Gerald Schatten

Energy Metabolism in Human Pluripotent Stem Cells and Their Differentiated Counterparts

Background Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells. Methodology/Principal Findings We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism. Conclusions/Findings Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH).

Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos

Abstract The strictly maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) in mammals is a developmental paradox promoted by an unknown mechanism responsible for the destruction of the sperm mitochondria shortly after fertilization. We have recently reported that the sperm mitochondria are ubiquitinated inside the oocyte cytoplasm and later subjected to proteolysis during preimplantation development (P. Sutovsky et al., Nature 1999; 402:371–372). Here, we provide further evidence for this process by showing that the proteolytic destruction of bull sperm mitochondria inside cow egg cytoplasm depends upon the activity of the universal proteolytic marker, ubiquitin, and the lysosomal apparatus of the egg. Binding of ubiquitin to sperm mitochondria was visualized by monospecific antibodies throughout pronuclear development and during the first embryonic divisions. The recognition and disposal of the ubiquitinated sperm mitochondria was prevented by the microinjection of anti-ubiquitin antibodies and by the treatment of the fertilized zygotes with lysosomotropic agent ammonium chloride. The postfecundal ubiquitination of sperm mitochondria and their destruction was not seen in the hybrid embryos created using cow eggs and sperm of wild cattle, gaur, thus supporting the hypothesis that sperm mitochondrion destruction is species specific. The initial ligation of ubiquitin molecules to sperm mitochondrial membrane proteins, one of which could be prohibitin, occurs during spermatogenesis. Even though the ubiquitin cross-reactivity was transiently lost from the sperm mitochondria during epididymal passage, likely as a result of disulfide bond cross-linking, it was restored and amplified after fertilization. Ubiquitination therefore may represent a mechanism for the elimination of paternal mitochondria during fertilization. Our data have important implications for anthropology, treatment of mitochondrial disorders, and for the new methods of assisted procreation, such as cloning, oocyte cytoplasm donation, and intracytoplasmic sperm injection.

Development: Ubiquitin tag for sperm mitochondria

Like other mammals, humans inherit mitochondria from the mother only, even though the sperm contributes nearly one hundred mitochondria to the fertilized egg. In support of the idea that this strictly maternal inheritance of mitochondrial DNA arises from the selective destruction of sperm mitochondria, we show here that sperm mitochondria inside fertilized cow and monkey eggs are tagged by the recycling marker protein ubiquitin. This imprint is a death sentence that is written during spermatogenesis and executed after the sperm mitochondria encounter the eggs cytoplasmic destruction machinery.

Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys.

Intracytoplasmic sperm injection has begun an era of considerable improvements in treating male infertility. Despite its success, questions remain about the dangers of transmitting traits responsible for male infertility, sex and autosomal chromosome aberrations and possible mental, physical and reproductive abnormalities. We report here the first births of rhesus monkeys produced by intracytoplasmic sperm injection at rates greater or equal to those reported by clinics. Essential assumptions about this process are flawed, as shown by results with the preclinical, nonhuman primate model and with clinically discarded specimens. Dynamic imaging demonstrated the variable position of the second meiotic spindle in relation to the first polar body; consequently, microinjection targeting is imprecise and potentially lethal. Intracytoplasmic sperm injection resulted in abnormal sperm decondensation, with the unusual retention of vesicle-associated membrane protein and the perinuclear theca, and the exclusion of the nuclear mitotic apparatus from the decondensing sperm nuclear apex. Male pronuclear remodeling in the injected oocytes was required before replication of either parental genome, indicating a unique G1-to-S transition checkpoint during zygotic interphase (the first cell cycle). These irregularities indicate that the intracytoplasmic sperm injection itself might lead to the observed increased chromosome anomalies.

Paternal Contributions to the Mammalian Zygote: Fertilization after Sperm-Egg Fusion

Mammalian fertilization has traditionally been regarded as a simple blending of two gametes, during which the haploid genome of the fertilizing spermatozoon constitutes the primary paternal contribution to the resulting embryo. In contrast to this view, new research provides evidence of important cytoplasmic contributions made by the fertilizing spermatozoon to the zygotic makeup, to the organization of preimplantation development, and even reproductive success of new forms of assisted fertilization. The central role of the sperm-contributed centriole in the reconstitution of zygotic centrosome has been established in most mammalian species and is put in contrast with strictly maternal centrosomal inheritance in rodents. The complementary reduction or multiplication of sperm and oocyte organelles during gametogenesis, exemplified by the differences in the biogenesis of centrosome in sperm and oocytes, represents an intriguing mechanism for avoiding their redundancy during early embryogenesis. New studies on perinuclear theca of sperm revealed its importance for both spermatogenesis and fertilization. Remodeling of the sperm chromatin into a male pronucleus is guided by oocyte-produced, reducing peptide glutathione and a number of molecules required for the reconstitution of the functional nuclear envelope and nuclear skeleton. Although some of the sperm structures are transformed into zygotic components, the elimination of others is vital to early stages of embryonic development. Sperm mitochondria, carrying potentially harmful paternal mtDNA, appear to be eliminated by a ubiquitin-dependent mechanism. Other accessory structures of the sperm axoneme, including fibrous sheath, microtubule doublets, outer dense fibers, and the striated columns of connecting piece, are discarded in an orderly fashion. The new methods of assisted fertilization, represented by intracytoplasmic sperm injection and round spermatid injection, bypass multiple steps of natural fertilization by introducing an intact spermatozoon or spermatogenic cell into oocyte cytoplasm. Consequently, the carryover of sperm accessory structures that would normally be eliminated before or during the entry of sperm into oocyte cytoplasm persist therein and may interfere with early embryonic development, thus decreasing the success rate of assisted fertilization and possibly causing severe embryonic anomalies. Similarly, foreign organelles, proteins, messenger RNAs, and mitochondrial DNAs, which may have a profound impact on the embryonic development, are propagated by the nuclear transfer of embryonic blastomeres and somatic cell nuclei. This aspect of assisted fertilization is yet to be explored by a focused effort.

Clonal propagation of primate offspring by embryo splitting

Primates that are identical in both nuclear and cytoplasmic components have not been produced by current cloning strategies, yet such identicals represent the ideal model for investigations of human diseases. Here, genetically identical nonhuman embryos were produced as twin and larger sets by separation and reaggregation of blastomeres of cleavage-stage embryos. A total of 368 multiples were created by the splitting of 107 rhesus embryos with four pregnancies established after 13 embryo transfers (31% versus 53% in vitro fertilization controls). The birth of Tetra, a healthy female cloned from a quarter of an embryo, proves that this approach can result in live offspring.

DNA Damage Responses in Human Induced Pluripotent Stem Cells and Embryonic Stem Cells

Background Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability. Methodology/Principal Findings Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB), and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G2 phase of the cell cycle, displaying a lack of the G1/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. Conclusions/Significance High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G1/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.

Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

Vesicular traffic and golgi apparatus dynamics during mammalian spermatogenesis: implications for acrosome architecture.

Abstract Vesicular membrane trafficking during acrosome biogenesis in bull and rhesus monkey spermatogenesis differs from the somatic cell paradigm as imaged dynamically using the Golgi apparatus probes β-COP, giantin, Golgin-97, and Golgin-95/GM130. In particular, sorting and delivery of proteins seemed less precise during spermatogenesis. In early stages of spermiogenesis, many Golgi resident proteins and specific acrosomal markers were present in the acrosome. Trafficking in both round and elongating spermatids was similar to what has been described for somatic cells, as judged by the kinetics of Golgi protein incorporation into endoplasmic reticulum-like structures after brefeldin A treatment. These Golgi components were retrieved from the acrosome at later stages of differentiation and were completely devoid of immature spermatozoa. Our data suggest that active anterograde and retrograde vesicular transport trafficking pathways, involving both β-COP- and clathrin-coated vesicles, are involved in retrieving Golgi proteins missorted to the acrosome and in controlling the growth and shape of this organelle.

Dynamic Imaging of the Metaphase II Spindle and Maternal Chromosomesin Bovine Oocytes: Implications for Enucleation Efficiency Verification, Avoidanceof Parthenogenesis, and Successful Embryogenesis

Abstract Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocytes meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4–7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 ± 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 ± 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 ± 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus “cloning” success, without compromising the oocytes developmental potential after activation.