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Ovarian Transplants to the Anterior Chamber of the Eye
Ovarian Transplants to the Anterior Chamber of the Eye Robert W. Noyes, M.D., * Aileen M. Yamate, A. B., and Thomas H. Clewe, M.D.t
undergo rapid maturational processes shortly before ovulation, and unless they are quickly fertilized, they degenerate soon thereafter. Many basic experiments in the field of mammalian reproductive physiology, such as, for example, fertilization of ova in vitro, suffer from the unavailability of fresh, fertile ova. Ova may be obtained either from the ovarian follicles, or from the fallopian tube, but neither of these sources is entirely satisfactory. The exact time of ovulation cannot be determined for most species, so that neither freshly ovulated nor ripe follicular ova are readily available. Follicular ova of mts 4 and rabbits 2 attain normal fertility a few hours before they are ovulated, and they probably retain their fertility for a time within the follicle if ovulation fails to occur. At least these retained ova appear to remain intact morphologically, though their actual fertility has not yet been tested. If a method could be developed to de-
MAMMALIAN OVA
From the Department of Obstetrics and Gynecology, Stanford University School of Medicine, San Francisco, Calif. Presented at the Thirteenth Annual Meeting of the American Society for the Study of Sterility, New York, N. Y., May 31-June 2, 1957. * Markle Fellow in Medical Science. t Present address: Department of Anatomy, Yale University School of Medicine, New Haven, Conn. This work was supported in part by a research grant from the Research Grants Division (RG4470C) of the National Institutes of Health, Public Health Service. The pregnant mare's serum gonadotrophin (Equinex) was graciously supplied by Dr. John B. Jewell, of Ayerst Laboratories. The chorionic gonadotrophin was kindly given by Dr. Edward Reifenstein, Jr., of E. R. Squibb and Sons (Follutein), and by Dr. R. W. Talley, of the Upjohn Company. Received for publication June 7, 1957. 99
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termine the maturity of follicular ova without damaging them, the problem of securing experimental ova would be solved. Follicles are maturing and undergoing atresia concomitantly in the mammalian ovary, and there is no way to distinguish a growing from an atretic follicle by inspecting the ovary at any particular time of the cycle. In order to correlate the growth rate of a follicle with the maturity of its contained ovum, it is essential that the follicle be observed continuously. Attempts to do this in tissue culture have failed, but in the anterior chamber of the eye, follicular growth, and occasionally ovulation, can be observed with ease. In a previous publication, 3 the technics for transplanting the ovarian fragments, for observing the follicles, and for testing the fertility of the follicular ova were given, and the literature was briefly reviewed. This report amplifies our previous qualitative observations by adding quantitative data on the relationship of follicular growth to the maturation and fertility of ova. Since preliminary experiments indicated good results with intraspecies transplants between genetically distinct strains, and since only this kind of transplant would give the freedom of experimental design ultimately desired, only the transfer of ovaries from a black strain into the eyes of an albino strain of rats was studied in detail.
MORPHOLOGIC OBSERVATIONS In a typical experiment, half an ovary from a 25-day-old black donor rat is inserted into the eye of a 25-day-old white male recipient rat. A section through such an ovary is shown in Fig. 1, and a more magnified view of a typical follicle in the stage of early antrum formation is shown in Fig. 2. After four days in the eye, all of the original antrum-containing follicles have degenerated, and the few surviving primordial follicles are located along the interface between the ovary and the iris (Fig. 3). The earliest of the new antrum-containing follicles appear on the fourth day (Fig. 4). At this time the recipient animal is castrated so that the effect of both intrinsic and extrinsic gonadotrophic hormones will be localized to the transplant rather than to the recipient gonad. On the fourth day, 15 international units of pregnant mare's serum gonadotrophin, a hormone in which follicle stimulating activity predominates, is injected subcutaneously into the recipient rat. Fifty-six hours later the follicles have enlarged greatly (Fig. 5) and the
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Fig. 1. Ovary from a 25-day-old rat. (x 20) Fig. 2. An early antrum follicle from Fig. 1. Volume 65 x 106 cubic p.. (X 90) Fig. 3. Half an ovary from a 25-day-old donor rat, 4 days after transfer to the anterior chamber of the eye of a 25day-old recipient rat of a different strain. (x 20) Fig. 4. An early antrum follicle from Fig. 3. Vol. = 4,6 x 106 cu, p.' (x 90) Fig. 5. Rat ovarian transplant 56 hours after injection of the recipient rat with 15 I.U. pregnant mare's serum gonadotrophin. (X 20) Fig. 6. Developing follicle from Fig. 5. Vol. = 1l 0 x 106 cu. p.. The ovum is unusual in that it has formed the first meiotic spindle. (x 90)
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Fig. 7. A developing follicle from an ovarian transplant 70 hours after injecting the recipient with PMS and 14 hours following the injection of 30 international units of human chorionic gonadotrophin. Volume = 90 x 106 cubic p.. The opening in the follicle
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OVARIAN TRANSPLANTS
103
ova have begun to separate from the mural granulosa. The nuclei in most of the ova remain in the immature or germinal vesicle stage (see Fig. 2) although occasional ova will form the first meiotic spindle (Fig. 6). Twentyfive international units of human chorionic gonadotrophin, a hormone in which luteinizing activity predominates, are then injected. These two hormones, hereafter referred to as PMS and CG, are given in order to duplicate Rowlands's5 results in superovulating immature rats. The time schedule was kept constant throughout the experiment, and although the dosage was varied somewhat, the doses given above seemed to give the best results. Fourteen hours following the injection of CG, the follicles have enlarged still further, and an occasional follicle ovulates (Fig. 7). Ova in the maturing follicles have completed meiosis and have separated completely from the mural granulosa, although mitotic activity in the cumulus cells is still abundant (Fig. 8). Corpora lutea have formed 22 hours after the administration of CG (Fig. 9), and ova that have not been ovulated are found compressed among the lutein cells (Fig. 10). The relationship of the transplant to the iris and to the cornea is shown in Fig. 9. In this particular section the connection between the iris and the ovary is narrow, but more frequently the iris is attached to a wide area of the transplant. Figure 11 is a photograph of a transplant taken through the cornea at the same magnification that was routinely used with the dissecting microscope. A freshly ovulated ovum, still surrounded by cumulus oophorus, is visible. Figure 12 shows a follicular ovum that has been stained in toto under a coverslip. This technic permits a rapid, accurate assessment of the state of maturity of the ovum nucleus and the cumulus cells. In this case, the crescentic first polar body is being abstricted from the horseshoe shaped telophase spindle. is possibly the point of ovulation. (x 90) Fig. 8. An ovum 14 hours following CG injection, showing the first polar body. Mitosis of a cumulus cell can be seen to the upper right. (X 370) Fig. 9. An ovarian transplant 78 hours following PMS and 22 hours following CG, showing early corpora lutea. The cornea is on the left and the attachment of the iris to the graft is shown to the right. (X 20) Fig. 10. An early corpus luteum from Fig. 9, showing a squeezed atretic ovum to the right. (X 90) Fig. 11. An ovarian transplant as seen through the dissecting microscope, 14 hours following CG, and showing, to the left, an ovulated ovum in front of the iris. (X 20) Fig. 12. A follicular rat ovum that has been fixed and stained in toto under a coverslip. The first polar body is being abstricted from the horseshoe-shaped late telophase spindle. (X 1300)
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RESULTS OF INTRAOCULAR TRANSFERS The data given in the subsequent paragraphs are based on 1115 intraspecies ovarian transfers to the anterior chamber of the eye of the rat, all having been performed subsequent to our previous publication. 3 Usually both eyes of the recipient were used. Technical failure, such as death of the recipient animal, or extrusion of the graft, occurred 19 times. There were 163 transplants (15 per cent) in which the grafts did not "take," i.e., the graft was not vascularized and rapidly degenerated. In 463 ovarian transplants, the development of follicles was observed from day to day, and the ova were then either removed from the follicles and stained in toto, or else the entire transplant was studied in serial sections. In the remaining 470 transplants, the fertility of the ova was tested, as outlined below. Ovulation occurred only 14 times, a lower incidence than was noted in our previous experience. Not all of the transplants were observed long enough to include the usual ovulation period, 12-18 hours after CG. Undoubtedly, several ovulated ova were overlooked during examination of the eye. The fact remains, however, that under the conditions of these experiments, ovulation in the eye is a rare phenomenon. On two occasions, 2 ova were ovulated from a single graft. THE RATE OF FORMATION OF MATURING FOLLICLES Daily sketches were made of 36 ovarian transplants between the fourth and seventh days following transplantation. The host animals to 30 of these transplants were subjected to PMS and CG injections, and 6 were untreated controls. The appearance rate of the follicles growing in treated animals did not differ from the controls. This suggests that the intrinsic pituitary gonadotrophin level of the castrate immature male recipient is high enough to stimulate all follicles that are mature enough to respond. Sixty-five follicles appeared (diameter 0.25 mm.) in the 36 transplants on the fourth, 60 on the fifth, 32 on the sixth, and 28 on the seventh postoperative day. The number of follicles that appear each day diminishes rapidly after the first 2 days, and this cannot be prevented by supplementary gonadotrophin injections. These direct observations are in agreement with the theory that the original stimulus for follicular maturation is independent of gonadotrophic hormone stimulus. In order to compare the performance of transplants with that of ovaries
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105
OVARIAN TRANSPLANTS
Vol. 9, No.2, 1958
in situ, 25-day-old female rats were injected with gonadotrophin on the same schedule that has been outlined above. Serial sections of these normal control ovaries showed that the average number of follicles that "appear" each day is five times that observed in the transplants. Probably ,the smaller original size of the transplant, plus the massive follicular degeneration that occurs before its new blood supply develops is sufficient to account for this difference. It is not likely that antigenic influences would be manifest so soon after transplantation. VOLUME CHANGES IN MATURING FOLLICLES The volume of follicles was estimated by measuring the diameters of each follicle in a given transplant each day. In the living follicles the I,oOOXIO
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DAV AFTER TRANSPLANTATION TO EYE Fig. 13. Growth of ovarian follicles in the anterior chamber of the eye as affected by pregnant mare's serum and human chorionic gonadotrophin injections. Each point represents the average volume of all of the grossly visible follicles in many transplants. The measurements were made on living follicles. The points that pertain to a group of follicles that first appeared on the same day are connected by lines. The time of injection and dosage of PMS and CG are given in the text.
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diameter was determined with an error not greater than 0.25 mm. by using an eyepiece micrometer, and the volume was calculated using the formula V = 1/6 7T d 3 • The volumes of 157 follicles in 36 transplants were recorded daily from the fourth through the seventh postoperative days. In 24 of the transplants, both PMS and CG were given to the recipient animal; in 6, only PMS was given; and in 6 no gonadotrophins were given. The average volume for each group of follicles that appeared on a given day, in each of the three groups of animals was plotted against time (Fig. 13). The average volume of 65 follicles that appeared on the fourth day, was 40 x 1()6 cubic JL, and one day later, following castration and PMS administration, the average volume had increased to 165 x 106 cubic JL. When the recipients were castrated, but no PMS was given, the average volume was slightly less, 120 x lO6 cubic JL, and although they continued to grow, the follicles in uninjected animals grew more slowly than those in the treated animals. When CG is given there is further increase in the follicular growth rate. The curve of growth is very similar for each group of follicles irrespective of the day on which they appeared. In the group of animals treated with PMS, the average volume of follicles that first appeared on the sixth day is much larger than that in the preceding groups. This artifact is caused by the sudden emergence into view of older follicles that had been growing deeply on the interface between the iris and the ovary. Although they appear on the sixth day many of these follicles are actually older, and thus larger, than the more superficial follicles. These observations were not continued long enough to include the declining growth of follicles as they become atretic, or the curves for those that formed corpora lutea. Similar data were plotted from the average volumes of the larger follicles in the serially sectioned transplants (Fig. 14). Here two diameters were measured at right angles to each other with an eyepiece micrometer, and the third diameter was obtained by counting the total number of sections in which the follicle appeared. Again, three groups of transplants were studied (castration only, PMS only, and PMS and CG), and a parallel series of three groups of normal immature rat ovaries in situ was studied for comparison. The average follicular volume in the normal untreated ovary does not increase between the twenty-ninth and the thirty-second day of age, but there is a steady rise in the follicular volume each day the ovarian transplant remains in the eye of the castrate male recipient. None of the follicles
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OVARIAN TRANSPLANTS
Vol. 9, No.2, 1958 6
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Fig. 14. The effect of gonadotrophin injections on the volumes of ovarian follicles in eye transplants and in normal immature ovaries in situ. Each point represents the average volume of many follicles from different ovaries. The measurements were made on fixed, sectioned tissue. The time of injection and dosage of PMS and CG are given in the text.
in untreated animals matures completely, although there is a tendency for ova in some atretic follicles to undergo early meiotic nuclear activity. The volume of both in situ and transplanted ovaries increases rapidly follOWing PMS treatment, and injection of CG causes still further growth after a short lag period. In the in situ ovaries, ovulation and regression follow the final dramatic growth spurt. Follicles in the eye transplants grow more slowly, and do not attain the large preovulatory volumes that follicles in normal ovaries do. However, the rate of nuclear maturation of the ova appears to be the same in the eye as in the normal ovary, and follows exactly the schedule outlined in the paragraph on morphology. The curve of follicular growth obtained in the normal ovary of adult
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female rats by Boling et aZ. 1 is very similar to the curve for superovulated ovaries in situ, although the time sequences cannot be directly compared. No doubt the lack of a final growth spurt in transplanted ovarian follicles is related in some way to their low ovulation rate. At these early stages there is no evidence that intraocular pressure is increased, or that moderate increases in extrinsic pressure would interfere with follicular growth or ovulation. In the sectioned transplants blood vessels are smaller and less numerous than in the normal immature ovary. Perhaps the failure of preovulatory growth and ov'ulation can be explained on the basis of inadequate blood supply. Shrinkage resulting from fixation of the tissue accounts for the smaller over-all follicular volumes in the sectioned material compared with the living transplants, but other than this, the data from the two series are quite comparable. More than 100 ova were stained in toto to correlate the growth rate of the follicle with the maturation of the contained ovum. The results were exactly the same as those for the serially sectioned ova. In each group of ova recovered from ovarian transplants, however, a few immature vesicular ova from the smaller follicles of an earlier generation were seen. Although these unripe ova were obviously unlike the maturing ova when they were fixed and stained, they were not easy to distinguish in the living state. THE FERTILITY OF FOLLICULAR OVA FROM OVARIAN TRANSPLANTS At various stages of development the ovarian transplants were removed from the eyes of the recipient animals, and the larger follicles were opened under saline. The cumulus encased ova were then extruded, counted, and drawn into a small pipet. From 470 transplants, 1154 ova were obtained, an average of 2.5 ova per transplant. More than twice this number of large follicles were counted under the dissecting microscope and in the sectioned material, so it is obvious that our recovery technic was imperfect. Approximately 6 follicular ova were pipetted into each of 184 ovarian bursas of previously mated albino recipient animals. The method for injecting ova is illustrated in Fig. 15. The ova are picked up by the oviduct within an hour and are fertilized at about the same time as the ovulated ova of the recipient. If the follicular ova are fertile, they develop into embryos with black iris pigment, which readily distinguishes them from the control embryos at the time of laparotomy 18 days after transfer.
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Vol. 9, No.2, 1958
Fig. 15.
OVARIAN TRANSPLANTS
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Method for transferring follicular ova into the ovarian bursa. The tip of the pipet (see arrow) is visible behind the bursal membrane.
In a previous experiment, l when 130 developing follicular ova were removed from the ovaries of normal adult animals six hours or less before the expected time of ovulation, and were then transferred into the bursas of 19 recipient animals, 44 (34 per cent) of the ova were fertilized and developed to term embryos. Our present experience with the fertility of follicular ova obtained from eye transplants is not nearly this encouraging (Table 1). When 809 maturing ova from eye transplants were transferred to 131 bursas, only 36 (4.5 per cent) developed into term embryos. The optimal stage of follicular development was between the fourteenth and sixteenth hours following the administration of CG (Fig. 16), but even at this time only 10 per cent of transferred ova survived. The results of individual experiments were quite variable, and the apparent high fertility of ova occurring 24 hours after CG was probably a chance occurrence. This was a very rigorous test for fertility, with many chances for ova to be lost and for inadequate conditions for fertilization to be present. However, the
110
Fertility & Sterility
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Fig. 16. The fertility of follicular rat ova taken from ovaries transplanted to the eyes of recipient rats of a different strain. The data are from Table l. The figures within the circles are the numbers of ova in each transfer in which one or more of the transferred ova developed to term. The figures in the squares represent the number of all ova transferred into recipient bursas at a given hour following chorionic gonadotrophin injection.
conditions in these eye transplant experiments were similar to those with normal follicular ova, yet only one-tenth as many of the ova from the eye transplants were fertile as compared with the preovulatory ova from ovarian follicles in situ.
INTERSPECIES OVARIAN TRANSPLANTS Fragments of immature rabbit ovaries were transplanted into the eyes of immature rats, and of 90 such transplants, 74 established a new blood supply. In all 16 failures, the donor rabbit was 59 days of age, while the donors in the successful takes were 24 days old. The recipient rat was not treated at all in 8 transplants, and received only PMS in 16 transplants. Various treatments were then tried in an attempt to diminish the recipient's immune reaction. In 8 transplants the recipient received 250r of total body x-irradiation two days before the ovarian transplantation, and then PMS was given. In another 8 rabbit ovarian transplants the recipient rat was injected with 1.4 mg. of cortisone 2 days prior
TABLE 1.
Follicular Ova Transferred into the Ovarian Bursas of Recipient Host Rats No. hrs. elapsed after chorionic gonadotrophin injection
10
6 Transfers in which follicular ova survived to term in the eXpel"imental uterine horn Transfers Ova Ova Surviving Transfers in which only control embryos survived in the experi. mental uterine horn Transfers Ova Subtotals Transfers in which the recipient was pregnant. but no embryo survived in the experimental uterine horn Transfers Ova Transfers in which the recipient failed to continue pregnancy Transfers Ova Totals Transfers Ova Ova surviving
2
0
65
36 9
42
10
65
47
67
8
32
46
6 13
21 0
65
24
5
6
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0
10
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0
7
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2
9
25
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9 35
1
10
7
1
17
19
15
2
0
0
6
0
5
809
133
207
22
4
3 0
632
29
0
138 184
19
61
16
79 3
3
0
2
56
255
0
0
0
7
17
59
16
105
0 19
59
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0
0
3 44 2
72
51
6
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116 7
221
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22
30
6
177 36
3
3
60
43
28 15
0
16
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5 185
4
20
31
95
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20
9 21
4
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105
153
112
24 11
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Totals
24
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8
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2 34
0
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23 21
86
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42
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5
19
36
21
22
21
2
6
2
8 54
52
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6
7
7
14
6
10
8
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20
19
6 16
31
32
4
0
2
6
5 15
0
6
18
17
16
15
14
12
1154
26 3
36
112
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Fertility & Sterility
to the transplantation, and then PMS was given in the same dose and schedule as used previously. Both x-ray and cortisone were given to the recipients of 16 transplants. Twenty-six rat embryos were injected on the fifteenth day of pregnancy with a suspension of rabbit white blood cells, the purpose being to inhibit the future development of rabbit antibodies. The survivors of this treatment who lived to the age of 25 days were used as hosts for 14 rabbit ovarian transplants. The results were identical following each of these treatment schemes. The transplants attained a new blood supply, and some grew slightly, but no normal ova were recovered. Twenty-six immature rat ovaries were transferred to as many rabbits' eyes, but all failed to «take." Thirty-four pieces of adult human ovaries were transferred to the eyes of rats, 10 to the eyes of guinea pigs, and 6 to rabbits' eyes. Despite various combinations of x-ray, cortisone, and PMS given to the recipient animals, none of the human ovarian grafts became vascularized. SUMMARY AND CONCLUSIONS
Transfers of immature rat ovarian tissue to the anterior chamber of the eyes of immature male recipient rats of a different strain produced vascularized, growing grafts in about 85 per cent of trials. Most of the antrumcontaining follicles of these grafts degenerate, but new ones form from surviving primordial follicles within 4 days following transplantation. One to 4 new follicles begin to grow each day, and an average of 6 mature within 4 days. The number of follicles that appear is independent of extrinsic gonadotrophin injections, but is dependent on the intrinsic rise of gonadotrophin level following castration of the host animal. When treated with pregnant mare's serum gonadotrophin, the follicles increase in volume at about the same rate that has been reported for follicles in the mature ovary in situ, while in untreated grafts, follicular growth lags. Following the injection of human chorionic gonadotrophin, the follicles in transplants do not grow so rapidly or become so large as those developing in ovaries in situ, and ovulation is rare. However, meiotic changes in the ovum's nucleus, and cumulus maturation of these ova, seem to progress at the normal rate despite their smaller volume. Only 4.5 per cent of ova removed from follicles that seemed to be maturing proved to be fertile. This is only one-tenth the fertility rate expected from previous experiments on follicular ova obtained from mature ovaries in situ.
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OVARIAN TRANSPLANTS
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Interspecies transplants of ovarian tissue from rabbit to rat, from rat to rabbit, and from human to rat, rabbit, or guinea pig, all failed to produce normal ova despite extensive and varied treatments aimed at reducing the antigenic response of the host. These experiments have been of great interest insofar as the study of follicular growth is concerned, but they have not resulted in the discovery of an ideal source for experimental ova. From an immunologic point of view it is interesting that in these acute experiments, ova can be brought to maturity despite the fact that these intraspecies grafts invariably degenerate a short time later. It is felt that inadequate blood supply, rather than antigenicity, may be the cause for the failure of rapid growth following the injection of chorionic gonadotrophin. Slow growth may in turn be responsible for the failure of ovulation, and still more remotely may decrease the fertility of these ova. Further advances in solving this problem will depend on better immunologic control in the recipient host, so that transplants may grow long enough to attain a normal blood supply before ovum maturation is attempted. Stanford Univ. School of Medicine Clay and Webster Sts. San Francisco, Calif.
REFERENCES 1. BOLING, J. L., et al. Growth of the graffian follicle and the time of ovulation in the albino rat. Anat. Rec. 79:313, 1941. 2. CHANG, M. C. Fertilization and normal development of follicular oocytes in the rabbit. Science 121 :867, 1955. 3. CLEWE, T. H., YAMATE, A. M., and NOYES, R. W. Maturation of ova in mammalian ovaries in the anterior chamber of the eye. Proceedings of the Second World Congress on Fertility and Sterility, Naples, Italy, 1956. To be published. 4. NoYES, R. W. Fertilization of follicular ova. Fertil. & Steril. 3:1, 1952. 5. ROWLANDS, I. W. The production of ovulation in the immature rat. ]. Endocrinol. 3:384, 1944.
undergo rapid maturational processes shortly before ovulation, and unless they are quickly fertilized, they degenerate soon thereafter. Many basic experiments in the field of mammalian reproductive physiology, such as, for example, fertilization of ova in vitro, suffer from the unavailability of fresh, fertile ova. Ova may be obtained either from the ovarian follicles, or from the fallopian tube, but neither of these sources is entirely satisfactory. The exact time of ovulation cannot be determined for most species, so that neither freshly ovulated nor ripe follicular ova are readily available. Follicular ova of mts 4 and rabbits 2 attain normal fertility a few hours before they are ovulated, and they probably retain their fertility for a time within the follicle if ovulation fails to occur. At least these retained ova appear to remain intact morphologically, though their actual fertility has not yet been tested. If a method could be developed to de-
MAMMALIAN OVA
From the Department of Obstetrics and Gynecology, Stanford University School of Medicine, San Francisco, Calif. Presented at the Thirteenth Annual Meeting of the American Society for the Study of Sterility, New York, N. Y., May 31-June 2, 1957. * Markle Fellow in Medical Science. t Present address: Department of Anatomy, Yale University School of Medicine, New Haven, Conn. This work was supported in part by a research grant from the Research Grants Division (RG4470C) of the National Institutes of Health, Public Health Service. The pregnant mare's serum gonadotrophin (Equinex) was graciously supplied by Dr. John B. Jewell, of Ayerst Laboratories. The chorionic gonadotrophin was kindly given by Dr. Edward Reifenstein, Jr., of E. R. Squibb and Sons (Follutein), and by Dr. R. W. Talley, of the Upjohn Company. Received for publication June 7, 1957. 99
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termine the maturity of follicular ova without damaging them, the problem of securing experimental ova would be solved. Follicles are maturing and undergoing atresia concomitantly in the mammalian ovary, and there is no way to distinguish a growing from an atretic follicle by inspecting the ovary at any particular time of the cycle. In order to correlate the growth rate of a follicle with the maturity of its contained ovum, it is essential that the follicle be observed continuously. Attempts to do this in tissue culture have failed, but in the anterior chamber of the eye, follicular growth, and occasionally ovulation, can be observed with ease. In a previous publication, 3 the technics for transplanting the ovarian fragments, for observing the follicles, and for testing the fertility of the follicular ova were given, and the literature was briefly reviewed. This report amplifies our previous qualitative observations by adding quantitative data on the relationship of follicular growth to the maturation and fertility of ova. Since preliminary experiments indicated good results with intraspecies transplants between genetically distinct strains, and since only this kind of transplant would give the freedom of experimental design ultimately desired, only the transfer of ovaries from a black strain into the eyes of an albino strain of rats was studied in detail.
MORPHOLOGIC OBSERVATIONS In a typical experiment, half an ovary from a 25-day-old black donor rat is inserted into the eye of a 25-day-old white male recipient rat. A section through such an ovary is shown in Fig. 1, and a more magnified view of a typical follicle in the stage of early antrum formation is shown in Fig. 2. After four days in the eye, all of the original antrum-containing follicles have degenerated, and the few surviving primordial follicles are located along the interface between the ovary and the iris (Fig. 3). The earliest of the new antrum-containing follicles appear on the fourth day (Fig. 4). At this time the recipient animal is castrated so that the effect of both intrinsic and extrinsic gonadotrophic hormones will be localized to the transplant rather than to the recipient gonad. On the fourth day, 15 international units of pregnant mare's serum gonadotrophin, a hormone in which follicle stimulating activity predominates, is injected subcutaneously into the recipient rat. Fifty-six hours later the follicles have enlarged greatly (Fig. 5) and the
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Fig. 1. Ovary from a 25-day-old rat. (x 20) Fig. 2. An early antrum follicle from Fig. 1. Volume 65 x 106 cubic p.. (X 90) Fig. 3. Half an ovary from a 25-day-old donor rat, 4 days after transfer to the anterior chamber of the eye of a 25day-old recipient rat of a different strain. (x 20) Fig. 4. An early antrum follicle from Fig. 3. Vol. = 4,6 x 106 cu, p.' (x 90) Fig. 5. Rat ovarian transplant 56 hours after injection of the recipient rat with 15 I.U. pregnant mare's serum gonadotrophin. (X 20) Fig. 6. Developing follicle from Fig. 5. Vol. = 1l 0 x 106 cu. p.. The ovum is unusual in that it has formed the first meiotic spindle. (x 90)
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Fig. 7. A developing follicle from an ovarian transplant 70 hours after injecting the recipient with PMS and 14 hours following the injection of 30 international units of human chorionic gonadotrophin. Volume = 90 x 106 cubic p.. The opening in the follicle
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.
Vol. 9, No.2, 1958
OVARIAN TRANSPLANTS
103
ova have begun to separate from the mural granulosa. The nuclei in most of the ova remain in the immature or germinal vesicle stage (see Fig. 2) although occasional ova will form the first meiotic spindle (Fig. 6). Twentyfive international units of human chorionic gonadotrophin, a hormone in which luteinizing activity predominates, are then injected. These two hormones, hereafter referred to as PMS and CG, are given in order to duplicate Rowlands's5 results in superovulating immature rats. The time schedule was kept constant throughout the experiment, and although the dosage was varied somewhat, the doses given above seemed to give the best results. Fourteen hours following the injection of CG, the follicles have enlarged still further, and an occasional follicle ovulates (Fig. 7). Ova in the maturing follicles have completed meiosis and have separated completely from the mural granulosa, although mitotic activity in the cumulus cells is still abundant (Fig. 8). Corpora lutea have formed 22 hours after the administration of CG (Fig. 9), and ova that have not been ovulated are found compressed among the lutein cells (Fig. 10). The relationship of the transplant to the iris and to the cornea is shown in Fig. 9. In this particular section the connection between the iris and the ovary is narrow, but more frequently the iris is attached to a wide area of the transplant. Figure 11 is a photograph of a transplant taken through the cornea at the same magnification that was routinely used with the dissecting microscope. A freshly ovulated ovum, still surrounded by cumulus oophorus, is visible. Figure 12 shows a follicular ovum that has been stained in toto under a coverslip. This technic permits a rapid, accurate assessment of the state of maturity of the ovum nucleus and the cumulus cells. In this case, the crescentic first polar body is being abstricted from the horseshoe shaped telophase spindle. is possibly the point of ovulation. (x 90) Fig. 8. An ovum 14 hours following CG injection, showing the first polar body. Mitosis of a cumulus cell can be seen to the upper right. (X 370) Fig. 9. An ovarian transplant 78 hours following PMS and 22 hours following CG, showing early corpora lutea. The cornea is on the left and the attachment of the iris to the graft is shown to the right. (X 20) Fig. 10. An early corpus luteum from Fig. 9, showing a squeezed atretic ovum to the right. (X 90) Fig. 11. An ovarian transplant as seen through the dissecting microscope, 14 hours following CG, and showing, to the left, an ovulated ovum in front of the iris. (X 20) Fig. 12. A follicular rat ovum that has been fixed and stained in toto under a coverslip. The first polar body is being abstricted from the horseshoe-shaped late telophase spindle. (X 1300)
104
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RESULTS OF INTRAOCULAR TRANSFERS The data given in the subsequent paragraphs are based on 1115 intraspecies ovarian transfers to the anterior chamber of the eye of the rat, all having been performed subsequent to our previous publication. 3 Usually both eyes of the recipient were used. Technical failure, such as death of the recipient animal, or extrusion of the graft, occurred 19 times. There were 163 transplants (15 per cent) in which the grafts did not "take," i.e., the graft was not vascularized and rapidly degenerated. In 463 ovarian transplants, the development of follicles was observed from day to day, and the ova were then either removed from the follicles and stained in toto, or else the entire transplant was studied in serial sections. In the remaining 470 transplants, the fertility of the ova was tested, as outlined below. Ovulation occurred only 14 times, a lower incidence than was noted in our previous experience. Not all of the transplants were observed long enough to include the usual ovulation period, 12-18 hours after CG. Undoubtedly, several ovulated ova were overlooked during examination of the eye. The fact remains, however, that under the conditions of these experiments, ovulation in the eye is a rare phenomenon. On two occasions, 2 ova were ovulated from a single graft. THE RATE OF FORMATION OF MATURING FOLLICLES Daily sketches were made of 36 ovarian transplants between the fourth and seventh days following transplantation. The host animals to 30 of these transplants were subjected to PMS and CG injections, and 6 were untreated controls. The appearance rate of the follicles growing in treated animals did not differ from the controls. This suggests that the intrinsic pituitary gonadotrophin level of the castrate immature male recipient is high enough to stimulate all follicles that are mature enough to respond. Sixty-five follicles appeared (diameter 0.25 mm.) in the 36 transplants on the fourth, 60 on the fifth, 32 on the sixth, and 28 on the seventh postoperative day. The number of follicles that appear each day diminishes rapidly after the first 2 days, and this cannot be prevented by supplementary gonadotrophin injections. These direct observations are in agreement with the theory that the original stimulus for follicular maturation is independent of gonadotrophic hormone stimulus. In order to compare the performance of transplants with that of ovaries
.
•.
105
OVARIAN TRANSPLANTS
Vol. 9, No.2, 1958
in situ, 25-day-old female rats were injected with gonadotrophin on the same schedule that has been outlined above. Serial sections of these normal control ovaries showed that the average number of follicles that "appear" each day is five times that observed in the transplants. Probably ,the smaller original size of the transplant, plus the massive follicular degeneration that occurs before its new blood supply develops is sufficient to account for this difference. It is not likely that antigenic influences would be manifest so soon after transplantation. VOLUME CHANGES IN MATURING FOLLICLES The volume of follicles was estimated by measuring the diameters of each follicle in a given transplant each day. In the living follicles the I,oOOXIO
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DAV AFTER TRANSPLANTATION TO EYE Fig. 13. Growth of ovarian follicles in the anterior chamber of the eye as affected by pregnant mare's serum and human chorionic gonadotrophin injections. Each point represents the average volume of all of the grossly visible follicles in many transplants. The measurements were made on living follicles. The points that pertain to a group of follicles that first appeared on the same day are connected by lines. The time of injection and dosage of PMS and CG are given in the text.
106
NOYES ET AL.
Fertility & Sterility
diameter was determined with an error not greater than 0.25 mm. by using an eyepiece micrometer, and the volume was calculated using the formula V = 1/6 7T d 3 • The volumes of 157 follicles in 36 transplants were recorded daily from the fourth through the seventh postoperative days. In 24 of the transplants, both PMS and CG were given to the recipient animal; in 6, only PMS was given; and in 6 no gonadotrophins were given. The average volume for each group of follicles that appeared on a given day, in each of the three groups of animals was plotted against time (Fig. 13). The average volume of 65 follicles that appeared on the fourth day, was 40 x 1()6 cubic JL, and one day later, following castration and PMS administration, the average volume had increased to 165 x 106 cubic JL. When the recipients were castrated, but no PMS was given, the average volume was slightly less, 120 x lO6 cubic JL, and although they continued to grow, the follicles in uninjected animals grew more slowly than those in the treated animals. When CG is given there is further increase in the follicular growth rate. The curve of growth is very similar for each group of follicles irrespective of the day on which they appeared. In the group of animals treated with PMS, the average volume of follicles that first appeared on the sixth day is much larger than that in the preceding groups. This artifact is caused by the sudden emergence into view of older follicles that had been growing deeply on the interface between the iris and the ovary. Although they appear on the sixth day many of these follicles are actually older, and thus larger, than the more superficial follicles. These observations were not continued long enough to include the declining growth of follicles as they become atretic, or the curves for those that formed corpora lutea. Similar data were plotted from the average volumes of the larger follicles in the serially sectioned transplants (Fig. 14). Here two diameters were measured at right angles to each other with an eyepiece micrometer, and the third diameter was obtained by counting the total number of sections in which the follicle appeared. Again, three groups of transplants were studied (castration only, PMS only, and PMS and CG), and a parallel series of three groups of normal immature rat ovaries in situ was studied for comparison. The average follicular volume in the normal untreated ovary does not increase between the twenty-ninth and the thirty-second day of age, but there is a steady rise in the follicular volume each day the ovarian transplant remains in the eye of the castrate male recipient. None of the follicles
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107
OVARIAN TRANSPLANTS
Vol. 9, No.2, 1958 6
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Fig. 14. The effect of gonadotrophin injections on the volumes of ovarian follicles in eye transplants and in normal immature ovaries in situ. Each point represents the average volume of many follicles from different ovaries. The measurements were made on fixed, sectioned tissue. The time of injection and dosage of PMS and CG are given in the text.
in untreated animals matures completely, although there is a tendency for ova in some atretic follicles to undergo early meiotic nuclear activity. The volume of both in situ and transplanted ovaries increases rapidly follOWing PMS treatment, and injection of CG causes still further growth after a short lag period. In the in situ ovaries, ovulation and regression follow the final dramatic growth spurt. Follicles in the eye transplants grow more slowly, and do not attain the large preovulatory volumes that follicles in normal ovaries do. However, the rate of nuclear maturation of the ova appears to be the same in the eye as in the normal ovary, and follows exactly the schedule outlined in the paragraph on morphology. The curve of follicular growth obtained in the normal ovary of adult
lOB
NOYES ET At.
Fertility & Sterility
female rats by Boling et aZ. 1 is very similar to the curve for superovulated ovaries in situ, although the time sequences cannot be directly compared. No doubt the lack of a final growth spurt in transplanted ovarian follicles is related in some way to their low ovulation rate. At these early stages there is no evidence that intraocular pressure is increased, or that moderate increases in extrinsic pressure would interfere with follicular growth or ovulation. In the sectioned transplants blood vessels are smaller and less numerous than in the normal immature ovary. Perhaps the failure of preovulatory growth and ov'ulation can be explained on the basis of inadequate blood supply. Shrinkage resulting from fixation of the tissue accounts for the smaller over-all follicular volumes in the sectioned material compared with the living transplants, but other than this, the data from the two series are quite comparable. More than 100 ova were stained in toto to correlate the growth rate of the follicle with the maturation of the contained ovum. The results were exactly the same as those for the serially sectioned ova. In each group of ova recovered from ovarian transplants, however, a few immature vesicular ova from the smaller follicles of an earlier generation were seen. Although these unripe ova were obviously unlike the maturing ova when they were fixed and stained, they were not easy to distinguish in the living state. THE FERTILITY OF FOLLICULAR OVA FROM OVARIAN TRANSPLANTS At various stages of development the ovarian transplants were removed from the eyes of the recipient animals, and the larger follicles were opened under saline. The cumulus encased ova were then extruded, counted, and drawn into a small pipet. From 470 transplants, 1154 ova were obtained, an average of 2.5 ova per transplant. More than twice this number of large follicles were counted under the dissecting microscope and in the sectioned material, so it is obvious that our recovery technic was imperfect. Approximately 6 follicular ova were pipetted into each of 184 ovarian bursas of previously mated albino recipient animals. The method for injecting ova is illustrated in Fig. 15. The ova are picked up by the oviduct within an hour and are fertilized at about the same time as the ovulated ova of the recipient. If the follicular ova are fertile, they develop into embryos with black iris pigment, which readily distinguishes them from the control embryos at the time of laparotomy 18 days after transfer.
..
Vol. 9, No.2, 1958
Fig. 15.
OVARIAN TRANSPLANTS
109
Method for transferring follicular ova into the ovarian bursa. The tip of the pipet (see arrow) is visible behind the bursal membrane.
In a previous experiment, l when 130 developing follicular ova were removed from the ovaries of normal adult animals six hours or less before the expected time of ovulation, and were then transferred into the bursas of 19 recipient animals, 44 (34 per cent) of the ova were fertilized and developed to term embryos. Our present experience with the fertility of follicular ova obtained from eye transplants is not nearly this encouraging (Table 1). When 809 maturing ova from eye transplants were transferred to 131 bursas, only 36 (4.5 per cent) developed into term embryos. The optimal stage of follicular development was between the fourteenth and sixteenth hours following the administration of CG (Fig. 16), but even at this time only 10 per cent of transferred ova survived. The results of individual experiments were quite variable, and the apparent high fertility of ova occurring 24 hours after CG was probably a chance occurrence. This was a very rigorous test for fertility, with many chances for ova to be lost and for inadequate conditions for fertilization to be present. However, the
110
Fertility & Sterility
NOYES ET At.
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Fig. 16. The fertility of follicular rat ova taken from ovaries transplanted to the eyes of recipient rats of a different strain. The data are from Table l. The figures within the circles are the numbers of ova in each transfer in which one or more of the transferred ova developed to term. The figures in the squares represent the number of all ova transferred into recipient bursas at a given hour following chorionic gonadotrophin injection.
conditions in these eye transplant experiments were similar to those with normal follicular ova, yet only one-tenth as many of the ova from the eye transplants were fertile as compared with the preovulatory ova from ovarian follicles in situ.
INTERSPECIES OVARIAN TRANSPLANTS Fragments of immature rabbit ovaries were transplanted into the eyes of immature rats, and of 90 such transplants, 74 established a new blood supply. In all 16 failures, the donor rabbit was 59 days of age, while the donors in the successful takes were 24 days old. The recipient rat was not treated at all in 8 transplants, and received only PMS in 16 transplants. Various treatments were then tried in an attempt to diminish the recipient's immune reaction. In 8 transplants the recipient received 250r of total body x-irradiation two days before the ovarian transplantation, and then PMS was given. In another 8 rabbit ovarian transplants the recipient rat was injected with 1.4 mg. of cortisone 2 days prior
TABLE 1.
Follicular Ova Transferred into the Ovarian Bursas of Recipient Host Rats No. hrs. elapsed after chorionic gonadotrophin injection
10
6 Transfers in which follicular ova survived to term in the eXpel"imental uterine horn Transfers Ova Ova Surviving Transfers in which only control embryos survived in the experi. mental uterine horn Transfers Ova Subtotals Transfers in which the recipient was pregnant. but no embryo survived in the experimental uterine horn Transfers Ova Transfers in which the recipient failed to continue pregnancy Transfers Ova Totals Transfers Ova Ova surviving
2
0
65
36 9
42
10
65
47
67
8
32
46
6 13
21 0
65
24
5
6
5
0
10
0
0
7
0
2
9
25
0
9 35
1
10
7
1
17
19
15
2
0
0
6
0
5
809
133
207
22
4
3 0
632
29
0
138 184
19
61
16
79 3
3
0
2
56
255
0
0
0
7
17
59
16
105
0 19
59
0
0
0
3 44 2
72
51
6
0
0
116 7
221
0
0
22
30
6
177 36
3
3
60
43
28 15
0
16
7
5 185
4
20
31
95
17
3
0
2
5
7
3
20
9 21
4
14
3
105
153
112
24 11
8
0
0
3
2 0
0
0
12
Totals
24
0
0
0
8
7
2 34
0
2
23 21
86
0
42
5
5
19
36
21
22
21
2
6
2
8 54
52
4
6
7
7
14
6
10
8
7
20
19
6 16
31
32
4
0
2
6
5 15
0
6
18
17
16
15
14
12
1154
26 3
36
112
NOYES ET At.
Fertility & Sterility
to the transplantation, and then PMS was given in the same dose and schedule as used previously. Both x-ray and cortisone were given to the recipients of 16 transplants. Twenty-six rat embryos were injected on the fifteenth day of pregnancy with a suspension of rabbit white blood cells, the purpose being to inhibit the future development of rabbit antibodies. The survivors of this treatment who lived to the age of 25 days were used as hosts for 14 rabbit ovarian transplants. The results were identical following each of these treatment schemes. The transplants attained a new blood supply, and some grew slightly, but no normal ova were recovered. Twenty-six immature rat ovaries were transferred to as many rabbits' eyes, but all failed to «take." Thirty-four pieces of adult human ovaries were transferred to the eyes of rats, 10 to the eyes of guinea pigs, and 6 to rabbits' eyes. Despite various combinations of x-ray, cortisone, and PMS given to the recipient animals, none of the human ovarian grafts became vascularized. SUMMARY AND CONCLUSIONS
Transfers of immature rat ovarian tissue to the anterior chamber of the eyes of immature male recipient rats of a different strain produced vascularized, growing grafts in about 85 per cent of trials. Most of the antrumcontaining follicles of these grafts degenerate, but new ones form from surviving primordial follicles within 4 days following transplantation. One to 4 new follicles begin to grow each day, and an average of 6 mature within 4 days. The number of follicles that appear is independent of extrinsic gonadotrophin injections, but is dependent on the intrinsic rise of gonadotrophin level following castration of the host animal. When treated with pregnant mare's serum gonadotrophin, the follicles increase in volume at about the same rate that has been reported for follicles in the mature ovary in situ, while in untreated grafts, follicular growth lags. Following the injection of human chorionic gonadotrophin, the follicles in transplants do not grow so rapidly or become so large as those developing in ovaries in situ, and ovulation is rare. However, meiotic changes in the ovum's nucleus, and cumulus maturation of these ova, seem to progress at the normal rate despite their smaller volume. Only 4.5 per cent of ova removed from follicles that seemed to be maturing proved to be fertile. This is only one-tenth the fertility rate expected from previous experiments on follicular ova obtained from mature ovaries in situ.
Vol. 9, No.2, 1958
OVARIAN TRANSPLANTS
113
Interspecies transplants of ovarian tissue from rabbit to rat, from rat to rabbit, and from human to rat, rabbit, or guinea pig, all failed to produce normal ova despite extensive and varied treatments aimed at reducing the antigenic response of the host. These experiments have been of great interest insofar as the study of follicular growth is concerned, but they have not resulted in the discovery of an ideal source for experimental ova. From an immunologic point of view it is interesting that in these acute experiments, ova can be brought to maturity despite the fact that these intraspecies grafts invariably degenerate a short time later. It is felt that inadequate blood supply, rather than antigenicity, may be the cause for the failure of rapid growth following the injection of chorionic gonadotrophin. Slow growth may in turn be responsible for the failure of ovulation, and still more remotely may decrease the fertility of these ova. Further advances in solving this problem will depend on better immunologic control in the recipient host, so that transplants may grow long enough to attain a normal blood supply before ovum maturation is attempted. Stanford Univ. School of Medicine Clay and Webster Sts. San Francisco, Calif.
REFERENCES 1. BOLING, J. L., et al. Growth of the graffian follicle and the time of ovulation in the albino rat. Anat. Rec. 79:313, 1941. 2. CHANG, M. C. Fertilization and normal development of follicular oocytes in the rabbit. Science 121 :867, 1955. 3. CLEWE, T. H., YAMATE, A. M., and NOYES, R. W. Maturation of ova in mammalian ovaries in the anterior chamber of the eye. Proceedings of the Second World Congress on Fertility and Sterility, Naples, Italy, 1956. To be published. 4. NoYES, R. W. Fertilization of follicular ova. Fertil. & Steril. 3:1, 1952. 5. ROWLANDS, I. W. The production of ovulation in the immature rat. ]. Endocrinol. 3:384, 1944.