Folliculogenesis in the Ovary of the Mature Mouse : A Radioautographic Study ' TERRELL R. HOAGE AND IVAN L. CAMERON Department of A n a t o m y , T h e University of T e x a s Health Science Center at Sun Antonio, 7703 Floyd Curl Drive, S u n Antonio, Texas 78284 ABSTRACT Studies on the growth sequence of follicles i n mature mice four to six months old were conducted by giving three injections of 16 wCi tritiated thymidine (3H-T)/gm body weight at four hour intervals over a period of eight hours. Subsequent radioautographic analysis on ovaries obtained 1, 3 , 5 , 9 and 17 days after the last injection showed an overall follicle growth time of 17 to 19 days. The duration of the different follicle stage times were also estimated from radioautographic data. Intense "-T incorporation was noted i n the developing antra and zona pellucida of follicles of animals sacrificed one and three days after treatment suggesting that these areas serve as precursor storage sites during development. Oocytes of follicles i n early antrum formation also showed juxtanucleolar 3H-T incorporation concomitantly with rapid and massive oocyte and follicular growth. The findings further indicate that the earliest follicular cells surrounding the oocyte originate at some distance from the developing primordial follicle. Through light microscopy studies i t has been established that primordial germ cells in mammals are differentiated in early embryonic life (Hertig et al., '56), 'and that after migration to the genital ridge, these germ cells are the source of all the oocytes in the adult ovary. During the twelfth day of gestation in the mouse, clusters of oogonia become incompletely surrounded by follicle cells and in subsequent days the number of follicle cells increases by mitosis. This results in the oogonial clusters being completely surrounded and follicular vascularization begins (Odor and Blandau, '69). Primordial follicles form a s individual oocytes are surrounded by squamous follicle cells (Odor and Blandau, '69) and develop so that in the prepubertal mouse multilaminar follicles consisting of as many as six to nine cellular layers develop in concert with the oocyte. This prepubertal follicular development does not appear to be under anterior pituitary control (Greep, '63) which later plays a regulatory role in synthesis, growth and degeneration of the oocyte. With the onset of estrus in the ovary of the mature mouse, marked changes occur in the number and sizes of follicles, as well as in the growth rate of the follicles ANAT. REC., 184: 699-710. (Mandl and Zuckerman, '52; Pederson, '70a). Growth rates of granulosa cells along with the growth of whole follicles have been extensively examined and categorized (Pederson and Peters, '68; Peters, '69; Pederson, '70a,b). The above studies utilized mice ranging in age from birth up to three months of age. The mice were killed from 1 to 48 hours after the last injection of tritiated thymidine (3H-T). The above data provided an excellent baseline for the present radioautographic time series studies on the proliferation of follicle cells using four to six month old mice injected with 48 ,Ci "-T/gm body weight. Peters ('69) and Pederson ('70a) have projected a follicle growth cycle duration of 17 to 19 days which is corroborated in the time series data of the present study. In addition to determining the granulosa cell labeling characteristics, we have also determined the duration of each follicular stage throughout the 17-day time period. MATERIALS AND METHODS Adult, female virgin mice of the A/Jax strain, four to six months old and weighReceived Aug. 18, '75. Accepted Oct. 29, '75. 1 Supported by USPHS grants NS08949-06 and CA16831-01, 699 700 TERRELL R. HOAGE AND IVAN L. CAMERON Two slides from each animal were seing between 22 and 28 gms, each received three subcutaneous injections of 16 lected at random and used for radioautopci /gm body weight of tritiated thymidine graphic analysis. (3H-T) (specific activity 1.9 Ci/mM in Differential counts o f follicles 0.9% saline, Schwartz-Mann) at four hour Follicles were counted in each section intervals. At 1, 3 , 5, 9 and 17 days after the initial injection three animals were anes- and scored for the developmental stage as thetized with amobarbital sodium and per- described by Peters ('69). Six stages were fused through the left ventricle with a enumerated in classes as follows: ( 1 ) cacodylate buffered mixture of formalde- Stage 2 , oocyte surrounded by a single hyde and glutaraldehyde modified from the layer of squamous follicle cells (fig. 1); procedure described by Brightman and ( 2 ) Stage 3 , oocyte surrounded by a single Reese ('69). A dilute solution of 1% form- layer of cuboidal cells (fig. 2 ) ; ( 3 ) Stage aldehyde and 1.25% glutaraldehyde was 4, oocyte surrounded by two to three cell administered at room temperature for two layers without antrum formation (fig. 3 ) ; minutes without prior rinse. A concen- ( 4 ) Stage 5-6, two or more layers of cells trated solution containing 4% formal- around oocytes with the onset of antrum dehyde and 5% glutaraldehyde was then formation (fig. 4 ) ; ( 5 ) Stage 7-8, oocyte perfused for 15 minutes following which with cumulus oophorus and corona radiata the animal was undisturbed for one to surrounded by a distinct antrum (fig. 5 ) ; three hours. Perfused whole animals were ( 6 ) atretic follicles were also counted and stored in buffered formalin until target tis- were considered degenerating when pyknosues were excised. Ovaries were removed tic nuclei formed a distinct layer on the and embedded in paraffin for light niicro- inner boundary of granulosa cells (fig. 6). scopy. Five micron sections were cut and The presence of these pyknotic cells in prepared as slide sets to include animals any stage caused it to be placed in the killed 1, 3 , 5, 9, and 17 days after treat- atretic class. Stage 2 follicles exhibited no ment as follows: label and were not counted in total oocyte 1. Sections of the ovary were placed on determinations. slides subbed in 0.05% agar, subsequently Silver grain count methods deparaffinized, and air dried. 2. One set of slides was subjected to Background counts were determined by treatment in 5 N HC1 for one hour, then counting ten of one hundred 0.03 mm exposed to Schiff's reagent for one hour. squares circumscribed by a calibrated These slides were evaluated for DNA stain- ocular graticule. Background counts were ing after the above Feulgen reaction. taken over open slide areas at the border 3. Four additional sets of the slide series of tissues. These counts were then extrawere dipped in Kodak NTB-3 liquid radio- polated to a standard area that could be autographic emulsion along with the Feul- used to compare the cytoplasm and nuclei gen stained slides mentioned above. observed in the study. All further deter4. One set of slides was digested in minations were made on counts above this DNase (Worthington Biochemical, 10 mg background level. Antrum counts were in 35 ml Tris buffer, pH 7.2) for eight made as described above and recorded in hours while a comparison set remained in terms of grains per unit area as in backTris buffer. ground determinations. Diameters were 5. All dipped slide sets were allowed to determined on follicles cut in sagittal secair dry and were placed in light tight slide tion and with oocytes showing nucleoli boxes and stored at 4 ° C for three weeks within the nucleoplasm. This insured a of exposure. relative maximum for both cell and nu6. After three weeks of exposure the clear diameter. The outside diameter of the slides were developed in Dektol and stained zona pellucida ( Z P ) was taken and used with hematoxylin and eosin, except for the along with the oocyte diameter to deterFeulgen stained slides which were counter- mine the area of the ZP on which total stained with Fast green. grain counts were taken. MOUSE OVARIAN FOLLICLES Fig. 1 Stage 2 primordial oocyte ( 0 ) of mouse surrounded by squamous follicle cells ( F ) , stroma cells ( S ) . x 1,400. Fig. 2 Mouse primary oocyte of stage 3 with a layer of cuboidal follicle cells ( F ) surrounding the oocyte ( 0 ) . x 1,200. Fig. 3 Follicle stage in transition from 3 to 4 with a differentiating thecal (T)layer at the periphery of the mouse follicle cells ( F ) , oocyte ( 0 ) . x 1,200. 70 1 702 TERRELL R. HOAGE AND IVAN L. CAMERON 703 MOUSE OVARIAN FOLLICLES Numbers of Follicles in Mouse Ovary Sections Expressed in Percent for Each Stage Scored r ~ + 100I 0 a - W 80v) W - 1 0 - 60- e - T _I 40F L 0 5 L - 20- w 0 - LT W L O / 0 & I I 2 I 1 4 I l l 6 I 8 I I 10 1 I 12 l l 14 I I 16 1 DAYS AFTER 3H-THYMIDINE INJECTION Fig. 7 Average frequencies of follicular stages of mouse ovaries at 1, 3, 5, 9 and 17 days after injection of 3H-T. Photomicrographs were taken on a Zeiss microscope using either Panatomic-x or high contrast copy film which was subsequently developed in Microdol-x or D-19 respectively. RESULTS Differential follicle counts The differential follicle count present without reference to labeled cells, in the stages described above, are recorded in table 1. Follicles in stage 3 were most numerous in sections obtained one and nine days after sacrifice. Stages 4 and 5-6 were not significantly different from each other while the stage 7-8 follicles were significantly less than all of the other classes in all sections counted. The average change in follicular types with time after 3H-T in~ ~~ Fig. 4 Stage 5-6 mouse follicle demonstrating concentrated "-T label i n the antrum ( A ) , zona pellucida ( Z ) and nuclear ( N ) label of actively synthesizing follicle cells and the oocyte (0). x 800. Fig. 5 Follicle i n stage 7-8 with fully developed oocyte ( O ) , Nucleus ( N ) , antrum ( A ) . x 250. Fig, 6 Atretic stage 7-8 follicle as noted by the layer of pyknotic nuclei (arrows) bordering the antrum ( A ) , oocyte (0). x 250. jection are shown in figure 7 indicating a decline in the proportional number of functional follicles over the 17 day test period, in the animals studied. I n conjunction with this decline there is a significant increase in atretic follicles over time (table 1). Follicle labeling patterns No labeled cell nuclei were observed in stage 2 follicles at any time. Of the remaining follicle stages, it was estimated that 90% of the observed follicles possessed one or more heavily labeled theca and granulosa cells from one to five days after "-T administration. The observed label varied from an estimated 150' grains per nucleus in cells of animals sacrificed one and three days after injection to the five grains or less observed per nucleus in animals sacrificed 17 days after JH-T injection. This reduction in grains per nucleus is expected i n rapidly proliferating cell populations such as found in oocyte follicular development. Beginning with 150' grains/nucleus it would require six doublings to reduce the grain count to less than 5 grains/nucleus. This dilution effect coupled with peak labeling percentages observed in granulosa cells (table 2, fig. 8 ) 704 TERRELL R. HOAGE AND IVAN L. CAMERON TABLE 1 Differential follicle counts o f four t o six m o n t h old m o u s e ovaries t a k e n at 1, 3, 5, 9 a n d 17 days a f t e r injection o f 48 Fci " - T / g m body weight over a n eight hour period Stage of follicle development Day(s) after YH-T injection 1 3 5 9 17 1 Mean Stage 3 Stage 4 Stage 5-6 Stage 7-8 Atretic 36.221.21 27.820.61 20.420.95 30.420.98 12.720.94 18.2t1.04 25.220.89 22.421.32 19.2'0.81 14.011.51 23.2k0.82 29.0'1.29 24.5k1.55 17.620.77 23.0e1.31 12.020.64 1O.OkO.59 8.8k0.91 7.620.67 3.820.85 10.520.71 8.3C1.25 37.222.73 25.2-tO.97 47.3%1.71 -I- S.E.M. TABLE 2 Percent o f granulosa cells labeled per follicle in ovaries o f f o u r to six m o n t h old mice injected with 48 yci 3 H - T / g m body weight over a n eight hour period a n d sacrificed I , 3, 5 , 9 a n d 17 days a f t e r treatment Stage of follicle development Day(s) after 3H-T injection 0% 1 3 5 9 17 1 Mean Stage 4 Stage 5-6 Stage 7-8 4 2 . 6 1 1.47 7 1 . 3 t 1.24 89.5 -C 1.81 94.3 2 0.62 35.72 1.35 41.22 1.55 59.2 t 2.03 7 8 . 4 r 1.34 100.0 2 0.00 21.620.75 51.1 &2.36 98.0 2 0.84 68.0-t 1.71 64.0 2 1.88 00.0t0.00 Stage 3 59.5? 3.14 80.0 2 1.56 70.72 0.92 2.4 t 0.82 S.E.M. a cn J B gE UJ V W rrm WQ CLJ 0 2 4 6 8 10 12 14 16 DAYS AFTER 3H-THYMIDINE INJECTION Fig. 8 Mouse ovary follicle granulosal label b y stage over a t i m e series o f 1, 3, 5, 9 and 17 days after SH-T injection. were used to estimate time periods for the development of the various follicle stages. For example, stage 3 follicles exhibited the highest frequency of labeled cells on the fifth day after 'H-T injection (table 2, fig. 8 ) and the number of grains per nucleus were approximately one half that observed at day 3 . This indicates division of cells either within the follicle or within a labeled cell population serving as precursors to the granulosa cells. The label over granulosa cells of all 705 MOUSE OVARIAN FOLLICLES follicular stages in animals sacrificed at 9 and 17 days was further reduced in grains/nucleus and the number of cells counted as labeled declined even more. Such a decline in the percentage of labeled cells would be expected by halving of grains nucleus over six or more cell cycles. Stages 4 and 5-6 showed a peak in percent of labeled cells on the ninth day (table 2 ) after isotope injection, which was interpreted as the result of proliferation from highly labeled cells present immediately after ,'H-T injection. This proliferation of labeled cells is confirmed by the reduced number ol grains per nucleus over cells at day nine. Further proliferative activity causes stages 4 and 5-6 follicles to grow into the mature stage 7-8 follicles. Stage 7-8 follicles counted on days 5, 9 and 17 decline in number of grains per nucleus and concomitantly in the number of nuclei designated as labeled (table 2, fig. 8 ) . Following the above observations on grains per nucleus and percent of labeled cells in each stage scored, it is concluded that stage 3 follicles stimulated to growth required from day 5 to 17 to complete growth of labeled follicles into stage 4 or later. Stage 4 follicles maintain a high percentage of labeled nuclei on days 3, 5 and 9 (fig. 8 ) and then decline to less than day 1 levels by day 17. Such a sequence indicates that stage 4 follicle cells labeled at day 1 are advanced to later stage 4's until eventually all stage 4's have moved on to stage 5-6. This dilution in percent of labeled cells by division which occurs after day 9 indicates a 7-day maximum duration for stage 4 to grow into stage 5-6. The increase in percent of labeled cells which occurs between three to past nine days of stage 4 follicles indicates a 6-day time duration for stage 4 follicles to grow into stage 5-6 follicles. Stage 5-6 follicles are somewhat more difficult to interpret due to the greatly reduced number of grains over nuclei on days 5 and 9. This rapid dilution of grains nucleus in labeled cells indicates that the cell populations of stage 5-6 seen on day 3 are different follicles than the stage 5-6 follicles seen on days 5, 9 and 17. Therefore those stage 5-6 follicles seen on days 5, 9 and 17 must have originated from a n earlier follicle stage which possessed a high grain count per nucleus on day 3 . Thus i t can be concluded that stage 5-6 follicles move into stage 7-8 between days 3 and 5 thus suggesting a duration of two days or less. The above is also true of mature stage 7-8 follicles that go to almost 100% labeled cells from day 1 to day 3. The loss of stage 7-8 follicles to ovulation results in immediate replacement from stage 5-6 with a concomitant loss in number of grains nucleus in these new stage 7-8 follicles until by day 17 the number of grains/nucleus in labeled cells is less than or equal to background label. This indicates a maximum time of less than two days for stage 5-6 follicles to move into stage 7-8 and then out. It also indicates that at least six cell cycles have occurred during the growth of early follicle stages to the mature stage. Thecal cell development In early follicular development the type of cells associated with the oocyte is limited to the squamous cell type which in the later follicular stages become either theca externa and theca interna or granulosa cells. The degree of labeling per follicle, ex- TABLE 3 Percent of thecal cells labeled per follicle i n sections of ovaries of f o u r to six m o n t h old m i c e injected w i t h 48 pci s H - T / g m body weight and sacrificed 1 , 3 , 5, 9 and 1 7 days a f t e r treatment Stage of follicle development D a y ( s ) after 3H-T injection 1 3 5 9 17 1 Mean & S.E.M. Stage 3 00.0 & 0.00 38.3 2 2.31 36.0t 2.44 54.7t2.42 00.0"0.00 Stage 4 37.71 1.30 42.8% 1.28 46.8% 2.70 85.0% 1.30 16.3% 1.57 Stage 5-6 38.811.50 40.8-C 1.48 58.6 -C 1 .80 64.7k 3.22 8.3 k 1.07 Stage 7-8 32.6t 1.70 51.822.31 59.72 2.30 88.0% 1.33 4.0 i 1.52 706 TERRELL R. HOAGE A N D IVAN L. CAMERON Stage 78lnterna W 0 Stage 4 a J Stage 7-8 Externa 0 5-6 [L I 5 3 9 17 DAYS AFTER 3H-THYMIDINE INJECTION Fig. 9 Percent labeled thecal cells in follicular development of four to six month old mice treated with 3H-T and sacrificed 1, 3, 5, 9 and 17 days after injection. TABLE 4 Follicle antrum label in sections of ovaries of four to six month old mice injected with 48 pci SH-T/gm body weight and sacrificed 1, 3, 5, 9 and 17 days after treatment Stage of follicle development Day(s) after 3H-T injection 1 3 5 9 17 1 Mean Stage 5-6 Stage 7-8 115% 1.73 39 i 1.48 29 f 1.55 3 1 1.11 27 f 0.86 146 C 2.32 58C2.18 27 t 0.78 2 9 r 1.33 23 t 0.58 * Background label 25 % 0.96 23 % 1.49 28 C 0.74 2 2 2 1.85 24 i0.89 f S.E.M. pressed in average percent of thecal cells labeled is given in table 3. Cellular proliferative activity expressed by the labeling index was not seen in stage 3 of day 1. Stage 3 thecal cells reached a labeled maximum on day 9. It appeared that those cells labeled in the various stages other than stage 3 increased slowly in label to a maximum on day 9 with rather severe drops in label by day 17 (table 3 , fig. 9 ) . This can be accounted for by the development of follicles stimulated to maturity within this 17-day test period. The subsequent ovulations (four estrous cycles within this test period) would account for the thecal label decline as would dilution through several division of cells labeled very early in the follicle cycle activities. Theca externa cells of stages 5-6 and 7-8 exhibit rather rapid dilution of label which is expected in rapidly proliferative cell populations. Of particular interest is the absence of label in the early stage 3 follicle cells one day after "-T injection and the subsequent appearance of labeled thecal cells in later stage 3 development which occurs several days after the 'H-T injection. Such a labeling pattern of early stage 3 follicle cells suggests migratory activity of labeled cells to the oocytes by either labeled stroma cells or by some other labeled cell from a population extrinsic to the ovary. Antrum development Antrum development is initiated in late stage 5 follicles. In those antral follicles active at the time of "-T injection very intense label is found at day 1 (table 4 ) . Both stages 5-6 and 7-8 exhibit the concen- MOUSE OVARIAN FOLLICLES i -\ I 3 5 9 I7 DAYS A F T E R 3H-THYMIDINE INJECTION Fig. 10 Graph showing radioautographic label lost per unit area of antrum in follicles of four to six month old mice 1, 3, 5, 9 and 17 days after 3H-T injection. tration of radioactive material at highly significant levels above background label. Similar counts are also observed in the ZP of these follicle types. The fate of this precursor is not obvious within this study but the 3-day antral follicles maintained a level of label that was still significantly above background although not as high as day 1 antra. Days 5, 9 and 17 did not show label above that observed for background (fig. 10). Enzymatic digestion with DNase did not significantly decrease the antrum label from 115 and 146 grains in stages 5-6 and 7-8 respectively. Since this level was not affected by DNase digestion, the 'H-T or its metabolites are bound in material other than DNA. DISCUSSION The frequency of specific follicle stages has been delineated by Pederson ('70a,b) and the follicles were generally categorized into small follicles (stages 1, 2 , 3 a ) , medium follicles (stages 3b, 4), large follicles (stages 5, 6 ) and preovulatory follicles (stages 7, 8). These categories correspond to the relative stages described in this study. It is deduced from this study that type 2 follicular cells have their origin in the vicinity of the type 2 follicles observed. The statement that type 2 follicles do not grow but are formed (Pederson, '70a) is apparent in previous studies from the lack 707 of incorporation of 'H-T into the squamous cells seen in primordial follicles (Pederson, '69). Peters and Pederson ('67) state that the origin of these cells would appear to be from the ovarian stroma although the site of label and interim location were not identified, Labeled stroma cells were observed by the previous authors but at great distances from the oocyte nests. Since the origin of both thecal cells and follicle cells cannot be ascertained with certainty, further studies are needed to identify the sources and to determine both the stimulus and migratory time to initiate a shift from the nongrowing stage 2 follicle type to the stage 3 which is a growing follicle type. Parabiosis with 'H-T radioautographic studies is in progress to clarify this phenomenon. Our follicular numbers, types, duration of developmental stages as well as the total follicle development time are within the same range as depicted by Pederson ('70a). There is, however, a distinct increase in atretic follicle number over the time span of this study and several things could account for this increase atresia. It has been shown that high levels of incorporation of 'H-T ( 1500 @/pregnant mouse over a 120 hour period) cause oocyte degeneration if incorporated during embryonic growth (Callebaut, '68). At four months of age these female mice had ovaries with no functional oocytes and many follicles that did not complete development. This then, suggests a degeneration of follicles if the oocyte is damaged. Such radiation damage to cellular function could account for the increased atresia of follicles noted in the latter days of this experiment. Also the fact that oocytes do incorporate 3H-T into juxtanucleolar DNA (Hoage and Cameron, unpublished observations; Crone and Peters, '68) could also account for the increased atresia on the basis of radioactive damage. It is also possible that the incorporation of 3H-T into secretory cells of the pituitary could affect hormone levels that lead to atresia in the small growing oocytes and follicles (Jones and Krohn, '61; Pavic, '63). Pederson ('69) concluded that only large follicles degenerate which was not the case in this study where pyknotic nuclei occurred in medium sized follicles comparable to stage 4. This variation can also be 708 TERRELL R. HOAGE AND IVAN L. CAMERON attributed to the effects of increased 'H-T incorporation in the nucleus of stage 5 oocytes (Hoage and Cameron, unpublished observations. It has been shown that baseline estimates of pulse labeled follicles established by Peters ('69) and Pederson ('70a) are accurate when determined over longer sampling times ( u p to 17 days after 'H-T application). The total time determined in this study for follicular growth from stimulated stage 3 through to ovulation was 17-19 days which agrees with previous studies (Pederson, '70a,b). Thecal cell development has been followed in this study and the findings indicate that the precursors to thecal cells originate from a population of labeled cells exterior to the primordial follicle. The source of the thecal cells is not apparent from this examination but the source appears to be drawn from either the ovarian stroma or another undifferentiated cell population with migratory capabilities. The ultrastructural similarity of lymphocytes and secretory thecal cells and granulosa cells (Weakly, '66) suggests that lymphocytes may be a source. Antrum development through fluid production has been well documented (Hertig and Barton, '72, for review) but the role of the antrum is not clear with respect to development of follicle cells and oocyte activities. The presence of extremely high levels of radioactive label in antra shortly after injection of 3H-T suggests a reservoir of precursors for the rapid proliferative activity that accompany antra and follicle growth in these stages. Concurrent with the growth phase there is an increase in oocyte nuclear synthetic activity that involves juxtanucleolar DNA synthesis (Hoage and Cameron, unpublished observations) that could draw on nucleoside reserves present in the antrum. That this antrum labeling is not DNase sensitive indicates that the material is not incorporated into DNA. Routine histological processing might be expected to extract soluble JH-T,however, it is conceivable that soluble ,'H-T was in some way fixed to higher molecular weight material at the time of glutaraldehyde fixation in a manner similar to the fixation of an amino acid to macromolecular material as shown by Peters and Ashley ('67). The state of the tritium label in the antrum is not clear but it must be highly compact and or complexed to ellicit the concentrated response seen in this study. The increased thecal development concurrent with the antrum label suggests a coordinated transport system to augment the rapid growth sequence of the maturing follicles. ACKNOWLEDGMENTS The authors wish to thank Dr. E. I<. Adrian for his cooperation and for generously supplying the animals used in this study. We also appreciate helpful discussions about the manuscript with Dr. E. G. Rennels and the excellent assistance of W. A. Pavlat. LITERATURE CITED Brightman, M. W., and T. S. Reese 1969 Junction between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol., 40: 648-677. Callebaut, M. 1968 Development of the gonads of mice after intense incorporation of tritiated thymidine during the period of oogenesis. Experientia, 24: 828-829. Crone, M., and H. Peters 1968 Unusual incorporation of tritiated thymidine into early diplotene oocytes of mice. Exp. Cell Res., 50: 664668. Greep, R. 0. 1963 Histology, histochemistry and ultrastructure of adult ovary. In: The Ovary. H. G. Grady and D. E. Smith, eds. Williams and Wilkins, Baltimore, pp. 48-68. Hertig, A. T., and B. R. Barton 1972 Fine structure of mammalian oocytes and ova. In: Handbook of Physiology-Endocrinology 11-Female Reproductive System Part I .Vol. 7. American Physiological Society, Washington, D.C., pp. 3 17-348. Hertig, A. T., J. Rock and E. C. Adams 1956 A description of 34 h u m a n ova within the first 17 days of development. Am. J. Anat., 98: 435-493. Jones, E. C., and P. L. Krohn 1961 The effect of hypophysectomy o n age changes in the ovaries of mice. J. Endocr., 21: 497-508. Mandl, A. M., and S . Zuckerman 1952 Cyclical changes in the number of medium and large follicles i n the adult r a t ovary. J. Endocr., 8: 34 1-346. Odor, D. L., and R. J. Blandau 1969 Ultrastructural studies on fetal and early postnatal mouse ovaries. I. Histogenesis and organogenesis. Am. J. Anat., 124: 163-186. Pavic, D. 1963 The effect of gonadotrophic hormones on young rat ovaries grown in organ culture. J. Endocr., 26: 531-538. Pederson, T. 1969 Follicle growth in the immature mouse ovary. Acta Endocrinologica, 62: 117-132. 1970a Follicle kinetics in the ovary of the cyclic mouse. Acta Endocrinologia, 64: 304323. MOUSE OVARIAN FOLLICLES 1970b Determination of follicle growth rate i n the ovary of the immature mouse. J . Reprod. Fertil., 21: 81-93. Pederson, 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 development of the mouse ovary f r o m birth to maturity. Acta Endocrinologia, 62: 98-116. Peters, T., and C. A. Ashley 1967 An artefact 709 i n radioautography d u e to binding of free amino acids to tissues by fixatives. J. Cell Biol., 33: 53-60. Peters, H., and T. Pederson 1967 Origin of follicle cells in the infant mouse ovary. Fertil. and Steril., 18: 309-313. Weakly, B. S. 1966 Electron microscopy of the oocyte and granulosa cells in the developing ovarian follicles of the golden hamster. J. Anat., 100: 503-534.