Progress 09/01/13 to 08/31/17
Outputs
Target Audience:Academic research community, in particular the animal breeding sector, and commercial animal breeding industry. Changes/Problems:We found that localized testicular irradiation was ineffective at preparing recipient bulls for donor spermatogonial stem cell transplantation. Thus, we devised a new strategy that involves CRISPR/Cas9 based gene editing. This strategy is technically challenging and requires significant optimization, especially in non-rodent species like cattle. We were able to develop an efficient pipeline for CRISPR/Cas9 gene editing in bovine embryos but ran out of time and money to produce animals from the embryos. What opportunities for training and professional development has the project provided?The project allowed for a Fullbright Scholar from Pakistan to conduct his PhD training at Washington State University. The project also allowed for the principal investigator and a graduate student to attend several confereces in the area of animal reprduction. How have the results been disseminated to communities of interest?One manuscript was published regarding cultured bovine spermatogonia and several others are in preparation. In addition, several abstracts of the research were presented as posters at science conferences and symposia. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1 was achieved by evaluating two different doses of localzied testicular irradiation (LTI) treatment with 4 month old bull calves. At puberty, the animals lacked spermatozoa in the ejaculate suggesting elimination of the germline. However, we discovered that testosterone concentration in the serum was significantly reduced compared to control bulls indicating damage to the steroid producing Leydig cell population. This collateral complication reduces the usefullness of LTI treated bulls to serve as recipients for germ cell transplantation becuase testosterone is required to sustain normal spermatogenesis. A manuscript detailing these findings is in preparation. For Goal 2, we assessed the capacity of LTI treated bulls to serve as recipients for spermatogonial transplantation. Despite having reduced testosterone production, we were able to measure engraftment of cultured spermatogonia 6 months after transplantation. However, complete spermatogenesis did not occur and was halted at the metiotic stage of germ cell maturation which is indicative of a reduced testosterone environment. Nevertheless, the findings demonstrate that primary cultures of bovine undifferentiated spermatogonia retain the capacity to function as stem cells by engrafting in a recipient testis following transplantation. Next, we developed a new strategy to prepare recipient bulls that circumvented the issue of inducing deleterious effects on the somatic support cell populations (i.e. Leydig cells). We chose to pursue a genetic engineering strategy to produce bulls that are germline sterile but retain normal somatic support cell function. To achieve this, we utilized the CRISPR/Cas9 gene editing strategy as a means to ablate the expression of a germ cell specific gene in zygotes. We desgined sgRNAs to target the NANOS2 gene that is known to be expressed specifically in male germ cells in all mammalian species. Importantly, ablation of the gene in mice leads to apoptosis of germ cells during early development and males are sterile in adulthood. We validated the efficacy of several sgRNAs for producing INDELS of the gene in primary cultures of bovine embryonic fibroblasts followed by confirming the editing efficiency in bovine embryos. The outcomes have demonstrated an efficiency of 80% in producing blastocysts stage embryos in which both NANOS2 alleles contain large deletions. At the end of the project period, we have created a large cryobank of edited embryos that are ready for transfer to recipient females for generating NANOS2 knockout bulls. We exect these animals to be ideal surrogates for transplantation with donor spermatogonial stem cells.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Oatley MJ, Kaucher AV, Yang QE, Waqas MS, Oatley JM. 2016. Conditions for Long-Term Culture of Cattle Undifferentiated Spermatogonia. Biol. Reprod. 95: 14.
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
Waqas MS, Kaucher AV, Oatley MJ, Tibary A, Oatley JM. Efficacy of localized testicular irradiation in preparing recipient bulls for spermatogonial stem cell transplantation. In Preparation.
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:Academic research community and animal breeding commercial industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Several manuscripts detailing the findings are in preparation. What do you plan to do during the next reporting period to accomplish the goals?In the coming reporting period, we are planning to generate bulls from embryos containing inactivated alleles for the targeted male germ cell specific gene of interest. Because cattle are a not litter bearing, to achieve this we must develop the means to conduct largescale microinjection of sgRNA and Cas9 RNA into bovine zygotes and transfer these to multiple recipient females. Withthis capability in place, weforesee beingable to generate at least one homozygous knockout bull that can be used as a recipient for transplantation of cultured spermatogonia. We anticipate that the outcomes will demonstratethe utility of usinggene editing technologiestoproduce germline sterile recipients for spermatogonial transplantation.
Impacts
What was accomplished under these goals?
Goal 1 was achieved by evaluating two different doses of localzied testicular irradiation (LTI) treatment with 4 month old bull calves. At puberty, the animals lacked spermatozoa in the ejaculate signifying elimination of the germline. However, we discovered that testosterone concentration in the serum was significantly reduced compared to control bulls indicating damage to the steroid producing Leydig cell population. This collateral complication reduces the usefullness of LTI treated bulls to serve as recipients for germ cell transplantation becuase testosterone is required to sustain normal spermatogenesis. A manuscript detailing these findgins is in preparation. Goal 2 is still in process. We assessed the capacity of LTI treated bulls to serve as recipients for spermatogonial transplantation. Despite having reduced testosterone production, we were able to measure engraftment of cultured spermatogonia 6 months after transplantation. However, complete spermatogenesis did not occur and was halted at the metiotic stage of germ cell maturation which is indicative of a reduced testosterone environment. Nevertheless, the findings demonstrate that primary cultures of bovine undifferentiated spermatogonia retain the capacity to function as stem cells by engrafting in a recipient testis following transplantation. We are now in the process of developing new strategies to prepare recipient bulls that will circumvent the issue of induing deleterious effects on the somatic support cell populations (i.e. Leydig cells). We have chosen to pursue a genetic engineering strategy to produce genetically modified bulls that are germline sterile but retain normal somatic support cell function. To achieve this, we are adapting the CRISPR/Cas gene editing strategy to cattle as a means to ablate the expression of a germ cell specific gene in zygotes. We have desgined sgRNAs to target a gene that is known to be expressed specifically in male germ cells in a range of eukaryotic organisms. Importantly, ablation of the gene in mice leads to apoptosis of germ cells during early developemnt and males are sterile in adulthood. We have validated the efficacy of several sgRNAs for producing INDELS of the gene in primary culturesof bovine embryonic fibroblasts. We have also assesed the utility of the sgRNAs in generating gene edited bovine emrbyosfollowing micorinjection into zygotes. The outcomeshave revelaed an efficiency of 65%in producing edited blastocysts.
Publications
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience: Academic research community and animal breeding commercial industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? An poster of the findings was presented at the annual meeting of the Society for the Study of Reproduction What do you plan to do during the next reporting period to accomplish the goals? We are actively developing a genetic engineering strategy to produce genetically modified bulls that are germline sterile but retain normal somatic support cell function. To achieve this, we are adapting the recently reported CRISPR/Cas gene editing strategy tocattle as a means to ablate the expression of a germ cell specific gene in zygotes. Mature bulls will then be produced from the edited zygotes and assessed for utility as recipients for transplantation with cultured spermatogonia. At present, we have designed and validated essential components for the CRISPR based editing and develop an in vitro system for producing bovine zygotes. In the coming reporting period we will be evaluating the efficiacy of the CRISPR based tools to induce editing of the targeted germ cell specific gene. If successful, this approach will provide ideal recipients fortransplantationof donor spermatogonial stem cells and will afford an immediately useful tool for the cattle industry to take advantage of superior genetics.
Impacts
What was accomplished under these goals?
Goal 1 was achieved by evaluating two different doses of localzied testicular irradiation (LTI) treatment with 4 month old bull calves. At puberty, the animals lacked spermatozoa in the ejaculate signifying elimination of the germline. However, we discovered that testosterone concentration in the serum was significantly reduced compared to control bulls indicating damage to the steroid producing Leydig cell population. This collateral complication reduces the usefullness of LTI treated bulls to serve as recipients for germ cell transplantation becuasetestosterone is required to sustain normalspermatogenesis. Goal 2 is still in process. We assessed the capacity of LTI treated bulls to serve as recipients for spermatogonial transplantation. Despite having reduced testosterone production, we were able to measure engraftment of cultured spermatogonia6 months after transplantation. However, complete spermatogenesis did not occur and was halted at the metiotic stage of germ cell maturation which is indicative of a reduced testosterone environment. Nevertheless, the findings demonstrate that primary cultures of bovine undifferentiated spermatogonia retain the capacity to function as stem cells by engrafting in a recipient testis following transplantation. We are now in the process of developing new strategies to prepare recipient bulls that will circumvent the issue of induing deleterious effects on the somatic support cell populations (i.e. Leydig cells). These strategies will hopefully provide an optimal recipient environment for both engraftment of transplanted spermatogonia and support continual spermatogenesis at alevel that confers fertility.
Publications
- Type:
Other
Status:
Accepted
Year Published:
2014
Citation:
Kaucher AV, Oatley MJ, Tibary A, Oatley JM. Evaluation of Localized Testicular Irradiation to Prepare Recipient Bulls for Spermatogonial Stem Cell Transplantation. Society for the Study of Reproduction Annual Meeting. July 2014. Poster Abstract.
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