Priscila Ramos-Ibeas

Sex-specific embryonic origin of postnatal phenotypic variability

Preimplantation developmental plasticity has evolved in order to offer the best chances of survival under changing environments. Conversely, environmental conditions experienced in early life can dramatically influence neonatal and adult biology, which may result in detrimental long-term effects. Several studies have shown that small size at birth, which is associated with a greater risk of metabolic syndrome, is largely determined before the formation of the blastocysts because 70%-80% of variation in bodyweight at birth has neither a genetic nor environmental component. In addition, it has been reported that adult bodyweight is programmed by energy-dependent process during the pronuclear stage in the mouse. Although the early embryo has a high developmental plasticity and adapts and survives to adverse environmental conditions, this adaptation may have adverse consequences and there is strong evidence that in vitro culture can be a risk factor for abnormal fetal outcomes in animals systems, with growing data suggesting that a similar link may be apparent for humans. In this context, male and female preimplantation embryos display sex-specific transcriptional and epigenetic regulation, which, in the case of bovine blastocysts, expands to one-third of the transcripts detected through microarray analysis. This sex-specific bias may convert the otherwise buffered stochastic variability in developmental networks in a sex-determined response to the environmental hazard. It has been widely reported that environment can affect preimplantation development in a sex-specific manner, resulting in either a short-term sex ratio adjustment or in long-term sex-specific effects on adult health. The present article reviews current knowledge about the natural phenotypic variation caused by epigenetic mechanisms and the mechanisms modulating sex-specific changes in phenotype during early embryo development resulting in sex ratio adjustments or detrimental sex-specific consequences for adult health. Understanding the natural embryo sexual dimorphism for programming trajectories will help understand the early mechanisms of response to environmental insults.

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.

An Efficient System to Establish Biopsy-Derived Trophoblastic Cell Lines from Bovine Embryos

ABSTRACT Trophoblastic cells play a crucial role in implantation and placentogenesis and can be used as a model to provide substantial information on the peri-implantation period. Unfortunately, there are few cell lines for this purpose in cattle because of the difficulty of raising successive cell stocks in the long-term. Our results show that the combination of a monolayer culture system in microdrops on a surface treated with gelatin and the employment of conditioned media from mouse embryonic fibroblasts support the growth of bovine trophoblastic cells lines from an embryo biopsy. Expression profiles of mononucleate- and binucleate-specific genes in established trophoblastic cells lines represented various stages of gestation. Moreover, the ability to expand trophoblastic cell lines for more than 2 yr together with pluripotency-related gene expression patterns revealed certain self-renewal capacity. In summary, we have developed a system to expand in vitro trophoblastic cells from an embryo biopsy that solves the limitations of using amplified DNA from a small number of cells for bovine embryo genotyping and epigenotyping and, on the other hand, facilitates the establishment of trophoblastic cell lines that can be useful as peri-implantation in vitro models.

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

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

Most regions of mouse epididymis are able to phagocytose immature germ cells

The role of the epididymis as a quality control organ in preventing infertile gametes entering the ejaculate has been extensively explored, and it has been suggested that a specific region of mammalian epididymis is able to phagocytose abnormal germ cells. This study examines whether the epithelium of certain zones of the mouse epididymis can act as a selection barrier by removing immature germ cells from the lumen by phagocytosis. To detect the presence of immature germ cells in the epididymis, we generated transgenic mice expressing enhanced green fluorescent protein under the deleted in azoospermia-like (mDazl) promoter to easily identify immature germ cells under fluorescence microscopy. Using this technique, we observed that during the first stage of spermatogenesis in prepuberal mice, a wave of immature germ cells is released into the epididymis and that the immature epididymis is not able to react to this abnormal situation. By contrast, when immature germ cells were artificially released into the epididymis in adult mice, a phagocytic response was observed. Phagosomes appeared inside principal cells of the epididymal epithelium and were observed to contain immature germ cells at different degradation stages in different zones of the epididymis, following the main wave of immature germ cells. In this paper, we describe how the epididymal epithelium controls sperm quality by clearing immature germ cells in response to their artificially induced massive shedding into the epididymal lumen. Our observations indicate that this phenomenon is not restricted to a given epididymis region and that phagocytic capacity is gradually acquired during epididymal development.

The role of prion protein in stem cell regulation.

Cellular prion protein (PrP(C)) has been well described as an essential partner of prion diseases due to the existence of a pathological conformation (PrP(Sc)). Recently, it has also been demonstrated that PrP(C) is an important element of the pluripotency and self-renewal matrix, with an increasing amount of evidence pointing in this direction. Here, we review the data that demonstrate its role in the transcriptional regulation of pluripotency, in the differentiation of stem cells into different lineages (e.g. muscle and neurons), in embryonic development, and its involvement in reproductive cells. Also highlighted are recent results from our laboratory that describe an important regulation by PrP(C) of the major pluripotency gene Nanog. Together, these data support the appearance of new strategies to control stemness, which could represent an important advance in the field of regenerative medicine.

Tet-mediated imprinting erasure in H19 locus following reprogramming of spermatogonial stem cells to induced pluripotent stem cells

Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. Currently, it is unclear whether artificial reprogramming induced by the expression of Yamanaka factors disrupts these marks and whether cell type of origin affects the dynamics of reprogramming. In this study, spermatogonial stem cells (SSC) that harbor paternalized imprinting marks, and fibroblasts were reprogrammed to iPSC (SSCiPSC and fiPSC). The SSCiPSC were able to form teratomas and generated chimeras with a higher skin chimerism than those derived from fiPSC. RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC. Likewise, reprogramming of SSC affected 26 of 41 imprinting gene clusters known in the mouse genome. A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC. Imprinting erasure in SSCiPSC was maintained even after in vivo differentiation into teratomas. Reprogramming of SSC from Tet1 and Tet2 double knockout mice however lacked demethylation of H19 ICR. These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus.

Potential Health Risks Associated to ICSI: Insights from Animal Models and Strategies for a Safe Procedure.

Artificial reproductive techniques are currently responsible for 1.7–4% of the births in developed countries and intracytoplasmatic sperm injection (ICSI) is the most commonly used, accounting for 70–80% of the cycles performed. Despite being an invaluable tool for infertile couples, the technique bypasses several biological barriers that naturally select the gametes to achieve an optimal embryonic and fetal development. In this perspective, ICSI has been associated with an increased risk for diverse health problems, ranging from premature births and diverse metabolic disorders in the offspring to more severe complications such as abortions, congenital malformations, and imprinting disorders. In this review, we discuss the possible implications of the technique per se on these adverse outcomes and highlight the importance of several experiments using mammalian models to truthfully test these implications and to uncover the molecular base that origins these health problems. We also dissect the specific hazards associated to ICSI and describe some strategies that have been developed to mimic the gamete selection occurring in natural conception in order to improve the safety of the procedure.

Elimination of methylation marks at lysines 4 and 9 of histone 3 (H3K4 and H3K9) of spermatozoa alters offspring phenotype

The contribution of the contents of spermatozoa to the development of the embryo is currently being considered wider than was previously thought. Recent findings point to the participation of epigenetic marks present in the retained histones of mature spermatozoa on embryo and fetal development. Here we created a novel conditional transgenic mouse that expresses lysine (K) demethylase 1a (Kdm1a) during spermatogenesis when the testicles are subjected to heat stress. Using these animals under these conditions we were able to reduce the methylation level of histone 3 at lysines 4 and 9 (H3K4 and H3K9, respectively) in mature spermatozoa. The offspring of these transgenic mice were followed for correct development and growth after birth. We found that the offspring of males expressing Kdm1a suffered 20% of reabsorptions at Day 15 after implantation (vs 0.3% in the control). In addition, 35% of the offspring sired by these males showed some kind of abnormality (suckling defects, lack of movement coordination, dropping forelimbs, abnormal body curvature, absence of eyes, gigantisms and neuromuscular defects) and 25% died before postnatal Day 21. Some abnormalities were maintained to adulthood. These results show that alteration of epigenetic marks present in the retained histones of mature spermatozoa affect fetal development and have phenotypic consequences in the newborn.

The oviduct: from sperm selection to the epigenetic landscape of the embryo

Abstract The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of preimplantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed. Summary Sentence Herein we review the influence of the oviduct on spermatozoa and embryo with special emphasis on the composition of the oviductal flow, sperm selection and remodelling of embryo epigenetics.