Renewal of Oviduct Cilia During the Menstrual Cycle of the Rhesus Monkey

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1 Renewal of Oviduct Cilia During the Menstrual Cycle of the Rhesus Monkey ROBERT M. BRENNER, PH.D. THE LITERATURE on THE CYCLIC BEHAVIOR of the ciliated cells of the primate oviduct is both diverse and contradictory. Shimoyama and Clyman both reported that no significant changes occur during the human menstrual cycle. However, Novak and Everett had earlier reported that the ciliated cells increase in height during the follicular phase and shrink during the luteal phase. Others have found that oviducal ciliated cells decrease in number during the luteal phase and increase during the follicular phase. This has been reported by Schultka for man, by Westmann for the rhesus monkey, and by Joachimovits for the Java monkey. I have recently shown 3 5 in the rhesus monkey that oophorectomy causes a dramatic atrophy of the oviducal epithelium, with complete loss of the cilia from the epithelial cells, and that estrogen treatment restores the epithelium to a fully ciliated state. Further, I have described the morphogenetic events that occur during such estrogen-driven ciliogenesis. Previously, I had briefly reported 4 5 that during the normal menstrual cycle in rhesus monkeys, an analogous loss and regeneration of cilia occurs during the luteal and follicular phases. The cytomorphogenetic processes involved were identical with those found during experimentally induced estrogen-driven ciliogenesis. The present paper reports these findings in more detail. MATERIALS AND METHODS Thirty-six mature female rhesus monkeys were laparotomized at various times during the menstrual cycle (Table 1). Both ovaries were removed, and the fimbriae and ampulla of each oviduct were biopsied. The ovaries From the Department of Electron Microscopy, Oregon Regional Primate Reseach Center, Beaverton, Ore. Supported by The Population Council, Grant 1\ , and by Grants HD to the author and FR00163 to the Oregon Regional Primate Research Center from the National Institutes of Health, U. S. Public Health Service. This is Publication No. 353 of the Oregon Regional Primate Research Center. 599

2 600 BRENNER FERTILITY & STERILITY TABLE 1. Number of Rhesus Monkeys Laparotomized on Various Cycle Days Cycle No. of Cycle No. of Cycle No. of Cycle No. of day animals day animals day animals day animals were embedded in paraffin and serially sectioned for light-microscopic analysis. The oviduct samples were fixed in the following medium for both light and electron microscopy: % Glutaraldehyde (Fisher's) Paraformaldehyde 3.00 Sucrose 4.50 Sodium cacodylate 1.00 CaCb (added immediately before use) 0.05 MgS Tissues were fixed for one-half hour and then washed a minimum of 24 hr. in an aqueous solution containing 4.5% sucrose and 1% sodium cacodylate. Blocks were postfixed for one and one-half hr. in a mixture of 1% o~mium tetroxide, 4.5% sucrose, and 1% sodium cacodylate. The osmicated tissues were dehydrated through an alcohol series and embedded in Araldite. * Bright gold sections were stained with uranyl acetate and lead citrate and examined in a Philipst EM-200 electron microscope. Light Microscopic Findings RESULTS The Fimbriae. Figures 1-8 illustrate the changes that occur in the epithelium of the fimbriae during the menstrual cycle. On Day 2, the epithelial cells are characteristically low and deciliated. All the cells are of uniform staining density so that it is impossible to distinguish between future ciliated and future secretory cells. These atrophied cells are also characterized by a high nucleocytoplasmic ratio. On Day 3 (Fig. 2), considerable mitotic activity occurs, and the epithelial cells begin to hypertrophy. At this time, two cell types appear: a lightstaining or "clear" cell and a dark-staining cell. Electron microscopy (vide infra) reveals that the clear cells are future ciliated cells; the "dark" cells, *Ciba Corporation, Summit, N. J. tphilips Electronic Instruments, Mount Vernon, N. Y.

3 VoL. 20, No. 4, 1969 RENEWAL OF OviDUCT CILIA Fig. 1. Day 2 of cycle. Epithelium is characteristically low. Ciliated cells are either absent or extremely rare. (X 1200) Fig. 2. Day 3 of cycle. Mitotic activity beeomes prevalent, and epithelium begins to hypertrophy. (X 1200) Fig. 3. Day 4 of cycle. Epithelial cells become maximally hypertrophied, and basal bodies are formed. Note basal bodies (arrow) in large clear cell at center. (X 1200) Fig. 4. Day 5-6 of cycle. Cilia are produced at this time. (X 1200) future secretory cells. By Day 4, the clear cells reach their maximum height and begin to manufacture basal bodies. In all such cells, the basal bodies migrate to the luminal cell surface and arrange themselves in an orderly pattern before ciliation (Fig. 3). On Days 5-6, the cilia grow out from the basal bodies and attain their normal length of p.. (Fig. 4). The ciliated cells undergo no further change until about Days 15-18,. when cellular atrophy begins (Fig. 5). Days 1&--27 are characterized by a dramatic atrophy of the epithelium, with eventual shedding of the entife ciliary apparatus from each cell. For example, on Day 18. (Fig. 6), the epithelium is maximally atrophied, but the cilia are still present. Ordjay 25, the tips of the ciliated cells are pinched off and shed (Fig. 7), so that by Day 27 (Fig. 8), the epithelium is almost completely devoid of Cilia~ Occasionally (Fig. 8), a few cells fail to shed their cilia, but roughly 95%

4 602 BRENNER FERTILITY & STERILITY Fig. 5. Day 15 of cycle. Ciliated cells are now somewhat atrophied. (X 1200) Fig. 6. Day 18 of cycle. Ciliated cells are maximally atrophied at this time. (X 1200) Fig. 7. Day 25. Tips of ciliated cells, including entire ciliary apparatus, are pinched off and shed by process like that in apocrine secretion. (X 1200) Fig. 8. Day 27 of cycle. Epithelium is deciliated and maximally atrophied. Occasionally, an atrophied ciliated cell remains. ( X 1200) of the cells become completely deciliated by the.end of the luteal phase. The Ampulla. Unlike the epithelium of the fimbriae, that of the ampulla does not deciliate so dramatically during the luteal phase of the menstrual cycle; the cells merely atrophy and, for the most part, retain their cilia. Those ampullary cells that lose their cilia do so exactly like those of the fimbriae, namely, by a pinching-off process. During the early follicular phase, the atrophied, ciliated epipthelium of the ampulla becomes hypertrophied, reaching its full height by Days 5-6. A small number of clear ciliogenic cells, identical with those of the fimbriae, can be found in the ampulla during Days 3-4. Thus, the ampulla is characterized by a waxing and waning in height of the ciliated cells, with minimal deciliation and ciliogenesis during each cycle. By contrast, almost all the ciliated cells of the fimbriae deciliate and ciliate during each cycle, as well as wax and wane in height. It is difficult to understand why such similar tissues should

5 VoL.20,No.4,1969 RENEWAL OF OviDUCT CILIA 603 respond so differently to the changing levels of hormones during the menstrual cycle. Electron Microscopic Findings All the electron micrographs (Fig. 9-16) are of cells of the fimbriae, not of the ampulla; however, ciliogenesis appears to be identical in both regions. The atrophied, deciliated cells characteristic of Day 2 are shown in Fig. 9; the hypertrophied, clear cells characteristic of Day 3, in Fig. 10. Note that the atrophied cells are characterized by a uniform density and a high nucleocytoplasmic ratio, whereas the hypertrophied cells appear as either light or dark cells. The dark cells contain small secretory granules; the apical cytoplasmic regions of the light cells contain the various precursors of the basal bodies (Fig. 10). The first of these precursors, the so-called proliferative elements, 8 which appears on Day 3 (Fig. 11), is an aggregate of small dense granules ( A), interwoven by a network of fine filaments ( A). In many cells, these proliferative elements are the only precursor forms evident on Day 3, whereas, in other cells, they are accompanied by larger dense granules (Fig. 12), known as condensation forms ( mil). Often, a central condensation form is seen (Fig. 12), with 2-4 nascent basal bodies (150 mil X 150 mil) arranged radially around it; these clusters are known as generative complexes. By Day 4, the generative complexes have disappeared, and the basal bodies have enlarged to their full size ( mil). Condensation forms are usually still present and may remain attached to some of the mature basal bodies (Fig. 13). During this period, the basal bodies migrate to the cell surface and become attached to the cell membrane. Condensation forms usually disappear by this phase, though some may still be attached to the proximal ends of some basal bodies (Fig. 14). On Days 4--5, ciliary buds develop from the basal bodies, and genesis of ciliary filaments occurs within the ciliary buds. In addition, each basal body produces a lateral satellite and a proximal rootlet (Fig. 16). Proliferative elements are often present near the proximal ends of some aligned basal bodies, and these may be involved in the formation of the rootlets. The cell membrane grows with the developing cilia and forms the ciliary membrane (Fig. 15). By Day 5, the cilia have reached their mature length of IL (Fig. 16). DISCUSSION This study shows that in the rhesus monkey's oviduct, the cilia are shed and regenerated during the menstrual cycle. The degree of renewal varies

6 Fig. 9 (left). Atrophied epithelium characteristic of Day 2 of cycle. Ciliary apparatus is completely absent, and cells are maximally atrophied. (X 4500) Fig. 10 (right). Hypertrophied epithelium, characteristic of Days 2-4 of cycle. The clear cell is future ciliated cell. Proliferative elements are present in distal regions of cytoplasm. Dark cells are future secretory cells. (X 3000)

7 Fig. 11 (left). Proliferative elements (PE) in cytoplasm of future ciliated cell on Day 3 of cycle. Arrows point to typical amorphous masses of dense material that represent proliferative elements. The latter tend to occur in small clusters, and very delicate filaments can occasionally be seen to traverse randomly throughout the clusters. Mitochondria ( M) are abundant in these cells. (X 50,000) Fig. 12 (right). A mixture of proliferative elements (PE), condensation forms (CF), and generative complexes (GC) in cytoplasm of future ciliated cell on Day.3 of cycle. Generative complex consists of two immature basal bodies in contact with condensation form. (X 47,000)

8 Fig. 13 (left). A group of basal bodies (BB) and condensation forms (CF; arrows) in cytoplasm of future ciliated cell on Day 4 of cycle. These basal bodies are longer and, consequently, more mature than those in Fig. 12. (X 20,000) Fig. 14 (right). Basal bodies migrate to luminal surface of future ciliated cells on Day 4 of cycle. Condensation forms may be still attached to some of the basal bodies (arrow). (X 22,000)

9 VoL. 20, No. 4, 1969 RENEWAL OF OviDUCT CILIA 607 with the region of the oviduct. In the fimbriae, the process is dramatic in that more than 95% of the cells shed and regenerate their cilia each cycle. In the ampulla, the ciliated cells show a definite waxing and waning in height each cycle, but only a small number of them show complete loss and renewal of their cilia. After oophorectomy, however, the ciliated cells of the ampulla eventually lose most of their cilia, and complete regeneration can be provoked by estrogen treatment. 3 5 It seems reasonable to assume, therefore, that the real difference between the fimbriae and ampulla is one of sensitivity and Fig. 15. Cilia grow out from basal body by Day 5 of cycle. (X 42,000) that the fimbriae are more responsive to the cyclic changes in estrogenprogesterone blood levels than the ampulla. Although we have no direct evidence of the role progesterone plays in the loss of cilia, a chance observation suggests that, during the luteal phase, progesterone inhibits the cilia-maintaining effects of estrogen. The ovaries of 2 animals, examined on Days 23 and 27, had large follicles with no corpora lutea, indicating that ovulation had not occurred. The epithelium

10 608 BRENNER FERTILITY & STERILITY Fig. 16. Fully developed ciliary apparatus, characteristic of Days 5-20 of cycle. Ciliary rootlets (R) and lateral satellites (S) are also present during this period. (X 30,000)

11 VoL. 20, No. 4, 1969 RENEWAL OF OviDUCT CILIA 609 of the fimbriae, unlike that of the other animals in the late luteal phase, was fully ciliated and resembled that of animals in midcycle. Apparently, the progesterone levels had not risen high enough or the estrogen levels had not fallen low enough to provoke the usual deciliation. Andrews has published other, more direct evidence on progesterone as an inhibitor of estrogen-stimulated ciliogenesis in the human oviduct. In a study of postparturient women, he reported that the oviducal epithelium is low and mostly deciliated at the time of delivery (average height, 16 fl) and becomes lower and more deciliated during the postpartum period (average height, 10 fl). Women who received diethylstilbestrol on the day of delivery ( 5 mg.jday for 5-9 days) showed an intense proliferation and hypertrophy of the ciliated epithelium (average height, fl). Women who received diethylstilbestrol with progesterone during the same period, failed to develop ciliated cells, and the epithelium remained the same as at the time of delivery. Treatment with diethylstilbestrol days before delivery did not stimulate ciliogenesis, and progesterone treatment alone had no effect. Andrews also found that in the late menopausal state, which is characterized by low levels of both estrogen and progesterone, the human oviducal epithelium is low and deciliated. It seems reasonable to conclude that in the rhesus monkey the cyclic loss and renewal of oviducal cilia is caused by the cyclic changes in the balance of progesterone and estrogen. Some of the contradictory reports on the cyclic renewal of human ciliated epithelium may be caused by various authors' sampling different regions of the oviduct. I have reviewed these discrepancies elsewhere. 5 Ciliogenesis has been the subject of several recent reports. In 1965, Dirksen and Crocker, who coined the terms, "proliferative elements," "generative complexes," and "condensation forms," reported on the mechanism of basal body replication in the oviduct of the fetal mouse. They suggested that the centrioles of the cell gave rise to the first cluster of proliferative elements and that, once basal bodies were formed, some of these were parent to new clusters, which then entered a new cycle of basal body formation. I have not found centrioles associated with clusters of proliferative elements on Days 2 and 3 of the cycle, perhaps because such associations are extremely rare. I have found clusters of proliferative elements associated with mature basal bodies present at the cell membrane on Day 5 of the cycle, but these appear to be associated with rootlet formation. In a recent study of ciliogenesis in the trachea and epidermis of the embryos of Xenopus laevis, Steinman found a series of basal body precursor forms, identical with those found in the oviduct, though he used a different system of nomenclature to describe them. For example, he referred to proliferative

12 610 BRENNER FERTILITY & STERILITY elements as "procentriole-precursor bodies" and to condensation forms as "procentriole organizers." He called the proliferative elements found with mature basal bodies at the cell membrane "axonemal precursor bodies," on the assumption that they contribute to the formation of the ciliary axoneme, rather than to a new cycle of basal body formation. Biava and Matsuura, who studied ciliogenesis in the rat's regenerating tracheal epithelium, found a similar series of basal body precursor forms. However, they suggested that the proliferative elements, which they called "ciliary precursor bodies, 11 originate by the transformation of large polyribosomes and made no comment on the possible progenitor role of the cell centriole. Sorokin studied ciliogenesis in the rat's fetal lungs and also found a similar series of precursor structures. He used the terms "fibrogranular aggregates" and "deuterosomes" for structures similar to proliferative elements and condensation forms, respectively. The deuterosomes are spheroids with a dense wall and a light center; unlike the condensation forms, whigh are uniformly dense spheroids. Also, the fibrogranular aggregates are more highly organized structures than the proliferative elements. Sorokin suggested that the old centriole, Golgi elements, and annulate lamellas might all play a role in the formation of the fibrogranular aggregates. Martinez Martinez and Daems recently described a completely different form of basal body formation in the choroid cells of adult rats and proposed a trellis-like arrangement of striated cytoplasmic structures as the progenitqr of basal bodies. It seems clear that a definitive, universally applicable explanation of the origin of the proliferative elements, of procentriole precursor material, or of other basal body progenitor structures will depend upon the findings from some kind of tracer study. In our laboratory, we are concentrating our efforts on the study of estrogen-driven ciliogenesis in oophorectomized animals, because, in this system, we can precisely control the events of basal body and cilia formation. Recently, we have grown oviducal cilia in organ culture from estrogen-treated rhesus monkey castrates. 6 We are now conducting in-vitro studies of the uptake and migration of radioactive amino acids and nucleic acid precursors during ciliogenesis by means of electronmicroscopic autoradiography. The effects of inhibitors of protein and nucleic acid synthesis on ciliogenesis in vitro are also being examined. These studies will tell us something about the chemical nature of the proliferative elements and of other basal body precursor structures and should eventually lead us to a fuller understanding of the developmental mechanisms that control ba~al body replication and cilia formation.

13 VoL. 20, No. 4, 1969 RENEWAL OF OviDUCT CILIA 6ll SUMMARY The oviducts of 36 mature rhesus monkeys were examined by light and electron microscopy. During the luteal phase of the cycle, the ciliated cells of the fimbriae atrophy and, for the most part, shed their cilia. In the ampulla, the cells atrophy but do not shed their cilia. In the early follicular phase, cellular hypertrophy and ciliogenesis occur, particularly in the fimbriae. The ultrastructural details of ciliogenesis are described. Department of Electron Microscopy Oregon Regional Primate Research Center 505 N.W. 185 Ave. Beaverton, Ore REFERENCES 1. ANDREWS, M. C. Epithelial changes in the puerperal fallopian tube. Amer I Obstet Gynec 62:28, BIAvA, C. G., and MATSUURA, S. Morphogenesis of cilia from polyribosomes in differentiating tracheal epithelium of rats. I Cell Biol 35: 13A, BRENNER, R. M. Electron microscopy of estrogen effects on ciliogenesis and secretory cell growth in rhesus monkey oviduct. Anat Rec 157:218, BRENNER, R. M. Ciliogenesis during the menstrual cycle in rhesus monkey oviduct. I Cell Biol 35: 16A, BRENNER, R. M. "The Biology of Oviductal Cilia." In The Mammalian Oviduct. Blandau, R., and Hafez, E. S. E., Eds. Univ. Chicago Press, Chicago, In press. 6. BRENNER, R. M., ANDERSON, R. A., and KAMM, M. J. Estrogen-driven ciliogenesis in organ culture of rhesus monkey oviduct. I Cell Biol 39: 16A, CLYMAN, M. J. Electron microscopy of the human fallopian tube. Fertil Steril17: 281, DIRKSEN, E. R., and CROCKER, T. T. Centriole replication in differentiating ciliated cells of mammalian respiratory epithelium. An electron microscopic study. I Micr 5:629, JoACIDMOVITS, R. Studien zur Menstruation, Ovulation, Aufbau und Pathologie desweiblichen Genitales bei Mensch und Affe (Pithecus fascicularis mordax) Eileiter und Ovar. Biol Generalis (Wien) 11:281, lo. MARTINEZ MARTINEZ, P., and DAEMS, W. T. Le phases precoces de Ia formation des cils et le probleme de l'origine du corpuscle basal. Z Zellforsch 87:46, NovAK, E., and EVERETT, H. S. Cyclical and other variations in the tubal epithelium. Amer I Obstet Gynec 16:499, ScHULTKA, R. Der Sekretionscyklus der Flimmerzellen der menschlichen Tube uterina auf Grund cytologischer und cytotopochemischer Untersuchung. Acta Histochem (lena) 15:285, :3. SmMOYAMA, T. Electron microscopic study of the epithelial cells of the fallopian tube mucosa in the mature woman. I Iap Obstet Gynec Soc 15:1237, SoROKIN, S. P. Reconstructions of centriole formation and ciliogenesis in mammalian lungs. I Cell Sci 3:201, STEINMAN, R. M. An electron microscopic study of ciliogenesis in developing epidermis and trachea in the embryo of Xenopus laevis. Amer I Anat 122:19, WESTMANN, A. E. Einige Bemerkungen aus Anlass des Aufsatzes von Jageroos: Die sexualzyklishen Umwandlungen in der Tube uterina beim Menschen und bei den niedrigen Primaten. Acta Obstet Gunec Scand 13:263, 1934.