Nile Red and BODIPY Staining of Lipid Droplets in Mouse Oocytes and Embryos.

Lipid droplets (LDs) are intracellular structures composed of hydrophobic lipids. Their amount in oocytes and embryos varies among the mammalian species and even among different strains of the same species. Here we describe a method to stain LDs, which can be applied to previously fixed mouse oocytes and embryos. This method is based on fluorescent dyes, Nile red and BODIPY, which allow visualization and quantification of LDs using conventional and confocal fluorescence microscopy.

Parental ageing and Assisted Reproduction Technologies: analysis of risk of chronic diseases in the progeny / Pózne rodzicielstwo i rozród wspomagany: analiza ryzyka wystapienia chorób przewleklych u potomstwa.

Conception of a child at advanced parental age (> 35 years) has been steadily increasing in recent decades, especially in developed countries. Socio-economic factors, effective contraceptives, and the availability of Assisted Reproduction Technologies (ART) have a direct impact on postponing the decision to have a baby. ART enables reproductive success for people diagnosed as infertile or with reduced possibilities of becoming pregnant due to concomitant pathologies. Epidemiological studies indicate that both advanced parental age and ART are associated with pathologies of pregnancy, such as gestational diabetes, risk of pre-eclampsia, miscarriage, placental abruption, preterm labor, stillbirth, neurodevelopmental disorders and chronic disease of the offspring. In our work, we will focus on the available information on metabolic changes that increase the risk of developing cardiovascular diseases in the offspring of parents at an advanced age and conceived through ART. Finally, we will address the sources of the observed disturbances at the gamete and embryo level, related to oxygen stress, epigenetic modifications and DNA damage, considering possible rescue actions.

Ribosomal DNA methylation in human and mouse oocytes increases with age.

An age-dependent increase in ribosomal DNA (rDNA) methylation has been observed across a broad spectrum of somatic tissues and the male mammalian germline. Bisulfite pyrosequencing (BPS) was used to determine the methylation levels of the rDNA core promoter and the rDNA upstream control element (UCE) along with two oppositely genomically imprinted control genes (PEG3 and GTL2) in individual human germinal vesicle (GV) oocytes from 90 consenting women undergoing fertility treatment because of male infertility. Apart from a few (4%) oocytes with single imprinting defects (in either PEG3 or GTL2), the analyzed GV oocytes displayed correct imprinting patterns. In 95 GV oocytes from 42 younger women (26-32 years), the mean methylation levels of the rDNA core promoter and UCE were 7.4±4.0% and 9.3±6.1%, respectively. In 79 GV oocytes from 48 older women (33-39 years), methylation levels increased to 9.3±5.3% (P = 0.014) and 11.6±7.4% (P = 0.039), respectively. An age-related increase in oocyte rDNA methylation was also observed in 123 mouse GV oocytes from 29 4-16-months-old animals. Similar to the continuously mitotically dividing male germline, ovarian aging is associated with a gain of rDNA methylation in meiotically arrested oocytes. Oocytes from the same woman can exhibit varying rDNA methylation levels and, by extrapolation, different epigenetic ages.

Species-Specific Paternal Age Effects and Sperm Methylation Levels of Developmentally Important Genes.

A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three (NFKB2, RASGEF1C, and RPL6) age-related differentially methylated regions (ageDMRs) in humans, four (CHD7, HDAC11, PAK1, and PTK2B) in bovines, and three (Def6, Nrxn2, and Tbx19) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80%) or hypomethylated (<20%). In humans and mice, ageDMRs lost methylation, whereas bovine ageDMRs gained methylation with age. Our results are in line with the hypothesis that sperm ageDMRs are in regions under epigenomic evolution and may be part of an epigenetic mechanism(s) for lineage-specific environmental adaptations and provide a solid basis for studies on downstream effects in the genes analyzed here.

DNA damage in preimplantation embryos and gametes: specification, clinical relevance and repair strategies.

BACKGROUND: DNA damage is a hazard that affects all cells of the body. DNA-damage repair (DDR) mechanisms are in place to repair damage and restore cellular function, as are other damage-induced processes such as apoptosis, autophagy and senescence. The resilience of germ cells and embryos in response to DNA damage is less well studied compared with other cell types. Given that recent studies have described links between embryonic handling techniques and an increased likelihood of disease in post-natal life, an update is needed to summarize the sources of DNA damage in embryos and their capacity to repair it. In addition, numerous recent publications have detailed novel techniques for detecting and repairing DNA damage in embryos. This information is of interest to medical or scientific personnel who wish to obtain undamaged embryos for use in offspring generation by ART. OBJECTIVE AND RATIONALE: This review aims to thoroughly discuss sources of DNA damage in male and female gametes and preimplantation embryos. Special consideration is given to current knowledge and limits in DNA damage detection and screening strategies. Finally, obstacles and future perspectives in clinical diagnosis and treatment (repair) of DNA damaged embryos are discussed. SEARCH METHODS: Using PubMed and Google Scholar until May 2021, a comprehensive search for peer-reviewed original English-language articles was carried out using keywords relevant to the topic with no limits placed on time. Keywords included 'DNA damage repair', 'gametes', 'sperm', 'oocyte', 'zygote', 'blastocyst' and 'embryo'. References from retrieved articles were also used to obtain additional articles. Literature on the sources and consequences of DNA damage on germ cells and embryos was also searched. Additional papers cited by primary references were included. Results from our own studies were included where relevant. OUTCOMES: DNA damage in gametes and embryos can differ greatly based on the source and severity. This damage affects the development of the embryo and can lead to long-term health effects on offspring. DDR mechanisms can repair damage to a certain extent, but the factors that play a role in this process are numerous and altogether not well characterized. In this review, we describe the multifactorial origin of DNA damage in male and female gametes and in the embryo, and suggest screening strategies for the selection of healthy gametes and embryos. Furthermore, possible therapeutic solutions to decrease the frequency of DNA damaged gametes and embryos and eventually to repair DNA and increase mitochondrial quality in embryos before their implantation is discussed. WIDER IMPLICATIONS: Understanding DNA damage in gametes and embryos is essential for the improvement of techniques that could enhance embryo implantation and pregnancy success. While our knowledge about DNA damage factors and regulatory mechanisms in cells has advanced greatly, the number of feasible practical techniques to avoid or repair damaged embryos remains scarce. Our intention is therefore to focus on strategies to obtain embryos with as little DNA damage as possible, which will impact reproductive biology research with particular significance for reproductive clinicians and embryologists.

Perturbations of the hepatic proteome behind the onset of metabolic disorders in mouse offspring developed following embryo manipulation.

Assisted Reproductive Technologies (ART) allowed the births of >8 million babies worldwide. Even if ART children are healthy at birth, several studies reported that ART may cause changes in foetal programming, leading to an increased predisposition to metabolic disorders in adulthood. Previous studies on mouse model showed obesity, glucose intolerance, and hepatic lipid accumulation in ART offspring. A cumulative effect of the different components of ART protocol has been previously described, for example, in the occurrence of epigenetic defects. Here, we investigated whether there is a cumulative effect of embryo transfer (ET), in vitro culture (IVC) and blastomere biopsy (BB) in the onset of metabolic disorders in mouse offspring vs those naturally conceived (Control - CTR). To this aim, proteomic analysis was performed on the livers from adult mouse offspring developed following ET, IVC and BB vs CTR. We observed deregulated expression of proteins involved in lipid, carbohydrate, energy metabolisms and cellular processes in ART offspring. Moreover, we found increased body weight in all ART offspring while i) insulin resistance in BB male, ii) females glucose intolerance and high level of triglycerides and cholesterol in BB females and iii) low levels of interleukin-6 in BB, IVC and ET males. In conclusion, our study suggests that the use of various embryo manipulations influences the metabolic health of adult offspring, resulting in an increased predisposition to hepatic diseases and metabolic syndrome in a sex-specific manner.

Lipid droplets in mammalian eggs are utilized during embryonic diapause.

Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally "nondiapausing" mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.

The effects of assisted reproduction technologies on metabolic health and disease†.

The increasing prevalence of metabolic diseases places a substantial burden on human health throughout the world. It is believed that predisposition to metabolic disease starts early in life, a period of great susceptibility to epigenetic reprogramming due to environmental insults. Assisted reproductive technologies (ART), i.e., treatments for infertility, may affect embryo development, resulting in multiple adverse health outcomes in postnatal life. The most frequently observed alteration in ART pregnancies is impaired placental nutrient transfer. Moreover, consequent intrauterine growth restriction and low birth weight followed by catch-up growth can all predict future obesity, insulin resistance, and chronic metabolic diseases. In this review, we have focused on evidence of adverse metabolic alterations associated with ART, which can contribute to the development of chronic adult-onset diseases, such as metabolic syndrome, type 2 diabetes, and cardiovascular disease. Due to high phenotypic plasticity, ART pregnancies can produce both offspring with adverse health outcomes, as well as healthy individuals. We further discuss the sex-specific and age-dependent metabolic alterations reflected in ART offspring, and how the degree of interference of a given ART procedure (from mild to more severe manipulation of the egg) affects the occurrence and degree of offspring alterations. Over the last few years, studies have reported signs of cardiometabolic alterations in ART offspring that are detectable at a young age but that do not appear to constitute a high risk of disease and morbidity per se. These abnormal phenotypes could be early indicators of the development of chronic diseases, including metabolic syndrome, in adulthood. The early detection of metabolic alterations could contribute to preventing the onset of disease in adulthood. Such early interventions may counteract the risk factors and improve the long-term health of the individual.

Increasing methylation of sperm rDNA and other repetitive elements in the aging male mammalian germline.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age-related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α-satellite and interspersed LINE1 repeats, marmoset α-satellite, bovine alpha- and testis satellite I, mouse minor and major satellite, and LINE1-T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age-related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo.

Mouse single oocyte imaging by MALDI-TOF MS for lipidomics.

Reproductive cells are a very special kind of material for the analysis. Depending on the species, their dimensions allow for the application of mass spectrometry imaging-based techniques to receive a reasonable data for interpretation of their condition without any additional sample preparation steps, except for typical sample preparation characteristic for IMS protocols. A comparison between lipid profiles of oocytes could answer the question of the overall quality of the cells in the function of time or conditions of storage. Even tiny differences in the lipid profiles, but still detectable by bioinformatic analysis, could be crucial for the estimation of the conditions of the cells in various stages of development or aging. In our study, MALDI-TOF/TOF MSI was used to analyze and visualize the single oocytes. We deposited the cells on the transparent indium-tin-oxide (ITO) glass and marked their positions, which allowed for the fast localization of the cells and precise laser targeting in the ion source. We also optimized the usage of different MALDI matrices and different approaches. The proposed way of measurement allows analyzing quite a significant quantity of oocytes in a reasonably short time. During the analysis, the lipid composition of the single cell was successfully estimated in a conventional usage of the MALDI ion source, and the localization of lipids was confirmed by imaging mass spectrometry (IMS) analysis. The observed quantity of the lipids allowed for the application of the LIFT™ technique to obtain MS/MS spectra sufficient for lipids' unambiguous identification. We hope that our idea of the oocyte analysis will help to elucidate chemical changes that accompany different processes in which oocytes are involved. There could be such fascinating phenomena as the oocyte maturation, changes in the lipid components during their storage, and much more.

Assessing the epigenetic risks of assisted reproductive technologies: a way forward.

Since the birth of the first baby conceived by in vitro fertilization (IVF), assisted reproductive technologies (ART) have been constantly evolving to accomodate needs of a growing number of infertile couples. Rapidly developing ART procedures are directly applied for human infertility treatment without prior long-term safety evaluation. Although the majority of ART babies are healthy at birth, a comprehensive assessment of the long-term risks associated with ART is still lacking. An increased risk of epigenetic errors has been associated with the use of ART, which may contribute to the onset of civilization disease later in adolescence/adulthood and/or in subsequent generations. Therefore, our investigations should not focus on (or be limited to) the occurrence of a few very rare imprinting disorders in ART children, which might be associated with parental age and/or the use of ART, but on the possibly increased disease susceptibilities later in life and their potential transmission to the subsequent generations. Retrospective studies do not offer exhaustive information on long-term consequences of ART. Animal models are useful tools to study long-term effects including transgenerational ones and the epigenetic risk of a given ART procedure, which could then be translated to the human context. The final goal is the establishment of common guidelines for assessing the epigenetic risk of ART in humans, which will contribute to two key objectives of the Horizon2020 programme, i.e. to improve our understanding of the causes and mechanisms underlying health and disease, and to improve our ability to monitor health and prevent/manage disease.

Anti-HERV-WEnv antibodies are correlated with seroreactivity against Mycobacterium avium subsp. paratuberculosis in children and youths at T1D risk.

Recent evidence points at the role that human endogenous retroviruses (HERVs) may play through the activation of genes integrated across the human genome. Although a variety of genetic/epigenetic mechanisms maintain most HERVs silenced, independent environmental stimuli including infections may transactivate endogenous elements favoring pathogenic conditions. Several studies associated exposures to Mycobacterium avium subsp. paratuberculosis (MAP) with increased anti-MAP seroreactivity in T1D patients. Here, we assessed humoral responses against HERV envelope antigens (HERV-KEnv and HERV-WEnv) and four MAP-derived peptides with human homologs in distinct populations: Sardinian children at T1D risk (rT1D) (n = 14), rT1D from mainland Italy (n = 54) and Polish youths with T1D (n = 74) or obesity unrelated to autoimmunity (OB) (n = 26). Unlike Sardinian rT1D, youths displayed increased anti-HERV-WEnv Abs prevalence compared to age-matched OB or healthy controls (24.32 vs. 11.54%, p = 0.02 for Polish T1D/OB and 31.48 vs. 11.90%, p = 0.0025 for Italian rT1D). Anti-HERV-KEnv responses showed variable trends across groups. A strong correlation between Abs levels against HERV-WEnv and homologous peptides was mirrored by time-related Abs patterns. Elevated values registered for HERV-WEnv overlaped with or preceded the detection of T1D diagnostic autoantibodies. These results support the hypothesis of MAP infection leading to HERV-W antigen expression and enhancing the production of autoantibodies in T1D.

Polychlorinated biphenyls (PCBs) alter DNA methylation and genomic integrity of sheep fetal cells in a simplified in vitro model of pregnancy exposure.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants ubiquitously detectable in the environment and in the food chain. Prenatal exposure to PCBs negatively affects fetal development and produces long-term detrimental effects on child health. The present study sought to evaluate the cytotoxic and genotoxic effects of chronic PCB exposure on fetal cells during pregnancy. To this aim, sheep embryonic fibroblasts (SEF) and amniocytes (SA) were cultured in vitro in the presence of low doses of PCBs for a period of 120days, comparable to the full term of ovine pregnancy. Cellular proliferation rates, global DNA methylation, chromosome integrity, and markers of DNA damage were evaluated at different time points. Moreover, SEF treated with PCBs for 60days were left untreated for one further month and then examined in order to evaluate the reversibility of PCB-induced epigenetic defects. PCB-treated SEF were more sensitive than SA treated with PCBs, in terms of low cell proliferation, and increased DNA damage and global DNA methylation, which were still detectable after interruption of PCB treatment. These data indicate that chronic exposure of fetal cells to PCBs causes permanent genomic and epigenetic instability, which may influence both prenatal and post-natal growth up to adulthood. Our in vitro model offer a simple and controlled means of studying the effects of different contaminants on fetal cells - one that could set the stage for targeted in vivo studies.

Developmental peculiarities in placentae of ovine uniparental conceptuses.

Genomic imprinting is an epigenetic phenomenon regulating mono-allelic expression of genes depending on their parental origin. Defective genomic imprinting is involved in several placental disorders, such as intrauterine growth restriction and pre-eclampsia. Uniparental embryos, having maternal-only or paternal-only genomes (parthenogenotes [PAR] and androgenotes [AND], respectively), are useful models to study placentation. The aim of this work was to reveal the effect of parental genome (maternal and paternal) on placentation. To do this, uniparental (AND and PAR) and biparental (CTR) in vitro produced sheep embryos transferred to recipient females were collected at day 20 of pregnancy and their placentae were analyzed. qPCR analysis showed that imprinted genes (H19, IGF2R and DLK1) were expressed accordingly to their parental origin while the expression f DNA methyltransferases () was disregulated, especially in PAR (P < 0.05). AND placentae were significantly hypomethylated compared to both PAR and CTR (P = 0.023). Chorion-allantoid of AND showed impaired development of vessels and reduced mRNA expression of vasculogenetic factors (ANG2 P = 0.05; VEGFR2 P< 0.001; TIE2 P < 0.001). Morphologically, PAR placentae were characterized by abnormal structure of the trophoectodermal epithelium and reduced total number (P<0.03) of Trophoblastic Binucleate Cells. A reduced implantation rate of both classes of uniparental embryos (P<0.03) was also noted. Our results provide new insights into the characterization of uniparental embryos and demonstrate the complementary role of parental genomes for the correct establishment of pregnancy. Thus, our findings may suggest new targets to improve our understanding of the origin of imprinting-related placental dysfunction.

Embryo biopsy and development: the known and the unknown.

Preimplantation genetic diagnosis (PGD) has been introduced in clinical practice as a tool for selecting 'healthy' embryos before their transfer in utero. PGD protocols include biopsy of cleaving embryos (blastomere biopsy (BB)) or blastocysts (trophectoderm biopsy (TB)), followed by genetic analysis to select 'healthy' embryos for transfer in utero. Currently, TB is replacing the use of BB in the clinical practice. However, based on the European Society of Human Reproduction and Embryology Preimplantation Genetic Diagnosis Consortium reports, BB has been used in >87% of PGD cycles for more than 10 years. An exhaustive evaluation of embryo biopsy (both BB and TB) risks and safety is still missing. The few epidemiological studies available are quite controversial and/or are limited to normalcy at birth or early childhood. On the other hand, studies on animals have shown that BB can be a risk factor for impaired development, during both pre- and postnatal life, while little is known on TB. Thus, there is an urgent need of focused researches on BB, as it has contributed to give birth to children for more than 10 years, and on TB, as its application is significantly growing in clinical practice. In this context, the aim of this review is to provide a complete overview of the current knowledge on the short-, medium- and long-term effects of embryo biopsy in the mouse model.

Cobalamin supplementation during in vitro maturation improves developmental competence of sheep oocytes.

Pregnancies obtained by Assisted Reproductive Technologies are at higher risk of miscarriage than those obtained naturally. Previously, we reported impaired placental vascular development of in vitro produced (IVP) sheep embryos and defective DNA methylation in the placentae of those embryos. One reason behind these observed defects may be an impaired One Carbon Metabolism (OCM) The present study was performed to test the hypothesis that Cobalamin (Vitamin B12, an important OCM co-factor) supplementation during IVM corrects DNA methylation of IVP embryos and, consequently, ameliorates placental vasculogenesis. To this aim, embryos derived from oocytes matured with Cobalamin (B12 group) or without (negative control group, -CTR) were transferred to synchronized recipient sheep. At day 20 of pregnancy, collected embryos were morphologically evaluated while placentae were subjected to qPCR and histological analysis. The positive control group (+CTR) consisted of conceptuses obtained from naturally mated sheep. Results showed an increased fertilization rate in the B12 group vs -CTR (69.56% vs 57.91% respectively, P = 0.006) not associated with quantitative improvement in blastocyst and/or implantation rate (44.32% vs 36.67% respectively, P > 0.05). Moreover, Cobalamin supplementation during oocyte IVM ameliorated resulting conceptuses quality, in terms of placental vascularization (vessels' maturity and vasculogenetic factors' expression). The expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was also improved in placentae from the B12 group. In conclusion, Cobalamin supplementation during oocyte IVM improves IVP embryo quality. These results suggest that Cobalamin should be included in standard IVM media.

Pregnancy at Advanced Maternal Age Affects Behavior and Hippocampal Gene Expression in Mouse Offspring.

There is growing evidence that advanced maternal age is a risk factor for neurological and neuropsychiatric disorders in offspring. However, it remains unclear whether the altered brain programming induced by advanced maternal age is mediated by pre- or postnatal factors. Here, a mouse model was used to investigate whether pregnancy at advanced age may provoke behavioral and brain gene expression changes in offspring. Swiss Albino mice conceived by 3-month-old males and either 15-18-month-old (n = 11) or 3-month-old control females (n = 5), were delivered by cesarean section, fostered after birth by 3-month-old dams and subjected to a battery of behavioral tests. Furthermore, genome-wide mRNA expression was analyzed in the hippocampi of 4-month-old males offspring using microarrays. Offspring conceived by old mothers exhibited increased ultrasound vocalization activity during separation from the foster mother, increased anxiety-like behaviors in adult life, and altered patterns of hippocampal gene expression, compared to controls. These effects were not reversed by the postnatal maternal care provided by the young foster mothers, suggesting that the altered brain programming is already established at birth, consistent with prenatal effects related to maternal aging.

Deregulated Expression of Mitochondrial Proteins Mfn2 and Bcnl3L in Placentae from Sheep Somatic Cell Nuclear Transfer (SCNT) Conceptuses.

In various animal species, the main cause of pregnancy loss in conceptuses obtained by somatic cell nuclear transfer (SCNT) are placental abnormalities. Most abnormalities described in SCNT pregnancies (such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium) suggest that placental cell degeneration may be triggered by mitochondrial failure. We hypothesized that placental abnormalities of clones obtained by SCNT are related to mitochondrial dysfunction. To test this, early SCNT and control (CTR, from pregnancies obtained by in vitro fertilization) placentae were collected from pregnant ewes (at day 20 and 22 of gestation) and subjected to morphological, mRNA and protein analysis. Here, we demonstrated swollen and fragmented mitochondria and low expression of mitofusin 2 (Mfn2), the protein which plays a crucial role in mitochondrial functionality, in SCNT early placentae. Furthermore, reduced expression of the Bcnl3L/Nix protein, which plays a crucial role in selective elimination of damaged mitochondria, was observed and reflected by the accumulation of numerous damaged mitochondria in SCNT placental cells. Likely, this accumulation of damaged organelles led to uncontrolled apoptosis in SCNT placentae, as demonstrated by the high number of apoptotic bodies, fragmented cytoplasm, condensed chromatin, lack of integrity of the nuclear membrane and the perturbed mRNA expression of apoptotic genes (BCL2 and BAX). In conclusion, our data indicate that deregulated expression of Mfn2 and Bcnl3L is responsible for placental abnormalities in SCNT conceptuses. Our results suggest that some nuclear genes, that are involved in the regulation of mitochondrial function, do not work well and consequently this influence the function of mitochondria.

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.

Evidence of Placental Autophagy during Early Pregnancy after Transfer of In Vitro Produced (IVP) Sheep Embryos.

Pregnancies obtained by Assisted Reproductive Technologies (ART) are associated with limited maternal nutrient uptake. Our previous studies shown that in vitro culture of sheep embryos is associated with vascularization defects in their placentae and consequent reduction of embryo growth. Autophagy is a pro-survival cellular mechanism triggered by nutrient insufficiency. Therefore, the goal of our present study was to determine if autophagy is involved in early placental development after transfer of in vitro produced (IVP) embryos. To do this, placentae obtained following transfer of IVP sheep embryos were compared with placentae obtained after natural mating (control-CTR). The placentae were collected on day 20 post-fertilization and post-mating, respectively, and were analyzed using molecular (qPCR), ultrastructural and histological/immunological approaches. Our results show drastically increased autophagy in IVP placentae: high levels of expression (p<0.05) of canonical markers of cellular autophagy and a high proportion of autophagic cells (35.08%; p<0.001) were observed. We conclude that high autophagic activity in IVP placentae can be a successful temporary counterbalance to the retarded vasculogenesis and the reduction of foetal growth observed in pregnancies after transfer of IVP embryos.