Impact of Superovulation And In Vitro Fertilization On LINE-1 Copy Number And Telomere Length In C57BL/6J Mice Blastocysts

Objective: Millions of babies have been conceived by IVF, yet debate about its safety to offspring continues. We hypothesized that superovulation and in vitro fertilization (IVF) promote genomic changes, including altered telomere length (TL) and activation of the retrotransposon LINE-1 (L1), and tested this hypothesis in a mouse model. Material and methods: Experimental study analyzing TL and L1 copy number in C57BL/6J mouse blastocysts in vivo produced from natural mating cycles (N), in vivo produced following superovulation (S), or in vitro produced following superovulation (IVF). We also examined the effects of prolonged culture on TL and L1 copy number in the IVF group comparing blastocysts cultured 96 hours versus blastocysts cultured 120 hours. TL and L1 copy number were measured by Real Time PCR. Results: TL in S (n=77; Mean: 1.50± 1.15; p=0.0007) and IVF (n=82; Mean: 1.72± 1.44; p<0.0001) exceeded that in N (n=16; Mean: 0.61± 0.27). TL of blastocysts cultured 120 hours (n=15, Mean: 2.14± 1.05) was significantly longer than that of embryos cultured for 96 hours (n=67, Mean: 1.63 ± 1.50; p=0.0414). L1 copy number of blastocysts cultured for 120 hours (n=15, Mean: 1.71± 1.49) exceeded that of embryos cultured for 96 hours (n=67, Mean: 0.95 ± 1.03; p=0.0162). Conclusions: Intriguingly ovarian stimulation, alone or followed by IVF, produced embryos with significantly longer telomeres compared to in vivo, natural cycle-produced embryos. The significance of this enriched telomere endowment for the health and longevity of offspring born from IVF merit future studies.

An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative-feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.

PSX-B-13 Effect of epidermal growth factor and prolactin on the quality of bovine oocytes aging in vitro

The evaluation of factors responsible for the protection of the oocytes attained the metaphase-II stage from aging is importance for successful in vitro embryo reproduction. The aim of the present research was to study dose-dependent effects of epidermal growth factor (EGF) and prolactin (PRL) on the quality of bovine oocytes after their aging in vitro. Bovine cumulus-enclosed oocytes (CEOs) were matured in vitro for 20 h in TCM 199 containing 0.2 mM sodium pyruvate, 10% fetal calf serum (FCS), 10 μg/ml FSH and LH. At the end of in vitro maturation, oocytes were transferred to TCM 199 supplemented with 10% FCS (aging medium) and cultured for additional 24 h in the absence (Control) and in presence of EGF (10 and 50 ng/ml) and PRL (20 and 50 ng/ml). After prolonged culture oocytes were used for apoptosis detection (TUNEL staining, n=251) and the state of chromosomes evaluation (Tarkowski’s cytogenetic method, n=359). The data from 3–4 replicates were analyzed by ANOVA. At the end of prolonged culture (24 h) the rate of apoptotic oocytes in the Control group was 47.4±8.5%. EGF at concentration of 10 ng/ml and PRL at both doses decreased this rate to 15.0–22.1% (p &lt; 0.05). Furthermore, PRL (not EGF) reduced the frequency of abnormal chromosome modifications (decondensation, adherence, clumping) at concentrations of 20–50 ng/ml from 58.7±2.1% (Control) to 41.2±1.9 and 45.6±2.7% respectively (p &lt; 0.01). Thus, EGF and PRL is able to maintain the apoptosis resistance of bovine oocytes during their prolonged in vitro culture as well as PRL have the decelerating effect on abnormal modifications of M-II chromosomes. The research was supported by RFBR (17-29-08035) and the Ministry of Science and Higher Education of Russia.

Using a micro-physiological system to prolong the preservation of ex vivo lung tissue

Current in vitro and in vivo disease models have been reported to lack sufficient translation to human. Precision-Cut Lung Slices (PCLS) are viable sections of lung tissue and have been described to be a translational model for the ex vivo assessment of pharmacological and toxicological compounds. In most studies PCLS were cultured under static conditions. These lung sections, however, suffer from the limited viability. Here we present a novel modular microphysiological system (MPS) to prolong the cultivation of ex vivo lung tissue. A tailored MPS setup was designed using the PDMS free modular plug&play MPS construction kit. PCLS from mice were cultivated for up to one week under static versus perfused conditions. Using the MPS technology enabled a prolonged culture period with improved viability as shown by lowered lactate dehydrogenase release and improved membrane integrity. Using this technology might allow us to use PCLS for longer culture periods such as e.g. repeated dose toxicity or pharmacology studies.

Differential Exoproteome and Biochemical Characterisation of Neoparamoeba perurans

Infection with the protozoan ectoparasite Neoparamoeba perurans, the causative agent of AGD, remains a global threat to salmonid farming. This study aimed to analyse the exoproteome of both an attenuated and virulent N. perurans isolate using proteomics and cytotoxicity testing. A disproportionate presence of proteins from the co-cultured microbiota of N. perurans was revealed on searching an amalgamated database of bacterial, N. perurans and Amoebozoa proteins. LC‑MS/MS identified 33 differentially expressed proteins, the majority of which were upregulated in the attenuated exoproteome. Proteins of putative interest found in both exoproteomes were maltoporin, ferrichrome-iron receptor, and putative ferric enterobactin receptor. Protease activity remained significantly elevated in the attenuated exoproteome compared with the virulent exoproteome. Similarly, the attenuated exoproteome had a significantly higher cytotoxic effect on rainbow trout gill cell line (RTgill W1) cells compared with the virulent exoproteome. The presence of a phosphatase and serine protease in the virulent exoproteome may facilitate AGD infection but do not appear to be key players in causing cytotoxicity. Altogether, this study reveals prolonged culture of N. perurans affects the exoproteome composition in favour of nutritional acquisition, and that the current culturing protocol for virulent N. perurans does not facilitate the secretion of virulence factors.

Assessment of bovine oocytes quality and their resistance to age-related changes after maturation in single-phase and two-phase culture systems

Existing approaches to in vitro maturation (IVM) of bovine oocytes do not take into account their specific demands during terminal phase of IVM including the need for increasing of their resistance to age-re-lated changes. In this work, we performed for the first time a comparative investigation of nuclear maturation and abnormal changes of MII chromosomes in bovine oocytes after their maturation in single-phase system and different two-phase systems and after the subsequent prolonged culture of the ova. When using the single-phase system, cumulus-oocyte complexes (COCs) were cultured for 24 h in the medium TCM 199 containing 10% fetal bovine serum (FBS), 10 μg/ml FSH, and 10 μg/ml luteinizing hormone (LH). In the two-phase system, oocytes matured in the same conditions for first 16 h and then in a new medium (TCM 199 containing 10% FBS (Control) or the same medium supplemented with 50 ng/ml progesterone) for the remaining 8 h of IVM. After maturation in the single-phase and the two-phase systems, a part of COCs were transferred to an aging medium (TCM 199 containing 10% FBS) and further cultured for 24 h. The state of the nuclear material (stage of meiosis and abnormal changes of MII chromosomes) in the matured and aged oocytes was assessed using cytogenetic analysis. The rate of oocytes being at the MII stage of meiosis after IVM in the single-phase and the two-phase systems was similar and amounted 82.7-86.3%. In addition, no effect of the culture system on the rate of MII oocytes with abnormal morphology of chromosomes was revealed. For the single-phase culture, this rate after the end of the IVM period was 32.2±0.5% and for the two-phase systems culture 38.5±4.0%. The prolonged culture of matured oocytes led to an increase the frequency of destructive changes in MII chromosomes to 56.9±2.9 and 68.4±3.0%, respectively. Progesterone in the IVM medium (during the last 8 hours) also did not effect on the rate of nuclear maturation, but reduced the rate of oocytes with abnormal changes in chromosomes: after IVM, as compared with the Control and the two-phase system; after 24-hours prolonged culture as compared to control (P <0.05). The results of the study suggest that the two-phase maturation of bovine oocytes may be used as an alternative to the conventional IVM protocol and that progesterone during the second phase of IVM causes an increase in the quality of ova and their resistance to subsequent age-related transformations.

An ERK5-KLF2 signalling module regulates early embryonic gene expression dynamics and stem cell rejuvenation

The ERK5 MAP kinase signalling pathway drives transcription of naive pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5-dependent ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative-feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern dynamic 2C gene transcription and mESC rejuvenation.

Stress Response of Mouse Embryonic Fibroblasts Exposed to Polystyrene Nanoplastics

Polystyrene (PS) nanoplastic exposure has been shown to affect the viability of neuronal cells isolated from mouse embryonic brains. However, the viability of mouse embryonic fibroblasts (MEFs) was not affected although PS nanoplastics accumulated in the cytoplasm. It is currently unknown whether MEFs do not respond to PS nanoplastics or their cellular functions are altered without compromising viability. Here, we found that PS nanoplastics entered the cells via endocytosis and were then released into the cytoplasm, probably by endosomal escape, or otherwise remained in the endosome. Oxidative and inflammatory stress caused by intracellular PS nanoplastics induced the antioxidant response pathway and activated the autophagic pathway. However, colocalization of the autophagic marker LC3B and PS nanoplastics suggested that PS nanoplastics in the cytoplasm might interfere with normal autophagic function. Furthermore, autophagic flux could be impaired, probably due to accumulation of PS nanoplastic-containing lysosomes or autolysosomes. Intriguingly, the level of accumulated PS nanoplastics decreased during prolonged culture when MEFs were no longer exposed to PS nanoplastics. These results indicate that accumulated PS nanoplastics are removed or exported out of the cells. Therefore, PS nanoplastics in the cytoplasm affect cellular functions, but it is temporal and MEFs can overcome the stress caused by PS nanoplastic exposure.

Age-Dependent Maturation of iPSC-CMs Leads to the Enhanced Compartmentation of β2AR-cAMP Signalling

The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β2AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following βAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-β-cyclodextrin (MβCD) to remove cholesterol as a method of decompartmentalising β2AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through β2AR (but not β1AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the β2AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the βAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the β2AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the β2AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.