Anex vivoovulation system enables the discovery of novel ovulatory pathways and nonhormonal contraceptive candidates

Abstract

Ovulation is an integral part of the women’s menstrual cycle and fertility. Understanding the mechanisms of ovulation has broad implications for the treatment of anovulatory diseases and development of novel contraceptives. Now, few studies have developed effective models that both faithfully recapitulate the hallmarks of ovulation and possess scalability. We established a 3D encapsulated in vitro follicle growth (eIVFG) system that recapitulates folliculogenesis and produces follicles that undergo ovulation in a controlled manner. Here, we determined whether ex vivo ovulation preserves molecular signatures of ovulation and demonstrated its use in discovering novel ovulatory pathways and non-hormonal contraceptive candidates through a high-throughput ovulation screening. Mature murine follicles from eIVFG were induced to ovulate ex vivo using human chorionic gonadotropin and collected at 0, 1, 4, and 8 hours post-induction. Phenotypic analyses confirmed key ovulatory events, including cumulus expansion, oocyte maturation, follicle rupture, and luteinization. Single-follicle RNA-sequencing analysis revealed the preservation of ovulatory genes and dynamic transcriptomic profiles and signaling. Soft clustering identified distinct gene expression patterns and new pathways that may critically regulate ovulation. We further used this ex vivo ovulation system to screen 21 compounds targeting established and newly-identified ovulatory pathways. We discovered that proprotein convertases activate gelatinases to sustain follicle rupture and do not regulate luteinization and progesterone secretion. Together, our ex vivo ovulation system preserves molecular signatures of ovulation, presenting a new powerful tool for studying ovulation and anovulatory diseases as well as for establishing a high-throughput ovulation screening to identify novel non-hormonal contraceptives for women.

Dose-response functional and transcriptomic effects of follicle-stimulating hormone onex vivomouse folliculogenesis

“…These genes have been implicated in inflammation during ovulation (96). Using the same eIVFG system, our published RNA-seq data also discovered a significant up-regulation of Nts and Tnfrsf9 by 4- and 195-fold, respectively, during ex vivo ovulation (45). These results suggest that excessive FSH promotes the expression of proinflammatory genes; despite that the induction of these transcripts is not sufficient to trigger ovulation, they may cause ovarian inflammation.…”

Ovarian disrupting effects and mechanisms of long- and short-chain per- and polyfluoroalkyl substances in mice

“…This procedure enabled us to obtain 150 follicles from 3-5 prepubertal mice. Isolated follicles were encapsulated for in vitro culture as we previously described [77][78][79][80][81]. Briefly, follicles were encapsulated in 0.5% (w/v) alginate hydrogel (Sigma-Aldrich, St. Louis, MO).…”

Vitrification preserves follicular transcriptomic dynamics during ex vivo ovulation

“…LH binds to its surface receptor in mural granulosa cells and theca cells of a preovulatory follicle, and activates a series of signaling molecules to drive key ovulatory events, including follicle rupture, cumulus cell expansion, and resumption of oocyte meiosis I. We have recently demonstrated that the eIVFG-based ex vivo ovulation not only morphologically recapitulates these key ovulatory events but also conserves the ovulatory gene regulatory pathways, such as those related to epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase 1/2 (ERK1/2), progesterone receptor (PGR), inflammation, and proteolysis [3]. Herein, we further determine whether vitrification of immature follicles preserves the ovulation potential at molecular levels when follicles mature to the preovulatory stage in eIVFG.…”

“…With fresh follicles as the control, vitrified follicles were warmed and cultured for 8 days in eIVFG [5, 6]. Vitrified and fresh follicles were grown to preovulatory stage and were freed from alginate encapsulation and treated with 1.5 IU/mL human chorionic gonadotropin (hCG) to induce ovulation ex vivo [3]. Follicles were collected at 0, 1, 4, and 8-h post-hCG for single-follicle ribonucleic acid sequencing (RNA-seq, GEO number: GSE227091) using an optimized SMART-seq2 protocol [7].…”

“…The fold changes of most DEGs at each time points were highly correlated between fresh and vitrified follicles with Pearson correlation coefficients (R) of 0.9443, 0.9589, and 0.9307 at 1, 4, and 8 h, respectively (Figure 1B). The majority of LH target genes are highly induced and peak in follicular cells at 4 h in both in vivo and in vitro ovulation models [3, 9]. We next selected the top 100 up-regulated ovulatory genes at 4-h post-hCG versus 0 h in fresh follicles and compared the temporal log-transformed expression patterns of these 100 genes at all four post-hCG time points.…”