Fish cell culture: Initiation of a line of pituitary cells from carp (Cyprinus carpio) to study the release of gonadotropin in vitro

Summary A cell line was initiated from carp pituitaries. Growth was best at 30° C at seeding levels of 6×10⁵ cells/ml. Primary cultures produced gonadotropin (GtH) in vitro and 1.45 ng of GtH was secreted by 6 × 10⁵ cells in 4 h at 25° C. The cell line at present is in 57th passage. The investigation was supported by the Deutsche Forschungsgemeinschaft.     

Nicotine stimulates maturational gonadotropin (GtH2) release from carp (Cyprinus carpio L.) pituitary cells

1. Perifusion of dispersed pituitary cells and pituitary cell culture was used to investigate the effects of cholinergic drugs on the secretion of maturational gonadotropin (GtH₂) in carp.2. Nicotine strongly, and in a dose dependent manner, stimulated GtH₂ release in male and in female carp (from 10⁻⁸M in the Perifusion and 10⁻¹⁰M in the cells cultures).3. Nicotine is 10 times more active in females than in males.4. The results suggest that in carp, nicotine stimulates GtH₂ release directly from the pituitary cells, indicating a possible involvement of a cholinergic system in the regulation of GtH₂ secretion in teleost fish.     

Bioactive forms of gonadotropin releasing hormone in the brain of an Indian major carp,Catla catla (Ham.)

Two forms of biologically active gonadotropin releasing hormones were isolated from the hypothalami ofCatla catla. Gonadotropin releasing hormone activity was studiedin vitro using enzymatically dispersed carp pituitary cell incubation system. Gonadotropin released into the medium was measured by carp gonadotropin-radio immuno assay. Acetic acid extracted hypothalamic material was subjected to acetone fractionation. Among the three protein pellets obtained at different time periods (ACI, ACII and ACIII), AC II exhibited the gonadotropin releasing hormone activity. Gel filtration of AC II through Sephadex G-25 column showed three protein peaks (SG I, SG II SGIII) and only S G II demonstrated strong gonadotropin releasing hormone activity. Elution of SG II through FPLC Mono Q column (an anion exchanger) in NaCl gradient programme showed one unadsorbed (MQ I) and three adsorbed (MQ II, MQ III and MQ IV) protein peaks. MQ III, which was eluted with 51% NaCl, exhibited gonadotropin releasing hormone activity. Surprisingly, unadsorbed fractions, MQ I, also showed gonadotropin releasing hormone activity. MQ 1 was therefore subjected to FPLC Mono S (a cation exchanger) column chromatography where a highly active gonadotropin releasing hormone enriched peak, i.e., MS III, could be eluted with 45% NaCl. These findings show thatCatla catla hypothalamus has two forms of gonadotropin releasing hormones one anionic (carp gonadotropin releasing hormone I) and another cationic (carp gonadotropin releasing hormone II). These two forms of gonadotropin releasing hormones were also active in heterologous carp species, rohu(Labeo rohita), mrigal(Cirrhinus mrigala) and an exotic common carp(Cyprinus carpio). Combined activity of two forms of gonadotropin releasing hormones was significantly greater as compared to any of the single form.     

Primary culture of normal rat adrenocortical cells

Summary Rat adrenocortical cells retiained their differentiated characteristics over 2 wk in culture without a specific requirement for additives other than inorganic salts, amino acids, vitamins, and fetal bovine serum. The cells were maintained free from fibroblast overgrowth by substitution ofd-valine in place ofl-valine in the medium. Corticotropin (ACTH) inhibited the growth of adrenocortical cells in this medium and the effect was reversible. The adrenocortical cells had a limited capacity for growth as reflected by total cell counts and [³H]thymidine uptake with cells from young animals demonstrated a greater potential for DNA synthesis than cells obtained from mature animals. A very sensitive assay for ACTH using a small number of cells in primary culture also is described. This work was supported by Grant CA-16417 from the National Cancer Institute.     

Trout sertoli cells and germ cells in primary culture: I. Morphological and ultrastructural study

In order to characterize trout Sertoli cells and germ cells obtained after testis dissociation and cell separation, we have studied their morphology, ultrastructure, survival, and ability to express differentiated activities in primary cultures. After dissociation, the fine structure of Sertoli cells does not differ from that observed in situ and only minor changes are shown for at least 13 days. Until they are flattened in a monolayer, they keep the ability to retain germ cells on their surface. When flattened, some of them are able to divide. At the opposite of meiotic germ cells, spermatogonia can develop independently of Sertoli cells. They are able to proliferate during at least 10 days. Spermatocytes and spermatids are obtained as single cells and multinucleated giant cells (symplasts). In the absence of somatic cells, their maximal viability is approximately 5 days, whereas spermatocytes adhering to Sertoli cells can survive at least 10–12 days, provided trout lipoproteins are present. Spermatocytes are able to differentiate to spermatids, although this process is impaired for some ceils. The adhesion of spermatogonia and spermatocytes to Sertoli cells is specific, mediated by desmosome-like junctions and favored by lipoproteins. These data are compared to what is known in mammals and in amphibians.     

Radioimmunoassay for fibroblast growth factor (FGF): release by the bovine anterior pituitary in vitro

A radioimmunoassay (RIA) was developed to measure fibroblast growth factor (FGF) using antiserum generated against a synthetic replicate of [Tyr¹⁰]FGF(1–10). The antisera, previously shown to be capable of inhibiting the biological action of FGF on bovine aortic arch endothelial cells in vitro [1], are highly specific for the amino-terminus of FGF. In the RIA, the antisera recognize the decapeptide antigen [Tyr¹⁰]FGF(1–10) and the intact mitogen on an equimolar basis and show less than 0.01% cross-reactivity with N-acetyl-[Tyr¹⁰]FGF(1–10).

Bovine adenohypophysial cells maintained in primary monolayer culture release and ir-FGF which is indistinguishable from the intact mitogen in as much as it is retained on heparin-Sepharose affinity columns and shows a dose-dependent and parallel displacement in RIA. The release of ir-FGF by the bovine adenohypophysis can be increased with forskolin (10⁻⁵ M) or KCl (50 mM). Preincubation of pituitary cells with 17β-estradiol has no measurable effects on basal ir-FGF, but increases the release after KCl treatment 2–3-fold. These results show that ir-FGF can be released by the bovine adenohypophysis in vitro and lend credence to the hypothesis that FGF plays a physiological role in the homeostatic mechanisms regulating mesoderm-derived cell growth.     

The effect of luteinizing hormone releasing hormone and anti-luteinizing hormone releasing hormone antibodies on chorionic gonadotropin and progesterone secretion by human placental villiin vitro

Gonadotropin releasing hormone has been located and found to be secreted by the human placenta in culture. Addition of the releasing hormone upto 1μg concentration in the placental cultures brings about stimulation of chorionic gonadotropin and progesterone secretion. Higher amounts of the decapeptide has an inhibitory influence on both the gonadotropin and the steroid production. The action of the releasing hormone on the placenta could be blocked by the anti-luteinizing hormone releasing hormone monoclonal antibodies indicating a possible site of action of the antibodies for control of fertility     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

The effects of gastric inhibitory polypeptide (GIP) on the release of anterior pituitary hormones

Several members of the secretin family of hormones have been demonstrated to alter anterior pituitary hormone secretion. Here we report the action of gastric inhibitory polypeptide (GIP) on gonadotropin and somatotropin release. Intraventricular injection of 1 microgram (0.2 nmole) GIP (2.5 microliters) produced a significant decrease in plasma FSH at 30 (p less than 0.02) and 60 min after its injection (p less than 0.01). The FSH-lowering effect of a higher dose of 5 micrograms (1 nmole) of GIP was already developed at 15 min (p less than 0.01) and was prolonged until the end of the experiment (60 min, p less than 0.05). No change in plasma LH was detected at any time during the experimental period. If 5 micrograms of estradiol-benzoate were given SC 48 hr prior to experiment, the initial values of FSH and LH were markedly decreased. In these animals GIP failed to influence plasma FSH and LH. When dispersed anterior pituitary cells from OVX rats were cultured overnight and incubated in vitro with GIP, the peptide was found to induce both FSH and LH release. Highly significant release occurred with the lowest dose tested of 10(-7) M and there was a dose-response effect for both hormones. The slope of the dose-response curve was similar for both FSH and LH release. GIP was less potent than LHRH which produced a greater stimulation of both FSH and LH release at a dose of 10(-9) M than did 10(-7) M GIP. The two peptides had an additive effect on the release of both FSH and LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum-free culture of AtT 20 pituitary cells: A system for neuroendocrine studies under defined conditions

Summary The growth of the mouse pituitary cell line AtT 20 was studied under different in vitro conditions. A completely defined, serum-free culture medium supported the survival of cells for a period of more than 2 mo. The medium, designed SFI, consisted of basal medium supplemented with transferrin, insulin, putrescine, and selenium. For maintenance of cells during long-term culture, no additional compounds were necessary. The time-dependent increases in cell number during culture with fetal bovine serum (FBS) and under serum-free conditions showed similar properties. Analysis of the effects of different substrata on cell growth demonstrated that polylysine supported adhesion and initial growth of cells to a greater extent than untreated plastic or FBS adsorbed to culture dishes. Synthesis and regulation of proopiomelanocortin (POMC)-mRNA, the precursor-mRNA of adrenocorticotropin (ACTH), could be detected by Northern blot analysis under basal conditions and after incubation with steroids and corticotropin-releasing hormone (CRH), indicating the serum-independent expression of important cellular properties.     

Establishment of functional human pituitary tumor cell cultures

Summary Five primary human pituitary tumor cell cultures were initiated from adenoma fragments obtained from patients with prolactin-secreting adenomas and acromegaly. Functional cell cultures were maintained and propagated in monolayer or suspension culture for up to 9 months. Optimal cell viability and growth were achieved using Ham’s F10 medium enriched with 20% fetal bovine serum, although cells from a patient with acromegaly also grew in serum-free, defined, hormone-containing medium. Bromocriptine (100 ng/ml) did not alter the growth curve of replicating cells derived from a patient with acromegaly. These cells initially secreted 5.5 μg human growth hormone/10⁶ cells, and hormone production diminished after 6 wk. Prolactin secretion by cells derived from prolactinomas (0.5 to 1.3 μg/10⁶ cells/24 h) was stimulated by thyrotropin-releasing hormone (10 ng/ml) in two of the cultures. Both dopamine (10 ng/ml) and nickel chloride (1 mM) suppressed PRL secretion. These studies demonstrate that responsive human pituitary tumor cell cultures can be initiated and maintained. This research was supported by VA Medical Research Funds and NIH Grant HD 7181.     

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 μg/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20–60 pg [¹²⁵I]CG bound/μg DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 μg/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.     

Inhibitory effect of gonadotrophin-releasing hormone (GnRH) on rat granulosa cell deoxyribonucleic acid synthesis

Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-β (TGF-β), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of ³H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGFβ (2.5 ng/ml), at concentrations as low as 5 × 10⁻¹¹ M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED₅₀ = 1.58 ± 0.22 × 10⁻¹⁰ M) and native GnRH (ED₅₀ = 1.4 ± 0.3 × 10⁻⁶ M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10⁻⁸ M could prevent the inhibition elicited by 10⁻⁷ M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development. Mol. Reprod. Dev. 47: 170–174, 1997. © 1997 Wiley-Liss, Inc.     

Perifusion system: its use in the study of the neuroendocrine control of human pituitary tumoral cells

Conclusion Taken together, these results show the usefulness of the perifusion technique both in the studyof hormone regulation and in the physiopathology of the human pituitary. It allows the studyof dynamic changes in hormone release, relationships between in vivo and in vitro responses, relationships between hormone response and receptor status. Furthermore, we could use this approach to demonstrate release of pituitary neuropeptides and the relation between secretoryprofiles of neuropeptides and those of pituitary hormones. It is another approach to all thesedifferent points than long term culture that needs enzymatic dispersion, and several days ofrecovery before any experiment can be performed on the cells.     

Ontogeny of gonadotropin releasing hormone and gonadotropin immunoreactivity in brain and pituitary of normal and estrogen-treated guppies,Poecilia reticulata Peters

Summary Gonadotropin releasing hormone (GnRH) and gonadotropic hormone (GTH) were identified by immunohistochemistry in the brains and pituitaries of neonate, juvenile and adult guppies. GTH was present in some cells of the pars intermedia (pi) and proximal pars distalis (ppd) of all animals. GnRH was found in the perikarya of the nucleus olfactoretinalis. In the pituitaries of juvenile 30-day-old guppies, GnRH-immunoreactive cells existed in a juvenile pattern, whereas in adult animals GnRH was recognized in only a few cells. GnRH-immunoreactive fibers were seen in the pituitaries of animals that were 30 days or older. In adult guppies, the ventral and lateral ppd (the gonadotropic region) contained a dense network of GnRH-immunoreactive fibers. Pituitary cells staining for either GnRH or GTH were located in different places. After immunohistochemical double staining of adult pituitaries, none of the GnRH-immunoreactive cells were LH-immunoreactive, although both cell types were often found in close proximity. After 20 days or more of ethinylestradiol treatment, less immunoreactive GnRH was detected in the pituitary cells of juvenile guppies, and fewer animals exhibited the juvenile pattern of GnRH-immunoreactive pituitary cells, when compared with untreated controls. The results indicate that GnRH-immunoreactive pituitary cells in the guppy are distinct from gonadotropes and that these cells are involved in regulatory processes along the juvenile brain-pituitary-gonad axis.     

Use of semipermeable polyurethane hollow fibers for pituitary organ culture

Summary A new model for organ culture of endocrine tissue is described. Rat anterior pituitary fragments were cultured for 4 wk within semipermeable polyurethane isocyanate hollow fibers. Growth hormone and prolactin, two of the anterior pituitary hormones, were released into the medium during the entire culture period. Electron microscopy of the pituitary fragments after 2 wk in in culture showed a rim of viable tissue in all specimens examined. Individual cells, from this outer rim, exhibited excellent organelle preservation and numerous secretory granules. Experiments involving potassium depolarization and 10⁻⁶ M dopamine provided evidence for the normal responsiveness of the cultured pituitary tissue to both stimulatory and inhibitory factors. These studies illustrate the potential utility of the described organ culture system for further investigations of endocrine physiology.     

Cloning and sequencing of the cDNA encoding the pituitary gonadotropin II β-subunit of yellowfin porgy (Acanthopagrus latus)

The complementary DNA (cDNA) encoding the gonadotropic hormone (GTH) II pre-β-subunit of yellowfin porgy ( Acanthopagrus latus ) was isolated from a pituitary gland cDNA phage library. The cDNA insert, 598 base pairs (bp), contained a 411 bp open reading frame with 35 bp and 152 bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequence revealed a putative signal peptide of 24 amino acid residues and a 113 amino acid mature β-subunit of GTH polypeptide. This pre-β-subunit polypeptide of the yellowfin porgy GTH II showed 86% sequence identity with that of bonito GTH II-β 73% with killifish GTH II-β, 63% with African catfish GTH II-β 61% with pike eel GTH-β, 60% with silver carp GTH-β, 59% with chum salmon GTH 11-β, 58% with common carp GTH-β 57% with chinook Pacific salmon GTH-β, and 53% with European eel GTH II-β.