Vitrification preserves follicular transcriptomic dynamics during ex vivo ovulation

Graphical Abstract

Dear Editor, Vitrification is an emerging cryopreservation approach for storing cells or tissues at ultra-low temperatures over an extended period of time.Compared with traditional slowfreezing, vitrification is more cost-effective due to the simple and short process.Vitrification is widely used in assisted reproductive technology to cryopreserve gametes, embryos, or gonad tissues for fertility preservation and infertility treatment.We previously demonstrated that a closed vitrification system preserves murine follicle viability, and vitrified follicles exhibited comparable functions to fresh follicles in a 3D encapsulated in vitro follicle growth (eIVFG) system, including follicle maturation, hormone secretion, ovulation, and resumption of oocyte meiosis [1].Our recent study further demonstrated the faithful preservation of molecular signatures of vitrified murine follicles during follicle-stimulating hormone (FSH)-stimulated maturation [2].However, it is unknown whether ovulation, another crucial gonadotropindependent follicular event, and the involved ovulatory genes and signaling are well conserved in vitrified follicles.
Ovulation is a complex dynamic process by which a surge of the luteinizing hormone (LH) from the anterior pituitary stimulates a fully grown preovulatory follicle to release a fertilizable oocyte.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.
Multilayered secondary follicles isolated from 16-day-old CD-1 mouse ovaries were vitrified as we previously described [1,2,4].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].Fastq files were generated using bcl2fastq and imported into the Partek Flow software.Reads were aligned to the whole mouse genome assembly-mm10 using the HISAT 2 align, quantified by Ensembl Transcripts release 99 using the Partek EM algorithm, and normalized using the Transcripts Per Million (TPM) method, and differential gene expression analysis was performed using the DESeq2(R).Principal component analysis (PCA) showed distinct follicle separation across post-hCG time points, but separation was not seen between fresh and vitrified follicles at each time (Figure 1A).The volcano plots in Supplemental Figure 1 and Supplemental Table 1 showed all identified differentially expressed genes (DEGs) with fold change >2 or <0.5 and false discovery rate adjusted P-value < 0.05 between vitrified and fresh follicles at each time points.There were 52, 130, 30, and 476 DEGs at 0, 1, 4, and 8-h post-hCG, respectively.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis over these DEGs using the functional enrichment tool WebGestalt [8] revealed that none or a few GO terms and signaling pathways were enriched at 0, 1, and 4 h (Supplemental Table 2).At 8 h, DEGs were primarily associated with Cell cycle, guanosine triphosphatases (GTPase) binding, and AMPactivated protein kinase (AMPK) signaling, etc.Whether these DEGs along with enriched GO terms and signaling pathways after vitrification are not sufficient to affect ovulation at the morphological and hormonal levels or they affect molecular events of ovulation and/or subsequent luteinization requires further investigations.
We further calculated different sets of DEGs at 1, 4, and 8 versus 0 h in fresh or vitrified follicles and examined correlations of DEGs between fresh and vitrified follicles at each time point.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.Both the expression levels and temporal patterns of these 100 genes were highly consistent between fresh and vitrified follicles, including many genes that have been reported to critically regulate ovulation (Figure 1C and Supplemental Table 3).In Figure 1D, we provided a more detailed illustration of the consistent transcriptional dynamics of several well-established ovulatory genes, including genes involved in the EGF signaling (Amphiregulin, Areg), cumulus cell expansion (Hyaluronan synthase 2, Has2), follicle rupture (Pgr), inflammation (Prostaglandin-endoperoxide synthase 2, Ptgs2), luteinization (Steroidogenic acute regulatory protein, Star), and proteolysis (Plasminogen activator, urokinase, Plau).
In summary, these results demonstrate that our closed vitrification system preserves follicular transcriptomic dynamics during ex vivo ovulation.Together with our previous findings that vitrification preserves FSH-stimulated follicle maturation [1, 2], the integration of vitrification of immature follicles and eIVFG has a great potential to serve as an additional fertility preservation approach for young cancer patients and endangered species.Moreover, follicle vitrification enables a highquality/content ovarian follicle biobank, which can greatly improve the throughput of eIVFG for studying ovulation biology, anovulatory disease, toxicology, and novel contraceptive development targeting ovulation.Along with follicle maturation, the follicle-enclosed oocyte produces and accumulates maternal mRNAs, proteins, and organelles to acquire meiotic and developmental competence for subsequent fertilization and early embryogenesis.This process, termed oogenesis, largely relies on the bidirectional communications between the oocyte and surrounding follicular somatic cells [10].In future studies, we will determine whether our closed vitrification system preserves oogenesis.
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‡ Jiyang Zhang and Daniela D. Russo equally contributed to this work.

Figure 1 .
Figure 1.Comparison of the follicular transcriptomes during ovulation between fresh and vitrified follicles using single-follicle RNA-seq.(A) PCA of the first two principal components between fresh and vitrified follicles at different time points post-hCG.Ellipses represent 95% confidence intervals.n = 3-18 follicles in each group.(B) Pearson correlation analysis of DEGs for fresh and vitrified follicles at 1, 4, and 8-h post-hCG, respectively.(C) mRNA expression levels (Log2 (TPM+1)) at all four time points post-hCG for the top 100 up-regulated genes identified at 4-h post-hCG.(D) mRNA expression levels (Log2 (TPM+1)) of representative ovulatory genes in fresh (F) and vitrified (V) follicles at different time points post-hCG.