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
2.1. Animals, follicles, oocytes, culture Bovine ovaries, obtained at a local slaughterhouse, were rinsed and collected in physio-
logical saline at 25–30
◦
C. The ovaries were transported to the laboratory within 1 h, rinsed briefly in ethyl alcohol and washed twice in saline. The follicles were dissected and separated
into two categories, medium M 3–6 mm in diameter and small S 1–2 mm in diameter, twice washed in saline and placed into 9 mm Petri dishes with a culture basic medium BM
of the following composition: TCM 199 Sevac, Praha, Czech Republic ×10 conc., 9.4 ml; NaHCO
3
7.5 solution Sevac, 2.1 ml; HEPES acid, 9.5 mM; Na-pyruvate, 1.82 mM; polyvinyl alcohol PVA, 3 mgml
− 1
; penicillin K-salt, 50 IUml
− 1
; streptomycin sulfate, 50 IUml
− 1
; amphotericin B, 125 ngml
− 1
all from Sigma supplemented with deionized and nanopure filtered H
2
O to 100 ml. The oocytes were isolated from the follicles with fine forceps and preparation needle, picked up and transferred to the 500 ml of BM supple-
mented with butyrolactone I Funakoshi, Tokyo, Japan, 100 mM, called meiosis-inhibiting medium MIM and cultured in 4-well Nunclon dishes Roskilde, Denmark under min-
eral oil Serva, Heidelberg, Germany for either for 24 or 70 h. For the second experi- ment the same MIM was supplemented with BSA Serva, 3 mgml
− 1
. For the second step of the culture, the COC were washed four times in BL I free BM and cultured 24 h in
meiosis-stimulating medium with gonadotropins MSM Pavlok et al., 1997. The control group of COC of S category was cultured directly after isolation in MSM for 48 and 72 h,
respectively. All cultures were carried out at 39
◦
C, in atmosphere of 5 CO
2
, 10 O
2
, and 85 N
2
. Instat software was used for statistical evaluation GraphPad Software. The effect of BL
I on meiotic maturation was evaluated by χ
2
analysis with Yates continuity correction. 2.2. Labeling of oocytes, autoradiography and electron microscopy
Immediately after COC isolation or after the appropriate time of their culture, the oocytes were transferred to the same culture medium enriched with 5-
3
H-uridine Amersham, Little Chalfont, England, spec activity 40 MBqmM
− 1
, 7.4 MBqml
− 1
for 30 min. At the end of labeling, the COC were washed three times in nonradioactive medium at 4
◦
C and fixed in 0.6 paraformaldehyde Sigma + 2.5 glutaraldehyde Sigma, mixture with cacodylate
Sigma, buffer pH 7.4. After washing in this buffer at 4
◦
C the oocytes were post-fixed for 60 min in 2 OsO
4
Sigma dehydrated in ethanol and embeded in Polybed Polysciences, Warrington, FL, USA. From individual oocytes, semi-thin sections 1 mm were prepared
for autoradiography and thin sections for electron microscopy.
4 A. Pavlok et al. Animal Reproduction Science 64 2000 1–11
Semi-thin sections were coated with nuclear liquid emulsion Ilford K5 Mobberley, Eng- land and exposed at 4
◦
C for 30 days. After development with Kodak solution D 19b Rochester, NY, USA at 19
◦
C for 4 min and fixation Baserga and Malamud, 1969 the autoradiograms were stained with methylene blue before examination.
The labeling intensity was examined by light microscopy. The images were captured with SenSys CCD camera Photometrics Ltd., Tuscon, AZ on Olympus AX70 microscope
Olympus Optical Co. Ltd., Tokyo, Japan using 100× objective. Ratio of grain to nuclear area expressed in percent units was measured using Olympus Micro Image software. The
sorting of oocytes according to the proportion of GV cowered with silver grains was as follows. The GV with 65–80 of grain area were evaluated as ++++, 45–64 as +++,
15–44 as ++, 3–14 as +. About 1–2 represented the background and was evaluated as below.
For electron microscopy, a few thin sections practically of all oocytes used for autora- diographic analysis were prepared on grids, contrasted with uranyl acetate for 30 min and
with lead citrate for 10 min and examined under JEOL 1 200 EX electron microscope at 80 kV. The most representative photos of S category are presented in the manuscript.
3. Results
