Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells

1998 ◽  
Vol 78 (4) ◽  
pp. 587-597 ◽  
Author(s):  
M. Y. Yang ◽  
R. Rajamahendran
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Culture System ◽  
Physiological Role ◽  
Serum Free ◽  
Igf I ◽  
The Media ◽  
Estradiol 17Β ◽  
Insulin Like Growth Factors

The objectives of this study were: 1) to develop a bovine granulosa cell (GC) culture system; and 2) to use this system to evaluate the effects of gonadotropins (FSH and LH) and insulin-like growth factors-I and -II (IGF-I and IGF-II) on steroidogenesis of bovine GC derived from small, medium, and large antral follicles (diameters ≤4, 5–8 and >8 mm, respectively). Granulosa cells were cultured (concentration, 5 × 105 cells per well) in serum-free medium for 48 h with variable doses of hormones and growth factors. Concentrations of progesterone (P4) and estradiol-17β (E2) in the media were determined by radioimmunoassay. Basal E2 production by GC from follicles of all sizes decreased with time of culture (P < 0.01) while basal P4 production increased (P < 0.01). Basal E2 and P4 production increased with increasing size of follicles (P < 0.01). Only very low concentrations of FSH stimulated E2 production from medium and large follicles. Follicle-stimulating hormone stimulated P4 production by GC of follicles of all sizes (P < 0.05). Luteinizing hormone inhibited E2 production by GC in medium and large follicles (P < 0.05), suggesting that LH is responsible for the rise in plasma E2 through effects on both theca cells and GC. A dose of 100 ng mL−1 of IGF-I increased E2 production by GC from medium and large follicles (P < 0.05). Progesterone production by GC from all categories of follicles was also stimulated by IGF-I (P < 0.05). Estradiol-17β production by GC from large follicles decreased in response to IGF-II (P < 0.05). The physiological role of IGF-II on steroidogenesis in the bovine ovary remains to be elucidated. In summary, these results demonstrate the development of a serum-free culture system for bovine GC, and that FSH, LH, IGF-I and IGF-II have different effects on steroidogenesis by bovine GC from different size follicles. Key words: Granulosa cells, gonadotropins, Insulin-like growth factors, progesterone, estradiol-17β, cows

scholarly journals GH/IGF e neoplasia: o que há de novo nesta associação

2005 ◽  
Vol 49 (5) ◽  
pp. 833-842 ◽  
Author(s):  
Angela M. Spinola e Castro ◽  
Gil Guerra-Júnior
Keyword(s):  
Growth Factors ◽  
Binding Proteins ◽  
De Novo ◽  
Igf Binding Proteins ◽  
Igf I ◽  
Ciclo Celular ◽  
Igf Binding ◽  
Insulin Like Growth Factors

Estudos in vitro e em animais sugerem que os membros do sistema insulin-like growth factors (IGFs), incluindo IGF-I, IGF-II, receptores de IGF-I e IGF-II (IGF-IR e IGF-IIR), e as IGF-binding proteins (IGFBPs) podem ter um importante envolvimento no desenvolvimento e na progressão de neoplasias. Mais especificamente, as IGFs promovem a progressão do ciclo celular e inibem a apoptose tanto por ação direta com outros fatores de crescimento como por ação indireta interagindo com outros sistemas moleculares intracelulares envolvidos na promoção e/ou progressão do câncer. Além disso, inúmeros estudos epidemiológicos têm sugerido que concentrações elevadas das IGFs, independente das alterações nas IGFBPs, podem estar associadas a um aumento no risco de desenvolver determinadas neoplasias. Esta revisão tem como objetivo apresentar o envolvimento do sistema IGF na regulação tumoral, os principais estudos epidemiológicos realizados e o risco de desenvolvimento de neoplasia em pacientes (com ou sem história pessoal de neoplasia prévia) que receberam hormônio de crescimento (rhGH). É importante salientar que o uso clínico de rhGH, nas indicações aprovadas internacionalmente, é seguro e não existem evidências, até o momento, da associação com o desenvolvimento de neoplasias.

scholarly journals Proteolytic conversion of insulin-like growth factors to an acidic form(s)

1984 ◽  
Vol 223 (1) ◽  
pp. 97-103 ◽  
Author(s):  
A D Kuffer ◽  
A C Herington
Keyword(s):  
Growth Factors ◽  
Human Serum ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Full Spectrum ◽  
Acidic Form ◽  
Igf I ◽  
Acid Gel ◽  
Insulin Like Growth Factors

The relative amounts of the various forms of bioassayable insulin-like growth factors (IGF) isolated from human serum or serum fraction Cohn IV-1 depend on the purification procedure. With acid gel filtration or acid/ethanol extraction as the initial step, IGF-II (pI approximately 6.5) was the most abundant (40-70%) followed by somatomedin A (pI approximately 7.4; 15-23%), an acidic form of insulin-like activity (ILA pI 4.8) (13-21%) and IGF-I (pI approximately 8.5; 5-27%). If, however, pH 5.5 ion-exchange chromatography on SP-Sephadex was used prior to acid gel filtration, the acidic pI 4.8 form was the major (greater than 90%) species recovered and was accompanied by a quantitative loss of the other IGF species. This suggested a possible conversion of IGF-I, somatomedin A and/or IGF-II to the acidic ILA pI 4.8 form(s) during the SP-Sephadex procedure. Further experiments indicated that differences in the yields of ILA pI 4.8 were not due simply to differences in the initial pH conditions of the various methods (i.e. acid versus neutral), although exposure to pH 9.7 (a pH experienced during elution of IGF activity from the SP-Sephadex) did appear to play a role. The involvement of the carrier protein in the conversion process was tested by subjecting carrier-free IGF-I and IGF-II to the SP-Sephadex procedure. No conversion of the free forms to ILA pI 4.8 occurred. To examine the possible role of proteinase in the conversion of IGFs to ILA pI 4.8, SP-Sephadex chromatography was performed in the presence of a broad spectrum proteinase inhibitor. The IGF distribution pattern obtained closely resembled the ‘normal’ pattern seen with acid gel filtration, indicating that proteinase inactivation had prevented conversion to ILA pI 4.8. These data suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to more acidic ILA pI 4.8 form(s) (i) occurs during SP-Sephadex chromatography, (ii) is not prevented simply by prior acid exposure, and (iii) takes place only when IGF-I and -II are in their high-Mr carrier-bound forms. Since IGF-I and IGF-II, although homologous, have unique amino acid sequences, the conversion of both IGFs implies that at least two acidic ILA forms exist. Nevertheless, because ILA pI 4.8 retains the full spectrum of IGF bioactivities in vitro, and significant quantities are present in normal human serum (21%), it would suggest that proteolytic conversion of IGF-I, somatomedin A and IGF-II to ILA pI 4.8 in vivo may be a physiologically significant event.

The effect of IGF-I on the growth of secondary hair follicles of the Cashmere goat in vitro

1997 ◽  
Vol 1997 ◽  
pp. 170-170
Author(s):  
H. Galbraith ◽  
D. Sims ◽  
D. Hazlerigg
Keyword(s):  
Growth Factors ◽  
Hair Follicle ◽  
Human Hair ◽  
Hair Follicles ◽  
Cashmere Goat ◽  
Igf I ◽  
Hair Follicle Cycle ◽  
Insulin Like Growth Factors

Factors regulating the growth of Cashmere fibre and the hair follicle cycle are poorly understood. Insulin-like growth factors (IGFs) or insulin at higher concentrations, have been shown to stimulate in vitro growth of human hair follicles (Philpott et al, 1994). The role of such mitogens in the production of cashmere fibre by the Cashmere goat has not been previously investigated. The objective the study reported here was to investigate the growth of hair follicles in the absence and presence of insulin or IGF-I using our established in vitro technique.

Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture

Reproduction ◽  
2000 ◽  
pp. 211-219 ◽  
Author(s):  
EM Shores ◽  
HM Picton ◽  
MG Hunter
Keyword(s):  
Granulosa Cells ◽  
Culture System ◽  
Theca Cell ◽  
Serum Free ◽  
Theca Cells ◽  
Plating Density ◽  
Factor I ◽  
Viable Cells ◽  
Igf I ◽  
Oestradiol Production

The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.

Effects of metabolic hormones and growth factors on forskolin and dibutyryl adenosine 3′,5′-cyclic monophosphate induced steroidogenic responses by porcine granulosa cells in vitro

1995 ◽  
Vol 75 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Y. Xu ◽  
R. N. Kirkwood ◽  
P. A. Thacker ◽  
K. Rajkumar
Keyword(s):  
Growth Factors ◽  
Granulosa Cells ◽  
Metabolic Hormones ◽  
Estradiol Secretion ◽  
Porcine Granulosa Cells ◽  
Igf I ◽  
The Face ◽  
Positive Effect

The present experiments were conducted to examine the influence of metabolic hormones and growth factors on progesterone and estradiol secretion by porcine granulosa cells in culture and to determine the possible site(s) of action of these agents on steroidogenesis. Porcine granulosa cells, obtained from medium-sized (4–6 mm) nonatretic follicles of prepubertal gilts slaughtered at a commercial abattoir, were cultured at a density of 2 × 106 viable cells well−1 in serum-free Eagle's Minumum Essential Medium for 72 h with 0, 10 or 100 ng mL−1 of GH, insulin or IGF-I or IGF-II. At 72 h, each treatment group was divided into three subgroups, one subgroup remained untreated (basal) while either 25 μM FK or 3 mM dbcAMP was added to the other two subgroups. Additionally, androstenedione (10−7 M) was provided to all cultures as a substrate for estradiol biosynthesis. Media were collected at 96 h for determination of progesterone and estradiol content. Both IGF-I and IGF-II increased (P < 0.01) basal progesterone and estradiol secretion and enhanced the FK- and dbcAMP-induced stimulation of progesterone and estradiol secretion (P < 0.01). Insulin, at 100 ng mL−1, had a modest but significant positive effect on both FK- and dbcAMP-stimulated steroidogenic responses. In contrast, GH inhibited basal, FK- and dbcAMP-induced progesterone and estradiol production. Based on the stimulatory effects of both IGFs above those seen with dbcAMP alone and the inhibitory effects of GH in the face of dbcAMP stimulation, we conclude that their effects are likely mediated by non-cAMP dependent pathways. Key words: Steroidogenesis, granulosa cells, growth hormone, IGF, insulin, pigs

scholarly journals Developmentally Regulated Responses of Human Granulosa Cells to Insulin-Like Growth Factors (IGFs):IGF-I and IGF-II Action Mediated Via the Type-I IGF Receptor1

1998 ◽  
Vol 83 (4) ◽  
pp. 1256-1259 ◽  
Author(s):  
Debbie S. Willis ◽  
Helen D. Mason ◽  
Hazel Watson ◽  
Stephen Franks
Keyword(s):  
Growth Factors ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Similar Degree ◽  
Type I ◽  
Steroid Production ◽  
Human Granulosa Cell ◽  
Igf I ◽  
Igf Receptor ◽  
Insulin Like Growth Factors

In experimental animal models, insulin-like growth factors (IGFs) have been found to be more potent stimulators of ovarian function than insulin. In human theca cells, however, insulin, IGF-I, and IGF-II have similar effects on androgen production. The relative effects of insulin and IGFs on human granulosa cell steroidogenesis is unknown. Furthermore, it is unclear whether effects of IGF-II on steroidogenesis are mediated by the type-I or type-II IGF receptor. The effects of insulin, IGF-I, and IGF-II on human granulosa cell steroidogenesis were compared in vitro. As expected, insulin, IGF-I, and IGF-II enhanced steroidogenesis. Previously, IGF-II has been shown to enhance granulosa cell steroid production after insulin preincubation. In this study, an effect of IGF-II, independent of insulin priming, also was observed. In granulosa cell cultures from small antral follicles (≤13 mm), insulin and IGF-I stimulated steroid production to a similar degree, whereas IGF-II was less effective. In contrast, IGFs were more effective than insulin (IGF-I &gt; IGF-II &gt; insulin) in granulosa cells isolated from preovulatory follicles. IGF-I and IGF-II actions were mediated via the type-1 IGF receptor. The increased responsiveness of mature granulosa cells to IGFs may be an important mechanism by which granulosa cells increase their steroidogenic output in the preovulatory follicle.

Effects of growth factors and hormones on basal and FSH-stimulated inhibin production by porcine granulosa cells in vitro

1991 ◽  
Vol 3 (2) ◽  
pp. 201 ◽  
Author(s):  
U Michel ◽  
S Ludemann ◽  
H Jarry ◽  
W Wuttke
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Type I ◽  
Tgf Beta ◽  
G Cells ◽  
Porcine Granulosa Cells ◽  
Igf I ◽  
Factor Type

The effect of several growth factors, protein and steroid hormones on follicle stimulating hormone (FSH)-stimulated and basal inhibin secretion by mature porcine granulosa cells (g-cells) in culture was examined in order to elucidate the putative role of growth factors and hormones in the regulation of inhibin secretion by porcine g-cells in vitro. Cells were incubated with the respective hormones over a timespan of 0-144 h and immunoreactive inhibin was measured with a radioimmunoassay against porcine inhibin. Epidermal growth factor (EGF) and human transforming growth factor type beta (TGF-beta) decreased basal and gonadotrophin-stimulated inhibin and progesterone in a dose-dependent manner. In the absence of insulin, insulin-like growth factor type I (IGF-I) caused a 4-fold enhancement of basal inhibin secretion, but inhibin secretion was elevated only to 20% above control in the presence of 500 nM insulin. Porcine platelet-derived growth factor (PDGF) had no significant effect on basal or FSH-induced inhibin secretion by g-cells. In addition, neither gonadotrophin-releasing hormone (GnRH) nor prolactin (PRL), arginine vasopressin (AVP) and oxytocin affected basal or FSH-stimulated inhibin release by porcine g-cells. Oestradiol caused a slight but significant (P less than 0.01) rise of basal inhibin production (158% of control) in the last 2 days of culture (96-144 h) and the effect of androstenedione on basal (158% of control) and FSH-stimulated (140% of control) inhibin release (P less than 0.01) was also only visible on Days 4-6 of culture. In contrast to androstenedione and oestradiol, progesterone did not show any effect during 6 days of culture in a dose range of 10(-5) to 10(-9) M. Like steroids, prostaglandin E2 (PGE2) had a stimulatory effect on basal inhibin production (250% of control) by porcine g-cells, visible on Days 3-6 of culture, but an inhibitory effect on FSH-stimulated release (less than 40% of control). Over all the experiments with different hormones and growth factors, tested in varying doses and over a time span of 0-144 h, there was a strong correlation between progesterone and inhibin secretion by g-cells (0-48 h = 0.78; 48-96 h = 0.92; 96-144 h = 0.92). These results suggest that EGF, TGF-beta, IGF-I, oestradiol and androstendione as well as PGE2 have para- and/or autocrine modulatory effects on basal and FSH-stimulated inhibin secretion by mature porcine g-cells in vitro and further demonstrate that the secretion of the proteohormone inhibin and the steroid progesterone are closely related.

Free and total insulin-like growth factors and insulin-like growth factor binding proteins during 14 days of growth hormone administration in healthy adults

1996 ◽  
Vol 135 (6) ◽  
pp. 672-677 ◽  
Author(s):  
Christian Skjæbæk ◽  
Jan Frystyk ◽  
Jens Møller ◽  
Jens Sandahl Christiansen ◽  
Hans Ørskov
Keyword(s):  
Growth Hormone ◽  
Growth Factors ◽  
Growth Factor ◽  
Healthy Adults ◽  
Insulin Like Growth Factor ◽  
Serum Free ◽  
Factor Binding ◽  
Hormone Administration ◽  
Igf I ◽  
Insulin Like Growth Factors

Skjærbæk C, Frystyk J, Møller J, Christiansen JS, Ørskov H. Free and total insulin-like growth factors and insulin-like growth factor binding proteins during 14 days of growth hormone administration in healthy adults. Eur J Endocrinol 1996;135:672–7. ISSN 0804–4643 The objective was to investigate the effect of growth hormone (GH) administration on circulating levels of free insulin-like growth factors (IGFs) in healthy adults. Eight healthy male subjects were given placebo and two doses of GH (3 and 61U/m2 per day) for 14 days in a double-blind crossover study. Fasting blood samples were obtained every second day. Free IGF-I and IGF-II were determined by ultrafiltration of serum. Total IGF-I and IGF-II were measured after acid–ethanol extraction. In addition, GH, insulin, IGF binding protein 1 (IGFBP-1) and IGFBP-3 were measured. Serum-free and total IGF-I increased in a dose-dependent manner during the 14 days of GH administration. After 14 days, serum-free IGF-I values were 610 ± 100 ng/l (mean±sem) (placebo), 2760 ± 190 ng/l (3IU/m2) and 3720 ± 240 ng/l(6 IU/m2) (p = 0.0001 for 3 and 6 IU/m2 vs placebo; p = 0.004 for 3 IU/m2 vs 6 IU/m2). Total IGF-I values were 190 ± 10 μg/l (placebo), 525 ± 10 (3 IU/m2), and 655 ± 40 μg/l (6 IU/m2) (p < 0.0001 for 3 and 6IU/m2 vs placebo; p = 0.04 for 3 IU/m2 vs 6 IU/m2). There were no differences in the levels of free or total IGF-II during the three study periods. Insulin-like growth factor binding protein 1 was decreased during GH administration (p = 0.04 for placebo vs 3IU/m2; p = 0.006 for placebo vs 6 IU/m2). In conclusion, fasting serum free IGF-I increased dose dependently during GH administration and free IGF-I increased relatively more than total IGF-I. This may partly be due to the decrease in IGFBP-1. Christian Skjærbæk, Institute of Experimental Clinical Research, Medical Research Laboratories, Aarhus Kommune Hospital, Norrebrogade 44, DK-8000 Aarhus C, Denmark

scholarly journals In Vitro Culture of Bovine Fibroblasts using Select Serum-Free Media Supplemented with Chlorella vulgaris Extract

2021 ◽  
Author(s):  
Galileo Defendi-Cho ◽  
Timothy Gould
Keyword(s):  
Cell Culture ◽  
Growth Factors ◽  
Cell Growth ◽  
Culture Media ◽  
Serum Free ◽  
The Media ◽  
Chlorella Algae ◽  
Free Media ◽  
Cells Cultured

Standard cell culture practices require addition of animal-derived serum to culture media to achieve adequate cell growth. Typically, 5-10% by volume of fetal bovine serum (FBS) is used, which accounts for a vast majority of the cost of media while also imposing environmental and ethical concerns associated with the use of animal serum. Here we tested the efficacy of culturing cells by replacing serum in the media with algae extract and select additives. Using LC-MS, we compared molecular signatures of FBS to Chlorella algae extracts and identified NAD(H)/NADP(H) as common and relatively abundant features in their characteristic profiles. Bovine fibroblasts, cultured in serum-free media supplemented with C. vulgaris extract and just two growth factors plus insulin, showed significant growth with enhanced viability compared to control cells cultured without serum, albeit still lower than that of controls cultured with 10% FBS. Moreover, C. vulgaris extract enhanced cell viability beyond that of cells cultured with the two growth factors and insulin alone. These results suggest that key components in serum which are essential for cell growth may also be present in C. vulgaris extract, demonstrating that it may be used at least as a partial alternative to serum for cell culture applications.

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