Synergy of Paracrine Signaling During Early-Stage Mouse Ovarian Follicle Development In Vitro

Abstract

Introduction-Paracrine signals, such as soluble cytokines and extracellular matrix cues, are essential for the survival and development of multicellular ovarian follicles. While it is well established that hydrogel-based culture systems successfully support the growth of late-stage follicles for fertility preservation, growing small, early-stage ovarian follicles still proves to be challenging. We hypothesized that paracrine factors secreted from neighboring follicles may be crucial for improving the survival of early-stage follicles in vitro. Methods-To test our hypothesis, we investigated the bidirectional crosstalk of the paracrine signals, such as cellsecreted cytokines, sex hormones and transcription factors (TFs), in follicles encapsulated and cultured for 12 days in alginate in groups of five (59) and ten (109). Results-The differential profiles of TF activity and secretome during folliculogenesis were analyzed using TRanscriptional Activity CEllular aRray (TRACER) and data-driven multivariate modeling approach. The mechano-and oxygenresponsive TFs, NF-jB and HIF1, exhibited a unique upregulation signature in 109 follicles. Consistently, levels of proangiogenic factors, such as VEGF-A and angiopoietin-2, were significantly higher in 109 follicles than those in 59 follicles, reaching 269.77 and 242.82 pg/mL on the last day of culture. The analysis of TRACER and secreted cytokines also revealed critical early interactions between cytokines and TFs, correlating with the observed phenotypical and functional differences between conditions. Conclusions-We identified unique signatures of synergism during successful early-stage ovarian follicle development. These findings bring us closer to understanding of mechanisms underlying the downstream effects of interactions between the extracellular microenvironment and early-stage folliculogenesis in vitro.

Capitalizing on transcriptome profiling to optimize and identify targets for promoting early murine folliculogenesis in vitro

“…They grew at different rates, reaching a mean diameter of 212.36 ± 10.04 and 300.02 ± 8.00 µm (mean ± SEM) respectively (Fig. 1 B, D), corroborating previous findings 11 , 21 . There were no statistical differences in follicle survival rate between 5 and 10X conditions (Fig.…”

Adipose-derived stem cell-secreted factors promote early stage follicle development in a biomimetic matrix

“…In contrast, the 3D CM profile shows consistent concentrations of these factors over time, which corresponds to clustering of all time points in the second quadrant of the scores plot. These factors have important roles in regulating folliculogenesis 13,40 and promoting angiogenesis 41,42 , and therefore association of these factors with the co-culture and 2D CM points may account for slightly increased growth in these conditions.…”

“…Alternatively, follicles co-cultured with ovarian stromal cells had improved folliculogenesis outcomes, possibly the result of bidirectional crosstalk between the follicles and stromal cells 11 . Follicles co-cultured in groups of five or ten also show improved follicle growth outcomes over follicles cultured individually in a number dependent manner 12 stressing the importance of crosstalk between follicles and changes in the secretome 13 . These studies suggest that depending on the type of feeder cells and the paracrine mediators they secrete, unidirectional or bidirectional communication may be effective in promoting folliculogenesis.…”

Adipose-derived stem cells promote survival, growth, and maturation of early-stage murine follicles

“…Recent reports of the use of mesenchymal stem cells for the paracrine mediated, restoration of ovarian function in animal models of primary ovarian insufficiency [ 39 – 41 ] provide the basis for our hypothesis that the paracrine signals from ADSCs can rescue individually cultured primary and early secondary ovarian follicles and promote folliculogenesis [ 38 ]. We recently analyzed the secretome of human ADSCs cultured in three-dimensional culture and found statistically significant increases in cytokine production [ 36 ] and gene expression [ 38 ] among genes and cytokines that have previously been established as vital to follicular survival, growth, and development. Upregulated genes included vascular endothelial growth factor A (VEGFA), transforming growth factor-beta 2 (TGF-β2), and hepatocyte growth factor (HGF), which are known to be beneficial for follicular angiogenesis, follicular regulation, and apoptotic suppression, respectively.…”

“…Upregulated genes included vascular endothelial growth factor A (VEGFA), transforming growth factor-beta 2 (TGF-β2), and hepatocyte growth factor (HGF), which are known to be beneficial for follicular angiogenesis, follicular regulation, and apoptotic suppression, respectively. Zhou et al demonstrated an increase in monocyte chemotactic protein-1 (MCP-1) (related to oocyte maturation), Follistatin (regulation of granulosa cell proliferation), and IL-6 (regulator of cumulus cell function) [ 36 ].…”