1 Departments of Anatomy & Histology and 2 Obstetrics and Gynaecology, The University of Sydney, Sydney, NSW 2006, Australia and 3 School of Anatomical Sciences, The University of the Witwatersrand Medical School, Parktown 2193, Johannesburg, South Africa
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Abstract |
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Key words: colloidal gold/human/pinocytosis/pinopods/rats
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Introduction |
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The first observations of apical surface projections on uterine epithelial cells were made in mice (Nilsson, 1958) and rats (Warren and Enders, 1964
). However, it was Nilsson who provided the first extensive discussion and illustration of apical projections, which he referred to simply as cytoplasmic protrusions and blebs (Nilsson, 1966
). Early speculation on the function of these projections included an apocrine function, but Nilsson, and Enders and Nelson proposed their function to be the absorption of luminal endometrial contents (Nilsson, 1972
; Enders and Nelson, 1973
). Enders and Nelson went on to prove their hypothesis by demonstrating electron dense tracer (ferritin) uptake in the apical protrusions of rats, and coined the term pinopod from the Greek drinking foot to signify their pinocytotic function (Enders and Nelson, 1973
). This term took hold and has subsequently been used invariably when referring to cytoplasmic projections in the uterus of many species.
Because of the considerable clinical interest in these human pinopods or pinopodes as biological markers for implantation in IVF settings, it is unfortunate that understanding of their function is so poor. However, in recognition of the structural differences between rodent and human apical protrusions, Murphy compared morphological characteristics of these structures and argued that the word pinopod was misleading when referring to the human structures; he therefore proposed the more general term uterodome as more appropriate (Murphy, 2000b).
Tracer studies determining pinocytotic activity of these apical protrusions in animals other than rats and mice are scarce. However, Parr and Parr using ferritin as tracer (Parr and Parr, 1982), and Guillomot and colleagues using horseradish peroxidase (Guillomot et al., 1986
), demonstrated that protrusions in rabbits and cows were not pinocytotic. These findings therefore alert us to the fact that not all endometrial protrusions have the same function, as already highlighted (Murphy, 2000b
).
The aim of this study was therefore to address the question of pinocytotic function in human uterine epithelial apical protrusions (uterodomes). We did this by modifying techniques utilized for determining pinocytotic uptake in rats, using colloidal gold of a small particle size as the tracer. An exposure time to the tracer of 10 min was selected, as it had been previously shown to be sufficient for pinopod uptake in the rat (Enders and Nelson, 1973; Parr and Parr, 1974
).
Due to the potential toxicity of all electron dense tracer particles in patients, it was decided to use fresh hysterectomy tissue. Care was taken to prevent avascularity and speed was used in administering the gold solutions. A rat model was run concurrently as a control for pinopod presence, abundance and pinocytotic ability for colloidal gold, with tracer-treated and non-tracer-treated horns. This rat model also included hysterectomy studies as a further control. Since it was not possible to establish uterodome presence in the human prior to hysterectomy, it was necessary to maximize the possibility of uterodome presence by hormone priming. Thus as the appearance of these structures is known to be stimulated by progesterone, all patients were primed with progesterone 14 days before surgery (Nikas, 1999a; Adams and Murphy, 2001
).
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Materials and methods |
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Hysterectomy specimens were obtained through the abdominal route to minimize devascularization to the uterus. Tissue was prepared within the operating suite during the hysterectomy procedure, with the patient under general anaesthesia. At surgery, a Pfannsteil incision was made and the abdominal cavity was entered through layers. Bilateral sides of uterine fundus were clamped by forceps to hold up the uterus and round ligament, and ovarian (or infundibulopelvic) ligaments were clamped, cut and tied. These procedures interrupted partial blood supply to the uterus through these ligaments. Then the peritoneal incision was extended in the broad ligament to open the retroperitoneal spaces and uterine arteries in both sides were exposed. Immediately after clamping the bilateral uterine arteries that mainly supply blood circulation to the uterus, the uterine body was quickly removed by cutting the isthmo-cervical junction. Gold solution was injected into the uterine cavity through the anterior cervical os within 3060 s of detachment. The duration of the clamping of the uterine arteries to the injection of the gold solution did not exceed 1 min. The cervical stump was then excised, thus completing the hysterectomy. Clamps were retained on the Fallopian tubes at the body of the uterus to prevent possible leakage and the uterus was supported upright for 10, 20 or 30 min to ensure maximum lavage of the luminal fundal epithelium. Timed uterine biopsies were retrieved using a fine, blunt Hurrocks uterine curette, rinsed in physiological saline and placed into 2.5% glutaraldehyde fixative (EM grade; TAAB, UK) in 0.1 mol/l phosphate buffer (PB), pH 7.4, for 2 h. The specimen was rinsed in PB and processed immediately.
Colloidal gold solution
Gold colloid EM grade (Sigma, USA) 5 nm particle size (4.56.5 nm) (monodispersive) was prepared from 0.01% gold chloride solution (0.01% HAuCl4 suspended in 0.01% tannic acid with 0.04% trisodium citrate, 0.26 mmol/l potassium carbonate and 0.02% sodium azide) with a final concentration of 5x1013 gold particles per ml.
Optimization of colloidal gold dilutions and exposure time
To determine the optimal time for exposure to, and potential uptake of particles, three different time sequences and gold concentrations were used. These were as follows: (i) four uteri were exposed to 100% colloidal gold concentration for 10, 20 and 30 min. The scanning electron microscopy (SEM) results demonstrated some tissue degradation at 30 min; therefore, subsequent biopsies were taken at 10 and 20 min only; (ii) three uteri were exposed to 10% colloidal gold (with sodium chloride), pH 7.4, and (iii) three uteri were exposed to 2% colloidal gold (with sodium chloride), pH 7.4. A volume of 1520 ml of gold solution was required per uterus. Uterodomes were not observed in the tissue of one patient and the patient was excluded from the study.
Colloidal gold standards
Three standard solutions of 2, 10 and 100% colloidal gold were prepared and 1 drop was placed on parlodion (Mallinckrodt Chemical Works, USA) coated copper grids and left to dry overnight. They were platinum sputter coated, viewed by transmission electron microscopy (TEM) using area analysis at scanning transmission electron microscopy (STEM) magnifications of 19 500, 53 000 and 110 000 for 100 live seconds, using fixed 0.4x0.4 µm raster windows, and the results used to visualize gold particles in the specimens.
Rat tissue: collection and preparation
Eleven sexually mature 1216 week old virgin female Hooded Wistar rats of inbred strain SPF were used. The animals were obtained from the Bosch Animal House, The University of Sydney. The animals were maintained in temperature-controlled quarters, 23°C, with a 12 h light-dark cycle, and received Forbes rat and mouse cubes (Doust and Babbidge, Forbes NSW, Australia) and water ad libitum. All rats in proestrus were placed in cages overnight with males of proven fertility. If sperm was found in the vaginal smear the following morning, this was deemed day 1 of pregnancy. Animals were killed at 09.00 on day 5 of pregnancy.
All animals were anaesthetized with an i.p. injection of 0.4 ml Nembutal sodium pentabarbitone (Abbott Pty Ltd, Sydney, Australia) and a central ventral incision was made to expose the uterine horns. The three concentrations (as per the human tissue) of colloidal gold electron microscopy (EM) grade 5 nm particle size were utilized and 1 ml was injected into the cranial (anterior) end of the left uterine tube until flushing appeared at the vaginal end. Care was taken to not overdistend the uterine tube. After 10 min, the left uterine horn was flushed with 1 ml normal saline. Both uteri were excised, pinned onto dental wax to ensure that the original length was maintained and placed immediately in 2.5% EM grade glutaraldehyde (AGAR Scientific, London, UK) in PB for 10 min. Two rats, used as hysterectomy controls, were anaesthetized as above, but both horns were dissected immediately and laid flat, whereby the left horn was injected with 100% colloidal gold for 10 min, then flushed with normal saline, and the right horn was injected with normal saline for 10 min. All tissue was fixed in 2.5% glutaraldehyde in PB for 1 h and prepared for electron microscopy. The uterine horns were cut into 23 mm pieces for TEM or 5 mm pieces for SEM and fixed for a further 40 min.
Further processing
Scanning electron microscopy
The tissue was rinsed in PB and post-fixed in 1% osmium tetroxide (OsO4; Johnson Matthey, Materials Technology, London, UK) in PB for 1 h and rinsed in PB. The tissue was further processed in 1% OsO4 for 30 min, rinsed in distilled water, dehydrated through graded ethanols 50100% with a final wash in absolute alcohol for critical point drying. Dried tissue was mounted onto aluminium stubs, edged with conductive carbon paint and sputter coated to 20 nm with platinum in a planar magnetron sputter coater and viewed using a Philips Scanning Electron Microscope 505 (Eindhoven, The Netherlands), operating at 20 kV.
Transmission electron microscopy
The tissue was prepared as for SEM, but did not undergo a second osmification prior to dehydration to 100% ethanol. The tissue was post-fixed in 1% OsO4 (Johnson Matthey) in PB for 1 h and rinsed in PB before routine dehydration through a series of graded analytical alcohol, infiltrated with Spurrs resin (Agar Scientific, Essex, UK) and embedded in flat cassettes (ProSci Tech, Australia) to allow for tissue orientation. Eight resin blocks per specimen were allowed to polymerize for 24 h in a 60°C oven.
Semi-thin sections of 0.51 µm were cut and stained with toluidine blue, in 0.5% ethanolamine, to determine the presence of epithelium prior to further trimming. Ribbons of ultra-thin 70 nm sections were cut using a diamond knife (Diatome, Switzerland) on a Reichert-Jung Ultracut E microtome (Germany), and placed onto 100 mesh copper grids (Agar Scientific). Sections were counterstained in 4% aqueous uranyl acetate for 45 min and lead citrate for 15 min (Reynolds, 1963). At least eight grids per specimen were examined using a Philips CM12 transmission electron microscope operating at 120 kV.
Data collection
Data collection was either by black and white 35 mm photography or digital imaging when applied to either TEM or SEM techniques. At the SEM level of observation, a standard series of magnifications were used, i.e. 525, 1050, 2100, 4200 and 8400. Images were also recorded of unique observations.
X-ray microanalysis (XRMA)
For XRMA, the tissue was prepared as for TEM. Energy dispersive spectroscopy (EDS) analyses were carried out using a Philips CM12 transmission electron microscope operating at 120 kV fitted with an energy dispersive X-ray detection system (EDAX 9900), in both nanoprobe (highly focused beam) and STEM modes. This technique was used to confirm the presence of gold particles in the cells. Copper, sodium, chlorine, osmium, uranium and lead were included in the analyses to provide a baseline for the samples, as they were metals used in the preparation techniques. All uterodome vacuoles and all large vacuoles in pinopods and large subapical vacuoles were analysed.
Field emission scanning electron microscopy (FESEM)
The following techniques were employed to assess the presence of gold particles remaining in the uterine lumen or adhering to the apical membrane in both rat and human tissue. Random SEM views of areas of epithelium containing fully developed uterodomes/pinopods were utilized. FESEM employed the JEOL 6000F (Tokyo, Japan) system operating at 20 kV which has a 1 nm resolution.
X-ray mapping
X-ray mapping was carried out using the EDX Map Software (EDAX, USA) on the FEI XL30 (Eindhoven, The Netherlands) operating at 25 kV, at a resolution of 128x100 pixels with a dwell time of 35 min livetime. The distribution of the elements gold and osmium were assessed using X-ray mapping.
Back scattered imaging
Back scatter compositional atomic contrast imaging mode was employed while using the system for X-ray mapping to also determine gold particles at the luminal surface.
TUNEL
Both human and rat tissue was prepared for sectioning by immersion fixation in 4% paraformaldehyde in phosphate buffered saline (PBS). Prior to freezing, the tissue was rinsed in PBS and placed in 15% sucrose for 12 h, embedded in moulds with OCT embedding compound (Tissue Tek, USA), quenched in super-cooled iso-pentane (Unilab) and stored under liquid nitrogen.
Sections of 10 µm were cut using a Reichert Jung 2800 Frigocut cryotome (Leica). Orientation was crucial for determining the presence of epithelium before proceeding further. When epithelium was confirmed, the blocks were further sectioned at 20 µm, sections collected onto slides previously coated with gelatin and poly-L-lysine (Sigma) and allowed to air dry for 1 h prior to antibody labelling.
The dried slides were immersed in 70% ethanol and rinsed in distilled water prior to equilibration with 1xterminal deoxytransferase (TdT) buffer, 3 mmol/l Trizma base (Sigma) with added 0.1 mmol/l cobalt chloride (Sigma) and 14 mmol/l sodium cacodylate (Alltech), at pH 7.2, for 10 min. Sections were then incubated for 1 h, in a humid chamber at 37°C, in a reaction mixture containing 0.04 nmol biotinylated dUTP (Boehringer-Mannheim, Mannheim, Germany) and 0.3 IU/µl TdT (Boehringer-Mannheim) in TdT buffer. The reaction was stopped by a 15 min incubation in SSC (0.3 mol/l NaCl, 0.03 mol/l sodium citrate at pH 7.4).
Non-specific binding was minimized by incubating the sections in 1% bovine serum albumin (BSA) in PBS for 15 min, followed by washes in 0.1 mol/l PBS. Sections were then incubated in streptavidin conjugate indocarbocyanine (Cy3, Jackson Immuno Research Inc./Amersham International P/C, PA, USA) diluted 1:500 in PBS for 1 h, and counterstained with bisbenzamide (BBZ) (Hoechst) to identify live cells.
Confocal microscopy and quantification
All immunolabelled sections of the colloidal gold-treated human uterine luminal epithelium were viewed with a Leica TCS-NT confocal laser scanning microscope equipped with argon-krypton and UV lasers. Images were obtained sequentially to maximize signal separation and photomultiplier tube settings held constant to ensure that the relative intensities were recorded accurately.
The number of TUNEL positive profiles in successive 800 µm lengths of epithelium extending the full length of four to eight sections from each specimen, were counted. A t-test was used to weigh the difference found between the control and gold treated tissue using a significance level of P < 0.05.
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Results |
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Due to the epithelial degradation observed in the longer gold exposure times, only samples exposed to gold (all concentrations) for 10 min were used for further analysis at the TEM and XRMA level.
Our selection criteria for analysis of cells displaying uterodomes at the TEM level dictated that they must be attached to the underlying stroma at the basement membrane and must display a nucleus to ensure a central representation of the cell was achieved. Uterodomes were observed visually for gold particles followed by XRMA for gold detection. A total of 134 uterodomes were analysed, of which 27 contained vacuoles (Figure 1B). Gold tracer was not observed in any of the uterodomes, nor detected by X-ray analysis at any of the concentrations used (Figure 1C
).
Rat
Observation with the SEM showed random groups of pinopods that were difficult to locate. However, pinopods were observed (Figure 1D) at all three concentrations of gold for 10 min exposure. Comparison with the non-treated uterine horns demonstrated pinopod abundance and vacuolization to be similar (Figure 1E
).
At the TEM level, a majority of cells with pinopods fitting the selection criteria for stromal attachment and presence of nucleus were observed to have vacuoles (43 of 54 pinopods observed). Of these, seven were observed visually to contain several gold particles (10% concentration), as referenced by the gold standard analyses, at a magnification of x53 000. However, due to the low count rate, XRMA was unable to differentiate these particles from background noise. Eleven pinopods were observed, at 100% gold, visually to display gold particles in large vacuoles either above or below the apical plasma membrane (Figure 2AD) and XRMA confirmed their presence (Figure 2E
). At 2% gold, no gold could be detected.
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Similar results were obtained with the rat hysterectomy tissue, demonstrating the presence of pinopods and uptake of gold in pinopod and sub apical vacuoles with 100% gold concentration.
TUNEL showed few positive cells in either human or rat uteri, thus confirming that both epithelia were viable.
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Discussion |
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One particular aspect of the plasma membrane transformation that has attracted much attention is the appearance of large rounded projections on the apical surface of uterine epithelial cells. While these protrusions have been widely reported to appear in rats and mice, their presence has also been noted in cows (Guillomot and Guay, 1982), camels (Abd-Elnaeim et al., 1999
) and humans (Johanisson and Nilsson, 1972; Martel et al., 1981
; Psychoyos and Martel, 1985
; Murphy et al., 1987
; Psychoyos and Nikas, 1994
; Nikas et al., 1995
, 1999
; Nikas, 1999b
; Murphy, 1995
, 2000a
for reviews). Since these protrusions have been implicated in the implantation process, continued study of these structures in the human is important because of their possible utility as biomarkers of implantation in clinical fertility programmes (Nikas and Psychoyos, 1997
; Nikas, 1999b
; Nikas et al., 1999
; Adams and Murphy, 2001
; Adams et al., 2001
).
In rats and mice, it has been established that the projections, termed pinopods, are truly pinocytotic and absorb fluid from the uterine lumen (Enders and Nelson, 1973; Parr and Parr, 1974
, 1977
). However, the term pinopod has also been used to describe other cellular projections of the apical plasma membrane in species where no pinocytotic function has been established. To highlight the disparity in terminology, there are morphological differences between the true pinopods observed in rats and mice, and the structures seen in the human uterus. Hence, in recognition of this difference, the more descriptive term of uterodome, has been proposed (Murphy, 2000b
). Thus to understand further the structures in the human uterus (uterodomes), the present study has used several approaches to determine whether they have a pinocytotic function.
Because of the potential toxicity in humans of conventional electron dense tracer material, human uterine epithelial tissue was obtained from hysterectomy patients. Cellular viability and integrity of the epithelium from hysterectomy tissue was supported by a negative TUNEL result and observation of cellular ultrastructure. The TUNEL technique allows early recognition of apoptosis (Gavrieli et al., 1992).
From earlier ferritin tracer studies of pinocytotic incorporation in rats, it has been shown that uptake is a rapid process, taking no more than 310 min, with no further uptake after 10 min (Enders and Nelson, 1973; Parr and Parr, 1974
). Based on this evidence, 10 min exposure to gold was taken as sufficient time for pinocytotic activity to be demonstrated in both rat and human tissue in our study.
To further validate the findings, we eliminated the possibility of elastic recoil causing tracer to be pulled into the cells when placed in fixative (Enders and Nelson, 1973), by using X-ray mapping and FESEM to detect clumping or aggregation of the gold particles at the cell surface. Both techniques gave a negative result, suggesting that luminal tracer had been washed away with the saline rinse prior to fixation.
Observation of human uterodome protrusions showed that they were numerous and appeared in clusters on adjacent cells. Ultrastructurally, these protrusions did not generally exhibit vacuoles and there was no evidence of pinocytotic uptake. Conversely, rat pinopods were found to be randomly dispersed, frequently displayed large vacuoles in their apical protrusion and were confirmed to have gold particles in these vacuoles, consistent with the known pinocytotic ability of these structures (Enders and Nelson, 1973).
However, no difference was detected in the pinocytotic activity of pinopods when comparing rat hysterectomy with in-situ perfused tissue. Uterodomes therefore did not display pinocytotic activity in human hysterectomy tissue, and our study shows that hysterectomy and ensuing tracer administration techniques did not compromise any possible endocytotic function of these structures, observations that were supported by the TUNEL assay.
Although the main point of this study was to examine the pinocytotic function or otherwise of uterodomes, we recognize the possibility that the results do not necessarily reflect the function of uterodomes formed in the mid-secretory phase, since it is known that hormonal priming affects the degree of pinocytocis in endometrial epithelial cells (Leroy et al., 1976). However, since we have not specifically attempted to correlate uterodome appearance with the window of receptivity, future work can be conducted to ascertain if our results can be reproduced in human tissue during the receptive phase of the cycle (say at 7 days progesterone). Certainly further examination is needed to determine the length of time uterodomes can be made to exist with continuous progesterone. While this determination was not the main focus of our study, some studies have reported uterodomes (pinopods) for longer periods of time up to 14 days progesterone (Singh et al., 1996
; Novotny et al., 1999
; Acosta et al., 2000
).
Our data presented here clearly confirm the endocytotic activity of rat pinopods in their ability to take up electron dense material as first demonstrated by Enders and Nelson using ferritin (Enders and Nelson, 1973). This ability to uptake material is not seen in the human uterodome, adding evidence to the view that these structures are not pinocytotic and further highlighting the need for an alternative to pinopod when referring to these human uterine protrusions.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 29, 2001; resubmitted on February 7, 2002; accepted on April 9, 2002.