mTOR hypoactivity leads to trophectoderm cell failure by enhancing lysosomal activation and disrupting the cytoskeleton in preimplantation embryo.

BACKGROUND: Metabolic homeostasis is closely related to early impairment of cell fate determination and embryo development. The protein kinase mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism in the body. Inhibition of mTOR signaling in early embryo causes postimplantation development failure, yet the mechanisms are still poorly understood. METHODS: Pregnancy mice and preimplantation mouse embryo were treated with mTOR inhibitor in vivo and in vitro respectively, and subsequently examined the blastocyst formation, implantation, and post-implantation development. We used immunofluorescence staining, RNA-Seq smart2, and genome-wide bisulfite sequencing technologies to investigate the impact of mTOR inhibitors on the quality, cell fate determination, and molecular alterations in developing embryos. RESULTS: We showed mTOR suppression during preimplantation decreases the rate of blastocyst formation and the competency of implantation, impairs the post implantation embryonic development. We discovered that blocking mTOR signaling negatively affected the transformation of 8-cell embryos into blastocysts and caused various deficiencies in blastocyst quality. These included problems with compromised trophectoderm cell differentiation, as well as disruptions in cell fate specification. mTOR suppression significantly affected the transcription and DNA methylation of embryos. Treatment with mTOR inhibitors increase lysosomal activation and disrupts the organization and dynamics of the actin cytoskeleton in blastocysts. CONCLUSIONS: These results demonstrate that mTOR plays a crucial role in 8-cell to blastocyst transition and safeguards embryo quality during early embryo development.

Three-Dimensional Visualization of Mouse Endometrial Remodeling After Superovulation.

Uterine status determines pregnancy success. Although it is well known that superovulation operations can disrupt uterine function, our understanding of the morphological changes in the uterine endometrium at the three-dimensional (3D) level is limited. Here, combining the tissue clearing with 3D deep imaging, we reveal an increase in epithelial density and angiogenesis after ovarian stimulation, which is accompanied by a circulating surge in P4 levels. Using an ovariectomized mouse model, we further detected the separate regulatory effects of P4 and E2 on the uterine endometrium, with P4 promoting endothelial cell growth and E2 inducing epithelial proliferation. Additionally, we observed that the effects of E2 can be partially neutralized by P4, and vice versa. By analyzing the 3D uterine imaging, we discovered an interesting phenomenon in which the growing blood vessels closely surround the remodeling uterine epithelium, indicating a close relationship between angiogenesis and epithelial growth. These findings provide new insight into the uterine epithelial changes and angiogenesis at the 3D level, and explain a potential reason for endometrial changes due to the low implantation rate in patients undergoing clinic super-ovulation.

BET bromodomain inhibitor JQ1 regulates spermatid development by changing chromatin conformation in mouse spermatogenesis.

As a BET bromodomain inhibitor, JQ1 has been proven have efficacy against a number of different cancers. In terms of male reproduction, JQ1 may be used as a new type of contraceptive, since JQ1 treatment in male mice could lead to germ cell defects and a decrease of sperm motility, moreover, this effect is reversible. However, the mechanism of JQ1 acting on gene regulation in spermatogenesis remains unclear. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse testes treated with JQ1 or vehicle control to determine the transcriptional regulatory function of JQ1 in spermatogenesis at the single cell resolution. We confirmed that JQ1 treatment could increase the numbers of somatic cells and spermatocytes and decrease the numbers of spermatid cells. Gene Ontology (GO) analysis demonstrated that differentially expressed genes which were down-regulated after JQ1 injection were mainly enriched in "DNA conformation change" biological process in early developmental germ cells and "spermatid development" biological process in spermatid cells. ATAC-seq data further confirmed that JQ1 injection could change the open state of chromatin. In addition, JQ1 could change the numbers of accessible meiotic DNA double-stranded break sites and the types of transcription factor motif that functioned in pachytene spermatocytes and round spermatids. The multi-omics analysis revealed that JQ1 had the ability to regulate gene transcription by changing chromatin conformation in mouse spermatogenesis, which would potentiate the availability of JQ1 in male contraceptive.

High-Efficiency Differentiation of Human Pluripotent Stem Cells to Hematopoietic Stem/Progenitor Cells in Random Positioning Machine Bioreactors.

Human pluripotent stem cells (PSCs) are known to differentiate into almost all the blood lineage cells in vitro and hold a great promise for studying human early hematopoietic development and have a huge potential in the treatment of hematological disorders. Although several methods of hematopoietic stem/progenitor cell (HSPC) differentiation have been developed, the HSPC yields achieved using these strategies are not yet available for clinical application. Recently, bioreactor-based devices and biochemical factors synergistically have been used to induce hematopoietic differentiation and showed a potential role in hematopoiesis. This chapter describes a protocol for using a random positioning machine bioreactor to culture human PSCs and the large-scale production of HPCs. Techniques for characterizing the differentiated cells and assessing the efficiency of hematopoietic differentiation in the bioreactor with immunostaining and flow cytometry are also presented.

Single-cell ATAC-Seq reveals cell type-specific transcriptional regulation and unique chromatin accessibility in human spermatogenesis.

During human spermatogenesis, germ cells undergo dynamic changes in chromatin organization/re-packaging and in transcriptomes. In order to better understand the underlying mechanism(s), scATAC-Seq of 5376 testicular cells from 3 normal men were performed. Data were analyzed in parallel with the scRNA-Seq data of human testicular cells. In all, 10 germ cell types associated with spermatogenesis and 6 testicular somatic cell types were identified, along with 142 024 peaks located in promoter, genebody and CpG Island. We had examined chromatin accessibility of all chromosomes, with chromosomes 19 and 17 emerged as the leading chromosomes that displayed high chromatin accessibility. In accessible chromatin regions, transcription factor-binding sites were identified and specific motifs with high frequencies at different spermatogenesis stages were detected, including CTCF, BORIS, NFY, DMRT6, EN1, ISL1 and GLI3. Two most remarkable observations were noted. First, TLE3 was specifically expressed in differentiating spermatogonia. Second, PFN4 was found to be involved in actin cytoskeletal organization during meiosis. More important, unique regions upstream of PFN4 and TLE3 were shown to display high accessibility, illustrating their significance in supporting human spermatogenesis.

Simulated Microgravity Potentiates Hematopoietic Differentiation of Human Pluripotent Stem Cells and Supports Formation of 3D Hematopoietic Cluster.

Microgravity has been shown to induces many changes in proliferation, differentiation and growth behavior of stem cells. Little is known about the effect of microgravity on hematopoietic differentiation of pluripotent stem cells (PSCs). In this study, we used the random position machine (RPM) to investigate whether simulated microgravity (SMG) allows the induction of hematopoietic stem/progenitor cell (HSPC) derived from human embryonic stem cells (hESCs) in vitro. The results showed that SMG facilitates hESCs differentiate to HSPC with more efficient induction of CD34+CD31+ hemogenic endothelium progenitors (HEPs) on day 4 and CD34+CD43+ HSPC on day 7, and these cells shows an increased generation of functional hematopoietic cells in colony-forming unit assay when compared with normal gravity (NG) conditions. Additionally, we found that SMG significantly increased the total number of cells on day 4 and day 7 which formed more 3D cell clusters. Transcriptome analysis of cells identified thousands of differentially expressed genes (DEGs) between NG and SMG. DEGs down-regulated were enriched in the axonogenesis, positive regulation of cell adhesion, cell adhesion molecule and axon guidance, while SMG resulted in the up-regulation of genes were functionally associated with DNA replication, cell cycle, PI3K-Akt signaling pathway and tumorigenesis. Interestingly, some key gene terms were enriched in SMG, like hypoxia and ECM receptor interaction. Moreover, HSPC obtained from SMG culture conditions had a robust ability of proliferation in vitro. The proliferated cells also had the ability to form erythroid, granulocyte and monocyte/macrophage colonies, and can be induced to generate macrophages and megakaryocytes. In summary, our data has shown a potent impact of microgravity on hematopoietic differentiation of hPSCs for the first time and reveals an underlying mechanism for the effect of SMG on hematopoiesis development.

Development of mouse preimplantation embryos in space.

The development of life beyond planet Earth is a long-standing quest of the human race, but whether normal mammalian embryonic development can occur in space is still unclear. Here, we show unequivocally that preimplantation mouse embryos can develop in space, but the rate of blastocyst formation and blastocyst quality are compromised. Additionally, the cells in the embryo contain severe DNA damage, while the genome of the blastocysts developed in space is globally hypomethylated with a unique set of differentially methylated regions. The developmental defects, DNA damage and epigenetic abnormalities can be largely mimicked by the treatment with ground-based low-dose radiation. However, the exposure to simulated microgravity alone does not cause major disruptions of embryonic development, indicating that radiation is the main cause for the developmental defects. This work advances the understanding of embryonic development in space and reveals long-term extreme low-dose radiation as a hazardous factor for mammalian reproduction.

Impacts of Caffeine during Pregnancy.

Epidemiological studies have revealed that caffeine consumption during pregnancy is associated with adverse gestational outcomes, yet the underlying mechanisms remain obscure. Recent animal studies with physiologically relevant dosages have begun to dissect adverse effects of caffeine during pregnancy with respect to oviduct contractility, embryo development, uterine receptivity, and placentation that jointly contribute to pregnancy complications. Interestingly, caffeine's effects are highly variable between individual animals under well-controlled experimental settings, suggesting the possibility of epigenetic regulation of these phenotypes, in addition to genetic variants. Moreover, caffeine exposure during sensitive windows of pregnancy may induce epigenetic changes in the developing fetus or even the germ cells to cause adult-onset diseases in subsequent generations. We discuss these research frontiers in light of emerging data.

Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production.

Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction. With the increasing demand for transfusion, however, there is an undersupply of donors to provide the platelet source. Thus, scientists have sought to design methods for deriving clinical-scale platelets ex vivo. Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells (SCs), the platelet yields achieved using these strategies have not been adequate for clinical application. In this review, we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo, recapitulate the key advances in the production of SC-derived platelets using several SC sources, and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.

Analysis of in vivo uterine peristalsis in the non-pregnant female mouse.

Uterine peristalsis due to spontaneous contractions of the myometrial smooth muscles has important roles in pre-implantation processes of intra-uterine sperm transport to the fertilization site, and then embryo transport to the implantation sites. We developed a new objective methodology to study in vivo uterine peristalsis in female mice during the pro-oestrus phase. The acquisition procedure of the uterine organ is remote without interfering with the organ function. The uniqueness of the new approach is that video images of physiological pattern were converted using image processing and new algorithms to biological time-dependent signals that can be processed with existing algorithms for signal processing. Using this methodology we found that uterine peristalsis in the pro-oestrus mouse is in the range of 0.008-0.029 Hz, which is about one contraction per minute and with fairly symmetric contractions that occasionally propagate caudally. This rate of contractions is similar to that of human uterine peristalsis acquired in vivo, which is important information for a popular animal model.

Caffeine consumption during early pregnancy impairs oviductal embryo transport, embryonic development and uterine receptivity in mice.

Caffeine consumption has been widely used as a central nervous system stimulant. Epidemiological studies, however, have suggested that maternal caffeine exposure during pregnancy is associated with increased abnormalities, including decreased fertility, delayed conception, early spontaneous abortions, and low birth weight. The mechanisms underlying the negative outcomes of caffeine consumption, particularly during early pregnancy, remain unclear. In present study, we found that pregnant mice treated with moderate (5 mg/kg) or high (30 mg/kg) dosage of caffeine (intraperitoneally or orally) during preimplantation resulted in retention of early embryos in the oviduct, defective embryonic development, and impaired embryo implantation. Transferring normal blastocysts into the uteri of caffeine-treated pseudopregnant females also showed abnormal embryo implantation, thus indicating impaired uterine receptivity by caffeine administration. The remaining embryos that managed to implant after caffeine treatment also showed increased embryo resorption rate and abnormal development at mid-term stage, and decreased weight at birth. In addition to a dose-dependent effect, significant variations between individual mice under the same caffeine dosage were also observed, suggesting different sensitivities to caffeine, similar to that observed in human populations. Collectively, our data revealed that caffeine exposure during early pregnancy impaired oviductal embryo transport, embryonic development, and uterine receptivity, which are responsible for abnormal implantation and pregnancy loss. The study raises the concern of caffeine consumption during early stages of pregnancy.

Effect of microgravity on proliferation and differentiation of embryonic stem cells in an automated culturing system during the TZ-1 space mission.

OBJECTIVE: Despite a great number of studies analysing the effects of microgravity on stem cell proliferation and differentiation, few of them have focused on real-time imaging estimates in space. Herein, we utilized the TZ-1 cargo spacecraft, automatic cell culture equipment and live cell imaging techniques to examine the effects of real microgravity on the proliferation and differentiation of mouse embryonic stem cells (mESCs). MATERIALS AND METHODS: Oct4-GFP, Brachyury-GFP mESC and Oct4-GFP mESC-derived EBs were used as experimental samples in the TZ-1 spaceflight mission. These samples were seeded into chambers, cultured in an automatic cell culture device and were transported into space during the TZ-1 mission. Over 15 days of spaceflight, bright field and fluorescent images of cell growth were taken in micrography, and the medium was changed every day. Real-time image data were transferred to the ground for analysis. RESULTS: Space microgravity maintains stemness and long-term survival of mESCs, promising 3D aggregate formation. Although microgravity did not significantly prevent the migration of EBs on the ECM substrate, it did prevent terminal differentiation of cells. CONCLUSIONS: This study demonstrates that space microgravity might play a potential role in supporting 3D cell growth and maintenance of stemness in embryonic stem cells, while it may negatively affect terminal differentiation.

Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs.

The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA 1 . Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress2,3 and metabolic disorders4-6. How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat-diet-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (m5C, m2G) in sperm 30-40 nt RNA fractions that are induced by a high-fat diet. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs and rRNA-derived small RNAs, which might be essential in composing a sperm RNA 'coding signature' that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated m5C contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.

Fighting against Skin Aging: The Way from Bench to Bedside.

As the most voluminous organ of the body that is exposed to the outer environment, the skin suffers from both intrinsic and extrinsic aging factors. Skin aging is characterized by features such as wrinkling, loss of elasticity, laxity, and rough-textured appearance. This aging process is accompanied with phenotypic changes in cutaneous cells as well as structural and functional changes in extracellular matrix components such as collagens and elastin. In this review, we summarize these changes in skin aging, research advances of the molecular mechanisms leading to these changes, and the treatment strategies aimed at preventing or reversing skin aging.

Silk fibroin/chitosan scaffold with tunable properties and low inflammatory response assists the differentiation of bone marrow mesenchymal stem cells.

The physical and chemical properties of the scaffold are known to play important roles in three-dimensional (3D) cell culture, which always determine the cellular fate or the results of implantation. To control these properties becomes necessary for meeting the requirements of a variety of tissue engineering applications. In this study, a series of silk fibroin/chitosan (SF/CS) scaffolds with tunable properties were prepared using freeze-drying method, and the rat bone marrow-derived mesenchymal stem cells (BM-MSCs) were seeded in these scaffolds to evaluate their availability of use in tissue engineering. The 3D structure, mechanical properties and degradation ability of SF/CS scaffold can be tuned by changing the total concentration of the precursor solution and the blending ratio between SF and CS. BM-MSCs cultured in the SF/CS scaffold exhibited excellent proliferation and multiple morphologies. The induction of osteogenic and adipogenic differentiation of BM-MSCs were successful in this scaffold when cultured in vitro. Subcutaneous implantation of the SF/CS scaffolds did not cause any inflammatory response within four weeks, which revealed good compatibility. Moreover, the implanted scaffold allowed host cells to invade, adhere, grow and form new blood vessels. With these excellent performance, SF/CS scaffold has great potential in preparing implants for tissue engineering applications.

Decidual Stromal Cell Necroptosis Contributes to Polyinosinic-Polycytidylic Acid-Triggered Abnormal Murine Pregnancy.

Infectious agents can reach the placenta either via the maternal blood or by ascending the genito-urinary tract, and then initially colonizing the maternal decidua. Decidual stromal cells (DSCs) are the major cellular component of the decidua. Although DSCs at the maternal-fetal interface contribute to the regulation of immunity in pregnancy in the face of immunological and physiological challenges, the roles of these DSCs during viral infection remain ill defined. Here, we characterized the response of DSCs to a synthetic double-stranded RNA molecule, polyinosinic-polycytidylic acid [poly(I:C)], which is a mimic of viral infection. We demonstrated that both transfection of cells with poly(I:C) and addition of extracellular (non-transfected) poly(I:C) trigger the necroptosis of DSCs and that this response is dependent on RIG-I-like receptor/IPS-1 signaling and the toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-ß pathway, respectively. Furthermore, following poly(I:C) challenge, pregnant mixed lineage kinase domain-like protein-deficient mice had fewer necrotic cells in the mesometrial decidual layer, as well as milder pathological changes in the uterine unit, than did wild-type mice. Collectively, our results establish that necroptosis is a contributing factor in poly(I:C)-triggered abnormal pregnancy and thereby indicate a novel therapeutic strategy for reducing the severity of the adverse effects of viral infections in pregnancy.