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The Macaque Ovary, with Special Reference to the Cynomolgus Macaque (Macaca fascicularis)
1 Covance Laboratories GmbH, Pathology, Müster, Germany Correspondence: Martina Zöller, Covance Laboratories GmbH—Research and Safety Assessment, Kesselfeld 29 Münster 48163 Germany; e-mail:martina.zoeller{at}covance.com.
Concerning functional and morphological aspects, the ovary of the cynomolgus macaque is representative for the conditions in higher primates like humans and is therefore of major relevance in toxicological research. Against this background, a comprehensive overview about the cynomolgus macaque ovary is given from its embryonic appearance, throughout the adolescent and adult development until old age. The overview includes morphologic characteristics, a description of the different cell types, comparisons between the expression of selected receptors, and some details on hormonal effects if considered necessary for understanding the unit of ovarian morphology and function. The close correlation of hormones and morphological characteristics of the ovary and of the other reproductive organs is emphasized by several schematic drawings and images. Special emphasis is also laid on the comparison to the human organism indicating the similarity of both species and hence underlining the importance of the cynomolgus macaque as a model in toxicological research. Competing Interests: This article was sponsored by Covance Inc. and Schering-Plough. Martina Zöller and Eberhard Buse are employed by Covance Inc. Eric Van Esch is employed by Schering-Plough. No other competing interests were declared.
Key Words: nonhuman primate • ovary • cynomolgus macaque • female reproductive system
The morphology of the cynomolgus macaque (Macaca fascicularis) ovary is presented with respect to its major biological functions—allocation, maturation, and delivery of oocytes—and to its hormone production supporting the intrinsic ovarian processes and the development and maturation of secondary reproductive organs. As a key figure in these functions, the ovary plays a central role in the endocrine regulatory loop, interacting with the CNS, the pituitary gland, and with further peripheral organs (e.g., uterus, vagina, heart) and metabolic pathways (e.g., lipid metabolism). Its temporary morphological status is considered as a mirror of the humoral and intrinsic sex hormone interactions. The cynomolgus macaque has increasingly gained in importance in toxicological research in view of its close phylogenetic relationship to man, which is likewise true for the female reproductive system. In the literature, there are extensive scientific treatises about the female reproductive organs from various primate species including the cynomolgus macaque, the rhesus macaque (Macaca mulatta), and the pig-tailed macaque (Macaca nemestrina). However, the most relevant information has been collected from rodents (Greenwald and Shyamal 1994; McGee and Hsueh 2000). Our current knowledge about the primate female reproductive system has certainly been merged from different species that exhibit close similarities. However, fundamental multispecies research also complicates a clear discrimination of the female reproductive characteristics between the different nonhuman primate species as well as between nonhuman primates and humans because of the considerable similarity in the reproductive organs in all these species (Dvorak and Tesarik 1990; Gougeon 1996; Hope 1965; Koering 1986; Peters and McNatty 1980; vanWagenen and Simpson 1973). The close similarity of the reproductive system in cynomolgus macaques and in women refers to the morphology, the endocrine system, hormone receptors, the control of unilateral single-egg ovulation, and the delivery of single offspring with the occasional exception of twin births. The reproductive cycle duration in cynomolgus macaques is twenty-eight to thirty days, which corresponds to the human cycle length. However, marked differences of potentially high toxicological relevance may be more or less apparent among various nonhuman primate species (e.g., the number of ovulations and siblings or certain physiological, histological, and molecular differences) and may raise the question of the applicability of nonhuman primate animal models in extrapolating to human reproductive medicine. A common feature of reproduction in the cynomolgus macaque, in the rhesus macaque, and in humans is the occurrence of a single ovulation per cycle differing from two to four ovulations and siblings in marmosets and even eight to twelve in rodents. In view of human-targeted toxicological research, the cynomolgus macaque so far seems to be the most representative animal model. It is the objective of this article to spotlight and clarify some characteristics of the cynomolgus macaque ovary with respect to the role of this species as a suitable animal model for man in toxicological research. Special emphasis is laid on estrogen (ER), progesterone (PR), and androgen receptors (AR) as special indicators of the intrinsic unit of morphology and function (Brenner, West, and McClellan 1990; Förster and Kietz 2006).
The data included in this article have been collected over more than thirty years and derive from hundreds of control and placebo cynomolgus macaques from toxicological pre-clinical safety studies. Their ages cover the whole scale from embryonic, fetal, juvenile, adult, to postmenopausal stages. Histological evaluations were performed on standard paraffin-embedded and hematoxylin and eosin (H&E) stained tissue sections. Further staining methods included Feulgen stain, Goldner stain, Azan stain, and Alizarin Red stain. Immunohistochemistry was performed with commercially available primary antibodies including anti-neuron specific enolase (NSE)-antibody (monoclonal mouse anti-human neuron specific enolase, DakoCytomation, Hamburg, Germany), anti-synaptophysin-antibody (polyclonal rabbit anti-human synaptophysin, DakoCytomation, Hamburg, Germany), anti-CD3-antibody (monoclonal mouse anti-human CD3, DakoCytomation, Hamburg, Germany), anti-CD20-antibody (monoclonal mouse anti-human CD20, DakoCytomation, Hamburg, Germany), anti-CD68-antibody (monoclonal mouse anti-human CD68, DakoCytomation, Hamburg, Germany), anti-Ki67-antibody (monoclonal mouse anti-human Ki67 antigen, DakoCytomation, Hamburg, Germany), anti-smooth muscle actin (SMA)-antibody (monoclonal mouse anti-human actin (smooth muscle), DakoCytomation, Hamburg, Germany), anti-progesterone receptor-antibody (polyclonal rabbit anti-human progesterone receptor, DakoCytomation, Hamburg, Germany), anti-estrogen receptor-antibody (monoclonal mouse anti-human estrogen receptor, DakoCytomation, Hamburg, Germany), anti-androgen receptor-antibody (monoclonal mouse anti-human androgen receptor, DakoCytomation, Hamburg, Germany), and anti-caspase-antibody (monoclonal rabbit anti-human caspase 3, Cell Signaling Technology, Massachusetts, USA). Antibodies were used according to the recommendations of the supplier with involvement of appropriate positive and negative controls. Hormone measures and evaluations were performed on serum samples of cynomolgus monkeys at Covance Laboratories GmbH, Münster, Germany. The following validated methods/test kits were used:
The cynomolgus macaque ovary is of typical primate morphology, differing from human anatomy only in size and in the number of follicles (Greenwald and Shyamal 1994) (Figure 1). The mature ovary measures approximately 1 x 0.8 x 0.8 cm in diameter (ovary without fresh luteal body) and weighs 0.42 mg on average (n = 99, SD = 0.14). There is no significant difference in weight between the right and the left ovary (right: 0.21 mg; left: 0.21 mg; n = 99).
The ovary derives from the urogenital ridge and is therefore located in a retroperitoneal position. After protruding into the abdominal cavity it remains retro-peritoneally, where it is attached by a complex system of ligaments to the abdominal wall (suspensory ligament of ovary), the uterus (broad ligament and utero-ovarian ligament), and the uterine tube (mesosalpinx). The broad ligament with the round ligament and the mesovarium are also part of the attachment apparatus. The suspensory ligament of the ovary contains the supplying vessels and nerves, such as the ovarian artery, the pampiniform venous plexus (ovarian vein), and the nervous ovarian plexus (containing nerves from the abdominal aortic plexus, the renal plexus, and the superior mesenteric plexus). Additional blood supply derives from the uterine artery (ovarian branch) and reaches the ovary via the utero-ovarian ligament. During pregnancy, these vessels dilate significantly and thus increase the ovarian blood and hormone supply. Lymphatic drainage of the ovary is into the superior lumbar lymph nodes. The mesosalpinx, located between the ovary and the uterine tube, contains the rudiments of the embryonal Wolffian duct, which is called Gärtner’s duct in the adult individual and represents the mesonephric duct. The pathological relevance of this structure becomes obvious in aged macaques because of its disposition to undergo mesothelial proliferation known as epoophoron proliferation in a more cranial and paroophoron proliferation in a more caudal site. The covering ovarian epithelium is termed "germinal epithelium." It represents the continuation of the pelvic mesothelium, which covers the whole genital tract. The anatomical similarities between nonhuman primates, humans, and other mammalian species allow the use of the same terminology.
The Ovary The basic structure of the primate and generally the mammalian ovary is the presence of a central core, the so-called "medulla," and a peripheral cortex containing the oocytes. While the number of oocytes in the cortex is very high in prenatal life, it continuously decreases with age. Part of the medulla is the hilum, where the supplying vessels and the nerves enter the ovary (Figure 2).
The stroma of the medulla predominantly contains connective tissue with collageneous fibres and myofibrocytes (Figure 3). It also contains a dense net of vessels and small sympathetic and parasympathetic nerve endings (Espey and Lipner 1994), which can be identified with anti-neuron specific enolase (NSE)-antibody and with anti-synaptophysin-antibody. The nerve endings are mainly located in the stromal tissue surrounding the follicles. Small accumulations of endocrine cells referred to as hilum interstitial cells or "ovarian hilar cells" are rarely observed during routine histological examinations. Such cells, however, have been demonstrated by immunohistochemical methods. They are regarded to represent testosterone producing equivalents to the male Leydig cells (Adashi 1990; Lüllmann-Rauch 2003). Individual T-cells (positive for anti-CD3-antibody) are scattered throughout the ovarian stroma and slightly increase in number in older animals. B-lymphocytes (positive for anti-CD20-antibody) are absent in the ovary of premature females, but can occasionally be detected in mature ovaries. Macrophages identified with anti-CD68-antibody are present in differing numbers depending on cycle status and ovarian activity.
The ovarian cortex is peripherally covered by a monolayered peritoneal epithelium (synonyms: germinal epithelium, mesothelium, serosa, peritoneum) and a thin layer of dense connective tissue, the tunica albuginea, which is free of oocytes and follicles (Figure 3). The cortical stroma contains densely packed basophilic spinocellular matrix cells with diffusely distributed oocytes in its periphery. They display the earliest follicle phenotypes. The oocytes are covered by a single layer of flat granulosa epithelial cells (granulosa cells). These early follicle stages are termed primordial follicles and are separated from the ovarian matrix by a basal lamina. With maturation the granulosa epithelial cells become cuboidal, and the corresponding follicles are then known as primary follicles. Subsequently, the cuboidal granulosa cells proliferate leading to the formation of a multilayered secondary follicle (preantral follicle). The final stage of follicle development is the tertiary follicle (antral follicle), which is characterized by the formation of an antrum among the granulosa cell layers. During maturation the follicles shift into a more centralized ovarian position. In prenatal life, oocytes significantly increase in number due to distinct mitotic activity (first meiotic division). However, this enormous proliferative activity is later followed by a similar degree of atresia. At birth, the proliferative activity has usually decreased significantly, a phenomenon described in all mammalian species. Whereas the number of oocytes before birth is calculated to be approximately 5 to 7 millions in macaques, only 1 to 2 million oocytes are present at birth and 200,000 to 450,000 are left at the time of menarche (Figure 4) (Baker 1986; Baker and Franchi 1972; Gougeon and Chainy 1987; Greenwald and Shyamal 1994). Most reports about oocyte numbers in nonhuman primates refer to rhesus macaques rather than to cynomolgus macaques. However, follicle numbers in both species are considered to be similar because of the comparable ovarian size and further similarities in reproductive physiology, and hence are summarized in Figure 4.
In the ovaries of the female cynomolgus macaque, the number of follicles appears to increase before birth, and atretic follicle stages are rare. However, there is a significant increase of atresia in the immediate postnatal period (e.g., from day 4 and day 12 onwards). The exhaustion of cynomolgus macaque follicles is reached after at least fifteen to twenty years and irrevocably represents the end of fecundity. Currently, there are only a few references available on menopausal ovaries with less than one thousand follicles left (Baker 1986; Gougeon 1996). Respective calculations on oocyte numbers in humans reveal approximately 6 to 7 million oocytes in gestational week 20 (Adashi 1990), 1 to 2 million oocytes at birth, and 300,000 to 500,000 oocytes at the time of menarche (Bresch and Hausmann 1972; Lüllmann-Rauch 2003; Nieschlag et al. 2005). The sizes of oocytes and follicles (measured in formalin-fixed, paraffin-embedded histological sections) increase during their development from the primary to the mature antral stage (Table 1). Mitotic oocytes in the perinatal cynomolgus macaque measure less than 15 µm in diameter, whereas primordial follicles measure approximately 30–40 µm in diameter. Oocytes of the secondary follicles show a rapid increase in diameter from 75 µm in early secondary follicles up to approximately 100 µm in advanced stages. Oocytes in tertiary follicles are up to 120 µm in diameter. The corresponding follicle diameters are listed in Table 1. The tertiary (antral) follicles increase their size by 1.0–2.0 mm, and the preovulatory (Graafian) follicle finally measures more than 2.5 mm. Diameters and numbers of cynomolgus tertiary follicles are slightly smaller compared to those of humans, whereas follicles in marmosets and rats display significantly smaller measures (Koering, Danforth, and Hodgen 1994).
The Primordial and Primary Follicle The primordial follicle represents a dormant oocyte stage (dictyotene prophase stage of meiosis) (Bresch and Hausmann 1972; Koering 1986; Wartenberg 1990) prepared to persist over years but ready for being stimulated in the process of "initial recruitment" to enter the developmental course towards the primary, secondary, and tertiary follicles of the menstrual cycle. In the view of follicle transience, the primary follicles are the initial stages of development. Primordial and primary follicles have been classified into three categories (Adashi 1990; Gougeon and Busso 2000) (Figure 5, Table 1):
The primordial oocytes contain a finely granulated karyoplasm and a prominent nucleolus. Immunostaining with anti-Ki67-antibody for evaluation of cellular proliferative activity does not reveal evidence of proliferation in the oocytes or in the covering granulosa cells. According to Adashi (1990), primordial oocytes start to develop as soon as they have exceeded a size of approximately 20 µm in diameter. In the primordial follicle of category 3 (advanced primary follicle), the oocyte expands from more than 20 µm up to 75–100 µm in diameter (human oocytes measures: 30–120 µm) (Lüllmann-Rauch 2003; Nieschlag et al. 2005). It is then superficially covered by a distinct prismatic cell layer (Figure 5). The granulosa epithelial cells show nuclear expression of progesterone receptors in a percentage of approximately 20% arguing for certain follicle sensitivity for progesterone. Expression of estrogen and androgen receptors, however, can not be demonstrated at this developmental stage. The different categories of the primordial and primary follicles can be observed in cynomolgus macaques of all ages (juvenile and mature). They are exclusively located in the peripheral cortex. In newborn female cynomolgus macaques, only few advanced primary follicles are present, whereas they can no longer be observed in females older than fifteen years. Interestingly, it is assumed that fifteen-year-old cynomolgus macaques correspond to thirty-five-year-old women (Gougeon 1986). However, such correlation should not necessarily apply for the ovarian age.
The Secondary (Pre-Antral) Follicle Early secondary follicles have been detected in the ovary on postnatal day 74. However, it is assumed that they are already present between day 30 and day 50 after birth. The sizes of secondary follicles increase from between 40 to 100 µm (oocyte) to between 100 to 250 µm (follicle) (Table 1). These measures correlate well with the given sizes in women (Dvorak and Tesarik 1990; Gougeon 1996; Lüllmann-Rauch 2003). Proliferating granulosa cells positive for anti-Ki67-antibody are rarely found in small secondary follicles, but their number increases significantly with follicle size (Figure 6). The advanced secondary follicle is surrounded by a connective tissue layer, the internal theca. It originates from matrix cells and is separated from the granulosa cells by a distinct basal membrane.
Progesterone receptors are strongly expressed by almost all epithelial granulosa cells as well as internal and external theca cells (Brenner, West, and McClellan 1990). They are equally present in young and mature females. Estrogen receptors are expressed by relatively few granulosa cells. They slowly increase in number with the size of secondary follicles, and in advanced secondary follicles about 10% of the granulosa cells exhibit estrogen receptor immunoreaction. Moreover, estrogen receptors are expressed by some internal theca cells in mature preantral follicles. Androgen receptors are not expressed by secondary follicles.
The Tertiary (Antral) Follicle
Progesterone receptors are expressed by about 50% of the granulosa cells in the tertiary follicle and persist throughout the subsequent luteal phase. They are furthermore expressed in internal and external theca cells at a relatively high density. Estrogen receptor expression increases with follicle growth, and in advanced tertiary follicles almost 100% of granulosa cells are positive. However, estrogen receptor expression rapidly declines, when the follicle granulosa cells transform into luteal epithelial cells. Estrogen receptor expression appears to correlate with the expression of Ki67 antigen. Androgen receptor expression now becomes evident, and receptors are expressed by more than 80% of the granulose cells of tertiary follicles and by almost 100% of the external theca cells. Androgen receptors are absent in the internal theca (Vendola et al. 1998). The internal theca is densely vascularized in advanced tertiary follicles and involved in supplying nutrition, oxygen, and growth moderating molecules, such as hormones and growth factors, via the blood to the granulosa cells, the ovarian cumulus, and the follicular liquor. Lipid droplets in the internal theca cells are conspicuous signs of active cholesterol metabolism in the synthesis of sex steroids. The external theca confines the follicle periphery and separates the follicle from the ovarian stroma. Besides the expression of progesterone and androgen receptors, it is characterized by distinct immunoreactivity with anti-smooth muscle actin (SMA)-antibody confirming its myofibrocyte differentiation. The majority of the tertiary follicles finally undergo degeneration in the form of atresia. However, although atresia mainly affects tertiary follicles, it can also be seen in secondary follicles. Initially few and later on an increasing number of granulosa cells become apoptotic and detach from the granulosa cell layer into the antrum. Finally, the follicle collapses and the epithelial remnants are almost completely eliminated by phagocytes (Figure 8). Rudiments of atretic follicles are the hypertrophic internal theca cells (synonyms: interstitial glands, theca organ, secondary interstitial cells). They are thought to be the source of androgens, that is, a substrate for aromatase-mediated estrogen production. A hyaline remnant of the follicle may persist for several months. The exact mechanism of apoptosis-mediated atresia of the follicle is not currently understood.
Among all follicle stages in both juvenile and adult cynomolgus macaques, there is a low incidence of follicles with two oocytes in one follicle each having its own pellucid zone (bioocyte, double-nucleated oocyte) (Papadaki 1978). In less frequency, two nuclei in one oocyte are observed both enclosed within a common pellucid zone (binuclear oocyte) (Figure 9).
Follicles with two nuclei are described in several mammalian species including humans and nonhuman primates (Kennedy and Donahue 1969; Koering 1986; Papadaki 1978; Ron-El et al. 1990). After in-vitro fertilization in hormone treated humans, bioocyte follicles and binuclear oocytes occur with an incidence of 0.25% to 0.3% in differently advanced follicles. These findings are interpreted to be twin anlagen. In humans, twins occur with a natural incidence of 0.6% to 4% (http://www.health.sa.gov.au/ppg/Default.aspx?PageContentID=780&tabid=75). In the cynomolgus macaques, the exact incidence of the presence of twin anlagen in the ovary has not been calculated. The frequency of twin births was five twin siblings in thirty-six hundred cynomolgus macaques (0.12%).
The Luteal Body If there is no oocyte fertilization, the luteal body involutes and becomes atrophic and disappears within a couple of weeks (Figure 10). By the time of the following ovulation, the previous luteal body may be reduced by half of its original size. Three to four luteal rest bodies in different stages of atrophy can be identified in regularly cycling ovaries. Therefore, a histological backtrack for up to eight cycles might be possible (four ovulations in each ovary). In its final stage, the luteal body condenses and persists as a scarred hyaline rest body, the corpus albicans.
Estrogen receptors and androgen receptors disappear from the granulosa cells shortly after ovulation. Progesterone receptors persist for a few weeks until they also slowly vanish. In case of oocyte fertilization and subsequent pregnancy, the luteal body retains its progesterone synthesizing function and persists for weeks/months as the corpus luteum graviditatis, until progesterone synthesis is finally taken over by the placenta. Luteolysis in the atrophic luteal body is characterized by degenerative vacuolization and increasing lipidosis accompanied by vascularization, invasion of macrophages, and finally increasing expression of the macrophage marker CD68 on the luteal cells (Figure 11). In older luteal bodies, CD68 is finally expressed by 100% of the luteal granulosa cells (Morales et al. 2000). Luteolysis is furthermore associated with increasing accumulation of CD3 positive T-lymphocytes.
Certain histological differences are obvious between the luteal bodies of cynomolgus macaques and of humans. In cynomolgus macaques, the luteal cells have a uniformly large appearance, whereas in humans, the majority of large luteal cells is surrounded by a rim of small internal theca derived luteal cells. Both phenotypes, however, share the same functional features (Figure 11) (Junqueira, Carneiro, and Kelley 2002; Lüllmann-Rauch 2003; Magoffin 2005).
The Prenatal Ovary This section briefly describes the morphological hallmarks of the prenatal and juvenile ovary with regard to the increasing use of fetal and juvenile cynomolgus macaques in toxicology during recent years. In the 35-day-old cynomolgus macaque embryo, the urogenital ridge represents the preliminary stage of the gonads and kidneys (Figure 12). On day 40, both primordial parts are separated from each other. The gonads show a faint peripheral condensation of the parenchyma with some large cells in between. These cells contain round and pale nuclei with prominent nucleoli. They are considered to be stem cells that have immigrated from the yolk sac. The immigration phase in the cynomolgus macaque is considered to be complete on day 40 of the embryonic development. In women, the colonization of the ridge with stem cells is finished on day 33 after conception (Baker 1986; Wartenberg 1990).
On gestational day 45 and day 50, the gonad gender is histologically recognized on the basis of a thin peripheral tunica albuginea in the ovary of the developing female and initial tubular organization in the testis of the developing male fetus. This stage morphologically corresponds to human embryos between day 46 and day 50 (vanWagenen and Simpson 1973; Wartenberg 1990). On day 70, the ovarian cortical parenchyma appears densely settled with oocytes. Oocytes occupy most of the ovarian tissue, and the peripheral tunica albuginea can hardly be recognized. Until gestational day 100 (a stage of toxicological relevance in view of developmental analyses that are usually performed on that day), the ovary has gained a weight of 10.85 mg (n = 189, SD = 4) and a size of approximately 0.5 x 0.3 x 0.3 mm. The cortex consists of polymorphic matrix cells, intermingled with spinocellular cells and contains peripherally located small mitotic oocytes (Figure 13). Central parts of the cortex are populated with oocytes containing large nuclei. At least parts of them are covered by early granulosa epithelial cells and are considered to be at the end of the first meiotic division (dictyotene prophase stage of dormant oocytes). Characteristically, they are surrounded by a distinct nuclear membrane. Their large nuclei have prominent nucleoli and contain condensed chromatin.
Early granulosa epithelial cells and interstitial cells originate from a common stem cell that undergoes further differentiation after migrating into the ovary. The process of stem cell invasion might be noteworthy as a critical process in folliculogenesis and as an objective for toxicological viewpoints. The organ growth on gestational day 100 correlates with a high number of mitotic figures. Receptors for estrogen and progesterone are not detectable by immunohistochemical analysis on gestational day 100. Lack of estrogen receptors is considered to correlate with the low fetal estrogen hormone serum level, which measures 158 pmol/L (n = 7) (Table 2) and is consistent with the rather insignificant role of placenta derived fetal estradiol-sulphate in follicle development. Serum progesterone levels, however, measure about 96 nmol/L (n = 12), a value similar to that of the mother animals. Progesterone is well known to be able to pass the placental barrier (human: 90% into maternal and 10% into fetal circulation) and to be predominantly utilized for the synthesis of adrenal mineral and glucocorticosteroids (Nieschlag et al. 2005). Progesterone synthesis by the fetus is of no relevance, and the hormone effects on the fetal macaque ovaries are considered to be minor in view of the low or absent nuclear receptor expression.
On day 150 (average duration of gestation in cynomolgus macaques is 160 days), the ovary has an average weight of 34 mg (n = 60, SD = 22). The cortex is extensively occupied by primordial follicles, whereas the medulla is small and might even be absent in histological sections (Figure 14).
The histomorphology indicates an inwards-directed pattern of folliculogenesis with a large peripheral population of mitotic oocytes and a centrally located population of post mitotic (dormant) primordial and primary follicles with a minority of atretic follicles (Baker and Franchi 1972; Gougeon 1996). Proliferating cells are prevalent and densely packed in the peripheral zone of the cortex, parts of them surrounded by a connective tissue sheath. Secondary and tertiary follicles are not present.
Estrogen receptors are not detectable, arguing for the insignificance of estrogen function on the ovary at this developmental stage. Immunoreactivity for progesterone receptors is displayed by approximately 20% of oocyte nuclei in variable intensity. Granulosa epithelial cells are negative for anti-progesterone receptor-antibody, but about 95% of the spinocellular matrix cell nuclei show distinct immunoreaction (Figure 14). The average serum progesterone level in twelve fetuses was 68.66 pg/mL (Table 2), which represents an expected value in view of the maternal placenta permeability for progesterone. Results from further immunohistochemical examinations with antibodies against salmon calcitonin (SCF), transforming growth growth factor
The Premature Ovary Substantial information about control mechanisms of initial recruitment has been collected from various mammalian species including rodents, cattle, cynomolgus macaques, and baboons, but the processes have not been completely clarified (Braw-Tal 2002; Gougeon and Busso 2000; Hirshfield 1991; Oktay, Schenken, and Nelson 1995; Parrott and Skinner 1999). It is assumed that there is only insignificant influence of follicle stimulating hormone (FSH), luteinizing hormone (LH), estrogen, and progesterone on initial recruitment, a hypothesis that is supported by the lack of estrogen receptors on primary follicles. Numerous experiments have shown that there is undisturbed regular follicle initiation and early development during complete absence of FSH, for example, after experimental inhibition of the GnRH-FSH-axis, in hypophysectomized rhesus macaques (Beier 1990), or in anencephalic humans (Adashi 1990; Dierich et al. 1998; Eppig and O’Brien 1996; Gougeon and Busso 2000; McGee and Hsueh 2000; Oktay, Briggs, and Gosden 1997; Oktay et al. 1998; Wandji et al. 1997). Evidence for early initial recruitment is easily obtained from studies of follicle development. In cynomolgus macaques, McGee and Hsueh (2000) observed signs of follicular development on gestational day 150. Our observations similarly indicate the presence of the first advanced primary oocytes between gestational day 100 and day 150, but most certainly close to day 150 (Table 3). Secondary follicles do not develop until day 12 after birth, but on day 74 both secondary and tertiary follicles are already present. It is assumed that early secondary follicles are present close to day 40 after birth. There is a similar duration of follicle development in both the macaque and in man (Adashi 1990; Wartenberg 1990). Due to different gestation times, secondary and tertiary follicles in humans develop during the intrauterine phase, whereas in the cynomolgus macaque, follicles appear after birth. Hence, duration of pregnancy needs to be kept in mind when comparing follicle development in both species.
Attempts to quantify activated primordial follicles in the process of initial recruitment and during the decrease of oocytes throughout life time have been assessed and were reviewed by Miller et al. (1999) (Figure 4). The authors implicate a steady logarithmic decrease of 10% of follicles from puberty until menopause in both humans and rhesus macaques. Conclusively, approximately 90% (80%–95%) of oocytes are in the stage of "follicle dormancy." However, even in dormant stages, RNA synthesis and nuclear growth have been postulated to continue. Oocytes with nuclei measuring 19 µm in diameter are considered to pass a critical size for entering the phase of further follicle development (Gougeon 1996; Gougeon and Chainy 1987). Hence, there is some evidence for slow and unidirectional progress even within the "dormant" primordial follicle. The selection processes controlling the continuing development of primordial follicles are not fully understood. They are certainly mediated by a variety of selecting and driving factors and have to be considered as key mechanisms in toxicological research. Three main hypotheses on initial recruitment have been postulated so far (Gougeon and Busso 2000): Hypothesis 1: Removal of an intrinsic inhibition factor and unblocking for continuous follicular recruitment. As a first step, the granulosa cells transform from the flat into a cuboidal phenotype (Braw-Tal 2002). This process takes place under the control of the transforming growth factor β2 (TGF β2), which contributes multiple effects on the follicle cell differentiation including inhibition of granulosa cell growth and promotion of DNA synthesis (Dorrington, Chuma, and Bendell 1988; Mulheron et al. 1992; Skinner et al. 1987). A second step of follicle growth might then be mediated by growth differentiation factor 9 (GDF 9/BMP 15, produced by the oocyte) and by a kit ligand (produced by granulosa cells). Hypothesis 3: Direct influence of the granulosa cells on an oocyte borne RNA factor. The granulosa cells are considered to have self-stimulating capabilities and to mediate follicle development. They are suspected to synthesize a protein that inhibits the development of the adjacent follicles and follicle cohorts (Balboni et al. 1987; Chegini and Williams 1992; Maruo et al. 1993; Shull and Doetschman 1994).
Course of Adolescent Development
The premature FSH/estrogen serum status appears to be of key relevance for the professional judgment of premature ovarian morphology. The FSH serum level in six-to twenty-four-month-old juvenile female macaques is less than 1 ng/ml, and thus considerably lower than in mature females (Table 5). Under the influence of low FSH levels the follicular estrogen synthesis is minimal, thus not supporting follicle maturation. Lack of estrogen receptors furthermore causes inhibition of follicular development. Low FSH levels are also reported to promote follicular atresia in newborn hypophysectomized rhesus macaques (Gulyas et al. 1977). A relatively high number of atretic follicles, however, is present in juvenile ovaries, that is, in nearly complete absence of FSH. The histological outcome of low FSH is schematically described in Figure 16.
On day 4 after birth, the ovarian cortex shows a polymorphic picture concerning cellular morphology (Figure 17). It contains a considerable number of oocytes with mitotic figures. Furthermore, there are high numbers of primordial and primary follicles, as well as few atretic follicles, all embedded in a spinocellular matrix. Secondary follicles are missing.
On day 12 after birth, oocytes of different developmental stages are present, part of them still displaying mitotic figures and being grouped in clusters (Figure 17). They clearly indicate that the post mitotic oocyte population is not yet complete at this age. The majority of oocytes, however, has apparently reached a dormant meiosis dictyotene prophase stage. A subset of them is already covered by cuboidal granulosa cells, and is probably ready for the selection in cyclic recruitment. A pellucid zone has not formed yet. Atresia appears to be increased and, compared to the other stages seen so far, reaches its highest values. Secondary follicles can now be observed. Secondary and antral follicles are present in the ovaries of three-month-old juvenile female cynomolgus macaques (e.g., day 70, week 10) (Baker 1986; vanWagenen and Simpson 1973). The antral follicles are, at this age, smaller than those in mature animals (Figures 15, 18). Ovarian cumuli have not yet developed, and "dominant follicles" cannot be identified. Remarkably, the antral follicles develop in a milieu of low or even absent FSH and under the obvious absence of estrogen receptors in the ovarian tissue with the exception of the germinal epithelium.
In the six-month-old ovary (one-half year, day 180 after birth), antral follicles remain relatively small do not have an ovarian cumulus, but show a considerable amount of atresia (Figure 18). Both internal and external theca are well developed. An increase in oocyte size up to 120 µm is visible in secondary follicles. Previous atretic follicles are recognizable by numerous eosinophilic hyaline rest bodies, which represent the remnants of swollen oocyte pellucid zones. Progesterone receptors are widely distributed, whereas estrogen receptors are exclusively expressed by the germinal epithelium. In the nine-month-old female, there is strong developmental progress towards maturity with intense antral follicle development. However, low hormone levels indicate slow maturation pace, which becomes obvious by the undeveloped oocyte cumuli. The ovarian morphology is characterized by high developmental activity correlating with numerous tertiary follicles (without cumulus), pellucid rest bodies, epithelial proliferations, and lymphoid cell accumulations (Figure 19).
In the ovaries of one-year-old macaques, follicle sizes increase significantly, without, however, reaching the final mature diameters. The follicles are still devoid of an ovarian cumulus. An obvious finding at this age is the presence of follicular cysts that may be lined by a thin granulosa cell layer or only by a fibrous capsule in the final stage. Follicular cysts appear frequently and occasionally become quite large (Figure 20). Remarkably, there are groups of luteinized cells in the ovarian stroma. They are considered to derive from transformed follicular granulosa cells and are supposed to represent small luteal bodies. In conclusion, the ovary shows the histological correlates of increased endocrine activity in terms of luteal body formation, numerous developing and atretic follicles, cyst formation, hyaline rest bodies, and so on.
Estrogen receptors are still expressed only by the germinal epithelium, whereas progesterone receptors are present on approximately 60% of the stromal cells, on internal and external theca cells, and on germinal epithelial cells. The serum progesterone level remains low, whereas serum estrogen measures indicate a slow increase of the hormone which is considered to mirror the approach towards sexual maturity (Table 5).
The ovary of the 1
The Mature Ovary FSH Control and Estrogen Allocation (Follicle Phase) Maturity is reached with the onset of regular ovulation and the first menstrual bleeding that determines the end of puberty. In the cynomolgus macaque, menstrual bleeding occurs at an age of approximately twenty-four to thirty months. The stimulation of menstrual bleeding derives from an initial event in the central nervous system and is transmitted via a factor/hormone/transmitter-cascade leading to FSH release from the basophilic pituitary cells. Central activation is equally described in both cynomolgus macaques and humans and impacts both organ morphology and function (Adams, Kaplan, and Koritnik 1985; Beier 1990) (Figure 22).
During the transition to maturity, the serum FSH level in the monkey elevates from the premature values of <1 ng/ml to adult average value of 15.24 ng/ml (Table 5). At the same time, the follicle estrogen machinery is prepared to react to the FSH stimulation as is morphologically obvious by the presence of well developed antral follicles with both FSH receptors (Wartenberg 1990) and estrogen receptors. Reacting to FSH, the granulosa cells intensify the estrogen synthesis. Estrogen becomes a key hormone (serum levels observed in individual mature cynomolgus macaques: >2,500 pmol/L), which is apparent from the serum levels, from the estrogen receptor expression by granulosa cells, and from the induction of the key process of dominant follicle selection (Figures 22, 23, 24) (Gougeon 1996; Zelinski-Wooten et al. 1998).
The whole functional machinery behind the follicle changes appears to be highly complex, and many involved factors (cytokines, growth factors, etc.) have probably not been discovered or functionally characterized yet. The mediating factors of follicle development probably represent significant targets for chemical compounds developed to influence the regulation and processes of follicle development and maturation (Table 4). The exact time of follicle development from the initial recruitment to antrum formation and atresia in juvenile cynomolgus macaques is unclear. In adult monkeys, the developmental period from the primordial to the mature antral ovulatory follicle (Graafian follicle) has been calculated to be 215 days (seven months, thirty weeks), which includes approximately seven hormonal cycles (Adashi 1990; Gougeon 1986, 1996). Interestingly, the corresponding period in humans is quite similar with 195 days (seven months or twenty-eight weeks) (Beier 1990; Lüllmann-Rauch 2003). Of the seven hormone cycles experienced by the developing follicle, four are spent in FSH independent period, whereas three are FSH dependent. The last two cycles include the late secondary to the advanced antral stage. Accordingly, only the last cycle mediates the selection and maturation of the dominant follicle (Figures 23–25). Only the most advanced follicle of the last, seventh cycle is valid to fit into the often used terminology of "follicle in the follicular phase." In view of follicle age and morphological status, a cycle dependant follicle categorization into five follicle classes has been suggested on the basis of antrum diameters, thus resulting in eight classes of follicle development if preantral stages are included (Gougeon 1996).
However, in routine histology it is difficult to identify individual follicle stages with the exception of the advanced mid-cycle dominant follicle. The remaining follicles are cohort members in certain stages (classes) of one of the seven alternative hormone cycles. In adult cynomolgus and rhesus macaques, the ovaries contain approximately two to five predominant antral follicles competing for dominance, whereas three to eleven follicles are regarded to be competitive in humans (Lüllmann-Rauch 2003; Pache et al. 1990). The mechanisms responsible for the number of maturing dominant follicles may differ from species to species. In the cynomolgus and rhesus macaque and in humans, usually one follicle ovulates, whereas there are up to four ovulating follicles in the marmoset and up to ten or even more in the dog, rabbit, mouse, and rat. Such differences are reflected by the morphology of macaque and human ovaries on one hand and marmosets and rat ovaries on the other (Figures 26, 27).
In view of the functional status of the mature cynomolgus macaque ovary, cellular proliferation and the expression of estradiol-, progesterone-, and androgen receptors were studied in the various ovarian tissues. Proliferative activity identified with the anti-Ki67 antibody appears to be minimal or almost absent in the granulosa cells of the primordial follicles, but it increases dramatically from the primary towards the advanced tertiary follicle (Figure 28). A decrease in proliferative activity appears from the enlarged dominant follicle stage onwards. Proliferation of granulosa epithelial cells is described to be reciprocal to the elevation of serum progesterone, which might explain the large follicle phenotype (Koering 1986; Zeleznik, Wildt, and Schuler 1980). Decreased proliferation continues during the luteal body development as growth of the luteal body depends on cellular hypertrophy rather than on mitotic division. Granulosa cells in atretic follicles temporarily keep their proliferative activity until they finally undergo degeneration.
In the secondary and early tertiary follicles, proliferation is clearly evident in the internal theca, whereas all surrounding tissues including the external theca remain proliferatively silent. Such a pattern suggests that the increase of granulosa cells is initiated by a local factor-mediated event rather than by migration of cells from peripheral tissues. The stromal cells, in contrast, display minimal proliferative activity. Estrogen receptor expression, at least in mature females, is considered to be indicative for the presence of estrogen as well as for cells and target tissues within the ovary sensitive for estrogen. Selected estrogen functions in the ovary are listed in Table 6. In the ovary of the mature cynomolgus macaque, estrogen receptors are expressed by the majority of germinal epithelial cells (which can be relevant in relation to oncogenesis), whereas stromal cells and granulosa cells of primordial and primary follicles are almost completely negative for estrogen receptors (Figure 29). Estrogen receptors develop in a small percentage (about 10%) of granulosa cells in medium-sized secondary follicles, and their number increases with follicle growth. At the same time, the number of estrogen receptors on internal theca cells also increases with follicle size.
In the most advanced antral follicles, approximately 50% to 70% of granulosa cells are positive for estrogen receptors. The receptors rapidly disappear in atretic follicles and after follicle transformation into the luteal body (Figure 30).
The distribution of progesterone receptors reflects the targets of the hormone within the ovary (selected progesterone functions; see Table 6). In the follicle phase, progesterone receptors are present on the majority of matrix cells, granulosa cells, internal and external theca cells, and the germinal epithelium (Figure 30). There is no apparent difference between adolescent and adult stages. Almost 100% of granulosa cells of small and large antral follicles express progesterone receptors. No correlation can be drawn between progesterone receptor expression and phases of hormonal activity. The progesterone receptors rapidly vanish in apoptotic granulosa epithelial cells but are preserved in the granulosa luteal cells of the luteal body for several weeks (Suzuki et al. 1994). The androgen receptors are especially sensitive to testosterone (Vendola et al. 1998) (selected androgen functions; see Table 6). Their distribution in the cynomolgus macaque ovary differs from that of estrogen and progesterone receptors in view of their absence in the tunica albuginea, the germinal epithelium, the granulosa cells of primordial, primary, and young secondary follicles, the luteal cells of the luteal body, and the internal theca (Figure 31). Androgen receptors, however, are expressed by granulosa cells of advanced secondary follicles and in large numbers by the external theca and the cortical stroma cells. Functionally, the internal theca synthesizes the androgens from cholesterol (Figure 24). The androgens are then released from the internal theca and diffuse into the follicle for binding the corresponding nuclear granulosa cell receptors. In the granulosa cells, they are either metabolized into estrogen or involved in the mediation of follicle atresia in cases of a low FSH/aromatase level (Birkhäuser 1994). The receptor expression in the external theca, however, is not fully understood but is assumed to prevent excess of androgen diffusion into other ovarian compartments.
The distribution of the steroid receptors is suspected to reflect the effects of the respective hormones in the ovarian tissue (Saunders et al. 2000). Functional and morphological correlates are highlighted in the corresponding figures and schemes. The evaluation of estrogen and progesterone receptors in view of the prospective leading versus nonleading follicle, of activated versus inhibited follicles, and of follicles in the ipsi versus the contra lateral ovaries reveal no apparent qualitative (staining intensity) and quantitative difference. The topography of receptor expression in the follicles of mature cynomolgus macaque is schematically summarized in Figure 32.
Ovulation The oocyte expulsion in Macaca fascicularis usually occurs on day 13 of the menstrual cycle (Dukelow, Fan, and Sacco 1986), but preparative processes begin a few days earlier. As midcycle approaches, there is a dramatic increase in estrogen levels (95% of estrogen is produced by the dominant follicle) (Dvorak and Tesarik 1990; Rebar 1995; Rebar, Kenigsberg, Hodgen 1995), followed by the progesterone and LH surge, which finally triggers ovulation of the dominant follicle (Chaffin, Dissen, and Stouffer 2000; Dubourdieu et al. 1991; Hibbert et al. 1996). LH stimulates a number of critical actions in the ovary, such as the release of histamine and the stimulation of local hyperemia and permeability of capillaries leading to a local preovulatory edema. It further stimulates the synthesis of proteases, the elevation of prostaglandins in the dominant follicle, and the release of collagenases from granulosa cells. Collagenases mediate the lysis of the follicular wall. Hence, the actual ovulatory rupture is shortly delayed after the LH surge. Weakening the follicular wall by local enzymatic interaction is considered to be more relevant for ovulation than a formerly suspected increased antral pressure induced by prostaglandin-mediated contraction of the myofibroblasts of the external theca (see Figure 7) (Dvorak and Tesarik 1990). Several further studies indicate that ovulation also depends on progesterone. Inhibition of progesterone, for example, with epostane, leads to the blockage of ovulation (Espey and Lipner 1994). In the cynomolgus macaque, superficial protrusion of the follicle above the ovarian surface has been observed by pelvic endoscopical examinations two to three days prior to ovulation (Dukelow 1975; Dukelow, Fan, and Sacco 1986) (compared to five to six days in humans). At this time, the steroid synthesis in the granulosa cells shifts from estrogen to predominantly progesterone production (Chaffin, Dissen, and Stouffer 2000; Chaffin, Schwinof, and Stouffer 2001) (Figure 33). The change of steroid predominance is completed within 36 to 38 hr in the cynomolgus macaque and within 40 hr in the Japanese macaque (Macaca fuscata) (Nigi 1978) and is characterized by an increase of the serum progesterone level. The ovulatory process, however, takes only 20 to 30 sec (Dukelow 1975; Espey and Lipner 1994) (Figure 26).
After ovulation, the follicular liquor transforms into a fibrin and blood rich coagulate (corpus rubrum; Figure 10) and the ovulation wound closes with an inflammatory like reaction with participation of plasminogen activator, histamines, vasoactive factors, eicosanoids, associated with invasion of fibroblasts. Factors discussed to be relevant in ovulation in nonhuman primates and several laboratory animals include kinins, prostaglandins, lipoxygenase, arachidonate, kallikreins, and collagenases (Espey and Lipner 1994; Oerke, Einspanier, and Hodges 1996). The majority of nonruptured antral follicles are considered to become either atretic (larger follicles) or to persist in a decelerated growth status exposed to low FSH stimulation. Endocrinologically, the follicular/estrogen phase has been replaced by the progesterone/luteal phase. The granulosa luteal cells persist in case of pregnancy or undergo atrophy and degeneration in case of no conception.
The Luteal Phase and the Luteal Body, The ovary during luteal phase and menstruation If pregnancy fails, cells regress spontaneously by atrophic degeneration (Dubourdieu et al. 1991). After 14 ± 2 days, progesterone decreases towards basic levels permitting central FSH stimulation initializing the next menstruation cycle. Five to eight cycles later, the luteal body is degraded, but remnants may persist as a hyaline scar referred to as corpus albicans. Uniparous animals (cynomolgus macaque, rhesus macaque, human) show a single luteal body on the ovulating ovary, whereas multiparous animals (marmoset, dog, rabbit, rat, mouse) reveal several luteal bodies of species specific size and shape on the ovaries (Figures 26 and 27). Species dependent differences like one versus several siblings or one versus several ovulations are considered to be mediated by significant physiologic, endocrine, and molecular mechanisms of high toxicological relevance.
The ovary during gravidity In the pregnant cynomolgus macaque, the size of the luteal body increases significantly and may measure more than 10 x10 x 10 mm. Progesterone production is taken over by the placenta after approximately one month irrespectively of persistence of the large luteal body (Dukelow, Fan, and Sacco 1986). Evidence of the placental function is supplied by experiments with ovariectomized female rhesus macaques. In this species, ovariectomy did not lead to abortion beyond day 25 after conception. In ovariectomized marmosets, pregnancy was independent from the ovarian luteal body after the first third of pregnancy and respectively after day 40–50 in humans (Koering 1986).
On Pregnancy Day 100
The luteal stroma appears markedly vascularised, and both cellular atrophy and proliferation are almost absent. Progesterone receptors are well expressed by almost all granulosa luteal cells correlating with a serum progesterone level of approximately 54 nmol/L (Table 2). Progesterone receptors are furthermore observed in all ovarian epithelial and mesenchymal tissue cells of the ipsi and contra lateral ovary. The intensity of receptor expression is similar to the profile in nonpregnant females. The distribution pattern of estrogen receptors in antral follicles is also similar to that in mature nonpregnant females. Blood estrogen measured in six pregnant females on day 100 revealed an average value of 4,385 pmol/L (Table 2). Fetus correlation on gestational day 100 (Table 2): Progesterone blood level: 96.36 nmol/L (n = 12). Progesterone receptors are slightly expressed in single cells. Estrogen blood level: 158 pmol/L (n = 7). Estrogen receptors are not expressed.
On Pregnancy Day 150 Progesterone receptors are still weakly expressed by the granulosa luteal cells, and the correlating serum progesterone level was 66.62 nmol/L (average of fourteen pregnant females). Estrogen receptors were comparably expressed by granulosa epithelial cells of antral follicles at day 100 of pregnancy and in mature nonpregnant females. Hence, the receptors could not be correlated with the elevated estrogen serum level of 2,851 pmol/L (Khattab and Jequier 1979). Fetus correlation on gestational day 150 (Table 2): Progesterone blood level: 68.66 nmol/L (n = 12). Progesterone receptors are increasingly expressed in comparison to day 100. Estrogen blood level: not available. Estrogen receptors are not expressed.
The Aged Ovary—Menopause
In animals with few remaining small follicles, the hormone profile still resembled that of a mature female (Table 7, Figure 35). These small follicles, that lack oocytes but have small fluid-filled spaces, appeared as enlarged primary and secondary follicle like structures with partly swollen and irregularly distributed granulosa epithelial cells (similar to Call-Exner bodies). Remaining tertiary follicular structures had particularly small antral cavities. Mitotic activity was low or missing. Sporadically, signs of true follicular development could be observed, and then, oocytes appeared to be of regular diameter and were covered by a pellucid zone.
Progesterone receptors were expressed as long as antral follicles and stromal remnants were preserved. In contrast, estrogen receptors could not be seen in granulosa cells and were only expressed by few cells of the germinal epithelium. In ovaries with complete replacement of functional tissue by fibrous tissue, a few germinal epithelial cells were positive for progesterone receptors, whereas estrogen receptors were generally lacking. Such ovaries coincided with a typical menopausal hormone profile (Figure 36, Table 7). Histologically, the aged cynomolgus macaque ovaries—premenopausal and menopausal—are characterized by
In consistence with further reports, there was complete follicle exhaustion in menopausal animals, which is described to occur beyond the twentieth year of life (Kavanagh, Williams, and Wagner 2005), and there was a clear correlation between ovarian follicle presence/activity and the well established menopausal hormone profile (high LH and low estradiol level) (Shideler et al. 2001). In the rhesus macaque, the follicle exhaustion period is reported to appear in twenty-four- to twenty-seven-year-old females and to be completed at the age of twenty-seven to thirty-four (Hodgen et al. 1977; Walker 1995). Human females, in comparison, become menopausal at the average age of fifty (Siegenthaler 1994). However, most data on menopause in humans are derived from serum hormone profiles rather than from histomorphological examinations.
Pharmaceutical drug safety testing requires the availability of reliable animal models in toxicological studies. Especially for compounds that interfere with the hypothalamus-pituitary-ovarian axis or for compounds that target the ovaries in another way, macaques are the species of choice because of their high similarity to man. With respect to the ovary, considerable histomorphological similarities between the cynomolgus macaque and man from fetal stages until menopause are documented in this article. However, a comparable anatomical structure does certainly not exclude the presence of significant differences on the molecular level between the two species. The presented steroid receptor expression and distribution mirror functional processes and can demonstrate hormonal conditions on a cellular basis. The interaction of tissue/cell and hormones is emphasized in schemes included in the article, and its complexity may be conducted from the number of influencing factors presented in Table 4, which again closely resembles cynomolgus monkey and man. However, minute chemical differences may be of major toxicological relevance. In addition to the morphological examinations of the ovary, the article includes hormone measurements in fetal, juvenile, and adult animals unless these are not part of the article "Physiology and Endocrinology of the Ovarian Cycle in Macaques" (Weinbauer et al. 2008, this issue, pp. 7S–23S).
The authors gratefully acknowledge the skilled and dedicated assistance of the technical and operational staff at Covance Laboratories GmbH in Münster, Germany, and Schering-Plough (formerly Organon), Oss, the Netherlands, during the experimental conduct and data collection and analysis.
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Abstract
Introduction
-year-old females for the first time. 
(TGF-
increase of progesterone; decrease of cytochrome P450
