The centriolar satellite protein Cfap53 facilitates formation of the zygotic microtubule organizing center in the zebrafish embryo.

In embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation.

Teratoma Assay for Testing Pluripotency and Malignancy of Stem Cells: Insufficient Reporting and Uptake of Animal-Free Methods-A Systematic Review.

Pluripotency describes the ability of stem cells to differentiate into derivatives of the three germ layers. In reporting new human pluripotent stem cell lines, their clonal derivatives or the safety of differentiated derivatives for transplantation, assessment of pluripotency is essential. Historically, the ability to form teratomas in vivo containing different somatic cell types following injection into immunodeficient mice has been regarded as functional evidence of pluripotency. In addition, the teratomas formed can be analyzed for the presence of malignant cells. However, use of this assay has been subject to scrutiny for ethical reasons on animal use and due to the lack of standardization in how it is used, therefore questioning its accuracy. In vitro alternatives for assessing pluripotency have been developed such as ScoreCard and PluriTest. However, it is unknown whether this has resulted in reduced use of the teratoma assay. Here, we systematically reviewed how the teratoma assay was reported in publications between 1998 (when the first human embryonic stem cell line was described) and 2021. Our analysis of >400 publications showed that in contrast to expectations, reporting of the teratoma assay has not improved: methods are not yet standardized, and malignancy was examined in only a relatively small percentage of assays. In addition, its use has not decreased since the implementation of the ARRIVE guidelines on reduction of animal use (2010) or the introduction of ScoreCard (2015) and PluriTest (2011). The teratoma assay is still the preferred method to assess the presence of undifferentiated cells in a differentiated cell product for transplantation since the in vitro assays alone are not generally accepted by the regulatory authorities for safety assessment. This highlights the remaining need for an in vitro assay to test malignancy of stem cells.

Classification of Atretic Small Antral Follicles in the Human Ovary.

The reproductive lifespan in humans is regulated by a delicate cyclical balance between follicular recruitment and atresia in the ovary. The majority of the small antral follicles present in the ovary are progressively lost through atresia without reaching dominance, but this process remains largely underexplored. In our study, we investigated the characteristics of atretic small antral follicles and proposed a classification system based on molecular changes observed in granulosa cells, theca cells, and extracellular matrix deposition. Our findings revealed that atresia spreads in the follicle with wave-like dynamics, initiating away from the cumulus granulosa cells. We also observed an enrichment of CD68+ macrophages in the antrum during the progression of follicular atresia. This work not only provides criteria for classifying three stages of follicular atresia in small antral follicles in the human ovary but also serves as a foundation for understanding follicular degeneration and ultimately preventing or treating premature ovarian failure. Understanding follicular remodeling in the ovary could provide a means to increase the number of usable follicles and delay the depletion of the follicular reserve, increasing the reproductive lifespan.

Humanised Mice and Immunodeficient Mice (NSG) Are Equally Sensitive for Prediction of Stem Cell Malignancy in the Teratoma Assay.

The use of human pluripotent stem cells (hPSCs) in regenerative medicine has great potential. However, it is important to exclude that these cells can undergo malignant transformation, which could lead to the development of malignant tumours. This property of hPSCs is currently being tested using the teratoma assay, through which cells are injected into immunodeficient mice. Transplantation of stem cells in immunocompromised recipient animals certainly has a much higher incidence of tumour formation. On the other hand, the results obtained in immunodeficient mice could indicate a risk of tumour formation that is practically not present in the human immunocompetent recipient. The presence of a humanised immune system might be more representative of the human situation; therefore, we investigated if the demonstrated malignant features of chosen and well-characterised stem cell lines could be retrieved and if new features could arise in a humanised mouse model. Hu-CD34NSGTM (HIS) mice were compared side by side with immunocompromised mice (NSG) after injection of a set of benign (LU07) and malignant (LU07+dox and 2102Ep) cell lines. Analysis of the tumour development, histological composition, pathology evaluation, and malignancy-associated miRNA expression levels, both in tumour and plasma samples, revealed no differences among mouse groups. This indicates that the HIS mouse model is comparable to, but not more sensitive than, the NSG immunodeficient model for studying the malignancy of stem cells. Since in vivo teratoma assay is cumbersome, in vitro methods for the detection of malignancy are urgently needed.

Reverse transcription priming methods affect normalisation choices for gene expression levels in oocytes and early embryos.

Mammalian oocytes and embryos rely exclusively on maternal mRNAs to accomplish early developmental processes. Since oocytes and early embryos are transcriptionally silent after meiotic resumption, most of the synthesised maternal mRNA does not undergo immediate translation but is instead stored in the oocyte. Quantitative RT-PCR is commonly used to quantify mRNA levels, and correct quantification relies on reverse transcription and the choice of reference genes. Different methods for reverse transcription may affect gene expression determination in oocytes. In this study, we examined the suitability of either random or oligo(dT) primers for reverse transcription to be used for quantitative RT-PCR. We further looked for changes in poly(A) length of the maternal mRNAs during oocyte maturation. Our data indicate that depending on the method of reverse transcription, the optimal combination of reference genes for normalisation differed. Surprisingly, we observed a shortening of the poly(A) tail lengths of maternal mRNA as oocytes progressed from germinal vesicle to metaphase II. Overall, our findings suggest dynamic maternal regulation of mRNA structure and gene expression during oocyte maturation and early embryo development.

Lysophosphatidic Acid Accelerates Bovine In Vitro-Produced Blastocyst Formation through the Hippo/YAP Pathway.

The segregation of trophectoderm (TE) and inner cell mass in early embryos is driven primarily by the transcription factor CDX2. The signals that trigger CDX2 activation are, however, less clear. In mouse embryos, the Hippo-YAP signaling pathway is important for the activation of CDX2 expression; it is less clear whether this relationship is conserved in other mammals. Lysophosphatidic acid (LPA) has been reported to increase YAP levels by inhibiting its degradation. In this study, we cultured bovine embryos in the presence of LPA and examined changes in gene and protein expression. LPA was found to accelerate the onset of blastocyst formation on days 5 and 6, without changing the TE/inner cell mass ratio. We further observed that the expression of TAZ and TEAD4 was up-regulated, and YAP was overexpressed, in LPA-treated day 6 embryos. However, LPA-induced up-regulation of CDX2 expression was only evident in day 8 embryos. Overall, our data suggest that the Hippo signaling pathway is involved in the initiation of bovine blastocyst formation, but does not affect the cell lineage constitution of blastocysts.

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep.

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus-oocyte-complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase-mediated dUTP nick-End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short-term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long-term carry-over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late-stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3-0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.

Bovine oocyte maturation: acquisition of developmental competence.

Although millions of oocytes are formed during embryo and fetal development in the cow, only a small fraction of these will form a developmentally competent oocyte and be fertilised. Development to competence relies on an intimate contact between the oocyte and the surrounding somatic cells in ovarian follicles, via both direct cell-cell contact and paracrine signalling. An important aspect of oocyte maturation is the segregation of homologous chromosomes and subsequently sister chromatids to form a haploid oocyte. Furthermore, the cytoplasm needs to be prepared for the formation of pronuclei and nuclear reprogramming to form a totipotent zygote. Conditions such as high levels of fatty acids or oxidative stress constrain the developmental competence of oocytes, and a better insight into these processes may help improve in vitro and in vivo oocyte maturation success. In addition, identification of the developmentally competent oocyte is useful for the efficiency of (artificial) reproduction.

Human-specific subcellular compartmentalization of P-element induced wimpy testis-like (PIWIL) granules during germ cell development and spermatogenesis.

STUDY QUESTION: What is the dynamics of expression of P-element induced wimpy testis-like (PIWIL) proteins in the germline during human fetal development and spermatogenesis? SUMMARY ANSWER: PIWIL1, PIWIL2, PIWIL3 and PIWIL4 were expressed in a sex-specific fashion in human germ cells (GC) during development and adulthood. PIWILs showed a mutually exclusive pattern of subcellular localization. PIWILs were present in the intermitochondrial cement and a single large granule in meiotic GC and their expression was different from that observed in mice, highlighting species-differences. WHAT IS KNOWN ALREADY: In mice, PIWIL proteins play prominent roles in male infertility. PIWIL mouse mutants show either post-meiotic arrest at the round spermatid stage (PIWIL1) or arrest at the zygotene-pachytene stage of meiosis I (PIWIL2 and PIWIL4) in males, while females remain fertile. Recent studies have reported a robust piRNA pool in human fetal ovary. STUDY DESIGN, SIZE, DURATION: This is a qualitative analysis of PIWILs expression in paraffin-embedded fetal human male (N = 8), female gonads (N = 6) and adult testes (N = 5), and bioinformatics analysis of online available single-cell transcriptomics data of human fetal germ cells (n = 242). PARTICIPANTS/MATERIALS, SETTING, METHODS: Human fetal gonads from elective abortion without medical indication and adult testes biopsies were donated for research with informed consent. Samples were fixed, paraffin-embedded and analyzed by immunofluorescence to study the temporal and cellular localization of PIWIL1, PIWIL2, PIWIL3 and PIWIL4. MAIN RESULTS AND THE ROLE OF CHANCE: PIWIL1, PIWIL2 and PIWIL4 showed a mutually exclusive pattern of subcellular localization, particularly in female oocytes. To our surprise, PIWIL1 immunostaining revealed the presence of a single dense paranuclear body, resembling the chromatoid body of haploid spermatocytes, in meiotic oocytes. Moreover, in contrast to mice, PIWIL4, but not PIWIL2, localized to the intermitochondrial cement. PIWIL3 was not expressed in GC during development. The upregulation of PIWIL transcripts correlated with the transcription of markers associated with piRNAs biogenesis like the TDRDs and HENMT1 in fetal GC. LARGE SCALE DATA: Non-applicable. LIMITATIONS, REASONS FOR CAUTION: This study is limited by the restricted number of samples and consequently stages analyzed. WIDER IMPLICATIONS OF THE FINDINGS: In the germline, PIWILs ensure the integrity of the human genome protecting it from 'parasitic sequences'. This study offers novel insights on the expression dynamics of PIWILs during the window of epigenetic remodeling and meiosis, and highlights important differences between humans and mice, which may prove particularly important to understand causes of infertility and improve both diagnosis and treatment in humans. STUDY FUNDING/COMPETING INTEREST(S): M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011]; N.H. by China Scholarship Council (CSC) [No. 201307040026] and F.W. by Medical Personnel Training Abroad Project of Henan Province [No. 2015022] and S.M.C.d.S.L. by the Netherlands Organization of Scientific Research (NWO) [ASPASIA 015.007.037] and the Interuniversity Attraction Poles-Phase VII [IUAP/PAI P7/14]. The authors have no conflicts of interest to declare.

Stearoyl-CoA desaturase activity in bovine cumulus cells protects the oocyte against saturated fatty acid stress.

Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid.

Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells.

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole-transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency-related gene network of the LIF- versus FGF-derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC-like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC-like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extent, in the piPSC-like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line-transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. 84: 229-245, 2017. © 2016 Wiley Periodicals, Inc.

Validating reference microRNAs for normalizing qRT-PCR data in bovine oocytes and preimplantation embryos.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that act as post-transcriptional regulators of gene targets. Accurate quantification of miRNA expression using validated internal controls should aid in the understanding of their role in epigenetic modification of genome function. To date, most studies that have examined miRNA expression levels have used the global mean expression of all expressed genes or the expression of reference mRNAs or nuclear RNAs for normalization. RESULTS: We analyzed the suitability of a number of miRNAs as potential expression normalizers in bovine oocytes and early embryos, and porcine oocytes. The stages examined were bovine oocytes at the germinal vesicle (GV) and metaphase II stages, bovine zygotes, 2, 4 and 8 cell embryos, morulae and blastocysts, as well as porcine cumulus oocyte complexes, GV, metaphase I and II oocytes. qRT-PCR was performed to quantify expression of miR-93, miR-103, miR-26a, miR-191, miR-23b, Let-7a and U6 for bovine samples and miR-21, miR-26a, miR-93, miR-103, miR-148a, miR-182 and miR-191 for porcine oocytes. The average starting material for each sample was determined using specific standard curves for each primer set. Subsequently, geNorm and BestKeeper software were used to identify a set of stably expressed miRNAs. Stepwise removal to determine the optimum number of reference miRNAs identified miR-93 and miR-103 as the most stably expressed in bovine samples and miR-26a, miR-191 and miR-93 in porcine samples. CONCLUSIONS: The combination of miR-93 and miR-103 is optimal for normalizing miRNA expression for qPCR experiments on bovine oocytes and preimplantation embryos; the preferred combination for porcine oocytes is miR-26a, miR-191 and miR-93.

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed. RESULTS: Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated. CONCLUSION: The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos.

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

The cumulus cell layer protects the bovine maturing oocyte against fatty acid-induced lipotoxicity.

Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report, we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate and unsaturated oleate) on oocyte maturation and quality. In the current study, the effects of elevated fatty acid levels on cumulus cells were investigated. In a dose-dependent manner, the three fatty acids induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3- to 6-fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of the three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of the ceramide-C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin-B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed.

Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos.

Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs ≥12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-coupling factor 6, mitochondrial-like (ATP-synth_F6)) and oxygen free radical scavenging (glutathione peroxidase 3 (GPX3)) were investigated in oocytes before and after in vitro maturation (IVM), and in early embryos. Expression of TFAM, mtPOLB and ATP-synth-F6 declined after IVM (P<0.05). However, maternal age did not affect oocyte ATP content or expression of genes involved in mitochondrial replication or function. Day 7 embryos from mares ≥12 years had fewer mtDNA copies (P=0.01) and lower mtDNA:total DNA ratios (P<0.01) than embryos from younger mares, indicating an effect not simply due to lower cell number. Day 8 IVP embryos had similar mtDNA copy numbers to Day 7 in vivo embryos, but higher mtPOLB (P=0.013) and a tendency to reduced GPX3 expression (P=0.09). The lower mtDNA number in embryos from older mares may compromise development, but could be an effect rather than cause of developmental retardation. The general down-regulation of genes involved in mitochondrial replication and function after IVM may compromise resulting embryos.

Free fatty acid levels in fluid of dominant follicles at the preferred insemination time in dairy cows are not affected by early postpartum fatty acid stress.

The fertility of high-yielding dairy cows has declined during the last 3 decades, in association with a more profound negative energy balance (NEB) during the early weeks postpartum. One feature of this NEB is a marked elevation in circulating free fatty acid (FFA) concentrations. During the early postpartum period (≤ d 42), circulatory FFA levels were measured weekly, and progesterone concentrations and the diameter of the dominant follicles were determined thrice weekly. Retrospectively, cows that ovulated within 35 d postpartum were grouped as "normal ovulating" cows (n = 5), and the others were grouped as "delayed ovulating" cows (n = 5). In both groups, high total FFA levels (>500 µM) were evident immediately postpartum. Interestingly, cows with delayed ovulation had higher plasma FFA concentrations in the first weeks postpartum compared with normal ovulating cows. In both cow groups, FFA decreased to control levels of non-NEB cows within 3 wk postpartum. The FFA compositions and concentrations in fluids from the dominant follicles of postpartum cows were not different between the normal and delayed ovulating cows when measured at potential insemination points: d 55, 80, and 105 postpartum. Interestingly, the concentration of monounsaturated oleic acid was higher and that of saturated stearic acid lower in follicular fluids of both groups compared with that in blood. The level of FFA in follicular fluid was correlated with the ratio of 17ß-estradiol (E2) to progesterone (P4) in follicular fluid, with a relatively high level of unsaturated FFA in follicles with a low E2:P4 ratio. Taken together, these results indicate that a more severe NEB early postpartum is related to a delay in the first postpartum ovulation and does not affect FFA composition in follicular fluid at the preferred insemination time. The high FFA level in dominant follicles with a low E2:P4 ratio may be due to a different FFA metabolism in these follicles. The diagnostic value of this observation for selective screening of dominant follicles needs further investigation.

Relationship between genome and epigenome--challenges and requirements for future research.

Understanding the links between genetic, epigenetic and non-genetic factors throughout the lifespan and across generations and their role in disease susceptibility and disease progression offer entirely new avenues and solutions to major problems in our society. To overcome the numerous challenges, we have come up with nine major conclusions to set the vision for future policies and research agendas at the European level.

Usefulness of bovine and porcine IVM/IVF models for reproductive toxicology.

Women presenting fertility problems are often helped by Assisted Reproductive Techniques (ART), such as in vitro fertilization (IVF) programs. However, in many cases the etiology of the in/subfertility remains unknown even after treatment. Although several aspects should be considered when assisting a woman with problems to conceive, a survey on the patients' exposure to contaminants would help to understand the cause of the fertility problem, as well as to follow the patient properly during IVF. Daily exposure to toxic compounds, mainly environmental and dietary ones, may result in reproductive impairment. For instance, because affects oocyte developmental competence. Many of these compounds, natural or synthetic, are endocrine disruptors or endocrine active substances that may impair reproduction. To understand the risks and the mechanism of action of such chemicals in human cells, the use of proper in vitro models is essential. The present review proposes the bovine and porcine models to evaluate toxic compounds on oocyte maturation, fertilization and embryo production in vitro. Moreover, we discuss here the species-specific differences when mice, bovine and porcine are used as models for human.