Timing and regulatory aspects of oocyte maturation in vitro in the tammar wallaby (Macropus eugenii)

Abstract

Oocytes from a marsupial, the tammar wallaby (Macropus eugenii), resemble those of eutherian mammals in their ability to resume meiosis in vitro when cultured under suitable conditions. Culture for 42–48 h in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal calf serum, and 10 g mL ^–1 porcine luteinizing hormone (pLH) was required in order for oocytes, collected from the large antral follicles (> 2 mm diameter) of tammar wallabies (primed with 6 mg of porcine follicle stimulating hormone twice daily for four days), to proceed to metaphase II (MII) of meiosis. Under these conditions, chromatin condensation was observed within 4–8 h of culture in 61% of oocytes; metaphase I (MI) chromosomes were observed from 18–30 h of culture (66%); and most oocytes (76%) progressed to MII by 42 h in vitro. The addition of cycloheximide, a protein synthesis inhibitor, at concentrations of 1–100 g mL ^–1 , prevented maturation of tammar wallaby oocytes in vitro. This effect was reversible, as oocytes washed free of cycloheximide after 4 h of incubation were able to progress to MII. The addition of cycloheximide to wallaby oocytes at MI of meiosis prevented normal progression to MII suggesting that proteins critical for nuclear maturation are synthesized throughout the maturation process. Genistein, a protein kinase inhibitor decreased maturation of wallaby oocytes in a dose dependent manner. However, the concentration required to significantly inhibit maturation of wallaby oocytes (60 g mL ^–1 ) was greater than that required for eutherian species. Most wallaby oocytes were able to undergo germinal vesicle breakdown (GVBD) in the presence of high concentrations of genistein but produced abnormal chromatin configurations and were unable to progress to MII. Future studies will examine whether cytoplasmic changes occur in marsupial oocytes in vitro and their temporal relationship to nuclear maturation.