Serous Cysts of the Aging Guinea Pig Ovary I. LIGHT MICROSCOPY AND ORIGIN STEVEN L. QUATTROPANI Department ofAnatomy, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 ABSTRACT Ovaries from guinea pigs of different ages have been examined by light microscopy. Serous cysts measuring between 1mm and 2.5 cm are present in all animals over one year of age. The cysts are fewer in number and size a t six months, are least developed a t ten weeks and are not apparent in the nearterm fetus. The cysts arise from the rete ovarii. They are lined with a simple cuboidal to columnar epithelium composed of cells bearing solitary cilia or tufts of cilia. The structure and origin of these cysts is discussed and compared with what is known of the human serous cystadenoma. The increased incidence of neoplasms in the aged ovary (Julian et al., '74) and decline in fertility in aged mammals (Albrecht et al., "75) are two problems which reflect the need for information on the biology of the ovary in aging mammals. The rat, one of the most commonly studied aged laboratory animals, is well known for its disposition toward irregular cycles and polycystic ovaries (Quadri et al., '73). The cysts of such ovaries are of follicular origin and appear to be structurally similar to the preovulatory follicles of younger animals. In humans the most common ovarian neoplasm is the serous cystadenoma which is frequently malignant (Bullock et al., '55; De Santo et al., '59). In a population studied by Bullock et al. ('55),the mean age of women with benign serous cysts was 41 years with a range of 16 to 84 years. Unlike the rat these cysts do not resemble ovarian follicles and several authors (Evans, '68; Hertig and Gore, '61; Novak and Woodruff, '74) have attributed their origin to the ovarian mesothelium. The present investigation is concerned with the appearance of cysts in the ovaries of guinea pigs. These cysts are more common in older animals, and are comparable structurally to the serous cystadenoma of the human rather than the follicular cyst of the rat. The light microscopic anatomy and origin of these cysts are the subject of this paper. MATERIALS AND METHODS Four groups of guinea pigs (Hartley strain) ANAT. REC., 288: 351-360. were used in this study: (1) 2-year-old retired breeders; (2) 1-year-old retired breeders; (3) 6-month-old females; (4) 10-week-old virgin females. Three females were examined in each of these groups. In addition, one near term female fetus was taken from one of the 6-month old females and examined. The animals were killed with sodium pentobarbital and the ovaries immediately removed and placed in an aldehyde fixative. This fixative was composed of 3% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Portions of some ovaries were postfixed in a 1%solution of OsO, with 0.1 M phosphate buffer (pH 7.41, dehydrated in alcohol, and embedded in Epon 812. Sections (0.75 p m thick) of this material were stained with toluidine blue, examined and photographed by light microscopy. The remaining tissues were transferred to fresh aldehyde fixative containing 5 ml of saturated picric acid per 100 ml of solution (It0 and Karnovsky, '68). The tissues were fixed in this solution for 18 hours a t 4"C, embedded in paraplast, serially sectioned a t 5 pm and stained with hematoxylin and eosin. Reconstructions of portions of the serially sectioned ovaries were made using camera lucida drawings. Photomicrographs of selected sections in these series also were made. RESULTS In the present study ovarian cysts measure Received July 30, '76. Accepted Dec. 10, "76. ' Supported by A. D. Williams Grant 3658 (5301, Medical College of Virginia, Virginia Commonwealth University. 351 352 STEVEN L. QUATTROPANI up to 2.5 cm in diameter. By gross observation these cysts are unilocular with a smooth inner surface and are filled with a clear fluid of low viscosity. The number of cysts, as well as their diameter varies greatly. When the cysts of a given ovary are less than 5 mm in diameter, several are clustered in the cephalic pole near the hilar region. In the specimens exhibiting the larger cysts (2 cm or greater) there is usually only one cyst and no other recognizable ovarian tissue. While the size and frequency of the cysts vary among animals of the same age group, the largest cysts are in the animals of the 1and 2-year-old groups. In each of the animals examined in these groups, there is a t least one cyst greater than 1mm in diameter. Three out of seven animals examined in these groups have one ovary with a cyst greater than one centimeter in diameter. In each of the animals of the 1- and 2-year-old age groups in which the left and right ovaries are identified (5 of 6 animals), the largest cysts are in the left ovary. In general cysts in the 10-week-old and 6-month-old groups are less than 0.1 mm in diameter. However, there is one cyst with a diameter of 1 mm in a 6-month-old animal. Cysts are least frequent in the 10-week-old animal and are absent in the near term fetus. Microscopicexamination of the ovary of the near term fetus indicates the presence of numerous follicles which are frequently joined to one another in linear configurations (fig. 1). Such configurations are also in continuity with the ovarian mesothelium. In the hilar region of this ovary, there are cords of basophilic cells (fig. 1). These cords extend into the more cortical regions of the ovary where their proximity to the follicles gives the appearance of continuity. A slit-like lumen is frequently present in these cords but there are no cystic dilations. Microscopic examination of the ovary of the adult guinea pig reveals large numbers of cystic structures which vary considerably in diameter but are composed of similar cell types (fig. 2). Serial reconstruction of ovaries indicates that the larger cysts are frequently multilocular (figs. 3, 4) and that the smaller cysts (less than 500 pm in diameter) form a network of interconnecting cysts. In figures 510, selected sections from such a series demonstrates the continuity of these cysts in the ovary of a 2-year-old guinea pig. In this series profile x of figure 5 is in continuity with profile y in figure 7. Profile y (fig. 10) measures 75 x 112~m and has been traced through 20- sections 5 pm in thickness. At each end of the 20-section series the profile ends blindly (fig. 5).The section in figure 5 is the most proximal of figures 5 through 10 to the hilar region of this ovary. The lumen of profile x is followed in sections closer to the hilar region for 300 pm. In this span this lumen becomes continuous with that of another cyst of slightly larger diameter and finally in the hilar region is continuous with the lumen of a cyst 200 p m in diameter. While such cysts penetrate throughout the ovary, they are most common in the hilar region. In this region there is a complex of branched tubules whose lumen is less obvious (fig. 2). Cysts are in continuity with these tubules. All cysts are lined with a simple epithelium which varies from low cuboidal to columnar. Two cell types can be identified in this epithelium (figs. 11,121. The most abundant of these cell types is characterized by the presence of a solitary cilium which projects into the lumen of the cyst. This cell contains a basophilic cytoplasm but no other cytoplasmic specialization is apparent by light microscopy. The apical surface of the cell is slightly domed with a few cytoplasmic projections. A second population of cells is identified by the presence of a tuft of cilia on the luminal surface of each cell. In the cytoplasm underlying these cilia is a row of basal bodies. The cilia of a given cell are all bent in the same direction. The surface of the ciliated cells may be flat or slightly domed. Cells with the domed appearance are generally more basophilic than those with a more flattened surface. Of the two types of tufted cells the less basophilic is the least numerous. The nuclei of all cell types are irregularly shaped and a t times exhibit deep clefts. They are generally central in location although in taller columnar cells they are situated more basally. The nuclei have a single nucleolus and a peripheral concentration of heterochromatin. The non-cystic tubular system found in the hilar region is also composed of a simple cuboidal to columnar epithelium which is not ciliated. The dilated or cystic portions of this system, however, do contain ciliated cells. No structural evidence of secretory activity was detected in any of the above cell types. DISCUSSION Descriptions of ovarian development in rats, humans and monkeys (Gillman, '48; SEROUS CYSTS OF THE AGING GUINEA PIG OVARY Merchant, ’75; Quattropani, ’75) indicate that cords of epithelial and primordial germ cells develop and grow in the stroma of the ovarian blastema. These cords are continuous with the ovarian mesothelium as well as the mesonephric tubules. Portions of these cords containing germ cells later fragment to become ovarian follicles; while other portions retain the cord-like appearance. In the hilar region of the ovary those cords which are derived from the mesonephric tubules (Merchant, ’75) are devoid of germ cells and are known as the rete ovarii. The cords of epithelial cells observed a t each age in the hilar region of the guinea pig ovary are the remnants of this rete ovarii. Continuity between the rete ovarii, follicles and ovarian mesothelium is observed in the guinea pig. The presence of the rete ovarii is not restricted to the fetus or neonate. It has been described in a variety of adult mammals including man (Sauramo, ’54). In the guinea pig the large serous cysts of the adult ovary develop from the rete ovarii. This conclusion is based on four factors: (1) the cysts are most concentrated in the hilar region of the ovary; (2) the smaller cysts are in continuity with one another; (3) the rete ovarii of young animals (and old animals when not obscured by large cysts) exhibits dilated or cystic regions and the epithelium of these cystic regions is similar in appearance to that of the larger cysts; (4) there is dissimilarity between the epithelium of the atretic follicles and the cysts. The cystic nature of the rete ovarii in the guinea pig was first noted by Mossman (‘73). However, the guinea pig is not unique in this property; the heifer (Archbald et al., ’71),pig (Wilkerson, ’23)and man (Sauramo, ’54) have also been reported as being cystic. Preliminary observations in this laboratory of a population of prairie deermice also indicates the presence of cysts similar to those seen in the guinea pig. In man it is commonly accepted that the serous cyst develops from the ovarian mesothelium (Novak and Woodruff, ’74). According to Hertig and Gore (‘61), this conclusion is based on the presence of germinal inclusion cysts and infoldings of the ovarian mesothelium. These investigators have listed the rete ovarii as a n “unlikely” origin. However, the similarities in ovarian organogenesis in mammals and the reports of cystic enlargement of the rete ovarii in humans (Sauramo, ’541, suggests that a re-evaluation of the origin of the serous cysts in man might be appropriate. Such a study is underway in this laboratory. 353 The mechanism of growth of these cysts has not been resolved. The expansion or dilation of the rete ovarii, an increase in the number of epithelial cells, or a combination of these two mechanisms are possible explanations. In the present study mitotic figures were not observed. Whether this is due to the fact that samples were taken only a t one time of the day or that the dividing cells are few and the division time is short has not yet been established. However, the presence of light and dark cells of the tufted type may be indicative of a cell cycle. There are several other questions pertaining to the growth of these cysts which remain to be resolved. No signs of secretory activity were observed in the epithelial cells of these cysts. This would suggest that the cyst fluid is a plasma filtrate. We are unable a t this time to state whether or not cystogenesis in the guinea pig is hormonally influenced. It has been reported, however, that in the heifer cysts vary with the estrus cycle (Archbald e t al., ’71). The cystadenoma of the guinea pig described herein bears striking resemblance to the benign serous cyst of the human (Gondos, ’71; Hertig and Gore, ’61). Both are composed of a simple epithelium with ciliated and nonciliated cells. The benign cystoma of humans also exhibits a smooth surface and may be unilocular or multilocular. In the human, however, there is a high frequency of malignancy among the serous cystomas (Hertig and Gore, ’61). Malignant forms exhibit papillary modification of the surface, stromal invasion, a decrease in the number of ciliated cells and irregularly shaped nuclei (Gondos, ”71).In the ages examined to date, no signs of malignant transformation have been observed in the ovarian cysts of the guinea pig. The increased size and number of cysts in older specimens suggest that cyst formation is a normal function of aging in the guinea pig. In humans (Sauramo (‘54) reports that the cystic appearance of the rete ovarii is more prominant in older women. The fact that small cysts are found in young animals suggests that cyst development is not incompatible with fertility. One 6-month-old female which was pregnant when killed did exhibit one serous cyst l m m in diameter. In this female each horn of the uterus contained a fetus; and the cystic ovary contained what appeared to be a functional corpus luteum. Cystic development in the guinea pig is, therefore, unlike the microcystic syndrome of rats 354 STEVEN L. QUATTROPANI in which there is ovulatory failure (Quadri et al., '73). The high incidence of cysts does not appear to be peculiar to the population of guinea pigs used in this study. Preliminary observations of ovaries from Hartley strain guinea pigs from a second supplier indicates similar cystogenesis. Several studies of the cystic ovary of the guinea pig are currently underway in this laboratory to determine the hormonal dependency, growth pattern, and potential for malignancy of these cysts. We believe that these studies will enable the establishment of a model for the study of the serous cystadenoma in humans. LITERATURE CITED Albrecht, E. D., R. D. Kws and W. B. Wehrenberg 1975 Ovarian A5-3P-hydroxysteroid dehydrogenase and cholesterol in the aged mouse during pregnancy. Biol. Reprod., 13: 158-162. Archbald, L. F., R. H. Schultz, M. L. Fahning, H. J. Kurtz and R. Zemjanis 1971 Rete ovarii in heifers: A preliminary study. J. Reprod. Fert.. 26: 413-414. Bullock, W. K., R. E. Houts and J. J. Gikane 1955 Ovarian tumors. AMA arch. Surg., 71:153-166. DeSanto, D. A., W. K. Bullock and F. J. Moore 1959 Ovarian cystomas. Arch. Surg., 78: 98-107. Gillman, J. 1948 The development of the gonads in man, with a consideration of the role of fetal endocrines and the histogenesis of ovarian tumors. Carnegie Inst. Contrib. Embryol., 32: 81-131. Gondos, B. 1971 Electron microscopic study of papillary serous tumors of the ovary. Cancer, 27: 1455-1464. Hertig, A. T., and H. Gore 1961 Tumors of the female sex organs. Part 3. Tumors of the ovary and fallopian tube. In: Atlas of Tumor Pathology. Sect. IX, Fasc. 33, AFIP, Washington, D.C. Ito, S., and M. J. Karnovsky 1968 Formaldehyde-glutaraldehyde fixatives containing trinitro compounda. J. Cell Biol., 39: 168a. Julian, C. G., J. Goss, K. Blanchard and J. D. Woodruff 1974 Biologic behavior of primary ovarian malignancy. Obstet. Gynec.. 44:873-884. Merchant, H. 1975 Rat gonadal and ovarian organogenesis with and without germ cells. An ultrastructural study. Dev. Biol., 44: 1-21. Mossman, H. W., and K. L. Duke 1973 Comparative Morphology . -. of the Mammalian Ovary. Univ. Wisconsin Press, Madison, 461 pp. Novak, E. R., and J. D. Woodruff 1974 Novak's Gynecologic and Obstetric Pathology. Saunders, New York. Quadri, S. K., G. S. Kledzik and J. Meites 1973 Reinitiation of estrous cycles in old constant estrus rats by central-acting drugs. Neuroendocrinology, 11: 248-255. Quattropani, S. L. 1975 Microscopy of ovarian cords and mesothelium in the fetal monkey (Macaca arctiods) and fetal human. Anat. Rec., 181: 454 (Abstract). Sauramo, H. 1954 Development, occurrence, function and pathology of the rete ovarii. Acta Obstet. Gynec. Scand. (Suppl. 2), 33: 29-46. Wilkerson, W.V. 1923 The rete ovarii a s a normal structure of the adult mammalian ovary. Anat. Rec., 26: 75-78. PLATE I EXPLANATION OF FIGURES 1 Mid-sagittal section of ovary of a near-term fetus showing a densely stained rete ova. rii (R) cords of follicles (F)continuity of follicles with mesothelium (black arrowhead) and with rete ovarii (white arrowhead). Paraplast, Hematoxylineosin. X 77. 2 Cysts of varying size tC) from the hilar reaon of the left ovary of a 1-year-oldguinea pig. A dilated portion of the rete ovarii (or branched tubules, is seen a t D. Paraplast. Hematoxylin-eosin. X 170. 3 This section is from the same specimen as figure 2, but is five sections removed and shows the smooth surface of two cysts in the hilar region. X 70. 4 This is from the same section a s is seen in figure 2 and shows continuity the two cysts shown in figure 3. X 170. tX)between SEROUS CYSTS OF THE AGING GUINEA PIG OVARY Steven L. Quattropani PLATE 1 355 PLATE 2 EXPLANATION OF FIGURES Figures 5-10 are sections from a serially sectioned ovary from a 2-year-old animal. The lumen of the cyst in figure 5 has been labeled X in figures 5-8. The lumen of the cyst in figure 10 has been labeled Y in figures 6-10. Paraplast, Hematoxylin-eosin. X 166. 5 This is the first of the series or section 1. 6 Section 3. 7 Section 8 8 Section 10. 9 Section 12. 10 Section 14. 356 SEROUS CYSTS O F THE AGING GUINEA PIG OVARY Steven L. Quattropani PLATE 2 357 PLATE 3 EXPLANATION OF FIGURES 11 This Epon section is from the ovary of a 1-year-old guinea pig. The different cell types which comprise the cystic epithelium are seen: cell with solitary cilium (S), hasophilic cell with tuft of cilia (TD), less basophilic cell with tuft of cilia (TL). X 1,800. 12 This micrograph was taken with Nomarski optics. The specimen was fixed in the aldehyde solution and a portion of a cyst was sliced and photographed in buffer. Note the tufted cell (TI and the cell with the solitary cilium (S). X 1,800. 358 SEROUS CYSTS OF THE AGING GUINEA PIG OVARY Steven L. Quattropani PLATE 3 359
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