Anat Embryol (1982) 163:367-370

Anatomy and Embryology 9 Springer-Verlag 1982

A Freeze-Fracture Electron Microscopic Study of Tight Junctions of Epithelial Cells in the Human Uterus C.R. Murphy*, J.G. Swift**, J.A. Need*, T.M. Mukherjee**, and A.W. Rogers* * Departments of Human Morphology and Obstetrics and Gynaecology,School of Medicine, The Flinders University of South Australia, Bedford Park, 5042 South Australia ** Electron MicroscopeUnit, Division of Tissue Pathology, Institute of Medical and VeterinaryScience, Frome Road, Adelaide, 5000 South Australia

Summary. Freeze-fracture electron microscopy has been used to study tight junctions of luminal epithelial cells of the human uterus. The junctions are deeper and more extensive in the middle of the menstrual cycle than they are later in the cycle. The results suggest that the contents of the uterine lumen may be more closely regulated at some times than at others. Key words: Tight junctions - Epithelium - Human uterus

Introduction Tight junctions form part of the junctional complex between adjacent cells in most vertebrate epithelia. By preventing movement of some molecules between the cells (i.e. via the paracellular route), these junctions serve to isolate, to varying degrees, the contents of the lumen from surrounding tissues, as has been shown by experiments with tracer molecules (Staehelin 1974, McNutt 1977, Gilula 1978). In recent freeze-fracture electron microscopic studies, we have shown that when ovariectomised rats are injected with ovarian hormones, the tight junctions between uterine luminal epithelial cells undergo dramatic changes in geometric organization and depth (Murphy et al. 1980, 1981a). In other studies, we have shown that similar changes in tight junction organization occur in uterine epithelial cells of rats during normal pregnancy (Murphy et al. 1981 b). These findings imply that tight junctions between uterine epithelial cells serve to regulate the contents of the uterine lumen during pregnancy in rats at least. We therefore thought it would be interesting to see if changes in the structure of tight junctions also occur in epithelial cells of the human uterus at different stages of the menstrual cycle. Offprint requests to." Dr. C.R. Murphy, Department of Human Morphology, School of Medicine, The Flinders Universityof South Australia, Bedford Park, 5042 South Australia

0340-2061/82/0163/0367/$01.00

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Materials and Methods Portions of endometrial tissue were taken from patients undergoing gynaecological procedures not related to menstrual disturbances. For the purpose of the study the menstrual cycle was divided into a middle (days 14-16) and later (days 24, 25) stage. Samples of endometrial tissue from 4 or 5 patients at each stage were used and were taken by curettage as near as practicable from the fundus of the uterus. Tissue was sliced into pieces about 2 m m 2 and fixed for 40 min in 2% glutaraldehyde - 21/2 formaldehyde in 0.1 M phosphate buffer, pH 7.4. Following three washes in fresh buffer the tissue was transferred to 30% glycerol in buffer a n d stored at 4 ~ C until used. Samples of 1 m m 3 were frozen in liquid freon 22 cooled in N~, transferred to liquid N2 and then fractured without etching at - l l 0 ~ in a Balzers Apparatus (Model BA 360 M). After platinum shadowing with an electron beam gun and carbon deposition, the replicas were separated from tissue in sodium hypochlorite, washed in water, placed on copper grids and examined with a Siemens Elmiscop 102 electron microscope operating at 80 KV. For morphometry, every picture obtained which showed well-shadowed tight junction was analysed. This number varied for each specimen (patient) since it was not always possible owing to the ever present difficulties of working with h u m a n material to obtain several good pictures from each. However, at least I and usually 2 regions of junction could be measured for each specimen. Measurement consisted of determining the distance the junctional region extended down the lateral m e m b r a n e from the cell apex and measures of this depth were made each 2 cm along the cell apex. The measures of depth were pooled to give a mean (_+ SD) depth of tight junction down the lateral m e m b r a n e for each of the two groups. These means were analysed with the 2-tailed t-Test. The n u m b e r of strands comprising the tight junction were also estimated by counting the n u m b e r of strands which ran approximately parailel to the cell apex. Only PF face m e m b r a n e (Branton et ai. i975) was used in this study.

Fig. 1. Freeze-fracture electron micrograph showing the tight junctional region of a uterine luminal epithelial cell in the middle o f the menstrual cycle. The junctional region extends about 0.4 g m down the lateral m e m b r a n e from the cell apex (A). x 75,000

Tight Junctions of Epithelial Cells in Human Uterus

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Fig. 2. Freeze-fractureelectron micrograph showing the tight junctional region of an uterine luminal epithelial ceil late in the menstrual cycle. The junctional stands do not extend as far down the lateral membrane from the cell apex (A) as in Fig. 1. x 75,000. (Shadowing direction is indicated by an arrow in the bottom right-hand corner)

Results

Tight junctions of epithelial cells from the middle of the menstrual cycle are shown in Fig. 1. They extend 0.43 +0.09 gm (mean • down the lateral cell m e m b r a n e and consist of 4-5 strands, those nearest to the lumen running more or less parallel to the apical membrane. Tight junctions of epithelial cells from later in the menstrual cycle are shown in Fig. 2. These junctions extend 0.21+0.10 gm down the lateral m e m b r a n e which is significantly less (p < 0.01; by 2-tailed t-test) than those shown in Fig. 1 and consist of 1 or 2 strands which show irregular orientation.

Discussion

Recent studies by Martinez-Palomo et al. (1980) (who also review much of the pertinent literature) have established that tight junctions are dynamic structures which can adjust their organization in response to various stimuli. We have shown that tight junctions between uterine luminal epithelial cells undergo characteristic changes both in structure and organization when exogenous ovarian hormones are administered to ovariectomised rats, and in rats

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in normal stages of pregnancy (Murphy et al. 1980, 1981 a, b). The present study shows that tight junctions in luminal epithelial cells of the human uterus also undergo significant changes in depth and organization during the menstrual cycle, being more extensive and deeper in the middle of the cycle than they are later in the cycle. Correlated freeze-fracture and physiological studies have established that the capacity of tight junctions to prevent movement of substances between cells is related to the complexity of organization and depth of the junctions as seen in freeze-fracture electron micrographs (Claude and Goodenough 1973, Staehelin 1974). In view of this, and the findings reported in the present study, it appears likely that tight junctions between epithelial cells in the human uterus are more effective in preventing movement of substances between the cells in the middle of the menstrual cycle than later. Thus, the contents of the uterine lumen may be more closely regulated in the middle of the menstrual cycle than they are at later times. Studies in other animals suggest that regulation of the luminal contents is an important factor in implantation (Pinsker et al. 1974, Denker 1977, Weitlauf 1978). At present however its significance in the human uterus awaits further study. Acknowledgements." This work was supported by an Australian N.H. and M.R.C. grant to Professor A.W. Rogers. Dr. T.M. Mukherjee wishes to acknowledge the N.H. & M.R.C. for providing funds for the purchase of the Balzers Apparatus. We are grateful to Miss J. Deer for expert secretarial assistance.

References Branton D, Bullivant S, Gilula NB, Karnovsky M J, Moor H, Muhlethaler K, Northcote DH, Packer L, Satir B, Satir P, Speth V, Staehelin LA, Steere RL, Weinstein RS (1975) Freeze-etching nomenclature. Science 190: 54-56 Claude P, Goodenough DA (1973) Fracture faces of zonulae occludentes from '~tight" and "leaky" epithelia. J Cell Biol 58:390-400 Denker H-W (1977) Implantation. The role of proteinases and blockage of implantation by proteinase inhibitors. Adv Anat Embryol Cell Biol 53(5) : 1 123 Gilula NB (1978) Structure of intercellular junctions. In: Feldman J, Gilula NB, Pitts JD (eds) Intercellular junctions and synapses. Chapman and Hall, London 1-22 Martinez-Palomo A, Meza I, Beaty G, Cerijido M (1980) Experimental modulation of occluding junctions in a cultured transporting epithelium. J Cell Biol 87 : 736 745 McNutt NS (1977) Freeze-fracture techniques and applications to the structural analysis of the mammalian plasma membrane. Cell Surface Reviews 3:75-126 Murphy CR, Rogers AW, Swift JG, Mukherjee TM (1980) Ovarian hormones after tight junction structure in uterine luminal epithelial cells. Micron 11:375-376 Murphy CR, Swift JG, Mukherjee TM, Rogers AW (1981a) Effects of ovarian hormones on ceil membranes in the rat uterus. I1. Freezefracture studies on tight junctions of the lateral plasma membrane of the luminal epithelium. Cell Biophys 3 : 57-69 Murphy CR, Swift JG, Mukherjee TM, Rogers AW (1981b) Changes in the structure of tight junctions between uterine luminal epithelial cells in pregnant rats. J Anat (in press) Pinsker MG, Sacco AG, Mintz B (1974) Implantation - associated proteinase in mouse uterine fluid. Dev Biol 38:285590 Staehelin LA (1974) Structure and functions of intercellular junctions. Int Rev Cytol 39:191 283 Weitlauf HM (i978) Factors in mouse uterine fluid that inhibit the incorporation of 3H-Uridine by blastocysts in vitro. J Reprod Fert 52:321 325 Accepted November 28, 1981