A critical role for hyaluronan in sheep embryo development and implantation

怀孕的建立需要一个好的胚胎和一个处于正确阶段的子宫来接受它。很多时候，胚胎不会植入，因为胚胎不正常或子宫没有为接受胚胎做好适当的准备。生育率低导致许多人遭受痛苦，并给农民带来重大经济损失。这个项目是关于确定在发育的关键时期，导致胚胎植入子宫壁的母亲和胚胎（所谓的胚泡）之间对话的性质。我们认为，一种复杂的糖分子，称为Hybryonan（HA），存在于大多数动物组织中，在母体-胚胎通讯系统中起着至关重要的作用。来自我们和其他人的信息表明，HA在这一过程中起着非常重要的作用，我们有证据表明，修饰的HA可以帮助绵羊胚胎着床。我们将研究其在植入中的作用。HA究竟如何向母亲发出胚胎正在植入的信号是我们将试图回答的另一个问题。我们认为孕激素（胚胎生长和着床期间的主导激素）对HA的产生和功能至关重要。为了回答第二个问题，我们将研究HA是如何产生的，以及它是如何在母体-胚胎通讯系统中变得活跃的。这项申请的目的是确保所需的资金，以进一步研究HA在胚胎植入过程中的功能。我们的研究需要用动物做实验。然而，我们也将在项目的很大一部分中使用来自屠宰场的动物组织，从而将项目所需的动物数量保持在绝对最低水平。我们计划进行以下实验：1.将使用两种方法来确定孕酮对HA产生的影响：A）在培养皿中用孕酮处理子宫细胞。B）羊的卵巢被切除（卵巢产生孕酮），因此被剥夺了孕酮，将接受孕酮替代。将收集培养物中的子宫细胞以及孕酮/给药绵羊的子宫和输卵管，并分析HA和HA修饰酶。2.将使用两种方法来产生胚胎并确定HA对胚胎发育至囊胚期和囊胚质量的影响：A）将允许用激素处理以诱导多次排卵的绵羊交配并自然怀孕。B）从屠宰场卵巢中获得卵母细胞，并与精子混合以生成胚胎（IVF）。通过任何一种方法产生的胚胎都将暴露于已知会影响HA水平的化学物质中。将定量监测胚胎发育至囊胚期，并在胚胎染色后评估囊胚质量。3.将使用两种方法来确定HA对胚泡植入的作用。A）囊胚，在实验室中产生，并将与子宫细胞一起培养。将在第14 - 17天期间添加改变HA水平的药剂，此时将在子宫中发生植入。将检查胚胎的发育和子宫细胞的HA和相关分子的产生。B）在预期胚胎附着日（交配后第15天）前一天，用类似试剂输注自然交配绵羊的子宫。在屠宰一半绵羊后（第17天）收集子宫组织，以评估胚泡附着并研究与胚胎着床相关的已知基因和蛋白质的表达。剩余绵羊将在第35天屠宰，以评估胎仔存活率和妊娠率。该项目将有可能导致体外受精或自然受孕后生育能力的改善。

The establishment of pregnancy needs both the presence of a good embryo and a uterus at the correct stage to receive it. Very often, the embryo will not implant because either the embryo is abnormal or the uterus is not properly prepared to receive the embryo. Poor fertility leads to much human suffering and a significant economic loss for farmers. This project is about identifying the nature of the dialogue between the mother and the embryo (so-called blastocyst) during the critical period of development that leads up to the implantation of the embryo in the wall of the uterus. We believe that a complex sugar molecule called Hyaluronan (HA) that is present in most animal tissues plays a vital role within the mother-embryo communication system. Information from ourselves and others suggests that HA has a very important role in this process, and we have evidence that modified HA can assist embryo implantation in sheep. We shall investigate its role in implantation. Exactly how HA works in signalling to the mother that an embryo is implanting is a further question that we will try to answer. We believe that progesterone (dominant hormone during the period of embryo growth and implantation), is vital for HA production and function. In order to answer this second question we will investigate how HA is made, and how it is processed to become active in the mother-embryo communication system. The purpose of this application is to secure the funding required to further our investigation into the function of HA in the embryo implantation process. Our investigation requires that we use animals for the experiments. However, we will also use animal tissues from abattoirs for a significant part of the project thus keeping the number of animals required for the project to an absolute minimum. We have planned the following experiments for these investigations: 1. Two methods will be used to determine the effects of progesterone on HA production: A) Uterine cells will be treated with progesterone in culture dish. B) Sheep which have had their ovaries removed (ovaries produce progesterone) and therefore have been deprived of progesterone, will receive progesterone replacement. Uterine cells from the culture, as well as the uterus and oviducts of the progesterone /treated sheep, will be collected and analysed for HA as well as HA modifying enzymes. 2. Two methods will be used to generate embryos and to determine the effects of HA on development of embryos to blastocyst stage and blastocyst quality: A) Sheep treated with hormones to induce multiple ovulations will be allowed to mate and become pregnant naturally. B) Oocytes will be obtained from abattoir ovaries and mixed with sperm to generate embryos (IVF). Embryos produced through either method will be exposed to chemicals that are known to affect the levels of HA. Embryo development to blastocyst stage will be quantitatively monitored and the quality of the blastocysts will be assessed after embryo staining. 3. Two methods will be used to determine the role of HA on blastocyst implantation. A) Blastocysts, produced in the lab and will be cultured together with uterine cells. Agents that modify levels of HA will be added during days 14-17, when implantation would occur in the uterus. The embryos will be examined for development and the uterine cells for their production of HA and associated molecules. B) Uterus in naturally mated sheep will be infused with similar agents one day before the expected day of embryo attachment (day 15 after mating). Uterine tissue will be collected after slaughter of half the sheep (day 17) to assess blastocyst attachment and to study of expression of known genes and proteins related to embryo implantation. The remainder of the sheep will be slaughtered on day 35 to assess foetal survival and pregnancy rate. This project will potentially lead to improvements in fertility following either in vitro fertilization or natural conception.