Projects

CONTENTS

Introduction
Projects


The uterus is the maternal tissue which has evolved to ensure that the considerable energy expended on pregnancy in mammals has a high probability of resulting in viable offspring. Notwithstanding the central importance of the uterus in mammalian reproduction, the actual processes of placentation in mammals are widely disparate. Considerable evidence however, indicates that the epithelial cells lining the uterus are centrally important in the events of early pregnancy and in particular, of blastocyst attachment during the earliest events of implantation and placentation. Among the many maternal preparations for pregnancy, the plasma membrane of the uterine epithelial cells - which is the first site of contact between maternal and foetal tissues - undergoes a unique, hormonally-programmed series of shape changes resulting in a complete morphological and biochemical transformation which we know is essential if the blastocyst is to attach and placentation and pregnancy begin. Such alterations are not seen in the membranes of other cells.

My major interest has been to understand the molecular-structural aspects of all regions - apical and basolateral - of the plasma membrane of uterine epithelial cells, especially during early pregnancy and the period of blastocyst attachment, with a view to understanding the changed molecular organization which takes place during early pregnancy and how this contributes to uterine receptivity for attachment. I have also been interested in this phenomenon from a purely cytological viewpoint, seeing it as an opportunity to contribute to understanding of fundamental plasma membrane phenomena. Mammals have been widely studied in this context and my own work has used rats as the experimental animal with human tissue providing direct clinical relevance and extending the work into another species. In recent years however, I have wondered if such fundamental structural alterations in a key organelle as the plasma membrane of uterine epithelial cells might not be a characteristic of viviparity (live birth) in general across species and so I have extended my investigations into viviparous Australian lizards. This is of considerable evolutionary interest because while viviparity has evolved in mammals only once, it has evolved separately in lizards many times and there are many of these species in Australia. Very recent work directly confirms the existence of similar structural alterations in the plasma membrane of lizard species during early pregnancy to those seen in mammals.

The peri-implantation role of human uterine epithelium
S M Adams, C R Murphy (collaborators: D Saunders, Dept of Obstetrics & Gynaecology, RNSH Sydney)

This project is a broadly-based one which has a direct objective, improvements in patient treatment for a variety of female reproductive disorders. Biopsies of human uterine epithelium are obtained from various fertility specialists with whom the Laboratory collaborates on several projects in human uterine biology. The major thrust of this work is to find a morphological biomarker for implantation in the uterus. We work closely with clinicians and their patients with previous failed attempts at IVF. Our work strongly suggests that there is an individual response to endogenous hormones and we are having considerable success of pregnancy by manipulating the luteal phase of the reproductive cycle with supplemental hormone treatment on an individual basis in these patients.

Investigations of hormonal manipulation and morphological response is also done on other animals and correlation of results helps us better understand the human menopausal reaction and importantly the uterine response to various HRT strategies.. Microscopic study of biopsies from patients on different hormonal replacement regimes is used to evaluate the value of these regimes in restoring normal function to patients with non-functional ovaries and in restoring other aspects of normal physiological function. Recently, this work has expanded into evaluating hormonal replacement therapies for post-menopausal women and promises to provide a sound scientific basis for this important clinical option.

Effects of superovulatory drugs on the uterus and vagina
S M Adams, M Hosie, C R Murphy

Superovulatory drugs such as clomiphene citrate are commonly used to obtain eggs for subsequent fertilization and implantation in infertile women (IVF) and are very effective in producing these eggs. We have little idea, however, about their effects on other hormone- sensitive reproductive tissues such as the vagina and ovary. This research has shown major morphological effects on both these tissues and may explain why pregnancy rates are lowered in cycles exposed to the superovulatory drugs as well as indicate side-effects of their use. Future work would assess and analyze the effects of GnRH-a on the human uterus with respect to their possible deleterious affect on the normal morphological response required in IVF clinics if pregnancy rates are to improve.

The evolution of viviparity
S M Adams, S Liu, R Stewart, J Biazik, C R Murphy in collaboration with A/Prof M Thompson of Biological Science

This project studies reptiles to examine how live birth evolves from egg-laying from the uterine prerspective and changes in the placental structures. We are particularly interested in the cell biological structures which are common to the placentas of mammals and other animals which give live birth. Our model animals are several species of Australian lizards because while live birth has evolved once in mammals, it has evolved over one hundred times separately in lizards and many of these lizards are native to Australia making Australia an ideal laboratory in which to study the evolution of live birth. Our work to date has shown that the plasma membrane transformation is present in several species of lizards during early pregnancy as it is in mammals and that more particularly the key structures called uterodomes are also present in some lizards. These findings suggest that common cellular structures are found in the uteri of lizards and mammals during early pregnancy and suggests key cellular commonalities exist across amniotes in the evolution of live birth.

The role of tight junctions and cytoskeleton in uterine epithelial cells
M Orchard, S Liu, C R Murphy

To understand the dramatic morphological changes seen in the uterine epithelium at the time of blastocyst implantation we have studied cytoskeletal structures such as microvilli, terminal web, adherens junctions, desmosomes and tight junctions. We use methods to study cytoskeletal structures, e.g. including myosin S1 decoration of actin filaments, TEM (transmission electron microscopy), SEM (scanning electron microscopy), freeze fracture of the plasma membrane, immunofluorescence and immunohistochemistry (to label cytoskeletal proteins), and confocal and deconvolution microscopy. Our previous studies showed that the terminal web and microvilli are under maternal hormonal control, and that these structures adopt a particular conformation to allow the blastocyst to attach to the apical surface of uterine epithelial cells.

Currently this project is focusing on the tight junctions of uterine epithelial cells which control the movement of fluids and solutes across the epithelial layer. Thus in the uterus tight junctions play a role in controlling the environment in which the implanting blastocyst attaches to the apical surface of uterine epithelial cells. Recent studies of tight junctions in other tissues have shown that tight junctions are size and ion selective. This ability to exclude large molecules and particular ions depends on the composition of claudins (a family of 20 proteins) in the tight junctions of a particular epithelial layer. Our current work on the tight junctions of uterine epithelial cells during early pregnancy aims to find the relationship between hormonal control of epithelial cell receptivity to blastocyst attachment and the molecular composition of uterine epithelial cell tight junctions.

In conjunction with the Evolution of Viviparity project we are also examining tight junctions in uterine cells of viviparous reptilian species. This project uses immunofluorescence and TEM to study the role of tight junctions in regulating the transport of solutes from the mother to the embryo.

Aquaporins
L Lindsay, CR Murphy

Implantation is a highly regulated process that involves dramatic changes in the uterine epithelial cells as well as the luminal contents. Previous studies have shown a dramatic decrease in the amount of uterine luminal fluid at the time of implantation. Hence the aim of this project is to determine the mechanisms of fluid transport occurring in the uterus. Currently we are concentrating on the location and hormonal regulation of water channel proteins (aquaporins) which are believed to play an important role in this fluid reabsorption. In order to address these issues we have developed a new immunogold technique used at the electron microscope level to enable precise localisation of these proteins. In addition we routinely use immunofluorescence techniques as well as hormonal manipulation of animals to determine the hormonal control of this fluid transport. In the next few years this project will involve the use of PCR technology and in situ hybridisation techniques to investigate changes in the regulation of the aquaporin genes.

Endometriosis
R Stewart, R Ilad, S Fleming, CR Murphy.

Endometriosis is a painful, chronic disease that affects ? million women and girls in Australia and millions more worldwide. It occurs when tissue like that which lines the uterus (the endometrium) is found outside the uterus - usually in the abdomen, on the ovaries, fallopian tubes, and ligaments that support the uterus; the area between the vagina and rectum; the outer surface of the uterus; and the lining of the pelvic cavity.

Presently we have little idea why endometrial tissue grows in these abnormal sites. Our project is taking innovative new approaches to understanding the causes of the abnormal growth and is also trying to understand which components of the endometriotic tissue are most damaging to the ectopic sites. We are using several molecular and morphological approaches to the disease and are especially working on cytoskeletal proteins, abnormal cell death, the role of ubiquitin and are developing a range of new markers of the disease.

Structural relationships of membrane molecules
F Png, J Isaacs, C R Murphy

Using a variety of manipulative tools including freeze-fracture electron microscopy and cytochemistry, this project seeks to understand at the molecular anatomical level how plasma membrane molecules are structurally related. It has shown that in this particular membrane, carbohydrates and proteins for example, are much more heterogeneously related than in the much-studied red blood cell membrane. There may thus be more glycolipids in this membrane than is usual. Studies with cholesterol binding cytochemicals have also revealed a highly fluctuating concentration of this molecule during the implantation period with the highest levels at the receptive time. Since cholesterol is a powerful modulator of membrane fluidity, an increase as we have shown adds to our understanding of the dynamism this membrane displays in preparation for implantation.