Culture of bovine oocytes from small antral follicles in meiosis-inhibiting medium with butyrolactone I: RNA synthesis, nucleolar morphology and meiotic competence

• Published in: Animal reproduction science 2000-12, Vol.64 (1-2), p.1-11
• Main Authors: Pavlok, A, Kaňka, J, Motlı́k, J, Vodička, P
• Format: Article
• Language: English
• Subjects: 4-Butyrolactone - analogs & derivatives; 4-Butyrolactone - pharmacology; 5-[formula omitted]-uridine; Animals; Autoradiography; Butyrolactone I; Cattle - physiology; Cattle-reproductive technology; Cell Nucleolus - ultrastructure; Enzyme Inhibitors - pharmacology; Female; Image Processing, Computer-Assisted; Meiosis; Meiosis - physiology; Methylene Blue - chemistry; Microscopy, Electron - veterinary; Oocyte; Oocytes - growth & development; Oocytes - physiology; Oocytes - ultrastructure; Ovarian Follicle - physiology; Protein Kinase Inhibitors; RNA - biosynthesis
• ISSN: 0378-4320; 1873-2232
• DOI: 10.1016/S0378-4320(00)00189-5

Bovine oocytes originated from follicles of two different size categories (medium (M), 3–6mm and small (S), 1–2mm) were cultured for 24 and 70h, respectively, in a meiosis-inhibiting medium (MIM) supplemented with 100μM butyrolactone I (BL I). At the end of culture, cumulus oocyte complexes (COC) from M and S follicles were labeled by 3H-uridine for 30min. The autoradiography (ARG) of semi-thin sections of COC showed the labeling of the germinal vesicles (GV) as: (1) The COC of the M category labeled immediately after isolation from follicles showed only weak labeling (+) of the GV. The COC of the S category labeled immediately after their isolation showed mostly intensive labeling (+++, +++) of the GV. (2) When the COC were labeled after 24 and 70h of culture in MIM, no labeling was observed in the M category. The S category of oocytes showed the slightly decreased labeling (+++, ++) after 24h and negligible labeling after 70h of culture. The pattern of very intensive labeling of granulosa cell nuclei of all mentioned groups was practically not changed during the whole 70h period of culture in both categories. The nucleolar ultrastructure of S category oocytes revealed time dependent changes from the reticular fibrillogranular structure present in freshly isolated oocytes. The several fibrillar centers before the culture changed to the fibrillogranular appearance with few large and a number of small vacuoles and an exclusively fibrillar area after 24h of culture. Finally, nucleoli acquired a mostly exclusively fibrillar structure with one large fibrillar center after 70h of culture. In the second experiment, the meiotic maturation of COC of S category was inhibited in MIM for 48h. The subsequent 24h culture in a medium with BOS and gonadotropins resulted in 81.0% oocytes matured to metaphase II (M II). Only 27.1 and 11.3% of the control S oocytes cultured in a medium, with BOS and gonadotropins directly after isolation, reached M II after 48 or 72h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles.