Implantation in the Domestic Ferret (Mustela Putorius)

该项目的目标是更好地了解胚胎在雪貂子宫内植入的过程。雪貂属于鼬科，鼬科还包括其他鼬类、熊类、浣熊、海豹和海象。许多熊纲动物的一个显著特征是严格控制着床时间。以黑熊为例，胚胎植入发生在受精几个月后。不幸的是，许多熊目动物，如大熊猫和黑足雪貂受到威胁或濒临灭绝。重要的是要更好地了解这些物种的繁殖，包括胚胎植入过程，以改进圈养繁殖技术。家雪貂数量众多，且已被驯化，是研究兽纲食肉动物胚胎着床的良好模型。初步研究表明，由卵巢产生的葡萄糖-6-磷酸异构酶（GPI）在雪貂胚胎着床过程中起着至关重要的作用。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，它会刺激神经细胞的生长。GPI还能刺激肿瘤细胞的运动和侵袭。这种作用可能类似于它对胚胎的作用，胚胎必须移动并侵入子宫并在那里植入。因此，通过研究GPI在胚胎着床中的作用，可以进一步了解GPI在肿瘤细胞中的作用，从而进一步了解癌症生物学。为了确定GPI在胚胎着床中的作用，我们将追求5个具体目标：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。首先，将通过手术切除怀孕雪貂的卵巢。然后给这些雪貂注射GPI，以确定GPI是否能触发着床。在第二个实验中，给怀孕的雪貂注射一种阻断GPI的抗体，以确定在没有GPI的情况下是否可以着床。2)怀孕的雪貂卵巢中GPI的表达模式是什么？我们将在不同的怀孕阶段切除雪貂的卵巢，以确定卵巢是否产生GPI，为植入做准备。我们将使用PCR来确定GPI基因作为mRNA的表达水平，并使用western blotting来确定GPI蛋白的合成量。3)妊娠期间卵巢分泌GPI吗？我们将从怀孕不同阶段的雪貂身上采集血液样本，以确定有多少GPI被分泌到血液中，并在那里被带到子宫。GPI的量可以通过测定其酶活性来测定。4)黄体GPI的靶组织是什么？为了确定GPI在哪里起作用，我们将在怀孕的各个阶段切除子宫和胚胎，并寻找GPI受体mRNA和蛋白质。我们还将标记GPI，并将其添加到子宫样本中并观察其结合的位置。5)黄体GPI的表达是否需要催乳素？雪貂胚胎着床需要垂体激素催乳素。因此，它可能是卵巢产生GPI的信号。为了验证这一点，我们将给怀孕的雪貂注射溴隐亭，这种药物可以阻断泌乳素。然后我们将确定雪貂卵巢是否仍然产生GPI蛋白和mRNA。这些研究将提高目前对雪貂和相关物种繁殖的认识，并增加对癌症生物学方面的了解。

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.