Implantation in the Domestic Ferret (Mustela Putorius)

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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.