Long-Term Maintenance and Meiotic Entry of Early Germ Cells in Murine Testicular Organoids Functionalized by 3D Printed Scaffolds and Air-Medium Interface Cultivation

Short-term germ cell survival and central tissue degeneration limit organoid cultures. Here, testicular organoids (TOs) were generated from two different mouse strains in 3D printed one-layer scaffolds (1LS) at the air-medium interface displaying tubule-like structures and Leydig cell functionality supporting long-term survival and differentiation of germ cells to the meiotic phase. Chimeric TOs, consisting of a mixture of primary testicular cells and EGFP+ germline stem (GS) cells, were cultured in two-layer scaffolds (2LSs) for better entrapment. They showed an improved spheroidal morphology consisting of one intact tubule-like structure and surrounding interstitium, representing the functional unit of a testis. However, GS cells did not survive long-term culture. Consequently, further optimization of the culture medium is required to enhance the maintenance and differentiation of germ cells. The opportunities TOs offer to manipulate somatic and germ cells are essential for the study of male infertility and the search for potential therapies.

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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

Human Reproduction ◽

10.1093/humrep/deab130.798 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

G Richer ◽

E Goossens ◽

R Hobbs ◽

K Loveland ◽

Y Baert

Keyword(s):

Stem Cells ◽

Germ Cells ◽

Functional Unit ◽

Germline Stem Cells ◽

Organ Cultures ◽

Additional Layer ◽

Testicular Cells ◽

3D Printed ◽

Air Medium

Abstract 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

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Contextualizing Autophagy during Gametogenesis and Preimplantation Embryonic Development

International Journal of Molecular Sciences ◽

10.3390/ijms22126313 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6313

Author(s):

Marcelo T. Moura ◽

Laís B. Latorraca ◽

Fabíola F. Paula-Lopes

Keyword(s):

Embryonic Development ◽

Germ Cells ◽

Physiological Role ◽

Degradation Pathway ◽

Environmental Stressors ◽

Preimplantation Embryos ◽

Environmental Cues ◽

Term Survival ◽

Assisted Reproduction Technologies

Mammals face environmental stressors throughout their lifespan, which may jeopardize cellular homeostasis. Hence, these organisms have acquired mechanisms to cope with stressors by sensing, repairing the damage, and reallocating resources to increase the odds of long-term survival. Autophagy is a pro-survival lysosome-mediated cytoplasm degradation pathway for organelle and macromolecule recycling. Furthermore, autophagy efflux increases, and this pathway becomes idiosyncratic depending upon developmental and environmental contexts. Mammalian germ cells and preimplantation embryos are attractive models for dissecting autophagy due to their metastable phenotypes during differentiation and exposure to varying environmental cues. The aim of this review is to explore autophagy during mammalian gametogenesis, fertilization and preimplantation embryonic development by contemplating its physiological role during development, under key stressors, and within the scope of assisted reproduction technologies.

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Genetic Regulation of Long-Term Nonprogression in E-55+ Murine Leukemia Virus Infection in Mice

Journal of Virology ◽

10.1128/jvi.73.11.9232-9236.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9232-9236

Author(s):

Vily Panoutsakopoulou ◽

Kathryn Hunter ◽

Thomas G. Sieck ◽

Elizabeth P. Blankenhorn ◽

Kenneth J. Blank

Keyword(s):

Bone Marrow ◽

Murine Leukemia Virus ◽

Genetic Locus ◽

Leukemia Virus ◽

Inbred Mouse ◽

Major Histocompatibility Complex Gene ◽

Mouse Strains ◽

Term Survival ◽

Murine Leukemia

ABSTRACT Certain inbred mouse strains display progression to lymphoma development after infection with E-55+ murine leukemia virus (E-55+ MuLV), while others demonstrate long-term nonprogression. This difference in disease progression occurs despite the fact that E-55+ MuLV causes persistent infection in both immunocompetent BALB/c–H-2k (BALB.K) progressor (P) and C57BL/10–H-2k (B10.BR) long-term nonprogressor (LTNP) mice. In contrast to immunocompetent mice, immunosuppressed mice from both P and LTNP strains develop lymphomas about 2 months after infection, indicating that the LTNP phenotype is determined by the immune response of the infected mouse. In this study, we used bone marrow chimeras to demonstrate that the LTNP phenotype is associated with the genotype of donor bone marrow and not the recipient microenvironment. In addition, we have mapped a genetic locus that may be responsible for the LTNP trait. Microsatellite-based linkage analysis demonstrated that a non-major histocompatibility complex gene on chromosome 15 regulates long-term survival and is located in the same region as the Rfv3 gene. Rfv3 is involved in recovery from Friend virus-induced leukemia and has been demonstrated to regulate neutralizing virus antibody titers. In our studies, however, both P and LTNP strains produce similar titers of neutralizing and cytotoxic anti-E-55+ MuLV. Therefore, while it is possible that Rfv3 influences the course of E-55+ MuLV infection, it is more likely that the LTNP phenotype in E-55+ MuLV-infected mice is regulated by a different, closely linked gene.

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Production of common carp donor-derived offspring from goldfish surrogate broodstock

10.1101/2020.08.11.245803 ◽

2020 ◽

Author(s):

Roman Franěk ◽

Vojtěch Kašpar ◽

David Gela ◽

Martin Pšenička

Keyword(s):

Stem Cells ◽

Body Size ◽

Common Carp ◽

Germ Cells ◽

Reproductive Performance ◽

Small Body ◽

Important Species ◽

Testicular Cells

AbstractBackgroundCommon carp is the fourth most-produced species in worldwide aquaculture. Significant efforts are invested in breeding and preservation of genetic integrity of this important species. However, maintaining carp gene bank in situ can be considered as demanding due to its big body size. Recent progress in reproductive biotechnologies in fish allows improving some unfavourable characteristics of a target species using surrogate reproduction. Germ stem cells (gamete precursors) from one species are transplanted into different surrogate species with small body size. After maturation, surrogates are producing donor-derived progeny. Efficient protocols for cryopreservation of carp male and female germ stem cells have been developed lately. Thus, the next logical goal was to assess the potential of goldfish surrogate to produce donor-derived gametes of common carp after intraperitoneal transplantation of testicular cells.ResultsHigh transplantation success was achieved when 44% of the surviving goldfish produced pure donor-derived gametes of common carp. More importantly, both viable eggs and sperm giving rise to pure common carp progeny were produced, witnessing sustainability of the presented method. Donor-derived identity of the offspring was confirmed by genotyping and typical phenotype corresponding to the donor species. Reproductive performance of chimeras was similar to goldfish controls. Assessment of gamete characteristics showed that the size of donor-derived eggs is between control carp and goldfish eggs. Interestingly, flagellum length in donor-derived spermatozoa was comparable to common carp flagellum and significantly shorter than goldfish flagellum.ConclusionsIn this study, we succeeded in the production of pure common carp progeny from surrogate goldfish recipients transplanted intraperitoneally by testicular germ cells. Here we reported production of viable eggs between most distant species up to date. Good reproductive performance of goldfish germline chimeras gives a promising prospect for further analysis about the long-term reproductive performance of surrogates, recovery of cryopreserved germ cells or production of monosex stocks. Presented technology is ready to ease needs for carp breeds preservation and their recovery using many times smaller goldfish surrogates.

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Deviation of the Fecal Stream in Colonic Bowel Segments Results in Increased Numbers of Isolated Lymphoid Follicles in the Submucosal Compartment in a Novel Murine Model of Diversion Colitis

BioMed Research International ◽

10.1155/2017/5265969 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Annabel Kleinwort ◽

Paula Döring ◽

Christine Hackbarth ◽

Claus-Dieter Heidecke ◽

Tobias Schulze

Keyword(s):

Murine Model ◽

Transverse Colon ◽

Mucosal Inflammation ◽

Mouse Strains ◽

Diversion Colitis ◽

Term Survival ◽

Long Term Survival ◽

Promising Tool ◽

Human Pathology

Introduction.Diversion colitis is a significant health problem due to its high incidence in patients with diverting enterostomy. This mucosal inflammation presents characteristic histopathological features allowing for the differentiation of this entity from other inflammatory bowel diseases. The pathophysiology of this disease remains ill-defined, in part due to the lack of appropriate animal models. The present study was performed in order to develop and characterize a murine model of diversion colitis.Methods.A diverting loop colostomy was performed in C57BL/6 mice either in the ascending colon or in the transverse colon. Animals were assessed for clinical and histopathological parameters during short-term and long-term survival.Results.Animals with a colostomy in the transverse colon showed a good long-term survival and developed a mild colitis in the bypassed bowel closely resembling the human pathology on a histopathological level.Conclusion.This model is a promising tool to further elucidate the pathomechanism leading to impaired mucosal homeostasis in bypassed colonic segments. Moreover, the establishment of the model in the C57BL/6 background allows the combination of this colitis model with various transgenic mouse strains to investigate the effect of locally deregulated mucosal immunity on systemic immune homeostasis and to develop specific therapeutic strategies.

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Stem Cell Therapy Post-AMI

10.1093/med/9780199544769.003.0010 ◽

2011 ◽

Author(s):

Philippe Menasché

Keyword(s):

Therapeutic Strategy ◽

Experimental Studies ◽

Left Ventricular ◽

Term Survival ◽

Paracrine Signalling ◽

Donor Cells ◽

Regeneration Of Cardiomyocytes ◽

Experimental Findings ◽

Cell Functionality

• Experimental studies suggest that bone marrow-derived stem cells can improve function of infarcted myocardium• This benefit seems to involve paracrine signalling and limitation of left ventricular remodelling rather than true regeneration of cardiomyocytes from donor cells• These experimental findings have been translated in the clinical setting into significant, although moderate, improvements in cardiac function and LV remodelling but the extent to which these benefits impact on event-free long term survival remains to be determined• Optimisation of this therapeutic strategy will require a more comprehensive characterisation of cell functionality and an improvement in the methods used in cell transfer, engraftment, survival and integration.

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Hydrogel-delivered brain-derived neurotrophic factor promotes tissue repair and recovery after stroke

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x16649964 ◽

2016 ◽

Vol 37 (3) ◽

pp. 1030-1045 ◽

Cited By ~ 82

Author(s):

Douglas J Cook ◽

Cynthia Nguyen ◽

Hyun N Chun ◽

Irene L Llorente ◽

Abraham S Chiu ◽

...

Keyword(s):

Tissue Repair ◽

Mouse Strains ◽

Systemic Delivery ◽

Behavioral Recovery ◽

Term Survival ◽

Neural Repair ◽

Stroke Models ◽

Hyaluronic Acid Hydrogel ◽

Human Primate

Stroke is the leading cause of adult disability. Systemic delivery of candidate neural repair therapies is limited by the blood–brain barrier and off-target effects. We tested a bioengineering approach for local depot release of BDNF from the infarct cavity for neural repair in chronic periods after stroke. The brain release levels of a hyaluronic acid hydrogel + BDNF were tested in several stroke models in mouse (strains C57Bl/6, DBA) and non-human primate ( Macaca fascicularis) and tracked with MRI. The behavioral recovery effects of hydrogel + BDNF and the effects on tissue repair outcomes were determined. Hydrogel-delivered BDNF diffuses from the stroke cavity into peri-infarct tissue over 3 weeks in two mouse stroke models, compared with 1 week for direct BDNF injection. Hydrogel delivery of BDNF promotes recovery of motor function. Mapping of motor system connections indicates that hydrogel-BDNF induces axonal sprouting within existing cortical and cortico-striatal systems. Pharmacogenetic studies show that hydrogel-BDNF induces the initial migration of immature neurons into the peri-infarct cortex and their long-term survival. In chronic stroke in the non-human primate, hydrogel-released BDNF can be detected up to 2 cm from the infarct, a distance relevant to human functional recovery in stroke. The hydrogel can be tracked by MRI in mouse and primate.

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Production of common carp donor-derived offspring from goldfish surrogate broodstock

10.21203/rs.3.rs-57467/v1 ◽

2020 ◽

Author(s):

Roman Franěk ◽

Vojtěch Kašpar ◽

David Gela ◽

Martin Pšenička

Keyword(s):

Stem Cells ◽

Body Size ◽

Common Carp ◽

Germ Cells ◽

Reproductive Performance ◽

Small Body ◽

Important Species ◽

Testicular Cells

Abstract Background: Common carp is the fourth most-produced species in worldwide aquaculture. Significant efforts are invested in breeding and preservation of genetic integrity of this important species. However, maintaining carp gene bank in situ can be considered as demanding due to its big body size. Recent progress in reproductive biotechnologies in fish allows improving some unfavourable characteristics of a target species using surrogate reproduction. Germ stem cells (gamete precursors) from one species are transplanted into different surrogate species with small body size. After maturation, surrogates are producing donor-derived progeny. Efficient protocols for cryopreservation of carp male and female germ stem cells have been developed lately. Thus, the next logical goal was to assess the potential of goldfish surrogate to produce donor-derived gametes of common carp after intraperitoneal transplantation of testicular cells. Results: High transplantation success was achieved when 44% of the surviving goldfish produced pure donor-derived gametes of common carp. More importantly, both viable eggs and sperm giving rise to pure common carp progeny were produced, witnessing sustainability of the presented method. Donor-derived identity of the offspring was confirmed by genotyping and typical phenotype corresponding to the donor species. Reproductive performance of chimeras was similar to goldfish controls. Assessment of gamete characteristics showed that the size of donor-derived eggs is between control carp and goldfish eggs. Interestingly, flagellum length in donor-derived spermatozoa was comparable to common carp flagellum and significantly shorter than goldfish flagellum. Conclusions: In this study, we succeeded in the production of pure common carp progeny from surrogate goldfish recipients transplanted intraperitoneally by testicular germ cells. Here we reported production of viable eggs between most distant species up to date. Good reproductive performance of goldfish germline chimeras gives a promising prospect for further analysis about the long-term reproductive performance of surrogates, recovery of cryopreserved germ cells or production of monosex stocks. Presented technology is ready to ease needs for carp breeds preservation and their recovery using many times smaller goldfish surrogates.

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Caspases and apoptosis

Essays in Biochemistry ◽

10.1042/bse0380009 ◽

2002 ◽

Vol 38 ◽

pp. 9-19 ◽

Cited By ~ 121

Author(s):

Guy S Salvesen

Keyword(s):

Cysteine Proteases ◽

Signalling Pathways ◽

Term Survival ◽

Mature Adult ◽

Intracellular Signalling Pathways ◽

Adult Cell ◽

Caspase Family ◽

Cell Fragments ◽

Pro Inflammatory Cytokines

The ability of metazoan cells to undergo programmed cell death is vital to both the precise development and long-term survival of the mature adult. Cell deaths that result from engagement of this programme end in apoptosis, the ordered dismantling of the cell that results in its 'silent' demise, in which packaged cell fragments are removed by phagocytosis. This co-ordinated demise is mediated by members of a family of cysteine proteases known as caspases, whose activation follows characteristic apoptotic stimuli, and whose substrates include many proteins, the limited cleavage of which causes the characteristic morphology of apoptosis. In vertebrates, a subset of caspases has evolved to participate in the activation of pro-inflammatory cytokines, and thus members of the caspase family participate in one of two very distinct intracellular signalling pathways.

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