Functional Significance of Selective Expression of ERα and ERβ in Mammary Gland Organ Culture

Thoracic pair of mammary glands from steroid hormone-pretreated mice respond to hormones structurally and functionally in organ culture. A short exposure of glands for 24 h to 7,12 Dimethylbenz(a)anthracene (DMBA) during a 24-day culture period induced alveolar or ductal lesions. Methods: To differentiate the functional significance of ERα and ERβ, we employed estrogen receptor (ER) knockout mice. We compared the effects of DMBA on the development of preneoplastic lesions in the glands in the absence of ERα (αERKO) and ERβ (βERKO) using an MMOC protocol. Glands were also subjected to microarray analyses. We showed that estradiol can be replaced by EGF for pretreatment of mice. The carcinogen-induced lesions developed under both steroids and EGF pretreatment protocols. The glands from αERKO did not develop any lesions, whereas in βERKO mice in which ERα is intact, mammary alveolar lesions developed. Comparison of microarrays of control, αERKO and βERKO mice showed that ERα was largely responsible for proliferation and the MAP kinase pathways, whereas ERβ regulated steroid metabolism-related genes. The results indicate that ERα is essential for the development of precancerous lesions. Both subtypes, ERα and Erβ, differentially regulated gene expression in mammary glands in organ cultures.

New Insights into the Host–Pathogen Interaction of Mycoplasma gallisepticum and Avian Metapneumovirus in Tracheal Organ Cultures of Chicken

Respiratory pathogens are a health threat for poultry. Co-infections lead to the exacerbation of clinical symptoms and lesions. Mycoplasma gallisepticum (M. gallispeticum) and Avian Metapneumovirus (AMPV) are two avian respiratory pathogens that co-circulate worldwide. The knowledge about the host–pathogen interaction of M. gallispeticum and AMPV in the chicken respiratory tract is limited. We aimed to investigate how co-infections affect the pathogenesis of the respiratory disease and whether the order of invading pathogens leads to changes in host–pathogen interaction. We used chicken tracheal organ cultures (TOC) to investigate pathogen invasion and replication, lesion development, and selected innate immune responses, such as interferon (IFN) α, inducible nitric oxide synthase (iNOS) and IFNλ mRNA expression levels. We performed mono-inoculations (AMPV or M. gallispeticum) or dual-inoculations in two orders with a 24-h interval between the first and second pathogen. Dual-inoculations compared to mono-inoculations resulted in more severe host reactions. Pre-infection with AMPV followed by M. gallispeticum resulted in prolonged viral replication, more significant innate immune responses, and lesions (p < 0.05). AMPV as the secondary pathogen impaired the bacterial attachment process. Consequently, the M. gallispeticum replication was delayed, the innate immune response was less pronounced, and lesions appeared later. Our results suggest a competing process in co-infections and offer new insights in disease processes.

Homo- and Dikaryons of the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis Differ in Life History Strategy

Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts that have the potential to improve crop yield. These multinucleate organisms are either “homokaryotic” or “dikaryotic”. In AMF dikaryons, thousands of nuclei originating from two parental strains coexist in the same cytoplasm. In other fungi, homokaryotic and dikaryotic strains show distinct life history traits (LHTs), such as variation in growth rates and fitness. However, how such traits compare between dikaryons and homokaryons of AMF is unknown. To address this, we measured 20 LHT of four dikaryons and five homokaryons of the model fungus Rhizophagus irregularis across root organ cultures of three host plants (carrot, chicory, and tobacco). Our analyses show that dikaryons have clearly distinct life history strategies (LHSs) compared to homokaryons. In particular, spores of homokaryons germinate faster and to a higher proportion than dikaryons, whereas dikaryons grow significantly faster and create a more complex hyphal network irrespective of host plant species. Our study links AMF nuclear status with key LHT with possible implications for mycorrhizal symbiotic functioning.

Mesenchymal stem cell secretome decreases the inflammatory response in annulus fibrosus organ cultures

Mesenchymal stem/stromal cell (MSC)-based therapies have been proposed for back pain and disc degeneration, despite limited knowledge on their mechanism of action. The impact of MSCs/their secretome on annulus fibrosus (AF) cells and tissue was analysed in bovine AF organ cultures (AF-OCs) exposed to upper-physiological cyclic tensile strain (CTS, 9 %, 1 Hz, 3 h/d) and interleukin (IL)-1β in a custom-made device. A 4 d treatment of the CTS + IL-1β-stimulated AF-OCs with MSC secretome downregulated the expression of inflammation markers [IL-6, IL-8, prostaglandin-endoperoxide synthase 2 (PTGS2)], complement system regulators [cluster of differentiation (CD)46, CD55, CD59] and matrix metalloproteinase 1 but also of tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) and collagen type I. At the protein level, it was confirmed that IL-6, MMP-3 and collagen content was decreased in AF-OCs treated with the MSC secretome compared to the CTS + IL-1β stimulation alone. 9 d after treatment, a biomechanical peel-force test showed that the annular adhesive strength was significantly decreased by the MSC secretome treatment. Overall, MSC secretome had a stronger impact on AF tissue than MSCs in co-culture. The secretome contributed to a decrease in the inflammatory and catabolic status of AF cells activated by CTS + IL-1β and played a role in the regulation of the complement system. However, it also contributed to a decrease in collagen at the gene/protein level and in AF mechanical strength compared to the CTS + IL-1β stimulation alone. Therefore, the use of MSC secretome requires further investigation regarding its influence on disc matrix properties.

P–799 Long-term maintenance and meiotic entry of early germ cells in functionalized murine testicular organoids mediated by 3D printed scaffolds and air-medium interphase cultivation

Study question Can improved culture conditions advance the functionality of murine testicular organoids (TOs)? Summary answer Testicular cells formed spheroidal TOs resembling the functional unit of the testis and supporting meiotic entry of germ cells during long-term culture in printed macropores. What is known already Organ cultures at the air-medium interphase have traditionally been used for in-vitro spermatogenesis (IVS) in rodents because they best preserve the testicular architecture, which is pivotal in achieving IVS. However, organ cultures do not offer the ability to access and manipulate single cells, making it an inefficient model for mechanistic studies. Culturing testicular cell suspensions into organoids offer these features. Previously, testicular organoids in immersion culture resulted in testicular architecture, but only supported short-term survival of germ cells. Moreover, millimeter-sized organoids show signs of degeneration due to insufficient nutrient and oxygen supply. Study design, size, duration First, we focused on recreating the testicular architecture at air-medium interphase and determined whether higher cell densities could improve our previously developed 3D printed culture model during long-term culture using different mouse strains. Afterwards, the focus was put on improving TO morphology by adapting the scaffold design. Moreover, to expand the potential of TOs, the possibility to cultivate chimeric mixtures of testicular cells and germ line stem cells expressing a reporter transgene (EGFP) was assessed. Participants/materials, setting, methods Prepubertal testicular cells from C57BL/6J (n = 5) or CBAB6F1 (n = 3) mice were cultured in the macropores of 3D printed squared 1-layered scaffolds (1LSs) composed of Cellink-RGD (8x104 cells/mm²). Next, 1LS was modified with an additional layer of alginate (2LS) to culture a chimeric mixture of testicular cells of prepubertal C57BL/6J mice and EGFP-expressing germline stem cells (2:1). Cell reorganization and differentiation were characterized by immunohistochemistry and testosterone was quantified by electrochemiluminescence. Main results and the role of chance During long-term cultures in 1LSs, testicular cells reorganized into organoids with restoration of testicular architecture and Leydig cell functionality supporting the differentiation of germ cells to the meiotic phase, regardless of the mouse strain. However, pore overgrowth and fusion of adjacent aggregates, resulted in irregularly shaped TOs. Based on these results, the design of 1LS was modified with an additional layer of alginate to entrap reorganizing cells (2LS). To non-invasively evaluate germ cell behavior, EGFP-expressing germline stem cells were mixed with testicular cells of prepubertal C57BL/6J mice in 2LS. This approach resulted in the formation of chimeric organoids with a more regular and spheroidal morphology. These improved TOs consisted typically of 1 tubule-like structure and surrounding interstitium, representing the functional unit of a testis. in contrast to primary germ cells, germline stem cells were not observed after the 3rd week of culture. Limitations, reasons for caution Candidate factors have to be tested in their ability to elevate the meiotic blockage of germ cells in TOs. In addition, the culture medium needs further optimization to enhance maintenance of germline stem cells in chimeric models. Finally, results obtained with rodents remain to be confirmed in further human studies. Wider implications of the findings: The opportunities testicular organoids offer to manipulate cells through genetic modification, inclusion and exclusion, are essential for the study of male infertility and the search for potential therapies. Moreover, they permit high-throughput screening of chemicals, thereby substantially reducing the number of animals for the high demanding reproductive toxicity studies. Trial registration number Not applicable