Preservation of ejaculated mouse spermatozoa

本申请的主要目的是开发一种简单有效的方法，通过使用保存的射出精子进行ICSI来保存有价值的小鼠基因组。本申请的假设是，射出的精子可以重复地从男性中取出，保存，然后用于ICSI，高效地产生活的后代。从小鼠中收集精子仅限于涉及安乐死的技术，并导致雄性死亡，阻止其进一步繁殖。一种重复获得精液的方法可以使雄性保持活力并能够繁殖。在具体目标1中，我们将测试通过ICSI将射出的保存精子注射到卵母细胞中是否可以高效地获得活的后代。我们将反复从几只C57 BL/6雄性动物中获得精子，并通过常规冷冻保存和简单冷冻（无冷冻保护）保存。我们将用ICSI将这些精子注射到C57 BL/6雌性的卵母细胞中， 胚胎我们将通过分析受精卵中的父系染色体来评估这些方法对精子DNA完整性的影响，并进行胚胎移植和产生活的后代。最后，我们将提出的技术应用于一个突变小鼠品系（azh -异常精子头部形状）与生育问题，以证明其在医学上重要的小鼠品系的保存有用。在具体目标2中，我们将评估该机制 精液保存的小鼠精子中染色体降解的研究。我们将探讨所观察到的现象，即精子暴露于子宫内容物更容易受到DNA损伤的生物学意义。我们将确定交配后子宫中存在的未知因素是否影响活精子或干扰无保护精子冷冻的条件。我们还将独立分析子宫内容物的两种化合物的作用：子宫液和精囊液。这项提议的意义在于，它将有力地测试使用保存的射出精子进行ICSI以维持有价值的小鼠基因组的新方法。这种方法将允许提高重建小鼠品系的总体效率，并将加速在科学界内共享给定的遗传物质（即新突变）。我们的期望是，本申请的结果将允许我们推荐使用保存的射出精子的ICSI作为保存有价值的小鼠基因组的简单而有效的方法。

The major goal of this application is to develop a simple and efficient method for preservation of valuable mouse genomes by using ICSI with preserved ejaculated spermatozoa. The hypothesis of this application is that ejaculated spermatozoa can be repeatedly retrieved from a male, preserved, and then used for ICSI yielding live offspring with high efficiency. The collection of spermatozoa from mice is limited to techniques that involve euthanasia and result in a death of the male, precluding him from further breeding. A method for repeatedly obtaining ejaculates allows for keeping the male alive and able to breed. In the Specific Aim 1 we will test if ejaculated preserved spermatozoa injected into the oocytes by ICSI allow obtaining live offspring with high efficiency. We will repeatedly obtain spermatozoa from several C57BL/6 males and preserve them by conventional eryopreservation and by simple freezing without cryoprotection. We will use ICSI to inject these spermatozoa into the oocytes from C57BL/6 females to produce embryos. We will evaluate the effects of these methods on sperm DNA integrity by analysis of patemal chromosomes in the zygotes and perform embryo transfer and produce live offspring. Finally, we will apply the proposed technology to one mutant mouse strain (azh - abnormal spermatozoon head-shape) with fertility problems to demonstrate its usefulness in preservation of a medically important mouse strain. In the Specific Aim 2 we will evaluate the mechanism of chromosome degradation in ejaculated preserved mouse spermatozoa. We will explore the biological significance of observed phenomenon that spermatozoa exposed to uterine content are more susceptible to DNA damage. We will establish if the unknown factor present in the uterus after mating affects live spermatozoa or rather interferes with the conditions of unprotected sperm freezing. We will also analyze independently the effects of two compounds of the uterine content: uterine fluid and seminal vesicle fluid. The significance of this proposal is that it will vigorously test the novel approach of using ICSI with preserved ejaculated spermatozoa to maintain valuable mouse genomes. This method will allow increasing the overall efficiency of reconstituting mouse strains and will accelerate sharing of given genetic material (i.e. a novel mutation) within the scientific community. Our expectations are that the results of this application will allow us to recommend ICSI with preserved ejaculated spermatozoa as a simple and efficient method for preservation of valuable mouse genomes.