Fertilization of Chinese Hamster Ova in Vitro and in Vivo and Their Subsequent Development in Culture

BIOLOGY OF REPRODUCTION Fertilization 39, 409-418 of Chinese TERRY (1988) Hamster Ova in Vitro and in Vivo Development in Culture A. PARKENING1 Departments and PAULINE of Anatomy and Their Subsequent L. CISNEROS2 & Neurosciences and Obstetrics University and Gynecology of Texas Medical Texas Branch 77550-2 772 ABSTRACT Oocytes (70% mented of from 112 with superovulated Chinese ova) or a modified 4 mg/mI of bovine hamsters designated serum albumin. can be fertilized in vitro using the culture medium BWW as MBWW (76% of 122 ova) when either medium is suppleOva fertilized in vitro will also cleave to the 2-cell stage in BWW either medium (52% in BWW, 87% in MBWW), but fail to develop any further in culture. Oocytes fertilized in vivo and recovered at the late 2-cell or early 4-cell stages from females on Day 3 of pregnancy have the capacity to develop into expanded blasto cysts in MBWW. When early embryos that developed into moruiae and early blastocysts in culture were transferred to surrogate females, eight normal young were born. INTRODUCTION The Chinese commercially laboratory smaller hamster available animal since animal (mean cellular (Cricetulus in the United 1952 (Yerganian, SEM ± has griseus) weight, been = 36.0 The ± tory 0.6 g, males = 41.4 ± 0.7, n 24) than the Syrian or golden hamster, it has not attained the same popularity as the Syrian hamster for scientific studies. This is - probably due the female presented mating in large Chinese means of of this species part of the aggressive nature Several papers the male during (Schwentker, Weihe, 1967; Porter and that may have restricted relatively number to hamster. protecting 1957; required 1967). of breeding maturity Syrian hamster has animal for reproductive little research has however, regarding have the Bel#{233}i#{233} and embryonic The species low become a popular laborastudies since zona-free in culturing other than determine for such fertilization (8-12 attempts blastocyst Scientifically, the Chinese hamster is probably most noted for its line of ovarian cells (CHO cells), which are cultured in vitro and widely used for Since there early embryos the mouse, it the potential use of purposes. The present of Chinese hamster subsequent testing including 1983). fertility in humans Relatively been done with the Chinese fertilization of oocytes development. success species, Lacey, 1969). Other factors the use of this species is the for studies. hamster oocytes can be used for many vertebrate male species, (Yanagimachi et a!., 1976; Rogers, small litter size (4.8, Avery, 1968), small litters (4.9, Parkening, 1982) and the length of time wk, Yerganian, cytogenetic chromosome number (2n = 22) and the ability to readily distinguish one chromosome from another have made it a good candidate for genetic research. States as a 1958). A females and to has hamster, and early been little of any rodent is of interest to the Chinese hamster study describes the oocytes in vitro and culture early zygotes to the stage. MATERIALS AND METHODS Animals Accepted March 23, 1988. Received December 28, 1987. ‘Reprint requests. 2 Present address: University of partment of Obstetrics and Gynecology, The derived Texas Health Houston, Science Center, TX 77030. Chinese from hamsters stock that Line Company existed at the De- since 409 1976, (Vineland, University is inbred used in this was purchased NJ). of except study from were Chick Our colony, which has Texas Medical Branch for the addition of 12 Galveston, 410 PARKENING males in acquired the fall bred in (sawdust and with Co., was from of Dr. 1984. G. The 29(L) X as litter) Yerganian hamsters 19(W) under (Boston, were AND MA) housed and X 13(H)-cm plastic cages uniform lighting 14L: 1OD temperature (21-23#{176}C). They were provided food (Formulab Chow #5008, Ralston Purina St. Louis, MO) and water ad libitum. Oatmeal provided 1-2 times weekly to supplement their diet. CISNEROS exuding sperm was medium incubator under oil had equilibrated gassed with 5% CO2 addition of perse for sperm. The new 200-tl /ml, Various hamsters protocols from 1-6 were used to induce ovulation in mo of age. The method yielding h after the diluted tate, the superovulated the consistent results oviducts IU pregnant animals 1-2 mare’s mo old later, 5 IU human i.p., and the animals after the injection yielded 15-30 Culture Medium The serum (15-2 i.p. injection of 3 gonadotropin (PMSG) in 3 gm). Forty-eight to 50 h chorionic gonadotropin were killed between of hCG. These was given 15 and 16 h procedures generally medium (Table 1) was a modification medium known developed as BWW) standard egg culture et al. (1971) (commonly culturing raising early mouse embryos. It was modified by the sodium pyruvate (Sigma #P-2256, Sigma Co., St. previously (1977) and by Louis, done lowering MO) by from Basler the sodium 0.028 and lactate by for to 0.040 R#{246}hrborn (Sigma #L-1375) from 2.416 to 2.03 5 g/l. Crystalline bovine serum albumin (BSA; Sigma #A-2153) was added at 4.0 mg/ml. The medium will be designated as modifiedBWW (MBWW) throughout this paper. The components of the medium were either purchased from Sigma Chemical Co. or Fisher Scientific Co. (Fair Lawn, NJ). The chromatography Inc. (Paris, KY). with the various medium was In Vitro Fertilization water a manner was high-performance liquid grade obtained from Malhinckrodt, After equilibration in the incubator gassing mixtures, the pH of the 7.3-7.5. A 5- to 8-mo-old the cauda epididymidis as to proven-fertile removed prevent than removed sterile filter oil in the under oil and paper. culture carefully Under a sterilized ampulla needles of each were oviduct, blotted were then containing dissecting in than 5X on placed the hold and the ova 2 to capaciand their a piece of in paraffin capacitated microscope, used to allowing dis- Approximately had been allowed hamsters were killed They dish sperm. pipetted higher 1 X 106 /ml. sperm to then (equilibrated in concentrations lower allowed and two heat- rupture within the their examination. of the Biggers Chemical g/l, as manner) but mm cumulus mass to exude into the oil. Each egg mass was then pushed into the droplet of medium containing sperm. The gametes were incubated for 7-10 h, and then the ova were removed and processed for oocytes/animal. culture fertiliza- blood male was killed and by dissection in such from adhering to the tissue. While the epididymidis was held with a fine forceps, a small incision was made with a pair of iridectomy scissors, and a droplet of the freshly Examination Each of Ova ovum pipetting adhering placed was several washed times to in fresh culture remove any med (...truncated)