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Biomedical subjects

Christopher R Murphy

Publications and source records attributed to Christopher R Murphy.

At least 19 recordsLinked to original sources

Uptake of dextran-FITC by epithelial cells of the chorioallantoic placentome and the omphalopleure of the placentotrophic lizard, Pseudemoia entrecasteauxii.

Placental nutrient provision has evolved in multiple lineages of squamate reptiles and although possible structural specializations for placentotrophy have been described in a variety of species, neither the pathways nor the mechanisms of placental transfer are known. Lizards of the Australian genus Pseudemoia are placentotrophic and have elaborate placental structures that are thought to enhance nutrient transfer. The chorioallantoic placenta, which occupies the embryonic hemisphere of the egg, is regionally diversified into a large area with low epithelial height and a smaller placentome with cuboidal or columnar epithelia. Both regions are underlain by an extensive vascular bed. The abembryonic hemisphere of the egg is covered by an omphaloplacenta, which is similar to the placentome in having cuboidal or columnar epithelia but with a different embryonic vascular supply. We tested the hypothesis that embryonic epithelial cells of the placentome and the omphaloplacenta of Pseudemoia entrecasteauxii are each capable of endocytosis. Embryos (stages 33-39) with intact extraembryonic membranes were surgically removed from the uterus and incubated in a solution containing fluorescein isothiocyanate-dextran (77,000 MW). The fluorescent label was detected in the cytoplasm of scattered populations of epithelial cells in both placental regions of all embryonic stages. We conclude that both the placentome and the omphaloplacenta of P. entrecasteauxii are sites of histotrophic nutrient transport. However, there are histological and cytological differences in the embryonic epithelia of these two placental regions. The histological differences reflect differences in the evolutionary precursors of each tissue. The cytological differences likely portray different functional specializations.

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Desmosomes in uterine epithelial cells decrease at the time of implantation: an ultrastructural and morphometric study.

Displacement of uterine epithelial cells is an important aspect of implantation in the rat and other species, allowing invasion of the blastocyst into the endometrial stroma. Desmosomes, which are part of the lateral junctional complex, function in cell-to-cell adhesion, and are therefore likely to be involved in displacement of uterine epithelial cells at the time of implantation. This study used transmission electron microscopy to study rat uterine epithelial cells during the peri-implantation period to investigate the change in the number of structural desmosomes along the lateral plasma membrane of uterine epithelial cells. We found a significant decrease in the number of desmosomes along the entire lateral plasma membrane as pregnancy progressed. Furthermore, there were also significant decreases in the number of desmosomes on the apical portion of the lateral plasma membrane between all days of pregnancy examined. In addition, on day 6 of pregnancy, the time of attachment, desmosomes were larger and seen as "giant desmosomes." For the first time, this study has shown that there is a significant reduction in cell height and actual number of ultrastructurally observable desmosomes at the time of implantation in the rat. It is proposed that this reduction in desmosome number leads to a decrease in lateral adhesion between uterine epithelial cells at the time of implantation, and hence is involved in the loss of uterine epithelial cells to facilitate blastocyst invasion.

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Redistribution of aquaporins 1 and 5 in the rat uterus is dependent on progesterone: a study with light and electron microscopy.

During early pregnancy in the rat there is a dramatic reduction in luminal fluid which is associated with uterine receptivity for blastocyst implantation. This study investigates the presence and distributional changes of several members of the aquaporin (AQP) family in the rat uterus in response to hormonal regime. An increase in apical AQP5 protein expression was found in response to progesterone alone or in combination with oestrogen, which is similar to that seen at the time of implantation. AQP1 was found in endothelial cells of the endometrium and in the inner circular layer of smooth muscle, with maximal protein expression seen after three doses of progesterone plus 8 hr of oestrogen treatment. These results, for the first time, show that the up-regulation of AQP5 in the apical plasma membrane of uterine epithelial cells and AQP1 in the inner circular layer of myometrium, is dependent on progesterone. Furthermore, unlike during normal pregnancy, there is no differential gradient of AQP5 expression between mesometrial and antimesometrial poles of the progesterone treated uterus. Hence it is suggested that the differential gradient of AQP5 is dependent on the presence of a blastocyst, in addition to the appropriate hormonal environment.

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Cyto-epitheliochorial placenta of the viviparous lizard Pseudemoia entrecasteauxii: a new placental morphotype.

The structural features of the uterine epithelium of the chorioallantoic placenta and omphalloplacenta in the viviparous Australian skink, Pseudemoia entrecasteauxii, were investigated using SEM and TEM techniques. In particular, the structural characteristics that would allow interpretation of function were analyzed, particularly those of gas exchange in the chorioallantoic placenta and histotrophy in the omphaloplacenta. Pseudemoia entrecasteauxii has a complex placenta consisting of a placentome, paraplacentome, and omphaloplacenta. The paraplacentome has a well-vascularized lamina propria in which projecting uterine capillaries displace the overlying uterine epithelial cells, reducing them to attenuated cytoplasmic extensions. Associated cell nuclei and organelles are lost from this region, to provide a capillary lumen to uterine lumen barrier of 0.5-1.0 microm. Hence, the paraplacentome is likely a prominent site for gaseous exchange via simple diffusion. The omphaloplacenta has a similar cytology to that of the placentome, but the uterine epithelial cells are hypertrophied and the apical plasma membrane actively secretes vesicles into the uterine lumen. The omphaloplacenta shows features that are associated with histotrophic transport of nutrients via vesicle secretion, very similar to that of lipid apocrine secretion. The placentome consists of cuboidal cells in the uterine epithelium, with large centrally located nuclei overlying the well-vascularized lamina propria. Although the placentome has a similar cytological structure to that of the omphaloplacenta, granules or active vesicle secretion were not observed. Thus, the placentome may be associated with histotrophy, but not via apocrine secretion. Squamate placentation is epitheliochorial; however, we propose a new term be used to describe the type of placentation in P. entrecasteauxii: "cyto-epitheliochorial," because of the extreme attenuation of uterine epithelial cells of the paraplacentome.

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Ubiquitin is associated with the survival of ectopic stromal cells in endometriosis.

BACKGROUND: Endometriosis is a condition that affects women of reproductive age, where the glandular and/or stromal tissues from the eutopic endometrium implant in ectopic locations. It is well established that the survival of ectopic implants is due to lower levels of apoptosis, but no consensus exists as to which pathway/s this is mediated by. The ubiquitin protein shares a similar sequence homology to an anti-apoptotic protein called BAG-1 and is expressed in the normal endometrium. Currently, no studies have been conducted to determine ubiquitin expression and its possible anti-apoptotic effects in endometriosis. METHODS: Archived endometrial tissues from endometriosis patients and women undergoing laparoscopic diagnosis (controls) from January 2000 to July 2003 at Westmead Hospital were examined, where 14 cases of endometriosis and 55 controls were included in the study. RESULTS: Both the ubiquitin protein and apoptosis were expressed in both glandular and stromal cells throughout the menstrual cycle of the eutopic endometrium, in which ubiquitin exhibited a cyclic expression, reaching a peak in late proliferative phase. In contrast, ubiquitin was predominantly expressed in cells of stromal origin in endometriosis, was no longer regulated by a cyclic pattern and was associated with an aberrant level of cell survival. CONCLUSIONS: For the first time, this study shows that ubiquitin is expressed in endometriotic cells and may contribute to a reduced sensitivity of ectopic endometrial tissue to apoptosis. These findings also suggest that stromal cells contribute differentially to the development of ectopic endometrial tissue.

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Endometrial response to IVF hormonal manipulation: comparative analysis of menopausal, down regulated and natural cycles.

BACKGROUND: Uterine luminal epithelial cell response to different hormonal strategies was examined to determine commonality when an endometrium attains a receptive, stimulated, morphological profile that may lead to successful implantation. METHODS: Endometrial biopsies from 3 cohorts of patients were compared. The tissue samples taken from these patients were categorized into 8 different groups according to their baseline and the hormone regime used. RESULTS: Pre-treatment natural cycle tissue was variable in appearance. Downregulation with a GnRH analogue tissue appeared menopausal in character. HRT after downregulation resulted in tissue uniformity. HRT in menopause resulted in a 'lush' epithelial surface. HST in the natural cycle improved the morphology with significant difference in secretion between the two regimes examined. CONCLUSIONS: Down regulation plus HRT standardized surface appearance but tissue response is significantly different from the natural cycle, natural cycle plus HRT or menopause plus HRT. HRT in menopause reinstates tissue to a state similar to a natural cycle but significantly different from a natural cycle plus HST. HST with a natural cycle is similar to tissue from the natural cycle but significant differences reflect the influence of the particular hormones present (at any point) within the cycle.

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Redistribution of aquaporins in uterine epithelial cells at the time of implantation in the rat.

The aim of this study was to investigate the presence of aquaporins 1, 4 and 5 in uterine epithelial cells during the early stages of pregnancy in the rat. Immunofluorescent and immunogold techniques showed that there was a shift of aquaporin 5 to the apical surface of uterine epithelial cells in the mesometrial pole of the uterus at the time of implantation. This study also revealed the absence of aquaporin 4 in the rat uterus during early pregnancy. Aquaporin 1 was observed in stromal blood vessels but there was no change in uterine epithelial cells during early pregnancy. These results suggest that aquaporin 5 plays an important role in the removal of uterine luminal fluid at the time of implantation in the rat and may contribute to the antimesometrial positioning of the implanting blastocyst.

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Progesterone treatment and the progress of early pregnancy reduce desmoglein 1&2 staining along the lateral plasma membrane in rat uterine epithelial cells.

Uterine epithelium undergoes dramatic changes during early pregnancy in preparation for implantation. We have studied distribution patterns of the desmosomal marker, desmoglein 1&2, in rat uterine epithelial cells during early pregnancy as well as in hormonally stimulated ovariectomised animals. On day 1 of pregnancy as well as in oestradiol treated rats, desmoglein 1&2 staining was localized along the entire length of the lateral plasma membrane. By day 3 and on subsequent days of pregnancy as well as in ovariectomised animals treated with progesterone alone or in combination with oestradiol, desmoglein 1&2 staining was concentrated at the apical portion of the lateral plasma membrane. We suggest that the reorganisation of these desmosomal cadherins is an important component of uterine epithelial receptivity and this relocation is under the control of the ovarian hormone progesterone.

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Aquaporin-1 increases in the rat myometrium during early pregnancy.

Immunofluorescence and immunogold techniques were used to determine the presence and distribution of aquaporin-1 (AQP1) within the rat uterus. Uterine tissue from non-pregnant (proestrus) as well as pregnant (days 1, 3, 6 and 7) rats were used. It was found that this water channel was present in the myometrium of the pregnant rat uterus with the intensity of AQP1 immunoreactivity increasing from day 1 to day 6 of pregnancy. In particular, an increase was also observed in mesometrial as compared to antimesometrial myometrium. Immunolocalization at the electron microscope level indicated that AQP1 was localized to the plasma membrane of smooth muscle cells found within the inner circular layer. It is suggested that AQP1 plays a role in stromal oedema, uterine closure and orientation of the blastocyst.

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Uterine receptivity and the plasma membrane transformation.

This review begins with a brief commentary on the diversity of placentation mechanisms, and then goes on to examine the extensive alterations which occur in the plasma membrane of uterine epithelial cells during early pregnancy across species. Ultrastructural, biochemical and more general morphological data reveal that strikingly common phenomena occur in this plasma membrane during early pregnancy despite the diversity of placental types--from epitheliochorial to hemochorial, which ultimately form in different species. To encapsulate the concept that common morphological and molecular alterations occur across species, that they are found basolaterally as well as apically, and that moreover they are an ongoing process during much of early pregnancy, not just an event at the time attachment, the term 'plasma membrane transformation' is suggested which also emphasises that alterations in this plasma membrane during early pregnancy are key to uterine receptivity.

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Viviparous lizard, Eulamprus tympanum, shows changes in the uterine surface epithelium during early pregnancy that are similar to the plasma membrane transformation of mammals.

The "plasma membrane transformation" describes a series of ultrastructural, biochemical, and morphological changes that occur in the uterus of many mammals at the time of blastocyst attachment. These changes, regardless of placental type or length of gestation, include alterations to microvillar length and density and the presence or absence of pinopods or uterodomes. Scanning electron microscopy (SEM) was used to 1) document the topographical ultrastructure of the uterus of Eulamprus tympanum, an eastern Australian viviparous skink with a simple chorioallantoic placenta, for the first time; and 2) determine whether changes identified as "plasma membrane transformation" in mammals occur in E. tympanum. Tissues collected over three seasons from nonreproductive subadult females, preovulatory, postovulatory, and early to mid-gestational females were examined. At low magnification the uterine epithelium of subadults displays a distinctive pattern of tissue folding that includes rectangular areas of tissue delineated by deep lateral and transverse folds. At higher magnification, the uterine epithelium surface is composed of two dominant cell types, i.e., those covered by microvilli and ciliated cells. The folding pattern observed in subadults is less evident in vitellogenic females and the cell surfaces appear highly secretory, with bulging cell apices. Tissue from postovulatory lizards has no distinctive folding pattern and cell surfaces are frequently smooth and lack microvilli. Uterine egg chambers lack ciliated cells at the embryonic pole, but display abundant secretory droplets. Thus, the uterus of E. tympanum undergoes a plasma membrane transformation. The scope of this transformation is not fully understood but may be related to the complexity of placental structure and the development of the embryo/fetus at parturition.

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Ultrastructural localisation of Muc-1 on the plasma membrane of uterine epithelial cells.

The presence and distribution pattern of Muc-1 was determined immunohistochemically in rat uterine epithelial cells and localised ultrastructurally on the plasma membrane of uterine epithelial cells. Rat uterine tissue was examined on days 1, 3 and 6 of pregnancy and it is shown to be expressed in uterine luminal epithelial cells on day 1 of pregnancy with decreasing expression towards the time of implantation on day 6. It was demonstrated ultrastructurally that Muc-1 is expressed at the external surface of the plasma membrane of uterine epithelial cells thus confirming that it is well positioned to act as an anti-adhesive molecule during the nonreceptive stage of pregnancy. Ultrastructural localisation also revealed that Muc-1 was not present on the outer surface of the plasma membrane during the receptive phase.

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Evolution of viviparity: what can Australian lizards tell us?

Historically, Australia has been important in the study of, and the development of hypotheses aimed at understanding, the evolution of viviparity in amniote vertebrates. Part of the importance of Australia in the field results from a rich fauna of skinks, including one of the broadest ranges of diversity of placental structures within one geographic region. During the last decade, we have focussed our studies on one lineage, the Eugongylus group of skinks of the subfamily Lygosominae because it contains oviparous species and some that exhibit complex placentae. Our specific objective has been to attempt to understand the fundamental steps required when viviparity, and ultimately complex placentae, evolve from oviparous ancestors. We have taken a three-prong approach: (1) detailed study of the morphology and ontogeny of the placentae of key species at the light microscope level; (2) study of changes in the uterus associated with pregnancy, or the plasma membrane transformation; and (3) measures of the net exchange of nutrients across the placenta or eggshell of key species. In turn, we have found that: (1) details of the morphology and ontogeny of placentae are more complex that originally envisaged, and that the early conclusions about a sequence in the evolution of complex placentae was naïve; (2) a plasma membrane transformation occurs in viviparous, but not oviparous lizards, and thus may be a fundamental feature of the evolution of viviparity in amniotes; and (3) species with more complex chorioallantoic placentae tend to transport more nutrients across the placenta during pregnancy than those with simpler chorioallantoic placentae but, because the correlation is not tight, the importance of the omphaloplacenta in transporting nutrients may have been overlooked. Also, the composition of yolk of highly matrotrophic species is broadly similar, but not identical, to the yolk of oviparous species. Some of the interpretation of our data within the context of our specific objective is not yet possible, pending the publication of a robust phylogeny of Eugongylus group skinks. Once such a phylogeny is available, we are in a position to propose specific hypotheses about the evolution of viviparity that can be tested using another lineage of amniotes, possibly Mabuya group skinks.

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Alterations in tight junction molecules of uterine epithelial cells during early pregnancy in the rat.

Distribution patterns of the tight junction associated proteins ZO-1, claudin-1 and occludin were investigated in rat uterine epithelial cells during early pregnancy. Light microscopy and immunohistochemical labelling were used to detect these proteins on days 1, 3, 6 and 7 of pregnancy. Intense staining of claudin-1 at the apical region of the lateral plasma membrane accompanied diffuse staining throughout the cytoplasm. ZO-1 was also localised in the apical region, but ZO-1 was not present in the lower two thirds of the lateral plasma membrane or in the cytoplasm. Occludin was present only on days 6 and 7 of pregnancy. Labelling was also localised in the apical region of the lateral plasma membrane where tight junctions are known to be present. Our results show that ZO-1, claudin-1 and occludin are present in the apical region of uterine epithelial cells, and appear to play a role in the very dynamic tight-junctional network of uterine epithelial cells during early pregnancy. In particular, occludin appears only during uterine receptivity for implantation.

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Cytoskeletal proteins in uterine epithelial cells only partially return to the pre-receptive state after the period of receptivity.

Immunohistochemical staining of 5 cytoskeletal proteins (actin, alpha-actinin, gelsolin, plectin and plakoglobin) was used to investigate changes in distribution patterns of these proteins after the period of uterine receptivity for blastocyst implantation in the rat. Actin was found throughout the cytoplasm but it was concentrated along the apical plasma membrane on day 1 of pregnancy, decreased by day 6 and then increased again at day 9. Alpha-actinin and gelsolin were localized in distinctive bands along the apical plasma membrane at day 6 of pregnancy but became diffusely distributed at day 9. Plectin was localized along the apical and basal plasma membranes at day 6 but in higher amounts apically and at day 9, it was concentrated in apical and basal zones in the cells. Plakoglobin was found along the lateral and basal membranes with increased intensity along the apical third of the lateral plasma membrane from day 6 to day 9 of pregnancy. These results show that all 5 cytoskeletal proteins redistributed after the period of uterine receptivity: some exhibited a similar pattern of labelling to that found during the prereceptive state, whereas others only partially returned to the pre-receptive state. This change in distribution patterns may reflect differences in the epithelial barrier function before and after the period of receptivity.

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Differences in muscle fiber growth in slow-twitch muscles of the forelimb and hindlimb of the rat: role of the pituitary and food intake.

This investigation tested the hypothesis that differences in the growth of fore- and hindlimb muscles in the rat are regulated by the pituitary and food intake. Using morphometric techniques, the growth of muscle fibers was compared in two slow-twitch muscles, the flexor carpi ulnaris (FCU) of the forelimb, and the soleus of the hindlimb, in male Wistar rats fed ad libitum, food restricted (FR) or hypophysectomized (hypox) from age 60 days. Growth was defined as an increase in fiber diameter and/or type 1 fiber percentage. The soleus had larger diameter fibers than the FCU in controls and FR, but not hypox rats. The growth in diameter, between 60 and 180 days, of both types 1 and 2 fibers in the soleus and type 2 fibers in the FCU was inhibited by hypox and, to a lesser extent, FR. Neither type 1 fiber diameter nor percentage of type 1 fibers in the FCU increased with age nor was it affected by hypox or FR. The percentage of type 1 fibers was higher in the soleus than the FCU and was further increased in the soleus of hypox rats. Food restriction produced a smaller rise than hypox in type 1 fiber percentage in the soleus. Thus, differences in fore- and hindlimb muscle fiber growth are modulated by pituitary hormones and, to a lesser extent, by food intake.

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Manipulation of the follicular phase: Uterodomes and pregnancy - is there a correlation?

BACKGROUND: Manipulation of the follicular phase uterine epithelium in women undergoing infertility treatment, has not generally shown differing morphological effects on uterine epithelial characteristics using Scanning Electron Microscopy (SEM) and resultant pregnancy rates have remained suboptimal utilising these manipulations. The present study observed manipulation of the proliferative epithelium, with either 7 or 14 days of sequential oestrogen (E) therapy followed by progesterone (P) and assessed the appearance of pinopods (now called uterodomes) for their usefulness as potential implantation markers in seven women who subsequently became pregnant. Three endometrial biopsies per patient were taken during consecutive cycles: day 19 of a natural cycle - (group 1), days 11/12 of a second cycle after 7 days E then P - (group 2), and days 19/22 of a third cycle after 14 days E then P - (group 3). Embryo transfer (ET) was performed in a subsequent long treatment cycle (as per Group 3). RESULTS: Seven pregnancies resulted in seven viable births including one twins and one miscarriage. Analysis of the individual regimes showed 5 days of P treatment to have a higher correlation for uterodomes in all 3 cycles observed individually. It was also observed that all 7 women demonstrated the appearance of uterodomes in at least one of their cycles. CONCLUSIONS: We conclude that manipulation of the follicular phase by shortening the period of E exposure to 7 days, does not compromise uterine epithelial morphology and we add weight to the conclusion that uterodomes indicate a receptive endometrium for implantation.

Journal Article↗

A successful pregnancy following SEM fine tuning of hormonal priming.

BACKGROUND: Manipulation of the uterine epithelium utilising standard dose exogenous oestrogen (E2) and progesterone (P4) has been shown to achieve a mature secretory morphological response. However, in an in vitro fertilisation (IVF) setting, frozen embryo transfer (ET) has had a low success rate. We propose that in patients with previously failed ET attempts, the uterine epithelium can be directly visualised by biopsy and Scanning Electron Microscopy (SEM) and that with an individualised fine tuning of the hormone supplementation regime, based on the SEM examination of sequential uterine biopsies, it is possible to provide a uterine environment conducive to successful ET. METHODS: A 47 year old women was chosen for endometrial biopsy, histopathological dating and endometrial observation utilising SEM to determine the integrity of her secretory uterine epithelium because of her age and several previously failed attempts at frozen ET. Exogenous E2 and P4 supplementation was administered in modified doses according to the SEM result, in consecutive cycles until the epithelial response appeared satisfactory for potential implantation. RESULTS: This case study demonstrates the dramatic change in epithelial characteristics that can be achieved as a response to these altered doses of E2 and P4. The uterine morphology changed from a hypotrophic to a mature, receptive epithelium such that ET resulted in the birth of healthy twin boys. CONCLUSION: The comparison between the consecutive biopsies in direct response to the SEM analysis and tailored modification of E2 and P4 dose clearly demonstrates, in this case, the effectiveness of individual morphological monitoring to maximise the successful outcome of ET.

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