Unveiling the Differentiation Potential of Ovarian Theca Interna Cells from Multipotent Stem Cell-like Cells.

RESEARCH QUESTION: Theca interna cells (TICs) are an indispensable cell source for ovarian follicle development and steroidogenesis. Recent studies have identified theca stem cells (TSCs) in both humans and animals. Interestingly, TSCs express mesenchymal stem cell (MSC)-related markers and can differentiate into mesenchymal lineages. MSCs are promising for tissue engineering and regenerative medicine due to their self-renewal and differentiation abilities. Therefore, this study investigated the potential origin of TICs from MSCs. DESIGN: Whole ovaries from postmenopausal organ donors were obtained, and their cortex was cryopreserved prior to the isolation of stromal cells. These isolated cells were differentiated in vitro to TICs using cell media enriched with various growth factors and hormones. Immunocytochemistry, an enzyme-linked immunosorbent assay, flow cytometry, and reverse transcription-quantitative polymerase chain were employed at different timepoints. Data were analyzed using one-way ANOVA. RESULTS: Immunocytochemistry showed an increase in TIC markers from day 0 to day 8 and a significant rise in MSC-like markers on day 2. This corresponds with rising androstenedione levels from day 2 to day 13. Flow cytometry identified a decreasing MSC-like cell population from day 2 onwards. The CD13+ cell population and its gene expression increased significantly over time. NGFR and PDGFRA expression was induced on days 0 and 2, respectively, compared to day 13. CONCLUSIONS: This study offers insights into MSC-like cells as the potential origin of TICs. Differentiating TICs from these widely accessible MSCs holds potential significance for toxicity studies and investigating TIC-related disorders like polycystic ovary syndrome (PCOS).

Tandem mass tag-based proteomic analysis of granulosa and theca interna cells of the porcine ovarian follicle following in vitro treatment with vitamin D3 and insulin alone or in combination.

This study was performed to investigate the proteomic basis underlying the interaction between vitamin D3 (VD) and insulin (I) within ovarian follicle using the pig as a model. Porcine antral follicles were incubated in vitro for 12 h with VD alone and I alone or in combination (VD + I) or with no treatment as the control (C). In total, 7690 and 7467 proteins were identified in the granulosa and theca interna compartments, respectively. Comparative proteomic analysis revealed 97 differentially abundant proteins (DAPs) within the granulosa layer and 11 DAPs within the theca interna layer. In the granulosa compartment, VD affected proteome leading to the promotion of cell proliferation, whereas I influenced mainly proteins related to cellular adhesion. The VD + I treatment induced granulosa cell proliferation probably via the DAPs involved in DNA synthesis and the cell cycle regulation. In the theca interna layer, VD alone or in co-treatment with I affected DAPs associated with cholesterol transport and lipid and steroid metabolic processes that was further confirmed by diminished lipid droplet accumulation. SIGNIFICANCE: The application of quantitative proteomics demonstrated for the first time the complexity of VD and I interactions in porcine ovarian follicle, providing a framework for understanding the molecular mechanisms underlying their cross-talk. Although identified DAPs were related to crucial ovarian processes, including the granulosa cell proliferation and cholesterol transport in the theca interna layer, novel molecular pathways underlying these processes have been proposed. The identified unique proteins may serve as indicators of VD and I interactions in both follicle layers, and could be useful biomarkers of ovarian pathologies characterized by impaired VD and I levels, such as polycystic ovary syndrome.

Integrated transcriptomic and metabolomic investigation of the genes and metabolites involved in swine follicular cyst formation.

Follicular cysts are a common reproductive disorder in mammals that is usually caused by stress. However, the pathogenesis of follicular cysts in sows remains unclear. To provide new insights into the mechanisms of follicular cyst formation in pigs, we conducted a combined transcriptomic and metabolomic analysis on theca interna and mural granulosa cells of follicular cysts and mature follicles. We identified 2,533 up-regulated and 1,355 down-regulated genes in follicular cysts, compared with mature follicles. These differentially expressed genes were mainly found in signaling pathways related to tumor formation and cortisol synthesis and secretion as shown by Ingenuity Pathway Analysis, which predicted 4,362 upstream regulatory factors. The combined gene expression and pathway analysis identified the following genes as potential biomarkers for porcine follicular cysts: cytochrome P450 family 2 subfamily C polypeptide 18, L-lactate dehydrogenase, carbamoyl-phosphate synthase, fibroblast growth factor 7, integrin binding sialoprotein, interleukin 23 receptor, prolactin receptor, epiregulin, interleukin 1 receptor type II, arginine vasopressin receptor 1A, fibroblast growth factor 10, claudin 7, G Protein Subunit Gamma 3, cholecystokinin B receptor and cytosolic phospholipase A2. Metabolomics analysis found significant differences in 87 metabolites, which were enriched in unsaturated fatty acid biosynthesis, and sphingolipid signaling pathways. These results provide valuable information on the molecular mechanisms of follicular cyst formation, which may facilitate the development of new therapeutics to prevent and treat follicular cysts.

Effects of interleukin 6 and tumor necrosis factor-α on the proliferation of porcine theca interna cells: Possible role of these cytokines in the pathogenesis of polycystic ovary syndrome.

OBJECTIVE: We studied the effects of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) on the proliferation of porcine theca interna (TI) cells and further elucidated the roles of IL-6 and TNF-α in the pathogenesis of polycystic ovary syndrome. MATERIALS AND METHODS: TI cells were treated with 10 pg/mL, 100 pg/mL, and 1000 pg/mL IL-6 or TNF-α. TI cell proliferation was then examined by carboxyfluorescein diacetate succinimidyl ester labeling and flow cytometry. RESULTS: Cell proliferation was not significantly different in TI cells cultured in medium alone (control) or in the presence of IL-6. At 72 hours of treatment, the mean fluorescence intensity was significantly lower in TI cells treated with 100 pg/mL and 1000 pg/mL TNF-α than in the control (p < 0.05). CONCLUSION: TNF-α, but not IL-6, was able to promote TI cell proliferation. Our results suggest that TNF-α might play a role in hyperandrogenism, cortex thickness, and the increased ovary volume observed in polycystic ovaries.

Expression of orexins and their precursor in the porcine ovary and the influence of orexins on ovarian steroidogenesis in pigs.

Orexins A and B are hypothalamic neuropeptides associated with homeostasis and the reproductive system. The aim of the study was to compare the expression of the prepro-orexin gene and the intensity of orexins immunoreactivity in the porcine ovary (corpora lutea, granulosa and theca interna cells) during four different stages of the oestrous cycle (days: 2-3, 10-12, 14-16 and 17-19) and to examine the in vitro effect of orexins on the secretion of steroid hormones by porcine luteal, granulosa and theca interna cells. The highest expression of prepro-orexin mRNA was observed in theca interna cells on days 17-19 of the oestrous cycle. The highest content of immunoreactive orexin A was noted in corpora lutea on days 10-12 and the highest level of immunoreactive orexin B on days 14-16 of the cycle. Immunoreactive orexin A concentrations were higher in theca interna cells than in granulosa cells, whereas similar levels of immunoreactive orexin B were observed in both cell types. Under in vitro conditions, at the concentration of 10 nM, orexins A and B inhibited FSH-induced oestradiol secretion by granulosa cells. The obtained results suggest that the pattern of orexin peptide expression in the porcine ovary is related to the animals' hormonal status. Our findings imply that orexins can affect porcine reproductive functions through modulation of ovarian steroidogenesis.