Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle

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Abstract

Blastocyst complementation (BC) systems have enabled in vivo generation of organs from allogeneic pluripotent cells, compensating for an empty germ cell niche in gene knockout (KO) animals. Here, we succeeded in producing chimeric beef cattle (Wagyu) by transferring allogenic germ cells into ovaries using somatic cell nuclear transfer and BC technology. The KO of NANOS3 ( NANOS3 ^−/−) in Wagyu bovine ovaries produced a complete loss of germ cells. Holstein blastomeres ( NANOS3 ^+/+) were injected into NANOS3 ^−/− Wagyu embryos. Subsequently, exogenous germ cells ( NANOS3 ^+/+) were identified in the NANOS3 ^−/− ovary. These results clearly indicate that allogeneic germ cells can be generated in recipient germ cell-free gonads using cloning and BC technologies.

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Most cited references 33

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Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development.

J.J. Parrish, A. Krogenaes, J.L. Susko-Parrish (1995)

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P 0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P 0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.

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Production of cattle lacking prion protein.

Isao Ishida, Joaquín Castilla, Francisco Vargas … (2006)

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.

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Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs.

Hiroshi Nagashima, Kazuhiro Umeyama, Masaki Nagaya … (2013)

In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.

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Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group

ISSN (Electronic): 2045-2322

Publication date (Electronic): 27 April 2016

Publication date Collection: 2016

Volume: 6

Electronic Location Identifier: 24983

Affiliations

[1 ]Research and Development Group, Zen-noh Embryo Transfer Center , Kamishihoro, Hokkaido 080-1407, Japan

[2 ]Biological Sciences Section, Central Research Institute for Feed and Livestock of Zen-noh , Tsukuba, Ibaraki 300-4204, Japan

[3 ]Research and Development Group, Zen-noh Institute of Animal Health , Sakura, Chiba 285-0043, Japan

[4 ]Pig Breeding Laboratory, Central Research Institute for Feed and Livestock of Zen-noh , Kamishihoro, Hokkaido 080-1406, Japan

Author notes

[a ] ideta-atsushi@ 123456zennoh.or.jp

[b ] sendai-yutaka@ 123456zennoh.or.jp

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Publisher Item ID: srep24983

DOI: 10.1038/srep24983

PMC ID: 4846992

PubMed ID: 27117862

SO-VID: 654a4160-4d39-42b6-94e7-2f86ac61e6a0

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Generation of exogenous germ cells in the ovaries of sterile NANOS3-null beef cattle

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Gamma Delta T cells

Most cited references 33

Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development.

Production of cattle lacking prion protein.

Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs.

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