Significant decrease of maternal mitochondria carryover using optimized spindle-chromosomal complex transfer.

Mutations in mitochondrial DNA (mtDNA) contribute to a variety of serious multi-organ human diseases, which are strictly inherited from the maternal germline. However, there is currently no curative treatment. Attention has been focused on preventing the transmission of mitochondrial diseases through mitochondrial replacement (MR) therapy, but levels of mutant mtDNA can often unexpectedly undergo significant changes known as mitochondrial genetic drift. Here, we proposed a novel strategy to perform spindle-chromosomal complex transfer (SCCT) with maximal residue removal (MRR) in metaphase II (MII) oocytes, thus hopefully eliminated the transmission of mtDNA diseases. With the MRR procedure, we initially investigated the proportions of mtDNA copy numbers in isolated karyoplasts to those of individual oocytes. Spindle-chromosomal morphology and copy number variation (CNV) analysis also confirmed the safety of this method. Then, we reconstructed oocytes by MRR-SCCT, which well developed to blastocysts with minimal mtDNA residue and normal chromosomal copy numbers. Meanwhile, we optimized the manipulation order between intracytoplasmic sperm injection (ICSI) and SCC transfer and concluded that ICSI-then-transfer was conducive to avoid premature activation of reconstructed oocytes in favor of normal fertilization. Offspring of mice generated by embryos transplantation in vivo and embryonic stem cells derivation further presented evidences for competitive development competence and stable mtDNA carryover without genetic drift. Importantly, we also successfully accomplished SCCT in human MII oocytes resulting in tiny mtDNA residue and excellent embryo development through MRR manipulation. Taken together, our preclinical mouse and human models of the MRR-SCCT strategy not only demonstrated efficient residue removal but also high compatibility with normal embryo development, thus could potentially be served as a feasible clinical treatment to prevent the transmission of inherited mtDNA diseases.

The press-assisted fusion scheme significantly mitigates the quantity of HVJ-E required in mitochondrial replacement techniques.

In brief: The impact of HVJ-E employed in mitochondrial replacement techniques (MRTs) on embryonic development remains uncertain. This study has exhibited the influence of HVJ-E utilized in MRTs on embryonic development and has devised a novel HVJ-E-induced fusion approach to curtail the amount of HVJ-E employed in MRTs. Abstract: Mitochondrial replacement techniques (MRTs) provide a viable option for women carrying pathogenic mitochondrial DNA (mtDNA) variants to conceive disease-free offspring with a genetic connection. In comparison to electrofusion, HVJ-E-induced fusion has been identified as the most promising approach for clinical translation of MRTs due to its absence of electrical interference. However, despite confirmation of the absence of RNA activity in HVJ-E, a reduction in blastocyst quality has been observed in various MRTs studies utilizing the HVJ-E-induced fusion scheme. Recent investigations have revealed a dose-dependent elevation of reactive oxygen species (ROS) levels in various cancer cells incubated with HVJ-E. However, the impact of HVJ-E as a sole determinant on embryonic development in MRTs remains unverified. This investigation establishes that the augmented concentration of HVJ-E utilized in the conventional HVJ-E fusion protocol is an autonomous variable that influences embryonic development in MRTs. This effect may be attributed to amplified DNA damage resulting from heightened levels of ROS in reconstructed embryos. To mitigate the presence of HVJ-E in reconstructed zygotes while maintaining optimal fusion efficiency in MRTs, a novel HVJ-E-induced fusion approach was devised, namely, press-assisted fusion. This technique offers potential advantages in reducing detrimental factors that impede embryo development in MRTs.

Pum3 is dispensable for mouse oocyte maturation and embryo development in vitro.

Pumilio3 (Pum3), an evolutionarily distant homologue of the classical RNA-binding protein PUF (PUMILIO and FBF) family member, is also involved in the process of RNA metabolism through post-transcriptional regulation. However, the functions of Pum3 in mouse oocyte maturation and preimplantation embryonic development have not been elucidated. By comparing RNA levels in different tissues, we found that Pum3 was widely expressed in multiple tissues, but moderately predominant in the ovary. Histochemical staining suggested that the PUM3 protein exhibits positive signals in oocytes, granulosa cells and theca cells of different follicle stages. Oocyte immunofluorescence results showed a slightly higher level of PUM3 protein in metaphase II compared with the germinal vesicle (GV) stage. After knockdown of Pum3 in GV oocytes using siRNA injection (siPUM3), no obvious defect was observed in the processes of GV breakdown and polar body extrusion during in vitro maturation (IVM) for the siPum3 oocytes. Compared with the control group, the siPUM3 group displayed no significant abnormality in the cleavage and blastocyst formation rate of these fertilized oocytes. Therefore, we can conclude that depletion of Pum3 does not affect mouse oocyte maturation and early embryonic development in vitro.

Long noncoding RNA ANRIL promotes the malignant progression of cholangiocarcinoma by epigenetically repressing ERRFI1 expression.

Long noncoding RNAs (lncRNAs) have recently been verified to have significant regulatory functions in many types of human cancers. The lncRNA ANRIL is transcribed from the INK4b-ARF-INK4a gene cluster in the opposite direction. Whether ANRIL can act as an oncogenic molecule in cholangiocarcinoma (CCA) remains unknown. Our data show that ANRIL knockdown greatly inhibited CCA cell proliferation and migration in vitro and in vivo. According to the results of RNA sequencing analysis, ANRIL knockdown dramatically altered target genes associated with the cell cycle, cell proliferation, and apoptosis. By binding to a component of the epigenetic modification complex enhancer of zeste homolog 2 (EZH2), ANRIL could maintain lysine residue 27 of histone 3 (H3K27me3) levels in the promoter of ERBB receptor feedback inhibitor 1 (ERRFI1), which is a tumor suppressor gene in CCA. In this way, ERRFI1 expression was suppressed in CCA cells. These data verified the key role of the epigenetic regulation of ANRIL in CCA oncogenesis and indicate its potential as a target for CCA intervention.

Highly conserved epigenetic regulation of BOULE and DAZL is associated with human fertility.

Separation of germ cells from somatic cells is a widespread feature of animal sexual reproduction, with a core set of germ cell factors conserved among diverse animals. It is not known what controls their conserved gonad-specific expression. Core components of epigenetic machinery are ancient, but its role in conserved tissue expression regulation remains unexplored. We found that promoters of the reproductive genes BOULE and DAZL exhibit differential DNA methylation, consistent with their gonad-specific expression in humans and mice. Low or little promoter methylation from the testicular tissue is attributed to spermatogenic cells of various stages in the testis. Such differential DNA methylation is present in the orthologous promoters not only of other mammalian species, but also of chickens and fish, supporting a highly conserved epigenetic mechanism. Furthermore, hypermethylation of DAZL and BOULE promoters in human sperm is associated with human infertility. Our data strongly suggest that epigenetic regulation may underlie conserved germ-cell-specific expression, and such a mechanism may play an important role in human fertility.-Zhang, C., Xue, P., Gao, L., Chen, X., Lin, K., Yang, X., Dai, Y., Xu, E. Y. Highly conserved epigenetic regulation of BOULE and DAZL is associated with human fertility.

The effect of an extended culture period on birth weight among singletons born after single or double vitrified embryo transfer.

Aim: To evaluate the effect of an extended culture period on birth weight among singletons born after vitrified-warmed embryo transfer. Methods: A retrospective cohort study was performed among 12400 women who gave birth to 1015, 1027, 687, and 9671 singletons after single blastocyst transfer, single cleavage-stage embryo transfer, double blastocyst transfer, and double cleavage-stage embryo transfer, respectively. Results: The unadjusted birth weight of singletons born after vitrified blastocyst transfer were heavier than those born after cleavage-stage transfer (ß=30.28, SE=13.17, P=0.022), as were the adjusted birth weights (ß=0.09, SE=0.03, P=0.007). In addition, there was a 37% increased odd of having an infant with high birth weight after vitrified blastocyst transfer compared with vitrified cleavage stage transfer (OR=1.37, 95% CI:1.07-1.77). Conclusion: The unadjusted and adjusted birth weight and odds of having an infant with high birth weight significantly increased after blastocyst transfer compared with cleavage-stage embryo transfer in vitrified-warmed cycles.

Earlier second polar body transfer and further mitochondrial carryover removal for potential mitochondrial replacement therapy.

The second polar body (PB2) transfer in assisted reproductive technology is regarded as the most promising mitochondrial replacement scheme for preventing the mitochondrial disease inheritance owing to its less mitochondrial carryover and stronger operability. However, the mitochondrial carryover was still detectable in the reconstructed oocyte in conventional second polar body transfer scheme. Moreover, the delayed operating time would increase the second polar body DNA damage. In this study, we established a spindle-protrusion-retained second polar body separation technique, which allowed us to perform earlier second polar body transfer to avoid DNA damage accumulation. We could also locate the fusion site after the transfer through the spindle protrusion. Then, we further eliminated the mitochondrial carryover in the reconstructed oocytes through a physically based residue removal method. The results showed that our scheme could produce a nearly normal proportion of normal-karyotype blastocysts with further reduced mitochondrial carryover, both in mice and humans. Additionally, we also obtained mouse embryonic stem cells and healthy live-born mice with almost undetectable mitochondrial carryover. These findings indicate that our improvement in the second polar body transfer is conducive to the development and further mitochondria carryover elimination of reconstructed embryos, which provides a valuable choice for future clinical applications of mitochondrial replacement.

Mammalian Pum1 and Pum2 Control Body Size via Translational Regulation of the Cell Cycle Inhibitor Cdkn1b.

Body and organ size regulation in mammals involves multiple signaling pathways and remains largely enigmatic. Here, we report that Pum1 and Pum2, which encode highly conserved PUF RNA-binding proteins, regulate mouse body and organ size by post-transcriptional repression of the cell cycle inhibitor Cdkn1b. Binding of PUM1 or PUM2 to Pumilio binding elements (PBEs) in the 3' UTR of Cdkn1b inhibits translation, promoting G1-S transition and cell proliferation. Mice with null mutations in Pum1 and Pum2 exhibit gene dosage-dependent reductions in body and organ size, and deficiency for Cdkn1b partially rescues postnatal growth defects in Pum1-/- mice. We propose that coordinated tissue-specific expression of Pum1 and Pum2, which involves auto-regulatory and reciprocal post-transcriptional repression, contributes to the precise regulation of body and organ size. Hence PUM-mediated post-transcriptional control of cell cycle regulators represents an additional layer of control in the genetic regulation of organ and body size.

Essential requirement of mammalian Pumilio family in embryonic development.

Mouse PUMILIO1 (PUM1) and PUMILIO2 (PUM2) belong to the PUF (Pumilio/FBF) family, a highly conserved RNA binding protein family whose homologues play critical roles in embryonic development and germ line stem cell maintenance in invertebrates. However, their roles in mammalian embryonic development and stem cell maintenance remained largely uncharacterized. Here we report an essential requirement of the Pum gene family in early embryonic development. A loss of both Pum1 and Pum2 genes led to gastrulation failure, resulting in embryo lethality at E8.5. Pum-deficient blastocysts, however, appeared morphologically normal, from which embryonic stem cells (ESCs) could be established. Both mutant ESCs and embryos exhibited reduced growth and increased expression of endoderm markers Gata6 and Lama1, making defects in growth and differentiation the likely causes of gastrulation failure. Furthermore, ESC Gata6 transcripts could be pulled down via PUM1 immunoprecipitation and mutation of conserved PUM-binding element on 3'UTR (untranslated region) of Gata6 enhanced the expression of luciferase reporter, implicating PUM-mediated posttranscriptional regulation of Gata6 expression in stem cell development and cell lineage determination. Hence, like its invertebrate homologues, mouse PUM proteins are conserved posttranscriptional regulators essential for embryonic and stem cell development.

Generation and functional characterization of a conditional Pumilio2 null allele.

The highly conserved RNA binding protein PUF (Pumilio/FBF) family is present throughout eukaryotes from yeast to mammals, with critical roles in development, fertility and the nervous system. However, the function of the mammalian PUF family members remains underexplored. Our previous study reported that a gene-trap mutation of Pum2 results in a smaller testis but does not impact fertility and viability. Although the gene-trap mutation disrupted the key functional domain of PUM protein-PUM-HD (Pumilio homology domain), but still produced a chimeric Pum2-ß-geo protein containing part of PUM2, raising a question if such a chimeric protein may provide any residual function or contribute to the reproductive phenotype. Here, we report the generation of a conditional PUM2 allele, when knocked out, producing no residual PUM2 and hence a complete loss-of-function allele. We also uncovered small but significant reduction of male fertility and viability in the mutants, suggesting requirement of PUM2 for male fertility and viability.