Impact of media supplements FGF2, LIF and IGF1 on the genome activity of porcine embryos produced in vitro.

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

Meiotic and developmental competence of growing pig oocytes derived from small antral follicles is enhanced in culture medium containing FGF2, LIF, and IGF1 (FLI medium).

BACKGROUND: Oocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles. METHODS: The growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed. RESULTS: The maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF. CONCLUSIONS: This work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

Porcine oocytes matured in a chemically defined medium are transcriptionally active.

The statement that fully-grown porcine oocytes (oocytes from follicles with diameter from 3 to 6 mm) are transcriptionally quiescent is not as strongly supported as it was before. Currently, we know that there is a difference between the transcription profile of germinal vesicle (GV) and metaphase II (MII) oocytes. The goal of our study was to compare the transcription profile of GV, germinal vesicle breakdown (GVBD), metaphase I (MI), and MII oocytes matured in the chemically defined medium FLI. Oocytes were sequenced, and the results were subsequently validated using quantitative reverse transcription polymerase chain reaction (RT-qPCR). We detected multiple differentially transcribed mRNAs, of which many were upregulated. Among them we found mRNAs necessary for protein production, mitochondrial functions and cytoplasmic maturation. Collectively, these data support the hypothesis that transcription activity in fully-grown porcine oocytes is necessary for key processes during their successful maturation in vitro in a chemically defined maturation medium.

Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids: the potential association with oocyte quality.

BACKGROUND: Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable microenvironment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV) miRNAs in porcine FFs and their potential association with oocyte quality. METHODS: Individual aspirated oocytes were stained with lissamine green B stain (LB), a vital stain for oocyte quality, and each oocyte was classified as high-quality (unstained; HQ) or low-quality (stained; LQ). FFs corresponding to oocytes were pooled together into HQ and LQ groups. Small-EVs were isolated from FFs, characterized, and their miRNA cargo was identified using the Illumina NovaSeq sequencing platform. Additionally, s-EVs from the HQ and LQ groups were utilized to investigate their effect on oocyte development after co-incubation during in vitro maturation. RESULTS: A total of 19 miRNAs (including miR-125b, miR-193a-5p, and miR-320) were significantly upregulated, while 23 (including miR-9, miR-206, and miR-6516) were downregulated in the HQ compared to the LQ group. Apoptosis, p53 signaling, and cAMP signaling were among the top pathways targeted by the elevated miRNAs in the HQ group while oocyte meiosis, gap junction, and TGF-beta signaling were among the top pathways targeted by the elevated miRNAs in the LQ group. The supplementation of small-EVs during maturation does not affect the oocyte developmental rates. However, LQ s-EVs increase the proportion of oocytes with homogeneous mitochondrial distribution and decrease the proportion of heterogeneous distribution. CONCLUSION: Our findings indicated that FF-EVs contain different miRNA cargos associated with oocyte quality and could affect the mitochondrial distribution patterns during oocyte maturation.

SCF Ligases and Their Functions in Oogenesis and Embryogenesis-Summary of the Most Important Findings throughout the Animal Kingdom.

SCF-dependent proteolysis was first discovered via genetic screening of budding yeast almost 25 years ago. In recent years, more and more functions of SCF (Skp1-Cullin 1-F-box) ligases have been described, and we can expect the number of studies on this topic to increase. SCF ligases, which are E3 ubiquitin multi-protein enzymes, catalyse protein ubiquitination and thus allow protein degradation mediated by the 26S proteasome. They play a crucial role in the degradation of cell cycle regulators, regulation of the DNA repair and centrosome cycle and play an important role in several diseases. SCF ligases seem to be needed during all phases of development, from oocyte formation through fertilization, activation of the embryonic genome to embryo implantation. In this review, we summarize known data on SCF ligase-mediated degradation during oogenesis and embryogenesis. In particular, SCFßTrCP and SCFSEL-10/FBXW7 are among the most important and best researched ligases during early development. SCFßTrCP is crucial for the oogenesis of Xenopus and mouse and also in Xenopus and Drosophila embryogenesis. SCFSEL-10/FBXW7 participates in the degradation of several RNA-binding proteins and thereby affects the regulation of gene expression during the meiosis of C. elegans. Nevertheless, a large number of SCF ligases that are primarily involved in embryogenesis remain to be elucidated.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Improving the Quality of Oocytes with the Help of Nucleolotransfer Therapy.

The nucleolus is an important nucleus sub-organelle found in almost all eukaryotic cells. On the one hand, it is known as a differentiated active site of ribosome biogenesis in somatic cells, but on the other hand, in fully grown oocytes, zygotes, and early embryos (up to the major embryonic genome activation), it is in the form of a particular homogenous and compact structure called a fibrillar sphere. Nowadays, thanks to recent studies, we know many important functions of this, no doubt, interesting membraneless nucleus sub-organelle involved in oocyte maturation, embryonic genome activation, rRNA synthesis, etc. However, many questions are still unexplained and remain a mystery. Our aim is to create a comprehensive overview of the recent knowledge on the fibrillar sphere and envision how this knowledge could be utilized in further research in the field of biotechnology and nucleolotransfer therapy.

Inhibition of miR-152 during In Vitro Maturation Enhances the Developmental Potential of Porcine Embryos.

Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes. The inhibition (Inh) of miR-152 during oocyte maturation does not affect the MII and cleavage rates, however it significantly enhances the blastocyst rate compared to the overexpression (OvExp) and control groups. Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFß, Hippo, FoxO, and Wnt signaling) that are enriched in the predicted target genes of miR-152. Gene expression analysis revealed that IGF1 was significantly up-regulated in the Inh group and downregulated in the OvExp group of oocytes. Moreover, IGF1R was significantly upregulated in the Inh oocyte group compared to the control one and IGFBP6 was downregulated in the Inh oocyte group compared to the other groups. Blastocysts developed from the OvExp oocytes exhibited an increase in miR-152 expression, dysregulation in some quality-related genes, and the lowest rate of blastocyst formation. In conclusion, our results demonstrate a negative correlation between miR-152 expression level and blastocyst rate in pigs. This correlation could be through targeting IGF system components during oocyte development.

Assessing the effect of interspecies oocyte nucleolar material dosage on embryonic development.

Sequence differences are considered to be the basic cause of developmental failure in interspecies embryos when more distant species are combined. However, other phenomena, such as insufficient or excessive quantity of specific cellular factors, might also influence the outcome. These effects are usually not considered. One of the organelles shown to contain different amount of proteins is the oocyte nucleolus-like body. Here we show that upon interspecies transfer, a single porcine nucleolus-like body is unable to support the development of a mouse parthenogenetic embryo derived from an enucleolated oocyte. However, when the amount of the porcine nucleolar material is increased to equalize the amount of mouse nucleolar material by transferring two nucleolus-like bodies, mouse embryos are able to pass the developmental block elicited by enucleolation. These embryos progress to the blastocyst stage at rates comparable to controls. Thus, using the model of an interspecies nucleolus-like body transplantation between mouse and pig oocytes, we show that an inadequate amount of nucleolar factors, rather than the species origin, affects the development. In a wider context of interspecies nuclear transfer schemes, the observed incompatibility between more distant species might not stem simply from sequence differences but also from improper dosage of key cellular factors.

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential in vitro.

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P<0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.

Intranuclear characteristics of pig oocytes stained with brilliant cresyl blue and nucleologenesis of resulting embryos.

Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus-oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB-) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB- oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB- embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB- embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB- oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.

Maternally inherited rRNA triggers de novo nucleolus formation in porcine embryos.

SummaryThe present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 µg/ml for inhibition of RPI; 2.0 µg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 µg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI inhibition, nucleolar precursors transformed into segregated fibrillogranular structures, however no fibrillar centres were observed. The localization of rDNA and clusters of rRNA were detected in 57.1% immunoprecipitated (IP) analyzed nucleoli and dispersed RPI; 30.5% of nuclei showed large deposits of nucleolar proteins. Embryos from the AD-2.0 group did not display any transcriptional activity. Nucleolar formation was completely blocked, however 39.4% of nuclei showed rRNA clusters; 85.7% of nuclei were co-localized with nucleolar proteins. Long-term transcriptional inhibition resulted in the lack of ARG and RPI labelling; 40% of analyzed nuclei displayed the accumulation of rRNA molecules into large foci. In conclusion, maternally inherited rRNA co-localized with rDNA and nucleolar proteins can initiate a partial nucleolar assembly, resulting in the formation of fibrilogranular structures independently on activation of RPI-mediated transcription.