All oocytes and embryos w ere cultured in an atm osphere of 5% CO2 in
air, at 37°C, u n d er paraffin oil (PSA laboratories, Loughborough, UK), in Falcon tissue culture dishes (Beckton Dickinson Labware, N ew Jersey, USA). The oocytes w ere exam ined 18-24 hours post insem ination (hpi) for signs of fertilisation. After stripping the cum ulus cells from the oocyte, by gently aspirating the oocyte up and dow n a flame polished pasteur pipette, the presence of pronuclei and polar bodies was recorded. The oocyte w as deem ed to be fertilised if two pronuclei and 2 polar bodies were seen. After exam ination all oocytes were transferred to fresh 100|il drops of m edia u n d er oil, w ithout sperm . Unfertilised oocytes w ere re-exam ined to ensure no pronuclei w ere present and, if patients agreed to research, could be entered into the activation protocol. Fertilised oocytes w ere cultured for a further 24 hours by which time they had usually reached the two to four
cell cleavage stage of development. On day 2 or 3 after egg collection, tw o or three em bryos w ith the 'best' m orphological appearance w ere chosen for em bryo replacem ent. Good m orphology consisted of regular blastom eres w ith little or no fragm entation. A Bourn-Wallace catheter (Wallace Ltd, Colchester, UK) w as used to transfer the em bryos transcervically into the uterine cavity. Following embryo transfer luteal sup p o rt was given in the form of intram uscular progesterone, 50mg twice weekly, (Gestone, Paines and Byrne, W est Byfleet, Surrey, U.K.), or progesterone pessaries, 400mg daily (Cyclogest, H oechst UK Ltd, Hounslow, UK) for tw o weeks. A pregnancy test w as done if the patient had not had a vaginal bleed two w eeks following the embryo transfer, and the first ultrasound scan w as perform ed 10-14 days following a positive test.
2.4 Culture of human fetal lung fibroblasts
N orm al hu m an fetal lung fibroblasts MRC-5 (European collection of anim al cell cultures catalogue, ECACC, a gift from A udrey M uggleton- H arris), w ere tested for m ycoplasma contam ination before being
subcultured in T-25 flasks to m aintain them in the log grow th phase. The fibroblasts h ad been cultured in Minimal Essential M edium (MEM, Gibco Life Technologies Ltd, Paisley, Scotland) or H am s F-10 (Gibco Life
Technologies Ltd, Paisley, Scotland), supplem ented w ith 10% fetal bovine serum (FBS), 28mM HEPES buffer and 5 0 |ig /m l penicillin and
streptom ycin, at 37°C in an atm osphere of 5% CO2 in hum idified air
(M uggleton-H arris and A roian 1982). Following trypsinisation, w ith 0.25% trypsin (Gibco Life Technologies Ltd, Paisley, Scotland) to loosen cells from the bottom of the culture flask, cells could be seeded onto tissue culture dishes or stored in aliquots at -80°C for subsequent use. A confluent single
layer of fibroblasts w as obtained betw een 24-48 hours after innoculation.
2.5 Parthenogenetic activation
All oocytes w ere exam ined for the presence of a first polar body and those at the germ inal vesicle or m etaphase I stage, together w ith any degenerate oocytes w ere excluded from the protocol. The oocytes w ere rem oved from their culture m edium of EBS or T6, supplem ented w ith 10% HIS or HSA, and w ashed through HEBS + BSA. The oocytes w ere then left for 30 m inutes in HEBS + BSA in a hum idified dish w ithout oil at 37°C (Winston, Johnson, et al. 1991). This step had been show n
previously to im prove the activation rate, the likely explanation being that ionophore is highly lipophilic and m ay partition out to any lipids present in the culture m edium , as w ould be the case w hen HIS supplem entation is used.
The fresh or aged oocytes w ere then exposed to a 5|xM solution of calcium ionophore A23187 (Sigma Chemical Co.; stored as a 2 m g /m l stock solution in dim ethyl sulphoxide, DMSO, at -70°C) for precisely 5 m inutes. After three w ashes through EBS + Alb, the oocytes w ere cultured at 37°C in 50 pL drops of the sam e m edium under oil, at an atm osphere of 5% CO2 in
air. Oocytes w ere scored for signs of activation by exam ining for extrusion of a second polar body, and the appearance of pronuclei.
2.6 Fixing
A ctivated hum an oocytes, fertilised m ouse em bryos and their respective control m etaphase II oocytes w ere fixed at varying intervals after activation or fertilisation, for analysis of their DNA content. Fixation w as achieved using a 4% solution of form aldehyde in PBS for 30-60
m inutes, followed by w ashing through an excess of PBS and the activated oocytes or parthenogenetic embryos w ere stored in drops of FBS u n d er oil at4°C .
Dishes of fibroblasts were fixed by flooding w ith 4% form aldehyde in PBS for 30 m inutes after which they were w ashed thoroughly w ith excess PBS. After covering the fibroblasts w ith fresh PBS the dishes w ere stored at 4°C until stained for DNA quantitation.
2.7 Dapi staining
N uclear DNA can be stained w ith the fluorochrom e 4'6'-diamidino-2- phenylindole.2H C l (DAPI, Boehringer M annheim , UK), w hich binds specifically to the A-T region of double stranded DNA (Otto and Tsou 1985a) and fluoresces w hen exposed to ultraviolet (UV) light, enabling nuclear m orphology to be assessed. The use of DAPI staining for
m icrospectrofluorom etric estim ation of cellular DNA content has been reported previously (Coleman, et al. 1981; Coleman and Goff 1985; W inston, et al. 1993). The fluorescence of DAPI stained DNA is directly p roportional to DNA quantity and does not appear to be affected by the state of the DNA being m easured (Coleman, M aguire, et al. 1981). D api w as stored at -80°C as a stock solution of 2 m g /m l in dim ethyl sulfoxide
(DMSO) (Sigma). All m ouse or hum an oocytes and em bryos w ere stained by transferring m aterial to drops of DAPI in PBS u n d er oil, usually at a concentration of 5 |ig /m l, for 15 m inutes. The stained m aterial w as then w ashed through drops of PBS and placed in fresh drops of PBS for exam ination u n d er ultraviolet light and DNA analysis.
Fibroblasts w ere stained by replacing the PBS covering the fixed cells w ith a 5 |ig /m l solution of DAPI in PBS for 15 m inutes. This was followed
by three w ashes w ith PBS and covering w ith fresh PBS for analysis. Each dish of fibroblasts served as its ow n control as it contained cells of three distinct phenotypes. The m ajority of fibroblasts w ere flattened onto the bottom of the dish, had typical long cytoplasmic processes (Plate 2.1) and on DAPI staining had interphase nuclei (Plate 2.3). O ther cells w hich w ere heaped u p as easily recognised, rounded 'mitotic spheres' (Plate 2.1), contained chrom osomes in m etaphase on DAPI staining (Plate 2.3). It w as also sim ple to identify pairs of cells which had just undergone cleavage, w hose chrom osomes were in anaphase or telophase (Plates 2.2 and 2.3). Thus each culture dish contained cells in m etaphase (presum ed 4C DNA content), cells w hich had recently undergone cleavage (presum ed 2C DNA content), and interphase nuclei w hich should have DNA contents
Plate 2.1: Photograph of human fetal lung fibroblasts in culture showing 2 round 'mitotic