Reproductive Strategies in Animal Species Emphasizing Developmental Biology

强调发育生物学的动物物种的生殖策略

基本信息

批准号：
7965028
负责人：
STEPHEN J O'BRIEN
金额：
$ 49.73万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7965028
关键词：
Animals Artificial Insemination Assisted Reproductive Technology Behavioral Biological Assay Cat Diseases Cell Line Cell membrane Cells Collaborations Comparative Physiology Cryopreservation DNA Development Developmental Biology Disease Disease model Embryo Embryo Transfer Family Felidae Feline Immunodeficiency Virus Felis catus Fertilization Fertilization in Vitro Fibroblasts Freezing Gene Targeting Generations Genes Genetic Variation Genome Genomics Germ Cells HIV Hereditary Disease Hormones Human Human Genetics In Vitro Incidence Injection of therapeutic agent Investigation Knock-out Laboratories Litter Size Maintenance Mediating Methods Modeling Monitor Mutation National Institute of Neurological Disorders and Stroke Nuclear Protein Nuclear Proteins Oncogenes Orthologous Gene Pennsylvania Play Procedures Protamines Relative (related person)Reproduction Reproductive system Research Research Personnel Resources Retroviral Vector Role Schools Seminal fluid Shapes Stem cells Stress System Techniques Temperature Testicular Germ Cell Tumor Testing Time Tissues Transgenic Organisms Universities Veterinary Medicine Work blastocyst egg fitness gain of function gene therapy homologous recombination human disease male men new technology nuclear transfer pluripotency reproductive research study sperm cell therapeutic target transcription factor transmission process

项目摘要

The reproductive system of model species such as the domestic cat and its non-domestic relatives offers an unusual opportunity for understanding the changes and adaptations of genes that mediate species isolation and survival. The Laboratory of Genomic Diversity (LGD) has undertaken a comparative physiology approach to describe the aspects of feline reproduction that discriminate between species and allow for behavioral co-adaptation. In addition, empirical methods to develop cryopreservation have been assessed to optimize assisted reproductive technologies in these species, including artificial insemination, in vitro fertilization (IVF) and embryo transfer between these closely related species. Models of how to use new technologies to assess reproductive fitness are emerging to help insure gene diversity and propagate endangered species. Non-invasive hormone metabolite monitoring assays, artificial insemination techniques and genome resource banking have been developed to aid in studies examining the adaptive differences among the Felidae. Significant discoveries include the finding that standard cooling techniques for cat sperm result in extensive cell membrane damage, allowing the creation of slower, more effective cooling procedures. Sperm from males producing many malformed cells are less likely to survive cooling-freezing-thawing stress. Egg freezing studies reveal that cat eggs are highly sensitive to cool temperatures. Investigations also continue on the transmission of Feline Immunodeficiency Virus (FIV) (related to HIV) in cat semen. A related challenge is the high incidence of abnormally shaped sperm found in the semen of some domestic cats and many endangered cat species. This condition, known as teratospermia and common in men, limits fertilization capacity. Although sperm from teratospermic cats were found to have the same amount of DNA as normal sperm, the former have decreased amounts of protamine, a class of nuclear proteins that play an important role in DNA stabilization. Semen has also been collected and cryopreserved from several wildtype and disease cat models for testing through IVF studies Cats have the short generation times and large litter sizes required for development of transgenic and knock-out research models. Successful adaptation of these techniques to the cat would greatly increase the ability of researchers to develop feline models of human genetic diseases. In collaboration with James Kehler (University of Pennsylvania, School of Veterinary Medicine), we have developed a new method to capture the genetic diversity of existing feline models of human genetic diseases. While working in collaboration with Dr. Ronald McKay at the NINDS, we have succeeded in using retroviral vectors to transduce feline fibroblasts to over-express 4 exogenous human genes sufficient to reprogram them into putative pluripotential stem cell (iPS) lines. These gain of function experiments demonstrate a conservation of function between the human and feline orthologs encoding the transcription factor Oct4 that is required for the maintenance of pluripotency. In addition, Oct4 has also been implicated as an oncogene required to maintain the pluripotentiality of some human testicular germ cell tumors (TGCTs) and is a potential therapeutic target. We are using this strategy of reprogramming to establish iPS lines from several of the unique cat models of genetic diseases being characterized as part of other projects at the LGD. Tissues differentiated from these iPS lines will be useful resources for studying these diseases in vitro. New gene therapies to correct these genetic defects in trans will be tested on these cells first in vitro. In addition, wildtype feline iPS cell lines will be used for gene targeting through homologous recombination and subsequent nuclear transfer or chimeric blastocyst injection experiments to develop new knock-out disease models of cats.

模型物种（例如家猫及其非家养近亲）的生殖系统为了解介导物种隔离和生存的基因的变化和适应提供了一个不寻常的机会。基因组多样性实验室 (LGD) 采用了比较生理学方法来描述猫科动物繁殖过程中区分物种和允许行为共同适应的各个方面。此外，还评估了开发低温保存的经验方法，以优化这些物种的辅助生殖技术，包括人工授精、体外受精 (IVF) 和这些密切相关物种之间的胚胎移植。 如何使用的模型评估生殖健康的新技术不断出现，以帮助确保基因多样性和繁殖濒危物种。非侵入性激素代谢物监测分析、人工授精技术和基因组资源库已被开发出来，以帮助研究猫科动物之间的适应性差异。重大发现包括发现猫精子的标准冷却技术会导致广泛的细胞膜损伤，从而可以创建更慢、更有效的冷却程序。产生许多畸形细胞的雄性精子不太可能在冷却-冷冻-解冻应激下存活。卵子冷冻研究表明，猫卵对低温高度敏感。对猫精液中猫免疫缺陷病毒 (FIV)（与 HIV 相关）传播的调查仍在继续。 一个相关的挑战是在一些家猫和许多濒临灭绝的猫科动物的精液中发现形状异常的精子的发生率很高。这种情况被称为畸形精子症，常见于男性，会限制受精能力。虽然畸形精子猫的精子被发现与正常精子具有相同数量的 DNA，但前者的鱼精蛋白含量减少，鱼精蛋白是一类核蛋白，在 DNA 稳定中发挥重要作用。还从几种野生型和疾病猫模型中收集并冷冻保存精液，用于通过体外受精研究进行测试 猫的世代时间短，窝产仔数大，这是开发转基因和敲除研究模型所需的。将这些技术成功应用于猫将大大提高研究人员开发人类遗传疾病猫模型的能力。我们与詹姆斯·凯勒（宾夕法尼亚大学兽医学院）合作，开发了一种新方法来捕获现有人类遗传疾病猫科动物模型的遗传多样性。在与 NINDS 的 Ronald McKay 博士合作时，我们成功地使用逆转录病毒载体转导猫成纤维细胞，使其过表达 4 个外源人类基因，足以将它们重新编程为假定的多能干细胞 (iPS) 系。这些功能获得实验证明了人类和猫科动物之间编码维持多能性所需的转录因子Oct4的功能保守性。此外，Oct4还被认为是维持某些人类睾丸生殖细胞肿瘤（TGCT）多能性所需的癌基因，并且是潜在的治疗靶点。我们正在使用这种重编程策略，从几种独特的遗传性疾病猫模型中建立 iPS 系，这些模型是 LGD 其他项目的一部分。从这些 iPS 系分化而来的组织将成为体外研究这些疾病的有用资源。纠正这些反式遗传缺陷的新基因疗法将首先在这些细胞上进行体外测试。此外，野生型猫iPS细胞系将用于基因打靶，通过同源重组和随后的核移植或嵌合囊胚注射实验，开发新的猫敲除疾病模型。