In Vitro Cultivation of Rabbit Ova Following In Vitro Fertilization in Tubal Fluid1

Transcription

1 416 Cytologia 31 In Vitro Cultivation of Rabbit Ova Following In Vitro Fertilization in Tubal Fluid1 Shuetu Suzuki2 Division of Reproductive Biology, Department of Obstetrics and Gynecology, School of Medicine, University of Pennsylvania, Philadelphia, Pa., U. S. A. Received December 11, 1965 In vitro fertilization and cultivation of mammalian ova has been singled out as one of the most challenging and frustration research projects in the field of reproductive biology. According to Austin (1961a, 1933), in spite of numerous attempts to fertilize mammalian ova in vitro, valid reports have appeared only sporadically. However, the cultivation of mammalian ova under conditions of various media have been reported to be successful since the pioneering works of Long (1912), Brachet (1913), Lewis and Gregory (1929), Pincus (1930), and Nicholas and Hall (1942). Most of the cultivated ova were started from two or more cell stages, and few attempts to cultivate fertilized ova following in vitro fertilization have been reported. In a previous experiment, Suzuki and Mastroianni (1965) have reported that rabbit ova can be fertilized in vitro in rabbit tubal fluid. A total of 337 ova were inseminated in vitro in rabbit tubal fluid under mineral oil using capacitated spermatozoa recovered from the uterus 12 hours after mating. The fresh tubal fluid was collected from ligated rabbit oviducts, diluted with Waymouth's medium and, for 124 of these, the mineral oil was pre. treated with 5% carbon dioxide in air. After four hours, ova were transferred to fresh tissue culture medium with added 10% rabbit serum and cultured for hours. Ova were examined for evidence of sperm penetration, pronuclear formation, polar bodies, and cleavage. The fertilization rate was 30.9% without prior carbon dioxide equilibration and 63.7% when the diluted, carbon dioxide treated, oil was used. The purpose of the present investigation was to extend this previous work by cultivat ing these fertilized rabbit ova in vitro to more cell stages in suitable conditions. Materials and methods New Zealand White rabbits weighing 3 to 5kg were used to collect tubal fluid. The oviducts were ligated just proximal to the fimbria and at the uterotubal junction. Only non-bloody, clear fluid accumulated between the ligatures was used after 4-5 days. An estrous rabbit was mated two or three times with fertile bucks. After 12 hours, fluid containing spermatozoa was aspirated from the uterine cavity. In most cases, ml of slightly turbid uterine fluid containing progressive, motile spermatozoa was recovered. One drop of uterine fluid was placed on a sterile watch glass and immediately mixed with 2-3ml of fresh tubal fluid. This suspension was placed in a tuberculin syringe at Ž for a few minutes before use. Ova were recovered from two does, injected 12 hours pre viously with 100 I. U. of chorionic gonadotropin3, by flushing the oviducts with fresh tubal fluid which had been warmed to Ž. The ova, in cumulus, were transferred to de 1 This work was supported by U. S. P. H. S. Grant HD from the National Institute of Child Health and Human Development and by the Ford Foundation. 2 On leave from the Department of Obstetrics and Gynecology, School of Medicine, University of Keio; Tokyo, Japan. 3 This was generously supplied as A. P. L. by the Ayerst Laboratories.

2 1966 In Vitro Cultivation of Rabbit Ova 417 pression slides with a fine pipette, and approximately three times the volume of sperm suspension was added. After thorough mixing, this suspension was covered with warmed mineral oil and incubated at Ž. The mineral oil had been first mixed with sterile culture medium in a ratio of 20 to 1, and the mixture equilibrated with 5% carbon dioxide in air before use. After four hours, ova were gently washed in a depression slide with tissue culture medium (36-37 Ž) and placed in groups of 5-7 in a small Carrel flask con taining 1ml of a medium consisting of 90% Medium 199 (Hyland Laboratory) and 10% rabbit serum and added lactic acid at 1.0mg/ml of medium. Antibiotics were not included. The cultures were incubated at 37 Ž in a humid atmosphere of 5% carbon dioxide in air Throughout this experiment, aseptic precautions were used, and all glassware was sterilized. and warmed before and during use. Individual ova were placed in center of four petroleum jelly spots on a slide. A coverslip was placed over each ovum and gently pressed down until structures within the ovum were clearly visible under the phase-contrast microscope (Chang 1955, Ohnuki 1959). Results and discussion The difficulty of settling on suitable criteria for in vitro fertilization has been stressed by previous investigators. Also, it has been found that the mammalian ovum is easily activated to various degrees by modifying the thermal, osmotic, and chemical factors in its environment. However, Smith (1949) has observed that when ova are incubated with scrapings of fallopian tube mucosa, cleavage of unfertilized rabbit ova in culture occurs much less Table 1. Cleavage intervals of successful cultivation of the in vitro fertilized rabbit ova frequently. In the present experiment, a second polar body, sperm in the perivitellien space, and normal cleavage were used, all together, as criteria for fertilization. In addition, representative ova believed to be fertilized in vitro have shown the same ultrastructural detail as seen in ova fertilized in vivo. A total of 207 ova were inseminated in vitro in rabbit tubal fluid under mineral oil using capacitated spermatozoa recovered from the uterus 12 hours

3 418 S. Suzuki Cytologia 31 after mating. When the ova were examined, about 20 hours after the transfer for cultivation, 123 (59.4%) of them were at the 2- or 4-cell stages, and they were classified as normally cleaved and definitely fertilized ova. Sixty-five of the fertilized ova were cultivated for two or three days after insemination in vitro. Details of the cleavage intervals of successful cultivation are given in Table 1. Fourteen (21.50%) of the 65 ova could be reached at over the 16-cell stage. A suitable environment is essential, both for the fertilization of ova in vitro and storage in culture for longer periods. Unfortunately, a medium which well supports the fertilization in vitro has not always been found. Figs , an ovum after culture for about 7 hours after in vitro insemination showing conjugation of pronuclei and two polar bodies in the perivitelline space ( ~600). 2, ova after culture for about 70 hours after in vitro insemination; 16-cell stage. See Fig. 3. Inasmuch as fertilization normally occurs within the fallopian tube, the effect in vitro, of tubal secretions is of some interest. In recent years various important components in tubal fluid have been studied biologically and bio chemically by several groups of workers. The relatively high success rate attained in the previous and present series suggest that tubal fluid contains all of the ingredients prerequisite for fertilization. A number of media listed by Austin (1961b) have been used for culture of mid-cleavage ova so as to permit further development in vitro, and many media have proved successful. The first important improvement in culture techniques for early ova was that of Whitten ( ), who cultivated mouse ova in saline, glucose, and egg albumin for a period of 48 hours, securing development up to the blastocyst stage from the 8-celled stage, and then he added calcium lactate or sodium lactate to the medium. More recently, he has attained more success with two-celled ova by incorporating L(+)-

4 1966 In Vitro Cultivation of Rabbit Ova 419 lactic acid. McLaren and Biggers (1958) have successfully transferred mouse ova cultivated for a period of 48 hours in simple, chemically defined media. Purshottam and Pincus (1961) reported the study of rabbit and mouse ova cultivated in certain synthetic media and concluded that fertilized rabbit and mouse ova could be cleaved up to the morula stage in Eagle's basal medium without serum and grown up to the blastocyst stage with 10% dialyzed horse serum. Brinster (1963) and Gwatkin (1963) used a modified Krebs-Ringer bicarbonate solution containing sodium lactate and crystalline bovine albumin, and several thousand two-celled mouse ova were cultivated successfully in vitro into normal blastocysts. Recently, Mintz (1964), in the mouse, has successfully used a medium consisting of 50% fetal calf serum and 50% Earle's balanced salt solution with lactic acid. Lutwak-Mann (1962) de monstrated that tubal and uterine fluids contained significant amounts of bicarbonate. Indeed, lactic acid, bicarbonate, and pyruvate are important Fig. 3. Ova after culture for about 70 hours after in vitro insemination showing over 16-cell stage. factors for the early development of fertilized ova (Vishwakarma 1962 and Braden 1963). Brinster (1965a, 1965b, 1965c) has reported that energy for development of two-celled mouse ova could be supplied by lactate, pyruvate, oxaloacetate, or phosphoenolpyruvate. Alternating the environmental conditions of developing zygotes may also effect the rate of cleavage. In vivo, rabbit ova can usually develop to the stage of blastocyst in about 70 hours after mating. But, in the present ex periment, the cleavage rate was slightly delayed. If various thioamino acids are added to the medium, cell division of rabbit zygotes in vitro has been shown to proceed normally (Pincus 1937, Pincus and Werthessen 1938, Miller and Reimann 1940). However, Medium 199, which we used in this experiment, is a Hanks' balanced salt solution enriched with many kinds of amino acids, various vitamins, and several other ingredients. In addition, lactic acid, which has been proved to be one of the most important exogenous energy sources, was included in the medium. It was impressed that delay in cleavage which had been seen in the present experiment, might be dependent on the delay in the first cleavage. Cytologia 31,

5 420 S. Suzuki Cytologia 31 It is not yet clear what components in tubal fluid are most important for the fertilization phenomenon and also for the early development of fertilized ova. These problems are worthy of continued attention in the future, through the study of in vitro fertilization. To attempt the transfer of cultivated ova in the stage of morula to the uterine horns of pseudopregnant rabbits is still in progress. Summary A total of 207 rabbit ova were inseminated in vitro in tubal fluid under mineral oil using capacitated spermatozoa recovered from the uterus 12 hours after mating. About 20 hours after the transfer for cultivation, 123 (59.4%) of them were at the 2- to 4-cell stages. Sixty-five of the fertilized ova were cultivated for two or three days after insemination in vitro, and 21.5% of them could be reached at over the 16-cell stage. Acknowledgment Grateful acknowledgment is made to Professor Mastroianni of the Department of Obstetrics and Gynecology, School of Medicine, University of Pennsylvania, Philadelphia, for his constant interest and guidance in this investigation. References Austin, C. R. 1961a. Fertilization of mammalian eggs in vitro. Int. Rev. Cytol. 12: b. The mammalian egg. Blackwell, Oxford. p Fertilization and Transport of the Ovum. In: Mechanism Concerned with Con ception. Ed. by C. G. Hartman. The Macmillan Company, New York: p Brachet, A Recherche sur le determinisme hereditaire de l'oeuf mammiferes de velopment "in vitro" de Jaunes vesicules blastodermique de lapin. Arch. Biol. 28: 477. Braden. A. W. H Properties of the membranes of rat and rabbit eggs. Aust. J. Sci. Res. 5: 460. Brinster, R. L A method for in vitro cultivation of mouse ova from two-cell to blastocyst. Expt. Cell Res. 32: a. Studies on the development of mouse embryos in vitro. I. The effect of osmo larity and hydrogen ion concentration. J. Exp. Zool. 158: b. Studies on the development of mouse embryos in vitro. II. The effect of energy source. J. Exp. Zool. 158: c. Studies on the development of mouse embryos in vitro. III. The effect of fixed nitrogen source. J. Exp. Zool. 158: 69. Chang, M. C The maturation of rabbit oocytes in culture and their maturation, fertilization and subsequent development in the fallopian tubes. J. Exp. Zool. 128: 379. Gwatkin, R. B. L Effect of viruses on early mammalian development. I. Action of meningo encephalitis virus on mouse ova culture in vitro. Proc. Nat. Acad. Sci. 50: 576. Lewis, W. H. and Gregory, P. W Cinematographs of living developing rabbit eggs. Sci. 69: 226. Long, J. A III. The living eggs of rats and mice, with a description of apparatus for obtaining and observing them. Univ. Calif. Pub. Zool. 9: 105. Lutwak-Mann, C Some properties of uterine and cervical fluid in the rabbit. Bio

6 1966 In Vitro Cultivation of Rabbit Ova 421 chem. Biophys. Acta. 58: 637. McLaren, A. C. and Biggers, J. D Successful development and birth of mice culti vated in vitro as early embryos. Nature 182: 877. Miller, B. J. and Reimann, S. P Effect of KL-methionine and L-cysteine on the cleavage rate of mammalian eggs. Arch. Path. 29: 181. Minz, B Formation of genetically mosaic mouse embryos, and early development of lethal (t12/t12)-normal mosaics. J. Exp. Zool. 157: 273. Nicholas, J. S. and Hall, B. V Experiments on developing rats. II. The develop ment of isolated blastomeres and fused eggs. J. Exp. Zool. 90: 441. Ohnuki, Y A phase microscopy study on the morphological and structural changes in living hamster eggs during ovulation, fertilization and early cleavage. Cytologia 24: 348. Pincus, G Observations on the living eggs of the rabbit. Proc. Roy. Soc., ser. B. 107, The metabolism of ovarian hormones, especially in relation to the growth of the fertilized ovum. Cold Spring Harbor Symposia Quant. Biol., 5: and Wethessen, N. T The comparative behavior of mammalian eggs in vivo. III. Factors controlling the growth of the rabbit blastocyst. J. Exp. Zool. 78:1. Purshottam, N. and Pincus, G In vitro cultivation of mammalian eggs. Anat. Rec. 140: 51. Smith, A. U Cultivation of rabbit eggs and cumuli for phase-contrast microscopy. Nature 164: Suzuki, S. and Mastroianni, L In vitro fertilization of rabbit ova in tubal fluid. Am. J. Obst. & Gynec. 93: 465. Vishwakarma, P The ph and bicarbonate-ion content of the oviduct and uterine fluids. Fertil. Steril. 13: 481. Whitten, W. K Culture of tubal mouse ova. Nature 177: Culture of tubal ova. Nature 179: *