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
3.1. RNA synthetic activity of S and M oocytes As seen from the Table 1, the oocytes from COC of the M category labeled with
3
H-uridine immediately after isolation showed only weak labeling + of the GV area. After 24 and
70 h of culture in the meiosis-inhibiting medium, the oocytes of the same category showed no labeling of the GV area. However, the nuclei of the granulosa cells were intensively
labeled in all groups. The oocytes of the S category labeled immediately after isolation before culture showed intensive labeling ++++, +++ of GV area Fig. 1. When the
COC of the same category were labeled after 24 h of culture, a slightly decreased tendency of labeling of GV area mostly +++, ++ was observed Figs. 2 and 3. After 70 h of
culture, the GV area was labeled only slightly above the background Fig. 4. Also the COC
Table 1 Labeling of nuclei of bovine oocytes with 5-
3
H-uridine before and after 24 and 70 h culture in a meiosis-inhibiting medium with butyrolactone I
a
Oocyte category Time of culture h
Labeling intensity and no. of oocytes Total no. of oocytes
++++ +++
++ +
M 3–6 mm 2
1 3
24 4
4 70
3 3
S 1–2 mm 4
3 1
8 24
2 2
1 1
6 70
1 2
3
a
Labeling intensity ++++ to + is represented by Figs. 1–4.
A. Pavlok et al. Animal Reproduction Science 64 2000 1–11 5
Fig. 1. Light microscopy radiography of oocytes from two size categories of follicles M, 3–6 mm and S, 1–2 mm in diameter. Semi-thin sections of oocytes labeled for 30 min with 5-
3
H-uridine after 0, 24 and 70 h of culture in meiosis-inhibiting medium supplemented with 100 mM BL I. Thirty days
,
exposition under the nuclear liquid emulsion ×600. Heavily labeled dark GV area of oocyte and surrounding granulosa cells. S category oocyte
before culture labeling intensity evaluated as ++++.
Fig. 2. Light microscopy radiography of oocytes from two size categories of follicles M, 3–6 mm and S, 1–2 mm in diameter. Semi-thin sections of oocytes labeled for 30 min with 5-
3
H-uridine after 0, 24 and 70 h of culture in meiosis-inhibiting medium supplemented with 100 mM BL I. Thirty days
,
exposition under the nuclear liquid emulsion ×600. Heavily labeled GV area mainly around nucleoli of 24 h cultured S category oocyte labeling
intensity evaluated as +++. The oocyte surrounding granulosa cells are also heavily labeled.
6 A. Pavlok et al. Animal Reproduction Science 64 2000 1–11
Fig. 3. Light microscopy radiography of oocytes from two size categories of follicles M, 3–6 mm and S, 1–2 mm in diameter. Semi-thin sections of oocytes labeled for 30 min with 5-
3
H-uridine after 0, 24 and 70 h of culture in meiosis-inhibiting medium supplemented with 100 mM BL I. Thirty days
,
exposition under the nuclear liquid emulsion ×600. Mild labeling of GV area of 24 h cultured S category oocyte labeling intensity evaluated as
++ . The granulosa cells are heavily labeled.
Fig. 4. Light microscopy radiography of oocytes from two size categories of follicles M, 3–6 mm and S, 1–2 mm in diameter. Semi-thin sections of oocytes labeled for 30 min with 5-
3
H-uridine after 0, 24 and 70 h of culture in meiosis-inhibiting medium supplemented with 100 mM BL I. Thirty days
,
exposition under the nuclear liquid emulsion ×600. Only significantly more grains than the background of GV area of 70 h cultured S category
oocyte labeling intensity evaluated as +. Most of the granulosa cells are heavily labeled.
A. Pavlok et al. Animal Reproduction Science 64 2000 1–11 7
Fig. 5. Fine structure of nucleoli in oocytes of S category before culture, after 24 and 70 h culture in meiosis-inhibiting medium MIM with 100 mM of BL I as seen in Material and methods. Nucleolus from the S
category oocyte of reticular structure with several fibrillar centers arrows fixed immediately after isolation from the follicle ×2500.
of S category showed the intensive labeling of granulosa cells nuclei during the whole 70 h of culture.
3.2. Nucleolar ultrastructure of S category oocytes: freshly isolated, 24 and 70 h cultured with BL I
Immediately after isolation of S category oocytes, the nucleoli possessed a reticular fibrillogranular structure with numerous fibrillar centers arrows Fig. 5. After 24 h culture
in MIM, fibrillogranular structure was shifted peripherally by several large vacuoles. A number of small vacuoles and exclusively fibrillar area arrow were observed Fig. 6.
After 70 h of culture in MIM, nucleoli were exclusively fibrillar with one large fibrillar center arrow Fig. 7.
3.3. Two-step culture of S category oocytes Comparing with the one-step maturation of S category oocytes, the two-step culture of
these oocytes in MIM and subsequently in MSM have the capability to increase signif- icantly P 0.01 their meiotic competence Table 2. While after the one-step 48 and
72 h maturation of control COC in MSM, only 27.1 and 11.3 matured to M II stage, respectively, two-step culture of experimental COC resulted in 81.0 maturing to M II. In
the control group, of COC matured 48 and 72 h, 67.8 and 54.7 did not proceed beyond the metaphase I M I–anaphase I block stage, respectively. However, in both the control
and experimental groups, some proportions of oocytes 5.1, 30.2 and 7.9, respectively remained either in the GV or late diakinesis LD stages.
8 A. Pavlok et al. Animal Reproduction Science 64 2000 1–11
Fig. 6. Fine structure of nucleoli in oocytes of S category before culture, after 24 and 70 h culture in meiosis-inhibiting medium MIM with 100 mM of BL I as seen in Material and methods. Nucleolus from the S
category oocyte fixed 24 h after culture in MIM. The fibrinogranullar structure is accompanied with some large vacuoles, more small vacuoles and one fibrillar area arrow ×3500.
Fig. 7. Fine structure of nucleoli in oocytes of S category before culture, after 24 and 70 h culture in meiosis-inhibiting medium MIM with 100 mM of BL I as seen in Material and methods. Nucleolus of the S
category oocyte fixed 70 h of culture in MIM. Compact fibrillar material is accompanied with a large single fibrillar center arrow ×8000.
A. Pavlok et al. Animal Reproduction Science 64 2000 1–11 9
Table 2 Meiotic competence of bovine oocytes from a small category of oocytes 1–2 mm in follicle diameter cultured
directly 48 h in meiosis-stimulating medium MSM with gonadotropins control group-C1, or in two-step pro- tocol, in the first step 48 h in MSM without gonadotropins and in the second step in MSM with gonadotropins
C2
a
Group Time of culture h in Total no.
of COC GV–LD
b
M I anaphase I block
Activated pronuclei
M II MIM MSM
C1 –
48 59
3 5.1 a 40 67.8 a
16 27.1 a C2
– 72
53 16 30.2 b
29 54.7 a 2 3.8
6 11.3 a Exp.
48 24
63 5 7.9 a
7 11.1 b 51 81.0 b
a
The experimental group Exp. was cultured in the first step 48 h in meiosis-inhibiting medium MIM and in the second step 24 h in MSM with gonadotropins. The data in the same column with different letters differ
significantly P 0.05.
b
Late diakinesis.
4. Discussion
