Fertilization of Follicular Ova

Transcription

1 Fertilization of Follicular Ova Robert W. Noyes, M.D. P..oGRESs in surmounting common blocks to fertility, such as anovulation, closed oviducts, and oligospermia, has slowed to the point where reconnaissance is in order. Since neither ovulation, nor the oviduct, nor more than.. one sperm per ovum is theoretically necess~ for reproduction, the possibility exists that an ovum from a donor ovary may be fertilized and transferred to the uterus of a recipient host, thus circumventing rather than overcoming the causes for sterility mentioned above. The groundwork for this eventuality has been in progress for many years. The technique for transferring fertilized ova into the uterus of the rabbit was worked out by Heape in A recent review and brilliant contribution concerning the conditions required for development of transferred rabbit ova has been published by Chang. Pincus and co-authors have reported maturation and activation of follicular ova of the rabbitl2 13 and the maturation of human follicular ova in vitro. 14 Menkin and Rock have reported the fertilization of human follicular ova in vitro, while Comer has reported failure. Umbaugh has transferred follicular ova of cattle into the oviducts of inseminated recipients, and Decker has aspirated the contents of human ovarian follicles through the culdoscope and has transferred this material into the previously inseminated uterus; but so far the results of these experiments are equivocal. Certain criteria, accepted by many authors From the Department of Obstetrics and Gynecology, Stanford University School of Medicine. Acknowledgments are gratefully made to Dr. C. E. McLennan for criticism, Dr. W. E. Castle and Dr. E. Dempster of University of California for inbred rat strains, V. Montescleros for expert care of animals, to E. Zapata for photomicrography, and to M. H.. Noyes for preparation of the manuscript. Presented, by invitation, at the Seventh Annual Conference of the American Society for the Study of Sterility, Atlantic City, New Jersey, June 9,

2 NOYES [Fertility & Sterility as being indicative of fertilization-formation of more than one polar body, more than one pronucleus, and cleavage of the ovum-are open to the serious criticism that peculiar parthenogenetic changes take place in disturbed mammalian ova which may mimic closely the corresponding phases of fertilization and cleavage. In summary it may be said that while follicular ova apparently undergo normal maturation in vitro, no normal offspring have thus far been produced by transfer of follicular ova into a recipient animal. FIGURE 1. Three experimental and five control fetuses at term showing contrast in eye pigment. FIGURE 2. Four control and four experimental young showing hybrid vigor of the latter. 2 This paper reports the successful transfer, fertilization and growth to normal young of ova removed from ovarian follicles of donor rats and transferred into the ovarian bursas of recipients. Briefly, this was accomplished by obtaining ova from the ovary of a black donor female rat by piercing the maturing ovarian follicles under saline solution. The ova were picked up with a capillary pipette, and were transferred into the ovarian bursa of a previously mated albino female. Successfully fertilized follicular ova could easily be distinguished from the ovulated albino ova when they matured into dark-eyed embryos and young as shown in Figs. 1 and 2.

3 Vol. 3, No. 1, 1952] FERTILIZATION OF FOLLICULAR OVA 3. The rat was chosen as an experimental animal because the estrus cycle is conveniently short and the timing of ovulation is possible, 5 because no albumin coat is formed around tubal ova as in the rabbit, and finally because it was felt that data obtained from a spontaneously ovulating species may more nearly parallel conditions in human ova. On crossmating, our Wistar albino strain proved to be of mixed genetic composition as far as hood and agouti characters were concerned, so a homozygous strain of Sprague Dawley animals was used for recipients. A carefully inbred homozygous, black, major-hooded, non-agouti strain was used for donors. - Attempts to transfer follicular ova with their surrounding granulosa directly into the oviduct of the recipient rat failed because of the inaccessibility of the tubal ostium and because the oviduct proximal to the ostium could not be pierced with a transferring pipette large enough to pass these relatively large egg masses. Likewise no successful results were obtained in eleven transfers into the ovarian bursa when the bursal membrane was pierced by a pipette, bleeding from fine vessels being the chief difficulty. METHOD The method of transfer which produced successful results is briefly as follows. Dark-eyed donor females were killed with ether, the ovaries were removed to normal saline in a watch glass at 37 C. and were rinsed free of blood. The larger follicles were pricked with a sharp pointed blade, the contents were expressed and then transferred to fresh saline. Each cumulus with its contained ovum was separated from its shell of mural granulosa by blunt dissection. Several of each group of ova were set aside for staining, and the remainder were counted and picked up in a capillary pipette of about 200,u. inside diameter. The pipette and ova were then stored in an incubator for about ten minutes while the previously mated, light-eyed recipient was being anesthetized with ether. One ovary was exposed through a flank incision and was drawn out onto a moistened sheet by a hook placed around the uterine cornu. Thus a foramen in the bursal membrane, recently discovered by Alden, was exposed. Through this the transferring pipette was passed, the contained ova being expelled into the bursal sac. The ovary was replaced in the abdomen and the wound closed. The entire procedure required about forty-five minutes. The sample of ova saved for staining was transferred to a clean slide between two capillary glass spacers, of about two-thirds the diameter of the

4 4 NOYES [Fertility & Sterility ovum mass, and was covered with a cover slip. The sides were then sealed with paraffin, and Bouin's fixative was injected under the edge of the cover slip, the excess being drawn through with small pieces of blotting paper. The fixative was followed by alcohol, water, hematoxylin, alcohol, xylol and balsam, the required time averaging one-half hour. The finished preparation is somewhat thick, especially for fine photography, but has the advantages of speed, ease, and visualization of the entire specimen in a single field of view. After transfer of ova the recipient female rats are allowed to go to term. Since the mother may occasionally destroy the newborn litter, it has been found necessary to laparotomize the recipient at term through a ventral midline incision. The dark-eyed fetuses developing from the transferred ova can be differentiated with ease and accuracy in utero from the light-eyed controls ovulated by the recipient. In Figure 1 the mature fetuses have been removed from the uterus for clarity of photography, and the dark eye pigment of experimental embryos 2, 4, and 8 (counting clockwise from the left) is in contrast to the lighter eyes of the control animals. The recipients are then allowed to deliver the litter as a recheck on the count and as a check on the viability of the young (Fig. 2). RESULTS Fifty experiments of this type were performed, in addition to four in which ovulated rather than follicular ova were transferred. Of these fiftyfour experiments, fourteen were considered failures: 1 because the recipient died postoperatively; 7 because the recipient, though plugged, did not become pregnant; and 6 because the recipient carried no fetuses, neither experimental nor control, in the experimental cornu. The results of the remaining forty experiments are shown in Table 1 and in its accompanying graph, Fig. 3. Since the order but not the rate of changes in maturing follicular ova is known, the groupings and the scale of the abscissa in Figure 2 are necessarily arbitrary. Figs. 4 through 9 are high power photomicrographs of whole ova, prepared as described above, indicating how the stage of development of the transferred ova is determined. Blandau has shown that these changes take place within the ten hours before ovulation. It must be remembered that the stage of development of a given group of transferred ova is judged by the appearance of but a small sample of stained ova (Table 1, column 1) and since a certain variability in maturation rate is to be expected,

5 Vol. 3, No. 1, 1952] FERTILIZATION OF FOLLICULAR OVA 5 TABLE 1. Development of Transferred Follicular Ova in Relation to Their Stage of Maturation No. af %of transtransferred ferred ova Stage of No. of ova No. of control young in developing maturation fmm donor young in recipient recipient to term Experimentamental Experi- Trans- Control Stained ferred cornu cornu cornu Unripe" Total, 17 transfers (?2) Tetrads Total, 8 transfers Total, 11 transfers Total, 4 transfers Total, 40 transfers The first line represents a summation of 16 transfers; the remaining lines of the table represent individual transfers.

6 6 NOYES [Fertility & Sterility all ova in a given clutch may not be in the same phase of development. This scattering probably accounts for the one fertilized ovum in the unripe group, which most likely had actually begun to mature before its companions. Otherwise the data show clearly, more particularly after considerations to be discussed below, that follicular ova before the onset of maturation division, when transferred to inseminated recipients do not develop into young. The first polar body is formed, according to Blandau, four to six hours ~ c 111:1 ~a uaw Ii-I- ~~ ~t... _ ~i,o. og... Z> 1&1111 1)0 cr 1&1 0. ".. 11z.trads Ovulatian FIGURE 3. 5TAGE OF MATUQATION Development of transferred follicular ova in relation to their stage of maturation, derived from Table 1. after the onset of heat, i.e., six to four hours before ovulation. The data herein reported (Table 1) indicate that more ova are successfully fertilized at this first polar body phase (Figures 7 through 9) than before or after FIGURE 4. Unripe follicular ovum removed in early estrus. X 500 FIGURE 5. Tetrad formation, early, nucleus central, loss of nuclear membrane. X 500 FIGURE 6. Tetrads, late, peripheral migration of nucleus. X 500 FIGURE 7. First meiotic spindle. X 500 FIGURE 8. First polar body. X 500 FIGURE 9. Maturation complete, first polar body and second meiotic spindle. X 500

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8 8 NOYES [Fertility & Sterility this time. Since the age of the ova reported in the ovulated group was calculated to be not more than five hours post ovulation, these few results confirm the previously held impression that ova deteriorate rapidly after ovulation. It is interesting that though the number of ova transferred varied from six to ten, the maximum number of young obtained after transfer was four. Considering only the optimally developed group in Table 1, that is the first polar body group, inspection of columns 3, 4, and 5 indicates that more young are carried in the experimental than in the control cornu, and that transferred ova have at least as good a chance as the ovulated ova of going to term. Thus it seems unlikely that damage to the transferred ova is the limiting factor. Nor does uterine capacity per se seem a likely limiting factor, since in superovulated animals more than double the average litter may be carried to term. The probable cause may be a relative progesterone deficiency, since with this technique there is always an unphysiologic preponderence of ova compared with corpora lutea. The question arises whether the unripe appearing follicular ova of the rat undergo maturation division in vitro as do those of man and the rabbit, and if so what factors prevent fertilization and/ or development to living young. Some preliminary investigations have been made by studying ova maintained in vitro, and some in vivo, for varying intervals. These results are not conclusive, but certain tendencies seem worth reporting. Unripe follicular ova kept at 37 C. for from 1 to 12 hours in saline, in half saline and half serum, in serum, or in serum mixtures containing penicillin underwent suggestive changes such as disintegration of the nuclear membrane and peripheral migration of the chromatin, but neither spindle nor polar body formation was noted. However, unripe follicular ova transferred into the bursas of recipients did mature as far as spindle formation (Fig. 10) though it is doubtful whether these represent arrested first spindles or second spindles, there being no normal appearing polar bodies evident. Undoubtedly the environment favorable to normal maturation is a rather critical factor in rat ova. Assuming that fertilization takes place only in the oviduct, the question of how long these ova remain in the bursa assumes importance. Premature fertilization may interfere with development, and on the other hand changes in the ovum taking place while it is lying in the bursa might- inhibit fertilization later. A few observations showed that

9 Vol. 3, No. 1, 1952] FERTILIZATION OF FOLLICULAR OVA 9 mature ova transferred to a recipient near the expected time of ovulation were found in the tubes within an hour, while a much smaller percentage FIGURE 10. Arrested spindle in transferred unripe ovum which was removed from recipient after 16 hours in vivo. X 500 FIGURE 11. Tetrads in transfen-ed unripe ovum which was removed from recipient after three hours in vivo. X 500 FIGURE 12. Same as Fig. 11, showing unripe granulosa. X 150 FIGURE 13. Same as Fig. 5, granulosa ripening. X of unripe ova had reached there at this time. In the post-ovulatory phase most of the unripe ova reached the oviduct rapidly, but in the preovulatory

10 10 NOYES [Fertility & Sterility phase only about one-third of ova were recovered from the oviduct, and the pick-up mechanism was slower. Analysis of the several factors involved here is difficult, and no conclusions may be drawn; but since the sixteen transfers of unripe ova recorded in Table 1 were performed in recipients throughout the entire range from before heat (in which case the recipient was mated postoperatively) to several hours post-ovulation, it seems justifiable to feel that at least some of these ova were present in an inseminated oviduct at all representative phases in their development-yet none was successfully fertilized. An observation that may be the key to this failure of fertilization is that the granulosa, which swells and becomes viscid in the maturing follicle undergoes no such change in vitro or in vivo when transferred to the ovarian bursa. Rather, the unripe granulosa cells seem to remain densely packed about the zona pellucida for several hours, then disintegrate leaving a nude ovum, irrespective of the presence or absence of sperm. For example, the ovum shown in Fig. 11 was one of a group of unripe follicular ova transferred to a recipient and then removed three hours later. The nucleus has undergone early maturation as shown by loss of nuclear membrane, tetrad formation, and migration of the nucleus peripherally, and one would expect about 22% of such ova to become fertilized. However, the granulosa remains dense and clumped; and, as has been shown, fertilization does not occur. Contrast this with Fig. 5, which is a follicular ovum at the same stage of nuclear maturation but which has a loose homogeneous granulosa. The difference in granulosa is more clearly shown in the low power photomicrographs of these same two ova, Figures 12 and 13 respectively. It is surmised that the much smaller volume of the unripe ovum mass and possibly the higher ph of the granulosa (roughly estimated here by use of the indicator dye bromothymol blue applied supra vitally) may be the important factors in decreased tubal pick up, and the increased density and ph of the granulosa may also prevent successful fertilization. Another possibility is that the unripe granulosa has disintegrated before the ovum has matured to a fertile state and that the sperm cannot penetrate a bald zona pellucida (Fig. 10). The young resulting from fertilized follicular ova are apparently normal in all respects, in fact in these experiments the transferred young demonstrated hybrid vigor by outgrowing their control litter mates (Fig. 2). Of 27 young developing from transferred ova in which the sex was noted, 15 were males and 12 were females.

11 Vol. 3, No. 1, 1952] FERTILIZATION OF FOLLICULAR OVA 11 DISCUSSION These experiments indicate that follicular ova which have matured to the earliest phase of meiosis are functionally intact, and that normal young result from their fertilization in vivo. Contrariwise, unripe follicular ova do not develop into young when transferred to a fertile recipient, though whether immaturity of the ovum or of the granulosa is at fault is uncertain. The fertile life span of the ovum is shown to be independent of ovulation, the total period of fertility extending from not more than ten hours before to not more than ten hours after ovulation. 4 Thus in the rat the ovum is fertile for about 20 of the 96 hours of the cycle, or roughly 20% of the cycle. Finding human ova within this fertile period may be expected to be difficult because the time relationships of human ovulation are as yet not clearly established, and because only one human ovum undergoes maturation per cycle. In a planned search for maturing human follicular ova, Allen, Pratt, Newell and Bland ( 1930) found only one such ovum though approximately 200 patients were examined. It may be that unripe human follicular ova, unlike those of the rat, may, as Pincus and Saunders' experiments suggest, undergo normal maturation after removal from the follicle. Or it may be that careful priming of human ovaries with gonadotropins shortly before operation may yield fertile follicular ova. Ultimately it may be found possi- \ ble to perfuse whole human ovaries in vitro and control growth and maturation of ova within the follicles. Given ova of known fertility, fascinating problems involving sperm, fertilization, growth and implantation remain before pregnancy can be successfully induced by transferring human ova. SUMMARY AND CONCLUSION Ovarian follicular ova of the rat when transferred to the ovarian bursa of mated recipients are fertilized and grow to become normal animals genetically the product of their parents. Maturation of these ova to the first stages of meiotic division is essential to fertilization and growth, and further maturation greatly enhances their fertility. No normal maturation of ova, and no granulosa! ripening was noted in small numbers of unripe ova maintained for varying periods of time in vivo or in vitro. Consistently successful results in transfer of unfertilized ova will depend either upon more accurate knowledge of the time of ovulation than is now known for most species, or

12 12 NOYES [Fertility & Sterility upon development of a method for the artificial maturation of ova in vivo or in vitro. REFERENCES I. Alden, R. H.: Anat. Rec. 83:421, Allen, E., Pratt, J.P., Newell, Q. U., and Bland, L. J.: Am. J. Anat. 46:1, Blandau, R. J.: Anat. Rec. 92:449, Blandau, R. J., and Jordan, E. S.: Am. J. Anat. 68:275, Boling, J. L., Blandau, R. J., Soderwall, A. L., and Young, W. C.: Anat. Rec. 79:313, Chang, M. C.: J. Exper. Zool. 114:197, Corner, G. W., Farris, E. J., and Corner, G. W., Jr.: Am. J. Obst. & Gynec. 59:514, Decker, A.: Personal communication. 9. Hammond, J.: J. Exper. Bioi. 11:140, Heape, W.: Proc. Roy. Soc. 48:457, Menkin, M. F., and Rock, J.: Am. J. Obst. & Gynec. 55:440, Pincus, G., and Enzmann, E. V.: J. Exper. Med. 62:665, Pincus, G.: J. Exper. Zool. 82:85, Pincus, G., and Saunders, B.: Anat. Rec. 75:537, Umbaugh, R.: Fertil. & Steril. 2:243, 1950.