Federica Zacchini

Post-implantation mortality of in vitro produced embryos is associated with DNA methyltransferase 1 dysfunction in sheep placenta

STUDY QUESTION Is DNA methyltransferase 1 (DNMT1) dysfunction involved in epigenetic deregulation of placentae from embryos obtained by assisted reproduction technologies (ARTs)? SUMMARY ANSWER DNMT1 expression in growing placentae of in vitro produced (IVP) embryos is compromised and associated with pregnancy loss. WHAT IS KNOWN ALREADY DNMT1 maintains the methylation profile of genes during cell division. The methylation status of genes involved in placenta development is altered in embryos obtained in vitro. Disturbances in the epigenetic regulation of gene expression during placentogenesis could be involved in the frequent developmental arrest and loss of IVP embryos. STUDY DESIGN, SIZE, DURATION Forty sheep were naturally mated (Group 1, CTR). IVP blastocysts (2-4 per ewe) were surgically transferred to the remaining 46 recipient sheep 6 days after oestrus (Group 2). Twenty-one recipients from Group 1 and 27 recipients from Group 2 were allowed to deliver in order to compare embryo survival in both groups at term (150 days). From the remaining recipients (n = 38), fetuses and placentae of both groups were recovered by paramedian laparotomy at Days 20, 22, 24, 26 and 28 of gestation. MATERIALS, SETTING, METHODS Immediately after collection, early placental tissues (chorion-allantois) were snap frozen in liquid nitrogen and DNMT1 expression and activity was evaluated. mRNA levels (for DNMT1, HDAC2, PCNA, DMAP1, MEST, IGF2, CDKN1C, H19) and the methylation status of H19 were also analyzed. Furthermore, embryo size and survival rate were measured. MAIN RESULTS AND THE ROLE OF CHANCE Our study shows that DNMT1 expression was reduced in early placentae from sheep IVP embryos. This reduction was associated with growth arrest and subsequent death of the sheep embryos. Conversely, normal levels of DNMT1 and its cofactors were observed in placentae from IVP embryos that survived this developmental bottleneck. Although DNA methylation machinery was severely compromised in IVP placentae only up to Day 24, the low DNMT1 enzymatic activity that persisted after this stage in IVP placentae was not lethal for the developing embryos. LIMITATIONS, REASONS FOR CAUTION The studied genes represent only a small fraction of genes regulating DNA methylation. Further studies are needed to evaluate changes in the expression and methylation status of other genes that may lead to developmental arrest of IVP embryos. As this is the only study evaluating the functionality of DNMT1 machinery in placentae from ART embryos, studies on other species are needed to confirm if our observation may be applicable to all mammalian embryos produced in vitro. WIDER IMPLICATIONS OF THE FINDINGS The knowledge about compromised activity of DNMT1 in placentae obtained from IVP embryos should stimulate detailed studies on the metabolic requirements of oocytes and embryos in order to adequately enrich the culture media.

A short exposure to polychlorinated biphenyls deregulates cellular autophagy in mammalian blastocyst in vitro

BACKGROUND Polychlorinated biphenyls (PCBs) are common environmental contaminants that represent an important risk factor of reproductive disorders in chronically exposed human populations. However, it is not known whether a short accidental exposure of embryos to PCBs before implantation might influence their further development and whether the effect might be reversible. METHODS AND RESULTS To this aim, in vitro-matured sheep blastocysts were incubated with 2 or 4 µg/ml Aroclor 1254 (A1254), a mixture of 60 PCB congeners for 48 h after which blastocyst proliferation and ability for outgrowth in vitro were assessed. Blastocysts exposed to A1254 showed: (i) reduced proliferation and cell number (particularly in the inner cell mass compartment); (ii) accumulation of vacuoles and lipid droplets, diffused mitochondrial damage and up-regulation of autophagy markers (ATG6 and LC3), all signs indicative of deregulated autophagy, and (iii) massive cell death. Although exposed embryos resumed growth following A1254 removal, their subsequent development remained severely perturbed. CONCLUSIONS These findings indicate that short exposure of blastocysts to PCBs leads to its damage characterized by deregulated autophagy and subsequent cell death.

Towards storage of cells and gametes in dry form

We review published data on cell/gamete lyophilization. Most studies have utilized the same established protocols for cryopreservation (storage in liquid nitrogen) as for cell lyophilization (dehydration of frozen samples by water sublimation). Surveying natural lyoprotectants, we suggest trehalose and late embryogenesis abundant (LEA) proteins as ideal candidates for the reversible desiccation of mammalian cells/gametes. We find that despite the numerous water subtraction techniques, scientists have relied almost exclusively on lyophilization. There is thus room for improvement in both medium formulation and water subtraction strategies for dry cell/gamete storage. We believe the development of dry processing protocols for use in biobanks of cells/gametes, at reduced cost and with minimal carbon footprint, is within our grasp.

Grand-paternal age and the development of autism-like symptoms in mice progeny

Advanced paternal age (APA) contributes to the risk of autism spectrum disorders (ASDs) in children. In this study, we used a mouse model to investigate the effects of APA on behavioral features related to autistic syndromes (that is, social deficits, communication impairments and stereotypic/repetitive behaviors). We also examined whether such effects are transmitted across generations. To do this, males aged 15 months (APA) and 4 months (control) were bred with 4-month-old females, and the resulting offspring (F1) and their progeny (F2; conceived by 4-month-old parents) were tested for the presence and severity of ASD-like behaviors. Our results indicate that APA resulted in offspring that displayed distinctive symptoms of ASD. We found that both F1 conceived from old fathers and F2 derived from old grandfathers displayed increased ultrasound vocalization (USV) activity, decreased sociability, increased grooming activity and increased anxiety-like responses. Moreover, such abnormalities were partially transmitted to the second generation of mice, having APA grandfathers. In conclusion, our study suggests that the risk of ASD could develop over generations, consistent with heritable mutations and/or epigenetic alterations associated with APA.

Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures

Summary Protamines confer a compact structure to the genome of male gametes. Here, we find that somatic cells can be remodeled by transient expression of protamine 1 (Prm1). Ectopically expressed Prm1 forms scattered foci in the nuclei of fibroblasts, which coalescence into spermatid-like structures, concomitant with a loss of histones and a reprogramming barrier, H3 lysine 9 methylation. Protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone TH2B exchange and developed into normal blastocyst stage embryos in vitro. Altogether, our findings present a model to study male-specific chromatin remodeling, which can be exploited for the improvement of somatic cell nuclear transfer.

Genomic stability of lyophilized sheep somatic cells before and after nuclear transfer.

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.

Efficient production and cellular characterization of sheep androgenetic embryos.

The production of androgenetic embryos in large animals is a complex procedure. Androgenetic embryos have been produced so far only in cattle and sheep using pronuclear transfer (PT) between zygotes derived from in vitro fertilization (IVF) of previously enucleated oocytes. PT is required due to the poor developmental potential of androgenotes derived from IVF of enucleated oocytes. Here we compare the developemt to blastocyst of androgenetic embryos produced by the standard pronuclear transfer and by fertilization of oocytes enucleated in Ca2+/Mg2+-free medium, without pronuclear transfer. The enucleation in Ca2+/Mg2+-free medium abolished almost completely the manipulation-induced activation, significantly improving the development to blastocyst of the androgenetic embryos (IVF followed by PT; 18.6%: IVF only; 17.7%, respectively). Karyotype analysis of IVF revealed a similar proportion of diploid embryos in androgenetic and control blastocysts (35% and 36%, respectively), although mixoploid blastocysts were frequently observed in both groups (64%). Androgenotes had lower total cell numbers than control and parthenogenetic embryos, but more cells in ICM cells comparing to parthenogenotes (30.42 vs. 17.15%). Higher expression of the pluripotency-associated gene NANOG, and trophoblastic-specific gene CDX2, were also observed in androgenotes compared to parthenogenotes and controls. The global methytion profile of androgenetic embryos was comparable to controls, but was lower than parthenogenetic embryos. The cell composition and methylation pattern we have detected in monoparental sheep monoparental embryos are unprecedented, and differ considerably from the standard reference mouse embryos. Altogether, these finding indicate significant differences across species in the molecular mechanisms regulating early development of monoparental embryos, and highlights the need to study postimplantation development of androgenetic embryos in sheep.

Impaired Placental Vasculogenesis Compromises the Growth of Sheep Embryos Developed In Vitro

ABSTRACT To evaluate how assisted reproductive technologies (ART) affect vasculogenesis of the developing conceptus, we analyzed placental and fetal development of in vitro-produced (IVP) sheep embryos. Pregnancies produced by ART carry increased risk of low birth weight, though what causes this risk remains largely unknown. We recently reported that developmental arrest of sheep conceptuses obtained by ART is most pronounced when the cardiovascular system develops (Days 20–30 of development). A total of 86 IVP blastocysts (2–4 per ewe) were surgically transferred to 30 recipient sheep 6 days after estrus; 20 sheep were naturally mated (control). Conceptuses were recovered from sheep at Days 20, 22, 26, and 30 of gestation and morphologically evaluated. Then, the conceptuses and part of their placentae (chorion-allantois) were fixed for histological and immunohistochemical analysis and snap-frozen in liquid nitrogen for subsequent mRNA expression analysis. Results demonstrate that the cardiovascular systems of sheep IVP conceptuses were severely underdeveloped. Pericardial and placental hemorrhages were noted in a majority (5/7) of the dead embryos. In the surviving IVP embryos, the expression of angiogenetic factors was reduced at Day 20. The placental vessels were underdeveloped on Days 20 and 22 (P < 0.05), though placental vasculogenesis was successfully completed on subsequent days. However, low vessel number persisted at Days 26 and 30 (4.6 vs. 5.9 and 6.64 vs. 8.70 per field, respectively; P < 0.05) together with reduced vessel diameter at Day 26 (46.89 vs. 89.92 μm; P < 0.05). In vitro production of sheep embryos induced severely impaired vasculogenesis early in gestation. This may lead to developmental programing problems, such as intrauterine growth restriction of the fetus, resulting in long-term health consequences for the offspring, such as cardiovascular diseases.

A simplified approach for oocyte enucleation in mammalian cloning.

Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.

Synergies between assisted reproduction technologies and functional genomics

This review, is a synopsis of advanced reproductive technologies in farm animals, including the discussion of their limiting factors as revealed by the study of offspring derived from embryos produced in vitro and through cloning. These studies show that the problems of epigenetic mis-programming, which were reported in the initial stages of assisted reproduction, still persist. The importance of whole-genome analyses, including the methylome and transcriptome, in improving embryo biotechnologies in farm animals, are discussed. Genome editing approaches for the improvement of economically-relevant traits in farm animals are also described. Efficient farm animal embryo biotechnologies, including cloning and the most recent technologies such as genome editing, will effectively complement the latest strategies to accelerate genetic improvement of farm animals.