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The Macaque Endometrium, with Special Reference to the Cynomolgus Monkey (Macaca fascicularis)
1 Department of Toxicology and Drug Disposition, Schering-Plough, the Netherlands Correspondence: Eric Van Esch, Schering-Plough (formerly Organon), Department of Toxicology and Drug Disposition, P.O. Box 20, 5340 BH Oss, The Netherlands; e-mail:eric.van.esch{at}spcorp.com
The macaque endometrium undergoes dramatic morphologic and functional changes during the menstrual cycle that are nearly identical to those of the human endometrium. The sequential events that take place in the endometrium are mainly driven by the ovarian steroids and their respective receptors. To be able to interpret the changes and effects induced by mammalian or synthetic hormones and other compounds that could have influence on the hormonal status of the animal, a thorough knowledge of the anatomy, physiology, and histology of the cyclic hormone-mediated processes within the endometrium is indispensable. In this paper we give an overview of uterine growth and development, anatomy, basic histology, aging, spontaneous pathology, and the techniques to study the endometrium in-life. In addtion, a comprehensive description of the receptor-mediated, hormone-driven morphological changes during the menstrual cycle in the cynomolgus monkey (Macaca fascicularis) is given. Where possible, differences between the macaque and human endometria are discussed. Competing Interests: This article was sponsored by Covance Inc. and Schering-Plough. Gerhard F. Weinbauer and Eberhard Buse are employed by Covance Inc. Eric Van Esch is employed by Schering-Plough. No other competing interests were declared.
Key Words: female reproduction • endometrium • histology • menstrual cycle • hormone receptors • endometrial dating • cynomolgus monkey
Regulatory authorities usually require that new drugs are tested for safety in two different animal species, a rodent and a nonrodent species. The reason for such testing is to elucidate possible intrinsic toxicity to minimize the potential risk to humans. It is therefore of major importance that the animal species of choice is able to predict this risk. Generally the rat or the mouse is used as the rodent species in toxicity studies, and the dog is commonly used as the nonrodent species. However, when clear differences in anatomy, physiology, metabolism, or the genome exist between the dog and man, scientists might choose to use nonhuman primates instead of dogs. For instance it is well known that the physiology of the reproductive cycle in the female dog differs significantly from that in humans, and that dogs have greater sensitivity to estrogens and progestogens. Thus the dog is less suitable for investigating the toxicity of such hormonal substances or substances that interfere with endogenous hormones. On the contrary, the physiology of the menstrual cycle of Old World monkeys (Cercopithecidea) closely resembles that of the human. For this reason, monkey species belonging to this group are preferred as experimental models in studies of human reproduction, pharmacology, and toxicology. Especially for studies with sex hormones, synthetic steroidal compounds or compounds that interfere with the hypothalamus–pituitary–ovarian (or testicular) axis, such monkey species are predictive (Cline et al. 2001). The rhesus monkey (Macaca mulatta) and the cynomolgus monkey (Macaca fascicularis) are the species most often used for these purposes. In our laboratories, purpose-bred cynomolgus monkeys are used for such studies. Besides their resemblance in menstrual cycle physiology, these monkeys are relatively small and easy to handle. Both the human and the macaque monkey endometria undergo dramatic morphologic and functional changes during the menstrual cycle. The sequential events that take place in the endometrium are mainly driven by the ovarian steroids estradiol and progesterone and their respective receptors. To be able to interpret the changes and effects induced by mammalian or synthetic hormones and other compounds that could have influence on the hormonal status of the animal, or that interfere with the regulatory processes within the endometrium, a thorough knowledge of the anatomy, physiology, and histology of cyclic hormone-mediated processes within the endometrium is indispensable. In this paper we give a brief overview of endometrial growth and development, anatomy, basic histology, and the effects of aging, and we briefly discuss the spontaneous pathology of the endometrium of macaques. The receptor-mediated, hormone-driven morphological changes that occur during the menstrual cycle are discussed in more detail with special reference to the cynomolgus monkey. Finally, the macaque endometrium is briefly compared with the human equivalent, and the techniques used to study the endometrium in-life are also illustrated and discussed.
As in the human, the uterus of the cynomolgus monkey arises from the fusion of the caudal parts of the Müllerian ducts, which in turn are mesodermal derivatives of the coelomic epithelium. In macaques, organogenesis begins on day 17 to 19 and is completed on day 45 to 46 of pregnancy (Allen et al. 1982). The luminal epithelium of the primitive uterus consists of cuboidal epithelial cells with large nuclei containing prominent nucleoli. This epithelium is surrounded by a layer of undifferentiated mesenchyme. The early development of the basic endometrial structures is illustrated in a longitudinal section through the uterus of a fifty-day-old cynomolgus fetus (Figure 1).
In a hundred-day-old fetus, the fetal endometrial stroma and myometrium are already clearly distinguishable (Figure 2). The smooth muscle cells within the fetal myometrium are arranged in a more or less haphazard fashion. The epithelium lining the fetal uterus at this time is columnar. The nuclei of the epithelial cells are located in the upper half of the cytoplasm. The lower half of the cells contain large vacuoles, giving the epithelium a more or less similar morphology to the glandular epithelium during the early luteal phase of a mature, cyclic animal. At this stage of uterine development, small blood vessels appear in the fetal endometrium.
Subsequently, invaginations of the epithelium begin to form the glands by downgrowth into the underlying stroma, a process that, in the human fetus, starts around week 19 of gestation (Robboy et al. 2002). In the cynomolgus monkey, down-growth of endometrial glands is clearly evident in the uterus of a 150-day-old cynomolgus monkey fetus (Figure 3). At this stage, the smooth muscle cells of the myometrium are arranged in bundles. The surface and glandular epithelial cells of the fetal endometrium store large amounts of glycogen. The primitive stroma contains significant amounts of acid mucopolysaccharides (Figure 4).
The trigger for the start of glandular morphogenesis is still unknown, although interactions between glandular epithelium and stroma are thought to be required for normal development (Bartol et al. 1999; Spencer et al. 1999). Nevertheless, the glands, endometrial stroma, and myometrium are intimately related during their parallel development. Using an antibody raised against the proliferation marker Ki67 (monoclonal mouse anti-Ki67, Clone MIB-1, M7240, Dako, Carpinteria, CA), we were able to visualize the proliferation of the primitive surface epithelium, glands, stroma, and myometrium that underlie the development process of the uterus in the 150-day-old cynomolgus fetus (Figure 5C).
In the uterus of the 150-day-old cynomolgus monkey fetus, estrogen and progesterone receptors could be visualized (depicted in Figures 5A and 5B, respectively) (see also "Factors and Hormone Receptors"). Despite the early presence of these hormone receptors, it is not known whether they are actively involved in the molecular cascade leading to proliferation and differentiation during the development of the different uterine compartments. During pregnancy, the fetus is exposed to high levels of progesterone, and to a lesser degree, estrogens. Nevertheless, in humans, there is clear evidence that shortly before birth, when all uterine structures are fully developed, progesterone receptors become active in the fetal uterus. In human fetuses eight months of age, tortuous glands, loose stroma, and stromal predecidual changes may be observed (the term "predecidual" is used to describe the change in morphology of the stromal cell in response to progesterone in the human). In the fetus, such morphologic changes are thought to be caused by maternal progesterone (Dallenbach-Hellweg 1987). Also, in a large study including 169 human female newborns (Ober and Bernstein 1955), evidence was found that newborn endometria were responsive to steroids. In this group of newborns, approximately two thirds had endometria with proliferative changes, whereas one third had endometria with secretory changes. In 5% of the newborns, predecidual stromal changes or even signs of menstrual shedding were observed. The presence of such proliferative and/or secretory morphologic characteristics in newborns strongly suggests the presence of functional estrogen and progesterone receptors (Brandenberger et al. 1997). After birth, the influence of the "exogenous," maternal hormones suddenly disappears and the newborn endometrium regresses. Until the onset of puberty, the endometrium stays thin and inactive, with rudimentary glands. During puberty the endometrium again comes under the influence of (endogenous) ovarian hormones. In macaques, this process happens between two and one half and four years of age (see paper by Weinbauer et al. 2008). After the mucosa develops under the influence of the increasing level of estradiol produced in the first maturating follicles, cyclic changes gradually start. The weight of the nongravid cynomolgus uterus increases rapidly in the first few years of life, then reaches a plateau around five to five and one half years of age (Watanabe et al. 2006). In the mature cynomolgus females we studied, the mean absolute uterine weight was 7.42 ± 2.98 g (N = 61). The individual absolute weights ranged from 2.83 to 16.37 g: because of the cyclic events in the uterus, uterine weights normally have a broad dispersion. In the recent study by Watanabe et al. (2006), the mean uterus weight per cycle phase was determined in a group of 103 cyclic cynomolgus monkeys. In this study, mean absolute uterine weights ranged from 5.07 g in the early follicular phase (range, 3.20–6.86 g) to 8.99 g in the early luteal phase (range, 4.93–14.51 g).
In higher primates, the uterus is a pear-shaped organ situated in the pelvic cavity between the urinary bladder and the rectum (Figure 6).
The uterus can grossly be divided into three parts; the fundus (the dome-shaped top), the corpus (or body), and the isthmus (or neck). The isthmus leads into the cervix. The uterus is supported by the round, cardinal, uterosacral, and utero-ovarian ligaments. The mucosa of the uterus, the endometrium, lines the uterine cavity. A thick muscular layer, the myometrium (tunica muscularis), is composed of a thick inner circular layer, a middle layer containing smooth muscular fibers arranged in random directions, and a thinner outer longitudinal layer. The smooth muscle fibers in the different layers are separated by collagen and elastic fibers (Figure 7B). The region between the two outer layers of smooth muscle contains the large blood vessels, the branches of the radial arteries and veins. The uterine serosa (tunica serosa) or perimetrium is composed of mesothelial cells and a thin, submesothelial layer of connective tissue (Figure 7A). This peritoneal layer forms a cul-de-sac caudally between the rectum and the cervix, but on the anterior surface reflects at a more cranial level onto the urinary bladder.
The vascular architecture of the uterus in macaques closely resembles that of the human uterus (Ramsey 1981). The uterus is supplied by the uterine arteries, which branch off the internal iliac arteries on each side. Both main uterine arteries supply the uterine body and continue to the fallopian tubes, where they form anastomoses with the ovarian arteries (Ginther et al. 1974). More caudally, branches of the uterine arteries form anastomoses with the vaginal artery. Along the lateral wall of the uterus, within the broad ligament, the utero-ovarian anastomosis forms a sinuous arterial channel. Several arcuate arteries branch off this channel and first supply the myometrium. From the arcuate arteries the radial arteries branch off. The radial arteries run within the myometrium and are directed to the central portion of the uterus. When the radial arteries pass the myometrial–endometrial junction, they are called spiral arteries. From these spiral arteries short, straight branches, the basal arteries, supply the zona basalis (the outer layer of the endometrium that is not shed during menstruation). The longer, coiled branches, which supply the zona functionalis (the temporary inner layer of the endometrium that to a large extent is shed during menses or menstruation) are the so-called spiral arterioles. These specialized spiral arterioles run parallel to the endometrial glands. From these spiral arterioles, capillary branches form a rich network throughout the stroma. Oxygen-poor blood follows the same way in opposite directions via the uterine veins. The veins are, however, less coiled compared to the arteries. The nerves innervating the uterus are derived from the hypogastric and uterovaginal plexuses and from the sacral nerves. The innervation of the myometrium of the cynomolgus monkey consists of bundles of unmyelinated nerve fibres running between the smooth muscle cells (Barbe and Taxi 1986).
Cellular Composition Surface and Glandular Epithelium The endometrial surface and glands are lined by a single layer of secretory type epithelium. The surface epithelium shows less cyclic variation than the glandular epithelium. Glandular epithelium consists of a number of different cell types, varying over the length of the glands and during the different phases of the cycle. The most abundant cell type is the "secretory cell," of which the morphology varies under the influence of the fluctuating estradiol and progesterone levels (see also "Cyclic Morphologic Changes") (Figure 8). In the cytoplasm of the secretory cells, proteins, mucopolysaccharides, glycogen, lipids, and various enzymes are synthesized. This function is reflected in ultrastructural features including free and bound ribosomes, rough endoplasmic reticulum, a Golgi complex, mitochondria, and varying amounts of glycogen granules. The formation of these organelles is stimulated by estradiol (Kaiserman-Abramof and Padykula 1989). These cells abundantly express alkaline phosphatase (thought to be involved in the processes of growth and proliferation, since its activity is greatest during the follicular phase), and carbohydrate metabolic enzymes such as glycogen synthetase (involved in synthesis of glycogen from glucose), glycogen phosphorylase and glucose-6-phosphatase (involved in the breakdown of glycogen to glucose) (Dallenbach-Hellweg 1987).
The second cell type is the "ciliated cell." During estrogen dominance, the number of ciliated cells increases. Ciliated cells are not equally distributed within the uterine endometrium; they are more common toward the endocervix. Ciliated cells have clear cytoplasm, and their round nuclei are often located above those of the neighboring secretory cells. Although difficult to recognize in routinely prepared paraffin sections, small cilia can be observed on the apical cell surface (Figure 9).
A less common cell type within the endometrial glands is the "clear cell." This cell type is most frequently seen during the follicular phase. The clear cell is thought to be the precursor of the ciliated cell that has not yet reached the luminal surface where it can extrude the cilia. As with the ciliated cell, the clear cells are easily recognized because of their more rounded appearance and their distinct perinuclear clear cytoplasm. Since they are most often seen during the follicular phase and are rarely seen in inactive and atrophic endometria, the clear and ciliated cells are believed to represent useful markers of estrogenic activity on the endometrium (Figure 10).
Although they have no typical morphological characteristics, the presence of stem cells is suggested by a number of authors (Padykula et al. 1984; Padykula et al. 1989; Padykula 1991; Prianishnikov 1978). Stem cells are detected using [3H]-thymidine labeling studies. Their location in the most basal parts of the endometrial glands protects them for being shed during menstrual bleeding. They persist throughout the whole reproductive period. Via the process of proliferation and differentiation, stem cells are thought to be responsible for the replenishment of the pool of different functional cell types in the glands (see also "Endometrial Proliferation and Differentiation").
Stromal Cells
The morphology of the endometrial stromal cells varies throughout the menstrual cycle (see also "Cyclic Morphologic Changes"). In the human endometrium, a typical change in morphology appears under progesterone dominance during the luteal phase. During this phase, the stromal cell enlarges and becomes decidualized. "Decidualization" is the process in which the stromal cells transform to large, polyhedral, cytoplasm-rich cells storing a large amount of glycogen (the decidua is the layer of the endometrium of the pregnant uterus, all of which, except the deepest layer, is shed at parturition). Ultrastructurally, these decidualized cells have numerous ribosomes, a pronounced rough endoplasmic reticulum, and varying amount of glycogen in their cytoplasm (Rhodin 1977). In general this process starts, and is most prominent, in the stroma adjacent to the well-developed spiral arteries. In case of pregnancy, the process of decidualization expands gradually and becomes uniform throughout the endometrium. Similar change in the absence of pregnancy is called pseudodecidualization. In the human endometrium, this process is much more pronounced compared to macaques and baboons (Kraemer et al. 1977). It is our experience that this process of (pseudo)decidualization is hard to recognize during the luteal phase in a normal cycling (nonpregnant) female cynomolgus monkey, but it is obviously present during pregnancy (Figure 12) and can also be induced by treatment with progestogens. At relative low but superphysiological levels of progestogens, decidual changes appear in the stroma localized around the spiral arteries (Figures 13A and 13B) and underneath the superficial epithelium, but at higher levels the whole of the stroma in the functionalis and even part of the basalis can become decidualized in the monkey (Wadsworth et al. 1980). In some cases this induced process of decidualization can be so prominent that the glands become constricted, causing obstruction and dilation of the lower parts of the glands (Figures 14A and 14B).
Endometrial Lymphocytes Endometrial lymphocytes appear mainly during the luteal phase of the cycle. They are round, often with clear cytoplasm with a centrally located round, kidney-shaped, or more segmented nucleus and variable shaped eosinophilic cytoplasmic granules. Because of their morphologic resemblance, endometrial lymphocytes were once mistaken for infiltrating leukocytes and erroneously named "endometrial granulocytes." The granules can be easily visualized using dyes as Chromotrope 2R used in the Chromotrope-Aniline blue methods (Churg and Prado 1956) (Figure 15) or with the phloxine-tartrazine staining method. Using flow cytometry (Slukvin et al 2001) or immunohistochemical techniques (Kämmerer et al. 1999), these cells have now been characterized in macaques and man as large granular lymphocytes with an unusual phenotype; these T-cells are CD56bright+, CD3–, and CD16– and have a natural killer function. They are derived from the peripheral blood and home into the endometrium. It is suggested that these cells may be uterus specific (Kämmerer et al. 1999; King et al. 1996).
Lymphocytes, Others Next to the above described CD3- endometrial lymphocytes, a low number of CD3+ T-cells can be found randomly throughout the endometrium (Slukvin et al. 2004). Also, aggregates of small lymphocytes can be present in the normal cyclic endometrium (Figure 16). The presence of a limited number of aggregates of lymphocytes does not have a pathological significance.
Macrophages Estrogens increase the number of endometrial macrophages (Bukowski et al. 1995). These macrophages produce various cytokines and growth factors that could affect endometrial growth and differentiation.
Blood Vessels
Nerves The presence of nerve fibers was demonstrated in the human endometrium (Dallenbach and Vonderlin 1973). Small nerve fibers were found to extend into the endometrium to various levels up to the zona functionalis.
Lymphatics
Noncellular Components
Between the cellular components and reticular network, the ground substance is present. With the alcian blue and periodic acid Schiff (PAS) staining method, neutral and acid mucopolysaccharides may be demonstrated (Figure 19). Depending on the amount of stromal edema, the concentration of the mucopolysaccharides varies during the course of the cycle (Runge et al. 1956). Matrix metalloproteinases are important enzymes within the stroma that play a major role in the degradation of most components of the extracellular matrix during the menstrual phase (Osteen et al. 1999). The pattern of expression of endometrial metalloproteinases during the menstrual cycle in macaques is similar to that reported for women. In both species, matrix metalloproteinases are strongly up-regulated during the decline of progesterone levels in the late luteal phase, and their expression declines after menstruation (Rudolph-Owen et al. 1998). Several metalloproteinases are highly expressed in areas where tissue breakdown occurs during the menstrual phase (Hulboy et al. 1997). In general the expression of metalloproteinases is much lower during the remaining phases of the cycle (and during pregnancy). MMP-1 (interstitial collagenase) is the key player in the process of tissue breakdown. Collagen types I, II, III, VII and X are all substrates for this enzyme. MMP-1 mRNA is found at or around arterioles and small vessels and in stromal cells in the zona functionalis during the menstrual phase (Hulboy et al. 1997). Segments of the spiral arteries running through the zona functionalis, but not the segments running through the zona basalis, show strong expression of metalloproteinases during the menstrual phase (Rudolph-Owen et al. 1998).
Endometrial Compartmentalization In humans, the endometrium usually is compartmentalized into three functional zones: the compacta, the spongiosa, and the basalis. The compacta and upper spongiosa are also referred to as the zona functionalis (the layer that is shed during menstruation). The zona functionalis is much more affected by fluctuations in circulating ovarian hormones than is the zona basalis. The zona basalis, which escapes the process of shedding, plays an important role in the renewal of the endometrium after menses. In addition to this commonly used classification system, Bartelmez et al. (1951) divided the human and rhesus endometrium into four histologically different horizontal zones, respectively Zones I, II, III, and IV (Figure 20).
Zones I and II are equivalent to the zona functionalis and consist of the upper approximately two thirds of the endometrium. Zone I, which is more or less equivalent to the zona compacta in the triple zonation system, consists of the superficial or luminal epithelium with both secretory cells and ciliated cells and the subjacent, densely packed stroma. The maturation of the cells in the surface epithelium does not always accurately reflect the phase of cycle, although mitotic figures (in the follicular phase) and subnuclear vacuolation (mainly in the luteal phase) are seen. Zone II, the thickest zone, contains mostly the unbranched portions of the glands that run perpendicular to the luminal surface. The portions of the glands in this zone are embedded in and widely separated by a relatively loose stroma with a well-developed vascular network. In general, the glands in Zone II display the strongest hormone-mediated morphological changes. Endometrial dating therefore largely depends upon the cycle-dependent morphological characteristics in this zone. Zone III contains the bodies of the endometrial glands, which, at this level, are frequently simply branched. This zone is characterized by a dense stroma. Zone IV is the most basal region of the endometrium where the blind ends of the endometrial glands terminate. The zone is situated just adjacent to the innermost layer of the myometrium. In Zone IV the branched glands sometimes have a budded or sacculated appearance (Figure 21). The stroma in Zone IV is compact and fibrous.
Zones III and IV are equivalent to the zona basalis, which, in the monkey, covers approximately one third of the height of the endometrium. The more prominent portions of the spiral arteries are located in the Zones III and IV. This quadripartite zonation had largely been ignored until functional differences were identified among the four zones using autoradiography (Padykula et al. 1984). The ultrastructural luminal and glandular epithelial characteristics in the four different functional zones of the primate endometrium during estrogen and progesterone dominance are well described by Kaiserman-Abramof and Padykula (1989) and support the relevance of the quadripartite zonation system proposed by Bartelmez in the early 1950s. The cycle-related morphologic changes in the different zones are described in more detail in the paragraph "Cyclic Hormone-mediated Morphologic Changes."
Hormone Receptors and Factors Using an anti-estrogen receptor antibody (mouse-anti-human estrogen receptor, Clone 1D5, M7047, Dako, Carpinteria, CA) and an anti-progesterone receptor antibody (rabbit-anti-progesterone receptor, Clone SP2, RM9102, LabVision/Neomarkers, Fremont, CA), we were able to detect the expression of estrogen and progesterone receptors in the endometrium of a 150-day-old cynomolgus fetus (see Figures 5A and 5B). In this fetal endometrium, focal formation of gland buds by downgrowth of the superficial epithelium represents the first sign of glandular development. The estrogen receptors are expressed within the nuclei of the surface epithelium and in the gland buds, the fetal endometrial stroma, and the developing myometrium. Progesterone receptors were clearly demonstrated in the epithelium and stroma of the fetal uterus as well. These results are partly in agreement with those reported earlier by Hochner-Celnikier et al. (1986), who found biochemical evidence for the presence of estrogen receptors in the uterus of a third-trimester cynomolgus monkey fetus. The authors, however, were not able to detect progesterone receptors. Although storage of large amounts of glycogen within the cytoplasm of the epithelial cells in a fetus of this age can be demonstrated using PAS methods, it is not clear whether the progesterone receptors are indeed already actively signaling and responsible for this "secretory-like" status. During the menstrual cycle in mature females, the endometrium is under the hormonal influence of cyclic fluctuations of estradiol and progesterone that are mainly produced in the ovaries. Estradiol and progesterone act on the endometrial components through binding to their respective receptors. Not only do the levels of estradiol and progesterone fluctuate during the cycle, but the expression levels of nuclear receptors also change (see "Receptor Expression Patterns in Cynomolgus Endometrium"). In general, the main regulatory function of the estrogen receptor is endometrial growth, whereas that of the progesterone receptor modulates endometrial maturation. The follicular phase is therefore also referred to as the proliferative phase, whereas the luteal phase is called the secretory phase, based on the two main processes that take place during these subsequent cyclic events. In general, during the follicular phase the amount of nuclear and cytoplasmic estrogen receptor mRNA and protein increases and is related to the increase in endometrial proliferation (Koji and Brenner 1993). In humans, estradiol is responsible for inducing its own receptor (Lessey et al. 1989), and there are no indications that this function would be different in macaques. Although cytosolic and membrane estrogen receptors have been described (Elsner et al. 1977), estrogen receptors are most abundant in the nucleus. The number of progesterone receptors increases during the follicular phase, since the synthesis of the progesterone receptor also is induced by estrogens (Kreitmann-Gimbal et al. 1979; Okulicz et al. 1989). In the luteal phase, the number of estrogen receptors declines steadily. The initial decline during the early luteal phase starts when estrogen levels decline because of the transformation of the ovulated follicle to a corpus luteum and the concomitant increase in serum progesterone levels. It is clear that the serum progesterone levels must rise above 1 ng/mL to initiate this effect (West and Brenner 1983). During the course of the luteal phase, the estrogen and progesterone receptor levels are further down-regulated by the opposing action of progesterone (Elsner et al. 1977; West and Brenner 1985; West et al. 1986; Okulicz and Balsamo 1993). In uteri of spayed cynomolgus monkeys that received estradiol-filled implants for fourteen days, after which progesterone implants were introduced additionally and left for another fourteen days, five-fold lower mean endometrial nuclear estrogen receptor levels were present, compared to spayed animals that only received estradiol-filled implants (McClellan et al. 1984). West and Brenner (1985) found a significant decrease in estradiol receptors in the cytosol and nucleus after six days of sequential E2 plus progesterone treatment in cynomolgus monkeys. This down-regulation was found to be zone and cell-type sensitive (Okulicz et al. 1993). Naturally cycling rhesus monkeys also show a decrease in the total (cytoplasmic and nuclear) concentration of both the estrogen and progesterone receptor when measured from days 2 to 6 postovulation. The level of nuclear estrogen receptors, however, remained unchanged over this period (Ghosh and Sengupta 1988). During natural menstrual cycles, the concentration of nuclear and cytoplasmic progesterone receptors decreases when the circulating progesterone levels are highest. This finding suggests a negative feedback mechanism of progesterone on its own receptor expression (Kreitmann-Gimbal et al. 1980). During the menstrual phase, it is the decline of serum progesterone that precedes the initiation of estrogen receptors in the endometrium, rather than the rise of the estrogen levels (West and Brenner 1983). In cynomolgus monkeys with an artificially induced changeover from menstruation to regeneration phase, the first cells found to express the estrogen receptor were stromal cells (McClellan et al. 1986). Glandular cells still remained negative for several days. Thereafter, the epithelial cells in the upper zones of the endometrium start to express the estrogen receptor, a process that coincides with the increase in mitotic activity in these glandular areas. Recently, a subtype of the estrogen receptor cloned from rat prostate tissue was identified (Kuiper et al. 1996). This subtype is known as the estrogen receptor beta (ER-β). This receptor occurs in a wide range of tissues in several species, including macaques (Pelletier et al. 1999) and man (Mosselman et al. 1996).
Pau et al. (1998) and Pelletier et al. (1999) investigated the uterus using reverse transcription-polymerase chain reaction and in situ hybridization, respectively, to localize ER-β mRNA expression. Although in both investigations a very limited number of monkeys were studied, labeling for ER-β mRNA was observed in the surface epithelium, glandular epithelium, and stromal cells. Weak labeling in the smooth muscle cells of the myometrium was noted in the study by Pelletier et al. (1999), but Pau et al. (1998) found only ER-
So far, studies with ER- In the early 1970s, two progesterone receptor binding isoforms were characterized, type PR-A and PR-B. It is now known that in humans both isoforms are products from a single gene (Giangrande and McDonnell 1999; Kastner et al. 1990). PR-A and PR-B have also be identified in the cynomolgus monkey (Wang et al. 2003).
Receptor Expression Patterns in Cynomolgus Endometrium If present, immunoreactivity of estrogen and progesterone receptors was mainly restricted to the nuclei of the different responding cell types. In some cases only faint cytoplasmic reactivity was observed with the antibodies used. This reactivity was often too weak for a reliable score, so the data presented are based on nuclear immunoreactivity of both antibodies.
The Estrogen Receptor
In the subsequent regenerative phase, the nuclei of the epithelial cells lining the glands in the deeper functionalis (Zone II) and the whole basalis start to express the estrogen receptor (Figure 22: 2F, 2B). The expression level of the estrogen receptor in the nuclei of the stroma and myometrium is comparable to that observed during the late menstrual phase. When the endometrium enters the early follicular phase, expression of the estrogen receptor is observed over the full thickness of the endometrium in superficial epithelium, glandular epithelium, stroma, and myometrium (Figure 22: 3F, 3B). Nevertheless, a gradual increase in expression over the full length of the glands toward the basalis is evident. Some endometrial components—such as blood vessels, scattered epithelial cells (mainly located in the glands in the deepest zona basalis) and scattered stromal cells—remain negative. In the glands of the zona basalis, the presence of scattered cells exhibiting negative nuclear staining could point to the fact that these cells are stem and/or progenitor cells, which are not (yet) under the control of estrogens (Figure 25).
During the further course of the follicular phase (the mid- and late follicular phase), the expression of the estrogen receptor in the nuclei of the epithelial cells lining the glands remains at high levels (Figure 22: 4F, 4B and 5F, 5B). In contrast, the expression in the superficial epithelial cells vanishes. The expression of estrogen receptors in the nuclei of the stromal cells and myometrium also diminishes; although the expression is still moderate during the mid-follicular phase, it decreases when the follicular phase progresses into the preovulatory period (the late follicular phase) and further into the period around ovulation (Figure 22: 6F, 6B). After ovulation, the endometrium is mainly under the influence of progesterone. This hormonal shift runs parallel to changes in receptor expression levels and consequently related morphologic events in the endometrium. During the early luteal phase, expression of the estrogen receptor is restricted to the epithelium lining the glands in the deep functionalis and the basalis (Figure 22: 7F, 7B). The stromal and smooth muscle cells of the myometrium are negative. The mid-luteal phase is characterized by the presence of estrogen receptors in the nuclei of the glands in the deep basalis (Zone IV) exclusively. The level of expression in this zone remains quite strong (Figure 22: 8F, 8B). During the late luteal phase, the whole endometrium and myometrium becomes devoid of estrogen receptors (Figure 22: 9F, 9B). During the whole menstrual cycle, estrogen receptors were not detected in the uterine vessels with the antibody used. The next cycle again starts with the initial expression of estrogen receptors in the stroma (and myometrium).
The Progesterone Receptor
When regeneration becomes evident (the regenerative phase), expression of the progesterone receptor decreases in the stroma of the upper functionalis, and a gradual increase in receptor expression starts to appear in the nuclei of the epithelial cells lining the glands in the deep functionalis and basalis (Figure 23: 2F, 2B). The whole follicular phase is characterized by high levels of expression in the nuclei of the superficial and glandular epithelial cells, stroma, and myometrium (Figure 23: 3F, 3B and 4F, 4B and 5F, 5B). A more or less comparable expression pattern is observed in the period around ovulation (Figure 23: 6F, 6B) and during the early luteal phase (Figure 23: 7F, 7B), with the exception that in the latter phase, areas with negative glandular epithelium lining the portions of the glands in the upper basalis (upper zone IV) can be observed (Figure 37). In the mid-luteal phase, a gradual decrease in the expression of the progesterone receptor is observed in the glands of the functionalis and upper basalis (Figure 23: 8F, 8B). This decrease progresses, and when the endometrium enters the late luteal phase, the portion of the glands in the functionalis becomes totally negative. Also, in the upper basalis the expression vanishes, whereas the expression persists in the epithelial cells in the deepest portion of the glands (Zone IV) up to the moment the endometrium shows the first signs of menstruation (Figure 23: 9F, 9B).
Figure 24 summarizes the expression of ER-  , PR, and Ki67 in the different compartments of the endometrium during the menstrual cycle of the mature cynomolgus monkey, as investigated using the mentioned antibodies. The scheme does not show the extent of expression, which normally has a gradual course.
The presence of the estrogen and progesterone receptors was also studied in menopausal and postmenopausal cynomolgus endometria. In both the menopausal and postmenopausal endometria, the expression of estrogen and progesterone receptors is high in all components (surface, glandular epithelium, stroma, and myometrium) (Figure 26). In the senile endometria, the number of receptors tends to decrease. Down-regulation of human progesterone receptor has been recently suggested to be isoform specific, suggesting that this staining pattern may not accurately reflect the complete picture of progesterone receptor changes (Mote et al. 1999).
Our results on endometrial and myometrial estrogen and progesterone receptor expression in natural cyclic cynomolgus monkeys very closely resemble those obtained for these receptors in natural cyclic animals (Brenner et al. 1991) and ovariectomized female cynomolgus monkeys implanted subcutaneously with estradiol and subsequent progesterone implants (McClellan et al. 1986; Okulicz et al. 1990; West and Brenner 1983) and rhesus monkeys (Okulicz and Scarrell 1998; Okulicz et al. 1993). The observation that some cells within the epithelium lining the most basal portions of the glands did not express the estrogen receptor is supported by the results obtained by McCllellan et al. (1986). These authors noted that such negative cells were located immediately adjacent to cells with pronounced nuclear reactivity. In general, the expression patterns of estrogen and/or progesterone receptors during the natural menstrual cycle of cynomolgus monkey are comparable to the expression patterns of both receptors reported in women (Bergeron et al. 1988; Garcia et al. 1988; Scharl et al. 1988). Garcia et al. (1988) studied the presence of estrogen and progesterone receptors in endometrial biopsies obtained from a selected number of healthy, normocyclic women during the mid-follicular to late luteal phase. However, the use of standard biopsy techniques in this study excluded the possibility to investigate the portions of the gland in the zona basalis. Nevertheless, in comparable studies (Coppens et al. 1993; Scharl et al. 1988) that were based on hysterectomy material, a relatively high expression of estrogen receptor was observed in the glands of the basalis, whereas expression of this receptor in the glands of the functionalis was weak. When comparing the results of our study to those of the studies of Garcia et al. (1988), Scharl et al. (1988), Lessey et al. (1988), and Coppens et al. (1993), the most important difference is the presence of estrogen receptors in the nuclei of stromal cells during the luteal phase. Although both investigators found a slight to moderate expression of estrogen receptor in the human endometrium during this phase, this reactivity was not observed in the cynomolgus. In the study done by Garcia et al. (1988), a few endometrial biopsies of oligomenorrheic women were included also. In these samples a relatively high expression of both estrogen and progesterone receptors was found in glands and stroma. These findings are similar to those observed in the inactive endometria of menopausal cynomolgus monkeys in our study. In studies that included material from postmenopausal women, high levels of estrogen receptor expression were observed (Scharl et al. 1988; Snijders et al. 1992). Comparable results were observed in aged cynomolgus monkeys (Cline et al. 2002). Although less pronounced, progesterone receptors also persist in the endometrium of postmenopausal women (Snijders et al. 1992).
Androgen Receptor
Vascular Endothelial Growth Factor
Relaxin Receptor Relaxin is a peptide hormone that is produced in the ovarian corpus luteum and endometrium (Hayes 2004) of humans and nonhuman primates. Although its role is not well understood, there is good evidence that relaxin is associated with the process of decidualization and implantation. The receptor for relaxin is located in the endometrial stromal cells closely related to the glands in the functionalis (Ivell et al. 2003). During the luteal phase, expression is stronger than during the follicular phase.
When ovariectomized rhesus monkeys with artificially induced menstrual cycles are treated with relaxin, uterine weight, angiogenesis, and the number of endometrial lymphocytes increase (Goldsmith et al. 2004). Other changes were observed in factors necessary to maintain early pregnancy, such as the number of estrogen receptor-
Cyclic Hormone-Mediated Morphologic Changes The menstrual cycle, with an average duration of 30.4 ± 4.7 days in the cynomolgus monkey (Weinbauer et al. 2008), can be divided roughly into four major phases, the follicular (or proliferative) phase, the luteal (or secretory) phase, the menstrual phase, and the regenerative phase. More often, a three-phase classification is used, leaving the regenerative phase out. We believe that the physiological events that take place during the regenerative phase are distinct from those observed during the follicular phase. Histologically, the follicular and luteal phases can each be further subdivided into three subphases, respectively, the early, mid-, and late follicular and luteal phases. To describe the chronologic morphologic events that appear during the different phases of the menstrual cycle, as mentioned previously, a selection was made out of approximately eighty mature, untreated and placebo female cynomolgus monkeys. From each phase of the cycle, a few representative cases were selected, and the morphological changes studied in microscopic detail. For this purpose, slides of a full transverse section through the body of the uterus were stained with hematoxylin and eosin (H&E), PAS, alcian blue, and Verhoef’s method for reticulin fibers. Because even within one phase variations exist in the grade to which the morphological changes appear, the average changes are described here. The cycle-related changes gradually appear and disappear, and no sharp distinction between the different subphases can be made. The fact that we used mature, naturally cyclic animals instead of ovariectomized females with artificial hormone-induced synchronized cycles is a strength of this report but also a source of variation, since the length of the menstrual cycle in macaques varies substantially (see Weinbauer et al., 2008). Therefore it is often difficult to characterize the morphological changes in the endometrium of the cynomolgus monkey on a day-by-day basis, as was done by Dallenbach-Hellweg (1987) for the changes during the luteal phase of the human menstrual cycle. For the follicular phase, such a detailed dating is, even in the human endometrium, impossible because of its variable length (the length may fluctuate between ten and twenty days). Although day 0 of a cycle is the day on which the first clinical signs of menstrual bleeding appear, for describing the morphologic changes during the cycle we chose to begin with the follicular phase when the buildup of the endometrium starts. On average, the follicular phase starts on cycle day 4. The major morphologic characteristics of the different subsequent phases in the macaque endometrium in relation to the cyclic fluctuating (pituitary and) ovarian hormone levels are depicted in Figure 28 (see also Weinbauer et al., 2008).
The Follicular Phase The follicular phase is a period in which controlled growth of the endometrium occurs to achieve a morphological basis for implantation. All tissue components, the superficial and glandular epithelium, the stroma, and the endometrial vasculature are in varying degrees involved in this physiological proliferation process.
The Early Follicular Phase Morphologically, the early follicular phase is characterized by a low, still quite inactive endometrium with sparse, narrow, and straight tubular glands within a loose stroma. The zona basalis can be easily recognized because of its more compact stroma (clearly noticeable in Figure 20). The portion of the glands in the zona functionalis are lined by low columnar epithelial cells. Most of the nuclei of the epithelial cells are round to slightly oval in shape and basally located, forming a line with the nuclei of the adjacent cells. The cytoplasm of the epithelial cells is quite homogeneous, although sporadically small cytoplasmic vacuoles may be found. In general, no glycogen can be demonstrated within the cytoplasm of these cells. During this phase, few to scattered mitotic figures are found in the glands of the zona functionalis. Clear and ciliated cells are observed. The cytology of the surface epithelial cells is slightly different compared to the secretory cells in that they have small projections on their surface (microvilli). The nuclei of these cells show slightly more nuclear pseudostratification, and nucleoli are found in most of the nuclei. Also, few to scattered mitotic figures are found within the surface epithelium. Spiral arteries are inconspicuous. A number of young, small vessels with large, active endothelial cells run parallel to the glands up to the surface, where they branch. The smaller branches run parallel to the surface epithelium. The glands and vessels are embedded in a loose stroma. The stromal cells are fusiform to stellate and have scant cytoplasm. Nucleoli, although present, are not conspicuous. Within the stroma, quite a number of mitotic figures can be observed. The portions of the glands in the zona basalis are inactive and small, often with only a very narrow, sometimes even obscured lumen. Remnants of inspissated secretory material can be found within the gland lumina. The cells lining the glands in this zone are cuboidal to low columnar with scant cytoplasm. The glands are surrounded by a compact, sometimes "fibrous"-looking stroma. Mitotic figures are absent in this part of the endometrium. Granular lymphocytes can be present in the stroma.
The Mid-Follicular Phase The portion of the glands in the zona functionalis are lined by medium to high columnar epithelial cells. These epithelial cells lie on a thick basement membrane that even on low magnification is obvious. According to Kaiserman-Abramof and Padykula (1989), who studied the ultrastructural epithelial zonation in the rhesus monkey, this is a hallmark of Zone II epithelium during this phase. The nuclei of the epithelial cells are oval and larger compared to those during the early follicular phase. One or two distinct nucleoli are present in most nuclei. Nuclear pseudostratification now becomes apparent, and mitotic figures are numerous in the surface epithelium, glands, and stroma of the zona functionalis. Slight intracytoplasmic glycogen production starts to become visible in the epithelial cells lining the portion of the glands in the deeper zona functionalis as areas with slight subnuclear cytoplasmic rarefaction. Stromal cells can have scant basophilic cytoplasm. Their nuclei are enlarged, and a single nucleolus is often present. In between the stromal cells, edematous fluid accumulates. Using alcian-blue staining, significant amounts of intercellular acid mucopolysaccharides can be demonstrated in the full thickness of the endometrium (see also Figure 19). The vasculature does not differ from that present during the early follicular phase, except that it becomes slightly more prominent. The morphologic characteristics of the basalis are more or less similar to those described for the early follicular phase. Mitoses are absent in the deepest portions of the glands and in the stroma. Spiral arteries are not prominent.
The Late Follicular Phase As a result of the decrease in intercellular fluid content, the total thickness of the endometrium decreases slightly. Although the stromal volume decreases, the epithelial cells of the zona functionalis continue to proliferate and mitotic figures are still frequently observed. The glands are coiled or tortuous. Pseudostratification and glandular crowding are prominent because the glands grow faster than the stromal component can expand. The stromal cells remain active, and the endometrial lymphocytes become more numerous. The portion of the glands in the zona basalis does not differ much from those described during the mid-follicular phase of the cycle, although in some cases early proliferative activity can already be observed. The physiologic (and morphologic) events associated with the maturation and ovulation of the Graafian follicle followed by its transformation to an early corpus luteum provide the support that some authors use to distinguish this as a separate phase, the "interval phase." Theoretically, this phase forms the transition between the follicular and luteal phases. Although physiologically significant changes play a major role, there are no outstanding morphologic features that characterize these events.
The Luteal Phase In contrast to the human endometrium, in which the morphologic changes during the luteal phase vary from day to day permitting accurate dating, this is less clear in the monkey. Therefore, in comparison to the follicular phase, the luteal phase is divided in three subphases, each with its own morphologic characteristics. After ovulation, the first sign of progestational development in cynomolgus macaques is an increase in sacculation (branching) of the glands in Zones III and IV of the endometrium (Brenner et al. 1983).
The Early Luteal Phase The stroma of the functionalis is loose, but acid mucopolysaccharides are not detectable in this phase. The cytoplasm of the stromal cells is inconspicuous. Nevertheless, the nuclei are still large, often contain one nucleolus, and mitoses are only sporadically observed. The typical endometrial lymphocytes are found scattered throughout the stroma. In the portion of the glands in the zona basalis, which up to this stage were quiescent, significant changes now start to appear. The glands show increased sacculation, and glandular epithelial cells become medium to high columnar. The nuclei are round to slightly oval, and small nucleoli are sometimes present. In the basalis III, sub- and supranuclear, PAS-positive cytoplasmic vacuoles containing glycogen can be observed. Few mitotic figures are observed in the epithelium of the basalis IV parts of the glands. The stroma surrounding the basalis glands is fibrous in appearance. The stromal cells have oval to slightly spindle-shaped nuclei. The spiral arteries start to develop and become prominent in this zone. Endometrial lymphocytes are found, although fewer than in the functionalis.
The Mid-Luteal Phase Apical secretory activity in the form of apical cytoplasmic blunt protrusions that detach from the cell surface is evident, and PAS-positive, homogeneous to granular secretory material accumulates within the gland lumina. Since the major constituent of this secretory material is made of cytoplasmic vesicles that bud off from the cell surface, often many round, partially empty structures resembling lysed erythrocytes are present within the lumen of the glands (see also Figure 8). The nuclei of the epithelial cells still can show some pseudostratification, but less pronounced than during the late follicular and early luteal phase. Mitotic activity is completely absent in the surface and glandular epithelium in the functionalis. The stroma in the functionalis is less loose compared to the early luteal phase. Sometimes remnants of interstitial edema are found. The stromal cells are fusiform and have medium-sized nuclei. Endometrial lymphocytes are abundant. The hallmark of the mid-luteal phase is a significant proliferative activity in the deepest part of the glands in the zona basalis. The epithelial cells in this zone are high columnar and have large oval to cigar-shaped nuclei with prominent nucleoli. Nuclear pseudostratification and mitotic figures are observed. The cells have a smooth surface. Significant amounts of glycogen can be demonstrated in these cells. The stroma of the basalis is dense and can sometimes show a fibrous appearance. Spiral arteries are most prominent at the basalis–functionalis junction.
The Late Luteal Phase During the late luteal phase, the spiral arteries fully develop. The stromal cells in the immediate vicinity of the spiral arteries become slightly hypertrophied, a process that is called decidual transformation or pseudodecidualization (real decidualization occurs during pregnancy). The borderline between the adventitia of the spiral arteries and the endometrial stroma becomes less distinct. This process of decidualization is much less extensive than in the human endometrium during the luteal phase and generally inconspicuous. In the human endometrium, the enlargement of stroma cells also starts around the spiral arteries, but during the course of the mid- and late luteal phase extends to the whole upper parts of the endometrium. Such an extent of pseudodecidualization can be induced in macaques using progestational compounds, but it does not occur during the normal menstrual cycle. The glands in the zona functionalis are somewhat less tortuous than during the mid-luteal phase and contain large amounts of homogeneous, slightly inspissated, PAS-positive secretory material. The epithelial cells lining the glands are medium columnar to cuboidal and show signs of exhausted secretory activity. The latter is observed as small apical cytoplasmic vacuoles, with diminished intracellular glycogen. The nuclei are round to slightly oval, and a clear nucleolus is often absent. The nuclei are located at the basal side of the cytoplasm and aligned in a row with the neighboring cells. The surface epithelium is low columnar with round to slightly oval nuclei with no nucleolus. Small cytoplasmic protrusions are noted on the luminal surface. The stroma of the functionalis is comparable to that found during the mid-luteal phase. However, in the area just underneath the surface epithelium, the stroma can be edematous. Small hemorrhages and fibrin leakage can be found intercellularly. Endometrial lymphocytes are numerous. Mitotic activity is absent in glands and stroma in the zona functionalis. The spiral arteries running through the functionalis are prominent. In the zona basalis, the blunt ends of the glands are distinct, and the lumen seems to be "punched out." The cells lining the glands are medium columnar with a sharp and smooth surface. The nuclei may still show some pseudostratification. The nuclei of the cells are oval and their chromatin is denser compared to those of the mid-luteal phase. Nucleoli are inconspicuous. Scattered signs of apoptosis of individual cells are obvious (nuclear debris) (see Figure 38). Also, in this zone the amount of intracellular glycogen decreases. Some glands are filled with eosinophilic, PAS-positive secretory material. The stroma is comparable to that found during the mid-luteal phase. Spiral arteries are still prominent.
The Menstrual Phase If no implantation occurs, the corpus luteum in the ovary regresses and the output of progesterone declines. The drop in circulating progesterone (and estrogen) inevitably leads the endometrium into the menstrual phase. During this process, the exhausted upper parts of the endometrium (the functionalis) regress and are shed to prepare the endometrium for a new cycle. Owing to vasoconstriction of the spiral arteries, the upper parts of the endometrium become ischemic (Chabbert Buffet et al. 1998). The release of proteolytic enzymes from endometrial and inflammatory cells is responsible for the tissue breakdown. Tissue necrosis, vascular thrombus formation, disruption of the microvasculature, and leukocyte infiltration are the main morphological features of the menstrual phase (Ferenczy 2003). Small hemorrhages within the dense superficial stroma just beneath the superficial epithelium are the first signs of the menstrual phase. The portion of the glands and stroma in the zona functionalis disintegrates, and the continuity between the epithelium lining the glands and surface epithelium is lost (Figure 29). The stroma of the functionalis becomes de-epithelialized. Parts of free endometrial tissue are shed and found within the uterine lumen (Figure 30). This whole process is accompanied by blood loss from the stromal vessels. In this stage blood and remnants of shed endometrium tissue can be found in the cervical canal and vagina (Figure 31).
The epithelium lining the remaining parts of the glands is low to sometimes medium columnar in shape. Within the cytoplasm in the upper part of the cells, vacuoles still can be present, as can the cytoplasmic protrusions on the apical surface (at least during the first days of menstruation). Remnants of intraluminal secretory material can be abundant. The nuclei of the cells are round to oval and located at the basal part of the cell. Small nucleoli can be observed. Scattered apoptotic bodies are seen within the glandular epithelial lining. Although the stroma across the full length of the endometrium is compact, the stroma in the zona functionalis is especially dense and deeply blue stained in H&E sections, with cellular clumping. The stromal cells in the upper regions have small, dark nuclei, and the cytoplasm is inconspicuous. Numerous endometrial lymphocytes are present in the stroma. In the deeper parts of the functionalis, the stromal cells have recognizable basophilic cytoplasm. The spiral arteries are prominent throughout all layers of the endometrium. Aggregates of lymphocytes can be found within the stroma. The zona basalis escapes the process of shedding. The glands are still sacculated and often contain remnants of secretory material. The cells are cuboidal to low columnar and have a smooth apical surface. The nuclei are round to oval and are located at the basal part of the cell. In Zone III of the basalis, sometimes areas with nuclear crowding are present, and nuclei are long, oval, and orientated perpendicular to the basement membrane. In the glands in the basalis, scattered apoptotic bodies still can be found in between, or within, the lining epithelial cells. The stroma in the basalis is "fibrous" in appearance, with spindle-shaped stromal cells. Also, in this region endometrial lymphocytes are found. It is our experience, that in the cynomolgus monkey, the entire zona functionalis is shed during the process of menstruation.
The Regeneration Phase The first signs of the rejuvenation of the sloughed endometrium are observed in the remaining upper parts of the glands of the functionalis. This regeneration process gradually starts already during the late menstrual phase. The nuclei of the exhausted secretory cells become larger, and a distinct nucleolus appears. Via proliferation and migration, cells from the remaining portion of the glands in the zona functionalis and upper basalis start to cover the denuded endometrial stroma. The epithelium starts to proliferate from the stumps of the remaining gland structures that are exposed to the lumen. Before spreading over the denuded stroma, the epithelial cells in the stump of the glands pile up against each other. From these areas, the epithelial cells spread laterally and finally close the defects, trapping the dissociated stromal cells as fish in a net (Figure 32). The newly formed surface epithelium first consists of flat epithelial cells.
In general, during this phase, the stromal cells are small, with pleiomorphic, hyperchromatic nuclei and scant cytoplasm. Scattered endometrial lymphocytes are still found within the stroma. Aggregates of lymphocytes also can be found within the compact stroma. Spiral arteries are much more prominent in the zona basalis compared to the newly formed zona functionalis. Compared to the late luteal phase, it would appear that the spiral arteries behave like a stretched spring that is released (possibly because the contact with the reticular network is lost because of its disintegration). The gland portions in the zona basalis are quiescent. From day 5 onward, there is a further increase in both nuclear DNA synthesis and the number of mitoses, in all components of the regenerated endometrium. These changes are accompanied by an increase in the plasma estradiol level, which strongly suggests that the proliferative activity within the endometrium is now under control of estradiol. The detailed subsequent epithelial and cytologic changes in the glandular portions of the zona functionalis during the menstrual cycle of the cynomolgus monkey are depicted in Figure 33.
A diagram including selected major or characteristic changes that, in our experience, are the most useful in evaluating and dating the macaque endometrium, is provided at the end of this paper (see Figure 43).
Endometrial Proliferation and Differentiation In our collection of cynomolgus endometria we studied proliferation during the different phases of the normal menstrual cycle using an antibody raised against the Ki67 antigen (monoclonal mouse anti-Ki67, Clone MIB-1, M7240, Dako, Carpinteria, CA). The Ki67 antigen is expressed in the nucleus during all active phases of the cell cycle (G1, postmitotic phase; S, DNA synthesis phase; G2, premitotic phase; and M, actual mitosis), with peak expression in the early S phase, and has been used extensively as a marker for cell proliferation. Cells in the G0 or proliferative resting or terminal phase of the cell cycle do not express the Ki67 antigen. During the menstrual phase, no clear positive immunoreactivity is observed throughout the endometrium. The (remaining) gland epithelium is completely negative, and only scattered positive stromal cells are noted. In contrast, during the regenerative phase a large number of cell nuclei within the upper parts of the glands show a positive reaction (Figure 34). This finding is in line with the numerous mitotic figures that are observed in these areas. Also, some positive cells are found within the newly formed superficial epithelium, but these are found to be related mostly to the neck of the glands. The newly formed flat surface epithelium did not show significant immunoreactivity to the antibody. The reaction in the stroma is comparable to that observed during the menstrual phase. With the exception of the stroma in the zona basalis, the stroma within the upper layers of the endometrium shows the presence of some Ki67-positive nuclei.
During the early follicular phase, numerous surface, glandular, and stromal cells in the zona functionalis exhibit a positive reaction for the Ki67 antigen, whereas in the zona basalis no significant positive reaction is observed. Only sporadic stromal cells have positive-stained nuclei. In the mid- and late follicular phase, the immunoreactivity extends toward the portion of the glands in the upper basalis, whereas meanwhile, the reactivity in the surface epithelium declines. During the shift from the late follicular to the early luteal phase, the immunoreactivity in the glands of the functionalis and upper basalis decreases abruptly. Immunoreactivity persists in the stroma cells, but few positive nuclei are present in the gland epithelium only. In contrast, in the deepest portions of the glands numerous positive nuclei are present at this time. This pattern persists during the mid-luteal phase. No positive immunoreactivity is observed in the endometrial glands during the late luteal phase. A varying number of positive stromal cell nuclei are present in the functionalis during the mid- and late luteal phase, but this number tends to decrease premenstrually. During the entire luteal phase, a positive reaction is observed in the nuclei of blood vessel cells. Based on these findings, and supported by the literature, it can be hypothesized that three controlled, subsequent waves of proliferative activity alternate in the surface and glandular epithelium in the cyclic cynomolgus endometrium. The first wave of proliferative activity, also observed in the human endometrium, takes place during the menstrual and regenerative phase: although epithelial proliferation is virtually absent during the start of menses, signs of epithelial proliferation readily appear during the further course of this phase (Ferenczy 1976). At that time, mitotic figures are found in the exposed ends of the glands in the upper layers of the remaining endometrium. The function of this wave of proliferative activity is to heal the defects that appeared as a consequence of the disintegration of the zona functionalis during menses. The cells that proliferate are well-differentiated epithelial cells in the stumps of the glands that escaped the disintegration process (Figure 35A). They form the new surface epithelium and support the growth of the length of the glands. The function of these cells shifts from secretory to progenitive. Within the newly formed surface epithelium, no mitotic activity is found in the human endometrium (Ferenczy 1976). In the cynomolgus monkey, however, some Ki67 labeling was observed within the regenerated surface epithelium (see Figure 34). This first wave seems not to be directly mediated by the ovarian hormones, since during this wave both circulating estradiol and progesterone levels are low. Additionally, within the glands throughout the endometrium, estrogen receptors (and progesterone receptors) are almost lacking (Figure 35B). These observations strongly suggest that the epithelial cells that participate in the regeneration process do not require estrogen receptors and are therefore not directly dependent on estradiol (Bhartiya and Bajpai 1995; Brenner and Slayden 1994; Ferenczy 1976; Ferenczy 1977; McClellan et al. 1986). A similar mechanism is proposed for the macaque endometrium (Brenner et al. 1991; Padykula 1991; Padykula et al. 1984; Okulicz et al. 1997). In contrast to the epithelial cells, stromal cells do express estrogen receptors during the regenerative phase. Comparable to our observations, Brenner et al. (1991) reported that even during the very early follicular phase, most of the estrogen and progesterone receptors were specifically localized in the endometrial stromal cells and not in the epithelial cells. The areas where glandular proliferation began were found in close relation to areas in the stroma where the stromal cells expressed the estrogen and progesterone receptors. Brenner et al. (1991) combined immunohistochemical technique for the estrogen receptor with [3H]-thymidine autoradiography on the same tissue sections. Using these combined techniques, they were able to prove that estradiol indeed indirectly stimulated DNA synthesis in epithelial cells in the zona functionalis that did not express the estrogen receptor. Based on these results, it is hypothesized that the endometrial stromal cells mediate the DNA synthesis during the very early follicular phase by paracrine factors released by the stromal cells.
However, the exact factors that initiated this wave of proliferation in the gland epithelium are still unknown. A supportive role is suggested for factors such as fibroblast growth factor, platelet-derived growth factor, transforming growth factor beta (Koji et al. 1994), keratinocyte growth factor (Koji et al. 1994; Slayden et al. 2000), and insulin-like growth factor (IGF-1) (Zhou et al. 1994). The remarkable regenerative potential of the rhesus endometrium was shown by the classical studies of Hartman in 1944. After drastic surgical removal of the entire endometrium, it regenerated sixteen to nineteen days post-surgery. Even in the absence of hormonal support, the endometrium has this capability to heal (Koji et al. 1994). From these observations it can be concluded that even small remnants of Zone IV, containing both the glandular stem cells and stroma, are able to reconstruct the whole endometrium. Recently, investigators (Leyendecker et al. 2002) suggested that the remarkable regenerative capacity of the basal endometrium also could form the basis of endometriosis. In the macaque endometrium, the first wave of endometrial proliferation gradually fades into a second proliferative event. This second wave represents direct, estrogen-dependent glandular proliferation (McClellan et al. 1990). In extent and severity, this is the strongest wave of endometrial proliferation (Ferenczy et al. 1979a; Brenner et al. 2003). The function of this proliferation is to create an appropriate mucosal thickness with a large pool of differentiated glandular secretory cells to provide an excellent morphological basis for blastocyst implantation. During the E2 surge, the average labeling index (measured using [3H]-thymidine autoradiography) in the zona functionalis of the rhesus monkey is 10% (Padykula et al. 1984; Padykula et al. 1989). This proliferative activity appears in the surface epithelium and in portions of the glands in the functionalis. The cells that proliferate are mainly the well-differentiated epithelial cells bearing estrogen receptors. When after ovulation the progesterone levels rise, the expression of estrogen receptors is down-regulated. The loss of stromal estrogen receptors coincides with the disappearance of glandular proliferative activity in the functionalis. However, expression of estrogen receptors is retained in the deepest portion of the glands in the zona basalis (Okulicz et al. 1993). The third wave of glandular proliferation takes place exclusively in the deep zona basalis (Zone IV). In cynomolgus and rhesus monkeys, mitotic activity increases in the glands in the deep basalis during the early luteal phase under progesterone dominance (Bensley 1951; Elsner et al. 1977; Okulicz et al. 1993; West and Brenner 1985; West et al. 1986). Also in the cynomolgus monkeys we studied, such an increase in proliferative activity was noted during the early and mid-luteal phase. In some animals, proliferation started already during the late follicular phase (see Figure 36).
Studies using [3H]-thymidine uptake have shown that during progesterone dominance, epithelial proliferation is strongly inhibited in the glands of the functionalis and upper basalis, but meanwhile an up to tenfold increase in proliferation occurs in the glands of the zona basalis of the rhesus monkey compared to the late follicular phase (Padykula et al. 1984; Padykula et al. 1989). Using a double immunofluorescent method for the simultaneous localization of both the estrogen receptor and the Ki67 antigen, Okulicz and Balsamo (1993) clearly demonstrated that under progesterone dominance, the nuclear estrogen receptor persisted in the epithelial cells in the deep basalis and that there was coincident proliferative activity. Under these circumstances, despite the presence of significant levels of nuclear estrogen receptors, a different control and regulatory mechanism for this last wave of proliferation is thought to exist. Studies in the rabbit have suggested that the glands in the basalis possess a regenerative capacity not mainly dependent on estradiol or progesterone (Hegele-Hartung 1992). Nevertheless, an indirect regulatory role for progesterone cannot be ruled out, since proliferation in the portion of the glands in the deep basalis of rhesus monkey endometrium during an artificial mid-luteal phase is inhibited when the animals are treated with RU-486, an antiprogestogen (Okulicz et al. 1997). Comparable results were obtained in rhesus monkeys by Slayden et al. (2001) using different antiprogestogens. Although the factor(s) that are directly involved in the regulation of the proliferation of the glands in the zona basalis are unknown, evidence for involvement of specific gene products such as the retinoblastoma gene is suggested. Expression of the retinoblastoma gene, which has an inhibitory effect on cell growth, is exclusively down-regulated in the deep zona basalis during progesterone dominance (Okulicz et al. 1993; Ace and Okulicz 1995). Recently, in studies using knockout mice lacking the progesterone receptor isoform PR-A (PRAKO), an unexpected progesterone-dependent enhanced proliferative effect in the epithelium was observed after treatment with estrogens (Conneely et al. 2001). This effect was not observed in wild-type and knockout mice lacking both isoforms. These results indicate that in these mice, expression of the progesterone receptor isoform-B alone results in a synergistic proliferative action of estrogen and progesterone. These studies also indicate that the presence of PR-A is required to inhibit the estrogen-induced proliferation and to limit the potentially adverse proliferation regulated via PR-B in the endometrium (Conneely et al. 2003). It would be very interesting to study the expression patterns of PR-A and PR-B in the different endometrial compartments of the macaque and human endometrium during the cycle, especially during the luteal phase. An (indirect) mitogenic role for progesterone was proposed as the mechanism supporting the specific wave of proliferation in the deepest portions of the glands in the basalis (Koji et al. 1994; Okulicz et al. 1993). Padykula et al. (1984) postulated that endometrial stem cells (undifferentiated cells that have the ability to divide and that are self-maintaining) populate the glands located in this region. Via proliferation and differentiation, progenitor cells develop from these stem cells. Progenitor cells have a high proliferative capacity and the ability to differentiate further into more mature glandular epithelial cells. The remarkable enhancement of proliferative activity in the portion of the glands in the deep basalis is therefore thought to reflect the process of replenishing the pool of stem and progenitor cells (Padykula et al. 1989). It can be hypothesized that the upper part of basalis Zone IV represents a transition zone, a temporary "nursery" for progenitor cells. When further differentiated, these cells shift up to the more active portions of the glands in Zone III. In the next menstrual cycle, this new generation of cells becomes involved in the process of endometrial proliferation and maturation. This hypothesis is largely supported by the observations made by Padykula et al. (1984, 1989). Figure 36 clearly demonstrates the proliferation dynamics within the cynomolgus endometrium during the late follicular phase. In the deepest parts of the glands (the blind ends of the glands) in Zone IV, stem cells start to proliferate and form progenitor cells that shift up in this zone. In the upper parts of Zone IV—the hypothesized transition zone—proliferative activity is very low. At the same time, the glandular proliferative activity in Zone III is very high. This finding suggests that the glandular epithelial cells of Zone III form a population of cells that coordinates the rapid outgrowth of the glands within the endometrium after menses and form the new zona functionalis (Zone II of the following menstrual cycle). Zone II, the zona functionalis of the actual cycle, is clearly distinguishable from the zona basalis because of its significant proliferation of stromal cells. The indication that during the luteal phase the upper Zone IV seems to be devoid of progesterone receptors (that at that time are expressed in the functionalis II, III, and the deep basalis IV) could also support the hypothesis; it could mean that the cells in this zone are shielded from the actions of progesterone (Figure 37). Further supportive evidence for the stem cell theory is based on the determination of stem cell markers. c-kit/CD117, a molecular marker of stem cells, is prominently expressed in the glands and stroma of the zona basalis of the human endometrium (Cho et al. 2004). Although c-kit/CD117 is not expressed in the fetal endometrium, it is found during the full reproductive period. Tanaka et al. (2003) found evidence for a monoclonal composition of most human endometrial glands. However, different glands were found to have different clonal constitutions, forming a mosaic pattern that was also expressed in the luminal epithelium. This fact suggests the possibility of more primitive cells that develop into stem cells in each gland during uterine development. Since the zona basalis escapes the process of menstruation, the maintenance of the pool of stem and progenitor cells must be controlled via the process of apoptosis. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) method, von Rango et al. (1998) indeed observed an increase in apoptosis in the basal portions of the glands of the human endometrium in the mid- and late luteal phase. During these phases, a decrease in the expression of the apoptosis-blocking pro-oncogene Bcl-2 protein was noted (Cho et al. 2004; Mertens et al. 2002; von Rango et al. 1998). No signs of significant glandular apoptosis were observed during the follicular phase, however. Interestingly, in our set of cynomolgus monkeys, increased apoptosis was observed in the glands of the basalis during the period progesterone levels declined, just after the wave of proliferation in these glands (Figure 38). This observation is in line with the findings of McClellan et al. (1986), who studied artificially menstruating macaques. We believe that this process plays a role mainly in the remodeling and regeneration of the gland epithelium.
Endometrial Aging
First, the glands and stroma become inactive. Inactive glands are morphologically comparable to glands during the very early follicular phase, but without any significant mitotic activity. The glands are narrow, straight, and without many branches. The epithelial cells lining the glands are cuboidal to low columnar. The nuclei of the cells are all arranged on the same level within the cells and located more to the basal side of the epithelium. There are no signs of glycogen storage and secretory activity. Also, the endometrial stroma in this period is devoid of any signs of proliferation and appears remarkably compact. The thickness of the endometrium is subsequently moderate to low. The expression of nuclear estrogen and progesterone receptors in glands and stroma remains very high during this period, however (see Figures 26A and 26B). A similar morphology is observed in the first period after ovariectomy or when animals are treated with estrogen-receptor antagonists (Wang et al. 2002). When, with increased age, hormonal stimulation discontinues, regression of the endometrium (and myometrium) becomes more advanced. The endometrium enters the stage of severe senile atrophy. The thickness of such atrophic endometrium is very low compared to cyclic animals and consists mainly of the zona basalis with only few scattered glands. Often, the glands in postmenopausal endometria are oriented parallel to the endometrial surface (Figure 40). This feature is probably caused by the lack of space in the now very compact, collagenized or fibrotic stroma. Cystically dilated glands are often a feature of atrophic endometria, and both are found in the macaque (Figure 41) and human endometrium (Ferenczy 1994). The dilation is probably caused by obstruction of the neck of the glands because of the condensation of the surrounding stroma. The glandular epithelial cells in the scarce glands are cuboidal, with very little cytoplasm and inactive nuclei. Mitoses are absent throughout all endometrial structures.
In the postmenopausal human and macaque, endometrium estrogen receptors persist in the epithelial and stromal cells for a long period. This explains why upon estrogenic stimulation the endometrium can be rejuvenated (Ferenczy and Bergeron 1991). Within a rejuvenated endometrium, progesterone receptors also can be reinduced, and in principle, a normal cyclic endometrium can be reestablished. After a prolonged period of destimulation, however, the estrogen receptors vanish (Press et al. 1984).
Spontaneous Pathology Since most of the monkeys used for regulatory toxicity studies are pubertal or young mature animals and their health is generally very well monitored and controlled, the incidence of spontaneous background pathology in the reproductive and other organ systems is low. Regarding the reproductive system, histopathological findings most frequently observed are those related to functional ovarian or hypophyseal disturbances. Monkeys suffering from hormonal imbalance or disturbances can have no, short, or relatively long cycles (see Weinbauer et al., 2008), depending on the cause. In such animals, morphological features suggestive for inactive endometrium, "deficient follicular phase," or "luteal phase defect" (or "deficient luteal phase"), respectively, can be recognized microscopically. Sometimes features of irregular shedding are found in animals suffering from early regressing corpora lutea. Degenerative and inflammatory changes, such as acute and chronic endometritis and myometritis, are rarely observed. The presence of small aggregates of lymphocytes within the endometrial stroma is not pathological per se and is observed with a rather high incidence. Vascular lesions such as infarcts and (peri)vasculitis are rare. However, hyalinization of the vessels in the myometrium is a common finding in monoparous or multiparous females. An interesting but rare spontaneous finding in the nulliparous primate is the "epithelial plaque." This "surrogate lesion" is observed in control cynomolgus monkeys (Kaspareit et al. 2004) and rhesus monkeys (Valerio 1989). Most different types of metaplasia recognized in the human endometrium are rare or even nonexistent in the nonhuman primate. One case of mucinous glandular metaplasia was observed in the endometrium of a cynomolgus monkey in our collection. "Ciliated cell metaplasia," although not a true metaplastic change because the ciliated cell is a normal constituent of the endometrium, can be observed in the macaque endometrium. In young animals the incidence of hyperplastic and neoplastic lesions is understandably low. For various reasons, older animals are sometimes kept for research purposes. Often such animals are used for breeding purposes or are kept as models for geriatric diseases. Although the described endometrial senile atrophy observed in postmenopausal animals is not a pathological but rather a physiological condition, various (histo)pathological uterine lesions, including neoplasia, can be observed in such aged animals. The most common findings in the cynomolgus monkey are adenomyosis, stromal fibrosis, and vascular wall hyalinization (in multiparous females) (Cline and Bane 1995). The most common neoplastic lesions are benign uterine (fibro)leiomyoma (Lapin 1982; Lowenstine 2003; Kaspareit et al. 2007; Takayama et al. 2000) and endometrial polyps. In general, primary uterine neoplasms are uncommon (Beniashvilli 1989; Kaspareit et al. 2007; Squire et al. 1978). In adult rhesus monkeys, lesions such as endometriosis, uterine adenomyosis, vascular fibrosis, endometrial polyps, leiomyoma, hemagioma, and chronic endometritis have been reported (DiGiacomo and McCann 1970). Because of its clinical consequences in women, endometriosis receives a lot of attention. This well-known condition in women also occurs in the rhesus and cynomolgus monkey (Fanton and Hubbard 1983; Lapin 1982; Rippy et al. 1996) and is most likely caused by the extrauterine implantation of endometrial tissue after retrograde transmission from the uterus through the fallopian tube into the pelvis. In more severe cases, fibrous adhesions between uterus, urinary bladder, omentum, and intestines, sometimes involving both ovaries, can be observed (DiGiacomo and McCann 1970).
Comparisons to the Human Endometrium In contrast to what happens in the zona functionalis during the luteal phase, results on the expression of estrogen receptors in the zona basalis of the human endometrium are contradictory. Some investigators, who reported on glandular estrogen receptor expression in the human endometrium during the normal menstrual cycle, noted a gradual decrease of such receptors during the luteal phase (Koshiyama et al. 1995; Lessey et al. 1988; Snijders et al. 1992; Vollmer et al. 1990). This finding is in accordance with the above mentioned description, since in this phase of the cycle there is progesterone dominance. Koshiyama et al. (1995) reported an immunohistochemical positivity index (the number of positive staining cells per hundred glandular epithelial cells) for the estrogen receptor in the zona basalis of 29% in the early luteal phase and respectively 0% and 0.2% in the mid-and late luteal phase (compared to 87.7% during the follicular phase). These results are in strong contrast to the results published by others (Pickartz et al. 1990; Press et al. 1984; Press et al. 1986), who, also using antibodies specific to the estrogen receptor, detected persistence of the estrogen receptor in the zona basalis during the whole luteal phase, despite a dramatic reduction in the number of estrogen receptors in the zona functionalis. Press et al. (1984) found up to 25% strongly estrogen receptor-positive nuclei in the zona basalis (compared to approximately 3% in the functionalis) during the mid- and late luteal phase in the human endometrium. Based on these results, the progesterone-mediated down-regulation of the estrogen receptor seems to be zone dependent. If the findings reported by these authors are valid, the epithelial cells in the zona basalis seem to escape the antagonistic action of progesterone. This finding is supported by some other authors (Coppens et al. 1993). Interestingly, a comparable escape mechanism is reported to exist in the rhesus monkey (Kaiserman-Abramof and Padykula 1989; Okulicz and Balsamo 1993; Okulicz et al. 1990; Okulicz et al. 1993; Okulicz et al. 1997), cynomolgus monkey (McClellan et al. 1986), and the baboon (Hild-Petito et al. 1992). The question remains whether the possible presence of significant estrogen receptor levels in the human zona basalis during progesterone dominance leads to significant proliferative activity. In that context, the studies performed by Ferenzcy et al. (1979b), Felix and Farahmand (1997), Koshiyama et al. (1995), Shiozawa et al. (1996), and Padykula et al. (1989) are of outstanding interest. Using [3H]-thymidine, Ferenzcy et al. (1979b) studied the proliferation kinetics of human endometrium during the normal menstrual cycle and demonstrated that during the luteal phase, proliferation in the zona basalis was low. The labeling index during the early, mid-, and late luteal phase was 2.51%, 0.22%, and 0.11%, respectively (for comparison, in the early, mid-, and late follicular phase this finding was 2.08%, 3.47%, and 2.56%, respectively). More or less comparable findings have been reported by others (Hild-Petito et al. 1992; Lessey et al. 1988; Mertens et al. 2002; Pickartz et al. 1990; von Rango et al. 1998). The proliferation rate of the glands in the basalis, as measured by MIB-1 (Ki67) expression, decreased significantly during the mid- and late luteal phase (Felix and Farahmand 1997; Koshiyama et al. 1995; Mertens et al. 2002; Shiozawa et al. 1996; von Rango et al. 1998). Comparable to the study by Ferenczy et al. (1979b), Padykula et al. (1989) studied the proliferation kinetics during the normal menstrual cycle in the rhesus monkey, also using [3H]-thymidine. In contrast to the findings reported in the human endometrium (Ferenczy et al. 1979b; Hild-Petito et al. 1992; Lessey et al. 1988; Pickartz et al. 1990), these authors found a clear enhancement of proliferation in the zona basalis during the luteal phase of the normal cycle in the rhesus monkey. The labeling index increased gradually to day 21 of the cycle, on which it reached its maximum of 11%. (In comparison, in the study by Ferenczy et al. (1979b), the labeling index in the period of day 19–22 was 0.22%.) The labeling index in the zona basalis rose steadily during the E2 surge and continued to increase in the presence of elevated plasma progesterone levels. The fact that epithelial expansion and nuclear crowding in the glands of the basalis is a normal event during the luteal phase in the monkey supports this finding. In some monkeys, this epithelial expansion results in significant sacculation (see Figure 21) and later, in the cycle overgrowth of the gland portions in the Zone IV. In contrast, such an extent of epithelial expansion and nuclear crowding is, as far as we know, not a feature of the human basalis during progesterone dominance, a view also supported by others (Brenner et al. 2003; Padykula et al. 1989). "Postovulatory intensification of the epithelial proliferation in the basalis of the rhesus monkey is at variance with the view that progesterone inhibits epithelial proliferation completely in the human endometrium" (Padykula et al. 1989). Brenner et al. (2003) found comparable differences, when comparing human and rhesus endometria for the presence of Phospho H3 using an antibody that identifies histone H3 on chromosomes during mitosis and that therefore represents a marker for proliferation. The indices calculated for the glands in the zona basalis of the monkey in the (artificial) luteal phase exceeded those in normal cyclic women. If the finding that estrogen receptors persist during the luteal phase in the zona basalis of the human endometrium (Koshiyama et al. 1995; Press et al. 1984; Vollmer et al. 1990) is valid, their presence does not seem to lead to significant proliferative activity during the normal cycle. From the above mentioned results it can be hypothesized that during progesterone dominance, the zona basalis of the monkey endometrium is more susceptible to the induction of proliferation than that of normal cyclic women. A second difference between the cynomolgus and human endometrium is the onset of subnuclear vacuolation in the glands of the functionalis. In the human endometrium, the appearance of subnuclear, PAS-positive cytoplasmic vacuoles is the first sign of the influence of progesterone on the endometrium. This event, therefore, is accepted as the morphological hallmark for the start of the luteal phase. However, it is our experience that in the cynomolgus monkey, subnuclear vacuoles in the epithelial cells lining the glands of the functionalis appear already during the late follicular phase (in rare cases, even in the mid-follicular phase), which has been confirmed by other authors (Bartelmez et al. 1951; Brenner et al. 1983; Cline 2001; Cline et al. 2001). This finding suggests that macaque glandular epithelial cells are possibly very susceptible to the low levels of circulating progesterone (the number of progesterone receptors in these animals is high during the late follicular phase). After ovulation, subnuclear vacuolation may become more pronounced than before ovulation. The extent is not as striking as is observed in the human endometrium. A third difference between the macaque and human endometria is the degree of periarteriolar stromal cell differentiation during the last week of the menstrual cycle (Brenner and Slayden 1994). As mentioned previously, in the human, the stromal predecidual changes start around the spiral arteries and extend to the upper two thirds of the functionalis. In contrast, the predecidual change in the macaque endometrium is not prominent. The stromal cells in the periarterial areas become somewhat hypertrophied but never reach the volume that is observed in the normal human endometrium under normal cyclic conditions. This does not mean that a true decidual change cannot appear in the macaque endometrium. Pregnancy or high doses of progesterone or synthetic progestagens can cause a dramatic hypertrophy of the stromal cells, starting around the spiral arteries and extending to the whole functionalis and even upper basalis. During the luteal phase, the stromal cells in the macaque seem therefore less susceptible to the predecidualizatory action of progesterone than in humans. This difference could possibly also be explained by the differences in the progesterone levels (AUC and peak levels) during the menstrual cycle in cynomolgus monkeys and women (Stabenfeldt and Hendrickx 1973). The progesterone levels in the rhesus are even lower compared to those in cynomolgus. The last difference is related to the presence of estrogen receptors in the stromal cells during the luteal phase. In the human endometrium, the stromal cells express the estrogen receptor during the whole luteal phase (Coppens et al. 1993; Garcia et al. 1988; Press et al. 1984; Snijders et al. 1992), whereas this is not the case in the natural cyclic cynomolgus monkey (our observation) and in macaques with an artificially induced luteal phase. In these cases, expression of the estrogen receptor was down-regulated in response to the rising progesterone levels (McClellan et al. 1986; Okulicz and Balsamo, 1993; Okulicz et al. 1993).
Ultrasonography Ultrasound imaging techniques can be used as a noninvasive method to study the cyclic changes in endometrial thickness in macaques (Foster et al. 1992; Morgan et al. 1987; Shiozawa et al. 1996). An example of a transverse ultrasound image taken from a mature cynomolgus monkey is shown in Figure 42.
The changes in endometrial thickness measured using ultrasonography correlated only to the day of the cycle during the menstrual and early follicular phase (Stabenfeldt and Hendrickx 1973). Also, the increase in endometrial thickness during the follicular phase correlated well with the increase in plasma estradiol. An increase in reflectivity, most likely related to the enlargement and increased tortuosity of the glands and the increase in mucin and glycogen products characterized the luteal phase. Additionally, pelvic ultrasonography can be used to detect signs of endometriosis in macaques (Fanton and Hubbard 1983; Rippy et al. 1996). However, the lesions have to be large enough and preferably cystic to be easily detected.
Biopsies
Withdrawal Bleeding
Functional Genomics The most widely used genomic techniques to study the endometrium are the mRNA differential display (DD)-based method and the more recently developed cDNA microarrays (Sherwin et al. 2006). mRNA differential display technology works by systematic amplification of the 3' terminal portions of mRNA and resolution of those fragments on a DNA sequencing gel. Okulicz et al. (2003) succeeded in identifying several (partial novel) progesterone-regulated genes in the rhesus monkey using this technique. Tynan et al. (2005) demonstrated by microarray experiments that the so-called "deleted in malignant brain tumors 1'' gene (DMBT1) is an estrogen-responsive gene in the (experimentally ovariectomized) cynomolgus monkey endometrium, and that it possibly plays a role in supporting epithelial cell proliferation. Also using microarrays, Ace and Okulicz (2004) identified a number of genes that were significantly regulated during the transition from the follicular to the luteal phase. Interestingly, more genes were down-regulated than up-regulated during this shift. Up-regulated genes included "secretory leukocyte protease inhibitor," an elastase inhibitor that possibly has a role in implantation and secretoglobulin/uteroglobulin. This progesterone-binding protein is present in the apical parts of the glandular epithelial cells during the early and mid-luteal phase. As an example of down-regulated genes, the gene encoding matrix metalloproteinase 11 is worth mentioning. Differences in gene expression are found not only during the successive phases of the cycle, but also within the different functional zones of the endometrium during one phase. Laser microdissection techniques, applied to harvest different endometrial structures from the zona functionalis and the zona basalis in rhesus monkeys, are particularly helpful in this respect. Using the differential display reverse transciptase polymerase chain reaction, cell-type or region-specific gene expression could be detected in samples obtained using this technique (Torres et al. 2002). It has long been known that estradiol induces its own receptor (Lessey et al. 1989; Okulicz 2006). Estradiol therefore plays a very important role in the complex molecular network involved in the regulation of the genes involved in the construction of its own receptor(s) (McDonnell and Norris 2002). The original model, whereby estrogen receptor agonists such as endogenous estradiol bind to the receptor and cause conformational changes to the receptor that subsequently lead to gene transcription regulation, nowadays is thought to be an oversimplification of the reality. More and more information is emerging on coregulators (coactivator and corepressor proteins) that can interact with the estrogen receptor, resulting in modulation of its function(s). The existence of a complex network of different coregulator proteins emphasizes the complexity of hormonal signaling regulation in target tissues. It also can explain the tissue-selective activities of estrogen receptors (Hall and McDonnell 2005).
As described herein, the endometrium of the cynomolgus monkey represents the general pattern of the development, structure, and function of the endometrium of Old World nonhuman primates and human beings. Species differences are generally minor, but they may have relevance to the interpretation of study outcomes. Correct interpretation of endometrial histology requires knowledge of the species under consideration; the possible confounding factors of immaturity, hypothalamic hypogonadism, or senescence; effects of seasonality in some species (e.g., the rhesus monkey); and familiarity with the spectrum of background changes occurring spontaneously, as described in this paper and elsewhere in this volume. In summary and as a succinct reference, a diagrammatic representation of major histologic changes in the endometrium of cynomolgus macaques is provided in Figure 43. This diagram summarizes selected major changes that in our experience are the most useful in evaluating the endometrium. Additional changes as described in the preceding text and by other authors will also hopefully be of use to the reader. Furthermore, the endometrium should be evaluated in concert with other findings whenever possible, including circulating sex steroid concentrations, menstrual bleeding pattern, and the histologic appearance of the ovaries and other segments of the reproductive tract.
We gratefully acknowledge Wilma van Ravesloot for all the excellent immunohistochemical techniques performed.
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Toxicologic Pathology, Vol. 36, No. 7 Suppl,
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Abstract
Introduction
