Cells responding to hedgehog signaling contribute to the theca of ovarian follicles

Cell-fate mapping was used to identify cells that respond to the hedgehog (HH) signaling pathway and that are incorporated into the theca cell layer during ovarian follicle development. Expression of Gli1 is increased by HH signaling and can be used as a marker of cells responsive to HH in reporter mice. In transgenic Gli1ERcre/tdT mice, injection of tamoxifen (TAM) induces cre-mediated recombination and expression of td tomato (tdT) which leads to permanent fluorescent marking of cells expressing Gli1 and their progeny. The identity of tdT-positive cells was determined by co-staining ovaries for endothelial cells (CD31), pericytes (CSPG4), vascular smooth muscle cells (VSMC; smooth muscle actin) and steroidogenic cells (cytochrome P450 17A1). Gli1ERcre/tdT mice were injected with TAM on the day of birth. Cells positive for tdT in 2-day-old mice were identified as pericytes, located primarily in the medulla of the ovary in close proximity to endothelial cells. In both prepubertal mice and adult mice treated with equine chorionic gonadotropin to induce the formation of preovulatory follicles, tdT-positive cells were located within the theca cell layer and were identified as pericytes, VSMC and steroidogenic theca cells. Granulosa cells are known to express two HH ligands, Indian HH and desert HH (DHH). In DHHcre/tdT reporter mice, endothelial cells were marked as tdT-positive indicating that endothelial cells, in addition to granulosa cells, express Dhh in the ovary. These findings suggest that HH signaling may stimulate the development of the vasculature along with steroidogenic capacity of the theca layer during follicle development.

In vitro development of mechanically and enzymatically isolated cat ovarian follicles

Graphical Abstract Isolation of ovarian follicles is a key step in culture systems for large mammalian species to promote the continued growth of follicles beyond the preantral stage in fertility preservation efforts. Still, mechanical isolation methods are user-skill dependent and time-consuming, whereas enzymatic strategies carry increased risk of damaging theca cell layers and the basement membranes. Here, we sought to determine an optimal method to rescue domestic cat (Felis catus) early antral and antral stage follicles from ovarian tissue and to evaluate the influence of isolation strategy on follicle development, survival, and gene expression during 14 days of in vitro culture in alginate hydrogel. Mechanical isolation was compared with 90 min digestion in 0.7 and 1.4 Wünsch units/mL Liberase blendzyme (0.7L and 1.4L, respectively). Mechanical isolation resulted in improved follicle growth and survival, and better antral cavity and theca cell maintenance in vitro, compared with 1.4L (P < 0.05) but displayed higher levels of apoptosis after incubation compared with enzymatically isolated follicles. However, differences in follicle growth and survival were not apparent until 7+ days in vitro. Expressions of CYP19A1, GDF9, LHR, or VEGFA were similar among isolation-strategies. Cultured follicles from all isolation methods displayed reduced STAR expression compared with freshly isolated follicles obtained mechanically or via 0.7L, suggesting that prolonged culture resulted in loss of theca cell presence and/or function. In sum, early antral and antral stage follicle development in vitro is significantly influenced by isolation strategy but not necessarily observable in the absence of extended culture. These results indicate that additional care must be taken in follicle isolation optimizations for genome rescue and fertility preservation efforts. Lay summary The ovary contains hundreds of eggs with only a select few developing from an immature stage through to ovulation over the course of an animal's lifetime. Rescue of eggs from this pool, and the ability to grow them in culture to a mature stage, would be incredibly valuable for fertility preservation efforts in both humans and endangered species. Currently, the isolation of ovarian follicles (eggs with their surrounding helper cells) is a key step in culture systems for large mammalian species, to promote continued growth. Yet, isolation methods may affect the follicle’s future developmental capacity. We evaluated two isolation strategies, mechanical micro-dissection (needle/scalpel blade) and enzymatic digestion (using Liberase blendzyme) on ovaries of domestic cats obtained via routine spay procedures. Mechanically isolated follicles displayed improved growth, survival, and indications of developmental competence in 14-day culture, compared with high concentration (1.4 Wünsch units/mL) enzyme-isolated follicles. However, mechanical isolation was not different from low (0.7 Wünsch units/mL) enzyme for these metrics, or for expression of key genes indicative of follicular cell functions. Further, differences in follicle growth/survival were not apparent until 7+ days in culture. Thus, ovarian follicle isolation strategies influence developmental potential in culture, and extended culture will be required to identify optimal methods for fertility preservation efforts.