A critical role for hyaluronan in sheep embryo development and implantation

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.

The overall aim of this project is to determine the effects of hyaluronan (HA) on embryo quality, elongation and attachment in a sheep model. Incorporating parallel in vivo and in vitro studies, we hypothesise that progesterone (P4) activates HAS-3 and Hyal-2 to generate small HA fragments which increase blastocyst survival and quality by reducing apoptosis, and improve embryo attachment through induction of COX-2 and synthesis of PGE/I. Experiment 1. Effects of P4 on HA biosynthesis and degradation. Ovariectomised sheep and in vitro cultured uterine cells will be treated with P4. Uterine and oviductal tissues as well as secretions will be collected to extract HA and to measure its size and concentration by HPLC. Expression of HAS-3 and Hyal-2 will be assessed by quantitative RT-PCR and Western blotting. Experiment 2. Effects of HA size on embryo development and blastocyst quality. Sheep embryos, produced in vitro or after superovulation and natural mating, will be exposed to small or large HA, 4-MU (an inhibitor of HA synthesis) or Hyal-2. Development to blastocyst stage will be recorded and quality of the blastocysts will be assessed by differential staining and by counting the number of apoptotic cells in the trophoblast and the ICM. Experiment 3. Role of small HA fragments on embryo elongation and attachment: Blastocysts, produced in vitro and co-cultured on uterine cells, or produced from natural mating will be exposed to small and large HA molecules, 4-MU, NS-398 (COX-2 inhibitor) and Hyal-2 by supplementation in vitro from day 6 or in vivo via uterine infusion from day 14. Blastocysts will be assessed morphologically and by TEM for elongation and attachment. Uterine tissue will be collected on day 17 to assess the rate of blastocyst attachment and to study the expression of CD44, COX-2, IL-1 by immunohistochemistry and in situ hybridisation. Some of the sheep will be slaughtered on day 35 to determine foetal survival and pregnancy rate.