NEO-OVOGENESIS I N THE ADULT MONKEY CONSEQUENCES O F ATRESIA O F OVOCYTES GERTRUDE J. VERMANDE-VAN ECE ' Department of Anatomy, Y a l e University School of Hedicine New Hrven, Connecticut T W O FIGURES New ova form from the germinal epithelium in some small adult mammals (Allen, '23 ; Cowperthwaite, '25 ; Evans and Swezy, '31 ; Allen and Creadick, '37; Schmidt and Hoffman, '41; Latta and Pedersen, '44). From the time of Waldeyer, 1870, it has been accepted that the human ovary contained at birth all the ovocytes for the future life. Waldeyer's estimate was 36,000-50,000 in the neonatal ovary. Von Hanseniann ('12)? counted every fifth section ,of a neonatal ovary and estimated the total number between 40,000 and 80,000. Hammar and Hellmann ( '20), counted the ovocytes in a girl 3 years and 8 months old who died of thyreoaplasia and found 194,283 ovocytes in one ovary. Haggstrom ('2l), counted the ovocytes in a young woman of 22 years and found 419,911 ovocytes in the two ovaries. From these counts the number of 400,OO ovocytes with which the young girl is born has been generally accepted. This theory of Waldeyer that no new ova are formed during postnatal life implies a possible life of the ovocytes of 45-50 years. More recently the formation of new ova throughout sexual maturity, as in the lower mammals, has been assumed (Evans and Swezy, '31 ; Simkins, '32 ; Schwarz and coworkers, '49). Ovogenesis may be either continuous or in waves related to ovulation, and may occur from the germinal epithelium or from a neogenic zone in the cortex of the ovary; the rate of formation may diminish progressively with age. 'This study was supported by a grant from the United States Public Health Service. 207 208 GERTRUDE J. VERMANDE-VAN ECK The follicle can degenerate at any stage of development. Atresia is assumed to be of short duration for the primordial and small primary follicles; the follicle is resorbed and its space in the ovary replaced with stroma. The zona pellucida from the growing follicle may persist for some time after disintegration of the ovocyte and follicle cells. The process of atresia of the growing follicle has never been ascertained. Atresia of the mature follicle was estimated by Sturgis ('49), to be complete after about 5 weeks; the corpus atreticum can then remain for months in the ovary. In the human, one follicle usually reaches maturity each month; all other larger follicles most likely degenerate (Allen and coworkers, '30). This suggests that atresia occurs in waves, at least as far as the large follicles are concerned. Evans and Swezy ( '31), described such waves in relation to the ovulatory cycle ; they mentioned very intensive atresia in the rat, guinea pig, cat and dog, and accepted formation of ova from the germinal epithelium in corresponding waves. Mandl and Zuckerman ( '49) could not confirm this hypothesis after counting the normal and atretic ovocytes in rats' ovaries. At early diestrus the number of normal ova was at a maximum, and at late diestrus at a minimum, whereas the other phases of the cycle did not show significant differences. These slight changes during the estrous cycle were negligible compared to changes in age and litter relationship. The number of ovocytes in the rat decreased with advancing age (Arai, '20; Mandl and Zuckerman, '50). Green and Zuckerman ('50) were the first to estimate the number of ovocytes of the ovaries of rhesus monkeys. The ovarian follicles of 13 monkeys were divided into 7 groups, representing different stages of development. I n each group both the normal and the atretic follicles were counted. Their analysis did not support the viewpoint of Evans and Swezy ('31) that the total number of ovocytes of the rhesus monkey fluctuates in phase with the menstrual cycle, and that its maximum coincides with ovulation. Only the number of larger follicles is at its peak at this time. These findings are similar NEO-OVOGENESIS I N THE ADULT MONKEY 209 to the results obtained by Mandl and Zuckerman ('50) in the rat. Ovogenesis in the adult mammal has been studied by different methods. Early investigators used the histological techniques : Winiwarter and Sainmont ( '08) in the cat ; Allen ( '23) in the rat; Cowperthwaite ( '25) in the rat; Evans and Swezy ( '31) in the dog, cat, guinea pig, monkey and human; and Simkins ( '32) in human ovaries. Experimental methods were used later. Allen and Creadick ('37) used the colchicine technique to arrest mitosis in the germinal epithelium of the rat. Schmidt and Hoffmann ( '41) did Simkins experiments with guinea pigs. Latta and Pedersen ('44) used India ink to mark the germinal epithelium of the rat in vivo. Jones ('49) also used India ink f o r vital staining of the germinal epithelium of the rat. Mandl and Zuckerman ( '50) used unilateral castration of the rat and destruction of the germinal epithelium by tannic acid and carbolic acid. Simpson and van Wagenen ('53) studied the ovaries of immature monkeys that had received high doses of follicle stimulating hormone. These experiments were contradictory or were interpreted differently. Schwarz and coworkers ( '49) concluded that between 20 and 40 years of age ova are formed from the germinal epithelium of human ovaries. The number of ova diminished rapidly at the onset of the climacterium and in the menopause only a few or no ova were present. The purpose of this paper is t o approach this problem from another point of view. The number of normal and atretic ovocytes in rhesus monkeys were estimated, giving special attention to the two possible mechanisms for the diminishing number of ovocytes; that is atresia alone or the atresia in combination with a decreased neogenesis. MATERIALS AND METHODS The ovaries were obtained from several sources. 1. Eight ovaries of immature monkeys from our own colony (Yale Series). 210 2. GERTRUDE J. VERMANDE-VAN ECK Six ovaries of immature monkeys from the Carnegie Colony (Carnegie Series C l ) . 3. Seven ovaries of mature monkeys from the Carnegie Colony (Carnegie Series C2). The ovaries of the Yale Series were fixed in Bouin's solution, embedded in paraffin, cut at 8 p and stained with hematoxylin and triosin. The ovocytes, normal and atretic, were counted at all stages of development. For this purpose we used the technique described by Green and Zuckerman ( '50). The follicles were classified in 7 groups in order to promote uniformity in publications and to enable us to compare our figures with theirs. The classification is as follows: 1. Primordial ova with complete cover of flattened cells or incomplete epithelial cover. 2. Ova completely surrounded by 1 layer of cuboidal cells. 3. Ova completely surrounded by 2 layers of cuboidal cells. 4. Ova completely surrounded by 3 layers of cuboidal cells. 5. Ova completely surrounded by 4 or 5 layers of cuboidal cells. 6. Ova completely surrounded by 6 or more layers of cuboidal cells. 7. Follicles with antra. The primordial ovocytes and the ovocytes in the small follicles were counted in every 40th section. F o r the large growing follicles (group 6) and the follicles with antra, every section was screened. Oocytes were counted only when the nucleolus was visible. The numbers of ova in follicles counted in groups 1-5 were multiplied by 40 and by a correction factor for the thickness of the section. The numbers of groups 6 and 7 were multiplied only by the correction factor. Abercrombie ('46) has shown that when the diameter of the object counted, e.g. the nucleus of a cell, is large in relation to the thickness of the section, the true nuclear population P will be approximately: section thickness diameter. section thickness + nuclear diameter x 211 NEO-OVOGENESIS I N T H E ADULT MONKEY The average size of the nucleoli of the different groups and the corresponding correction factors for 10 p section thickness were determined by Green and Zuckerman ( '50). We repeated the measurements on our ovaries, which were fixed the same way. The average of 20 follicles of group 1 differed less than 3% from the figures given by Green and Zuckerman. The correction factor for 8 ~-r section thickness was applied to our series (table 1). TABLE 1 Average size of nucleoli of group 1-7 follicles, and the correspondhg correction factor for sections of 8 and l o p thickness QROUP NUCLEOLAR SIZE IN MICRONS CORRECTIOON FACTOR CORREOTION PACTOR 10 B Bfi 0.79 0.77 0.76 0.73 0.71 0.69 0.66 0.74 0.73 0.72 2.7 3.0 3.1 3.6 4.1 4.6 5.8 0.69 0.66 0.63 0.58 Figures from Green and Zuekerman. Duplicate counts were made from 5 ovaries. The counting error was lower than 876, and was mainly due to differences in group 1follicles. F o r determination of the duration of the atresia, three monkeys were x-irradiated with 1200 r, divided into two doses on subsequent days.2 One ovary was removed from each of two monkeys on day 7, 10, and 14 after irradiation. The criteria of atresia were : chromatolysis of the nucleus, and shrinkage, and more intense eosinophilic staining of the cytoplasm of the ovocyte. RESULTS 1. Yale series The monkeys of this series had had one or more menstrual periods but had not yet ovulated. Their weights ranged from * 200 kilo volts; 15 milli Amperes; 50 em target distance; filter 3 mm Cu Al; half value layer 1.1 mm Cu. + 1m m 212 GERTRUDE J. VERMANDE-VAN E C K 2870-3780gm7 and their estimated ages ranged from two t o two and one-half years. The total number of ovocytes per ovary differed widely; the lowest was 22,900 and the highest 117,970, the average number was 50,120 10,600. The total atresia per ovary was between 2.4 and 6.876, with an average of 4.5 _t 0.5% (table 2). TABLE 2 Number of ovocytes in normal monkey ovaries Yale Series Immature animals MONKEY NUMBER TOTAL OVOCYTES ATRETIC OVOCYTES 12R 15R 2OR 74L 75L 95R 103R 103L 22,900 38,110 117,970 40,050 26,630 56,070 53,810 48,130 550 1380 3140 2380 1450 3800 2730 1840 Average Carnegie Series C1 Immature animals 201R 201L 202R 202L 209R 209L Average Carnegie Series C2 Mature animals Average 124R 137R 137L 206R 206L 219R 219L %J 2.4 3.9 2.7 5.9 5.4 6.8 5.1 3.8 50,120 2140 4.5 2 10,600 r 375 2 0.5 26,480 30,820 124,460 97,520 40,290 45,300 1100 970 4020 3290 2480 1990 4.2 3.1 3.2 3.4 6.1 4.4 60,810 2310 4.1 ? 16,470 C 490 5 0.5 76,220 24,030 18,160 58,810 128,930 58,800 58,910 3660 1180 960 1320 5680 4030 3220 4.8 4.9 5.3 2.2 4.4 6.8 5.6 60,550 2960 4.8 2 10,900 & 660 k 0.5 213 NEO-OVOGENESIS IN T H E ADULT MONKEY The distribution of ovocytes over the different stages of f ollicular development was relatively constant, and independent of the total number of ova (table 3, fig. 1). The percentage of atretic ova was constant for each group; the widest variation occurred in the follicles with antra. The fact that these monkeys were not selected for periods of the menstrual cycle may be responsible. The percent of atresia was low in the primordial ovocytes, and increased gradually TABLE 3 Distribution of ovocytes according to the size of the follicle expressed in percentage of total ovocytes Yale Series Immature rhesus monkey 12R 15R 20R 74L 75L 95R 103R 103L Average Carnegie Series C1 Immature rhesus monkey 201R 201L 202R 202L 209R 209L Average Carnegie Series C2 Adult rhesus monkey Average 124R 137R 137L 206R 206L 219R 219L 93.7 84.7 91.0 82.3 91.0 89.4 89.9 90.2 4.8 9.3 7.8 10.6 5.7 5.8 6.8 7.5 1.1 5.6 1.1 6.8 3.2 4.6 3.3 2.2 .16 .23 .02 .06 .07 .06 .02 .03 .10 .23 .06 .21 .06 .04 .04 89.0 7.3 3.5 .08 .10 77.3 74.2 84.9 83.0 67.4 62.1 14.9 17.2 11.2 12.2 17.9 23.0 7.2 8.3 3.8 5.6 14.4 14.5 .11 .17 .04 .05 .05 .09 .37 .34 .04 .07 .19 .25 74.8 16.1 9.0 .08 .21 83.8 82.6 80.2 87.6 93.3 76.0 83.2 12.7 12.2 13.4 8.2 3.8 13.9 10.0 3.4 5.0 5.7 4.0 3.4 9.7 6.6 .02 .08 .09 .06 .07 .13 .12 .11 .17 .23 .06 .06 .18 .19 83.8 10.6 5.4 .08 .14 .lo 214 GERTRUDE J. VERMANDE-VAN ECK during the process of maturation t o attain an average of 38 3.576 in the follicles with antra. Since both ovaries of the same monkey were studied in only one instance, the number of ovocytes in the right and left ovaries of this group could not be compared (table 4,fig. 2). 100 80 0 Y a l e series 0 Garnegie I 0 Cornegie 2 - In al e h > 60 - 0 0 c 0 c u- 40- 8 20 - Groups I 2 3 -5 6 7 Fig. 1 Uistribut,ion of ovocytes of the rhesus monkey according to the size of follicle. 2. CarNegie series C l The monkeys of this group were ovariectomized 6 weeks after joining the colony. All had shown one o r more menstrual periods, and the day of cycle at which the ovaries were removed was known. The ovaries contained small and medium sized follicles with antra, but showed no signs of ovulation. The average total number of ovocytes was 60,810 16,470, with an average atresia of 4.1 2 0.5% (table 2). 215 NEO-OVOGENESIS I N THE ADULT MONKEY The distribution of follicles of different size among the different groups was constant (table 3, fig. 1) as was the percentage of atresia per group (table 4,fig. 2). The total number of ovocytes varied widely from one monkey to the other. The right and left ovaries of the same monkey contained nearly equal numbers of ovocytes. TABLE 4 Percentages of atretic ova in normal monkey ovaries MONKEY NUMBER Yale Series: Immature rhesus monkeys 12R 15R 20R 74L 75L 95R 103R 103L DAY OIP O Y O ~ E GROUP 1 25 19 19 55 58 61 .. . . Average Carnegie Series C l : Immature rhesus monkeys 201R 20111 202R 202L 209R 209L 23 23 12 12 18 18 Average Carnegie Series C2: Adult rhesus monkeys Average 124R 137R 137L 206R 20GL 219R 219L .. 57 57 23 23 22 22 1.4 2.9 1.2 5.9 5.4 6.8 4.9 3.8 GROUP 2 13.5 5.6 12.5 13.7 11.2 12.5 6.3 4.1 Gs: GROUP 6 23.1 9.4 4.6 11.7 7.1 7.8 6.8 5.8 4.0 9.8 9.5 rt .7 -C 1.4 k 2.1 4.0 2.7 2.5 3.0 3.4 3.2 6.3 4.7 8.8 2.6 5.3 7.3 3.8 2.5 8.9 7.5 16.5 68 9 14 17 21 11 12 19 3.1 5.8 6.5 zk .9 & 2.5 3.3 4.0 3.5 2.0 3.8 5.6 5.3 10.8 7.5 9.0 5.4 9.9 11.6 5.5 17.4 12.5 14.6 ... 13.2 8.5 8.1 3.9 f .5 8.5 10.6 t .9 & 2.1 42 28 39 31 50 27 53 32 21 38 t 6.8 t 3.5 27 9 4 26 21 24 26 23 30 27 25 34 .. k .2 GROUP 7 18 22 rt 4.0 t 2.9 40 10 19 20 34 42 12 38 39 32 27 20 48 34 25 41 rt 5.0 -C 4.4 Dated from the time of onset of the preceding vaginal bleeding. 216 GERTRUDE J. VERMANDE-VAN E C K 3. Carszegie series C2 The monkeys of this group, with the exception of M206, were observed during longer periods in the colony. They had had several menstrual periods which were regular, but with greater intervals during the summer. The average total num100 PJ Yale series 0 Cornegie I 8 Cornegie 2 80 ' o 60 2 L b Groups I 2 3-5 6 7 Fig. 2 The percentages of atretic ova in the rhesus monkey. Distribution over groups according t o size of follicle. ber of ovocytes was 60,550 t 10,900, the average atresia of 4.8 f 0.5%. Again the number of ovocytes differed from animal to animal. The numbers of ova in the right and left ovaries were similar with the exception of monkey 206 (table 2 ) . Distribution over the groups was not different from the other series of monkeys (table 3, fig. 1). The days of the cycle on which the ovaries were removed are marked in table 4;the 217 NEO-OVOGENESIS I N T H E ADULT MONKEY presence of one or more corpora lutea in the ovaries indicated that these animals had reached full maturity. Duratiolz of the atresia. The duration of the atresia of the follicles was determined by x-irradiation of the ovary with doses adequate to destroy the growing follicles. Three young adult monkeys, weighing 36504200 gm, two and one-half to three years of age received calculated tissue doses of 1200 r TABLE 5 Normal and atretic ova in x-rayed monkeys 7-14 days after 12OOr MONKEY NUYBPR TOTAL OVA ATRETIC OVA % ' ATRPSIA ~ 87R 92L 98R 98L Average 69,750 50,420 77,880 77,520 3,340 3,830 5,760 4,820 4.8 7.6 7.4 6.2 68,890 4,440 6.5 TABLE 6 Per cent atresia in x-rayed monkeys 7-14 days after 1300r MONKEY NUMBER DAY 03" CYCLE 87R 92L 98R 98L 19 19 22 22 Average GROUP 2 DAYS 3.6 5.0 6.4 5.8 58 45 59 79 7 10 10 14 5.2 60 GROUP 1 (600 r on two successive days) between the 19th and 22nd day of the menstrual cycles. They were irradiated through portals of 10cm2 over the ovarian area. They were ovariectomized after 7 , 10, and 14 days. The irradiated ovaries had no follicles larger than group 2. All growing and mature follicles were transformed into corpora atretica. Only in the ovaries removed 7 days after irradiation, and in one ovary removed 10 days after the irradiation, a few atretic ova, which had derived from secondary follicles, were still recognizable. 218 GERTRUDE J. VERMANDE-VAN ECK After 14 days no ova surrounded with more than one layer of cells were present, and the atresia of all other follicles was complete. Four of these ovaries were suitable for counting (table 5). The total number of ovocytes was relatively high (av. 68,890, with an atresia of 6.5%). It was remarkable that the atresia in the primordial ovocytes was not higher than in the normal ovary, whereas the atresia of follicles in group 2 averaged 60% (table 6). DISCUSSION The number of ovocytes differed greatly in individual monkeys; even monkeys belonging to the same age group showed these differences. Mandl and Zuckerman ('50) found f o r rats that litter relationship and age were associated with significant differences in the number of ovocytes. Rats from the same litter did not differ much, whereas rats of different litters did. I n addition, they found, in agreement with Arai ( '20), that the number of ovocytes decreased with age. I n our experiments nothing was known about the relationship of the monkeys used, as all were imported from India. For the Yale series the estimates of age were based upon stage of dentition and body weight (van Wagenen), as well as onset of menstrual periods, and the continuing dnovulatory cycles. I n the Carnegie series the age was unknown; the immature monkeys had not ovulated and had shown only one menstrual period ; the mature group had shown regular menstrual periods and had corpora lutea in their ovaries. The mature monkeys, as a group, had the same average total number of ovocytes as the immature monkeys. The number of ova in 13 adult monkeys averaged 55,500 for the right ovaries, and 53,800 for the left (Green and Zuckerman). The total number of ovocytes of the different groups was: 90% primordial or primary follicles with one layer of cells around them (group 1 and 2) ; the number of larger follicles decreased inversely and exponentially with the size. The atresia from the ovocytes with two or more layers alone was 4.6 5.1% of the total number. This does not include the atretic ova with one NEO-OVOGENESIS I N THE ADULT MONKEY 219 layer of cells around them, so the percentage was actually higher. Sturgis ('49) divided the atretic follicles into three stages. I n the first stage, the disorganization and breakdown of the granulosa layer occurs, the ovum may still appear normal, or show early degenerative changes. I n the early second stage, there are no normal granulosa cells left, the ovocyte is dense, amorphous and irregular; in the late second stage, the ovocyte deteriorates rapidly, the vitelline membrane breaks around which there is a loose organization of fibroblasts and wandering blood cells ; the hyaloid membrane of the theca is well developed. I n the third stage, there is a progressive shrinkage of the follicle, a further infolding of the hyaloid membrane, and a complete absorption of the thin fibroblastic meshwork filling the cavity until eventually nothing remains but the many hyaloid bands which may or may not enclose an irregular central cavity. The first and second stages were included as atretic follicles in this study, the third stage was regarded as corpora atretica. Rhesus monkeys had comparable numbers of ovocytes in the pre- and postmenstrual period (Green and Zuckerman, '50). Only at the time of ovulation is the number of follicles with antra higher, but this small number cannot influence the total. Unknown factors determine the number of ovocytes in the ovary. The ovocytes in one monkey were equally divided between both ovaries. The numbers of oocytes in the right and the left ovary differed greatly in only one monkey. Careful attention was paid to the atresia of the follicles of the different stages of development, For the primordial ovocytes it is sometimes difficult to determine whether the cell is atretic or normal. I n every case where any doubt existed, the cell was counted as normal. Our estimates of atresia may therefore be somewhat low. Comparison of the separate groups shows that the number of atretic follicles increased in relation to the size of the follicle. I n all series this increase was relatively constant for each group (table 2 ) . Thus, during the process of growth and 220 GERTRUDE J. VERMANDE-VAN ECK maturation of the follicle the rate of atresia increases constantly. The largest fluctuations between the number of normal and atretic ova existed in the follicles with antra. Because the ovariectomies were performed at different stages of menstrual cycle, we could expect fluctuations in this group. During the secretory phase of the cycle, 66% of the follicles with antra were normal; 34% were atretic. This would indicate that in the monkey not all follicles with antra degenerate at the time of ovulation. The total atresia of all three series was low and remarkably constant: the averages were 4.5 f 0.5%; 4.1 2 0.5%; and 4.8 2 0.5%. This means that at any time in the ovary of the monkey, about 4.5% of the existing ovocytes are in the process of acute degeneration. Seven days after irradiation practically no ova larger than group 2 were recognizable, and after 14 days only ova surrounded with one layer of epithelial cells were present, and all of the larger follicles were young corpora atretica. These facts give us some insight into the rapidity of the atresia. Once atresia occurs in a follicle the process was completed within two weeks; for the small growing follicles the process required less than one week. Sturgis ('49) estimated the first stage of atresia to take place within one or two days, the second stage were seen from the second and third days until the 10th day; the third stage after about three weeks. Our findings after x-rays correspond with those of Sturgis, who dated his estimates on the age of the corpora lutea and the presence of luteinized tissue in the atretic follicles. Atresia occurs after damage of the ovocyte. Whether the damage is caused physiologically (from hitherto unknown factors) or artificially (by x-rays) makes no difference in the rapidity of the process. The constant atresia of 6 5 % of the total ovocytes would result in a steady decrease of their number if no formation of new ova from whatever source occurred. I f the duration of the atresia is known, and the percentage of atresia is known by counting, a regression line for the number of ovocytes in relation to the time can be plotted, using the formula t, = a(l-r)n, in which t,= the number of ovocytes NEO-OVOGENESIS I N T H E ADULT MONKEY 221 after n periods of atresia; a = the original number of ovocytes, and r the percentage atresia. The percentage of atresia was 6 5 % in all series ; this gives a decline in the curve which is much stronger than the decrease of the number of ovocytes in reality. The ovaries would be practically exhausted within two years, which is in contradiction to the findings. We even found a balance between atresia and new formation by comparing the mature and immature monkeys. Both had the same number, and the same death rate of ovocytes ( 6 5 % per week). The average life of the ovocyte can then be calculated to range between 21 and 26 weeks, which gives us an average life of the ovocyte of the rhesus monkey of about half a year. We have already calculated that the ovary, without new formation, would be deprived of ovocytes within two years, which gives us the theoretical maximum life of the ovocyte. Only a limited number of follicles with antra were degenerating at any time of the menstrual cycle (av. 33%). This degeneration was complete after 10-14 days. Consequently the average time required for the growth of the mature follicle from the large follicle without antrum is about 4-6 weeks; the whole process of development is apparently much longer than hitherto assumed. The total length of the process in the monkey is unknown, but the last step requires 4-6 weeks. For the human, it is often accepted that the time required from primordial to mature follicle is 2-4 weeks. Several textbooks of Obstetrics and of Histology give this statement. I n the literature, references are often made t o the original publications of Robert Schroeder ( '15) and of Robert Meyer ( '13). However, in their original publication such statements were not noted. Our figures imply that continuous formation of new ova occurs throughout sexual life. The number forming probably decreases with aging of the monkey and finally ceases. The human ovary is said to be devoid of ovocytes a few years after the menopause. For the monkey it would be possible to follow the rate of new formation by counting ovaries at different 222 GERTRUDE J. VERMANDE-VAN ECK known ages. It would be helpful to study ovaries from monkeys that have attained advanced ages. SUMMARY The problem of postnatal ovogenesis was approached by analyzing the number of normal and atretic ova in the rhesus monkey. Eight immature monkey ovaries (Yale Colony), 6 immature, and 7 mature monkey ovaries (Carnegie Colony) were counted. The counting technique described by Green and Zuckerman ( '50) was used. The follicles were grouped in 7 stages ranging from primordial to mature follicles. The normal and atretic ova were determined separately. The number of ovocytes differed widely among animals of similar ages. The right and left ovaries of one monkey usually contained about equal numbers of ovocytes. The averages of the total number of ovocytes for the three groups ranged between 50,120 2 10,600 and 60,810 116,470 per ovary, with an average atresia of 4.1 2 0.5 to 4.8 I+ 0.5%. The distribution in the different stages of follicle development was constant. The percentage of atretic ova was also constant for each group. The duration of the atresia was determined by x-irradiation of the ovary with doses (1200 r) adequate to destroy all growing follicles. Atresia after irradiation occurred in 7 days. The average and maximum life of the ovocytes were calculated (about 6 months and two years respectively) as was the average time required for the growth of the mature follicle from the large primary follicle (4 to 6 weeks). ACKNOWLEDGMENTS I should like t o express my gratitude to Dr. William U. Gardner, Chairman of the Department of Anatomy, Yale University School of Medicine, for his guidance and stimulating comments ; to Dr. George Corner, Carnegie Institution of NEO-OVOGENESIS I N T H E ADULT MONKEY 223 Washington, Department of Embryology, Baltimore, for the opportunity given me to study his material ; and to Dr. Colin White, Department of Public Health, Yale University, for his help in the statistical evaluation of the data. LITERATURE CITED ABERCROMBIE, M. 1946 Estimation of nuclear population from microtome scetions. Anat. Rec., 9 4 : 238-247. ALLEN,E. 1923 Ovogenesis during sexual maturity. Am. J. Anat., 31 :439-481. ALLEN,E., AND R. N. CREADICK1937 Ovogenesis during sexual maturity. The first stage, mitosis in the germinal epithelium, as shown by the colchicine technique. Anat. Rec., 69: 191-195. ALLEN,E., J. P. PRATT, Q. N. NEWELLAND L. J. BLAND 1930 Human ova from large follicles; including a search f o r maturation divisions and observations on atresia. Am. J. Anat., 46: 1-53. ARAI, H. 1920 On the postnatal development of the ovary (albino r a t ) , with especial reference to the number of ova. Am. J. Anat., 87: 405-462. COWPERTHWAITE, M. M. 1925 Observations on pre and post pubertal oogenesis in the white rat : Mus norvegicus albinus. Am. J. Anat., 36 : 69-89. EVANS, H. M., AND 0.SWEZY 1931 Ovogenesis and the normal follicular cycle in adult mammalian. Memoirs o f Univ. of Calif., 9: 119-188. GREEN, S. H., AND S. ZUCKERMAN1950 The number of oocytes in the mature rhesus monkey (Macaca mulatta). J. Endocrin., 7: 194-202. HAGGSTROM,P. 1921 Zahlenmassige Analyse der Ovarien eines 22 jahrigen gesunden Weibes. Upsala l k . Forh., $6: XV, 1-52. 1920 Ein Fall von Thyreoaplasia unter HANMAR, J. A., AND T. J. HELLMANN Beriicksichtigung gewisser innersekrctorischer und lymphoiden Organe. Z. angew. Anat., 5: 218-267. JONES, R. McC. 1949 The use of vital staining in the study of the origin of germ cells in the female rat, Mus norvegicus. J. Morphol., 84: 293-334. LATTA,J. S., AND E. S. PEDERSON 1944 The origin of ova and follicle cells from the germinal epithelium of the ovary of the albino r a t as demonstrated by selective intravital staining with India ink. Anat. Rec., 90: 23-35. MEYER,R. 1913 Ueber die Beziehung der Eizelle und des befruchteten Eies zum Follikelapparat, sowie des corpus luteum zur Menstruation. Ein Beitrag zur normalen und pathologischen Anatomie und Physiologie des Ovariums. Arch. f. Gyn., ZOO: 1-19. MANDL,A. M., AND S. ZUCKERMAN1949 The number of normal and atretic ova in the mature rat. J. Endocrin., 6: 426-435. 1950 Number of normal and atretic oocytes in unilaterally spayed rats. J. Endocrin., 7 : 112-119. 1950 The effect of destruction of the germinal epithelium on the number of oocytes. J. Endocrin., 7 : 103-111. - -___ 1950 The relation of age to numbers of oocytes. J. Endocrin., 7: 190-1 93. 224 GERTRUDE J. VERMANDE-VAN ECK MANDL,A. M., AND S. ZUCKERMAN1950 Changes in ovarian structure following the injection of carbolic acid into the ovarian bursa. J. Endocrin., 7: 227-235. SCHROEDER, R. 1915 Anatomische Studien zur normalen und pathologisehen Physiologie des Menstruationszyklus. Arch. f. Gyn., 104 : 27-102. SCHMIDT, I. G., AND F. G. HOFFMAN 1941 Proliferation and ovogenesis in the germinal epithelium of the normal mature guinea pig, as shown by the colchicine technique. Am. J. Anat., 68: 263-273. SCHWARZ, 0. H., J. C. YOUNGAND J. C. CROUSE 1949 Ovogenesis in the adult human ovary. Am. J. Obstet. Gynaec., 58: 54-64. SIMKINS,C. 8. 1932 Development of the human ovary from birth t o sexual maturity. Am. J. Anat., 51 : 465-505. SIMPSON,M. E., AND G. VAN WACENEN 1953 Response of the ovary of the monkey (Macaca mulatta) t o the administration of pituitary follicle stimulating hormone (F.S.H.). Anat. Rec., 115: 370. STURGIS,S. H. 1949 Rate and significance of atresia of the ovarian follicle of the rhesus monkey. Contr. to Embryol., 33: 67-80. SWEZY,O., AND M. N. EVANS 1929 Ovogenesis in the mammalia. Proc. SOC. Exp. Biol. and Med., 2'7: 11. VAN WAGENEN, G. 1954 Personal communication. VON HANSEMANN, S. 1912 Ueber den Kampf der Eier in den Ovarien. Arch. f. Entw. Mech., 35: 223-235. WALDEYER, W. 1870 Eierstoek und Ei. Wilhelm Engelmann, Leipzig. WINIWARTER, H. VON, AND G. SAINMONT 1908 Ueber die ausschliesslich post fetale Bildung der dcfinitiven Eier bei der Katze. Anat. Anz., 32: 61 3-616. ZUCKERNAN, S., AND A. M. MANDL 1949 The number of normal and atretic ova in the mature rat. J. Endoerin., 6 : 426-435. S., S . H. GREENAND A. M. MANDL 1951 The proportion of ovarian ZUCKERMAN, follicles in different stages of development in rats and monkeys. J. Anat., 85: 325-329.