V. Havlicek

Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions

ABSTRACT Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro- and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENEs bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows.

Infertility in dairy cattle is a multifactorial problem that may be linked to follicle development and the quality of the ovulated oocyte, to sperm transport and fertilization, to the reproductive tract environment, or to a combination of these factors. Using a state-of-the-art endoscopic embryo transfer technique, the aim of this study was to compare the ability of the reproductive tract of postpartum dairy cows and nulliparous heifers to support the development of early embryos to the blastocyst stage. Bovine embryos of 2 to 4 cells (n=1,800) were produced by in vitro maturation and fertilization of oocytes derived from the ovaries of slaughtered cattle. The estrus cycles of nulliparous Holstein heifers (n=10) and postpartum Holstein cows (n=8, approximately 60 d postpartum) were synchronized using an 8-d controlled internal drug release device coupled with prostaglandin injection. On d 2, one hundred 2- to 4-cell embryos were endoscopically transferred to the oviduct ipsilateral to the corpus luteum. Five days later, on d 7, the oviduct and uterus were flushed nonsurgically to recover the embryos. The number of embryos developing to the blastocyst stage was recorded immediately at recovery and following overnight culture in vitro. A representative number of blastocysts from heifers and cows were stained to assess cell number. Progesterone concentrations were lower in cows than in heifers on d 5, 6, and 7 (d 7=2.39+/-0.33 vs. 5.34+/-0.77ng/mL, respectively). More embryos were recovered from heifers than cows (79.0+/-7.0 vs. 57.2+/-11.4%). Of the embryos recovered, 33.9+/-3.6% had developed to the blastocyst stage in the heifer oviduct compared with 18.3+/-7.9% in the postpartum cow oviduct. There was no evidence of a difference in blastocyst quality as evidenced by total cell number in the blastocysts (71.2+/-5.7 vs. 67.0+/-5.3, respectively). In conclusion, the reproductive tract of the postpartum lactating dairy cow may be less capable of supporting early embryo development than that of the nonlactating heifer, and this may contribute to the lower conception rates observed in such animals.

Effect of Elevated Circulating Progesterone Concentration on Bovine Blastocyst Development and Global Transcriptome Following Endoscopic Transfer of In Vitro Produced Embryos to the Bovine Oviduct

Elevated concentrations of circulating progesterone in the immediate postconception period have been associated with an increase in embryonic growth rate, interferon-tau production, and pregnancy rate in cattle and sheep. Much of this effect is likely mediated via downstream effects of progesterone-induced changes in gene expression in the uterine tissues. Using state-of-the-art endoscopic techniques, this study examined the effect of elevated progesterone on the development of in vitro produced bovine zygotes transferred to the oviducts of heifers with high or normal circulating progesterone concentrations and on the transcriptome of blastocysts developing under such conditions. Simmental heifers (n = 34) were synchronized using a controlled internal drug release (CIDR) device for 8 days, with a prostaglandin F2alpha analogue administered 3 days before removal of the CIDR device. Only animals exhibiting a clear standing estrus (Day 0) were used. To produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the estrous cycle; the PRID was left in place until embryo recovery. All animals were sampled for blood daily from Day 0 to Day 7. Cleaved embryos were transferred by endoscopy to the ipsilateral oviduct of each recipient on Day 2 and then recovered by nonsurgically flushing the oviduct and the uterus on Day 7. The number of embryos developing to the blastocyst stage was recorded at recovery and following overnight culture in vitro. Potential effects of elevated progesterone on transcript abundance were examined using the Affymetrix GeneChip Bovine Genome Array. Insertion of a PRID on Day 3 resulted in a significant elevation of progesterone concentration (P < 0.05) from Day 3.5 until Day 6. Elevated progesterone did not affect the proportion of embryos developing to the blastocyst stage. Genomewide gene expression analysis identified 194 differentially expressed genes between embryos collected from heifers with normal or elevated progesterone, and quantitative real-time PCR validation with a subset of selected genes and an independent sample confirmed the microarray results. Interaction network analysis indicated a significant interaction between progesterone-regulated genes in the blastocyst and in the maternal endometrium. These results suggest that elevated concentrations of progesterone do not affect the ability of the early embryo to reach the blastocyst stage in vivo but do result in subtle changes to the transcriptome of the embryo that may be associated with advanced elongation posthatching.

Effect of reproductive tract environment following controlled ovarian hyperstimulation treatment on embryo development and global transcriptome profile of blastocysts: implications for animal breeding and human assisted reproduction

BACKGROUND In mammals, the reproductive tract plays a crucial role in the success of early reproductive events and provides an optimal microenvironment for early embryonic development. However, changes in the reproductive tract environment associated with controlled ovarian hyperstimulation and the influence on the embryo transcriptome profile have not been investigated. Therefore, we investigated differences in the development rate and the transcriptome profile of bovine blastocysts developing in the reproductive tract of unstimulated or superovulated heifers. METHODS Nineteen Simmental heifers were synchronized, superovulated and artificially inseminated; nine heifers were flushed on Day 2 after insemination and 2-4-cell stage embryos were recovered and endoscopicaly transferred to the ipsilateral oviduct of unstimulated (i.e. single-ovulating) synchronized recipients (n= 4 recipients; 25-50 embryos per recipient). The remaining 10 superovulated heifers and the unstimulated recipients were then non-surgically flushed on Day 7 to collect embryos. The blastocyst transcriptome profile was examined using the Affymetrix GeneChip Bovine Genome Array. RESULTS The proportion of embryos, which developed to the blastocyst stage, was lower in superovulated heifers than unstimulated heifers (P< 0.05). Blastocysts that developed under the abnormal endocrine conditions associated with ovulation induction showed higher cellular and metabolic activities, as genes involved in the oxidative phosphorylation pathway, different metabolic processes and translation and transcription processes, in addition to genes expressed in response to stress, were highly expressed compared with embryos that developed in the oviduct of unstimulated animals. CONCLUSIONS The environment in which the embryo develops in the oviduct/uterus significantly alters gene expression patterns, especially those genes that regulate metabolic activity in the embryo.

Influence of lactation on metabolic characteristics and embryo development in postpartum Holstein dairy cows

The aim of this study was to examine the direct effect of lactation on the ability of the reproductive tract of postpartum dairy cows to support early embryo development. Twenty-one primiparous Holstein heifers were used. Immediately after calving, half of the cows were dried off (i.e., never milked), and the other half entered the milking herd and were milked twice daily. Jugular blood samples were taken twice per week from 15 d before calving to approximately 100 d postpartum to measure nonesterified fatty acids, β-hydroxybutyrate, glucose, insulin, and insulin-like growth factor-I. At the same time, body weight and body condition score were recorded for each cow. At approximately 60 d postpartum (experiment 1), approximately 65 two- to four-cell embryos, produced by in vitro maturation and fertilization, were endoscopically transferred to the oviduct ipsilateral to the corpus luteum of all cows on d 2 of the estrous cycle. Five days later (d 7), the oviduct and uterus were flushed nonsurgically and the number of embryos developing to the blastocyst stage was recorded. At approximately 90 d postpartum (experiment 2), the estrous cycles of the same cows were resynchronized and 15 to 20 in vitro-produced blastocysts were transferred to the uterus of each recipient on d 7. All cows were slaughtered on d 14 to assess embryo survival and dimensions. Body weight and body condition score were significantly different between groups for the entire postpartum period of the study. Concentrations of nonesterified fatty acids and β-hydroxybutyrate were higher and concentrations of glucose, insulin, and insulin-like growth factor-I were lower in lactating compared with nonlactating cows. Embryo recovery rates from lactating and dry cows were similar. In experiment 1, fewer embryos developed to the blastocyst stage in the lactating cows compared with the nonlactating cows. In experiment 2, embryo survival and conceptus dimensions were not different between lactating and nonlactating cows. In conclusion, the data indicate that the reproductive tract of the lactating dairy cow is compromised in its ability to support early embryo development compared with that of matched dry cows and this may contribute to early embryo mortality observed in such animals.

Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street?

ABSTRACT This study examined the effect of the presence of single or multiple embryos on the transcriptome of the bovine oviduct. In experiment 1, cyclic (nonbred, n = 6) and pregnant (artificially inseminated, n = 11) heifers were slaughtered on Day 3 after estrus, and the ampulla and isthmic regions of the oviduct ipsilateral to the corpus luteum were separately flushed. Oviductal epithelial cells from the isthmus region, in which all oocytes/embryos were located, were snap-frozen for microarray analysis. In experiment 2, heifers were divided into cyclic (nonbred, n = 6) or pregnant (multiple embryo transfer, n = 10) groups. In vitro-produced presumptive zygotes were transferred endoscopically to the ipsilateral oviduct on Day 1.5 postestrus (n = 50 zygotes/heifer). Heifers were slaughtered on Day 3, and oviductal isthmus epithelial cells were recovered for RNA sequencing. Microarray analysis in experiment 1 failed to detect any difference in the transcriptome of the oviductal isthmus induced by the presence of a single embryo. In experiment 2, following multiple embryo transfer, RNA sequencing revealed 278 differentially expressed genes, of which 123 were up-regulated and 155 were down-regulated in pregnant heifers. Most of the down-regulated genes were related to immune function. In conclusion, the presence of multiple embryos in the oviduct resulted in the detection of differentially expressed genes in the oviductal isthmus; failure to detect changes in the oviduct transcriptome in the presence of a single embryo may be due to the effect being local and undetectable under the conditions of this study.

Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern.

Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase II oocytes with (PB(+)) and without (PB(-)) first polar body and zygotes were subjected to DEP at 4 MHz and 450 mum electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of post-activation, the blastocyst rate of very slow dielectrophoretic PB(+) and PB(-) oocytes was significantly (P < 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P < 0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB(+) oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB(+) oocytes and zygotes respectively. In conclusion, dielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.

Expression of mRNA, before and after freezing, in bovine blastocysts cultured under different conditions

Production methods and culture systems have been shown to affect blastocyst mRNA expression and cryopreservability, which may serve as sensitive indicators of embryo quality and developmental competence. In the present study, the impact of four established culture conditions for producing bovine blastocysts (in vitro production, IVP; gamete intra-fallopian transfer, GIFT; transfer of cleaved stages into the oviduct, CLVT; multiple ovulation embryo transfer, MOET) was assessed, in terms of both cryosurvival and levels of mRNA expression of several selected genes (occludin, desmocollin 2, solute carrier family 2 member 3, BAX, BCL-XL, heat shock protein 1A, aquaporin 3, DNA methyltransferase 1a) detected with RT-qPCR. At 24 hours post-thawing, blastocysts derived from in vitro production showed a significantly higher re-expansion rate compared to the other groups. At later times, this difference was no longer significant. Before freezing, embryos of the MOET group showed significantly more desmocollin 2 mRNA compared to embryos produced using other culture methods. After freezing, significant upregulation was found in transcripts of heat shock protein 1A in embryos of all groups; of solute carrier family 2 member 3, only in IVP derived embryos; of BAX, BCL-XL, occludin, desmocollin 2, only in the MOET and IVP groups. Aquaporin 3 and DNA methyltransferase 1a were neither up- nor downregulated in blastocysts of any group. In conclusion, these findings suggest that, after freezing, embryos seem to have switched on mRNA synthesis, an active metabolism, operational cell connections, and are prepared for hatching and beyond.

Expression of Apoptosis Regulatory Genes and Incidence of Apoptosis in Different Morphological Quality Groups of In Vitro-produced Bovine Pre-implantation Embryos

Apoptosis occurs during early development in both in vivo- and in vitro-produced embryos, and is considered as one of the causes of embryonic loss. The objectives of this study were, therefore, investigating stage-specific expression profiles of apoptosis regulatory genes in three quality groups of in vitro-produced bovine pre-implantation embryos; and analysing the relationship between cell number and DNA fragmentation with expressions of those genes. The relative abundance of mRNA of 9 pro- (Bax, caspase-9, Bcl-xs, P53, Caspase-3 and Fas) and anti- (Bcl-w and Mcl-1) apoptotic genes was analysed. Differential cell staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling were performed to analyse the variation in cell numbers and detect apoptotic nuclei respectively. Expression of Bax and Caspase-3 genes was significantly (p < 0.05) higher in poor quality pre-implantation embryos as compared with that of morphologically good quality embryos of the same developmental stages. Moreover, Mcl-1 expression was significantly higher in good quality immature oocytes than that in the poor quality group. Moreover, higher DNA fragmentation was evidenced in morphologically poor quality blastocysts. In conclusion, our study demonstrates that Bax, caspase-3 and Mcl-1 can be used as potential markers of embryo quality to evaluate in vitro-produced bovine embryos. Further studies are required to investigate specific molecular signatures that can be used in evaluating in vivo-derived embryos.

Stochastic modelling, Bayesian inference, and new in vivo measurements elucidate the debated mtDNA bottleneck mechanism

Dangerous damage to mitochondrial DNA (mtDNA) can be ameliorated during mammalian development through a highly debated mechanism called the mtDNA bottleneck. Uncertainty surrounding this process limits our ability to address inherited mtDNA diseases. We produce a new, physically motivated, generalisable theoretical model for mtDNA populations during development, allowing the first statistical comparison of proposed bottleneck mechanisms. Using approximate Bayesian computation and mouse data, we find most statistical support for a combination of binomial partitioning of mtDNAs at cell divisions and random mtDNA turnover, meaning that the debated exact magnitude of mtDNA copy number depletion is flexible. New experimental measurements from a wild-derived mtDNA pairing in mice confirm the theoretical predictions of this model. We analytically solve a mathematical description of this mechanism, computing probabilities of mtDNA disease onset, efficacy of clinical sampling strategies, and effects of potential dynamic interventions, thus developing a quantitative and experimentally-supported stochastic theory of the bottleneck. DOI: http://dx.doi.org/10.7554/eLife.07464.001