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.

scholarly journals Interaction of bovine granulosa and theca cells in a novel serum-free co-culture system

Reproduction ◽  
2003 ◽  
pp. 527-538 ◽  
Author(s):  
C Allegrucci ◽  
MG Hunter ◽  
R Webb ◽  
MR Luck
Keyword(s):  
Cell Survival ◽  
Culture System ◽  
Close Contact ◽  
Cell Phenotype ◽  
Serum Free ◽  
Theca Cells ◽  
Plating Density ◽  
Igf I ◽  
Defined Culture ◽  
Cell Plating

The objective of this study was to develop a defined culture system in which bovine follicular and granulosa cells are grown in close contact with each other and with the extracellular matrix (ECM) component laminin. Granulosa and theca cells from follicles 4-6 mm in diameter were cultured on either side of laminin-coated BioCoat cell culture inserts in a serum-free medium containing 10 ng insulin ml(-1) at plating densities of 10(5) and 3 x 10(5) cells per membrane side. The cells adopted a clumped arrangement, maintained steroidogenic activity for at least 7 days and demonstrated paracrine communication by increased steroidogenesis and enhanced cell survival compared with cells in mono-culture. Co-cultured theca cells secreted significantly more androstenedione compared with cells in mono-culture. Granulosa cell viability was doubled by co-culture with theca cells. Co-cultures at both cell plating densities were responsive to treatment with physiological combinations of either FSH, LH and LR3 insulin-like growth factor I (IGF-I) (treatment A) or FSH, LR3 IGF-I and androstenedione (treatment B). Significantly more androstenedione was secreted in the presence of treatment A compared with controls. In contrast, oestradiol secretion was increased only by treatment B. Progesterone secretion was unaffected by treatment and did not increase during culture. Co-cultures at the higher plating density demonstrated higher theca cell survival and better maintenance of the follicular cell phenotype. In conclusion, this novel co-culture system provides a unique model for the study of paracrine communication between ovarian somatic cells and cell-ECM interactions during follicle growth.

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro

1994 ◽  
Vol 143 (1) ◽  
pp. 157-164 ◽  
Author(s):  
J G Gong ◽  
D McBride ◽  
T A Bramley ◽  
R Webb
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Dependent Manner ◽  
Serum Free ◽  
Factor I ◽  
Igf I ◽  
Serum Free Conditions ◽  
Dose Dependent ◽  
Bovine Somatotrophin ◽  
Cells Cultured

Abstract Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, <5 mm; medium-sized, 5–10 mm and large, >10 mm in diameter. Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10–1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1–1000 ng/ml) and IGF-I (10–1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml). In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions. However, unlike their effects on cell proliferation, the minimal effective concentrations of these factors required to stimulate granulosa cell steroidogenesis were higher than those observed in our previous studies in vivo. Journal of Endocrinology (1994) 143, 157–164

scholarly journals Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells

2011 ◽  
Vol 48 (1) ◽  
pp. 49-60 ◽  
Author(s):  
J M Young ◽  
A S McNeilly
Keyword(s):  
Culture System ◽  
Regulatory Protein ◽  
Theca Cell ◽  
Cell Culture System ◽  
Inhibitory Effects ◽  
Enzyme Expression ◽  
Serum Free ◽  
Theca Cells ◽  
Thecal Cells ◽  
Steroidogenic Acute Regulatory

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1–100 ng/ml) suppressed androstenedione production. Inhibin (1–100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.

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

Longitudinal bone growth in vitro: effects of insulin-like growth factor I and growth hormone

1991 ◽  
Vol 124 (5) ◽  
pp. 602-607 ◽  
Author(s):  
Ben A. A. Scheven ◽  
Nicola J. Hamilton
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Bone Growth ◽  
Long Bone ◽  
Long Bones ◽  
Insulin Like Growth Factor ◽  
Serum Free ◽  
Factor I ◽  
Igf I

Abstract. Longitudinal growth was studied using an in vitro model system of intact rat long bones. Metatarsal bones from 18- and 19-day-old rat fetuses, entirely (18 days) or mainly (19 days) composed of chondrocytes, showed a steady rate of growth and radiolabelled thymidine incorporation for at least 7 days in serum-free media. Addition of recombinant human insulin-like growth factor-I to the culture media resulted in a direct stimulation of the longitudinal growth. Recombinant human growth hormone was also able to stimulate bone growth, although this was generally accomplished after a time lag of more than 2 days. A monoclonal antibody to IGF-I abolished both the IGF-I and GH-stimulated growth. However, the antibody had no effect on the growth of the bone explants in control, serum-free medium. Unlike the fetal long bones, bones from 2-day-old neonatal rats were arrested in their growth after 1-2 days in vitro. The neonatal bones responded to IGF-I and GH in a similar fashion as the fetal bones. Thus in this study in vitro evidence of a direct effect of GH on long bone growth via stimulating local production of IGF by the growth plate chondrocytes is presented. Furthermore, endogenous growth factors, others than IGFs, appear to play a crucial role in the regulation of fetal long bone growth.

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