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Enhancing Oocyte Competence With Milrinone as a Phosphodiesterase 3A Inhibitor to Improve the Development of Porcine Cloned Embryos.

ABSTRACT: The objective of this study was to investigate the effect of milrinone supplementation as a phosphodiesterase 3A inhibitor during in vitro maturation (IVM) to coordinate the cytoplasmic and nuclear maturation of porcine oocytes and subsequent development of porcine cloned embryos. Brilliant cresyl blue (BCB)-stained (BCB +) oocytes, classified as well-developed, and BCB- oocytes were used in parthenogenesis (PA) and cloning, and their preimplantation development was compared. In PA embryos, BCB + oocytes had significantly higher rates of development than BCB- oocytes in terms of maturation (87.5 vs. 71.3%), cleavage (88.6 vs. 76.3%), and blastocyst development (34.3 vs. 25.3%) and also had higher cell numbers (46.9 vs. 38.9%), respectively (p < 0.05). In cloned embryos, the BCB + group also had a significantly higher blastocyst formation rate than the BCB- group (30.6 vs. 20.1%; p < 0.05). Supplementation with 75 μM milrinone during IVM of BCB- oocytes showed improvement in maturation and blastocyst development rates, which may be due to the coordinated maturation of the cytoplasm with the nucleus as an effect of milrinone. Moreover, the analysis of nuclear reprogramming via the examination of the expression levels of the reprogramming-related genes POU5F1, DPPA2, and NDP52IL in milrinone-supplemented BCB- oocytes showed higher expression levels than that in non-treated BCB- oocytes. These findings demonstrate that milrinone is useful in improving developmental competence in less competent oocytes during IVM and for proper nuclear reprogramming in the production of porcine cloned embryos by coordinating cytoplasmic and nucleus maturation.

SUBMITTER: Roy PK

PROVIDER: S-EPMC8120234 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe ovulatory surge of gonadotropins triggers oocyte maturation and rupture of the ovarian follicle. The resumption of nuclear maturation in the oocyte from the prophase stage is characterized by germinal vesicle breakdown (GVBD). It has previously been shown that specific inhibition of cAMP degradation by PDE3 prevents the resumption of oocyte meiosis. However, no report has characterized the activity of PDE3 in the porcine oocyte, or the implication of the cAMP-PDE3 pathway in the entire nuclear maturation process. In this study, PDE3 activity in the oocyte was assessed during in vitro maturation (IVM) and the possible roles of the cAMP-PDE3 pathway in the resumption and progression of meiosis were investigated in terms of different models of oocyte maturation.ResultsCyclic AMP-degrading PDE activity was detected in the cumulus-oocyte complex (COC) and was partially inhibited by a specific PDE3 inhibitor, cilostamide. When measured only in the denuded oocyte, PDE activity was almost completely inhibited by cilostamide, suggesting that cAMP-PDE3 activity is the major cAMP-PDE in porcine oocytes. PDE3A mRNA was detected by RT-PCR. PDE3 activity did not vary significantly during the early hours of IVM, but a maximum was observed at 13 hours. In cumulus-oocyte complexes, meiosis resumed after 20.81 hours of culture. PDE3 inhibition no longer maintained meiotic arrest if sustained beyond 17.65 hours of IVM, 3 hours prior to resumption of meiosis. Thereafter, PDE3 inhibition progressively lost its efficacy in GVBD. When the protein phosphatase 1 and 2A inhibitor okadaic acid was continuously or transiently (3 hours) present during IVM, meiosis resumed prematurely; PDE3 inhibition was unable to prevent GVBD. However, PDE3 inhibition in COC treated with OA for 3 hours significantly delayed meiosis at the intermediate stage.ConclusionThe present investigation has demonstrated that PDE3A is the major cAMP-degrading PDE in the oocyte. It regulates the resumption of meiosis until 3 hours prior to GVBD and transiently affects meiotic progression.

Project description:The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

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Enhancing Oocyte Competence With Milrinone as a Phosphodiesterase 3A Inhibitor to Improve the Development of Porcine Cloned Embryos.

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