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BIOLOGY

50,

531-536 (1976)

Relationship

RALPH

between Growth and Meiotic of the Mouse Oocytel A. SORENSEN’

The Departments of Anatomy and Biological Reproductive Biology, Harvard

AND

PAUL

Maturation

M. WASSARMAN”

Chemistry, and the Laboratory of Human Reproduction Medical School, Boston, Massachusetts Ml15

and

Accepted December 22, 1975 Oocytes of various sizes were isolated from trypsinized ovaries ofjuvenile mice, cultured in a chemically defined medium, and scored for the resumption and completion of meiotic maturation. Oocytes recovered from mice younger than 15 days remained in the germinal vesicle stage, whereas those from mice 15 days or older resumed meiosis at a frequency which increased with the age of the mice. The mean diameter of the oocytes recovered also increased with the age of the mice. Within individual litters, the mean diameter of oocytes which failed to mature (incompetent oocytesl was significantly less than that of oocytes which matured (competent oocytes). The frequency of premature metaphase I arrest decreased markedly as the age of the mice and oocyte volume increased. These results suggest that the ability to resume meiosis is acquired at a specific stage of oocyte growth in the juvenile mouse, and that the ability to complete meiotic maturation is acquired subsequently. These oocytes provide an in vitro system with which to study the control of meiosis in the mammal. INTRODUCTION

Shortly after birth, the mouse ovary is populated with thousands of small oocytes arrested in late prophase of meiosis. Commencement of oocyte growth is apparently regulated within the ovary, the number of oocytes entering the growth phase being a function of the size of the pool of nongrowing oocytes (11, 15). The oocyte and its surrounding follicle grow coordinately, progressing through a series of definable morphological stages (14). The oocyte completes its growth in the adult mouse before the formation of the follicular antrum; consequently, the majority of follicle growth occurs after the oocyte has stopped growing (4). Growth is continuous, ending in the ovulation of a mature oocyte, or degen’ A preliminary account of some of this work was presented at the Meeting of the Federation of American Societies for Experimental Biology, Minneapolis, Minnesota, June, 1974. e Present address: Department of Biological Sciences, De Paul University, 1036 W. Belden Avenue, Chicago, Illinois 60614. x To whom inquiries and reprint requests should be directed.

eration (atresia) of the oocyte and its follicle (13). In the sexually mature female, fully grown oocytes in Graafian follicles resume meiosis and complete the first meiotic division just prior to ovulation. This process is characterized by dissolution of the nuclear (germinal vesicle) membrane, condensation of the diffuse dictyate chromatin into distinct bivalents, separation of the homologous chromosomes and emission of the first polar body, and arrest of meiotic progression at metaphase II. The completion of meiosis is triggered by fertilization or parthenogenetic activation. Progression of the oocyte from the germinal vesicle stage to metaphase II is termed “meiotic maturation.” Maturation of mouse oocytes will occur in vitro if the oocytes are physically removed from large follicles of adult ovaries and cultured in a chemically defined medium (2, 6, 17). Mouse oocytes matured and fertilized in vitro have developed into viable fetuses following transplantation to the uteri of foster mothers (5). It has been reported that oocytes re531

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved

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covered from mice younger than l&days postpartum are not competent to resume meiosis in vitro (18). Similarly, a lower proportion of oocytes recovered from prepubertal rabbits (70-90 days of age) matured to metaphase II irz uitro, as compared to adult controls (19). Experiments reported here suggest that the capacity to resume meiosis is acquired at a specific stage of oocyte growth in the juvenile mouse, and that the ability to complete meiotic maturation is acquired subsequently. The availability of mammalian oocytes of different sizes isolated in culture, permits biochemical studies not only of oocyte growth (12), but also of the control of meiosis. MATERIALS

AND

METHODS

Randomly bred Swiss albino mice (CD1, Charles River Labs) were used in all experiments. Oocytes were obtained from juvenile mice by trypsinization of ovaries according to the method described by Szybet (18). “Adult oocytes” were obtained by physical disruption of the larger follicles of 6- to lo-week-old females (6). Oocytes were collected with a micro-pipet, washed, and transferred to microdrops of chemically defined culture medium under oil (3). Culture was carried out at 37°C in an atmosphere of 5% CO, in air. At the end of the culture period, the oocytes were scored for the presence or absence of a germinal vesicle and first polar body, and were classified as germinal vesicle (GV), metaphase I, or metaphase II. These classifications were confirmed in representative cultures by examination of chromosomes in fixed and stained preparations (6). Oocyte diameters (exclusive of zona pellucida) were measured with an ocular micrometer attached to an inverted microscope. The diameters of adult oocytes measured by this method were significantly larger than those obtained from histological sections of whole ovaries (4). This may be due to the effects of fixation, and/or to the flattening of the oocyte on the bottom of the culture dish.

VOLUME 50, 1976 RESULTS

Figure 1 shows representative oocytes isolated from 9-, 12-, 15, and 18-day-old mice. It should be noted that, although the trypsinization procedure completely removed adhering follicle cells from the oocytes, it did not grossly affect the zona pellucida. Oocytes recovered from juvenile mice 9, 11, and 13 days postpartum failed to resume meiosis when cultured overnight in vitro, as evidenced by the retention of intact germinal vesicles. On the other hand, oocytes recovered from mice 15 days of age or older resumed meiosis at a frequency which increased with the age of the mice (Fig. 2B). Of the oocytes, 10, 52, 62, and 87% recovered from mice 15, 17, 19, and 21 days of age, respectively, resumed meiosis, as compared to 94% for oocytes from adult mice. Within individual l&day litters, the mean diameter of oocytes which retained their germinal vesicle (incompetent oocytes) was significantly less than that of oocytes which underwent germinal vesicle breakdown (competent oocytes) (Table 1). These results correlate with the increase in mean diameter of oocytes recovered over the age range investigated. Between 9 and 21 days, the mean oocyte diameter increased from less than 50 pm to nearly 75 pm, corresponding to an approx fourfold increase in oocyte volume (Fig. 2A). Of the oocytes which resumed meiosis in those from younger animals exvitro, hibited a high frequency of incomplete meiotic maturation with arrest at metaphase I (Fig. 2C). Accordingly, the ratio of metaphase II to metaphase I arrest changed markedly with the age of the mice, increasing from 0.16 for the 15-dayold animal to 9.0 for the adult. DISCUSSION

In the adult mouse, the oocyte grows from a diameter of 13 pm (in histological section) to a terminal diameter of 70 pm (4). Each oocyte is contained within a cellular follicle which grows concomitantly

BRIEF NOTES

533

FIG. 1. Photograph of oocytes recovered from trypsinized ovaries of juvenile mice 9, 12, 15, and 18 days postpartum (A-D, respectively). x 160. Photographs taken using a Nikon inverted microscope (Model MS) equipped with Hoffman Modulation Contrast Optics.

with the oocyte, from a single layer of flattened cells to three layers of cuboidal granulosa cells by the time the oocyte has completed its growth. The theta is first distinguishable, outside of and separated by a basement membrane from the granu-

losa cells, when the granulosa layer is two cell layers thick. The follicular antrum first appears when the follicle is several cell layers thick and after the oocyte has reached its maximum diameter. Oocytes recovered from early and late

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Adult

Pm

FIG. 2. Relationship between oocyte size and competence and the age of the donor mice. (A) Mean diameters of oocytes recovered from 9-, 12-, 15-, 18-, 21-day-old, and adult mice. Two hundred or more oocytes were examined in each age group; the vertical lines represent the standard error of the measurement. (B) Percentage germinal vesicle breakdown (GVBD) as a function of the age of the donor mice. Oocytes were cultured for approx 16 hr as described in Materials and Methods, and then were examined. Two hundred or more oocytes were examined in each age group; the vertical lines represent the standard error of the measurement. (Cl Percentage metaphase I (open bar) and metaphase II (closed bar) arrest for those oocytes which resumed meiosis as a function of the age of the donor mice. The data are taken directly from Fig. 2B. TABLE

1

(pm) OF OOCYTES RECOVERED MEAN DIAMETERS FROM THREE INDIVIDUAL IS-DAY LITTERS” Litter 1

2 3

Incompetent? oocytes 59.9 k 2.3 (20)’ 59.4 t 1.9 (15) 61.2 + 1.9 (20)

Competent6 oocytes 68.5 ? 1.4 (20) 67.8 k 1.3 (15) 70.2 f 2.1 (20)

” Measurements were made after 4 hr of oocyte culture as described in Materials and Methods. b After 4 hr of culture, those oocytes which retained a GV were termed “incompetent” and those which underwent GV breakdown were termed “competent.” I’ The value in parentheses is the actual number of oocytes measured.

antral follicles of the adult mouse ovary resume meiosis at a high frequency !8391%) in vitro, whereas oocytes recovered from small, preantral follicles (two or

three cell layers thick) fail to resume meiosis (8). These results suggest that the oocyte develops the capacity to initiate meiotic maturation just prior to or at the same time as antrum formation. As expected from Brambell’s (4) descriptions of the relationship between oocyte and follicle growth, incompetent oocytes recovered from small follicles were significantly smaller than competent oocytes recovered from larger antral follicles, indicating a correlation between maturation competence and the termination of oocyte growth. Similarly, Tsafriri and Channing (20) have demonstrated a graded increase in the competence of oocytes to resume meiosis in vitro according to the stage of follicle growth in the adult pig ovary; however, they did not report any measurements of oocyte diameters. Furthermore,

BRIEF

incipient antrum formation is the earliest follicle stage that meiotic maturation figures have been described in oocytes of degenerating follicles of the mouse (7, 16). More follicles begin to grow, and at a faster rate, in the early (7 and 14 days) than in the late (21, 28, and 35 days) juvenile mouse ovary (13). At 15 days of age (the youngest mouse from which competent oocytes could be obtained in this study) the largest follicles present in the ovary are composed of 4 to 5 cell layers, while the first follicle with an incipient antrum is seen at 16 days. Larger antra are observed in the increasing number of follicles with 6 or more cells layers at 17, 18, and 19 days; fully developed Graafian follicles are first seen at 20 days (10). The large number of follicles which initiate growth in the early juvenile ovary results in an unusually large number of multilayered follicles in the ovaries of 21-day-old mice. Accordingly, Gates (9) obtained maximal oocyte yields upon superovulation of 21-day-old mice. If we assume that oocytes recovered from juvenile ovaries by the trypsinization procedure were released from the largest follicles present (supported by the size data), then those oocytes resuming meiosis were probably from follicles composed of at least 4 cell layers. Szybet (18) did not, however, report a distinct correlation between the occurrence of competent oocytes in one ovary and a particular stage of follicle development in the contralateral ovary. It is surprising that the mean diameter of the competent oocyte population observed in this study was significantly smaller than that of the imcompetent oocytes recovered from small, preantral follicles of the adult mouse (8). A possible explanation of this observation may be that oocytes of juvenile mice terminate their growth at a smaller volume than do oocytes of the adult. The difference in the growth rate of follicles in early juvenile ovaries, as compared to adults, and the fact that the first wave of growing follicles

NOTES

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is normally destined to degenerate before the mouse attains sexual maturity (13), are bases for anticipating a difference in the growth patterns of oocytes from juvenile and adult mice. In any event, it is clear that there is not a strict correlation between absolute oocyte size and the capacity to resume meiosis when comparing oocytes from juvenile and adult mice. The high percentage of incomplete maturation (arrest at metaphase I) observed in oocytes from juvenile mice, suggests that the development of the capacity to mature is acquired in a two-step process. It is tempting to speculate that this further development of the oocyte’s capacities may involve components of the spindle apparatus. Oocytes within cultured mouse ovarian fragments can be induced to mature by the addition of gonadotropins to the culture medium; oocytes within preantral follicles, however, do not proceed beyond metaphase I (1). The low percentage of metaphase I arrest observed by Donahue (6) and reported in this study with oocytes from adult mice may, therefore, represent oocytes recovered from small follicles. Erickson and Sorensen (8) did not observe premature arrest in their study, probably due to the failure to examine late preantral and early postantral follicles. On the other hand, Tsafriri and Channing (20) found that the frequency of metaphase I arrest and the stage of follicle development were related in the pig. This research was supported by grants from the National Institute of Child Health and Human Development (HD-069161, the National Science Foundation (GB-435281, and the Rockefeller Foundation. REFERENCES 1. BAKER, T. G., and NEAL, P. (1972). “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 377-396. University Press, Baltimore. 2. BIGGERS, J. D., WHITTINGHAM, D. G., and DONAHUE, R. P. (19671. Proc. Nat. Acad. Sci. USA 58, 560-567. 3. BIGGERS, J. D. (1971). In “The Biology of the Blastocyst” (R. J. Blandau, ed.), pp. 319-327. University of Chicago Press, Chicago. 4. BRAMBELL, F. W. R. (1928). Proc. ROY. SOC.

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Lond. (Ser. B) 103, 258-272. 5. CROSS, P. C., and BRINSTER, R. L. (1970). Biol. Reprod. 3, 298-307. 6. DONAHUE, R. P. (1968). J. Ezp. Zool. 169, 237250. 7. ENGLE, E. T. (1927). Amer. J.Anat. 39,187-203. 8. ERICKSON, G. F., and SORENSEN, R. A. (1974). J. Exp. Zool. 190, 123-127. 9. GATES, A. H. (1971). In “Methods in Mammalian Embryology” (J. C. Daniel, ed.), pp. 64-75. W. H. Freeman, San Francisco. 10. KENT, J. (1972). J. Reprod. Fert. 31, 323-326. 11. KRARUP, T., PEDERSEN, T., and FABER, M. (1969). Nature (London) 224, 187-188. 12. MANGIA, F., and EPSTEIN, C. J. (1975). Deu. Biol. 45, 211-220.

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13. PEDERSEN, T. (1969). Actu Endocrin. 62,117-132. 14. PEDERSEN, T., and PETERS, H. (1968). J. Reprod. Fertil. 17, 555-557. 15. PETERS, H., BYSKOV, A. G., LINTERN-MOORE, S., FABER, M., and ANDERSEN, M. (1973). J. Reprod. Fertil. 35, 139-141. 16. PINCUS, G. (1936). “The Eggs of Mammals.” Macmillan, New York. 17. SORENSEN, R. A. (1973). Amer. J. Anat. 136, 265-276. 18. SZYBEC, K. (1972). J. Endocrinol. 54,527-528. 19. THIBAULT, C. G. (1972). In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 397-411. University Park Press, Baltimore. 20. TSAFRIRI, A., and CHANNING, C. P. (1975). J. Reprod. Fert. 43, 149-152.

Relationship between growth and meiotic maturation of the mouse oocyte.

DEVELOPMENTAL BIOLOGY 50, 531-536 (1976) Relationship RALPH between Growth and Meiotic of the Mouse Oocytel A. SORENSEN’ The Departments of Ana...
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