Efecto del origen y las condiciones de cultivo en la heterogeneidad de poblaciones celulares pluripotentes

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Fisiología Animal, leída el 04-07-2014Sección Deptal. de Fisiología (Veterinaria)Fac. de VeterinariaTRUEunpu

In vitro culture of ovine embryos up to early gastrulating stages

Developmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes - trophoblast and hypoblast - and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated 'The people behind the papers' interview

An ancient testis-specific IQ motif-containing H gene regulates specific transcript isoform expression during spermatogenesis

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications – principally increased intron retention – resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biologyThis study was funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (PID2021-122507OB-I00 and PID2020-117491GB-I00) and the European Union NextGenerationEU/PRTR. P.N.-L. was supported by a pre-doctoral fellowship from the Ministerio de Ciencia e Innovación (PRE2019-088813) and M.L. was supported by a Juan de la Cierva postdoctoral contract (FJC2019-040385-I) from the Ministerio de Ciencia e Innovación. Open access funding provided by Consejo Superior de Investigaciones Cientıficas. Deposited in PMC for immediate releas

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior

Pluripotency and X chromosome dynamics revealed in pig pre-gastrulating embryos by single cell analysis.

High-resolution molecular programmes delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are largely inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and of epiblast and hypoblast in late blastocysts. We show that following an emergent short naive pluripotent signature in early embryos, there is a protracted appearance of a primed signature in advanced embryonic stages. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras

Zrsr2 and functional U12-dependent spliceosome are necessary for follicular development

SUMMARY ZRSR2 is a splicing factor involved in recognition of 30 -intron splice sites that is frequently mutated in myeloid malignancies and several tumors; however, the role of mutations of Zrsr2 in other tissues has not been analyzed. To explore the bio logical role of ZRSR2,we generated threeZrsr2 mutantmouse lines. AllZrsr2 mutant lines exhibited blood cell anomalies, and in two lines, oogenesis was blocked at the secondary follicle stage. RNA-seq of Zrsr2mu secondary follicles showed aberrations in gene expression and showed altered alternative splicing (AS) events involving enrichment of U12-type intron retention (IR), supporting the functional Zrsr2 action in minor spliceosomes. IR events were preferentially associated with centriole repli cation, protein phosphorylation, and DNA damage checkpoint. Notably, we found alterations in AS events of 50 meiotic genes. These results indicate that ZRSR2 mu tations alter splicing mainly in U12-type introns, which may affect peripheral blood cells, and impede oogenesis and female fertility

Intracytoplasmic Sperm Injection Using DNAFragmented Sperm in Mice Negatively Affects Embryo- Derived Embryonic Stem Cells, Reduces the Fertility of Male Offspring and Induces Heritable Changes in Epialleles

<p>Intracytoplasmic sperm injection (ICSI) in mice using DNA-fragmented sperm (DFS) has been linked to an increased risk of<br>genetic and epigenetic abnormalities both in embryos and offspring. This study examines: whether embryonic stem cells<br>(ESCs) derived from DFS-ICSI embryos reflect the abnormalities observed in the DFS-ICSI progeny; the effect of DFS-ICSI on<br>male fertility; and whether DFS-ICSI induces epigenetic changes that lead to a modified heritable phenotype. DFS-ICSIproduced<br>embryos showed a low potential to generate ESC lines. However, these lines had normal karyotype accompanied<br>by early gene expression alterations, though a normal expression pattern was observed after several passages. The fertility<br>of males in the DFS-ICSI and control groups was compared by mating test. Sperm quantity, vaginal plug and pregnancy<br>rates were significantly lower for the DFS-ICSI-produced males compared to in vivo-produced mice, while the number of<br>females showing resorptions was higher. The epigenetic effects of DFS-ICSI were assessed by analyzing the phenotype<br>rendered by the Axin1Fu allele, a locus that is highly sensitive to epigenetic perturbations. Oocytes were injected with<br>spermatozoa from Axin1Fu/+ mice and the DFS-ICSI-generated embryos were transferred to females. A significantly higher<br>proportion of pups expressed the active kinky-tail epiallele in the DFS-ICSI group than the controls. In conclusion: 1) ESCs<br>cannot be used as a model of DFS-ICSI; 2) DFS-ICSI reduces sperm production and fertility in the male progeny; and 3) DFSICSI<br>affects the postnatal expression of a defined epigenetically sensitive allele and this modification may be inherited across<br>generations.</p> <p> </p

The people behind the papers - Priscila Ramos-Ibeas and Pablo Bermejo-Álvarez

3 Pág.Symmetry breaking in ungulates occurs in a flat embryonic disc similar to that of humans, making it a potential model for studying early human development. However, it has previously been impossible to develop ungulate blastocysts in vitro to the symmetry-breaking stages. Now, a new paper in Development describes culture conditions that allow ungulate embryos to be developed in vitro through to early gastrulation. We caught up with corresponding authors, Priscila Ramos-Ibeas, Científico Titular at El Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), and Pablo Bermejo-Álvarez, Científico Titular at INIA, to find out more about their research and their future plans.Peer reviewe

Solving the "X" in embryos and stem cells

X-chromosome inactivation (XCI) is a complex epigenetic process that ensures that most X-linked genes are expressed equally for both sexes. Female eutherian mammals inactivate randomly the maternal or paternal inherited X-chromosome early in embryogenesis, whereas the extra-embryonic tissues experience an imprinting XCI that results in the inactivation of the paternal X-chromosome in mice. Although the phenomenon was initially described 40 years ago, many aspects remain obscure. In the last 2 years, some trademark publications have shed new light on the ongoing debate regarding the timing and mechanism of imprinted or random XCI. It has been observed that XCI is not accomplished at the blastocyst stage in bovines, rabbits, and humans, contrasting with the situation reported in mice, the standard model. All the species present 2 active X-chromosomes (Xa) in the early epiblast of the blastocyst, the cellular source for embryonic stem cells (ESCs). In this perspective, it would make sense to expect an absence of XCI in undifferentiated ESCs, but human ESCs are highly heterogeneous for this parameter and the presence of 2 Xa has been proposed as a true hallmark of ground-state pluripotency and a quality marker for female ESCs. Similarly, XCI reversal in female induced pluripotent stem cells is a key reprogramming event on the path to achieve the naïve pluripotency, and key pluripotency regulators can interact directly or indirectly with Xist. Finally, the presence of 2 Xa may lead to a sex-specific transcriptional regulation resulting in sexual dimorphism in reprogramming and differentiation. © Copyright 2012, Mary Ann Liebert, Inc