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Nuclear-cytoplasm interaction and microtubule dynamics during rat oocyte maturation in vitro
280
Theriogenology
NUCLEAR-CYTOPLASM INTERACTION AND MICROTUBULE DYNAMICS DURING RAT OOCYTE MATURATION I/V VITRO Li Meng’, Mary M. Fahy2, Don P. Wolf”2 and David T. Armstrong3. Oregon Regional Primate Research Center, Beaverton OR 97006’; Dept. of Ob/Gyn and Physiology, Oregon Health Sciences University, Portland, OR 97201 ; Dept. of OblGxn and Physiology, The University of Western Ontario, London ON N6A 5A5 Canada We previously demonstrated that, during rat oocyte maturation in vitro, protein synthesis is controlled by cytoplasmic regulators rather than by intrinsic nuclear components (Biol. Reprod. 1994 Suppl 1:731). The current objective was to define further the role of the germinal vesicle (GV), in this case, by monitoring the activity of maturation promoting factor (MPF) and microtubule organization. Oocyte-cumulus cell complexes were collected from ovaries of immature rats (25-28 day-old) 48 h after PMSG injection. Oocytes were denuded and intact GVs were removed by micromanipulation in TALP-HEPES + 10% BSA + 0.2 mM 3-isobutyl-1 methylxanthine (IBMX). Enucleated (GV ) and control (Gv’) oocytes were cultured in KSOM+lO% neonatal calf serum for up to 16 h. GV+ or GVoocytes (20) at each stages of maturation (0, 2, 4, 6, 8, 10, 12, 14, 16 h) were lysed in histone Hi kinase (Hl K) extraction buffer for Hl K assays (2 replicates), as an indirect indicator of MPF activity, or were fixed in 2% paraformaldehyde+0.05% picric acid in PBS on poly-L-lysine coated coverslides after zona removal for immunocytochemistry. Microtubules were localized by fluorescence microscopy using mouse anti-Stubulin monoclonal IgG antibody followed by goat anti-mouse IgG conjugated to crystalline tetramethylrhodamine isothiocyanate (TRITC). DNA was stained by Hoechst 33342. Hl K activity in intact oocytes showed a biphasic pattern with low levels at the GV stage increasing to the first peak at M I (7 h) followed by a decrease to baseline at anaphase and telophase I (10 h), and finally reaching a second peak at M II (after 12 h). The HI K activity in GV oocytes displayed the same biphasic pattern. Taxol, a potent microtubule assembly and stabilization agent, was used to probe the requirements for microtubule aster formation at the end of each culture period (10 uM for 15 min). Initially, a microtubule network without microtubular foci (aster) was observed in both GV+ and GV oocytes regardless of taxol treatment. Within 3 h of culture, GVB occurred in all GV’ oocytes. At 4-5 h, initiation of spindle formation was evident and microtubuies radiating from the condensing chromosomes were visible in 68% of GV+ oocytes. No detectable microtubules were observed in the GV oocytes at the same culture period. However, when treated with taxol, microtubular foci appeared in both GV’ and GV oocytes. At 7 h, 97% of GV+ oocytes reached late prometaphase I or metaphase I (M I). Anaphase I and telophase I in GV+ oocytes appeared at 9-11 h. 74% of GV’ oocytes were in metaphase II (M II) after 12 h culture increasing to 93Ohat 16 h. During the transition from M I to M II (7-12 h of culture), the meiotic spindles (cone-shaped with distinct poles) were the only antitubulin-labelled structures apparent in GV’ oocytes; no visible microtubules were observed in GV oocytes. When treated with taxol, multiple microtubular foci were detected near the cortex in both GV’ and GVoocytes, which were not spindle associated (GV’ oocytes). These results indicate that after GVB in rat oocytes cultured in vitro, microtubules are restricted to the spindle of GV’ oocytes, although aster formation can be induced by taxol exposure in both GV+ and GV. Hl K activity was independent of GV presence. (supported by HD 28484 and RR 00163)
Theriogenology
NUCLEAR-CYTOPLASM INTERACTION AND MICROTUBULE DYNAMICS DURING RAT OOCYTE MATURATION I/V VITRO Li Meng’, Mary M. Fahy2, Don P. Wolf”2 and David T. Armstrong3. Oregon Regional Primate Research Center, Beaverton OR 97006’; Dept. of Ob/Gyn and Physiology, Oregon Health Sciences University, Portland, OR 97201 ; Dept. of OblGxn and Physiology, The University of Western Ontario, London ON N6A 5A5 Canada We previously demonstrated that, during rat oocyte maturation in vitro, protein synthesis is controlled by cytoplasmic regulators rather than by intrinsic nuclear components (Biol. Reprod. 1994 Suppl 1:731). The current objective was to define further the role of the germinal vesicle (GV), in this case, by monitoring the activity of maturation promoting factor (MPF) and microtubule organization. Oocyte-cumulus cell complexes were collected from ovaries of immature rats (25-28 day-old) 48 h after PMSG injection. Oocytes were denuded and intact GVs were removed by micromanipulation in TALP-HEPES + 10% BSA + 0.2 mM 3-isobutyl-1 methylxanthine (IBMX). Enucleated (GV ) and control (Gv’) oocytes were cultured in KSOM+lO% neonatal calf serum for up to 16 h. GV+ or GVoocytes (20) at each stages of maturation (0, 2, 4, 6, 8, 10, 12, 14, 16 h) were lysed in histone Hi kinase (Hl K) extraction buffer for Hl K assays (2 replicates), as an indirect indicator of MPF activity, or were fixed in 2% paraformaldehyde+0.05% picric acid in PBS on poly-L-lysine coated coverslides after zona removal for immunocytochemistry. Microtubules were localized by fluorescence microscopy using mouse anti-Stubulin monoclonal IgG antibody followed by goat anti-mouse IgG conjugated to crystalline tetramethylrhodamine isothiocyanate (TRITC). DNA was stained by Hoechst 33342. Hl K activity in intact oocytes showed a biphasic pattern with low levels at the GV stage increasing to the first peak at M I (7 h) followed by a decrease to baseline at anaphase and telophase I (10 h), and finally reaching a second peak at M II (after 12 h). The HI K activity in GV oocytes displayed the same biphasic pattern. Taxol, a potent microtubule assembly and stabilization agent, was used to probe the requirements for microtubule aster formation at the end of each culture period (10 uM for 15 min). Initially, a microtubule network without microtubular foci (aster) was observed in both GV+ and GV oocytes regardless of taxol treatment. Within 3 h of culture, GVB occurred in all GV’ oocytes. At 4-5 h, initiation of spindle formation was evident and microtubuies radiating from the condensing chromosomes were visible in 68% of GV+ oocytes. No detectable microtubules were observed in the GV oocytes at the same culture period. However, when treated with taxol, microtubular foci appeared in both GV’ and GV oocytes. At 7 h, 97% of GV+ oocytes reached late prometaphase I or metaphase I (M I). Anaphase I and telophase I in GV+ oocytes appeared at 9-11 h. 74% of GV’ oocytes were in metaphase II (M II) after 12 h culture increasing to 93Ohat 16 h. During the transition from M I to M II (7-12 h of culture), the meiotic spindles (cone-shaped with distinct poles) were the only antitubulin-labelled structures apparent in GV’ oocytes; no visible microtubules were observed in GV oocytes. When treated with taxol, multiple microtubular foci were detected near the cortex in both GV’ and GVoocytes, which were not spindle associated (GV’ oocytes). These results indicate that after GVB in rat oocytes cultured in vitro, microtubules are restricted to the spindle of GV’ oocytes, although aster formation can be induced by taxol exposure in both GV+ and GV. Hl K activity was independent of GV presence. (supported by HD 28484 and RR 00163)