Degeneration of Mouse Oocytes in Response to Polycyclic Aromatic Hydrocarbons BELA J. GULYAS ' AND DONALD R. MATTISON Endocrinology and Reproduction Research Branch, Section on Endocrinology, National Institute of Child Health and Human Development and Laboratory of Chemical Pharmacology, Biochemical Pharmacology Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014
ABSTRACT Destruction of mouse oocytes in primordial' and small primary follicles in response to treatment with 3-methylcholanthrene (MC) was studied at the ultrastructural level. Four-week old C57B1/6N (B6) strain mice received a single injection of 80 mg/Kg MC in corn oil intraperitoneally. Controls received only corn oil. Ovaries from animals were prepared for light and electron microscopic examination a t specified intervals after treatment: The number of primordial follicles remained constant in control animals. In contrast, their number decreased significantly (P < 0.01) by three days, and they were depleted by seven days after MC treatment. Subtle degenerative modifications were noted in the ooplasm of primordial follicles two days after treatment. These changes consisted of vesiculation of mitochondrial cristae, increased electron density of the mitochondrial matrix, myelin structures in lipid droplets and in mitochondria. More advanced stages of degeneration of primordial follicles were characterized by further vesiculation or disappearance of mitochondrial cristae, chromatin clumping, and increased density of the ooplasm. Small primary follicles had undergone similar initial degeneration as primordial follicles. In more advanced stages of degeneration nuclear and cytoplasmic contents condensed, endoplasmic reticulum, Golgi complex and mitochondria swelled, small vesicles and multivesicular bodies appeared. In the most advanced stages of degeneration of small primary follicles it appeared that small portions of the oocyte were engulfed by the surrounding follicular cells. It is concluded t h a t exposure of B6 mice t o a single dose of MC results in atresia of oocytes in primordial and small primary follicles. Ultrastructurally, these degenerating oocytes of treated mice looked much like the spontaneously atretic oocytes in untreated animals. The mammalian ovary has a dual function in that it not only produces steroid hormones essential in the establishment and maintenance of reproductive function, but also serves as a repository of oocytes, the female gametes. Toxins acting on the ovary may impair reproductive function by interfering with steroidogenesis, direct destruction of oocytes or more subtle alterations of gamete DNA, RNA or proteins. In most mammals oogenesis ceases during intrauterine or early postnatal development so that destruction of oocytes will impair reproductive function in an irreversible manner (Zuckerman and Baker, '77). At the completion of oogenesis oocytes enter meiosis and proceed to the diplotene (or dictyANAT. REC. (1979) 193: 863-882.
ate) stage of the first meiotic division; meiosis is arrested at this point and does not resume until just prior to, or after ovulation (Zuckerman and Baker, '77). A majority of oocytes never progress beyond the diplotene stage, however, because they are destroyed within the ovary by a poorly understood process of atresia (Ingram, '62). The human ovary, for example contains approximately 7,000,000 oocytes a t four months gestational age, Received June 29, '78. Accepted Oct. 20, '78. 'To whom correspondence and reprint requests should be addressed: National Institutes of Health, Building 18, Rome 101, Bethesda, Maryland 20014. *Present address: Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
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BELA J. GULYAS AND DONALD R. MATTISON
1,000,000 a t birth, 50,000 a t puberty and none a t menopause a t age 50 (Block, '51). Only 400 to 600 oocytes are lost by ovulation, indicating the major role played by atresia in determining the reproductive life span of the human female. Although the mechanism(s1of atresia is not understood, certain modulating factors have been discovered. In the mouse the rate of atresia can be decreased by hypophysectomy (Jones and Krohn, '61) and markedly increased by the polycyclic aromatic hydrocarbons (PAH); benzo(a1pyrene (BPI, 3-methylcholanthrene (MC) and 7,12-dimethylbenz(a)anthracene (DMBA) (Mattison and Thorgeirsson, unpublished). These P A H s are also carcinogenic and will initiate ovarian granulosa cell tumors (Jull, '73). The interrelationship between ovarian carcinogenicity and oocyte destruction is of interest, as no tumors appear until after the oocytes are completely destroyed (Krarup, '67). Current concepts of PAH carcinogenesis indicate that these compounds are not directly carcinogenic, but require metabolic activation by the microsomal mixed function monooxygenase system to reactive electrophilic metabolites, capable of covalent binding to tissue macromolecules of DNA, RNA and protein (Sims and Grover, '74). The relationship between metabolism and ovotoxicity has been investigated using two inbred strains of mice that differ by a single gene in PAH induction of the microsomal monooxygenase arylhydrocarbon hydroxylase (AHH, EC. 1.14.14.21, the enzyme responsible for the first step in the metabolic activation of PAH. After treatment with MC, ovarian AHH increases threefold in PAH responsive C57B1/6N (B6) mice, but no change occurs in ovarian AHH in PAH nonresponsive DBA/2N mice (Mattison and Thorgeirsson, '77). Subsequent experiments have demonstrated the microsomal location and biochemical characteristics of rodent ovarian AHH (Mattison and Thorgeirsson, '78). The rate of destruction of primordial oocytes in the mouse ovary by PAH is proportional to the carcinogenicity of the PAH with DMBA > MC > BP (Jull, '73; Mattison and Thorgeirsson, '77) and the rate of metabolism with destruction occurring more regularly in responsive B6 than nonresponsive DBAI2N mice (For review see Nebert and Felton, '76). Noncarcinogenic PAH and noncarcinogenic inducers of AHH do not increase the rate of primordial oocyte destruction and a-naphtoflavone,a known competitive inhibitor of PAH metabo-
lism by AHH, will inhibit PAH ovotoxicity (Mattison and Thorgeirsson, unpublished). Preliminary light microscopic observations suggest that PAH treatment destroyed oocytes of primary and primordial follicles without an apparent change in follicular or stroma1 cells. The objective of this study was to characterize PAH induced atresia of small follicles in B6 mice at the fine structural level. MATERIALS AND METHODS
Inbred mice of the C57B1/6N strain were obtained from the NIH Veterinary Resources Branch. The animals were housed in plastic cages with hardwood bedding, fed laboratory chow (Purina, St. Louis, Missouri) and received water ad libitum. Four-week-old animals received a single intraperitoneal injection of 3-methylcholanthrene (MC) (80 mg/ Kg; Sigma, St. Louis, Missouri) dissolved in corn oil. Control animals received corn oil only. Animals were killed by cervical dislocation a t specified intervals after treatment. For light microscopic studies both ovaries from each mouse were fixed in Bouin's solution, serially sectioned at 5 pm and stained with hematoxylin and eosin. The number of primordial follicles in each ovary was determined by counting every fortieth section, summing and multiplying the total by 40 (Mattison and Thorgeirsson, '78). For electron microscopic observations a combination of the following fixatives and buffers was used: 3% or 4% glutaraldehyde (Ladd) in 0.1 M phosphate, or cacodylate, or s-collidine buffer, pH 7.4, for one and one-half to two hours. In addition, another fixative, consisting of 2.25% glutaraldehyde, 0.25% formaldehyde in 0.1 M s-collidine buffer, was also used. Tissue blocks were washed in the appropriate buffer twice for 15 minutes prior to postfixation in 1%Oso, in 0.1 M of either phosphate, cacodylate or s-collidine buffer, at 4°C for one hour. Tissues were washed after osmication, stained en bloc in 0.5%uranyl acet a t e in Verona1 acetate buffer (Farquhar and Palade, '65) for one to one and one-half hours. Then the tissue blocks were dehydrated in an increasing concentration of ethanol and embedded in Epon 812 (Luft, '61). Thick Epon sections were stained with neutral toluidine blue. Gold to silver thin sections were picked up on Parlodion-coated, carbon-reinforced copper grids. The sections were stained with uranyl acetate (Watson, '59) and lead citrate (Reynolds, '63) prior to examination.
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PAH INDUCED ATRESIA
0
1
2
3
4
5
6
7
DAYS -t
Fig. 1 Primordial oocyte number in control ( O ) ,corn oil treated ( A ) and MC treated ( 0 )B6 mice (Mean SEM). Numbers in parenthesis refer to the ovaries counted.
RESULTS
TABLE I
Results discussed below pertain only to the primordial and the small primary follicles, because those are the ones reported to have been affected by PAH (Krarup, '67; Mattison and Thorgeirsson, "77). The equivalent and more precise descriptive nomenclature for these follicles is type 1, 2 and 3A, respectively (Pedersen and Peters, '68). Figure 1 illustrates the rate of disappearance of primordial oocytes from the ovaries of B6 mice in response to a single treatment with MC. Whereas the number of primordial oocytes remained constant in the control animals, their number has decreased significantly (P < 0.01) by three days after treatment, in the experimental animals.
Number offollicles studied in thick and thin sections
Electron microscopic observations
Ultrastructure of oocytes and follicles from the mouse ovary has been the subject of numerous publications, (for reviews see Zamboni, '70, '72). For this reason, we shall describe the ultrastructure only of the small follicles of the untreated B6 mouse, pertinent to the comparison of fine structural changes in response to MC treatment. The number of ovaries that were fixed for electron microscopy, and the number of small follicles which were studied in thick or thin sections is provided in table 1. By definition, a type-1 follicle consists of a
Group
Number of ovaries
Number of follicles (1, 2 and 3A combined)
Untreated Oil control 2-day 3-day MC treated 2-day 3-day
6
97
3 3
45 58
10 10
140 124
small oocyte with no follicular cells attached to its surface (Pedersen and Peters, '68). In the 4-week-old B6 mouse follicular development has progressed to the point that only a small percentage of the follicles were type- 1, which were embedded in the outer cortex (fig. 2). Furthermore, many follicles that were classified as type-1 with the light microscope, were found to be in a transitional stage between type-1 and type-2 after electron microscopic examination. In these instances, although the nucleus of a follicular cell was not always in view, the oocyte was partially enclosed by an attenuated follicular cell, and formation of the basal lamina could be detec ted . Several cytoplasmic characteristics set these oocytes apart from the cortical and
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BELA J. GULYAS AND DONALD R. MATTISON
stromal cells, providing reliable identification. Type-1 oocytes were three to four times the size of the cortical cells; they were spherical or slightly elongated and had a large nucleus (fig. 2). The structural organization of the oocyte cytoplasm was relatively simple. Mitochondria were spherical or oval, had a light matrix and a few irregularly arranged lamelliform cristae. Several short profiles of granular endoplasmic reticulum (ER) were closely associated with mitochondria and an occasional lipid droplet (fig. 2). Numerous free ribosomes were scattered throughout the cytoplasm. Type-2 follicle (primordial) consists of a small oocyte which has a few follicular cells attached to its surface (Pedersen and Peters, '68). These oocytes were located in the middle and the inner portion of the cortex (fig. 31, and a thin basal lamina separated the stromal cells from the follicular cells (fig. 4). In some areas the follicular cells were attenuated. Neither the nucleus nor the cytoplasm of the oocyte differed considerably from those of type-1 follicles. By definition, in type-3A (small primary) follicles the oocyte is completely surrounded by a single layer of cuboidal follicular cells (Pedersen and Peters, '68). These follicles were found in groups near the inner region of the cortex (fig. 5). A gradual cytodifferentiation occurred in these small follicles going from the follicles with a few elongated follicular cells, to follicles with a full complement of cuboidal cells. A basal lamina separated the follicular cells from the stromal cells, and granulosa cells of other follicles (fig. 6). Occasionally macula adherentes were present between follicular cells and the oocyte (figs. 6, 7). The plasma membrane of the oocyte lacked microvilli. Mitochondria were enlarged and most of them were ovoid. Their matrix was light and the cristae in some of them were arranged in parallel arrays. Granular ER and free ribosomes were distributed throughout the cytoplasm. Golgi complexes, aggregates of small vesicles, as well as, a few multivesicular bodies were present in small groups in a juxtanuclear position. A few membrane-limited structures, with a homogeneous dense matrix (dense bodies), were associated with the Golgi complexes (fig. 6). The fine structure of ovarian follicles of corn oil-treated animals showed no morphological deviation from that of the untreated animals described above.
UDtake of corn oil into the ovaries In untreated animals the surface epithelial cells and the cells of the outer cortex had only a few small lipid inclusions in their cytoplasm. In corn oil treated animals and in experimental animals, the oil, and presumably the MC dissolved in it, was taken up readily into the ovaries. The corn oil either penetrated between cells (fig. 8, inset) or it was taken up by the epithelial cells in large globules (fig. 8). Once in the cells, the large lipid droplets were broken down into smaller inclusions and they could no longer be differentiated from endogenous lipid droplets. Lipid uptake by oocytes was seldom observed. It should be noted that difficulties were encountered in the fixation procedures of ovaries from MC treated mice. Although we used various combinations of fixation procedures, all of which gave satisfactory preservation of oocytes from untreated and control animals, we were not satisfied with the fine structural appearance of oocytes from treated animals. In follicles of MC treated mice, plasma membranes of follicular cells and oocytes, and membranes of cytoplasmic organelles were broken and the mitochondria were extremely swollen. Cells of the epithelium and the cortex from treated mice were always highly osmiophilic. Because all of these features are characteristic of poorly fixed tissue, as well as of necrotic cells, more than usual caution was exercised in interpreting our observations. It should, however, be reemphasized that we had not encountered these difficulties with ovaries of intact or oil control animals.
Type-1 and 1 -to-2transition stage oocytes after MC treatment Most of the smallest follicles which were examined with the electron microscope were in transition from type-1 to type-2. Whereas some oocytes appeared normal ultrastructurally, others showed subtle signs of degeneration after two days of treatment. The subtle degenerative modifications were exemplified by myelin structures associated with lipid droplets (fig. 9) and found in the matrix of mitochondria (fig. 10); vesicular configuration of mitochondria1 cristae (fig. lo), and increased electron density of the mitochondrial matrix (fig. 10). Oocytes in more advanced stages of degeneration could be recognized in thick sections (fig. 11).The nucleus was irregular in shape and the electron den-
PAH INDUCED ATRESIA
sity of the ooplasm had increased (figs. 11,121. Mitochondria1 cristae were vesiculated, or they were absent (fig. 13). The perinuclear space was swollen and the peripheral chromatin showed extensive clumping (fig. 13). Degeneration of type-2 and type-3Afollicles
The early subtle degenerative changes observed in the type-2 and the small primary follicles after two days of treatment, were similar to those observed in type-1 oocytes (fig. 14). In addition to mitochondrial changes, numerous dense bodies were present in the cytoplasm (fig. 15). Some of these were similar to Golgi associated dense bodies in untreated primordial follicles, whereas others resembled lysosomes. In more advanced stages of degeneration, the nuclear and cytoplasmic contents of the oocytes were condensed, causing an increased electron density (fig. 16). The cytoplasm contained clusters of small vesicles and multivesicular bodies. The perinuclear cisterna, Golgi complex and mitochondria were all swollen in these oocytes. The surrounding follicular cells were unaltered (figs. 14-16). At the most advanced stage of degeneration of small primary follicles both the nucleoplasm and the cytoplasm of the oocyte were condensed (fig. 17). Mitochondria of the oocytes had only fragments of their cristae remaining. The follicular cell cytoplasm was abundant in digestive vacuoles and residual bodies, and it appeared that small portions of the oocyte might have been engulfed by the follicular cells. After three days of MC treatment in a few type-3 follicles necrosis of follicular cells occurred at a time when only subtle degenerative changes of the oocyte were noticed (fig. 18).Lysosomes and residual bodies accumulated in these follicular cells, and their nuclea r chromatin and mitochondrial matrix were condensed. DISCUSSION
Small oocytes are selectively destroyed and eliminated from ovaries after treatment with carcinogenic PAH. The rate of oocyte destruction is rapid because only 2-3%of the primordial and small primary follicles remain three days after MC treatment. That degeneration of an overwhelming number of oocytes occurred in response to MC treatment, is supported by the lack of a significant decline in
867
the number of oocytes in control animals during the interval of our experiment. The rate of primordial oocyte destruction by carcinogenic PAHs in mice is dependent upon the rate of PAH metabolism and its carcinogenicity (Mattison and Thorgeirsson, unpublished). Similar effects of carcinogenic PAH's have been observed on sperm morphology, indicating the vulnerability of the gonads and gametes to environmental influences (Wyrobek and Bruce, '75). Atresia is a poorly understood process which may account for the major loss of oocytes from the ovary throughout life in laboratory animals and man. Although atresia can occur a t any stage of oocyte development, it is most extensive in the prefollicular stage of differentiation (Gondos e t al., '71); that is, in oogonia undergoing mitosis and oocytes in meiotic prophase (Baker and Franchi, '67). Degeneration is first apparent during fetal life with a wave of extensive atresia which continues into early postnatal life (Byskov and Rasmussen, '73; Gondos and Zamboni, '69). Although a few primordial follicles are occasionally seen a t birth (depending on strain of mice studied), marked follicular growth is apparent only during, and after, the second week after birth (Peters, '69). In order to assure that our observations were not masked by the early massive spontaneous atresia, we used 4-week-old mice. Although the process of degeneration in treated mice is not necessarily the same as that occurring spontaneously, the morphological appearance of damaged oocytes in ovaries of treated animals is similar to spontaneously atretic oocytes observed by others in untreated animals (Byskov and Rasmussen, '73; Odor and Blandau, '73). Although advanced stages of atresia are recognizable with the light microscope, the early morphological alterations can be detected only a t the ultrastructural level. Appearance of myelin configurations in lipid droplets and in mitochondria (regardless of the nature of the fixative), increased density of the mitochondrial matrix and vesiculation of mitochondrial cristae, were the earliest noticeable changes in oocytes of treated mice. Myelin figures have been demonstrated in degenerating rat (Franchi and Mandl, '621, hamster (Weakley, '66), and human oocytes. Moreover, similar membrane configurations have been found to be acid phosphatase positive (Anderson, '72) and were thought to be indicative of atresia. Swelling of mitochondria (Vaz-
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BELA J. GULYAS AND DONALD R. MAlTISON
quez-Nin and Sotelo, '67) and vesiculation of Franchi, '67; Gondos and Hobel, '71; Gondos et their cristae have previously been considered al., '71; Byskov and Rasmussen, '73). Exposure of B6 mice to a single dose of MC significant early signs of degeneration of dramatically accelerated the loss of oocytes, small oocytes. A more advanced stage of degeneration was causing complete depletion within one week indicated by nuclear infoldings, peripheral through the process of atresia. Although the clumping of the chromatin, dilation of the ER factors causing oocyte degeneration in our exand the nuclear envelope, and further swell- perimental animals may be different from ing and vesiculation of the mitochondria. In those in spontaneously atretic oocytes, morprimary follicles "dense bodies" and lysosomes phologically, atretic oocytes induced experiappeared in the ooplasm. Nuclear changes ob- mentally in our work appeared similar to served in developing gametes have been con- spontaneously atretic oocytes reported by sidered as the first easily recognizable sign of others (Byskov and Rasmussen, '73; Odor and degeneration in rat gonocytes (Roosen-Runge Blandau, '73). Extrusion through the surface and Leik, '68), monkey oocytes (Baker and epithelium is another process by which ooFranchi, '67, '72), and human fetal testis and cytes are eliminated from mouse ovary during ovaries (Gondos and Hobel, '71, '73; Gondos, the first two weeks of life of intact animals '76; Baker and Franchi, '67). In our work, (Byskov and Rasmussen, '73; Hiura and Futhe nuclear infoldings occurring concurrent jita, '77). This mode of oocyte depletion does with chromatin condensation are similar to not occur in MC treated mice. It is reasonable to suggest from our observathose observed in spontaneously degenerating mouse oocytes (Byskov and Rasmussen, tions that exposure of B6 mice to MC shortens '73). Even a t the most advanced stages of the reproductive life span of the ovary, beatresia the nuclear envelope remained intact cause MC accelerates the depletion of oocytes. until complete breakdown of the oocyte. The Extrapolation to the human from this study dilation of the ER and perinuclear space, and on mice should be made with extreme caution, loss of mitochondria1 cristae were also similar if a t all. Nevertheless, because toxic PAHs to observations on atretic mouse oocytes are found in the environment, and because re(Byskov and Rasmussen, '73; Beltermann, cent epidemiological data support the notion that human oocytes are also sensitive to some '65) and rat (Vazquez-Nin and Sotelo, '67). The origin of "dense bodies" and multivesic- PAH's (Lingerman, '74; Jick e t al., '771, the efular bodies is not clear; nevertheless, these fect of these toxins on human oocytes deserves structures, along with primary lysosomes and close a t tent ion. large ooplasmic vesicles are associated with ACKNOWLEDGMENTS the degenerative process of oocytes (Odor and The authors are indebted to Mrs. Lydia '69, '73). Blandau, For the most part, follicular cells of primor- Yuan for her technical assistance and to Ms. dial and small primary follicles showed no Diane Zodikoff and Miss Debra Livant for signs of necrosis during the early stages of their clerical help. oocyte degeneration. However, in a few inLITERATURE CITED stances, follicular cells of primary follicles Anderson, E. 1972 The localization of acid phosphatase were necrotic at a time when only subtle deand the uptake of horseradish peroxidase in the oocyte generative changes were noticed in the oocyte. and follicle cells of mammals. In: Oogeneais. J.D. Biggers and A. W. Schuetz, eds. University Park Press, Baltimore, This observation deviates from previous rePp. 87-117. ports that the follicular cells remain intact T. G., and L. L. Franchi 1967 The fine structure of even a t the most advanced stages of spon- Baker, oogonia and oocytes in human ovaries. J. Cell Scien., 2: taneous atresia of oocytes in small follicles 213-224. (Gondos and Hobel, '71). 1972 The tine structure of oogonia and oocytes in the rhesus monkey (Macaca rnulattai. 2.Zelforsch., In advanced stages of degeneration of small 126: 53-74. primary follicles i t appeared that portions of Beltermann, R. 1965 Electronenmikroskopische Befunde the degenerating oocyte might have been bei beginnender Follikelatresia im Avor der Maus. Arch. G ~ n a k .200: , 601-609. engulfed by the follicular cells. Phagocytosis of atretic oocytes by follicular cells has been Block, E. 1951 Quantitative morphological investigations of the follicular system in women. Acta. Anat., 12: reported to occur in ovaries of various mam267-285. malian species (Franchi et al., '62; Baker and Byskov, A. G., and G. Rasmussen 1973 Ultrastructural
PAH INDUCED ATRESIA studies of t h e developing follicle. In: The Development and Maturation of t h e Ovary and its Function. H. Peters, ed. Excerpta Medica, pp. 55-62. Farquhar, M. G., and G. E. Palade 1965 Cell junctions in amphibian skin. J. Cell Biol., 26: 263-291. 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. Franchi, L. L.,A. M. Mandl and S. Zuckerman 1962 The development of the ovary and t h e progress of oogenesis. In: The Ovary. S. Zuckerman, ed. Academic Press, New York, VOl. 1, pp. 2-88. Gondos, B. 1976 Differentiation and growth of cells in the gonads. In: Differentiation and Growth of Cells. G. Goldspink, ed. John Wiley and Sons, New York, pp. 169-208. Gondos, B., P. Bhiraleus and C. J. Hobel 1971 Ultrastructural observations on germ cells in human fetal ovaries. h e r . J. Obstet. Gynecol., 110: 644-652. Gondos, B., and C. J. Hobel 1971 Ultrastructure of germ cell development in the human fetal testis. Z. Zellforsch., 119: 1-20. 1973 Germ cell degeneration and phagocytosis in t h e human foetal ovary. In: The Development and Maturation of t h e Ovary and its Function. H. Peters, ed. Excerpta Medica, pp. 77-83. Gondos, B., and L. Zamboni 1969 Ovarian development: The functional importance of germ cell interconnections. Fertil. Steril., 20: 176-189. Hiura, M., and J. Fujita 1977 Electron microscopic observations on the elimination of the oocyte through the peritoneal epithelium in t h e neonatal mouse ovary. Cell Tiss. Res., 182: 73-79. Ingram, D. L. 1962 Atresia. In: The Ovary. S. Zuckerman, ed. Academic Press, New York. Vol. 1, pp. 247-273. Jick, H., J. Porter and A. S. Morrison 1977 The effect of smoking on t h e age of menopause. Lancet, 1: 1354-1355. Jones, E. C., and P. L. Krohn 1961 The effect of hypophysectomy on age changes in t h e ovaries of mice. J. Endocrin., 21: 497-509. Jull, J. W. 1973 Ovarian tumorigenesis. In: Methods in Cancer Research. H. Busch, ed. Academic Press, 7: 131-186. Krarup, T. 1967 9:lO-Dimethyl-l:2-benzanthracene induced ovarian tumors in mice. Acta Path. Microbiol. Scandinav., 70: 241-248. Lingerman, C. H. 1974 Etiology of cancer of the human ovary: A review. J. Natl. Cancer Inst., 53: 1603-1618. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414.
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Mattison, D. R., and S. S. Thorgeirsson 1977 Genetic differences in mouse ovarian metabolism of benzo[alpyrene and oocyte toxicity. Biochem. Pharmac., 26: 906-912. 1978 Gonadal aryl hydrocarbon hydroxylase in rats and mice. Canc. Res., 38: 1368-1373. Nebert, D. W., and J. S. Felton 1976 Importance of genetic factors influencing the metabolism of foreign compounds. Fed. Proc., 35: 1133-1141. Odor, D. L.,a n d R . J. Blandau 1969 Ultrastructural studies on fetal and early postnatal mouse ovaries. 11. Cytcdifferentiation. Am. J. Anat., 125: 177-216. 1973 Ultrastructural observations on atresia in whole organ cultures of foetal mouse ovaries. In: The Development and Maturation of the Ovary and its Function. H. Peters, ed. Excerpta Medica, pp. 63-76. Pedersen, T., and H. Peters 1968 Proposal for a classification of oocytes and follicles in the mouse ovary. J. Reprod. Fertil., 17: 555-557. Peters, H. 1969 The derelopment of the mouse ovary from birth to maturity. Acta. Endocrin., 62: 98-116. Reynolds, E. S. 1963 The use of lead citrate a t high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Roosen-Runge, E. G., and J. Leik 1968 Gonocyte degeneration in the postnatal male rat. Am. J. Anat., 122: 275-300. Sims, P., and P. L. Grover 1974 Epoxides in polycyclic aromatic hydrocarbon metabolism and carcinogenesis. Adv. in Canc. Res., 20: 165-274. Vazquez-Nin, G. H., and J. R. Sotelo 1967 Electron microscope study of t h e atretic oocytes of t h e rat. Z. Zellforsch., 80: 518-533. Watson, M. 1959 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478. Weakley, B. S. 1966 Electron microscopy of the oocyte and granulosa cells in t h e developing ovarian follicles of the golden hamster (Mesocricetus auratus). J. Anat., 100: 503-534. Wyrobek, A. J., and W. R. Bruce 1975 Chemical induction of sperm abnormalities in mice. Proc. Natl. Acad. Sci., 72: 4425-4429. Zamboni, L. 1970 Ultrastructure of Mammalian oocytes and ova. Biol. Reprod., Suppl. 2: 44-63. 1972 Comparative studies on the ultrastructure of mammalian oocytes. In: Oogenesis. J. D. Biggers and A. W. Schuetz, &. University Press, Baltimore, pp. 5-45. Zuckerman, S., and T. G. Baker 1977 The development of the ovary and t h e process of oogenesis. In: The Ovary. S. Zuckerman and B. J. Weir, eds. Academic Press, New York, Vol. 1, pp. 41-67.
PLATE 1 EXPLANATION OF FIGURES
2 T w o neighboring primordial oocytes in the cortex of intact mouse ovary beneath the surface epithelium (El.The nucleus (N)is slightly elongated. Oval mitochondria (M), Lipid (L) and short segments of the granular endoplasmic reticulum (GER)are X 9,000. scattered in the ooplasm (0). 3 One-micron section of type-2 (primordial) follicle in the cortex. 4
870
X
860.
Electron micrograph of two adjacent type-2 (primordial) follicles. The organization of the ooplasm (0) appears similar to that of type-I oocytes. Attenuated portions of follicular cells (FC)surround the oocyte and a thin basal lamina (BL)separated developing follicles from each other and the stromal cells. x 9,000.
PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattiaon
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
5 One-micron section illustrating two type-3A follicles (arrows) from a control mouse ovary. X 750. 6 Electron micrograph of type-3A follicles from a control mouse. The nucleus (N) is large and spherical; mitochondria (MI are numerous in the ooplasm (0).The oocyte is enclosed mostly by cuboidal follicular cells (FC). Occasionally macula adherentes (MA) are found between t h e oocyte and a follicular cell. Golgi complex ( G O , aggregates of small vesicles (V) and a few membrane-limited dense bodies (DB)are also present. Basal lamina (BL). x 11,000.
7 A macula adherens between an oocyte (0)and a follicular cell (FC).
872
X
24,500.
PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 2
PLATE 3 EXPLANATION OF FIGURES
8 Light micrograph (inset) and electron micrograph illustrating uptake of lipid (L) by the surface epithelium of a mouse ovary two days after MC treatment. x 540; X 7,700. 9 Follicle in transition from type-1 to type-2 in a mouse ovary 2 days after MC treatment. Note myelin figure (arrow) associated with lipid (L). x 9,000. 10 Vesicular cristae and myelin figures (arrows) in mitochondria of type-1 oocytes after two days of MC treatment. X 16,700.
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PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 3
PLATE 4 EXPLANATION OF FIGURES
11 One-micron section of a n ovary three days after MC treatment. Degenerating type1 (l),type-2 (2) and type-3A (3A) follicles have denser ooplasm than unaffected oocytes. X 860. 12 Degenerating oocyte (transient from type-1 to type-2) after three days of MC treatment. Mitochondria1 (M) cristae are vesiculated, ooplasm and nucleoplasm show increased electron density. X 9,000. 13 Oocytes in advanced stage of degeneration three days after MC treatment. Nuclear chromatin (CH)is clumped, perinuclear space swollen and vesicles (V)fill the 00plasm (0). Follicular cells (FC) are unaltered. X 9.000.
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PAN INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 4
PLATE 5 EXPLANATION OF FIGURES
14 Small primary follicle (type-3A) two days after MC treatment. Note vesiculation of cristae and increased electron density of mitochondria1 matrix (M)of the oocyte (0).Follicular cell (FC). X 24,500. 15 Numerous dense bodies (DB) and some lysosomes (LY)appear in the ooplasm (0) two days after treatment. Nucleus (N); Golgi complex (GO; follicular cell (FC) X
7,700.
16 Small primary follicle at advanced stages of degeneration three days after MC treatment. Mitochondria (M) are swollen having only a few cristae, and both t h e nucleoplasm (N) and ooplasm (0) show increased electron density. Clusters of small vesicles (V)and multivesicular bodies (MVB) are numerous. Follicular cell (FC) is unchanged. X 13,500.
878
PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 5
PLATE 6 EXPLANATION OF FIGURES
17 Portions of a degenerating small primary follicle appeared to be engulfed by surrounding follicular cells (FC). Three cells are abundant in digestive vacuoles and X 11.000. residual bodies (RBI. Oocyte (0). 18 Necrosis of follicular cells (FC)in a type-3A follicle. Lysosomes (LY)and residual mitochonbodies (RB)are observed in their follicular cells. Cristae in oocyte (0) dria (M)are vesicular. X 7,700.
880
PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 6
ABSTRACT Destruction of mouse oocytes in primordial' and small primary follicles in response to treatment with 3-methylcholanthrene (MC) was studied at the ultrastructural level. Four-week old C57B1/6N (B6) strain mice received a single injection of 80 mg/Kg MC in corn oil intraperitoneally. Controls received only corn oil. Ovaries from animals were prepared for light and electron microscopic examination a t specified intervals after treatment: The number of primordial follicles remained constant in control animals. In contrast, their number decreased significantly (P < 0.01) by three days, and they were depleted by seven days after MC treatment. Subtle degenerative modifications were noted in the ooplasm of primordial follicles two days after treatment. These changes consisted of vesiculation of mitochondrial cristae, increased electron density of the mitochondrial matrix, myelin structures in lipid droplets and in mitochondria. More advanced stages of degeneration of primordial follicles were characterized by further vesiculation or disappearance of mitochondrial cristae, chromatin clumping, and increased density of the ooplasm. Small primary follicles had undergone similar initial degeneration as primordial follicles. In more advanced stages of degeneration nuclear and cytoplasmic contents condensed, endoplasmic reticulum, Golgi complex and mitochondria swelled, small vesicles and multivesicular bodies appeared. In the most advanced stages of degeneration of small primary follicles it appeared that small portions of the oocyte were engulfed by the surrounding follicular cells. It is concluded t h a t exposure of B6 mice t o a single dose of MC results in atresia of oocytes in primordial and small primary follicles. Ultrastructurally, these degenerating oocytes of treated mice looked much like the spontaneously atretic oocytes in untreated animals. The mammalian ovary has a dual function in that it not only produces steroid hormones essential in the establishment and maintenance of reproductive function, but also serves as a repository of oocytes, the female gametes. Toxins acting on the ovary may impair reproductive function by interfering with steroidogenesis, direct destruction of oocytes or more subtle alterations of gamete DNA, RNA or proteins. In most mammals oogenesis ceases during intrauterine or early postnatal development so that destruction of oocytes will impair reproductive function in an irreversible manner (Zuckerman and Baker, '77). At the completion of oogenesis oocytes enter meiosis and proceed to the diplotene (or dictyANAT. REC. (1979) 193: 863-882.
ate) stage of the first meiotic division; meiosis is arrested at this point and does not resume until just prior to, or after ovulation (Zuckerman and Baker, '77). A majority of oocytes never progress beyond the diplotene stage, however, because they are destroyed within the ovary by a poorly understood process of atresia (Ingram, '62). The human ovary, for example contains approximately 7,000,000 oocytes a t four months gestational age, Received June 29, '78. Accepted Oct. 20, '78. 'To whom correspondence and reprint requests should be addressed: National Institutes of Health, Building 18, Rome 101, Bethesda, Maryland 20014. *Present address: Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
863
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BELA J. GULYAS AND DONALD R. MATTISON
1,000,000 a t birth, 50,000 a t puberty and none a t menopause a t age 50 (Block, '51). Only 400 to 600 oocytes are lost by ovulation, indicating the major role played by atresia in determining the reproductive life span of the human female. Although the mechanism(s1of atresia is not understood, certain modulating factors have been discovered. In the mouse the rate of atresia can be decreased by hypophysectomy (Jones and Krohn, '61) and markedly increased by the polycyclic aromatic hydrocarbons (PAH); benzo(a1pyrene (BPI, 3-methylcholanthrene (MC) and 7,12-dimethylbenz(a)anthracene (DMBA) (Mattison and Thorgeirsson, unpublished). These P A H s are also carcinogenic and will initiate ovarian granulosa cell tumors (Jull, '73). The interrelationship between ovarian carcinogenicity and oocyte destruction is of interest, as no tumors appear until after the oocytes are completely destroyed (Krarup, '67). Current concepts of PAH carcinogenesis indicate that these compounds are not directly carcinogenic, but require metabolic activation by the microsomal mixed function monooxygenase system to reactive electrophilic metabolites, capable of covalent binding to tissue macromolecules of DNA, RNA and protein (Sims and Grover, '74). The relationship between metabolism and ovotoxicity has been investigated using two inbred strains of mice that differ by a single gene in PAH induction of the microsomal monooxygenase arylhydrocarbon hydroxylase (AHH, EC. 1.14.14.21, the enzyme responsible for the first step in the metabolic activation of PAH. After treatment with MC, ovarian AHH increases threefold in PAH responsive C57B1/6N (B6) mice, but no change occurs in ovarian AHH in PAH nonresponsive DBA/2N mice (Mattison and Thorgeirsson, '77). Subsequent experiments have demonstrated the microsomal location and biochemical characteristics of rodent ovarian AHH (Mattison and Thorgeirsson, '78). The rate of destruction of primordial oocytes in the mouse ovary by PAH is proportional to the carcinogenicity of the PAH with DMBA > MC > BP (Jull, '73; Mattison and Thorgeirsson, '77) and the rate of metabolism with destruction occurring more regularly in responsive B6 than nonresponsive DBAI2N mice (For review see Nebert and Felton, '76). Noncarcinogenic PAH and noncarcinogenic inducers of AHH do not increase the rate of primordial oocyte destruction and a-naphtoflavone,a known competitive inhibitor of PAH metabo-
lism by AHH, will inhibit PAH ovotoxicity (Mattison and Thorgeirsson, unpublished). Preliminary light microscopic observations suggest that PAH treatment destroyed oocytes of primary and primordial follicles without an apparent change in follicular or stroma1 cells. The objective of this study was to characterize PAH induced atresia of small follicles in B6 mice at the fine structural level. MATERIALS AND METHODS
Inbred mice of the C57B1/6N strain were obtained from the NIH Veterinary Resources Branch. The animals were housed in plastic cages with hardwood bedding, fed laboratory chow (Purina, St. Louis, Missouri) and received water ad libitum. Four-week-old animals received a single intraperitoneal injection of 3-methylcholanthrene (MC) (80 mg/ Kg; Sigma, St. Louis, Missouri) dissolved in corn oil. Control animals received corn oil only. Animals were killed by cervical dislocation a t specified intervals after treatment. For light microscopic studies both ovaries from each mouse were fixed in Bouin's solution, serially sectioned at 5 pm and stained with hematoxylin and eosin. The number of primordial follicles in each ovary was determined by counting every fortieth section, summing and multiplying the total by 40 (Mattison and Thorgeirsson, '78). For electron microscopic observations a combination of the following fixatives and buffers was used: 3% or 4% glutaraldehyde (Ladd) in 0.1 M phosphate, or cacodylate, or s-collidine buffer, pH 7.4, for one and one-half to two hours. In addition, another fixative, consisting of 2.25% glutaraldehyde, 0.25% formaldehyde in 0.1 M s-collidine buffer, was also used. Tissue blocks were washed in the appropriate buffer twice for 15 minutes prior to postfixation in 1%Oso, in 0.1 M of either phosphate, cacodylate or s-collidine buffer, at 4°C for one hour. Tissues were washed after osmication, stained en bloc in 0.5%uranyl acet a t e in Verona1 acetate buffer (Farquhar and Palade, '65) for one to one and one-half hours. Then the tissue blocks were dehydrated in an increasing concentration of ethanol and embedded in Epon 812 (Luft, '61). Thick Epon sections were stained with neutral toluidine blue. Gold to silver thin sections were picked up on Parlodion-coated, carbon-reinforced copper grids. The sections were stained with uranyl acetate (Watson, '59) and lead citrate (Reynolds, '63) prior to examination.
865
PAH INDUCED ATRESIA
0
1
2
3
4
5
6
7
DAYS -t
Fig. 1 Primordial oocyte number in control ( O ) ,corn oil treated ( A ) and MC treated ( 0 )B6 mice (Mean SEM). Numbers in parenthesis refer to the ovaries counted.
RESULTS
TABLE I
Results discussed below pertain only to the primordial and the small primary follicles, because those are the ones reported to have been affected by PAH (Krarup, '67; Mattison and Thorgeirsson, "77). The equivalent and more precise descriptive nomenclature for these follicles is type 1, 2 and 3A, respectively (Pedersen and Peters, '68). Figure 1 illustrates the rate of disappearance of primordial oocytes from the ovaries of B6 mice in response to a single treatment with MC. Whereas the number of primordial oocytes remained constant in the control animals, their number has decreased significantly (P < 0.01) by three days after treatment, in the experimental animals.
Number offollicles studied in thick and thin sections
Electron microscopic observations
Ultrastructure of oocytes and follicles from the mouse ovary has been the subject of numerous publications, (for reviews see Zamboni, '70, '72). For this reason, we shall describe the ultrastructure only of the small follicles of the untreated B6 mouse, pertinent to the comparison of fine structural changes in response to MC treatment. The number of ovaries that were fixed for electron microscopy, and the number of small follicles which were studied in thick or thin sections is provided in table 1. By definition, a type-1 follicle consists of a
Group
Number of ovaries
Number of follicles (1, 2 and 3A combined)
Untreated Oil control 2-day 3-day MC treated 2-day 3-day
6
97
3 3
45 58
10 10
140 124
small oocyte with no follicular cells attached to its surface (Pedersen and Peters, '68). In the 4-week-old B6 mouse follicular development has progressed to the point that only a small percentage of the follicles were type- 1, which were embedded in the outer cortex (fig. 2). Furthermore, many follicles that were classified as type-1 with the light microscope, were found to be in a transitional stage between type-1 and type-2 after electron microscopic examination. In these instances, although the nucleus of a follicular cell was not always in view, the oocyte was partially enclosed by an attenuated follicular cell, and formation of the basal lamina could be detec ted . Several cytoplasmic characteristics set these oocytes apart from the cortical and
866
BELA J. GULYAS AND DONALD R. MATTISON
stromal cells, providing reliable identification. Type-1 oocytes were three to four times the size of the cortical cells; they were spherical or slightly elongated and had a large nucleus (fig. 2). The structural organization of the oocyte cytoplasm was relatively simple. Mitochondria were spherical or oval, had a light matrix and a few irregularly arranged lamelliform cristae. Several short profiles of granular endoplasmic reticulum (ER) were closely associated with mitochondria and an occasional lipid droplet (fig. 2). Numerous free ribosomes were scattered throughout the cytoplasm. Type-2 follicle (primordial) consists of a small oocyte which has a few follicular cells attached to its surface (Pedersen and Peters, '68). These oocytes were located in the middle and the inner portion of the cortex (fig. 31, and a thin basal lamina separated the stromal cells from the follicular cells (fig. 4). In some areas the follicular cells were attenuated. Neither the nucleus nor the cytoplasm of the oocyte differed considerably from those of type-1 follicles. By definition, in type-3A (small primary) follicles the oocyte is completely surrounded by a single layer of cuboidal follicular cells (Pedersen and Peters, '68). These follicles were found in groups near the inner region of the cortex (fig. 5). A gradual cytodifferentiation occurred in these small follicles going from the follicles with a few elongated follicular cells, to follicles with a full complement of cuboidal cells. A basal lamina separated the follicular cells from the stromal cells, and granulosa cells of other follicles (fig. 6). Occasionally macula adherentes were present between follicular cells and the oocyte (figs. 6, 7). The plasma membrane of the oocyte lacked microvilli. Mitochondria were enlarged and most of them were ovoid. Their matrix was light and the cristae in some of them were arranged in parallel arrays. Granular ER and free ribosomes were distributed throughout the cytoplasm. Golgi complexes, aggregates of small vesicles, as well as, a few multivesicular bodies were present in small groups in a juxtanuclear position. A few membrane-limited structures, with a homogeneous dense matrix (dense bodies), were associated with the Golgi complexes (fig. 6). The fine structure of ovarian follicles of corn oil-treated animals showed no morphological deviation from that of the untreated animals described above.
UDtake of corn oil into the ovaries In untreated animals the surface epithelial cells and the cells of the outer cortex had only a few small lipid inclusions in their cytoplasm. In corn oil treated animals and in experimental animals, the oil, and presumably the MC dissolved in it, was taken up readily into the ovaries. The corn oil either penetrated between cells (fig. 8, inset) or it was taken up by the epithelial cells in large globules (fig. 8). Once in the cells, the large lipid droplets were broken down into smaller inclusions and they could no longer be differentiated from endogenous lipid droplets. Lipid uptake by oocytes was seldom observed. It should be noted that difficulties were encountered in the fixation procedures of ovaries from MC treated mice. Although we used various combinations of fixation procedures, all of which gave satisfactory preservation of oocytes from untreated and control animals, we were not satisfied with the fine structural appearance of oocytes from treated animals. In follicles of MC treated mice, plasma membranes of follicular cells and oocytes, and membranes of cytoplasmic organelles were broken and the mitochondria were extremely swollen. Cells of the epithelium and the cortex from treated mice were always highly osmiophilic. Because all of these features are characteristic of poorly fixed tissue, as well as of necrotic cells, more than usual caution was exercised in interpreting our observations. It should, however, be reemphasized that we had not encountered these difficulties with ovaries of intact or oil control animals.
Type-1 and 1 -to-2transition stage oocytes after MC treatment Most of the smallest follicles which were examined with the electron microscope were in transition from type-1 to type-2. Whereas some oocytes appeared normal ultrastructurally, others showed subtle signs of degeneration after two days of treatment. The subtle degenerative modifications were exemplified by myelin structures associated with lipid droplets (fig. 9) and found in the matrix of mitochondria (fig. 10); vesicular configuration of mitochondria1 cristae (fig. lo), and increased electron density of the mitochondrial matrix (fig. 10). Oocytes in more advanced stages of degeneration could be recognized in thick sections (fig. 11).The nucleus was irregular in shape and the electron den-
PAH INDUCED ATRESIA
sity of the ooplasm had increased (figs. 11,121. Mitochondria1 cristae were vesiculated, or they were absent (fig. 13). The perinuclear space was swollen and the peripheral chromatin showed extensive clumping (fig. 13). Degeneration of type-2 and type-3Afollicles
The early subtle degenerative changes observed in the type-2 and the small primary follicles after two days of treatment, were similar to those observed in type-1 oocytes (fig. 14). In addition to mitochondrial changes, numerous dense bodies were present in the cytoplasm (fig. 15). Some of these were similar to Golgi associated dense bodies in untreated primordial follicles, whereas others resembled lysosomes. In more advanced stages of degeneration, the nuclear and cytoplasmic contents of the oocytes were condensed, causing an increased electron density (fig. 16). The cytoplasm contained clusters of small vesicles and multivesicular bodies. The perinuclear cisterna, Golgi complex and mitochondria were all swollen in these oocytes. The surrounding follicular cells were unaltered (figs. 14-16). At the most advanced stage of degeneration of small primary follicles both the nucleoplasm and the cytoplasm of the oocyte were condensed (fig. 17). Mitochondria of the oocytes had only fragments of their cristae remaining. The follicular cell cytoplasm was abundant in digestive vacuoles and residual bodies, and it appeared that small portions of the oocyte might have been engulfed by the follicular cells. After three days of MC treatment in a few type-3 follicles necrosis of follicular cells occurred at a time when only subtle degenerative changes of the oocyte were noticed (fig. 18).Lysosomes and residual bodies accumulated in these follicular cells, and their nuclea r chromatin and mitochondrial matrix were condensed. DISCUSSION
Small oocytes are selectively destroyed and eliminated from ovaries after treatment with carcinogenic PAH. The rate of oocyte destruction is rapid because only 2-3%of the primordial and small primary follicles remain three days after MC treatment. That degeneration of an overwhelming number of oocytes occurred in response to MC treatment, is supported by the lack of a significant decline in
867
the number of oocytes in control animals during the interval of our experiment. The rate of primordial oocyte destruction by carcinogenic PAHs in mice is dependent upon the rate of PAH metabolism and its carcinogenicity (Mattison and Thorgeirsson, unpublished). Similar effects of carcinogenic PAH's have been observed on sperm morphology, indicating the vulnerability of the gonads and gametes to environmental influences (Wyrobek and Bruce, '75). Atresia is a poorly understood process which may account for the major loss of oocytes from the ovary throughout life in laboratory animals and man. Although atresia can occur a t any stage of oocyte development, it is most extensive in the prefollicular stage of differentiation (Gondos e t al., '71); that is, in oogonia undergoing mitosis and oocytes in meiotic prophase (Baker and Franchi, '67). Degeneration is first apparent during fetal life with a wave of extensive atresia which continues into early postnatal life (Byskov and Rasmussen, '73; Gondos and Zamboni, '69). Although a few primordial follicles are occasionally seen a t birth (depending on strain of mice studied), marked follicular growth is apparent only during, and after, the second week after birth (Peters, '69). In order to assure that our observations were not masked by the early massive spontaneous atresia, we used 4-week-old mice. Although the process of degeneration in treated mice is not necessarily the same as that occurring spontaneously, the morphological appearance of damaged oocytes in ovaries of treated animals is similar to spontaneously atretic oocytes observed by others in untreated animals (Byskov and Rasmussen, '73; Odor and Blandau, '73). Although advanced stages of atresia are recognizable with the light microscope, the early morphological alterations can be detected only a t the ultrastructural level. Appearance of myelin configurations in lipid droplets and in mitochondria (regardless of the nature of the fixative), increased density of the mitochondrial matrix and vesiculation of mitochondrial cristae, were the earliest noticeable changes in oocytes of treated mice. Myelin figures have been demonstrated in degenerating rat (Franchi and Mandl, '621, hamster (Weakley, '66), and human oocytes. Moreover, similar membrane configurations have been found to be acid phosphatase positive (Anderson, '72) and were thought to be indicative of atresia. Swelling of mitochondria (Vaz-
868
BELA J. GULYAS AND DONALD R. MAlTISON
quez-Nin and Sotelo, '67) and vesiculation of Franchi, '67; Gondos and Hobel, '71; Gondos et their cristae have previously been considered al., '71; Byskov and Rasmussen, '73). Exposure of B6 mice to a single dose of MC significant early signs of degeneration of dramatically accelerated the loss of oocytes, small oocytes. A more advanced stage of degeneration was causing complete depletion within one week indicated by nuclear infoldings, peripheral through the process of atresia. Although the clumping of the chromatin, dilation of the ER factors causing oocyte degeneration in our exand the nuclear envelope, and further swell- perimental animals may be different from ing and vesiculation of the mitochondria. In those in spontaneously atretic oocytes, morprimary follicles "dense bodies" and lysosomes phologically, atretic oocytes induced experiappeared in the ooplasm. Nuclear changes ob- mentally in our work appeared similar to served in developing gametes have been con- spontaneously atretic oocytes reported by sidered as the first easily recognizable sign of others (Byskov and Rasmussen, '73; Odor and degeneration in rat gonocytes (Roosen-Runge Blandau, '73). Extrusion through the surface and Leik, '68), monkey oocytes (Baker and epithelium is another process by which ooFranchi, '67, '72), and human fetal testis and cytes are eliminated from mouse ovary during ovaries (Gondos and Hobel, '71, '73; Gondos, the first two weeks of life of intact animals '76; Baker and Franchi, '67). In our work, (Byskov and Rasmussen, '73; Hiura and Futhe nuclear infoldings occurring concurrent jita, '77). This mode of oocyte depletion does with chromatin condensation are similar to not occur in MC treated mice. It is reasonable to suggest from our observathose observed in spontaneously degenerating mouse oocytes (Byskov and Rasmussen, tions that exposure of B6 mice to MC shortens '73). Even a t the most advanced stages of the reproductive life span of the ovary, beatresia the nuclear envelope remained intact cause MC accelerates the depletion of oocytes. until complete breakdown of the oocyte. The Extrapolation to the human from this study dilation of the ER and perinuclear space, and on mice should be made with extreme caution, loss of mitochondria1 cristae were also similar if a t all. Nevertheless, because toxic PAHs to observations on atretic mouse oocytes are found in the environment, and because re(Byskov and Rasmussen, '73; Beltermann, cent epidemiological data support the notion that human oocytes are also sensitive to some '65) and rat (Vazquez-Nin and Sotelo, '67). The origin of "dense bodies" and multivesic- PAH's (Lingerman, '74; Jick e t al., '771, the efular bodies is not clear; nevertheless, these fect of these toxins on human oocytes deserves structures, along with primary lysosomes and close a t tent ion. large ooplasmic vesicles are associated with ACKNOWLEDGMENTS the degenerative process of oocytes (Odor and The authors are indebted to Mrs. Lydia '69, '73). Blandau, For the most part, follicular cells of primor- Yuan for her technical assistance and to Ms. dial and small primary follicles showed no Diane Zodikoff and Miss Debra Livant for signs of necrosis during the early stages of their clerical help. oocyte degeneration. However, in a few inLITERATURE CITED stances, follicular cells of primary follicles Anderson, E. 1972 The localization of acid phosphatase were necrotic at a time when only subtle deand the uptake of horseradish peroxidase in the oocyte generative changes were noticed in the oocyte. and follicle cells of mammals. In: Oogeneais. J.D. Biggers and A. W. Schuetz, eds. University Park Press, Baltimore, This observation deviates from previous rePp. 87-117. ports that the follicular cells remain intact T. G., and L. L. Franchi 1967 The fine structure of even a t the most advanced stages of spon- Baker, oogonia and oocytes in human ovaries. J. Cell Scien., 2: taneous atresia of oocytes in small follicles 213-224. (Gondos and Hobel, '71). 1972 The tine structure of oogonia and oocytes in the rhesus monkey (Macaca rnulattai. 2.Zelforsch., In advanced stages of degeneration of small 126: 53-74. primary follicles i t appeared that portions of Beltermann, R. 1965 Electronenmikroskopische Befunde the degenerating oocyte might have been bei beginnender Follikelatresia im Avor der Maus. Arch. G ~ n a k .200: , 601-609. engulfed by the follicular cells. Phagocytosis of atretic oocytes by follicular cells has been Block, E. 1951 Quantitative morphological investigations of the follicular system in women. Acta. Anat., 12: reported to occur in ovaries of various mam267-285. malian species (Franchi et al., '62; Baker and Byskov, A. G., and G. Rasmussen 1973 Ultrastructural
PAH INDUCED ATRESIA studies of t h e developing follicle. In: The Development and Maturation of t h e Ovary and its Function. H. Peters, ed. Excerpta Medica, pp. 55-62. Farquhar, M. G., and G. E. Palade 1965 Cell junctions in amphibian skin. J. Cell Biol., 26: 263-291. 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. Franchi, L. L.,A. M. Mandl and S. Zuckerman 1962 The development of the ovary and t h e progress of oogenesis. In: The Ovary. S. Zuckerman, ed. Academic Press, New York, VOl. 1, pp. 2-88. Gondos, B. 1976 Differentiation and growth of cells in the gonads. In: Differentiation and Growth of Cells. G. Goldspink, ed. John Wiley and Sons, New York, pp. 169-208. Gondos, B., P. Bhiraleus and C. J. Hobel 1971 Ultrastructural observations on germ cells in human fetal ovaries. h e r . J. Obstet. Gynecol., 110: 644-652. Gondos, B., and C. J. Hobel 1971 Ultrastructure of germ cell development in the human fetal testis. Z. Zellforsch., 119: 1-20. 1973 Germ cell degeneration and phagocytosis in t h e human foetal ovary. In: The Development and Maturation of t h e Ovary and its Function. H. Peters, ed. Excerpta Medica, pp. 77-83. Gondos, B., and L. Zamboni 1969 Ovarian development: The functional importance of germ cell interconnections. Fertil. Steril., 20: 176-189. Hiura, M., and J. Fujita 1977 Electron microscopic observations on the elimination of the oocyte through the peritoneal epithelium in t h e neonatal mouse ovary. Cell Tiss. Res., 182: 73-79. Ingram, D. L. 1962 Atresia. In: The Ovary. S. Zuckerman, ed. Academic Press, New York. Vol. 1, pp. 247-273. Jick, H., J. Porter and A. S. Morrison 1977 The effect of smoking on t h e age of menopause. Lancet, 1: 1354-1355. Jones, E. C., and P. L. Krohn 1961 The effect of hypophysectomy on age changes in t h e ovaries of mice. J. Endocrin., 21: 497-509. Jull, J. W. 1973 Ovarian tumorigenesis. In: Methods in Cancer Research. H. Busch, ed. Academic Press, 7: 131-186. Krarup, T. 1967 9:lO-Dimethyl-l:2-benzanthracene induced ovarian tumors in mice. Acta Path. Microbiol. Scandinav., 70: 241-248. Lingerman, C. H. 1974 Etiology of cancer of the human ovary: A review. J. Natl. Cancer Inst., 53: 1603-1618. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414.
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Mattison, D. R., and S. S. Thorgeirsson 1977 Genetic differences in mouse ovarian metabolism of benzo[alpyrene and oocyte toxicity. Biochem. Pharmac., 26: 906-912. 1978 Gonadal aryl hydrocarbon hydroxylase in rats and mice. Canc. Res., 38: 1368-1373. Nebert, D. W., and J. S. Felton 1976 Importance of genetic factors influencing the metabolism of foreign compounds. Fed. Proc., 35: 1133-1141. Odor, D. L.,a n d R . J. Blandau 1969 Ultrastructural studies on fetal and early postnatal mouse ovaries. 11. Cytcdifferentiation. Am. J. Anat., 125: 177-216. 1973 Ultrastructural observations on atresia in whole organ cultures of foetal mouse ovaries. In: The Development and Maturation of the Ovary and its Function. H. Peters, ed. Excerpta Medica, pp. 63-76. Pedersen, T., and H. Peters 1968 Proposal for a classification of oocytes and follicles in the mouse ovary. J. Reprod. Fertil., 17: 555-557. Peters, H. 1969 The derelopment of the mouse ovary from birth to maturity. Acta. Endocrin., 62: 98-116. Reynolds, E. S. 1963 The use of lead citrate a t high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Roosen-Runge, E. G., and J. Leik 1968 Gonocyte degeneration in the postnatal male rat. Am. J. Anat., 122: 275-300. Sims, P., and P. L. Grover 1974 Epoxides in polycyclic aromatic hydrocarbon metabolism and carcinogenesis. Adv. in Canc. Res., 20: 165-274. Vazquez-Nin, G. H., and J. R. Sotelo 1967 Electron microscope study of t h e atretic oocytes of t h e rat. Z. Zellforsch., 80: 518-533. Watson, M. 1959 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478. Weakley, B. S. 1966 Electron microscopy of the oocyte and granulosa cells in t h e developing ovarian follicles of the golden hamster (Mesocricetus auratus). J. Anat., 100: 503-534. Wyrobek, A. J., and W. R. Bruce 1975 Chemical induction of sperm abnormalities in mice. Proc. Natl. Acad. Sci., 72: 4425-4429. Zamboni, L. 1970 Ultrastructure of Mammalian oocytes and ova. Biol. Reprod., Suppl. 2: 44-63. 1972 Comparative studies on the ultrastructure of mammalian oocytes. In: Oogenesis. J. D. Biggers and A. W. Schuetz, &. University Press, Baltimore, pp. 5-45. Zuckerman, S., and T. G. Baker 1977 The development of the ovary and t h e process of oogenesis. In: The Ovary. S. Zuckerman and B. J. Weir, eds. Academic Press, New York, Vol. 1, pp. 41-67.
PLATE 1 EXPLANATION OF FIGURES
2 T w o neighboring primordial oocytes in the cortex of intact mouse ovary beneath the surface epithelium (El.The nucleus (N)is slightly elongated. Oval mitochondria (M), Lipid (L) and short segments of the granular endoplasmic reticulum (GER)are X 9,000. scattered in the ooplasm (0). 3 One-micron section of type-2 (primordial) follicle in the cortex. 4
870
X
860.
Electron micrograph of two adjacent type-2 (primordial) follicles. The organization of the ooplasm (0) appears similar to that of type-I oocytes. Attenuated portions of follicular cells (FC)surround the oocyte and a thin basal lamina (BL)separated developing follicles from each other and the stromal cells. x 9,000.
PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattiaon
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
5 One-micron section illustrating two type-3A follicles (arrows) from a control mouse ovary. X 750. 6 Electron micrograph of type-3A follicles from a control mouse. The nucleus (N) is large and spherical; mitochondria (MI are numerous in the ooplasm (0).The oocyte is enclosed mostly by cuboidal follicular cells (FC). Occasionally macula adherentes (MA) are found between t h e oocyte and a follicular cell. Golgi complex ( G O , aggregates of small vesicles (V) and a few membrane-limited dense bodies (DB)are also present. Basal lamina (BL). x 11,000.
7 A macula adherens between an oocyte (0)and a follicular cell (FC).
872
X
24,500.
PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 2
PLATE 3 EXPLANATION OF FIGURES
8 Light micrograph (inset) and electron micrograph illustrating uptake of lipid (L) by the surface epithelium of a mouse ovary two days after MC treatment. x 540; X 7,700. 9 Follicle in transition from type-1 to type-2 in a mouse ovary 2 days after MC treatment. Note myelin figure (arrow) associated with lipid (L). x 9,000. 10 Vesicular cristae and myelin figures (arrows) in mitochondria of type-1 oocytes after two days of MC treatment. X 16,700.
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PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 3
PLATE 4 EXPLANATION OF FIGURES
11 One-micron section of a n ovary three days after MC treatment. Degenerating type1 (l),type-2 (2) and type-3A (3A) follicles have denser ooplasm than unaffected oocytes. X 860. 12 Degenerating oocyte (transient from type-1 to type-2) after three days of MC treatment. Mitochondria1 (M) cristae are vesiculated, ooplasm and nucleoplasm show increased electron density. X 9,000. 13 Oocytes in advanced stage of degeneration three days after MC treatment. Nuclear chromatin (CH)is clumped, perinuclear space swollen and vesicles (V)fill the 00plasm (0). Follicular cells (FC) are unaltered. X 9.000.
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PAN INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 4
PLATE 5 EXPLANATION OF FIGURES
14 Small primary follicle (type-3A) two days after MC treatment. Note vesiculation of cristae and increased electron density of mitochondria1 matrix (M)of the oocyte (0).Follicular cell (FC). X 24,500. 15 Numerous dense bodies (DB) and some lysosomes (LY)appear in the ooplasm (0) two days after treatment. Nucleus (N); Golgi complex (GO; follicular cell (FC) X
7,700.
16 Small primary follicle at advanced stages of degeneration three days after MC treatment. Mitochondria (M) are swollen having only a few cristae, and both t h e nucleoplasm (N) and ooplasm (0) show increased electron density. Clusters of small vesicles (V)and multivesicular bodies (MVB) are numerous. Follicular cell (FC) is unchanged. X 13,500.
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PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 5
PLATE 6 EXPLANATION OF FIGURES
17 Portions of a degenerating small primary follicle appeared to be engulfed by surrounding follicular cells (FC). Three cells are abundant in digestive vacuoles and X 11.000. residual bodies (RBI. Oocyte (0). 18 Necrosis of follicular cells (FC)in a type-3A follicle. Lysosomes (LY)and residual mitochonbodies (RB)are observed in their follicular cells. Cristae in oocyte (0) dria (M)are vesicular. X 7,700.
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PAH INDUCED ATRESIA Bela J. Gulyas and Donald R. Mattison
PLATE 6
