Cloned Embryos from Semen. Part 1: In Vitro Proliferation of Epithelial Cells on Embryonic Fibroblasts after Isolation from Semen by Gradient Centrifugation

Department of Animal Sciences, LSU Agricultural Center, Baton Rouge, Louisana 70803, USA.
Cloning and Stem Cells (Impact Factor: 2.66). 04/2008; 10(1):143-60. DOI: 10.1089/clo.2007.0067
Source: PubMed


Although epithelial-like somatic cells have been previously isolated from semen, cell proliferation rates were low. Culture of whole semen samples resulted in loss of potentially valuable spermatozoa. The aims of the present study were to: (1) isolate somatic cells from semen, while preserving sperm viability, and (2) optimize in vitro culture conditions for semen-derived epithelial cells. Density gradient centrifugation of washed ejaculates of two rams (Ovis aries) (n = 24) and one eland bull (Taurotragus oryx) (n = 4) was performed using a three-layer discontinuous Percoll column consisting of 90% (P-90), 50% (P-50), and 20% (P-20) Percoll. In vitro culture and Trypan Blue staining indicated that live somatic cells settled in the P-20 layer. Nonmotile spermatozoa were recovered at the P-50 and P-90 interfaces, whereas motile spermatozoa were collected in the pellet from the P-90 layer. Subsequently, somatic cells isolated from the P-20 layer were plated either on inactivated 3T3 mouse embryonic fibroblast feeder layers, collagen-coated plates with 3T3 feeder cell inserts, or on collagen-coated plates. Initial somatic cell plating was similar among treatments, but proliferation significantly increased when cocultured with 3T3 cells (feeder or insert). Furthermore, two different types of epithelial cells were obtained. The exact origin of the cells in the male reproduction system is uncertain and probably variable. The present method of cell isolation and in vitro culture may be of value for preserving endangered species. Specifically, cells isolated and cultured from cryopreserved semen of nonliving males could be used for producing embryos by somatic cell nuclear transfer.

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    • "In the second method, somatic cells were isolated using a procedure described earlier with modifications [9]. Briefly, fresh semen was diluted 1∶5 in DPBS and 2.5 ml of it was layered over a column of 20, 50, and 90% Percoll (2.5 ml each) in a 15 ml Falcon tube. "
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    ABSTRACT: Somatic cells were isolated from cryopreserved semen of 4 buffalo bulls, 3 of which had died over 10 years earlier, and were established in culture. The cells expressed cytokeratin-18, keratin and vimentin indicating that they were of epithelial origin. The cells were used as nuclear donors for hand-made cloning for producing buffalo embryos. The blastocyst rate and quality, as indicated by apoptotic index, were comparable among embryos produced using cells obtained from fresh or frozen-thawed semen or those obtained from conventional cell sources such as skin. Examination of the epigenetic status revealed that the global level of H3K27me3 but not that of H3K9/14ac and H4K5ac differed significantly (P<0.05) among cloned embryos from different bulls. The relative mRNA abundance of HDAC1, DNMT1, P53 and CASPASE 3 but not that of DNMT3a differed in cells and in cloned embryos. Following transfer of 24 cloned embryos produced from fresh semen-derived cells to 12 recipients, one calf weighing 55 kg, which is now 6 months of age and is normal, was born through normal parturition. Following transfer of 20 embryos produced from frozen-thawed semen-derived cells to 10 recipients, 2 became pregnant, one of which aborted in the first trimester; the calf born was severely underweight (17 kg), and died 12 h after birth. The ability of cells derived from fresh and frozen-thawed semen to produce live offspring confirms the ability of these cells to be reprogrammed. Our findings pave the way for restoration of highly precious progeny-tested bulls, which has immense economic importance, and can also be used for restoration of endangered species.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "Then, the semen sample was placed in a Styrofoam cooler with an ice pack in the bottom, and separated by a paper towel, for transport (~15 min) and subsequent preparation for processing in the laboratory. Somatic cells were isolated and cultured as described in Liu et al. (2009) and Nel-Themaat et al. (2008a; b) with minor modifications. Briefly, each semen sample was gently layered over a 3-layer gradient column (2.5 mL each of 20%, 50%, and 90% Percoll®) in a 15 mL conical tube and centrifuged at 400g for 20 min. "
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    ABSTRACT: Somatic cells in mammalian semen can be a potential source of nuclei for nuclear transfer to produce cloned animals. Somatic cells recovered from avian semen could also provide a source of cells for use in chimera formation or cloning of threatened and endangered birds. This type of assisted reproductive technology is especially important when a genetically unique animal has died and the only viable genetic material available is semen cryopreserved for artificial insemination and in vitro fertilization purposes. The usefulness of somatic cells obtained from fresh and frozen mammalian semen for nuclear transfer has already been evaluated, but still remains to be accomplished for avian semen. A non-invasive, or minimally invasive, technique to conserve avian genetic diversity via somatic cell collection from fresh avian semen, to our knowledge, has not been described. The present study investigated the use of fresh semen samples from domestic chickens (Gallus domesticus) (n = 7), i.e., white leghorn (n = 4) and silkie chickens (n = 3), as a source of somatic cells, specifically fibroblast-like cells and epithelial cells, for cytological analysis and somatic cell gene banking. Ultimately, somatic cell culture was successful for two of six selected samples (33.3%), with two of four white leghorn semen samples yielding cell cultures compared to 0 of 2 silkie chicken semen samples. Further research may include applying this technique to other avian species or address the effects of cryopreservation on the viability and DNA integrity of somatic cells derived in this manner.
    Full-text · Article · Dec 2013 · Avian biology research
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    ABSTRACT: Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.
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