Factors Controlling Proliferation and Progesterone Production by Bovine Granulosa Cells in Serum-Free Medium*

Savion, Naphtali; Lui, Ge Ming; Laherty, Richard; Gospodarowicz, Denis

doi:10.1210/endo-109-2-409

Cited by 209 publications

(66 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…These data demonstrate that synthetic IGF-I can stimulate the growth of either adrenal cortical or granulosa cells maintained under serum-free conditions. The range of concentrations of synthetic IGF-I to which both cell types responded is similar to that observed when adrenal cortical or granulosa cells maintained under serum-free conditions were exposed to SM-C (26,30).…”

Section: Resultssupporting

confidence: 64%

See 1 more Smart Citation

Total synthesis of insulin-like growth factor I (somatomedin C).

Li¹,

Yamashiro²,

Gospodarowicz³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Human insulin-like growth factor I (somatomedin C) with 70 amino acid residues and three disulfide bridges has been synthesized by the solid-phase method. The synthetic product behaves as a pure polypeptide in paper electrophoresis, isoelectric focusing, and high-performance liquid chromatography. Its elution behavior in Sephadex G-50, isoelectric point, amino acid composition, and growth-stimulating activity in bovine adrenal cortical cells or granulosa cells are comparable to those reported for the natural product. In radioimmunoassay, the synthetic product is indistinguishable from the natural hormone when either synthetic or natural product is used as the labeled ligand.Production of somatomedins (SMs) and insulin-like growth factors (IGFs) is known to be stimulated by growth hormone (somatotropin) in animals (1, 2) and human subjects (3, 4). SM-C (5) and IGFs (6) have been purified to homogeneity from human plasma. Complete amino acid sequences for IGF-I (7) and IGF-II (8) have been proposed, but only limited sequence data for SM-C are known (5). Various bioassay and radioimmunoassay (RIA) data indicate that IGF-I and SM-C are identical (5, 9, 10). IGF-I consists of 70 amino acid residues with three disulfide bridges, and its structure is homologous to that of proinsulin (7, 11). Fig. 1 presents the primary structure of IGF-I as proposed by Rinderknecht and Humbel (7). Because chemical synthesis of any known growth factor has not been reported, this communication describes the total synthesis of IGF-I (SM-C). MATERIALS AND METHODSProtected IGF-I Resin. 4-(Boc-alanyloxymethyl)-phenylacetamidomethyl-resin (1.60 g, 0.60 mmol) prepared as described (12) was placed in a Beckman model 990 peptide synthesizer and carried through procedures of solid-phase synthesis (13) consisting of (i) 15-min deprotection with 50% (vol/vol) trifluoroacetic acid/CH2Cl2, (ii) neutralization with 5% (vol/vol) diisopropylethylamine (DIEA)/CH2Cl2 (two times). Coupling was effected with preformed symmetrical anhydrides (1.8 mmol, 3 eq) of for 20 min in CH2Cl2 except for Boc-Met(O)-OH, Boc-Arg(Tos)-OH, and Boc-Gln-OH, for which 40-min coupling was allowed and sufficient N,N-dimethylformamide was employed to give concentrations of 20, 15, and 20%, respectively. Boc-Asn-OH was coupled with dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (15). In all cases a second coupling stage was performed (20 min): (i) for residues 69 through 59 and all subsequent couplings in which dimethylformamide was used 1 eq of DIEA was added (16); (ii) for all other residues 1 eq of N-methylmorpholine and sufficient trifluoroethanol to give a concentration of 20% were added (16). The following side-chain blocking groups were used: Asp, cyclopentyl (17); Thr, Ser, and Glu, benzyl; Cys, 3,4-dimethylbenzyl (18); Met, sulfoxide (19); Tyr and Lys, 2-bromobenzyloxycarbonyl; Arg, tosyl. The yield of protected peptide resin was 7.92 g (weight gain was 87% of theory).IGF-I. Protected IGF-I resin (1.336 g, 0.101 mmol) was treated with 50% trifluoroacetic ac...

show abstract

Section: Resultssupporting

confidence: 64%

“…Primary cultures of bovine adrenal cortex cells (25) and granulosa cells (26) were prepared as described. For cell growth measurement, cell monolayers from stock plates were dissociated by exposure (2-3 min, 24°C) to a solution containing 0.9% NaCl, 0.01 M sodium phosphate (pH 7.4), 0.05% trypsin, and 0.02% EDTA (STV solution, Difco).…”

mentioning

confidence: 99%

Total synthesis of insulin-like growth factor I (somatomedin C).

Li¹,

Yamashiro²,

Gospodarowicz³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Human follicular fluid was collected from women who were undergoing in vitro fertilization procedures at the Department of Obstetrics and Gynecology of this university. The cell culture method was essentially as reported (18,19), and briefly described as follows: Granulosa cells in the follicular fluid were pooled from different sized follicles and separated by centrifugation at 500 x g in a table-top clinical centrifuge for 10 min. The cell suspension was washed three times with 5 ml of sterile phosphate-buffered saline (PBS) and resuspended in 10 ml of fresh PBS containing 10 pug of deoxyribonuclease, 2.5 mg of collagenase, and 5 mg of Dispase, grade II.…”

mentioning

confidence: 99%

Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells.

Ramasharma¹,

Li²

1987

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-H). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-H into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-ll secretion from these cells. When synthetic lutropin-releasing hormone and c-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-H into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin.The insulin-like growth factors (IGF-I and IGF-II) are a family of low Mr single-chain peptides with considerable structural and functional similarities to proinsulin (1-3). These growth factors appear to follow a divergent pattern of regulatory mechanisms. The IGF-I levels in plasma are low at birth and gradually increase with age, thus closely following the actions of growth hormone (4-6). Conversely, IGF-II is considered a fetal hormone and appears partially dependent on growth hormone (7,8). Although the liver is considered the primary site of synthesis of these factors, recent studies have shown that several other tissues including the testes and the ovary (9-11) are also capable of producing IGFs.The exact role of IGFs in gonadal function is not clear. Attempts to demonstrate the production of IGF-I by cultured granulosa and Sertoli cell (12, 13) have been inconclusive. We have recently identified significant amounts of IGF-II in human follicular and seminal fluids (14). We report the production of IGF-II by human granulosa cells under serumfree conditions and present evidence for multihormonal regulation of IGF-II release by these cells. MATERIALS AND METHODSIGF-II was synthesized by the solid-phase method as described (15 (75 cm2) and 24-well culture plates were from Coming Glass Works.Human Granulosa Cell Cultures. Human follicular fluid was collected from women who were undergoing in vitro fertilization procedures at the Department of Obstetrics and Gynecology of this university. The cell culture method was essentially as reported (18,19), and briefly described as follows: Granulosa cells in the follicular fluid were pooled from different sized follicles and separated by centrifugation at 500...

show abstract

“…In the present study, granulosa cells obtained from small antral follicles could also be luteinized when they were incubated with insulin plus forskolin. It is well demonstrated that insulin or insulin-like growth factor-I (IGF-I) stimulates proliferation and progesterone production in granulosa cells [16][17][18][19][20][21][22][23] It is of interest to note that some of the large luteal-like cells (which were alive) were obviously smaller than other cells. In the present study, androstenedione production by granulosa cell culture was negligible (data not shown), indicating no contamination by theca cells.…”

Section: Discussionmentioning

confidence: 99%