Summary: Mammalian oocyte maturation is achieved when oocytes reach metaphase II (MII) stage, and accumulate mRNA and proteins in the cytoplasm following fertilization. It has been shown that oocytes investigated before and after in vitro maturation (IVM) differ significantly in transcriptomic and proteomic profiles. Additionally, folliculogenesis and oogenesis is accompanied by morphogenetic changes, which significantly influence further zygote formation and embryo growth. This study aimed to determine new transcriptomic markers of porcine oocyte morphogenesis that are associated with cell maturation competence. An Affymetrix microarray assay was performed on an RNA template isolated from porcine oocytes before (n = 150) and after (n = 150) IVM. The brilliant cresyl blue (BCB) staining test was used for identification of cells with the highest developmental capacity. DAVID (Database for Annotation, Visualization, and Integrated Discovery) software was used for the extraction of the genes belonging to a cell morphogenesis Gene Ontology group. The control group consisted of freshly isolated oocytes. In total, 12,000 different transcripts were analysed, from which 379 genes were downregulated and 40 were upregulated in oocytes following IVM. We found five genes, SOX9, MAP1B, DAB2, FN1, and CXCL12, that were significantly upregulated in oocytes after IVM (in vitro group) compared with oocytes analysed before IVM (in vivo group). In conclusion, we found new transcriptomic markers of oocyte morphogenesis, which may be also recognized as significant mediators of cellular maturation capacity in pigs. Genes SOX9, MAP1B, DAB2, FN1, and CXCL12 may be involved in the regulation of the MII stage oocyte formation and several other processes that are crucial for porcine reproductive competence.

The key mechanisms responsible for achievement of full reproductive and developmental capability in mammals are the differentiation and transformation of granulosa cells (GCs) during folliculogenesis, oogenesis, and oocyte maturation. Although the role of 17 beta-estradiol (E2) in ovarian activity is widely known, its effect on proliferative capacity, gap junction connection (GJC) formation, and GCs-luteal cells transformation requires further research. Therefore, the goal of this study was to assess the real-time proliferative activity of porcine GCs in vitro in relation to connexin (Cx), luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), and aromatase (CYP19A1) expression during short-term (168 h) primary culture. The cultured GCs were exposed to acute (at 96 h of culture) and/or prolonged (between 0 and 168 h of culture) administration of 1.8 and 3.6 μM E2. The relative abundance of Cx36, Cx37, Cx40, Cx43, LHR, FSHR, and CYP19A1 mRNA was measured. We conclude that the proliferation capability of GCs in vitro is substantially associated with expression of Cxs, LHR, FSHR, and CYP19A1. Furthermore, the GC-luteal cell transformation in vitro may be significantly accompanied by the proliferative activity of GCs in pigs.

Maturation of cumulus–oocyte complexes (COCs) is crucial for further successful monospermic fertilization, embryo growth, and implantation. All these events are accompanied by proliferation and differentiation of cumulus cells. The migration of COCs to the oviduct after ovulation and the interaction between female gametes and/or embryos with maternal tissues are still poorly recognized on the molecular level. This study was aimed to first demonstrate the mRNA expression profile of cell migration markers during different stages of porcine oocytes maturation and developmental capability in vitro. The COCs were collected from a total of 45 pubertal crossbred Landrace gilts, brilliant cresyl blue (BCB) stained, and analyzed before (n = 150) or after (n = 150) in vitro maturation (IVM). Using the Affymetrix^® Porcine Gene 1.1 ST Array, the expression profile of 12,258 porcine transcripts was examined. We found nine genes involved in cell migration mechanisms, that is, PLD1, KIT, LAMA2, MAP3K1, VEGFA, TGFBR3, INSR, TPM1, and RTN4. These genes were upregulated in porcine oocytes before IVM as compared with post-IVM expression analysis. Moreover, important mechanisms of biological interaction between VEGFA–KIT and VEGFA–INSR were also observed. The upregulation and/or downregulation of selected mRNAs expression after microarray assays was checked and approved by real-time quantitative polymerase chain reaction. We suggest that several genes, including LAMA2 or TPM1, encode proteins participating in the formation of the oocyte's protein architecture such as microtubules and kinetochore reorganization. As the expression of all “migration regulatory genes” investigated in this study was significantly upregulated in oocytes before IVM, we conclude that they may contribute to the maturational capability of porcine oocytes. However, migration potency of COCs is not accompanied by achievement of the MII stage by porcine oocytes in vitro. The investigated genes such as PLD1, KIT, LAMA2, MAP3K1, VEGFA, TGFBR3, INSR, TPM1, and RTN4 may be recognized as a new marker of porcine oocytes maturational competence during in vitro culture.

Proper oocyte maturation in mammals produces an oocyte capable of monospermic fertilization and embryo preimplantation. The cumulus-oocyte complexes (COCs), surrounding an oocyte, play a significant role in oocyte maturation. During this process, when the COCs undergo cumulus expansion wherein tightly compact cumulus cells (CCs) form a dispersed structure, permanent biochemical and molecular modifications occur in the maturing oocytes, indicating that the gene expression between immature and mature oocytes differs significantly. This study focuses on the genes responsible for the cellular components of morphogenesis within the developing oocyte. Brilliant cresyl blue (BCB) was used to determine the developmental capability of porcine oocytes. The immature oocytes (GV stage) were compared with matured oocytes (MII stage), using microarray and qRT-PCR analysis to track changes in the genetic expression profile of transcriptome genes. The data showed substantial upregulation of genes influencing oocyte’s morphology, cellular migration and adhesion, intracellular communication, as well as plasticity of nervous system. Conversely, downregulation involved genes related to microtubule reorganization, regulation of adhesion, proliferation, migration and cell differentiation processes in oocytes. This suggests that most genes recruited in morphogenesis in porcine oocyte in vitro, may have cellular maturational capability, since they have a higher level of expression before the oocyte’s matured form. It shows the process of oocyte maturation and developmental capacity is orchestrated by significant cellular modifications during morphogenesis.