which secrete (1) ACTH only, (2) prolactin only, (3) prolactin and GH or (4) ACTH, prolactin and GH. None of the subclones produced detectable amounts of thyrotrophic or gonadotrophic hormones. Subclones which secrete a single hormone have shown no change in the type of hormone produced, indicating that these subclones were each derived from a committed cell. The cells of all subclones exhibit a normal diploid karyotype and show good growth characteristics. The cells of the different subclones can be classified by phase contrast microscopy into four categories. However, no clear-cut ultrastructural features have been observed which can be correlated with the different categories of subclones. On the basis of the results a hypothesis is proposed relative to the functional cytodifferentiation of anterior pituitary cells.
Adrenocorticotropic Hormone↗
Effect of the isolated removal of the median eminence (ME) and pituitary stalk (PS) on the immunohistology and hormone release of the anterior pituitary gland grafted into the hypophysiotrophic area (HTA) and/or of the in situ pituitary gland.
Isolated removal of the median eminence (ME) and pituitary stalk (PS) of female rats was performed under visual control, using a new instrument to open up the 3rd ventricle. Atrophy of the uterus, the follicles and the intersitial tissue in the ovaries was accompanied by persistent corpora lutea and persistent diestrous vaginal smears in rats which had undergone a successful removal of ME and PS. No change was, however, detected in the weight of the thyroid and adrenal glands at the end of the six weeks experimental period. An adenohypophysis implanted in the place of the ME at the time of the surgery, could not prevent these changes. In animals, in which the removal of the ME was not complete, the changes of the gonadal system were less pronounced. Immunocytology of the pituitary LH-, FSH, TSH- and prolactin-cells in animals with completely removed ME and PS showed inactive LH- and FSH-cells both in the grafted and in situ pituitaries, while the TSH- and prolactin-cells appeared to be in a stimulated state. In animals with ME remnant, LH-RH axon terminals were localized only on the blood vessels of the remnant. The part of the pituitary graft in contact with these blood vessels, as well as some areas of the in situ pituitary gland, contained active LH cells as judged from their size and immunohistological appearance. Since in the absence of the ME, the hypophysiotrophic area is not able to exert its regulatory effect on the gonadotrophs of the pituitary implant in this area, the authors suggest that this effect is mediated by the blood circulation of the ME which is rich in releasing hormones and is drained toward both the anterior pituitary and the medial basal hypothalamus.
Animals↗