Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope

2007 ◽  
Vol 19 (4) ◽  
pp. 576 ◽  
Author(s):  
L. Nel-Themaat ◽  
M. C. Gómez ◽  
P. Damiani ◽  
G. Wirtu ◽  
B. L. Dresser ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cell Attachment ◽  
Gulf Coast ◽  
Somatic Cells ◽  
Cell Types ◽  
Ovis Aries ◽  
Sheep Milk ◽  
Immunocytochemical Detection ◽  
Culture Surface ◽  
Semen Donor

Semen and milk are potential sources of somatic cells for genome banks. In the present study, we cultured and characterised cells from: (1) cooled sheep milk; (2) fresh, cooled and frozen–thawed semen from Gulf Coast native (GCN) sheep (Ovis aries); and (3) fresh eland (Taurotragus oryx) semen. Cells attached to the culture surface from fresh (29%), cooled (43%) and slow-frozen (1°C/min; 14%) ram semen, whereas no attachment occurred in the fast-frozen (10°C/min) group. Proliferation occurred in fresh (50%) and cooled (100%) groups, but no cells proliferated after passage 1 (P1). Eland semen yielded cell lines (100%) that were cryopreserved at P1. In samples from GCN and cross-bred milk, cell attachment (83% and 95%, respectively) and proliferation (60% and 37%, respectively) were observed. Immunocytochemical detection of cytokeratin indicated an epithelial origin of semen-derived cells, whereas milk yielded either fibroblasts, epithelial or a mixture of cell types. Deoxyribonucleic acid microsatellite analysis using cattle-derived markers confirmed that eland cells were from the semen donor. Eland epithelial cells were transferred into eland oocytes and 12 (71%), six (35%) and two (12%) embryos cleaved and developed to morulae or blastocyst stages, respectively. In conclusion, we have developed a technique for obtaining somatic cells from semen. We have also demonstrated that semen-derived cells can serve as karyoplast donors for nuclear transfer.

63 IMPROVEMENT OF SEMEN-DERIVED EPITHELIAL CELL PROLIFERATION BY FIBROBLAST CO-CULTURE

2006 ◽  
Vol 18 (2) ◽  
pp. 140
Author(s):  
L. Nel-Themaat ◽  
M. C. Gómez ◽  
G. Wirtu ◽  
A. Cole ◽  
K. R. Bondioli ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Attachment ◽  
Gulf Coast ◽  
Growth Stimulation ◽  
Ovis Aries ◽  
Fibroblast Cells ◽  
Immunohistochemical Detection ◽  
3T3 Cells ◽  
Vimentin Expression ◽  
Feeder Layers

We previously isolated epithelial-like cells (ELC) from sheep (Ovis aries) and eland (Taurotragus oryx) semen (Nel-Themaat et al. 2004 Reprod. Fertil. Dev. 16, 152) and subsequently developed a system to separate ELC before plating (Nel-Themaat et al. 2005 Reprod. Fertil. Dev. 17, 314). Cells attached and proliferated in only 50% and 31% of the attempts, respectively. Therefore, the purposes of the present study were to improve ELC proliferation by co-culture with inactivated 3T3 mouse fibroblasts and to characterize the obtained cells. Semen fractions from two mature Gulf Coast Native rams (n = 20 ejaculates) and one common eland bull (n = 2 ejaculates) were plated on feeder layers (A), on collagen with feeder cell inserts (B), or on collagen alone (C). For B and C, cell attachment and division were assessed; proliferation and passage 1 (P1) confluence were evaluated for A, B, and C. No difference in attachment rates between B (80%) and C (70%) were found for ovine cells, but (P < 0.05) cells divided more times in B (80%) than in C (35%). All colonies in A (60%) and B (70%) reached P1 confluence and no difference was detected between A and B, but less proliferation (10%) and P1 confluence (5%) were observed in C. Therefore, contact between epithelial cells and feeders was not necessary for growth stimulation by the feeder cells. No difference among A, B, and C was detected for eland ELC proliferation (100%), but no P1 confluence was observed in C. Ram cells were subsequently characterized by immunohistochemical detection of keratin and vimentin, as well as morphology. The 3T3 cells cross-reacted with keratin, and characterization was thus performed mainly on morphology and vimentin expression. Distribution of vimentin microfilaments differed between different epithelial morphologies and fibroblasts. Expression in epithelial cells was faint and patchy in confluent colonies and located around cytoplasmic extremities in semi-confluent colonies. In 3T3 cells, expression was very prominent throughout the cytoplasm and around the nucleus. For treatments A and B, 63 and 57%, respectively, were characterized as only epithelial cells; 25 and 36%, respectively, appeared to contain a mixture of epithelial and fibroblast cells; and 13 and 7%, respectively, contained only fibroblast cells. Only one sample was evaluated from treatment C and only keratin was detected in the epithelial-like colony. We conclude that culture of semen-derived ELC is markedly improved by 3T3 fibroblast co-culture. Further research on conditioned media may simplify the system and reduce chances of 3T3 cell contamination.

In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Susa Shin ◽  
Kwang Sung Ahn ◽  
Seong-Jun Choi ◽  
Soon Young Heo ◽  
Hosup Shim
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Somatic Cells ◽  
Cell Types ◽  
Neural Precursor ◽  
Blastocyst Development ◽  
Donor Cells

SummaryUndifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.

INTERACTION OF S PROTEIN/VITRONECTIN WITH CULTURED ENDOTHELIAL CELLS: PROMOTION OF ATTACHMENT AND SPECIFIC BINDING

1987 ◽  
Author(s):  
K T Preissner ◽  
E Anders ◽  
G Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Cell Attachment ◽  
Specific Binding ◽  
Umbilical Vein ◽  
Attachment Site ◽  
Cell Types ◽  
S Protein ◽  
Specific Association ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Proteins

The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.

Cloned blastocysts produced by nuclear transfer from somatic cells in cynomolgus monkeys (Macaca fascicularis)

Primates ◽  
2007 ◽  
Vol 48 (3) ◽  
pp. 232-240 ◽  
Author(s):  
Junko Okahara-Narita ◽  
Hideaki Tsuchiya ◽  
Tatsuyuki Takada ◽  
Ryuzo Torii
Keyword(s):  
Nuclear Transfer ◽  
Macaca Fascicularis ◽  
Somatic Cells ◽  
Cynomolgus Monkeys

Quantitative Studies of Fibronectin Adsorption on Submicron Scaffolds

2012 ◽  
Author(s):  
Shawn Regis ◽  
Manisha Jassal ◽  
Sina Youssefian ◽  
Nima Rahbar ◽  
Sankha Bhowmick
Keyword(s):  
Surface Functionalization ◽  
Cultured Cells ◽  
Cell Attachment ◽  
Md Simulations ◽  
Cell Types ◽  
Biological Processes ◽  
Integrin Receptors ◽  
Tissue Engineering Scaffolds ◽  
Quantitative Studies

Fibronectin plays a crucial role in adhesion of several cell types, mainly due to the fact that it is recognized by at least ten different integrin receptors. Since most cell types can bind to fibronectin, it becomes involved in many various biological processes. The interaction of cells with ECM proteins such as fibronectin provides the signals affecting morphology, motility, gene expression, and survival of cells [1]. Fibronectin exists in both soluble and insoluble forms; soluble fibronectin is secreted by cells and exits in cell media or body fluids, whereas insoluble fibronectin exists in tissues or the extracellular matrix of cultured cells [2]. The ability to control adsorption of fibronectin on tissue engineering scaffolds would therefore play a huge role in controlling cell attachment and survival in vivo. This can be achieved through surface functionalization of the scaffolds. The goal of these studies is to use molecular dynamics (MD) simulations to mechanistically understand how fibronectin adsorption is enhanced by surface functionalization of submicron scaffolds.

Somatic cells of goat and sheep milk: Analytical, sanitary, productive and technological aspects

2007 ◽  
Vol 68 (1-2) ◽  
pp. 126-144 ◽  
Author(s):  
K. Raynal-Ljutovac ◽  
A. Pirisi ◽  
R. de Crémoux ◽  
C. Gonzalo
Keyword(s):  
Somatic Cells ◽  
Sheep Milk

Cloned rabbits produced by nuclear transfer from adult somatic cells

2002 ◽  
Vol 20 (4) ◽  
pp. 366-369 ◽  
Author(s):  
Patrick Chesné ◽  
Pierre G. Adenot ◽  
Céline Viglietta ◽  
Michel Baratte ◽  
Laurent Boulanger ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Somatic Cells

Nuclear transplantation and the control of gene activity in animal development

1970 ◽  
Vol 176 (1044) ◽  
pp. 303-314 ◽  
Keyword(s):  
Nuclear Transfer ◽  
Somatic Cells ◽  
Gene Activity ◽  
Protein Fractions ◽  
Nuclear Transplantation ◽  
Animal Development ◽  
Cytoplasmic Proteins ◽  
Intracellular Movement ◽  
Selective Accumulation ◽  
Structure And Metabolism

The transplantation of nuclei from differentiated or determined somatic cells to enucleated frogs’ eggs consistently leads to a complete and clearly recognizable change of gene activity. Within 1 to 2 h of nuclear transfer, somatic nuclei have come to resemble in structure and metabolism the zygote nuclei of fertilized eggs. The change in gene activity therefore takes place very soon after nuclear transfer and results from an effect of egg cytoplasm. The induced change in gene activity is associated with a selective accumulation of cytoplasmic proteins in transplanted nuclei. Examples are given of various ways in which nuclear transplantation and microinjection can be used to elucidate the intracellular movement of proteins and the effect of known protein fractions on gene activity.

scholarly journals 57EMBRYO DEVELOPMENT FOLLOWING INTERSPECIES NUCLEAR TRANSFER OF AFRICAN BUFFALO (SYNCERUS CAFFER), BONTEBOK (DAMALISCUS DORCUS DORCUS) AND ELAND (TAUROTRAGUS ORYX) SOMATIC CELLS INTO BOVINE CYTOPLASTS

2004 ◽  
Vol 16 (2) ◽  
pp. 150 ◽  
Author(s):  
M. Matshikiza ◽  
P. Bartels ◽  
G. Vajta ◽  
F. Olivier ◽  
T. Spies ◽  
...  
Keyword(s):  
South Africa ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Critically Endangered ◽  
African Buffalo ◽  
Syncerus Caffer ◽  
Cell Stage ◽  
Wildlife Species ◽  
Interspecies Nuclear Transfer

Wildlife conservation requires traditional as well as innovative conservation strategies in order to preserve gene and species diversity. Interspecies nuclear transfer has the potential to conserve genes from critically endangered wildlife species where few or no oocytes are available from the endangered species, and where representative cell lines have been established for the wildlife population while numbers were still abundant. The purpose of this study was to investigate the developmental ability of embryos reconstructed with transfer of somatic cells from the African buffalo (Syncerus caffer), bontebok (Damaliscus dorcus dorcus) and eland (Taurotragus oryx) to enucleated domestic cattle (Bos taurus) oocytes. Skin tissue from the three wildlife species were collected by surgically removing approx. 1.0×1.0cm ear skin notches from animals immobilized with a combination of etorphine hydrochloride (M99; South Africa) and azaperone (Stressnil, South Africa). The biopsies were placed into physiological saline and transported to the laboratory at 4°C within 2h, cleaned with chlorohexidine gluconate and sliced finely in Minimal Essential Medium supplemented with 10% fetal calf serum. The resultant tissue explants were treated as previously described (Baumgarten and Harley 1995 Comp. Biochem. Physiol. 110B, 37–46) and actively growing fibroblast cultures made available for the nuclear transfer process. Nuclear transfer was performed using the HMC technique (Vajta et al., 2003 Biol. Reprod. 68, 571–578) using slaughterhouse-derived bovine oocytes. Culture was performed in SOFaaci (Vajta et al., 2003 Biol. Reprod. 68, 571–578) medium supplemented with 5% cattle serum using WOWs (Vajta et al., Mol. Reprod. Dev. 50, 185–191). Two identical replicates were made with somatic cells of each species. After successful reconstruction, 57, 42 and 48 nuclear transferred and activated buffalo, bontebok and eland embryos were cultured, respectively. All except for 2 buffalo embryos cleaved; 22 (39%) developed to or over the 8-cell stage, and 2 (3.5%) of them to the blastocyst stage. All but 3 bontebok embryos cleaved, 17 (40%) developed to or over the 8-cell stage, but none of them reached the compacted morula or blastocyst stage. Sixteen (33%) of the eland embryos developed to or over the 8-cell stage with one (2%) reaching the blastocyst stage. In conclusion, buffalo, bontebok and eland embryos developed from reconstruction using their respective somatic cells combined with bovine cytoplasts, however, in vitro developmental ability to the blastocyst stage was limited. Additional basic research that establishes the regulative mechanisms involved with early preimplantation development together with optimising nuclear transfer techniques may have the potential to one day play a role in the conservation of critically endangered wildlife species.

The developmentally regulated ECM glycoprotein ISG plays an essential role in organizing the ECM and orienting the cells of Volvox

2000 ◽  
Vol 113 (24) ◽  
pp. 4605-4617
Author(s):  
A. Hallmann ◽  
D.L. Kirk
Keyword(s):  
Critical Role ◽  
Somatic Cells ◽  
Cell Types ◽  
Model System ◽  
Swimming Behavior ◽  
Truncated Form ◽  
Developmentally Regulated ◽  
Gene Encoding ◽  
Ecm Architecture ◽  
Multicellular Organisms

Volvox is one of the simplest multicellular organisms with only two cell types, yet it has a surprisingly complex extracellular matrix (ECM) containing many region-specific morphological components, making Volvox suitable as a model system for ECM investigations. ECM deposition begins shortly after inversion, which is the process by which the embryo turns itself right-side-out at the end of embryogenesis. It was previously shown that the gene encoding an ECM glycoprotein called ISG is transcribed very transiently during inversion. Here we show that the developmentally controlled ISG accumulates at the bases of the flagella right after inversion, before any morphologically recognizable ECM structures have yet developed. Later, ISG is abundant in the ‘flagellar hillocks’ that encircle the basal ends of all flagella, and in the adjacent ‘boundary zone’ that delimits the spheroid. Transgenic Volvox were generated which express a truncated form of ISG. These transgenics exhibit a severely disorganized ECM within which the cells are embedded in a highly chaotic manner that precludes motility. A synthetic version of the C-terminal decapeptide of ISG has a similar disorganizing effect, but only when it is applied during or shortly after inversion. We postulate that ISG plays a critical role in morphogenesis and acts as a key organizer of ECM architecture; at the very beginning of ECM formation ISG establishes an essential initial framework that both holds the somatic cells in an adaptive orientation and acts as the scaffold upon which the rest of the ECM can be properly assembled, assuring that somatic cells of post-inversion spheroids are held in orientations and locations that makes adaptive swimming behavior possible.

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