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Paracrine Regulation of Steroidogenesis in Theca Cells by Granulosa Cells Derived from Mouse Preantral Follicles.

Liu X, Qiao P, Jiang A, Jiang J, Han H, Wang L, Ren C - Biomed Res Int (2015)

Bottom Line: Interaction partners of follicular cells play a significant role in steroidogenesis, follicular formation, and development.Thus, GCs were capable of promoting steroidogenesis and LH responsiveness in TCs.This study provided a basis for further exploration of ovarian endocrine mechanism and pathologies.

View Article: PubMed Central - PubMed

Affiliation: Clinical Center of Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261031, China.

ABSTRACT
Interaction partners of follicular cells play a significant role in steroidogenesis, follicular formation, and development. Androgen secreted by theca cells (TCs) can initiate follicle development and ovulation and provide precursor materials for estrogen synthesis. Therefore, studies on ovarian microenvironment will not only lead to better understanding of the steroidogenesis but also have clinical significance for ovarian endocrine abnormalities such as hyperandrogenism in polycystic ovary syndrome (PCOS). This study applied the Transwell coculture model to investigate if the interaction between granulosa and theca cells may affect androgen production in theca cells. Concentrations of testosterone and androstenedione in the spent medium were measured by radioimmunoassay and enzyme linked immunosorbent assay, respectively. The results show that the coculture with granulosa cells (GCs) increases steroidogenesis in TCs. In addition, testosterone and androstenedione productions in response to LH stimulation were also increased in the coculture model. Significantly increased mRNA expressions of steroidogenic enzymes (Star, Cyp11a1, Cyp17a1, and Hsd3b2) were observed in the cocultured TCs. Thus, GCs were capable of promoting steroidogenesis and LH responsiveness in TCs. This study provided a basis for further exploration of ovarian endocrine mechanism and pathologies.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence staining of CYP17A1 and FSHR in theca cells. Scale bar = 100 μm. The purified cells were examined with immunofluorescence method after 24 h. By a series of titration experiments, the density of TCs was adjusted to approximately 70% confluence. FITC (green) staining CYP17A1 indicated the TCs origin. Hoechst staining (blue) indicated the location of cell nuclei. More than 90% of the cells were stained positive for CYP17A1, confirming the purity of TC cells population. Weak staining with FSHR antibody indicated that these TCs cultures were largely free of GCs contamination.
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fig1: Immunofluorescence staining of CYP17A1 and FSHR in theca cells. Scale bar = 100 μm. The purified cells were examined with immunofluorescence method after 24 h. By a series of titration experiments, the density of TCs was adjusted to approximately 70% confluence. FITC (green) staining CYP17A1 indicated the TCs origin. Hoechst staining (blue) indicated the location of cell nuclei. More than 90% of the cells were stained positive for CYP17A1, confirming the purity of TC cells population. Weak staining with FSHR antibody indicated that these TCs cultures were largely free of GCs contamination.

Mentions: After overnight incubation, the wells coated with TCs were washed with medium to remove unattached cells. The cellular morphology and confluence of TCs were observed under the microscope. Typically, the attached cells looked thin and dendritic, indicating their TC origin. By a series of titration experiments, the density of TCs was adjusted to approximately 70% confluence. The purity of TCs was further verified with immunofluorescence staining using a TC-specific marker Cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1, also known as 17a-hydroxylase/17, 20-lyase), and the GC-specific marker Follicle Stimulating Hormone Receptor (FSHR) for reverse identification. The results in Figure 1 demonstrated that more than 90% of the cells were stained positive for CYP17A1, confirming these cells' TC origin. As FSHR is a GC-specific marker not TCs, we used it to confirm that the extracted cells were TCs and contain almost no GCs by reverse identification. Results from control experiments using FSHR antibody did not show significant fluorescence signal, indicating that these TCs cultures were largely free of GC contamination.


Paracrine Regulation of Steroidogenesis in Theca Cells by Granulosa Cells Derived from Mouse Preantral Follicles.

Liu X, Qiao P, Jiang A, Jiang J, Han H, Wang L, Ren C - Biomed Res Int (2015)

Immunofluorescence staining of CYP17A1 and FSHR in theca cells. Scale bar = 100 μm. The purified cells were examined with immunofluorescence method after 24 h. By a series of titration experiments, the density of TCs was adjusted to approximately 70% confluence. FITC (green) staining CYP17A1 indicated the TCs origin. Hoechst staining (blue) indicated the location of cell nuclei. More than 90% of the cells were stained positive for CYP17A1, confirming the purity of TC cells population. Weak staining with FSHR antibody indicated that these TCs cultures were largely free of GCs contamination.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556819&req=5

fig1: Immunofluorescence staining of CYP17A1 and FSHR in theca cells. Scale bar = 100 μm. The purified cells were examined with immunofluorescence method after 24 h. By a series of titration experiments, the density of TCs was adjusted to approximately 70% confluence. FITC (green) staining CYP17A1 indicated the TCs origin. Hoechst staining (blue) indicated the location of cell nuclei. More than 90% of the cells were stained positive for CYP17A1, confirming the purity of TC cells population. Weak staining with FSHR antibody indicated that these TCs cultures were largely free of GCs contamination.
Mentions: After overnight incubation, the wells coated with TCs were washed with medium to remove unattached cells. The cellular morphology and confluence of TCs were observed under the microscope. Typically, the attached cells looked thin and dendritic, indicating their TC origin. By a series of titration experiments, the density of TCs was adjusted to approximately 70% confluence. The purity of TCs was further verified with immunofluorescence staining using a TC-specific marker Cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1, also known as 17a-hydroxylase/17, 20-lyase), and the GC-specific marker Follicle Stimulating Hormone Receptor (FSHR) for reverse identification. The results in Figure 1 demonstrated that more than 90% of the cells were stained positive for CYP17A1, confirming these cells' TC origin. As FSHR is a GC-specific marker not TCs, we used it to confirm that the extracted cells were TCs and contain almost no GCs by reverse identification. Results from control experiments using FSHR antibody did not show significant fluorescence signal, indicating that these TCs cultures were largely free of GC contamination.

Bottom Line: Interaction partners of follicular cells play a significant role in steroidogenesis, follicular formation, and development.Thus, GCs were capable of promoting steroidogenesis and LH responsiveness in TCs.This study provided a basis for further exploration of ovarian endocrine mechanism and pathologies.

View Article: PubMed Central - PubMed

Affiliation: Clinical Center of Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261031, China.

ABSTRACT
Interaction partners of follicular cells play a significant role in steroidogenesis, follicular formation, and development. Androgen secreted by theca cells (TCs) can initiate follicle development and ovulation and provide precursor materials for estrogen synthesis. Therefore, studies on ovarian microenvironment will not only lead to better understanding of the steroidogenesis but also have clinical significance for ovarian endocrine abnormalities such as hyperandrogenism in polycystic ovary syndrome (PCOS). This study applied the Transwell coculture model to investigate if the interaction between granulosa and theca cells may affect androgen production in theca cells. Concentrations of testosterone and androstenedione in the spent medium were measured by radioimmunoassay and enzyme linked immunosorbent assay, respectively. The results show that the coculture with granulosa cells (GCs) increases steroidogenesis in TCs. In addition, testosterone and androstenedione productions in response to LH stimulation were also increased in the coculture model. Significantly increased mRNA expressions of steroidogenic enzymes (Star, Cyp11a1, Cyp17a1, and Hsd3b2) were observed in the cocultured TCs. Thus, GCs were capable of promoting steroidogenesis and LH responsiveness in TCs. This study provided a basis for further exploration of ovarian endocrine mechanism and pathologies.

No MeSH data available.


Related in: MedlinePlus