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

• 批准号: 8157175
• 负责人: STEPHEN J O'BRIEN
• 金额: $ 45.14万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/8157175
• 关键词: Animals; Developmental Biology; reproductive

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 studiesCats 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.

家猫及其非家养亲属等模式物种的生殖系统为理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）已经采取了比较生理学的方法来描述猫科动物繁殖的各个方面，区分物种和允许行为的共同适应。此外，开发冷冻保存的经验方法已被评估，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）和这些密切相关的物种之间的胚胎移植。如何使用新技术来评估生殖健康的模型正在出现，以帮助确保基因多样性和繁殖濒危物种。非侵入性的激素代谢物监测分析，人工授精技术和基因组资源库已开发，以帮助研究检查猫科动物之间的适应性差异。重要的发现包括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-解冻压力下存活。卵子冷冻研究表明，猫卵对低温非常敏感。对猫精液中猫免疫缺陷病毒（FIV）（与艾滋病毒有关）传播的调查也在继续。一个相关的挑战是在一些家猫和许多濒危猫科动物的精液中发现的畸形精子的高发生率。这种情况被称为畸形精子症，在男性中很常见，限制了受精能力。虽然畸形精子症猫的精子与正常精子的DNA含量相同，但前者的鱼精蛋白含量减少，鱼精蛋白是一类在DNA稳定中起重要作用的核蛋白。精液也从几个野生型和疾病猫模型中收集和冷冻保存，用于通过IVF研究进行测试。猫具有开发转基因和基因敲除研究模型所需的短世代时间和大窝仔数。如果这些技术能成功地应用于猫，将大大提高研究人员开发人类遗传疾病猫模型的能力。 与宾夕法尼亚大学兽医学院的James Kehler合作，我们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的罗纳德McKay博士合作的同时，我们成功地使用逆转录病毒载体使猫成纤维细胞过度表达4种外源性人类基因，足以将其重编程为推定的多能干细胞（iPS）系。这些功能获得实验证明了编码多能性维持所需的转录因子Oct 4的人和猫直系同源物之间的功能保守性。此外，Oct 4也被认为是维持某些人睾丸生殖细胞肿瘤（TGCT）多能性所需的癌基因，是潜在的治疗靶点。我们正在使用这种重新编程的策略，从几种独特的遗传疾病猫模型中建立iPS系，这些模型被描述为LGD其他项目的一部分。从这些iPS系分化的组织将是体外研究这些疾病的有用资源。新的基因疗法，以纠正这些基因缺陷的反式将测试这些细胞首先在体外。此外，野生型猫iPS细胞系将通过同源重组和随后的核移植或嵌合囊胚注射实验用于基因打靶，以开发新的猫基因敲除疾病模型。