Huabin Zhu

Recovery of mitochondrial function and endogenous antioxidant systems in vitrified bovine oocytes during extended in vitro culture

This study was designed to examine the recovery of mitochondrial function and endogenous antioxidant systems in vitrified oocytes during extended incubations. After 16 hr of in vitro maturation, bovine meiosis‐II oocytes were vitrified, and then surviving oocytes were cultured an additional 8 hr. ATP content, ATP synthase activity, expression of ATP synthase F0 subunit 6 (ATP6) and 8 (ATP8) genes, and reactive oxygen species (ROS) levels were investigated in the vitrified oocytes during this additional period (4 or 8 hr). The results showed that: (1) the ATP content and ATP synthase activities in vitrified oocytes at 8 hr post‐warming (754.6 fmol, 25.9 nmol NADH/min/mg) were significantly higher than in oocytes immediately warmed (568.3 fmol, 8.7 nmol NADH/min/mg), but still lower than in control oocytes (901.5 fmol, 30.7 nmol NADH/min/mg); (2) the relative expression of ATP6 and ATP8 was initially down‐regulated in oocytes when they were first warmed, increased by 4 hr post‐warming, and were again down‐regulated by 8 hr post‐warming; (3) ROS levels in oocytes at 0, 4, and 8 hr post‐warming were significantly higher than in control oocytes; and (4) after parthenogenetic activation, the blastocyst rate of oocytes at 8 hr post‐warming (26.7%) was significantly higher than that of oocytes immediately warmed (16.9%). These results indicated that mitochondrial function and endogenous antioxidant systems recovered significantly better in vitrified–thawed bovine oocytes with 8 hr of additional incubation, but they did not achieve the activity levels found in fresh oocytes. Mol. Reprod. Dev. 78:942–950, 2011.

Effect of vitrification on promoter methylation and the expression of pluripotency and differentiation genes in mouse blastocysts.

The present study was designed to determine the effects of vitrification on promoter methylation and the expression levels of pluripotency and differentiation genes in mouse blastocysts. Promoter region CpG methylation patterns and the expression levels of octamer‐binding transcription factor (Oct4), Nanog homeobox (Nanog), caudal‐type homeobox 2 (Cdx2), and heart and neural crest derivatives‐expressed transcript 1 (Hand1) were analyzed in fresh and vitrified mouse blastocysts. Methylation was measured by bisulphate mutagenesis and sequencing; gene expression was determined by real‐time reverse transcription‐PCR. The results showed that vitrification significantly reduced the methylation levels of the Oct4 (85% vs. 62.5%), Nanog (77.5% vs. 55%), and Cdx2 promoters (4.6% vs. 1.4%; P < 0.05) in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog in vitrified blastocysts. Hand1 promoter methylation was not significantly different in the fresh (17.9%) versus vitrification group (21.4%; P > 0.05). The expression levels of Cdx2 and Hand1 were not significantly different in fresh and vitrified blastocysts. In conclusion, vitrification significantly decreased Oct4, Nanog, and Cdx2 promoter methylation in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog. Mol. Reprod. Dev. 79:445–450, 2012.

Effect of cyclosporine pretreatment on mitochondrial function in vitrified bovine mature oocytes.

Vitrification had a significantly negative impact on the mitochondrial function of bovine oocytes. However, 40 μg/mL cyclosporine pretreatment before vitrification contributed greatly to maintaining mitochondrial membrane potential and adenosine triphosphate content, decreasing reactive oxygen species level, and thereby increasing the developmental ability of vitrified oocytes.

Melatonin inhibits apoptosis and improves the developmental potential of vitrified bovine oocytes.

Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10−9 m melatonin. We analyzed the ROS, mitochondrial Ca2+ (mCa2+) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase‐3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa2+, Bax mRNA, and caspase‐3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa2+, Bax mRNA expression, and caspase‐3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10−9 m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential.

Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN) embryos.

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Δψ) and microtubule distribution in mouse 2‐PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2‐PN embryos was significantly lower than in fresh ones (67.3 ± 3.0% vs. 84.9 ± 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2‐PN embryos without mitochondrial rings (61.7 ± 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 ± 2.8%). (3) Following staining by 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbo‐cyanine iodide (JC‐1), most red‐colored mitochondria (high Δψ) were distributed peripherally around pronuclei and along cell membranes of fresh 2‐PN embryos. Conversely, red‐colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Δψ) evenly distributed throughout the cytoplasm. The proportion of fresh 2‐PN embryos with obvious aggregation of high Δψ mitochondria (84.2 ± 2.2%) was significantly higher than that of vitrified embryos (26.7 ± 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 ± 3.4%) was similar to that of vitrified embryos (74.7 ± 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2‐PN embryos, events which may affect subsequent developmental viability of such embryos. Mol. Reprod. Dev. 76: 1056–1063, 2009.

Identification of differentially expressed proteins between bull X and Y spermatozoa

Differential expression of genes leads to variation in phenotypes of X and Y sperm, even though some differential gene products are shared through an intercellular bridge. Differentially expressed proteins between X and Y sperm sorted from semen of nine bulls were compared using two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis. Overall, 663±12 and 647±22 protein spots were detected in X sperm and Y sperm, respectively, and 42 significant protein spots were differentially expressed between them (P<0.05). Sixteen of these protein spots were successfully identified by MS and tandem MS and were found to be closely relevant to energy metabolism, stress resistance, cytoskeletal structure and the activity of serine proteases. Expression levels of two of these proteins, CAPZB and UQCRC1, were verified by Western blot. We propose that these differentially expressed proteins may affect the phenotype of X and Y sperm, binding and fusion of sperm/oocyte and development of the zygotic embryo. Our preliminary results provide an overview of differential expression in total protein levels between X and Y spermatozoa. Identification of these altered proteins may provide a theoretical basis for understanding the biological differences between the two types of sperm.

Effect of vitrification on promoter CpG island methylation patterns and expression levels of DNA methyltransferase 1o, histone acetyltransferase 1, and deacetylase 1 in metaphase II mouse oocytes

OBJECTIVE To investigate the effect of vitrification on Dnmt1o, Hat1, and Hdac1 promoter CpG island methylation patterns and messenger RNA (mRNA) expression levels in mouse metaphase II (MII) oocytes. DESIGN In vitro study. SETTING Academic institution. ANIMAL(S) Kunming white mice. INTERVENTION(S) After vitrification, surviving mouse MII oocytes subjected to methylation and expression analysis with fresh oocytes used as a control. MAIN OUTCOME MEASURE(S) Expression levels of mRNA as measured by real-time reverse-transcriptase polymerase chain reaction of methylation patterns of the CpG islands in the Dnmt1o, Hat1, and Hdac1 promoters analyzed by bisulfite mutagenesis and sequencing. RESULT(S) The methylation patterns of the promoter CpG islands in Dnmt1o, Hat1, and Hdac1 were not statistically significantly when comparing vitrified oocytes and fresh oocytes. The expression levels of Hat1 and Hdac1 mRNA were not statistically significantly different in comparing in vitrified oocytes and fresh oocytes. The expression of Dnmt1o mRNA was statistically significantly lower in vitrified oocytes compared with fresh oocytes. CONCLUSION(S) Vitrification did not statistically significantly alter the methylation patterns of the promoter CpG islands in Dnmt1o, Hat1, or Hdac1, but did statistically significantly decrease the expression of Dnmt1o mRNA in mouse MII oocytes.

Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos.

Preimplantation embryos are sensitive to oxidative stress‐induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ‐induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real‐time quantitative polymerase chain reaction analysis, we found that melatonin pre‐incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro‐apoptotic caspase‐3 and Bax, while the expression of the anti‐apoptotic Bcl‐2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ‐induced damage in a p38‐dependent manner, but extracellular signal‐regulated kinase (ERK) and c‐JUN N‐terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions.

Melatonin enhances the in vitro maturation and developmental potential of bovine oocytes denuded of the cumulus oophorus.

This study was designed to determine the effect of melatonin on the in vitro maturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with 10-9 M melatonin (DOs + MT), cumulus-oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6 and ATP8), bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ), Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) and AQP3 mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%; P < 0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s; P < 0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %; P < 0.05) and moderated the reduction of relative mRNA levels of ATP6, ATP8, BMP-15 and GDF-9 caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%; P < 0.05), and expression levels of IFN-τ, Na+/K+-ATPase, CTNNBL1 and AQP3 mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality.

Identification and characterization of genes differentially expressed in X and Y sperm using suppression subtractive hybridization and cDNA microarray

Differential expression of genes leads to variations in the phenotypes of X and Y sperm, although some differentially expressed gene products are shared through intercellular bridges. Genes differentially expressed in bovine X and Y sperm were identified by a combination of suppression subtractive hybridization (SSH), cDNA microarray, and sequence‐homology analysis. Microarray data and Significance Analysis of Microarrays software were used to identify 31 differentially expressed genes, only four of which were previously identified. These genes are involved in fundamental life processes of mature sperm, and may be associated with the differences between X and Y sperm since 27 versus 4 were upregulated in X versus Y sperm, respectively. The levels of expression of seven genes—including the known genes UTY, DPH3, CYTB, and ISCU, and the unknown genes X + Y contig 41, X + Y contig 18, and Y + X contig 16—were validated by quantitative real‐time PCR, and some genes were clearly differentially expressed by X and Y sperm, despite the presence of intercellular bridges among spermatids. These results provide a theoretical basis for research on gene expression during sperm development, as well as on sex control at the level of sperm. Mol. Reprod. Dev. 81: 908–917, 2014.