L-Carnitine sustainably affects bioenergetic profile of bovine blastocysts and transcriptome profile of elongation-stage embryos.

L-Carnitine (LC) is known to play key roles in lipid metabolism and antioxidative activity, implicating enhanced cryotolerance of bovine blastocysts. However, sustainability of LC supplementation during culture period on preimplantation development beyond the blastocyst stage has not been investigated so far. Therefore, all embryos were cultured under fatty acid free conditions, one group with LC (LC-Embryos) and the control group without LC (Control) supplementation. Transfer to recipients was conducted on day 6. Elongation-stage embryos were recovered on day 14; metrics of embryo recollection, developmental rates to early elongation-stage as well as mean embryo length did not differ between the groups. Gene expression analysis via NGS revealed 341 genes to be differentially regulated between elongation-stage embryos derived from LC supplementation compared to controls. These played mainly a role in molecular functions and biological processes like oxidoreductase activity, ATP dependent activity, cellular stress and respiration. Pathways like oxidative phosphorylation and thermogenesis, ECM receptor signaling, PI3K-Akt and focal adhesion were affected by differentially regulated genes. Moreover, all DEGs located on the mitochondria were significantly downregulated in the LC-Embryos, being in line with lower mitochondrial copy number and mtDNA integrity compared to the control group. Finally, we uncovered alterations of the bioenergetic profile on day 7 as a consequence of LC supplementation for the first time, revealing significantly higher oxygen consumption rates, ATP linked respiration and spare capacity for LC-embryos. Conclusively, we uncovered direct effects of LC supplementation during the culture period on the bioenergetic profile going along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos.

Machine-learning methods applied to integrated transcriptomic data from bovine blastocysts and elongating conceptuses to identify genes predictive of embryonic competence.

Early pregnancy loss markedly impacts reproductive efficiency in cattle. The objectives were to model a biologically relevant gene signature predicting embryonic competence for survival after integrating transcriptomic data from blastocysts and elongating conceptuses with different developmental capacities and to validate the potential biomarkers with independent embryonic data sets through the application of machine-learning algorithms. First, two data sets from in vivo-produced blastocysts competent or not to sustain a pregnancy were integrated with a data set from long and short day-15 conceptuses. A statistical contrast determined differentially expressed genes (DEG) increasing in expression from a competent blastocyst to a long conceptus and vice versa; these were enriched for KEGG pathways related to glycolysis/gluconeogenesis and RNA processing, respectively. Next, the most discriminative DEG between blastocysts that resulted or did not in pregnancy were selected by linear discriminant analysis. These eight putative biomarker genes were validated by modeling their expression in competent or noncompetent blastocysts through Bayesian logistic regression or neural networks and predicting embryo developmental fate in four external data sets consisting of in vitro-produced blastocysts (i) competent or not, or (ii) exposed or not to detrimental conditions during culture, and elongated conceptuses (iii) of different length, or (iv) developed in the uteri of high- or subfertile heifers. Predictions for each data set were more than 85% accurate, suggesting that these genes play a key role in embryo development and pregnancy establishment. In conclusion, this study integrated transcriptomic data from seven independent experiments to identify a small set of genes capable of predicting embryonic competence for survival.

The extent of the abundance of exosomal and non-exosomal extracellular miRNAs in the bovine follicular fluid.

Follicular fluid (FF) plays an important role during follicular development and it contains several bioactive molecules including extracellular microRNAs (ECmiRNAs) that may mediate cell-cell communication during follicular development. Yet, the distribution patterns of ECmiRNAs in FF is not well characterized. This study aims to investigate the distribution of ECmiRNAs in two major fractions, namely exosomal and non-exosomal, of bovine follicular fluid (bFF). Exosomal and non-exosomal fractions from bFF were separated using Exoquick™ exosomes precipitation kit. miRNA expression was evaluated using the human miRCURY LNA™ Universal RT miRNA PCR array system. Transmission electron microscopy and immunoblotting revealed that the isolated vesicles were exosomes. The real-time PCR-based expression analysis revealed that 516 miRNAs were detected in the exosomal fraction of bFF, while 393 miRNAs were detected in the non-exosomal fraction. Among the detected miRNAs, a total of 370 miRNAs were detected in both fractions, while 145 miRNAs and 23 miRNAs were solely detected in exosomal and non-exosomal fractions, respectively. Exploratory pathway analysis showed that the genes targeted by exosomal and non-exosomal miRNAs to be involved in MAPK, Wnt, FoxO, TGF-beta, Oxytocin, ErbB, PI3K-Akt, Neurotrophin signalling pathways which are believed to be involved in follicular development, cell proliferation, and meiotic resumption. The results of our study demonstrated that besides the exosomal fraction, non-exosomal fractions can carry a significant amount of miRNAs in bFF where the exosomal fraction carries a significantly higher number of detectable miRNAs.

α/ß-Hydrolase D16B Truncation Results in Premature Sperm Capacitation in Cattle.

Recently it was shown that a specific form of male infertility in Holstein cattle was caused by a nonsense variant in the α/ß-hydrolase domain-containing 16B (ABHD16B) gene resulting in a protein truncation at amino acid position 218 (p.218Q*) and loss of function. Lipidomics showed that the absence of ABHD16B influenced the content of phosphatidylcholine (PC), ceramide (Cer), diacylglycerol (DAG), and sphingomyelin (SM) in variant carrier sperm membranes. However, the exact cause of infertility in affected sires has remained unclear until now. To elucidate the cause of infertility, we analyzed (i) standard sperm parameters (i.e., total sperm number, morphological intact sperm, total sperm motility), (ii) in vitro fertilizability and effects on early embryonic development, and (iii) sperm survival rates (i.e., capacitation time). The affected spermatozoa showed no changes in the usual sperm parameters and were also capable of fertilization in vitro. Furthermore, the absence of ABHD16B did not affect early embryonic development. Based on these results, it was concluded that the affected spermatozoa appeared to be fertilizable per se. Consequently, the actual cause of the inability to fertilize could only be due to a time- and/or place-dependent process after artificial insemination and before fertilization. A process fundamental to the ability to fertilize after insemination is capacitation. Capacitation is a biochemical maturation process that spermatozoa undergo in the female genital tract and is inevitable for the successful fertilization of the oocyte. It is known that the presence and concentration of certain sperm membrane lipids are essential for the correct course of capacitation. However, precisely these lipids are absent in the membrane of spermatozoa affected by the ABHD16B truncation. Since all other causes of fertilization inability were excluded in the previous experiments, consequently, the only remaining hypothesis was that the loss of function of ABHD16B leads to a capacitation disruption. We were able to show that heterozygous and homozygous affected spermatozoa exhibit premature capacitation and therefore decay before fertilization. This effect of the loss of function of ABHD16B has not been described before and our studies now revealed why sires harboring the variant in the ABHD16B gene are infertile.

The global gene expression outline of the bovine blastocyst: reflector of environmental conditions and predictor of developmental capacity.

BACKGROUND: Morphological evaluation of embryos has been used to screen embryos for transfer. However, the repeatability and accuracy of this method remains low. Thus, evaluation of an embryo's gene expression signature with respect to its developmental capacity could provide new opportunities for embryo selection. Since the gene expression outline of an embryo is considered as an aggregate of its intrinsic characteristics and culture conditions, we have compared transcriptome profiles of in vivo and in vitro derived blastocysts in relation to pregnancy outcome to unravel the discrete effects of developmental competence and environmental conditions on bovine embryo gene expression outlines. To understand whether the gene expression patterns could be associated with blastocyst developmental competency, the global transcriptome profile of in vivo (CVO) and in vitro (CVT) derived competent blastocysts that resulted in pregnancy was investigated relative to that of in vivo (NVO) and in vitro (NVT) derived blastocysts which did not establish initial pregnancy, respectively while to unravel the effects of culture condition on the transcriptome profile of embryos, the transcriptional activity of the CVO group was compared to the CVT group and the NVO group was compared to the NVT ones. RESULTS: A total of 700 differentially expressed genes (DEGs) were identified between CVO and NVO blastocysts. These gene transcripts represent constitutive regions, indel variants, 3'-UTR sequence variants and novel transcript regions. The majority (82%) of these DEGs, including gene clusters like ATP synthases, eukaryotic translation initiation factors, ribosomal proteins, mitochondrial ribosomal proteins, NADH dehydrogenase and cytochrome c oxidase subunits were enriched in the CVO group. These DEGs were involved in pathways associated with glycolysis/glycogenesis, citrate acid cycle, pyruvate metabolism and oxidative phosphorylation. Similarly, a total of 218 genes were differentially expressed between CVT and NVT groups. Of these, 89%, including TPT1, PDIA6, HSP90AA1 and CALM, were downregulated in the CVT group and those DEGs were overrepresented in pathways related to protein processing, endoplasmic reticulum, spliceasome, ubiquitone mediated proteolysis and steroid biosynthesis. On the other hand, although both the CVT and CVO blastocyst groups resulted in pregnancy, a total of 937 genes were differential expressed between the two groups. Compared to CVO embryos, the CVT ones exhibited downregulation of gene clusters including ribosomal proteins, mitochondrial ribosomal protein, eukaryotic translation initiation factors, ATP synthases, NADH dehydrogenase and cytochrome c oxidases. Nonetheless, downregulation of these genes could be associated with pre and postnatal abnormalities observed after transfer of in vitro embryos. CONCLUSION: The present study provides a detailed inventory of differentially expressed gene signatures and pathways specifically reflective of the developmental environment and future developmental capacities of bovine embryos suggesting that transcriptome activity observed in blastocysts could be indicative of further pregnancy success but also adaptation to culture environment.

NRF2-mediated signaling is a master regulator of transcription factors in bovine granulosa cells under oxidative stress condition.

Transcription factors (TFs) are known to be involved in regulating the expression of several classes of genes during folliculogenesis. However, the regulatory role of TFs during oxidative stress (OS) is not fully understood. The current study was aimed to investigate the regulation of the TFs in bovine granulosa cells (bGCs) during exposure to OS induced by H2O2 in vitro. For this, bGCs derived from ovarian follicles were cultured in vitro till their confluency and then treated with H2O2 for 40 min. Twenty-four hours later, cells were subjected to various phenotypic and gene expression analyses for genes related to TFs, endoplasmic reticulum stress, apoptosis, cell proliferation, and differentiation markers. The bGCs exhibited higher reactive oxygen species accumulation, DNA fragmentation, and endoplasmic reticulum stress accompanied by reduction of mitochondrial activity after exposure to OS. In addition, higher lipid accumulation and lower cell proliferation were noticed in H2O2-challenged cells. The mRNA level of TFs including NRF2, E2F1, KLF6, KLF9, FOS, SREBF1, SREBF2, and NOTCH1 was increased in H2O2-treated cells compared with non-treated controls. However, the expression level of KLF4 and its downstream gene, CCNB1, were downregulated in the H2O2-challenged group. Moreover, targeted inhibition of NRF2 using small interference RNA resulted in reduced expression of KLF9, FOS, SREBF2, and NOTCH1 genes, while the expression of KLF4 was upregulated. Taken together, bovine granulosa cells exposed to OS exhibited differential expression of various transcription factors, which are mediated by the NRF2 signaling pathway.

The regulatory role of miR-20a in bovine cumulus cells and its contribution to oocyte maturation.

Dynamic changes in microRNAs in oocyte and cumulus cells before and after maturation may explain the spatiotemporal post-transcriptional gene regulation within bovine follicular cells during the oocyte maturation process. miR-20a has been previously shown to regulate proliferation and differentiation as well as progesterone levels in cultured bovine granulosa cells. In the present study, we aimed to demonstrate the function of miR-20a during the bovine oocyte maturation process. Maturation of cumulus-oocyte complexes (COCs) was performed at 39°C in an humidified atmosphere with 5% CO2 in air. The expression of miR-20a was investigated in the cumulus cells and oocytes at 22 h post culture. The functional role of miR-20a was examined by modulating the expression of miR-20a in COCs during in vitro maturation (IVM). We found that the miR-20a expression was increased in cumulus cells but decreased in oocytes after IVM. Overexpression of miR-20a increased the oocyte maturation rate. Even though not statistically significant, miR-20a overexpression during IVM increased progesterone levels in the spent medium. This was further supported by the expression of STAR and CYP11A1 genes in cumulus cells. The phenotypes observed due to overexpression of miR-20a were validated by BMP15 supplementation during IVM and subsequent transfection of BMP15-treated COCs using miR-20a mimic or BMPR2 siRNA. We found that miR-20a mimic or BMPR2 siRNA transfection rescued BMP15-reduced oocyte maturation and progesterone levels. We concluded that miR-20a regulates oocyte maturation by increasing cumulus cell progesterone synthesis by simultaneous suppression of BMPR2 expression.

MicroRNA-Mediated Gene Regulatory Mechanisms in Mammalian Female Reproductive Health.

Mammalian reproductive health affects the entire reproductive cycle starting with the ovarian function through implantation and fetal growth. Various environmental and physiological factors contribute to disturbed reproductive health status leading to infertility problems in mammalian species. In the last couple of decades a significant number of studies have been conducted to investigate the transcriptome of reproductive tissues and organs in relation to the various reproductive health issues including endometritis, polycystic ovarian syndrome (PCOS), intrauterine growth restriction (IUGR), preeclampsia, and various age-associated reproductive disorders. Among others, the post-transcriptional regulation of genes by small noncoding miRNAs contributes to the observed transcriptome dysregulation associated with reproductive pathophysiological conditions. MicroRNAs as a class of non-coding RNAs are also known to be involved in various pathophysiological conditions either in cellular cytoplasm or they can be released to the extracellular fluid via membrane-bounded extracellular vesicles and proteins. The present review summarizes the cellular and extracellular miRNAs and their association with the etiology of major reproductive pathologies including PCOS, endometritis, IUGR and age-associated disorders in various mammalian species.

Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality.

Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality.

Transcriptome profile and association study revealed STAT3 gene as a potential quality marker of bovine gametes.

The present study was aimed to investigate differences in molecular signatures in oocytes derived from Holstein-Friesian heifers with different genetic merit for fertility, euthanized during day 0 or day 12 of the estrous cycle. Moreover, association between single nucleotide polymorphisms (SNPs) of ODC1 and STAT3 genes and bull fertility traits was investigated. The gene expression patterns were analyzed using cDNA array and validated with quantitative real-time polymerase chain reaction (PCR). The result revealed that several genes have shown not only to be regulated by fertility merit but also by the day of oocyte recovery during the estrous cycle. The STAT3 gene was found to be upregulated in oocytes recovered from animals with high fertility merit at both day 0 and day 12. Some other genes like PTTG1, ODC1 and TUBA1C were downregulated at day 0 and upregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In contrast, the transcript abundance of TPM3 was upregulated at day 0 and downregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In addition, ODC1 and STAT3 were found to be associated (P < 0.05) with sperm quality traits as well as flow cytometry parameters. Therefore, the expression of several candidate genes including ODC1 and STAT3 was related to the genetic merit of the cow. In addition polymorphisms in these two genes were found to be associated with bull semen quality.

Quercetin supports bovine preimplantation embryo development under oxidative stress condition via activation of the Nrf2 signalling pathway.

Nrf2 is a master regulator for antioxidant machinery against oxidative stress in bovine preimplantation embryos. The endogenous or exogenous modulation of Nrf2-KEAP1 system in bovine embryos may contribute to the understanding of the mechanisms behind the response of embryos to stress conditions. Therefore, here we aimed to investigate the protective effect of quercetin on bovine preimplantation embryos exposed to higher atmospheric oxygen concentration. For that, blastocysts, which were developed from zygotes cultured in media supplemented with or without quercetin under high oxygen level (20%), were subjected intracellular ROS level and mitochondrial analysis, and determining blastocyst formation rate and total cell number. Moreover, mRNA and protein expression level of Nrf2 and selected downstream antioxidant genes were investigated in the resulting blastocysts. Quercetin supplementation in vitro culture did not affect cleavage and blastocyst rate until day 7. However, quercetin supplementation resulted in higher blastocyst total cell number and reduction of intracellular ROS level accompanied by increasing mitochondrial activity compared with control group in both day 7 and day 8 blastocysts. Moreover, quercetin supplementation induced mRNA and protein of Nrf2 with subsequent increase in the expression of downstream antioxidants namely: NQO1, PRDX1, CAT and SOD1 antioxidants. In conclusion, quercetin protects preimplantation embryos against oxidative stress and improves embryo viability through modulation of the Nrf2 signalling pathway.

The Role of MicroRNAs in Mammalian Fertility: From Gametogenesis to Embryo Implantation.

The genetic codes inscribed during two key developmental processes, namely gametogenesis and embryogenesis, are believed to determine subsequent development and survival of adult life. Once the embryo is formed, its further development mainly depends on its intrinsic characteristics, maternal environment (the endometrial receptivity), and the embryo-maternal interactions established during each phase of development. These developmental processes are under strict genetic regulation that could be manifested temporally and spatially depending on the physiological and developmental status of the cell. MicroRNAs (miRNAs), one of the small non-coding classes of RNAs, approximately 19-22 nucleotides in length, are one of the candidates for post-transcriptional developmental regulators. These tiny non-coding RNAs are expressed in ovarian tissue, granulosa cells, testis, oocytes, follicular fluid, and embryos and are implicated in diverse biological processes such as cell-to-cell communication. Moreover, accumulated evidences have also highlighted that miRNAs can be released into the extracellular environment through different mechanisms facilitating intercellular communication. Therefore, understanding miRNAs mediated regulatory mechanisms during gametogenesis and embryogenesis provides further insights about the molecular mechanisms underlying oocyte/sperm formation, early embryo development, and implantation. Thus, this review highlights the role of miRNAs in mammalian gametogenesis and embryogenesis and summarizes recent findings about miRNA-mediated post-transcriptional regulatory mechanisms occurring during early mammalian development.

Sexual dimorphic expression and release of transcription factors in bovine embryos exposed to oxidative stress.

Sexually dimorphic differences in genome activity, which is orchestrated by transcription factors (TFs), could explain the differential response of male and female embryos to environmental stressors. To proof this hypothesis, the expression of cellular and extracellular TFs was investigated in male and female bovine embryos in vitro cultured either under low (5%) or high (20%) oxygen levels. The intracellular reactive oxygen species (ROS), total cell number, expression of nuclear factor (erythroid-derived 2) factor 2 (NFE2L2), Krüppel-like factor 4 (KLF4), notch receptor 1 (NOTCH1), E2F transcription factor 1 (E2F1), and SREBF2 along with extracellular vesicles (EVs) biogenesis genes were assessed at the blastocyst stage and their released EVs. Low blastocyst rate in both sexes due to oxidative stress (OS) was accompanied by increased ROS accumulation and reduced cell number in female embryos. The messenger RNA and protein levels of NFE2L2, as well as KLF4 expression, were higher in male embryos exposed to OS compared with female embryos. However, the expression of NOTCH1 and E2F1 was higher in female embryos cultured in high oxygen level. Male embryos exposed to OS released more EVs enriched with NFE2L2, superoxide dismutase 1, and NOTCH1 accompanied by elevated expression of EVs biogenesis genes. Accordingly, differential expression of TFs and their release into spent media could partially explain the sexual dimorphic response of bovine embryos to environmental stresses.

Supplementing rumen-protected methionine to lactating multiparous dairy cows did not improve reproductive performance.

There is evidence that supplementing methionine has positive effects on uterine environment, oocyte quality and embryo development in cattle. Thus, the objective of this study was to evaluate reproductive traits of cows supplemented with rumen-protected methionine (RPM) during early to mid-lactation in comparison with an untreated control group (CON). An additional focus was on the effect of puerperal diseases on reproductive performance parameters in RPM-supplemented group MET and in CON. A total of 1,709 multiparous Holstein-Friesian cows were enrolled in this field trial conducted on a commercial dairy farm in Slovakia. Cows were allocated at approximately 12 days post-partum (dpp) to either CON or MET, the latter supplemented with 25.0 g-27.2 g RPM per cow per day incorporated into the total mixed ration (TMR) until leaving the study pen at approximately 140 dpp. The amount of RPM was calculated based on individual feed ingredients analysis and adjusted during the study period when TMR changed. Cows were monitored during the post-partum period by vaginal examination (day 5 pp), measuring of beta-hydroxybutyrate in blood (3, 5, and 8 dpp) and by vaginal examination, uterine cytology and measuring of back fat thickness by ultrasound (all at 31 ± 3 dpp). Compared with CON, cows supplemented with RPM did not show better reproduction performance parameters (first service submission rate, days to first service, conception risk, days open 140). Results from binary logistic regression model for the risk of conception showed that metritis had a significant effect, but the supplementation of methionine had not. Results of Cox regression analysis for the odds of conception within 140 dpp revealed only metritis and clinical endometritis as significant factors. In conclusion, supplementation of RPM had no beneficial effect on reproductive performance in this study farm compared with an untreated control group.

Endogenous and Exogenous Modulation of Nrf2 Mediated Oxidative Stress Response in Bovine Granulosa Cells: Potential Implication for Ovarian Function.

Nrf2 is a redox sensitive transcription factor regulating the expression of antioxidant genes as defense mechanism against various stressors. The aim of this study is to investigate the potential role of noncoding miRNAs as endogenous and quercetin as exogenous regulators of Nrf2 pathway in bovine granulosa cells. For this cultured granulosa cells were used for modulation of miRNAs (miR-28, 153 and miR-708) targeting the bovine Nrf2 and supplementation of quercentin to investigate the regulatory mechanisms of the Nrf2 antioxidant system. Moreover, cultured cells were treated with hydrogen peroxide to induce oxidative stress in those cells. Our results showed that, oxidative stress activated the expression of Nrf2 as a defense mechanism, while suppressing the expression of those miRNAs. Overexpression of those miRNAs resulted in downregulation of Nrf2 expression resulted in higher ROS accumulation, reduced mitochondrial activity and cellular proliferation. Quercetin supplementation showed its protective role against oxidative stress induced by H2O2 by inducing the expression of antioxidant enzymes. In conclusion, this study highlighted the involvement of miR-153, miR-28 and miR-708 in regulatory network of Nrf2 mediated antioxidant system in bovine granulosa cells function. Furthermore, quercetin at a low dose played a protective role in bovine granulosa cells against oxidative stress damage.

Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation.

BACKGROUND: Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. RESULTS: The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. CONCLUSION: In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

Correction to: Sulforaphane protects granulosa cells against oxidative stress via activation of NRF2-ARE pathway.

There is an error in the original publication of this paper. Figures 1-6 were shown in the wrong version, thus corrected figures provided were in this article.

Sulforaphane protects granulosa cells against oxidative stress via activation of NRF2-ARE pathway.

Sulforaphane (SFN) has been considered as an indirect antioxidant and potential inducer of the Nrf2-ARE pathway. This study was conducted to investigate the protective role of SFN against oxidative stress in bovine granulosa cells (GCs). GCs were collected from antral follicles (4-8 mm) and cultured according to the experimental design where group 1 = control, group 2 = treated with SFN, group 3 = treated with hydrogen peroxide (H2O2), group 4 = pretreated with SFN and then with H2O2 (protective) and group 5 = treated with H2O2 followed by SFN treatment (rescuing). Results showed that SFN pretreatment significantly increases cell viability and reduces cytotoxicity in GCs under oxidative stress. Following H2O2 exposure, expression of NRF2 was found to be significantly increased (p < 0.05) in SFN-pretreated cells, while no significant differences were observed between group 3 and group 5, although the expression was significantly increased compared to the control group. Moreover, the relative abundance of the NRF2 downstream target antioxidant genes (CAT, PRDX1, SOD1 and TXN1) were higher (fold change ranged from 7 to 14, p < 0.05) in sulforaphane pretreated GCs. Low level of ROS and lipid accumulation and higher mitochondrial activity were observed in GCs pretreated with SFN, whereas no such changes were observed in GCs treated with SFN after exposure to oxidative stress (group 5). Thus, we suggest that SFN pretreatment effectively protects GCs against oxidative damage through the activation of the NRF2-ARE pathway, whereas addition of SFN during oxidative insult failed to rescue GCs.

MicroRNAs: tiny molecules with a significant role in mammalian follicular and oocyte development.

The genetic regulation of female fertility (follicular development, oocyte maturation and early preimplantation embryo development) involves the spatio-temporal regulation of those genes that play key roles in various stages of the female reproductive axis. MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to regulate the expression of a large proportion of such genes. In recent decades, multiple studies have aimed to determine the roles of these non-coding RNAs in mammalian follicular development, oocyte growth and embryo development. These studies have applied a variety of approaches, including conditional knockout of miRNA biogenesis genes, high-throughput sequencing technologies for pattern recognition in miRNA expression and loss- and gain-of-function of miRNAs in various animal models. In addition to the cellular miRNAs, a large variety of RNAs are found in circulation, being coupled with extracellular vesicles, proteins and lipids. Because of their potential as diagnostic markers for abnormal physiologies, there is increasing interest in the identification of extracellular miRNAs in various biological fluids and spent in vitro culture media. This review focuses on studies addressing the expression and potential role of cellular and extracellular miRNAs in mammalian follicular cell physiology and subsequent ovarian functionality and oocyte maturation.

Clinical and subclinical endometritis induced alterations in bovine endometrial transcriptome and miRNome profile.

BACKGROUND: Clinical and subclinical endometritis are known to affect the fertility of dairy cows by inducing uterine inflammation. We hypothesized that clinical or subclinical endometritis could affect the fertility of cows by disturbing the molecular milieu of the uterine environment. Here we aimed to investigate the endometrial molecular signatures and pathways affected by clinical and subclinical endometritis. For this, Holstein Frisian cows at 42-60 days postpartum were classified as healthy (HE), subclinical endometritis (SE) or clinical endometritis (CE) based on veterinary clinical examination of the animals and histological evaluation the corresponding endometrial biopsies. Endometrial transcriptome and miRNome profile changes and associated molecular pathways induced by subclinical or clinical endometritis were then investigated using GeneChip® Bovine Genome Array and Exiqon microRNA PCR Human Panel arrays, respectively. The results were further validated in vitro using endometrial stromal and epithelial cells challenged with subclinical and clinical doses of lipopolysaccharide (LPS). RESULT: Transcriptome profile analysis revealed altered expression level of 203 genes in CE compared to HE animals. Of these, 92 genes including PTHLH, INHBA, DAPL1 and SERPINA1 were significantly upregulated, whereas the expression level of 111 genes including MAOB, CXCR4, HSD11B and, BOLA, were significantly downregulated in CE compared to the HE animal group. However, in SE group, the expression patterns of only 28 genes were found to be significantly altered, of which 26 genes including PTHLH, INHBA, DAPL1, MAOB, CXCR4 and TGIF1 were common to the CE group. Gene annotation analysis indicated the immune system processes; G-protein coupled receptor signaling pathway and chemotaxis to be among the affected functions in endometritis animal groups. In addition, miRNA expression analysis indicated the dysregulation of 35 miRNAs including miR-608, miR-526b* and miR-1265 in CE animals and 102 miRNAs including let-7 family (let-7a, let-7c, let-7d, let-7d*, let-7e, let-7f, let-7i) in SE animals. Interestingly, 14 miRNAs including let-7e, miR-92b, miR-337-3p, let-7f and miR-145 were affected in both SE and CE animal groups. Further in vitro analysis of selected differentially expressed genes and miRNAs in endometrial stroma and epithelial cells challenged with SE and CE doses of LPS showed similar results to that of the array data generated using samples collected from SE and CE animals. CONCLUSION: The results of this study unraveled endometrial transcriptome and miRNome profile alterations in cows affected by subclinical or clinical endometritis which may have a significant effect on the uterine homeostasis and uterine receptivity.