Human endometrial fluid kinetics as observed by scanning electron microscopy E.
S.
H.
LUDWIG,
H.
METZGER
Detroit,
E.
HAFEZ,
Michigan,
PH.D. M.D.
and Essen,
West Germany
Segments of human endometrium, obtained during difierent stages of the menstrual cycle, were fixed in glutaraldehyde, processed by critical point drying, coated with carbon and gold, then observed with a scanning electron microscope under magnifications varying from 20 to 200,000. The endometrium was basically made of two different types of cells: secretory nonciliated cells and ciliated cells. Different types of secretory cells at different stages of secretory cycles were observed. The endometrial secretion is apocrine: the apical cell membrane of the endometrial cell ruptures, releasing secretory material. The rupture of cells within a given segment of the endometrium is asynchronous. Development of aptical microvilli, synthesis, storage, and release of endometrial secretory granules, and ciliogenesis are hormone dependent. The response of the ciliated and secretory cells of the endometrium varies throughout the menstrual cycle. This is particularly noted in cell specialization, the development of apical microcilli, and ciliogenesis.
T H E E N D 0 M E T R I A L secretion, which is under endocrine control, provides an optimal environment for the transport and capacitation of spermatozoa and the nutrition of the preimplantation blastocyst. Extensive investigations have been carried out on the embryology, ultrastructure, physiology, and biochemistry of the human endometrium.‘5, ?“. ?s A few studies were done on the scanning electron microscopy of the uterus.“, i, lo, I2 The human endometrium From the Department of and C. S. Mott Center for and Development, Wayne of Medicine, Detroit, and of Obstetrics-Gynecology, School of Medicine.
is an organ with a high rate of structural change brought about by the influence of ovarian hormones. These changes are probably mediated by the lysosomes associated with uterine tissue. The endometrial cells have a microvillous surface which is strongly influenced by the action of ovarian hormones, and microvilli are sensitive indicators of the degree of ovarian hormone stimulation. After ovariectomy in the mouse the endometrial microvilli practically disappear, but can be re-established following suitable estrogen replacement therapy.l” Prior to implantation, there are remarkable changes in number and shape of these microvilli.‘” Histochemical techniques have been applied to study the enzyme systems, mucins,’ and carcinoma”” in the human endometrium. Radioactive tracers in conjunction with autoradiography and electron microscopy have been used to investigate endometrial secretion. There are cyclical variations in glycogen content and incorporation of radioactive sulfate by endometrial cells.17* I8 The secretions of the human endometrium, as judged by histologic and cytochemical criteria, are
Gynecology-Obstetrics Human Growth State University School Frauenklinik, Department University of Essen
This investigation was supported by Ford Foundation Grant No. 710-02874 and the National Institute of Child Health and Human Development Grant No. HD-02634. Received Accepted
for publication October
August
21, 1974.
18, 1974.
Reprint requests: Dr. E. S. E. Hafer, Department of Gynecology-Obstetrics, Medical Research Building, 550 E. Canfield, Detroit, Michigan 48201. 929
930
Hafez,
Ludwig,
and
Metzger
Fig. 1. S.E.M. photographs of the human endometrium. Note the general distribution ciliated cells in clusters within the secretory cells covered with microvilli. B, C, D, cells after release of their secretory material. Note the remnants of cells covered generating microvilli. A, ~1,000; B, ~5,000; C, ~5,000; D, ~10,000.
maximal at days 17 to 22 of the menstrual cycle, coinciding with the time of initiation of implantation of blastocyst. Several attempts have been made to collect the uterine luminal fluid by means of a permeable chamber irrigating the lumen, with the use of double-lumen cannula, establishing a uterine fistula, or absorbing the secretion on absorbent paper.” All these techniques have disadvantages with respect to biochemical analysis and physiologic intcrpretations due to contamination of uterine fluid with blood, oviductal secretions, cervical mucus, and peritoneal fluid. The purpose of this investigation is to study the nature and physiologic mechanisms affecting the secretion of the endometrial fluid as observed by scanning electron microscopy. Materials
and
methods
Specimens of uteri were taken from women in the reproductive age at different stages of the re-
of (A) Secretory with de-
productive cycle. One patient was wearing a Lippes loop whereas the rest of the patients were not taking contraceptive measures. Endometrial tissues (approximately 8 by 8 mm. of epithelial surface) were pinned to a cork plate of 2 to 3 mm. in thickness. Specimens attached to cork plates were Boated in freshly prepared, chilled, 2.5 per cent glutaraldehyde. A stock solution of 25 per cent in phosphate buffer of pH 7.4 was kept in the refrigerator. After fixation, two or more pieces of -I by -1 mm. were cut from the specimen. The side to be examined was placed upward and was pinned to a thin cork plate ( 1 mm. thickness). A solution of 5 per cent glutaraldehyde in 0.1M phosphate buffer of pH 7.2 with an osmolarity of 699 mOsm. or 2.5 per cent glutaraldehyde in the same buffer with osmolarity of 399 mOsm. gave excellent results. The higher percentage of glutaraldehyde offered the best fixation, although there is a greater risk of artifacts, especially in very thin biopsies. A good indication of the optimal osmolarity
Volume Number
122 8
Endometrial
fluid
kinetics
931
Fig. 2. S.E.M. photographs of human endometrium showing different stages of ciliogenesis. Note ciliary buds of various length and two ruptured secretory cells in (A). A, ~2,000;B, C, D, ~1,000.
is provided by the adjoining red corpuscles: if they maintain their biconcave shape the higher osmolarity has not adversely influenced them. Before immersing the tissues in the fixative, it was necessary to rinse them gently with an isotonic solution of Krebs-Ringer-glucose or physiologic saline. The tissues were kept in the fixative for 24 to 48 hours. Tissue specimens covered with large quantities of mucus were then transferred to a 0.2M sucrose solution in O.lM phosphate buffer of pH 7.2 for another 24 hours to dissolve and remove any mucus. Severe shrinkage and deformation of tissue was inevitable if fixed tissues were dried without previous dehydration. To minimize these artifacts, the specimens were floated with their surface downward in ascending concentrations of ethyl alcohol, The tissues were dehydrated for 20 to 30 minutes in each of 30, 50, 70, and 90 per cent alcohol and for 10 to 20 minutes in each of 96 per cent and absolute alcohol. Occasionally the tissue was moved slightly. During the critical point drying, the tissue was
placed in amylic acetate for at least 30 minutes with occasional moving. Coating with carbon and gold was made in a vacuum evaporator or in a Hummer Sputtering device. I” The specimens were observed by a Cambridge scanning electron microscope. Details of this technique were described by Ludwig and associates.‘G Specimens were also processed for transmission electron microscopy and light microscopy with Masson trichrome stain. Fresh specimens were immersed in tissue culture medium TC 199 and cilia beat observed 37O C. with interference Normoski optics. Results Much of the endometrial epithelium was shed at each menstrual bleeding. Following the restoration of the integrity of the endometrium, the cells appear poorly differentiated except for those lining the deepest endometrial glands. The endometrium, as observed by scanning electron microscopy, was essentially made of secretory nonciliated cells in different
932
Hafez,
Ludwig,
and
Metzger
Fig. 3. Human endometrium from a patient wearing The endometrium is composed primarily of nonciliated of ciliated cells. Nonciliated cells are heavily coated formation and some stubby cilia. A, ~2,000. B, ~5,000;
stages of development and a few ciliated cells (Figs. 1 and 2). Secretory cells. Both ciliated and secretory gland cells have microvilli (Fig. 1). These microvilli are fine filaments which probably function to stabilize the configuration of the cell surface. At midcycle, coinciding with presumed ovulation, the microvilli became larger. This was preceded by a period associated with an increase in the cytoplasmic content of secretory cells and a rise in the intracellular accumulations of glycogen. There was considerable variation in the morphology of the cells, even within the same region. After ovulation the secretory cells showed extensive hypertrophy. In the middle of the luteal phase the endometrial secretions were discharged through the rupture of apical surface of the cell into the lumen. There was no striking difference between the surface ultrastructure of the lumen and glandular epithelium of the endometrium. Large
a Lippes loop during day 13 of the cycle. secretory cells with interspersed clusters with secretory material. Note rosette-like C, D, ~10,000.
cytoplasmic projections were noted at the apical membrane of the nonciliated cells. The abundance, length, and shape of apical microvilli varied throughout the cycles and the interbranching of microvilli occurred during the menstrual cycle. Fig. 5 is a diagrammatic illustration of different stages of secretory cells. A few degenerating secretory cells could be recognized in all stages of the menstrual cycle. It is not known whether the degenerating cells are exhibiting senescence or they represent changes which precede the release of the secretory material through the ruptured membrane. Ciliated cells. Ciliated cells were less abundant than in the tubal epithelium (Fig. 2). They were found in clusters and around the openings of the endometrial glands. This suggests that the action of kinocilia is to facilitate the release and distribution of the endometrial glands. Ciliogenesis was particularly active around the expected time of
Volume Number
122
Endometrial
a
fluid
kinetics
933
Fig. 4. Human endometrium from the corpus portion on day 13 of the cycle from a patient wearing a Lippes loop. Note the cilia generated at the cell periphery where they are the longest. At the center of the cell note microvilli and shorter cilia in the process of ciliogenesis. A, ~5,000; B, ~10,000; C, ~20,000.
ovulation. A few ciliated cells may also persist from one menstrual cycle to the other. Before the onset of menstruation, ciliated cells with well-developed kinocilia were noted. The microvilli covering the secretory cells became shorter and less branched. The cilia of the endometrium are typical kinocilia (motile) with nine peripheral and two central filaments. The endometrium of the patient wearing the Lippes loop showed the same ultrastructural characteristics of normal endometrium (Figs. 3 and 4). In patients with atrophic endometrium, the cells become flattened and were covered with abundant but short microvilli. Ciliated cells are less abundant and have few kinocilia. The method of fixation had more marked effects on the subsequent S.E.M. image than any of the dehydration routines subsequently used. Different dehydration techniques gave good results at low to medium magnification. The technique of critical
point drying magnifications
becomes valuable (over x20,000).
at low
and
high
Comment
Endometrial cells have several mechanisms of transport processes across the plasma membrane. These processes regulate cellular volume, nutrition, excretion, and communication along the surface of the cell as well as the intracellular and extracellular communication. The endometrial secretions play a major role in the capacitation of spermatozoa and the nutrition of the blastocyst (Figs. 5 and 6). The endometrial fluid is made of ( 1) components from the transudation of the blood serum and (2) protein, carbohydrate, and other metabolites synthesized within the endometrial cells and discharged through the apical cell membrane. The sequence of events of synthesis, release, and distribution of secretory granules is illustrated diagrammatically m Fig. 7. The translation by the ribosomes of a
T>L
iFaiez,
Ludwig,
and
Kefzger
Fig. 5. Diagrammatic illustration different stages of secretory activity.
of the
nonciliated
message is transformed by a molecule of messenger RNA into a polypeptide with a characteristic aminoacid sequence. The protein is assembled on the rough surface of the endoplasmic reticulum. The synthesized polypeptides are transported to the Golgi apparatus where the immature secretory granules undergo maturational changes and coalesce before they are discharged into the lumen. The release of secretory material from the secretory cells seems to occur over several days during the menstrual cycle. This is achieved by asynchronous discharge of adjacent cells in the same region. In the preparatory stages of endometrial secretions? the mitochondria and Golgi reach maximal activity. This is associated with the formation of various vesicles from the Golgi and accumulation of the glycogen particles beneath the plasma membrane, and the attachment of bundles of microfilaments with desmosomes.3, !‘* L’4 Large aggregates of glycogen and various organelles accumulate in the apical portion of the cndometrial cells, causing protrusion of the cytoplasm and subsequent rupture of
cells
of the
human
endometrium
during
the cell membrane and release of the secretions into the uterine lumen. This apocrine type of secretion reaches its maximal activity during days 19 and 21 of the cycle.“, 2b Apocrine secretions have been also demonstrated between days 20 and 24 of the cycle, as shown by scanning electron microscopy.” Degenerated cells may or may not be secretory cells which have already discharged their contents. It is not known whether each cell discharges secretions only once or more during its lifetime. The secretory material is not simply a nonspecific deposit of luminal secretion product for it is absent from the surface of cilia. The physiologic mechanisms which control the synthesis of secretory material are different from those which control discharge of secretions into the uterine lumen. Endometrial epithelium is not generally considered to be absorptive but there are occasions when absorption may play a part in its function. In some species, the preimplantation changes in the uterus are only completed when the embryo is transported to the uterus. If the stimulation from the blastocyst
Endometrial
fluid
kinetics
935
Fig. 6. Diagrammatic illustration of the distrihution pattern of ciliated cells, and the initiation of ciliary huds during the ciliogenesis in the human endometrium. is neurohumoral or chemical, then the substance may be absorbed by the endometrium before reaching the target cells. However, the functional significance of this phenomenon is unknown. The endometrium selectively retains necessary metabolites during the process of filtration, and regulates the composition of endometrial fluid through facultative absorptive and secretory processes (see Keynes’ ’ ) This active transport applies to both secretory and absorptive functions, depending on the net direction of movement of the substrate molecules to and from the cell across the cell membrane via the carrier protein. Throughout the menstrual cycle, the cyclical differentiation and regression of cytoplasmic organelles in the endometrium may be associated with self digestion by lysomes (autolysomes) . Lysomes are membrane-bound vesicles containing several acid hydrolases (digestive enzymes)
Lysomes appear in higher concentrations in the cells of endometrial glands during the late follicular phase of the menstrual cycle. They contain cellular debris in several stages of digestion, some arising from the ingestion of adjacent epithelial cells, leukocytes, and from portions of the cell’s own cytoplasm which have been released in the 1umen.l” The increase in lysosomal activity may be associated with focal cytoplasmic membranes due to the effect of progesterone in the luteal phase, or with autoregulation of excess secretory material.“, ?‘, 25 The degeneration of endometrial cells and subsequent expulsion of the cellular debris into the lumen increase with approaching menstrual period.g’ The Golgi apparatus is involved both in the elaboration of secretory granules (Fig. 1) and in transferring enzymes to lysomes. In actively synthesizing endometrial cells, the Golgi apparatus, the immature secretory granules, and the lysomes are
Fig. 7. Hypothetical fluid. Secretion material. Active
representation of two models through the rupture of the apical transport across the cell membrane.
aggregated in the apical portion of the cell. Most of the carbohydrates in the endometrial fluid are assembled and ester sulfate groups are added to the sugars in the Golgi apparatus of the secretory cell.” Endometrial cells, like most other epithelial cells, possess a variety of cytoplasmic fi1aments.l Microvilli seem to give some rigidity to cell membrane and may be concerned with some forms of movement under the influence of the contractile protein action. Cell membranes are hormonally controlled as judged by the cyclical changes in the distribution, shape, and number of apical microvilli seen in the secretory cells throughout the menstrual cycle. In young female mice, the endometrial cells are completely covered by densely packed microvilli. The tissue from old females are consistently characterized by a conspicuous reduction in number of microvilli,
showing the kinetics cell membrane and
of human the release
endometrial of secretory
some cells being completely denuded while the rernainder show a very sparse covering as a result of reduced ovarian estrogen and/or reduced response of the endometrium to this hormone.“’ In the inactive endometrium, there is a decrease in the ergastoplasm,’ cytoplasmic RNA, and apical alkaline phosphatase.4 The reduction in total surface area as a result of the decrease in the number of microvilli in the aged female may have an adverse effect on the secretory functions of the endometrium. The luminal and glandular epithelium show differential response to circulating levels of estrogen and progesterones. The cyclical changes in the endometrial, cervical, and vaginal cytology also show differential response to steroids. Under the influence of estrogens, rapid cell multiplication is associated with accelerated DNA-dependent RNA synthesis
Endometrial
glyrogen and phospholipid metabolism, the synthesis of alkaline and acid phosphatase, and mitotic activity.” Under the influence of progesterone there is an increase in the biosynthesis and release of mucopolysaccharides, lipids, glycogen, and hydrolytic enzyme activity.‘l Endometrial ciliogenesis may begin as early as day 2 or 3 of the menstrual cycle as judged by the presence of well-developed ciliated cells in this period.’ Future
research
Scanning electron microscopy is a useful investigative tool which provides additional data on uterine physiology and pathology. The following areas are recommended for future investigation: (1) the role of cytoplasmic receptors of estrogens in the control of cyclic changes in endometrial fluid kinetics, and
REFERENCES
1.
364, 3.
GYNECOL.
9%
833,
1967.
Dallenbach-Hellweg, G.: Histopathology of the Endometrium, New York, 1971, Springer-Verlag. L., Israelstam, D., Nino, 5. Edwards, R. G., Talbert, H. V., and Johnson, M. H.: Diffusion chamber for exposing spermatozoa to human uterine secretion, AM. J. OBSTET. GYNECOL. 102: 388, 1968. 6. Ferenczy, A., and Richart, R. M.: Scanning and transmission electron microscopy of the human endometrial surface epithelium, J. Clin. Endocrinol. Metab. 36: 4.
7.
8.
9. 10. 11.
13.
14.
1959.
Cavazos, F., Green, J. A., Hall, D. G., and Lucas, F. V.: Ultrastructure of the human endometrial glandular cell during menstrual cycle, AM. J. OBSTET.
999, 1973. Ferenczy. A., Richart, R. M., Agate, F. J.. Jr., Purkerson, M. L., and Dempsey, E. W.: Scanning electron microscopy of the human endometrial surface epithelium, Fertil. Steril. 23: 515, 1972. Filipe, M. I., and Dawson, I. M. P.: Qualitative and quantitative enzyme histochemistry of the human endometrium and cervix in normal and pathological conditions, J. Pathol. Bact. 95: 243, 258, 1968. Gompel, C.: The ultrastructure of the human endometrial cell studied by electron microscopy, AM. J. OBSTET. GYNECOL. 84: 1000, 1962. Hafez, E. S. E., editor: S.E.M. Atlas of Mammalian Reproduction, Tokyo, 1975, Igaku Shoin, Ltd. Henzl, M. R., Smith, R. E., Boost, G., and Tyler, E. T.: Lysosomal concept of menstrual bleeding in humans, J. Clin. Endocrinol. Metab. 34: 360, 1972.
kinetics
937
in different uterine pathologies, e.g., hyperplasianeoplasia, and adenocarcinoma; (2) the effect of oral steroid contraceptives on the physiology, storage, release, and distribution of endometrial secretions; and (3) the role of endometrial fluid in the transport, maturation, survival, and “capacitation” of spermatozoa. Future research is also needed to delineate the cytochemical changes which the secretory granules undergo during their maturity before they are released in the lumen. Attempts should be made to distinguish between active and passive transports, with emphasis on functional polarity of epithelia, and diffusion potentials with electroneutral active transport. The flux-ratio equation is often expressed as a relation between the gradient of electrochemical potential and the ratio of two unidirectional fluxes.
12.
Adelman, M. R., Borisy, G. G., Shelanski, M. L., Weisenberg, R. C., and Taylor, E. W.: Cytoplasmic filaments and tubules, Fed. Proc. 27: 1186, 1968. 2. Borelli, U., Nilsson, O., and Westman, A.: The cyclical changes occurring in the epithelium lining the endometrial glands. An electron microscopical study in the human being, Acta Obstet. Gynecol. Stand. 38:
fluid
15.
Johannisson,
E., and Nilsson, L.: Scanning
electron
microscopic study of the human endometrium, Fertil. Steril. 23: 613, 1972. Jones, E. C.: The endometrium and the effects of aging (mouse), in Hafez, E. S. E., editor: S.E.M. Atlas of Mammalian Reproduction, Tokyo, 1975, Igaku Shoin, Ltd. Keynes, R. D.: From frog skin to sheep rumen: A survey of transport of salts and water across multicellular structures, Q. Rev. Bio-phys. 2: 177, 1969.
Lawn, A. M.: The ultrastructure
of the endometrium
during
M.
sexual
cycle,
in
Advances in Reproductive
Bishop,
W.
H.,
Physiology, London,
editor:
1973,
Elek. Science, chap. 2. 16. Ludwig, H., Patek, E. E., Nilsson, L., Metzger, H., and Hafez, E. S. E.: Methodology of scanning electron microscopy, in Hafez, E. S. E., editor: S.E.M. Atlas of Mammalian Reproduction, Tokyo, 1975, Igaku Shoin, Ltd., chap. 1. F., and Moricard, R.: Hormonal deter17. Moricard, mination of uterine morphogenesis, Excerpta Med. Int. Congr. Series No. 133, 483, 1967. R., Moricard, F., Gothie, S., Carter, R., 18. Moricard, and Hugon, J.: Modifications ultrastructurales et change en glycogene dans les cellules cylindriques de la muqueuse uterine humaine par action de l’oestradiol et de la progesterone chez la femme ovariectomisee, C. R. Seanc. Sot. Biol. 155: 1831, 1961. 19. Nilsson, 0.: Ultrastructure of mouse uterine surfaces epithelium under different estrogenic influences. 1. Spayed animals and estrus animals, J. Ultrastruct. Res. 1: 375, 1958. 0.: Electron microscopy of the glandular 20. Nilsson, epithelium in the human uterus. 1. Follicular phase, J. Ultrastruct. Res. 6: 413, 1962. 21. O’Malley, B. W.: Mechanisms of action of steroid hormones, N. Engl. J. Med. 284: 370, 1971. 22. Peterson, M., and Leblond, C. P.: Synthesis of complex carbohydrates in the Golgi region, as shown by
25.
24.
25.
raclit ~nuto~raphy alter injecti
S.
H.
LUDWIG,
H.
METZGER
Detroit,
E.
HAFEZ,
Michigan,
PH.D. M.D.
and Essen,
West Germany
Segments of human endometrium, obtained during difierent stages of the menstrual cycle, were fixed in glutaraldehyde, processed by critical point drying, coated with carbon and gold, then observed with a scanning electron microscope under magnifications varying from 20 to 200,000. The endometrium was basically made of two different types of cells: secretory nonciliated cells and ciliated cells. Different types of secretory cells at different stages of secretory cycles were observed. The endometrial secretion is apocrine: the apical cell membrane of the endometrial cell ruptures, releasing secretory material. The rupture of cells within a given segment of the endometrium is asynchronous. Development of aptical microvilli, synthesis, storage, and release of endometrial secretory granules, and ciliogenesis are hormone dependent. The response of the ciliated and secretory cells of the endometrium varies throughout the menstrual cycle. This is particularly noted in cell specialization, the development of apical microcilli, and ciliogenesis.
T H E E N D 0 M E T R I A L secretion, which is under endocrine control, provides an optimal environment for the transport and capacitation of spermatozoa and the nutrition of the preimplantation blastocyst. Extensive investigations have been carried out on the embryology, ultrastructure, physiology, and biochemistry of the human endometrium.‘5, ?“. ?s A few studies were done on the scanning electron microscopy of the uterus.“, i, lo, I2 The human endometrium From the Department of and C. S. Mott Center for and Development, Wayne of Medicine, Detroit, and of Obstetrics-Gynecology, School of Medicine.
is an organ with a high rate of structural change brought about by the influence of ovarian hormones. These changes are probably mediated by the lysosomes associated with uterine tissue. The endometrial cells have a microvillous surface which is strongly influenced by the action of ovarian hormones, and microvilli are sensitive indicators of the degree of ovarian hormone stimulation. After ovariectomy in the mouse the endometrial microvilli practically disappear, but can be re-established following suitable estrogen replacement therapy.l” Prior to implantation, there are remarkable changes in number and shape of these microvilli.‘” Histochemical techniques have been applied to study the enzyme systems, mucins,’ and carcinoma”” in the human endometrium. Radioactive tracers in conjunction with autoradiography and electron microscopy have been used to investigate endometrial secretion. There are cyclical variations in glycogen content and incorporation of radioactive sulfate by endometrial cells.17* I8 The secretions of the human endometrium, as judged by histologic and cytochemical criteria, are
Gynecology-Obstetrics Human Growth State University School Frauenklinik, Department University of Essen
This investigation was supported by Ford Foundation Grant No. 710-02874 and the National Institute of Child Health and Human Development Grant No. HD-02634. Received Accepted
for publication October
August
21, 1974.
18, 1974.
Reprint requests: Dr. E. S. E. Hafer, Department of Gynecology-Obstetrics, Medical Research Building, 550 E. Canfield, Detroit, Michigan 48201. 929
930
Hafez,
Ludwig,
and
Metzger
Fig. 1. S.E.M. photographs of the human endometrium. Note the general distribution ciliated cells in clusters within the secretory cells covered with microvilli. B, C, D, cells after release of their secretory material. Note the remnants of cells covered generating microvilli. A, ~1,000; B, ~5,000; C, ~5,000; D, ~10,000.
maximal at days 17 to 22 of the menstrual cycle, coinciding with the time of initiation of implantation of blastocyst. Several attempts have been made to collect the uterine luminal fluid by means of a permeable chamber irrigating the lumen, with the use of double-lumen cannula, establishing a uterine fistula, or absorbing the secretion on absorbent paper.” All these techniques have disadvantages with respect to biochemical analysis and physiologic intcrpretations due to contamination of uterine fluid with blood, oviductal secretions, cervical mucus, and peritoneal fluid. The purpose of this investigation is to study the nature and physiologic mechanisms affecting the secretion of the endometrial fluid as observed by scanning electron microscopy. Materials
and
methods
Specimens of uteri were taken from women in the reproductive age at different stages of the re-
of (A) Secretory with de-
productive cycle. One patient was wearing a Lippes loop whereas the rest of the patients were not taking contraceptive measures. Endometrial tissues (approximately 8 by 8 mm. of epithelial surface) were pinned to a cork plate of 2 to 3 mm. in thickness. Specimens attached to cork plates were Boated in freshly prepared, chilled, 2.5 per cent glutaraldehyde. A stock solution of 25 per cent in phosphate buffer of pH 7.4 was kept in the refrigerator. After fixation, two or more pieces of -I by -1 mm. were cut from the specimen. The side to be examined was placed upward and was pinned to a thin cork plate ( 1 mm. thickness). A solution of 5 per cent glutaraldehyde in 0.1M phosphate buffer of pH 7.2 with an osmolarity of 699 mOsm. or 2.5 per cent glutaraldehyde in the same buffer with osmolarity of 399 mOsm. gave excellent results. The higher percentage of glutaraldehyde offered the best fixation, although there is a greater risk of artifacts, especially in very thin biopsies. A good indication of the optimal osmolarity
Volume Number
122 8
Endometrial
fluid
kinetics
931
Fig. 2. S.E.M. photographs of human endometrium showing different stages of ciliogenesis. Note ciliary buds of various length and two ruptured secretory cells in (A). A, ~2,000;B, C, D, ~1,000.
is provided by the adjoining red corpuscles: if they maintain their biconcave shape the higher osmolarity has not adversely influenced them. Before immersing the tissues in the fixative, it was necessary to rinse them gently with an isotonic solution of Krebs-Ringer-glucose or physiologic saline. The tissues were kept in the fixative for 24 to 48 hours. Tissue specimens covered with large quantities of mucus were then transferred to a 0.2M sucrose solution in O.lM phosphate buffer of pH 7.2 for another 24 hours to dissolve and remove any mucus. Severe shrinkage and deformation of tissue was inevitable if fixed tissues were dried without previous dehydration. To minimize these artifacts, the specimens were floated with their surface downward in ascending concentrations of ethyl alcohol, The tissues were dehydrated for 20 to 30 minutes in each of 30, 50, 70, and 90 per cent alcohol and for 10 to 20 minutes in each of 96 per cent and absolute alcohol. Occasionally the tissue was moved slightly. During the critical point drying, the tissue was
placed in amylic acetate for at least 30 minutes with occasional moving. Coating with carbon and gold was made in a vacuum evaporator or in a Hummer Sputtering device. I” The specimens were observed by a Cambridge scanning electron microscope. Details of this technique were described by Ludwig and associates.‘G Specimens were also processed for transmission electron microscopy and light microscopy with Masson trichrome stain. Fresh specimens were immersed in tissue culture medium TC 199 and cilia beat observed 37O C. with interference Normoski optics. Results Much of the endometrial epithelium was shed at each menstrual bleeding. Following the restoration of the integrity of the endometrium, the cells appear poorly differentiated except for those lining the deepest endometrial glands. The endometrium, as observed by scanning electron microscopy, was essentially made of secretory nonciliated cells in different
932
Hafez,
Ludwig,
and
Metzger
Fig. 3. Human endometrium from a patient wearing The endometrium is composed primarily of nonciliated of ciliated cells. Nonciliated cells are heavily coated formation and some stubby cilia. A, ~2,000. B, ~5,000;
stages of development and a few ciliated cells (Figs. 1 and 2). Secretory cells. Both ciliated and secretory gland cells have microvilli (Fig. 1). These microvilli are fine filaments which probably function to stabilize the configuration of the cell surface. At midcycle, coinciding with presumed ovulation, the microvilli became larger. This was preceded by a period associated with an increase in the cytoplasmic content of secretory cells and a rise in the intracellular accumulations of glycogen. There was considerable variation in the morphology of the cells, even within the same region. After ovulation the secretory cells showed extensive hypertrophy. In the middle of the luteal phase the endometrial secretions were discharged through the rupture of apical surface of the cell into the lumen. There was no striking difference between the surface ultrastructure of the lumen and glandular epithelium of the endometrium. Large
a Lippes loop during day 13 of the cycle. secretory cells with interspersed clusters with secretory material. Note rosette-like C, D, ~10,000.
cytoplasmic projections were noted at the apical membrane of the nonciliated cells. The abundance, length, and shape of apical microvilli varied throughout the cycles and the interbranching of microvilli occurred during the menstrual cycle. Fig. 5 is a diagrammatic illustration of different stages of secretory cells. A few degenerating secretory cells could be recognized in all stages of the menstrual cycle. It is not known whether the degenerating cells are exhibiting senescence or they represent changes which precede the release of the secretory material through the ruptured membrane. Ciliated cells. Ciliated cells were less abundant than in the tubal epithelium (Fig. 2). They were found in clusters and around the openings of the endometrial glands. This suggests that the action of kinocilia is to facilitate the release and distribution of the endometrial glands. Ciliogenesis was particularly active around the expected time of
Volume Number
122
Endometrial
a
fluid
kinetics
933
Fig. 4. Human endometrium from the corpus portion on day 13 of the cycle from a patient wearing a Lippes loop. Note the cilia generated at the cell periphery where they are the longest. At the center of the cell note microvilli and shorter cilia in the process of ciliogenesis. A, ~5,000; B, ~10,000; C, ~20,000.
ovulation. A few ciliated cells may also persist from one menstrual cycle to the other. Before the onset of menstruation, ciliated cells with well-developed kinocilia were noted. The microvilli covering the secretory cells became shorter and less branched. The cilia of the endometrium are typical kinocilia (motile) with nine peripheral and two central filaments. The endometrium of the patient wearing the Lippes loop showed the same ultrastructural characteristics of normal endometrium (Figs. 3 and 4). In patients with atrophic endometrium, the cells become flattened and were covered with abundant but short microvilli. Ciliated cells are less abundant and have few kinocilia. The method of fixation had more marked effects on the subsequent S.E.M. image than any of the dehydration routines subsequently used. Different dehydration techniques gave good results at low to medium magnification. The technique of critical
point drying magnifications
becomes valuable (over x20,000).
at low
and
high
Comment
Endometrial cells have several mechanisms of transport processes across the plasma membrane. These processes regulate cellular volume, nutrition, excretion, and communication along the surface of the cell as well as the intracellular and extracellular communication. The endometrial secretions play a major role in the capacitation of spermatozoa and the nutrition of the blastocyst (Figs. 5 and 6). The endometrial fluid is made of ( 1) components from the transudation of the blood serum and (2) protein, carbohydrate, and other metabolites synthesized within the endometrial cells and discharged through the apical cell membrane. The sequence of events of synthesis, release, and distribution of secretory granules is illustrated diagrammatically m Fig. 7. The translation by the ribosomes of a
T>L
iFaiez,
Ludwig,
and
Kefzger
Fig. 5. Diagrammatic illustration different stages of secretory activity.
of the
nonciliated
message is transformed by a molecule of messenger RNA into a polypeptide with a characteristic aminoacid sequence. The protein is assembled on the rough surface of the endoplasmic reticulum. The synthesized polypeptides are transported to the Golgi apparatus where the immature secretory granules undergo maturational changes and coalesce before they are discharged into the lumen. The release of secretory material from the secretory cells seems to occur over several days during the menstrual cycle. This is achieved by asynchronous discharge of adjacent cells in the same region. In the preparatory stages of endometrial secretions? the mitochondria and Golgi reach maximal activity. This is associated with the formation of various vesicles from the Golgi and accumulation of the glycogen particles beneath the plasma membrane, and the attachment of bundles of microfilaments with desmosomes.3, !‘* L’4 Large aggregates of glycogen and various organelles accumulate in the apical portion of the cndometrial cells, causing protrusion of the cytoplasm and subsequent rupture of
cells
of the
human
endometrium
during
the cell membrane and release of the secretions into the uterine lumen. This apocrine type of secretion reaches its maximal activity during days 19 and 21 of the cycle.“, 2b Apocrine secretions have been also demonstrated between days 20 and 24 of the cycle, as shown by scanning electron microscopy.” Degenerated cells may or may not be secretory cells which have already discharged their contents. It is not known whether each cell discharges secretions only once or more during its lifetime. The secretory material is not simply a nonspecific deposit of luminal secretion product for it is absent from the surface of cilia. The physiologic mechanisms which control the synthesis of secretory material are different from those which control discharge of secretions into the uterine lumen. Endometrial epithelium is not generally considered to be absorptive but there are occasions when absorption may play a part in its function. In some species, the preimplantation changes in the uterus are only completed when the embryo is transported to the uterus. If the stimulation from the blastocyst
Endometrial
fluid
kinetics
935
Fig. 6. Diagrammatic illustration of the distrihution pattern of ciliated cells, and the initiation of ciliary huds during the ciliogenesis in the human endometrium. is neurohumoral or chemical, then the substance may be absorbed by the endometrium before reaching the target cells. However, the functional significance of this phenomenon is unknown. The endometrium selectively retains necessary metabolites during the process of filtration, and regulates the composition of endometrial fluid through facultative absorptive and secretory processes (see Keynes’ ’ ) This active transport applies to both secretory and absorptive functions, depending on the net direction of movement of the substrate molecules to and from the cell across the cell membrane via the carrier protein. Throughout the menstrual cycle, the cyclical differentiation and regression of cytoplasmic organelles in the endometrium may be associated with self digestion by lysomes (autolysomes) . Lysomes are membrane-bound vesicles containing several acid hydrolases (digestive enzymes)
Lysomes appear in higher concentrations in the cells of endometrial glands during the late follicular phase of the menstrual cycle. They contain cellular debris in several stages of digestion, some arising from the ingestion of adjacent epithelial cells, leukocytes, and from portions of the cell’s own cytoplasm which have been released in the 1umen.l” The increase in lysosomal activity may be associated with focal cytoplasmic membranes due to the effect of progesterone in the luteal phase, or with autoregulation of excess secretory material.“, ?‘, 25 The degeneration of endometrial cells and subsequent expulsion of the cellular debris into the lumen increase with approaching menstrual period.g’ The Golgi apparatus is involved both in the elaboration of secretory granules (Fig. 1) and in transferring enzymes to lysomes. In actively synthesizing endometrial cells, the Golgi apparatus, the immature secretory granules, and the lysomes are
Fig. 7. Hypothetical fluid. Secretion material. Active
representation of two models through the rupture of the apical transport across the cell membrane.
aggregated in the apical portion of the cell. Most of the carbohydrates in the endometrial fluid are assembled and ester sulfate groups are added to the sugars in the Golgi apparatus of the secretory cell.” Endometrial cells, like most other epithelial cells, possess a variety of cytoplasmic fi1aments.l Microvilli seem to give some rigidity to cell membrane and may be concerned with some forms of movement under the influence of the contractile protein action. Cell membranes are hormonally controlled as judged by the cyclical changes in the distribution, shape, and number of apical microvilli seen in the secretory cells throughout the menstrual cycle. In young female mice, the endometrial cells are completely covered by densely packed microvilli. The tissue from old females are consistently characterized by a conspicuous reduction in number of microvilli,
showing the kinetics cell membrane and
of human the release
endometrial of secretory
some cells being completely denuded while the rernainder show a very sparse covering as a result of reduced ovarian estrogen and/or reduced response of the endometrium to this hormone.“’ In the inactive endometrium, there is a decrease in the ergastoplasm,’ cytoplasmic RNA, and apical alkaline phosphatase.4 The reduction in total surface area as a result of the decrease in the number of microvilli in the aged female may have an adverse effect on the secretory functions of the endometrium. The luminal and glandular epithelium show differential response to circulating levels of estrogen and progesterones. The cyclical changes in the endometrial, cervical, and vaginal cytology also show differential response to steroids. Under the influence of estrogens, rapid cell multiplication is associated with accelerated DNA-dependent RNA synthesis
Endometrial
glyrogen and phospholipid metabolism, the synthesis of alkaline and acid phosphatase, and mitotic activity.” Under the influence of progesterone there is an increase in the biosynthesis and release of mucopolysaccharides, lipids, glycogen, and hydrolytic enzyme activity.‘l Endometrial ciliogenesis may begin as early as day 2 or 3 of the menstrual cycle as judged by the presence of well-developed ciliated cells in this period.’ Future
research
Scanning electron microscopy is a useful investigative tool which provides additional data on uterine physiology and pathology. The following areas are recommended for future investigation: (1) the role of cytoplasmic receptors of estrogens in the control of cyclic changes in endometrial fluid kinetics, and
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GYNECOL.
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Dallenbach-Hellweg, G.: Histopathology of the Endometrium, New York, 1971, Springer-Verlag. L., Israelstam, D., Nino, 5. Edwards, R. G., Talbert, H. V., and Johnson, M. H.: Diffusion chamber for exposing spermatozoa to human uterine secretion, AM. J. OBSTET. GYNECOL. 102: 388, 1968. 6. Ferenczy, A., and Richart, R. M.: Scanning and transmission electron microscopy of the human endometrial surface epithelium, J. Clin. Endocrinol. Metab. 36: 4.
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Cavazos, F., Green, J. A., Hall, D. G., and Lucas, F. V.: Ultrastructure of the human endometrial glandular cell during menstrual cycle, AM. J. OBSTET.
999, 1973. Ferenczy. A., Richart, R. M., Agate, F. J.. Jr., Purkerson, M. L., and Dempsey, E. W.: Scanning electron microscopy of the human endometrial surface epithelium, Fertil. Steril. 23: 515, 1972. Filipe, M. I., and Dawson, I. M. P.: Qualitative and quantitative enzyme histochemistry of the human endometrium and cervix in normal and pathological conditions, J. Pathol. Bact. 95: 243, 258, 1968. Gompel, C.: The ultrastructure of the human endometrial cell studied by electron microscopy, AM. J. OBSTET. GYNECOL. 84: 1000, 1962. Hafez, E. S. E., editor: S.E.M. Atlas of Mammalian Reproduction, Tokyo, 1975, Igaku Shoin, Ltd. Henzl, M. R., Smith, R. E., Boost, G., and Tyler, E. T.: Lysosomal concept of menstrual bleeding in humans, J. Clin. Endocrinol. Metab. 34: 360, 1972.
kinetics
937
in different uterine pathologies, e.g., hyperplasianeoplasia, and adenocarcinoma; (2) the effect of oral steroid contraceptives on the physiology, storage, release, and distribution of endometrial secretions; and (3) the role of endometrial fluid in the transport, maturation, survival, and “capacitation” of spermatozoa. Future research is also needed to delineate the cytochemical changes which the secretory granules undergo during their maturity before they are released in the lumen. Attempts should be made to distinguish between active and passive transports, with emphasis on functional polarity of epithelia, and diffusion potentials with electroneutral active transport. The flux-ratio equation is often expressed as a relation between the gradient of electrochemical potential and the ratio of two unidirectional fluxes.
12.
Adelman, M. R., Borisy, G. G., Shelanski, M. L., Weisenberg, R. C., and Taylor, E. W.: Cytoplasmic filaments and tubules, Fed. Proc. 27: 1186, 1968. 2. Borelli, U., Nilsson, O., and Westman, A.: The cyclical changes occurring in the epithelium lining the endometrial glands. An electron microscopical study in the human being, Acta Obstet. Gynecol. Stand. 38:
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E., and Nilsson, L.: Scanning
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Lawn, A. M.: The ultrastructure
of the endometrium
during
M.
sexual
cycle,
in
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Bishop,
W.
H.,
Physiology, London,
editor:
1973,
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raclit ~nuto~raphy alter injecti
