Fertilization and cleavage of mouse oocytes exposed to the conditions of human oocyte retrieval for in vitro fertilization*

Transcription

1 FERTILITY AND STERILITY Copyright <> 1989 The American Fertility Society Vol. 51, No.4, April1989 Printed in U.S.A. Fertilization and cleavage of mouse oocytes exposed to the conditions of human oocyte retrieval for in vitro fertilization* Katharine V. Jackson, B.S. Ann A. Kiessling, Ph.D. t Department of Obstetrics, Gynecology and Reproductive Biology and Laboratory for Human Reproduction and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts Ova from two strains of mice (a hybrid-inbred strain, BJ) 2F1o and a random-bred strain, CDl) were shocked by exposure to environmental conditions possibly encountered by human oocytes retrieved for in vitro fertilization (IVF). Shocked and control mouse ova were fertilized in vitro in either simple or complex media and zygote development to morulae and blastocyst stages compared with that of zygotes fertilized in vivo. Development of the hybrid-inbred zygotes following fertilization in the simple media of shocked and control ova was essentially the same as for ova fertilized in vivo (84 ± 6.6, 89 ± 1.6, 87 ± 6.0% to the 2-cell stage and 89 ± 5.2, 94 ± 2.3, 99 ± 1.0% of two cells to blastocysts, respectively); development in the complex media also was the same following fertilization of shocked and control ova (80 ± 8.7, 90 ± 2.7% to two cells and 33 ± 3.5, 35 ± 4.5% of two cells to blastocysts, respectively) but lower than that of in vivo zygotes (92 ± 4.6 to two cells, 58± 4.7 two cells to blastocysts). In contrast, the fertilization and development in the simple media of shocked and control random-bred ova was lower and more variable (90 ± 5.8, 67 ± 11.1% to two cells and 17 ± 8.3, 42 ± 13.6% two cells to blastocysts, respectively) than the development of in vivo zygotes (96 ± 1.5% to two cells, 51 ± 5.5 two cells to blastocysts). Thus, in these studies, fertilization and development of ovulated mouse ova were more dependent on the strain of mouse and the culture media conditions than on transient environmental shocks encountered before fertilization. Fertil Steri151: 675,1989 Human oocytes recovered for in vitro fertilization (IVF) are subjected to environmental changes not normally encountered by oocytes ovulated spontaneously into the oviduct. The changes, including fluctuations in ph, temperature, and exposure to light, are potentially deleterious to the viability and subsequent developmental potential of the oocytes. Studies in animal systems indicate Received July 25, 1988; revised and accepted November 17, * Supported in part by grants HD and HD from the Public Health Service. t Reprint requests: Ann A. Kiessling, Ph.D., Director, IVF Laboratory, Brigham and Women's Hospital, 250 Longwood Avenue, Room 204, Boston, Massachusetts that phs outside the normal physiologic range decrease successful fertilization and development in mouse oocytes, l-a and that exposure to high levels of light also inhibit development of fertilized hamster oocytes. 4 If the laboratory conditions during insemination and early cleavage of human oocytes are well controlled, the oocyte pertubations would be transitory, limited to the length of time of oocyte retrieval and transfer to physiologically equilibrated culture conditions. That fertilization of human oocytes may be resilient to transient physiologic shock is evidenced by the greater than 60% fertilization in vitro of human oocytes reported by many IVF groups. s-a The relatively low percentage of fertilized oocytes that actually give rise to off-. Vol. 51, No.4, April1989 Jackson and Kiessling Fertilization and cleavage of shocked mouse oocytes 675

2 spring, however, indicate that, although fertilization may occur with human oocytes, subsequent "normal" development applies to only a small percentage of oocytes recovered. A number of factors, including the extent of oocyte maturation before human chorionic gonadotropin (hcg) administration and adequacy of the endometrium, contribute to successful embryo development. However, more precise information about which environmental shocks to otherwise competent oocytes are deleterious to subsequent embryonic development would facilitate planning for oocyte retrieval and patient care with respect to, for instance, the equipment used for oocyte recovery. To begin to understand the sensitivities of unfertilized oocytes, we have systematically examined the outcome of exposure of ovulated mouse oocytes to environmental extremes patterned after the rigors possibly encountered during routine retrieval of human oocytes. Mouse oocytes from two strains of females were exposed to extremes of temperature, ph, and vacuum, then fertilized and monitored for development to the blastocyst stage in vitro. Development of the in vitro fertilized zygotes was compared with development of in vivo fertilized zygotes from the same two strains of females: a random-bred Swiss (CD1) and a hybrid-inbred (B 6D 2F 1) of C57B1/6 XDBA. MATERIALS AND METHODS Superovulation and Embryo and Gamete Collection Zygotes were recovered from superovulated female B 6D 2FdJ mice as previously described.9-11 Ovulated ova were collected for fertilization in vitro essentially as previous described11 from 9- to 16-week-old virgin BaD 2FdJ (Jackson Labs, Bar Harbor, ME) and CD1 (Charles River Breeding Labs, Portage, MI) female mice that were superovulated with intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG; Sigma, St. Louis, MO) followed 48 to 52 hours later with 5 IU human chorionic gonadotrophin (hcg, Sigma). Briefly, the animals were killed by cervical dislocation 13 hours after hcg administration and cumulus-enclosed oocytes were collected into Dulbecco's phosphate-buffered saline (DPBS; Gibco, Grand Island, NY) containing 4 mg/ml bovine serum albumin (BSA; Fraction V, Sigma) at 35"C in. organ culture dishes (Falcon 3037; Fisher Scientific, Boston, MA). The cumulus masses from each oviduct were placed into separate pools and washed twice through DPBS/BSA before being subjected to the test condition. Sperm for each experiment were collected from one mature B 6D 2FdJ male (7 to 52 weeks old) selected from the stud colony and killed by cervical dislocation. Each cauda epididymis was excised, punctured with a small gauge needle, and placed into a 250 ~l microdrop of Ham's F- 10 (HF10, Gibco) containing 4 mg/ml BSA, that had equilibrated for 18 hours in 5.5% C0 2 in air at 37"C under 3.0 ml silicone oil (Aldrich Chemical, Milwaukee, WI) in a Falcon 3001 dish. The sperm were capacitated for at least 1 hour in the microdrop before being mixed with the oocytes. Medium Preparation Ham's F-10 and Earle's balanced salts solution (EBSS) were prepared weekly with freshly distilled type 1 (18 megaohm water) as previously described.s-11 Briefly, the Ham's F-10 (powder, Gibco) was supplemented with 25 mm sodium bicarbonate (Mallinckrodt, Paris, NY) and 1.2 mm calcium lactate (Calbiochem, La Jolla, CA), and the EBSS (powder, Gibco) was supplemented with 22 mmol/l sodium bicarbonate (Fisher), 0.2 mmolf l calcium lactate (Mallinckrodt), 26.4 mmol/l sodium lactate (Sigma), and 0.32 mmolfl sodium pyruvate (Sigma). Penicillin G (Calbiochem), 95 mg/ land streptomycin sulfate (Calbiochem), 50 mg/l also were added to both media. The osmolarities of the Ham's F-10 and EBSS were adjusted to 285 to 295 mosm and 295 to 305 mosm, respectively, by removing 5% of the Ham's F-10 and 7.5% of the EBSS and replacing it with freshly distilled water. The diluted concentrations of the media components are listed in Table 1. The medium designated BWW" is actually more similar to Whitten's. 12 Ham's F-10 also contains amino acids (alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine at 95 ~molfl; methionine, phenylalanine, threonine, and valine at 30 ~mol/l; tryptophane at 3 ~mol/l; tyrosine at 10 ~mol/1; leucine and isoleucine at 20 ~mol/l; lysine at 152 ~molfl; glutamine at 190 ~molfl; and cysteine at 19 ~molfl and vitamins and cofactors (biotin, choline chloride, folic acid, inositol, niacinamide, pantothenic acid, pyridoxine, riboflavin, and thiamine at the concentrations listed (Gibco)), and metallic salts (Cu2(S0 4)a at 10 nm; Fe S0 4 at 3 ~M; ZnS0 4 at 90 nm). The media then were sterile-filtered through 676 Jackson and Kiessling Fertilization and cleavage of shocked mouse oocytes Fertility and Sterility

3 Table 1 Composition of Modified Media for IVF of Human and Mouse Ova a Concentration Component HF-10 EBSS mm(l mm(l NaCl KCl KH2P Na2HP MgS CaCl Ca lactate Nalactate 24.4 Napyruvate Glucose NaH2COs Phenol red Hypoxanthine 0.03 Thymidine BWW mm/l a Values listed are final concentrations of components following osmolality adjustments as described in Materials and Methods. 0.2-micron nylon filters (Nalgene, Fisher), saturated with a 5% C02, 5% 0 2, balance N2 gas mixture, and stored in T75 culture flasks (Corning, Fisher) at 4 oc. For this study, all media used were <2 weeks old. Human fetal cord sera were collected, heat inactivated, and sterile filtered as previously described.10 All cord sera and BSA lots were previously tested with the routine pronuclear stage mouse embryo test employed in this laboratory as a quality control assay for human in vitro fertilization (IVF). Only lots that allowed >85% of the pronuclear embryos to cleave to the 2-cell stage and >53% of the 2-cell embryos to progress to morulae and blastocysts after 92 hours in culture were used; these conditions support >85% development to blastocysts of embryos explanted at the 2-cell stage. The concentrations of cord blood and BSA used in this study were 10% (vol/vol) and 4 mg/ml, respectively. Ten micromolar ethylenediamine tetraacetic acid (EDT A, Sigma) was added to the media where indicated, using a 1:10 dilution of a 0.10 mmol/1 stock made up in type I water. Initially, controls for these experiments with no EDTA were a 1:10 dilution with the Type I water. No change in embryo development was observed with the diluted media. Environmental Shock Conditions All test conditions were conducted on two randomly selected cumulus masses from a pool of three females (six oviducts). Except as noted, all oocytes were subjected to test conditions while in 4 mg/ml BSA in DPBS at ph After exposure to the test conditions, the unfertilized oocytes were transferred into pre-equilibrated insemination dishes. Determinations of ph were performed using a Corning ph/ion meter 150 with a Corning X-EL probe that was calibrated in standard Fisher ph buffers 7 and 8 and National Bureau of Standards ph buffer (ph 7.41), as previously described. 10 Readings of ph were taken in replicate sterile conicals containing 5 ml of media that had equilibrated for at least 12 hours. Alkaline ph conditions were created by using nongassed Ham's F-10 without protein supplement that was equilibrated to 35oC in room air. The ph of these dishes was between 8.43 and The acidic ph conditions were created by using BSA/DPBS equilibrated overnight at 37oC in 5.5% C02/air. The ph in these conditions was 6.46 to The 12 to 15oC temperature shock was achieved by placing the culture dish in a glass chamber suspended in an ice water bath. The temperature of the media in the dish remained between 12oC and 15oC for 30 minutes. The control for the low temperature experiments was a culture dish placed on a 35oC slide warmer. Oocytes were subjected to severe light by placing the culture dish onto the dissecting microscope stage plate and adjusting the mirror to direct all transmitted light from the illuminator (Starlight, AO, Fisher) onto the center of the dish. Controls for the light experiments were parallel culture plates placed on the back of the microscope stage away from the transmitted light. Suction methods were tested using DeLee suction catheters and mucus traps (Argyle, Sherwood Medical, St. Louis, MO) rinsed with 5.0 ml BSA/DPBS and connected to sterile three-way stopcocks (Mallinckrodt). Suction then was applied either using a 20.0 cc syringe (BD, Fisher) or a vacuum pump set for 600 mm Hg. In both cases, the stopcock remained closed until the maximum suction force was achieved. As a control, oocytes were pipetted into a rinsed DeLee catheter for 10 minutes and then removed by pouring into a petri dish (Falcon 3002, Fisher). For the combination test conditions, oocytes were placed in unequilibrated Ham's F-10 (ph 8.43 to 8.7) at 12 to 15oC for 10 minutes followed by 10 minutes of severe light and then suctioned into a DeLee catheter under 600 mm Hg vacuum. The effect of marking pens on the oocytes was tested by applying a 1.0 em strip of black ink (Sharpie, Fisher) around the perimeter Vol. 51, No.4, April1989 Jackson and Kiessling Fertilization and cleavage of shocked mouse oocytes 677

4 of the inner well of the organ culture dishes (outside surface) of both the insemination and growth media culture plates. Embryo Insemination and Culture All inseminations and cultures were conducted as previously described 11 in the center well of organ culture dishes containing 1.0 ml of culture media under 1.0 ml of silicone oil that had been prewashed with Type I water and heat sterilized at llooc for 2 hours. The outer well contained 3.0 ml of culture media (without protein) for humidification. The dishes were equilibrated for 18 to 24 hours in a 5.5% C02/ air incubator at 3TC prior to use. Under these conditions, the phs of the culture conditions in EBSS, Ham's F-10, and Ham's F-10 plus cord serum were 7.2, 7.3, and 7.4, respectively. Cumulus masses were transferred from test plates into insemination dishes using fire-polished, glass 50 ~l micro-pipet (Fisher). All gamete manipulations were performed quickly to insure minimal ph changes in the culture dishes. Five to 15 ~l of the sperm suspension were added to the insemination plates to bring the sperm concentration to 5 to 10 X 10 5 /ml. After 4 hours at 37oC, 5.5% C0 2 in air, the fertilized oocytes were transferred into fresh plates {growth media) and cultured undisturbed (except for the 24-hour observation) for the duration of the experiment. Fertilization rates were obtained by assessing 2-cell embryo development after 24 hours in culture. Embryos were assessed for development to morulae and blastocysts with an inverted phase microscope (magnification = 200X) following 96 hours in culture. RESULTS Our previous studies 9 employed Biggers, Whitten and Whittingham (BWW) as simple culture medium. More recent experiments have shown that a similar simple medium, EBSS, supports equally well the development to blastocysts of pronuclear mouse zygotes fertilized in vivo, as shown in Table 2. All of the hybrid-inbred (B 6D 2F 1) embryos that developed to the morula stage went on to form blastocysts in the simple medium. The inclusion of low concentrations of the chelating agent, EDT A, allowed blastocyst development in the simple medium of nearly all of the B 6D 2F 1 zygotes that cleaved to the 2-cell stage. The development of these zygotes in the complex medium, with Table 2 Development in Simple and Complex Media of Hybrid-inbred and Random Bred Mouse Zygotes Fertilized In Vivo Embryo developmentb Culture conditions" Two-cell Morula Blastocyst Zygotes from B 6D 2F 1 females EBSS/BSA (5) 92 ± ± ± 10.0 (6) 87±6.0 99± ± 1.0 HFlO (23) 87 ± ± ± 4.3 HFlO/BSA (6) 92 ±4.6 70± ± 4.7 HFlO/CS (10) 92 ± ± ± 4.8 Zygotes from CDl females EBSS/BSA (3) 94 ± ± ± 3.5 (4) 96 ± ± ± 5.5 Number in parenthesis is replicates of embryos per culture dish from different pools of embryos recovered from three females. b Numbers (±SE of the mean) are percent of normally appearing oocytes with one or two polar bodies that developed to two-cell embryos in 24 hours and percent of two-cell embryos that developed to morulae (~12 cells) or fully cavitated blastocysts in hours in vitro. or without the added protein, was significantly (f < 0.003) reduced at both the morula and blastocyst stages relative to the simple medium. Development of the in vivo fertilized zygotes from random-bred Swiss females (CD1) was more markedly enhanced (P < 0.001) by the inclusion of the EDT A into the EBSS/BSA culture condition (Table 2). In contrast with the hybrid-inbred embryos, however, not all of the random-bred morulae developed into blastocysts even in the presence of EDT A. (Swiss pronuclear stage embryos were not cultured in the Ham's F-10 because previous studies 9 have shown that they do not develop in the complex medium.) The development of B 6D 2F 1 ova fertilized in vitro in the simple media with EDTA was equivalent to the development of the in vivo fertilized zygotes (Table 3) cultured in parallel conditions. The same was true for the embryos fertilized in Ham's F-10 plus BSA and cultured in Ham's F-10 with no added protein (Table 3). Development in the Ham's F-10 plus cord serum was the lowest and at approximately the same levels whether the oocytes were fertilized in vivo or in vitro. The most marked change in development of in vitro versus in vivo fertilized embryos was in the Ham's F -10 plus BSA condition, in which approximately 60% of the in vivo embryos developed to blastocysts, whereas only 35% of embryos fertilized in vitro in this condition developed to blastocysts. The inclusion of 10 ~M EDTA in the Ham's F-10 plus BSA did not 678 Jackson and Kiessling Fertilization and cleavage of shocked mouse oocytes Fertility and Sterility

5 Table 3 Development of Mouse Ova Fertilized In Vitro in Simple and Complex Media Embryo development' Culture conditions a Fertilization Morulae Blastocysts Ova from B 6D 2F 1 females EBSS/BSA (10) 95± ± ± 3.9 (14) 89± ± ± 2.3 HFlO (6) 89 ± ± ± 5.7 HFlO/BSA (16) 90± ± ± 4.5 HFlO/BSA + EDTA (3) 82 ± ± ± 1.2 HFlO/CS (4) 87± ± ± 7.1 Ova from CDl females (8) 67± ± ± 13.6 EBSS/CS + EDTA (4) 76± 3.8 0± 0 0± 0 HFlO/BSA (4) 84± ± ± 1.25 a Numbers in parentheses are numbers of separate experiments, with 19 to 23 embryos each. Fertilization was measured as a percentage of normal-appearing ova with one or two polar bodies that developed to two cells within 24 hours of insemination. 'Development was scored as described in Table 2. Values are means ± standard errors. have the beneficial effect noted in the EBSS plus BSA culture conditions. The experiments to test the effects of environmental shocks were conducted initially with B 6D 2F 1 ova in Ham's F -10 with BSA. The high vacuum, the solvents in marking pens, alkaline or acidic ph shocks, and exposure to light in DPBS, did not appreciably affect fertilization or development to morulae and blastocysts (Table 4). Combining all of the possible environmental shocks associated with retrieval of human ova (an alkaline ph shock plus cooling and exposure to light and high vacuum conditions) also did not significantly reduce the numbers of B 6D 2F 1 ova that underwent fertilization or development to the morulae stage, in either the Ham's F-10 with BSA or the simple medium, EBSS with BSA and EDTA (Table 4). Studies of the responses of ova from the randombred Swiss mice (CD1) revealed that even in EBSS/BSA plus EDTA, fertilization and cleavage to the 2-cell stage was more variable than with the in vivo Swiss zygotes, although development of ova not exposed to the environmental shock conditions that cleaved to the 2-cell stage was not statistically significantly different (P < 0.3) from the development of in vivo zygotes (Table 3). Development to the blastocyst stage of shocked ova was significantly reduced relative to development of pronuclear stage embryos fertilized in vivo, but not reduced relative to the control in vitro fertilized ova. Although 50% of the normally appearing shocked ova underwent cleavage to the morula stage, only 17% of the 2-cell embryos developed to blastocysts (Table 4). Substituting cord serum for the BSA in the EBSS plus EDT A resulted in no significant difference in fertilization of the CD1 ova, but development was arrested in the premorula stages. Fertilization of the CD1 ova and cleavage to the 2-cell stage also proceeded normally in the Ham's F-10 plus BSA, but in agreement with previous studies, 9 development to morulae and blastocysts was blocked. DISCUSSION As background information for the IVF studies of shocked ova, we have compared the development Table4 Effects of Environmental Shocks on Fertilization and Development of Mouse Ova Embryo development Shock" Fertilization Morulae Blastocysts Ova from B 6D 2F 1 females HFlO/BSA (10) 90± ± ± 4.5 DeLee/no suction (3) 90± ± ± 2.1 DeLee/600 mm Hg (3) 98± ± ± 7.0 DeLee/syringe (3) 95± ± ± 6.1 Black Sharpie pen (3) 98± ± ± 5.4 Alkaline ph shock X lorn (3) 84± ± ± 5.7 Acidic ph shock X lorn (3) 96± ± ± Light in DPBS X lorn (3) 87± ± ± "C in DPBS X lorn (4) 84± ± ± 5.0 Alk ph/15"c/light 600mmHg(4) 80± ± ± 3.5 (14) 89± ± ± 2.3 Alk ph/15"c/light 600mmHg(3) 84± ± ± 5.2 Ova from CDl females (8) 67 ± ± ± 13.6 Alk ph/15"c/light 600mmHg(3) 90± ± 0 17 ± 8.3 a Environmental shocks were conducted as described in Materials and Methods. Fertilization and cleavage was carried out in Ham's F-10 with BSA or in. Numbers in parentheses are replicates with embryos each. Embryo development was scored as described in Table 3. Data are means ± standard errors of the mean. Vol. 51, No.4, April1989 Jackson and Kiessling Fertilization and cleavage of shocked mouse oocytes 679

6 in simple and complex media of in vivo pronuclear stage embryos recovered from hybrid-inbred (B 6 D 2 F 1 ) and random-bred Swiss (CD1) female mice. As indicated by previous reports,9-11 the hybrid-inbred embryos develop in either media, particularly well if high molecular weight protein is omitted from the Ham's F-10 and EDTA is added to the simple medium. The decrease in development to morulae and blastocysts if BSA or cord serum is added to the Ham's F-10 is not understood. We have previously shown 11 that 48 hours of exposure of fertilized B 6D 2F 1 ova to Ham's F-10 containing 10% cord serum substantially decreases embryo viability, although there is no decrease in fertilization and cleavage to the 4-cell stage. Thus, the deleterious effect of this medium on zygotes is manifested at later cleavage stages. We have previously reported9 the in vitro development to blastocysts in BWW of random-bred Swiss zygotes that were formerly considered to block at the 2-cell stage The random-bred embryos develop less well in vitro and are more dependent on the presence of the EDT A. The beneficial effect of low concentrations of EDT A has been previously reported9 14 and may be due to chelation of trace heavy metals (not Fe++, cu++, or zn++)9 contaminating the components of the culture media. Development to morulae and blastocysts of B 6D 2F 1 ova following fertilization in vitro was the same as development of in vivo zygotes in the simple media and in the Ham's F-10 with no added protein. By these criteria, our laboratory conditions for fertilization and the first 12 hours of culture of ova from this strain of females are equivalent to the comparable stages in vivo. That this is not entirely true has been shown by lower fetal weights and fewer live pups born following transfer of in vitro fertilized embryos at the 4-cell stagey The decreased development to morulae and blastocysts following the addition of protein to the Ham's F-10 is consistent with our earlier observations.9-11 Since addition of the same BSA to the simple medium does not have an inhibitory effect, studies are in progress to determine which component of Ham's interacts with the BSA to inhibit development. Development to morulae and blastocysts of the more sensitive CD1 ova following fertilization in vitro also was not significantly different from development of in vivo zygotes in the simple medium, but development was blocked at premorula stages in serum or in Ham's F-10, in agreement with earlier studies.9 The lower overall development of this strain of mice indicates either that the ova and zygotes are fundamentally less viable, or that the laboratory conditions are not optimal for development of this strain from the pronuclear stage. The B 6D 2F 1 ova apparently were not adversely affected by the multiple environmental shock conditions imposed in these studies. The conditions tested were more extreme than those encountered by human oocytes. Since the nature of conducting the environmental shock experiments involved short re-equilibration times for the ova before the sperm were added, it would appear as though any deleterious effects to the ova were quickly compensated once they were transferred to their fertilization conditions. In contrast, the more sensitive random-bred Swiss ova did not tolerate the environmental shock conditions well, resulting in decreased rates of fertilization and blastocyst formation. The overall indications from this study are that the rigors encountered by human oocytes may not adversely affect fertilization and cleavage in some cases. The decreased development of the shocked random-bred Swiss ova, however, suggests the possibility that some human oocytes may be much more sensitive than others to similar environmental shocks. Information about the responses of ova from other mouse strains and other mammalian species will be helpful to fully understand the range of responses possible, the shocks that are the most deleterious, and possibly the intracellular enzyme systems most critical during fertilization and early cleavage. These results also raise the possibility that development to blastocysts of pronuclear stage hybrid-inbred embryos is not the best quality control assay for human IVF programs. Acknowledgments. We wish to thank John Biggers, Ph.D., for helpful discussions, and Richard Crowell, M.S., and Hyuck Dong Han, M.D., for technical assistance. REFERENCES 1. Puissant F, Degueldre L, Buisson L, Leroy F: Effects of carbon dioxide acidification of mouse oocytes before in vitro fertilization, culture, and transfer. Gamete Res 13:223, Miyamoto H, Toyoda Y, Chang MC: Effect of hydrogenion concentration on in vitro fertilization of mouse, golden hamster, and rat eggs. Biol Reprod 10:487, Brinster RL: Studies on the development of mouse embryos 680 Jackson and Kiessling Fertilization and cleavage of shocked mouse oocytes Fertility and Sterility

7 in vitro. I. The effect of osmolarity and hydrogen ion concentration. J Exp Zool158:49, Hirao Y, Y anagimachi R: Detrimental effect of visible light on meiosis of mammalian eggs in vitro. J Exp Zool 206:365, Marrs RP, Saito H, Yee B, Sato F, Brown J: Effect of variation of in vitro culture techniques upon oocyte fertilization and embryo development in human in vitro fertilization procedures. Fertil Steril41:519, Fishel SB, Edwards RG, Purdy JM: Analysis of 25 infertile patients treated consecutively by in vitro fertilization at Bourn Hall. Fertil Steril42:191, Meldrum DR, Chetkowski R, Steingold KA, de Ziegler D, Cedars Ml, Hamilton M: Evolution of a highly successful in vitro fertilization-embryo transfer program. Fertil Steril 48:86, Kiessling AA, Loutradis D, McShane PM, Jackson KV: Fertilization in trypsin-treated oocytes. Ann NY Acad Sci 541:614, Loutradis D, John D, Kiessling AA: Hypoxanthine causes a 2-cell block in random-bred mouse embryos. Bioi Reprod 37:311, John DP, Kiessling AA: Improved pronuclear mouse embryo development over an extended ph range in Ham's F-10 medium without protein. Fertil Steril49:150, Han HD, Kiessling AA: In vivo development of transferred mouse embryos conceived in vitro in simple and complex media. Fertil Steril50:159, Whitten WK, Biggers JD: Complete development in vitro of the preimplantation stages of the mouse in a simple chemically defined medium. J Reprod Fertil17:399, Whittingham DG, Biggers JD: Fallopian tube and early cleavage in the mouse. Nature 213:942, Abramczuk J, Solter D, Koprowski H: The beneficial effect of EDTA on development of mouse one-cell embryos in chemically defined medium. Dev Biol61:378, 1977 Vol. 51, No.4, April1989 Jackson and Kiessling Fertilization and cleavage of shocked mouse oocytes 681