Abstract
Simple SummaryIn vitro production (IVP) of embryos is an essential element of many reproductive biotechnologies. Although some remarkable achievements have been obtained for ovine IVP, the overall efficiency is still low. A limiting factor of the technique is the great variability of the oocyte quality used in IVP. The current selection criterion of oocytes is based on a morphological factor, which is insufficient to determine oocytes’ competence. Therefore, the identification of biomarkers for the selection of oocytes of high developmental competence is a major research goal. The purpose of this study was to evaluate the morphometric parameters of oocytes and the relative gene expression of oocytes and cumulus cells (CCs) as biomarkers of oocyte quality after individually culturing them in order to increase the final efficiency of the IVP technique in sheep. We observed that higher blastocyst rates were obtained from oocytes classified as intermediate and large according to total diameter, oocyte diameter, and zona pellucida thickness. Moreover, the expression of genes related to oocyte quality was higher in oocytes classified as large and in CCs from oocytes classified as large and thus able to reach the blastocyst stage. Oocyte morphometric assessments and gene expression in CCs may be used as biomarkers of oocyte quality.Oocyte quality is crucial for subsequent embryo development and so it is a major challenge in assisted reproductive technologies. The aim of the present work was to evaluate the morphometric parameters of oocytes (experiment 1) and the relative gene expression of oocytes and cumulus cells (CCs) (experiment 2) as biomarkers of oocyte quality after individually culturing them (one oocyte or embryo/drop). In experiment 1, individually matured oocytes were measured and classified into small, intermediate, and large oocytes after a cluster analysis, based on total diameter (with zona pellucida, ZP), oocyte diameter (without ZP), and ZP thickness. These oocytes were individually fertilized in vitro and cultured. The embryo development was evaluated up to the blastocyst stage. According to the total diameter, oocyte diameter, and ZP thickness, the blastocyst rate decreased in the small oocytes group (3.1 ± 3.1, 14.1 ± 9.4, and 26.7 ± 3.9, respectively) compared to the intermediate (29.4 ± 5.2, 30.5 ± 10.1, and 28.6 ± 9.6, respectively) and large oocytes groups (54.2 ± 13.5, 44.4 ± 3.9, and 67.6 ± 12.4, respectively). In addition, the probability of reaching the blastocyst stage was positively related to the total diameter (p < 0.001), oocyte diameter (p < 0.05), and ZP thickness (p < 0.001). Furthermore, the relative gene expression of BAX, BCL2, GDF9, and GJA1 was lower in oocytes classified as large. In experiment 2, the mRNA transcript relative abundance pattern of genes in CCs was evaluated according to oocyte total diameter and developmental stage reached. CCs from oocytes classified as large and oocytes capable of developing to the blastocyst stage had a lower relative expression of BAX, STAR, and PTGS2, while a higher expression of HAS2 and SDC2 transcript was observed for those oocytes. In conclusion, oocyte morphometric parameters and gene expression analysis in oocytes and CCs provide methods for the identification of the most competent oocytes for assisted reproductive technologies in sheep.
Highlights
In vitro production (IVP) of embryos is an essential instrument for many reproductive biotechnologies such as nuclear transfer, cloning, gene editing, and stem cell research.IVP in sheep is of great interest to accelerate breeding and improve the productivity and profitability of livestock
Individual in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) did not affect the developmental competence of oocytes (Table 2)
The results from our study showed that the expression of steroidogenic acute regulatory protein (STAR) and prostaglandin-endoperoxide synthase 2 (PTGS2) was lower in cumulus cells (CCs) of oocytes of high quality
Summary
In vitro production (IVP) of embryos is an essential instrument for many reproductive biotechnologies such as nuclear transfer, cloning, gene editing, and stem cell research.IVP in sheep is of great interest to accelerate breeding and improve the productivity and profitability of livestock. One of the main reasons for the low blastocyst rates is the high variability in the quality of oocytes used for in vitro maturation (IVM). The IVP technique includes three successive procedures: IVM, a crucial step to provide high-quality oocytes, in vitro fertilization (IVF), and in vitro culture (IVC) of embryos. Oocytes selected for IVM are retrieved from follicles at different stages of folliculogenesis; most, meiotically competent, have not undertaken all essential cytoplasmic changes to support further development. The selection criteria currently used are based on subjective morphological parameters such as the homogeneity of the cytoplasm or the number of cumulus cell layers, and these may be insufficient to accurately differentiate oocytes of high or low quality [6,7,8,9]. In bovine and caprine livestock, a minimum oocyte diameter of 115 μm is necessary for full meiotic competence, and full developmental competence is acquired at 120 μm [12,14,15]
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