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

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.

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

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.

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

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

scholarly journals Assessment of the Growth and Reproductive Performance of Cloned Pietrain Boars

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2053
Author(s):  
Junsong Shi ◽  
Baohua Tan ◽  
Lvhua Luo ◽  
Zicong Li ◽  
Linjun Hong ◽  
...  
Keyword(s):  
Growth Rate ◽  
Body Size ◽  
Growth Performance ◽  
Reproductive Performance ◽  
Semen Quality ◽  
Small Body ◽  
Economic Benefits ◽  
Large Animal ◽  
Significant Difference ◽  
F1 Progeny

How to maximize the use of the genetic merits of the high-ranking boars (also called superior ones) is a considerable question in the pig breeding industry, considering the money and time spent on selection. Somatic cell nuclear transfer (SCNT) is one of the potential ways to answer the question, which can be applied to produce clones with genetic resources of superior boar for the production of commercial pigs. For practical application, it is essential to investigate whether the clones and their progeny keep behaving better than the “normal boars”, considering that in vitro culture and transfer manipulation would cause a series of harmful effects to the development of clones. In this study, 59,061 cloned embryos were transferred into 250 recipient sows to produce the clones of superior Pietrain boars. The growth performance of 12 clones and 36 non-clones and the semen quality of 19 clones and 28 non-clones were compared. The reproductive performance of 21 clones and 25 non-clones were also tested. Furthermore, we made a comparison in the growth performance between 466 progeny of the clones and 822 progeny of the non-clones. Our results showed that no significant difference in semen quality and reproductive performance was observed between the clones and the non-clones, although the clones grew slower and exhibited smaller body size than the non-clones. The F1 progeny of the clones showed a greater growth rate than the non-clones. Our results demonstrated through the large animal population showed that SCNT manipulation resulted in a low growth rate and small body size, but the clones could normally produce F1 progeny with excellent growth traits to bring more economic benefits. Therefore, SCNT could be effective in enlarging the merit genetics of the superior boars and increasing the economic benefits in pig reproduction and breeding.

In situ generation of human brain organoids on a micropillar array

Lab on a Chip ◽  
2017 ◽  
Vol 17 (17) ◽  
pp. 2941-2950 ◽  
Author(s):  
Yujuan Zhu ◽  
Li Wang ◽  
Hao Yu ◽  
Fangchao Yin ◽  
Yaqing Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Brain ◽  
Pluripotent Stem Cells ◽  
3D Culture ◽  
In Situ Generation ◽  
Induced Pluripotent ◽  
High Throughput Manner

We present a simple and high throughput manner to generate brain organoids in situ from human induced pluripotent stem cells on micropillar arrays and to investigate long-term brain organogenesis in 3D culture in vitro.

Effects of Obesity and Small Body Size on Operative and Long-Term Outcomes of Coronary Artery Bypass Surgery: A Propensity-Matched Analysis

2005 ◽  
Vol 79 (6) ◽  
pp. 1976-1986 ◽  
Author(s):  
Robert H. Habib ◽  
Anoar Zacharias ◽  
Thomas A. Schwann ◽  
Christopher J. Riordan ◽  
Samuel J. Durham ◽  
...  
Keyword(s):  
Body Size ◽  
Coronary Artery ◽  
Coronary Artery Bypass ◽  
Coronary Artery Bypass Surgery ◽  
Bypass Surgery ◽  
Artery Bypass ◽  
Small Body ◽  
Small Body Size ◽  
Long Term Outcomes

scholarly journals Self-repopulating recipient bone marrow resident macrophages promote long-term hematopoietic stem cell engraftment

Blood ◽  
2018 ◽  
Vol 132 (7) ◽  
pp. 735-749 ◽  
Author(s):  
Simranpreet Kaur ◽  
Liza J. Raggatt ◽  
Susan M. Millard ◽  
Andy C. Wu ◽  
Lena Batoon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Key Points ◽  
Bone Marrow Engraftment

Key Points Recipient macrophages persist in hematopoietic tissues and self-repopulate via in situ proliferation after syngeneic transplantation. Targeted depletion of recipient CD169+ macrophages after transplant impaired long-term bone marrow engraftment of hematopoietic stem cells.

Reproductive performance in a phyletic dwarf, the spider crab Petramithrax pygmaeus (Bell, 1836) (Decapoda: Brachyura: Mithracidae)

2020 ◽  
Vol 40 (3) ◽  
pp. 325-329
Author(s):  
Joshua T Fields ◽  
Hayden K Mullen ◽  
Clayr M Kroenke ◽  
Kyla A Salomon ◽  
Abby J Craft ◽  
...  
Keyword(s):  
Body Size ◽  
Reproductive Performance ◽  
Related Species ◽  
Egg Production ◽  
Marine Invertebrates ◽  
Small Body ◽  
Reproductive Investment ◽  
Spider Crab ◽  
Closely Related Species ◽  
Body Sizes

Abstract The spider crab Petramithrax pygmaeus (Bell, 1836), a phyletic dwarf, was used to test predictions regarding reproductive performance in small marine invertebrates. Considering the disproportional increase in brooding costs and the allometry of egg production with increasing body size, it was expected that this minute-size species would produce large broods compared to closely related species that attain much larger body sizes. Fecundity in P. pygmaeus females carrying early and late eggs varied, respectively, between 17 and 172 eggs crab–1 (mean ± SD = 87.97 ± 48.39) and between 13 and 159 eggs crab–1 (55.04 ± 40.29). Females did not experience brood loss during egg development. Egg volume in females carrying early and late eggs varied, respectively, between 0.13 and 0.40 mm3 (0.22 ± 0.07) and between 0.15 and 0.42 mm3 (0.26 ± 0.06 mm3). Reproductive output (RO) varied between 0.91 and 8.73% (3.81 ± 2.17%) of female dry body weight. The RO of P. pygmaeus was lower than that reported for closely related species with larger body sizes. The slope (b = 0.95 ± 0.15) of the line describing the relationship between brood and parental female dry weight was not statistically significant from unity. Overall, our results disagree with the notion that the allometry of gamete production and increased physiological costs with increased brood size explain the association between brooding and small body size in marine invertebrates. Comparative studies on the reproductive investment of brooding species belonging to monophyletic clades with extensive differences in body size are warranted to further our understanding about disparity in egg production in brooding marine invertebrates.

Xenogeneic and endogenous spermatogenesis following transplantation of rat germ cells into testes of immunocompetent mice

2012 ◽  
Vol 24 (2) ◽  
pp. 337 ◽  
Author(s):  
Ning Qu ◽  
Munekazu Naito ◽  
Jun Li ◽  
Hayato Terayama ◽  
Shuichi Hirai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Immune Response ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Recipient Mouse ◽  
Immunocompetent Mice ◽  
Rat Spermatogenesis ◽  
Privileged Site

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and are characterised by their ability to self-renew and to produce differentiated progeny that form spermatozoa. It has been demonstrated that rat spermatogenesis can occur in the seminiferous tubules of congenitally immunodeficient recipient mice after transplantation of rat SSCs. However, the testis is often viewed as an immune-privileged site in that autoimmunogenic antigens on germ cells do not normally elicit an immune response in situ. In the present study, we tried to transplant rat SSCs into immunocompetent mice after depletion of their own germ cells by means of busulfan. The results showed that some transplanted SSCs could undergo complete spermatogenesis in recipient mouse testes, the rat spermatozoa being detected in 7 of 28 recipient epididymides. A significant increase in mouse spermatozoa was also noted in all 28 epididymides of recipient mice regardless of whether rat spermatozoa were concurrently present or not. These results suggest that transplanted rat SSCs can be tolerated in the testes of immunocompetent mice and that the transplantation of rat SSCs stimulates endogenous spermatogenesis in the recipient mice.

285 VIABILITY AND GENE EXPRESSION OF MESENCHYMAL STEM CELLS CRYOPRESERVED WITH DIFFERENT CRYOPROTECTANTS

2008 ◽  
Vol 20 (1) ◽  
pp. 222 ◽  
Author(s):  
M. K. Kim ◽  
S. A. Ock ◽  
B. G. Jeon ◽  
J. H. Cho ◽  
G. J. Rho
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Expression Patterns ◽  
Long Term Storage ◽  
Expression Of Genes ◽  
Cell Tissue ◽  
Fetal Bovine

Long-term storage of stem cells with self-renewal plays a pivotal role in cell tissue engineering, which could be a novel option for improving regenerative diseases. However, the choice of a selective cryoprotectant is still to be addressed. Dimethyl sulfoxide (DMSO), which in current practice is the most widely used as a cryoprotectant for cell freezing, is known to have toxic side effects (Wang et al. 2007 Cryobiology 55, 60–65). In this study, therefore, the effect of two different cryoprotectants, used alone or in combination, on frozen–thawed porcine mesenchymal stem cells (MSCs) was investigated by evaluating their viability, apoptosis, and gene expression patterns. MSCs isolated from bone marrow were cultured in advanced Dulbecco's modified Eagle medium (ADMEM) supplemented with 10% fetal bovine serum (FBS) and characterized by the expression of alkaline phosphatase (AP) activity and cell-surface antigen profiles (CD105 as positive, CD45 and CD133 as negative markers). Subconfluent cultures of MSCs (1 � 106 cells mL–1) at 4–5 passages were equilibrated in different cryoprotectants: (1)ADMEM supplemented with 10% DMSO, (2) ADMEM supplemented with 1.5 m ethylene glycol (EG), and (3) ADMEM supplemented with 0.75 m EG and 5% DMSO, and frozen in a programmable freezer. The straws were cooled at –4�C min–1 from 25�C to –7�C. After being seeded, the straws were further cooled to –35�C at a –0.6�C min–1 ramp rate, and then immediately plunged into LN2 and stored at least a week. After thawing at 37�C in a water bath, viability and apoptosis of cells were analyzed by flow cytometry using an In Situ Cell Death Detection kit (Roche, Mannheim, Germany). Expression of HSP70, Nanog, and β-actin was analyzed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), and compared to that of non-cryopreserved MSCs. Doubling time of cryopreserved MSCs was higher than for non-cryopreserved MSCs. There were no significant differences among cryopreserved groups. The rates of viability and apoptosis of non-cryopreserved MSCs (91% and 4.71%, respectively) was significantly (P < 0.05) higher than for MSCs cryopreserved with 10% DMSO, 1.5 m EG, or the 0.75 m EG and 5% DMSO mixture (71.78% and 3.45%, 70.09% and 3.22%, 68.97% and 2.42%, respectively), but the rates among different cryopreserved treatments did not differ. After thawing, expression of HSP70, Nanog, and β-actin in cryopreserved MSCs showed patterns similar to those of non-cryopreserved MSCs. In conclusion, the present study indicates that EG is an alternative cryoprotectant for cryopreservation of porcine MSCs. Further studies are needed to evaluate the possible effects on the expression of genes related to viability and apoptosis in MSCs cryopreserved with different cryoprotectants.

229 LONG-TERM PROPAGATION AND CRYOPRESERVATION OF CAT SPERMATOGONIAL STEM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 246
Author(s):  
L. M. Vansandt ◽  
M. Dickson ◽  
R. Zhou ◽  
L. Li ◽  
B. S. Pukazhenthi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Domestic Cat ◽  
Feeder Cell ◽  
Cell Clusters ◽  
Fetal Fibroblasts

Spermatogonial stem cells (SSC) are unique adult stem cells that reside within the seminiferous tubules of the testis. As stem cells, SSC maintain the ability to self-replicate, providing a potentially unlimited supply of cells and an alternate source for preservation of the male genome. While self-renewing, long-term SSC culture has been achieved in mice, there is virtually no information regarding culture requirements of felid SSC. Therefore, the objectives of this study were to (1) evaluate the ability of 3 feeder cell lines to support germ cell colony establishment in domestic cats (Felis catus), and (2) assess long-term culture using the best feeder(s). Cells isolated enzymatically from peripubertal cat testes (n = 4) and enriched by differential plating were cultured on mouse embryonic fibroblasts (STO line), mouse-derived C166 endothelial cells, and primary cat fetal fibroblasts (cFF). Colony morphology was assessed every other day and immunocytochemistry (ICC) was performed to investigate expression of SSC markers. At 5 days in vitro (DIV), a cluster forming activity assay was used to estimate the number of SSC supported by each feeder cell line. Differences among treatments were compared using Tukey-Kramer adjustment for pair-wise mean comparisons. Data were expressed as mean cluster number ± SE per 105 cells input. When cultured on STO feeders, cat germ cells were distributed as individual cells. On both C166 cells and cFF feeders, germ cell clumps (morphologically consistent with SSC colonies in other species) were observed. Immunocytochemistry revealed that the single germ cells present on STO feeders were positive for UCHL1 and weakly expressed PLZF and OCT4. Cells within the germ cell clumps on C166 cells and cFF co-expressed all 3 SSC markers. The C166 cells supported a higher number of germ cell clusters (77.4 ± 13.8) compared with STO (3.5 ± 1.1, P = 0.0003) or cFF (22.7 ± 1.0, P = 0.0024). Therefore, subsequent subculture experiments were performed exclusively with C166 feeder layers. Cultures from 2 donors were passaged at 12 DIV and periodically as needed thereafter. Germ cell clumps consistently reestablished following each subculture and immunocytochemistry analysis confirmed maintenance of all 3 SSC markers. Cells were also positive for alkaline phosphatase activity. Cells that had been cryopreserved in culture medium with 5% (vol/vol) dimethyl sulphoxide after144 DIV (7 passages) were thawed and cultured for an additional 18 days. These cells continued to express SSC markers and form germ cell clusters. Taken together, these data demonstrate that C166 feeder cells can facilitate colony establishment and in vitro propagation of germ cell clumps in the domestic cat. This represents an important first step towards attainment and optimization of a long-term SSC culture system in the cat. This system would provide a mechanism to explore regulation of spermatogenesis, test species-specific drugs, and produce transgenic biomedical models.

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