A Scanning Electron Microscopic Study of the Luteo-Follicular Complex 11. EVENTS LEADING TO OVULATION PIETRO MOTTA AND JONATHAN VAN BLERKOM 2,3 Department of A n a t o m y , University of R o m e , R o m e , Italy; 2 Depai'tment of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80302, U.S.A. a n d 3 B. F . Stolinsky Laboratories, Departm e n t of Pediatrics, University of Colorado Medical Center, Denver, Colorado 80220 U.S.A.
ABSTRACT Morphological changes on the ovarian surface of different mammals both before and during ovulation have been examined by scanning electron microscopy. Preovulatory follicles were blisterlike structures that protruded markedly from the ovarian surface. Basal areas of preovulatory follicles were covered with polyhedral cells containing numerous microvilli, whereas on the lateral surfaces, superficial cells were elongated and possessed few microvilli. At the apex of the follicle, cells were very flattened and possessed few microvilli, which were present only in regions of intercellular contact. In some apical areas, cells appeared to be degenerating, whereas in other regions, groups of cells had "sloughed off." In addition, a fluidlike material was observed to exude from intercellular spaces of the superficial epithelium and to cover some apical cells. By transmission electron microscopy, the same fluidlike material was observed to (1) infiltrate the connective tissue of the tunica albuginea, (2) accumulate under the basal lamina, and ( 3 ) distend intercellular spaces of the superficial epithelium. Just prior to ovulation, large, irregular areas of the apex were ruptured and the oocyte, covered with a large amount of fluid, appeared to emerge from the follicle. At ovulation, the oocyte was not completely covered with follicle cells and the zona pellucida was clearly evident. The surface of the zona was quite irregular and contained numerous infoldings, channels and crypts. Follicle cells had polyhedral or star shapes and possessed large cytoplasmic evaginations that obliquely penetrated the zona. Both the zona peZ2ucida and corona cells were covered with a fine layer of granular material. The SEM results and parallel TEM observations suggest that a local increase of fluids (edema) may be an important factor in the final decomposition of the distended and weakened apex of the preovulatory follicle. In addition, the participation of follicle cells, smooth muscle cells and the oviduct in the escape of the oocyte from the ruptured follicle is discussed.
Mammalian ovulation would seem to be
a relatively simple phenomenon if regarded only as a dynamic process. However, this simplistic view is complicated by the fact that ovulation involves a series of timed hormonal, biochemical and morphological events. Although the hormonal steps that prepare the follicle and effect ovulation are well known, the concomitant biochemical and morphological processes are not comAM. J. ANAT., 143: 241-264.
pletely understood (Blandau, '66; Rondell, '70; Espey, '74). Recent transmission (TEM) and scanning (SEM) electron microscopic studies have described the morphological processes occurring at the apex of the preovulatory follicle (Espey, '67; Byskov, '69; Motta et al., '71; Nilsson and Munshi, '73; Cherney Accepted February 3, '75. 3Present address: See 3, above.
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et al., ’74). These studies demonstrate that a progressive disruption of the follicle wall, superficial epithelium, and the connective tissue of the ovarian cortex takes place a t the apex of the preovulatory follicle. The present study provides new information regarding three-dimensional changes occurring on the surface of the preovulatory follicle and demonstrates, by SEM, the actual process of ovulation. These SEM observations, if correlated with previous biochemical and morphological results, may provide a more complete understanding of the ovulatory process. MATERIALS AND METHODS
Only ovaries from healthy young adult mice, rats (albino) and rabbits (DutchBelted) were examined. I n order to study ovulation under normal conditions, ovaries were removed from animals that had not received exogenous hormone to induce ovulation. For mice and rats, the phase of the estrous cycle was determined by vaginal smears. Ovaries from a total of 35 mice and 15 rats (all i n late proestrous or early estrous) were examined. Rabbits, which are reflex ovulators, ovulate approximately 12 hours post c o i t u m (Austin and Braden, ’54). Ovaries from 15 rabbits were removed between 8 and 12 hours post c o i t u m and prepared for electron microscopy. Ovaries were perfused or fixed by immersion in a solution of 2.5% glutaraldehyde in 0.18 M cacodylate buffer, pH 7.3 (Sabatini et al., ’63). After a period of from one to several days in this solution, the tissues were washed in the same buffer for one-half hour and then cut into small blocks with a razor blade. Great care was exercised at this stage in order not to disturb obvious preovulatory or ovulating follicles. Dehydration was carried out rapidly through graded concentrations of acetone. After dehydration, the specimens were transferred to liquid COXfor critical-point drying (Porter et al., ’72). The dried samples were mounted on aluminum studs using conductive siIver paint and coated with a thin layer of carbon and gold in a high vacuum evaporator (DV-502, Denton Vacuum) under continuous rotation and with the appropriate tilt of the stud. The distance between the specimens and the
gold i n the basket of the vacuum evaporator was approximately 12 cm. The current was slowly raised from 0 to 18 amperes, and in about 60 seconds, all the gold was evaporated. All specimens were examined and photographed using a Cambridge Stereoscan Model S4 operated at 10 to 20 kV. For transmission electron microscopy, tissues were fixed overnight in either the same fixative used for SEM or i n Karnovsky’s (’65) formaldehyde-glutaraldehyde solution. The specimens were then postfixed i n 1.3% osmium tetroxide (in 0.18 M cacodylate buffer) for two hours, washed in the same buffer and dehydrated through graded concentrations of ethanol. The blocks were embedded in Epon 812 (Luft, ’61), sectioned with glass knives in a Porter-Blum MT-I or MT-I1 ultramicrotome and stained with uranyl acetate (Watson, ’58) followed by lead citrate (Reynolds, ’63). Sections were examined with either a Zeiss EM 9 or Philips EM 300 electron microscope. RESULTS
By scanning electron microscopy, and without great variation among the mammals studied, preovulatory follicles were blisterlike structures that protruded markedly from the ovarian surface (figs. 1, 2). I n the rabbit ( a n animal in which the number of ovarian “papillae” is considerably larger than observed in the mouse or rat, Motta, ’74), numerous “papillae” were distributed around some preovulatory follicles, forming a n intriguing and elegantlooking “flowerlike bouquet” (fig. 3 ) . The superficial epithelium of the ovary normally consisted of cuboidal or polyhedral cells that possessed numerous microvilli (fig. 4 ) . This type of epithelium appeared in a n unaltered form around the base of preovulatory follicles (figs. 1, 2 ) , whereas on the lateral surfaces, superficial cells, straightened by tension, were elongated and generally had a reduced number of microvilli (fig. 5 ) . The apices of preovulatory follicles contained large areas of flattened cells (squamous) that possessed few, relatively short microvilli (fig. 10). Many other cells in these apical regions had lost contact with one another and seemed to be undergoing a marked alteration or degeneration (fig. 6 ) . As a conse-
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quence of this process, the superficial epithelium a t the apices of the follicles appeared to “slough off” in amounts ranging from large to rather small groups of cells (figs. 6, 7). Desquamation of the superficial epithelium revealed a n amorphous connective tissue stroma that underlay the apex of the follicle (tunica albuginea) (figs. 8 , 9 ) . Many spherical or ovoidal blebs were observed on the surface of the apices of preovulatory follicles (figs. 10, 11). These blebs were either free on the surface of the superficial epithelium (fig. 1 0 ) or appeared to bulge from the cytoplasm of these cells (fig. 11). When these particular apical regions were examined by transmission electron microscopy, a large accumulation of a n amorphous, fluidlike material was observed in the following areas: (1) the connective tissue of the tunica albuginea, ( 2 ) the intercellular spaces of the superficial cells, and (3) under the basal lamina (figs. 12, 13). This amorphous material was so abundant in some regions that the intercellular spaces were completely filled and distended by its presence (figs. 12, 13). I n addition, the cytoplasm of many superficial cells was filled with large vacuoles containing an amorphous, dense material (fig. 14). At ovulation, large, irregular areas of the apex of the follicle are ruptured. The ruptured areas of ovulating follicles examined in this study were frequently covered with large amounts of fluid (liquor folliculi and intercellular fluids) and consequently, it was rather difficult to observe the ovulating oocyte directly. In only a few instances (and after a detailed examination of 130 ovaries from estrous mice, rats, and post coitum rabbits) was it possible to obtain satisfactory images of oocytes, ovulated under normal conditions (i.e.,without exogenous hormonal intervention). The stereo pair presented in figure 15 probably demonstrates the process of ovulation. Because the rupture was quite large, the oocyte was visible in this specimen. Large quantities of a fluidlike material and cellular debris were present around the opening of the rupture. When viewed in stereo, the oocyte appears to emerge from within the cavity of the follicle (fig. 15), and because this oocyte is not completely covered with
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follicle cells, the surface of the zoaa pellucida is clearly evident a t higher magnification (fig. 16). When present, fluids surrounding the oocyte appeared in a condensed form (possibly precipitated) in large, irregular masses that bound together groups of free follicle cells (fig. 1 7 ) and partially obscured their surfaces. Follicle cells of the corona radiata had a n irregular, polyhedral or star shape which resulted from the presence of large, cytoplasmic evaginations (figs. 18, 19). By SEM, the surface of the corona cells appeared rather rough owing to a layer of fine granules (figs. 18, 19). The zona pellucida was a dense, compact and amorphous membrane that contained many infoldings, channels and crypts (figs. 20, 21). The entire surface of the zona pellucida was covered with fine granules (fig. 21) similar to those observed on the surface of the cells of the corona radiata. DISCUSSION
Numerous theories have been proposed to explain the process of mammalian ovulation (for a review, see Blandau, ’66; Rondell, ’70; Espey, ’74). Among the phenomena considered to play a role in ovulation are intrafollicular pressure (Rondell, ’64), contraction of smooth muscle cells (OShea, ’70; Fumagalli et al., ’71), enzymatic digestion (Espey and Lipner, ’65), vascular changes (Burr and Davies, ’51; Motta et al., ’71), and nervous control (Bahr et al., ’74). At present, no single phenomenon is widely accepted as being the “actual” cause of ovulation. However, from a strictly morphological point of view, what has become increasingly evident is that prior to ovulation, a progressive degeneration and decomposition of cells occurs at the apex of the preovulatory follicle (Espey, ’67; Byskov, ”69; Motta et al., ’71). This process of decomposition, a s viewed by TEM, consists primarily of a gradual alteration of the intercellular ground substance of the connective tissue with a parallel dissociation of the fibrillar and cellular components. The superficial epithelium, thecal, and granulosa cells in areas surrounding the apex of the preovulatory follicle and fibrocytes present in the cortical areas of the follicle all appear to undergo a progressive alteration and
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degeneration prior to ovulation (Espey, ’67; Byskov, ’69; Motta et al., ’71). It has also been demonstrated that the dissociation of fibers and cells and the depolymerization of the intercellular ground substance in the connective tissue of the tuncia albuginea are increased by fluid infiltrating the perifollicular zones; this fluid accumulates mostly under the surface of the apex of the preovulatory follicle (Motta et al., ’71; Cherney et al., ’74). The present SEM study of preovulatory follicles has revealed that i n some apical regions of the follicle, cells of the superficial epithelium become progressively flattened, lose their microvilli, and slough off. However, in other regions these cells appear relatively unaltered. The presence of droplets (blebs) of a dense, fluidlike material on the surface of the superficial epithelium of some preovulatory follicles suggests that this material may arise from underlying structures. These results are in agreement with observations of Nilsson and Munshi (’73) who, by SEM, observed a thin, fluid layer covering the surface of the stigma of some preovulatory follicles in the mouse. By TEM, our observations show that owing to the presence of large amounts of a fluidlike material, the apices of preovulatory follicles are almost completely dissociated. This fluidlike material, which apparently mixes with, or dissociates, the ground substance, accumulates largely underneath the basal lamina and infiltrates into the intercellular spaces present in the superficial epithelium, This process of infiltration transforms the intercellular spaces into dilated channels and may cause cells to be pushed outward. After examining the submicroscopic changes in preovulatory follicles, it seems evident that the increase i n fluids (edema) may be a major cause of the progressive dissociation and labilization of tissues present in the apical zone of the preovulatory follicle. The increase i n fluid content of perifollicular areas just prior to ovulation may be related to a n increase in the vascularization of the follicle known to occur before ovulation (Burr and Davies, ’51; Blandau, ’66; Jewett and Dukelow, ’71). Vascular changes (primarily a n increase in the permeability and fragility of the endothelial wall) in perifollicular areas may depend
upon the synthesis and local release of estrogen by follicular components (theca interna) leading to a local accumulation of fluids with a consequent edema and hemostasis (Rona, ’63; Szego and Gitin, ’64). Thus, because of the edema, the apex of the follicle, which is known to be devoid of blood vessels (Motta et al., ’71) and is distended by a n increase in the fluid volume of the follicle, becomes so fragile that the rupture of the follicular wall appears to be not only possible, but inevitable (Cherney et al., ’74). Rather than a n explosive phenomenon, ovulation seems to be a gradual process in which the oocyte, immersed in a gel-like material, escapes from the ruptured surface of the ovary. This gel-like material is composed of liquor folliculi mixed with fluids that had accumulated in apical regions of the preovulatory follicle. The interpretation of morphological changes observed in the present study are consistent with the microcinematographic demonstration that in vivo, ovulation is more similar to a “blister that bursts’” than to a n explosive event (Blandau, ’66). If ovulation consists simply of the rupture of the follicle due to the decomposition of the apical region (as it seems), then one question central to a n understanding of the entire process of ovulation is what forces are involved in the escape of the oocyte from the follicular cavity? Possible mechanisms involved in the emergence of the oocyte following rupture of the follicular wall are presented in the following discussion. At the moment of ovulation, or just before, the oocyte and follicle cells of the corona radiata are, for the most part, already detached from the granulosa layer (cumulus oophorus). The oocyte, at this point, lies free in the liquor folliculi contained within the follicular cavity. Detachment of the oocyte from the granulosa layer may be related to the passive infiltration of liquoT folliculi, which may mechanically dissociate the cells, or may depend upon ameboid movements of the granulosa cells (Motta and DiDio, ’74). The presence of contractile microfilaments within the ameboid evaginations of granulosa cells may indirectly contribute to the expulsion of the oocyte. I n addition, the contractile, ameboid movements of cells of the corona
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radiata may be a contributing factor in the transport of the oocyte along the short distance separating the stigma of the follicle from the fimbriae of the oviduct. The presence of smooth muscle cells throughout the ovarian tissue and especially in perifollicular regions (for older references, see Fumagalli et al., ’71; Espey, ’74) may very well be the source of the “squeezing” of the follicle (and ovary itself) that occurs a t ovulation (Rocereto et al., ’69; Palti and Freund, ’72). Furthermore, the contractile, peristaltic activity of the oviduct and ciliary movement of the fimbriae (Blandau, ’68) may have a significant role in creating local currents and thus ensuring the complete expulsion of the oocyte and contents of the follicular cavity. A final point concerns the presence of granular material on the surface of corona cells and zona pellucida. These granules are also encountered within the zona, and it has been suggested that they might originate from follicle cell processes that traverse the zona pellucida (Motta and Van Blerkom, ’74). These granules have been observed previously by Zamboni and Mastroianni (’66) and seem to be comparable to granules thought to arise as small surface protrusions (“micropapillae”) of the plasmalemma of rabbit oocytes and adjacent follicle cell processes (Pedersen and Seidel, ’72). The composition and function of these granules remain obscure. Zamboni and Mastroianni (’66) suggested that they might be glycogen, but Pedersen and Seidel (’72) concluded that their fine structural appearance and staining reactions indicated that the granules were entirely different from glycogen. Although the present observations confirm the occurrence of the granules, they do not contribute to a n elucidation of either their significance or composition. Possibly, some relationship exists between the release and appearance of these granules and the complex events occurring in the oocyte as it prepares for fertilization. ACKN OWLEDCMENTS
This work was partially supported by a Fulbright grant to Dr. Pietro Motta. I n addition, funds from a grant to Dr. Cole Manes, Department of Pediatrics, University of Colorado Medical Center, Denver,
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Colorado, were made available to Dr. Jonathan Van Blerkom (Grant HD-04274 from the National Institutes of Health, United States Public Health Service). This study was made i n the Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado. The authors are grateful to Professor Keith R. Porter for his generosity i n making laboratory facilities available (to P. M.) and to Dr. Cole Manes for his helpful suggestions i n the preparation of the manuscript. The special support of the Italian Foreign Office is also greatly appreciated. LITERATURE CITED Austin, C. R., and A. W. H. Braden 1954 Time relationships and their significance in the ovulation and penetration of eggs in rats and rabbits. Aust. J. Biol. Sci., 7: 179-194. Bahr, J., L. Kao and A. V. Nalbandov 1974 The role of catecholamines and nerves in ovulation. Biol. Reproduct., 10: 273-290. Blandau, R. J. 1966 The mechanisms of ovulation. In: Ovulation: Stimulation Suppression Detection. R. B. Greenblatt, ed. J. B. Lippincott Co., Philadelphia, p. 3. 1968 Gamete transport. Comparative aspects. In: The Mammalian Oviduct, Comparative Biology and Methodology. E. S. E. Hafez and R. J. Blandau, eds. The University of Chicago Press, Chicago and London, pp. 129163. Burr, J. M., and J. I. Davies 1951 The vascular system of the rabbit ovary and its relationship to ovulation. Anat. Rec., 1 1 1 : 273-297. Byskov, A. G. 1969 Ultrastructural studies on the preovulatory follicle in the mouse ovary. Z. Zellforsch., 100: 285-299. Cherney, D. D., L. J. A. DiDio and P. Motta 1974 The development of rabbit ovarian follicles following copulation. Fertil. & Steril., (in press). Espey, L. L. 1967 Ultrastructure of the apex of the rabbit Graafian follicle during the ovulatory process. Endocrinology, 81 : 267-276. 1974 Ovarian proteolytic enzymes and ovulation. Biol. Reprod., 10: 216-235. Espey, L. L., and H. Lipner 1965 Enzymeinduced rupture of rabbit Graafian follicle. Amer. J. Physiol., 208: 208-213. Fumagalli, Z., P. Motta and S. Calvieri 1971 The presence of smooth muscular cells in the ovary of several mammals as seen under the electron microscope. Experientia, 27: 682-683. Jewett, D. A., and W. R. Dukelow 1971 Follicular morphology in m ~ c u c a fascicularis. Fed. Proc., 30: 216-220. Karnovskv. M. .T. 1965 A formaldehyde-glutarfixaliveof high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A.
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Luft, J. A. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. Motta, P. 1974 Fine structure of ovarian cortical crypts and cords in mature rabbits: A transmission and scanning electron microscopic study. Acta Anatomica. (Basel), 90: 3&64. Motta, P., D. D. Cherney and L. J. A. DiDio 1971 Scanning and transmission electron microscopy of the ovarian surface in mammals with special reference to ovulation. J. Submicr. Cytol., 3: 85-100. Motta, P., and L. J. A. DiDio 1974 Microfilaments in granulosa cells during the follicular development and transformation in the corpus luteum in the rabbit ovary. J. Submicr. Cytol., 6 : 15-27. Motta, P., and J. Van Blerkom 1974 A scanning electron microscopic study of the luteofollicular complex. I. Follicle and oocyte. J. Submicr. Cytol., 6: 297-310. Nilsson, O., and S. F. Munshi 1973 Scanning electron microscopy of mouse follicles at ovulation. J. Submicr. Cytol., 5 : 1-6. O’Shea, J. D. 1970 A n ultrastructural study of smooth muscle-like cells in the theca externa of ovarian follicles in the rat. Anat. Rec., 167: 127-139. Palti, Z., and M. Freund 1972 Spontaneous contractions of the human ovary in vitro. J . Reprod. Fert., 28: 113-115. Pedersen, H., and G . Seidel 1972 Micropapillae: A local modification of the cell surface observed in rabbit oocytes and adjacent follicular cells. J. Ultrastruct. Res., 39: 540-548.
Porter, K. R., D. Kelley and P. M. Andrews 1972 The preparation of cultured cells and soft tissues for scanning electron microscopy. In: Proceedings of the 5th Annual Stereoscan Colloquium, Chicago, Kent Cambridge Scientific co., pp. 1-19. Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Rocereto, T., D. Jacobowitz and E. E. Wallach 1969 Observations o n spontaneous contractions of the cat ovary “in vitro.” Endocrinology, 84: 1336-1340. Rona, G. 1963 The role of vascular mucopolysaccharides in the hemostatic action of estrogens. Am. J. Obst. Gynec., 87: 434-444. Rondell, P. 1964 Follicular pressure and distensibility in ovulation. Amer. J. Physiol., 207: 590-594. 1970 Biophysical aspects of ovulation. Biol. Reprod. (Suppl.), 2 : 64-89. Sabatini, D. D., K. G. Bensch and R. J. Barrnett 1963 Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19-58. Szego, C. M., and E. S. Gitin 1964 Ovarian histamine depletion during acute hyperaemic response to luteinizing hormone. Nature, 201 : 682-684. Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 175-177. Zamboni, L., and L. Mastroianni 1966 Electron microscopic studies o n rabbit ova. I. The follicular oocyte. J. Ultrastruct. Res., 14: 95-117.
Note added in proof: Subsequent to the acceptance of the present study, several reports on induced ovulation and the mechanism of follicle rupture by L. Bjersing and S. Cajander have appeared (Cell. Tiss. Res., 149: 287337. 1974). Results from those studies are i n general agreement with the observations presented in this communication.
PLATE 1 EXPLANATION O F FIGURES
1
Some preovulatory follicles bulge markedly from the surface of the ovary. Base of follicle (B), lateral surface ( L ) , and apex of preovulatory follicle ( A ) . (Rat ovary, from animal in the estrous phase, X 185.)
2
The apex (A) of a preovulatory follicle containing very flattened cells. Cells on the lateral surface ( L ) are elongated, whereas those at the base of the follicle (B) are polyhedral. (Mouse ovary, from animal in late proestrous, x 340.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
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3
A number of “papillae” (arrows) are present around the protruding surface of a preovulatory follicle, forming a “flower-like bouquet” ( F ) . (Rabbit ovary, 8 hours post coitum, x 170.)
4
Typically, the superficial epithelium of the ovary is composed of cuboidal or polyhedral cells containing numerous microvilli and isolated cilia (arrows). (Rabbit ovary, 10 hours post coitum, x 4,250.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
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5
Cells of the superficial epithelium (Se) present o n the lateral surface of the preovulatory follicle are flattened and possess a reduced number of microvilli. The microvilli are mainly restricted to regions of intercellular contact (arrows). (Rat ovary, from animal in the estrous phase, x 2,620.)
6
Large areas of cellular disruption and “sloughing o f f of the superficial epithelium on the surface of a preovulatory follicle are demonstrated in this scanning electron micrograph. (Rabbit ovary, 9 hours post coitum, x 1,450.)
7
The apex ( A ) of a preovulatory follicle is not completely covered by the superficial epithelium (Se); zones of desquamation are evident (arrows). (Rabbit ovary, 9 hours post coitum, X 125.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 3
25 1
PLATE 4 EXPLANATION O F FIGURES
8,9
In areas near the apex of a preovulatory follicle, the disruption and “sloughing off’ of the superficial epithelium (Se) is so great that the connective stroma which composes (fig. 8 ) and underlies (fig. 9 ) the tunica albuginea ( T a ) is revealed. (Rabbit ovary, 10 hours post coitum, x 1,610 and x 1,410.)
10,11 On the apex of some preovulatory follicles, a coagulated, fluidlike material is observed either free on the surface of the flattened, superficial epithelium (Se) (arrows, fig. 10) or as spherical (or irregular) droplets (white triangle, fig. 11) that bulge from the cytoplasm of some cells. The black arrows in figure 11 point to “blebs” that may be either free on the surface or bulging out from the cytoplasm. In addition, figure 10 illustrates the appearance of epithelial cells at the apex of the preovulatory follicle. These cells are quite flattened and possess few, relatively short microvilli. (Mouse ovary, from a n animal i n the estrous phase, x 3,100; rabbit ovary, 11 hours p o s t coitum, X 5,900.)
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SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
12,13 Areas of the apex of a preovulatory follicle similar to those illustrated in figures 10 and 11. As can be seen in these transmission electron micrographs, a fluidlike material accumulates in the connective tissue of the tunica albuginea ( T a ) , under the basal lamina (BL) and in the spaces between cells of the superficial epithelium (Se). This material is so abundant that the intercellular spaces ( I S ) in some areas are dramatically distended by its presence. Microvilli ( M ) . (Rabbit ovary, 10 hours post coitum, x 6,810 and x 13,200.)
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SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 5
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PLATE 6 EXPLANATION OF FIGURES
14 Transmission electron micrograph of the apex of a preovulatory follicle. The cytoplasm of some superficial cells contains large droplets filled with a dense, amorphous, fluidlike material ( F d ) . M, microvilli. (Rabbit ovary, 8 hours post coiturn, x 14,150.)
15 This stereo pair probably illustrates the process of ovulation. The apex of the follicle is ruptured and the oocyte, immersed in a large amount of fluid and cytoplasmic debris, appears to emerge from within the follicular cavity (the oocyte is visible i n the center of the micrograph). (Mouse ovary, from an animal in the estrous phase, X 230. )
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SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan V a n Blerkom
PLATE 6
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PLATE 7 EXPLANATION OF FIGURE
16
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This scanning electron micrograph illustrates on oocyte ( O o ) , possibly at the mount of ovulation. The surface of the egg is only partially covered with corona cells ( G ) . A large quantity of fluid and cellular debris (F) surrounds the ruptured area through which the oocyte emerges. (Mouse ovary, from an animal in the estrous phase, X 3,500.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 7
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PLATE 8 E X P L A N A T I O N OF F I G U R E S
17 This scanning electron micrograph shows the ruptured area of a follicle just after ovulation ( R ) . Large numbers of follicle cells immersed in fluid ( F ) cover regions adjacent to the ruptured follicle ( R ) . The egg (Oo), which has just escaped from the follicle and is covered by a mass of corona cells, is visible just under the ruptured area. (Rabbit ovary, 9 hours post coitum, x 195.) A
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This figure is a higher magnification of the area (00) indicated in figure 17. The oocyte is not directly visible because it is covered by a mass of follicle cells mixed with spermatozoa (Sp). The spermatozoa (Sp). The spermatozoa have traveled through the reproductive tract and reached the surface of the ovary i n approximately nine hours. x 1,720.
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PLATE 8
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PLATE 9 EXPLANATION OF FIGURES
18
The appearance of follicle cells ( F ) in areas adjacent to the oocyte at ovulation is illustrated in this electron micrograph. These cells are irregular or polyhedral in shape and, due to the presence of a fine, granular material, appear to have rough surfaces (arrows). (Mouse ovary, from an animal in the estrous phase, x 5,350.)
19 This scanning electron micrograph shows a cluster of follicle cells ( F ) attached to the zona pellucida (Zp) which covers the oocyte (00). The irregular surface of the zona pellucida is also clearly evident. (Mouse ovary, from a n animal i n the estrous phase, x 2,380.)
20,21
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These electron micrographs show the surface of the zona pellucida at higher magnification. The zona pellucida is a dense, compact and amorphous membrane containing infoldings, channels and crypts that are often in continuity within the zona. The surface of the zona (fig. 21) is covered with a fine, granular material (arrows). (Mouse ovary, from an animal in the estrous phase, x 5,800 and x 10,800.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
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ABSTRACT Morphological changes on the ovarian surface of different mammals both before and during ovulation have been examined by scanning electron microscopy. Preovulatory follicles were blisterlike structures that protruded markedly from the ovarian surface. Basal areas of preovulatory follicles were covered with polyhedral cells containing numerous microvilli, whereas on the lateral surfaces, superficial cells were elongated and possessed few microvilli. At the apex of the follicle, cells were very flattened and possessed few microvilli, which were present only in regions of intercellular contact. In some apical areas, cells appeared to be degenerating, whereas in other regions, groups of cells had "sloughed off." In addition, a fluidlike material was observed to exude from intercellular spaces of the superficial epithelium and to cover some apical cells. By transmission electron microscopy, the same fluidlike material was observed to (1) infiltrate the connective tissue of the tunica albuginea, (2) accumulate under the basal lamina, and ( 3 ) distend intercellular spaces of the superficial epithelium. Just prior to ovulation, large, irregular areas of the apex were ruptured and the oocyte, covered with a large amount of fluid, appeared to emerge from the follicle. At ovulation, the oocyte was not completely covered with follicle cells and the zona pellucida was clearly evident. The surface of the zona was quite irregular and contained numerous infoldings, channels and crypts. Follicle cells had polyhedral or star shapes and possessed large cytoplasmic evaginations that obliquely penetrated the zona. Both the zona peZ2ucida and corona cells were covered with a fine layer of granular material. The SEM results and parallel TEM observations suggest that a local increase of fluids (edema) may be an important factor in the final decomposition of the distended and weakened apex of the preovulatory follicle. In addition, the participation of follicle cells, smooth muscle cells and the oviduct in the escape of the oocyte from the ruptured follicle is discussed.
Mammalian ovulation would seem to be
a relatively simple phenomenon if regarded only as a dynamic process. However, this simplistic view is complicated by the fact that ovulation involves a series of timed hormonal, biochemical and morphological events. Although the hormonal steps that prepare the follicle and effect ovulation are well known, the concomitant biochemical and morphological processes are not comAM. J. ANAT., 143: 241-264.
pletely understood (Blandau, '66; Rondell, '70; Espey, '74). Recent transmission (TEM) and scanning (SEM) electron microscopic studies have described the morphological processes occurring at the apex of the preovulatory follicle (Espey, '67; Byskov, '69; Motta et al., '71; Nilsson and Munshi, '73; Cherney Accepted February 3, '75. 3Present address: See 3, above.
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PIETRO MOTTA AND JONATHAN VAN BLERKOM
et al., ’74). These studies demonstrate that a progressive disruption of the follicle wall, superficial epithelium, and the connective tissue of the ovarian cortex takes place a t the apex of the preovulatory follicle. The present study provides new information regarding three-dimensional changes occurring on the surface of the preovulatory follicle and demonstrates, by SEM, the actual process of ovulation. These SEM observations, if correlated with previous biochemical and morphological results, may provide a more complete understanding of the ovulatory process. MATERIALS AND METHODS
Only ovaries from healthy young adult mice, rats (albino) and rabbits (DutchBelted) were examined. I n order to study ovulation under normal conditions, ovaries were removed from animals that had not received exogenous hormone to induce ovulation. For mice and rats, the phase of the estrous cycle was determined by vaginal smears. Ovaries from a total of 35 mice and 15 rats (all i n late proestrous or early estrous) were examined. Rabbits, which are reflex ovulators, ovulate approximately 12 hours post c o i t u m (Austin and Braden, ’54). Ovaries from 15 rabbits were removed between 8 and 12 hours post c o i t u m and prepared for electron microscopy. Ovaries were perfused or fixed by immersion in a solution of 2.5% glutaraldehyde in 0.18 M cacodylate buffer, pH 7.3 (Sabatini et al., ’63). After a period of from one to several days in this solution, the tissues were washed in the same buffer for one-half hour and then cut into small blocks with a razor blade. Great care was exercised at this stage in order not to disturb obvious preovulatory or ovulating follicles. Dehydration was carried out rapidly through graded concentrations of acetone. After dehydration, the specimens were transferred to liquid COXfor critical-point drying (Porter et al., ’72). The dried samples were mounted on aluminum studs using conductive siIver paint and coated with a thin layer of carbon and gold in a high vacuum evaporator (DV-502, Denton Vacuum) under continuous rotation and with the appropriate tilt of the stud. The distance between the specimens and the
gold i n the basket of the vacuum evaporator was approximately 12 cm. The current was slowly raised from 0 to 18 amperes, and in about 60 seconds, all the gold was evaporated. All specimens were examined and photographed using a Cambridge Stereoscan Model S4 operated at 10 to 20 kV. For transmission electron microscopy, tissues were fixed overnight in either the same fixative used for SEM or i n Karnovsky’s (’65) formaldehyde-glutaraldehyde solution. The specimens were then postfixed i n 1.3% osmium tetroxide (in 0.18 M cacodylate buffer) for two hours, washed in the same buffer and dehydrated through graded concentrations of ethanol. The blocks were embedded in Epon 812 (Luft, ’61), sectioned with glass knives in a Porter-Blum MT-I or MT-I1 ultramicrotome and stained with uranyl acetate (Watson, ’58) followed by lead citrate (Reynolds, ’63). Sections were examined with either a Zeiss EM 9 or Philips EM 300 electron microscope. RESULTS
By scanning electron microscopy, and without great variation among the mammals studied, preovulatory follicles were blisterlike structures that protruded markedly from the ovarian surface (figs. 1, 2). I n the rabbit ( a n animal in which the number of ovarian “papillae” is considerably larger than observed in the mouse or rat, Motta, ’74), numerous “papillae” were distributed around some preovulatory follicles, forming a n intriguing and elegantlooking “flowerlike bouquet” (fig. 3 ) . The superficial epithelium of the ovary normally consisted of cuboidal or polyhedral cells that possessed numerous microvilli (fig. 4 ) . This type of epithelium appeared in a n unaltered form around the base of preovulatory follicles (figs. 1, 2 ) , whereas on the lateral surfaces, superficial cells, straightened by tension, were elongated and generally had a reduced number of microvilli (fig. 5 ) . The apices of preovulatory follicles contained large areas of flattened cells (squamous) that possessed few, relatively short microvilli (fig. 10). Many other cells in these apical regions had lost contact with one another and seemed to be undergoing a marked alteration or degeneration (fig. 6 ) . As a conse-
SCANNING ELECTRON MICROSCOPY OF OVULATION
quence of this process, the superficial epithelium a t the apices of the follicles appeared to “slough off” in amounts ranging from large to rather small groups of cells (figs. 6, 7). Desquamation of the superficial epithelium revealed a n amorphous connective tissue stroma that underlay the apex of the follicle (tunica albuginea) (figs. 8 , 9 ) . Many spherical or ovoidal blebs were observed on the surface of the apices of preovulatory follicles (figs. 10, 11). These blebs were either free on the surface of the superficial epithelium (fig. 1 0 ) or appeared to bulge from the cytoplasm of these cells (fig. 11). When these particular apical regions were examined by transmission electron microscopy, a large accumulation of a n amorphous, fluidlike material was observed in the following areas: (1) the connective tissue of the tunica albuginea, ( 2 ) the intercellular spaces of the superficial cells, and (3) under the basal lamina (figs. 12, 13). This amorphous material was so abundant in some regions that the intercellular spaces were completely filled and distended by its presence (figs. 12, 13). I n addition, the cytoplasm of many superficial cells was filled with large vacuoles containing an amorphous, dense material (fig. 14). At ovulation, large, irregular areas of the apex of the follicle are ruptured. The ruptured areas of ovulating follicles examined in this study were frequently covered with large amounts of fluid (liquor folliculi and intercellular fluids) and consequently, it was rather difficult to observe the ovulating oocyte directly. In only a few instances (and after a detailed examination of 130 ovaries from estrous mice, rats, and post coitum rabbits) was it possible to obtain satisfactory images of oocytes, ovulated under normal conditions (i.e.,without exogenous hormonal intervention). The stereo pair presented in figure 15 probably demonstrates the process of ovulation. Because the rupture was quite large, the oocyte was visible in this specimen. Large quantities of a fluidlike material and cellular debris were present around the opening of the rupture. When viewed in stereo, the oocyte appears to emerge from within the cavity of the follicle (fig. 15), and because this oocyte is not completely covered with
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follicle cells, the surface of the zoaa pellucida is clearly evident a t higher magnification (fig. 16). When present, fluids surrounding the oocyte appeared in a condensed form (possibly precipitated) in large, irregular masses that bound together groups of free follicle cells (fig. 1 7 ) and partially obscured their surfaces. Follicle cells of the corona radiata had a n irregular, polyhedral or star shape which resulted from the presence of large, cytoplasmic evaginations (figs. 18, 19). By SEM, the surface of the corona cells appeared rather rough owing to a layer of fine granules (figs. 18, 19). The zona pellucida was a dense, compact and amorphous membrane that contained many infoldings, channels and crypts (figs. 20, 21). The entire surface of the zona pellucida was covered with fine granules (fig. 21) similar to those observed on the surface of the cells of the corona radiata. DISCUSSION
Numerous theories have been proposed to explain the process of mammalian ovulation (for a review, see Blandau, ’66; Rondell, ’70; Espey, ’74). Among the phenomena considered to play a role in ovulation are intrafollicular pressure (Rondell, ’64), contraction of smooth muscle cells (OShea, ’70; Fumagalli et al., ’71), enzymatic digestion (Espey and Lipner, ’65), vascular changes (Burr and Davies, ’51; Motta et al., ’71), and nervous control (Bahr et al., ’74). At present, no single phenomenon is widely accepted as being the “actual” cause of ovulation. However, from a strictly morphological point of view, what has become increasingly evident is that prior to ovulation, a progressive degeneration and decomposition of cells occurs at the apex of the preovulatory follicle (Espey, ’67; Byskov, ”69; Motta et al., ’71). This process of decomposition, a s viewed by TEM, consists primarily of a gradual alteration of the intercellular ground substance of the connective tissue with a parallel dissociation of the fibrillar and cellular components. The superficial epithelium, thecal, and granulosa cells in areas surrounding the apex of the preovulatory follicle and fibrocytes present in the cortical areas of the follicle all appear to undergo a progressive alteration and
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PIETRO MOTTA AND JONATHAN VAN BLERKOM
degeneration prior to ovulation (Espey, ’67; Byskov, ’69; Motta et al., ’71). It has also been demonstrated that the dissociation of fibers and cells and the depolymerization of the intercellular ground substance in the connective tissue of the tuncia albuginea are increased by fluid infiltrating the perifollicular zones; this fluid accumulates mostly under the surface of the apex of the preovulatory follicle (Motta et al., ’71; Cherney et al., ’74). The present SEM study of preovulatory follicles has revealed that i n some apical regions of the follicle, cells of the superficial epithelium become progressively flattened, lose their microvilli, and slough off. However, in other regions these cells appear relatively unaltered. The presence of droplets (blebs) of a dense, fluidlike material on the surface of the superficial epithelium of some preovulatory follicles suggests that this material may arise from underlying structures. These results are in agreement with observations of Nilsson and Munshi (’73) who, by SEM, observed a thin, fluid layer covering the surface of the stigma of some preovulatory follicles in the mouse. By TEM, our observations show that owing to the presence of large amounts of a fluidlike material, the apices of preovulatory follicles are almost completely dissociated. This fluidlike material, which apparently mixes with, or dissociates, the ground substance, accumulates largely underneath the basal lamina and infiltrates into the intercellular spaces present in the superficial epithelium, This process of infiltration transforms the intercellular spaces into dilated channels and may cause cells to be pushed outward. After examining the submicroscopic changes in preovulatory follicles, it seems evident that the increase i n fluids (edema) may be a major cause of the progressive dissociation and labilization of tissues present in the apical zone of the preovulatory follicle. The increase i n fluid content of perifollicular areas just prior to ovulation may be related to a n increase in the vascularization of the follicle known to occur before ovulation (Burr and Davies, ’51; Blandau, ’66; Jewett and Dukelow, ’71). Vascular changes (primarily a n increase in the permeability and fragility of the endothelial wall) in perifollicular areas may depend
upon the synthesis and local release of estrogen by follicular components (theca interna) leading to a local accumulation of fluids with a consequent edema and hemostasis (Rona, ’63; Szego and Gitin, ’64). Thus, because of the edema, the apex of the follicle, which is known to be devoid of blood vessels (Motta et al., ’71) and is distended by a n increase in the fluid volume of the follicle, becomes so fragile that the rupture of the follicular wall appears to be not only possible, but inevitable (Cherney et al., ’74). Rather than a n explosive phenomenon, ovulation seems to be a gradual process in which the oocyte, immersed in a gel-like material, escapes from the ruptured surface of the ovary. This gel-like material is composed of liquor folliculi mixed with fluids that had accumulated in apical regions of the preovulatory follicle. The interpretation of morphological changes observed in the present study are consistent with the microcinematographic demonstration that in vivo, ovulation is more similar to a “blister that bursts’” than to a n explosive event (Blandau, ’66). If ovulation consists simply of the rupture of the follicle due to the decomposition of the apical region (as it seems), then one question central to a n understanding of the entire process of ovulation is what forces are involved in the escape of the oocyte from the follicular cavity? Possible mechanisms involved in the emergence of the oocyte following rupture of the follicular wall are presented in the following discussion. At the moment of ovulation, or just before, the oocyte and follicle cells of the corona radiata are, for the most part, already detached from the granulosa layer (cumulus oophorus). The oocyte, at this point, lies free in the liquor folliculi contained within the follicular cavity. Detachment of the oocyte from the granulosa layer may be related to the passive infiltration of liquoT folliculi, which may mechanically dissociate the cells, or may depend upon ameboid movements of the granulosa cells (Motta and DiDio, ’74). The presence of contractile microfilaments within the ameboid evaginations of granulosa cells may indirectly contribute to the expulsion of the oocyte. I n addition, the contractile, ameboid movements of cells of the corona
SCANNING ELECTRON MICROSCOPY OF OVULATION
radiata may be a contributing factor in the transport of the oocyte along the short distance separating the stigma of the follicle from the fimbriae of the oviduct. The presence of smooth muscle cells throughout the ovarian tissue and especially in perifollicular regions (for older references, see Fumagalli et al., ’71; Espey, ’74) may very well be the source of the “squeezing” of the follicle (and ovary itself) that occurs a t ovulation (Rocereto et al., ’69; Palti and Freund, ’72). Furthermore, the contractile, peristaltic activity of the oviduct and ciliary movement of the fimbriae (Blandau, ’68) may have a significant role in creating local currents and thus ensuring the complete expulsion of the oocyte and contents of the follicular cavity. A final point concerns the presence of granular material on the surface of corona cells and zona pellucida. These granules are also encountered within the zona, and it has been suggested that they might originate from follicle cell processes that traverse the zona pellucida (Motta and Van Blerkom, ’74). These granules have been observed previously by Zamboni and Mastroianni (’66) and seem to be comparable to granules thought to arise as small surface protrusions (“micropapillae”) of the plasmalemma of rabbit oocytes and adjacent follicle cell processes (Pedersen and Seidel, ’72). The composition and function of these granules remain obscure. Zamboni and Mastroianni (’66) suggested that they might be glycogen, but Pedersen and Seidel (’72) concluded that their fine structural appearance and staining reactions indicated that the granules were entirely different from glycogen. Although the present observations confirm the occurrence of the granules, they do not contribute to a n elucidation of either their significance or composition. Possibly, some relationship exists between the release and appearance of these granules and the complex events occurring in the oocyte as it prepares for fertilization. ACKN OWLEDCMENTS
This work was partially supported by a Fulbright grant to Dr. Pietro Motta. I n addition, funds from a grant to Dr. Cole Manes, Department of Pediatrics, University of Colorado Medical Center, Denver,
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Colorado, were made available to Dr. Jonathan Van Blerkom (Grant HD-04274 from the National Institutes of Health, United States Public Health Service). This study was made i n the Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado. The authors are grateful to Professor Keith R. Porter for his generosity i n making laboratory facilities available (to P. M.) and to Dr. Cole Manes for his helpful suggestions i n the preparation of the manuscript. The special support of the Italian Foreign Office is also greatly appreciated. LITERATURE CITED Austin, C. R., and A. W. H. Braden 1954 Time relationships and their significance in the ovulation and penetration of eggs in rats and rabbits. Aust. J. Biol. Sci., 7: 179-194. Bahr, J., L. Kao and A. V. Nalbandov 1974 The role of catecholamines and nerves in ovulation. Biol. Reproduct., 10: 273-290. Blandau, R. J. 1966 The mechanisms of ovulation. In: Ovulation: Stimulation Suppression Detection. R. B. Greenblatt, ed. J. B. Lippincott Co., Philadelphia, p. 3. 1968 Gamete transport. Comparative aspects. In: The Mammalian Oviduct, Comparative Biology and Methodology. E. S. E. Hafez and R. J. Blandau, eds. The University of Chicago Press, Chicago and London, pp. 129163. Burr, J. M., and J. I. Davies 1951 The vascular system of the rabbit ovary and its relationship to ovulation. Anat. Rec., 1 1 1 : 273-297. Byskov, A. G. 1969 Ultrastructural studies on the preovulatory follicle in the mouse ovary. Z. Zellforsch., 100: 285-299. Cherney, D. D., L. J. A. DiDio and P. Motta 1974 The development of rabbit ovarian follicles following copulation. Fertil. & Steril., (in press). Espey, L. L. 1967 Ultrastructure of the apex of the rabbit Graafian follicle during the ovulatory process. Endocrinology, 81 : 267-276. 1974 Ovarian proteolytic enzymes and ovulation. Biol. Reprod., 10: 216-235. Espey, L. L., and H. Lipner 1965 Enzymeinduced rupture of rabbit Graafian follicle. Amer. J. Physiol., 208: 208-213. Fumagalli, Z., P. Motta and S. Calvieri 1971 The presence of smooth muscular cells in the ovary of several mammals as seen under the electron microscope. Experientia, 27: 682-683. Jewett, D. A., and W. R. Dukelow 1971 Follicular morphology in m ~ c u c a fascicularis. Fed. Proc., 30: 216-220. Karnovskv. M. .T. 1965 A formaldehyde-glutarfixaliveof high osmolality for use in electron microscopy. J. Cell Biol., 27: 137A.
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Luft, J. A. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. Motta, P. 1974 Fine structure of ovarian cortical crypts and cords in mature rabbits: A transmission and scanning electron microscopic study. Acta Anatomica. (Basel), 90: 3&64. Motta, P., D. D. Cherney and L. J. A. DiDio 1971 Scanning and transmission electron microscopy of the ovarian surface in mammals with special reference to ovulation. J. Submicr. Cytol., 3: 85-100. Motta, P., and L. J. A. DiDio 1974 Microfilaments in granulosa cells during the follicular development and transformation in the corpus luteum in the rabbit ovary. J. Submicr. Cytol., 6 : 15-27. Motta, P., and J. Van Blerkom 1974 A scanning electron microscopic study of the luteofollicular complex. I. Follicle and oocyte. J. Submicr. Cytol., 6: 297-310. Nilsson, O., and S. F. Munshi 1973 Scanning electron microscopy of mouse follicles at ovulation. J. Submicr. Cytol., 5 : 1-6. O’Shea, J. D. 1970 A n ultrastructural study of smooth muscle-like cells in the theca externa of ovarian follicles in the rat. Anat. Rec., 167: 127-139. Palti, Z., and M. Freund 1972 Spontaneous contractions of the human ovary in vitro. J . Reprod. Fert., 28: 113-115. Pedersen, H., and G . Seidel 1972 Micropapillae: A local modification of the cell surface observed in rabbit oocytes and adjacent follicular cells. J. Ultrastruct. Res., 39: 540-548.
Porter, K. R., D. Kelley and P. M. Andrews 1972 The preparation of cultured cells and soft tissues for scanning electron microscopy. In: Proceedings of the 5th Annual Stereoscan Colloquium, Chicago, Kent Cambridge Scientific co., pp. 1-19. Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Rocereto, T., D. Jacobowitz and E. E. Wallach 1969 Observations o n spontaneous contractions of the cat ovary “in vitro.” Endocrinology, 84: 1336-1340. Rona, G. 1963 The role of vascular mucopolysaccharides in the hemostatic action of estrogens. Am. J. Obst. Gynec., 87: 434-444. Rondell, P. 1964 Follicular pressure and distensibility in ovulation. Amer. J. Physiol., 207: 590-594. 1970 Biophysical aspects of ovulation. Biol. Reprod. (Suppl.), 2 : 64-89. Sabatini, D. D., K. G. Bensch and R. J. Barrnett 1963 Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19-58. Szego, C. M., and E. S. Gitin 1964 Ovarian histamine depletion during acute hyperaemic response to luteinizing hormone. Nature, 201 : 682-684. Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 175-177. Zamboni, L., and L. Mastroianni 1966 Electron microscopic studies o n rabbit ova. I. The follicular oocyte. J. Ultrastruct. Res., 14: 95-117.
Note added in proof: Subsequent to the acceptance of the present study, several reports on induced ovulation and the mechanism of follicle rupture by L. Bjersing and S. Cajander have appeared (Cell. Tiss. Res., 149: 287337. 1974). Results from those studies are i n general agreement with the observations presented in this communication.
PLATE 1 EXPLANATION O F FIGURES
1
Some preovulatory follicles bulge markedly from the surface of the ovary. Base of follicle (B), lateral surface ( L ) , and apex of preovulatory follicle ( A ) . (Rat ovary, from animal in the estrous phase, X 185.)
2
The apex (A) of a preovulatory follicle containing very flattened cells. Cells on the lateral surface ( L ) are elongated, whereas those at the base of the follicle (B) are polyhedral. (Mouse ovary, from animal in late proestrous, x 340.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
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3
A number of “papillae” (arrows) are present around the protruding surface of a preovulatory follicle, forming a “flower-like bouquet” ( F ) . (Rabbit ovary, 8 hours post coitum, x 170.)
4
Typically, the superficial epithelium of the ovary is composed of cuboidal or polyhedral cells containing numerous microvilli and isolated cilia (arrows). (Rabbit ovary, 10 hours post coitum, x 4,250.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
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5
Cells of the superficial epithelium (Se) present o n the lateral surface of the preovulatory follicle are flattened and possess a reduced number of microvilli. The microvilli are mainly restricted to regions of intercellular contact (arrows). (Rat ovary, from animal in the estrous phase, x 2,620.)
6
Large areas of cellular disruption and “sloughing o f f of the superficial epithelium on the surface of a preovulatory follicle are demonstrated in this scanning electron micrograph. (Rabbit ovary, 9 hours post coitum, x 1,450.)
7
The apex ( A ) of a preovulatory follicle is not completely covered by the superficial epithelium (Se); zones of desquamation are evident (arrows). (Rabbit ovary, 9 hours post coitum, X 125.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 3
25 1
PLATE 4 EXPLANATION O F FIGURES
8,9
In areas near the apex of a preovulatory follicle, the disruption and “sloughing off’ of the superficial epithelium (Se) is so great that the connective stroma which composes (fig. 8 ) and underlies (fig. 9 ) the tunica albuginea ( T a ) is revealed. (Rabbit ovary, 10 hours post coitum, x 1,610 and x 1,410.)
10,11 On the apex of some preovulatory follicles, a coagulated, fluidlike material is observed either free on the surface of the flattened, superficial epithelium (Se) (arrows, fig. 10) or as spherical (or irregular) droplets (white triangle, fig. 11) that bulge from the cytoplasm of some cells. The black arrows in figure 11 point to “blebs” that may be either free on the surface or bulging out from the cytoplasm. In addition, figure 10 illustrates the appearance of epithelial cells at the apex of the preovulatory follicle. These cells are quite flattened and possess few, relatively short microvilli. (Mouse ovary, from a n animal i n the estrous phase, x 3,100; rabbit ovary, 11 hours p o s t coitum, X 5,900.)
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SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
12,13 Areas of the apex of a preovulatory follicle similar to those illustrated in figures 10 and 11. As can be seen in these transmission electron micrographs, a fluidlike material accumulates in the connective tissue of the tunica albuginea ( T a ) , under the basal lamina (BL) and in the spaces between cells of the superficial epithelium (Se). This material is so abundant that the intercellular spaces ( I S ) in some areas are dramatically distended by its presence. Microvilli ( M ) . (Rabbit ovary, 10 hours post coitum, x 6,810 and x 13,200.)
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SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 5
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PLATE 6 EXPLANATION OF FIGURES
14 Transmission electron micrograph of the apex of a preovulatory follicle. The cytoplasm of some superficial cells contains large droplets filled with a dense, amorphous, fluidlike material ( F d ) . M, microvilli. (Rabbit ovary, 8 hours post coiturn, x 14,150.)
15 This stereo pair probably illustrates the process of ovulation. The apex of the follicle is ruptured and the oocyte, immersed in a large amount of fluid and cytoplasmic debris, appears to emerge from within the follicular cavity (the oocyte is visible i n the center of the micrograph). (Mouse ovary, from an animal in the estrous phase, X 230. )
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SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan V a n Blerkom
PLATE 6
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PLATE 7 EXPLANATION OF FIGURE
16
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This scanning electron micrograph illustrates on oocyte ( O o ) , possibly at the mount of ovulation. The surface of the egg is only partially covered with corona cells ( G ) . A large quantity of fluid and cellular debris (F) surrounds the ruptured area through which the oocyte emerges. (Mouse ovary, from an animal in the estrous phase, X 3,500.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 7
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PLATE 8 E X P L A N A T I O N OF F I G U R E S
17 This scanning electron micrograph shows the ruptured area of a follicle just after ovulation ( R ) . Large numbers of follicle cells immersed in fluid ( F ) cover regions adjacent to the ruptured follicle ( R ) . The egg (Oo), which has just escaped from the follicle and is covered by a mass of corona cells, is visible just under the ruptured area. (Rabbit ovary, 9 hours post coitum, x 195.) A
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This figure is a higher magnification of the area (00) indicated in figure 17. The oocyte is not directly visible because it is covered by a mass of follicle cells mixed with spermatozoa (Sp). The spermatozoa (Sp). The spermatozoa have traveled through the reproductive tract and reached the surface of the ovary i n approximately nine hours. x 1,720.
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 8
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PLATE 9 EXPLANATION OF FIGURES
18
The appearance of follicle cells ( F ) in areas adjacent to the oocyte at ovulation is illustrated in this electron micrograph. These cells are irregular or polyhedral in shape and, due to the presence of a fine, granular material, appear to have rough surfaces (arrows). (Mouse ovary, from an animal in the estrous phase, x 5,350.)
19 This scanning electron micrograph shows a cluster of follicle cells ( F ) attached to the zona pellucida (Zp) which covers the oocyte (00). The irregular surface of the zona pellucida is also clearly evident. (Mouse ovary, from a n animal i n the estrous phase, x 2,380.)
20,21
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These electron micrographs show the surface of the zona pellucida at higher magnification. The zona pellucida is a dense, compact and amorphous membrane containing infoldings, channels and crypts that are often in continuity within the zona. The surface of the zona (fig. 21) is covered with a fine, granular material (arrows). (Mouse ovary, from an animal in the estrous phase, x 5,800 and x 10,800.)
SCANNING ELECTRON MICROSCOPY OF OVULATION Pietro Motta and Jonathan Van Blerkom
PLATE 9
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