2.3. Tres historias sublevantes: el libro
2.3.3. Fénix
5.1 INTRODUCTION
In the GDI mouse strain, XY (male) embryos form a blastocoel earlier, on average, than females (Tsunoda et al., 1985). More recently Valdivia et a/.(1993) have shown that, for the C57BL/6 strain, this XY advantage is seen following in vitro fertilisation (IVF), thus ruling out the possibility that the difference is due to Y-bearing sperm arriving earlier than X-bearing sperm. Burgoyne (1993) has demonstrated that this XX-XY difference is also manifest as a cell number difference at 3.5 dpc. This implies that blastocoel formation occurs at the same cell number in XX and XY embryos but the XY embryos simply achieve this cell number earlier. Burgoyne also formally demonstrated that this preimplantation XX-XY difference is due to an accelerating effect of the Y-chromosome that is quite separate from the effect of X- chromosome constitution (described in Chapter 3) which influences later development. The experiments below had two main aims:
(/) To assess the efficacy of FISH (Fluorescent In-Situ Hybridisation) for sexing interphase nuclei from blastocyst spreads, initially using a single probe to identify the Y-chromosome and, in later experiments, using dual colour FISH for the simultaneous identification of X and Y-chromosomes. FISH is offered as an alternative approach for sexing embryos rather than the karyotyping method described by Burgoyne (1993) which is time-consuming and technically difficult. Initially, MFl and RIE derived Y-chromosomes were investigated to validate the FISH approach, since their effect on preimplantation development is known (Burgoyne, 1993).
(zz) To assess the influence on preimplantation development of the Y-chromosomes used in the crosses in Chapters 2, 3, and 4. From the results in Chapter 3, it appeared that the Y ^" chromosome (present in the Patchy-fur stock) conferred a developmental advantage to XY fetuses over X^O sibs at 10.5 6pc, which might represent the ‘carry-over’ of a preimplantation advantage. It was therefore necessary to test for a preimplantation effect using the Y^^" chromosome. In Chapter 4, the ^z^-negative Y^^ chromosome was shown to confer a developmental advantage to 'XXYSry over XX fetuses at 10.5 dpc and was therefore tested for a preimplantation accelerating effect (in conjunction with a normal Y^^ chromosome as a control).
5.2 MATERIALS AND METHODS
5.2.1 Mice
A series of crosses were set up for the production of male and female embryos. In all cases the females used were from a colony of random bred albino MFl mice from the NIMR, Mill Hill (originally established with mice obtained from Olac, UK). The females were allowed to ovulate naturally, since the size of the preimplantation XX-XY difference may be reduced following superovulation (Zwingman et al, 1993). The males used were as follows:
(a) M Fl males
(b) M Fl, males
The Y™ chromosome has been backcrossed to MFl for more than 10 generations and is unusual in that it produces no XY advantage (in the context of an MFl background).
(c) X“ '‘y “ " males.
These mice were produced by mating males from the Patchy-fur (Paf) stock to MFl females in order to replace the fa/"X-chromosome with an MFl X. The subsequent mating to MFl females produces embryos in which all X-chromosomes are of MFl origin. The Permutation arose spontaneously in a subline of the C3H/HeSnJ strain (Lane and Davisson, 1990), thus the Y-chromosome in the PeTstock is of C3H origin (referred to as Y^^" throughout).
(d) XY^^^N/yY-del^ males
These males were produced by mating XXY^^^&y' females (Mahadevaiah et al, 1993) to XY-del™ males (Conway et al, 1994). In both cases, the Y-chromosomes have been backcrossed to MFl for more than ten generations. The 6%y-negative chromosome originated in the 129 strain (Lovell-Badge and Robertson, 1990). The Y-del™ chromosome is identical to the Y™ chromosome used above (cross 2), except for a deletion of two-thirds of the long arm (Conway et al, 1994). The majority of these ‘XYY’ males is initially sterile (due to the presence of three sex chromosomes) but most become fertile in later life due to the generation
breeding data have established that they produce almost exclusively XX and XYSry female offspring (Mahadevaiah et al, 1993).
In fact, test matings involving five separate XYY studs (including those used in the present study) sired a total of 325 livebom offspring, all of which were female (P. S. Burgoyne, pers. comm ). It is thought that sperm fi-om the XY-del™ component are ‘competed out’ during passage through the female tract, since sperm fi-om XY-del™ males have an increased incidence of sperm abnormalities (Conway et al, 1994) and have been shown to be less efficient than those fi"om XY™ males in heterospermic competition experiments (B.Peitz and P.S.Burgoyne, pers. comm ).
(e) M Fl, males
These studs served as a control for the previous experiment, since the &y-negative Y- chromosome is derived fi-om the Y^^^ chromosome (Y^^ has been backcrossed onto an MFl background for 8 generations).
Pregnant females were killed in the morning (1000-1200) of the third day after the vaginal plug was recorded (3.5 dpc). Embryos were flushed from both uterine horns with a 25 gauge needle using a solution of phosphate buffered saline (PBS) supplemented with bovine serum albumin (BSA 4 mg/ml). The BSA is needed to prevent embryos from adhering to the pipette and each other.
5.2.2 Cytogenetic preparation (Blastocyst spreading)
Embryos were processed according to a modification of the method of Burgoyne (1993) - the main difference being that groups of embryos rather than single embryos were processed together on the same slide. After washing through several droplets of PBS/BSA, the embryos were placed in a drop of hypotonic solution (1% w/v trisodium citrate) for between
1.5 (late morulae) and 2.5 minutes (blastocysts). After this time, embryos were transferred to a clean glass slide in a fine glass pipette (drawn to a diameter just bigger than that of the swollen embryo) containing 0.75% w/v trisodium citrate. The embryos on the slide were viewed continuously under a stereo dissection microscope (Wild M5A, x 250) as two drops of aqueous fixative (9:3:2 methanol:acetic acid: distilled water) were applied to the citrate
drop just before it dried. As the aqueous fixative dissolved the zona pellucida, the embryos began to collapse visibly. The acetic acid component removed most of the cytoplasm, thus exposing the nuclei for the later FISH procedure. At this stage, a drop of 3:1 methanol:acetic acid fixative was applied between the embryos. This served to wash away cytoplasmic debris and traces of the aqueous fix. The preparations were then blown dry and aged overnight at 37°C before being processed for FISH. A number of slides were stained with fluorescent dye (DAPI) prior to FISH to allow accurate cell number counts in case of blastomere loss during the FISH procedure (crosses 4 and 5).
5.2.3 Fluorescent in-situ hybridisation (FISH)
FISH was carried out on the interphase nuclei of mouse embryos using the method described by GriflBn et a/.,(1991). In the single colour FISH experiments (crosses 1-4 above) a biotinylated DNA probe, pY353B, was used to detect a series of repetitive sequences on the long arm of the mouse Y-chromosome (Bishop et al, 1985). In the dual colour experiments (later litters fi’om cross 4 and all litters fi-om cross 5), the X-chromosome was detected using biotinylated DXSmhlS, which is located in multiple copies just below the centromere on the mouse X-chromosome (Nasir et al, 1990), and the Y-chromosome with digoxygenin- labelled pY353B (Plate 5.1.2). Details of DNA probes, probe labelling and the FISH procedure are given in Appendix V.
Briefly, slides with fixed blastocysts were pretreated with RNase and proteinase K and fixed in 1% formaldehyde. The labelled probe(s) was dissolved in hybridisation buffer, applied to the slide under a coverslip and sealed. Simultaneous dénaturation of probe and genomic DNA by heating to 80°C, preceded overnight incubation. Post-hybridisation washes were followed by probe detection by sequential incubations in avidin-FITC, biotinylated anti-avidin D and avidin-FITC (for biotinylated pY353B or DXSmhl5) with the inclusion of anti- digoxygenin conjugated to rhodamine, in the final step for detection of both X and Y probes in the dual colour experiments. Slides were washed in PBS and mounted in antifade medium containing 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI - 0.5 pg/ml) and propidium iodide (4 pg/ml) as counterstain. For dual colour detection, DAPI alone was used
5.2.4 Determination of cell number and sexing of blastocysts
Following the FISH procedure, individual fixed blastocysts were located under a xlO objective. Cell counts were performed on blastocyst spreads under a xlOO oil immersion objective on a Reichert Polyvar microscope fitted for fluorescence. Nuclei (either interphase or metaphase) were counted using the U1 filter set (excitation 365 nm/ emission 480 nm) for detection of the DAPI counterstain, to avoid bias and for practical convenience (since FITC signals are prone to fading). After cell counts were performed, the same nuclei were sexed (scored for presence or absence of the Y-chromosome) using the B1 (excitation 490 nm/emission 525 nm) and G2 filter set (excitation 520 nm/emission 610 nm) simultaneously to detect FITC signals and the propidium iodide (PI) counterstain, which provides a better contrast for the FITC signal than DAPI (Plate 5.1.1). Dual colour experiments (Plate 5.1.2) were analysed using DAPI, FITC and Texas red filters on a Zeiss Axioscope with a cooled CCD camera attachment (Photometries) and IPLab software (Apple Computer, Inc.).
5.2.5 Quantitative analysis
The speed of preimplantation embryo development is both genetically and environmentally determined. By studying embryos from within the same litters, many of the environmental factors may be standardised or removed. Grossly abnormal embryos (morphologically abnormal or with fewer than 8-cells at 3.5 6pc) were not processed. Embryos with cell numbers falling more than 2.6 standard deviations from the litter mean were excluded from the analysis as ‘outliers’. Cell numbers were log transformed (since embryos are growing exponentially at this stage) before comparisons were made within litters between mean XX and XY logio cell numbers using the mean weighted difference, r-tests were used to determine the statistical significance of XX-XY differences. Chi-squared tests were used to assess the sex ratios of offspring obtained from each cross and as a crude comparison of the numbers of deviations falling above and below litter means for XX and XY genotypes from different crosses (Appendix ET).
5.3 RESULTS
Validation o f FISH procedure using M F o n d M F l Ÿ ^ crosses.
Of 294 embryos obtained from the 4th day of pregnancy, 4 were excluded because of gross abnormalities (either dead or fragmenting blastomeres) or missing zonae pellucidae. Generally between 3 and 6 embryos were processed simultaneously on the same slide resulting in a slightly higher frequency (18/290 = 6.2%) of embryo loss from preparations compared with the published loss (10/535 = 2%) observed when individual embryos were spread on separate slides (Burgoyne, 1993).
A number of embryos were damaged either during spreading, in-situ hybridisation or a combination of both procedures (16/290 = 5.5%) making either accurate cell counts or diagnosis of sex impossible. However, sexing and cell number counts were successfully performed for all but one of the embryos which were intact on the slide following the FISH procedure, giving an overall success rate of 88 per cent (255/290) which compares well with a published success rate of 78 per cent (Burgoyne, 1993).
A small number (10/255 = 3.9%) of the embryos scored for both sex and cell numbers were excluded from analysis on the basis of their eccentric cell numbers which were more than 2.6 standard deviations from the litter mean. The outliers with a low cell number may have been embryos with an abnormal karyotype (Burgoyne, 1993), since neither the full sex chromosome nor autosome complement was monitored in the present experiments. One such embryo appeared to have two Y-specific hybridisation signals in all blastomeres and was presumably either 41,XYY or 60,XYY. Outliers with a high cell number could occur as a result of coalescence of adjacent embryos during spreading.
The means for actual cell numbers and estimates of mitotic indices for XX and XY embryos from each of the crosses are presented in Table 5.1. Mitotic index is an indicator of developmental stage reached, peaking at the late morula stage and decreasing towards implantation in murine (Copp, 1978) and bovine embryos (Yadav et a l, 1993). In these data,
cells on average) and a lower mitotic index is not consistent. This is not surprising for a number of reasons. First, the divisions between morulae and blastocyst stages (16-32-64 cells) are still very synchronous making it difficult to observe differences in numbers of actively dividing cells between embryos when looking at a single time point. Second, some metaphases would not be identified as such due to poor fixation. Finally, this procedure misses those cells which would have arrested in metaphase, during a period of culture in colcemid.
The sex-ratio (or XY.XX ratio) was calculated for both crosses and, using a chi-squared analysis, it was clear that neither cross showed a significant distortion fi-om the expected sex ratio of 1.0.
Mean weighted differences between XX and XY embryos are shown in Table 5.2. Burgoyne (1993) previously demonstrated a significant cell number advantage of XY embryos over XX littermates fi-om crosses involving the MFl Y-chromosome, and this is confirmed in the pooled MFl data obtained here. However, in the present study it was observed that no XX- XY difference was apparent for the embryos fi-om one MFl stud male (#45), and this continued to be the case, despite collecting a substantial amount of data (see Tables 5.1 and 5.2). Data are therefore also presented for the pooled MFl studs excluding MFl #45 and these show a more marked XX-XY difference - the mean cell number of XY blastocysts is increased by approximately 6 cells compared with XX litter mates which compares with the estimate of ~8 cells by Burgoyne (1993).
It has previously been established that the Y™ chromosome on MFl background produces no XX-XY difference (Burgoyne, 1993). Mean cell numbers for XX and XY embryos fi-om this cross were equivalent, in agreement with this observation. The data fi-om these two crosses are illustrated in histogram form (Figures 5. la and d).
Crosses involving Ÿ^^Sry and
Of 331 embryos obtained fi-om the 4th day of pregnancy, 6 were excluded because of gross abnormalities, and the fi-equency of embryo loss fi-om preparations was slightly lower (16/325 = 4.9%) than in earlier experiments, although a similar proportion of embryos was damaged
during the experimental procedures (26/325 = 8.0%). Reliable sexing could not be performed in 5 cases, owing to a failure of hybridisation or subsequent detection. The overall success rate (278/325 = 86%) was similar to that achieved in the validation experiments, with only 2 outliers excluded from analysis.
In Chapter 3, the chromosome present in the Paf stock, was shown to be associated with increased fetal weights at 10.5 (XY^^^ fetuses were significantly larger than X^O fetuses). It was suggested that this might be due to the ‘carry-over’ of a preimplantation Y- effect. Here this is tested by comparing cell numbers of XX and XY^^ embryos at 3.5 dpc. In this comparison, the X-chromosomes are all MFl since the X-chromosome was of MFl strain in a number of earlier comparisons (Burgoyne, 1993) and during the validation of the FISH technique (see above). The results show that XY^^ embryos do indeed have significantly more cells (~ 8 cells on average) than XX embryos from the same litters (Tables 5.3 and 5.4). This difference is illustrated in histogram form in Figure 5.1c. XY deviations are markedly skewed above the litter mean while XX deviations are slightly skewed below the litter mean.
In Chapter 4, XXY^^&y" fetuses were shown to be significantly larger than XX fetuses at 10.5 dpc and, once again, it was suggested that this was due to the ‘carry-over’ of a Y-effect from the preimplantation period. It is particularly important to test this directly, because Sry (which is deleted in these XXY’ fetuses) is expressed in the preimplantation period (Zwingman et al, 1993) and has been proposed to be responsible (at least in part) for the accelerating effect of the Y. The results show that XYSry embryos have more cells, on average, than XX embryos but the difference is not significant (Tables 5.3 and 5.4). A similar difference, which again is not significant, between XX and XY cell numbers is seen for embryos from the control cross in which the male has a Y-chromosome of 129 origin (this time with the Sry gene present). In fact the magnitude of the XX-XY difference (~ 2.2 cells) is not significantly different from that seen in the Y^^^Sry cross (i^O.8). For this reason the two data sets have been pooled to strengthen the XX-XY comparison. It is clear from the pooled Y^^^ data that, despite the apparently small size of the XX-XY cell number difference
The extent of the XX-XY differences in the crosses involving the Y^^Sry and chromosomes is illustated in the form of histograms, in which individual embryo log cell numbers are plotted as deviations from the litter means (Figures 5. \d and 5. le). The finding that the XX-XY^^^ difference, although small, is significant is supported by the skewing of deviations about the litter means for XX and XY embryos from the pooled Y^^ and Y^^^Sry crosses (x^= 4.92; P<0.05).
Table 5.1 Estimated cell numbers and mitotic indices for XX and XY embryos at 3.5 dpc (Y^^ and Y ^ )
Genotypes compared
No.embryos** Mean cell number/embryo^
Range (cell no.)*
Mean metaphase count/embryo
Mitotic Index® Sex Ratio XY/XX IPooled A 90 42.5 21-66 1.9 4.4 0.99 0.01 XX 91 38.0 20-66 1.8 4.7 40 41.9 21-64 1.4 3.4 1.11 0.10 XX 36 40.4 27-64 1.8 4.4 IPooledB 50 42.9 22-66 2.5 5.9 0.91 0.12 XX 55 36.6 20-66 1.5 4.1 XY™ 37 42.3 16-69 2.1 5.0 1.32 0.62 XX 28 42.4 24-69 2.5 5.9
Table 5.2 Mean log cell numbers and XX-XY differences for XX and XY embryos at 3.5 dpc and Y ^ ) Cross Numbers of embryos® XY XX Mean ± SEM logio cell number’’
XY XX Mean ± SEM^ weighted difference Significance (f) MFl X MFl (Pooled A) 90 91 1.63+0.02 1.58 + 0.02 0.037 + 0.016 0.025-0.01^ MFl X MFl (#45) 40 36 1.62 + 0.02 1.61+0.02 0.003+0.026 NS MFl xM Fl (Pooled B) 50 55 1.63+0.03 1.56 + 0.02 0.061+0.019 0.0025-0.001^ MFl xM Fl.Y™ 37 28 1.63+0.04 1.63+0.03 -0.002 + 0.029 NS
a (as for Table 5.1) b Mean of litter means
c The error for the mean weighted difference is calculated using the variance within genotypes within htters t Indicates significant difference, (using a one-tailed t-test)
Table 5.3 Estimated cell numbers and mitotic indices for XX and XY embryos at 3.5 (Y^^ or Y^^^
Genotypes compared
No.embryos^ Mean cell number/embryo^
Range (cell no.)*
Mean metaphase count/embryo
Mitotic Index ^ Sex Ratio XY/XX XY^3H 26 47.6 17-67 2.4 5.0 1.04 0.01 XX 25 40.1 16-65 2.7 6.8 65 40.2 16-65 1.3 3.3 1.30 0.98 XX 50 37.3 17-64 2.0 5.3 Xyl29 60 42.9 20-64 2.1 4.9 0.83 0.54 XX 72 40.7 22-73 2.2 5.3
Table 5.4 Mean log cell numbers and XX-XY difTerences for XX and XY embryos at 3.5 dpc (Y^^ or Y^^^ Cross Numbers of embryos® XY XX Mean + SEM"" logio cell number
XY XX Mean ± SEM^ weighted difference Significance (P) MFl xM Fl, 26 25 1.68 ±0.05 1.60 ±0.06 0.070 ±0.025 0.005-0,0025^ M F lxM Fl, Y '^% - 65 50 1.60 ±0.03 1.57 ±0.03 0.028 ± 0.021 NS MFl X MFl. 60 72 1.63 ±0.02 1.61 ±0.02 0.021 ±0.019 NS M FlxM Fl.Y ™ (Pooled data) 125 122 1.62 ±0.02 1.59 ±0.02 0.024 ±0.014 0.05-0.025’
Figure 5.1 Histograms illustrating XX-XY log cell number difTerences at 3.5 Ape
Comparisons involved the following Y-chromosomes:
(a) MFlPooled B
(b)
Rm
(c) C3H (P«/)
(d) 1295iy'
V n S T f
In each comparison the data for 3.5 àpc embryos are plotted as individual deviations from the average of the XX and XY ‘litter means’, i.e.
N = number of embryos
C = log cell number (deviations)
(n) Figures in parentheses indicate the number of deviations falling above or below the litter mean
For each cross XX data are plotted above the line: H
(a) (b) 1---1---1--- 1---- 1---r -0.3 -0.2 -0.1 0 +0.1 +0.2 +0.3 C (12) J (16) ( 17) ( 20) 1 1 1 1 1 1--- -OJ -O j -0.1 0 +0.1 +0.2 +0.3 C (c) (d) (e) (14) (7) (26)
H
WÊ (24) (39)I
I
(33) (19) (26) (39) (23) (37)Plate 5.1.1 Sexing preimplantation mouse embryos (single colour FISH)
(a) 29-cell early blastocyst spread after culture in colcemid and subsequent fixation. Chromosome number and the presence or absence of the Y-chromosome can be scored in each mitotic metaphase plate fi'om air-dried giemsa stained preparations (karyotype method).
(b) 32-cell early blastocyst spread stained with DAPI (x40 objective).
(c) 32-cell early blastocyst (seen in 5.1.16) counterstained with propidium iodide (red) following fluorescent in-situ hybridisation using a Y-specific FITC labelled probe (signal appears greenish-yellow). The blastocyst is presumed 40, XY.