Fluorescence in situ Hybridisation and Immunofluorescence of Mitotic and Meiotic

Chromosomes or Analysis of Chromosome Structure

Angel Allen

Abstract
Prior to the advent of molecular cytogenetic techniques, only rudimentary observations of chromosome
structure were possible for cytogenetic analysis. Methods such as fluorescence in situ hybridisation (FISH)
and immunostaining have allowed for remarkable progress in our understanding of genome function and
organisation, allowing the visualisation of molecular details in the context of the whole genome. Here,
experiments to observe various aspects of chromatin structure and the morphology of sex chromosomes
were performed. Staining of polytene chromosomes produced a banding pattern that differentiated
heterochromatin from euchromatin. FISH of human metaphase chromosomes allowed identification of the
sex chromosomes, including those in interphase nuclei. The location of the inactive X chromosome was visible
due to increased DAPI intensity, indicating the heterochromatic Barr body. Finally, immunolocalisation of
SCP3 in mouse spermatocytes allowed analysis of the different stages of prophase I, in addition to the
identification of an XY body - the product of meiotic sex chromosome inactivation.

Introduction heterochromatin and euchromatin respectively,

Molecular cytogenetic techniques allow the
investigation of chromosome structure and
evolution (1,2). Chromosome banding,
fluorescence in situ hybridisation (FISH), and
immunofluorescence were utilised here to analyse
chromatin structure, chromosome morphology,
and characteristics of sex chromosome
inactivation.
DNA is packaged as chromatin
The diploid human genome contains over 6
billion nucleotides, approximately two metres of
DNA, that need to fit into the nucleus of every cell
(3). To achieve this, DNA is packaged with
histones and other proteins as chromatin (4). This
fibre can be folded and condensed further, and the
degree of chromatin condensation is strongly
related to gene expression (5–7). If chromatin is
dense and tightly compacted then transcriptional
machinery will not be able to access the DNA to
initiate gene expression. On the other hand, looser
chromatin allows access to the DNA for the
binding of transcription factors, activators, and co-
activators that can further remodel chromatin in a
way that promotes gene expression (5). These two
contrasting states of chromatin are referred to as
terms originally proposed by Emil Heitz back in
1928 after he found differences in chromosome
condensation in moss (8,9).
Heterochromatin and euchromatin - banding
patterns
There are key characteristics of each chromatin
state that provide the molecular basis of
chromosome banding (specifically G-banding)
(10,11). Heterochromatin is transcriptionally
repressive, late replicating, gene-poor, AT-rich,
and is associated with increased protein
concentration and stability (through disulfide
bonds). Euchromatin is transcriptionally active,
early replicating, GC-rich, and takes on a looser,
more accessible conformation (10,12). Generally,
dyes for banding either target AT base pairs, or
precipitate in heterochromatic regions due to the
retention of hydrophobic proteins following
protease pretreatment (10). The open
conformation of euchromatin allows for easier
removal of hydrophobic proteins and less
retention of stains (10,13). Replication banding by
incorporation of a DNA base analogue (such as
BrdU) after early-replicating DNA has been
synthesised also produces G-bands. There is
conservation of this banding pattern, as certain
parts of the genome are constitutively
heterochromatic, and it can be used for the
identification of chromosomes (14).
Mitosis and meiosis
The somatic cell cycle consists of phases of cell
growth (G1), DNA replication (S), preparation for
cell division (G2), and finally the production of two
daughter cells through mitosis (M) (15). Mitosis
can be further broken down into stages of
chromosome condensation (prophase), alignment
of chromosomes and dissolution of the nuclear
membrane (metaphase), segregation of sister
chromatids (anaphase), reforming of the nuclear
envelope, and relaxing of chromosomes into their
original state (telophase) (16–18).
Meiosis allows cells to produce haploid gametes
by undergoing two rounds of cell division
following a single DNA replication (19). In meiosis
I, homologous chromosomes pair, synapse, and
undergo recombination before reductive cell
division. The second meiotic division (meiosis II)
is similar to mitosis in that sister chromatids are
segregated (19).
Synaptonemal complex
The joining of homologous chromosomes is
mediated by a protein lattice called the
synaptonemal complex (SC) (Figure 1). SC
formation begins in prophase I with leptotene. In
this stage, double-stranded breaks are introduced
to the DNA, and homologous chromosomes begin
to pair (20–22). Synaptonemal complex protein 3
(SCP3) begins to accumulate along the length of
each homologue, forming the axial element. The
central element of the SC forms in zygotene,
connecting axial elements of homologous pairs. By
the pachytene stage, the SC is fully formed and the
axial elements have become the lateral elements
(20).
Figure 1. Structure of the synaptonemal complex.
Axial elements (AE) from homologous
chromosomes are connected by transverse
filaments (TF) to form the SC. The lateral elements
(LE, former AE) consist of SCP2/3 and the central
element (CE) of SCP1 in mammals. Figure adapted
from Zhang et al. (2021) (23).
Sex chromosomes
Two phenomena involving sex chromosomes are
explored here - X chromosome inactivation (XCI),
and meiotic sex chromosome inactivation (MSCI).
X-chromosome inactivation
Dosage compensation by XCI occurs in female
therian mammals (24). During early embryo
development, random inactivation of all but one X
chromosome occurs in each cell. The inactive X
chromosome (Xi) is transcriptionally repressed
and has been found to localise to the nuclear
periphery during interphase as a heterochromatic
structure named the Barr body (24–27).
Meiotic sex chromosome inactivation
As sex chromosomes only share homology in the
pseudoautosomal region (PAR), they remain
largely asynaptic during spermatogenesis (28).
MSCI involves transcriptional repression and
localisation of the X/Y chromosomes to a
heterochromatic XY body, preventing illegitimate
recombination between nonhomologous regions
(28,29).
 Here, polytene chromosomes were stained to
observe banding patterns related to chromatin
structure. The morphology of chromosomes in
human metaphase spreads were observed, and
sex chromosomes were identified using FISH.
Finally, immunofluorescence of SCP3, the main
component of the axial/lateral element of the SC
(20,30), was performed here to observe SC
formation for homologous recombination in
meiotic prophase I.
Results & Discussion
Chromatin structure and banding patterns in
polytene chromosomes
Salivary glands were extracted from Drosophila
melanogaster third instar larvae to prepare
polytene chromosomes which were stained with
orcein. Alternating dark and light bands were
visible, representing heterochromatin and
euchromatin respectively (Figure 2).
Figure 2. Polytene chromosome from the salivary
glands of a Drosophila melanogaster third instar
larva, fixed with acetic acid and stained with
orcein (100x magnification). The predicted
location of the chromocentre is indicated by the
arrow. Bands of dark (heterochromatin) and light
(euchromatin) chromatin are visible
(arrowheads).
One key heterochromatic structure found in
chromosomes is the centromere - an
epigenetically defined region involved in sister
chromatid cohesion during cell division (31). The
predicted location of the chromocentre, where
nonhomologous chromosomes are joined by their
centromeric regions (32), was also identified in
Figure 2.
The chemical mechanisms of orcein staining are
currently unknown (33). As there was no
differential removal of protein, the stain is
unlikely to be precipitating due to the retention of
hydrophobic proteins in heterochromatin. There
was also no wash step following the addition of
the dye, so retention of colour based on chromatin
condensation is also unlikely. The higher protein
content of heterochromatin (6,10) may increase
its hydrophobicity over that of euchromatin, but
research supporting this could not be found.
Sex chromosomes were identified in mitotic
chromosomes
Fluorescence in situ hybridisation (FISH) was
utilised to identify the sex chromosomes in human
metaphase chromosome spreads prepared from
peripheral lymphocytes. DNA was stained with
DAPI (a fluorescent dye that associates with AT
base pairs (34)), and fluorescent probes specific to
centromeric repeat sequences in either the X or Y
chromosome were used (35). Clear signals were
seen in both the female (Figure 3A, 4) and male
(Figure 3B) spreads, allowing successful
identification of the sex chromosomes.
Figure 3. FISH of centromeric repeats in X/Y chromosomes in a female (A) and male (B) human metaphase
chromosome spread. DNA has been DAPI stained, scale bars 20 μm. The X-specific probe shows a green signal
and the Y-specific probe a red signal. The predicted Xi in the female spread is indicated by the arrow (A). In
the male spread, the Y chromosome has a strong DAPI signal representing the heterochromatic long arm
(arrow) and an interphase nucleus is visible in the top left (B).

chromosomes were similar in appearance, so the

Chromosome morphology
Chromosomes in Figure 2 appeared longer and
thinner than expected for metaphase and are
suspected to be earlier in the cell cycle. There are
45 distinguishable chromosomes in the female
spread (Figure 2A) with many segments
overlapping. Two distinct signals are present
indicating the locations of the X chromosomes. In
the male spread (Figure 3B) an interphase nucleus
is visible at the top left with a low signal for the X
chromosome and no Y signal visible. The spread is
likely incomplete as only 44 chromosomes were
counted. Clear signals are seen for the mitotic X
and Y chromosomes. Stronger DAPI signals are
visible at the centromeres due to the higher
affinity of DAPI to AT-rich heterochromatin (34).
A strong DAPI signal was observed on the
majority of the labelled Y chromosome (Figure
3B), attributable to the highly heterochromatic
nature of its long arm (36).
One of the X chromosomes in Figure 3A appeared
to be shorter and more condensed, which suggests
that it is the Xi. In Figure 4, both mitotic X
Xi was unable to be identified. However, there was
a region of intense DAPI staining around one of
the X chromosomes in the interphase nucleus,
indicating that it may be the Xi. While the other X
appears to be on the nuclear periphery, there is
low DAPI intensity suggesting this region is
euchromatic. The identified Xi appears to be
toward the centre of the nucleus, but could still be
the periphery in 3D space. To confirm the position
of the inactive X, RNA FISH of Xist, a long
noncoding RNA involved in silencing Xi (24), could
be performed.
Cells were in prophase-prometaphase
The chromosomes in both Figures 3 and 4
appeared to be pre-metaphase as individual
chromosome arms were not visible, indicating
cohesion of sister chromatids. During mitosis,
cohesin is removed from the chromosome arms to
resolve entangling following DNA replication and
to allow sufficient condensation of chromosomes
for segregation (37,38). This occurs during
prophase and by metaphase, only centromeric
cohesin remains which gives the chromosomes
their characteristic X shape (39–41).
Figure 4. FISH of the X chromosomes in a female
human metaphase chromosome spread. DNA was
DAPI stained, scale bar 20 μm. Two clear green
signals are seen in both the metaphase spread and
the interphase nucleus (top right), indicating the
locations of the X chromosomes. Stronger staining
by DAPI of heterochromatin in the centromeres is
evident here. The predicted location of the Xi is
indicated by the arrow.
The chromosomes were prepared from
peripheral lymphocytes that were cultured with
phytohaemagglutinin to induce mitosis (11).
Incubation for 72 hours is often stated as optimal
for cytogenetic analysis, particularly for
chromosome banding (42–44). When cells are
harvested at this stage, chromosomes have usually
reached early metaphase and a favourable degree
of condensation (Figure 5). The duration of
incubation following the addition of colchicine (a
spindle poison that causes depolymerisation of
microtubules, preventing progression to
anaphase) also has effects on chromosome
condensation and resolution of the chromosome
arms (11,42). If cells are allowed to culture for
longer after the addition of colchicine they will be
allowed to sit at metaphase and reach full
condensation. Complete removal of sister
chromatid cohesion along the chromosome arms
also occurs in cells arrested in metaphase (37).
Based on the appearance of chromosomes
prepared by Bangs and Donlon (Figure 5) (44), the
chromosomes in Figure 3 are likely in prophase,
whereas those in Figure 4 appear to be in
prometaphase.
Figure 5. Degree of chromosome condensation at
different stages of mitosis. Chromosomes were G-
banded to demonstrate band resolution and the
level of condensation. Reprinted from Bangs and
Donlon (2005) (44).
Identification of prophase I stages in mouse
spermatocytes
Immunofluorescence of SCP3 in primary
spermatocytes from juvenile mouse testes was
performed to allow visualisation of SC formation
and the identification of different stages of
prophase I. Figure 6 shows cells in leptotene and
zygotene, and Figure 7 shows cells in zygotene,
pachytene, and diplotene.
Figure 6. Immunolocalisation of SCP3 in mouse primary spermatocytes. Nuclei stained with DAPI, scale bar
20 μm (left image). (A) Leptotene/early zygotene, 10 μm scale bar. (B) Zygotene. SCP3 layer only, 20 μm scale
bar.

Figure 7. Immunolocalisation of SCP3 in mouse primary spermatocytes during prophase I. Nucleus stained
with DAPI, scale bars 20 μm. Proposed stages of prophase I: (A) zygotene, (B) pachytene, (C) diplotene, (D)
diplotene. The predicted locations of the sex chromosomes are labelled in (B), and indicated by the arrow in
(D).
Axial elements (AE) of the SC begin forming on all zygotene is relatively early in prophase,
chromosomes during leptotene, leading to many chromosomes will be minimally condensed and
faint SCP3 signals as assembly begins (46). This the axial elements (AE) will be elongated. Cells in
can be observed in Figure 6A. During zygotene, pachytene (Fig 7B) had strong and distinct SCP3
SCP3 signals were widespread and consisted of signals that appeared as thick lines between
long, partially broken lines (Figures 6 and 7A). As homologous chromosome pairs. The X and Y
chromosomes are able to be identified here as
they had not synapsed. Two thin lines indicating
their separate AE were present, and there was a
noticeable size difference between the two.
Diplotene (Fig 7C) was identified due to the
appearance of loops and "split ends", indicative of
SC disassembly and separation of the AE (47).
After the SC is removed, homologous
chromosomes remain tethered at recombination
nodules (chiasmata) until anaphase (21,28).
Identification of the cell in Figure 7D was
uncertain. On first observation, the disperse and
disorganised signals indicate zygotene. However,
an area of higher DAPI intensity with a strong line
for the SCP3 signal was observed which is
suggested to be an XY body from MSCI. They
appear to be unsynapsed with an accumulation of
SCP3, as seen by de la Fuenta et al. (20).
Zygotene and diplotene were difficult to
differentiate
While pachytene was easily identifiable (Fig 7B),
zygotene and diplotene were difficult to
differentiate (Fig 7A, C, and D). During both stages,
some regions will be unsynapsed because the SC
will be currently assembling or disassembling,
respectively. While SCP3 signals in zygotene are
expected to appear scattered and disordered as
the AE assemble (46), there are reports of strong
signals, similar to that seen in pachytene, forming
during mid-to-late zygotene (20,48). Strong SCP3
signals during diplotene have also been observed
(46,47).
Both Page et al. (2012) and Gil-Fernandez et al.
(2020) had very similar patterns of SCP3
localisation during zygotene and diplotene, with
only additional immunostaining allowing proper
identification of these stages (47,48). Lee et al.
(2003) did see differences between these stages
though - SCP3 during zygotene was much more
dispersed while diplotene had strong signals (46).
Immunolocalisation of SCP1 (central element of
the SC), RAD51 (marker of DSBs), or MLH1
(marker of recombination) could be performed to
better identify each stage of prophase I (20,28,47).
Synapsis of homologues could be confirmed by the
detection of SCP1, as the presence of SCP3 does
not directly indicate this (21,47). Furthermore, the
sex chromosomes will likely be easier to identify
due to a lack of synapsis (49). Histone variants
such as H3K4me1 and γH2AX have been shown to
differentiate the XY body during late pachytene
and diplotene (47) and could be also be used to
confirm the location of the sex chromosomes.
Alternatively, FISH using the same XY probes as
the previous experiment could be performed to
easily confirm the locations of the sex
chromosomes.
Materials and Methods
Methods were as described in the Genetics III
practical handbook (2022, S1).
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