Vitrification of immature follicles is an emerging approach for studying ovarian biology, toxicology, fertility preservation, and female contraception. We previously used a 3D encapsulated in vitro ...follicle growth (eIVFG) model to demonstrate that cryopreserved follicles using a closed vitrification system exhibit normal follicle development, hormone secretion, and ovulation, and vitrified follicles preserve follicle-stimulating hormone (FSH)-stimulated follicular transcriptome. Here, we investigated whether the molecular signatures of ovulation, another gonadotropin-dependent follicular event, are conserved in vitrified follicles. Preovulatory follicles grown from fresh or vitrified immature follicles in eIVFG were treated with human chorionic gonadotropin (hCG) to trigger ovulation. Follicles were collected at 0, 1, 4, and 8-hour for single-follicle RNA sequencing. Principal component analysis showed that fresh and vitrified follicles were separated into distinct clusters by post-hCG hours, but fresh and vitrified follicles at the same time points largely overlapped. Gene Ontology (GO) and KEGG analysis using identified differentially expressed genes (DEGs) revealed a few enriched GO terms and signaling pathways, which may affect molecular aspects of ovulation and/or subsequent luteinization. Pearson's correlation analysis showed that the fold changes of most DEGs at 1, 4, or 8-hour VS. 0-hour were highly correlated between fresh and vitrified follicles. Moreover, both the expression levels and temporal patterns of well-established ovulatory genes were highly consistent between fresh and vitrified follicles. Taken together, our study demonstrates that the closed vitrification system preserves follicular transcriptomic dynamics during ex vivo ovulation, enabling a high-quality follicle biobank for fertility preservation, endangered species conservation, and studies of ovulation biology, anovulatory disease, toxicology, and novel contraceptive development.
Seq-Well is a high-throughput, picowell-based single-cell RNA-seq technology that can be used to simultaneously profile the transcriptomes of thousands of cells (Gierahn et al. Nat Methods ...14(4):395-398, 2017). Relative to its reverse-emulsion-droplet-based counterparts, Seq-Well addresses key cost, portability, and scalability limitations. Recently, we introduced an improved molecular biology for Seq-Well to enhance the information content that can be captured from individual cells using the platform. This update, which we call Seq-Well S
(S
: Second-Strand Synthesis), incorporates a second-strand-synthesis step after reverse transcription to boost the detection of cellular transcripts normally missed when running the original Seq-Well protocol (Hughes et al. Immunity 53(4):878-894.e7, 2020). This chapter provides details and tips on how to perform Seq-Well S
, along with general pointers on how to subsequently analyze the resultant single-cell RNA-seq data.
