computerised image analysis system
4.3 d Live versus fixed material
H aving show n that there w as an exponential loss of corrected nuclear
intensity in the first hour following DAPI staining using fixed m aterial (see 4.2a), live m aterial was exam ined to determ ine w hether this exhibited the sam e
phenom enon. A live and fixed 2-cell m ouse em bryo w ere both stained for 15 m inutes in 5|Xg/ml DAPI and captured over 3 hours (figure 4.6).
corrected nuclear intensity value 800 700 600- 500- 400- 300 200 1 0 0-
□
o
□o
□o
□o
25 50 75 100 125 time (mins) 150 175 200 Figure 4.6Scatter plot to dem onstrate the change in corrected nuclear intensity values of DAPI stained live and fixed 2 cell m ouse embryos with time. The live cells (O and □ ) show very little change with time, while the fixed cells
(A
and ^ ) show the exponential fall previously observed in the first hour (fig 3). Fixed cells stain much more brightly initially, but after 100 minutes the nuclear values plateau to a similar level se en in the live material.intensities as observed previously, b u t although the live cells stained at a m uch low er level of intensity initially, they rem ained stable throughout repeated m easurem ents over 3 hours. The corrected nuclear intensities of the fixed cells plateaued after 100 m inutes at a similar value to the stable level observed in the live cells.
4.3e D ifferent concentrations of DAPI
To exclude a concentration dependent effect on the corrected nuclear intensity values, hu m an m etaphase II oocytes w ere stained in DAPI concentrations of 5 |ig /m l, 2 5 p g /m l or 50pg/m l and captured 7 times over 6 hours. Exactly the same p attern w as seen w ith time for the higher concentrations of DAPI as had been previously observed w ith 5 |ig /m l. The level at w hich the corrected nuclear intensities plateaued out w as the same for each concentration (figure 4.7). Thus stoichiom etric binding of DAPI to DNA w as dem onstrated.
C orrected N uclear Intensity 400 DAPI concentration O 5 |ig /m l/1 0 0 0 □ 25^ig/ml/1000 A 50ng/m l/1000 350 300 250 200 150 100 50 TIME [mins] Figure 4.7
Graph to dem onstrate the effect of different concentrations of DAPI on the corrected nuclear intensity (Seescan) score. The sam e pattern of loss of intensity was observed with time for three different DAPI concentrations, 5|ig/ml, 25pg/ml and 50|ig/ml, confirming stoichiometric binding of DAPI to DNA.
4.3f M easurem ent of the DNA content of hum an fetal lung fibroblasts
Since it is know n that cells w ith a diploid num ber of chromosomes (2N) generally replicate their DNA content from 2C to 4C before cleaving, fibroblasts show ing the phenotypical appearance of a cell about to undergo mitosis (mitotic spheres; plates 2.1 and 2.3) would be expected to have a 4C DNA content, twice that of the cells that have just completed telophase (2C; plates 2.2 and 2.3).
S tan d ard ised corrected nuclear intensity m e asu rem en t of DNA content
±
Mitotic sp h e re s (n=20) Interphase nuclei (n=61) T elo p h ase cells (n=14) Figure 4.8Box plots to d em onstrate the stan d ardised, corrected nuclear intensity (S e e sc a n m easurem ents of nuclear DNA content of human fetal lung fibroblasts. The upper and lower limit of each box are the 25th and 75th percentiles respectively, while the median is the bar lying betw een these. The upper and lower whiskers indicate the 90th and 10th percentiles and any individual values lying outside this range is marked. Three cellular phenotypes for the fibroblasts were m easured; (i) mitotic spheres with chrom osom es in mitotic m etaphase ready for cleavage which had a 4C DNA content; (ii) recently cleaved cells in telophase with a 2C DNA content; and (iii) interphase nuclei with a DNA content between 2C and 4C.
In order to facilitate direct comparison of the nuclear intensity values of DNA content obtained for the different phenotypic groups of fibroblasts, the mean nuclear intensity of the mitotic spheres was standardised to 4 (4C DNA) by dividing by the mean value and m ultiplying by 4 (mean = 4.0 ± 1.02sd), as described by Angell (Angell, Sumner, et al. 1987). By using the same arithmetic
m anipulation for all the results, the values obtained for cells that w ere in telophase or anaphase w ere show n to be half this and equivalent to
approxim ately 2C (mean = 2.05 ± 0.41sd). Interphase cells had nuclear values betw een 2C and 4C (mean = 2.78 ± 0.98sd) (figure 4.8). Therefore Seescan m easurem ents could be used to distinguish gross orders of ploidy.
4.3g M easuring the DNA content of carefully tim ed fertilised m ouse oocytes and em bryos
Fertilised m ouse oocytes were cultured and fixed at the following stages, (hours po st insem ination, hpi)
- early pronuclei (9hpi) - late pronuclei (14hpi) - early two cell (18hpi) - late two cell (33hpi) - early four cell (38hpi)
The oocytes and embryos w ere stained w ith DAPI (5 |ig /m l for 15 m inutes) (plates 4.1 and 4.2), and captured onto the Seescan after 90 minutes. Each image w as m easured three times. The num erical value obtained from the Seescan m easurem ents of DNA content for the control unfertilised m etaphase 11 oocytes, the fertilised oocytes and the cleavage stages were divided by the m ean value obtained for the control oocytes and m ultiplied by two. This sim ple arithm etic m anipulation resulted in the m ean of the control group of oocytes, w hich should have a 2C DNA content, being standardised to a num erical value equivalent to the gross ploidy i.e. 2. This facilitated direct com parison of DNA content w ith the fertilised oocytes and embryos ( figure 4.9).
Plate 4.1
Two mouse unfertilised m etaphase II oocytes stained with DAPI and viewed under UV light. These oocytes were used a s a control for a 2C DNA content.
Plate 4.2
stan d ard ised corrected nuclear 5 intensltiy m e asu rem en ts of DNA content (C) 0
1
0_L
1
T
- | 1 1 1 1 1 1---control activated early late syngam y early late m etap h asell oocytes pronuclei pronuclei [n=2] 2 cell 2 cell
oocytes [n=5] [n=11] [n=16] [n=12] [n=16] [n=16]
1 1 1---
3 cell, 3 cell: early large cell small cell 4 cell [n=2] [n=4] [n=16]
Figure 4.9
B ox p lo ts of s ta n d a r d is e d c o r r e c te d n u c le a r in te n s itie s for DAPI s ta in e d m o u s e o o c y te s a n d e m b ry o s . All v a l u e s a r e c o m p a r a b le to th e co n tro l u n fertilised m e t a p h a s e II o o c y te s w h ich h a v e a 2 C DNA c o n te n t. S p o n ta n e o u s ly a c tiv a te d o o c y te s a n d e a rly individual p ro n u c le i h a v e half th e DNA c o n te n t of th e c o n tro l o o c y t e s i.e. 1C . L ate p ro n u c le i a n d th e n u clei from e a rly tw o cell e m b ry o s , e a rly fo u r cell e m b r y o s a n d th e sm a ll c e lls in th r e e cell e m b ry o s h a v e a 2 0 DNA c o n te n t. C e lls a t s y n g a m y , n u clei fro m la te tw o cell e m b r y o s a n d th e la rg e c e lls in a th r e e cell e m b ry o h a v e a DNA c o n te n t th a t is a p p r o x im a te ly e q u iv a le n t to 4 C .
(G r o u p s w h e r e n < 5 h a v e b e e n s h o w n w ith o u t b o x e s s o th a t individual p o in ts c a n b e s e e n . T h e c e n tr a l b a r r e p r e s e n t s th e 5 0 th c e n tile , to p a n d b o tto m w h is k e rs th e 9 0 th a n d 1 0 th c e n tile s r e s p e c tiv e ly )
On DAPI staining five of the control oocytes were found to be in anaphase having undergone spontaneous activation during handling. They were therefore
grouped separately as a control for a 1C DNA content. Two uncleaved cells fixed at the tim e other fertilised oocytes had undergone cleavage to tw o cells, w ere found to be in m etaphase after syngamy. Two embryos w ith three cells w ere also fixed and the m easurem ents from the sm aller 4 cell sized blastom eres recorded separately from the larger cells.
The range and distribution of m easurem ents in each group is dem onstrated using box plots in figure 4.9. W here the num ber of cells m easured w as less th an 5 (i.e. at syngam y, and 3 cell embryos) rather than show ing boxes individual points are show n w ith a central bar representing the 50th centile and w hiskers denoting the 10th and 90th centiles. The m easurem ents conform ed to the expected order of DNA content at each stage of developm ent, w ith activated oocytes and early pronuclei having a 1C DNA content, late pronuclei, early tw o and four cell blastom eres having a 2C content, while at syngam y and the late 2 cell stage nuclear m easurem ents w ere approxim ately equivalent to 4C.
H aving show n that the DNA content for hum an fibroblasts and m ouse oocytes and embryos conformed to that expected for developm ental stage, the sam e technique w as applied to parthenogenetically activated hum an oocytes and em bryos.