Effects of preovulatory aging on the developmental competence of mouse oocytes

Preovulatory aging of oocytes is caused by a delay in ovulation and it is known to impair postimplantation embryonic development. However, hardly anything is known about the molecular mechanisms associated with preovulatory aging. To investigate several aspects of RNA dynamics in the preovulatory-aged oocytes and possible consequences on early preimplantation development, the present study used a mouse model in which ovulation was postponed with the GnRH antagonist cetrorelix. Preovulatory aging led to a lower number of ovulated oocytes. Furthermore, preovulatory-aged oocytes were more difficult to fertilize, as was demonstrated by a decrease in 2-cell embryo rate after mating compared to controls. These results are in accordance with previous studies on preovulatory aging in several vertebrate models including rats and humans. As an indicator for transcriptional silencing and genome stability during oocyte growth, the epigenetic histone modification H3K9 trimethylation was analyzed using immunofluorescence. No effect of preovulatory aging on H3K9 trimethylation levels was observed. Before transcriptional silencing, mRNAs are stored in the oocyte until recruitment for protein translation later during oocyte maturation. One major key player in the storage and recruitment of maternal transcripts is the RNA-binding protein Ybx2. Immunofluorescent staining showed a significant decrease in Ybx2 protein abundance after preovulatory aging, which might have severe implications for RNA dynamics in the oocyte. Since the RNA-storage potential seemed impaired, transcript levels of selected maternal effect genes were analyzed by qRT-PCR. Maternal effect genes are of crucial importance for oocyte developmental potential because they function in the early embryo before transcription initiates again. Transcript levels of 2 out of 10 investigated ME genes (Smarca4 and Tet3) decreased significantly after preovulatory aging. Additionally, oligo(dT) priming allowed investigation of poly(A) tail length of mRNAs of the candidate maternal effect genes by qRT-PCR. The poly(A) tail determines the translation efficiency of transcripts in the oocyte and early embryo. The majority of genes showed elongation of poly(A) tail length after preovulatory aging. Similar results were also found after in vitro preovulatory aging of oocytes grown in follicle culture, although not the same genes were affected than in vivo. Loss of Ybx2 protein and polyadenylation of maternal effect gene transcripts should occur later during oocyte maturation. Therefore, the results imply premature recruitment of mRNAs for protein translation. This assumption is supported by experiments determining the start of transcription at the 2-cell stage of embryonic preimplantation development in the course of embryonic genome activation. Embryos were incubated in BrUTP, which intercalates into nascent RNA and can by visualized by immunofluorescence. Compared to controls, in embryos derived from preovulatory-aged oocytes an increase in transcription was observed, indicating a precocious start of embryonic genome activation. Last, DNA methylation maintenance of three imprinted genes (H19, Snrpn, Igf2r) during epigenetic reprogramming in the preimplantation embryo was analyzed. Deep amplicon bisulfite sequencing of single 8-cell embryos showed stable maintenance of DNA methylation at the investigated loci. Overall, the results show that the investigated processes are distinctly regulated in the oocyte and early embryo and not all of them are susceptible to preovulatory aging. Nevertheless, preovulatory aging impairs oocyte quality with possible long-term effects on embryonic development and reproductive success.