107 Insights from roe deer oocyte transcriptome across embryonic diapause

Abstract

Embryonic developmental arrest, known as diapause, has been reported in more than 130 species. However, its mechanisms are still not completely understood. In the roe deer, the only known ungulate that exhibits this phenomenon, diapause lasts for approximately 5 months, starting after the rut period in mid-July to early August and ending with embryo elongation and implantation in December/January. Little is known regarding oocyte characteristics during this period. Here, we analysed the roe deer oocyte transcriptome as a model to understand diapause effects on oocyte features during embryonic developmental arrest and reactivation. During regular hunting, immature oocytes were obtained by ovary slicing from diapause and nondiapause stages, and classified according to morphological characteristics. Only oocytes with >2 layers of compact cumulus cells and cytoplasm from 30 hunted females were used for analyses. Immature oocytes were denuded and snap frozen. Additional oocytes were cultured in maturation medium for 20-24 h. Matured oocytes with a present polar body were snap frozen. Two pools of 10 immature and mature oocytes for both diapause and nondiapause stages were included (at least 4 donors/pool). Oocyte pools were processed using the Smart-seq 2 single-cell protocol (Illumina Inc., San Diego, CA, USA) for full-length cDNA and library preparation. We performed RNA-seq on an Illumina sequencer. The obtained Fastq files were clipped and analysed with a locally installed version of the Galaxy platform. Sequences were mapped against the roe deer transcriptome (unpublished data) and annotated against human and bovine transcripts. Differentially expressed genes (DEG; false discovery rate <1%) were identified using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) in immature (IMM) and mature (M) oocytes from diapause (D) and nondiapause (ND) stages. Additionally, to evaluate the effects of maturation on oocyte transcript abundancy, DEG between IMM and M oocytes for D and ND were identified. Multidimensional scaling resulted in clustering according to oocyte types. Gene ontology terms for biological processes were assigned using ToppCluster tools. A total of 23 066, 23 022, 22 438, and 22 532 transcripts were detected for IMM D, M D, IMM ND and M ND oocytes, respectively. Using false discovery rate filtering, we found 333 and 288 DEG in immature and mature oocytes, respectively. Furthermore, maturation changed the oocyte expression profiles during D (2233 DEG) and ND periods (2589 DEG). Gene ontology classification revealed that most of the DEG in immature oocytes were involved in macromolecule catabolic process and oxidation-reduction process. For mature oocytes, DEG were mostly related to regulation of chromosome condensation and ER and Golgi vesicle-mediated transport. Additionally, the top 20 DEG from IMM D v. M D oocytes were related to mRNA splicing, mRNA metabolic process, and mRNA processing. These processes were not identified in the top 20 DEG from IMM ND v. M ND oocytes. These preliminary results suggest that oocyte transcriptome analysis could disclose new pathways implicated in oocyte competence and embryonic developmental arrest and activation stages.