Serial Culture Is Critical for In Vitro Development of Parthenogenetic Embryos in the Golden Syrian Hamster

Abstract

Unlike oocytes of many other mammalian species, parthenogenetically activated hamster oocytes have not been reported to develop beyond the two-cell stage. This study investigated the in vitro development into blastocysts of parthenogenetic embryos of Golden Syrian hamsters. We observed that hamster oocytes could easily be artificially activated (AA) by treatment with ionomycin plus 6-dimethylaminopurine + cycloheximide + cytochalasin B as assessed by embryo cleavage in HECM-9 (63.15%) or HECM-10 (63.82%). None of the cleaved embryos developed beyond the two-cell stage when cultured in either of the two media. However, some of the embryos overcame the two-cell block and developed to the blastocyst stage (26.45%) when they were first cultured in HECM-10 for 24 hours and then in HECM-9 (serial culture media HECM-10-9) for 72 hours. Blastocyst development was further significantly (66.2%) improved when embryos were cultured in HECM-10 supplemented with ethylenediaminetetraacetic acid for 24 hours, then in HECM-9 supplemented with glucose for 72 hours (serial culture media HECM-11a-b). Hamster oocytes activated with ionomycin, ethanol, or a combination of the two treatments would develop to the blastocyst stage in serial culture media HECM-11a-b, whereas none of the spontaneously activated oocytes cleaved (0% vs. 86.93%, p < 0.05). DNA and microtubule configurations of spontaneously activated and AA oocytes were assessed by immunocytochemical staining and fluorescence microscopy. The results indicate that serial culture and the method of activation are critical for overcoming the in vitro developmental block of hamster parthenogenetic embryos. This study is the first to report blastocyst development from parthenogenetically activated hamster oocytes.

Keywords: blastocyst; hamster oocytes; parthenogenetic activation; serial cultural media.

FIG. 1.

Flowchart of experimental design.

FIG. 2.

Representative figures of spontaneously activated hamster oocytes compared with those of bovine oocytes. After 3 hours incubation in vitro, 59.0% (n = 39) of hamster oocytes were spontaneously activated, and 100% (n = 42) of the oocytes were activated after 6 hours incubation. The figures show a spontaneously activated hamster oocytes at anaphase II (3 hours) and an oocyte at the pronuclear stage (6 hours). None of the mature bovine oocytes were spontaneously activated after either 3 (n = 22) or 6 hours (n = 35) incubation. Figures show two bovine metaphase II oocytes (3 and 6 hours).

FIG. 3.

Representative figures of blastocysts produced by artificial activation. Oocytes were activated by ionomycin plus 6-DMAP + CHX + CB treatments. Parthenogenetic embryos were incubated in serial culture media HECM-11a-b. Blastocysts were photographed under bright field (A) or UV light after DNA staining with Hoechst (B). (A) is a group of early or expanding blastocysts at 84 hours of in vitro culture. (B) represents the cell nuclei of a blastocyst. Details of the preparation are described in Materials and Methods section. CHX, cycloheximide; 6-DMAP, 6-dimethylaminopurine; CB, cytochalasin B.

FIG. 4.

Representative figures of the DNA and microtubule status in the early stages of artificially activated hamster oocytes. Before activation, a metaphase II stage oocyte has chromosomes with condensed DNA arranged on the spindle (0 hours of activation). At 45 minutes postactivation, oocytes are at anaphase of the second meiosis. The chromatids are pulled by microtubules to separate and move to opposite poles (45 minutes). As the activated oocytes progress, DNA is now becoming decondensed, microtubules disappear, and two pronuclei form (1.5, 3 hours). Then the two pronuclei move toward each other to form a diploid nucleus (9 hours).