Abstract
BackgroundThis study investigated the effect of melatonin (MT) on cell cycle (G1/S/G2/M) of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II (MII) oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos.ResultsAfter vitrification and warming, oocytes were parthenogenetically activated (PA) and in vitro cultured (IVC). Then the spindle morphology and chromosome segregation in oocytes, the maternal mRNA levels of genes including Miss, Doc1r, Setd2 and Ythdf2 in activated oocytes, pronuclear formation, the S phase duration in zygotes, mitochondrial function at G1 phase, reactive oxygen species (ROS) level at S phase, DNA damage at G2 phase, early apoptosis in 2-cell embryos, cleavage and blastocyst formation rates were evaluated. The results indicated that the vitrification/warming procedures led to following perturbations 1) spindle abnormalities and chromosome misalignment, alteration of maternal mRNAs and delay in pronucleus formation, 2) decreased mitochondrial membrane potential (MMP) and lower adenosine triphosphate (ATP) levels, increased ROS production and DNA damage, G1/S and S/G2 phase transition delay, and delayed first cleavage, and 3) increased early apoptosis and lower levels of cleavage and blastocyst formation. Our results further revealed that such negative impacts of oocyte cryopreservation could be alleviated by supplementation of warming, recovery, PA and IVC media with 10− 9 mol/L MT before the embryos moved into the 2-cell stage of development.ConclusionsMT might promote cell cycle progression via regulation of MMP, ATP, ROS and maternal mRNA levels, potentially increasing the first cleavage of parthenogenetic zygotes developed from vitrified–warmed mouse oocytes and their subsequent development.
Highlights
This study investigated the effect of melatonin (MT) on cell cycle (G1/S/Gap 2 (G2)/M) of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II (MII) oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos
With the foregoing question in mind, in the present study, we investigated the effect of MT on: 1) the cleavage rate of parthenogenetic zygotes from vitrified-warmed mouse oocytes and their subsequent development, 2) the mitochondrial function in Gap 1 (G1) phase, Reactive oxygen species (ROS) level in S phase, DNA damage level in G2 phase, and the duration and transition of S phase in zygotes, and 3) the pronuclear formation of vitrified-warmed oocytes after parthenogenetic activation by detecting the stability of spindle morphology and chromosome segregation in vitrified oocytes and the mRNA level of key maternal genes i.e., Btg4, Orc2, Eif4e, Doc1r, Miss, Setd2 and Ythdf2 in activated oocytes
During the in vitro culture of activated oocytes, 50% of them were calculated for early cleavage at 15.47 ± 0.14 h in vitrification group and at 14.76 ± 0.22 h in the control group post activation, respectively (Fig. 2), and it was observed that both groups differed significantly (P < 0.05), indicating that oocyte vitrification can induce a delay in the initiation of the first cleavage
Summary
This study investigated the effect of melatonin (MT) on cell cycle (G1/S/G2/M) of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II (MII) oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos. The maternal mRNA degradation and translation are essentially required for maternal-to-zygotic transition and are regulated by transformation licensing factor BTG4 [22] and translation initiation factor EIF4E protein [23] respectively, before zygotic genome activation (ZGA) takes place. It has been reported that oocyte cryopreservation can lead to down-regulation of mRNA levels [29], alterations of mRNA expression of genes related to maternal-to-zygote transition in 2-cell embryos [10] and 1575 differentially expressed genes in 2-cell embryos compared to those in the control group [30] These alterations are believed to inevitably affect the quality of vitrified-warmed oocytes and their subsequent embryonic development. It will be of great value to further elucidate the potential mechanism underlying the reduced developmental competence of vitrifiedwarmed oocytes after fertilization by investigating the mRNA levels of essential genes including Miss, Doc1r, Btg, Eif4e, Ythdf and Setd
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