Sequential Observations of Oocyte Maturation using Microtubule and Chromosome Labelling

To examine changes in the distribution of microtubules during maturation, oocytes had to be fixed at specific times after the maturation process had been initiated The number of time points required to obtain a complete investigation of the

processes of maturation made the use of in vivo matured oocytes impractical.

Consequently, oocytes were matured in vitro by incubation in coelomic fluid with

maturation inducing activity (i.e. coelomic fluid with Coelomic Maturation Factor [CMF] activity) as described in section-3.2.

Experimental Design

Pooled oocytes from oocyte donors were incubated in coelomic fluid with maturation inducing activity at 10°C under natural illumination. Five microlities of oocytes were removed at the following time intervals after incubation had begun; 0, 10, 20, 40, 60, 80, 100, 120, 140, 160 and 180 minutes. Once removed, oocytes were fixed immediately, processed for microtubule and chromosome labelling and examined as described below. Fifty oocytes were also assessed for their maturational state at each time interval.

Immunofluorescence Labelling

Fixation of Oocytes and Embryos

Oocytes andembryos were fixedat the required developmental stage or time in fonnaldehyde fix (see appendix) for 30 minutes at 4°C. To prevent the foiinaldehyde in the fixation solution interfering with the antibody labelling and to facilitate storage of the embryos at 4®C, without deterioration of the microtubule structures, 95% of the fixative was removed and replaced with non-fixing PIPES buffer (see appendix).

Preparation of Coverslips for use in Labelling Procedures

Round 13 mm diameter coverslips, thickness No. 1 (Chance Ltd.) were used to mount the oocytes. Circular coverslips reduced the loss of embryos during the labelling process, were more easÜy rinsed and easier to handle than square coverslips. The first step in the procedure was to reduce the negative charge of the glass to facilitate adherence of the oocytes and embryos to the coverslips. To do this it was

necessary to add 75 pX of poly-L-lysine solution (Sigma Co.) to the coverslip. The

poly-L-lysine solution was then allowed to evaporate by placing the coverslips under a normal 60 W light for approximately 30 minutes. This leaves a layer of poly-L-lysine solution with a slight positive charge to the coverslip and allowed the oocytes and embryos to adhere to the coverslip even during washing.

Addition of Specimens

Coverslips were placed on parafilm squares (2 cm) in plastic petri dishes (50

mm diameter x 13 mm depth). The specimen suspension (250 pi) was pipetted on to

the coverslip. In some cases, where the specimens had been stored for a number of weeks, gentle mixing before applying the specimens broke up any aggregates that had formed during storage. The specimen suspension was left for 10 minutes to allow the embryos to adhere, 2 ml of PBS buffer (see appendix) was then pipetted around the edges of the parafilm. This gently immersed the specimens and rinsed off any traces

of formaldehyde. The rinse was left on for 15 minutes and then aspirated off. Coverslips were further dried with blotting paper, although they were never allowed to dry out completely.

Labelling with the Primary Antibody

The primary antibody used was monoclonal antibody (clone no. DM lA)

raised in mouse (Sigma Co.). Aliquots of 2 p\ were frozen at -20°C and defrosted as

required. The antibody was diluted to the required concentration in 1% sheep serum (Scottish Antibody Production Unit) in PBS buffer. The sheep serum was frozen in

10 ]Â aliquots and defrosted immediately before being diluted with the PBS buffer.

The final primary antibody concentration used was 1/1000 as this concentration was found to give the optimal labelling of microtubule stiuctures.

The primary antibody 1/1000 dilution (30 p\) was pipetted on to the coverslip

and left for 4 hours. This amount of primary antibody prevented the specimens from diying out over the 4 hour period and made sure that the meniscus covered all the embryos' surface. Lids of the petri dishes were replaced to maintain a humid atmosphere and the dishes were then covered to prevent direct sunlight heating or diying out the specimens. Although primaiy antibody attachment and detachment reaches equilibrium, 4 hours was found to be the optimum time for incubation, any longer increased evaporation and shorter times increased the incidence of poor labelling of specimens on a coverslip. After 4 hours the rinsing procedure described above was repeated.

Labelling with the Secondary Antibody and Hoechst 33258

After immersion in the rinse for 15 minutes the PBS buffer was aspirated off

and 30 p\ of the secondary antibody was added as described for the primary antibody

and then left for 2 hours before rinsing. The secondary antibody used was anti-mouse and raised in sheep with the fluorescent label (FTTC) conjugated to it (Scottish

1/100 and it was diluted with 1% sheep serum in PBS buffer. After the rinse had

been aspirated off, 20 pi of Hoechst 33258 (1 pg,mH in distilled water) was added

for 2 minutes then aspirated off.

Mounting the Coverslips

Duiing the 2 minute incubation of the specimens with the Hoechst 33258, 6 pi

of mounting fluid (see appendix) was placed on a clean microscope slide. After the Hoechst stain was aspirated off, the coverslips were dried with blotting paper and then placed, specimen side down, on to the mounting fluid. To prevent drying, each coverslip was then sealed by careful painting of nail varnish to overlap the edge of the coverslip by approximately 1 mm. Once the nail varnish was dry, slides could be stored for a niunber of weeks in the daik at 4°C until ready for viewing.

Controls

For all inmiunolabelling series a control was included. This involved the replacement of the primaiy antibody with 1% sheep semm in PBS buffer. Negative staining when viewing the specimens confirmed that the labelling observed in the

series was as a result of the recognition of the a tubulin by the primary antibody.

Visualisation of the Microtubule Structures

The following 3 methods were utilised:

1. A Leitz Dialux 20 microscope as described in Section 2.2.2

2. Nikon Diaphot 2 inverted microscope with a HBO 50 W/AC mercury short arc lamp with filter block A, exciting at 340-380 nm and suppressing at 430 nm and NPL FLUOTAR objectives of magnification of x20/0.55 and x60/0.7. Photographs were taken using monochrome 400 ASA TMY film.

3. Confocal images: Confocal Laser Scanning Microscope with krypton/argon lasers (Bio-rad Microscience Ltd; MRC-600, Nikon Diaphot 2 inverted microscope as

above). Data sets were stored on 5 V4" optical disks (Phillips) and copies of data were downloaded using a colour video printer (Sony VP5000) onto colour printer paper.

2,2.4 The Effect of Maturation Method on Microtubule