Since the experiments of Pincus and Enzmann(1935), a number of
investigators(shown in Table 1) have attempted to induce maturation of mammalian oocytes in vitro. It is now generally accepted that resumption of the arrested meiotic division of the follicular oocytes occurs in vivo when they are stimulated by luteinizing hormone in early estrus, or when they are liberated from graafian follicles into the appropriate medium. Edward(1962) found that mouse follicular oocytes could show meiotic chromosomes even though they wer cultured in saline without any nutrient. Other researchers also observed that a high proportion of varian oocytes from various inds of mammals resumed their meiosis outside the follicles, in the media or in the eye chamber. Thus, the follicular oocytes have an ability to resume their maturation soon after coming out of the follicles. This implies that the follicles contain some kind of inhibitors against the activation of the oocytes. Edwards assumed that the follicular fluid would contain such factors.
Recently foote and Thibault(1969) thought that the arrested condition of oocytes was mainly due to the presence of the inhibitory substance which is produced by the granuloss cells in the follicle. At this time it is introduced the results of an investigation on ovum maturation in the presence or absence of follicular fluid in the culture medium in order to clarify the in vitro effects of follicular fluid, and it is also hoped that experiments currently under way in our laboratory will clearify whether heating follicular fluid destroys a toxic substance or releases additional nutrient material.
Materials and Methods
Human ovaries or pieces of ovary removed during gynecological operations for various clinical conditions were used in the present studies. Immediately after the excision they were washed in saline solution and were kept in medium 199 for between one and three hours before collection of the oocytes from the follicles. When they were of an adequate size the oocytes were dissected with a find needle form the follicles, and were washed twice in fresh medium.
Culture of the oocytes was performed using the procedures of Brinster(1963). Each group of oocytes was cultured in three different kinds of media;
1) TC medium 199 supplemented with 0.4% bovine serum albumin(BSA, Pentex), 2) same as I, but mixed with equal amount of non-heated follicular fluid, and 3) same as 1, but mixed with follicular fluid heated at 60℃ for ten minutes before use. The follicular fluid was taken from the graafian follicles of the same ovary by aspiration with a syringe connected with a 27G needle. All occytes were introduced in samll drop of the medium suspended under mineral oil contained in plastic petri dishes and were incubated at 37℃ in an atmosphere of moisturized 95% air and 5% carbon dioxide for 48 hours.
After incubation the oocytes were fixed with acid-alcohol and stained with 0.5% acid-lacmoid for microscopic examination of the nuclear phases, Most of the oocytes cultured were surrounded by cumulus cells.
All instruments and glasswares were sterilized in a hot air sterilizer and the medium was filtered just before the experiment.
Results and Discussion
Table 2 shows the results of the experiment. When the oocytes were cultured in medium Tc-199 supplemented with BSA, only 18.4% resumed their maturation division producing metaphase Ⅰ chromosomes or extruding a polar body but nearly 50% of them underwent degeneration and the remainder (33.0%) were left at dictyotene or late prophase Ⅰ.
When we cultured the oocytes in the medium mixed with heated or non-heated, follicular fluid, more proceeded to further meiosis and fewer degenerated. Among the oocytes cultured in the medium cantaining the non-heated follicular fluid. 45.7% of them matured to the metaphase Ⅰ or metaphase Ⅰ, 23.7% remained at the diplotene or late prophase, while 30.5% degenerated. When the oocytes were maintained in the medium containing the heated follicular fluid, nearly 50% of them showed maturation and only 19.3% degenerated.
These studies show that the follicular fluid does not function as an inhibitor of maturation of human oocyte in vitro but it rather seems to be stimulant. Moreover, the follicular fluid is likely to contain a factor which suppresses degeneration of the ovum. Foote and Thibault(1969) found that the addition of follicular fluid to oocytes did not improve ovum maturation, although the supplement of natural serum to the medium produced more oocytes undergoing meiosis during cultivation. They assumed that follicular fluid might contain some kind of inhibitor originating from the granulosa cells in the graafian follicle. In the studies with rabbit follicles (Cho and Kim, 1969), the results were largely inconsistent with the observations of Foote and Thibault. The studies with intact follicles of rabbit showed that about 30% of the oocytes resumed their maturation division were when they cultured in the isolated follicles in Eagle medium supplemented with 0.5% BSA for 48 hours. Edwards (1962) also postulated that the fact that the oocytes were arrested in the
dictyotene stage in the follicles was due to the fact that follicular fluid contains an unknown factor which inhibits the resumption of meiosis.
Kennedy and Donahue(1969), who carried out investigation on the human ovum maturation in vitro in a chemically defined medium (Ham's F10) and medium Tc-199, found that the oocytes with the cumulus cells attached matured in large numbers than those denuded at the beginning of the incubation. they finally assumed that the cumulus cells detoxify substances in the follicular fluid. According to the work of Caravaglios and Cilotti(1957) who analyzed proteins contained in the follicular fluid of the cow, the protein components in the fluid are qualitatively similar to those of blood serum althouth there are some quantitative differences between the two, and there are a little more albumin and mucopolysaccharides in serum. Our work suggests that the follicular fluid itself does not inhibit maturation of ova, just as blood serum does not.
In the present studies, the oocytes resumed their maturation division with higher frequency in the medium mixed with the follicular fluid and such a tendency was enhanced by heating the follicular fluid compared with the medium without any additional fluid. Thus heating of the fluid brought a remarkable increase in the production of viable oocytes during cultivation. If it is true, as stated by Kennedy and Donahue, that the cumulus cells detoxify substances in the follicular fluid, the heating treatment of the fluid just before it is added to the culture medium could also be interpreted as detoxifying substances which finally produce ovum degeneration. This suggests that the toxic agents in the fluid would supposedly be one of proteins which are easily destroyed by heat. An alternative theory is that the follicular fluid contains a vital nutrient or nutrients which
promotes viability on addition to oocyte culture and that heating of follicular fluid makes more of this material available. In our recent studies with heated homologous follicular fluid (unpublished), the proportion of ovum maturation is not significantly different from that obtained in the present experiment with autologous fluid.
With reference to those results, it assumes that the follicular fluid in the intact follicles contains two kinds of materials; one being a toxic substance leading to oocyte degeneration, and the other inhibitors which arrest ovum meicsis. These factors remain in an active state as long as they are in the follicular fluid of the intact follicles. However, if the follicles are isolated from ovarian tissues, or the follicular fluid is drained from the follicles, these factors are inactivated or consumed during incubation. Even though the toxic materials still remain in the drained fluid, they are probably degraded by heating. Thus, follicular fluid removed from the follicle or with elimination of the toxic materials by heating may rather provide better a environment to the oocytes during cultivation.
From the present studies Ⅰ advance the following hypotheses for further testing. First, that follicular fluid contains both heat labile toxic substances and nutrient substances, and alternatively, that follicular fluid contains only nutrient substances, some of which require heating for activation.
In order to investigate the role of the follicular fluid in the human ovum maturation in vitro, the present studies were carried out. The follicular oocytes matured in large numbers when mixed with an equal amount of follicular fluid rather than when cultured in the medium without the fluid. If they were incubated in the medium with heated follicular fluid, there was a further increase in the production of viable oocytes. From the present studies, it is postulated that follicular fluid in the intact follicles contains two kinds of factors one being toxic substances causing oocytes to degeneratie, and the other inhibitor arresting the meiosis of the oocytes, these factors become inactive when the follicles are removed from the ovarian tissues or the follicular fluid is drained from the intact follicles, and such fluid provides better conditions for the maturation of the oocytes in vitro. These results have led us to postulate the existence of both a nutrient factor and a heat labile toxic factor in follicular fluid.
this study was supported by the Population Council, New York, N.Y. U.S.A., Grant No. M 66.69, given to W.K. Cho and, No. M 69.184, and M 71.0136 C to S.O. Chung. My thanks are due to Professor John D. Biggers at the Harvard Medical School, Boston,
Massachusetts, U.S.A. Professor W.K. Cho at College of Liberal Arts and Sciences, Seoul National University for their cordinal advice and to Dr. Hyun Mo Kwak, chairman of Department of Obstetrics and Gynecology, Yonsei University College of Medicine, Seoul, Korea and to Dr. Suk-whan Kim, Director of Cheong-ang Cancer Research Institute, Seoul, Korea. for their cooperation in supplying the materials.