Surface Epithelium of the Fetal Guinea-pig Ovary : A Light and Electron Microscopic Study TH. JEPPESEN Institute of A n a t o m y B , University of Aarhus, Denmark
ABSTRACT The surface epithelium in fetal guinea-pig ovaries was examined from the time of early sexual differentiation, about 34 days, until approximately ten days before birth. At day 34 the epithelium varied greatly in appearance as seen in the light microscope and possessed a superficial layer of flattened or cuboidal cells. By day 58 the epithelium had changed into one layer of regularly arranged columnar cells. During the same period the number of germinal cells decreased. Connections between the germinal cords and the surface epithelium were observed from day 34, being especially numerous and broad at days 34 and 42 and decreasing in number and size from day 46 onwards. The basement membrane beneath the epithelium gradually increased in thickness. In the electron microscope two types of somatic cells could be distinguished. One type formed a superficial single layer connected by junctional complexes and exhibited intracellular bundles of 60 A microfilaments running straight through the apical part of the cell, attached to junctional complexes on either side. These bundles were found frequently between days 34 and 42, but were rarely seen after the forty-sixth day. Microvillous projections into the coelomic cavity were especially numerous from day 34 to day 46. The other type of somatic cells lay in close proximity to the germinal cells: microfilaments or junctional complexes were not observed. The subepithelial basement lamina was continuous with that surrounding the connections and the germinal cords.
The role of the surface epithelium in the organogenesis of the mammalian ovary has been a matter of debate since the beginning of this century. While there is general agreement as regards the formation of the multilayered surface epithelium by proliferation of the mesothelial cells of the coelomic epithelium, considerable differences of opinion exist as regards the further development of the surface epithelium. Most authors (de Winiwarter, '01, '10; Sainmont, '06; Allen, '04; Simkins, '28; van Wagenen and Simpson, '65) have concluded that germinal cords originate from the surface epithelium, and many have claimed that the somatic cells of the germinal cords are the source of the follicular cells (Mossman, '38; Harrison and Matthews, '51; Boyd and Hamilton, '55; Arey, '65; Franchi and Mandl, '62). On the other hand others have found a solid cortex without clearly demarcated ANAT. REC., 183: 499-516.
germinal cords (Brambell, '27; Torrey, '45; Witschi, '51; Gropp and Ohno, '66) and have suggested that follicular cells develop from a common embryonic blastema, situated deep in the central part of the undifferentiated ovary. Bookhout ('45 j has described the development of the surface epithelium i n the fetal guinea-pig ovary. He found a first and second proliferation from the surface epithelium resulting in two separate sets of germinal cords (medullary and cortical ) . Previous work on the ultrastructure of the surface epithelium of the ovary is scanty (Yamada et al., '57; Wischnitzer, '65; Weakley, '67, '69). Only the latter author has examined the appearance of the surface epithelium in the fetal period and only from the last prenatal day, in the hamster. Thus there exist no published Received Feb. 10, '75. Accepted May 27, '75.
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ultrastructural data on the ontogenesis of the surface epithelium. The aim of this examination was to study the development of the surface epithelium in the guinea-pig ovary during the fetal period as seen in the light and electron microscope in order to obtain new information on the role of the surface epithelium in the morphogenesis of the ovary. MATERIAL AND METHODS
Female fetuses from albino guinea-pigs of the short-haired British type were examined at the following ages : gestational days 34, 38, 42, 46, 54, and 58. The gestational age was ascertained from the presence of sperm in vaginal smears within 24 hours after delivery. The crownrump length of the fetuses was measured and found to be in accordance with the findings of Draper ('20). Preparation. Pregnant guinea-pigs were anesthetized with nembutal, and the fetuses delivered by cesarean section. Each fetus, with the umbilical cord still intact, was then perfused with a fixative at 100 m m Hg pressure through the left ventricle, the right atrium being opened after the insertion of the needle. The fixative was 2% glutaraldehyde i n cacodylate buffer (Sabatini et al., '63) with 2% dextran T 40 (Bohrnan and Maunsbach, '70) at 2-4°C (pH 7.4). Ovaries were removed and fixed for a further three hours at the same temperature by immersion in the same fixative. The ovaries were then cut under a dissecting microscope, first longitudinally into two halves, and each half was then thinly sliced. Further processing included postfixation with 1% osmium tetroxide i n Veronal-acetate buffer, pH 7.2-7.4 (Sjostrand, '67) and block-staining for one hour with 0.5% Uranyl-acetate in the same buffer, pH 5.9 (Kellenberger et al., '58). After dehydration in alcohol and propylene oxide or in acetone the tissue was embedded in Epon 812 or Vestopal. Sections 1 EL thick were stained with 1% toluidine blue and examined in the light microscope. Suitable sections were taken as those cut approximately at right angles to the epithelial surface. After trimming, grey-silver sections were cut on a n LKB-microtome, stained with led citrate
(Reynolds, '63) and then examined in a Siemens Elmiscop 1 A. RESULTS
Light-microscope observations Day 34 The thickness of the surface epithelium in sections cut approximately at right angles to the surface varied considerably so that between one and four cell layers could be identified (fig. 1) , The cells themselves were also heterogeneous, both as regards shape and orientation. The surface of the epithelium consisted of one continuous layer of cells, of which most were flattened, but occasionally small areas with columnar cells were seen. Some of the superficial cells were cone shaped with their bases facing the coelomic cavity. At other places, especially where cone shaped or columnar cells were seen, the superficial cells bulged into the coelomic cavity. Beneath the superficial cell layer numerous germinal cells, as recognized from their globoid shape, were seen. The germinal cells lay either singularly or in small groups of 2-3. They were surrounded by somatic cells of variable appearance and orientation. Most of these latter cells were sickle shaped or irregularly polygonal, and lay in close proximity to the germinal cells, often enveloping the germinal cells with long, thin cytoplasmic processes. The size of the somatic cells, both in the superficial layer and the ones beneath, varied between 6 x 5 and 4 x 3 p, The shape of the somatic cell nuclei corresponded in general to that of the cell outline and their size varied between 4.5 x 3.5 and 3 x 3 p. Commonly one or two peripherally located nucleoli (size 1 X 1 to 1.3 X 0.6 were seen. Beneath the epithelium a thin layer of mesenchyme was observed. Stretching down from the epithelium into the gonadal anlage, loosely arranged cellular strands were observed, connecting the epithelium to the germinal cords (arrow in fig. 1 ) . These cellular strands contained germinal cells and somatic cells, of the same appearance as in the surface epithelium. At day 38 (fig. 2) one striking change was observed: the cells were much more tightly packed, both in the epithelium and
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY
in the cellular streaks, resulting in a very clear delimitation between the epithelium and the underlying loose mesenchyme. The connecting cellular strands were numerous (arrows in fig. 2 ) . As regards epithelial thickness, cellular shape, orientation and size these were the same as for day 34. The appearance of the surface epithelium at days 42, 46, 54, and 58 (figs. 3, 4, 5) changed gradually. The changes were as follows: The connecting cellular strands at day 42 were very numerous and in some projections very broad (maximally 5-6 cells broad) (arrows in fig. 3 ) . At day 46 they were not so numerous and were more slender (fig. 4 ) , while at days 54 and 58 (fig. 5) only occasional and very slender connecting cellular strands were seen. At the sites in the epithelium, where connecting cellular strands were seen, one could still observe great variability as regards cellular shape and orientation as described above, but in the continuously growing areas of surface epithelium, where there were no connecting cellular strands, the cellular arrangement in the superficial layer became progressively more regular, with the accumulation of columnar epithelial cells. The proportion of germinal cells in the surface epithelium was great at day 42, but steadily decreased from day 46, and germinal cells were rarely seen at day 58 (compare figs. 1, 2, 3, 4, 5 ) . The thickness of the epithelium i n sections oriented approximately at right angle to the surface varied from 2-4-cell layers at day 42 to predominantly 2-cell layers at day 46, and 1-2-cell layers at day 54. At day 58 the surface epithelium appeared predominantly as a one-layered reguIar epithelium, composed of columnar cells. Connecting cellular strands were very rarely seen and the number of germinal cells in the epithelium was very small. Throughout the examined period a subepithelial basement membrane was identified (figs. 1-5). This basement membrane increased steadily in thickness to a maximum of about 2 p. The basement membrane was continuous with a basement membrane surrounding the connecting cellular strands and the germinal cords.
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Mitoses of the somatic cells were only rarely observed in the surface epithelium.
Electron microscope observations Day 34 At low magnification a consistent difference could be observed between the continuous superficial layer of cells and the somatic cells lying beneath in the surface epithelium: the superficial cells were invariably connected to each other by junctional complexes about 0.3 to 0.4 in length (figs. 6-9), situated close to the coelomic cavity, The superficial cell membrane exhibited a moderate number of microvillous projections, about 0.3 in width and up to about 0.8 in length (fig. 6 ) . Associated with the junctional complexes almost linear filament bundles of varying length were frequently observed, running, as a rule, parallel to the superficial cell membrane (arrows in fig. 6 ) . At a magnification of X 30,000 (fig. 7) the filament bundles appeared more clearly, running through the apical parts of cells. I n some sections the filaments were observed running parallel to the superficial cell membrane attached to junctional complexes on either side )fig. 7 ) . The thickness of the filament bundles was 0.1 to 0.2 p. In other cells filament bundles of varying length were seen, presumably arising from different angles of sectioning, and originating from the junctional complexes. As a rule similar filament bundles were seen in adjacent cells. At high magnification (fig. 8 ) the filaments within the bundles exhibited a thickness of about 60 A. Some filaments appeared to end in or close to the membrane of the junctional complexes. Where filaments were only seen i n one cell, the junctional complexes were curved inwards in the direction of the attached filament bundle (fig. 7 ) . I n favourable sections the most luminal and basal part of the junctional complex appeared a s a five-layered structure (arrows in fig. 8 ) . The luminal and basal parts, at least, constituted zonulae occludentes or tight junctions (Farquhar and Palade, '63). The filament bundles spread out fan-like near the junction and the filaments inserted in or at the inner
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leaflet of the trilaminar cell membrane (fig. 8 ) . The inset to the left in figure 8 shows microfilaments in cross-section (arrows). The inset to the right in figure 8 shows another junctional complex, appearing as a zonula occludens or tight junction with tiny gaps i n it. Zonulae adhaerentes (intermediate junctions) or maculae adhaerentes (desmosomes) were not observed. In the somatic cells lying beneath the continuous superficial layer no desmosome-like structures or filament bundles were observed. Apart from this the ultrastructure of the somatic cells (fig. 1 0 ) did not differ from that of the superficial cells (fig. 9 ) . Thus, the mitochondria were spherical to elongate in shape. Sparse amounts of granular rough-surfaced endoplasmic reticulum and small Golgi-regions were occasionally seen. Neither smoothsurf aced endoplasmic reticulum nor lipid inclusions were observed. The nuclei contained dispersed chromatin and one or two nucleoli. Under the epithelium, delimiting it from the mesenchyme, a basement lamina was seen. It was about 200 A thick and consisted of loose granular material.
Day 38 The cells were much more tightly packed, as already observed in the light microscope, and only few intercellular gaps were seen. The intercellular space was mostly 2-300 A in width (fig. 11). The epithelium was now clearly delimited from the underlying mesenchyme by a basal lamina, about 100 A thick, consisting of homogenous material. Outside this there was a zone 1-2 thick, containing looser fibrillar material. The surface of the superficial cells exhibited more microvillous processes, there being up to ten or more in a single section. These processes varied i n length u p to about 0.8 and in width up to about 0.4 p. Cells with many processes invariably showed the same filament bundles as seen at day 34. The morphology of the superficial cells as regards filament bundles (arrows), junctional complexes and organelles were apparently unchanged from day 34 (fig. 11). The somatic cells in the surface epithelium lying beneath the super-
fical cell layer were also unchanged. The latter lay in intimate relation to the germinal cells (fig. 11). Connecting cellular strands were seen, surrounded by a continuation of the subepithelial basal lamina, and containing germinal cells and somatic cells of the same morphology as observed in the surface epithelium. There was direct continuity between the epithelium and the connecting cellular strands.
Day 42 The surface epithelium had the same appearance as at day 38. Numerous microvillous processes and filament bundles were seen in the superficial cell layer as well as many connecting cellular strands. Day 46 The most striking development by day 46 was the virtual absence of filament bundles. Microvillous processes were also scarce. Other ultrastructural features remained unchanged. Thus, junctional complexes were still present, occasionally with a length of several p , and the basal lamina was about 4,000 A in thickness.
Day 54 The cells were tightly packed with the superficial cells forming a continuous layer of predominantly columnar cells showing a few microvillous projections, but no filament bundles, Adjoining cells were connected by junctional complexes. Some of the nuclei exhibited deep indentations. I n many places the epithelium contained only the superfiicial cells, that is it was only a single cell layer, while in other places small sub-epithelial nests of somatic cells occurred. Very few germinal cells were seen. Day 58 At this stage the epithelium occurred as a single layer of columnar cells. Junctional complexes were still seen, but no microfilament bundles and only very few microvillous projections were seen. Some of the cells showed several junctional complexes along their adjoining membranes. The basal lamina was 1 thick. No germinal cells were seen in the epithelium.
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY DISCUSSION
The main findings of the present study may be summarized as follows: ( 1 ) In the superficial cells straight bundles of fiIaments and intercellular junctional complexes were identified, features which clearly distinguished these cells from those in the underlying cell layers. (2) The subepithelial basal lamina was continuous with the basil lamina surrounding the germinal cords via the attachments of the latter to the epithelium. The nature and function of the observed microfilaments is as yet undetermined. From their size and straightness they resemble the microfilaments or actin-like filaments identified in many other cell types (Cloney, '66; Giacomelli et al., '70; Goldman and Knipe, '73; Newstead, '71; Ross and Reith '70; Rostgaard, '72; Wessells et al., '71; Wren and Wessells, '69; Yamada et al., '70) and where they have been presumed to serve a contractile role. Bundles of thin microfilaments have often been described during embryogenesis: Yamada et al. ('70) found 50 A filaments in growing axons, Cloney ('66) found 50-70 A filaments in contracting epidermal cells in ascidia during their metamorphosis and Wrenn and Wessells ('69) found microfilaments in the apical regions of invaginating cells in the lens placode. Further, when tubular glands are formed in the chick oviduct by invagination from the epithelial surface, this is accompanied by the appearance of 50 A filaments in the apical region of the epithelial cells (Wessells et al., '71). Similar filaments are seen during the formation of salivary glands. In summary, it has been repeatedly observed that at the time of invagination of an epithelial surface bundles of linearly running filaments appear in the apical part of the invaginating cells, concurrent with a contraction of their apical parts, transforming the cells into a cone-like shape. In the light of these findings it seems likely that the bundles of filaments described here may serve a contractile role during the development of the surface epithelium. What this role may be remains obscure, but one possibility is that the filaments serve to contract the superficial cell
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layer with the added result that the underlying cells are forced downward into the gonadal anlage. The change in shape of the superficial cells from a squamous to a columnar form might also be expected as a result of the contraction of this cell layer. A contraction of the superficial cells does not, however, explain immediately why all the cells beneath are forced downwards. This must involve other mechanisms such as active amoeboid movement of the germ cells themselves as has been suggested by Witschi ('51) as the means of migration of the g e m cells from their extragonadal origin to the gonadal anlage. A similar mechanism may be responsible for their subsequent movements down into the germinal cords. As indicated in the opening paragraphs of this paper, light microscope studies of the fetal development of the mammalian ovary has given rise to conflicting interpretations as regards the differentiation and functional role of the surface epithelium. Histochemical examinations of cattle (Gropp and Ohno, '66) were interpreted as showing that a central blastema was first formed, presumably from the mesonephros, and the follicular cells then originated from this central blastema. Similarly from a study of human embryos and fetuses (Pinkerton et al., '61) it was claimed that a central blastema of mesenchymal cells first developed, and that this gave rise to the follicular cells. Both these latter authors claimed that the surface epithelium is inactive as regards the formation of germinal cords and the follicular cells. Previous studies of ovaries in hamster (Weakley, '67) and in mice (Wischnitzer, '65; Yamada et al., '57) have shown that the surface epithelium consisted of a single layer of flattened cells separated from the underlying tissue by a basal lamina. In one case (Weakley, '67) attachment zones between the cells and also microvillous projections were observed but none of these authors noted the presence of intracellular filaments. It is noteworthy that in the present study no filaments were found at day 54 and 58. It seems that they are only present at the time that changes in cell shape take place (between days 34 and 42, and
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- to a small degree - at day 46). The somatic cells that are located beneath the superficial cell layer, but i n the surface epithelium, i.e. on the epithelial side of the basal lamina, are according to their morphology and localization prefollicular cells. They are more or less identical to the follicular cells that Odor and Blandau ('69) describe in the fetal mouse ovary and that Gondos ('70) found in the newborn rabbit ovary, in close proximity to the germinal cells, and enveloping the latter with their thin cytoplasmic processes. The origin of these cells cannot be determined from present data. It is probable, however, that they arise from the superficial cells, since they lie on the epithelial side of the basal lamina. Possibly, some of the superficial cells invaginate and transform into follicular cells. An alternative explanation is that these somatic cells acconipany the germinal cells from their extragonadal site of origin and invade the surface epithelium together with the germinal cells. It is a fact that the germinal cells and their accompanying prefollicular cells are located within the surface epithelium (i.e. superficial to the subepithelial basal lamina) and are surrounded by a basal lamina common to the germinal cords and the connexions between the surface epithelium and the cords. This clearly indicates that the germinal cords are derived from the surface epithelium and that the prefollicular cells are of epithelial origin. Recently, however, Byskov and LinternMoore ('73) have demonstrated free communications between tubules belonging to the rete ovarii and the medullary end of the germinal cords indicating that the rete ovarii contributes to the formation of the germinal cords. LITERATURE CITED Allen, B. M. 1904 The embryonic develop ment of the ovary and testis of the mammals. Am. J. Anat., 3: 89-146. Arey, L. B. 1965 Developmental Anatomy. Seventh ed. W. B. Saunders Co., London. Bohman, S . O., and A. B. Maunsbach 1970 Effect on tissue fine structure of variations in colloid osmotic pressure of glutaraldehyde fixatives. J. Ultrastruct. Res., 30: 195-208. Bookhout, C. G. 1945 The development of the guinea-pig ovary from sexual differentiation to maturity. J. Morph., 77: 233-263.
Boyd, J. D., and W. J. Hamilton 1955 The cellular elements of the human ovary. In: Modern Trends in Obstetrics and Gynecology (Scand. series). K. Bowes, ed. Butterworth and Co. Ltd., London, pp. 50-78. Brambell, F. W. R. 1927 The development and morphology of the gonads of the mouse. Proc. roy. SOC. B, 101: 391-409. Byskov, A. G . S., and S., and S. Lintern-Moore 1973 Follicle formation in the immature mouse ovary: the role of the rete ovarii. J. Anat., 116: 207-217. Cloney, R. A. 1966 Cytoplasmic filaments and cell movements: Epidermal cells during ascidian metamorphosis. J. Ultrastruct. Res., 14: 300-328. Draper, R. L. 1920 The prenatal growth of the guinea-pig. Anat. Rec., 18: 369-392. Farquhar, M. G . , and G . E. Palade 1963 Junctional complexes in various epithelia. J. Cell Biol., 17: 375-412. Franchi, L. L., and A. M. Mandl 1962 The ultrastructure of oogonia and oocytes in the foetal and neonatal rat. Proc. roy. SOC.B, 157: 99-114. Giacomelli, F., J. Wiener and D. Spiro 1970 Cross-striated arrays of filaments i n endothelium. J. Cell Biol., 45: 188-192. Goldman, R. D., and D. M. Knipe 1973 Functions of cytoplasmic fibers in non-muscle cell motility. In: Symposia on Quantitative Biology. Vol. 37. Cold Spring Harbor Laboratory, pp. 523-534. Gondos, B. 1970 Granulosa cell-germ cell relationship i n the developing rabbit ovary. J. Embryol. exp. Morph., 23: 419426. Gropp, A,, and S. Ohno 1966 The presence of a common embryonic blastema for ovarian a n d testicular parenchymal (follicular, interstitial and tubular) cells in cattle, Bos Taurus. 2. Zellforsch., 74: 505-528. Harrison, R. J., and L. H. Matthews 1951 Subsurface crypts in the cortex of the mammalian ovary. Proc. zool. SOC.(London), 120: 699-712. Kellenberger, E., A. Ryter and J. Sechaud 1958 Electron microscope study of DNA containing plasm. J. biophys. biochem. Cytol., 4: 671. Mossman, H. W. 1938 The homology of the vesicular ovarian follicles of the mammalian ovary with the coelom. Anat. Res., 7 0 : 643-652. Newstead, J. D. 1971 Filaments in renal parenchymal and interstitial cells. J. Ultrastruct. Res., 34: 316-328. Odor, D. S., and R. J. Blandau 1969 Ultrastructural studies on fetal and early postnatal mouse ovaries. I. Histogenesis and organogenesis. Am. J. Anat., 124: 163-186. Pinkerton, J. H. M., D. G. McKay, E. C. Adams and A. T. Hertig 1961 Development of the human ovary - a study using histochemical technics. Obstet. and Gynec., 18: 152-181. Reynolds, E. S. 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 2 7: 208-212. Ross, M. H., and E. J . Keith 1970 Myoid elements in the mammalian nephron and their relationship to other specializations in the
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY basal part of kidney cells. Am. J. Anat., 129: 399-416. RostgBrd, J. 1972 Electron microscopy of filaments in the basal part of rat kidney tubule cells, and their in situ interaction with heavy meromyosin. 2. Zellforsch, 132: 497-521. Sabatini, D. D., K. Bensch and R. J. Barrnett 1963 Cytochemistry and electronmicroscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19-58. Sainmont, G. 1906 Recherches relatives B l’organogen6se du testicule et de l’ovaire chez le chat. Arch. Biol. (Paris), 22: 71. Sjostrand, F. S. 1967 Electron Microscopy of Cells and Tissues. Academic Press Inc., New York and London, pp 144-146. Simkins, C. S. 1928 Origin of the sex cells in man. Am. J. Anat., 41: 249-293. Torrey, T. W. 1945 The development of the urinogenital system of the albino rats. 11. The gonads. Am. J. Anat., 76: 375-397. Wagenen, G. van, and M. E. Simpson 1965 Embryology of the ovary and testis. Homo sapiens and macaca mulatta. Yale University Press, New Haven and London. Weakley, B. S. 1967 Light and electron microscopy of developing germ cells and follicle cells i n the ovary of the golden hamster: twenty-four hours before birth to eight days post partum. J. Anat., 107: 435-459. 1969 Differentiation of the surface epi-
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thelium of the hamster ovary. A n electron microscopic study. J. Anat., 105: 129-147. Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. Luduena, E. L. Taylor, J. T. Wrenn and K. M. Yamada 1971 Microfilaments in cellular and developmental processes. Science, 171: 135-143. Winiwarter, H. de 1901 Recherches sur l’ovogengse et l’organogen6se de l’ovaires des mammiferes (lapin et homme). Arch. Biol. (Paris), 17: 33-199. 1910 Contribution 2 l’etude de l’ovaire humain. Arch. Biol. (Paris), 25: 683-756. Wischnitzer, S. 1965 The ultrastructure of the germinal epithelium of the mouse ovary. J. Morph., 117: 387-400. Witschi, E. 1951 Gonad development and function. Embryogenesis of the adrenal and the reproductive glands. Recent Progr. Hormone Res., 6: 1-27. Wrenn, J. T., and N. K. Wessells 1969 A n ultrastructural study of lens invagination in the mouse. J. Exp. Zool., 171: 359-368. Yamada, E., T. Muta, A, Motomura and H. Koga 1957 The fine structure of the oocyte in the mouse ovary studied with the electron microscope. Kurume med. J., 4: 148-160. Yamada, K. M., B. S. Spooner and N. K. Wessells 1970- Axon growth: Roles of microfilaments and microtubules. Proc. nat. Acad. Sci., 66: 1206-1212.
PLATE 1 EXPLANATION O F FIGURES
1
Light microscopy of fetal guinea-pig ovary, day 34, showing the variability in thickness of the epithelium and of cell shape and orientation. Germinal cells ( G ) are present in the epithelium. Connecting cellular strands are seen running obliquely down into the gonadal anlage (arrows). x 300.
2
Light microscopy of fetal guinea-pig ovary, day 38. Numerous germ cells in the epithelium and many connecting cellular strands, continuous with the germinal cords, are seen. The cells in the epithelium and the connecting cellular strands (arrow) are considerably more compact as compared to day 34. x 300.
3 Light microscopy of fetal guinea-pig ovary, day 42. Numerous germinal cells are seen in the surface epithelium as well as many broad connecting cellular strands. The superficial cells are in part flattened, in part cuboidal. The basement membrane is clearly seen (arrow). X 300. 4
Light microscopy of fetal guinea-pig ovary, day 46. Only a few germinal cells may be seen in the surface epithelium and there are few and slender connecting cellular strands. The surf ace epithelium consists mainly of three layers of cuboidal cells. A basal membrane is seen under the epithelium and is continuous with that surrounding the connecting cellular strand (arrow). x 300.
5 Light microscopy of fetal guinea-pig ovary, day 58. The surface epithelium appears as a single-layered regular cylindrical sheet, separated from the underlying mesenchyme by a thick basal membrane. No germinal cells are seen in the surface epithelium. Only one very slender connecting cellular strand is seen (arrow). x 300.
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?LATE 2 EXPLANATION O F FIGURE
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Surface epithelium, guinea-pig ovary, day 34. The continuous layer of superficial cells is seen with their junctional complexes and associated bundles of filaments (arrows). A few microvillous projections are seen. EL, basal lamina. x 6,700.
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PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
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Superficial cell of the surface epithelium, day 34. A bundle of filaments runs straight through the apical part of the cell, attached to junctional complexes (arrows), one of which is curved inwards. X 30,000.
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Junctional complex and filaments a t high magnification. The insertion of the filaments i n or at the junctional complex as well as the straight course of the single filament (arrows) is apparent. Individual filaments are about 60 A in thickness. x 120,000. Inset to the right: Junctional complex at day 46. Small gaps within the tight junction are seen ( G ) . x 120,000. Inset to the left: Shows the microfilaments in cross-section (arrow). x 120,000.
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PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
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Superficial cell in the surface epithelium showing the junctional complexes and the straight bundles of filaments originating from them. x 11,000.
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Somatic cell in the surface epithelium, located beneath the superficial cell layer. No junctional complexes or filaments are seen. x 11,000.
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PLATE 5 EXPLANATION OF FIGURE
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Surface epithelium. day 38. The superficial cells still show junctional complexes and filament bundles (arrows). There are numerous microvillous projections. The compactness of the epithelium is increased. Beneath the superficial layer somatic cells (prefollicular cells) are seen i n close proximity to the germinal cells. B. basement lamina. x 4,050.
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ABSTRACT The surface epithelium in fetal guinea-pig ovaries was examined from the time of early sexual differentiation, about 34 days, until approximately ten days before birth. At day 34 the epithelium varied greatly in appearance as seen in the light microscope and possessed a superficial layer of flattened or cuboidal cells. By day 58 the epithelium had changed into one layer of regularly arranged columnar cells. During the same period the number of germinal cells decreased. Connections between the germinal cords and the surface epithelium were observed from day 34, being especially numerous and broad at days 34 and 42 and decreasing in number and size from day 46 onwards. The basement membrane beneath the epithelium gradually increased in thickness. In the electron microscope two types of somatic cells could be distinguished. One type formed a superficial single layer connected by junctional complexes and exhibited intracellular bundles of 60 A microfilaments running straight through the apical part of the cell, attached to junctional complexes on either side. These bundles were found frequently between days 34 and 42, but were rarely seen after the forty-sixth day. Microvillous projections into the coelomic cavity were especially numerous from day 34 to day 46. The other type of somatic cells lay in close proximity to the germinal cells: microfilaments or junctional complexes were not observed. The subepithelial basement lamina was continuous with that surrounding the connections and the germinal cords.
The role of the surface epithelium in the organogenesis of the mammalian ovary has been a matter of debate since the beginning of this century. While there is general agreement as regards the formation of the multilayered surface epithelium by proliferation of the mesothelial cells of the coelomic epithelium, considerable differences of opinion exist as regards the further development of the surface epithelium. Most authors (de Winiwarter, '01, '10; Sainmont, '06; Allen, '04; Simkins, '28; van Wagenen and Simpson, '65) have concluded that germinal cords originate from the surface epithelium, and many have claimed that the somatic cells of the germinal cords are the source of the follicular cells (Mossman, '38; Harrison and Matthews, '51; Boyd and Hamilton, '55; Arey, '65; Franchi and Mandl, '62). On the other hand others have found a solid cortex without clearly demarcated ANAT. REC., 183: 499-516.
germinal cords (Brambell, '27; Torrey, '45; Witschi, '51; Gropp and Ohno, '66) and have suggested that follicular cells develop from a common embryonic blastema, situated deep in the central part of the undifferentiated ovary. Bookhout ('45 j has described the development of the surface epithelium i n the fetal guinea-pig ovary. He found a first and second proliferation from the surface epithelium resulting in two separate sets of germinal cords (medullary and cortical ) . Previous work on the ultrastructure of the surface epithelium of the ovary is scanty (Yamada et al., '57; Wischnitzer, '65; Weakley, '67, '69). Only the latter author has examined the appearance of the surface epithelium in the fetal period and only from the last prenatal day, in the hamster. Thus there exist no published Received Feb. 10, '75. Accepted May 27, '75.
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ultrastructural data on the ontogenesis of the surface epithelium. The aim of this examination was to study the development of the surface epithelium in the guinea-pig ovary during the fetal period as seen in the light and electron microscope in order to obtain new information on the role of the surface epithelium in the morphogenesis of the ovary. MATERIAL AND METHODS
Female fetuses from albino guinea-pigs of the short-haired British type were examined at the following ages : gestational days 34, 38, 42, 46, 54, and 58. The gestational age was ascertained from the presence of sperm in vaginal smears within 24 hours after delivery. The crownrump length of the fetuses was measured and found to be in accordance with the findings of Draper ('20). Preparation. Pregnant guinea-pigs were anesthetized with nembutal, and the fetuses delivered by cesarean section. Each fetus, with the umbilical cord still intact, was then perfused with a fixative at 100 m m Hg pressure through the left ventricle, the right atrium being opened after the insertion of the needle. The fixative was 2% glutaraldehyde i n cacodylate buffer (Sabatini et al., '63) with 2% dextran T 40 (Bohrnan and Maunsbach, '70) at 2-4°C (pH 7.4). Ovaries were removed and fixed for a further three hours at the same temperature by immersion in the same fixative. The ovaries were then cut under a dissecting microscope, first longitudinally into two halves, and each half was then thinly sliced. Further processing included postfixation with 1% osmium tetroxide i n Veronal-acetate buffer, pH 7.2-7.4 (Sjostrand, '67) and block-staining for one hour with 0.5% Uranyl-acetate in the same buffer, pH 5.9 (Kellenberger et al., '58). After dehydration in alcohol and propylene oxide or in acetone the tissue was embedded in Epon 812 or Vestopal. Sections 1 EL thick were stained with 1% toluidine blue and examined in the light microscope. Suitable sections were taken as those cut approximately at right angles to the epithelial surface. After trimming, grey-silver sections were cut on a n LKB-microtome, stained with led citrate
(Reynolds, '63) and then examined in a Siemens Elmiscop 1 A. RESULTS
Light-microscope observations Day 34 The thickness of the surface epithelium in sections cut approximately at right angles to the surface varied considerably so that between one and four cell layers could be identified (fig. 1) , The cells themselves were also heterogeneous, both as regards shape and orientation. The surface of the epithelium consisted of one continuous layer of cells, of which most were flattened, but occasionally small areas with columnar cells were seen. Some of the superficial cells were cone shaped with their bases facing the coelomic cavity. At other places, especially where cone shaped or columnar cells were seen, the superficial cells bulged into the coelomic cavity. Beneath the superficial cell layer numerous germinal cells, as recognized from their globoid shape, were seen. The germinal cells lay either singularly or in small groups of 2-3. They were surrounded by somatic cells of variable appearance and orientation. Most of these latter cells were sickle shaped or irregularly polygonal, and lay in close proximity to the germinal cells, often enveloping the germinal cells with long, thin cytoplasmic processes. The size of the somatic cells, both in the superficial layer and the ones beneath, varied between 6 x 5 and 4 x 3 p, The shape of the somatic cell nuclei corresponded in general to that of the cell outline and their size varied between 4.5 x 3.5 and 3 x 3 p. Commonly one or two peripherally located nucleoli (size 1 X 1 to 1.3 X 0.6 were seen. Beneath the epithelium a thin layer of mesenchyme was observed. Stretching down from the epithelium into the gonadal anlage, loosely arranged cellular strands were observed, connecting the epithelium to the germinal cords (arrow in fig. 1 ) . These cellular strands contained germinal cells and somatic cells, of the same appearance as in the surface epithelium. At day 38 (fig. 2) one striking change was observed: the cells were much more tightly packed, both in the epithelium and
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY
in the cellular streaks, resulting in a very clear delimitation between the epithelium and the underlying loose mesenchyme. The connecting cellular strands were numerous (arrows in fig. 2 ) . As regards epithelial thickness, cellular shape, orientation and size these were the same as for day 34. The appearance of the surface epithelium at days 42, 46, 54, and 58 (figs. 3, 4, 5) changed gradually. The changes were as follows: The connecting cellular strands at day 42 were very numerous and in some projections very broad (maximally 5-6 cells broad) (arrows in fig. 3 ) . At day 46 they were not so numerous and were more slender (fig. 4 ) , while at days 54 and 58 (fig. 5) only occasional and very slender connecting cellular strands were seen. At the sites in the epithelium, where connecting cellular strands were seen, one could still observe great variability as regards cellular shape and orientation as described above, but in the continuously growing areas of surface epithelium, where there were no connecting cellular strands, the cellular arrangement in the superficial layer became progressively more regular, with the accumulation of columnar epithelial cells. The proportion of germinal cells in the surface epithelium was great at day 42, but steadily decreased from day 46, and germinal cells were rarely seen at day 58 (compare figs. 1, 2, 3, 4, 5 ) . The thickness of the epithelium i n sections oriented approximately at right angle to the surface varied from 2-4-cell layers at day 42 to predominantly 2-cell layers at day 46, and 1-2-cell layers at day 54. At day 58 the surface epithelium appeared predominantly as a one-layered reguIar epithelium, composed of columnar cells. Connecting cellular strands were very rarely seen and the number of germinal cells in the epithelium was very small. Throughout the examined period a subepithelial basement membrane was identified (figs. 1-5). This basement membrane increased steadily in thickness to a maximum of about 2 p. The basement membrane was continuous with a basement membrane surrounding the connecting cellular strands and the germinal cords.
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Mitoses of the somatic cells were only rarely observed in the surface epithelium.
Electron microscope observations Day 34 At low magnification a consistent difference could be observed between the continuous superficial layer of cells and the somatic cells lying beneath in the surface epithelium: the superficial cells were invariably connected to each other by junctional complexes about 0.3 to 0.4 in length (figs. 6-9), situated close to the coelomic cavity, The superficial cell membrane exhibited a moderate number of microvillous projections, about 0.3 in width and up to about 0.8 in length (fig. 6 ) . Associated with the junctional complexes almost linear filament bundles of varying length were frequently observed, running, as a rule, parallel to the superficial cell membrane (arrows in fig. 6 ) . At a magnification of X 30,000 (fig. 7) the filament bundles appeared more clearly, running through the apical parts of cells. I n some sections the filaments were observed running parallel to the superficial cell membrane attached to junctional complexes on either side )fig. 7 ) . The thickness of the filament bundles was 0.1 to 0.2 p. In other cells filament bundles of varying length were seen, presumably arising from different angles of sectioning, and originating from the junctional complexes. As a rule similar filament bundles were seen in adjacent cells. At high magnification (fig. 8 ) the filaments within the bundles exhibited a thickness of about 60 A. Some filaments appeared to end in or close to the membrane of the junctional complexes. Where filaments were only seen i n one cell, the junctional complexes were curved inwards in the direction of the attached filament bundle (fig. 7 ) . I n favourable sections the most luminal and basal part of the junctional complex appeared a s a five-layered structure (arrows in fig. 8 ) . The luminal and basal parts, at least, constituted zonulae occludentes or tight junctions (Farquhar and Palade, '63). The filament bundles spread out fan-like near the junction and the filaments inserted in or at the inner
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TH. JEPPESEN
leaflet of the trilaminar cell membrane (fig. 8 ) . The inset to the left in figure 8 shows microfilaments in cross-section (arrows). The inset to the right in figure 8 shows another junctional complex, appearing as a zonula occludens or tight junction with tiny gaps i n it. Zonulae adhaerentes (intermediate junctions) or maculae adhaerentes (desmosomes) were not observed. In the somatic cells lying beneath the continuous superficial layer no desmosome-like structures or filament bundles were observed. Apart from this the ultrastructure of the somatic cells (fig. 1 0 ) did not differ from that of the superficial cells (fig. 9 ) . Thus, the mitochondria were spherical to elongate in shape. Sparse amounts of granular rough-surfaced endoplasmic reticulum and small Golgi-regions were occasionally seen. Neither smoothsurf aced endoplasmic reticulum nor lipid inclusions were observed. The nuclei contained dispersed chromatin and one or two nucleoli. Under the epithelium, delimiting it from the mesenchyme, a basement lamina was seen. It was about 200 A thick and consisted of loose granular material.
Day 38 The cells were much more tightly packed, as already observed in the light microscope, and only few intercellular gaps were seen. The intercellular space was mostly 2-300 A in width (fig. 11). The epithelium was now clearly delimited from the underlying mesenchyme by a basal lamina, about 100 A thick, consisting of homogenous material. Outside this there was a zone 1-2 thick, containing looser fibrillar material. The surface of the superficial cells exhibited more microvillous processes, there being up to ten or more in a single section. These processes varied i n length u p to about 0.8 and in width up to about 0.4 p. Cells with many processes invariably showed the same filament bundles as seen at day 34. The morphology of the superficial cells as regards filament bundles (arrows), junctional complexes and organelles were apparently unchanged from day 34 (fig. 11). The somatic cells in the surface epithelium lying beneath the super-
fical cell layer were also unchanged. The latter lay in intimate relation to the germinal cells (fig. 11). Connecting cellular strands were seen, surrounded by a continuation of the subepithelial basal lamina, and containing germinal cells and somatic cells of the same morphology as observed in the surface epithelium. There was direct continuity between the epithelium and the connecting cellular strands.
Day 42 The surface epithelium had the same appearance as at day 38. Numerous microvillous processes and filament bundles were seen in the superficial cell layer as well as many connecting cellular strands. Day 46 The most striking development by day 46 was the virtual absence of filament bundles. Microvillous processes were also scarce. Other ultrastructural features remained unchanged. Thus, junctional complexes were still present, occasionally with a length of several p , and the basal lamina was about 4,000 A in thickness.
Day 54 The cells were tightly packed with the superficial cells forming a continuous layer of predominantly columnar cells showing a few microvillous projections, but no filament bundles, Adjoining cells were connected by junctional complexes. Some of the nuclei exhibited deep indentations. I n many places the epithelium contained only the superfiicial cells, that is it was only a single cell layer, while in other places small sub-epithelial nests of somatic cells occurred. Very few germinal cells were seen. Day 58 At this stage the epithelium occurred as a single layer of columnar cells. Junctional complexes were still seen, but no microfilament bundles and only very few microvillous projections were seen. Some of the cells showed several junctional complexes along their adjoining membranes. The basal lamina was 1 thick. No germinal cells were seen in the epithelium.
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY DISCUSSION
The main findings of the present study may be summarized as follows: ( 1 ) In the superficial cells straight bundles of fiIaments and intercellular junctional complexes were identified, features which clearly distinguished these cells from those in the underlying cell layers. (2) The subepithelial basal lamina was continuous with the basil lamina surrounding the germinal cords via the attachments of the latter to the epithelium. The nature and function of the observed microfilaments is as yet undetermined. From their size and straightness they resemble the microfilaments or actin-like filaments identified in many other cell types (Cloney, '66; Giacomelli et al., '70; Goldman and Knipe, '73; Newstead, '71; Ross and Reith '70; Rostgaard, '72; Wessells et al., '71; Wren and Wessells, '69; Yamada et al., '70) and where they have been presumed to serve a contractile role. Bundles of thin microfilaments have often been described during embryogenesis: Yamada et al. ('70) found 50 A filaments in growing axons, Cloney ('66) found 50-70 A filaments in contracting epidermal cells in ascidia during their metamorphosis and Wrenn and Wessells ('69) found microfilaments in the apical regions of invaginating cells in the lens placode. Further, when tubular glands are formed in the chick oviduct by invagination from the epithelial surface, this is accompanied by the appearance of 50 A filaments in the apical region of the epithelial cells (Wessells et al., '71). Similar filaments are seen during the formation of salivary glands. In summary, it has been repeatedly observed that at the time of invagination of an epithelial surface bundles of linearly running filaments appear in the apical part of the invaginating cells, concurrent with a contraction of their apical parts, transforming the cells into a cone-like shape. In the light of these findings it seems likely that the bundles of filaments described here may serve a contractile role during the development of the surface epithelium. What this role may be remains obscure, but one possibility is that the filaments serve to contract the superficial cell
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layer with the added result that the underlying cells are forced downward into the gonadal anlage. The change in shape of the superficial cells from a squamous to a columnar form might also be expected as a result of the contraction of this cell layer. A contraction of the superficial cells does not, however, explain immediately why all the cells beneath are forced downwards. This must involve other mechanisms such as active amoeboid movement of the germ cells themselves as has been suggested by Witschi ('51) as the means of migration of the g e m cells from their extragonadal origin to the gonadal anlage. A similar mechanism may be responsible for their subsequent movements down into the germinal cords. As indicated in the opening paragraphs of this paper, light microscope studies of the fetal development of the mammalian ovary has given rise to conflicting interpretations as regards the differentiation and functional role of the surface epithelium. Histochemical examinations of cattle (Gropp and Ohno, '66) were interpreted as showing that a central blastema was first formed, presumably from the mesonephros, and the follicular cells then originated from this central blastema. Similarly from a study of human embryos and fetuses (Pinkerton et al., '61) it was claimed that a central blastema of mesenchymal cells first developed, and that this gave rise to the follicular cells. Both these latter authors claimed that the surface epithelium is inactive as regards the formation of germinal cords and the follicular cells. Previous studies of ovaries in hamster (Weakley, '67) and in mice (Wischnitzer, '65; Yamada et al., '57) have shown that the surface epithelium consisted of a single layer of flattened cells separated from the underlying tissue by a basal lamina. In one case (Weakley, '67) attachment zones between the cells and also microvillous projections were observed but none of these authors noted the presence of intracellular filaments. It is noteworthy that in the present study no filaments were found at day 54 and 58. It seems that they are only present at the time that changes in cell shape take place (between days 34 and 42, and
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- to a small degree - at day 46). The somatic cells that are located beneath the superficial cell layer, but i n the surface epithelium, i.e. on the epithelial side of the basal lamina, are according to their morphology and localization prefollicular cells. They are more or less identical to the follicular cells that Odor and Blandau ('69) describe in the fetal mouse ovary and that Gondos ('70) found in the newborn rabbit ovary, in close proximity to the germinal cells, and enveloping the latter with their thin cytoplasmic processes. The origin of these cells cannot be determined from present data. It is probable, however, that they arise from the superficial cells, since they lie on the epithelial side of the basal lamina. Possibly, some of the superficial cells invaginate and transform into follicular cells. An alternative explanation is that these somatic cells acconipany the germinal cells from their extragonadal site of origin and invade the surface epithelium together with the germinal cells. It is a fact that the germinal cells and their accompanying prefollicular cells are located within the surface epithelium (i.e. superficial to the subepithelial basal lamina) and are surrounded by a basal lamina common to the germinal cords and the connexions between the surface epithelium and the cords. This clearly indicates that the germinal cords are derived from the surface epithelium and that the prefollicular cells are of epithelial origin. Recently, however, Byskov and LinternMoore ('73) have demonstrated free communications between tubules belonging to the rete ovarii and the medullary end of the germinal cords indicating that the rete ovarii contributes to the formation of the germinal cords. LITERATURE CITED Allen, B. M. 1904 The embryonic develop ment of the ovary and testis of the mammals. Am. J. Anat., 3: 89-146. Arey, L. B. 1965 Developmental Anatomy. Seventh ed. W. B. Saunders Co., London. Bohman, S . O., and A. B. Maunsbach 1970 Effect on tissue fine structure of variations in colloid osmotic pressure of glutaraldehyde fixatives. J. Ultrastruct. Res., 30: 195-208. Bookhout, C. G. 1945 The development of the guinea-pig ovary from sexual differentiation to maturity. J. Morph., 77: 233-263.
Boyd, J. D., and W. J. Hamilton 1955 The cellular elements of the human ovary. In: Modern Trends in Obstetrics and Gynecology (Scand. series). K. Bowes, ed. Butterworth and Co. Ltd., London, pp. 50-78. Brambell, F. W. R. 1927 The development and morphology of the gonads of the mouse. Proc. roy. SOC. B, 101: 391-409. Byskov, A. G . S., and S., and S. Lintern-Moore 1973 Follicle formation in the immature mouse ovary: the role of the rete ovarii. J. Anat., 116: 207-217. Cloney, R. A. 1966 Cytoplasmic filaments and cell movements: Epidermal cells during ascidian metamorphosis. J. Ultrastruct. Res., 14: 300-328. Draper, R. L. 1920 The prenatal growth of the guinea-pig. Anat. Rec., 18: 369-392. Farquhar, M. G . , and G . E. Palade 1963 Junctional complexes in various epithelia. J. Cell Biol., 17: 375-412. Franchi, L. L., and A. M. Mandl 1962 The ultrastructure of oogonia and oocytes in the foetal and neonatal rat. Proc. roy. SOC.B, 157: 99-114. Giacomelli, F., J. Wiener and D. Spiro 1970 Cross-striated arrays of filaments i n endothelium. J. Cell Biol., 45: 188-192. Goldman, R. D., and D. M. Knipe 1973 Functions of cytoplasmic fibers in non-muscle cell motility. In: Symposia on Quantitative Biology. Vol. 37. Cold Spring Harbor Laboratory, pp. 523-534. Gondos, B. 1970 Granulosa cell-germ cell relationship i n the developing rabbit ovary. J. Embryol. exp. Morph., 23: 419426. Gropp, A,, and S. Ohno 1966 The presence of a common embryonic blastema for ovarian a n d testicular parenchymal (follicular, interstitial and tubular) cells in cattle, Bos Taurus. 2. Zellforsch., 74: 505-528. Harrison, R. J., and L. H. Matthews 1951 Subsurface crypts in the cortex of the mammalian ovary. Proc. zool. SOC.(London), 120: 699-712. Kellenberger, E., A. Ryter and J. Sechaud 1958 Electron microscope study of DNA containing plasm. J. biophys. biochem. Cytol., 4: 671. Mossman, H. W. 1938 The homology of the vesicular ovarian follicles of the mammalian ovary with the coelom. Anat. Res., 7 0 : 643-652. Newstead, J. D. 1971 Filaments in renal parenchymal and interstitial cells. J. Ultrastruct. Res., 34: 316-328. Odor, D. S., and R. J. Blandau 1969 Ultrastructural studies on fetal and early postnatal mouse ovaries. I. Histogenesis and organogenesis. Am. J. Anat., 124: 163-186. Pinkerton, J. H. M., D. G. McKay, E. C. Adams and A. T. Hertig 1961 Development of the human ovary - a study using histochemical technics. Obstet. and Gynec., 18: 152-181. Reynolds, E. S. 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 2 7: 208-212. Ross, M. H., and E. J . Keith 1970 Myoid elements in the mammalian nephron and their relationship to other specializations in the
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY basal part of kidney cells. Am. J. Anat., 129: 399-416. RostgBrd, J. 1972 Electron microscopy of filaments in the basal part of rat kidney tubule cells, and their in situ interaction with heavy meromyosin. 2. Zellforsch, 132: 497-521. Sabatini, D. D., K. Bensch and R. J. Barrnett 1963 Cytochemistry and electronmicroscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19-58. Sainmont, G. 1906 Recherches relatives B l’organogen6se du testicule et de l’ovaire chez le chat. Arch. Biol. (Paris), 22: 71. Sjostrand, F. S. 1967 Electron Microscopy of Cells and Tissues. Academic Press Inc., New York and London, pp 144-146. Simkins, C. S. 1928 Origin of the sex cells in man. Am. J. Anat., 41: 249-293. Torrey, T. W. 1945 The development of the urinogenital system of the albino rats. 11. The gonads. Am. J. Anat., 76: 375-397. Wagenen, G. van, and M. E. Simpson 1965 Embryology of the ovary and testis. Homo sapiens and macaca mulatta. Yale University Press, New Haven and London. Weakley, B. S. 1967 Light and electron microscopy of developing germ cells and follicle cells i n the ovary of the golden hamster: twenty-four hours before birth to eight days post partum. J. Anat., 107: 435-459. 1969 Differentiation of the surface epi-
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thelium of the hamster ovary. A n electron microscopic study. J. Anat., 105: 129-147. Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. Luduena, E. L. Taylor, J. T. Wrenn and K. M. Yamada 1971 Microfilaments in cellular and developmental processes. Science, 171: 135-143. Winiwarter, H. de 1901 Recherches sur l’ovogengse et l’organogen6se de l’ovaires des mammiferes (lapin et homme). Arch. Biol. (Paris), 17: 33-199. 1910 Contribution 2 l’etude de l’ovaire humain. Arch. Biol. (Paris), 25: 683-756. Wischnitzer, S. 1965 The ultrastructure of the germinal epithelium of the mouse ovary. J. Morph., 117: 387-400. Witschi, E. 1951 Gonad development and function. Embryogenesis of the adrenal and the reproductive glands. Recent Progr. Hormone Res., 6: 1-27. Wrenn, J. T., and N. K. Wessells 1969 A n ultrastructural study of lens invagination in the mouse. J. Exp. Zool., 171: 359-368. Yamada, E., T. Muta, A, Motomura and H. Koga 1957 The fine structure of the oocyte in the mouse ovary studied with the electron microscope. Kurume med. J., 4: 148-160. Yamada, K. M., B. S. Spooner and N. K. Wessells 1970- Axon growth: Roles of microfilaments and microtubules. Proc. nat. Acad. Sci., 66: 1206-1212.
PLATE 1 EXPLANATION O F FIGURES
1
Light microscopy of fetal guinea-pig ovary, day 34, showing the variability in thickness of the epithelium and of cell shape and orientation. Germinal cells ( G ) are present in the epithelium. Connecting cellular strands are seen running obliquely down into the gonadal anlage (arrows). x 300.
2
Light microscopy of fetal guinea-pig ovary, day 38. Numerous germ cells in the epithelium and many connecting cellular strands, continuous with the germinal cords, are seen. The cells in the epithelium and the connecting cellular strands (arrow) are considerably more compact as compared to day 34. x 300.
3 Light microscopy of fetal guinea-pig ovary, day 42. Numerous germinal cells are seen in the surface epithelium as well as many broad connecting cellular strands. The superficial cells are in part flattened, in part cuboidal. The basement membrane is clearly seen (arrow). X 300. 4
Light microscopy of fetal guinea-pig ovary, day 46. Only a few germinal cells may be seen in the surface epithelium and there are few and slender connecting cellular strands. The surf ace epithelium consists mainly of three layers of cuboidal cells. A basal membrane is seen under the epithelium and is continuous with that surrounding the connecting cellular strand (arrow). x 300.
5 Light microscopy of fetal guinea-pig ovary, day 58. The surface epithelium appears as a single-layered regular cylindrical sheet, separated from the underlying mesenchyme by a thick basal membrane. No germinal cells are seen in the surface epithelium. Only one very slender connecting cellular strand is seen (arrow). x 300.
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SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY
PLATE 1
Th. Jeppesen
507
?LATE 2 EXPLANATION O F FIGURE
6
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Surface epithelium, guinea-pig ovary, day 34. The continuous layer of superficial cells is seen with their junctional complexes and associated bundles of filaments (arrows). A few microvillous projections are seen. EL, basal lamina. x 6,700.
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY Th. Jeppesen
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
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7
Superficial cell of the surface epithelium, day 34. A bundle of filaments runs straight through the apical part of the cell, attached to junctional complexes (arrows), one of which is curved inwards. X 30,000.
8
Junctional complex and filaments a t high magnification. The insertion of the filaments i n or at the junctional complex as well as the straight course of the single filament (arrows) is apparent. Individual filaments are about 60 A in thickness. x 120,000. Inset to the right: Junctional complex at day 46. Small gaps within the tight junction are seen ( G ) . x 120,000. Inset to the left: Shows the microfilaments in cross-section (arrow). x 120,000.
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY Th. Jeppesen
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
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9
Superficial cell in the surface epithelium showing the junctional complexes and the straight bundles of filaments originating from them. x 11,000.
10
Somatic cell in the surface epithelium, located beneath the superficial cell layer. No junctional complexes or filaments are seen. x 11,000.
SURFACE EPITHELIUM, FETAL GUINEA-PIG OVARY Th. Jeppesen
PLATE 4
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PLATE 5 EXPLANATION OF FIGURE
11
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Surface epithelium. day 38. The superficial cells still show junctional complexes and filament bundles (arrows). There are numerous microvillous projections. The compactness of the epithelium is increased. Beneath the superficial layer somatic cells (prefollicular cells) are seen i n close proximity to the germinal cells. B. basement lamina. x 4,050.
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PLATE 5
515
