Ovarian stimulation and collection of germinal vesicle (GV) phase oocytes

GV oocytes were collected from patients undergoing intracytoplasmic sperm injection (ICSI) treatment for male factors or combined with oviduct infertility from December 2007 to November 2008 at Center for Reproductive Medicine, Shandong Provincial Hospital, Shandong University. Ovarian stimulation was performed using standard protocols in our center, including downregulation of the pituitary gland with a gonadotrophin-releasing hormone agonist (GnRHa; Decapeptyl; Ferring, Kiel, Germany), followed by ovarian stimulation with exogenous follicle stimulating hormone (FSH; Gonal-F; Serono Laboratories, Aubonne, Switzerland). Ultrasound examination was carried out during the process. When 2-3 follicles of 18-20 mm in diameter were observed, and 17β-oestradiol concentrations reached 200 pg/mL per follicle more than 14 mm in diameter, ultrasound-guided follicle drilling and aspiration were performed 36 hours after the administration of 10,000 IU human chorionic gonadotrophin (HCG; Profasi; Serono Laboratories, Aubonne, Switzerland).

Ovarian stimulation protocols generated predominantly mature metaphase II (MII) oocytes. However, GV stage oocytes were frequently obtained, which, when cultured in appropriate medium, showed a high incidence of resumption and completion of maturation. In the current study, those GV oocytes from ICSI cycles were collected after informed, written consent. The study was approved by the ethics committee at Shandong Provincial Hospital, Shandong University. In all, 207 patients in 220 cycles donated their GV oocytes. The age of the patients was 28.7 ± 1.9 years (mean ± SD; range, 25–34 years).

In vitro maturation

GV oocytes were cultured for 24-48 hours at 37°C under conditions of 5% CO ₂ in air. The maturation medium adopted in the current study was similar to previous study with slight modifications. It was composed of commercially available G-2 medium (Vitrolife, Kungsbacka, Sweden), supplemented with 10% (v/v) human serum albumin (HSA; Vitrolife), 50 IU/mL penicillin (Sigma-Aldrich, St. Louis, MO), 50 μg/mL streptomycin (Sigma-Aldrich), 0.15 IU/mL HCG, 0.075 IU/mL recombinant FSH, 10 ng/mL epidermal growth factor (EGF; Sigma-Aldrich), and 10 μg/mL oestradiol (Sigma-Aldrich). The extrusion of the first polar body was used as the criterion for nuclear maturation.

Treatments

IVM was performed once GV oocytes were collected. They were divided randomly into control and study groups. More than 50 IVM procedures were performed for each group. Oocytes in control group were cultured in drug-free maturation medium for 24-48 hours. Oocytes in study group were cultured in maturation medium containing 10 μM microtubule polymerization inhibitor, colchicine (Sigma-Aldrich), or 10 μM microfilament polymerization inhibitor, cytochalasin B (CB; Sigma-Aldrich) for 48 hours. Chemicals of this concentration can disrupt cytoskeleton polymerization specifically without doing harm to oocytes. Then the chromatin state and position, spindle formation and migration, CG migration, and mitochondria redistribution during IVM of human oocytes were evaluated.

Immunofluorescence staining of microtubules and microfilaments

Oocytes were fixed with 2% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at 37°C. Then they were washed in PBS containing 5 mg/mL HSA for 3 times at 37°C, each for 5 minutes. The oocytes were extracted in PBS containing 5 mg/mL HSA and 0.1% (v/v) Triton X-100 for 45 minutes at 37°C, followed by 3 washes in PBS. For the immunostaining of microtubules, the oocytes were incubated in a mouse monoclonal antibody against α-tubulin (T-5168; Sigma-Aldrich) diluted by PBS (1:100) for 90 minutes at 37°C. Then they were washed with PBS and incubated in fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse Ig G (F-4018; Sigma-Aldrich) diluted with PBS (1:300) for 60 minutes at 37°C. Microfilaments labeling was performed by incubation of permeabilized oocytes with 1 μg/mL FITC-phalloidin (P-5282; Sigma-Aldrich) for 60 minutes at 37°C. Chromosomes were stained by exposing the oocytes to 10 μg/mL Hoechst 33342 for 10 minutes.

Immunofluorescence staining of CG and mitochondria

For the detection of CG, oocytes were first exposed to acidic Tyrode’s solution to remove the zona pellucida. Then they were fixed and permeabilized as described previously. After being washed in PBS, the oocytes were stained with 10 μg/mL FITC-labeled Lens Culinaris Agglutinin (LCA) (L-9262; Sigma-Aldrich) in PBS for 30 minutes. For the staining of mitochondria, zona intact oocytes were used. Oocytes were stained by 0.5 μM MitoTracker Green FM (Molecular Probes, Eugene, OR) for 30 minuts at 37°C. After 3 washes in PBS, they were fixed as described previously. Chromosomes were stained using the same method as mentioned previously. Finally, the oocytes were mounted on a glass slide with a drop of antifade medium (P-36934; Invitrogen, Carlsbad, CA) and covered with a coverslip. Then the oocytes were stored at −20°C until confocal microscopy was performed.

Confocal microscopy

The oocytes were observed using laser scanning confocal microscopy (TCS SP2; Leica, Wetzlar, Germany). The confocal protocol was performed as described previously. For CG, mitochondria, and microfilaments, sequential scanning with 2 μm step size was performed from top to bottom of the oocyte. Representative pictures were taken from the equatorial plane. For spindle, pictures were taken from the plane containing spindle and chromosomes. For observation of FITC and MitoTracker Green FM staining, a 488-nm excitation line and a 514-564 nm emission filter were used. The 355-nm line together with a 465-nm emission filter were used to excite Hoechst 33342 and visualize DNA. Data were saved to a disk and processed using Photoshop 7.0 software (Adobe, Mountain View, CA).

Statistical analysis

Data were analyzed by χ ² test. Oocytes that had degenerated or fragmented were excluded. The images were analyzed 4 times, on separate occasions at least a week apart. The determinations were made by 1 observer who was blinded to the treatments. Differences in the values were considered significant when P < .05.

Effects of microtubules and microfilaments on the condensation and movement of chromosomes, formation, and migration of spindle

Spindles were located in the cortical region and oriented vertically to the cell surface in most of the human oocytes matured in drug-free maturation medium ( Table 1 ; Figure 1 , A). In most of the oocytes incubated in 10 μM colchicine for 48 hours, GVBD could occur, chromosomes condense, and translocate to the cortex. However, meiotic spindles did not form and the meiosis process was arrested in MI stage ( Figure 1 , B). In CB group, neither GVBD nor MI spindle formation was influenced. However, spindle and condensed chromosomes could not migrate, locating in the central of the cytoplasm ( Figure 1 , C).

TABLE 1

Effects of colchicine and CB on the meiotic process during maturation of human oocytes

Groups	No. of oocytes	Developmental stages
		GV, n (%)	MI, n (%)	MII, n (%)
Control	76	12 (15.79)	12 (15.79)	52 (68.42)
Colchicine 10 μM	99	22 (22.22)	77 (77.78) a	0
CB 10 μM	96	10 (10.42)	80 (83.33) a	6 (6.25)

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