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Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro.

Brankin V, Mitchell MR, Webb B, Hunter MG - Reprod. Biol. Endocrinol. (2003)

Bottom Line: In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002).SCF has a role in modulating this local interaction.In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK. victoria.brankin@nottingham.ac.uk

ABSTRACT
Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2-6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0-10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids. Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization.

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Mean (± sed) log10 progesterone production by granulosa cells after (A) 48 hours and (B) 144 hours in serum free culture supplemented with either 0, 5, 10 or 15 oocytes per well, 1 ng/ml pFSH and 100 ng/ml Long R3 IGF-1. Values are from 3 independent cultures, each treatment having 4 replicates. There was a significant effect of treatment (P < 0.001) on progesterone production at both time points. Bars with different superscripts are significantly different (P < 0.05). Morphological appearance of the organisation of granulosa cells around the oocytes when co-cultured under serum-free conditions: (C) granulosa cells supplemented with 10 oocytes per well and (D) granulosa cells supplemented with 5 oocytes. Scale bar represents 100 μm.
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Figure 3: Mean (± sed) log10 progesterone production by granulosa cells after (A) 48 hours and (B) 144 hours in serum free culture supplemented with either 0, 5, 10 or 15 oocytes per well, 1 ng/ml pFSH and 100 ng/ml Long R3 IGF-1. Values are from 3 independent cultures, each treatment having 4 replicates. There was a significant effect of treatment (P < 0.001) on progesterone production at both time points. Bars with different superscripts are significantly different (P < 0.05). Morphological appearance of the organisation of granulosa cells around the oocytes when co-cultured under serum-free conditions: (C) granulosa cells supplemented with 10 oocytes per well and (D) granulosa cells supplemented with 5 oocytes. Scale bar represents 100 μm.

Mentions: Granulosa cells cultured with OCM showed a significant increase in viable cell number (P < 0.001) which was amplified with the addition of LR3 IGF-1 (Figure 1A). In the presence of OCM, large interconnected cell clusters developed compared to controls. These cell clusters were viable as shown by neutral red assay (Figure 1B). OCM inhibited progesterone production both after 48 (P < 0.001) and 144 (P < 0.001) hours in culture (Figure 2A and 2B). A 100-fold dilution of OCM was still potent enough to inhibit progesterone production after 48 hours (P < 0.001). OCM in combination with LR3 IGF-1 also elicited a significant increase overall in oestradiol production after both 48 (P = 0.03) and 144 (P < 0.001) hours (Figure 2C and 2D). After 144 hours, this increase in oestradiol production was evident when OCM was diluted 1000-fold (P = 0.041). In keeping with these findings, oocyte co-culture also elicited both a reduction in progesterone production after 48 hours (P < 0.001) and 144 hours (P < 0.001) (Figure 3A and 3B), and an increase in oestradiol synthesis after 48 hours (P < 0.001) where cells were supplemented with 5 and 10 oocytes per well. Interestingly, after 96 hours in culture, the clusters of granulosa cells (which were expected to be largely mural cells and not cumulus cells) had restructured around the oocytes (Figure 3C and 3D).


Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells in vitro.

Brankin V, Mitchell MR, Webb B, Hunter MG - Reprod. Biol. Endocrinol. (2003)

Mean (± sed) log10 progesterone production by granulosa cells after (A) 48 hours and (B) 144 hours in serum free culture supplemented with either 0, 5, 10 or 15 oocytes per well, 1 ng/ml pFSH and 100 ng/ml Long R3 IGF-1. Values are from 3 independent cultures, each treatment having 4 replicates. There was a significant effect of treatment (P < 0.001) on progesterone production at both time points. Bars with different superscripts are significantly different (P < 0.05). Morphological appearance of the organisation of granulosa cells around the oocytes when co-cultured under serum-free conditions: (C) granulosa cells supplemented with 10 oocytes per well and (D) granulosa cells supplemented with 5 oocytes. Scale bar represents 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC184512&req=5

Figure 3: Mean (± sed) log10 progesterone production by granulosa cells after (A) 48 hours and (B) 144 hours in serum free culture supplemented with either 0, 5, 10 or 15 oocytes per well, 1 ng/ml pFSH and 100 ng/ml Long R3 IGF-1. Values are from 3 independent cultures, each treatment having 4 replicates. There was a significant effect of treatment (P < 0.001) on progesterone production at both time points. Bars with different superscripts are significantly different (P < 0.05). Morphological appearance of the organisation of granulosa cells around the oocytes when co-cultured under serum-free conditions: (C) granulosa cells supplemented with 10 oocytes per well and (D) granulosa cells supplemented with 5 oocytes. Scale bar represents 100 μm.
Mentions: Granulosa cells cultured with OCM showed a significant increase in viable cell number (P < 0.001) which was amplified with the addition of LR3 IGF-1 (Figure 1A). In the presence of OCM, large interconnected cell clusters developed compared to controls. These cell clusters were viable as shown by neutral red assay (Figure 1B). OCM inhibited progesterone production both after 48 (P < 0.001) and 144 (P < 0.001) hours in culture (Figure 2A and 2B). A 100-fold dilution of OCM was still potent enough to inhibit progesterone production after 48 hours (P < 0.001). OCM in combination with LR3 IGF-1 also elicited a significant increase overall in oestradiol production after both 48 (P = 0.03) and 144 (P < 0.001) hours (Figure 2C and 2D). After 144 hours, this increase in oestradiol production was evident when OCM was diluted 1000-fold (P = 0.041). In keeping with these findings, oocyte co-culture also elicited both a reduction in progesterone production after 48 hours (P < 0.001) and 144 hours (P < 0.001) (Figure 3A and 3B), and an increase in oestradiol synthesis after 48 hours (P < 0.001) where cells were supplemented with 5 and 10 oocytes per well. Interestingly, after 96 hours in culture, the clusters of granulosa cells (which were expected to be largely mural cells and not cumulus cells) had restructured around the oocytes (Figure 3C and 3D).

Bottom Line: In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002).SCF has a role in modulating this local interaction.In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK. victoria.brankin@nottingham.ac.uk

ABSTRACT
Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2-6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0-10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids. Oocyte secreted factors were shown to stimulate both granulosa cell proliferation (P < 0.001) and oestradiol production (P < 0.001) by granulosa cells throughout culture. In contrast, oocyte secreted factors suppressed granulosa cell progesterone production after both 48 and 144 hours (P < 0.001). Thecal cell numbers were increased by oocyte secreted factors (P = 0.02), together with a suppression in progesterone and androstenedione synthesis after 48 hours (P < 0.001) and after 144 hours (P = 0.02), respectively. Oocyte secreted factors also increased viable cell numbers (P < 0.001) in co-cultures together with suppression of progesterone (P < 0.001) and oestradiol (P < 0.001). In granulosa cell only cultures, SCF increased progesterone production in a dose dependent manner (P < 0.001), whereas progesterone synthesis by theca cells was reduced in a dose dependent manner (P = 0.002). Co-cultured cells demonstrated an increase in progesterone production with increasing SCF dose (P < 0.001) and an increase in oestradiol synthesis at the highest dose of SCF (100 ng/ml). In summary, these findings demonstrate the presence of a co-ordinated paracrine interaction between somatic cells and germ cells, whereby oocyte derived signals interact locally to mediate granulosa and theca cell function. SCF has a role in modulating this local interaction. In conclusion, the oocyte is an effective modulator of granulosa-theca interactions, one role being the inhibition of luteinization.

Show MeSH
Related in: MedlinePlus