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- Original Article -
Characterization of nucleohistone and nucleoprotamine components in the mature human sperm nucleus
Yan Li1,2, Claudia
Lalancette1,2, David Miller3, Stephen A.
Krawetz1,2,4
1Center for Molecular Medicine and Genetics,
2Department of Obstetrics and Gynecology, Wayne State University
School of Medicine, Wayne 48201, MI, USA
3Reproduction and Early Development Group, University of Leeds, Institute of Genetics, Health and Therapeutics, Leeds LS29JT, UK
4Institute for Scientific Computing, Wayne State University, Wayne 48201, MI, USA
Abstract
Aim:To simultaneously determine the localization of histones and protamines within human sperm nuclei.
Methods: Immunofluorescence of the core histones and protamines and fluorescence
in situ hybridization of the telomere region
of chromosome 16 was assessed in decondensed human sperm
nuclei. Results: Immunofluorescent localization of
histones, protamine 1 (PRM1) and protamine 2 (PRM2) along with fluorescence
in situ hybridization localization of chromosome 16 telomeric sequences revealed a discrete distribution in sperm nuclei. Histones localized to the
posterior ring region (i.e. the sperm nuclear annulus), whereas PRM1 and PRM2 appeared to be dispersed throughout the
entire nucleus. Conclusion: The co-localization of the human core sperm histones with the telomeric regions of
chromosome 16 is consistent with the reorganization of specific non-protamine regions into a less compacted state.
(Asian J Androl, 2008 Jul; 10: 535_541)
Keywords: human sperm nucleus; histone; protamine; telomere
Correspondence to: Prof. Stephen A. Krawetz, 253 C.S. Mott Center; 275 East Hancock; Detroit, MI 48201, USA.
Tel: +1-313-577-0765 Fax: +1-313-577-8554
E-mail: steve@compbio.med.wayne.edu
Received 2008-01-09 Accepted 2008-03-07
DOI: 10.1111/j.1745-7262.2008.00410.x
1 Introduction
Spermatozoa are the product of a complex cellular program of differentiation initiating from the spermatogonia [1,
2]. This is characterized by a myriad of changes including chromosomal recombination and segregation culminating
in the post-meiotic reorganization of the genome into a highly condensed transcriptionally inactive form [3]. These
dynamic global changes are keyed through a variety of mechanisms including DNA methylation, phosphorylation,
acetylation, methylation and ubiquitination of the histone component [4, 5]. This sets the stage for the ordered
substitution of histones with transition proteins then subsequently with sperm-specific protamines yielding a
condensed state that is a hallmark of this transition.
Although most of the histones are replaced with protamines, the human male gamete retains approximately 15% of
its genome in a histone-bound state [6]. This is in direct contrast to other species such as the mouse
where replacement exceeds 98% [7]. Association of
histone and protamines with the spermatozoon's genome is not random [6].
Structural sequences, such as Alu repeats and telomeres, associate with protamines and histones, respectively, whereas
centromeric regions are partitioned between the histone and protamine compartments [8] and are part of the loop DNA
[9].
Some gene-rich regions are specifically distributed between the two nucleoprotein components. In human
spermatozoa, the embryo-specific ε-globin and γ-globin
and the paternally imprinted IGF2 genes are
histone-associated, like the promoter and nuclear matrix
attachment regions of the PRM1→PRM2→TNP2 locus.
In contrast, the postnatally activated β-globin and
δ-globin genes are protamine-associated [8, 10]. However, the
majority of the PRM1→PRM2→TNP2 locus
shows no enrichment for histone or protamine association [8]. Even
though specific examples have been delineated, the
sub-cellular distribution of histones and protamines in the sperm
nucleus has yet to be defined. To begin to address this
issue the distribution of histones and protamines within
the mature human sperm nucleus was examined by immunofluorescence. The core histones and chromosome
16 telomeric regions were localized at the base of the sperm
nuclei. In contrast, both protamines were distributed more
evenly throughout the nucleus. The significance of this
distribution as a means to partition the genome is discussed.
2 Materials and methods
2.1 Semen samples, preparation of nuclei and slides
Human semen samples were randomly collected from
the donor pool in accordance with the Wayne State
University Human Investigation Committee protocol 095701MP2F, processed then frozen in FSB, Frozen
Storage Buffer at _80ºC as described [11].
Briefly, frozen human spermatozoa were quickly thawed at room temperature, then washed once with cold
50 mmol/L Tris-HCl buffer, pH 7.4 containing a complete
protease inhibitor cocktail (CPI, Complete Mini
tablets; Roche Applied Science, Indianapolis, IN, USA). After
centrifugation at 2 000 × g for 5 min at
4ºC, the cells were resuspended to a concentration of
1 × 107 cells/mL in 50 mmol/L Tris-HCl buffer, pH 7.4, containing 0.5%
sodium lauryl sulfate (SDS) and nuclei isolated as described
[12]. Briefly, resuspended spermatozoa were transferred
in a type A glass dounce homogeneizer to dislodge the tails
from the heads. Dissociation was verified under bright
field microscopy. The heads were then pelleted by
centrifugation at 2 000 × g for 10 min at
4ºC. The supernatant was discarded and the heads were resuspended in a
sucrose solution (2.2 mol/L sucrose, 50 mmol/L Tris
pH 7.4, 5 mmol/L MgOAc) to a final concentration of
5 × 106 cells/mL and transferred to a type C glass dounce
homogeneizer. After homogenizing with three
strokes, the suspension of nuclei was layered onto sucrose/CsCl
cushions (2.2 mol/L sucrose, 10 mmol/L Tris pH 7.4,
5 mmol/L MgCl2, 0.89 mol/L
CsCl) and centrifuged at 113 000 ×
g for 90 min at 4ºC in a Beckman XL-90
Ultracentrifuge using a SW41 Ti rotor (Beckman Coulter Inc.,
Fullerton, CA, USA). The nuclei recovered from
centrifugation were then resuspended in FSB to a final
concentration of
2 × 107/mL and stored at
_80ºC.
2.2 Decondensation of sperm nuclei and cells
Sperm nuclei were mixed with 0.5 mL of cold
phosphate buffered saline (PBS), pH 7.4, containing CPI. An
aliquot containing 500 nuclei was then subjected to
centrifugation onto a slide at room temperature for 5 min at
2 000 × g using a cytospin 2 cytofuge (Thermo Shandon,
Pittsburgh, PA, USA). The attached nuclei were then
swollen by gently overlaying 50 μL of a solution
containing 0.05 mg/mL heparin and 10 mmol/L dithiothreitol
followed by incubation on ice for 30 min, as described [13].
Alternatively, the samples were treated with 2.5 mmol/L
DTT, 0.2% Triton X-100 and 100 U of heparin in PBS,
for 30 min [14]. The slides were then washed for 5 min
in PBS and the cells fixed in 4% formaldehyde. Samples
were then dehydrated through a 1 min series of graded
cold ethanol washes of 50%, 70%, 95% and 100%, then
fixed in _20ºC methanol for 20 min.
2.3 Immunolocalization
Anti-core histone sheep polyclonal (1.15 mg/mL)
antibody was purchased from Abcam (Cambridge, UK).
Mouse monoclonal anti-human protamine 1 (PRM1) and
protamine 2 (PRM2) antibodies were a generous gift from
Dr Rod Balhorn, Lawrence Livermore National Laboratory (Livermore, CA, USA).
Non-specific epitopes to mouse or goat antibodies
revealed by nuclear swelling were blocked with a
solution of PBS containing 5% BSA or 5 % rabbit serum for
1 h at room temperature. Before use, the anti-core
histone antibody was diluted 1:300; anti-PRM1 and PRM2,
1:200, in their blocking buffer. The slides were then
overlayed with their respective primary antibody
solution then hybridized overnight at 4ºC. The following
day the hybridized samples were washed using a series of 10 min
PBS washes to remove unreacted antibody. PRM1 and
PRM2 immunoreactive species were revealed following a
1 h, room temperature incubation with 1:500 diluted
CY5-conjugated goat anti-mouse antibody (Biomeda, Foster
City, CA, USA). Histone immunoreactive species were
first incubated for 1 h at room temperature, with biotin
conjugated rabbit anti-sheep antibody (PIERCE, Rockford,
IL, USA), followed by a 30 min incubation with Alexa
Fluor 488-conjugated streptavidin (Molecular Probes,
Carlsbad, CA, USA). The nuclei were then counterstained
by overlaying 10 μL of 0.5 μg/mL
4'-6-Diamidino-2-phenylindole (DAPI) containing Vectashield (Vector Labs
Inc, Burlingame, CA, USA).
2.4 Fluorescence in situ hybridization (FISH)
FISH was essentially as described by Zalensky
et al. [13]. Briefly, following immunolocalization, nuclei were
fixed in 4% formaldehyde for 10 min at room temperature. The samples were then dehydrated
through a series of 50%, 70%, 95% and 100% ethanol washes then denaturated in 70% deionized
formamide buffered with 2 × SSC that contained
300 mmol/L NaCl, 30 mmol/L sodium citrate buffer,
pH 7.0, at 70ºC for 3 min. The chromosome 16
telomeric probe was labeled with dUTP-biotin by
polymerase chain reaction (PCR) using specific forward
and reverse primers 5'-AAAGCTCTCAGAACCTCCCC-3', 5'-AGAGGTTCCCATGTAGTTCC-3' respectively.
The PCR reaction was carried out for 35 cycles of 30 s
at 94ºC, 45 s at 55ºC and 40 s at
72ºC using HotStarTaq DNA polymerase (Qiagen, Valentia, CA, USA). After
synthesis, the probe was purified by FlexiPrep, as
suggested by the manufacturer (Amersham Bioscience,
Piscataway, NJ, USA), then denatured in hybridization
solution containing 50 % deionized formamide buffered
with 2 × SSC at 75ºC for 5 min. Hybridization was then
carried out at 37ºC in a humidified chamber for 16 h. The
post-hybridized sample was then washed at room temperature in 2 × SSPE (300 mmol/L NaCl, 20 mmol/L
NaH2PO4, 2 mmol/L EDTA, pH 7.0) for 10 min, followed
by 1 × SSPE for 10 min, then 2 × SSPE at
45ºC for 10 min and, finally, 2 × SSPE at room temperature for 10 min.
The signal was then visualized with Alexa Fluor
594-conjugated streptavidin by fluorescence microscopy.
2.5 Image capture and processing
Images were captured using a Leica DMRA2 fluorescence microscope (100 × oil immersion objective) then
processed using Image Pro-express 5.1
(MediaCybernetics, Bethesda, MD, USA). Images were pseudocolored then
merged. DNA stained with DAPI was colored blue whereas immunofluorescence signals from the various
antibodies were colored yellow, green or red. A random
set of 10 fields that contained at least 50 nuclei were
selected for immunofluorescence signal frequency analysis. The number of spermatozoa or nuclei showing
the observed pattern in each field examined was assessed.
3 Results
3.1 Distribution of histones and protamines in the sperm
nucleus
An immunofluorescence assay using an anti-core histone antibody and antibodies prepared against PRM1
and PRM2 was used to immunolabel histones and
protamines respectively in human sperm nuclei. Epitopes were
revealed by decondensation prior to the application of
the antibodies [19]. Two decondensing protocols
essentially differing in the concentration of dithiothreitol
(DTT) and heparin, were assessed [13, 14]. As shown
in Figure 1A and 1B, both histones and protamines were
distributed towards the surface of the nuclei when the
nuclei were treated with 2.5 mmol/L DTT plus 100 U of
heparin. In contrast, when the nuclei were first treated
with 10 mmol/L DTT plus 0.05 mg/mL heparin prior to
the application of the anti-histone antibody, the
distribution of the core histones appeared altered. Surprisingly,
as shown in Figure 1C and 1D, 95% of the images
analyzed showed that the histones were radically
distributed from the posterior in what appeared as a ring-like
structure to the postacrosomal region. As summarized
in Table 1, this pattern of the histone antibody was
observed in the majority of the fields examined. In contrast,
at least 98% of images showed that the PRM1 and
PRM2 protamines were distributed throughout the nucleus.
To examine whether this distribution resulted from
the physical preparation of nuclei, dual immunolabeling
and differential interference contrast (DIC) microscopy
of intact spermatozoa using the histone antibody together
with either the PRM1 antibody or the PRM2 antibody
was undertaken. The results are summarized in Figure 2A
and 2B and confirm that the histones are distributed from
the posterior ring to the postacrosomal region. Perhaps
this reflects the sequence-specific compartmentalization
of a histone-enriched territory well apart from the PRM1
and PRM2 regions distributed throughout the nucleus. The
question arises, what does this unique packaging reflect?
3.2 Histone-Telomere
Previous studies report the association of telomere
regions with sperm histones, and those of chromosome 16
using PCR analysis [8, 15, 16]. Co-localization of the
chromosome 16 telomere together with the core histones within
the human sperm nuclei was determined. This employed
fluorescence in situ hybridization analysis of the
chromosome 16 telomere along with the immunofluorescent
detection of the core histones. The results of this analysis
in human sperm nuclei are shown in Figure 3. As
summarized in Table 2 the chromosome16 telomere is
histone-bound.
4 Discussion
Unlike spermatozoa from other species, those from
man are characterized by their somewhat incomplete
replacement of histones with protamines. Approximately
15% of the human sperm genome remains histone-associated [7],
in comparison with 1%_2% of the mouse sperm genome. Immunofluorescence analysis has
previously shown that the histones are peripherally
distributed around the mouse spermatozoon nucleus [14].
Their corresponding distribution in human spermatozoa is less well-characterized, although histone H2B has
been shown to localize specifically to the telomeres and
is observed as a punctuate structure [17]. This
punctuate distribution is reiterated in specific regions of
human gene clusters enriched in the histone complement
[8, 10]. We have yet to garner a global understanding of
the unique arrangement of these nuclear proteins in
human spermatozoa.
The physical organization of the nucleoprotamine and
nucleohistone components of human sperm nuclei was
assessed by immunolocalization and in situ hybridization.
Sperm nuclei present a highly compact structure that is
only accessible to probing when relaxed and the
membrane has been compromised. This is typically
facilitated with the use of DTT and heparin [18]. As
demonstrated from the above, the concentration of the
reducing agent, i.e., the use of 2.5 mmol/L or 10 mmol/L DTT
can markedly affect the outcome. It is clear that
independent of this factor, the histones localize towards the
posterior region of the nucleus and can be observed at
the periphery when mild reducing conditions are employed. Interestingly, when higher concentrations of
reducing agent are used, the protamines are distributed
throughout the nucleus, appearing well-separated from
the dominant histone fraction. A uniform distribution of
protamines within human sperm nuclei is also reported
by Zalensky and collegues [19]. The results obtained
using mild decondensation conditions are comparable to
those previously observed with intact mouse sperm
nuclei [14]. Together, these studies emphasize the
importance of sperm nuclei decondensation to permit the
antibodies to recognize their epitopes.
It is clear that human sperm chromatin is distributed
between histone and protamines in a non-random manner [8], and that these two components localize to
distinct regions of the sperm nucleus. These reiterate the
independent uniform distribution of PRM1 [19] and postacrosomal localization of histones [17, 20] that were
previously shown in human spermatozoa.
The unique packaging of the sperm nucleus might
be essential for proper unpacking of the male genome
during fertilization [8, 21]. For example, the
nucleohistone component of the nucleus could serve as a template
for the replacement of protamines by histones after
fertilization [22]. This could be initiated by the binding of
chromatin remodelers at acetylated histones, such as
H4K8ac and H4K12ac, which are present in the nuclei
prior to fertilization [22]. Others have suggested that the
histones in the sperm nuclei could influence which genes
are to be transcribed after fertilization [7, 10].
Further characterization of the human sperm
chromatin components revealed the co-localization of
chromosome 16 telomere with the core histones. Previous
studies report that histones associate with specific
regions that include the telomeres, promoter and repeating
regions and certain small gene-rich regions [6, 8, 23].
The nucleohistone components localized in the posterior
region of the nuclei, and extend to the region occupied
by the sperm nuclear annulus, likely provide an anchor
[24, 25].
Taken together, the data presented in this study show
the physical organization of the nucleoprotamine and
nucleohistone components within human sperm nuclei.
These two components are not randomly distributed,
but are generally organized into specific regions, with
the protamine-compacted chromatin splayed throughout the nucleus and equatorial regions of sperm nuclei,
and the histone-compacted chromatin localized in the
postacrosomal region, extending to the sperm nuclear
annulus. It is not known to what extent the histone
replacement with protamines is precise or stochastic.
However, studies of the human protamine locus show
that in human spermatozoa the promoter region remains
histone-bound, whereas the coding region of PRM1 is
protamine-bound [8]. Perhaps this unique packaging is
essential for appropriate decondensation and
reorganization of the paternal genome following fertilization.
Acknowledgment
This work was supported by National Institute of
Child Health and Development Grant HD36512 to Stephen
A. Krawetz, and a Biotechnology and Biological Sciences
Research Council (BBSRC), UK, grant to David Miller.
Claudia Lalancette is supported inpart by a Wayne State
University postdoctoral recruiting award.
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