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

Abstract

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.

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

“…It has been used previously for the non-invasive morphological assessment of porcine oocyte quality since it enables the detection of oocytes in the pre-apoptotic stage, expressing high levels of TP53, but still with low levels of pro-apoptotic genes [ 18 ]. Moreover, in another study from our group, Bartkova et al [ 17 ] reported LB staining as a non-invasive oocyte selection method that can detect cellular membrane damage in porcine COCs. Although oocyte staining with such stains is considered a non-invasive method, it is still not the optimal approach for oocyte selection, since further treatment and incubation steps need to be incorporated to evaluate the oocytes.…”

“…Each COC and its corresponding FF were allocated in separate wells in a 96-well plate. COCs were washed once in PXM-HEPES (HEPES buffered porcine X medium [ 16 ]) and then stained for 15 min at room temperature with 0.5% lissamine green B stain (LB), a vital synthetic stain for determining oocyte quality and competence [ 17 , 18 ]. Each COC was classified separately according to the oocyte stain into high-quality (unstained; HQ) and low-quality (stained; LQ) as presented in Fig.…”

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

“…Protein digestion was performed by adding 2 µg of trypsin (Trypsin Gold, Promega) in 0.05 M NH 4 HCO 3 and incubating at 37 °C overnight. Peptides were recovered by spinning the filter plates upside down at 14,000× g for 40 min The mixture was dried under vacuum and the pellet was resuspended in 50 µl of mobile phase (97% water, 3% acetonitrile) 11 .…”

“…For the spectral score, the required values were at least 8, 7, and 9. In addition, a maximum intensity value of at least 60% was required 11 .…”

“…Roughly 40% of presumed zygotes develop to the blastocyst stage for embryo transfer, with fewer cells observed in these embryos compared to those that progressed naturally. To ensure the developmental ability of in vitro embryos, it is imperative to examine their transcriptional profile during pre-implantation stages and identify possible factors that may affect their competence 9 , 11 , 12 .…”