FERTILITY AND STERILITY Copyright~ 1975 The American Fertility Society Vol. 26, No.7, July 1975 Printed in U.SA. IN VITRO FERTILIZATION OF RABBIT EGGS IN OVIDUCT SECRETIONS FROM DIFFERENT DAYS BEFORE AND AFTER OVULATION* RAYMOND D. LAMBERT, PH. D.,t AND CHARLES E. HAMNER, D.V.M., PH.D. Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Virginia School of Medicine, Charlottesville, Virginia 22901 In vitro fertilization of rabbit eggs has been achieved in several different media. L 2 It is interesting that only a few studies of in vitro fertilization in oviduct secretions have been reported, 3 4 since in most mammalian species fertilization occurs in the fallopian tube. The purpose of this investigation was to explore the influence of tubal secretions from different parts of the rabbit oviduct (ampulla and whole oviduct), collected on different days before and after ovulation, on the in vitro fertilization process. MATERIALS AND METHODS Collection of Oviduct Fluid. New Zealand White rabbits weighing 4 to 5 kg were used for collection of the tubal fluid with the continuous collection flask of Hamner and Williams. 5 6 The fluid was collected every 24 hours and stored at -20 C until used. Ovulation was induced in estrus rabbits by an intravenous injection of 75 IU of human chorionic gonadotropin (HCG) (Follutein; E. R. Squibb and Sons, Princeton, N. J.). Capacitation of Sperm. Semen was collected from mature Dutch Belted and New Received July 25, 1974. *Supported by United States Public Health Service Grant HD06573 from the National Institute of Child Health and Human Development, Research Career Development Award HD12760, and the World Health Organization. tpresent address: Department of Obstetrics and Gynecology, Universite Laval, Ste-Foy, Quebec, Canada. 660 Zealand White rabbits by artificial vagina and was filtered through two layers of cheesecloth, pooled, and diluted one time with saline (0.9% NaCl) at 37 C. This sperm preparation was used to artificially inseminate an estrus rabbit which had received an intravenous injection of 75 IU of HCG just prior to the insemination. Sixteen hours after the estrus doe was artificially inseminated, the uterine horns were flushed with 2.0 ml of Brackett's medium 7 or with centrifuged (1000 x g for 2 minutes) oviduct fluid to recover the capacitated sperm. The culture medium used did not contain glucose or bovine serum albumin but contained 20% rabbit blood serum heated to 56 C for 30 minutes (HBS). These sperm suspensions were kept at 37 C under paraffin oil (125/ 135 viscosity) until used. Collection of Ova for in Vitro Fertilization. Ova were recovered from estrus does, 14 hours after an intravenous injection of HCG (75 IU), by flushing the oviducts under oil with Brackett's medium containing 20% HBS. The eggs in cumulus were covered with paraffin oil and treated with hyaluronidase (200 IU/mg) for 10 minutes before being transferred to the capacitated sperm suspension in a volume as small as possible. The eggs were incubated with the capacitated sperm for a period of 5 hours. At the end of the 5-hour period, the in vitro-fertilized eggs were transferred to an incubation medium of either heated rabbit blood serum or oviduct fluid.
Vol. 26, No. 7 IN VITRO FERTILIZATION BEFORE AND AFTER OVULATION 661 TABLE 1. In Vitro Fertilization in Oviduct Secretions from Different Days before and after Ovulation in the Rabbit Ova studied No. of Fertilization medium experiments % P,' P' Total Fertilized Fertilized SD ' Brackett's medium + 20% HBSC 9 63 47 75 17 <0.005 Oviduct fluid Estrus 20 170 61 36 32 <0.005 1 day PSPa 16 157 71 45 36 <0.05 NSe 2-14 days PSP 11 71 58 81 18 NS <0.001 "P,, probability from t-test when compared with Brackett's medium+ 20% HBS. bp2, probability from t-test when compared with oviduct fluid collected during the estrous period. chbs, rabbit blood serum heated to 56" C for 30 minutes. apsp, pseudopregnant. ens, not significant. All of the fluid (HBS and oviduct fluid) and paraffin oil used were equilibrated with 5% C0 2, 8% 0 2, and 87% N 2 and kept at 37 C. In all of the experiments described, the sperm had good motility before, and at the end of, incubation. Our general criteria for fertilization were the appearance of the pronuclei and the occurrence of normal cleavage, as ------L-.!--...1 '--~,:.:....:_....:... _ i::l~\,;~.1-lli::l.lj..l~u uy.lj..l.l\,;.lu~v}ju,.; CA.CJ.J..l.l.l.l.lQ.l.t.lV.l.l (X 40 to X 250). RESULTS The results of in vitro fertilization in the oviduct secretions from different days before and after ovulation are presented in Table 1. Although sperm were able to penetrate the egg, the success rate for pronuclei formation and cleavage into two cells was very low in oviduct secretions from the estrous cycle (preovulation) and day 1 of the pseudopregnancy cycle (first 24 hours after the HCG injection; contained 10 hours' secretion of preovulatory fluid). Even with a very high standa-rrl rl.,n7latlnn tho -ra.anlta a.,..o al m,.iflpant lu... - --"......,...,... _......,-...... o... J different (p < 0.05) when compared with the results obtained in Brackett's medium. Fertilization rates were also significantly lower (P < 0.01) in oviduct fluid from the estrus secretions than they TABLE 2. Comparison of In Vitro Fertilization in Whole Oviduct Secretions with Secretions from the Ampulla Portion of the Oviduct Ova studied Fertilization No. of P," P./ P{ medium experiments % Total Fertilized Fertilized SD Estrus Oviduct fluid 13 114 40 35 33 NSd Ampulla fluid 7 56 21 37 29 NS NS 1 day PSP' Oviduct fluid 10 96 38 40 34 NS NS Ampulla fluid 6 55 30 55 38 NS NS NS 2-14 days PSP Oviduct fluid 6 41 30 75 14 <0.025 <0.05 Ampulla fluid 6 39 33 85 18 NS <0.01 <0.05 "P, probability when the results obtained in the ampulla portion of the oviduct are compared with the results obtained with whole oviduct secretions. bp 2, probability when compared with estrous oviduct fluid. cp 3, probability when compared with estrous ampulla fluid. dns, not significant. cpsp, pseudopregnant.
662 LAMBERT AND HAMNER July 1975 TABLE 3. In Vitro Fertilization in Brackett's Medium Containing 17{3-Estradiol or Progesterone Eggs recovered Fertilization No. of mediuma experiments % Total Fertilized Fertilized 17 {3-Estradiol, 1 13 12 92 10 ~-tglml Progesterone, 1 13 11 85 5~-tglml a consisted of Brackett's medium plus 20% HBS. were in oviduct fluid from days 2 through 14 of pseudopregnancy. A comparison of fertilization levels in the secretions from the whole oviduct and the ampulla portion of the oviduct (ampulla fluid) did not indicate any significant difference (Table 2). However, the percentage of fertilization was significantly lower in fluids from both the ampulla and the whole oviduct during the estrous period of the cycle than it was during days 2 to 14 of pseudopregnancy. To determine whether the effect on the fertilization process of oviduct secretions from the estrous period of the cycle was due to the estrogen or progesterone level in the oviduct secretions, 8 we did the vitro fertilization in Brackett's medium plus 20% HBS to which 17{3- estradiol or progesterone had been added at pharmacologic levels (Table 3). Under those conditions and at concentrations as high as 10 J.tg/ml of 17{3-estradiol and 5 J.tg/ml of progesterone, the fertilization rates were 92 and 85%, respectively. DISCUSSION Fertilization can occur in the oviduct during the entire pseudopregnant period in the rabbit. 9 16 Reduced fertilization rate in pseudopregnant rabbits has been attributed to failure of sperm transport,9 10 to rapid egg transport,tl 12 and to the production of an environment in the female reproductive tract which is hostile to the sperm and embryo. 12 16 However, the oviduct environment appears to be less hostile to the sperm than is the uterus during mid-pseudopregnancy or progestin treatment, 13 since capacitation is not inhibited in the rabbit oviduct.12 13 15 16 Results from in vitro fertilization in the oviduct secretions from psuedopregnant animals indicate that a very high percentage of fertilization can be obtained. The use of an in vitro system in the study of fertilization has the advantage of eliminating the problem of sperm or egg transport. These results support the assertion of Chang, 12 15 Bedford, 16 and Brown and Hamner13 that the failure of fertilization in the oviduct during mid-pseudopregnancy is due to the effect on gamete transport. On the other hand, our results concerning the significant inhibition of the appearance of the pronuclei in the rabbit embryos or inhibition of normal cleavage, when in vitro fertilization was carried out in oviduct secretions from estrus rabbits, are surprising (Tables 1 and 2). The fact that sperm motility remains good 18 hours after the end of in vitro fertilization indicates that the estrous oviduct secretions are favorable media for sperm survival. In eggs considered unfertilized we could see sperm in the perivitelline space. Consequently, it seems likely that the inhibition noted during estrus is not due to some effect on the sperm. It is important to realize that, with the oviduct fluid collection technique utilized, the products of ovulation are excluded from the fluid collected. One must wonder how important a role these products may play in pronuclear development and early cleavage of the embryo. The effect could be due to the hormone level in the oviduct fluid, but steroids are not known to act directly on the process of fertilization.17 18 The present results with in vitro fertilization in Brackett's medium containing 17{3-estradiol or progesterone are in agreement with that finding. These hormones, at levels as high as 10 J.tg/ml for 17{3-estradiol and 5 J.tg/ml for progesterone (which in both cases represent
Vol. 26, No. 7 IN VITRO FERTILIZATION BEFORE AND AFI'ER OVULATION 663 pharmacologic doses 8 ), did not inhibit fertilization. The results indicate that, under the estrous endocrine state, something is present in the oviduct fluid that is not favorable to the sperm-ovum relation during the process of fertilization and cleavage. It has already been shown that the oviduct secretions collected during the estrous period in the rabbit are much less favorable to the development of one-cell embryos than are the fluids collected from the first half of the pseudopregnancy period. 19 Preliminary research in this laboratory indicates that a specific protein factor can be extracted from the oviduct secretions collected during estrus and day 1 of pseudopregnancy. This protein factor was not found later in pseudopregnancy. Our preliminary results indicate that this factor may inhibit both the formation of the pronuclei when added to the in vitro fertilization medium and embryo development when added to the culture medium. 20 Because sperm wt:n.~e observed on the vitelline membrane, in eggs classified as unfertilized, this observation strengthens the conclusion that estrous oviduct secretions primarily affect the egg during the process of fertilization and early cleavage. SUMMARY In vitro fertilization of rabbit eggs in oviduct secretions has been studied through different days of the pseudopregnant cycle. The appearance of the pronuclei and cleavage into two cells, which were the criteria of fertilization, occurred significantly less frequently when in vitro fertilization was attempted in the oviduct secretions obtained during the estrous (preovulatory) period of the cycle than it was in the secretions obtained during pseudopregnancy or in Brackett's medium plus 20% heated rabbit blood serum. Whether fertilization was attempted in ampulla secretions or in whole oviduct secretions had no effect on the success rate. The effect of oviduct secretions from the estrous period of the cycle is probably due mainly to an effect on the egg, involving the very first development processes during and after fertilization. REFERENCES 1. Bedford JM: Techniques and criteria used in the study of fertilization. In Methods in Mammalian Embryology, Edited by JC Daniel. San Francisco, W H Freeman and Co, 1971, p37 2. Brackett BG, Williams WL: In vitro fertilization of rabbit ova. J Exp Zool 160:271, 1965 3. Suzuki S, Mastroianni L: In vitro fertilization of rabbit ova in tubal fluid. Am J Obstet Gynecol 93:465, 1965 4. Suzuki S, Mastroianni L: In vitro fertilization of rabbit follicular oocytes in tubal fluid. Fertil Steril 19:716, 1968 5. Hamner CE, Williams WL: Effect of the female reproductive tract on sperm metabolism in the rabbit and fowl. J Reprod Fertil 5:143, 1963 6. Hamner CE, Williams WL: Composition of rabbit oviduct secretions. Fertil Steril 16:170, 1965 7. Brackett BG: In vitro fertilization in mammalian ova. In Advances in Biosciences, Vol 4, Edited by G Raspe. New York, Pergamon Press, 1970, p 73 8. Hilliard J, Eaton LWM: Estradiol-17/3, progesterone and 20-a-hydroxypregn-4-en-3-one in the rabbit ovarian venous plasma. ll. From mating through implantation. Endocrinology 89:522, 1971 9. Austin CR: Fertilization and the transport of gametes in the pseudopregnant rabbit. J Endocrinol 6:63, 1949 10. Murphee RL, Black WG, Otto G, Casida LE: Effect of insemination upon the fertility of gonadotrophin-treated rabbits of different reproductive stages. Endocrinology 49:474, 1951 11. Chang MC: Effects of progesterone and related compounds on fertilization, transportation and development of rabbit eggs. Endocrinology 81:1251, 1967 12. Chang MC: Fertilization, transportation and degeneration of eggs in pseudopregnant or progesterone-treated rabbits. Endocrinology 84:356, 1969 13. Brown SM, Hamner CE: Capacitation of sperm in the female reproductive tract of the rabbit during estrus and pseudopregnancy. Fertil Steril 22:92, 1971
664 LAMBERT AND HAMNER July 1975 14. Hamner CE, Wilson LA: Inhibition of capacitation in the rabbit. Fertil Steril 23:196, 1972 15. Chang MC: Capacitation of rabbit spermatozoa in the uterus with special reference to the reproductive phase of the female. Endocrinology 63:619, 1958 16. Bedford JM: The influence of oestrogen and progesterone on sperm capacitation in the reproductive tract of the female rabbit. J Endocrinol 46:191, 1970 17. Jackson H: Antifertility substances. Pharmacol Rev 11:135, 1959 18. Saunders EJ: Effects of sex steroids and related compounds on pregnancy and on development of the young. Physiol Rev 48:601, 1968 19. Kille JW, Hamner CE: The influence of oviduct fluid on the development of one-cell rabbit embryos in vitro. J Reprod Fertil 35:415, 1973 20. Lambert RD, Stone SL, Hamner CE: Protein fractionation of the oviductal secretion from the rabbit before and after ovulation with special reference to an estrus modulated protein. In preparation