Not long ago, the IVM of porcine oocytes was improved markedly, which also helped to improve the production of porcine embryos [1]. Yuan et al. (2017) [1] developed a chemically defined medium containing fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) instead of fetal bovine serum (FBS), so-called FLI medium. Thanks to this, previously used undefined sources of proteins, for example in the study Prochazka et al. (2011) [2], were removed from the medium. This improvement opens up a lot of new questions and creates the need to re-check the quality of this new class of oocytes from a different point of view.

While pig oocytes become meiotically competent in early antral follicles, they only achieve full developmental competence after the resumption of meiosis triggered by the pre-ovulatory luteinizing hormone surge. During this period, called cytoplasmic maturation, dramatic changes in organelle distribution and oocyte metabolism occur, oocytes accumulate both mRNAs and proteins and become competent to be fertilized and fully support early embryo development [3].

It was generally accepted that after the growing period of mammalian oocytes (oocytes from follicles from 1.5 to 1.9 mm in diameter), in which they are transcriptionally active, the nucleus of an oocyte becomes transcriptionally inactive. When they reach the fully-grown stage (oocytes from follicles from 3 to 6 mm in diameter), they become transcriptionally quiescent and only utilize transcripts stored during early development. This is characterized by the presence of highly condensed chromatin, encapsulating an electron-dense fibrillar nucleolus [4,5]. Rodman and Bachvarova (1976) [6] have already demonstrated that according to prolonged staining using 3H uridine, there could be some transcription activity in fully-grown mouse oocytes. The same results were published on porcine [7] and human [8] oocytes. However, prolonged autoradiographic labeling might increase the problem with the detection of migrating RNAs and thus provide false positive results [7]. Only relatively recently have there been publications showing transcription activity in porcine oocytes during maturation in which modern methods have been used (microarrays, sequencing). One such study is Budna et al. (2017) [9], in which the authors used Brilliant Cresyl Blue (BCB) staining, which can distinguish BCB negative (BCB-) oocytes that are still growing from BCB positive (BCB+) oocytes, which are fully grown. In that study, only BCB- oocytes were analyzed. Therefore, that study cannot be taken as proof of transcription activity in fully-grown porcine oocytes. In a newer study, Yang et al. (2021) [10], reported many upregulated mRNAs when comparing the transcription profile of fully-grown germinal vesicle (GV) porcine oocytes and those after 44 h of maturation (MII). That study confirmed signs of possible transcription activity even in fully-grown oocytes which were denoted in previous studies as being transcriptionally silenced. In the study by Yang et al. (2020) [11], they were working on a hypothesis that long noncoding RNA (lncRNA) 2193 can regulate the maturation of porcine oocytes. They found that this lncRNA actually regulates germinal vesicle breakdown (GVBD), first polar body (PB1) extrusion and meiotic spindle organization. Therefore, not only the transcriptomic profile of mRNAs, but also the profile of lncRNAs in porcine oocytes plays an important role, as in the study by Jiao et al. (2020) [12]. In that study, they found new lncRNA transcripts that differed between the GV and MII developmental stages.

In our experiments, we measured changes in transcript abundance in fully-grown porcine oocytes collected at four different time points (0 h, 12 h, 28 h, and 44 h) of in vitro maturation in the FLI medium. Each of these time points represents a different developmental stage of porcine oocytes (GV, GVBD, MI, and MII), and therefore helped us to indicate the transcription activity of oocytes throughout the maturation period using next-gen sequencing (NGS) and RT-qPCR. To demonstrate the viability of the culture system, we measured MII ratio and cleavage/blastocyst ratios. To our knowledge, no such study has been published until now, since the previous studies only analyzed the GV and MII stages.