Note: Descriptions are shown in the official language in which they were submitted.
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DETECTION OF HUMAN CYSTEINE-RICH SECRETORY PROTEIN (CRISPI) IN SEMEN
AND MEDICAL APPLICATIONS RELATED THERETO
FIELD OF THE INVENTION
The invention relates to the field of biology. More particularly, it concerns
an epididymal
specific protein which is detectable in human semen.
BACKGROUND OF THE INVENTION
In the USA, more than 525 000 vasectomies are performed yearly on men aged
between 29
and 45 years. Worldwide, more than 100 million men rely on vasectomy for
contraception
purposes. However, due to changes in their personal life, an increasing number
of men
undergo surgical vasectomy reversal (vasovasostomy). In the USA only, this
surgical
procedure is performed on 250-300 000 men yearly. Therefore, there is a need
for efficient,
sensitive and non-invasive methods and kits for evaluating successfulness of
vasectomy
and of vasovasostomy in men.
Azoospermia is a medical condition of a male not having any measurable level
of sperm in
his semen. Azoospermia has two forms: (i) non-obstructive azoospermia, where
there is a
problem with spermatogenesis, and; (ii) obstructive azoospermia, where
spermatozoa are
created, but cannot be mixed with the rest of the ejaculatory fluid due to a
physical
obstruction, generally an obstruction of the epididymis or of the deferent
duct (vas deferens).
Unfortunately, it is currently very difficult to discriminate between both
forms: it requires
either a biopsy of the testicle which is invasive and painful; or an
ultrasonography of the
scrotum which is a complex medical procedure that can only be performed by
doctors in
hospitals or clinics. There is thus an urgent need for a quick and non-
invasive method for
discriminating between obstructive and non-obstructive azoospermia.
Human cysteine-rich secretory protein (CRISP1) is one of the major epididymal
secreted
protein. It plays important functions in sperm physiology (Gibbs et ai.,
Endocr Rev
2008;29:865-97; Cohen et al., Asian J Androl 2007;9:528-32; Roberts et al.,
Mol Cell
Endocrinol 2006;250:122-7) and expression of this protein in the epididymis is
greatly
affected by vasectomy (Thimon et al., Biol Reprod 2008;79:262-73). However,
there is
contradictive evidence as to the specificity of Crispl gene expression.
Although it has been
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reported that the human Crispl gene is expressed only in the epididymis
(Kratzschmar et
al., Eur. J. Biochem. 1996; 236, 827-836; Hayashi et al., Genomics 1996;32:367-
74), some
authors have shown expression of human Crisp1 not only in the epididymis, but
also in the
vas deferens and seminal vesicles by using more sensitive detection methods
(Nolan et al.,
Biol. Reprod. 2006; 74, 984-991). Accordingly, it was uncertain before the
present invention
that the absence of the CRISPI protein in human semen could be an indicator of
obstructive
azoospermia. Moreover, it was unknown that the absence or presence of human
CRISP1 in
the semen could serve as a reliable marker for evaluating in men
successfulness of
vasectomy or vasovasostomy.
Many mammalian members of the CRISP family of genes are known, including the
rat and
mouse members which have been the subject of a U.S. patent application
published under
No. US 2006/027803.
As it will be apparent from a review of the disclosure, drawings and
description of the
features of the invention below, the present invention addresses these needs
and other
needs by providing methods and kits which are based on the detection of
CRISPI.
BRIEF SUMMARY OF THE INVENTION
The invention relates to the detection of CRISPI as a marker for the presence
or absence of
epididymal secretions in a mammalian ejaculate.
According to one particular aspect, the invention relates to a method for
assessing the
presence of epididymal secretions in a human semen sample. The method
comprises
detecting in the semen sample presence or absence of human cysteine-rich
secretory
protein (CRISPI). Absence of CRISPI in the seminal plasma is indicative of an
absence of
epididymal secretions in the ejaculate.
The invention also relates to a method for evaluating successfulness of
vasectomy in a man.
The method comprises obtaining a semen sample from a vasectomized man and
detecting
in the semen sample the presence or absence of human cysteine-rich secretory
protein
(CRISPI). Absence of CRISP1 is indicative of a successful vasectomy while
presence of
CRISPI is indicative of an unsuccessful vasectomy.
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The invention also relates to a method for evaluating successfulness of
vasovasostomy in a
man. The method comprises obtaining a semen sample from a vasovasostomized man
and
detecting in the sample the presence or absence of human cysteine-rich
secretory protein
(CRISPI). Presence of CRISP1 is indicative of a successful vasovasostomy while
absence
of CRISPI is indicative of an unsuccessful vasovasostomy.
The invention further relates to a method for discriminating between
obstructive and non-
obstructive azoospermia in men. The method comprises obtaining a sample from
an
ejaculate of an azoospermic man and detecting in the sample the presence or
absence of
human cysteine-rich secretory protein (CRISP1). Presence of CRISPI is
indicative of a non-
obstructive azoospermia condition while absence of CRISPI is indicative of an
obstructive
azoospermia condition.
The invention further relates to a kit for evaluating successfulness of
vasectomy and
vasovasostomy in men. The kit comprises a user manual or instructions and at
least one
detection component for detecting in a human ejaculate the presence or absence
of human
cysteine-rich secretory protein (CRISPI ).
An advantage of the present invention is that it provides efficient, sensitive
and non-invasive
means for evaluating successfulness of vasectomy and of vasovasostomy in men.
The
invention further provides quick and non-invasive methods for discriminating
between
obstructive and non-obstructive azoospermia.
Additional aspects, advantages and features of the present invention will
become more
apparent upon reading of the following non-restrictive description of
preferred embodiments
which are exemplary and should not be interpreted as limiting the scope of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 comprises pictures showing immunohistological localization of CRISP1
in the caput
(A, D), corpus (B, E), and cauda (C, F) epididymides from normal (A, B, C) and
vasectomized men (D, E, F). C* and F* are negative controls. Arrows show lumen
positively
stained for CRISPI.
Figure 2 comprises pictures showing in situ localization of the CrispI mRNA
along normal
(A, B, C) and vasectomized (D, E, F) human epididymides. Histological sections
of human
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Caput (A, D), corpus (B, E), and cauda (C, F) segments of the epididymis were
probed with
DIG-labeled antisense or sense (negative control) (B*, E*) CRISP1 RNA probes..
Arrows
indicate principal cells positively stained for CRISPI mRNA.
Figure 3A is a dot graph representing densitometric determination of the
quantity of
CRISPI associated with 107 spermatozoa of fertile and vasovasostomized men.
CRISP1 is
expressed as a ratio of TUBA used as a ubiquitous protein.
Figure 3B is a panel showing examples of Western blots of CRISPI detection
performed on
protein extracted from 107 spermatozoa of fertile and vasovasostomized men.
Figures 4A and 4B are pictures showing immunolocalization of CRISP1 on
ejaculated
spermatozoa of fertile (Fig. 4A) and vasovasostomized (Fig. 4B) men. A' and B'
are negative
control using goat IgGs. Immune complexes appear as a dark staining. Arrows
indicate the
acrosomal region.
Figure 5 is a dot graph showing relative amount of CRISPI in seminal plasma
from normal,
vasectomized and vasovasostomized men. CRISP1 amounts are expressed as a ratio
of an
internal standard (acid phosphatase). Bars indicate average and "n" indicates
the number
of samples.
Figure 6 is a panel showing Western blot detection of CRISPI in tissues
homogenates from
testis (1), epididymis (2), vas deferens (3), prostate (4), and seminal
vesicles (5).
Figure 7 displays the amino acid sequence of CRISP1.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention is based on the identification of CRISP1 in semen.
Surprisingly, the
present inventors have found that CRISP1 is highly specific to the epididymis
and that
absence of CRISP1 in human semen correlates with a complete absence of any
epididymal
secretions in an ejaculate. This epididymal specificity makes CRISP1 a
reliable marker for
evaluating in men successfulness of vasectomy or vasovasostomy and/or for
discriminating
between obstructive and non-obstructive azoospermia.
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As used herein, the term "ejaculate" or "semen" generally refers to the
organic fluid which is
ejected from the penis following an ejaculation by a subject. Depending of the
subject's
condition, the semen may or may not comprise spermatozoa.
As used herein, the term "seminal plasma" or "seminal fluid" generally refers
to the liquid
supernatant, after sedimentation or centrifugation of semen, which has been
contributed by
the testis and the various sex glands (e.g. the seminal vesicles, the prostate
and the
bulbourethral glands).
As used herein, the term "epididymal secretions" generally refers to the
biological
materials which have been contributed by the epididymis.
As used herein, the term "CRISPI" generally refers to the human cysteine-rich
secretory
protein. CRISP1 is an epididymal protein (248 amino acids) which is known to
those skilled
in the art since it has been the subject of many scientific publications. The
GeneBankTM
(NCBI) accession number of human CRISPI is D38451, the UniProtTM accession
number is
P54107 and the UniGeneTM accession number is Hs.109620. Figure 7 provides the
complete amino acid sequence of CRISPI in humans (SEQ ID NO: 1). The methods
of the
invention encompass detection of the full-length CRISPI protein, and also the
detection of
fragments thereof comprising at least 10, 25, 50, 100, 150, 200, 225, 240,
245, or 247
contiguous amino acids of SEQ ID NO: 1.
Medical applications
As demonstrated in the Examplification section, CRISPI is a protein highly
specific to the
epididymis such that absence of CRISPI in semen is indicative of a complete
absence of
epididymal secretions in the ejaculate. This surprising finding opens
different avenues for
research, diagnostic and medical applications.
In one embodiment, detecting absence or presence of epididymal secretions in
the ejaculate
is especially useful for discriminating between obstructive and non-
obstructive azoospermia.
Therefore, the methods described herein may be useful to replace and/or
complement
various infertility assessment procedures, including but not limited to,
physical examination,
sperm count, biopsy of the testicle, ultrasonography. Detecting in semen the
presence or
absence of CRISPI may also be useful to the medical profession when
investigating
conditions such as azoospermia, sexual dysfunction, congenital defects of the
sperm, and
hypogonadism. Therefore, the methods described herein may also be carried out
in the
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course of various assisted reproduction procedures, including but not limited
to in vitro
fertilization (IFV), artificial insemination (AI), gamete intrafallopian
transfer (GIFT), embryo
transfer, intracytoplasmic sperm injection (ICSI), etc. The invention further
encompasses
diagnosis and management of human male fertility (e.g. fertility clinics)
using any of the
methods, techniques and kits described herein.
As described hereinbefore, detecting the presence or absence of CRISPI in a
semen
sample is useful for assessing successfulness of vasectomy or successfulness
of
vasovasostomy. According to a particular embodiment, a semen sample from a
vasectomized man is obtained and the sample is assessed for detecting therein
the
presence or absence of CRISP1. Since in vasectomized men the vasa deferentia
have been
severed, and then tied or sealed, epididymal proteins and sperm should not be
present in
the ejaculate. Therefore, absence of CRISP1 in the ejaculate would be
indicative of a
successful vasectomy while presence of CRISPI would be indicative of an
unsuccessful
vasectomy. Similarly, according to another embodiment, if the semen sample is
obtained
from a vasovasostomized man (i.e. the vasa deferentia have been reconnected
for reversing
the vasectomy), epididymal proteins and sperm should be present in the
ejaculate.
Accordingly, presence of CRISPI would be indicative of a successful
vasovasostomy while
absence of CRISPI would be indicative of an unsuccessful vasovasostomy.
Detection techniques
Different methods and tools can be used for measuring the presence or absence
of CRISP1
in semen. A preferred agent for detecting CRISP1 is an antibody capable of
binding to
CRISP1 (and/or to a fragment thereof). More preferably, the antibody is highly
specific to
CRISPI. Antibodies can be polyclonal, or more preferably monoclonal. An intact
antibody,
or a fragment thereof (e.g., Fab or F(ab')2) can be used. According to some
embodiments,
the antibody is labeled. The term "labeled", with regard to an antibody, is
intended to
encompass direct labeling of the antibody by coupling (i.e., physically
linking) a detectable
substance to the antibody, as well as indirect labeling of the antibody by
reactivity with
another reagent that is directly labeled. Examples of indirect labeling
include, but are not
limited to, detection of a primary antibody using a fluorescently labeled
secondary antibody.
Specific antibodies directed against the human CRISP1 are available and
include those
commercialized by Santa Cruz Biotechnology (CRISP-1 (H-75) antibody (rabbit
IgG, Catalog
# sc-33802; CRISP-1 (N-15) antibody, goat IgG, Catalog # sc-21276), Abnova
(mouse
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polyclonal antibody raised against a full-length human CRISP1 protein; Catalog
#
H00000167-B01); Sigma (anti-CRISPI antibody produced in rabbit, Catalog #
AV53580 ),
and Abcam (CRISPI rabbit polyclonal antibody, Catalog #ab74785).
Therefore, the present invention encompasses various CRISPI immunodetection
and/or
CRISP1 quantification methods, including but not limited to ELISA, flow
cytometry and
densitometric analysis of Western blots.
Those skilled in the art know how to use specific antibodies to make different
tools useful in
the immunodetection and quantification, including but not limited to antibody
coated colored
beads, antibody coated magnetic beads, strips and dip sticks coated with
antibodies, etc.
It is also conceivable according to the invention, that the biological
activity of CRISP1 be
measured in a semen sample. Therefore, in some embodiments, it is an activity
of CRISP1
which is assessed using methods and techniques known to those skilled in the
art. For
instance, published patent application US 2006/275803 suggests that CRISPI may
have a
protease activity.
Kits
Another aspect of the invention pertains to commercial packages or kits for
carrying out the
methods and assays of the invention. Kits according to the invention may be
used for
discriminating between obstructive and non-obstructive azoospermia and/or for
evaluating
successfulness of vasectomy and successfulness of vasovasostomy.
Kits according to the invention will employ, unless otherwise indicated,
conventional
techniques for protein detection. A kit of the invention may comprise one or
more of the
following elements: a buffer for the homogenization of a sperm sample;
purified sperm
fertility proteins (and/or a fragment thereof), including but not limited to
CRISP1 to be used
as controls; buffers (e.g. incubation buffer(s), substrate and assay
buffer(s), modulator
buffer(s)); detection materials (e.g. antibodies, fluorescein-labelled
derivatives, luminogenic
substrates, detection solutions, scintillation counting fluid, antibody coated
beads, etc.);
laboratory supplies (e.g. reaction tubes or microplates (e.g. 96- or 384-well
plates)); a user
manual or instructions, etc. The kit and methods of the invention may be
configured such as
to permit a quantitative detection or measurement of the protein(s) of
interest (e.g.
cytofluorimetry, LuminexTM, ECL PlexTM Western Blotting Detection System,
etc.).
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In one preferred embodiment, the kit is adapted for home-based uses. Indeed,
home
assessment of the presence or absence of sperm in an ejaculate is desirable,
for instance,
for confirming absence of sperm in the semen after a vasectomy procedure. Home-
based
diagnostic provides the benefits of an easier and faster diagnostic than an
evaluation of the
semen under a microscope or than obtaining a sperm count at a hospital or a
clinic.
In a particular embodiment, the kit comprises a dip stick, or a strip
comprising (e.g. coated or
impregnated with) one or more antibodies (e.g. labeled or non-labeled
antibodies) specific
for CRISPI. For instance, in one particular embodiment, a user would contact a
semen
sample or ejaculate with the dip stick or strip and a positive signal (e.g. a
coloration) would
appear if CRISPI is detected. The dip stick or strip could also comprise
control antibodies
for detecting control proteins (e.g. seminal plasma proteins).
The kits of the invention may also be particularly useful for diagnostic
applications in
humans according to the evaluation methods described hereinbefore. More
particularly, the
kits disclosed herein may be helpful for laboratory and diagnostic purposes in
humans
during infertility diagnostic procedures (e.g. for discriminating between
obstructive and non-
obstructive azoospermia). The kits and methods of the invention may be used in
combination with previously described assay kits.
Those skilled in the art will recognize, or be able to ascertain using no more
than routine
experimentation, numerous equivalents to the specific procedures, embodiments,
claims,
and examples described herein. Such equivalents are considered to be within
the scope of
the invention and covered by the claims appended hereto. The invention is
further illustrated
by the following example, which should not be construed as further limiting.
EXAMPLE
EXAMPLE 1: CRISPI expression in context to vasectomy surgical reversal
MATERIAL AND METHODS
Ethic consent
These studies were conducted with the approval of the ethic committee for
research on
human subjects of the Universite Laval.
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Biological material
Human epididymides were obtained from a local organ transplantation program.
After
obtaining family consent, testicles were removed while artificial circulation
was maintained to
preserve tissues assigned for transplantation. Donors were 26-50 years of age
with no
medical pathologies that could affect reproductive function, except vasectomy.
Organs were
brought to the lab on ice and processed within 3 hours after orchidectomy.
Vasectomy was
determined while dissecting the scrotal segment of the vas deferens.
Epididymal tissues
were dissected in caput, corpus and cauda and minced in small tissue pieces.
Tissues were
immediately snap frozen in liquid nitrogen for subsequent RNA extraction or
preparation of
protein extracts. For in situ hybridization, pieces of tissue were fixed in
freshly prepared 4%
paraformaldehyde in phosphate buffered saline (PBS), embedded in OCT and kept
at -80 C
until used. Other pieces of tissue were fixed by immersion in 4%
paraformaldehyde solution,
dehydrated and embedded in paraffin for immunohistological localization of
CRISP1.
Other tissues of the reproductive tract were used for Western blot
determination of CRISPI
expression. Testicular tissues were obtained when epididymides were dissected
from organ
donors. Segments of the vasa deferentia were obtained from healthy patients of
proven
fertility who were undergoing vasectomy or vasovasostomy. Prostate and seminal
vesicle
tissues were obtained from patients who were undergoing prostatectomy. This
surgical
procedure was performed by laparoscopy under general anesthesia.
Semen samples were obtained by masturbation from healthy control donors or
from
vasovasostomized men undergoing post-surgical spermogram follow-up performed
at the
clinical andrology laboratory of our institution. The time period elapsed
between vasectomy
and vasovasostomy varied from 3 to 9 years. Azoospermic and
oligoasthenospermic
samples from vasovasostomized men were not included in this study. After
liquefaction,
spermatozoa were pelleted and washed twice by centrifugation at 800g in
Dulbecco's
phosphate-buffered saline. Seminal plasma and sperm pellets were frozen at -80
C until
used.
In situ hybridization
In situ hybridization and tissue section preparations were performed as
previously described
(Legare et at., J Androl 2004;25:30-43). Briefly, CrispI complementary DNA
(cDNA) was
generated by RT-PCR using polyA RNA from normal human epididymis. The
oligonucleotides used as primers were 5'-GAA-GCC-TGC-CCA-AGT-AAC-TG-3' (SEQ ID
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NO:2) and 5'-GGG-AGT-TAA-GGT-CTC-CAG-CA-3' (SEQ ID NO:3) and the PCR product
was subcloned into pGEM-T (Promega, Madison, Wis, USA). The plasmid was
digested and
the mRNA was transcribed using SP6 and T7 RNA polymerase (Roche) in the
presence of
Digoxigenin-1 1-uridin-triphosphate (DIG)-UTP.
Epididymis cryosections were fixed with 4% paraformaldehyde for 5 min,
incubated for 10
min in 95% ethanol/5% acetic acid at -20 C, and rehydrated. Target mRNAs were
unmasked by enzymatic digestion with 10 g/mL proteinase K in PBS for 10 min
at 37 C.
Sections were incubated in 0.2% glycine, post-fixed for 5 min with 4%
paraformaldehyde,
acetylated with 0.25% acetic anhydride, OA M triethanolamine, pH 8 for 10 min,
and washed
in PBS.
Tissue sections were prehybridized for 2 hours at 42 C with 250 g/mL of
salmon sperm
DNA preheated in a hybridization buffer and incubated overnight at 42 C, under
cover slips,
with 25 L of 5 gg/ml heat-denatured antisense of sense cRNA probed with DIG.
Nonspecific staining was blocked by incubation for 1 hour with 5% heat-
inactivated sheep
serum in Tris buffer. Hybridization reactions were detected with an alkaline
phosphatase-
conjugated anti-DIG antibody diluted 1:1000 in a blocking buffer. The
hybridization signal
was visualized after incubation with the phosphatase substrate, nitroblue
tetrazolium
chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (GIBCO-BRL,
Gaithersburg, Mo, USA). Epididymis sections from normal and vasectomized men
were
processed in parallel to allow comparison.
Protein preparation
Spermatozoa
Sperm pellets (107) were resuspended in SDS-PAGE sample buffer (Laemmli UK.
Nature
1970;227:680-5) prepared without reducing reagent. After 10 min, spermatozoa
were
pelletted by centrifugation, the protein supernatant recovered and reduced by
addition of 2%
13-mercaptoethanol followed by 5 min heating in a boiling water bath.
Seminal plasma
Seminal plasma samples were centrifuged two times at 3000g for 10 min to
eliminate
spermatozoa and other cellular constituents. Supernatants were precipated with
MEOH/CHCI3, proteins were resuspended in sample buffer and submitted to SDS-
PAGE
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(Laemmli., 1970). Protein concentrations were determined by amido black
staining of dot
blots (Chapdelaine et al., 2001, Biotechniques 31: 478-480).
Tissues
Tissues were homogenized with a PolytronTM (InterSciences, Markham, ON) in a
homogenization buffer (0.01M Tris, 0.1mM EDTA, 1%SDS, and 1mM PMSF). Extracts
were
then centrifuged at 3000g for 15min at 4 C. Supernatants were precipitated
with
MEOH/CHCI3, proteins were resuspended in sample buffer and submitted to SDS-
PAGE
(Laemmli, 1970). Protein concentrations were determined by amido black
staining of dot
blots (Chapdelaine et al., 2001).
Antibodies
Goat polyclonal antibody against CRISPI was purchased from Santa Cruz
Biotechnologies
Inc. (Santa Cruz, CA, USA) and used at 1 g/ml for Western blot analysis, 5
g/ml for
immunohistochemistry on epididymis, and 15 g/ml on sperm smears. Rabbit
polyclonal
antibody against prostatic phosphatase acid (PAP) purified from human seminal
plasma was
used at 1/1000 (vol/vol). This antiserum was a generous gift of Dr RR Tremblay
and was
used as a marker of prostatic protein secretions. Mouse monoclonal antibody
against a-
tubulin (Sigma, ON, Canada) was used at 1/50000 (vol/vol). Mouse monoclonal
antibody
against Actin-0 (Sigma, ON, Canada) was used at 1/20 000 (v/v). Monoclonal
anti-
phosphotyrosine antibody (clone 4G10, Upstate Biotechnology) was used at
1/10000.
Rabbit anti-goat IgG, goat anti-rabbit and a goat anti-mouse conjugated to
horseradish
peroxidase were purchased from BIO/CAN Scientific (Mississauga, ON, Canada)
and used
respectively at 1/5000 (vol/vol). A biotinylated rabbit-anti-goat secondary
antibody was
obtained from Dako Diagnostics (Mississauga, ON, Canada) and used at 1/400
(vol/vol).
Immunohistochemical staining
Paraffin sections were prepared from fixed epididymal tissues. Ejaculated
spermatozoa from
normal and vasovasostomized men were washed, smear on microscopic slides,
fixed with
cold ethanol, and processed for CRISPI localization. Endogenous peroxidase
activity was
quenched with 3% H202 (v/v) in PBS for 30 min. Non-specific binding sites were
then
blocked with 10% goat serum in PBS for 1 h. The CRISPI-specific antibodies
were diluted in
PBS and applied overnight at 4 C. In control sections, the primary antibodies
were replaced
by the corresponding non-specific IgG and processed in parallel. Sections were
subsequently incubated with biotinylated rabbit anti-goat antibody for 30 min,
and with
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ABCTM reagent for 30 min. Immunostaining was revealed using 3-amino-9-
ethylcarbazole
(AEC). Harris hematoxylin was used for counterstaining, and mounted under
cover slip using
an aqueous mounting medium (Sigma). Slides were observed under a Zeiss
Axioskop2
PIUSTM microscope (Toronto, ON, Canada) linked to a digital camera from
Diagnostics
Instruments (Sterling Heights, MI). Images were captured using the SpotT""
software
(Diagnostics Instruments).
Western blotting
Proteins were separated on a 12% SDS-polyacrylamide gels and transferred to
nitrocellulose membranes (Towbin et al., Proc Nati Acad Sci USA 1979;76:4350-
4.). After
saturation with 5% milk in PBS-0.1% Tween-20TH, membranes were incubated with
an anti-
CRISP1, an anti-PAP, an anti-tubulin or anti-phosphotyrosine. Rabbit anti-
goat, goat anti-
rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase was used
for
chemiluminescent detection of proteins (ECLTM reagent: Amersham, Baie d'Urfe,
QC,
Canada). CRISPI, PAP, a-tubulin and phosphotyrosine-containing proteins
signals were
quantitated by densitometry in the linear range of film exposure and expressed
as arbitrary
units.
Statistical analysis
Statistical analysis was performed by analysis of variance using Super ANOVATM
software
(ABACUS Concepts, Berkeley, CA). Results were compared by Student-Newman-Keuls
test. Differences were considered to be significant at P <0.05.
RESULTS
Cellular localization of CRISPI mRNA and protein along the epididymis from
normal
and vasectomized men
The epididymal origin of CRISP1 was investigated by immunohistochemistry.
CRISP1 was
undetectable in the caput epididymidis, and an increasing amount of this
protein was
revealed in the corpus and cauda of normal tissues. As already published
(Legare et at.,
Endocrinology 1999;140:3318-27), vasectomy has major consequences on the
height of the
epididymal epithelium and the diameter of the lumen (Figure 1). As deduced
from Western
blots, a huge amount of CRISPI was accumulating in the lumen of cauda
epididymidis of
vasectomized men. In both normal and vasectomized tissues, CRISP1 was
undetectable in
the interstitial tissues of the epididymis.
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In situ hybridization localization of CrispI mRNA was in agreement with the
immunohistological localization of the translational product (Figure 2). In
both normal and
vasectomized tissues, the CrispI mRNA was expressed by the epithelium in the
corpus and
cauda segments. mRNA was undetectable in the caput. The staining was
restricted to the
epithelium as no staining was detected in interstitial tissues.
Effect of vasectomy on CRISPI associated with spermatozoa
Western blot analyses were performed on protein extracted from a constant
number of
spermatozoa from different men. CRISPI was detected at -32kDa. The
commercially
available antibody was specific to CRISP1 as shown by the absence of
immunodetectable
band revealed when Western blots were probed with a control serum (data not
shown).
When expressed as a ratio of a constitutive protein TUBA, the quantity of
CRISPI
associated with a constant number of ejaculated spermatozoa was significantly
higher in
vasovasostomized men when compared to normal donors (Figure 3) with a P>0.05.
CRISPI was associated with the acrosomal region of ejaculated spermatozoa from
both
normal and vasovasostomized men. The immune detection signal varied
significantly from
one sperm cell to the other in both normal and vasovasostomy samples. By
opposition to
Western blot analyses; it was not possible to quantitate the amount of CRISP1
immunodetected on smears of spermatozoa (Figure 4). The preimmune serum did
not stain
the acrosomal region but the equatorial segment was unspecifically stained for
an unknown
reason (Figure 4).
Vasectomy effect on CRISPI in seminal plasma
CRISPI was also found as a soluble form in the seminal plasma. When Western
blots were
performed on 20 g of seminal plasma proteins, CRISPI was detectable in semen
samples
of both normal and vasovasostomized men. As for CRISPI associated to
spermatozoa,
CRISPI was significantly (P>0.05) more concentrated in seminal plasma of
vasovasostomized men when compared to samples from normal men. Interestingly,
CRISP1
was undetectable in seminal plasma of vasectomized men (Figure 5). This
correlated well
with the fact that CRISPI was undetectable on Western blots of 20 g of
protein extracted
from human seminal vesicles and prostate tissues (Figure 6).
Conclusions
As shown herein, in human semen, CRISPI is found both associated to sperm and
in the
soluble fraction. Both forms are in higher concentrations in vasovasostomized
men if the vas
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CA 02693546 2010-02-18
Our ref.: 273859.90
deferens were repermeabilized. CRISPI remains localized to the acrosomal
region of
spermatozoa in both normal and vasovasostomized. This suggests that the
association of
this epididymal protein to spermatozoa is not a simple coating phenomenon and
that the
specificity of the interaction remains even though CRISP1 is secreted in
excess in some
vasovasostomized men.
The present study demonstrates for the first time that CRISPI is not
detectable in
vasectomized men, that CRISPI expression is restricted to the epididymis only
and that it is
not synthesized by accessory glands of the male reproductive. The presence of
CRISPI can
thus be considered as a good marker of excurrent duct permeability and can be
used in the
clinic to discriminate between secretory and obstructive azoospermy.
Headings are included herein for reference purposes and to aid in locating
certain sections
These headings are not intended to limit the scope of the concepts described
thereinunder,
and these concepts may have applicability in other sections throughout the
entire
specification. Thus, the present invention is not intended to be limited to
the embodiments
shown herein but is to be accorded the widest scope consistent with the
principles and novel
features disclosed herein.
It is understood that the examples and embodiments described herein are for
illustrative
purposes only and that various modifications or changes in light thereof will
be suggested to
persons skilled in the art and are to be included within the present invention
and scope of
the appended claims.
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