morula stages and at Day 5, all embryos reached the blastocyst stage. ... mastomys developed to the blastocyst stage in WM (13%, 13/71), indicating the ...
Journal of Reproduction and Development, Vol. 43, No. 1, 1997
Preimplantation Embryo Development in Mastomys (Praomys coucha) In Vivo and In Vitro Atsuo OGURA, Junichiro MATSUDA, Keiji MOCHIDA, Osamu SUZUKI, Kazuhiro TAKIMOTO, Kazue NAKAYAMA and Masaharu NAIKI Department of Veterinary Science, National Institute of Health, 1-23-1, Toyama, Shinjuku, Tokyo 162, Japan
Abstract. Mastomys (Praomys coucha) is a small murine rodent native in Africa. The present study was undertaken to investigate the developmental time course of mastomys embryos in vivo and the rate of development in vitro. Mastomys embryos developing in vivo exhibited a long 2-cell stage, which lasted from late Day 1 through Day 3. At Day 4, most embryos developed to the 8-cell or morula stages and at Day 5, all embryos reached the blastocyst stage. The attachment of embryos to the endometrium began at Day 5, as revealed by histological examinations. The development in vitro was investigated in different media with three inbred strains and an F1 hybrid. When 1-cell embryos from inbred mastomys were cultured in Whitten’s medium (WM), none of them (n=61) developed beyond the 2-cell stage. Embryos of the same strains collected at Day 2 (2-cells) reached the blastocyst stage (46%, 31/67) in the same medium. Meanwhile, 1-cell embryos from F1 hybrid mastomys developed to the blastocyst stage in WM (13%, 13/71), indicating the presence of a strong heterosis effect. CZB medium, which lacks glucose and contains glutamine and EDTA, supported the development of 1-cell inbred embryos to the blastocyst stage (24%, 17/70). Supplementation of glutamine, but not EDTA, to WM stimulated the development of 1-cells to the blastocyst stage (22%, 11/51). These findings indicate that the development of mastomys embryos in vitro is both strain and medium dependent and that glutamine may play a crucial role in early development of mastomys embryos. Key words: Mastomys, Embryo culture, Glutamine. (J. Reprod. Dev. 43: 65–71, 1997)
(the multimammate mouse, Praomys M astomys coucha) is an African rodent which is intermediate in size between the mouse and rat. This species provides a good model for cancer research because it develops a wide variety of neoplastic lesions [1]. Mastomys has also received attention as a new laboratory rodent with great phenotypic variations found in many organs [2–4]. In the field of reproductive biology, mastomys is probably most noted for the presence of a well-developed Accepted for publication: December 18, 1996 Correspondence: A. Ogura
ventral prostate in the female and an excessive number (7–9 each side) of nipples (the origin of its common name) [5]. It shows as good a reproductive performance as the laboratory mouse and rat. Under our conventional breeding condition with a photoperiod of 14 h/10 h (light/dark), female mastomys have relatively irregular 5- to 7-day estrous cycles and 25- or 26-day gestation. As they usually exhibit a fertile postpartum estrus, most strains of mastomys can be efficiently propagated. Little research has been done with mastomys, however, regarding early embryos. It is important to know the characteristics of preimplantation development
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in vivo and in vitro to determine the potential use of mastomys for study of developmental biology. In this study we investigated the developmental time course of mastomys embryos in vivo and their developmental ability in vitro.
Materials and Methods Animals Three inbred strains of mastomys, MWC (wild color) [2], MST (wild color) [3], MCC (diluted coat color and pink eyes) [4], and F1 hybrid mastomys (MST × MCC) were used in this study. Females were 60–150 days of age, and males 100–250 days of age. Animals were kept under a controlled lighting condition (lights on 0700–2100 h). They were provided with water and commercial laboratory mouse chow ad libitum. Culture Whitten’s medium (WM) [6] and CZB [7] were used for embryo culture. Bovine serum albumin (BSA fraction V; Sigma Chemical Co., St. Louis, MO, U.S.A.) was added immediately before each experiment (3 mg/ml). All reagents were of analytical grade (Wako Pure Chemicals, Osaka, Japan) and solutions were prepared using a Milli-Q reagent water system (Millipore Co., Bedford, MA). Collection of embryos Induction of superovulation is relatively easy in this species [8], but the superovulation treatment often causes a low incidence of successful mating and a decreased fertilization rate in vivo. In vitro fertilization using mature oocytes and epididymal spermatozoa is extremely difficult in mastomys [8]. To obtain preimplantation embryos of mastomys, therefore, females without any gonadotrophin administration were used. Females were placed into cages of fertile-proven inbred males, with one or two females to one male. Vaginal smears were examined every morning and the day on which spermatozoa were found was designated Day 1 of pregnancy. Plugs occasionally remained in the vagina, but often spontaneously dropped before examination of the vagina. When females were at 60–80 days of age, they usually mated with males within a week after caging together. Older females showed a lower incidence of successful mating. At 1500 h of each day of pregnancy (Day 1–5),
females were sacrificed with an overdose of ether and cervical dislocation. Embryos were collected from the oviducts and/or uteri by flushing with WM. Embryos were cultured in a 100- µl drop of the medium overlaid with liquid paraffin (Merck, Darmstadt, Germany) in a plastic culture dish. They were incubated at 37 C in 5% CO 2 in air.
Statistical analysis One-way ANOVA or Fisher’s exact probability test was carried out with Stat 123 for Windows. Differences with a probability of P
