MEK-inhibitors decrease Nfix in muscular dystrophy but induce unexpected calcifications, partially rescued with Cyanidin diet.

Muscular dystrophies (MDs) are incurable genetic myopathies characterized by progressive degeneration of skeletal muscles. Dystrophic mice lacking the transcription factor Nfix display morphological and functional improvements of the disease. Recently, we demonstrated that MAPK signaling pathway positively regulates Nfix in muscle development and that Cyanidin, a natural antioxidant molecule, strongly ameliorates the pathology. To explore a synergistic approach aimed at treating MDs, we administered Trametinib, a clinically approved MEK inhibitor, alone or combined with Cyanidin to adult Sgca null mice. We observed that chronic treatment with Trametinib and Cyanidin reduced Nfix in myogenic cells but, unexpectedly, caused ectopic calcifications exclusively in dystrophic muscles. The combined treatment with Cyanidin resulted in histological improvements by preventing Trametinib-induced calcifications in Diaphragm and Soleus. Collectively, this first pilot study revealed that Nfix is modulated by the MAPK pathway in MDs, and that Cyanidin partly rescued the unexpected ectopic calcifications caused by MEK inhibition.

Transcriptome-wide mapping of milk somatic cells upon subclinical mastitis infection in dairy cattle.

BACKGROUND: Subclinical intramammary infection (IMI) represents a significant problem in maintaining dairy cows' health. Disease severity and extent depend on the interaction between the causative agent, environment, and host. To investigate the molecular mechanisms behind the host immune response, we used RNA-Seq for the milk somatic cells (SC) transcriptome profiling in healthy cows (n = 9), and cows naturally affected by subclinical IMI from Prototheca spp. (n = 11) and Streptococcus agalactiae (S. agalactiae; n = 11). Data Integration Analysis for Biomarker discovery using Latent Components (DIABLO) was used to integrate transcriptomic data and host phenotypic traits related to milk composition, SC composition, and udder health to identify hub variables for subclinical IMI detection. RESULTS: A total of 1,682 and 2,427 differentially expressed genes (DEGs) were identified when comparing Prototheca spp. and S. agalactiae to healthy animals, respectively. Pathogen-specific pathway analyses evidenced that Prototheca's infection upregulated antigen processing and lymphocyte proliferation pathways while S. agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle, and carbohydrate and lipid metabolism. The integrative analysis of commonly shared DEGs between the two pathogens (n = 681) referred to the core-mastitis response genes, and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells (r2 = 0.72), followed by the udder health (r2 = 0.64) and milk quality parameters (r2 = 0.64). Variables with r ≥ 0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cytohubba plug-in. The genes in common between DIABLO and cytohubba (n = 10) were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals (sensitivity > 0.89, specificity > 0.81, accuracy > 0.87, and precision > 0.69). Among these genes, CIITA could play a key role in regulating the animals' response to subclinical IMI. CONCLUSIONS: Despite some differences in the enriched pathways, the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response. The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.

Characterization of BoHV-4 ORF45.

Bovine herpesvirus 4 (BoHV-4) is a Gammaherpesvirus belonging to the Rhadinovirus genus. The bovine is BoHV-4's natural host, and the African buffalo is BoHV-4's natural reservoir. In any case, BoHV-4 infection is not associated with a specific disease. Genome structure and genes are well-conserved in Gammaherpesvirus, and the orf 45 gene and its product, ORF45, are one of those. BoHV-4 ORF45 has been suggested to be a tegument protein; however, its structure and function have not yet been experimentally characterized. The present study shows that BoHV-4 ORF45, despite its poor homology with other characterized Rhadinovirus ORF45s, is structurally related to Kaposi's sarcoma-associated herpesvirus (KSHV), is a phosphoprotein, and localizes in the host cell nuclei. Through the generation of an ORF45-null mutant BoHV-4 and its pararevertant, it was possible to demonstrate that ORF45 is essential for BoHV-4 lytic replication and is associated with the viral particles, as for the other characterized Rhadinovirus ORF45s. Finally, the impact of BoHV-4 ORF45 on cellular transcriptome was investigated, an aspect poorly explored or not at all for other Gammaherpesvirus. Many cellular transcriptional pathways were found to be altered, mainly those involving p90 ribosomal S6 kinase (RSK) and signal-regulated kinase (ERK) complex (RSK/ERK). It was concluded that BoHV-4 ORF45 has similar characteristics to those of KSHV ORF45, and its unique and incisive impact on the cell transcriptome paves the way for further investigations.

CpG DNA methylation changes during epididymal sperm maturation in bulls.

BACKGROUND: During epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions. RESULTS: Caput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda. CONCLUSIONS: Specific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Amniotic Mesenchymal-Derived Extracellular Vesicles and Their Role in the Prevention of Persistent Post-Breeding Induced Endometritis.

Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose-response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 106 EVs with 10 × 106 spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

Extracellular vesicles from seminal plasma to improve fertilizing capacity of bulls.

Seminal plasma contains extracellular vesicles (EVs) that vehicle RNA, proteins, and other molecules able to influence the biological function of sperm. The aim of this study was to improve the fertilizing capacity of male gametes of low-fertility bulls using EVs isolated by ultracentrifugation from the seminal plasma of a bull of proven fertility. After dose-response curve, 10×106 sperm of low-fertility bulls were co-incubated for an hour with 400×106 EVs/ml. In addition, it has been verified that the incorporation of EVs, which takes place in the sperm midpiece, is maintained for 5 hours and even after cryopreservation. Subsequently, the spermatozoa of low-fertility bulls, with EVs incorporated, were used for the in vitro production of embryos. The rate of blastocyst at seventh day yield in vitro, with the use of sperm with EVs incorporated, increased by about twice the yield obtained with the same sperm in the absence of EVs: bulls having an average embryonic yield of 6.41±1.48%, 10.32±4.34% and 10.92±0.95% improved their yield to 21.21±1.99%, 22.17±6.09% and 19.99±5.78%, respectively (P<0.05). These encouraging results suggest that it might be possible to keep breeding bulls with poor fertility. Further studies will be needed to evaluate the in vivo fertility of sperm treated with EVs and understand how the content of EVs is involve in the sperm-vesicle interaction and in the improved sperm performance.

Endometrial and oviduct extra-cellular vescicles for in vitro equine sperm hyperactivation and oocyte fertilization.

Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs. Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. In vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies.

Variations of follicular fluid extracellular vesicles miRNAs content in relation to development stage and season in buffalo.

In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one of the major factors affecting farming profitability. Follicular fluid (FF) contains extracellular vesicles (EVs) playing an important role in modulating oocyte developmental competence and carrying microRNAs (miRNAs) essential for in vitro fertilization outcomes. The aim of this work was to characterize the FF-EVs-miRNA cargo of antral (An) and preovulatory (pO) follicles collected in the breeding (BS) and non-breeding (NBS) seasons, to unravel the molecular causes of the reduced oocyte competence recorded in buffalo during the NBS. In total, 1335 miRNAs (538 known Bos taurus miRNAs, 324 homologous to known miRNAs from other species and 473 new candidate miRNAs) were found. We identified 413 differentially expressed miRNAs (DE-miRNAs) (FDR < 0.05) between An and pO groups. A subset of the most significant DE-miRNAs between An and pO groups targets genes which function is related to the lipid and steroid metabolism, response to glucocorticoid and oestradiol stimulus. Comparison between BS and NBS showed 14 and 12 DE-miRNAs in An-FF-EVs and pO-FF-EVs, which regulate IL6 release and cellular adhesion, respectively. In conclusion, these results demonstrated that the miRNA cargo of buffalo FF-EVs varies in relation to both follicular development and season.

Maternal undernutrition during periconceptional period affects whole-genome ovine muscle methylation in adult offspring.

Experimental and epidemiological studies suggest that maternal nutritional status during early pregnancy, including the period around the time of conception, may induce long-lasting epigenetic changes in the offspring. However, this remains largely unexplored in livestock. Therefore, the objective of this study was to evaluate if modification of the maternal diet of sheep (CTR: control; UND: 50% undernutrition) during the periconceptional period (42 d in total: -14/+28 from mating), would impact CpG methylation in muscle tissue (Longissimus dorsi) of adult offspring (11.5 mo old). Reduced representation bisulfite sequencing identified 262 (Edge-R, FDR < 0.05) and 686 (logistic regression, FDR < 0.001) differentially methylated regions (DMRs) between the UND and CTR groups. Gene ontology analysis identified genes related to development, functions of the muscular system, and steroid hormone receptor activity within the DMRs. The data reported here show that nutritional stress during early pregnancy leads to epigenetic modifications in the muscle of the resulting offspring, with possible implications for cardiac dysfunction, muscle physiology, and meat production.

The formation of the epigenetic pattern of an organism is highly sensitive to environmental factors, especially during early mammalian development, when epigenetic reprogramming of the whole genome takes place. In utero adverse conditions experienced during early pregnancy, such as maternal undernutrition, may induce long-lasting epigenetic changes in the resulting offspring. This study investigated the CpG methylation variations in muscle tissue of adult offspring induced by differences in the diet of their mothers during pregnancy. Our data show that undernutrition during pregnancy leads to epigenetic alterations in the muscle of the offspring, with a potential impact on animal health and productivity.

Application of Perinatal Derivatives in Ovarian Diseases.

Reproductive diseases could lead to infertility and have implications for overall health, most importantly due to psychological, medical and socio-economic consequences for individuals and society. Furthermore, economical losses also occur in animal husbandry. In both human and veterinary medicine, hormonal and surgical treatments, as well as assisted reproductive technologies are used to cure reproductive disorders, however they do not improve fertility. With ovarian disorders being the main reproductive pathology in human and bovine, over the past 2 decades research has approached regenerative medicine in animal model to restore normal function. Ovarian pathologies are characterized by granulosa cell and oocyte apoptosis, follicular atresia, decrease in oocyte quality and embryonic development potential, oxidative stress and mitochondrial abnormalities, ultimately leading to a decrease in fertility. At current, application of mesenchymal stromal cells or derivatives thereof represents a valid strategy for regenerative purposes. Considering their paracrine/autocrine mode of actions that are able to regenerate injured tissues, trophic support, preventing apoptosis and fibrosis, promoting angiogenesis, stimulating the function and differentiation of endogenous stem cells and even reducing the immune response, are all important players in their future therapeutic success. Nevertheless, obtaining mesenchymal stromal cells (MSC) from adult tissues requires invasive procedures and implicates decreased cell proliferation and a reduced differentiation capacity with age. Alternatively, the use of embryonic stem cells as source of cellular therapeutic encountered several ethical concerns, as well as the risk of teratoma formation. Therefore, several studies have recently focussed on perinatal derivatives (PnD) that can be collected non-invasively and, most importantly, display similar characteristics in terms of regenerating-inducing properties, immune-modulating properties and hypo-immunogenicity. This review will provide an overview of the current knowledge and future perspectives of PnD application in the treatment of ovarian hypofunction.

Controlled spermatozoa-oocyte interaction improves embryo quality in sheep.

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture.

A Combined Flow Cytometric Semen Analysis and miRNA Profiling as a Tool to Discriminate Between High- and Low-Fertility Bulls.

Predicting bull fertility is one of the main challenges for the dairy breeding industry and artificial insemination (AI) centers. Semen evaluation performed in the AI center is not fully reliable to determine the level of bull fertility. Spermatozoa are rich in active miRNA. Specific sperm-borne miRNAs can be linked to fertility. The aim of our study is to propose a combined flow cytometric analysis and miRNA profiling of semen bulls with different fertility to identify markers that can be potentially used for the prediction of field fertility. Sperm functions were analyzed in frozen-thawed semen doses (CG: control group) and high-quality sperm (HQS) fraction collected from bulls with different field fertility levels (estimated relative conception rate or ERCR) by using advanced techniques, such as the computer-assisted semen analysis system, flow cytometry, and small RNA-sequencing. Fertility groups differ for total and progressive motility and in the abnormality degree of the chromatin structure (P < 0.05). A backward, stepwise, multiple regression analysis was applied to define a model with high relation between in vivo (e.g., ERCR) and in vitro (i.e., semen quality and DE-miRNA) fertility data. The analysis produced two models that accounted for more than 78% of the variation of ERCR (CG: R 2 = 0.88; HQS: R 2 = 0.78), identifying a suitable combination of parameters useful to predict bull fertility. The predictive equation on CG samples included eight variables: four kinetic parameters and four DNA integrity indicators. For the HQS fraction, the predictive equation included five variables: three kinetic parameters and two DNA integrity indicators. A significant relationship was observed between real and predicted fertility in CG (R 2 = 0.88) and HQS fraction (R 2 = 0.82). We identified 15 differentially expressed miRNAs between high- and low-fertility bulls, nine of which are known (miR-2285n, miR-378, miR-423-3p, miR-191, miR-2904, miR-378c, miR-431, miR-486, miR-2478) while the remaining are novel. The multidimensional preference analysis model partially separates bulls according to their fertility, clustering three semen quality variable groups relative to motility, DNA integrity, and viability. A positive association between field fertility, semen quality parameters, and specific miRNAs was revealed. The integrated approach could provide a model for bull selection in AI centers, increasing the reproductive efficiency of livestock.

Methylome Patterns of Cattle Adaptation to Heat Stress.

Heat stress has a detrimental impact on cattle health, welfare and productivity by affecting gene expression, metabolism and immune response, but little is known on the epigenetic mechanisms mediating the effect of temperature at the cellular and organism level. In this study, we investigated genome-wide DNA methylation in blood samples collected from 5 bulls of the heat stress resilient Nellore breed and 5 bulls of the Angus that are more heat stress susceptible, exposed to the sun and high temperature-high humidity during the summer season of the Brazilian South-East region. The methylomes were analyzed during and after the exposure by Reduced Representation Bisulfite Sequencing, which provided genome-wide single-base resolution methylation profiles. Significant methylation changes between stressful and recovery periods were observed in 819 genes. Among these, 351 were only seen in Angus, 366 were specific to Nellore, and 102 showed significant changes in methylation patterns in both breeds. KEGG and Gene Ontology (GO) enrichment analyses showed that responses were breed-specific. Interestingly, in Nellore significant genes and pathways were mainly involved in stress responses and cellular defense and were under methylated during heat stress, whereas in Angus the response was less focused. These preliminary results suggest that heat challenge induces changes in methylation patterns in specific loci, which should be further scrutinized to assess their role in heat tolerance.

The Biological Function of Extracellular Vesicles during Fertilization, Early Embryo-Maternal Crosstalk and Their Involvement in Reproduction: Review and Overview.

Secretory extracellular vesicles (EVs) are membrane-enclosed microparticles that mediate cell to cell communication in proximity to, or distant from, the cell of origin. Cells release a heterogeneous spectrum of EVs depending on their physiologic and metabolic state. Extracellular vesicles are generally classified as either exosomes or microvesicles depending on their size and biogenesis. Extracellular vesicles mediate temporal and spatial interaction during many events in sexual reproduction and supporting embryo-maternal dialogue. Although many omic technologies provide detailed understanding of the molecular cargo of EVs, the difficulty in obtaining populations of homogeneous EVs makes difficult to interpret the molecular profile of the molecules derived from a miscellaneous EV population. Notwithstanding, molecular characterization of EVs isolated in physiological and pathological conditions may increase our understanding of reproductive and obstetric diseases and assist the search for potential non-invasive biomarkers. Moreover, a more precise vision of the cocktail of biomolecules inside the EVs mediating communication between the embryo and mother could provide new insights to optimize the therapeutic action and safety of EV use.

Seasonal effects on miRNA and transcriptomic profile of oocytes and follicular cells in buffalo (Bubalus bubalis).

Season clearly influences oocyte competence in buffalo (Bubalus bubalis); however, changes in the oocyte molecular status in relation to season are poorly understood. This study characterizes the microRNA (miRNA) and transcriptomic profiles of oocytes (OOs) and corresponding follicular cells (FCs) from buffalo ovaries collected in the breeding (BS) and non-breeding (NBS) seasons. In the BS, cleavage and blastocyst rates are significantly higher compared to NBS. Thirteen miRNAs and two mRNAs showed differential expression (DE) in FCs between BS and NBS. DE-miRNAs target gene analysis uncovered pathways associated with transforming growth factor ß (TGFß) and circadian clock photoperiod. Oocytes cluster in function of season for their miRNA content, showing 13 DE-miRNAs between BS and NBS. Between the two seasons, 22 differentially expressed genes were also observed. Gene Ontology (GO) analysis of miRNA target genes and differentially expressed genes (DEGs) in OOs highlights pathways related to triglyceride and sterol biosynthesis and storage. Co-expression analysis of miRNAs and mRNAs revealed a positive correlation between miR-296-3p and genes related to metabolism and hormone regulation. In conclusion, season significantly affects female fertility in buffalo and impacts on oocyte transcriptomic of genes related to folliculogenesis and acquisition of oocyte competence.

Maternal peri-conceptional undernourishment perturbs offspring sperm methylome.

The genotype of an organism is stable throughout its life; however, its epigenome is dynamic and can be altered in response to environmental factors, such as diet. Inheritance of acquired epigenetic modifications by the next generation occurs through the germline, although the precise mechanisms remain to be elucidated. Here, we used a sheep model to evaluate if modification of the maternal diet (CTR; control, UND: undernutrition; FA: undernutrition and folic acid supplementation) during the peri-conceptional period affects the genome-wide methylation status of the gametes of male offspring. Sperm DNA methylation, measured by Reduced Representation Bisulfite Sequencing (RRBS), identified Differentially Methylated Regions (DMR) in offspring that experienced in utero undernutrition, both in UND (244) and FA (240), compared with CTR. Gene ontology (GO) analysis identified DMRs in categories related to sperm function, therefore we investigated whether the fertilizing capacity of the semen from the three groups differed in an in vitro fertilization assay. Spermatozoa from the undernourished groups showed lower motility and sperm chromatin structure abnormalities, represented by a higher percentage of DNA fragmentation and an increased number of immature cells, compared with CTR. While good quality blastocysts were obtained from all three groups, the proportion of embryos reaching the blastocyst stage was reduced in the UND vs CTR, an effect partially rescued by the FA treatment. The data reported here show that nutritional stress during early pregnancy leads to epigenetic modifications in the semen of the resulting offspring, the effects of which in next generation remain to be elucidated.

Amniotic microvesicles impact hatching and pregnancy percentages of in vitro bovine embryos and blastocyst microRNA expression versus in vivo controls.

Embryo development and implantation are dynamic processes, responsive to external signals, and can potentially be influenced by many environmental factors. The aims of this study were to evaluate the effects of a culture medium supplemented with amniotic-derived microvesicles (MVs) on in vitro embryo hatching after cryopreservation, and pregnancy rate following embryo transfer. In addition, miRNA profiling of blastocysts produced in vitro, with or without (control; CTR) amniotic MV supplementation, was also evaluated using blastocysts produced in vivo. In vitro embryos were cultured with and without amniotic MV supplementation. In vivo blastocysts were obtained from superovulated cows. Samples for RNA isolation were obtained from three pools of 10 embryos each (in vivo, in vitro-CTR and in vitro + MVs). Our results show that the hatching percentage of cryopreserved in vitro + MVs embryos is higher (P < 0.05) than in vitro-CTR embryos and the pregnancy rate with fresh and cryopreserved in vitro + MVs embryos is higher than in vitro-CTR embryos. In addition, the analysis of differently expressed (DE) microRNAs showed that embryos produced in vivo are clearly different from those produced in vitro. Moreover, in vitro-CTR and in vitro + MVs embryos differ significantly for expression of two miRNAs that were found in higher concentrations in in vitro-CTR embryos. Interestingly, these two miRNAs were also reported in degenerated bovine embryos compared to good quality blastocysts. In conclusion, MV addition during in vitro production of embryos seems to counteract the adverse effect of in vitro culture and partially modulate the expression of specific miRNAs involved in successful embryo implantation.

Epigenetic analysis of high and low motile sperm populations reveals methylation variation in satellite regions within the pericentromeric position and in genes functionally related to sperm DNA organization and maintenance in Bos taurus.

BACKGROUND: Sperm epigenetics is an emerging area of study supported by observations reporting that abnormal sperm DNA methylation patterns are associated with infertility. Here, we explore cytosine-guanine dinucleotides (CpGs) methylation in high (HM) and low motile (LM) Bos taurus sperm populations separated by Percoll gradient. HM and LM methylation patterns were investigated by bisulfite sequencing. RESULTS: Comparison between HM and LM sperm populations revealed that methylation variation affects genes involved in chromatin organization. CpG Islands (CGIs), were highly remodelled. A high proportion of CGIs was found to be methylated at low/intermediate level (20-60%) and associated to the repetitive element BTSAT4 satellite. The low/intermediate level of methylation in BTSAT4 was stably maintained in pericentric regions of chromosomes. BTSAT4 was hypomethylated in HM sperm populations. CONCLUSIONS: The characterization of the epigenome in HM and LM Bos taurus sperm populations provides a first step towards the understanding of the effect of methylation on sperm fertility. Methylation variation observed in HM and LM populations in genes associated to DNA structure remodelling as well as in a repetitive element in pericentric regions suggests that maintenance of chromosome structure through epigenetic regulation is probably crucial for correct sperm functionality.

Genome sequencing of Prototheca zopfii genotypes 1 and 2 provides evidence of a severe reduction in organellar genomes.

Prototheca zopfii (P. zopfii, class Trebouxiophyceae, order Chlorellales, family Chlorellaceae), a non-photosynthetic predominantly free-living unicellular alga, is one of the few pathogens belonging to the plant kingdom. This alga can affect many vertebrate hosts, sustaining systemic infections and diseases such as mastitis in cows. The aim of our work was to sequence and assemble the P. zopfii genotype 1 and genotype 2 mitochondrial and plastid genomes. Remarkably, the P. zopfii mitochondrial (38 Kb) and plastid (28 Kb) genomes are models of compaction and the smallest known in the Trebouxiophyceae. As expected, the P. zopfii genotype 1 and 2 plastid genomes lack all the genes involved in photosynthesis, but, surprisingly, they also lack those coding for RNA polymerases. Our results showed that plastid genes are actively transcribed in P. zopfii, which suggests that the missing RNA polymerases are substituted by nuclear-encoded paralogs. The simplified architecture and highly-reduced gene complement of the P. zopfii mitochondrial and plastid genomes are closer to those of P. stagnora and the achlorophyllous obligate parasite Helicosporidium than to those of P. wickerhamii or P. cutis. This similarity is also supported by maximum likelihood phylogenetic analyses inferences. Overall, the P. zopfii sequences reported here, which include nuclear genome drafts for both genotypes, will help provide both a deeper understanding of the evolution of Prototheca spp. and insights into the corresponding host/pathogen interactions.