Implantation in the Domestic Ferret (Mustela Putorius)

该项目的目标是更好地了解雪貂胚胎植入子宫的过程。雪貂属于弓形目，也包括其他黄鼠狼、熊、浣熊、海豹和海象。许多弓形类物种的一个显着特征是严格控制植入的时间。例如，在黑熊中，胚胎植入发生在受精后几个月。不幸的是，许多北极物种，如大熊猫和黑脚雪貂，都受到了威胁或濒临灭绝。为了改进圈养繁殖技术，更好地了解这些物种的繁殖，包括胚胎植入过程，是很重要的。家雪貂数量丰富，家养驯化，是研究弓形目食肉动物胚胎着床的良好模型。初步数据表明，卵巢产生的葡萄糖-6-磷酸异构酶(GPI)是雪貂胚胎着床的关键。众所周知，GPI具有多种功能。这种蛋白质是将糖转化为能量所必需的，它还能刺激神经细胞的生长。GPI还可以刺激肿瘤细胞的运动和侵袭。这一作用可能类似于它对胚胎的影响，胚胎必须移动并侵入子宫才能在那里植入。因此，通过研究GPI在胚胎着床中的作用，可以对GPI在肿瘤细胞中的功能有更深入的了解，从而加深对肿瘤生物学的认识。为了确定GPI在胚胎着床中的作用，我们将追求5个特定的目标：1)雪貂着床是否需要GPI？我们将用两个实验来回答这个问题。在第一个实验中，将通过手术切除怀孕雪貂的卵巢。然后，这些雪貂将被注射GPI，以确定GPI是否可以触发植入。在第二个实验中，怀孕的雪貂将被给予一种阻断GPI的抗体，以确定是否可以在没有GPI的情况下植入。2)怀孕的雪貂卵巢中GPI的表达模式是什么？我们将在怀孕的不同阶段摘除雪貂的卵巢，以确定卵巢是否会产生GPI，为植入做准备。我们将使用聚合酶链式反应来确定GPI基因作为mRNA的表达水平，并用Western blotting来确定GPI蛋白的量。3)GPI在怀孕期间是从卵巢分泌的吗？我们将在怀孕的不同阶段采集雪貂的血液样本，以确定有多少GPI被分泌到血液中，在那里它可以被带到子宫。GPI的量可以通过测量其酶活性来测量。4)黄体GPI的靶组织是什么？为了确定GPI在哪里起作用，我们将移除怀孕不同阶段的子宫和胚胎，并寻找GPI受体的mRNA和蛋白。我们还将标记GPI，并将其添加到子宫样本中，观察它与哪里结合。5)黄体GPI的表达是否需要催乳素？垂体腺激素催乳素是雪貂胚胎着床所必需的。因此，它可能是向卵巢发出信号，使其产生GPI。为了测试这一点，我们将用抑制催乳素的药物溴隐亭来治疗怀孕的雪貂。然后我们将确定雪貂卵巢是否仍然制造GPI蛋白和mRNA。这些研究将改善目前对雪貂及其相关物种繁殖的了解，并增加对癌症生物学方面的理解。

The goal of this project is to understand better the process by which embryos implant in the uterus in ferrets. Ferrets are classified in the Arctoideae that also includes other weasels, bears, raccoons, seals and walruses. A remarkable characteristic of many arctoidean species is tight control of the timing of implantation. In the black bear, for example, embryo implantation occurs months after fertilization. Unfortunately, many arctoidean species, such as the giant panda and the black-footed ferret are threatened or endangered. It is important to gain a better understanding of reproduction in these species, including the process of embryo implantation, in order to improve captive breeding techniques. Domestic ferrets are an excellent model for the study of embryo implantation in arctoidean carnivores because they are abundant and domesticated. Preliminary data indicate that the protein glucose-6-phosphate isomerase (GPI), produced by the ovary, is crucial for embryo implantation in ferrets. It is already known that GPI has multiple functions. The protein is necessary to convert sugars to energy, and it stimulates nerve cell growth. GPI also stimulates movement and invasion by tumor cells. This action is probably similar to its effect on the embryo, which must move and invade the uterus to implant there. Therefore, by studying how GPI functions in embryo implantation, insights may be gained about GPI function in tumor cells, thus furthering knowledge of cancer biology. To determine the role of GPI in embryo implantation, we will pursue 5 specific aims:1) Is GPI required for implantation in the ferret? We will use two experiments to answer this question. In the first, ovaries will be surgically removed from pregnant ferrets. These ferrets will then be injected with GPI to determine whether GPI can trigger implantation. In the second experiment, pregnant ferrets will be given an antibody that blocks GPI to determine whether implantation can occur without GPI.2) What is the pattern of expression of GPI by the pregnant ferret ovary? We will remove ovaries from ferrets at different stages of pregnancy to determine whether the ovaries make GPI in preparation for implantation. We will use PCR to determine expression levels of the GPI gene as mRNA, and western blotting to determine how much GPI protein is being made.3) Is GPI secreted from the ovary during pregnancy? We will take blood samples from ferrets at different stages of pregnancy to determine how much GPI is being secreted into the blood, where it can be carried to the uterus. The amount of GPI can be measured by measuring its enzyme activity. 4) What are the target tissues for luteal GPI? To determine where GPI is acting, we will remove the uterus and embryos at various stages of pregnancy, and look for the GPI receptor mRNA and protein. We will also label GPI, and add it to uterine samples and observe where it binds. 5) Is prolactin required for the expression of luteal GPI? The pituitary hormone prolactin is needed for embryo implantation in ferrets. Therefore, it may be signaling the ovaries to make GPI. To test this, we will treat pregnant ferrets with the drug bromocriptine, which blocks prolactin. We will then determine whether the ferret ovaries still make GPI protein and mRNA.These studies will improve present knowledge of reproduction in ferrets and related species, and increase understanding of aspects of cancer biology.