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Effect of vitrification temperature and cryoprotectant concentrations on the mRNA transcriptome of bovine immature oocytes

ABSTRACT: The present study mainly aimed to investigate the effect of vitrification temperature (VT) and cryoprotective agent concentrations (CPAs) on the mRNA transcriptome of bovine immature oocytes (BIOs). Cumulus oocyte complexes (COCs) were randomly divided into the following five groups: fresh oocytes (control), oocytes vitrified in liquid helium (LHe; -269℃) with 5.6 M CPAs (LHe 5.6 M), oocytes vitrified in LHe with 6.6 M CPAs (LHe 6.6 M), oocytes vitrified in liquid nitrogen (LN; -196℃) with 5.6 M CPAs (LN 5.6 M), and oocytes vitrified in LN with 6.6 M CPAs (LN 6.6 M). mRNA transcriptomes of each sample were analyzed by Smart-seq4, and the diﬀerentially expressed genes (DGEs) were detected by edgeR (p≤0.05; fold-change≥2). Compared with the control group, 505 DGEs were detected with 342 up-regulated and 163 down-regulated genes in LHe 5.6 M; 609 DGEs were detected with 493 up-regulated and 116 down-regulated gene in LHe 6.6 M; 218 DGEs were determined with 101 up-regulated and 117 down-regulated genes in LN 5.6 M; and 221 DGEs were detected with 104 up-regulated and 117 down-regulated genes in LN 6.6 M. LHe vitrification affected the mRNA transcriptome of vitrified BIOs mainly by up-regulating gene expression. Reduced CPAs (5.6 M) decreased the effect of vitrification on mRNA transcriptome when LHe vitrification was used. Among all the DGEs closely related to bovine immature oocytes, the genes possibly related to VT were ND2, MPV17L2, PIF1, LPIN1, IMP3, BRD1, DCTN3, DERA, ATP7B, NEK5, HVCN1 and MARK2. The gene that may be associated with CPAs is CC2D2A. Genes that may be affected by both VT and CPAs were PGK1, SLC7A3, FITM2, NPM3, ISCU, CWC15 and PSAP. DERA was up-regulated when reduced VT (-269 °C) was used, which may have contributed to the recovery and stress prevention of vitrified oocyte.

ORGANISM(S): Bos taurus

PROVIDER: GSE133550 | GEO | 2022/04/02

REPOSITORIES: GEO

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Project description:Vitrification is a method for long-term biological sample cryopreservation without causing intra- and extra-cellular ice formation. We recently established a novel closed vitrification system to cryopreserve mouse ovarian follicles. Using the 3D alginate hydrogel encapsulated in vitro follicle growth (eIVFG) method, we have demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes. Our recent study further demonstrated the faithful preservation of molecular signatures of follicle-stimulating hormone (FSH)-stimulated follicle maturation in vitrified follicles. However, it is unknown whether ovulation, another crucial gonadotropin-dependent follicular event, and involved ovulatory gene regulatory pathways are well conserved in vitrified follicles. Fresh and vitrified follicles grown for 8 days by eIVFG were collected at 0, 1, 4 and 8-hour post human chorionic gonadotropin (hCG) treatment for the single-follicle RNA sequencing. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Furthermore, the expression of several genes essential for ovulation were comparable between vitrified and fresh follicles. 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, the integration of vitrification of immature follicles and eIVFG has a great potential to serve as an additional fertilization preservation approach for young cancer patients and endangerers species. Moreover, follicle vitrification enables a high-content ovarian follicle biobank, which can greatly improve the throughput of eIVFG for studying ovulation biology, anovulatory disease, toxicology, and novel contraceptive drug development targeting ovulation.

Project description:Vitrification is a method for long-term biological sample cryopreservation that transforms cells into a glass-like state by cooling without causing intra- and extra-cellular ice formation, which is a major driver of cell cryoinjury. Using an alginate hydrogel encapsulated in vitro follicle growth (eIVFG) system, we have recently demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes as well as comparable expression levels of several genes that are essential for gonadotropin-dependent folliculogenesis and oogenesis. However, it is unknown whether vitrification preserves the molecular signatures of folliculogenesis at the whole transcriptomic level, which is the primary research focus in this study. Multilayered secondary follicles with a diameter of 130 -160 µm were mechanically isolated from 16-day-old CD-1 female mice and vitrified using the closed vitrification method. After a 2-week storage in liquid nitrogen, vitrified follicles were warmed, and together with freshly-harvested follicles, cultured for 8 days using the eIVFG method. Next, mature antral follicles from day 8 were collected for single-follicle RNA sequencing using the SMART-seq RNA-seq method. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Using the criterion of fold change >2 or <0.5 and a false discovery rate (FDR) adjusted p-value < 0.05, there were 35 differentially expressed genes out of a total of 11,806 detected genes between vitrified and fresh follicles, among which 17 were up-regulated and 18 were down-regulated in vitrified follicles. To the best of our knowledge, none of those differentially expressed genes identified between vitrified and fresh follicles have been shown to be critical to folliculogenesis and oogenesis. Meanwhile, gene ontology (GO) and KEGG pathway analysis over these 35 genes using the functional annotation tool DAVID revealed that no GO terms or signaling pathways were significantly enriched. Furthermore, the expression of several genes essential for the gonadotropin-dependent folliculogenesis and oogenesis were comparable between vitrified and fresh follicles, including granulosa cell-specific genes (Cyp19a1, Amh, Inha, Inhba, Inhbb, and Fshr), theca cell-specific genes (Cyp17a1, Col1a2, and Col3a1), and oocyte-specific genes (Gdf9, Bmp15, Zp1, Zp2, and Zp3). Equally important, Lhcgr, the gene that is primarily expressed in theca cells of preantral follicles and then greatly induced in mural granulosa cells in antral follicles to prepare for the luteinizing hormone (LH) surge and ovulation, also had comparable mRNA expression levels between vitrified and fresh follicles. Taken together, these results demonstrate that vitrification preserves the follicular cell transcriptome and molecular signatures of gonadotropin-dependent folliculogenesis in the eIVFG system. In conclusion, our study re-evaluates individual follicle cryopreservation by using the closed vitrification method in a more in-depth perspective and proves that vitrification preserves the follicular cell transcriptome in the eIVFG system, thus providing a robust model for fertility preservation, conservation of endangered species, and also establishing a high-content ovarian follicle biobank for studying ovarian biology and female reproductive toxicology.

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Effect of vitrification temperature and cryoprotectant concentrations on the mRNA transcriptome of bovine immature oocytes

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