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.

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 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

scholarly journals Stimulatory effect of insulin-like growth factor I on proliferation of mouse pituitary cells in serum-free culture

1998 ◽  
Vol 157 (1) ◽  
pp. 53-62 ◽  
Author(s):  
S Oomizu ◽  
S Takeuchi ◽  
S Takahashi
Keyword(s):  
Culture System ◽  
Specific Inhibitor ◽  
Secretory Cells ◽  
Pituitary Cells ◽  
Rt Pcr ◽  
Serum Free ◽  
Igf I ◽  
Pituitary Glands ◽  
Lh Cells ◽  
Mouse Pituitary

IGF-I is synthesized in the human and rat anterior pituitary glands. The present study was designed to clarify the growth-promoting action of IGF-I on mouse pituitary cells in a primary serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of bromodeoxyuridine (BrdU). BrdU labelling in the nucleus was found in all types of secretory cells: corticotrophs, thyrotrophs, gonadotrophs (LH cells and FSH cells), somatotrophs and mammotrophs. IGF-I (75 ng/ml) stimulated the proliferation of corticotrophs and mammotrophs among the pituitary secretory cells. IGF-I receptor mRNA was detected in the cultured pituitary cells using reverse transcription (RT)-PCR, indicating that mouse pituitary cells expressed IGF-I receptors. Insulin (100 ng/ml) or IGF-I (7.5 ng/ml) failed to increase the percentage of BrdU-labelled cells. However, treatment with insulin (100 ng/ml) plus IGF-I (7.5 ng/ml) increased the percentage of BrdU-labelled cells in a synergistic-like manner. Genistein, a tyrosine kinase specific inhibitor, decreased the IGF-I-induced cell proliferation, indicating that IGF-I acts through IGF-I receptors. IGF-I mRNA was also detected in the cultured pituitary cells by RT-PCR, and its peptides were immunocytochemically detected. The present results demonstrate that all types of pituitary secretory cells have the ability to proliferate in our serum-free culture system. IGF-I synthesized in the pituitary gland may stimulate the growth of pituitary cells, in particular corticotrophs and mammotrophs, by an autocrine or paracrine mechanism.

scholarly journals Ascorbic acid can promote the generation and expansion of neuroepithelial-like stem cells derived from hiPS/ES cells under chemically defined conditions through promoting collagen synthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rui Bai ◽  
Yun Chang ◽  
Amina Saleem ◽  
Fujian Wu ◽  
Lei Tian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Collagen Synthesis ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Induction System ◽  
Defined Culture

Abstract Introduction Spinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably. Methods We induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors. Results HESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genes PAX6, NESTIN, SOX1, and SOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express β-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway. Conclusions This new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons. Graphical abstract

Growth factor-extracellular matrix synergy in the control of trophoblast invasion

2000 ◽  
Vol 28 (2) ◽  
pp. 199-202 ◽  
Author(s):  
J. D. Aplin ◽  
H. Lacey ◽  
T. Haigh ◽  
C. J. P. Jones ◽  
C.-P. Chen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
First Trimester ◽  
Trophoblast Invasion ◽  
Cell Phenotype ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Igf I

At the periphery of the human placenta, trophoblast attaches to the uterine wall. The tissue interface contains many anchoring sites, with cytotrophoblast columns that form bridges between the overlying extraembryonic (villous) mesenchyme and the maternal decidual stroma beneath. From the periphery of these columns, large numbers of trophoblast cells detach, migrate through the decidua and eventually colonize and transform maternal arteries. In this way the placenta increases and gives priority to the maternal blood supply to the conceptus. We have shown that when early villous tissue is explanted on a collagen gel in serum-free medium, anchoring-site morphogenesis occurs. Thus, in the presence of placental mesenchyme but in the absence of maternal cells, contact with a permissive extracellular matrix (ECM) is necessary and sufficient for cytotrophoblast column development. Proliferation of trophoblast occurs, followed by differentiation into a columnar cell phenotype in which cells remain attached to one another and to the ECM. At this stage, interaction between fibronectin and integrin α5β1 at the cell surface stabilizes the column and the cells remain as a contiguous multilayered sheet. However, the addition of serum-free conditioned medium from first-trimester placental fibroblasts stimulates cytotrophoblast to detach from the distal column and migrate in streams across the ECM. The removal of insulin-like growth factor I (IGF-I) from the fibroblast medium decreases streaming activity, whereas the addition of exogenous IGF-I (10 ng/ml) to serum-free medium produces a streaming phenotype. In contrast, transforming growth factor β1 (10 ng/ml) maintains the cells in a tight sheet. These results suggest the possibility of a paracrine interaction between villous mesenchyme and cytotrophoblast in anchoring sites to stimulate the infiltration of the maternal ECM by trophoblast. Such a mechanism would be self-limiting because the signal diminishes with distance from the placenta.

OR9,1 Chimeric vitronectin: IGF-I proteins enhance cell growth and provide a novel defined culture system for stem cells

2008 ◽  
Vol 18 ◽  
pp. S18
Author(s):  
D. Van Lonkhuyzen ◽  
S. Richards ◽  
B. Hollier ◽  
K. Manton ◽  
G. Shooter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Growth ◽  
Culture System ◽  
Enhance Cell Growth ◽  
Igf I ◽  
Defined Culture
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