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J. Anat. (1992) 181, pp. 301-312, with 7 figures. Printed in Great Britain
Zonal differentiation of the marmoset (Callithrix jacchus) endometrium G. M. RUNE', U. LEUCHTENBERG', C. SCHROTER-KERMANF2 AND H.-J. MERKER"12 Departments of Anatomy and 2 Toxicology and Prenatal Pharmacology, Free University of Berlin, Germany
(Accepted 3 August 1992)
ABSTRACT
The differentiation of the marmoset (Callithrix jacchus) endometrium under different steroid hormone levels was investigated by electron microscopy and by the binding of different antibodies directed against collagen types. Based on differences in the glandular and interglandular compartments, the endometrium of sexually mature common marmosets consists of 3 zones: basal, adluminal and luminal. Hormone-dependent appearances are characterised. With low steroid concentrations, intercellular spaces between glandular epithelial cells occurred in the adluminal and the luminal areas. Epithelial cells of the basal region exhibited coated pits and phagolysosomes together with large apical protrusions. Under oestrogen dominance, phagolysosomes, fat vesicles and apical protrusions were evident in epithelial cells in the adluminal region. Secretory granules and concentric glycogen accumulations were a characteristic feature in epithelial cells of the adluminal and basal regions. With high progesterone concentrations, large empty vesicles were found with a higher frequency in adluminal than in basal epithelial cells. Using FITC-labelled antibodies against types V and VI collagen, binding was apparent adluminally in close vicinity to basement membranes, whereas reactivity was seen in the entire interglandular region of the basal area during this phase. Our findings indicate specific microenvironments with distinctive structural characteristics in the marmoset endometrium that are hormone dependent during all phases of the endometrial cycle. They are not related to menstruation, appear to be characteristic for primates and could reflect epithelial-mesenchymal interaction.
INTRODUCTION
The endometria of humans and menstruating primates (Rhesus) are subcompartmentalised. This was shown histologically by Bartelmez et al. (1951), ultrastructurally by Kaiserman-Abramof & Padykula (1989), and through [3H]thymidine uptake studies by Padykula et al. (1984). Based on differences between glandular epithelial cells a zonation of the endometrium was described as follows: zone I, luminal epithelium; zone II, uppermost gland segments; zone III, middle gland segments; and zone IV, basal gland segments. This definition was thought to provide a key to deciphering the mechanisms that underlie repetitive cyclicity. It is not known whether this kind of endometrial zonation is also valid for the interglandular compartment. Collagen, however, is known
to undergo considerable alterations during the menstrual cycle (Woessner, 1982; Aplin et al. 1988). An extremely dynamic state of the extracellular matrix (ECM) participates in the process of cyclic endometrial renewal. Therefore, with respect to endometrial cyclicity it appears essential to investigate the binding of various antibodies against different constituents of the ECM in association with studies on the ultrastructure of the glandular and interglandular compartment. For our experiments we used common marmosets (Callithrixjacchus) which are valuable primate models in reproductive-biological research (Holt & Moore, 1984; Jackson & Edmunds, 1984; Hiller et al. 1987; Rune & Heger, 1987; Rune et al. 1988, 1991). In addition, these New World monkeys are much more suitable for experimentation than other primates: e.g.
Correspondence to Dr G. M. Rune, Department of Anatomy, Free University of Berlin, Konigin-Luise-Strasse 15, D-1000 Berlin 33, Germany.
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low maintenance costs, 3-5 offspring per year and mother. However, marmosets do not menstruate (Hearn & Lunn, 1975) and thus, according to Padykula et al. (1984) and Kaiserman-Abramof & Padykula (1989) are not expected to show endometrial zonation or to be suitable as primate models for the study of uterine cyclicity. This study asks whether endometrial zonation is evident in both glandular and interglandular compartments and whether endometrial zonation is necessarily followed by menstruation. As it is well established that the uterus is controlled by varying levels of oestradiol and progesterone during the reproductive cycle, we studied the marmoset endometrium in relation to oestrogen and progesterone concentrations in urine (at defined stages of the reproductive cycle).
were placed in a metabolism cage and urine samples were collected at room temperature on sheets of aluminium foil between 11 a.m. and 2 p.m. The urine was sucked up with a disposable syringe and immediately stored at -20 °C until hydrolysis. Urinary 6,f-hydroxypregnanolone and oestrogen excretions were used for the determination of the cycle stage and were measured by means of high performance thinlayer chromatography, as fully described by Heger & Neubert (1983). Three cycles were monitored before the females were killed at a defined stage of the following cycle (see Fig. 1). Only regularly cycling monkeys were used. For this study we investigated monkeys at d 1 (2 monkeys), d 3 (1 animal), d 4 (1 animal), d 8 (3 animals), d 11 (2 animals), d 15, 18, and 20 (1 animal each), and d 28/29 (3 animals) of the reproductive cycle.
MATERIAL AND METHODS
Animal maintenance
Morphology
Marmosets were kept in our colony in single family groups of 1 female, 1 male and their offspring aged less than 120 d. The room temperature was at 27+1 °C and the humidity was maintained at 55 + 5 %. The animals were kept under a constant day/night cycle (light from 6 a.m. to 6 p.m.) and were fed a standardised Altromin Marmoset Diet, water ad libitum, and about 30 g fruit and 5 g boiled egg 3 times a week.
The animals were anaesthetised and killed by an overdose of thiopental (Byk van Gulden, Konstanz, Germany) at 10 a.m. The uteri of the animals were immediately removed, cut into small pieces and either fixed by immersion according to Karnovsky (1965) for 24 h or frozen in liquid nitrogen and stored at -20°C until use. The fixed specimens were then thoroughly washed in 0.1 M phosphate buffer, pH 7.4, and postfixed for 1 h in phosphate-buffered (0.1 M) 1 % OS04 at pH 7.4 for 1 h. After dehydration in acetone they were embedded in Epon 812. Sections were cut on an ultramicrotome (Reichert-Jung OmU 3, Vienna, Austria).
Monitoring the reproductive cycle
For the determination of the cycle stage, females were isolated from the male marmoset. Each morning they
Light microscopy. Semithin sections (1 gm) were mounted on slides and stained with methylene blue/azure II according to Richardson et al. (1960).
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Electron microscopy. Ultrathin sections were stained with uranyl acetate followed by lead citrate (Reynolds, 1963). The sections were examined with a Zeiss electron microscope (EM 10, Oberkochen, Germany).
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For preparation of the antibodies the following antigens were used. Type I collagen was obtained from fetal mouse skin according to Trelstad et al. (1976), type III collagen from fetal mouse skin according to Smith & Niles (1980), type IV collagen from human placenta according to Sage et al. (1979). Type V collagen from human placenta was purchased
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from Heyl (Berlin, Germany). Laminin was prepared from EHS sarcoma according to Timpl et al. (1979). Antisera were prepared in rabbits and purified by
immunoadsorption according to Kittelberger-Ewert et al. (1988). The monospecificity of the antibodies had been established by the ELISA technique
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Fig. 2. Low steroid level (L, glandular lumen; S, stroma). a, Cytoplasm of glandular epithelial cells with microvilli at the apical membrane and junctional complexes. Many mitochondria of the crista type, abundant rough ER and an extensive Golgi apparatus are present. Cells are in close contact with each other with invaginated plasma membranes ( x 16000). b, Enlargement of intercellular spaces between neighbouring epithelial cells (arrows) are apparent in the luminal and adluminal areas. Part of a lymphocyte is seen in the mid right of the micrograph ( x 18000). c, Coated pits (arrows) and lysosome-like structures- (arrowheads) are seen in epithelial cells of the myometrium ( x 30000). d, Cellular surfaces with short microvilli are smooth in the adluminal region of the endometrium ( x 17000). e, Large apical cytoplasmic protrusions are found in epithelial cells at the base of the glands ( x 6000).
(Gosslau & Barrach, 1979) on microtitre plates, coated with laminin, nidogen, fibronectin and types I, II, III, IV, V and VI collagen. All purified antibodies reacted only with their respective antigen. The binding of the antibodies to unfixed cryostat sections was followed
by incubation with fluoroisothiocyanate (FITC)labelled antibodies against IgG. The sections were evaluated under a Zeiss (Oberkochen, Germany) fluorescence microscope.
Differentiation of the marmoset endometrium
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Steroid levels As seen in Fig. 1 and based on steroid levels in urine, the reproductive cycles of the investigated marmosets lasted about 28-29 d. They were characterised by high levels of oestrogens after roughly one third of the
cycle, indicating the time of ovulation. Concentrations of 6,8-hydroxypregnanolone were low during the first 8-9 d of the cycle and increased after ovulation. The peripheral level of this steroid metabolite remained high up to 18-19 d on average after ovulation. The rise of oestrogens preceded the rise of the progesterone metabolite during the phase before ovulation and a
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3(e)
Fig. 3. High oestrogen/low progesterone level (L, glandular lumen; S, stroma). a and b, Large phagolysosomes together with fat vacuoles, apical cellular protrusions and apical secretory vesicles (arrow) are seen in the adluminal zone (a; x 3200). Except for apical secretory vesicles (arrow), these features are lacking in the basal region of the endometrium (b; x 4000). c and d, The apical secretory vesicles are highly electron dense in the adluminal area of the endometrium (c; x 15600) and appear as dense core-like vesicles (arrows) in epithelial cells at the base of the glands (d; x 20000). e andf, Glycogen accumulations (f; x 8000; arrows) mostly with a concentric arrangement (e; x 20000) together with dilated elements of rough ER (arrowheads) are typical epithelial features in the adluminal and basal regions.
continuous decrease of oestrogen was perceptible after the ovulatory oestrogen peak.
Morphology
During all 3 phases of the reproductive cycle investigated, zonation of the endometrium was evident based on the organisation of the stroma and morphological differences between the epithelial cells. Three zones were identified. A basal zone bordering on the myometrium could be distinguished from an upper adluminal area and the luminal epithelium. However, the appearance of each of these 3 zones was hormone-dependent. Low steroid level (d 1, 3 and 4 of the reproductive
cycle) The endometrium contained long narrow glands which consisted of tall columnar cells with nuclei in the majority of the cells located near the basement
membrane in all areas. Mitochondria, glycogen particles, abundant rough endoplasmic reticulum (RER) and an extended Golgi apparatus were present. Microvilli at the cell surfaces, junctional complexes between the apical lateral membranes, and microfilaments running parallel and beneath the apical plasma membrane were common characteristics of all epithelial cells (Fig. 2a). Enlargement of intercellular spaces between neighbouring epithelial cells particularly in the basolateral region, was found in the luminal and adluminal area (Fig. 2b). In the basal region, dilatations of the glandular lumen filled with necrotic cellular material were occasionally seen. Coated pits, lysosome-like structures (Fig. 2 c), and phagolysosomes were specific features of epithelial cells in this zone. In the adluminal area, the apical surfaces of the epithelial cells were smooth with only few and short microvilli (Fig. 2d), whereas large apical protrusions with numerous and long microvilli were typical in the basal region (Fig. 2e).
Fig. 4. Progesterone dominance (L, lumen; S, stroma). a, During the late secretory phase, large vesicles dominate the supranuclear cytoplasm in close association with an extensive Golgi apparatus (arrows). Some of the vesicles are filled with membranous material. They also occur in the glandular lumen ( x 5000). b and c, In the adluminal part of the endometrium these vesicles occur also in cytoplasmic areas of epithelial cells at the basement membrane (arrowheads) (b; x 3200). These vesicles are lacking at the basement membrane (arrowheads) in epithelial cells of the basal region of the endometrium (c; x 3200).
Differentiation of the marmoset endometrium
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The stroma surrounding the endometrial glands was highly cellular, mostly fibroblastic with more collagen bundles in the basal than in the upper zone.
High oestrogen/low progesterone levels (d 8 and 11 of the cycle) The width of the endometrium had clearly increased. Generally, the narrow glandular lumen was filled with cellular debris, which obviously originated from epithelial cells shed into the lumen. No regional differences were found for the distribution, shape or size of mitochondria, microvilli, and junctional complexes. All epithelial cells were closely apposed and large areas of invaginating plasma membranes were seen. In all epithelial cells the Golgi apparatus occupied large areas in the cytoplasm. Phagolysosomes of striking size and mostly in a supranuclear position together with fat vesicles were typical features of adluminal epithelial cells (Fig. 3 a). Apical protrusions were seen in the adluminal area (Fig. 3 a) but not in epithelial cells at the base of glands (Fig. 3 b). Secretory granules in the apical region of epithelial cells, filled with highly electron dense material were characteristic (Fig. 3 a, d). At the base of glands, these secretory granules appeared as dense core-like vesicles surrounded by a halo (Fig. 3d). Elements of RER frequently appeared dilated and glycogen accumulations were arranged concentrically (Fig. 3 e, f) in the adluminal and basal zones. Differences in cellular density between the adluminal and basal interglandular compartment became evident. Basally, cells were more closely packed than adluminally. Progesterone dominance (d 15, 18, 20, and 28 of the cycle) An increase of endometrial height and of glandular diameter was observed. Certain glandular segments were extremely widened and filled with necrotic cellular material. The widening of the glandular lumen started obviously in the basal area and progressed in the upper zone. Progesterone dominance did not influence the ultrastructure of mitochondria, RER, microvilli or junctional complexes. During early progesterone dominance concentrically arranged glycogen accumulations were still occasionally visible. Few phagolysosomes were seen in adluminal epithelial cells. Dilated elements of RER and intercellular spaces between the basolateral plasma membranes were found basally. In the adluminal and basal areas
in a supranuclear position were numerous large vesicles, some of which were filled with membranous material (Fig. 4a). They were more frequent in the adluminal than in the basal epithelial cells but were absent in the luminal epithelium. In the course of progesterone dominance they increased in number so that at the end of the cycle the entire supranuclear space was completely filled. Adluminally, they could also be observed in the basal part of the epithelial cell (Figs 4b, c). Generally, they were located in close vicinity to the extended Golgi apparatus. They had an irregular outline and varied in size. The largest appeared to flow together. Regional differences in the organisation of connective tissue between the endometrial glands were most obvious during the dominating progesterone influence (Figs 5, 6). In the area bordering on the myometrium the cells were closely packed, and were oriented with their longitudinal -cellular axis parallel to the glandular basement membranes. Thick collagen bundles appear to run between the cells. Connective tissue of the upper area appeared loosened. Intercellular spaces were clearly enlarged and collagen fibrils were arranged in a network-pattern instead of being oriented in parallel.
Immunohistochemistry The distribution pattern of collagen immunoreactivity revealed that types I and III (Fig. 7a) collagens were diffusely distributed in the connective tissue of the interglandular compartment with no regional or collagen type differences throughout the endometrial cycle. Type IV collagen and laminin were exclusively observed in a fine linear fashion along the blood vessels and along the uterine glands, thus showing the delineation of the basement membranes throughout the cycle (Fig. 7b). With low steroid levels as well as under dominant oestrogen influence, the interglandular region of the endometrium exhibited a strong fluorescent meshwork of fibres showing binding of antibodies against types V and VI collagen. An association with basement membranes was evident for type V collagen. Zonation of the endometrium in the adluminal zone and the basal area was only perceptible under the influence of high progesterone levels. With antibodies against type V collagen the fluorescence in the adluminal part was only detectable in close association with the basement membranes of vessels, uterine glands (Fig. 7c) and of the luminal epithelium. In this area, the remaining interglandular connective tissue was devoid of any reactivity. In contrast, the staining was comparable to
Differentiation of the marmoset endometrium
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Figs 5 and 6. Interglandular compartment under high progesterone concentrations. Semithin sections. The cellular density of the interglandular compartment is lower in the adluminal (4; x 380) than in the basal region of the endometrium (5; x 400).
the other cycle phases in the basal part of the endometrium (Fig. 7d, e). Using antibodies against type VI collagen, binding also was apparent adluminally in close vicinity to basement membrane (Fig. 7f), whereas reactivity was seen in the entire interglandular region of the basal area (Fig. 7g, h). DISCUSSION
Our electron microscope investigations have shown that, independent of the cycle, the marmoset endometrium exhibits zonal differentiation of glands and of the stromal compartment, despite the fact that these monkeys do not menstruate. Zonation of the endometrial stroma was also revealed by the binding of antibodies against types V and VI collagen. Zonal differentiation has also been shown in the human endometrium by histological investigations (Bartelmez et al. 1951) and in the Rhesus monkey (Padykula et al. 1984; Kaiserman-Abramof & Padykula, 1989) from the epithelial ultrastructure and mitotic rate. However, Padykula et al. (1984) were able to distinguish 4 zones in the Rhesus monkey, 21
whereas we could detect only 3 in the marmoset: a basal area, bordering on the myometrium, an adluminal region and the luminal zone. Thus the subdivision of the basal area into 2 zones, seen in the human and Rhesus endometrium seems to be absent in the marmoset. In the marmoset, zonal differentiation of epithelial cells was mainly based on the presence of intercellular spaces, phagolysosomes, secretory granules, coated pits, and widening of the glandular lumina. As in the Rhesus monkey (KaisermanAbramof & Padykula, 1989), the zonation was permanently evident throughout the cycle, despite the fact that single zones differed in their appearance at certain stages of the cycle. Thus finding implies that the existence of zonation per se in the marmoset endometrium is an integral feature that is independent of hormonal influences, whereas the differential behaviour of these zones throughout the cycle is due to varying steroid levels (e.g. secretory electron dense granules under the influence of oestrogen or empty vesicles under the influence of progesterone in the adluminal area). Consequently, zonation of the endometrium is not a unique feature of menstruating ANA 181
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Differentiation of the marmoset endometrium
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primates, as proposed by Kaiserman-Abramof and Padykula (1989) and thus is not directly related to menstruation. Instead, it could be a specific phenomenon of menstruating as well as of nonmenstruating primates. To our knowledge, it has never been described for other mammals. With regard to our morphological findings (zonation) we cannot exclude the possibility that the high osmolality and the slow and nonlinear penetration of the fixative could have resulted in some degree of artefactual change. Shrinkage artefacts are, however, unusual after immersion fixation of tissue fragments (Karnovsky, 1965) and our histochemical findings support the relevance of the conclusions based on the morphological findings obtained from fixed tissue. In addition to regional differences in the ultrastructure of epithelial cells, our results show that the zonation of primate endometria is also apparent in the interglandular compartment: (1) by different cellular density in the basal and adluminal regions and (2) by the different binding behaviour of types V and VI collagen antibodies in the interglandular compartment. In contrast to the ultrastructural zonation, the latter was seen exclusively under the influence of progesterone. In spite of rather well-established knowledge concerning the biochemical nature of types V and VI collagen their functions are not yet clarified (Timpl & Engel, 1987). From the close association of both collagen types to type I collagen, basement membranes and other ECM constituents, it has been suggested that they interconnect the different ECM components. For type VI collagen, Trueb & Bornstein (1984) have suggested an adaptor function with type VI collagen serving as a link for fibrous and globular proteins with cells. It appears that both types VI and V collagen act as binding or attachment proteins, linking and integrating different components of the ECM (Karkavelas et al. 1988). With regard to our findings concerning the different distribution of types V and VI collagen in the basal and adluminal compartments in the presence of high progesterone levels, it follows that during this phase this link is lost adluminally except for those areas beneath basement membranes of glands and vessels. This is partly
consistent with findings of Aplin et al. (1988), as in the human endometrium type VI collagen is abundant in the endometrium during the proliferative phase, but is progressively lost during the secretory phase. However, it is difficult to compare the results of that study with the findings of our investigation since these authors used material that was obtained from curettage operations. For methodological reasons it is impossible or at least very difficult to detect any zonation with such material. Furthermore, our findings support biochemical investigations which have clearly demonstrated that the menstrual cycle affects the deposition and removal of collagen (Pastore et al. 1989). Both steroid hormones, oestrogens and to a less extent progesterone, have been shown to be effective (Woessner, 1982). However, reports about the influence of oestrogen, for example, on collagen metabolism in the uterus, are rather conflicting. It has been suggested that oestrogens increase (Cullen & Harkness, 1964), have no effect (Ryan & Woessner, 1972) or even stimulate both the synthesis and breakdown of collagen (Pastore et al. 1989; Dyer et al. 1980). In our investigation the disappearance of any reactivity between the adluminal glands as soon as the progesterone level had risen, indicates that progesterone plays a regulative role in the metabolism of types V and VI collagen, which had already been found in women by Aplin et al. (1988). Neither regional nor hormone dependent differences were detectable when we used antibodies against types I and III collagen and against basement membrane components such as laminin and type IV collagen. Regional differences in the glandular and interglandular compartment in the marmoset endometrium may result from mesenchymal-epithelial interaction. It is known that the stroma is required for normal epithelial development (Cunha et al. 1985) and such an interaction has been demonstrated in man (Roberts et al. 1988). We could not find similar morphological correlations (e.g. lamina densa disruptions by epithelial projections) for such an interaction in the marmoset endometrium. Nevertheless, in our investigation this interaction could be mediated by the
Fig. 7. Immunoreactivity of anticollagen antibodies. Progesterone dominance. a, Endometrium stained with type II collagen antibody. An evenly stained network of interstitial immunoreactivity is evident ( x 250). b, Immunofluorescence of type IV collagen antibody. Only glandular and vascular basement membranes carry the antigen (x 120). c-e, Immunofluorescence of type V collagen antibody. c, In the adluminal part of the endometrium immunoreactivity is associated to the glandular and vascular basement membranes (x 230). On approaching the myometrium an additional fluorescent network becomes apparent. d, Transitional part between the adluminal and basal regions ( x 270). e, Basal region of the endometrium ( x 250). f-h, Immunofluorescence of type VI collagen antibody. f, In the adluminal part strong staining is seen throughout the walls of blood vessels and weaker immunoreactivity at the glandular basement membranes ( x 250). Immunoreactivity becomes additionally visible in the stroma of areas directed towards the myometrium. g, Transitional part between the adluminal and basal regions ( x 230). h, Basal region bordering on the myometrium ( x 160). 21-2
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composition of stromal ECM, at least under progesterone dominance.
ACKNOWLEDGEMENT
This investigation was supported by the Deutsche Forschungsgemeinschaft (Sfb 174).
REFERENCES
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854-861. RUNE GM, HEGER W (1987) Histochemical localization of 3,/hydroxysteroid dehydrogenase in marmoset ovaries during proand diestrus with special reference to substrate specificity. Histochemistry 86, 621-625. RUNE GM, HEGER W, MERKER HJ (1988) Development of the cellular basis for reproduction in female marmosets. II. Uterus. In Non-human Primates: Developmental Biology and Toxicology (ed. D. Neubert, H. J. Merker & C. Hendrickx), pp. 303-326. Vienna: Ueberreuther Wissenschaft. RUNE GM, DE SOUZA PH, MERKER HJ (1991) Ultrastructural and histochemical characterization of marmoset (Callithrix jacchus) Leydig cells during postnatal development. Anatomy and Embryology 183, 179-191. RYAN JN, WOESSNER JF (1972) Oestradiol inhibits collagen breakdown in the involuting rat uterus. Biochemical Journal 127, 705-713. SAGE H, WOODBURRY RG, BORNSTEIN P (1979) Structural studies on human type IV collagen. Journal of Biological Chemistry 254, 9893-9900. SMITH BD, NILES R (1980) Characterization of collagen synthesized by normal and chemically transformed rat liver epithelial cell lines. Biochemistry 19, 1820-1825. TIMPL R, ROHDEM H, GERONM RP, RENNARD SJ, FOIDART JM, MARTIN GR (1979) Laminin -a glycoprotein from basement membranes. Journal of Biological Chemistry 254, 9933-9937. TIMPL R, ENGEL J (1987) Type VI collagen. In Structure and Function of Collagen Types (ed. R. Mayne, R. Burgeon), pp. 105-143. New York: Academic Press. TRELSTAD RL, CATANESE VM, RUBIN DF (1976) Collagen fractionation: separation of native types I, II and III by differential precipitation. Annals of Biochemistry 71, 114-118. TRUEB B, BORNSTEIN P (1984) Characterization of the precursor form of type VI collagen. Journal of Biological Chemistry 259, 8597-8604. WOESSNER JF (1982) Uterus, cervix and ovary. In Collagen in Health and Disease (ed. J. B. Weiss & M. I. V. Jayson), pp. 183-214. Edinburgh: Churchill Livingstone.
J. Anat. (1992) 181, pp. 301-312, with 7 figures. Printed in Great Britain
Zonal differentiation of the marmoset (Callithrix jacchus) endometrium G. M. RUNE', U. LEUCHTENBERG', C. SCHROTER-KERMANF2 AND H.-J. MERKER"12 Departments of Anatomy and 2 Toxicology and Prenatal Pharmacology, Free University of Berlin, Germany
(Accepted 3 August 1992)
ABSTRACT
The differentiation of the marmoset (Callithrix jacchus) endometrium under different steroid hormone levels was investigated by electron microscopy and by the binding of different antibodies directed against collagen types. Based on differences in the glandular and interglandular compartments, the endometrium of sexually mature common marmosets consists of 3 zones: basal, adluminal and luminal. Hormone-dependent appearances are characterised. With low steroid concentrations, intercellular spaces between glandular epithelial cells occurred in the adluminal and the luminal areas. Epithelial cells of the basal region exhibited coated pits and phagolysosomes together with large apical protrusions. Under oestrogen dominance, phagolysosomes, fat vesicles and apical protrusions were evident in epithelial cells in the adluminal region. Secretory granules and concentric glycogen accumulations were a characteristic feature in epithelial cells of the adluminal and basal regions. With high progesterone concentrations, large empty vesicles were found with a higher frequency in adluminal than in basal epithelial cells. Using FITC-labelled antibodies against types V and VI collagen, binding was apparent adluminally in close vicinity to basement membranes, whereas reactivity was seen in the entire interglandular region of the basal area during this phase. Our findings indicate specific microenvironments with distinctive structural characteristics in the marmoset endometrium that are hormone dependent during all phases of the endometrial cycle. They are not related to menstruation, appear to be characteristic for primates and could reflect epithelial-mesenchymal interaction.
INTRODUCTION
The endometria of humans and menstruating primates (Rhesus) are subcompartmentalised. This was shown histologically by Bartelmez et al. (1951), ultrastructurally by Kaiserman-Abramof & Padykula (1989), and through [3H]thymidine uptake studies by Padykula et al. (1984). Based on differences between glandular epithelial cells a zonation of the endometrium was described as follows: zone I, luminal epithelium; zone II, uppermost gland segments; zone III, middle gland segments; and zone IV, basal gland segments. This definition was thought to provide a key to deciphering the mechanisms that underlie repetitive cyclicity. It is not known whether this kind of endometrial zonation is also valid for the interglandular compartment. Collagen, however, is known
to undergo considerable alterations during the menstrual cycle (Woessner, 1982; Aplin et al. 1988). An extremely dynamic state of the extracellular matrix (ECM) participates in the process of cyclic endometrial renewal. Therefore, with respect to endometrial cyclicity it appears essential to investigate the binding of various antibodies against different constituents of the ECM in association with studies on the ultrastructure of the glandular and interglandular compartment. For our experiments we used common marmosets (Callithrixjacchus) which are valuable primate models in reproductive-biological research (Holt & Moore, 1984; Jackson & Edmunds, 1984; Hiller et al. 1987; Rune & Heger, 1987; Rune et al. 1988, 1991). In addition, these New World monkeys are much more suitable for experimentation than other primates: e.g.
Correspondence to Dr G. M. Rune, Department of Anatomy, Free University of Berlin, Konigin-Luise-Strasse 15, D-1000 Berlin 33, Germany.
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low maintenance costs, 3-5 offspring per year and mother. However, marmosets do not menstruate (Hearn & Lunn, 1975) and thus, according to Padykula et al. (1984) and Kaiserman-Abramof & Padykula (1989) are not expected to show endometrial zonation or to be suitable as primate models for the study of uterine cyclicity. This study asks whether endometrial zonation is evident in both glandular and interglandular compartments and whether endometrial zonation is necessarily followed by menstruation. As it is well established that the uterus is controlled by varying levels of oestradiol and progesterone during the reproductive cycle, we studied the marmoset endometrium in relation to oestrogen and progesterone concentrations in urine (at defined stages of the reproductive cycle).
were placed in a metabolism cage and urine samples were collected at room temperature on sheets of aluminium foil between 11 a.m. and 2 p.m. The urine was sucked up with a disposable syringe and immediately stored at -20 °C until hydrolysis. Urinary 6,f-hydroxypregnanolone and oestrogen excretions were used for the determination of the cycle stage and were measured by means of high performance thinlayer chromatography, as fully described by Heger & Neubert (1983). Three cycles were monitored before the females were killed at a defined stage of the following cycle (see Fig. 1). Only regularly cycling monkeys were used. For this study we investigated monkeys at d 1 (2 monkeys), d 3 (1 animal), d 4 (1 animal), d 8 (3 animals), d 11 (2 animals), d 15, 18, and 20 (1 animal each), and d 28/29 (3 animals) of the reproductive cycle.
MATERIAL AND METHODS
Animal maintenance
Morphology
Marmosets were kept in our colony in single family groups of 1 female, 1 male and their offspring aged less than 120 d. The room temperature was at 27+1 °C and the humidity was maintained at 55 + 5 %. The animals were kept under a constant day/night cycle (light from 6 a.m. to 6 p.m.) and were fed a standardised Altromin Marmoset Diet, water ad libitum, and about 30 g fruit and 5 g boiled egg 3 times a week.
The animals were anaesthetised and killed by an overdose of thiopental (Byk van Gulden, Konstanz, Germany) at 10 a.m. The uteri of the animals were immediately removed, cut into small pieces and either fixed by immersion according to Karnovsky (1965) for 24 h or frozen in liquid nitrogen and stored at -20°C until use. The fixed specimens were then thoroughly washed in 0.1 M phosphate buffer, pH 7.4, and postfixed for 1 h in phosphate-buffered (0.1 M) 1 % OS04 at pH 7.4 for 1 h. After dehydration in acetone they were embedded in Epon 812. Sections were cut on an ultramicrotome (Reichert-Jung OmU 3, Vienna, Austria).
Monitoring the reproductive cycle
For the determination of the cycle stage, females were isolated from the male marmoset. Each morning they
Light microscopy. Semithin sections (1 gm) were mounted on slides and stained with methylene blue/azure II according to Richardson et al. (1960).
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Electron microscopy. Ultrathin sections were stained with uranyl acetate followed by lead citrate (Reynolds, 1963). The sections were examined with a Zeiss electron microscope (EM 10, Oberkochen, Germany).
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For preparation of the antibodies the following antigens were used. Type I collagen was obtained from fetal mouse skin according to Trelstad et al. (1976), type III collagen from fetal mouse skin according to Smith & Niles (1980), type IV collagen from human placenta according to Sage et al. (1979). Type V collagen from human placenta was purchased
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from Heyl (Berlin, Germany). Laminin was prepared from EHS sarcoma according to Timpl et al. (1979). Antisera were prepared in rabbits and purified by
immunoadsorption according to Kittelberger-Ewert et al. (1988). The monospecificity of the antibodies had been established by the ELISA technique
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Fig. 2. Low steroid level (L, glandular lumen; S, stroma). a, Cytoplasm of glandular epithelial cells with microvilli at the apical membrane and junctional complexes. Many mitochondria of the crista type, abundant rough ER and an extensive Golgi apparatus are present. Cells are in close contact with each other with invaginated plasma membranes ( x 16000). b, Enlargement of intercellular spaces between neighbouring epithelial cells (arrows) are apparent in the luminal and adluminal areas. Part of a lymphocyte is seen in the mid right of the micrograph ( x 18000). c, Coated pits (arrows) and lysosome-like structures- (arrowheads) are seen in epithelial cells of the myometrium ( x 30000). d, Cellular surfaces with short microvilli are smooth in the adluminal region of the endometrium ( x 17000). e, Large apical cytoplasmic protrusions are found in epithelial cells at the base of the glands ( x 6000).
(Gosslau & Barrach, 1979) on microtitre plates, coated with laminin, nidogen, fibronectin and types I, II, III, IV, V and VI collagen. All purified antibodies reacted only with their respective antigen. The binding of the antibodies to unfixed cryostat sections was followed
by incubation with fluoroisothiocyanate (FITC)labelled antibodies against IgG. The sections were evaluated under a Zeiss (Oberkochen, Germany) fluorescence microscope.
Differentiation of the marmoset endometrium
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Steroid levels As seen in Fig. 1 and based on steroid levels in urine, the reproductive cycles of the investigated marmosets lasted about 28-29 d. They were characterised by high levels of oestrogens after roughly one third of the
cycle, indicating the time of ovulation. Concentrations of 6,8-hydroxypregnanolone were low during the first 8-9 d of the cycle and increased after ovulation. The peripheral level of this steroid metabolite remained high up to 18-19 d on average after ovulation. The rise of oestrogens preceded the rise of the progesterone metabolite during the phase before ovulation and a
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3(e)
Fig. 3. High oestrogen/low progesterone level (L, glandular lumen; S, stroma). a and b, Large phagolysosomes together with fat vacuoles, apical cellular protrusions and apical secretory vesicles (arrow) are seen in the adluminal zone (a; x 3200). Except for apical secretory vesicles (arrow), these features are lacking in the basal region of the endometrium (b; x 4000). c and d, The apical secretory vesicles are highly electron dense in the adluminal area of the endometrium (c; x 15600) and appear as dense core-like vesicles (arrows) in epithelial cells at the base of the glands (d; x 20000). e andf, Glycogen accumulations (f; x 8000; arrows) mostly with a concentric arrangement (e; x 20000) together with dilated elements of rough ER (arrowheads) are typical epithelial features in the adluminal and basal regions.
continuous decrease of oestrogen was perceptible after the ovulatory oestrogen peak.
Morphology
During all 3 phases of the reproductive cycle investigated, zonation of the endometrium was evident based on the organisation of the stroma and morphological differences between the epithelial cells. Three zones were identified. A basal zone bordering on the myometrium could be distinguished from an upper adluminal area and the luminal epithelium. However, the appearance of each of these 3 zones was hormone-dependent. Low steroid level (d 1, 3 and 4 of the reproductive
cycle) The endometrium contained long narrow glands which consisted of tall columnar cells with nuclei in the majority of the cells located near the basement
membrane in all areas. Mitochondria, glycogen particles, abundant rough endoplasmic reticulum (RER) and an extended Golgi apparatus were present. Microvilli at the cell surfaces, junctional complexes between the apical lateral membranes, and microfilaments running parallel and beneath the apical plasma membrane were common characteristics of all epithelial cells (Fig. 2a). Enlargement of intercellular spaces between neighbouring epithelial cells particularly in the basolateral region, was found in the luminal and adluminal area (Fig. 2b). In the basal region, dilatations of the glandular lumen filled with necrotic cellular material were occasionally seen. Coated pits, lysosome-like structures (Fig. 2 c), and phagolysosomes were specific features of epithelial cells in this zone. In the adluminal area, the apical surfaces of the epithelial cells were smooth with only few and short microvilli (Fig. 2d), whereas large apical protrusions with numerous and long microvilli were typical in the basal region (Fig. 2e).
Fig. 4. Progesterone dominance (L, lumen; S, stroma). a, During the late secretory phase, large vesicles dominate the supranuclear cytoplasm in close association with an extensive Golgi apparatus (arrows). Some of the vesicles are filled with membranous material. They also occur in the glandular lumen ( x 5000). b and c, In the adluminal part of the endometrium these vesicles occur also in cytoplasmic areas of epithelial cells at the basement membrane (arrowheads) (b; x 3200). These vesicles are lacking at the basement membrane (arrowheads) in epithelial cells of the basal region of the endometrium (c; x 3200).
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The stroma surrounding the endometrial glands was highly cellular, mostly fibroblastic with more collagen bundles in the basal than in the upper zone.
High oestrogen/low progesterone levels (d 8 and 11 of the cycle) The width of the endometrium had clearly increased. Generally, the narrow glandular lumen was filled with cellular debris, which obviously originated from epithelial cells shed into the lumen. No regional differences were found for the distribution, shape or size of mitochondria, microvilli, and junctional complexes. All epithelial cells were closely apposed and large areas of invaginating plasma membranes were seen. In all epithelial cells the Golgi apparatus occupied large areas in the cytoplasm. Phagolysosomes of striking size and mostly in a supranuclear position together with fat vesicles were typical features of adluminal epithelial cells (Fig. 3 a). Apical protrusions were seen in the adluminal area (Fig. 3 a) but not in epithelial cells at the base of glands (Fig. 3 b). Secretory granules in the apical region of epithelial cells, filled with highly electron dense material were characteristic (Fig. 3 a, d). At the base of glands, these secretory granules appeared as dense core-like vesicles surrounded by a halo (Fig. 3d). Elements of RER frequently appeared dilated and glycogen accumulations were arranged concentrically (Fig. 3 e, f) in the adluminal and basal zones. Differences in cellular density between the adluminal and basal interglandular compartment became evident. Basally, cells were more closely packed than adluminally. Progesterone dominance (d 15, 18, 20, and 28 of the cycle) An increase of endometrial height and of glandular diameter was observed. Certain glandular segments were extremely widened and filled with necrotic cellular material. The widening of the glandular lumen started obviously in the basal area and progressed in the upper zone. Progesterone dominance did not influence the ultrastructure of mitochondria, RER, microvilli or junctional complexes. During early progesterone dominance concentrically arranged glycogen accumulations were still occasionally visible. Few phagolysosomes were seen in adluminal epithelial cells. Dilated elements of RER and intercellular spaces between the basolateral plasma membranes were found basally. In the adluminal and basal areas
in a supranuclear position were numerous large vesicles, some of which were filled with membranous material (Fig. 4a). They were more frequent in the adluminal than in the basal epithelial cells but were absent in the luminal epithelium. In the course of progesterone dominance they increased in number so that at the end of the cycle the entire supranuclear space was completely filled. Adluminally, they could also be observed in the basal part of the epithelial cell (Figs 4b, c). Generally, they were located in close vicinity to the extended Golgi apparatus. They had an irregular outline and varied in size. The largest appeared to flow together. Regional differences in the organisation of connective tissue between the endometrial glands were most obvious during the dominating progesterone influence (Figs 5, 6). In the area bordering on the myometrium the cells were closely packed, and were oriented with their longitudinal -cellular axis parallel to the glandular basement membranes. Thick collagen bundles appear to run between the cells. Connective tissue of the upper area appeared loosened. Intercellular spaces were clearly enlarged and collagen fibrils were arranged in a network-pattern instead of being oriented in parallel.
Immunohistochemistry The distribution pattern of collagen immunoreactivity revealed that types I and III (Fig. 7a) collagens were diffusely distributed in the connective tissue of the interglandular compartment with no regional or collagen type differences throughout the endometrial cycle. Type IV collagen and laminin were exclusively observed in a fine linear fashion along the blood vessels and along the uterine glands, thus showing the delineation of the basement membranes throughout the cycle (Fig. 7b). With low steroid levels as well as under dominant oestrogen influence, the interglandular region of the endometrium exhibited a strong fluorescent meshwork of fibres showing binding of antibodies against types V and VI collagen. An association with basement membranes was evident for type V collagen. Zonation of the endometrium in the adluminal zone and the basal area was only perceptible under the influence of high progesterone levels. With antibodies against type V collagen the fluorescence in the adluminal part was only detectable in close association with the basement membranes of vessels, uterine glands (Fig. 7c) and of the luminal epithelium. In this area, the remaining interglandular connective tissue was devoid of any reactivity. In contrast, the staining was comparable to
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Figs 5 and 6. Interglandular compartment under high progesterone concentrations. Semithin sections. The cellular density of the interglandular compartment is lower in the adluminal (4; x 380) than in the basal region of the endometrium (5; x 400).
the other cycle phases in the basal part of the endometrium (Fig. 7d, e). Using antibodies against type VI collagen, binding also was apparent adluminally in close vicinity to basement membrane (Fig. 7f), whereas reactivity was seen in the entire interglandular region of the basal area (Fig. 7g, h). DISCUSSION
Our electron microscope investigations have shown that, independent of the cycle, the marmoset endometrium exhibits zonal differentiation of glands and of the stromal compartment, despite the fact that these monkeys do not menstruate. Zonation of the endometrial stroma was also revealed by the binding of antibodies against types V and VI collagen. Zonal differentiation has also been shown in the human endometrium by histological investigations (Bartelmez et al. 1951) and in the Rhesus monkey (Padykula et al. 1984; Kaiserman-Abramof & Padykula, 1989) from the epithelial ultrastructure and mitotic rate. However, Padykula et al. (1984) were able to distinguish 4 zones in the Rhesus monkey, 21
whereas we could detect only 3 in the marmoset: a basal area, bordering on the myometrium, an adluminal region and the luminal zone. Thus the subdivision of the basal area into 2 zones, seen in the human and Rhesus endometrium seems to be absent in the marmoset. In the marmoset, zonal differentiation of epithelial cells was mainly based on the presence of intercellular spaces, phagolysosomes, secretory granules, coated pits, and widening of the glandular lumina. As in the Rhesus monkey (KaisermanAbramof & Padykula, 1989), the zonation was permanently evident throughout the cycle, despite the fact that single zones differed in their appearance at certain stages of the cycle. Thus finding implies that the existence of zonation per se in the marmoset endometrium is an integral feature that is independent of hormonal influences, whereas the differential behaviour of these zones throughout the cycle is due to varying steroid levels (e.g. secretory electron dense granules under the influence of oestrogen or empty vesicles under the influence of progesterone in the adluminal area). Consequently, zonation of the endometrium is not a unique feature of menstruating ANA 181
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primates, as proposed by Kaiserman-Abramof and Padykula (1989) and thus is not directly related to menstruation. Instead, it could be a specific phenomenon of menstruating as well as of nonmenstruating primates. To our knowledge, it has never been described for other mammals. With regard to our morphological findings (zonation) we cannot exclude the possibility that the high osmolality and the slow and nonlinear penetration of the fixative could have resulted in some degree of artefactual change. Shrinkage artefacts are, however, unusual after immersion fixation of tissue fragments (Karnovsky, 1965) and our histochemical findings support the relevance of the conclusions based on the morphological findings obtained from fixed tissue. In addition to regional differences in the ultrastructure of epithelial cells, our results show that the zonation of primate endometria is also apparent in the interglandular compartment: (1) by different cellular density in the basal and adluminal regions and (2) by the different binding behaviour of types V and VI collagen antibodies in the interglandular compartment. In contrast to the ultrastructural zonation, the latter was seen exclusively under the influence of progesterone. In spite of rather well-established knowledge concerning the biochemical nature of types V and VI collagen their functions are not yet clarified (Timpl & Engel, 1987). From the close association of both collagen types to type I collagen, basement membranes and other ECM constituents, it has been suggested that they interconnect the different ECM components. For type VI collagen, Trueb & Bornstein (1984) have suggested an adaptor function with type VI collagen serving as a link for fibrous and globular proteins with cells. It appears that both types VI and V collagen act as binding or attachment proteins, linking and integrating different components of the ECM (Karkavelas et al. 1988). With regard to our findings concerning the different distribution of types V and VI collagen in the basal and adluminal compartments in the presence of high progesterone levels, it follows that during this phase this link is lost adluminally except for those areas beneath basement membranes of glands and vessels. This is partly
consistent with findings of Aplin et al. (1988), as in the human endometrium type VI collagen is abundant in the endometrium during the proliferative phase, but is progressively lost during the secretory phase. However, it is difficult to compare the results of that study with the findings of our investigation since these authors used material that was obtained from curettage operations. For methodological reasons it is impossible or at least very difficult to detect any zonation with such material. Furthermore, our findings support biochemical investigations which have clearly demonstrated that the menstrual cycle affects the deposition and removal of collagen (Pastore et al. 1989). Both steroid hormones, oestrogens and to a less extent progesterone, have been shown to be effective (Woessner, 1982). However, reports about the influence of oestrogen, for example, on collagen metabolism in the uterus, are rather conflicting. It has been suggested that oestrogens increase (Cullen & Harkness, 1964), have no effect (Ryan & Woessner, 1972) or even stimulate both the synthesis and breakdown of collagen (Pastore et al. 1989; Dyer et al. 1980). In our investigation the disappearance of any reactivity between the adluminal glands as soon as the progesterone level had risen, indicates that progesterone plays a regulative role in the metabolism of types V and VI collagen, which had already been found in women by Aplin et al. (1988). Neither regional nor hormone dependent differences were detectable when we used antibodies against types I and III collagen and against basement membrane components such as laminin and type IV collagen. Regional differences in the glandular and interglandular compartment in the marmoset endometrium may result from mesenchymal-epithelial interaction. It is known that the stroma is required for normal epithelial development (Cunha et al. 1985) and such an interaction has been demonstrated in man (Roberts et al. 1988). We could not find similar morphological correlations (e.g. lamina densa disruptions by epithelial projections) for such an interaction in the marmoset endometrium. Nevertheless, in our investigation this interaction could be mediated by the
Fig. 7. Immunoreactivity of anticollagen antibodies. Progesterone dominance. a, Endometrium stained with type II collagen antibody. An evenly stained network of interstitial immunoreactivity is evident ( x 250). b, Immunofluorescence of type IV collagen antibody. Only glandular and vascular basement membranes carry the antigen (x 120). c-e, Immunofluorescence of type V collagen antibody. c, In the adluminal part of the endometrium immunoreactivity is associated to the glandular and vascular basement membranes (x 230). On approaching the myometrium an additional fluorescent network becomes apparent. d, Transitional part between the adluminal and basal regions ( x 270). e, Basal region of the endometrium ( x 250). f-h, Immunofluorescence of type VI collagen antibody. f, In the adluminal part strong staining is seen throughout the walls of blood vessels and weaker immunoreactivity at the glandular basement membranes ( x 250). Immunoreactivity becomes additionally visible in the stroma of areas directed towards the myometrium. g, Transitional part between the adluminal and basal regions ( x 230). h, Basal region bordering on the myometrium ( x 160). 21-2
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composition of stromal ECM, at least under progesterone dominance.
ACKNOWLEDGEMENT
This investigation was supported by the Deutsche Forschungsgemeinschaft (Sfb 174).
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