2 A. Pavlok et al. Animal Reproduction Science 64 2000 1–11
directly after isolation, reached M II after 48 or 72 h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles.
© 2000 Elsevier Science B.V. All rights reserved.
Keywords: Cattle-reproductive technology; Meiosis; Oocyte; Butyrolactone I; 5-
3
H-uridine; Autoradiography
1. Introduction
The meiotic competence of the mammalian oocyte develops in close correlation with follicular growth. The formation of the antral cavity seems to be the stage at which the rodent
oocytes acquire their full size and concomitant with their meiotic competence Erickson and Sorensen, 1974. In cattle, the small antral follicles less than 1 mm in diameter possess the
growing oocytes usually not exceeding the size of 110 mm. Only a limited portion of these oocytes resume meiosis in vitro for review see Motl´ık, 1989; Fair et al., 1995. The fully
grown oocytes about 120 mm in size with both, meiotic and developmental competence could be obtained from oocytes above 3 mm in size Pavlok et al., 1992; Fair et al., 1995.
The growing bovine oocytes synthesize very intensively ribosomal RNA rRNA and the nucleolar morphology is characterized by fibrillogranular elements, fibrillar centers and
discrete vacuoles. In oocytes, the gradual acquisition of their full size is accompanied by the distinct compactisation of nucleolar fibrillar material and formation of a single,
large fibrillar center Crozet et al., 1986. These changes are accompanied by termination of rRNA synthesis and reduction of heterogeneous nuclear RNA hnRNA synthesis. The
above mentioned morphological phenomena are correlated not only with the size of follicles and oocytes but also with the meiotic and developmental competence of oocytes cultured
in vitro Pavlok et al., 1992, 1997.
To simulate more precisely the in vivo maturation pattern of fully grown bovine oocytes, a two-step in vitro culture protocol was tested. In the first step, the oocytes were cultured in
a meiosis-inhibiting medium Lonergan et al., 1997; Avery and Greve, 1997; Saeki et al., 1997; Motl´ık et al., 1998 to prolong the time of the transcriptional and translational activity
specified for the GV stage Kastrop et al., 1990; Pavlok et al., 1997. Hyttel et al. 1997 proposed for this first period of culture under the technical term “capacitation”. After this
meiosis-inhibiting period, the oocytes are transferred for 24 h to the classical maturation medium.
A special prolonged culture protocol for in vitro growth and subsequent maturation of oocytes from preantral follicles was first used in a mouse Eppig and Schroeder, 1989. The
subsequent fertilization, embryo culture and their transfer resulted in some newborn mice. Quite recently, Yamamoto et al. 1999 cultured bovine oocytes isolated from early antral
follicles by the modified method of Harada et al. 1997 and obtained a new born calf after their fertilization and embryo transfer to a recipient. A new methodological approach to the
culture of ovine early antral follicles was developed by Newton et al. 1999. However, the proportion of successfully maturing oocytes was still very low.
In this study, RNA synthesis of bovine meiotically competent and incompetent oocytes was analyzed before and after their culture in a meiosis-inhibiting medium with butyro-
lactone I BL I as already described by Motl´ık et al. 1998 and Kubelka et al. 2000.
A. Pavlok et al. Animal Reproduction Science 64 2000 1–11 3
Concomitantly, ultrastructural changes of nucleoli from meiotically incompetent oocytes were followed. To increase the meiotic competence of oocytes from 1 to 2 mm follicles S
category, the two-step culture, in the meiosis-inhibiting medium with BL I and subse- quently in the meiosis-stimulating medium was tested.
2. Material and methods
