Sexual dimorphic miRNA-mediated response of bovine elongated embryos to the maternal microenvironment.

A skewed male-to-female ratio in cattle is believed to be due to the biased embryo losses during pregnancy. The changes in biochemical secretion such as miRNAs by the embryo due to altered maternal environment could cause a sex biased selective implantation resulting in a skewed male to female ratio at birth. Nevertheless, it is still not clear whether the male and female embryos could modify their miRNA expression patterns differently in response to altered physiological developmental conditions. Therefore, this study was focused on identifying sex specific miRNA expression patterns induced in the embryo during the elongation period in response to the maternal environment. For this, in vitro produced day female and male embryos were transferred to Holsteins Frisian cows and heifers. The elongated female and male embryos were then recovered at day 13 of the gestation period. Total RNA including the miRNAs was isolated from each group of elongated embryo samples were subjected to the next generation miRNA sequencing. Sequence alignment, identification and quantification of miRNAs were done using the miRDeep2 software package and differential miRNA expression analyses were performed using the edgeR bioconductor package. The recovery rate of viable elongating embryos at day 13 of the gestation period was 26.6%. In cows, 2.8 more viable elongating male embryos were recovered than female embryos, while in heifers the sex ratio of the recovered elongating embryos was close to one (1.05). The miRNA analysis showed that 254 miRNAs were detected in both male and female elongated embryos developed either in cows or heifers, of which 14 miRNAs including bta-miR-10b, bta-miR-148a, bta-miR-26a, and bta-miR-30d were highly expressed. Moreover, the expression level of 32 miRNAs including bta-let-7c, bta-let-7b, bta-let-7g, bta-let-7d and bta-let-7e was significantly different between the male and female embryos developed in cows, but the expression level of only 4 miRNAs (bta-miR-10, bta-mR-100, bta-miR-155 and bta-miR-6119-5p) was different between the male and female embryos that were developed in heifers. Furthermore, 19 miRNAs including those involved in cellular energy homeostasis pathways were differentially expressed between the male embryos developed in cows and heifers, but no significantly differentially expressed miRNAs were detected between the female embryos of cows and heifers. Thus, this study revealed that the sex ratio skewed towards males in embryos developed in cows was accompanied by increased embryonic sexual dimorphic miRNA expression divergence in embryos developed in cows compared to those developed in heifers. Moreover, male embryos are more sensitive to respond to the maternal reproductive microenvironment by modulating their miRNA expression.

The interaction between the environment and embryo development in assisted reproduction.

It can be assumed that the natural processes of selection and developmental condition in the animal provide the best prerequisites for embryogenesis resulting in pregnancy and subsequent birth of a healthy neonate. In contrast, circumventing the natural selection mechanisms and all developmental conditions in a healthy animal harbors the risk of counteracting, preventing or reducing the formation of embryos or substantially restricting their genesis. Considering these facts, it seems to be obvious that assisted reproductive techniques focusing on early embryonic stages serve an expanded and unselected germ cell pool of oocytes and sperm cells, and include the culture of embryos outside their natural habitat during and after fertilization for manipulation and diagnostic purposes, and for storage. A significant influence on the early embryonic development is seen in the extracorporeal culture of bovine embryos (in vitro) or stress on the animal organism (in vivo). The in vitro production per se and metabolic as well as endocrine changes in the natural environment of embryos represent adequate models and serve for a better understanding. The purpose of this review is to give a brief presentation of recent techniques aimed at focusing more on the complex processes in the Fallopian tube to contrast in vivo and in vitro prerequisites and abnormalities in early embryonic development and serve to identify potential new ways to make the use of ARTs more feasible.

Maternal metabolic status and in-vitro culture conditions during embryonic genome activation deregulate the expression of energy-related genes in the bovine 16-cells embryo.

The molecular consequences of the metabolic stress caused by milk production of dairy cows in the early embryo are largely unknown. The objective was to determine the impact of dam metabolic status or in vitro culture during embryonic genome activation (EGA) on the transcriptomic profiles of bovine 16-cell stage embryos. Two days after synchronized oestrus, in vitro produced 2- to 4-cell stage embryos were endoscopically transferred in pools of 50 into the oviduct ipsilateral to the corpus luteum of lactating (LACT, n = 3) or nonlactating (i.e. dried off immediately at calving; DRY, n = 3) dairy cows. On Day 4, the oviducts were flushed to recover the embryos. Pools of five Day-2 embryos (n = 5) and Day-4 16-cell stage embryos obtained in vitro (n = 3) or from LACT or DRY cows were subjected to RNAseq. Temporally differentially expressed genes (DEG; FDR<0.05) between Day-2 and Day-4 embryos were determined considering the differences between the three conditions under which EGA occurred. Also, DEG between Day-4 embryos derived from the three conditions were identified. Functional analysis of the temporal DEG demonstrated that genes involved in ribosome, translation and oxidative phosphorylation in the mitochondria were strongly more expressed in Day-4 than Day-2 embryos. Comparison of Day-4 embryos that underwent EGA in vitro, or in LACT or DRY cows, identified DEG enriching for mitochondrial respiration and protein translation, including the mTOR pathway. In conclusion, exposure of the embryo to an unfavourable maternal metabolic status during EGA influences its transcriptome and potentially the competence for pregnancy establishment.

Asynchrony between the early embryo and the reproductive tract affects subsequent embryo development in cattle.

The aim of this study was to determine the effect of maternal-embryonic asynchrony in the reproductive tract (oviduct and uterus) on subsequent embryo development in cattle. Fifty Day 1invitro-produced zygotes were transferred endoscopically into the oviduct ipsilateral to the corpus luteum of heifers (n=40) that were either synchronous with the embryos (Day 1 after ovulation) or asynchronous and ahead of the embryo (Day 3 after ovulation). A subset of heifers was killed in a commercial abattoir 3, 6 or 14 days after embryo transfer. Location within the reproductive tract, developmental stage and the quality of embryos were recorded. Transfer of embryos to an advanced (asynchronous) oviduct resulted, on Day 4, in fewer embryos at the expected location (oviduct), and a greater number of degenerated and retarded embryos with a lower total cell number than for embryos in the synchronous group. Similarly, on Day 7, asynchrony led to a greater number of degenerated and retarded embryos compared with the synchronous group. Total embryo cell number was similar among groups. Although Day 15 conceptuses were longer following asynchronous transfer, only 50% of the asynchronous heifers yielded conceptuses, compared with 100% in the synchronous group. In conclusion, asynchrony between the developing embryo and the reproductive tract has a negative effect on embryo development.

A novel approach to study the bovine oviductal fluid proteome using transvaginal endoscopy.

The oviduct provides the optimal micro milieu for early embryo development. However, accessing the bovine oviductal fluid in vivo for analysis is still challenging and therefore the oviductal fluid is usually collected post mortem. In the study presented here we introduce a novel approach to gain minimal invasive access to the bovine oviductal fluid proteome in vivo by transvaginal endoscopy at different stages of the estrous cycle. The first experiment aimed at transferring C4 derivatised magnetic beads to bind the oviductal fluid proteome in situ. Protein carrying beads were recovered by flushing the oviduct and proteins were eluted. In the second experiment a flushing solution was injected into and aspirated from the oviduct repeatedly. The flushing solution was centrifuged to separate the fluid from the cellular debris. Proteins were identified by nano-LC-MS/MS. Two different stages of the estrous cycle (Day 1 and Day 3) were analyzed in samples from 30 heifers. Both methods were applied successfully and in total, more than 3000 proteins were identified, so far representing the most comprehensive OF proteome published. This new minimal invasive approach to access the bovine oviductal fluid proteome facilitates future innovative experimental designs to study the role of the oviductal micro environment during early embryo development.

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre-Fertilization Processes by Quantitative Proteomics.

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable-isotope dimethyl labeling prior to nanoLC-MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N-glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.

An oviduct-on-a-chip provides an enhanced in vitro environment for zygote genome reprogramming.

Worldwide over 5 million children have been conceived using assisted reproductive technology, and research has concentrated on increasing the likelihood of ongoing pregnancy. However, studies using animal models have indicated undesirable effects of in vitro embryo culture on offspring development and health. In vivo, the oviduct hosts a period in which the early embryo undergoes complete reprogramming of its (epi)genome in preparation for the reacquisition of (epi)genetic marks. We designed an oviduct-on-a-chip platform to better investigate the mechanisms related to (epi)genetic reprogramming and the degree to which they differ between in vitro and in vivo embryos. The device supports more physiological (in vivo-like) zygote genetic reprogramming than conventional IVF. This approach will be instrumental in identifying and investigating factors critical to fertilization and pre-implantation development, which could improve the quality and (epi)genetic integrity of IVF zygotes with likely relevance for early embryonic and later fetal development.

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.

Endoscopy-mediated intratubal insemination in the cow - Development of a novel minimally invasive AI technique.

Conventionally inseminated spermatozoa suffer a dramatic reduction in numbers during their long journey until fertilization. In addition sperm survival seems to be strongly affected by the reconstitution of the female reproductive tract in the post partum period. The purpose of this study was to develop a novel AI technique for cattle that allows the deposition of spermatozoa directly into the ampulla in the immediate vicinity of the fertilization site. This new reproductive biotechnique was investigated with focus on semen origin, sperm dosage, semen preparation and time of insemination. Finally, a first practical application was carried out by inseminating superovulated heifers with sex-sorted semen. In total, 49 Simmental heifers were used for 65 intratubal inseminations (ITI) with single ovulation and 8 ITIs after superovulation, respectively. Insemination into the oviduct was performed under epidural anesthesia via transvaginal endoscopy using a curved glass capillary loaded with semen. Two days later the oviduct and the adjacent uterine horn were endoscopically flushed and embryos or unfertilized oocytes were collected for determination of fertilization success. Across all experimental groups, tubal insemination successfully resulted in the collection of embryos; however, first tubal AI attempts and ITIs close to ovulation led to low recovery rates. In total, 109 complexes were flushed from ITIs in superstimulated heifers (n = 8) using sex sorted semen, of which 24 (22%) were at the embryo stage. In conclusion, it was shown that intratubal insemination can be successfully used for semen deposition, thus bypassing the lower female genital tract. Factors such as time of insemination, semen processing and semen quantity for superovulatory use should be further investigated.

Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos.

STUDY QUESTION: Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? SUMMARY ANSWER: There is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos. WHAT IS KNOWN ALREADY: CIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown. STUDY DESIGN, SIZE, DURATION: Because human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF). PARTICIPANTS/MATERIALS, SETTING, METHODS: Single blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos. MAIN RESULTS AND THE ROLE OF CHANCE: The genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P < 0.0001), and the frequency of whole chromosome or segmental aberrations was higher in embryos produced in vitro than in embryos derived in vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P < 0.01) and 84.6% of IVM-IVF embryos (P < 0.001). LARGE SCALE DATA: Genotyping data obtained in this study has been submitted to NCBI Gene Expression Omnibus (GEO; accession number GSE95358). LIMITATIONS REASONS FOR CAUTION: There were two main limitations of the study. First, animal models may not always reflect the nature of human embryogenesis, although the use of an animal model to investigate CIN was unavoidable in our study. Second, a limited number of embryos were obtained, therefore more studies are warranted to corroborate the findings. WIDER IMPLICATIONS OF THE FINDINGS: Although CIN is also present in in vivo-developed embryos, in vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be applied with care. Additionally, our results encourage to refine and improve in vitro culture conditions and assisted reproduction technologies. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Agency for Innovation by Science and Technology (IWT) (TBM-090878 to J.R.V. and T.V.), the Research Foundation Flanders (FWO; G.A093.11 N to T.V. and J.R.V. and G.0392.14 N to A.V.S. and J.R.V.), the European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, EU324509 to J.R.V., T.V., O.T, A.D., A.S. and A.K.) and Horizon 2020 innovation programme (WIDENLIFE, 692065 to J.R.V., O.T., T.V., A.K. and A.S.). M.Z.E., J.R.V. and T.V. are co-inventors on a patent application ZL913096-PCT/EP2014/068315-WO/2015/028576 ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies'), licensed to Cartagenia (Agilent Technologies).

Semen modulated secretory activity of oviductal epithelial cells is linked to cellular proteostasis network remodeling: Proteomic insights into the early phase of interaction in the oviduct in vivo.

The oviductal epithelium is crucial for the integrity of the female organ. Previously we got evidence that the surface proteome of oviductal epithelial cells (Oecs) is promptly altered in response to insemination and thus suggested that this early phase plays a notable regulatory role in maintaining cellular function. This study further aimed to assess the effect of semen on the cellular and molecular mechanisms in rabbit Oecs. A quantitative gel-based proteomic approach was applied to analyze changes at three time points (0h, 1h, 2h) after intrauterine insemination (IUI) compared to time matched controls. Within two hours the abundance of 22 protein species was evidently altered in the intracellular fraction. Functional analysis revealed that the proteins were primarily involved in proteostasis as well as metabolic processes. The analysis of phosphoproteins specified a role of mitogen-activated protein kinase (MAPK) signaling molecules. Concurrently, semen increased oviduct-specific glycoprotein (OVGP1) secretion. A correlation between OVGP1 abundance and microtubule-associated proteins 1A/1B-light chain 3 lipidation was observed. The localization and changes in abundance of selected proteins were corroborated by antibody-based methods. These results clearly show that the early phase of interaction acts as a trigger for cellular adaptation to meet an altered demand in the female organ. SIGNIFICANCE: The oviductal epithelium and its secreted proteins exert a pivotal role in reproductive processes, including the final maturation of male gametes. Thereby, the regulation and subsequently the functionality of the oviductal epithelial cell layer are important factors for the establishment of the appropriate milieu in the female reproductive tract. Notably, male gametes themselves have been shown to be an extrinsic modulatory factor of the oviductal epithelium. Accordingly a comprehensive knowledge about the underlying cellular and molecular mechanisms in the epithelial cells is of interest, also with regard to in vitro purposes. So far, the role of the early phase of interaction in the female organ has not been considered in detail. To get a further insight into the underlying cellular and molecular mechanisms, herein we analyzed the effect of semen on oviductal epithelial cells (Oecs) on the intracellular proteome level within the first two hours after insemination. The present study revealed a directed response of Oecs in vivo and disclosed intracellular pathways that are affected by the interplay between semen and the female reproductive tract. The prompt adaptation of the secretory activity and remodeling of the oviductal epithelium was accompanied by the concerted alterations of protein species that are primarily involved in the maintenance of cellular homeostasis. Besides emphasizing the importance of the early interaction phase for subsequent reproductive processes, the gained knowledge might further be implemented for in vitro applications as well.

Exploring the oviductal fluid proteome by a lectin-based affinity approach.

The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein, we assessed the combined use of Concanavalin A and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel-based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) further allowed the reliable identification of 134 nonbound as well as 130 lectin-bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin-bound fraction were known glycoproteins (p-value [FDR] = 1.45E-31). The low variance of the number of peptide spectrum matches for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3% for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin-based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF.

Lactation-induced changes in metabolic status and follicular-fluid metabolomic profile in postpartum dairy cows.

The aim was to investigate the effect of lactation on the composition of pre-ovulatory follicular fluid (FF). Forty in-calf primiparous heifers and 20 maiden heifers were enrolled. Immediately after calving, half of the cows were dried off while the remainder were milked twice daily. Serum samples were collected twice weekly from two weeks pre- to 84 days postpartum (dpp). FF was analysed by gas chromatography-mass spectrometry. Serum concentrations of non-esterified fatty acids and ß-hydroxybutyrate were higher, while glucose, insulin and Insulin-like growth factor 1 (IGF1) concentrations were lower in lactating cows compared with non-lactating cows and heifers (P<0.01). Principal component analysis of FF metabolites revealed a clear separation of the lactating group from both non-lactating cows and heifers. The amino acids tyrosine, phenylalanine and valine and fatty acids heneicosanoic acid and docosahexaenoic acid were all lower in FF from lactating compared with dry cows (P<0.05). FF from lactating cows was higher in aminoadipic acid, α-aminobutyric acid, glycine and serine while histidine, leucine, lysine, methionine and ornithine were all lower than in dry cows and heifers (P<0.05). The ratio of n6:n3 was higher in lactating cows compared with both non-lactating cows and heifers, whereas total n3 polyunsaturated fatty acids, pentadecanoic, linolenic, elaidic and arachidonic acids were all lower in the FF of lactating cows than both non-lactating cows and heifers (P<0.05). In conclusion, lactation induces distinct changes in the overall metabolic status of postpartum lactating dairy cows which are associated with divergent metabolite profiles in FF.

Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

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

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.

A proteomic approach to monitor the dynamic response of the female oviductal epithelial cell surface to male gametes.

Sophisticated strategies to analyze cell surface proteins are indispensable to study fundamental biological processes, such as the response of cells to environmental changes or cell-cell communication. Herein, we describe a refined mass spectrometry-based approach for the specific characterization and quantitation of cell surface proteins expressed in the female reproductive tract. The strategy is based on in situ biotinylation of rabbit oviducts, affinity enrichment of surface exposed biotin tagged proteins and dimethyl labeling of the obtained tryptic peptides followed by LC-MS/MS analysis. This approach proved to be sensitive enough to analyze small sample amounts (<1µg) and allowed further to trace the dynamic composition of the surface proteome of the oviductal epithelium in response to male gametes. The relative protein expression ratios of 175 proteins were quantified. Thirty-one of them were found to be altered over time, namely immediately, 1h and 2h after insemination compared to the time-matched control groups. Functional analysis demonstrated that structural reorganization of the oviductal epithelial cell surface was involved in the early response of the female organ to semen. In summary, this study outlines a workflow that is capable to monitor alterations in the female oviduct that are related to key reproductive processes in vivo. BIOLOGICAL SIGNIFICANCE: The proper interaction between the female reproductive tract, in particular, the oviduct and the male gametes, is fundamental to fertilization and embryonic development under physiological conditions. Thereby the oviductal epithelial cell surface proteins play an important role. Besides their direct interaction with male gametes, these molecules participate in signal transduction and, thus, are involved in the mandatory cellular response of the oviductal epithelium. In this study we present a refined LC-MS/MS based workflow that is capable to quantitatively analyze the expression of oviductal epithelial cell surface proteins in response to insemination in vivo. A special focus was on the very early interaction between the female organ and the male gametes. At first, this study clearly revealed an immediate response of the surface proteome to semen, which was modulated over time. The described methodology can be applied for studies of further distinct biological events in the oviduct and therefore contribute to a deeper insight into the formation of new life.

Transcriptome profile of bovine elongated conceptus obtained from SCNT and IVP pregnancies.

In the present study we analyzed the gene expression changes induced by somatic cell nuclear transfer (SCNT) and in vitro production (IVP) in bovine elongated embryos using Affymetrix bovine genome array. For this, Day-16 bovine embryos from SCNT, IVP, and artificial insemination (AI) were recovered from recipients and used for transcriptome analysis. Despite comparable in vivo development rates, considerable reduction in elongation size was observed in SCNT compared to non-cloned embryos (93.3 mm for SCNT vs. 186.6 mm and 196.3 mm for IVP and AI embryos, respectively). Gene expression analysis revealed that the transcript levels of 477 genes, which are involved in various pathways including arginine and proline or glycerolipid and fatty acid metabolism, were significantly altered in SCNT compared to AI embryos. Similarly, 365 genes were differentially expressed in IVP embryos compared to AI. Thus, several pathways including TNRF-1 signaling and tight junction pathways were affected. To predict whether the altered transcripts were associated with culture condition or errors in transcriptional reprogramming, unique or common differentially expressed genes were analyzed in SCNT and IVP embryos compared to AI or fibroblast donor cells. Accordingly, 71 transcripts were found to be not transcriptionally reprogrammed, as their expression resembled the donor cells more than AI embryos; the remaining transcripts were either partially or incompletely reprogrammed. In conclusion, the present study identified deviations in elongation size, gene expression, and the corresponding molecular pathways in Day-16 SCNT and IVP conceptuses compared to their AI counterparts, which may subsequently be associated with the outcome of fetal development.

Comparison of gel-based phosphoproteomic approaches to analyse scarce oviductal epithelial cell samples.

The reversible change of the phosphorylation state of proteins regulates key cellular processes. In the present study, three different gel-based approaches were compared with regard to their applicability to quantitatively analyse the phosphoproteome of scarce biological material obtained ex vivo. Our results show that the phosphoproteome characterisation of oviductal epithelial cells isolated from the female reproductive tract requires affinity enrichment and pre-electrophoretic labelling using fluorescence dyes. Using this approach, 30 µg of enriched phosphoproteins proved to be sufficient for the phosphoproteome characterisation. In contrast, sequential fluorescence staining of 2D-separated total cell lysates as well as sequential staining in conjunction with a pre-enrichment step led to detection discrepancies and excluded further analysis steps. Information gained from this study provides a successful approach for the phosphoproteome analysis of scarce samples. In addition, the cellular processes taking place in the female reproductive tract can be monitored ex vivo.

Molecular mechanisms and pathways involved in bovine embryonic genome activation and their regulation by alternative in vivo and in vitro culture conditions.

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 EmbryoGENE's 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.

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity.

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.