ISSN: 2047-2919 ANDROLOGY

ORIGINAL ARTICLE

Correspondence
Ashok Agarwal, Andrology Center, and American Treatment of semen samples with a-
Center for Reproductive Medicine, Cleveland
Clinic, 10681 Carnegie Avenue, Cleveland, OH chymotrypsin alters the expression
44195, USA.
E-mail: agarwaa@ccf.org pattern of sperm functional
proteins—a pilot study
Keywords:
hyperviscosity, protein expression, sperm proteins,
1
spermatozoa, a-chymotrypsin M. K. Panner Selvam, 1A. Agarwal , 1R. Sharma and 1,2L. Samanta
1
American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA, and 2School of
Received: 13-Nov-2017 Life Sciences, Ravenshaw University, Cuttack, Odisha, India
Revised: 13-Dec-2017
Accepted: 19-Dec-2017

doi: 10.1111/andr.12466

SUMMARY
Semen hyperviscosity delays the liquefaction of semen sample and is subjected to limited proteolysis by addition of a-chymotryp-
sin to reduce the viscosity. a-Chymotrypsin is a proteolytic enzyme involved in degradation of the proteins and polypeptides. Even
though a-chymotrypsin improves the handling of hyperviscous samples, its effect on the sperm proteins is not clear. This study was
aimed to evaluate the alteration in the expression of sperm functional proteins in samples treated with a-chymotrypsin. Among all
the proteins examined in both donor and patient samples, HSPA2 (70 KDa), BAG6 (150 KDa), HIST1H2BA (14 KDa), SPA17 (17 KDa
formed after cleavage of C-terminal calmodulin-binding domain), and OXPHOS complexes were undetectable in a-chymotrypsin-
treated samples, while the expression of the native SPA17 (20 KDa) was significantly decreased in the a-chymotrypsin-treated sam-
ples in comparison with controls. The use of a-chymotrypsin for liquefaction of hyperviscous samples degrades functional proteins
of spermatozoa. Intracellular proteins, such as OXPHOS complexes and HIST1H2BA, and sperm surface proteins (HSPA2, BAG6, and
SPA17) were degraded in all treated samples. Whether treatment of samples with a-chymotrypsin affects the global proteomic out-
come is unclear. More in-depth calibration studies are required to determine the appropriate concentration of a-chymotrypsin for
processing hyperviscous semen samples without compromising its protein expression and function. Similarly, the effects of altered
protein function on assisted reproductive techniques (ART), such as intrauterine insemination (IUI) and in vitro fertilization (IVF)
outcome, are not known and require further research.

INTRODUCTION
Human semen coagulates immediately after ejaculation and
entraps the spermatozoa in a matrix formed by the seminal
plasma proteins. However, it usually liquefies in about 15 to
20 min both in vitro and in vivo (Robert & Gagnon, 1999). The
process of liquefaction is affected by the partial or incomplete
proteolysis of seminal plasma proteins present in the coagulum
(Emami et al., 2009). Semen hyperviscosity (SHV) is addressed
as one of the factors pertaining to male infertility. It affects about
12% to 29% of patients with male infertility (Du Plessis et al.,
2013). SHV has adverse effects on the physical and biochemical
characteristics of seminal fluid (Gonzales et al., 1993). It has also
a negative effect on vital sperm parameters such as motility and
morphology. Incomplete liquefaction of the semen exhibits a
trapping effect that hinders the forward sperm movement (Elza-
naty et al., 2004) having a negative impact on fertility (Du Plessis
et al., 2013).
Routine semen analysis is the first step in the laboratory evalu-
ation of male infertility. Complete liquefaction of the semen
sample is important as it ensures that the ejaculate can be accu-
rately tested for various semen parameters and second that the
spermatozoa are not trapped in the coagulum in case the sample
fails to liquefy completely. Among the various causes of SHV,
oxidative stress and reactive oxygen species play a major role
and pose a challenge during the preparation of sperm samples
for IUI and IVF resulting in the complete absence or poor sperm
recovery (Honea et al., 1993; Elia et al., 2009; Du Plessis et al.,
2013). Although different methods are proposed to treat SHV,
generally hyperviscous samples are subjected to limited proteol-
ysis by the addition of a mucolytic agent, a-chymotrypsin (EC
3.4.21.1) (5%) to reduce the viscosity (Honea et al., 1993; Zavos
& Correa, 1997). It is a well-known fact that proteolytic property
of a-chymotrypsin breaks the proteins and polypeptides (Tsiat-
siani & Heck, 2015; Giansanti et al., 2016). Human spermatozoa

© 2018 American Society of Andrology and European Academy of Andrology Andrology, 2018, 6, 345–350 345

M. K. Panner Selvam et al.
exhibit a-chymotrypsin-like activity during the acrosome reac-
tion (Morales et al., 1994). Several studies have reported that a-
chymotrypsin does not have a harmful effect on sperm motility
or viability (Chen et al., 2006; Xu et al., 2006; Agarwal et al.,
2016) and that it does not alter the concentration of the total
proteins present in the seminal plasma (Mendeluk et al., 2000).
Still, one cannot ignore the fact that treatment with a-chymo-
trypsin may damage or degrade the sperm proteins. However,
no study has examined the effect of a-chymotrypsin at the sub-
cellular level, especially on the sperm surface and intracellular
proteins. Thus, the aim of our study was to check the effect of a-
chymotrypsin on the expression of sperm proteins involved in
sperm function, such as acrosome reaction, sperm–egg recogni-
tion, and oocyte–zona pellucida binding, along with mitochon-
drial electron transport chain proteins involved in ATP synthesis
required for sperm motility.
MATERIALS AND METHODS
Study samples
Following the approval of the study by the Institutional Review
Board (IRB) of Cleveland Clinic, semen samples were obtained
from healthy male donors (n = 8) of unproven fertility present-
ing with normal semen parameters (WHO, 2010) and infertile
patients (n = 3). They were the partners of spouses who were
being evaluated by the gynecologist and sent to the Andrology
Center for initial semen evaluation before being referred to a
male infertility specialist. Semen samples were collected by mas-
turbation after sexual abstinence of at least 48 h. Round cell con-
centration was determined in all samples on a wet smear as per
WHO 2010 guidelines. Samples with round cell concentration
>1 9 106/mL were tested for white blood cell count by the per-
oxidase or the Endtz test (WHO, 2010). The inclusion criteria
were semen sample volume ≥1.5 mL and negative for leukocy-
tospermia (<1 9 106 white blood cells/mL).
Measurement of semen viscosity
Viscosity of each sample was measured qualitatively as per
WHO 2010 criteria. Each sample was collected in a wide-mouth
sterile collection cup and kept at 37 °C for complete liquefac-
tion. The viscosity was examined before transferring the sample
from the collection cup into a graduated 15-mL centrifuge tube
by gently aspirating the sample into a wide-bore, 5-mL serologi-
cal pipette and allowing the semen to drop by gravity. If, at the
end of incubation, the sample was completely liquefied, that is it
did not form threads >2 cm, the sample was graded as com-
pletely liquefied with a normal viscosity. If the sample was not
completely liquefied, it was categorized as incomplete liquefac-
tion and considered as viscous. Viscosity was categorized as fol-
lows: slight viscosity: drops with thread formation (length of
thread >2 cm and ≤4 cm); moderate viscosity: thread length
(>4 cm and ≤6 cm); and high viscosity: sample fails to drip at all
and stays as a coagulum. Samples displaying moderate and high
viscosity were categorized as hyperviscous.
a-Chymotrypsin treatment
Experiment 1
Eight semen samples from four donors collected on two differ-
ent days (~1 month apart) showed different grades of viscosity.
346 Andrology, 2018, 6, 345–350
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They were categorized into hyperviscous (n = 4) and normal
samples (n = 4). Hyperviscous samples were treated with 5 mg
of lyophilized a-chymotrypsin powder (viscosity treatment sys-
tem; VTS, Vitrolife, San Diego, CA). The entire content of the vial
was added to the collection cup. The sample was swirled and
vortexed quickly and then incubated for additional 20 min for
complete liquefaction.
Non-viscous samples were processed according to standard
protocols.
Experiment 2
Each non-viscous semen sample from four donors and three
infertile patients was split into two aliquots (control and treated)
after natural liquefaction. Semen samples from treated group
were incubated with 5 mg of lyophilized a-chymotrypsin (Vitro-
life, San Diego, CA, USA) for 20 min at 37 °C. The concentration
of a-chymotrypsin was used according to manufacturer’s
instructions for hyperviscous samples as mentioned in experi-
ment 1.
Protein selection criteria
A thorough literature search was carried out to select the
sperm surface proteins most commonly related to infertility,
HSPA2, BAG6, and SPA17, the latter being localized over the
acrosome region of the head (Kong et al., 1995; Bromfield et al.,
2015; Intasqui et al., 2017). We selected HIST1H2BA and four
mitochondrial proteins involved in oxidative phosphorylation
(OXPHOS) along with the cytoskeletal protein a-tubulin involved
in sperm motility to check the effect of a-chymotrypsin on the
intracellular proteins. All these proteins play an important role
in sperm function.
Protein extraction
After the incubation period, all samples were centrifuged at
300g for 7 min and the seminal plasma was removed. The sperm
pellet was then washed twice with PBS (phosphate buffer saline)
to remove the residual a-chymotrypsin and seminal plasma
from the sample. Subsequently, the sperm pellet was resus-
pended in 1 mL of PBS and sperm concentration was deter-
mined prior to storage. Finally, all samples were stored at
80 °C for protein expression analysis.
Frozen samples were thawed at 37 °C for 20 min and cen-
trifuged at 1000g for 10 min. Based on the predetermined sperm
concentration in each vial, spermatozoa were lysed overnight at
4 °C with radioimmunoprecipitation assay (RIPA) lysis buffer
containing the complete protease inhibitor cocktail (Roche,
Mannheim, Germany) using 100 lL of buffer for 100 9 106 sper-
matozoa. After centrifugation at 10,000g for 30 min at 4 °C,
supernatant was transferred to fresh microcentrifuge tube and
protein concentration was estimated by the bicinchoninic acid
assay (BCA) kit (Thermo Fisher Scientific, Rockford, IL, USA).
SDS-PAGE and Western blotting analysis
After quantification, each sample was mixed with sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
sample buffer, denatured at 100 °C for 5 min and resolved
(20 lg/well) on a 4%–15% gel (Bio-Rad, Hercules, CA, USA).
Electrophoresis was performed at constant voltage (90 V) for
120 min at 4 °C. The gels were briefly washed with distilled
water and proteins were transferred to polyvinylidene difluoride
© 2018 American Society of Andrology and European Academy of Andrology
 ANDROLOGY

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EFFECT OF a-CHYMOTRYPSIN ON SPERM PROTEINS

(PVDF) membranes (Thermo Fisher, Rockford, IL, USA) at 20 V Figure 1 Effect of treatment of a-chymotrypsin (5 mg, 20 min, 37 °C) on
for 50 min using a Trans-Blotâ SD Semi-Dry Electrophoretic expression profile of HSPA2 in spermatozoa from hyperviscous sample. Lane
1-4: control (normal liquefaction); Lane 5–8: hyperviscous samples treated
Transfer Cell (Bio-Rad, Hercules, CA, USA). PVDF membranes
with a-chymotrypsin.
were blocked with TBST containing 5% BSA and used for
immunodetection of sperm proteins. For each protein analysis,
specific primary antibodies were incubated at 4 °C overnight
(Table 1). Next, the membranes were washed 4 times with Tris-
buffered saline–Tween-20 (TBST) for 10 min each and incubated
with the secondary antibodies at room temperature for 1 h Figure 2 Effect of treatment of a-chymotrypsin (5 mg, 20 min, 37 °C) on
(Table 1). Membranes were washed again 4 times with TBST expression profile of sperm proteins. (A): BAG6, (B): HSPA2, (C): SPA17,
(10 min each) and finally treated with enhanced chemilumines- (D): HIST1H2BA, and (E): a-tubulin. CD: control donor; TD: treated donor;
CP: control patient; TP: treated patient.
cence (ECL) reagent (GE Healthcare, Marlborough, MA, USA) for
1 min. ECL-reacted blots were exposed to ChemiDoc (Chemi-
DocTM MP Imaging System, Bio-Rad, Hercules, CA, USA) to
detect the chemiluminescence signals.

RESULTS
The four donors who provided semen samples on two differ-
ent occasions were normozoospermic according to the WHO
2010 criteria. Two of the three patients were normozoospermic
and one was asthenozoospermic (motility 34%). None of these
patients presented with hyperviscous sample. The main goal of
our pilot study was to demonstrate the effect of a-chymotrypsin
on the expression of sperm functional proteins in hyperviscous
samples. In experiment 1, complete degradation of HSPA2 was
observed in the hyperviscous donor samples treated with 5 mg
of a-chymotrypsin, whereas its expression was prominent in the
normally liquefied samples (Fig. 1).
In experiment 2 in both donor and patient samples, expression
profile of sperm surface proteins (HSPA2 and BAG6), mem-
brane-bound protein SPA17, and intracellular proteins
(HIST1H2BA, OXPHOS complexes and a-tubulin) revealed the
degradation of proteins in the aliquots treated with a-chymo-
trypsin when compared with the untreated aliquots (Figs. 2&3).
DISCUSSION
At the molecular level, physiological functions of spermatozoa
are regulated by proteins, so alterations in their levels will affect
sperm function. In the sample aliquots treated with a-chymo-
trypsin (experiment 2), the expression of all proteins was non-
detectable, except SPA17 (20 KDa isoform) that was decreased
(Figs. 2 & 3). A similar change in the expression of cellular pro-
teins has been reported in the mammalian cells (breast cancer
cell line MCF-7) treated with 0.05% EDTA–trypsin (Huang et al.,
2010), which exhibits same kind of enzymatic action on the cells.
We analyzed the expression of two surface proteins HSPA2
and BAG6. HSPA2 belongs to the family of heat-shock proteins
and is essential not only for the spermatozoa to undergo
acrosome reaction, but also for sperm–oocyte recognition and
binding (Redgrove et al., 2012; 2013). BAG6 is located in the
anterior region of the sperm head and is important to maintain
the stability of HSPA2. Knockdown of BAG6 protein influences
the decreased expression of HSPA2 (Sasaki et al., 2008) and
results in failure of sperm–zona pellucida binding (Bromfield
et al., 2015). In the present study, both HSPA2 and BAG6 pro-
teins were non-detectable in the a-chymotrypsin-treated sam-
ples (Fig. 2A and b), implying their proteolytic degradation by
the enzyme. The absence of these fertility-related proteins might
affect the fertilizing ability of the spermatozoa at the molecular
level and compromise assisted reproductive techniques (ART)
outcome, including in vitro fertilization (IVF) or intrauterine
insemination (IUI).
The expression of the mannose-binding protein called sperm
protein 17 (SPA17) was also examined. This protein, predomi-
nantly present in the fibrous sheath of the tail region and local-
ized to some extent within the cytoplasm of the spermatozoa, is
proposed to be a zona pellucida interacting protein (Frayne &
Hall, 2002; Chiriva-Internati et al., 2009). SPA17 is also involved
in sperm maturation, capacitation, acrosome reaction, and

Table 1 List of antibodies used in this study

Protein Primary Secondary

Antibody Manufacturer Dilution Antibody Manufacturer Dilution

HSPA2 Anti-human Rabbit IgG Abcam ab154374 1 : 5000 Anti-rabbit Goat IgG Abcam ab97051 1 : 10,000
a-Tubulin Abcam ab4074 1 : 1000 1 : 10,000
SPA17 Abcam ab172626 1 : 1000 1 : 20,000
HIST1H2BA Abcam ab178426 1 : 1000 1 : 20,000
BAG6 Anti-human Mouse IgG Abcam ab88292 0.5 lg/mL Anti-mouse Goat IgG Santa Cruz sc-2005 1 : 10,000
OXPHOS cocktail Abcam ab11041 1 : 1000 Abcam ab97051 1 : 10,000

© 2018 American Society of Andrology and European Academy of Andrology Andrology, 2018, 6, 345–350 347

M. K. Panner Selvam et al.
Figure 3 Effect of treatment of a-chymotrypsin (5 mg, 20 min, 37 °C) on
expression profile of OXPHOS complexes (ATP5A, UQCRC, COXII, and
NDUFB8). CD: control donor; TD: treated donor; CP: control patient; TP:
treated patient.
fertilization process (Chiriva-Internati et al., 2009). Its N-termi-
nus shares a similar sequence with cAMP-dependent protein
kinase A regulatory subunit II (PKA RII), thus interacting with A-
kinase anchoring proteins (AKAPs) localized in the sperm flag-
ella and having an important role in sperm motility (Frayne &
Hall, 2002; Chiriva-Internati et al., 2009). On the other hand, its
C-terminus has an IQ calmodulin-binding motif and the central
portion of the protein has carbohydrate binding motifs and plays
a likely role in cell–cell adhesion (Wen et al., 1999). Our results
demonstrated a decrease in the expression of SPA17 (20 KDa) in
the samples treated with a-chymotrypsin when compared with
the untreated samples (Fig. 2C). In general, the C-terminal
calmodulin domain of SPA17 (20 KDa) is proteolytically cleaved
to 17 KDa after the acrosome reaction and binds to the extracel-
lular matrix of the oocyte (Ying et al., 2001; Frayne & Hall, 2002).
Under in vitro conditions, cleavage of SPA17 is executed by tryp-
sin, which in turn regulates the downstream signaling pathways
by modulating release of calmodulin (Wen et al., 1999). We also
observed a decrease in the expression levels of the native form of
SPA17 (20 KDa) as a result of the proteolytic action of a-chymo-
trypsin. Contrastingly, the spermatozoa treated with a-chymo-
trypsin revealed complete degradation of the cleaved form
(17 KDa) of protein (Fig. 2C), which might be due to the ability
of a-chymotrypsin to cleave at multiple sites. Although this is
the first study to illustrate the degradation of SPA17 (17 KDa) in
spermatozoa, it is in agreement with another study that showed
the complete degradation of purified insulin by a-chymotrypsin
when compared with trypsin alone (Schilling & Mitra, 1991).
Expression of testis/sperm-specific nuclear histone
HIST1H2BA and four proteins of the mitochondrial OXPHOS—a
regulator of sperm motility (Sudjarwo et al., 2012) was also
assessed to demonstrate the effect of a-chymotrypsin on protein
degradation at intracellular level. Our results showed a complete
degradation of HIST1H2BA protein (Fig. 2D) in semen samples
treated with a-chymotrypsin. The nucleoprotein HIST1H2BA is a
testis-specific histone involved in chromatin packaging, and its
expression was altered in male infertility conditions, such as
enhanced oxidative stress and varicocoele that may lead to
increased DNA damage (Zini et al., 2008; Sharma et al., 2013;
Agarwal et al., 2015). In fact, a previous report by Chatterjee &
Walker (1973) showed that a-chymotrypsin completely degrades
deoxyribonucleohistone proteins. It is also reported that hyper-
viscosity of semen reduces the sperm chromatin integrity
(Gopalkrishnan et al., 2000), thus increasing its susceptibility to
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damage. Therefore, degradation of HIST1H2BA will expose the
sperm DNA making it more vulnerable to fragmentation. On the
other hand, a report by Esfandiari et al. (2014) showed that when
the hyperviscous samples were liquefied mechanically by the
use of 5-mL syringe fitted with a sterile 18G needle, it did not
affect the sperm DNA fragmentation rate, thus validating the fact
that it is the a-chymotrypsin-induced damage to the nuclear his-
tone that may induce DNA fragmentation in the spermatozoa of
hyperviscous sample and not the viscosity. Similarly, using
OXPHOS human cocktail (Abcam, Cambridge, MA, USA) anti-
bodies, we identified four proteins of the OXPHOS system
(ATP5A, UQCRC, COXII, and NDUFB8) in the untreated control
samples, whereas they were totally absent in the treated samples
(Fig. 3). The temperature-sensitive complex II (SDHB) could not
be detected as the sample preparation step was carried out at
100 °C. Our study demonstrates that a-chymotrypsin not only
affects the surface-bound proteins (HSPA2 and BAG6) but also
degrades intracellular proteins (HIST1H2BA and OXPHOS com-
plexes). Furthermore, SPA17 is primarily a membrane protein
involved in cell–cell interaction and it is also present in soluble
fraction of SDS-treated spermatozoa lysates (Wen et al., 1999).
We observed a degradation of SPA17 by a-chymotrypsin. These
results demonstrate the deleterious effect of the currently rec-
ommended concentration of the enzyme that is available com-
mercially on both intracellular and surface proteins.
Even though there are certain beneficial outcomes of a-chy-
motrypsin treatment in viscous semen samples, including better
liquefaction and increase in sperm motility (Daw et al., 2011),
patients with seminal hyperviscosity demonstrated significantly
lower fertilization, implantation, and pregnancy rates (Esfandiari
et al., 2008). These negative effects of a-chymotrypsin on repro-
ductive function may be attributed to the alterations in the
expression pattern of sperm proteins that are vital for their nor-
mal function. Altogether, analysis of the expression of sperm
functional proteins explains the dramatic impact of a-chymo-
trypsin treatment (Figs. 2 and 3). A decrease in the expression of
a-tubulin, a major component of the cytoskeleton and centro-
some of spermatozoa, was observed in the present study
(Fig. 2E). This may be due to the damage induced to the sperm
membrane by a-chymotrypsin (Moody et al., 2017).
Another important aspect addressed by our pilot study is
regarding the suitability of the hyperviscous samples treated
with a-chymotrypsin for global proteomic studies. The overall
decrease in the protein expression suggests that enzymatic
action of a-chymotrypsin either degrades or digests the proteins,
making the sample unsuitable for further expressional pro-
teomic studies. Therefore, the hyperviscosity of semen samples
has to be reduced mechanically or by milder treatment with
lower concentrations of a-chymotrypsin for proteomic studies.
One of the most important implications of our study is that the
spermatozoa obtained by semen processing with a-chymotryp-
sin may drastically affect the outcome of ART.
One of the limitations of our study is a small sample size,
which is related to the small incidence (12–20%) of hyperviscous
sample in a population of infertile men. Therefore, most of the
protein expression profile examined in our study used normally
liquefied semen. Another limitation is the use of a-chymotrypsin
at a concentration (5 mg) as recommended by the manufacturer
for prescribed incubation period (20 min) at 37 °C. A dose- and
time-dependent analysis of the effect of chymotrypsin on
© 2018 American Society of Andrology and European Academy of Andrology

EFFECT OF a-CHYMOTRYPSIN ON SPERM PROTEINS
spermatozoa protein will further reveal the intensity and out-
come of damage to the spermatozoa.
To the best of our knowledge, this is the first report that
describes the significant alterations in sperm proteins, specifically
HSPA2, BAG6, SPA17, HIST1H2BA, OXPHOS complexes and
a-tubulin after a-chymotrypsin treatment as demonstrated by Wes-
tern blot analysis. The addition of this enzyme to semen samples
degrades the spermatozoal proteins, thereby making their expres-
sion non-detectable by immunoblotting. Therefore, spermatozoa
recovered from hyperviscous samples after treatment with a-chy-
motrypsin may affect other sperm functional parameters such as
capacitation, acrosome reaction, and DNA fragmentation, thereby
affecting ART outcome. This may be especially true in patients
undergoing intrauterine insemination where spermatozoa are pre-
pared on a double-density gradient separation to recover the highly
motile, morphologically normal spermatozoa for insemination.
This however needs further investigation.
ACKNOWLEDGEMENT
The authors wish to thank Tania Dias, MSc. (Cleveland Clinic),
and Ana Martins, MSc. (Cleveland Clinic), for their helpful com-
ments on our manuscript. The research in this study was sup-
ported by the funding from the American Center for
Reproductive Medicine, Cleveland Clinic.
AUTHORS’ CONTRIBUTIONS
AA, RS, and MP conceived and designed the study. MP carried
out the experiment. AA, RS, LS and MP involved in the analysis
and interpretation of the data. All authors have read and
approved the final version of the manuscript, and agree with the
order of presentation of the authors.
COMPETING INTERESTS
None of the authors declare competing financial interests.
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