Developmental progression continues during embryonic diapause in the roe deer.

Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.

Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching.

Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5ß-THF, 3ß,5α-THF, 3ß,5ß-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5ß-THE, 3ß,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20ß-DHP, 3α-DHP, 3ß-DHP, 5α-DHP, 5ß-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5ß-THP, 3ß,5α-THP, 3ß,5ß-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 µL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL-1 was achieved for all steroids suitable for the analysis of authentic samples.

One-week storage of refrigerated bovine milk does not affect the size, concentration, or molecular properties of extracellular vesicles.

Milk extracellular vesicles (EV) have gained extensive attention as promising diagnostic and therapeutic tools. Pre-analytical raw milk storage at low temperatures is an ordinary and usually necessary step after sample collection. It is known that direct freezing of unprocessed whole milk contaminates the native pool of milk EV with other cell structures. However, less evidence is available regarding prolonged cooling at 4°C. The current study assessed whether pre-analytical storage of bovine raw milk for several days affected EV isolation and further analysis. To confirm the independence from the health status of the mammary gland, we analyzed milk samples stored at 4°C for 1, 2, 3, and 7 d past collection, respectively, from 2 quarters of the same cow with different somatic cell counts (SCC). Seven days of refrigeration did not change the milk EV size, concentration, or morphology. We did not detect any changes in the EV cargo regarding the amount of protein and RNA, nor in the specific EV markers TSG101, CD9, and CD81 in milk from quarters with high and low SCC. Overall, we observed fewer CD81 and CD9 markers in quarters with high SCC. Moreover, we found no reduction in the mastitis-related miRNA bta-miR-223-3p, suggesting that refrigeration for several days up to 1 wk is a possible storage option compatible with further EV analyses. The findings of this study enhance the confidence that milk EV are highly stable in the raw milk matrix.

Transcriptomic signature of luteinized cumulus cells of oocytes developing to live birth after women received intracytoplasmic sperm injection.

OBJECTIVE: To compare the transcriptome of human cumulus cells (CCs) from oocytes with different outcomes (pregnancy yes/no, live birth [LB] yes/no), to identify noninvasive biomarkers for oocyte selection as well as new therapeutic targets to increase LB rates from assisted reproductive technologies (ART). DESIGN: Retrospective observational study. SETTINGS: This study was conducted at a University Hospital in Switzerland. PATIENTS: Subfertile couples undergoing controlled ovarian superstimulation and intracytoplasmic sperm injection with subsequent unbiopsied embryo transfer below the female age of 43 years. INTERVENTION(S): RNA sequencing of CCs from oocytes results in a pregnancy, no pregnancy, LB, or no LB. MAIN OUTCOME MEASURES: Differential gene expression (DEG) between CCs of oocytes results in "no pregnancy" vs. "pregnancy" and "pregnancy only" vs. "live birth." RESULTS: Although RNA sequencing did not reveal DEGs when comparing the transcriptomic profiles of the groups "no pregnancy" with "pregnancy," we identified 139 DEGs by comparing "pregnancy only" with "live birth," of which 28 belonged to clusters relevant to successful ART outcomes (i.e., CTGF, SERPINE2, PCK1, HHIP, HS3ST, and BIRC5). A functional enrichment analysis revealed that the transcriptome of CCs associated with LB depicts pathways of extracellular matrix, inflammatory cascades leading to ovulation, cell patterning, proliferation, and differentiation, and silencing pathways leading to apoptosis. CONCLUSION: We identified a CCs transcriptomic profile associated with LB after embryo transfer that, after further validation, could serve to predict successful ART outcomes. The definition of relevant pathways of CCs related to oocyte competency contributes to a broader understanding of the cumulus oocyte complex and helps identify further therapeutic targets for improving ART success.

Review: Embryonic diapause in the European roe deer - slowed, but not stopped.

Embryonic diapause in mammals describes a transient reduction of proliferation and developmental progression occurring at the blastocyst stage. It was first described in the European roe deer (Capreolus capreolus) in the 19th century, and later found to occur in at least over 130 mammalian species across several taxa. Diapause is often displayed as an interruption, a halt, or an arrest of embryonic development. In this review, we explore reduced, but not stopped pace of growth, proliferation and developmental progression during embryonic diapause and revisit early embryonic proliferation and continued slow development as peculiar phenomenon in the roe deer.

Circadian Rhythm Does Not Affect the miRNA Cargo of Bovine Raw Milk Extracellular Vesicles.

Extracellular vesicles (EVs) and their microRNA (miRNA) cargo have been proposed as possible mammary gland health biomarkers in cattle. However, throughout the day, the biologically active milk components, such as miRNAs, may change due to the dynamic nature of milk. The current study aimed to evaluate the circadian fluctuation of milk EVs miRNA cargo to assess the feasibility of milk EVs as future biomarkers for mammary gland health management. Milk from four healthy dairy cows was collected for four consecutive days in the two daily milking sessions in the morning and the evening. The isolated EVs were heterogeneous, intact, and carried the EV protein markers CD9, CD81, and TSG101, as shown by transmission electron microscopy and western blot. The miRNA sequencing results demonstrate that the abundance of miRNA cargo in milk EVs remained stable, unlike other milk components, such as somatic cells, that changed during milking sessions. These findings indicated that the miRNA cargo within milk EVs remains stable irrespective of the time of day, suggesting their potential utility as diagnostic markers for mammary gland health.

Machine learning analysis of humoral and cellular responses to SARS-CoV-2 infection in young adults.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces B and T cell responses, contributing to virus neutralization. In a cohort of 2,911 young adults, we identified 65 individuals who had an asymptomatic or mildly symptomatic SARS-CoV-2 infection and characterized their humoral and T cell responses to the Spike (S), Nucleocapsid (N) and Membrane (M) proteins. We found that previous infection induced CD4 T cells that vigorously responded to pools of peptides derived from the S and N proteins. By using statistical and machine learning models, we observed that the T cell response highly correlated with a compound titer of antibodies against the Receptor Binding Domain (RBD), S and N. However, while serum antibodies decayed over time, the cellular phenotype of these individuals remained stable over four months. Our computational analysis demonstrates that in young adults, asymptomatic and paucisymptomatic SARS-CoV-2 infections can induce robust and long-lasting CD4 T cell responses that exhibit slower decays than antibody titers. These observations imply that next-generation COVID-19 vaccines should be designed to induce stronger cellular responses to sustain the generation of potent neutralizing antibodies.

Reconsidering "low-dose"-Impacts of oral estrogen exposure during preimplantation embryo development.

Perturbations of estrogen signaling during developmental stages of high plasticity may lead to adverse effects later in life. Endocrine-disrupting chemicals (EDC) are compounds that interfere with the endocrine system by particularly mimicking the action of endogenous estrogens as functional agonists or antagonists. EDCs compose synthetic and naturally occurring compounds discharged into the environment, which may be taken up via skin contact, inhalation, orally due to contaminated food or water, or via the placenta during in utero development. Although estrogens are efficiently metabolized by the liver, the role of circulating glucuro- and/or sulpho-conjugated estrogen metabolites in the body has not been fully addressed to date. Particularly, the role of intracellular cleavage to free functional estrogens could explain the hitherto unknown mode of action of adverse effects of EDC at very low concentrations currently considered safe. We summarize and discuss findings on estrogenic EDC with a focus on early embryonic development to highlight the need for reconsidering low dose effects of EDC.

Endometrial extracellular matrix components do not change over the course of embryonic diapause and reactivation in the roe deer (Capreolus capreolus).

The modification of the endometrial extracellular matrix (ECM) is a crucial step for embryo implantation in many mammalian species. The embryo of the European roe deer (Capreolus capreolus) displays a 4-5 months long temporary reduction of developmental pace termed embryonic diapause. A reduction of epithelial cell height during diapause has previously been described. Co-occurring ECM modifications may contribute to the changes of the intra-uterine milieu during reactivation at which the embryo regains developmental velocity. We assessed the localization of five ECM proteins (collagen I and IV, fibronectin, laminin, and extracellular matrix protein 1) using immunohistochemistry in animals with early, late, and post-diapause (elongating) embryos. While our results confirmed the reduction of epithelial height during diapause, we only detected marginal differences in localization and staining intensities of the selected ECM proteins. Major ECM remodelling events in the roe deer endometrium are thus likely to occur only at implantation.

Novel role for conceptus signals in mRNA expression regulation by DNA methylation in porcine endometrium during early pregnancy†.

During early pregnancy, porcine conceptuses (the embryos with associated membranes) secrete estradiol-17ß (E2)-their major signal for maternal recognition of pregnancy-and prostaglandin E2 (PGE2). Both hormones induce prominent changes of the endometrial transcriptome in vivo. Studies on endometrial pathologies have shown that E2 affects gene expression by epigenetic mechanisms related to DNA methylation. Herein, we determined the effects of E2 and PGE2 alone, and a combined E2 + PGE2 treatment administered into the uterine lumen in vivo on the expression and activity of DNA-methyltransferases (DNMTs) and on CpG methylation patterns of selected genes in porcine endometrium. To compare the effect of treatment with the physiological effect of pregnancy, endometria from day 12 pregnant/cyclic gilts were included. Both E2 and PGE2 significantly reduced the expression of DNMTs. Likewise, the expressions of DNMT1 and DNMT3A were decreased on day 12 of pregnancy compared to the estrous cycle. DNMT activity increased in endometrial samples following E2 treatment and in gilts on day 12 of pregnancy. Treatment with E2 alone and/or simultaneously with PGE2 altered endometrial DNA methylation of CpG sites of ADAMTS20, ADH1C, BGN, PSAT1, and WNT5A. Different CpG methylation patterns of ADAMTS20, BGN, DMBT1, RASSF1, and WNT5A were found in the endometrium on day 12 of pregnancy compared to day 12 of the estrous cycle. Significant correlations were detected between CpG methylation and gene expression for ADAMTS20, ADH1C, BGN, DMBT1, PSAT1, and WNT5A. Our results indicate that CpG methylation induced by embryonic signals may contribute to regulating endometrial gene expression during pregnancy establishment.

Luminal and Glandular Epithelial Cells from the Porcine Endometrium maintain Cell Type-Specific Marker Gene Expression in Air-Liquid Interface Culture.

Two different types of epithelial cells constitute the inner surface of the endometrium. While luminal epithelial cells line the uterine cavity and build the embryo-maternal contact zone, glandular epithelial cells form tubular glands reaching deeply into the endometrial stroma. To facilitate investigations considering the functional and molecular differences between the two populations of epithelial cells and their contribution to reproductive processes, we aimed at establishing differentiated in vitro models of both the luminal and the glandular epithelium of the porcine endometrium using an air-liquid interface (ALI) approach. We first tested if porcine luminal endometrium epithelial cells (PEEC-L) reproducibly form differentiated epithelial monolayers under ALI conditions by monitoring the morphology and the trans-epithelial electrical resistance (TEER). Subsequently, luminal (PEEC-L) and glandular epithelial cells (PEEC-G) were consecutively isolated from the endometrium of the uterine horn. Both cell types were characterized by marker gene expression analysis immediately after isolation. Cells were separately grown at the ALI and assessed by means of histomorphometry, TEER, and marker gene expression after 3 weeks of culture. PEEC-L and PEEC-G formed polarized monolayers of differentiated epithelial cells with a moderate TEER and in vivo-like morphology at the ALI. They exhibited distinct patterns of functional and cell type-specific marker gene expression after isolation and largely maintained these patterns during the culture period. The here presented cell culture procedure for PEEC-L and -G offers new opportunities to study the impact of embryonic signals, endocrine effectors, and reproductive toxins on both porcine endometrial epithelial cell types under standardized in vitro conditions. Created with BioRender.com .

Inflammatory Response of Primary Cultured Bovine Mammary Epithelial Cells to Staphylococcus aureus Extracellular Vesicles.

In dairy cows, Staphylococcus aureus (S. aureus) is among the most prevalent microorganisms worldwide, causing mastitis, an inflammation of the mammary gland. Production of extracellular vesicles (EVs) is a common feature of S. aureus strains, which contributes to its pathogenesis by delivering bacterial effector molecules to host cells. In the current study, we evaluated the differences between five S. aureus mastitis isolates regarding their EV production. We found that different mastitis-related S. aureus strains differ in their behaviour of shedding EVs, with M5512VL producing the largest amount of EVs containing alpha-haemolysin, a strong cytotoxic agent. We stimulated primary cultured bovine mammary epithelial cells (pbMECs) with EVs from the S. aureus strain M5512VL. After 24 h of incubation, we observed a moderate increase in gene expression of tumour necrosis factor-alpha (TNF-α) but, surprisingly, a lack of an associated pronounced pro-inflammatory response. Our results contribute to understanding the damaging nature of S. aureus in its capacity to effectively affect mammary epithelial cells.

Determining extracellular vesicles properties and miRNA cargo variability in bovine milk from healthy cows and cows undergoing subclinical mastitis.

BACKGROUND: Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively. RESULTS: We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals. CONCLUSIONS: Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Influence of the farrowing process and different sow and piglet traits on uterine involution in a free farrowing system.

An inadequate uterine involution can lead to postpartal disorders in sows and thereby negatively affects the reproductive cycle and performance of the animals. Therefore, the aim of the study was to evaluate the influence of the farrowing process and different sow and piglet traits on the uterine involution in a free farrowing system. In total three data sets of 48 crossbred sows (Large White x Landrace) from the peripartal period were synthesised and analysed. The uterine diameter of three uterine horns per sow was measured daily during the first two weeks postpartum using ultrasonography and then the uterine regression was calculated. Furthermore, the following sow and piglet traits before and during parturition were assessed: parity, body condition score, backfat thickness, gestation length, number of live born piglets, number of stillborn piglets, farrowing duration, duration of placenta expulsion, litter weight, placenta weight, number of placenta parts, faecal score, and birth induction. In addition, the following parameters were recorded five days after farrowing: body temperature, severity and amount of vaginal discharge, pathological colour of vaginal discharge, and lack of appetite. Spearman's correlation test was used to examine the association between the parameters within the group and uterine regression. In addition, a linear multiple regression was used to model the relationship between the uterine involution and explanatory variables from the peripartal period. The mean diameter of the uterus decreased from 32.5 mm on day 2 post-partum to 11.4 mm on day 12 post-partum, which is a relative regression of 66%. In the linear multiple regression model, a significant influence of the body condition score (p = 0.046) and the presence of high body temperature (p = 0.022) on uterine involution was detected. When the BCS increases by one unit (from BCS 3 to 4), the relative regression of the uterine involution decreases by approximately 8% and when fever is present, uterine involution is almost 6% slower. Furthermore, a significant correlation between the gestation length and the uterine involution (r = 0.31; p = 0.035) was identified. However, no correlation between the farrowing process and piglets' traits on the uterine involution was detected. This study showed that the body condition score, the gestation length and fever in the first five days after farrowing had significant effects on uterine involution in sows in a free farrowing system. Therefore, these parameters should be routinely monitored in the farrowing management to assess reproductive health post-partum and warrant early intervention.

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries.

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 µg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 µg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.

Moderate Differences in Plasma Leptin in Mares Have no Effect on Either the Amino Acid or the Fatty Acid Composition of the Uterine Fluid.

Female mammalian reproductive functions are closely linked to body condition and metabolic status. Energy homeostasis is regulated by endocrine hormones such as insulin, IGF-I, leptin, and adiponectin via the hypothalamic-pituitary-adrenal axis. These metabolic hormones and their receptors are also expressed in reproductive tissues and the embryo. We investigated the relationship between circulating leptin and the fatty acid (FA) and amino acid (AA) composition of the equine uterine fluid (UF) and peripheral blood plasma (BP) by using a mass spectrometry-based approach. UF and BP were collected from ten broodmares on days 6 and 7 post ovulation, respectively. The mares were retrospectively assigned to two groups according to their BP leptin concentrations (high leptin [> 1.6 ng/mL] versus low leptin [<0.8 ng/mL]). Specific AA and FA compositions for BP and UF were found with different levels of respective metabolite abundances. The main FAs in BP were stearic, palmitic and linoleic acid. In UF, the three most abundant FAs were eicosapentaenoic, arachidonic and stearic acid. The AA profile of BP was dominated by glycine, glutamine, serine and alanine, which were likewise among the highly abundant AAs in UF. In UF, glutamic acid had by far the highest concentration. Therefore, BP leptin concentration within a physiological range does not seem to affect the specific FA nor the AA composition of the UF. The composition of the UF may therefore be mediated by local rather than by peripheral metabolic hormones.

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17ß signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues.

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos.

BACKGROUND: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts. RESULTS: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts. CONCLUSIONS: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Preferential Partitioning of Rumen-Protected n-3 and n-6 Fatty Acids into Functionally Different Adipose Tissues.

Lipids are stored at various sites inside the body as adipose tissue (AT). These include subcutaneous, abdominal, and intermuscular locations. The AT substantially differ in their metabolic function. It is, however, unclear whether AT have specific requirements for individual essential n-3 and n-6 fatty acids (FA). If so, control mechanisms would partition FA from the blood. To investigate the hypothesis of a selective FA incorporation, 18 beef heifers were fed diets supplemented with 60 g/kg diet with lipids from either fish oil (FO) or sunflower oil (SO). The lipids had partially been rumen-protected to ruminal biohydrogenation of n-3 and n-6 FA. The AT analyzed for n-3 and n-6 FA by gas chromatography were obtained from pericardial, longissimus thoracis (LT) intermuscular, perirenal, and subcutaneous sites. The greatest proportions of n-3 and n-6 FA were found in the pericardial AT. Despite generally low abundance, n-3 FA proportions increased with FO compared to SO supplementation in all AT, but to a different extent. No such partitioning was found for the n-6 FA when supplementing SO. Concomitantly, the n-6/n-3 FA ratio was reduced with FO in all AT, except in the pericardial AT. The latter has specific metabolic functions and thus appears to be quite resistant to diet-induced changes in FA profile in order to maintain its function. The present findings showed the special role of specific n-3 and n-6 FA in bovine AT.