Lei An

High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells

It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of “4N-ON” and the corresponding “4N-OFF” iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10−6 m or 10−8 m, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10−10 m melatonin‐treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10−9 m or 10−12 m melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis‐related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7‐fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7‐fold and 23.2‐fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53‐mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor‐independent, possibly through melatonins ability to directly scavenge free radicals.

Comparative Analysis of Dynamic Proteomic Profiles between in Vivo and in Vitro Produced Mouse Embryos during Postimplantation Period

Assisted reproductive technology (ART) increasingly is associated with long-term side-effects on postnatal development and behaviors. High-throughput gene expression analysis has been extensively used to explore mechanisms responsible for these disorders. Our study, for the first time, provides a comparative proteomic analysis between embryos after in vivo fertilization and development (IVO, control) and in vitro fertilization and culture (IVP). By comparing the dynamic proteome during the postimplantation period, we identified 300 and 262 differentially expressed proteins (DEPs) between IVO and IVP embryos at embryonic day 7.5 (E7.5) and E10.5, respectively. Bioinformatic analysis showed many DEPs functionally associated with post-transcriptional, translational, and post-translational regulation, and these observations were consistent with correlation analysis between mRNA and protein abundance. In addition to altered gene expression due to IVP procedures, our findings suggest that aberrant processes at these various levels also contributed to proteomic alterations. In addition, numerous DEPs were involved in energy and amino acid metabolism, as well as neural and sensory development. These DEPs are potential candidates for further exploring the mechanism(s) of ART-induced intrauterine growth restriction and neurodevelopmental disorders. Moreover, significant enrichment of DEPs in pathways of neurodegenerative diseases implies the potentially increased susceptibility of ART offspring to these conditions as adults.

Effect of Different Parthenogenetic Activation Methods on the Developmental Competence of in vitro Matured Porcine Oocytes

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% ∼ 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 μM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% ∼ 57.3%; 22.3% vs 7.4% ∼ 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% ∼ 46.2%; 18.0% vs 7.1% ∼ 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8–74.4% vs 56.5–57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% ∼ 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3–27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.

Melatonin improves the reprogramming efficiency of murine‐induced pluripotent stem cells using a secondary inducible system

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all‐iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10−6, 10−7, 10−8, 10−9, or 10−10 m) or with vitamin C (50 μg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10−8 (0.81 ± 0.04%), and 10−9 m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (P < 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT2 was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT1 and MT2 receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis‐related genes p53 and p21 was lower in the group treated with 10−9 m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53‐mediated apoptotic pathway.

Efficient and Safe Recipient Preparation for Transplantation of Mouse Spermatogonial Stem Cells: Pretreating Testes with Heat Shock

Recipient preparation is of prime importance for the successful transplantation of spermatogonial stem cells (SSCs). Busulfan destroys endogenous germs cells and is commonly used for recipient preparation. However, busulfan produces significant side effects, including systemic toxicity, and it is lethal in certain species. The side effects associated with busulfan compromise the efficiency of SSC transplantation and threaten the safety of recipients. Here, we show that heat shock treatment of testes can be used as an alternative to busulfan treatment. Fourteen days after heat shock treatment, mice received a testicular injection of donor germ cells expressing enhanced green fluorescent protein (EGFP). Busulfan-treated mice were used as controls. Two months after transplantation, the number (12 ± 1 mm) and length (30.46 ± 5.23 mm) of EGFP-expressing testicular colonies in heat shock-treated recipients were not significantly different from those in busulfan-treated recipients. Furthermore, healthy EGFP-expressing offspring were obtained after intracytoplasmic injection of round spermatids recovered from heat shock-treated recipients. This result indicates that donor SSCs undergo complete spermatogenesis in the heat shock-treated testes of recipients. Our findings demonstrate the feasibility of using heat shock for the preparation of recipients before SSC transplantation in mice. Heat shock may prove to be useful for recipient preparation in mammalian species in which busulfan produces significant toxicity.

Dynamic comparisons of high-resolution expression profiles highlighting mitochondria-related genes between in vivo and in vitro fertilized early mouse embryos.

STUDY QUESTION Does in vitro fertilization (IVF) induce comprehensive and consistent changes in gene expression associated with mitochondrial biogenesis and function in mouse embryos from the pre- to post-implantation stage? SUMMARY ANSWER IVF-induced consistent mitochondrial dysfunction in early mouse embryos by altering the expression of a number of mitochondria-related genes. WHAT IS KNOWN ALREADY Although IVF is generally safe and successful for the treatment of human infertility, there is increasing evidence that those conceived by IVF suffer increased health risks. The mitochondrion is a multifunctional organelle that plays a crucial role in early development. We hypothesized that mitochondrial dysfunction is associated with increased IVF-induced embryonic defects and risks in offspring. STUDY DESIGN, SIZE, DURATION After either IVF and development (IVO groups as control) or IVF and culture (IVF groups), blastocysts were collected and transferred to pseudo-pregnant recipient mice. Both IVO and IVF embryos were sampled at E3.5, E7.5 and E10.5, and the expression profiles of mitochondria-related genes from the pre- to post-implantation stage were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS ICR mice (5- to 6-week-old males and 8- to 9-week-old females) were used to generate IVO and IVF blastocysts. Embryo day (E) 3.5 blastocysts were transferred to pseudo-pregnant recipient mice. Both IVO and IVF embryos were sampled at E3.5, E7.5 and E10.5 for generating transcriptome data. Mitochondria-related genes were filtered for dynamic functional profiling. Mitochondrial dysfunctions indicated by bioinformatic analysis were further validated using cytological and molecular detection, morphometric and phenotypic analysis and integrated analysis with other high-throughput data. MAIN RESULTS AND THE ROLE OF CHANCE A total of 806, 795 and 753 mitochondria-related genes were significantly (P < 0.05) dysregulated in IVF embryos at E3.5, E7.5 and E10.5, respectively. Dynamic functional profiling, together with cytological and molecular investigations, indicated that IVF-induced mitochondrial dysfunctions mainly included: (i) inhibited mitochondrial biogenesis and impaired maintenance of DNA methylation of mitochondria-related genes during the post-implantation stage; (ii) dysregulated glutathione/glutathione peroxidase (GSH/Gpx) system and increased mitochondria-mediated apoptosis; (iii) disturbed mitochondrial β-oxidation, oxidative phosphorylation and amino acid metabolism; and (iv) disrupted mitochondrial transmembrane transport and membrane organization. We also demonstrated that some mitochondrial dysfunctions in IVF embryos, including impaired mitochondrial biogenesis, dysregulated GSH homeostasis and reactive oxygen species-induced apoptosis, can be rescued by treatment with melatonin, a mitochondria-targeted antioxidant, during in vitro culture. LIMITATIONS, REASONS FOR CAUTION Findings in mouse embryos and fetuses may not be fully transferable to humans. Further studies are needed to confirm these findings and to determine their clinical significance better. WIDER IMPLICATIONS OF THE FINDINGS The present study provides a new insight in understanding the mechanism of IVF-induced aberrations during embryonic development and the increased health risks in the offspring. In addition, we highlighted the possibility of improving existing IVF systems by modulating mitochondrial functions.

Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs.

We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence of in vitro matured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence of in vitro matured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP alphaT). No differences were observed in COMP alphaT values among GSH-treated and control groups. COMP alphaT values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.

Dynamic Proteomic Profiles of In Vivo- and In Vitro-Produced Mouse Postimplantation Extraembryonic Tissues and Placentas

ABSTRACT As the interface between the mother and the developing fetus, the placenta is believed to play an important role in assisted reproductive technology (ART)-induced aberrant intrauterine and postnatal development. However, the mechanisms underlying aberrant placentation remain unclear, especially during extraembryonic tissue development and early stages of placental formation. Using a mouse model, this investigation provides the first comparative proteomic analysis of in vivo (IVO) and in vitro-produced (IVP) extraembryonic tissues and placentas after IVO fertilization and development, or in vitro fertilization and culture, respectively. We identified 165 and 178 differentially expressed proteins (DEPs) between IVO and IVP extraembryonic tissues and placentas on Embryonic Day 7.5 (E7.5) and E10.5, respectively. Many DEPs were functionally associated with genetic information processing, such as impaired de novo DNA methylation, as well as posttranscriptional, translational and posttranslational dysregulation. These novel findings were further confirmed by global hypomethylation, and a lower level of correlation was found between the transcriptome and proteome in the IVP groups. In addition, numerous DEPs were involved in energy and amino acid metabolism, cytoskeleton organization and transport, and vasculogenesis and angiogenesis. These disturbed processes and pathways are likely to be associated with embryonic intrauterine growth restriction, an enlarged placenta, and impaired labyrinth morphogenesis. This study provides a direct and comprehensive reference for the further exploration of the placental mechanisms that underlie ART-induced developmental aberrations.

Impaired imprinted X chromosome inactivation is responsible for the skewed sex ratio following in vitro fertilization.

Significance Sex ratio is an important indicator of reproductive health, and its skewing reflects disturbed embryonic development. This study focused on sex skewing, which has recently identified in human in vitro fertilization (IVF) babies. We reported herein that the skewed sex ratio in mouse IVF offspring was due to the impaired imprinted X chromosome inactivation via suppressing the ring finger protein 12 (Rnf12)/X-inactive specific transcript (Xist) pathway; the sex skewing can be corrected by overexpressing Rnf12 or by supplementation of retinoic acid in embryo culture medium. Hence, our study not only identified a major epigenetic error responsible for sex skewing in IVF offspring, but also implicated a potential strategy for preventing sex skewing and IVF-associated complications by targeting erroneous epigenetic modifications induced by IVF. Dynamic epigenetic reprogramming occurs during normal embryonic development at the preimplantation stage. Erroneous epigenetic modifications due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF) are linked to various short- or long-term consequences. Among these, the skewed sex ratio, an indicator of reproductive hazards, was reported in bovine and porcine embryos and even human IVF newborns. However, since the first case of sex skewing reported in 1991, the underlying mechanisms remain unclear. We reported herein that sex ratio is skewed in mouse IVF offspring, and this was a result of female-biased peri-implantation developmental defects that were originated from impaired imprinted X chromosome inactivation (iXCI) through reduced ring finger protein 12 (Rnf12)/X-inactive specific transcript (Xist) expression. Compensation of impaired iXCI by overexpression of Rnf12 to up-regulate Xist significantly rescued female-biased developmental defects and corrected sex ratio in IVF offspring. Moreover, supplementation of an epigenetic modulator retinoic acid in embryo culture medium up-regulated Rnf12/Xist expression, improved iXCI, and successfully redeemed the skewed sex ratio to nearly 50% in mouse IVF offspring. Thus, our data show that iXCI is one of the major epigenetic barriers for the developmental competence of female embryos during preimplantation stage, and targeting erroneous epigenetic modifications may provide a potential approach for preventing IVF-associated complications.