Suppl. Figure 1. (A) Immunostaining for ADNP and β-Catenin on serial sections of two colon cancers. (B) Staining intensities of ADNP in colon cancer cells (n = 500, 5 different tumors) with high or low nuclear β- Catenin expression. P value is t test result. (C) Immunostaining for ADNP in normal mucosa. Right panels aremagnifications of areas boxed in left panel.Scale bars, 100 μm. Suppl. Figure 2. (A-B) Immunoblotting of indicated proteins on whole cell lysates in ADNP wild-type (WT) or knockout (KO) cell lines. Numbers below immunoblots indicate fold change by densitometry. (C) Representative micrographs (left panels) and quantification (right panels) of migrated or invaded tumor cells in transwell assays for indicated ADNP WT and KO cell lines, treated with indicated siRNAs or control siRNA (siCtrl). (D) Representative proliferation kinetics based on cell quantification by impedance measurements. Data are mean, n {greater than or equal to} 3. β-Cat = β-Catenin. Suppl. Figure 3. Representative micrographs (left panels) and quantification (right panels) of migrated or invaded HCT116 or SW1222 tumor cells with transient ADNP overexpression by transfection with p4.2-hADNP compared to empty p4.2 vector for 24 h in transwell assays. Suppl. Figure 4. (A) Immunoblotting of indicated proteins on whole cell lysates of SW1222, HCT116 and primary (P-Tu) colon cancer cells. Numbers below immunoblots indicate fold change by densitometry. (B) Immunostaining of SW1222, HCT116 and primary colon cancer (P-Tu) xenografts for β-Catenin. Scale bars, 50 μm. Suppl. Figure 5. (A-B) Dual-luciferase assays for indicatedADNPwild-type (WT) or knockout (KO) colon cancer cells transfected with TOPflash reporter constructs. Treatments with PBS, ketamine or the WNT inhibitor XAV939, as indicated. (C) Representativemicrographs (left panels) and quantification (right panels) ofmigrated or invaded HCT116 or SW1222 tumor cells in transwell assays, treated with PBS or 100 μM ketamine for 24 h, as indicated.Data aremean {plus minus}SD, Pvalues are t test results, n {greater than or equal to} 3. Suppl. Figure 6. Assessment of nuclear β-catenin staining in primary human colorectal cancers. Tumors were assigned scores from 0 (no nuclear β-catenin) to 3 (most tumor cells with strong nuclear β-catenin) and accordingly categorized as β-catenin low (score 0-1) and β-catenin high (score 2-3). Scale bars, 100 μm.

Supplementary Tables 1-7 Supplementary Materials and Methods Suppl. Table 1. WNT target gene sets used for GSEA Suppl. Table 2. Gene Expression (real-time RT-PCR) primers used in this study. Primers were selected from the Universal Probe Library (Roche). Suppl. Table 3. Antibodies used for immunoblotting and immunohistochemistry. Suppl. Table 4. Fold change (F.C.) of consistently deregulated genes in three gene expression data sets of colon cancer cells with high vs. low WNT activity, and F.C. in differential expression of these genes in TCGA data in colon cancer vs. normal mucosa. P values are t test results. Suppl. Table 5. Proteome analysis results upon ADNP depletion. Proteins with significant up- or downregulation are listed (P < 0.05; abs. fold change >1.5) Suppl. Table 6. Clinical/pathological data and ADNP expression in colorectal cancer. Suppl. Table 7. Multivariate analysis of cancer specific survival.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal muscles and heart. Animal models are essential for preclinical evaluation of novel diagnostic procedures and treatment strategies. Gene targeting/editing offers the possibility of developing tailored pig models for monogenic diseases. The first porcine DMD model was generated by deletion of DMD exon 52 (DMDΔ52) in cultured kidney cells, which were used for somatic cell nuclear transfer to produce DMDΔ52 offspring. The animals resembled clinical, biochemical, and pathological hallmarks of DMD, but died before sexual maturity, thus preventing their propagation by breeding. This limitation was overcome by the generation of female heterozygous DMDΔ52 carrier pigs, which allowed the establishment of a large breeding colony. In this overview, we summarize how porcine DMD models have been used for dissecting disease mechanisms, for validating multispectral optoacoustic tomography as an imaging modality for monitoring fibrosis, and for preclinical testing of a CRISPR/Cas9 based approach to restore an intact DMD reading frame. Particular advantages of porcine DMD models include their targeted design and the rapid disease progression with early cardiac involvement, facilitating translational studies in reasonable time frames.

Adipose tissue (AT) is no longer considered to be responsible for energy storage only but is now recognized as a major endocrine organ that is distributed across different parts of the body and is actively involved in regulatory processes controlling energy homeostasis. Moreover, AT plays a crucial role in the development of metabolic disease such as diabetes. Recent evidence has shown that adipokines have the ability to regulate blood glucose levels and improve metabolic homeostasis. While AT has been studied extensively in the context of type 2 diabetes, less is known about how different AT types are affected by absolute insulin deficiency in type 1 or permanent neonatal diabetes mellitus. Here, we analyzed visceral and subcutaneous AT in a diabetic, insulin-deficient pig model (MIDY) and wild-type (WT) littermate controls by RNA sequencing and quantitative proteomics. Multi-omics analysis indicates a depot-specific dysregulation of crucial metabolic pathways in MIDY AT samples. We identified key proteins involved in glucose uptake and downstream signaling, lipogenesis, lipolysis and β-oxidation to be differentially regulated between visceral and subcutaneous AT in response to insulin deficiency. Proteins related to glycogenolysis, pyruvate metabolism, TCA cycle and lipogenesis were increased in subcutaneous AT, whereas β-oxidation-related proteins were increased in visceral AT from MIDY pigs, pointing at a regionally different metabolic adaptation to master energy stress arising from diminished glucose utilization in MIDY AT. Chronic, absolute insulin deficiency and hyperglycemia revealed fat depot-specific signatures using multi-omics analysis. The generated datasets are a valuable resource for further comparative and translational studies in clinical diabetes research.

Aging of human testis and associated cellular changes is difficult to assess. Therefore, we used a translational, non-human primate model to get insights into underlying cellular and biochemical processes. Using proteomics and immunohistochemistry, we analyzed testicular tissue of young (age 2 to 3) and old (age 10 to 12) common marmosets (Callithrix jacchus). Using a mass spectrometry-based proteomics approach, we identified 63,124 peptides, which could be assigned to 5924 proteins. Among them, we found proteins specific for germ cells and somatic cells, such as Leydig and Sertoli cells. Quantitative analysis showed 31 differentially abundant proteins, of which 29 proteins were more abundant in older animals. An increased abundance of anti-proliferative proteins, among them CDKN2A, indicate reduced cell proliferation in old testes. Additionally, an increased abundance of several small leucine rich repeat proteoglycans and other extracellular matrix proteins was observed, which may be related to impaired cell migration and fibrotic events. Furthermore, an increased abundance of proteins with inhibitory roles in smooth muscle cell contraction like CNN1 indicates functional alterations in testicular peritubular cells and may mirror a reduced capacity of these cells to contract in old testes.

In the past years, the research focus on the effects of MP on aquatic organisms extended from marine systems towards freshwater systems. An important freshwater model organism in the MP field is the cladoceran Daphnia, which plays a central role in lacustrine ecosystems and has been established as a test organism in ecotoxicology. To investigate the effects of MP on Daphnia magna, we performed a chronic exposure experiment with polystyrene MP under strictly standardized conditions. Chronic exposure of D. magna to PS microparticles led to a significant reduction in body length and number of offspring. To shed light on underlying molecular mechanisms induced by microplastic ingestion in D. magna, we assessed the effects of PS-MP at the proteomic level, as proteins, e.g., enzymes, are especially relevant for an organism's physiology. Using a state-of-the-art mass spectrometry based approach, we were able to identify 28,696 different peptides, which could be assigned to 3784 different proteins. Using a customized bioinformatic workflow, we identified 41 proteins significantly altered in abundance (q-value <0.05) in the PS exposed D. magna. Among the proteins increased in the PS treated group were several sulfotransferases, involved in basic biochemical pathways, as well as GABA transaminase catalyzing the degradation of the neurotransmitter GABA. In the abundance decreased group, we found essential proteins such as the DNA-directed RNA polymerase subunit and other proteins connected to biotic and inorganic stress and reproduction. Strikingly, we further identified several digestive enzymes that are significantly downregulated in the PS treated animals, which could have interfered with the affected animal's nutrient supply. This may explain the altered morphological and life history traits of the PS exposed daphnids. Our results indicate that long-term exposure to PS microplastics, which are frequently detected in environmental samples, may affect the fitness of daphnids.

Background Many genetically multi-modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno-organs may impair their long-term function after orthotopic transplantation in non-human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR-KO) to reduce the size of donor pigs in one step. Methods Heart weight and proteome profile of myocardium were investigated in GHR-KO and control pigs. GHR-KO mutations were introduced using CRISPR/Cas9 in an alpha 1,3-galactosyltransferase (GGTA1)-deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple-modified (4GM) pigs. Results At age 6 months, GHR-KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin-like growth factor 1 (IGF1) levels (24 +/- 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 +/- 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi-modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes. Conclusion GHR-KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno-organ overgrowth in preclinical studies.

Large animal models for Duchenne muscular dystrophy (DMD) are crucial for preclinical evaluation of novel diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 ( DMD Δ52) resemble molecular, clinical and pathological hallmarks of DMD, but cannot be propagated by breeding due to death before sexual maturity. Therefore, female DMD +/- carriers were generated. A single founder animal had 11 litters with 29 DMD Y/- , 34 DMD +/- as well as 36 male and 29 female wild-type (WT) offspring. Breeding with F1 and F2 DMD +/- carriers resulted in additional 114 DMD Y/- piglets. The majority of them survived for 3-4 months, providing large cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance of human disease mechanisms. Importantly, DMD Y/- pigs reveal progressive fibrosis of myocardium and increased expression of connexin-43, associated with significantly reduced left ventricular fractional shortening and ejection fraction already at age 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability of DMD Y/- pigs. Our breeding cohort of DMD Δ52 pigs and standardized tissue repositories from DMD Y/- pigs, DMD +/- carriers, and WT littermate controls provide important resources for studying DMD disease mechanisms and for testing novel diagnostic procedures and treatment strategies.

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC-MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins was identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition - and hence independent of transcription - were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell-cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.

Sorafenib represents the current standard of care for patients with advanced-stage hepatocellular carcinoma (HCC). However, acquired drug resistance occurs frequently during therapy and is accompanied by rapid tumor regrowth after sorafenib therapy termination. To identify the mechanism of this therapy-limiting growth resumption, we established robust sorafenib resistance HCC cell models that exhibited mitochondrial dysfunction and chemotherapeutic crossresistance. We found a rapid relapse of tumor cell proliferation after sorafenib withdrawal, which was caused by renewal of mitochondrial structures alongside a metabolic switch toward high electron transport system (ETS) activity. The translation-inhibiting antibiotic tigecycline impaired the biogenesis of mitochondrial DNA-encoded ETS subunits and limited the electron acceptor turnover required for glutamine oxidation. Thereby, tigecycline prevented the tumor relapse in vitro and in murine xenografts in vivo. These results offer a promising second-line therapeutic approach for advanced-stage HCC patients with progressive disease undergoing sorafenib therapy or treatment interruption due to severe adverse events.

Age-related changes in the human testis may include morphological alterations, disturbed steroidogenesis, and impaired spermatogenesis. However, the specific impact of cell age remains poorly understood and difficult to assess. Testicular peritubular cells fulfill essential functions, including sperm transport, contributions to the spermatogonial stem cell niche, and paracrine interactions within the testis. To study their role in age-associated decline of testicular functions, we performed comprehensive proteome and secretome analyses of repeatedly passaged peritubular cells from Callithrix jacchus. This nonhuman primate model better reflects the human testicular biology than rodents and further gives access to young donors unavailable from humans. Among 5095 identified proteins, 583 were differentially abundant between samples with low and high passage numbers. The alterations indicate a reduced ability of senescent peritubular cells to contract and secrete proteins, as well as disturbances in nuclear factor (NF)-κB signaling and a reduced capacity to handle reactive oxygen species. Since this in vitro model may not exactly mirror all molecular aspects of in vivo aging, we investigated the proteomes and secretomes of testicular peritubular cells from young and old donors. Even though the age-related alterations at the protein level were less pronounced, we found evidence for impaired protein secretion, altered NF-κB signaling, and reduced contractility of these in vivo aged peritubular cells.

Testicular peritubular cells (TPCs) are smooth muscle-like cells, which form a compartment surrounding the seminiferous tubules. Previous studies employing isolated human testicular peritubular cells (HTPCs) indicated that their roles in the testis go beyond sperm transport and include paracrine and immunological contributions. Peritubular cells from a non-human primate (MKTPCs), the common marmoset monkey, Callithrix jacchus, share a high degree of homology with HTPCs. However, like their human counterparts these cells age in vitro and replicative senescence limits in-depth functional or mechanistic studies. Therefore, a stable cellular model was established. MKTPCs of a young adult animal were immortalized by piggyBac transposition of human telomerase (hTERT), that is, without the expression of viral oncogenes. Immortalized MKTPCs (iMKTPCs) grew without discernable changes for more than 50 passages. An initial characterization revealed typical genes expressed by peritubular cells (androgen receptor (AR), smooth-muscle actin (ACTA2), calponin (CNN1)). A proteome analysis of the primary MKTPCs and the derived immortalized cell line confirmed that the cells almost completely retained their phenotype. To test whether they respond in a similar way as HTPCs, iMKTPCs were challenged with forskolin (FSK) and ATP. As HTPCs, they showed increased expression level of the StAR protein (StAR) after FSK stimulation, indicating steroidogenic capacity. ATP increased the expression of pro-inflammatory factors (e.g. IL1B; CCL7), as it is the case in HTPCs. Finally, we confirmed that iMKTPCs can efficiently be transfected. Therefore, they represent a highly relevant translational model, which allows mechanistic studies for further exploration of the roles of testicular peritubular cells.

RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site - contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

The global prevalence of diabetes mellitus and other metabolic diseases is rapidly increasing. Animal models play pivotal roles in unravelling disease mechanisms and developing and testing therapeutic strategies. Rodents are the most widely used animal models but may have limitations in their resemblance to human disease mechanisms and phenotypes. Findings in rodent models are consequently often difficult to extrapolate to human clinical trials. To overcome this 'translational gap', we and other groups are developing porcine disease models. Pigs share many anatomical and physiological traits with humans and thus hold great promise as translational animal models. Importantly, the toolbox for genetic engineering of pigs is rapidly expanding. Human disease mechanisms and targets can therefore be reproduced in pigs on a molecular level, resulting in precise and predictive porcine (PPP) models. In this short review, we summarize our work on the development of genetically (pre)diabetic pig models and how they have been used to study disease mechanisms and test therapeutic strategies. This includes the generation of reporter pigs for studying beta-cell maturation and physiology. Furthermore, genetically engineered pigs are promising donors of pancreatic islets for xenotransplantation. In summary, genetically tailored pig models have become an important link in the chain of translational diabetes and metabolic research.

Objective: The liver is a central target organ of growth hormone (GH), which stimulates the synthesis of insulin-like growth factor 1 (IGF1) and affects multiple biochemical pathways. A systematic multi-omics analysis of GH effects in the liver has not been performed. GH receptor (GHR) deficiency is a unique model for studying the consequences of lacking GH action. In this study, we used molecular profiling techniques to capture a broad spectrum of these effects in the liver of a clinically relevant large animal model for Laron syndrome. Methods: We performed holistic proteome and targeted metabolome analyses of liver samples from 6-month-old GHR-deficient (GHR-KO) pigs and GHR-expressing controls (four males, four females per group). Results: GHR deficiency resulted in an increased abundance of enzymes involved in amino acid degradation, in the urea cycle, and in the tricarboxylic acid cycle. A decreased ratio of long-chain acylcarnitines to free carnitine suggested reduced activity of carnitine palmitoyltransferase 1A and thus reduced mitochondrial import of fatty acids for beta-oxidation. Increased levels of short-chain acylcarnitines in the liver and in the circulation of GHR-KO pigs may result from impaired beta-oxidation of short-chain fatty acids or from increased degradation of specific amino acids. The concentration of mono-unsaturated glycerophosphocholines was significantly increased in the liver of GHR-KO pigs without morphological signs of steatosis, although the abundances of several proteins functionally linked to non-alcoholic fatty liver disease (fetuin B, retinol binding protein 4, several mitochondrial proteins) were increased. Moreover, GHR-deficient liver samples revealed distinct changes in the methionine and glutathione metabolic pathways, in particular, a significantly increased level of glycine N-methyltransferase and increased levels of total and free glutathione. Several proteins revealed a sex-related abundance difference in the control group but not in the GHR-KO group. Conclusions: Our integrated proteomics/targeted metabolomics study of GHR-deficient and control liver samples from a clinically relevant large animal model identified a spectrum of biological pathways that are significantly altered in the absence of GH action. Moreover, new insights into the role of GH in the sex-related specification of liver functions were provided.

Over the last decades, fertility of dairy cows has declined due to selection strategies focusing on milk yield. To study the effect of genetic merit for fertility on the proteome of the bovine uterine luminal fluid, Holstein heifers with low- and two groups of heifers with high-fertility index (high-fertility Holstein and Montbeliarde) were investigated. To focus on the maternal effect, heifers from all groups were synchronized and received on Day 7 high-quality embryos. Uterine luminal fluid from Day 19 pregnant heifers was analyzed in a holistic proteomic approach using nano-LC-MS/MS analysis combined with a label-free quantification approach. In total, 1737 proteins were identified, of which 597 differed significantly in abundance between the three groups. The vast majority of proteome differences was found comparing both high-fertility groups to the low-fertility Holstein group, showing that the genetic predisposition for fertility is prevalent regarding the uterine luminal fluid proteome. Evaluation of this dataset using bioinformatic tools revealed an assignment of higher abundant proteins in low-fertility Holstein to several metabolic processes, such as vitamin metabolic process, which comprises folate receptor alpha (FOLR1) and retinol-binding protein, indicating an involvement of disturbed metabolic processes in decreased fertility. Moreover, immune system-related proteins - lactotransferrin and chromogranin A - were enriched in low-fertility cows together with interferon tau 3 h and interferon tau-2. Our results indicate that the genetic merit for fertility leads to substantial quantitative differences at the level of proteins in uterine fluid of pregnant animals, thus altering the microenvironment for the early conceptus. The proteome of bovine uterine fluid, which is crucial for conceptus development prior to implantation, is significantly altered by the genetic merit for fertility.

The corpus luteum (CL) is a transient endocrine organ, yet molecular mechanisms resulting in its demise are not well known. The presence of phosphorylated mixed lineage kinase domain-like pseudokinase pMLKL(T357/S358) in human and nonhuman primate CL samples ( Macaca mulatta and Callithrix jacchus ) implied that necroptosis of luteal cells may be involved. In M. mulatta CL, pMLKL positive staining became detectable only from the mid-late luteal phase onwards, pointing to necroptosis during regression of the CL. Cell death, including necroptosis, was previously observed in cultures of human luteal granulosa cells (GCs), an apt model for the study of the human CL. To explore mechanisms of necroptotic cell death in GCs during culture, we performed a proteomic analysis. The levels of 50 proteins were significantly altered after 5 days of culture. Interconnectivity analysis and immunocytochemistry implicated specifically the ceramide salvage pathway to be enhanced. M. mulatta CL transcriptome analysis indicated in vivo relevance. Perturbing endogenous ceramide generation by fumonisin B1 (FB1) and addition of soluble ceramide (C2-CER) yielded opposite actions on viability of GCs and therefore supported the significance of the ceramide pathway. Morphological changes indicated necrotic cell death in the C2-CER treated group. Studies with the pan caspase blocker zVAD-fmk or the necroptosis blocker necrosulfonamid (NSA) further supported that C2-CER induced necroptosis. Our data pinpoint necroptosis in a physiological process, namely CL regression. This raises the possibility that the primate CL could be rescued by pharmacological inhibition of necroptosis or by interaction with ceramide metabolism.

The early developing embryo faces a continuously changing microenvironment to supports its growth. In the European roe deer, this environment accompanies embryonic diapause, a period of up to 4 months in which fertilization and subsequent implantation are decoupled. Diapause is characterised by a deceleration of embryonic growth. In most ruminants such as cattle and sheep, interferon tau (IFNt) plays a major role in maternal recognition of pregnancy. Uniquely to ruminants, the roe deer embryo does not secrete IFNt. The roe deer was used as a model species to gain insights into the changing uterine environment devoid of IFNt that supports prolonged decelerated embryo development, resumption of developmental velocity, and subsequent implantation. Uterine fluid samples from 188 female does were collected during regular huntings between September and January, and 4 developmental stages-blastocysts at early, mid, and late diapause and elongated embryos (16, 57, 97, and 18 does per developmental stage, respectively)-were defined. The developmental stages were assigned based on morphological characteristics of the embryo and the embryonic genomic DNA content. For the analysis of amino acids (AA), all 188 uterine fluid samples were subjected to targeted liquid chromatography-tandem mass spectrometry. Almost all AA increased over the course of embryo development. Although most AA showed developmental stage-specific concentration peaks, serine, glycine, alanine, glutamate, and glutamine were most abundantly present irrespective of the developmental progression. For the analysis of the protein abundances in the uterine fluid in a selected subset of samples (n=5 per developmental stage), holistic liquid chromatography-tandem mass spectrometry identified and quantified a total of 819 proteins with a false discovery rate of <1%. Comparison between the developmental stages revealed 106 differentially abundant proteins. Most changes in protein abundance that occurred related to embryo elongation. Interestingly, 713 proteins remained stable during embryo development, indicating that these proteins may contribute to prolonged embryo survival during embryonic diapause. The differentially abundant proteins were clustered with DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). The most enriched clusters were cell-cell adhesion, biosynthesis of AA and carbon metabolism, microtubule, structural molecule activity, and chaperone binding. The ongoing detailed identification of stably abundant proteins will advance our basic understanding of the embryos’ needs for sustained survival during prolonged decelerated development. In addition, a comparison with the protein abundances around the time of maternal recognition of pregnancy in other species could advance our knowledge on conserved proteins that support embryo development and establishment of pregnancy in mammals. Our findings may contribute to defining optimal in vitro embryo culture conditions in a species-independent manner and potentially identify factors capable of halting embryo development.

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. (C) 2019 Elsevier Inc. All rights reserved.

OBJECTIVE:The liver regulates the availability of insulin to other tissues and is the first line insulin response organ physiologically exposed to higher insulin concentrations than the periphery. Basal insulin during fasting inhibits hepatic gluconeogenesis and glycogenolysis, whereas postprandial insulin peaks stimulate glycogen synthesis. The molecular consequences of chronic insulin deficiency for the liver have not been studied systematically. METHODS:We analyzed liver samples of a genetically diabetic pig model (MIDY) and of wild-type (WT) littermate controls by RNA sequencing, proteomics, and targeted metabolomics/lipidomics. RESULTS:Cross-omics analyses revealed increased activities in amino acid metabolism, oxidation of fatty acids, ketogenesis, and gluconeogenesis in the MIDY samples. In particular, the concentrations of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and of retinol dehydrogenase 16 (RDH16), which catalyzes the first step in retinoic acid biogenesis, were highly increased. Accordingly, elevated levels of retinoic acid, which stimulates the expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK1), were measured in the MIDY samples. In contrast, pathways related to extracellular matrix and inflammation/pathogen defense response were less active than in the WT samples. CONCLUSIONS:The first multi-omics study of a clinically relevant diabetic large animal model revealed molecular signatures and key drivers of functional alterations of the liver in insulin-deficient diabetes mellitus. The multi-omics data set provides a valuable resource for comparative analyses with other experimental or clinical data sets.