CLEAVAGE OF HUMAN OVA IN VITRO*

Transcription

1 FERTILITY AND STERn.1TY Copyright., 1971 by The WiUiams & Wilkins Co. Vol. 22, No.4, April 1971 Printed in U.S.A. CLEAVAGE OF HUMAN OVA IN VITRO* H. M. SEITZ, JR., M.D., G. ROCHA, M.D., B. G. BRACKETI, PH.D., AND L. MASTROIANNI, JR., M.D. Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania A knowledge of the environmental conditions necessary for maturation of follicular oocytes, fertilization, and subsequent normal cleavage of human ova is of fundamental importance in the study of human reproduction. The purpose of this investigation was to develop an in vitro system in which human follicular oocytes can complete the maturation process, and undergo cleavage following exposure to spermatozoa. MATERIALS AND METHODS Follicular oocytes were recovered from surgical specimens in which the indicated procedure included ovarian resection, or removal. Ovaries or wedges of ovarian tissue were obtained at random times (whenever available) throughout the menstrual cycle. No medications were given to stimulate ovarian activity. Ovarian tissue from pregnant, postpartum, or postmenopausal patients, or those taking oral contraceptives, was not used in this study. Surgically removed ovarian tissue was immediately placed into a thermal flask containing Ringer's solution, U.S.P., and transported to a C. tissue culture room in the laboratory. The tissue was washed free of blood by passing it through two or three small beakers of warm Ringer's solution. After cleansing, the specimen was immersed in Ringer's solution in a Petri dish and, while viewing through a * This work was supported by United States Public Health Service Grants FROO and HD , National Institutes of Health Contract , and a grant from the Ford Foundation. 255 dissecting microscope, the larger, grossly more mature follicles were incised with a No. 11 surgical blade (Fig. 1). This means of freeing follicular oocytes was considered superior to puncturing follicles with an 18- gauge needle since in the latter instance, used in preliminary experiments, many of the surrounding cumulus cells were lost. As each oocyte was removed from the follicle it was transferred to culture medium in a small tissue culture dish (sealable type, 12 X 30 mm. size, Bellco Glass, Inc.). The number of recovered oocytes ranged from 2-15 in each experiment (Fig. 2). The culture medium used was Ham's1 F-lO with 20% v/v of heated (55 0 C. for 20 min.) female human serum, and 25 JLg. of sodium estrone sulfate ("Premarin," Ayerst Laboratories) added per ml. (F-lO serum-estrogen medium). Oocytes were placed in 4.0 ml. of the culture medium and 5% CO 2 in air-equilibrated paraffin oil, N.F., was added to completely fill the dish which was then closed and incubated in the dark at C. under a 5% CO 2 in air atmosphere. Twenty-four hours after removal from follicles the oocytes were inseminated or incubated without spermatozoa in monkey uterine washings. Semen was obtained from fertile volunteers. Spermatozoa were washed free of seminal plasma by dilution to 5.0 ml. with warm calcium-free Krebs-Ringer phosphate solution followed by a 4-min. centrifugation at a maximum force of approximately 1146 X g at the bottom of the centrifuge tube. The supernatant solution was discarded and the cells were resuspended

2 256 SErrz ET AL. Vol. 22 FIG. 1. vvedge of human ovary containing several mature follicles. Four ova were recovered from this specimen after incising the follicles. and washed again. The cells were finally resuspended in 1.0 ml. of F-IO medium, and 0.5 ml. (approximately 10 6 spermatozoa) of this suspension was incubated in the uterus of a midcycle rhesus monkey. Mature, cycling rhesus monkeys between Day 11 and Day 15 of their menstrual cycles were used for uterine incubation of spermatozoa. The monkeys were anesthetized with phencyclidine HCI ("Sernylan," Parke-Davis & Company) given intramuscularly. Each monkey was opened by a midline incision, and a right angle gall bladder clamp was placed across the cervical canal (Fig. 3). Oviducts were occluded near the uterotubal junctions with small hemostats. A figure of eight suture using 4- o silk was placed in the uterine fundus at the site of the injection. A 20-gauge needle was introduced into the uterine lumen through the fundus and 0.5 ml. of sperm suspension was injected. The suture was tightened as the needle was removed, thereby preventing the escape of spermatozoa and bleeding. The monkey was temporarily closed and maintained under anesthesia during a 4-5 hr. sperm incubation interval. Spermatozoa were recovered by flushing the uterine cavity with 10 ml. of water F-IO medium introduced from a lo-cc. syringe through a 20-gauge needle. After flushing the uterine cavity, if the hemostats are removed and the cavity again flushed with warm saline, tubal patency can be re-established. The right angle clamp on the cervical canal did not

3 April 1971 CLEAVAGE OF HUMAN OVA 257 FIG. 2. Ova, as they appear immediately following recovery from the follicles shown in Fig. 1 seem to harm the animal as normal cyclic menstruation continued to occur in months following the procedure. The sperm F-lO suspension was centrifuged in the warm room for 5 min. at a maximum force of approximately 400 X g. The supernatant was removed and the spermatozoa were diluted to 4.0 ml. with F-lO serum-estrogen medium for incubation with ova. Approximately one-third of the spermatozoa used for incubation in the monkey uterus were recovered. Motility decreased from approximately 80-25% as a result of this treatment. The ova, now cultured for 24 hr., were transferred to the sperm suspension, and the medium was covered with 5% CO 2 in air-equilibrated paraffin oil and incubated at C. under a 5% CO 2 in air atmosphere. After hr. of incubation with spermatozoa, ova were transferred to fresh F-lO serumestrogen medium and reincubated. Ova were examined at 18, 40, 52-54, 72, and 96 hr. after insemination. Control experiments were carried out using twice-washed ejaculated spermatozoa, or washings from the monkey uterus without spermatozoa for incubation with ova. RESULTS Seventy human follicular oocytes were used in this investigation. Although a systematic search for polar bodies was not carried out, first polar bodies were seen in many ova, usually still surrounded by corona radiata cells, at the time of transfer from the sperm suspension, i.e., 18 hr. after insemination or 42 hr. after their recovery from follicles. In one ovum, two polar bodies were seen at this time. A thorough examination was not made for pronuclei or sperm remnants in the vitellus, since cleavage was the desired endpoint of these experiments.

4 \ 258 SEITZ ET AL. Vol. 22 FIG. 3. A, right angle clamp placed across cervical canal (black arrow). Small hemostats occlude oviduct at uterotubal junction (white arrows), Large white arrow shows purse string sutur, in place at insemination site; B, spermatozoa injected into fundus of monkey uterus; C, uterine cavity flushed with culture medium. Arrows indicate direction of flow (syringe barrel serves as a reservoir).

5 April 1971 CLEAVAGE OF HUMAN OVA 259. In experiments in which ovum cleavage occurred, the ovum donor was in either the immediately preovulatory or the postovulatory phase of the menstrual cycle. Cleavage was observed with oocytes removed on cycle Days 12, 13, 16, 20, and 21; no ovum cleavage occurred with oocytes recovered on cycle Days 2, 7, and 9. Of 50 ova inseminated with monkey uterine-incubated spermatozoa, 8 cleaved (Table 1). None of 15 inseminated with washed ejaculated spermatozoa and none of 5 incubated in monkey uterine washings without spermatozoa had cleaved, when examined 72 hr. later. Normal appearing 2, 3, 4, 6, and 12 cell stage ova were observed (Fig. 4). One dubious morula which might also have been a degenerated ovum of an earlier cleavage stage was seen 96 hr. after insemination. A 2 cell ovum was observed 40 hr. after insemination. Two cell, 3 cell, 4 cell, and two 6 cell stage ova were seen, when examined between 52 and 54 hr. after insemination. The 12 cell stage ovum observed at 72 hr. after insemination was stained with lacmoid (Fig. 5), and chromatin was seen in three of the blastomeres. Spermatozoa were observed adherent to and within the zona pellucida in many of the ova, and sperm motility was noted to persist for 3 days at 37 C. under these conditions. DISCUSSION There are several reports of in vitro fertilization of human ova This subject has TABLE 1. Cleavage of Human Ova in Vitro Experimental Ova cleaved/ Normal treatment ova recovered cleavage Insemination with sperm in- 8/50 o '" 16 cubated 4-5 hr. in monkey uterus Insemination with twice 0/15 0 washed ejaculated sperm Incubation with monkey 0/5 0 uterine washings (no sperm) been recently reviewed The relative inaccessibility of the human reproductive tract has retarded progress in this field. Oocytes recovered from ovarian follicles provide the most abundant source of research material. Complete in vitro maturation of follicular oocytes to metaphase II of meiosis requires approximately hr.4, 13, 15, 16 In the present study oocytes removed from mature follicles were selected. It is likely that these follicular oocytes were either already in the incipient stages of maturation at the time of recovery, or that their maturation was completed in the presence of spermatozoa following insemination. By initiating ovum maturation in vivo with gonadotropin treatment, Edwards, Steptoe, and PurdyS have recently decreased their in vitro incubation of follicular oocytes to 1-4 hr. prior to insemination. Ovum cleavage was reported, and the time intervals between cleavage stages were roughly comparable to those observed in the present study. Although Edwards, Steptoe, and PurdyS observed ovum cleavage and claimed the cleaved ova were fertilized in vitro, no documentation of the role of a spermatozoon in the process was provided, and no evidence was reported by which the possibility of parthenogenetic development of ova could be ruled out. Follicular ova removed near the time of ovulation from gonadotropin-stimulated rabbit ovaries and cultured in the absence of spermatozoa, frequently undergo cleavage which is morphologically indistinguishable from that resulting from fertilization.17 Hayashi6 has described parthenogenetic cleavage of human ova following liberation of oocytes from ovarian follicles. This report represents our current progress directed toward isolation of the human fertilization process in vitro. The question as to whether or not the ovum cleavage reported here resulted from in vitro fertilization cannot as yet be answered with absolute certainty. The evidence for the oc-

6 FIG. 4. A, cleaved human ova. Two-cell stage observed 40 hr. after in vitro insemination; B, C, and D, three-cell, 4-cell, and 6-cell stage, respectively, observed hr. after in vitro insemination. Spermatozoa can be seen stuck on the zona pellucidae in C and D. 260.

7 April 1971 CLEAVAGE OF HUMAN OVA 261 FIG. 5. A, twelve-cell stage observed 72 hr. after in vitro insemination stained with lacmoid; B, chromatin in a blastomere of the 12-cell stage ovum. currence of fertilization includes observations of spermatozoa in and around the zona pellucida, two polar bodies, progressive cleavage stages, chromatin in some of the blastomeres, and failure of control ova to undergo cleavage. Failure of cleavage after exposure of ova to washed ejaculated spermatozoa and normal cleavage following exposure of ova to monkey-incubated spermatozoa suggests a need for human sperm capacitation, but the data are insufficient to justify a fixed conclusion at this time. Anticipated data from experiments in progress should provide further insight into our understanding of the human fertilization process. SUMMARY Efforts were made to develop a repeatable in vitro system for the maturation and cleavage of human ova in a controlled environment. Follicular oocytes were recovered from fresh surgical specimens obtained at various times in the menstrual cycle. The oocytes were incubated for 24 hr. at 37 C. in a 5% CO 2 in air atmosphere in F-lO culture medium containing 20% human serum, and 25 J.Lg. of sodium estrone sulfate per cc. of medium. Human spermatozoa which had been incubated in the uterine cavity of a midcycle rhesus monkey for 4 hr. were used for insemination. The gametes were incubated further and examined at intervals for cleavage. Seventy ova were studied. Eight displayed cleavage from the 2-12 cell stage. In control experiments no cleavage was observed when ova were exposed to washed ejaculated spermatozoa or incubated in sperm-free washings from the monkey uterus. An understanding of in vitro conditions under which human ova are activated will afford the scientist

8 262 SEITZ ET AL. Vol. 22 opportunities to evaluate factors which retard as well as enhance fertility. Acknowledgments. The authors wish to thank Miss Eva Kelly for her technical assistance in the animal operating room, and Messrs. Chuck Meltzer, Tom Henry, Fred Norman, and Fred Granby for their valuable assistance in the care and preparation of the animals used throughout this investigation. REFERENCES 1. HAM, R. G. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp Cell Res 29:515, BAVISTER, B. D., EDWARDS, R. G., AND STEPrOE, P. C. Identification of the midpiece and tail of the spermatozoon during fertilization of human eggs in vitro. J Reprod FertiI20:159, EDwARDS, R. G., DoNAHUE, R. P., BARAMKI, T. A., AND JONES, H. Preliminary attempts to ferti. lize human oocytes matured in vitro. Amer J Obstet Gynec 96:192, EDWARDS, R. G., BAVISTER, B. G., AND STEPrOE, T. C. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (London) 221:632, EDwARDS, R. G., STEPrOE, P. C., AND PmmY, J. M. Fertilization and cleavage in vitro of preovulatory human oocytes. Nature (London) 227:1307, HAYASHI, M. "Fertilization in vitro using human ova." In Proceedings of the Seventh International Conference Planned Parenthood Federation, Sin gapore, Excerpta Medica Int Congr Ser 72, Am sterdam, 1963, pp MENKIN, M. S., AND RoCK, J. In vitro fertilization and cleavage of human ovarian eggs. Amer J Obstet Gynec 55:440, RoCK, J., AND MENKIN, M. F. In vitro fertilization and cleavage of human ovarian eggs. Science 100: 105, SHETTLES, L. B. Observations on human follicular and tubal ova. Amer J Obstet Gynec 66:235, SHETl'LES, L. B. A morula stage of human ova developed in vitro. Fertil SteriI6:287, BRACKETT, B. G. "Recent progress in investigations of fertilization in vitro." In Biology of the Blastocyst, Blandau, R. J., Ed. University of Chicago Press, Chicago, CHANG, M. C. In vitro fertilization of mammalian eggs. J Anim Sci 27:15, MAsTROIANNI, L., JR., AND NORIEGA, C. Observations on human ova and the fertilization process. AmerJ Obstet Gynec 107:682, THIBAULT, C. In vitro fertilization of the mammalian egg. In Fertilization (Vol. 2), Metz, C. B., and Monroy, A., Eds. Academic Press, New York, 1969, Chap. 9, p EDwARDS, R. G. Maturation in vitro of human ovarian oocytes. Lancet 2:926, KENNEDY, J. F., AND DONAHUE, R. P. Human oocytes: Maturation in chemically defmed media. Science 164:1292, BRACKETT, B. G., AND PEACE, M.D. In vitro fertilization of rabbit ova obtained from ovarian follicles. Unpublished data, 1970.