Hypoxanthine Causes a 2-Cell Block in Random-Bred Mouse Embryos

BIOLOGY OF REPRODUCTION 37, 311-3 Hypoxanthine 16 (1987) Causes a 2-Cell D. LOUTRADIS, Departments of Block in Random-Bred D. JOHN, Obstetrics, and and Medical Reproductive Biology School Massachusetts Boston, Embryos’ A. A. KIESSLING2 Gynecology, Harvard Mouse 02115 ABSTRACT Ham’s F-lU, a chemically as well bred Swiss mice contrast, 85% and added Ham’s as animal BWW, 100% (CD-i), the random-bred ethylenediaminetetraacetic CD-i but a simple, of 2-cell to the BWW F-b components from defined, oocytes, mothers did modified embryos was not block affected by medium, at the development of and of the into to blastocysts 1949) Kreb’s- or in Ringer block experiments was the paternal and Biggers, 2-cell stage sequent complex rarely that 2-celled when the with lactate Brinster, from 2 1963), cells procedure in from medium partially showed to embryos culture to laboratories. 1-celled developed simple medium was or pyruvate (Whitten, allowed the blastocysts many the mainly of Mc Laren at zygote to the is generally blocked (Whitten and by Accepted February 25, 1987. Received December 1, 1986. ‘This work was supported in part a NATO Fellowship award to DL. 2 Reprint and requests: Reproductive Building, 250 A. Biology, Longwood A. Ave., Kiessling, Harvard Boston, by Depts. Medical MA USPHS into 1970, 1982; 1985), medium mouse mice development As little as 15% of random- (BDF1). to blastocysts (v/v) Ham’s Supplementing the for the developmental in of F-JO BWW with block to mothers) and hybrid-inbred in Ham’s F-b (Ham, used for in vitro human embryos (BDF2) 1963), a fertilization (Edwards and et a!., 1980; Trounson et al., 1981; Laufer et a!., 1984; Quigley, and in BWW (Biggers et a!., Lopata 1985; 1968), et a!., Veeck, a simple in general embryos. development use for MATERIALS stage 1968; #HD16561 in vitro of C. and Female strains: mice were CD-i (Charles AND METHODS Breeding Swiss hybrid 5- to 10-wk-old virgins River, Wilmington, strain, of two of two MA), a and BDF1 (Jackson Labs, inbred strains, C57B1/6 X DBA/2J. Breeding males (5 to 50 weeks old) were of the same two strains and sources as the females, or were C57B1/6 (Jackson Labs, ME) males of the same age range. Females were superovulated with intraperitoneal injections of 5 IU pregnant mare’s serum gonadotropin (Sigma Chemical Co., St. Louis, and MO), followed in 48 h with 5 IU human chorionic gonadotropin (hCG; Sigma Chemical Co.), and were housed individually overnight with males. The presence Gynecology, Seeley Animals random-bred ME), an Fl Breeding and Pratt, (Muggle- of Obstetrics, School, from develop- Biggers, grant fertilization (40%) reversed by adding the chelating agent, that the hypoxanthine sensitivity of embryos complex medium early cleavage of sup1957; blastocyst Biggers, 1971; Shire and Whitten, 1980). experiments (MacLaren, 1981; Goddard 1983) and cytoplasmic transfer experiments in vitro of embryos hybrid-inbred embryos. responsible the Sub- in vitro remains limited to certain inbred strains of mice and several F-i hybrids; embryos from randombred strains explanted at the 1-ce!! stage undergo the first cleavage to the 2-ce!! stage, but further development from bred (from CD-i embryos cultured of mouse embryos become a routine However, for 92% ton-Harris et a!., 1982) have shown that the “2-cell block” is due to oocyte cytoplasmic factors and that it is not influenced by the paternal genome. We have examined embryo development of random- in (Hammond, but embryos explanted underwent a second cleavage. 1958), reports blastocysts plemented ment in of> genome. develop fertilized mouse ova that mouse morulae developed simple medium consisting bicarbonate (Whitten, 1956; used stage embryos random-bred (6-30 p.M) was INTRODUCTION Early attempts vitro demonstrated commonly 2-cell bicarbonate medium, supported BDF1 females, respectively. development that hypoxanthine The hypoxanthine acid. Breeding not culture development Kreb’s-Ringer from CD1 blocked the revealed embryos. complex blocked Mudd 02115. 311 human 312 LOUTRADIS of a copulation post-hCG) females plug the identified were always following Day bred females exception were of usually bred one experiment females were bred bred to to C57B1/6 morning (17-18 1 of pregnancy. to BDF1 males; to CD-i ET h BDF1 CD-i BDF1 males, with the in which some CD-i males and others were males. AL. concentration in Ham’s F-bO; 6) hypoxanthine (Sigma Chemical Co.) was added at 30 tiM, concentration in Ham’s F-b, or at the concentration in 10%, BSA, as BSA, 15%, or indicated. plus described the buffered saline (DPBS, were collected from Dulbecco’s phosphate- Grand Island Biological Co., Grand Island, NY) with 4 mg/mi bovine serum albumin (BSA, Frac. V; Sigma Chemical Co.) and were treated 1-2 mm with 67 IU/ml hyaluronidase (Sigma groups Chemical of 12-23. Co.), rinsed twice, and Embryos with 2 polar cultured bodies in and were prepared as described fresh (Biggers biweekly. et a!., NY) and 23 mM adjusted to 280-285 Na bicarbonate. milliosmols. Components BWW + BSA Ham’s F-b were following groups: of in the ions, FeSO4, 3jiM;CuSO4, (Sigma Chemical Co.); BWW 1968) 2) Osmolality was added to the 1) heavy metal iOnM;ZnSO4, amino acids, lOOnM BME non- and essential amino acids (Grand Island were added in concentrations equivalent F-b formulation, with the exception of (absent the from Ham’s concentration leucine, lysine, F-b), glutamine (1-10 F-b), and and isotyrosine, in Ham’s methionine, which were at 0.2-2 times the concentration in Ham’s F-10; 3) vitamins, BME vitamins were added at 0.3-3 times the concentrations in Ham’s, with the exception of biotin, which was 40 times the concentration; thymidine 4) BWW BWW + + components collected at Caesarean any serum. Culture at CO2 = 5% and was heated used protein for to 56#{176}C for embryo supplementation media in air, were or 95% either with pre-equilibrated 37#{176}C, 30 mm. culture 10% overnight humidity with pH 7.35-7.44. The results and were Fisher’s statistically Least analyzed Significant by Student’s Difference Test. RESULTS with freshly distilled Type 1(18 megaohm) water and BSA added at 4 mg/ml prior to sterile filtration. Ham’s F-b (Ham, 1963) (powdered, Grand Island Biological Co.) was prepared with freshly distilled, Type I water, and supplemented with i.35 mM calcium lactate (Calbiochem-Behring Corp., San Diego, CA), 0.095 mg/ml penicillin (Pfizer, New York, essential Biological) to Ham’s of blood, Culture Culture media formulated times leucine, F-b without t-test cystine cord filter), Ham’s to culture was fetal nitrocellulose The Statistics Embryo groups five in was s (...truncated)