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Ovarian Development: The Functional Importance of Germ Cell Interconnections
Ovarian Development: The Functional Importance of Germ Cell Interconnections BERNARD CONDOS, M.D., and LUCIANO ZAMBONI, M.D. With the technical assistance of Inga Jansen
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VARIAN DEVELOPMENT in mammals progresses through a regular sequence of formative stages. The first stage is that of active multiplication of oogonia which become arranged in cords extending into the stroma beneath the germinal epithelium. The mitotic stage is followed by a meiotic stage during which the oogonia go through the prophase of the first reductional division and mature into primordial oocytes. The meiotic stage is followed by extensive degeneration involving the vast majority of the primordial oocytes. By the time the degeneration ceases, the gonad has reached the final stage of development and is populated by numerous unilaminar follicles, each consisting of an oocyte surrounded by a single layer of follicle cells. The mechanism by which these oocytes escape degeneration remains obscure. It is known from previous studies 2 - 5 • 13. 16, 22. 25-27 that ovarian differentiation evolves in a synchronized pattern, in that there is a direct correspondence between stage of development and predominant stage (mitotic, meiotic, and degenerative) of cellular activity. We have recently demonstrated27 that the synchronization of ovarian differentiation is of a higher degree still; the germ cells, in fact, mature in groups, and each group consists of elements which are at an identical stage of differentiation. We also observed that germ cells are connected to one another by a diffuse network of intercellular bridges; we concluded that these bridges represent the structural basis for the synchronous pattern of cell differentiation. The purpose of the present report is to describe additional observations From the Division of Reproductive Biology, Department of Pathology, Harbor General Hospital, Torrance, Calif., and the University of California School of Medicine, Los Angeles, Calif. Supported by a Ford Foundation Grant in Reproductive Biology. Presented at the 24th Annual Meeting of The American Fertility Society, San Francisco, Calif., Mar. 27-30, 1968. The electron micrograph (Fig. 12) was provided by Dr. M. Baca.
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on these intercellular connections, to further evaluate their importance in relation to synchronization of oogenesis, and to consider other aspects of ovarian development which, in our judgment, are related to the presence of these bridges. MATERIAL AND METHODS
This study is concerned with the events that occur in the developing ovary of the rabbit within the period that extends from the third week of fetal life to Day 30 after birth. Fragments of ovarian tissue from 42 New Zealand white rabbit embryos and newborns were fixed in 1% OSO.I with salts added,29 and embedded in Epon 812.19 For orientation and light microscopic examination, sections 0.5-1.0 J.L in thickness were obtained from the blocks and were stained with toluidine blue. Micrographs of these sections were taken with a Zeiss Ultraphot on Plux-X-pan films. The thin sections for electron microscopy were stained with lead hydroxide 17 and examined with Hitachi HU -l1A and HU -11 C electron microscopes. RESULTS Stages of Cell Differentiation
The ovarian rudiment consists of germinal epithelium and sex cords with interposed stroma (Fig. 1). The cells of the germinal epithelium have morphologic characteristics typical of highly undiHerentiated elements. In places, the germinal epithelium is continuous with the cells of the cords which extend deep into the stroma (Fig. 1). From the third week of embryonal life until the end of gestation, the cords are short and consist mostly of mitotic oogonia. During the first few days after birth, the cords increase in length and become divided into distinct areas--an outer portion below the germinal epithelium which still contains oogonia mostly in mitotic activity, and an inner segment crowded by oocytes in meiotic prophase (Fig. 2). Around Day 5 postpartum, the oocytes located in the deepest portions of the cords begin to undergo degeneration. Thus, the cords can now be distinguished into an outer segment of mitotic oogonia, an intermediate portion of meiotic oocytes, and an inner segment composed of numerous oocytes in degeneration. By the end of the first week of life, the number of mitotic oogonia has decreased to the extent that only meiotic and degenerating cells are found in the cords (Fig. 3). During the second week, meiotic activity decreases, degeneration becomes extensive, and the first unilaminar follicles appear at
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Iig. 1 (top). Section of ovary from 24-hr.-old newborn rabbit showing germinal epithelium (at left) and cords with interposed vascular stroma. Arrow points to cluster of four mitotic oogonia at tip of cord. Section ca. 1 /L thick. (Toluidine blue, X 550) Fig. 2 (bottom). Section of ovary from 4-day-old rabbit showing portions of three cords. Upper cord includes oogonia in mitosis. Lower cord shows numerous oocytes in meiotic prophase. Section ca. 1 /L thick. (Toluidine blue, X 1000)
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Fig. 3 (top). Section of ovary from 7-day-old rabbit. In upper p~'rtirn of cortex (at left) are 'groups of meiotic oocytes. Lower portion shows extenshe ('ezcneration as indicated by confluence and hyalinosis of oocyte cytoplasm. Sectirn (a 1 fJ. thick. (Toluidine blue, X 400) Iig. 4 (bottom). Section of ovary from 19-day-c]Jla:J:.it. Note numerous primordial follicles and increased stroma. Portions of germinal epithelium and a few remaining cords are seen at upper left. Sectiw ca. 1 p. thick. (T ,Juid~ne blue, X 450)
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the tips of the cords. Ovarian development after the second week is characterized by arrest of meiotic activity, subsiding of degeneration, and increased follicle formation. Around the end of the period considered in this study, the cords have disappeared, and the ovarian cortex appears crowded by a large number of unilaminar follicles (Fig. 4). It is evident that a predominant type of cell activity characterizes any given period of ovarian development. For example, mitotic activity is predominant in the period from Days 2-5 postpartum, while the interval between Days 5 and 9 is characterized by a high number of meiotic cells. It is also apparent that there is a close correspondence between given areas of the cords and particular stages of cell differentiation. Just before the end of the first week, for example, the outer segments of the cords consist mostly of mitotic oogonia, the intermediate portions contain meiotic oocytes, and the deepest segments are filled by oocytes in degeneration. Finally, mitosis, meiosis, and degeneration consistently involve groups of cells each composed of elements at an identical stage of maturation (e.g., all in the same phase of mitosis). Intercellular Bridges
Intercellular bridges (Fig. 5-10) are found between germ cells in mitosis and in meiosis, as well as between resting cells. The bridges appear as cylindrical portions of cytoplasm connecting the bodies of adjacent cells. The plasma membrane of the bridge is thicker than the membrane of the cells with which it is continuous, and shows an increased electron opacity. The cytoplasm of the bridge has a normal population of ribosomes and contains organelles such as, among others, mitochondria (Fig. 5-9) and endoplasmic reticulum (Fig. 10). Germ cells connected to two or more elements by multiple bridges are common (Fig. 5 and 6). The conjoined cells always show identical morphologic characteristics or are in the same stage of mitosis or meiosis. There is a direct correspondence between the number of bridges and incidence of mitosis. The bridges appear shortly after the onset of mitotic activity and progressively increase-in number to reach a peak between Days 2 and 5 postpartum, coincidentally with the peak of mitosis. As degeneration becomes extensive at the end of the second week, the number of bridges gradually decreases. Only a few oocytes in early stages of degeneration are connected by intercellular bridges. In more advanced degeneration, bridges are not observed; the oocytes, however, show confluence of cytoplasm (Fig. 3).
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ig. 5.. Typical cytoplasmic bridges (arrows) connecting interphase oogonia within an varhm cord. One of the bridges contains two mitochondria. (X 10,500)
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Fig. 6 (top). One oogonimn (A) connected to two adjacent oogonia (B and C) by two intercellular bridges, one of which is traversed by an elongated mitochondrion. ( X 17,500) Fig. 7 (bottom). Plasma membrane of bridge is continuous with membranes of connected cells, but is thicker and more electron opaque. (X 6500)
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10 Fig.. 8. Bridge connecting two meiotic oocytes. Mitochondrion (arrow) appears to be
passing from one cell to the other. (X 13,000) Fig. 9. Intercellular bridge with mitochondria, vesicles, and ribosomes in the cytoplasm. (X 22,000) Fig. 10. Element of endoplasmic reticulum in cytoplasm of intercellular bridge. (X 14,000)
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Synchronous oogenetic patterns have been described in the rat,4.13 mouse,5 rabbit/ 5 • 22. 25 golden hamster,26 and to a lesser degree in the guinea pig,16 monkey,3 and man. 2 These oogenetic processes are considered to be synchronous because of a direct correlation between predominant stage of cellular activity and stage of development. vVe believe that ovarian development in mammals is characterized by a synchronization of cell differentiation that goes beyond such a simple correspondence and that finds expression in the zonal distribution of clusters of cells in identical stages of differentiation. 27 Our opinion is based on the finding of mitotic, meiotic, and degenerating cells in definite areas of the developing ovarian cords. In their respective sites, these cells are clustered together to form groups, each consisting of elements at the same stage of maturation. Such a high degree of synchronization is unusual, since it is not found even in tissues characterized by a high rate of mitotic activity, such as bone marrow. It is apparent that the synchronous pattern of differentiation observed in the ovary must have a structural basis peculiar to this organ. vVe believe that such a structural basis is provided by the presence of intercellular bridges connecting adjacent germ cells. These bridges are true intercellular connections 10 characterized by continuity of the cytoplasm of the conjoined cells. (Electron microscopy has revealed that the vast majority of the so-called intercellular bridges described in classic histology are merely points of close cellular apposition without any continuity between the cells.) A review of the literature reveals that this type of intercellular connection is not an exclusive feature of the developing rabbit ovary, since bridges were found in the embryonal ovaries of the golden hamster,26 rat/ 3 and man. 23 . 24 Intercellular connections identical to those observed in mammals have been seen also in the ovaries of lower animal species such as Artemia salina l and Drosophila melanogaster. 8 • 18 The only other organ where true intercellular bridges have been found is the male gonad. Presence of bridges has been described, in fact, between spermatogenetic cells in the adult testes of invertebrates,12 birds,21 and mammals,9. 11, 12 as well as in the embryonal testes of the rabbit. 20 The frequency with which we found these bridges in thin sections of the rabbit ovary indicates that their actual number is very high and that they form a network of intercellular connections which results in the organization of the germ cells into multiple syncytial groups. How can such a network of intercellular bridges develop? In agreement with the opinion of other investigators (see below), we believe that this network is the
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result of a sequence of mitotic divisions characterized by incomplete cell separation. The body of a cell at telophase of mitotic division normally becomes constricted, and the two forming daughter cells remain conjoined for a short time by a cylindrical cytoplasmic formation referred to as midbody, Zwischenkorper, or Flemming body (Fig. 11). The disappearance NORMAL CELL DIVISION
INCOMPLETE CELL DIVISION
~
Fig. 11. Diagrammatic representation of mitosis with complete (left) and incomplete (right) separation of daughter cells.
of this bridge at late telophase is responsible for the separation and independence of the newly formed cells. What we believe occurs in the developing ovary is that the mid-bodies of dividing oogonia persist, resulting in permanent connections of the daughter cells (Fig. 11). The incomplete and simultaneous mitotic division of two connected oogonia would lead to the formation of a syncytium of four oogonia. Incomplete cell separation, repeating itself at each mitotic division, would bring about the formation of larger syncytial groups localized in definite areas of the cords and consisting of populations of cells of the same age. The presence of organelles in the cytoplasm of the intercellular connections strongly suggests that material and information are exchanged between the connected cells. It is thus that they acquire common and synchronous patterns of differentiation. In this we fully concur with the hypothesis originally formulated by Fawcett and co-workers9 - 12 to explain the synchronous differentiation exhibited by groups of cells of the spermatogenetic line. These authors thought that incomplete separation of the
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bodies of spermatogonia and spermatocytes at each mitotic and meiotic division results in the formation of syncytial clusters of spermatog'netic cells consisting of elements of identical age and stage of differentiation. There are examples of syncytia in which the germ cells, connected by true intercellular bridges, exhibit asynchronous differentiation and have different potentialities. In Artemia salina, in fact, the bridges connect germ cells in different mitotic stages. 1 In this invertebrate, the germ cells have different potentialities, as indicated by the fact that the elements of each syncytial group differentiate into nursing cells and oocytes. Bridges connecting oocytes and nurse cells were found in Drosophila melanogaster. 1S Even more striking is the finding of syncytial groups consisting of spermatocytes in different stages of meiosis in a human testis showing abnormal spermatogenesis. ~H Here, the asynchrony obviously cannot be related to multiple potentialities of the spermatogenetic cells. All morphologic findings on mammalian oogenesis, however, point to the presence of a high degree of synchronization in association with the presence of intercellular connections. Despite the above-described conflicting observations made on invertebrate oogenesis and abnormal mammalian spermatogenesis, the hypothesis that these connections are the structural basis for synchronization of cell differentiation in mammalian oogenesis remains valid. The vast majority of primordial oocytes undergo degeneration either during or shortly after meiosis. 4 On the basis of their synchronous maturation, it is probable that meiotic oocytes connected by bridges undergo degeneration simultaneously, thus explaining the similarity of the regressive changes previously noted in adjacent degenerating oocytes. ~7 While bridges are still recognizable in oocytes in early degeneration, they are absent in later stages. Cells in advanced degeneration are characterized by confluent cytoplasm-a further indication of syncytial organization. The degenerative waves that mark the final stages of ovarian development in mammals contribute to one of the most obscure aspects of oogenesis. It has been demonstrated 6 and is generally accepted 14 that, in most species, degeneration eliminates the majority of oocytes that have matured meiotically during embryonal life, but that a sufficient number of these cells remain to constitute the finite stock of oocytes present in the adult mammalian ovary. The mechanisms by which these oocytes escape degeneration, whereas the majority do not, remain to be understood. In a preliminary note on this subject,27 we formulated the hypothesis that the key to survival could be complete cell division. In other words, only those cells in which karyokinesis is followed by cytokinesis would remain independent and survive the degeneration that affects the majority (syncy-
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ig. 12' (top). Electron micrograph of binucleated oocyte in follicle of adult human vary. (X 8000) Fig. 13 (bottom). Biovular follicle in adult mouse ovary. Section a. 1 p. thick. (Toluidine blue, X 550)
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tial) of the oocyte population. Such a differential cellular behavior does not find correspondence in the male gonad, however, where a syncytial organization identical to that of the ovary exists, but where the spermatogenetic cells are all conjoined and remain connected through the spermatid stage. 10 It is more likely, thus, that the surviving oocytes are those which remain in a syncytial organization through meiotic prophase, but separate from one another prior to being affected by the extensive degeneration. Anomalies such as binucleated follicular oocytes and polyovular follicles are not infrequently observed in adult ovaries of a variety of mammalian species. 7 We concur with the hypothesis of Stegner 23 that these defects may be related to persistence of oocyte connections at the time of follicle formation. If the incompletely separated oocytes in the follicle undergo cytoplasmic fusion, the result is a follicle containing a multinucleated oocyte (Fig. 12). On the other hand, in the absence of cytoplasmic fusion, a multiovular follicle develops (Fig. 13). Similarly, persistence of intercellular bridges in spermatids which fail to separate is thought to be responsible for such sperm anomalies as double heads and double tails. 10 Harbor General Hospital 1000 West Carson St. Torrance, Calif. 90509
REFERENCES 1. ANTEUNIS, A., FAUTREZ-FIRLEFYN, N., and FAUTREZ, J. La structure de ponts intercellulaires "obtures" et "ouverts" entre oogonia et oocytes dans l'ovaire d'Artemia salina. Arch Bioi (Uege) 77:645, 1966. 2. BAKER, T. C. A quantitative and cytological study of germ cells in human ovaries. Proc Roy Soc [Bioi] 158:417, 1963. 3. BAKER, T. C. A quantitative and cytological study of oogenesis in the rhesus monkey. ] Anat 100:761, 1966. 4. BEAUMONT, R. M., and MAKDL, A. M. A quantitative and cytological study of oogonia and oocytes in the foetal and neonatal rat. Proc Roy Soc [Biol] 155:557, 1962. 5. BORUM, K. Oogenesis in the mouse. Exp Cell Res 24:495, 1961. 6. BORUM, K. Oogenesis in the mouse: A study of the origin of the mature ova. Exp Cell Res 45:39, 1967. 7. BRAMBELL, F. W. R. "Ovarian Changes." In Physiology of Reproduction (Vol. 1), Parkes, A. S., Ed. Longmans, London, 1952, p. 397. 8. BROWN, E. R., and KING, R. C. Studies on the events resulting in the formation of an egg chamber in Drosophila melanogaster. Growth 28:41, 1964. 9. BURGOS, M. R., and FAWCETT, D. W. Studies on the fine structure of the mammalian testis: I. Differentiation of the spermatids in the cat (Felix domestica). ] Biophys Biochem Cytol1:287, 1955. 10. FAWCETT, D. W. Intercellular bridges. Exp Cell Res 8 (Suppl.):174, 1961. 11. FAWCETT, D. W., and BURGOS, M. R. 1956 Ciba Foundation Symposium: Aging of Transient Tissues. Little, Boston, 1956, p. 86. 12. FAWCETT, D. "V., ITo, S., and SLAUTTERBACK, D. The occurrence of intercellular
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13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
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bridges in groups of cells exhibiting synchronous differentiation. J Biophys Biochem Gytol 5:453, 1959. FRANCHI, L. L., and MANDL, A. M. The ultrastructure of oogonia and oocytes in the foetal and neonatal rat. Proc Roy Soc [Bioi] 157:99, 1962. FRANCHI, L. L., MANDL, A. M., and ZUCKERMAN, S. "The Development of the Ovary and the Process of Oogenesis." In The Ovary, Zuckerman, S., Mandl, A. M., and Eckstein, P., Eds. Acad. Press, London, 1962, chap. 1. CONDOS, B., and ZAMBONI, L. Electron microscopic studies of the embryogenesis and postnatal development of the rabbit ovary. J Ultrastruct Res 21: 162, 1967. IOANNOU, J. M. Oogenesis in the guinea pig. J Embryol Exp Morph 12:673, 1964. KARNOVSKY, M. J. Simple methods for "staining with lead" at high pH in electron microscopy. J Biophys Biochem Gytolll:729, 1961. KING, R C., and DEVINE, R L. Oogenesis in adult Drosophila melanogaster: VII. The submicroscopic morphology of the ovary. Growth 22:299, 1958. LUFT, J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Gytol 9:409, 1961. MATHUR, R, and1:AMBoNI, L. Unpublished observation. NAGANO, T. The structure of cytoplasmic bridges in dividing spermatocytes of the rooster. Anat Rec 141:73, 1961. PETERS, H., LEVY, K, and CRONE, M. Oogenesis in rabbits. J Exp Zool 158: 169, 1965. STEGNER, H. E. Die elektronenmikroskopische Struktur der Eizelle. Ergebn Anat Entwicklungsgesch 39:6, 1967. STEGNER, H. K, and WARTENBERG, H. Elektronenmikroskopische Untersuchungen an Eizellen des Menschen in verschiedenen Stadien der Oogenese. Arch Gynaek 199: 151, 1963. TEPLITZ, R, and OHNO, S. Postnatal induction of ovogenesis in the rabbit (Oryctolagus cuniculus). Exp Gell Res 31:183,1963. WEAKLEY, B. S. Light and electron microscopy of developing germ cells and follicle cells in the ovary of the golden hamster: twenty-four hours before birth to eight days post partum. J Anat 101:435, 1967. ZAMBONI, L., and CONDOS, B. Intercellular bridges and synchronization of germ cell differentiation during oogenesis in the rabbit. J Gell Bioi 36:276, 1968. ZAMBONI, L., and STEFANINI, M. Unpublished observations. ZETTERQVIST, H. The ultrastructural organization of the columnar absorbing cells of the mouse jejunum. Ph.D. Thesis. Karolinska Institutet, Stockholm, 1956.
Contract Program, Center for Population Research, NICHD An expanded contract program in population research will be launched this year by the new Center for Population Research of the National Institute of Child Health and Human Development, Bethesda, Maryland. This research effort will be directed toward the development of new contraceptive methods and population research in the social sciences, and will supplement research performed in NIH laboratories and supported by NIH grants. Certain critical research areas have already been identified. These areas are not exclusive; research on other subjects will also be considered. In contraceptive development, special areas include: (1) maturation and fertilizing capacity of spermatozoa; (2) oviduct function and gamete transport; (3) corpus luteum function and implantation; and (4) the biology of the preimplantation ovum. Four special areas in the social sciences are: (1) trends in fertility and related variables, to document changes in patterns of childbearing and fertility control; (2) the antecedents, processes, and consequences of population structure, distribution, and change; (3) the effects of explicit or implicit government population policies and data needed to help formulate population policies; and (4) the effects on fertility of family structure and patterns of sexual behavior. Advisory panels composed of outstanding scientists will be appointed to develop and oversee the programs for these topics. Further information may be obtained from PHILIP A. CORFMAN, M.D., Director, Center for Population Research, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014. Tel. (301) 496-1101.
Second Postgraduate Course Offere
Russell Malinak, M.D., and Paige Besch, Ph.D. Session VI. Surgical Techniques in Human Reproduction. lJfoderator: Robert H. Barter, M.D. Participants: Celso Ramon Garcia, M.D., and H. E. Riva, M.D. Session VII. Intrauterine Physiology and Fetal Salvage. Moderator: John W. Greene, M.D. Participants: Raymond Kaufman, M.D., and Elsie Carrington, M.D. Session VIII. Ovulation. Moderator: Alvin F. Goldfarb, M.D. Participants: Daniel L. Mishell, M.D., and C. Donald Christian, M.D. Each session will be limited to 75 registrants. The registration fee will be $50 per session. Further details and registration applications may be obtained from WINSTON H. WEESE, M.D., Registration Chairman, 944 South 18th St., Birmingham, Ala. 35205.
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VARIAN DEVELOPMENT in mammals progresses through a regular sequence of formative stages. The first stage is that of active multiplication of oogonia which become arranged in cords extending into the stroma beneath the germinal epithelium. The mitotic stage is followed by a meiotic stage during which the oogonia go through the prophase of the first reductional division and mature into primordial oocytes. The meiotic stage is followed by extensive degeneration involving the vast majority of the primordial oocytes. By the time the degeneration ceases, the gonad has reached the final stage of development and is populated by numerous unilaminar follicles, each consisting of an oocyte surrounded by a single layer of follicle cells. The mechanism by which these oocytes escape degeneration remains obscure. It is known from previous studies 2 - 5 • 13. 16, 22. 25-27 that ovarian differentiation evolves in a synchronized pattern, in that there is a direct correspondence between stage of development and predominant stage (mitotic, meiotic, and degenerative) of cellular activity. We have recently demonstrated27 that the synchronization of ovarian differentiation is of a higher degree still; the germ cells, in fact, mature in groups, and each group consists of elements which are at an identical stage of differentiation. We also observed that germ cells are connected to one another by a diffuse network of intercellular bridges; we concluded that these bridges represent the structural basis for the synchronous pattern of cell differentiation. The purpose of the present report is to describe additional observations From the Division of Reproductive Biology, Department of Pathology, Harbor General Hospital, Torrance, Calif., and the University of California School of Medicine, Los Angeles, Calif. Supported by a Ford Foundation Grant in Reproductive Biology. Presented at the 24th Annual Meeting of The American Fertility Society, San Francisco, Calif., Mar. 27-30, 1968. The electron micrograph (Fig. 12) was provided by Dr. M. Baca.
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on these intercellular connections, to further evaluate their importance in relation to synchronization of oogenesis, and to consider other aspects of ovarian development which, in our judgment, are related to the presence of these bridges. MATERIAL AND METHODS
This study is concerned with the events that occur in the developing ovary of the rabbit within the period that extends from the third week of fetal life to Day 30 after birth. Fragments of ovarian tissue from 42 New Zealand white rabbit embryos and newborns were fixed in 1% OSO.I with salts added,29 and embedded in Epon 812.19 For orientation and light microscopic examination, sections 0.5-1.0 J.L in thickness were obtained from the blocks and were stained with toluidine blue. Micrographs of these sections were taken with a Zeiss Ultraphot on Plux-X-pan films. The thin sections for electron microscopy were stained with lead hydroxide 17 and examined with Hitachi HU -l1A and HU -11 C electron microscopes. RESULTS Stages of Cell Differentiation
The ovarian rudiment consists of germinal epithelium and sex cords with interposed stroma (Fig. 1). The cells of the germinal epithelium have morphologic characteristics typical of highly undiHerentiated elements. In places, the germinal epithelium is continuous with the cells of the cords which extend deep into the stroma (Fig. 1). From the third week of embryonal life until the end of gestation, the cords are short and consist mostly of mitotic oogonia. During the first few days after birth, the cords increase in length and become divided into distinct areas--an outer portion below the germinal epithelium which still contains oogonia mostly in mitotic activity, and an inner segment crowded by oocytes in meiotic prophase (Fig. 2). Around Day 5 postpartum, the oocytes located in the deepest portions of the cords begin to undergo degeneration. Thus, the cords can now be distinguished into an outer segment of mitotic oogonia, an intermediate portion of meiotic oocytes, and an inner segment composed of numerous oocytes in degeneration. By the end of the first week of life, the number of mitotic oogonia has decreased to the extent that only meiotic and degenerating cells are found in the cords (Fig. 3). During the second week, meiotic activity decreases, degeneration becomes extensive, and the first unilaminar follicles appear at
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Iig. 1 (top). Section of ovary from 24-hr.-old newborn rabbit showing germinal epithelium (at left) and cords with interposed vascular stroma. Arrow points to cluster of four mitotic oogonia at tip of cord. Section ca. 1 /L thick. (Toluidine blue, X 550) Fig. 2 (bottom). Section of ovary from 4-day-old rabbit showing portions of three cords. Upper cord includes oogonia in mitosis. Lower cord shows numerous oocytes in meiotic prophase. Section ca. 1 /L thick. (Toluidine blue, X 1000)
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Fig. 3 (top). Section of ovary from 7-day-old rabbit. In upper p~'rtirn of cortex (at left) are 'groups of meiotic oocytes. Lower portion shows extenshe ('ezcneration as indicated by confluence and hyalinosis of oocyte cytoplasm. Sectirn (a 1 fJ. thick. (Toluidine blue, X 400) Iig. 4 (bottom). Section of ovary from 19-day-c]Jla:J:.it. Note numerous primordial follicles and increased stroma. Portions of germinal epithelium and a few remaining cords are seen at upper left. Sectiw ca. 1 p. thick. (T ,Juid~ne blue, X 450)
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the tips of the cords. Ovarian development after the second week is characterized by arrest of meiotic activity, subsiding of degeneration, and increased follicle formation. Around the end of the period considered in this study, the cords have disappeared, and the ovarian cortex appears crowded by a large number of unilaminar follicles (Fig. 4). It is evident that a predominant type of cell activity characterizes any given period of ovarian development. For example, mitotic activity is predominant in the period from Days 2-5 postpartum, while the interval between Days 5 and 9 is characterized by a high number of meiotic cells. It is also apparent that there is a close correspondence between given areas of the cords and particular stages of cell differentiation. Just before the end of the first week, for example, the outer segments of the cords consist mostly of mitotic oogonia, the intermediate portions contain meiotic oocytes, and the deepest segments are filled by oocytes in degeneration. Finally, mitosis, meiosis, and degeneration consistently involve groups of cells each composed of elements at an identical stage of maturation (e.g., all in the same phase of mitosis). Intercellular Bridges
Intercellular bridges (Fig. 5-10) are found between germ cells in mitosis and in meiosis, as well as between resting cells. The bridges appear as cylindrical portions of cytoplasm connecting the bodies of adjacent cells. The plasma membrane of the bridge is thicker than the membrane of the cells with which it is continuous, and shows an increased electron opacity. The cytoplasm of the bridge has a normal population of ribosomes and contains organelles such as, among others, mitochondria (Fig. 5-9) and endoplasmic reticulum (Fig. 10). Germ cells connected to two or more elements by multiple bridges are common (Fig. 5 and 6). The conjoined cells always show identical morphologic characteristics or are in the same stage of mitosis or meiosis. There is a direct correspondence between the number of bridges and incidence of mitosis. The bridges appear shortly after the onset of mitotic activity and progressively increase-in number to reach a peak between Days 2 and 5 postpartum, coincidentally with the peak of mitosis. As degeneration becomes extensive at the end of the second week, the number of bridges gradually decreases. Only a few oocytes in early stages of degeneration are connected by intercellular bridges. In more advanced degeneration, bridges are not observed; the oocytes, however, show confluence of cytoplasm (Fig. 3).
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ig. 5.. Typical cytoplasmic bridges (arrows) connecting interphase oogonia within an varhm cord. One of the bridges contains two mitochondria. (X 10,500)
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Fig. 6 (top). One oogonimn (A) connected to two adjacent oogonia (B and C) by two intercellular bridges, one of which is traversed by an elongated mitochondrion. ( X 17,500) Fig. 7 (bottom). Plasma membrane of bridge is continuous with membranes of connected cells, but is thicker and more electron opaque. (X 6500)
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8
10 Fig.. 8. Bridge connecting two meiotic oocytes. Mitochondrion (arrow) appears to be
passing from one cell to the other. (X 13,000) Fig. 9. Intercellular bridge with mitochondria, vesicles, and ribosomes in the cytoplasm. (X 22,000) Fig. 10. Element of endoplasmic reticulum in cytoplasm of intercellular bridge. (X 14,000)
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Synchronous oogenetic patterns have been described in the rat,4.13 mouse,5 rabbit/ 5 • 22. 25 golden hamster,26 and to a lesser degree in the guinea pig,16 monkey,3 and man. 2 These oogenetic processes are considered to be synchronous because of a direct correlation between predominant stage of cellular activity and stage of development. vVe believe that ovarian development in mammals is characterized by a synchronization of cell differentiation that goes beyond such a simple correspondence and that finds expression in the zonal distribution of clusters of cells in identical stages of differentiation. 27 Our opinion is based on the finding of mitotic, meiotic, and degenerating cells in definite areas of the developing ovarian cords. In their respective sites, these cells are clustered together to form groups, each consisting of elements at the same stage of maturation. Such a high degree of synchronization is unusual, since it is not found even in tissues characterized by a high rate of mitotic activity, such as bone marrow. It is apparent that the synchronous pattern of differentiation observed in the ovary must have a structural basis peculiar to this organ. vVe believe that such a structural basis is provided by the presence of intercellular bridges connecting adjacent germ cells. These bridges are true intercellular connections 10 characterized by continuity of the cytoplasm of the conjoined cells. (Electron microscopy has revealed that the vast majority of the so-called intercellular bridges described in classic histology are merely points of close cellular apposition without any continuity between the cells.) A review of the literature reveals that this type of intercellular connection is not an exclusive feature of the developing rabbit ovary, since bridges were found in the embryonal ovaries of the golden hamster,26 rat/ 3 and man. 23 . 24 Intercellular connections identical to those observed in mammals have been seen also in the ovaries of lower animal species such as Artemia salina l and Drosophila melanogaster. 8 • 18 The only other organ where true intercellular bridges have been found is the male gonad. Presence of bridges has been described, in fact, between spermatogenetic cells in the adult testes of invertebrates,12 birds,21 and mammals,9. 11, 12 as well as in the embryonal testes of the rabbit. 20 The frequency with which we found these bridges in thin sections of the rabbit ovary indicates that their actual number is very high and that they form a network of intercellular connections which results in the organization of the germ cells into multiple syncytial groups. How can such a network of intercellular bridges develop? In agreement with the opinion of other investigators (see below), we believe that this network is the
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result of a sequence of mitotic divisions characterized by incomplete cell separation. The body of a cell at telophase of mitotic division normally becomes constricted, and the two forming daughter cells remain conjoined for a short time by a cylindrical cytoplasmic formation referred to as midbody, Zwischenkorper, or Flemming body (Fig. 11). The disappearance NORMAL CELL DIVISION
INCOMPLETE CELL DIVISION
~
Fig. 11. Diagrammatic representation of mitosis with complete (left) and incomplete (right) separation of daughter cells.
of this bridge at late telophase is responsible for the separation and independence of the newly formed cells. What we believe occurs in the developing ovary is that the mid-bodies of dividing oogonia persist, resulting in permanent connections of the daughter cells (Fig. 11). The incomplete and simultaneous mitotic division of two connected oogonia would lead to the formation of a syncytium of four oogonia. Incomplete cell separation, repeating itself at each mitotic division, would bring about the formation of larger syncytial groups localized in definite areas of the cords and consisting of populations of cells of the same age. The presence of organelles in the cytoplasm of the intercellular connections strongly suggests that material and information are exchanged between the connected cells. It is thus that they acquire common and synchronous patterns of differentiation. In this we fully concur with the hypothesis originally formulated by Fawcett and co-workers9 - 12 to explain the synchronous differentiation exhibited by groups of cells of the spermatogenetic line. These authors thought that incomplete separation of the
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bodies of spermatogonia and spermatocytes at each mitotic and meiotic division results in the formation of syncytial clusters of spermatog'netic cells consisting of elements of identical age and stage of differentiation. There are examples of syncytia in which the germ cells, connected by true intercellular bridges, exhibit asynchronous differentiation and have different potentialities. In Artemia salina, in fact, the bridges connect germ cells in different mitotic stages. 1 In this invertebrate, the germ cells have different potentialities, as indicated by the fact that the elements of each syncytial group differentiate into nursing cells and oocytes. Bridges connecting oocytes and nurse cells were found in Drosophila melanogaster. 1S Even more striking is the finding of syncytial groups consisting of spermatocytes in different stages of meiosis in a human testis showing abnormal spermatogenesis. ~H Here, the asynchrony obviously cannot be related to multiple potentialities of the spermatogenetic cells. All morphologic findings on mammalian oogenesis, however, point to the presence of a high degree of synchronization in association with the presence of intercellular connections. Despite the above-described conflicting observations made on invertebrate oogenesis and abnormal mammalian spermatogenesis, the hypothesis that these connections are the structural basis for synchronization of cell differentiation in mammalian oogenesis remains valid. The vast majority of primordial oocytes undergo degeneration either during or shortly after meiosis. 4 On the basis of their synchronous maturation, it is probable that meiotic oocytes connected by bridges undergo degeneration simultaneously, thus explaining the similarity of the regressive changes previously noted in adjacent degenerating oocytes. ~7 While bridges are still recognizable in oocytes in early degeneration, they are absent in later stages. Cells in advanced degeneration are characterized by confluent cytoplasm-a further indication of syncytial organization. The degenerative waves that mark the final stages of ovarian development in mammals contribute to one of the most obscure aspects of oogenesis. It has been demonstrated 6 and is generally accepted 14 that, in most species, degeneration eliminates the majority of oocytes that have matured meiotically during embryonal life, but that a sufficient number of these cells remain to constitute the finite stock of oocytes present in the adult mammalian ovary. The mechanisms by which these oocytes escape degeneration, whereas the majority do not, remain to be understood. In a preliminary note on this subject,27 we formulated the hypothesis that the key to survival could be complete cell division. In other words, only those cells in which karyokinesis is followed by cytokinesis would remain independent and survive the degeneration that affects the majority (syncy-
i,
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ig. 12' (top). Electron micrograph of binucleated oocyte in follicle of adult human vary. (X 8000) Fig. 13 (bottom). Biovular follicle in adult mouse ovary. Section a. 1 p. thick. (Toluidine blue, X 550)
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tial) of the oocyte population. Such a differential cellular behavior does not find correspondence in the male gonad, however, where a syncytial organization identical to that of the ovary exists, but where the spermatogenetic cells are all conjoined and remain connected through the spermatid stage. 10 It is more likely, thus, that the surviving oocytes are those which remain in a syncytial organization through meiotic prophase, but separate from one another prior to being affected by the extensive degeneration. Anomalies such as binucleated follicular oocytes and polyovular follicles are not infrequently observed in adult ovaries of a variety of mammalian species. 7 We concur with the hypothesis of Stegner 23 that these defects may be related to persistence of oocyte connections at the time of follicle formation. If the incompletely separated oocytes in the follicle undergo cytoplasmic fusion, the result is a follicle containing a multinucleated oocyte (Fig. 12). On the other hand, in the absence of cytoplasmic fusion, a multiovular follicle develops (Fig. 13). Similarly, persistence of intercellular bridges in spermatids which fail to separate is thought to be responsible for such sperm anomalies as double heads and double tails. 10 Harbor General Hospital 1000 West Carson St. Torrance, Calif. 90509
REFERENCES 1. ANTEUNIS, A., FAUTREZ-FIRLEFYN, N., and FAUTREZ, J. La structure de ponts intercellulaires "obtures" et "ouverts" entre oogonia et oocytes dans l'ovaire d'Artemia salina. Arch Bioi (Uege) 77:645, 1966. 2. BAKER, T. C. A quantitative and cytological study of germ cells in human ovaries. Proc Roy Soc [Bioi] 158:417, 1963. 3. BAKER, T. C. A quantitative and cytological study of oogenesis in the rhesus monkey. ] Anat 100:761, 1966. 4. BEAUMONT, R. M., and MAKDL, A. M. A quantitative and cytological study of oogonia and oocytes in the foetal and neonatal rat. Proc Roy Soc [Biol] 155:557, 1962. 5. BORUM, K. Oogenesis in the mouse. Exp Cell Res 24:495, 1961. 6. BORUM, K. Oogenesis in the mouse: A study of the origin of the mature ova. Exp Cell Res 45:39, 1967. 7. BRAMBELL, F. W. R. "Ovarian Changes." In Physiology of Reproduction (Vol. 1), Parkes, A. S., Ed. Longmans, London, 1952, p. 397. 8. BROWN, E. R., and KING, R. C. Studies on the events resulting in the formation of an egg chamber in Drosophila melanogaster. Growth 28:41, 1964. 9. BURGOS, M. R., and FAWCETT, D. W. Studies on the fine structure of the mammalian testis: I. Differentiation of the spermatids in the cat (Felix domestica). ] Biophys Biochem Cytol1:287, 1955. 10. FAWCETT, D. W. Intercellular bridges. Exp Cell Res 8 (Suppl.):174, 1961. 11. FAWCETT, D. W., and BURGOS, M. R. 1956 Ciba Foundation Symposium: Aging of Transient Tissues. Little, Boston, 1956, p. 86. 12. FAWCETT, D. "V., ITo, S., and SLAUTTERBACK, D. The occurrence of intercellular
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13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
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bridges in groups of cells exhibiting synchronous differentiation. J Biophys Biochem Gytol 5:453, 1959. FRANCHI, L. L., and MANDL, A. M. The ultrastructure of oogonia and oocytes in the foetal and neonatal rat. Proc Roy Soc [Bioi] 157:99, 1962. FRANCHI, L. L., MANDL, A. M., and ZUCKERMAN, S. "The Development of the Ovary and the Process of Oogenesis." In The Ovary, Zuckerman, S., Mandl, A. M., and Eckstein, P., Eds. Acad. Press, London, 1962, chap. 1. CONDOS, B., and ZAMBONI, L. Electron microscopic studies of the embryogenesis and postnatal development of the rabbit ovary. J Ultrastruct Res 21: 162, 1967. IOANNOU, J. M. Oogenesis in the guinea pig. J Embryol Exp Morph 12:673, 1964. KARNOVSKY, M. J. Simple methods for "staining with lead" at high pH in electron microscopy. J Biophys Biochem Gytolll:729, 1961. KING, R C., and DEVINE, R L. Oogenesis in adult Drosophila melanogaster: VII. The submicroscopic morphology of the ovary. Growth 22:299, 1958. LUFT, J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Gytol 9:409, 1961. MATHUR, R, and1:AMBoNI, L. Unpublished observation. NAGANO, T. The structure of cytoplasmic bridges in dividing spermatocytes of the rooster. Anat Rec 141:73, 1961. PETERS, H., LEVY, K, and CRONE, M. Oogenesis in rabbits. J Exp Zool 158: 169, 1965. STEGNER, H. E. Die elektronenmikroskopische Struktur der Eizelle. Ergebn Anat Entwicklungsgesch 39:6, 1967. STEGNER, H. K, and WARTENBERG, H. Elektronenmikroskopische Untersuchungen an Eizellen des Menschen in verschiedenen Stadien der Oogenese. Arch Gynaek 199: 151, 1963. TEPLITZ, R, and OHNO, S. Postnatal induction of ovogenesis in the rabbit (Oryctolagus cuniculus). Exp Gell Res 31:183,1963. WEAKLEY, B. S. Light and electron microscopy of developing germ cells and follicle cells in the ovary of the golden hamster: twenty-four hours before birth to eight days post partum. J Anat 101:435, 1967. ZAMBONI, L., and CONDOS, B. Intercellular bridges and synchronization of germ cell differentiation during oogenesis in the rabbit. J Gell Bioi 36:276, 1968. ZAMBONI, L., and STEFANINI, M. Unpublished observations. ZETTERQVIST, H. The ultrastructural organization of the columnar absorbing cells of the mouse jejunum. Ph.D. Thesis. Karolinska Institutet, Stockholm, 1956.
Contract Program, Center for Population Research, NICHD An expanded contract program in population research will be launched this year by the new Center for Population Research of the National Institute of Child Health and Human Development, Bethesda, Maryland. This research effort will be directed toward the development of new contraceptive methods and population research in the social sciences, and will supplement research performed in NIH laboratories and supported by NIH grants. Certain critical research areas have already been identified. These areas are not exclusive; research on other subjects will also be considered. In contraceptive development, special areas include: (1) maturation and fertilizing capacity of spermatozoa; (2) oviduct function and gamete transport; (3) corpus luteum function and implantation; and (4) the biology of the preimplantation ovum. Four special areas in the social sciences are: (1) trends in fertility and related variables, to document changes in patterns of childbearing and fertility control; (2) the antecedents, processes, and consequences of population structure, distribution, and change; (3) the effects of explicit or implicit government population policies and data needed to help formulate population policies; and (4) the effects on fertility of family structure and patterns of sexual behavior. Advisory panels composed of outstanding scientists will be appointed to develop and oversee the programs for these topics. Further information may be obtained from PHILIP A. CORFMAN, M.D., Director, Center for Population Research, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014. Tel. (301) 496-1101.
Second Postgraduate Course Offere
Russell Malinak, M.D., and Paige Besch, Ph.D. Session VI. Surgical Techniques in Human Reproduction. lJfoderator: Robert H. Barter, M.D. Participants: Celso Ramon Garcia, M.D., and H. E. Riva, M.D. Session VII. Intrauterine Physiology and Fetal Salvage. Moderator: John W. Greene, M.D. Participants: Raymond Kaufman, M.D., and Elsie Carrington, M.D. Session VIII. Ovulation. Moderator: Alvin F. Goldfarb, M.D. Participants: Daniel L. Mishell, M.D., and C. Donald Christian, M.D. Each session will be limited to 75 registrants. The registration fee will be $50 per session. Further details and registration applications may be obtained from WINSTON H. WEESE, M.D., Registration Chairman, 944 South 18th St., Birmingham, Ala. 35205.