Thomas Kolbe

Cellular senescence plays key roles in tissue repair, tumour suppression and ageing. Here we identify a rapid, transcription‑independent senescence response in skin following injury. Within minutes to hours after wounding, skin cells at the edge of injury display hallmark features of senescence. This response involves the utilization of pre‑existing Cdkn1a mRNA through the removal of nuclear export inhibitors, which enables Cdkn1a transcript translation and rapid p21 protein accumulation. These cells enter stable cell‑cycle arrest and secrete pro‑migratory and pro‑inflammatory factors that promote tissue repair, including re‑epithelialization. Experimental suppression of this rapid senescence, either genetically or pharmacologically, markedly delays wound closure, whereas inhibition during later phases of repair has no effect. Our findings establish rapid‑onset senescence as a mechanistic requirement for efficient tissue regeneration.

The health status of laboratory animals plays a decisive role not only for the health and welfare of the animals but also for the validity of study results. In recent years, there has been an increasing number of publications on environmental health monitoring (EHM), which uses molecular biological methods to detect nucleic acids of infectious agents in individually ventilated cage systems, e.g. in exhaust air dust. This monitoring strategy can reduce the number of mice used for health monitoring in conformity with the 3Rs. Numerous studies have shown that EHM is reliable and sensitive and is, therefore, a useful method for health monitoring of mice. An online survey was created to assess the prevalence of the use of EHM in Germany, Austria, and Switzerland and to better understand the factors influencing its use in animal facilities. The survey revealed that the majority of facilities primarily equipped with individually ventilated cage systems already use EHM to varying degrees, replacing between 8 and 1200 animals per year and facility. However, the predominant strategy is still the use of (sentinel) animals for health monitoring. Beliefs on factors such as cost, reliability and the number of false-positive results differ significantly between facilities that predominantly use either animals or EHM. Additionally, the choice of monitoring strategy may be influenced by the existing cage system and the availability of a decontamination option for the equipment. The evaluation of the survey showed that there is still a gap in knowledge and a demand for specific training on the topic of health monitoring and especially on EHM.

2.1 Animals: Typo: C57bl/6JTac must be C57BL/6JTac. (purchased at Taconic) is redundant, already visible in the name C57BL/6JTac. Please stick to the ARRIVE guidelines: Caging system is missing, cage size, food and water, bedding. Please indicate t...

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)¹, which influences offspring development and adult phenotypes^1–7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood⁸. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

FAM3C/ILEI is an important factor in epithelial-to-mesenchymal transition (EMT) induction, tumor progression and metastasis. Overexpressed in many cancers, elevated ILEI levels and secretion correlate with poor patient survival. Although ILEI’s causative role in invasive tumor growth and metastasis has been demonstrated in several cellular tumor models, there are no available transgenic mice to study these effects in the context of a complex organism. Here, we describe the generation and initial characterization of a Tet-ON inducible Fam3c/ILEI transgenic mouse strain. We find that ubiquitous induction of ILEI overexpression (R26-ILEI^ind) at weaning age leads to a shortened lifespan, reduced body weight and microcytic hypochromic anemia. The anemia was reversible at a young age within a week upon withdrawal of ILEI induction. Vav1-driven overexpression of the ILEI^ind transgene in all hematopoietic cells (Vav-ILEI^ind) did not render mice anemic or lower overall fitness, demonstrating that no intrinsic mechanisms of erythroid development were dysregulated by ILEI and that hematopoietic ILEI hyperfunction did not contribute to death. Reduced serum iron levels of R26-ILEI^ind mice were indicative for a malfunction in iron uptake or homeostasis. Accordingly, the liver, the main organ of iron metabolism, was severely affected in moribund ILEI overexpressing mice: increased alanine transaminase and aspartate aminotransferase levels indicated liver dysfunction, the liver was reduced in size, showed increased apoptosis, reduced cellular iron content, and had a fibrotic phenotype. These data indicate that high ILEI expression in the liver might reduce hepatoprotection and induce liver fibrosis, which leads to liver dysfunction, disturbed iron metabolism and eventually to death. Overall, we show here that the novel Tet-ON inducible Fam3c/ILEI transgenic mouse strain allows tissue specific timely controlled overexpression of ILEI and thus, will serve as a versatile tool to model the effect of elevated ILEI expression in diverse tissue entities and disease conditions, including cancer.

Surgical embryo transfer in mice is a key technique in assisted reproduction and applied for different purposes in biomedical research. Due to its frequent application in rodent facilities across the world, further improvement of the procedure can substantially contribute to fulfil the principles of the 3Rs. Here, we investigated the effect of bilateral and unilateral left- or right-sided oviduct transfers on the success of embryo transfers. In total, we performed 223 embryo transfers (56 unilateral left, 56 unilateral right, 111 bilateral), in which we transferred 10–14 two-cell embryos each. We found that the type of transfer significantly influenced both the pregnancy rate of recipients and the survival rate of transferred embryos. Bilateral transfers yielded higher pregnancy and survival rates than left-sided unilateral transfers. Right-sided unilateral transfers yielded higher pregnancy rates than left-sided unilateral transfers and did not differ in embryo survival rates from bilateral transfers. We found no evidence that the number of transferred embryos affected the pregnancy rate. However, the number of born pups increased with the number of transferred embryos. In conclusion, unilateral embryo transfers into the right reproductive tract yield equally high pregnancy and embryo survival rates as bilateral transfers. Given that a second abdominal incision can be prevented and the time of surgery can be reduced, we recommend applying unilateral right-sided transfers, as this would reduce postoperative pain and lower the impact on recipients.

Ambient temperature is an important non-biotic environmental factor influencing immunological and oncological parameters in laboratory mice. It is under discussion which temperature is more appropriate and whether the commonly used room temperature in rodent facilities of about 21 °C represents a chronic cold stress or the 30 °C of the thermoneutral zone constitutes heat stress for the animals. In this study, we selected the physiological challenging period of lactation to investigate the influence of a cage temperature of 20 °C, 25 °C, and 30 °C, respectively, on reproductive performance and stress hormone levels in two frequently used mouse strains. We found that B6D2F1 hybrid mothers weaned more pups compared to C57BL/6N mothers, and that the number of weaned pups was reduced when mothers of both strains were kept at 30 °C. Furthermore, at 30 °C, mothers and pups showed reduced body weight at weaning and offspring had longer tails. Despite pronounced temperature effects on reproductive parameters, we did not find any temperature effects on adrenocortical activity in breeding and control mice. Independent of the ambient temperature, however, we found that females raising pups showed elevated levels of faecal corticosterone metabolites (FCMs) compared to controls. Peak levels of stress hormone metabolites were measured around birth and during the third week of lactation. Our results provide no evidence of an advantage for keeping lactating mice in ambient temperatures near the thermoneutral zone. In contrast, we found that a 30 °C cage temperature during lactation reduced body mass in females and their offspring and declined female reproductive performance.

Ambient temperature is an important non-biotic environmental factor influencing immunological and oncological parameters in laboratory mice. It is under discussion which temperature is more appropriate and whether the commonly used room temperature in rodent facilities of about 21°C represents a chronic cold stress or the 30°C of the thermoneutral zone constitutes heat stress for the animals. In this study we selected the physiological challenging period of lactation to investigate the influence of a cage temperature of 20°C, 25°C, and 30°C, respectively, on reproductive performance and stress hormone levels in two frequently used mouse strains. We found that more pups were weaned from B6D2F1 hybrids compared to C57BL/6N mothers and that the number of weaned pups was strongly reduced if mothers of both strains were kept at 30°C. Furthermore, at 30°C mothers and pups showed reduced body weight at weaning and offspring had longer tails. Despite pronounced temperature effects on reproductive parameters, we did not find any impact on adrenocortical activity in breeding and control mice. Independent of the ambient temperature however, we found that females raising pups showed elevated levels of fecal corticosterone metabolites (FCMs) compared to controls. Increased levels of stress hormone metabolites were measured specially around birth and during the third week of lactation. Our results provide no evidence for reduced or improved wellbeing of lactating mice at different ambient temperatures, but we found that a 30°C cage temperature impairs reproductive performance.

Cell competition is emerging as a quality-control mechanism that eliminates unfit cells in a wide range of settings from development to the adult. However, the nature of the cells normally eliminated by cell competition and what triggers their elimination remains poorly understood. In mice, 35% of epiblast cells are eliminated before gastrulation. Here we show that cells with mitochondrial defects are eliminated by cell competition during early mouse development. Using single-cell transcriptional profiling of eliminated mouse epiblast cells, we identify hallmarks of cell competition and mitochondrial defects. We demonstrate that mitochondrial defects are common to a range of different loser cell types and that manipulating mitochondrial function triggers cell competition. Moreover, we show that in the mouse embryo, cell competition eliminates cells with sequence changes in mt-Rnr1 and mt-Rnr2, and that even non-pathological changes in mitochondrial DNA sequences can induce cell competition. Our results suggest that cell competition is a purifying selection that optimizes mitochondrial performance before gastrulation.

Heteroplasmic mice represent a valuable tool to study the segregation of different mtDNA haplotypes (mtDNAs with differing alleles) in vivo against a defined nuclear background. We describe two methods for the creation of such models, differing in the resulting initial heteroplasmy levels: (a) transfer of ooplasm and (b) fusion of two blastomeres. These methods result in typical heteroplasmy of 5% and 50% donor mtDNA, respectively. The choice of method depends on the aim of the study. By means of breeding even 100% donor mtDNA can be reached within a few generations.