ORIGINAL ARTICLES: ANDROLOGY

Cigarette smoking is associated with

abnormal histone-to-protamine
transition in human sperm
Bolan Yu, Ph.D.,a,b Yanbin Qi, B.S.,c,d,e Dan Liu, B.S.,a,b Xingcheng Gao, Ph.D.,a,b Hui Chen, B.S.,c
Chuan Bai, Ph.D.,c and Zhaofeng Huang, Ph.D.c,d,e
a
Key Laboratory for Major Obstetric Diseases of Guangdong Province, Third Affiliated Hospital of Guangzhou Medical
University, Guangzhou; b Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes,
Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, People's Republic of China; c Institute of Human
Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou; d Department of Biochemistry, Zhongshan
School of Medicine, Sun Yat-sen University, Guangzhou; and e Key Laboratory of Tropical Diseases Control of Ministry of
Education, Sun Yat-sen University, Guangzhou, People's Republic of China

Objective: To investigate the association between smoking, semen quality, and the histone-to-protamine transition ratio in mature
sperm.
Design: Biochemical and molecular analysis in human samples and a cell line.
Setting: Andrology laboratory in a university-affiliated hospital.
Patient(s): Semen samples from 147 heavy smokers and 175 nonsmokers receiving infertility treatment.
Intervention(s): None.
Main Outcome Measure(s): Basic semen parameters, histone-to-protamine ratios, and number of sperm cells with abnormal histone
transition were calculated. The relative messenger RNA (mRNA) expression levels of protamine 1 and protamine 2 were assayed in
human sperm and in TM3 cells exposed to cigarette smoke condensate. T tests, Spearman tests, and nonparametric Mann-Whitney
U tests were used to detect significant differences.
Result(s): Normozoospermic smokers had significantly higher abnormalities than their nonsmoking counterparts. Sperm histone
replacement abnormalities were found to be closely correlated with sperm motility, viability, concentration, counts, and cotinine levels.
The ratios of protamine 1 to protamine 2 mRNA expression significantly increased in heavy smokers and in TM3 cells treated with ciga-
rette smoke condensate.
Conclusion(s): Smoking is strongly associated with abnormalities in histone-to-protamine
transition and with alteration of protamine mRNA expression in human sperm. (Fertil SterilÒ Use your smartphone
2014;101:51–7. Ó2014 by American Society for Reproductive Medicine.) to scan this QR code
Key Words: Smoking, protamine, histone replacement, defective spermatogenesis and connect to the
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I
nfertility affects 10%–15% of cou- (2–4). Cigarette smoke is considered especially prone to oxidative damage
ples worldwide and has become a a risk factor for male infertility (5). caused by smoking, owing to high
public health problem in recent It contains high concentrations of concentrations of polyunsaturated
decades (1). Male infertility accounts free radicals and can potentially fatty acids on their plasma membranes
for approximately half of these prob- induce the production of cellular (8). However, the mechanisms of
lems, and decreased semen quality has reactive oxygen species in the human cigarette smoke–associated damage to
been widely reported in recent decades body (6, 7). Human spermatozoa are human spermatogenesis are still
largely unknown.
Received March 18, 2013; revised August 23, 2013; accepted September 4, 2013; published online
October 7, 2013. Protamines are the most abundant
B.Y. has nothing to disclose. Y.Q. has nothing to disclose. D.L. has nothing to disclose. X.G. has nothing nucleoproteins in mature sperm. In
to disclose. H.C. has nothing to disclose. C.B. has nothing to disclose. Z.H. has nothing to disclose.
This study was supported by the National Natural Science Foundation of China (grants 81370751 and humans there are two protamines: prot-
31170831) and the Guangdong innovative R&D team program (grant 2009010058). amine 1 (SP1) and protamine 2 (SP2)
Reprint requests: Zhaofeng Huang, Ph.D., Institute of Human Virology, Zhongshan School of Medi-
cine, Sun Yat-sen University, 74 Zhongshan 2nd Rd., Building no. 10, Room N1311, Guangzhou
(9). These proteins are highly basic and
510080, People's Republic of China (E-mail: hzhaof@mail.sysu.edu.cn). contain large amounts of arginine and
cysteine residues (10). The arginine-
Fertility and Sterility® Vol. 101, No. 1, January 2014 0015-0282/$36.00
Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. rich core has a net positive charge,
http://dx.doi.org/10.1016/j.fertnstert.2013.09.001

VOL. 101 NO. 1 / JANUARY 2014 51

ORIGINAL ARTICLE: ANDROLOGY
and the cysteine residues can form multiple inter- and intra-
protamine disulfide bonds, both of which can make DNA
packaging in sperm cells more compact than in somatic cells
(11). During spermatogenesis, histones are first replaced by
testis-specific histone variants, which are then replaced by
transition proteins. The transition proteins in the condensing
chromatin are then replaced with protamines during the elon-
gating spermatid stage (9). Protamine genes are expressed in
the round spermatid stage, and the transcription products
are stored in early haploid cells and activated in elongated
spermatids (9, 12). This stage-specific pattern of protamine
gene expression is essential to correct sequential nucleopro-
tein exchange and complete differentiation of round sperma-
tids into mature spermatozoa.
Abnormalities in histone transition and protamine expres-
sion in humans have been found to be associated with male
infertility (13–18). In human sperm the histone-to-protamine
exchange rate is approximately 85% complete, and the SP1
and SP2 ratio is estimated to be approximately 1:1 (19). In
infertile men, elevated histone-to-protamine ratios have
been reported and altered protamine expression is
widespread (14, 15). An aberrant SP1/SP2 ratio is also linked
with decreased sperm parameters, such as sperm
concentration, motility, and morphology (16, 17). This is in
addition to decreased fertilization ability in assisted
reproductive technology (16, 18, 20).
The reasons for abnormal protamine expression are still
under investigation. Gene polymorphisms in protamine genes,
abnormal transcript retention, and dysfunction of transcrip-
tional factors have been suggested in previous studies
(21–23). It was recently reported that smoking might affect
protamine levels in humans, and the SP1/SP2 protein ratio
was abnormally elevated in smokers (24). This is interesting
because associations between smoking and decline in semen
quality and DNA damage have been reported previously, but
smoking's effects on histone transition are unclear (25–27).
These effects may be important for both DNA stability and
gene imprinting in human sperm.
The present study was designed to investigate the associ-
ation between smoking, sperm quality, and histone transition
in mature human sperm. The histone-to-protamine ratio in
147 heavy smokers and 175 nonsmokers was examined, the
relative messenger RNA (mRNA) expression levels of SP1
and SP2 were assayed, and histone transition was compared
between groups divided by smoking status (heavy smokers
and nonsmokers) and by semen quality (normozoospermic
males and abnormal males). Decreases in sperm protamine
levels have previously been shown to be associated with
smoking (24). The present study further validated this associ-
ation in a Chinese population and investigated possible mech-
anisms underlying abnormal protamine expression.
MATERIALS AND METHODS
Study Subjects
This study was approved by the institutional review board
of Guangzhou Medical University and the medical ethics
committee of Zhongshan School of Medicine of Sun Yat-
Sen University. All studies involving human subjects were
52
conducted in accordance with guidelines laid down in the
Declaration of Helsinki. Participants in this study were sched-
uled for interviews after written informed consents were
obtained, and the interviewers collected the subjects' data
on medical history, lifestyle, and smoking status with a struc-
tured questionnaire. Current smokers with smoking dose R1
pack per day over 10 years or smoking dose R2 packs per day
over 5 years were defined as heavy smokers. Individuals who
had never smoked were defined as nonsmokers. Study sub-
jects were ethnic Han Chinese from Guangzhou City and its
surrounding regions in South China. They were men who
visited the Reproductive Medicine Center in the Third Affili-
ated Hospital of Guangzhou Medical University for infertility
treatment during November 2011 to October 2012. Non-
smokers were randomly selected from patients who were
age-matched to heavy smokers. All subjects underwent phys-
ical examinations and at least two semen analyses. Men who
were unhealthy or had a known cause of defective spermato-
genesis, such as varicocele, infection, obstruction of the vas
deferens, chromosomal abnormalities, or microdeletions of
the azoospermia factor region on the Y chromosome, were
excluded. Patients who were diagnosed with azoospermia,
severe oligozoospermia (sperm concentration <5
106
cells/mL), hemospermia, leukospermia, and necrozoospermia
were also excluded owing to concerns for genetic or other
medical reasons for abnormal spermatogenesis. Finally, 147
heavy smokers and 175 nonsmokers were recruited. Among
them, 198 men were normozoospermic, and 124 men were
patients who had abnormal sperm parameters, including
decreased sperm concentration and motility. In the 124 men
with abnormal spermatogenesis, 18 smokers and 14 non-
smokers were oligoasthenozoospermic patients, and the
others were asthenozoospermic patients.
Sperm Collection and Determination of Clinical
Parameters
Semen samples were collected in sterile containers from
patients by masturbation after 2–7 days of sexual abstinence.
Samples were allowed to liquefy for at least 30 minutes at
room temperature. Analyses of semen volume, pH, sperm
concentration, vitality, and motility and computer-assisted
semen analysis were carried out according to World Health
Organization guidelines (28). Histone-to-protamine replace-
ment was examined immediately using a portion of each
sample. The other portion was centrifuged at 1,000
g for
10 minutes and stored as separate seminal plasma and cell
pellets at 80 C.
Cell Culture and Cigarette Smoke Condensate
Treatment
Murine testicular TM3 cells were cultured in Dulbecco's Modi-
fied Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (In-
vitrogen) supplemented with 10% fetal bovine serum, 100 U/
mL penicillin, and 100 mg/mL streptomycin (Invitrogen).
Cigarette smoke condensate (CSC) was prepared according
to the literature (29) and resuspended at a concentration of
50 mg/mL in dimethyl sulfoxide (DMSO) as stock solution.
VOL. 101 NO. 1 / JANUARY 2014
 Fertility and Sterility®

For smoke-exposure experiments, cells were cultured in me- Aniline Blue Staining
dium with DMSO and 0.1 mg/mL or 10 mg/mL of CSC. After The ratio of histone-to-protamine in sperm nuclei was
exposure to CSC for 6 hours, cells were lysed, and total measured using a Nucleoprotein Transition Test Kit (Hua-
RNA was extracted with TRIzol reagent (Invitrogen) and kang). Briefly, 5 mL of prepared spermatozoa were spread
was reverse-transcribed using the Reverse Transcription Sys- onto a glass slide and allowed to dry. The smears were fixed
tem (Promega). in 3% buffered glutaraldehyde in 0.2 M phosphate buffer
(pH 7.2) for 30 minutes. The slides were then stained with
Cotinine Measurement 5% aqueous aniline blue mixed with 4% acetic acid (pH 3.5)
The levels of cotinine in seminal plasma were measured using for 5 minutes. Positive staining indicates the abnormal
the Cotinine Direct ELISA Kit (Calbiotech), which is designed histone-to-protamine transition. A total of 400 sperm cells
to detect cotinine in serum, urine, and other fluids. Assay were evaluated under microscopy, and the percentage of
procedures were applied according to the manufacturer's stained sperm heads was calculated.
guidelines. Briefly, standards, controls, and seminal plasma
were pipetted into ELISA wells in duplicate, then enzyme Statistical Analysis
conjugate was added to each well and the plates were incu- The independent-sample t test was used to analyze numeric
bated for 60 minutes in the dark at room temperature. After data in two groups. Spearman's test was used to calculate cor-
washing with distilled water, the substrate reagent was added relations. The nonparametric Mann-Whitney U test was used
to each well. After a 30-minute incubation, stop solution to analyze differences in semen parameters and mRNA
was added and absorbance was read at 450 nm. The concen- expression between two groups. All analyses were performed
tration of cotinine (ng/mL) was calculated against the stan- using GraphPad Prism 4.0 for Windows (GraphPad Software)
dard curve generated from the standards supplied in the kit. and PASW Statistics 18 (SPSS). A two-sided P value of .05
Samples with concentrations >100 ng/mL were diluted for was considered statistically significant.
measurement.
RESULTS
Human Sperm RNA Extraction Age, Smoking Status, and Semen Parameters of
Total RNA was isolated from the sperm pellet of each subject Study Subjects
(20
106 spermatozoa) using an RNeasy Plus Universal Mini The mean age, smoking status, and semen parameters of 175
Kit (Qiagen) according to the manufacturer's protocol. The nonsmokers and 147 heavy smokers are given in Table 1. The
RNA concentration was measured using a NanoDrop spectro- two groups had similar average age (33.6 years vs. 35.6 years).
photometer (Thermo Fisher) and stored at 80 C until prepa- Heavy smokers had an average seminal cotinine level of 203
ration of complementary DNA (cDNA). ng/mL, which was 5 ng/mL in nonsmokers (P< .01). The
sperm counts (229
106 cells vs. 188
106 cells, P< .05)
Quantitative Real-Time Reverse Transcription– between the two groups were significantly different, but
Polymerase Chain Reaction other basic semen parameters, including sperm concen-
tration (69.0
106/mL vs. 57.5
106/mL), semen volume
Approximately 800 ng RNA was reverse-transcribed into
cDNA (Takara). Then quantitative polymerase chain reaction
(PCR) was performed using SYBR Green Super-mix and
iCycler (Bio-Rad). This cDNA (50 ng per sample) was used TABLE 1
for amplification of target genes with the primers of human
Age, smoking status, and semen parameters of study subjects.
GAPDH (F-ctgtccagttaatttctgacc; R-ctttgtacatggtattcaccac),
human SP1 (F-cggagctgccagacacgga; R-ctacatctcggtctg Parameter Nonsmokers Heavy smokers
tacctggg), human SP2 (F-aagacgctcctgcaggcac; R-gcct N 175 147
tctgcatgttctctt), mouse GAPDH (F-tgacgtgccgcctggagaaa; Age (y) 33.6  4.3 35.6  5.4
R-agtgtagcccaagatgcccttcag), mouse SP1 (F-tcaaaactcctgcgt Smoking-yearsa 0 10.6  3.6
Smoking dose 0 1.3  0.5
gagaa; R-gacaggtggcattgttcctt), and mouse SP2 (F-tgcaggtg Seminal cotinine (ng/mL) 53 203  83**
caggaaatgtag; R-cctcacatgatgttgcttgg). The thermal cycling Abnormality in histone transitionb 18.9  8.6 22.4  10.0**
program included an initial incubation at 95 C for 2 minutes, Sperm concentration (
106 69.0  52.6 57.5  40.9
cells/mL)
followed by 49 cycles of 95 C for 5 seconds and 60 C for Sperm volume (mL) 3.7  1.2 3.5  1.3
20 seconds. A final 65 C to 95 C step was used to form the Sperm count (
106 cells per 229  164 188  133*
melt curve. Values recorded for quantification were the frac- ejaculate)
tional cycle numbers (CT) for which the background-corrected Sperm progressive motility (%) 43  18 40  18
Sperm immotility (%) 51  20 55  20
amplification curves crossed a threshold value. The threshold Sperm vitality (%) 84  9 83  9
value was set within the log-linear phase of the amplification Note: Data are presented as mean  SD unless otherwise noted.
a
curves. Three replicates of each reaction were performed, and b
Information of smoking dose (packs per day) and years smoked was acquired from subjects.
The abnormality in histone transition was the percentage of sperm cells that had elevated
the CT values were averaged. The 2DCT was calculated to histone-to-protamine ratios.
*P< .05 and **P< .01 compared with nonsmokers.
represent the levels of genes after normalization to that of
GAPDH, where DCT ¼ (CTgenes  CTGAPDH). Yu. Smoking and sperm histone transition. Fertil Steril 2014.

VOL. 101 NO. 1 / JANUARY 2014 53

ORIGINAL ARTICLE: ANDROLOGY
(3.7 mL vs. 3.5 mL), sperm progressive motility (43% vs. 40%),
sperm immotility (51% vs. 55%), and sperm vitality (84% vs.
83%) were not significantly different (Table 1).
Sperm Nuclear Histone-to-Protamine Ratios in
Different Populations
The sperm nuclear histone-to-protamine ratio was assayed
using analine blue staining, as shown in Supplemental
Figure 1 (available online). Positively stained sperm had erro-
neous histone-to-protamine transition. This abnormality in
histone transition is defined as the percentage of sperm cells
that had elevated histone-to-protamine ratios. The data
show that heavy smokers had significantly higher abnormal-
ity than nonsmokers (P< .01), with values of 22.4 and 18.9,
respectively (Table 1). In addition, asthenozoospermic and
oligoasthenozoospermic patients (including smokers and
nonsmokers) had significantly elevated histone-to-protamine
ratios than normozoospermic men (P< .001), with values of
24.5 and 18.0, respectively.
Effects of Smoking on Sperm Nuclear Histone-to-
Protamine Ratios in Stratified Populations
All subjects were classified by both smoking status and semen
quality to produce a total of six groups: normozoospermic
men, men with low sperm motility (including asthenozoo-
spermic and oligoasthenozoospermic patients), and men
with low sperm concentration (including oligozoospermic
and oligoasthenozoospermic patients) in heavy smokers or
in nonsmokers. Smoking men with low sperm motility
or low sperm concentration had significantly higher abnor-
mality rates in their histone-to-protamine ratio than smoking
normozoospermic men (26.0 vs. 19.6 and 29.4 vs. 19.6, both
P< .01; Table 2). The same held true for groups of low
motility, low concentration, and normozoospermia in non-
smokers (22.9 vs. 16.8 and 24.9 vs. 16.8, both P< .01;
Table 2). In all patients with low sperm motility, smokers
had higher rates of histone transition abnormalities than their
nonsmoking counterparts, but the difference did not reach
statistical significance (26.0 vs. 22.9, P¼ .0755; Table 2). Like-
wise, in men with low sperm concentration, smokers had
higher rates of histone transition abnormalities compared
with nonsmokers, but this difference was not statistically
TABLE 2
Effects of smoking on the histone-to-protamine transition in different populations.
Heavy smokers
Parameter S-NS S-LM
Subjects 83 64
Abnormalitya 19.6  9.1b,c 26.0  10.2d
Note: S ¼ heavy smokers; NS ¼ normozoospermic subjects; LM ¼ subjects with low sperm motility; LC ¼ subjects with low sperm concentration; N ¼ nonsmokers.
a
The abnormality in histone transition was the percentage of sperm cells that had elevated histone-to-protamine ratios.
b
S-NS vs. S-LM: P< .01 (significant); S-NS vs. S-LC: P< .01 (significant).
c
S-NS vs. N-NS: P< .05 (significant).
d
S-LM vs. N-LM: P¼ .0755.
e
S-LC vs. N-LC: P¼ .2962.
f
N-NS vs. N-LM: P< .01 (significant); N-NS vs. N-LC: P< .01 (significant).
Yu. Smoking and sperm histone transition. Fertil Steril 2014.
54
significant (29.4 vs. 24.9, P¼ .2962; Table 2). In all normozoo-
spermic men, smokers had a significantly higher rate of
abnormality than their nonsmoking counterparts (19.6 vs.
16.8, P< .05; Table 2).
Correlations between Sperm Nuclear Histone-to-
Protamine Ratios and Age, Semen Parameters, and
Cotinine Levels
Spearman correlation analysis showed the sperm nuclear his-
tone-to-protamine ratio to be closely correlated with most
sperm parameters, including motility, vitality, concentration,
and count (all P< .01; Table 3). The abnormality rate was also
closely associated with the seminal cotinine level (P< .01;
Table 3).
Messenger RNA Expression Levels of SP1 and SP2
Genes in Spermatozoa
Relative quantitative reverse transcription (RT)-PCR analysis
was performed to determine the level of SP1 and SP2 gene
mRNA expression in spermatozoa from nonsmokers and
heavy smokers. The mRNA levels in spermatozoa of 40 non-
smokers and 38 heavy smokers were successfully measured
using real-time RT-PCR. Because the ratio of SP1 to SP2
mRNA expression might be affected by smoking and be
related to the sperm histone-to-protamine transition, subjects
were divided into the following four groups: nonsmokers with
20% or fewer of sperm cells that had elevated histone-to-
protamine ratios (low group of nonsmokers), nonsmokers
with more than 20% of sperm cells that had elevated his-
tone-to-protamine ratios (high group of nonsmokers), heavy
smokers with 20% or fewer of sperm cells that had elevated
histone-to-protamine ratios (low group of heavy smokers),
and heavy smokers with more than 20% of sperm cells that
had elevated histone-to-protamine ratios (high group of
heavy smokers). The threshold of sperm histone transition ab-
normalities is 20%, which was used for predicting IVF
outcome previously (30).
The results showed that there was no significant differ-
ence between the abnormalities of histone-to-protamine ra-
tios in the low groups of nonsmokers and heavy smokers,
but the high group in heavy smokers had significantly higher
rates of abnormalities than that in nonsmokers (P< .05;
Nonsmokers
S-LC N-NS N-LM N-LC
18 115 60 14
29.4  10.2e 16.8  7.8f 22.9  8.7 24.9  7.0
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 Fertility and Sterility®

TABLE 3 FIGURE 1

Correlation coefficients of age, sperm parameters, and cotinine level

with the histone-to-protamine transition.
Parameter ra P value a
Age 0.196 .215
Sperm motility 0.479 .001**
Sperm vitality 0.430 .004**
Sperm concentration 0.462 .002**
Sperm count 0.478 .001**
Seminal cotinine level 0.657 .000**
a
Analyzed by Spearman's correlation analysis.
**P<0.01.
Yu. Smoking and sperm histone transition. Fertil Steril 2014.

Fig. 1A). In the two low groups, smokers had significantly

higher SP1 to SP2 mRNA ratio than nonsmokers (1.678 vs.
1.0, P< .05; Fig. 1B), although they had similar abnormalities
of histone-to-protamine ratios (Fig. 1A). In the two high
groups, both smokers and nonsmokers had higher ratios
than the low group of nonsmokers (both P< .05; Fig. 1B),
with values of 1.681, 1.711, and 1.0, respectively. However,
there was no significant difference between the high groups
in smokers and nonsmokers (Fig. 1B).

Messenger RNA Expression Levels of SP1 and SP2

Genes in TM3 Cells after CSC Treatment
TM3 cells are murine testicular Leydig cells, with abundant
mRNA expression of P1 and P2 in them. The 10 mg/mL of
CSC was found to be able to alter antioxidant gene mRNA
expression in THP-1 cells (31). Therefore, this concentration
was chosen to examine the effects of CSC on P1/P2 gene tran-
scription in TM3 cells. In our pilot study, the antioxidant gene
expressions in TM3 cells could be affected upon CSC stimula-
tion (unpublished data). The SP1/SP2 mRNA levels were
analyzed by RT-PCR in murine testicular TM3 cells, after
they were treated with DMSO (control), 0.1 mg/mL CSC, and
10 mg/mL CSC for 6 hours. The ratios of SP1 to SP2 mRNA
expression were significantly higher in cells treated with
10 mg/mL CSC compared with control (P< .01; Fig. 1C),
whereas there were no significant differences between cells
treated with 0.1 mg/mL CSC and control (Fig. 1C).

DISCUSSION
Histone replacement by protamine is critical to DNA conden-
sation in mature sperm. Diminished spermatogenesis and
cigarette smoke have both been reported to be associated
with abnormal protamine expression. In the present work, a
case–control study was conducted in heavy smokers and non-
smokers, and these subjects were further stratified into nor-
mozoospermic men and patients with low sperm motility
and low sperm concentration (Tables 1 and 2). In this way,
the effects of smoking and spermatogenesis on protamine
expression were analyzed separately. The comparison of his-
tone-to-protamine transition among these groups supported
the hypothesis that smoking and defective spermatogenesis
The relative SP1 to SP2 mRNA ratios in human sperm and in TM3 cells.
(A) Columns show the percentage of sperm cells with elevated
histone-to-protamine ratios in nonsmokers and heavy smokers.
Subjects were divided into a high abnormality group and a low
abnormality group. *P<.05 compared with the high group of
nonsmokers. (B) Columns show the relative SP1/SP2 ratios in low
and high groups of nonsmokers and heavy smokers. *P<.05
compared with the low group of nonsmokers. (C) Columns show
the relative SP1 to SP2 mRNA ratios in TM3 cells exposed to
control, 0.1 mg/mL, and 10 mg/mL of CSC. *P<.01 compared with
the control.
Yu. Smoking and sperm histone transition. Fertil Steril 2014.

VOL. 101 NO. 1 / JANUARY 2014 55

ORIGINAL ARTICLE: ANDROLOGY
are associated with aberrant histone-to-protamine transition
independently (Table 2). Consistent with this, the nonsmoking
normozoospermic men had the lowest rate of abnormalities in
histone transition, and heavy-smoking men with low sperm
concentration had the highest rate (Table 2).
This association between diminished spermatogenesis and
altered protamine expression has been demonstrated in
numerous studies (9, 13, 17, 32–35). In mice, haploin-
sufficiency of SP1 or SP2 can cause male infertility (36). In
humans, altered protamine expression is associated with low
sperm quality and decreased fertility (16, 17, 37). The data
from this study also show that men with defective semen
quality have higher ratios of abnormalities in histone-to-
protamine transition (Table 2). This ratio was tightly linked
to basic semen parameters, including sperm motility,
viability, concentration, and count (Table 3). In this way, the
results of the present and previous studies consistently
support a tight linkage between semen quality and sperm
histone transition (16, 17, 37).
Our data also showed that heavy smoking was associated
with a significant increase in histone-to-protamine ratios in
the normozoospermic men (Table 2), suggesting that prot-
amine expression abnormalities might be affected by
smoking-related reactive oxygen species. Hammadeh et al.
(24) once demonstrated that, in a European population, there
was an abnormal elevation of the SP1/SP2 protein ratio in
smokers and that oxidative stress induced by cigarette smok-
ing may have a significant inverse effect on the protamine
replacement process (24). The present study proved similar
observations in a Chinese population.
The molecular mechanisms underlying smoking-induced
protamine abnormality are not clear yet. Our data demon-
strate that smoking can alter the expression of SP1 to SP2
mRNA ratio in human spermatozoa, regardless of whether
the abnormality in histone transition is high or low (Fig. 1A
and B). This observation was further verified in TM3 cell
models treated with CSC, which shows that the treatment of
cigarette smoke leads to alteration in protamine mRNA
expression (Fig. 1C). Therefore, both results suggest that
smoking may interfere with protamine expression at the
mRNA level. In previous studies it has been reported that pro-
longed cigarette smoke exposure can alter the mRNA expres-
sion of HO-1, NRF2, and BACH1 in human macrophages and
that heavy smoking can decrease the mRNA expression of
NRF2 in human peripheral blood mononuclear cells and
plasma (31, 38). In addition, smoke leads to S-nitrosylation
of cysteine residues in histone deacetylase 2 from smokers
with chronic obstructive pulmonary disease, which affects
the transcriptional profiles of glucocorticoid receptor (39).
In a similar way, cigarette smoke may impair the mRNA
expression of protamine, which may partially contribute to
the deficient replacement of histones by protamines in
sperm. However, the involved mechanisms of smoke-
induced alterations in protamine mRNA expression warrants
further investigation.
Smoking has been demonstrated to increase DNA frag-
mentation and damage in sperm (27, 40, 41). Considering
that protamines play important roles in DNA integrity (9),
one possible consequence of abnormal protamine
56
expression is a susceptibility to DNA damage. It was known
that patients with low SP1/SP2 ratios had significantly
elevated DNA fragmentation relative to patients with
normal and high SP1/SP2 ratios (42, 43). We indeed
observed an increase of DNA damage in men with abnormal
histone transition (unpublished data). Additionally, smoking
may affect the traits of progeny via disruption of protamine
expression, because protamines may be regulators of
genomic imprinting and epigenetics (44). A recent study has
suggested that men with abnormal protamines displayed
significantly altered methylation patterns across a large
number of CpGs in offspring DNA (45). Because imprinted
regions were more prone to deregulation than the genome
at large, the potential consequences on the epigenome
might be important for defective spermatogenesis and
embryo development related to smoking.
In conclusion, this study demonstrates that both smoking
and defective semen quality are strongly associated with the
histone-to-protamine transition in mature human sperm.
Smoking may interfere with the transcription of protamine
mRNA, and these effects were also observed in cell models
exposed to CSC. Altogether, sperm histone transition could
be affected by cigarette smoking at the level of protamine
mRNA transcription.
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57
ORIGINAL ARTICLE: ANDROLOGY

SUPPLEMENTAL FIGURE 1

Detection of the histone-to-protamine ratio in sperm nucleus by

aniline blue staining. Arrows indicate the sperm cells with elevated
histone-to-protamine ratios in (A) normozoospermic nonsmokers
and (B) asthenozoospermic heavy smokers.
Yu. Smoking and sperm histone transition. Fertil Steril 2014.

57.e1 VOL. 101 NO. 1 / JANUARY 2014