F ertilizability of Rabbit Ova after Removal of the Corona Radiata

Transcription

1 F ertilizability of Rabbit Ova after Removal of the Corona Radiata M. C. CHANG, Ph.D., and J. M. BEDFORD, M.R.C.V.S." FRESHLY ovulated rabbit ova are surrounded by a mass of follicular cells in a mucous clot, known as the cumulus oophorus. The follicular cells immediately attached to the zona pellucida, which are arranged in a compact radial pattern, compose the corona radiata. In vitro, the follicular cells of the cumulus oophorus can be dispersed by the presence of a large number of sperm or by treatment with hyaluronidase; the corona radiata, however, cannot be dispersed by sperm or hyaluronidase, and the denudation of ova has been attributed to a tubal factor. 7 Chang, 4 in a report subsequently verified by Dickmann, 5 showed that dissolution of the cumulus oophorus by means of hyaluronidase does not impair fertilization. An experimental study of the role that the corona radiata may play in the fertilization of rabbit ova is reported here. METHOD The method employed in this study was essentially the same as reported previously. 4 The unfertilized ova were :Hushed from the uterine tubes with a mixture of rabbit serum and Ringer's solution ( 1: 1) at 12, 14, or 16 hours after intravenous injection of about 30 I.U. of LH (i.e., at about 2, 4, or 6 hours after ovulation). Immediately, about 0.1 ml. of a 0.1% hyaluronidase solution was added to dissolve the cumulus oophorus. Usually, the ova of 2 animals were pooled and divided into 2 groups. One group of ova were suspended in 2 ml. of a culture medium, T.C. 199, in a small test tube which was shaken vigorously by hand for 5 min. to remove the corona radiata. 1 The other group of ova served as control. Both groups were then suspended From the Worcester Foundation for Experimental Biology, Shrewsbury, Mass. and the Department of Biology, Boston University, Boston, Mass. This work was supported by grants from the Population Council, Inc., and Grant RG-6944 from the U. S. Public Health Service. Sincere thanks are due Miss Dorothy M. Hunt for assistance, and to Dr. G. Pincus for reading the manuscript. *Present address: Royal Veterinary College, London, N. W. 1, England. 421

2 422 CHANG & BEDFORD FERTILITIY & STERILITY in 50% serum and kept in a culture plate at 30 C. for about 1 hour. The ova without corona radiata were transferred into the left tube and the control ova into the right tube of an animal mated with three males hours previously. Six or 24 hours after the operation the recipient animal was sacrificed. Ovulation spots were carefully counted and the recovered ova were mounted in toto and examined before and after fixation according to the method of Chang. 4 To distinguish between the transferred ova and those produced by the recipient, careful consideration was made of the number transferred, the number of ovulation spots in the recipient, the thickness of the mucin coat (expected to be thinner in the transferred ova),, the nuclear configurations of the ova (expected to be less advanced in those transferred), and the number of sperm attached on the zona pellucida (expected to be less in transferred ova). Where there was doubt, the ovum in question was discounted. RESULTS AND DISCUSSION The ova recovered 2 or 4 hours after the estimated time of ovulation were all in the cumulus oophorus, though this was more compact in some animals (Fig. 1) than in others. Of the ova recovered 6 hours after ovulation, some were free from the cumulus, which was rather loose at this time, and a few were denudated, i.e., without corona radiata. Since the dispersion of follicular cells in the tubes is related to the time interval after ovulation, the actual time of ovulation may have been slightly later (if the cumulus was still compact and all ova invested in the thick corona radiata) or earlier (if the ova were free or denudated) than the estimated time of ovulation. Or, the dispersion of follicular cells in the tubes may be more rapid in one animal than another. By treatment of freshly recovered ova with hyaluronidase, the cumulus clot was dispersed in a few minutes, but the corona radiata remained attached to the zona (Fig. 2.) Vigorous shaking removed nearly all the coronal cells (Fig. 3). Practically all fertilized ova examined 6 hours after transfer were at the pronuclear stage, though some ova shed by the recipient animals had cleaved or showed pronuclear configurations slightly advanced over those of transferred ova. In no case was an enlarged sperm head found in the ooplasm. Since the enlargement of the sperm head in the vitellus occurs soon after sperm penetration, it appears that penetration into the transferred ova takes place soon after transfer if the ova are able to be fertilized. The unfertilized ova (those containing the first polar body and the second maturation spindle) must have lost their fertilizability before their encounter

3 VoL. 13,No.5,1962 OvA FERTILIZATION 423 with sperm, although a few sperm were found on the zona pellucida of some. When examined 24 hours after transfer, the majority of ova ovulated by the recipients were in the 2- to 12-cell stage, while the transferred ova were at 4 to 8 cells (Fig. 4). Ova classified as fertilized were not partheno Fig. 1. Ovum in compact cumulus oophorus, 4 hours after ovulation. Fig. 2. Two ova removed 4 hours after ovulation and treated with hyaluronidase. Fig. 3. Group of ova, removed 4 hours after ovulation and treated with hyaluronidase. Corona radiata has been shaken off. Fig. 4. Eight fertilized ova obtained following the transfer of 19 ova, 6 hours after ovulation, into the tubes of a mated rabbit. Mter 7 of them were transferred into the tube of a second recipient rabbit, only 1 normal young was produced. (50% rabbit serum; phase-contrast; X 125, approximately) genetically cleaved, as shown by the presence of a second polar body, although the rate of cleavage was slow, especially in those transferred 6 hours after ovulation. From the data presented in Table 1, it appears that 2-4 hours after ovulation, fertilization could occur in about 85 per cent of ova with an intact corona radiata, but in only about 50 per cent of those without the corona radiata. At 6 hours after ovulation, 60 per cent of ova with corona radiata and only 12 per cent of those without were fertilized. It is obvious that the

4 424 CHANG & BEDFORD FERTILITY & STERILITY TABLE 1. Fertilization of Rabbit Ova after Removal of the Corona Radiata Ova without corona radiata Ova with corona radiata Age of ova (hr. No. of No. No. after recipient trans- No. ez- Fertilized trans- No. ez- Fertilized ovulation) rabbits ferred amined No. % ferred amined No. % corona radiata plays a major role in the fertilization of rabbit ova or in the maintenance of their fertilizability. The results can be interpreted in two ways. First, the penetration of sperm may be dependent on the presence of the corona radiata. That is, with no corona cell on the zona pellucida, no sperm is able to penetrate it. Thus the fertilized ova found following the removal of the corona may be the result of the effect of a few corona cells that were not shaken from the zona (Fig. 3). The second possibility is that the fertilizability of ova is protected by the presence of the corona, and disappears within a short time following its removal. Since after removal of the corona radiata, 50 per cent of ova can be fertilized 2-4 hours after ovulation, when their fertilizability was still at its maximum, 4 and since only 12 per cent of ova can be fertilized 6 hours after ovulation, when their fertilizability has declined, the second interpretation is more probably correct. This interpretation implies that the chemical components that determine the fertilizability of ova are very unstable, with rapid "decomposition" after the removal of the corona radiata either in vivo or in vitro. By counting the number of young produced following inseminations at various times before and after ovulation, Hammond 6 concluded that rabbit ova are capable of being fertilized for about 6 hours after ovulation. By counting the number of fertilized ova following transfer into the tubes of mated animals of ova ovulated at various times, Chant reported that rabbit ova can be fertilized up to 8 hours after ovulation. Since fertilized ova, especially those fertilized when they are aged would not necessarily develop into normal young, it is quite possible that Hammond's estimate is too short for fertilization per se. In order to determine whether the fertilized ova can develop into normal young, 29 fertilized ova obtained by the transfer of 58 six-hour ova into the uterine tubes of mated animals, were transferred again into the tubes of 3 unmated recipient animals that had ovulated 12 hours previously. At examination days later, only 6 normal young and 8 pieces of fetal

5 VoL. 13, No. 5, 1962 OVA FERTILIZATION 425 placentae were found. Thus only about 21 per cent of the ova fertilized 6 hours after ovulation are able to develop into normal young, and the other fertilized ova degenerate either before or after implantation. SUMMARY Rabbit ova recovered from the uterine tubes were treated with hyaluronidase to disperse the cumulus oophorus and divided into 2 groups. The corona radiata of one group of ova were shaken off. Of the ova recovered 2-4 hours after ovulation and transferred into the tubes of mated animals, per cent without corona radiata and per cent with corona radiata were fertilized. Of ova recovered 6 hours after ovulation and transferred, 12 per cent without and 60 per cent with corona radiata were fertilized. It appears that the cumulus oophorus does ~ot play a part in fertilization, but that the corona radiata plays an important role in protecting the fertilizability of ova. When 29 ova, obtained following fertilization 6 hours after ovulation, were transferred into recipient rabbits, only 6 normal young (21 per cent) were obtained. It seems that a high proportion of ova fertilized late degenerate either before or after implantation. The Worcester Foundation for Experimental Biology Shrewsbury, Mass. REFERENCES 1. BEDFORD, J. M., and CHANG, M. C. Nature 193:898, BRADEN, A. W. H. Aust.J. Sci. Res. B., 5:460, CHANe, M. C. Ann. N.Y. Acad. Sci. 52:1192, CHANe, M. C./. Exp. Zool. 121:351, DxcHMANN, Z. Proc. Fourth Int. Cong. Anim. Reprod., The Hague, June 5-9, HAMMOND, J. /. Exp. Biol. 11:140, SWYER, G. I. M. Nature 195:813, 1947.