Acta Biochimica et Biophysica Sinica Advance Access published September 20, 2016

Acta Biochim Biophys Sin, 2016, 1–8

doi: 10.1093/abbs/gmw096
Original Article

Original Article

Low levels of PRSS37 protein in sperm are

associated with many cases of unexplained
male infertility
Jianbing Liu1,2, Chunling Shen1,*, Weimin Fan3, Yan Chen2, Aijun Zhang3,

Downloaded from http://abbs.oxfordjournals.org/ at Cornell University Library on September 25, 2016

Yun Feng3, Zheng Li4, Ying Kuang5, and Zhugang Wang1,2,5,*
1
State Key Laboratory of Medical Genomics, Research Center for Experimental Medicine, Ruijin Hospital Affiliated
to Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025, China, 2Model Organism
Division, E-Institutes of Shanghai Universities, SJTUSM, Shanghai 200025, China, 3Reproductive Medical Center of
Ruijin Hospital, Shanghai 200025, China, 4Shanghai Human Sperm Bank of Shanghai Renji Hospital, Shanghai
200025, China, and 5Shanghai Research Center for Model Organisms, Shanghai 201203, China

*Correspondence address. Tel: +86-21-58951591; Fax: +86-21-54656097; E-mail: zhugangw@shsmu.edu.cn (Z.W.)/

Tel: +86-21-64370045; Fax: +86-21-58955923; E-mail: clshen@139.com (C.S.)
Received 11 May 2016; Accepted 25 August 2016

Abstract
PRSS37, a putative trypsin-like serine protease, is highly conserved during mammalian evolution
as revealed by multiple sequence alignment. Mice deficient for Prss37 gene exhibit male infertility,
but their mating behavior, spermatogenesis, sperm morphology, and motility remain unaffected,
similar to a situation called unexplained male infertility (UMI) in men (human being). Here, we
demonstrated that PRSS37 is restrictively expressed in human testis, where it is mainly located in
the elongating and elongated spermatids during spermiogenesis as shown by immunohistochem-
ical analysis of normal human testicular sections. In mature sperm, PRSS37 appears in the acro-
some region and diminishes during acrosome reaction. Further examination reveals that PRSS37
contents in sperm from patients with UMI are dramatically lower than those in sperm from men
with proven fertility or from sperm donors. Sperm with low PRSS37 contents exhibit abnormal
activation of the proacrosin/acrosin system and premature proteolysis of ADAM2, which may
impair the functional competence of human sperm in vivo. However, the in vitro fertilization out-
comes of sperm with low PRSS37 contents are not affected. Together, these data implicate an
important role of PRSS37 for male fertility. PRSS37 can be used as a potential molecular bio-
marker for evaluating sperm fertilization capability in vivo but not in vitro.

Key words: PRSS37, ACROSIN, ADAM2, semen analysis, unexplained male infertility

Introduction concentration and/or reduced semen total volume), decreased sperm

Infertility is a complex disorder that affects approximately 15% of
couples worldwide, resulting in significant financial and emotional
costs [1,2]. Male factor infertility, either isolated or combined with
female factor disorders, accounts for approximately half of all infer-
tility cases [3,4]. Infertility in males is mainly caused by insufficient
number of sperm present in the ejaculate (deficient sperm
viability, low sperm motility (or progressive motility) and abnormal-
ity in the morphology of the sperm [5], as revealed by clinical semen
analysis under the guidelines suggested by the World Health
Organization (WHO) [6]. However, conventional semen analysis is
only of limited value for predicting sperm fertility [7,8]. Male infer-
tility occurs even with all normal semen parameters; it accounts for

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2 PRSS37 associated unexplained male infertility
more than 10% of infertile males in clinical practice, after ruling out
female infertility factors [9], and is referred to as unexplained male
infertility (UMI) in this article. Actually, it is the functional compe-
tence of sperm but not the absolute number of motile sperm that
determines fertility, and many efforts have been made to develop
new diagnostic tests to provide more accurate information on the
fertilizing potential of human sperm. These tests are designed to
monitor various aspects of sperm function including cervical mucus
penetration and motility, capacitation, hyperactivation, zona recog-
nition, acrosome reaction, and sperm–oocyte fusion [10]. However,
most of these tests are only performed correctly in specialized aca-
demic institutions and do not meet the necessary requirements to be
adopted in clinical practice.
PRSS37, also known as TRYX2, is a probable inactive serine
protease 37 containing peptidase S1 domain. The human PRSS37
gene is located on chromosome 7q34 with nine exons. Several tran-
scripts have been identified due to alternative splicing. However, lit-
tle is known about its physiological function. Previously, PRSS37
was reported as one of the candidate predisposing genes in at least
two familial colorectal cancer cases [11]. And our work on a knock-
out mouse model demonstrates that Prss37 is required for male fer-
tility in the mouse [12]. Prss37 is exclusively expressed in the mouse
testis tissue, especially in the spermatids at steps 9–14 during sper-
miogenesis. Prss37 deficiency in mice results in male infertility but
does not affect the mating behavior, sperm count, morphology, and
motility, resembling a situation called UMI in men. Further investi-
gation reveals that Prss37 deficiency causes the absence of mature
Adam3 in sperm and a defect in sperm migration from uterus into
oviduct in vivo. Considering the high homology between mouse and
human orthologues, we wonder whether human PRSS37 plays a
similar role in male fertility and its clinical relevance to UMI.
In the present study, we determine the tissue expression profile
of PRSS37 and its distribution in testicle cells by using normal
human samples, compare the sperm PRSS37 protein level between
sperm from patients with UMI and those from normal controls, and
retrospectively analyze the in vitro fertilization (IVF) outcomes of
sperm with high or low PRSS37 contents.
Materials and Methods
Patients and controls
A total of 116 males with UMI and 118 males with proven fertility
(PF) were recruited from the Reproductive Medical Center of Ruijin
Hospital, affiliated to Shanghai Jiao Tong University School of
Medicine (Shanghai, China) between February 2011 and March
2013. Semen samples were obtained by masturbation after 2–7 days
of sexual abstinence. Semen analysis was carried out within 1 h of
sample collection using a computer-assisted sperm analysis system
(Sperm Class Analyzer, SCA5.0; Microptic, Barcelona, Spain). All
males with unexplained infertility were required to meet the follow-
ing four criteria: (i) all semen parameters were normal according to
the reference values of the WHO (WHO, 2010); (ii) their female
partners had normal ovulation; (iii) at least one side of their female
partners’ fallopian tubes was patent and the uterus was normal, as
shown by hysterosalpingogram; and (iv) antisperm antibodies were
undetected by immunosorbent or mixed antiglobulin reaction in the
blood and semen of infertile couples, as well as an absence of anti-
endotrium antibodies in females. In addition, no other causes of infer-
tility were identified during the treatment process. All males with PF
had fathered at least one child. Moreover, 46 sperm specimens from
sperm donors were included as another control group. These sperm
had been stored in the sperm bank of Renji Hospital (Shanghai,
China) and had made at least two women pregnant through intra-
uterine insemination (IUI). Approval for this study was granted by
the Ethics Committee of Ruijin Hospital. All participants signed con-
sent forms prior to sample collection.
Tissue expression profile
The relative expression levels of PRSS37 in different human tissues
were assayed by real-time PCR using cDNA preparations (Human
MTCTM Panel II, cat#636743; Clontech, Mountain View, USA) or
cDNA samples prepared from normal human tissues. Normal
human tissue specimens were obtained from surgery or from aut-
opsy, with institutional review board approval. Total RNA was
extracted by Trizol RNA isolation reagent (Invitrogen, Carlsbad,
Downloaded from http://abbs.oxfordjournals.org/ at Cornell University Library on September 25, 2016
USA) adhering to the manufacturer’s instruction and quantified by
UV spectrophotometry. First strand cDNA was synthesized from
2 μg of total RNA using Primescript Reverse transcriptase (Takara,
Tokyo, Japan). Real-time PCR was performed with an Eppendorf
Mastercycler ep realplex using the double-stranded DNA-specific
fluorophore SYBR Green (Takara) with the following primer sets:
5′-CTCTGCCCCCTCTGCTGAT-3′ (forward) and 5′-CAGTCTTC
TGGGTGGCAGTGA-3′ (reverse) for GAPDH; and 5′-CCCTGAC
TTGCGGCAGAA-3′ (forward) and 5′-TCGATTCCCTGGAGC
TTGTC-3′ (reverse) for PRSS37. Resolution of the product of inter-
est from nonspecific product amplification was achieved by melting
curve analysis. The expression level of PRSS37 was normalized to
GAPDH content.
Immunohistochemistry
Paraffin sections of normal human testis tissue were dewaxed, rehy-
drated and subject to microwave antigen retrieval. After pretreat-
ment with 3% H2O2 in methanol at room temperature for 10 min
to block the endogenous peroxidase activity, tissue sections were
blocked in 10% normal goat serum in phosphate-buffered saline
(PBS) at room temperature for 1 h and incubated overnight at 4°C
with diluted anti-PRSS37 rabbit antiserum (1:200, HPA020541;
Sigma, St Louis, USA). After three washes with PBS for 5 min each
time, sections were detected using a VECTASTAIN® ABC Kit
(Vector Laboratories, Burlingame, USA) according to the manufac-
turer’s instructions.
Tri-fluorescent staining of human sperm
Semen specimens from males with PF were collected by masturba-
tion and liquefied at 37°C for 30 min, and were washed twice in
8 ml of PBS. Sperm were capacitated at a concentration of 20 × 106
sperm/ml for 4 h in human tubal fluid (HTF) medium including
10% serum substitute supplement (SSS) in 5% CO2 at 37°C.
Acrosome reaction was induced by addition of calcium ionophore
A23187 (Sigma) at final concentration of 10 μM and incubated for
another 30 min. Capacitated sperm both before and after A23187
induction were washed with PBS, and smeared onto glass micro-
scope slides and allowed to air-dry. Slides were then fixed with 4%
paraformaldehyde (PFA) in PBS for 10 min and stored at −20°C.
For immunofluorescence, sperm smear slides were washed and incu-
bated in PBS containing 0.1% Triton X-100 for 10 min. Then they
were blocked with 5% normal goat serum in PBS at room tempera-
ture for 1 h and then incubated with diluted anti-PRSS37 rabbit
antiserum (1:100; Sigma) at room temperature for 1 h. After three
PRSS37 associated unexplained male infertility
washes in PBS for 5 min each, sections were incubated in the dark
with Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular Probes,
Eugene, USA) and Alexa Fluor 647-conjugated lectin peanut agglutinin
(PNA; Molecular Probes) at room temperature for 1 h. After additional
washes, slides were counterstained with 4′,6-diamino-2-phenylindole
(DAPI; Invitrogen), mounted in fluorescence mounting medium
(DAKO), coverslipped and examined under a confocal microscope.
Protein extraction and western blot analysis
Human sperm were washed twice with PBS and incubated in DIGE
lysis buffer consisting of 7 M urea, 2 M thiourea, 4% (w/v)
CHAPS, 2% (w/v) dithiothreitol (DTT), and a protease inhibitor
cocktail (Roche, Indianapolis, USA) for 1 h at 4°C. Samples were
then homogenized using an Ultra Turrax homogenizer (IKA,
Königswinter, Germany) at 11,000 rpm for 5 min on ice. After cen-
trifugation at 9391 g at 4°C for 30 min, the supernatant was col-
lected and stored at −80°C until use. The concentration of
extracted protein was determined by Bradford microprotein assay
using bovine serum albumin as the standard protein. Protein sam-
ples were refluxed for 5 min in sodium dodecyl sulfate (SDS)-PAGE
sample buffer consisting of 2% (v/v) mercaptoethanol, 2% (w/v)
sucrose in 0.1875 M Tris, pH 6.8, with bromophenol blue, and
then equal amounts of proteins (50 µg) were separated by 10%
SDS-PAGE and transferred onto nitrocellulose membranes (catalog
no. 162–0112; Bio-Rad, Hercules, USA). Membranes were blocked
with 5% nonfat milk for 1 h, followed by incubation with primary
antibodies as indicated overnight at 4°C. Antibodies used were
rabbit polyclonal antibody raised against PRSS37 (1:1000 dilution,
code HPA020541; Sigma), ADAM2 (1:1000 dilution, code
HPA024621; Sigma), ACROSIN (1:1000 dilution, code SC-67151;
Santa Cruz Biotechnology, Santa Cruz, USA), and mouse mono-
clonal antibody against GAPDH (1:10,000 dilution, code KC-5G4;
Kangchen, Shanghai, China). After being washed with Tris-
buffered saline and Tween 20, the membranes were then incubated
with anti-rabbit or anti-mouse secondary antibodies for 2 h at
room temperature. Secondary antibodies conjugated with
IRdye800CW (LI-COR, Lincoln, USA) were used to visualize the
specific protein bands using an Odyssey Infrared Imager (LI-COR).
Relative expression levels of PRSS37 were estimated by densitom-
etry and normalized to GAPDH.
In vitro fertilization
All control couples underwent IVF due to female tube obstruc-
tion; all female partners were <40 years of age and had day-3
follicle-stimulating hormone (FSH) level < 10 IU l−1. Semen prep-
aration, ovarian stimulation, oocyte retrieval, embryo culture,
embryo transfer, and pregnancy outcome follow-ups were per-
formed as previously described [13,14]. The oocyte was consid-
ered fertilized if the second polar body was extruded or if
pronuclei were seen 18 h after insemination. Those with two pro-
nuclei (2PN) and a second polar body were identified as normally
fertilized. The oocytes with a single pronucleus or three pronuclei
were considered to be abnormally fertilized. Oocytes without vis-
ible pronuclei were considered unfertilized. Embryo morphology
was evaluated on day 3 by measuring the number and symmetry
of the blastomeres and the percentage of fragmentation. Embryos
were characterized as viable by the presence of at least five blasto-
meres after insemination, the absence of multinucleated blasto-
meres and <30% cellular fragments. A good embryo was defined
as having eight or more than eight blastomeres with ≤10%
3
cellular fragmentation. Embryo transfers were performed 72 h
after oocyte retrieval, and only good viable embryos were selected
for transfer.
Statistical analysis
Results for the age and semen parameters between patients with
UMI and men with PF were presented as the mean ± SD and evalu-
ated with Student’s t-test. Tests for the frequency of low PRSS37
expression among different groups were carried out by Fisher exact
test. IVF outcomes were evaluated by chi-squared test. P < 0.05 was
considered of statistically significant difference.
Results
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Tissue expression profile of PRSS37 and its distribution
in testicle cells
Real-time PCR assay was adopted to elucidate the relative expres-
sion levels of PRSS37 in different human tissues including brain,
heart, lung, liver, spleen, pancreas, kidney, gall bladder, stomach,
small intestine, colorectum, skeletal muscle, skin, uterus, ovary,
epididymis, and testis. cDNA was obtained as described in
section Materials and Methods. PRSS37 mRNA displayed a testis-
specific pattern (Fig. 1A), similar to its mouse orthologue. In order
to clarify its distribution in testicle cells, immunohistochemistry
analysis was applied on paraffin sections of normal human testis
tissue. Compared with the negative control without primary anti-
body, a cell type-specific distribution of PRSS37 signal was seen,
which aggregated in the luminal margin of the seminiferous
tubules (Fig. 1B) corresponding to haploid spermatids during spe-
miogenesis. However, no PRSS37 signal was observed in cells near
the basement membrane, such as spermatogonia, primary and sec-
ondary spermatocytes, or Leydig cells. To further distinguish the
distribution of PRSS37 immunoreactive signals, higher magnifica-
tion images representing the six stages of the cycle of the semin-
iferous epithelium in man were taken (Fig. 1C). PRSS37
immunoreactive signals were obviously detected in both elongat-
ing (Sb2 and Sc) and elongated spermatids (Sd1). However, more
evidence is still required to clarify whether PRSS37 is also located
in round spermatids (Sa and Sb1) and mature sperm (Sd2 and eja-
culated sperm).
PRSS37 appears in the acrosome region of ejaculated
sperm and diminishes after induction of acrosome
reaction
The presence of PRSS37 in mature human sperm was evaluated by
tri-fluorescent staining and confocal microscopy. PNA, which binds
to the outer acrosomal membrane of non-acrosome-reacted sperm,
was used as a probe to evaluate the status of the acrosome. PRSS37
was detected in the entire acrosome of the human sperm, in-
cluding the anterior acrosomal region and the equatorial segment.
Interestingly, PRSS37 protein diminished after the induction of acro-
some reaction by calcium ionophore A23187 (Fig. 2A), which was
further demonstrated by western blot analysis (Fig. 2B), implying
that PRSS37 might play a role in human fertilization after the induc-
tion of acrosome reaction. However, details of the process of the
detachment of PRSS37 during acrosome reaction and its role in
human fertilization remained to be elucidated.
4 PRSS37 associated unexplained male infertility

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Figure 1. Tissue expression profile of PRSS37 and its distribution in testicle cells (A) qRT-PCR analysis of PRSS37 mRNA expression in 17 human tissues.
GAPDH was used as an internal control. (B) Immunohistochemistry analysis of PRSS37 protein distribution on paraffin sections of normal adult human testis tis-
sue. Black arrows indicate positive PRSS37 signals. The negative control image is taken from an identical assay without primary antibody. Scale bar, 200 μm.
(C) High magnification images showing PRSS37 signals along the six stages of the seminiferous epithelial cycle in human testis, as denoted by Roman numer-
als. Sa, Sb1, Sb2, Sc, Sd1, and Sd2 were six types of maturing spermatids. Sa, round; Sb1, round with flagellum; Sb2, elongating; Sc, elongating, nucleus fully
elongated; Sd1, elongated, head still not separated from mid-piece; Sd2, mature, large cytoplasmic sheath in mid-piece. Scale bar, 20 μm.

Figure 2. PRSS37 appears in the acrosome region of ejaculated sperm and diminishes after induction of acrosome reaction (AR) by A23187. (A) Tri-
fluorescent staining and confocal microscopy analysis of PRSS37 in human sperm before and after AR. Alexa Fluor 647-conjugated PNA was used to stain the
sperm acrosome and sperm DNA was counterstained by DAPI. Scale bar, 5 μm. (B) Western blot analysis of PRSS37 level in human sperm before and after AR.
Sperm were incubated in HTF medium in 5% CO2 at 37°C before adding A23187.
PRSS37 associated unexplained male infertility 5

PRSS37 contents in sperm from patients with UMI are
dramatically lower than those in sperm from men with
PF or from sperm donor
The testis-specific tissue expression profile and the probable
AR-related function of PRSS37 in human fertilization prompted us
to investigate its association with male fertility. Moreover, our previ-
ous work suggested that Prss37-knockout male mice exhibited a
phenotype similar to UMI in men. Thus, we decided to compare the
PRSS37 protein level in sperm from men with UMI to that from
normal controls. PRSS37 content in sperm was calculated as the
ratio of the densitometry of PRSS37 band to the GAPDH band. In
total, 116 sperm samples from patients with UMI and 118 sperm
samples from males with PF as well as 46 sperm samples from
sperm donor (SD) were examined and analyzed. Representative
images were shown in Fig. 3A. Both inter- and intra-assay coeffi-
cients of variability (CV) were less than 15% (data not shown).
significant difference in the frequency of low PRSS37 expression
among these three groups (Fig. 3C). However, no change in PRSS37
mRNA level was found between sperm with low PRSS37 protein
(n = 13) and sperm with high PRSS37 protein (n = 14) as assayed
by real-time PCR (Fig. 3D). These data suggested that low PRSS37
protein level in human sperm was, at least in part, associated with
UMI, supporting a potential role of PRSS37 as a molecular bio-
marker for the diagnosis of male infertility.
Sperm with low PRSS37 contents exhibit abnormal
activation of the proacrosin/acrosin system and
premature proteolysis of ADAM2
Acrosin is synthesized and stored in the sperm acrosome matrix as
proacrosin, an enzymatically inactive zymogen form, which is pro-
cessed into intermediate and mature forms through autoactivation

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PRSS37 contents in sperm from patients with UMI (mean ratio of
PRSS37/GAPDH was 1.84, n = 116) were dramatically lower than
those in sperm from men with PF (mean ratio of PRSS37/GAPDH
was 2.93, n = 118, P < 0.05) or those in sperm from SD (mean ratio
of PRSS37/GAPDH was 3.37, n = 46, P < 0.01) (Fig. 3B), although
no statistically significant differences existed in age and semen para-
meters between patients with UMI and men with PF (Table 1). If the
ratios of PRSS37/GAPDH < 0.2 and > 0.4 were regarded as low and
high PRSS37 expression, respectively, then there was a statistically
at a basic pH during acrosome reaction. It has been reported that
abnormalities in the activation of proacrosin/acrosin system corre-
lates with unexplained infertility [15]. Human proacrosin activation
involves the processing of the 55 kDa band into 39 and 35 kDa
enzymatically active intermediates, followed by conversion into low-
er relative molecular weight mass forms [16]. In the present study,
the activation process of human proacrosin was re-illustrated using
sperm from males with PF, which showed high PRSS37 protein
level, by western blotting (lanes N1, N2, and N3 in Fig. 4).

Figure 3. PRSS37 contents in sperm from patients with UMI are dramatically lower than those in sperm from men with PF or from SD (A) Representative
western blot analysis of PRSS37 and GAPDH in sperm from 24 patients with UMI (P1–P24) and from four men with PF (N1–N4), in which GAPDH was used as
internal controls. PRSS37 content in sperm was calculated as the ratio of the densitometry of PRSS37 band to the GAPDH band. M, protein molecular weight
marker. (B) PRSS37 contents in 116 sperm samples from patients with UMI, in 118 sperm samples from males with PF, and in 46 sperm samples from SD were
examined, analyzed and compared. Data were presented as the mean ± SEM and evaluated with t-test. (C) Comparison the frequency of low PRSS37 content
(ratio of PRSS37/GAPDH < 0.2) among sperm samples from patients with UMI, men with PF and SD. (D) Real-time PCR analysis of PRSS37 mRNA level in sperm
with low PRSS37 content (ratio of PRSS37/GAPDH < 0.2, n = 13) and in sperm with high PRSS37 content (ratio of PRSS37/GAPDH > 0.4, n = 14).
6 PRSS37 associated unexplained male infertility

Table 1. Characteristics for patients with UMI and men with PF in this study

Characteristics Patients with UMI mean (95% CI) Men with PF mean (95% CI)

Cases 116 118

Age 31.6 ± 3.7 32.6 ± 4.8
Semen volume (ml) 3.6 ± 1.5 3.4 ± 1.7
Sperm concentration (106 sperm/ml) 74.3 (68.5–81.2) 79.6 (71.1–92.9)
Total sperm number (106 sperm/ejaculate) 248.5 (204.7–293.2) 263.9 (218.8–301.1)
Progressive motility (%) 48.2 ± 13.1 54.1 ± 15.7
Total motility (%) 60.1 ± 13.6 65.6 ± 15.9
Normal morphology (%) 14.3 (12.9–15.5) 15.5 (13.5–16.5)

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Figure 4. Sperm with low PRSS37 contents exhibit abnormal activation of the proacrosin/acrosin system and premature proteolysis of ADAM2 ACROSIN
immunoblotting detected proacrosin bands (55 kDa) in sperm from men with PF with high PRSS37 content (N1–N3) but not in sperm from patients with UMI
with low PRSS37 content (L1–L4). ADAM2 immunoblotting detected only the precursor form (98 kDa or 72 kDa) in sperm from men with PF with high PRSS37
content (N1–N3), whereas only the mature form (44 kDa) existed in sperm from patients with UMI with low PRSS37 content (L1–L4). The sizes of specific protein
bands are marked on the right.

However, the 55 kDa proacrosin band was almost undetectable in
sperm from males with UMI, which showed low PRSS37 protein
level (lanes L1, L2, L3, and L4 in Fig. 4), implying that sperm with
low PRSS37 contents had abnormal activation of the proacrosin/
acrosin system.
Since Prss37 deficiency in mice causes the absence of mature
Adam3 in sperm, we wonder whether human ADAMs are affected
due to low PRSS37 level in human sperm. ADAMs are biosynthe-
sized as multidomain transmembrane preproproteins consisting of a
signal peptide, a prodomain, a metalloproteinase domain, a disinte-
grin domain, a cysteine-rich domain, an epidermal growth factor-
like domain, a transmembrane domain, and a cytoplasm domain.
Currently, the ADAM gene family has 29 members which act in a
highly diverse set of biological processes, including fertilization,
neurogenesis, myogenesis, embryonic TGF-α release, and the inflam-
matory response. ADAM1, ADAM2, and ADAM3 are best studied
ADAMs that play roles in fertilization. However, human ADAM1
and ADAM3 are pseudo genes and have been demonstrated to be
non-functional [17–19]. Human ADAM2 gene has a testis-specific
transcription and is generated as a precursor protein [20], which
undergoes successive proteolytic cleavages during post-translational
modification. The signal peptide, the prodomain and the metallopro-
teinase domain are cleaved off, leaving the disintegrin domain
N-terminal exposed during sperm maturation [21]. Western blot
analysis showed either a 98 or 72 kDa band in sperm from males
with PF, which had high PRSS37 protein level. However, a 44 kDa
band appeared in sperm from males with UMI, which had low
PRSS37 protein level (Fig. 4). These data implied that sperm with
low PRSS37 contents had premature proteolysis of ADAM2.
IVF outcomes of sperm with low PRSS37 contents are
not affected
Eleven UMI patients with low sperm PRSS37 contents (ratio of
PRSS37/GAPDH < 0.2) and thirty patients with normal sperm
PRSS37 contents (ratio of PRSS37/GAPDH > 0.4) underwent IVF
therapy in the Reproductive Medical Center of Ruijin Hospital due
to 3–5 cycles of IUI failure and fallopian tube obstruction of their
PRSS37 associated unexplained male infertility
Table 2. IVF outcomes of sperm with low and high PRSS37
contents
Characteristics Ratio of PRSS37/GAPDH Ratio of PRSS37/GAPDH
< 0.2, n = 11 > 0.4, n = 30
Female age 32.7 ± 4.7 31.4 ± 5.1
FSH (IU l−1) 7.7 ± 1.3 7.5 ± 1.6
Oocytes 16 ± 6.7 14.6 ± 5.1
retrieved
Fertilization rate 73 ± 18 75 ± 15
(%)
Good embryos 62 ± 27 57 ± 24
(%)
female partners, respectively. The IVF outcomes were retrospectively
analyzed and summarized in Table 2. No significant differences in
female age, basal FSH level, number of oocytes retrieved, fertiliza-
tion rate and good embryo rate were found between the two groups
(P > 0.05).
Discussion
Mammalian fertilization, a process of combining two germ cells
(oocyte and sperm) and creating a new life, is complex and finely
regulated. Ejaculated sperm must capacitate within the female repro-
ductive tract, migrate from the uterus to the oviduct ampulla before
they meet the oocyte–cumulus complex, penetrate through the granu-
lar cell layer and zona pellucida and finally fuse with the oocyte [22].
The oocyte was considered fertilized if the second polar body was
extruded or the two pronuclei (2PN) were formed. In recent years,
the molecular mechanism of fertilization has been studied extensively
in mice and, to a lesser degree, in other mammals, including humans.
The ability to introduce genes into mice (transgenic mice) and to dis-
rupt specific genes by targeted mutagenesis in mice (knockout mice)
made this system particularly useful for identifying gamete proteins
that may participate in the fertilization process [23–25]. However,
research findings obtained in mice can not be fully applied to human
beings, since differences in the fertilization mechanism remain exist
between mouse and human being. In a previous study, we demon-
strated that Prss37 was essential for male fertility in mice [12].
Whether PRSS37 plays a similar role in male fertility in humans is
unknown, even though mouse and human PRSS37 orthologues dis-
play high homology. The current study focuses on the expression
characteristics of PRSS37 in human and its clinical relevance in UMI.
PRSS37 appears in the cytoplasm of elongating and elongated
spermatids in the human testis, which is consistent with the expres-
sion specificity of its mouse orthologue, i.e. mouse Prss37 is exclu-
sively expressed in the spermatids at steps 9–14 of spermiogenesis.
PRSS37 is also located in the acrosome region of ejaculated human
sperm and is released from this region following the acrosome reac-
tion, which differs from its mouse orthologue in that no or a negli-
gibly small amount of Prss37 is detected in the mature mouse sperm.
This difference may be due to the diverse species differences between
human and mouse. This distribution of PRSS37 suggests that it is
important not only during spermiogenesis, but also in processes cru-
cial for human fertilization, such as sperm capacitation, sperm
migration in the female reproductive tract, and acrosome reaction.
We investigated the relative PRSS37 contents in sperm from
116 patients with UMI, in sperm from 118 males with PF and in
46 sperm samples from SD, and found that the former had
7
dramatically lower PRSS37 contents than the other two groups. The
incidence of low PRSS37 expression in patients with UMI was 21%,
much higher than that in males with PF (4.2%). There was almost
no low PRSS37 expression in clinically meticulously selected sperm
donors. We compared the activation of the proacrosin/acrosin sys-
tem in sperm with low PRSS37 expression with that in sperm with
high PRSS37 expression by western blotting. The 55 kDa proacrosin
band was almost undetectable in sperm with low PRSS37 expres-
sion. Instead, the 39- and 35-kDa enzymatically active intermediates
could be obviously detected, implying abnormal activation of the
proacrosin/acrosin system. Despite this, the morphology of sperm
from UMI samples was comparable to that of normal controls. The
morphology of sperm from UMI samples was analyzed by H&E
staining and observation under a light microscope. This method is
often used to assess the gross morphology. Of course, subtle abnor-
malities that could not be detected by this method should not be
Downloaded from http://abbs.oxfordjournals.org/ at Cornell University Library on September 25, 2016
excluded. The morphology of the acrosome needs to be evaluated
by transmission electron microscopy, which is inapplicable in clin-
ical practice. Moreover, western blot analysis showed either a 98 or
72 kDa ADAM2 band in sperm with high PRSS37 protein level,
whereas only a 44 kDa ADAM2 band appeared in sperm with low
PRSS37 protein level, implying premature proteolysis of ADAM2 in
sperm with low PRSS37 contents. In contrast, our previous study in
Prss37-knockout mouse model suggests that Prss37 deficiency
causes the absence of mature Adam3 in sperm, but irrelevant to
Adam2. This inconsistency might be another species difference
between human and mouse, since human ADAM3 is a pseudo gene
and has been demonstrated to be non-functional [18,19]. Taken
together, the above mentioned changes reflect the differences
between sperm from patients with UMI and sperm from normal
controls. These alterations might be the underlying cause of some
unexplained cases of human infertility, although the causal relation-
ship among them needs to be further elucidated. From a certain
point of view, each of acrosin, ADAM2 and PRSS37 could be
regarded as a biomarker for UMI. However, both acrosin and
ADAM2 have pro- or precursor form and active or mature form
under different sperm status. If we use western blotting method to
compare sperm samples from normal controls with those from UMI
patients, we may detect different protein bands, just as what we
show in Fig. 4. The advantage of using PRSS37 as a biomarker is
that there exists only one form with high or low level under different
sperm status. Besides western blotting method, PRSS37 is more suit-
able for developing a new easier handle assay for routine analysis.
IUI, with or without controlled ovarian stimulation, is a com-
mon first-line empiric treatment for unexplained infertility [26–28],
but the success rate is relatively low compared with IVF or gamete
intra-fallopian transfer [27,29]. The IVF outcomes are comparable
between sperm with low PRSS37 contents and sperm with high
PRSS37 contents, suggesting that low PRSS37 contents in sperm
probably affect sperm migration in the female reproductive tract, a
phenomenon similar to that observed in Prss37-knockout mice.
PRSS37 might be used as a potential molecular marker for evaluat-
ing the migration ability of sperm in the female reproductive tract.
Thus, for UMI patients with low PRSS37 contents in sperm, IVF
rather than IUI should be recommended as the better choice, avoid-
ing excessive inappropriate medical treatment. In addition, for
sperm donated in IUI treatment, it would be better to examine the
PRSS37 contents, so as to improve the success rate and protect the
sperm from being excessively wasted. With the accumulation of
more clinical data, the use of PRSS37 as a new molecular biomarker
in sperm may improve the diagnosis and management of UMI.
8 PRSS37 associated unexplained male infertility
Acknowledgements
We would like to thank the anonymous reviewers for their helpful
suggestions. We are grateful to Prof. Jingsheng Feng for staging of
the cycle of seminiferous epithelium of human testis.
Funding
This work was supported by the grants from National Natural Science
Foundation of China (No. 81430028), Ministry of Science and
Technology of China (No. 2011BAI15B02), Science and Technology
Commission of Shanghai Municipality (Nos. 13DZ2280600 and
15DZ2290800), and E-Institutes of Shanghai Municipal Education
Commission (No. E03003).
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