The Simulated Physiological Oocyte Maturation (SPOM) System Enhances Cytoplasmic Maturation and Oocyte Competence in Cattle

Abstract

In vitro embryo production is a widely applied technique that allows the expansion of genetics and accelerated breeding programs. However, in cattle, this technique still needs improvement in order to reach quality and pregnancy rates comparable to in vivo-derived embryos. One of the limitations of this technique is related to in vitro maturation, where a heterogeneous population of oocytes is harvested from follicles and cultured in vitro in the presence of gonadotropic hormones to induce maturation. As a result, oocytes with different degrees of competence are obtained, resulting in a decrease in the quality and quantity of embryos obtained. A novel system based on the use of cyclic adenosine monophosphate (cAMP) modulators was developed to enhance bovine oocyte competence, although controversial results were obtained depending on the in vitro embryo production (IVP) system used in each laboratory. Thus, in the present work, we employed a reported cAMP protocol named Simulated Physiological Oocyte Maturation (SPOM) under our IVP system and analysed its effect on cytoplasmic maturation by measuring levels of stress-related genes and evaluating the activity and distribution of mitochondria as a marker for cytoplasmic maturation Moreover, we studied the effect of the cAMP treatment on nuclear maturation, cleavage, and blastocyst formation. Finally, we assessed the embryo quality by determining the hatching rates, total cell number per blastocyst, cryopreservation tolerance, and embryo implantation. We found that maturing oocytes in the presence of cAMP modulators did not affect nuclear maturation, although they changed the dynamic pattern of mitochondrial activity along maturation. Additionally, we found that oocytes subjected to cAMP modulators significantly improved blastocyst formation (15.5% vs. 22.2%, p < 0.05). Blastocysts derived from cAMP-treated oocytes did not improve cryopreservation tolerance but showed an increased hatching rate, a higher total cell number per blastocyst and, when transferred to hormonally synchronised recipients, produced pregnancies. These results reflect that the use of cAMP modulators during IVM results in competent oocytes that, after fertilisation, can develop in more blastocysts with a better quality than standard IVM conditions.

Keywords: bovine; cAMP modulators; cytoplasmic maturation; embryo development; in vitro maturation; mitochondrial activity; oocyte competence.

Figure 1

Illustration of the experimental design. Figure was created in BioRender.com.

Figure 2

Representative confocal image showing how the area was selected in each oocyte to measure fluorescent intensity.

Figure 3

In vitro production of bovine embryos following standard IVM or IVM in the presence of cAMP modulators. Maturation rate was calculated considering the number of oocytes that showed the presence of the first polar body over the total number of oocytes subjected to maturation (n = 4, a total of 306 and 232 oocytes were analysed for control or cAMP modulators condition, respectively). Cleavage rate was calculated by determining the number of 2/4-cells embryos over initial oocytes (n = 9; a total of 729 and 634 oocytes were analysed for control or cAMP modulators condition, respectively). Blastocyst rate was determined by considering the total number of blastocysts over initial oocytes (n = 9; a total of 729 and 634 oocytes were analysed for control or cAMP modulators condition, respectively). * indicates significant differences (Wilcoxon test was performed, p < 0.05).

Figure 4

Relative quantification of stress-related genes in mature oocytes. Relative expression of hsp70, psmb5, atf-6, bip, and bax genes was analysed in oocytes subjected to 22 h maturation under standard conditions (control) or under the use of cAMP modulators. Expression of each gene was calculated using the standard curve method and normalised to hmbs housekeeping gene. Data were collected from three independent replicates from pools of 10 matured oocytes. Different letters indicate significant differences (t-test, p < 0.05). Graphic shows the fold change normalised to the housekeeping gene ± SEM.

Figure 5

Evaluation of mitochondrial activity and distribution during oocyte maturation. (A) Confocal microscopy images showing mitochondrial distribution in an oocyte at 0, 7, 15, and 22 h of maturation in standard IVM (control) or in IVM with the presence of cAMP modulators. (B) Mitochondrial activity at 0, 7, 15, and 22 h of in vitro maturation in control conditions or under the use of cAMP modulators. Alive oocytes were stained with Mitotracker Red CMXROS and imaged using confocal microscopy. Mitochondrial activity was measured in each oocyte derived from each treatment at different maturation times by determining fluorescence intensity. Different letters in each group and * between groups indicate significant differences (two-way ANOVA and Bonferroni’s multiple comparisons test, p < 0.05). Data were collected from 3 independent IVM procedures, where at least 10 oocytes were imaged at each time point. Graphic shows the mean of mitochondrial activity ± SEM.

Figure 6

Determination of embryo quality of blastocysts derived from standard IVM or IVM with cAMP modulators. (A) Hatching rate of embryos that derived from standard IVM or IVM with cAMP modulators. Hatching rate was calculated considering hatched embryos over the initial number of oocytes. Data were collected after 9 independent IVP procedures. ** indicates significant differences (Wilcoxon test, p < 0.05). Graphic shows the hatching rate mean ± SEM. (B) Day 7 blastocysts derived from control IVM or IVM with cAMP modulators were stained with Hoescht 33,342 and imaged to determine total cell number. A total of 3 blastocysts from 3 independent IVP procedures (total of 9 blastocysts per experimental group) were stained to determine total cell number. **** indicates significant differences (t-test, p < 0.05). Graphic shows the mean of total cell number per blastocyst ± SEM. (C) Day 7 blastocysts derived from control IVM or IVM with cAMP modulators were cryopreserved and thawed for evaluating cryotolerance. Embryo recovery and expansion were evaluated after 24 h of IVC. Between 30 and 50 embryos in total from 5 independent IVP procedures were cryopreserved and thawed to evaluate cryotolerance. Graphic shows the mean of recovery rate ± SEM (ns; Wilcoxon test).