Granulosa cell pyknosis in the dominant follicle of monkeys

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1 FERTJTY AND STERLTY Copyright o 1982 The American Fertility Society Vol. 37, No.6, June 1982 Printed in U.S.A. Granulosa cell pyknosis in the dominant follicle of monkeys Marilyn J. Koering, Ph.D.* Arnold L. Goodman, Ph.D. t+ Robert F. Williams, Ph.D.+ Gary D. Hodgen, Ph.D.:j: George Washington University Medical Center, Washington, D.C., and National nstitute of Child Health and Human Development, National nstitutes of Health, Bethesda, Maryland One of the primary morphologic criteria used as the first sign of degeneration of an antral follicle is the presence of a pyknotic nucleus in granulosa cells lining the lumen. This study was undertaken to determine whether granulosa cells with a pyknotic nucleus are also present in the putative dominant follicle. Dominance was verified by elevations of estradiol either following luteectomy or during the later stages of the follicular phase of the menstrual cycle. Some pyknosis is normal even in the viable dominant follicle at cycle day 8 or 12. Therefore; when evaluating the morphologic status ofa follicle, one should examine several areas to assure proper evaluation. Fertil Steril37:837, 1982 n the primate menstrual cycle, perhaps there is no greater mystery of ovarian function than the means by which a single follicle is selected and destined for ovulation while others of that cohort fail by atresia. Changes in the hormonal patterns that occur during this period of selection are valuable in providing.insight into this mechanism, but these changes should be correlated with morphologic variations in the follicle. However, precise structural changes of the follicle cells are not known. The presence of granulosa cells having pyknotic nuclei are regarded as a morphologic indication of the atretic status of ovarian follicles.1-3 Although such nuclei do characterize de- Received December 24, 1981; revised and accepted February 17, *Reprint requests: Dr. Marilyn J. Koering, Department of Anatomy, George Washington University Medical Center, 2300 Street, NW, Washington, D.C tpresent address: Department of Physiology, University of Alabama, Birmingham, Alabama. :f:pregnancy Research Branch, National nstitute of Child Health and Human Development, National nstitutes of Health, Bethesda, Maryland. Vol. 37, No.6, June 1982 generating cells, 4 in rodents the visualization of one or two granulosa cells manifesting nuclear pyknosis has been interpreted as an indication of early follicular atresia. 5 6 Evidence supporting this strict perspective, however, is less than com pelling. 7-9 n the monkey, which has a lengthy (about 14 days) follicular phase, much variability occurs between granulosa cells during the earlier stages of atresia,3 requiring that extensive evaluation be made of granulosa cells constituting the largest maturing follicles. Selection of the dominant follicle necessarily creates an asymmetry of ovarian function, manifested by differential estradiol (E2) secretion within 5 to 7 days after the onset ofmenses, despite the presence of several large growing follicles which co-exist bilaterally with the authentic dominantfollicle. 10 Clearly, the ultimate fate of all such follicles, except the one destined for ovulation, is atresia Thus, the questions arise: (1) To what extent can pyknosis normally occur in the dominant follicle? (2) How does the pattern of granulosa-cell nuclear pyknosis change as a function of normal maturation? (3) Does nuclear pyknosis of granulosa cells within large developing Koering et al. Granulosa cell pyknosis 837

2 follicles provide a reliable index for differentiating the ovulatory versus the degenerative status of ovarian follicles? The present study was undertaken to answer these questions by determining the pyknotic and mitotic indices in the putative dominant follicle (largest visible one) at 8 or 12 days after the onset of new follicle growth in Macaca monkeys. These primate surrogates serve to overcome the ethical constraints that limit comprehensive assessments in the human ovary. To provide greater surety of an appropriate evaluation of the granulosa cells in these follicles, a systematic sampling method was devised. These findings are correlated with prevailing levels of the principal ovarian steroids in peripheral serum and in the ovarian steroid effluent as indicators ofthe locus of the putative dominant follicle. Furthermore, to assure that the follicles examined were the putative dominant follicles, the following criteria were used on day 8 or day 12. A single large follicle, grossly visible and accompanied by significant asymmetrical secretion of E 2, had to be observed. Additionally, on day 12, an E 2 surge, typical of the surge antecedent to the midcycle gonadotropin surge and ovulation, had to have occurred. Previous observations have indicated that day-8 and day-12 follicles meeting these criteria are the follicles that ovulate. Therefore, follicles meeting these requirements can be designated as the putative dominant follicles and would have ovulated if the cycle had not been perturbed by ovariectomy. MONKEYS MATERALS AND METHODS The husbandry practices and menstrual recordings have been described previouslyy Beginning with the onset of menses (cycle day 1), normal intact adult rhesus and cynomolgus monkeys (Macaca mulatta and Macaca fascicularis) were bled daily (8:00 A.M.) from the femoral vein, under ketamine hydrochloride ( -15 mg/kg) anesthesia. SURGCAL PROCEDURES A total of eight monkeys were ovariectomized or hemiovariectomized 8 or 12 days after the initiation of new follicle growth. n the first set, three monkeys (928, 789, and 795) had both ovaries removed 12 days after the onset of menses, 838 Koering et al. Granulosa cell pyknosis whereas two others (413 and 404) were hemiovariectomized 12 days after luteectomy in the midluteal phase. n the second set, an additional two monkeys (788 and 812) were bilaterally ovariectomized on day 8, and one (598) was hemiovariectomized 8 days after luteectomy. n four of the monkeys, we collected bilateral ovarian venous effluents just prior to ovariectomy; the levels of E 2 served to indicate the side of the developing dominant follicle Luteectomy was used in three monkeys as a means of synchronizing the onset of new follicle growth, because in previous studies we had shown that surgical removal of the corpus luteum during the midluteal phase resulted in prompt initiation of a new follicular phase and that the subsequent ovulation was likely to occur on the ovary contralateral to the side of luteectomy Accordingly, these techniques were employed in the study design to increase the control over events in folliculogenesis. RADOMMUNOASSAYS Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), E 2, and progesterone (P) were measured by radioimmunoassay systems employed previously. 13 MORPHOLOGC ANALYSS Complete evaluations were performed on the largest follicles in ovaries taken from the eight monkeys. n the ovary of one monkey (598), two large follicles were evaluated because of initial ambiguity regarding identification of the putative dominant follicle. All ovaries were fixed in Bouin's fluid, dehydrated, embedded, and serially sectioned at 7 J.m. All sections were mounted and scanned under x 25 magnification. The crossdiameters were determined for each antral follicle, and the number of sections that composed each follicle were noted. Previously, morphologic observations on monkey ovaries 3 revealed that signs of atresia are not uniformly distributed throughout large follicles. Therefore, it was necessary to apply a representative sampling process to enhance the reliability of such evaluations. The total number of sections for each follicle were divided into five equal portions. The granulosa cell nuclei on the first section of divisions 2 to 5 were inspected with an ocular grid at x 400. Among smaller follicles, about 14,000 granulosa cell nuclei were counted on the four sections, up to 29,000 in the larger follicles. Nuclear pyknosis was recognized by the nucleus be- Fertility and Sterility

3 <Q,..:. -~"E Q- ce> <(0. a: f- (/) 100 UJ p el / 4040' 8,..0 o----cr'' DAYS <Q,..:. _~- 0 E -" c Cl <(a. 200 a: f- (/) 100 UJ DAYS OVAX ' 8 9 Figure 1 Levelsofestradiol-17[3duringthe late follicular phase for five monkeys bilaterally ovariectomized on day 12. For two monkeys (- - -) day 0 was the day of Luteectomy, and for the remaining three monkeys (-) day 1 was the first day of the menses. Figure 2 Levels of estradiol-1713 during the midfollicular phase for three monkeys. Monkeys 788 and 812 were bilaterally ovariectomized on day 8 of the menstrual cycle. Monkey 598 was hemiovariectomized 8 days following luteectomy. coming dense, decreasing in size, and eventually fragmenting. 14 Cells in this state are distinguishable from other cell types in that they lack a characteristic nuclear chromatin pattern. Also, the nuclei undergoing mitosis were recorded. Mitotic nuclei were characterized by their density and unique shapes representing the various stages of mitosis. The pyknotic index (P) was determined by calculation of the ratio of pyknotic nuclei to normal nuclei present in each section evaluated, and the mitotic index (M) was similarly derived. 8 RESULTS Figure 1 depicts the levels of circulating estradiol-17~ for five monkeys during the late follicular phase, illustrating their E 2 patterns at the time that ovaries were removed on day 12. Monkeys 928, 789, and 795, having completed the preovulatory midcycle estrogen surge, were very near the expected time of follicular rupture. Two additional monkeys, 413 and 404, were luteectomized 12 days earlier. Monkey 413 had achieved the preovulatory estradiol-17~ elevation, but the dominant follicle was about 48 hours from ovulation. The four monkeys that had E 2 elevations in peripheral serum manifested LH surges as well; the exception was monkey 404, whose peripheral serum estradiol-17~ levels also never reached 100 pg/ml. These findings enhance the possibility of the viable dominant follicle being present. n ad- Vol. 37, No. 6, June 1982 dition, ovaries were removed from three monkeys on day 8 (Fig. 2). The E 2 levels in the peripheral circulation were near 100 pg/ml at the time of ovariectomy, consistent with a less advanced state of follicular maturation. The morphologic assessment of pyknosis and mitosis in the granulosa cells of each putative dominant follicle were considered in relation to their hormonal status and other morphologic observations. Usually, the first sign of pyknosis was seen in the cells lining the lumen. Monkeys 928 and 789, Cycle Day 12, Ovulation mminent. Both monkeys manifested preovulatory estrogen peaks (Fig. 1) and the impending ovulatory status of monkey 789 was further substantiated by elevated E 2 levels, which had already reached maximal concentration, and by preovulatory Pin the left ovarian vein (Table 1). The ovaries of both contained an abundance of interstitial gland tissue surrounding numerous atretic follicles, a pattern not seen in ovaries containing less mature preovulatory follicles The wall of the putative dominant follicle in some areas was composed of eight layers of granulosa cells (Fig. 3). There was a separation or loosening of the granulosa cells composing these layers, probably due to an accumulation of antral fluid and concurrent distention of the follicular architecture. Pyknotic nuclei were evident in some of the granulosa cells (Fig. 4), and the P was similar in the two monkeys (about 0.70). Mitosis was minimal or had ceased (Tables 1 and 2). The theca Koering et al. Granulosa cell pyknosis 839

4 interna was variable in thickness. t was up to four cell layers deep in some areas with an abundant blood supply (Fig. 3). Monkey 795, Cycle Day 12, Near Ovulation. This putative dominant follicle was less advanced than in monkeys 928 or 789,judged on the basis ofthe absence of interstitial gland tissue. However, the greater concentration of E 2 and preovulatory P in the ovarian vein effluent was present on the side supporting the dominant follicle (Table 1). The wall of this large follicle varied in thickness, and the granulosa cells were more compact, resembling cells of monkey 413 (Fig. 5). The P varied within this follicle; one section had no identifiable pyknosis, whereas another section had an index of 0.2 (Table 1). Monkey 413, Day 12, About 48 Hours Before Ovulation. The wall of this putative dominant follicle, which had a preovulatory E 2 elevation (Fig. Figure 3 A portion of the wall of the largest follicle in the ovary from monkey 928, 1 day after the preovulatory E 2 peak. The granulosa layer is several cell layers in thickness in this area, and the granulosa cells are separating from one another. The theca interna (between arrows) is about four cell layers thick and has a large capillary (C) ( x 400). Figure 4 A portion of the wall of the largest follicle in the ovary from monkey 789, 1 day after the preovulatory E 2 peak. n this part of the follicle, the granulosa and theca interna layers are thinner than seen in other portions of this follicle. Two granulosa cells (arrows) with pyknotic nuclei are lining the lumen. The dark material in the lumen is precipitated follicular fluid ( x 400). 1), was typical of most large follicles, in that the wall on one side was composed of several layers of granulosa cells; the theca interna was 3 to 4 cell layers in thickness. The opposite wall had only 2 to 3 layers of granulosa cells (Fig. 5). Granulosa cells that had a pyknotic nucleus were seen in all sections sampled, resulting in a mean P of However, the distribution of pyknosis was not uniform (Table 2); this heterogeneity did not correlate with variability in the number of granulosa cells or theca interna layers. Mitosis of the granulosa cells was continuing in some cells (Table 2). Monkey 404, Day 12, Atresia of the Largest Follicle. Like the other large follicles, the wall varied in thickness, with some areas having up to eight layers of granulosa cells with mitosis (Fig. 6). However, granulosa cells with pyknotic nuclei lining the lumen were abundant in one area (Fig. Table. Relationships Between Follicle Size, Pyknosis (P), Mitosis (M), and Ovarian Venous Steroid Levels in Cynomolgus Monkeys 12 and 8 Days After the Onset of Menses Follicle Section Ovarian Vein Day Monkey size ndex Mean mm pg/ml pg/ml La 5.5 p R M L L 5.1 p R M L L 4.1 p R M L R 3.2 p R 727 < 0.1 M L 130 < 0.1 anenotes side of ovary or ovarian vein. be = estradiol cp = progesterone. 840 Koering et al. Granulosa cell pyknosis Fertility and Sterility

5 Table 2. Pyknotic (P) and Mitotic (M) ndices for Four Equidistant Sections Through the Largest Antral Follicle in Rhesus Monkeys 12 and 8 Days After the nitiation of New Follicle Growth. Follicle Day Monkey size ndex mm p 0.53 M p 0.20 M p 3.70 M (A) 2.6 p (B) M p 0.14 M Section Projected 3 4 Mean status Viable Viable Atretic (anovulation) Viable Atretic 7). The mean P from the four areas counted was 1.24, but values ranged from 3.70 in section 1 to 0.26 in section 4 (Table 2). Mitosis was seen throughout the follicle, even though it was undergoing the initial stages of atresia. The lack of elevated E 2-17~ levels in serum through day 12 (Fig. 1) confirmed that this largest follicle was not likely to achieve timely ovulation. Monkeys 788 and 812, Cycle Day 8. Each monkey had a single large follicle, although the follicle for monkey 788 was slightly larger, and its overall P was 1.24 (Table 1). Also, the ovarian venous effluent from the ovary supporting this follicle contained elevated E 2, but the granulosa cells with pyknotic nuclei lining the lumen were more abundant (Fig. 8) than those seen in monkey 812. Notably, monkey 788 demonstrated that viable follicular status and an elevated P can be concurrent (Table 1). The next largest developing follicle in either ovary was only 0.75 mm in diameter. Monkey 812 had elevated ovarian vein estrogen from the ovary with the large follicle and a mean P of0.5 (Table 1); no other developing follicle had achieved 1 mm in diameter. Monkey 598, Day 8. This ovary was removed from a monkey that had been luteectomized 8 days earlier. t contained two follicles (designated A and B) of equal size (2.6 mm). The number of granulosa layers of both follicles was variable, as was the thickness of the theca interna (Figs. 9 and 10). The question arose as to which follicle was the authentic viable dominant follicle. Morphologic analysis of the granulosa cells from the four sections evaluated for each follicle showed quantitative and qualitative differences. Follicle A had some granulosa cells with pyknotic nuclei (Fig. 9), particularly in one area (section 4, Table 2). ts M was elevated when compared with follicle B; further, cell divisions were unevenly dis- Vol. 37, No.6, June 1982 tributed. n contrast, follicle B had a greater incidence of unevenly distributed granulosa cells having pyknotic nuclei. The mean M was lower than that of follicle A (Table 1). Accordingly, it seems that follicle A was the viable one, selected for ultimate ovulation, whereas it is likely that follicle B was becoming atretic. nterestingly, follicles A and B were adjacent, their theca externa being contiguous in part. The pattern of E 2 in peripheral serum was similar to that of other members of the day-8 group (Fig. 2), and the next LH surge occurred 23 days after ovariectomy (versus 5 days if the dominant follicle had been in the ovary remaining in situ). DSCUSSON The data presented make two principal points: (1) Pyknosis normally does occur to some degree even within the granulosa cells of the viable dominant follicle during its emergence in the late follicular phase. Therefore, the presence of one or two pyknotic nuclei, found by cursory inspection, gives no surety of the onset of atresia. (2) Of equal importance, the method of assessment of granulosa cell morphology must provide data representative of the follicle as a whole. Since granulosa cell pyknosis is not uniformly distributed within large antral follicles, inadequate sampling may result in misleading determinations of the follicular status. Conventionally, the observation of pyknotic nuclei in one or two granulosa cells of a given section has been regarded by some investigators as a sign of early follicular atresia in rodents Other reports in sheep have been slightly less restrictive, in that the identification of "occasional" pyknotic nuclei was not regarded as an indication of the onset of follicular degenera- Koering et al. Granulosa cell pyknosis 841

6 Figure 5 A portion of the wall of the largest follicle seen in an ovary from monkey 413, 12 days after luteectomy. The granulosa is thinner than in some other parts of the follicle, and several cells (arrows) with pyknotic nuclei are seen lining the lumen. The theca intema is thin and many of its cells are indistinguishable from the surrounding connective tissue ( x 400). Figure 6 A portion of the wall from the largest follicle seen in an ovary from monkey 404, 12 days after luteectomy. The granulosa is several cell layers thick in this area, and mitotic figures are present in two cells (circles) ( x 400). Figure 7 A portion of the wall from another area of the same follicle seen in Figure 6. The granulosa and theca interna are slightly thinner, and several granulosa cells (arrows) with pyknotic nuclei are present. Also, mitosis (circle) is evident in a cell lining the lumen (X400). tion n adult human ovaries, studies have been limited to the examination of tissue obtained from biopsy and resection and from a portion of excised follicles, and have not permitted a thorough evaluation of the whole follicle. Additionally, when the wall of aspirated follicles was examined, a P of 1.0 was permitted for a healthy follicle. 22 This index could possibly be higher if the follicle were intact, assuring no loss of cells, which would probably occur during aspiration. Results from this study reveal that much variability exists in the P within a large follicle and between the largest follicles in monkeys. When these data were correlated with the E 2 concentratiqn, a follicle with an overall P as high as 1.20 may be considered developing due to elevations of E 2 in the ovarian vein (Table 1). n contrast, a follicle from a monkey at day 12 with a mean P of 1.24 was classified as atretic because of a low peripheral E 2 level and a localized area with a P of Another follicle from a monkey luteectomized 8 days earlier had a mean P of0.75 and was accompanied by a wide variability in the P throughout the follicle. This follicle was also considered atretic because of the presence of an adjacent follicle of similar size that was clearly developing (low P and elevated Ml). Because such 842 Koering et al. Granulosa cell pyknosis variability exists in the numbers of granulosa cells with a pyknotic nucleus within a developing follicle, more systematic techniques should be utilized when one is assessing the morphologic status of a follicle. Applications of a method providing representative analysis of preselected sections, as used in this study, is more likely to reveal the actual status of an ovarian follicle. Although the overall mean P may be equivocal as an absolute indicator of atresia, because of the nonuniformity of the degenerative process striking elevations in the incidence of pyknotic nuclei in any one section may justify classification as atretic. Equally crucial is the method used to determine the proportion of pyknotic nuclei in a given section. Frequently, the wall of a large antral follicle is variable in both thickness and the distribution of granulosa cells (Figs. 6 and 7). The uneven number of granulosa cell and theca interna layers is an established characteristic of the monkey ovary 23 and does not indicate the developmental or degenerative status of a follicle. Similarly, in the human ovary granulosa cells can aggregate along the basement membrane, leaving other areas sparsely populated. 21 Total direct counts of each section assure more precision than approaches based on counting a given Fertility and Sterility ' i

7 Figure 9 A portion of the wall of follicle A seen in the ovary from monkey 598, 8 days after luteectomy. This area has a granulosa cell (arrow) with a pyknotic nucleus and several cells in mitosis (circles) (x400). Figure 10 A portion of follicle B from the same ovary seen in Figure 9. Three granulosa cells (arrows) with pyknotic nuclei are lining the lumen; other cells have mitotic figures (circles) ( x 400). Figure 8 A portion of the wall from the largest follicle seen in an ovary from monkey 488, 8 days after the onset of menses. This area of the wall has four granulosa cells (arrows) with pyknotic nuclei lining the lumen ( x 400). area irrespective of its proximity to the lamina basalis or the number of granulosa cell layers. 7 9 The second index we employed to evaluate the status of growing follicles is mitosis. 24 Others have used the M to estimate follicular kinetics n the late follicular phase of the human menstrual cycle mitosis is known to cease in the mature follicle prior to its rupture. 21 Moreover, granulosa cell division within antral follicles of laboratory primates is seldom observed in the larger follicles during the luteal phase of the menstrual cycle. 3 Therefore, lack of mitosis alone is not a reliable indicator of atresia in the late follicular phase; its value comes when it is used in conjunction with the P, as well as a function of time after menses. Although changes in follicular fluid are likely to be a better indicator of the onset of atresia, there are times when such a procedure is not feasible. n this study removal of follicular fluid would have disrupted the follicular architecture, and the true status of its follicular form could not have been determined. Using serially sectioned ovaries, we classified the large antral follicles as viable or atretic on the basis of prevailing levels of principal ovarian steroids in serum during the cycle studied as well as the morphologic assessments of granulosa cells. n one series of monkeys, we relied upon the asymmetry of ovarian Vol. 37, No. 6, June 1982 vein steroids as an indicator of the presence and status ofthe dominant follicle. 10 Admittedly, it is not possible to establish without equivocation that the status assigned these largest follicles would have been their destiny (Tables 1 and 2). However, profound ovarian asymmetry in the late follicular phase is surely an indication that ovulation was likely to have occurred Unilateral elevation of both E 2 and P implies a state of impending ovulation. We have shown that some nuclear pyknosis of granulosa cells is seen in the viable dominant follicle of these monkeys. Accordingly, classifying a follicle as atretic when it contains pyknotic nuclei in only a few granulosa cells may be overly restrictive. Misleading interpretations about the degenerative or viable status of an ovarian follicle can be minimized by a thorough evaluation of preselected areas throughout the follicle and relating of that information to time in cycle and the ovarian architecture. Because of the limitations on undertaking such a study of human follicles, findings from this investigation should assist in the better interpretation of human follicular tissue obtained from biopsy and wedge resections. Acknowledgments. The authors acknowledge the excellent technical assistance of Messrs. Don Barber, Adrian Coleman, Charles Turner, and Almorris Lynch, and the skillful secretarial assistance of Ms. Linda Baldwin. Koering et al. Granulosa cell pyknosis 843

8 REFERENCES 1. Sturgis SH: Rate and significance of atresia of the ovarian follicle of the rhesus monkey. Contrib Embryo! 33:69, Deane HW: Histochemical observations on the ovary and oviduct of the albino rat during the estrous cycle. Am J Anat 91:363, Koering MJ: Cyclic changes in ovarian morphology during the menstrual cycle in Macaca mulatta. Am J Anat 126:73, Alfert M: Changes in the staining capacity of nuclear components during cell degeneration. Bioi Bull 109:1, Peters H: Some aspects of early follicular development. n Ovarian Follicular Development and Function, Edited by AR Midgley, WA Sadler. New York, Raven Press, 1979, p1 6. Hirshfield AN, Midgley AR: Morphometric analysis of follicular development in the rat. Bioi Reprod 19:597, Turnbull KE, Branden A WH, Mattner PE: The pattern of follicular growth and atresia in the ovine ovary. Aust J Bioi Sci 30:229, Draincourt MA: Follicular kinetics in the mare ovary. Ann Bioi Anim Biochim Biophys 19:1443, Gougeon A: Qualitative changes in medium and large antral follicles in the human ovary during the menstrual cycle. Ann Bioi Anim Biochim Biophys 19:1461, dizerega GS, Hodgen GD: Folliculogenesis in the primate ovary. Endocrine Reviews 2:27, dizerega GS, Marut EL, Turner CK, Hodgen GD: Asymmetrical ovarian function during recruitment and selection of the dominant follicle in the menstrual cycle of the rhesus monkey. J Clin Endocrinol Metab 51:698, dizerega GS, Lynch A, Hodgen GD: nitiation of asymmetrical ovarian estradiol secretion in the primate ovarian cycle after luteectomy. Endocrinology 108:1233, Goodman AL, Nixon WE, Johnson DK, Hodgen GD: Regulation of folliculogenesis in the cycling rhesus monkey: selection of the dominant follicle. Endocrinology 100:155, Leuchtenberger C: A cytochemical study of pyknotic nuclear degeneration. Chromosoma (Berlin) 3:449, Hay MF, Moor RM, Cran DG, Dott HM: Regeneration of atretic sheep ovarian follicles in vitro. J Reprod Fertil 55:195, Braw RH, Tsafriri A: Effect ofpmsg on follicular atresia in the immature rat ovary. J Reprod Fertil 59:267, Koering MJ: Comparative morphology of the primate ovary. n Contributions to Primatology, Vol ll, Edited by WP Luckett. Basel, S. Karger, 1974, p Byskov AG: Atresia. n Ovarian Follicular Development and Function, Edited by AR Midgley, WA Sadler. New York, Raven Press, 1979, p Brand A, dejong WHR: Qualitative and quantitative micromorphological investigations of the tertiary follicle population during oestrous cycle in sheep. J Reprod Fertil 33:431, Moor RM, Hay MF, Dott HM, Cran DG: Macroscopic identification and steriodogenic function of atretic follicles in sheep. J Endocrinol 77:309, Bomsel-Helmreich 0, Gougeon A, Thibault A, Saltarelli D, Milgrom E, Frydman R, Papiernik E: Healthy and atretic human follicles in the preovulatory phase: differences in ovulation of follicular morphology and steroid content of follicular fluid. J Clin Endocrinol Metab 48:686, Brailly S, Gougeon A, Milgrom E, Bomsel-Helmreich 0, Papiernik E: Androgens and progestins in the human ovarian follicle: differences in the evolution of preovulatory, healthy nonovulatory and atretic follicles. J Clin Endocrinol Metab 53:128, Hartman CG, Corner GW: The first maturation division of the macaque ovum. Contrib Embryo! 29:1, Byskov AGS: Cell kinetics studies of follicular atresia in the mouse ovary. J Reprod Fertil 37:277, McNatty KP, Smith DM, Makris A, Osathanondh R, Ryan KJ: The microenvironment of the human Graafian follicle: interrelationship among the steroid levels in antral fluid, the population of granulosa cells and the status of the oocyte in vivo and in vitro. J Clin Endocrinol Metab 49:851, Koering et al. Granulosa cell pyknosis Fertility and Sterility