doi:10.1093/humrep/dem385
Human Reproduction Vol.23, No.3 pp. 573–580, 2008
Advance Access publication on December 18, 2007
The role of sperm proteasomes during sperm aster formation
and early zygote development: implications for fertilization
failure in humans
Vanesa Y. Rawe1, Emilce S. Dı́az2, Roger Abdelmassih3, Cezary Wójcik4, Patricio Morales2,
Peter Sutovsky5 and Héctor E. Chemes6,7
1
7
Correspondence address. E-mail: [email protected]
BACKGROUND: Sperm aster organization during bovine and human fertilization requires a paternally-derived
centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the
26S proteasome in this process. METHODS: Proteasome localization and enzymatic activity were studied in normal
and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes
during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with
anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes
discarded after ICSI failures (n 5 28) were also examined. RESULTS: Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities
were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were
found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as
well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS: Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a
functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal
proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.
Keywords: proteasomes; sperm aster; early zygote; fertilization; sperm pathology
Introduction
Paternally contributed centrosomal components play a crucial
role in zygotic development of non-rodent mammals. In
bovine and human zygotes, the microtubular network responsible for syngamy and mitotic spindle formation requires
microtubule nucleation by the centrosome, a structure derived
from the sperm centriole and oocyte pericentriolar material
(Sutovsky and Schatten, 2000). The mechanism by which the
complex structure of the sperm tail connecting piece disintegrates to release the proximal centriole after sperm entry into
the oocyte is not fully understood, but sperm proteasomes
have been implicated (Sutovsky et al., 2004; Rawe, 2005).
The ubiquitin-proteasome pathway is the main cytosolic proteolytic system responsible for the regulated substrate specific
degradation of most cellular proteins in eukaryotic cells
(Glickman and Ciechanover, 2002). It takes part in the important events of fertilization in both invertebrates and vertebrates,
including mammals. Fertilization is inhibited by anti-ubiquitin
and anti-proteasome antibodies or by small molecule, peptidebased inhibitors of specific proteasome proteolytic activities
(Matsumura et al., 1991; Sawada et al., 2002a,b; Wang et al.,
2002; Sutovsky et al., 2003).
The present study explores the participation of sperm-borne
proteasomes during early development of bovine and human
zygotes. Experimental evidence is provided for their role during
the post-fertilization disassembly of the sperm tail connecting
piece, sperm aster formation, pronuclear development and apposition. Proteasomes have been localized by immunofluorescence
in human and bovine spermatozoa, and their specific enzymatic
# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
All rights reserved. For Permissions, please email: [email protected]
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Centro de Estudios en Ginecologı́a y Reproducción (CEGyR), Buenos Aires, Argentina; 2Faculty of Health Sciences, Department
of Biomedicine, University of Antofagasta, Antofagasta, Chile; 3Clı́nica e Centro de Pesquisa em Reprodução Humana Roger
Abdelmassih, Sao Paulo, Brazil; 4Department of Anatomy and Cell Biology, Indiana University School of Medicine, Evansville,
IN, USA; 5Division of Animal Science, Department of Obstetrics and Gynecology, University of Missouri, Columbia, MO, USA;
6
Laboratory of Testicular Physiology and Pathology, Center for Research in Endocrinology, National Research Council (CONICET),
Endocrinology Division, Buenos Aires Children’s Hospital, Buenos Aires, Argentina
Rawe et al.
activities were measured in sperm samples from men suffering
from centriolar deficiencies and abnormal head–tail attachment
(Chemes et al., 1999; Rawe et al., 2002). Here, functional blockage of sperm or zygote proteasomes was induced by specific antibodies or pharmacological proteasome-inhibitor MG132.
Finally, proteasome localization, pronuclear development and
sperm aster formation rates were examined in abnormal human
zygotes displaying post-ICSI fertilization failure.
Materials and Methods
Immunocytochemistry of ejaculated bull and human spermatozoa
Frozen bull semen (Juan de Bernardi Co., Buenos Aires, Argentina)
was thawed, layered over a 45290% Percoll gradient and centrifuged
at 300g for 15 min to isolate live sperm fraction. Cryopreserved fertile
human semen samples had been donated by consenting adult men at
Centro de Estudios en Ginecologı́a y Reproducción, washed with
phosphate buffer (0.1 M, pH 7.4), pelleted and resuspended in
buffer within 30 min of ejaculation. Human and bull spermatozoa
were sedimented on poly-L-lysine-coated coverslips and fixed for
1 h in 2% formaldehyde in phosphate-buffered saline (PBS). Following fixation, the samples were permeabilized for 60 min in PBS containing 1% Triton X-100, and non-specific reactions were blocked
by further incubation in PBS containing 2 mg/ml bovine serum
albumin (BSA) and 100 mM glycine. The primary antibodies used
to localize proteasomes in ejaculated spermatozoa were E446,
anti-20S proteasome a/b subunit antibody, LMP2, MECL-1 and
anti-p31 antibody at appropriate dilutions. Various anti-proteasomal
antibodies were tested in spermatozoa, eggs and zygotes because
free 20S and complete 26S proteasomes as well as other proteasomal
forms are ubiquitous and exist in different proportions according to
cell type, cell cycle phase and metabolic conditions (Russell et al.,
1999; Brooks et al., 2000; Tanahashi et al., 2000). In each case, the
antibody best suited for immunohistochemical localization was
selected. After incubation with the first antibody, the samples were
extensively washed in PBS containing 0.1% Triton X-100, and
sequentially labeled with Alexa Fluor 488 or Alexa Fluor
574
Transmission electron microscopy
Semen samples were obtained by masturbation and studied when
liquefaction was complete, approximately at 30 min after ejaculation,
according to methods previously reported (Chemes et al., 1999).
Briefly, the samples were diluted (1:3) with phosphate buffer
(0.1 M, pH 7.4) and spermatozoa were separated by centrifugation
at 1000g for 10 min. The pellets were fixed for 2–4 h with 3% glutaraldehyde in the same buffer, post-fixed for 2 h in 1.3% Osmium Tetroxide and embedded in Epon-Araldite. The blocks were cut in a RMC
MT-7000 automatic ultramicrotome (RMC Inc. Tucson, AZ, USA).
Thick 1 mm sections were cut with glass knives and thin sections
(70–80 nm) displaying pale golden interference colors were obtained
using diamond knives. Thin sections were double stained with uranyl
acetate and lead citrate and studied on a Zeiss EM 109 electron microscope (Zeiss Obercochen, Germany).
Biochemical determination of sperm proteasome
enzymatic activities
Semen samples were donated by two consenting teratozoospermic men
suffering from sperm centriolar deficiencies and abnormalities in the
development of the sperm head–tail attachment (Chemes et al.,
1999; Rawe et al., 2002). Semen samples from six fertile men were
used as controls. The samples were kept frozen and used to prepare
sperm extracts to study sperm proteasomal enzymatic activities. The
samples were thawed and diluted with 5 ml of modified Tyrode’s
medium consisting of 117.5 mM NaCl, 0.3 mM NaH2PO4, 8.6 mM
KCl, 25 mM NaHCO3, 2.5 mM CaCl2, 0.5 mM MgCl2, 2 mM
glucose, 0.25 mM Na pyruvate, 19 mM Na lactate, 70 mg/ml of both
streptomycin and penicillin, phenol red and 0.3% BSA (Sigma,
A7030), centrifuged at 300g for 10 min and then resuspended in homogenization buffer (50 mM Hepes, 10% glycerol, pH 7.4), supplemented with 100 mM Na-p-Tosyl-L-Lysine-Chloromethyl-Ketone
(TLCK, an acrosin inhibitor) at a concentration of 25 107 sperm/
ml. The sperm suspension was then sonicated (Virsonic. Gardiner,
NY, USA) with six 60 watt bursts for 20 sec each, followed by centrifugation for 30 sec at 5000g in a Beckman microfuge to remove nuclear
and flagellar material. The supernatant was used as the enzyme stock
preparation. All these procedures were performed at 48C. The
protein concentration in sperm extracts, obtained using the Bradford
method (Bradford, 1976), ranged between 0.3 and 0.8 mg/ml.
The chymotrypsin-like (ChTL) and peptidylglutamylpeptide hydrolyzing (PGPH) activity of the proteasome were assayed in duplicate
using the fluorogenic substrates N-Succinyl-Leu-Leu-Val-Tyr-7-amido4-methylcoumarin (Suc-LLVY-AMC, from Biomol International,
PA, USA), and Carbobenzoxy-Leu-Leu-Leu-AMC (Z-LLL-AMC,
from Peptides International, Louisville, KY, USA).
Aliquots of 50 ml of enzyme extract were incubated in a final
volume of 1 ml containing 10 mM CaCl2, 50 mM Hepes, pH 7.4,
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Chemicals and antibodies
All chemicals were obtained from Sigma Chemical Co (St Louis, MO,
USA), unless otherwise stated. Free 20S and complete 26S proteasomes as well as other proteasomal forms are ubiquitous and exist
in different proportions according to cell type, cell cycle phase and
metabolic conditions. Because of this heterogeneity various antibodies
were tested and those best suited for proteasome localization in a particular cell type (spermatozoa, oocytes, zygotes) were selected in pilot
experiments. Rabbit polyclonal antibodies raised against multiple 20S
proteasomal core subunits of a and b-type (anti-a/b), anti-LMP2
(inducible 20S subunit PSMB9/b-1i) and anti-MECL-1 (inducible
20S core subunit PSMB10/b-2i) were obtained from Biomol International, PA, USA, and used to label proteasomes in bovine and
human oocytes and zygotes. To investigate the presence of proteasomes in bull and human spermatozoa, rabbit polyclonal antibody
p31 against one of the subunits of PA700 was kindly donated by
Dr George DeMartino (The Southwestern Medical Center at Dallas).
For antibody-transfection experiments, a sodium azide-free rabbit
antibody against purified 20S proteasomal core was used (E446,
Wigley et al., 1999, donated by Dr DeMartino). Affinity purified
sheep anti-tubulin antibody was obtained from Cytoskeleton, Inc.
(Denver, CO, USA). Nuclear pore complexes (NPCs) of bovine
zygotes were labeled with mouse monoclonal antibody MAb411
(Covance Research Products Inc., Richmond, CA, USA).
594-conjugated secondary antibodies (Molecular Probes, Eugene,
OR, USA) for 1 h and the DNA stained with 4’,6-diamino2-phenylindole (DAPI, Molecular Probes). Coverslips were mounted
in VectaShield mounting medium (Vector Labs, Burlingame, CA,
USA) and sealed with nail polish. Samples were examined with an
Olympus BX40 epifluorescence-equipped microscope operated with
a DS-5Mc Nikon camera and ACT-2U software, or with a Nikon
Eclipse 800 microscope equipped with CoolSnap CCD camera and
MetaMorph software. Negative controls were performed by omitting
the first antibody or by incubation with a non-immune rabbit serum
followed by a fluorescently conjugated anti-rabbit immunoglobulin
(Ig) G. Image acquisition times were similar in control and labeled
samples. All experiments were repeated at least three times.
Proteasomes in early zygotic development
and 5 mM substrate. The assay was performed at 378C and the fluorescence was monitored with excitation at 380 nm and emission at
460 nm in a Shimadzu 1501 (Kyoto, Japan) spectrofluorometer.
IVF of bovine oocytes, antibody transfection of zygotes
and pharmacological inhibition of proteasomes
Bovine oocytes obtained from a slaughterhouse were matured and fertilized in vitro according to standard protocols (Sirard et al., 1988).
Mature bovine oocytes were placed into drops of Tyrode’s
albumin-lactate-pyruvate (TALP) culture medium under mineral oil
and inseminated with live bull spermatozoa at 398C under 5% CO2
until the desired stages in zygote development. In some experiments,
bull sperm mitochondria were prelabeled with the mitochondrial
probe MitoTracker Green FM (400 nM) to identify the male pronucleus by its association with the sperm tail mitochondria.
Antibody transfection was carried out at 8 h post-insemination,
TM
around the time of sperm aster formation, using the Chariot reagent
(Active Motif) according to Payne et al. (2003) and to manufacturer’s
recommendations. To conduct transfections in live zygotes, the
sodium-azide-free E446 antibody was used. Chariot–antibody binding
was accomplished by incubating a 20 ml volume mix containing a
TM
1:10 dilution of Chariot reagent and 1:5 dilution of pre-immune IgG
or 1:2 dilution of E446 antibody. For additional control experiments,
Chariot reagent and antibody E446 were also used alone. Following
this binding step, each 20 ml volume was added to one well of a
96-well plate containing 20 zonae- and cumulus-free zygotes suspended
in 80 ml of serum-free TALP medium. The samples were incubated at
398C for 1 h, after which an additional 100 ml of serum-containing
TALP medium was added to the well. Zygotes were cultured and
fixed for immunocytochemistry 10 h later (18 h post-insemination).
Pharmacological inhibition of proteasomal activities during fertilization was achieved by using MG132, a specific and reversible inhibitor of proteasomal proteolytic activity (Biomol International, PA,
USA). IVF was performed as described earlier. Specific proteasomal
inhibitor MG132 (10 mM) was added either to oocytes at the time of
insemination or by pretreating oocytes or spermatozoa with MG132
before insemination. Control IVF was performed without the inhibitor,
by using vehicle solution (100% ethanol) at concentration identical to
vehicle concentration in MG132-treated cells. Samples were further
cultured until 20– 24 h post-insemination, at which time they were
fixed and analyzed by immunofluorescence.
For immunocytochemistry, bovine cumulus cells and zonae pellucidae were removed from oocytes and zygotes by short incubations with
1 mg/ml hyaluronidase and 2 mg/ml pronase, respectively. Zona-free
bovine oocytes, zygotes and embryos were then gently pipetted onto
poly-L-lysine-coated coverslips in Ca2þ-free TALP medium, fixed
for 40 min in 2% formaldehyde and permeabilized in 10 mM
ICSI of human oocytes: injection with p31 antibody-loaded
human spermatozoa and spontaneous fertilization failures
A total of 12 metaphase II (MII) human oocytes, donated for this
research by consenting patients, were injected with SLO-permeabilized spermatozoa. Eight of them received SLOþp31 antibody-treated
spermatozoa (see above). The other four oocytes were used as controls
and injected with human spermatozoa permeabilized with SLO but
without antibodies. All injected human oocytes were fixed at 16 –
18 h after ICSI, processed by immunocytochemistry and studied by
epifluorescence or confocal microscopy as described earlier.
Twenty-eight discarded human oocytes (no pronuclei visualized
18– 20 h after sperm injection) were also studied. These zygotes
with post-ICSI fertilization failure were fixed, processed for immunocytochemistry and studied by confocal microscopy.
All supernumerary human oocytes were donated for research with
informed written consent by couples undergoing ICSI at the Clinic of
Human Reproduction Roger Abdelmassih (Sao Paulo, Brazil). No part
of work with human gametes or zygotes was performed at the University
of Missouri-Columbia, USA. Normal and abnormal human zygotes that
failed to cleave within 40 h after ICSI and supernumerary oocytes not
used for infertility treatment or cryopreservation were donated for
research by consenting, adult female donors (less than 35 years old)
with medical indication of ICSI because of male factor. All volunteer
donors remained anonymous to researchers involved in this project.
None of the presumed normal ova donated for this project were
allowed to cleave in vitro or be transferred to recipients after ICSI.
These ova were going to be discarded if not donated for research. All
animal and human procedures were approved by CEGYR and Abdelmassih Internal Review Board and Ethic Committee accordingly.
Statistical analysis
Each experiment was repeated at least three times. To compare percentages of fertility between control and treated groups, contingency
tables were constructed and analyzed by means of Chi-squared test.
Odds ratios were calculated and 95% confidence intervals were
obtained. Software used was InfoStat (Universidad Nacional de
Córdoba, Argentina). In all cases, less than 0.01error probability
was considered highly significant. For each experiment, representative
images are shown for oocytes, zygotes and embryos.
Results
Acrosomal and flagellar localization of proteasomes
in bull and human spermatozoa
Figure 1 depicts mature bull (Fig. 1A) and human spermatozoa (Fig. 1B) labeled with p31 anti-proteasome antibody. The
labeling reveals that each spermatozoon is labeled at two discrete
locations: there is a larger labeling in the acrosome and a smaller
punctuate pattern overlying the sperm proximal centriole and
the flagellar connecting piece. In electron micrographs, the
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Streptolysin O-permeabilization and sperm transfection
with anti-proteasome antibody
After swim-up, sperm concentration was adjusted to 5–10 106/ml.
Permeabilization was accomplished as described by Yunes et al.
(2000). Briefly, washed spermatozoa were resuspended in PBS containing 0.4 unit/ml of Streptolysin O (SLO) for 15 min at 488C. Cells were
washed once with PBS and resuspended in human tubal fluid (Irvine
Scientific, Santa Ana, CA, USA) supplemented with 15% of synthetic
serum supplement (Irvine Scientific, Santa Ana, CA, USA). SLOpermeabilized sperm were preloaded with p31 anti-proteasome antibodies for at least 1 h at 378C, washed and used for ICSI. Two aliquots
of the SLO-permeabilized antibody loaded spermatozoa were analyzed
to assess antibody penetration (by immunocytochemistry) and viability
(by the hyposmotic swelling test, Jeyendran et al., 1984).
PBSþ0.1% Triton X-100 for an additional 40 min. After fixation
and permeabilization, oocytes and zygotes were processed for
immunocytochemistry with anti-proteasome antibodies and imaged
using an Olympus spectral confocal microscope, with laser lines at
488, 568, 594 and 633 nm wavelengths, or by using a conventional epifluorescence Nikon Eclipse 800 microscope, equipped with CoolSnap
CCD camera operated by MetaMorph software. Negative controls
included preimmune sera or anti-MECL-1 antibody immunosaturated
with a synthetic MECL-1 peptide. Nuclear envelopes were labeled
with anti-nucleoporin antibody MAb414 (Covance).
Rawe et al.
connecting piece appears as a dense structure formed by the capitulum and the striated columns that enclose the sperm proximal
centriole, a cylindrical organelle formed by nine microtubular triplets deeply embedded in the center of the dense connecting piece
(Fig. 1C). In head–tail junction-defective human spermatozoa
(Chemes et al., 1999) acephalic forms exist in which the proximal
centriole and proximal centriolar adjunct display normal architecture and are also embedded in the center of the dense structures of
the connecting piece (Fig. 1C’).
Decreased enzymatic proteasomal activity in head – tail
junction-defective human spermatozoa
Specific proteasomal enzymatic activities were quantified in
six fertile (control) men and two infertile patients with centriolar anomalies and defects of the head – tail attachment. The
semen of these men displayed a mixture of acephalic spermatozoa and forms in which heads and tails were not aligned
along the same axis. In both patients, contamination with
round spermatogenic or somatic cells was negligible. Spermatozoa from these two patients exhibited a marked decrease in
both ChTL and PGPH activities of the proteasome below the
20% and 40% values of control samples (Fig. 2).
Localization of proteasomes in the pronuclei
of bovine zygotes
Specific immunolocalization of proteasomal subunits LMP2,
MECL1 and other a-type and b-type proteasomal subunits
was performed in bovine pronuclear zygotes at various stages
of pronuclear development. Early after sperm incorporation
into ooplasm, the fully condensed sperm nucleus was free of
detectable proteasomes (Fig. 3A). Proteasomal subunits were
first detected in the male and female pronuclei at the initial
stage of sperm nuclear decondensation. Proteasomal labeling
was present within both pronuclei prior to pronuclear apposition
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Figure 2: Proteasome activity of head–tail attachment defective
human spermatozoa
Extracts of human sperm were incubated with synthetic substrates for
ChTL and PGPH activity. Control sperm were obtained from six
normal donors. Specific activities are expressed per protein content
and as percentages of control values considered as 100%
and reached highest density inside the apposed, full-sized pronuclei (Fig. 3B and C). No nuclear/chromosomal labeling
with anti-proteasomal antibodies was observed in the ova that
remained unfertilized 20 h after insemination (not shown).
Negative control using anti-MECL1 antibody saturated with
synthetic MECL1 peptide did not show any signal (Fig. 3D).
Pharmacological inhibition of bovine zygotic
proteasomes using MG132
The role of zygote proteasomes in early post-fertilization events
was explored in bovine oocytes subjected to IVF using bull
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Figure 1: Ejaculated bull and human spermatozoa labeled with p31 antibody
(A and B) Overview of bull and human spermatozoa, respectively. Two populations of proteasomes can be observed on the ejaculated spermatozoa: in the acrosomal region (arrows) and in the sperm tail connecting piece (arrowheads). Unlike bull sperm, non-acrosomal human sperm
proteasomes seem to be more prominent in the connecting piece and on the mitochondrial sheath. (C) Normal configuration of the connecting
piece in a patient with defective sperm head –tail attachment. The tail is lodged in the concave implantation fossa (arrowheads). The microtubuletriplets of the proximal centriole (arrow) are surrounded by the striated columns (SC) of the connecting piece. (C’) Connecting piece of an acephalic spermatozoon. Note the lack of sperm head (arrowheads) and typical organization of the centriolar adjunct (double asterisk) fully embedded
in the dense striated columns (SC) of the connecting piece. Scale bars in A and B represent 5 mm, in C 0.15 mm and in C’ 0.13 mm
Proteasomes in early zygotic development
spermatozoa and treated with 10 mM MG132 (a specific proteasome inhibitor) after fertilization. The average fertilization rate
in ethanol-treated control zygotes was 75.7%. Incubation with
10 mM MG132 significantly reduced fertilization down to
38.2% (P , 0.0001, Table I). Pre-incubation of ova with
100 mM MG132 prior to fertilization had no effect on the
import of proteasomal subunits into pronuclei (not shown).
Pronuclei inside the fertilized ova were visualized by the
monoclonal antibody mAb 414 recognizing a group of related
NPC proteins. Nucleoporin labeling was chosen based on our
previous observation that it is an informative marker of nuclear
envelope integrity and functionality throughout all stages of pronuclear/zygotic development (Sutovsky et al., 1998).
The patterns of pronuclear development in the ova that were
fertilized in the presence of 10 mM MG132 were abnormal.
Table I. Percentages of fertilized bovine oocytes in control conditions and
under treatment with MG132 or Chariot-antibody complex.
Type of treatment
Control
Treated
Significance
MG132
Chariot-antibody complex
75.7 (n ¼ 87)
67.2 (n ¼ 299)
38.2 (n ¼ 107)
21.6 (n ¼ 315)
P , 0.0001
P , 0.0001
Differences in percentages were analyzed by the Chi-squared test. n, total
number of oocytes in each experimental condition.
Failed pronuclear formation due to premature chromosome
condensation (PCC) was the most common abnormality
(Fig. 3E and F, respectively). Partial scattering of chromosomes in PCC and the assembly of nucleoporins around the
condensed chromatin were observed. Such a pool of nucleoporins was likely not derived from a paternal contribution because
bull and human spermatozoa have only a residual amount of
nucleoporins located in the redundant nuclear envelopes of
the sperm tail connecting piece (Fig. 3G).
Immunological inhibition of bovine zygotic proteasomes by
Chariot-mediated antibody transfection
To explore the possible role of proteasomes in the late fertilization events leading to sperm aster formation, we transfected
newly fertilized bovine oocytes with E446 anti-proteasome
antibodies coupled to the Chariot reagent. Antibodies were
introduced into bovine zygotes at 8 h post-insemination, at
the time pronuclei begin to form in most IVF zygotes. These
were allowed to develop until 20 h after insemination when
full pronuclear formation is achieved under control conditions.
Co-localization of proteasomes (green fluorescence), tubulin
(red fluorescence) and DNA (TOTO-3; blue fluorescence)
was verified by immunocytochemistry.
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Figure 3: Proteasome localization during normal development of bovine zygotes and after inhibition by MG132. Localization of nucleoporins in
sperm.
A, B and C: immunolocalization of proteasomal subunits LMP2 in bovine pronuclear zygotes at various stages of zygotic development (red fluorescence). Early after incorporation into ooplasm, sperm nuclei were free of detectable proteasomes (A). Proteasomal subunits infiltrated male
pronucleus at the initial stage of sperm nuclear decondensation. Both the male and the female pronuclei became colonized by proteasomes as they
reached full size (B) and apposition. High density of proteasomes was observed inside the apposed, full size pronuclei (C) (MP, male pronucleus;
FP, female pronucleus). Negative control using anti-MECL1 antibody saturated with synthetic MECL-1 peptide is shown in D. DNA in all panels
was counterstained with DAPI. Sperm tail is visible due to the pre-incubation of spermatozoa prior to fertilization with mitochondrial dye MitoTracker Green FM (green). Panels E and F show abnormal pronuclear development and PCC (arrow in E) in bovine ova fertilized in the presence
of 10 mM MG132, a condition partially permissive to sperm penetration. Pronuclei were visualized by the combination of monoclonal antibody
against NPC (red), and the DNA stain DAPI (blue). Defects induced by MG-132 treatment included a combination of PCC (arrow in E) and failed
apposition (F). Note the partial scattering of chromosomes in PCC (E) and the clustering of NPC proteins (arrow head in F) around the condensed
chromatin. Bull spermatozoa carry only a residual amount of nucleoporins located in the redundant nuclear envelopes in the sperm tail connecting
piece (red, G)
Rawe et al.
aster that originated close to the male pronucleus (Fig. 4A0 ).
Inset shows the intranuclear localization of proteasomes in a
control human zygote with fully formed pronuclei and sperm
aster. After transfection with antibody E446 (incubation of
zygotes with the Chariot-E446 antibody complex), only
21.6% of inseminated bovine oocytes fertilized (P , 0.0001,
Table I). In affected zygotes, pronuclei did not form or were
insufficiently developed and the pronuclear proteasome labeling was either absent or aberrant (Fig. 4B). Cytoplasmic proteasomes were heavily concentrated over the sperm tail
connecting piece in a cloud-like configuration (Fig. 4B0 and
inset). There was also incomplete polymerization of tubulin
(absence or abnormalities of sperm aster formation, Fig. 4B0 )
and failure of pronuclear apposition.
Figure 4A and A0 (control) show normal pronuclear development and apposition after using either Chariot alone or Chariot
pre-incubated with non-immune IgG (fertilization rate 67.2%).
High density of proteasomes was observed inside the apposed
pronuclei and in lesser amounts throughout the cytoplasm
(Fig. 4A). Tubulin was organized in a fully developed sperm
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Proteasome localization in the discarded human zygotes
that failed to cleave after ICSI
As described in the previous section, injection of p31 antibodyloaded human spermatozoa into human oocytes or incubation
of bovine zygotes with either Chariot-E446 antibody complexes or MG 132 resulted in post-fertilization failures. To
test whether a similar phenomenon could be traced after spontaneous ICSI-failure in humans, 28 zygotes discarded because
of
fertilization
failure
were
processed
for
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Figure 4: Inhibition of proteasomal activity by immunological neutralization during bovine IVF (ChariotTM ), SLO experiments and in
human fertilization failures after ICSI
(A) Normal pronuclear development after the incubation of bovine
zygotes with Chariot alone (control). (A0 ) Proteasomes (green) were
assembled inside male and female pronuclei. Normal sperm aster formation is seen (red, tubulin) coinciding with fully apposed pronuclei.
(B) After zygote transfection with Chariot coupled to anti-proteasome
antibody E446, oocytes show failure of sperm aster formation and lack
of pronuclear apposition. An intense green labeling of proteasomes is
observed close to the sperm tail connecting piece in the majority of
transfected zygotes (arrow in boxed area). (B0 ) Absent sperm aster formation and aberrant pronuclear development. The inset shows the
merging of B and B0 boxed areas. Note the condensed sperm
nucleus (blue) and proteasome recruitment over the sperm tail connecting piece (green). In C and D, high magnification details of the
sperm nucleus region of two zygotes can be appreciated: (C) ICSI
zygote obtained by injection of a SLO-permeabilized human spermatozoon loaded with p31 anti-proteasomal antibody. Note proteasome
accumulation (green, arrows) to one side of the sperm pronucleus
(blue). Absence of the sperm aster formation (red) and pronuclear
development/apposition. Black and white panels to the right show
proteasome accumulations (upper insets) and paternal DNA labeling
(lower insets). (D) After human ICSI failure, proteasomes (green,
arrow) accumulated near the sperm nucleus (blue). Complete lack of
sperm aster formation (red). Black and white insets showing the
area of proteasomal ‘cloud’ (upper inset) next to the sperm nucleus
(lower inset) can be discerned separately. Scale in A, A0 , B and B0
is 20 mm (represented in A). Scale bar in C and D correspond to 5 mm
Immunological inhibition of sperm proteasome functions
in SLO-permeabilized spermatozoa
The above data demonstrated that antibody transfection of the
bovine fertilized ova efficiently inhibited the function of proteasomes involved in the disassembly of the sperm tail connecting piece and formation of the zygotic sperm aster. However,
when zona pellucida-free bovine oocytes were incubated with
bull spermatozoa pretreated with the Chariot-p31 antibody
complex, normal rates of pronuclear development, apposition
and sperm aster formation were observed (data not shown).
These negative results raised the question of the lack of penetration of the antibody complex into spermatozoa. In order
to ensure antibody accessibility to the connecting piece, we
used human spermatozoa that were first permeabilized with
SLO and subsequently loaded with function-blocking p31 antibodies. After microinjection of SLOþp31 antibody-treated
spermatozoa into eight MII human oocytes, only three of
them developed two pronuclei (37.5% fertilization rate) compared with a fertilization rate of 100% in four control
oocytes (injected with sperm treated with SLO but no antibodies). Control zygotes showed a fully developed sperm
aster and proteasomes localized over the well formed pronuclei. Conversely, non-fertilized oocytes (5/8, 62.5%) completely lacked sperm asters and pronuclear development/
apposition. Proteasomes did not assemble inside the pronuclei
and were concentrated as a dense cloud around the sperm tail
connecting piece on one side of the sperm nucleus (Fig. 4C).
The effects of E446 antibodies transfected into bovine
zygotes using the chariot reagent and those of p31 antibodies
introduced into SLO-permeabilized spermatozoa previous to
ICSI suggest that both p31 and E446 interfere with normal
proteasome function and therefore can be referred to as
function-blocking antibodies.
Proteasomes in early zygotic development
immunocytochemistry. Sixteen out of 28 (57%) of the studied
fertilization failures displayed dense accumulations of proteasomes close to the non-decondensed male pronucleus (proteasomal cloud). Failure of pronuclear development/apposition
and lack of sperm aster formation were also observed in
these 16 abnormal zygotes (Fig. 4D). This pattern was identical
to the pattern we had previously documented in bovine zygotes
treated with Chariot-E446 antibody, MG132 or in human ICSI
with SLO-permeabilized p31 antibody-loaded spermatozoa.
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Discussion
Cytoskeletal organization in bovine and human zygotes is
similar as both require the participation of a paternally
derived centriole that organizes the sperm aster responsible
for progression of pronuclear apposition and syngamy. The
mechanism by which this centriole is released from the dense
structures of the tail-connecting piece has not been clarified
to date, but it has been suggested that proteasomes may be
involved in this process (Sutovsky et al., 2004; Rawe, 2005).
Proteasomes are macromolecular protease complexes found
in most eukaryotic cells where they participate in regulated,
substrate-specific proteolysis of polyubiquitinated substrates.
Proteasomes take part in multiple steps of fertilization such
as acrosome exocytosis, zona pellucida penetration, sperm
mitochondrial sheath degradation and chromatin remodeling
in the zygote (Wójcik et al., 2000; Bialy et al., 2001; Sutovsky
et al., 1996, 2003; Haraguchi et al., 2004; Berruti and Martegani, 2005). Recent reports show the presence and proteolytic
activity of proteasomes in the nuclear compartment of somatic
cells and porcine zygotes (Chen et al., 2002; Sutovsky et al.,
2004). Our present findings extend these observations to
bovine and human zygotes and demonstrate that proteasome
translocation from the cytoplasm to pronuclei is efficiently
blocked by specific proteasomal inhibitors such as the MG132.
While proteasomes are abundant in nuclei and cytoplasm,
they are particularly enriched at the centrosomes of somatic
cells, where they form the ‘proteolytic center’ (Wojcik et al.,
1996; Wojcik, 1997a,b; Wigley et al., 1999; Fabunmi et al.,
2000). To date, little is known about the functional significance
of proteasomes associated with the centrioles of the sperm tail
connecting piece (Lin et al., 2000; Fabunmi et al., 2000;
Mochida et al., 2000; Wójcik et al., 2000; Berruti and Martegani, 2005). This location suggests novel functions for
centriole-associated proteasomes. Failure of converting the
reduced sperm centriole into an active zygotic centrosome
may be one of the reasons for post-fertilization developmental
arrests affecting couples treated at IVF clinics (Chemes et al.,
1999; Hewitson et al., 2000; Chemes and Rawe, 2003). This
possibility became very clear to us after the study of a case
of fertilization failure due to centriolar dysfunction in patients
with abnormal head –tail connecting piece attachment (Chemes
et al., 1999; Rawe et al., 2002). Since proximal centrioles are
structurally normal in these patients, one of the objectives of
the present study was to elucidate the nature of the centrosomal
dysfunction. The diminished sperm proteasome enzymatic
activity reported here in two patients with abnormalities of
the head – tail attachment indicates that they are endowed
with abnormal proteolytic machineries and are probably
unable to disassemble the connecting piece and therefore
release the functional sperm centriole into the oocyte cytoplasm. To further test this hypothesis, we studied proteasome
localization in human and bovine spermatozoa, oocytes and
early zygotes, carried out pharmacological or immunological
inhibition of proteasomes and examined proteasomal distribution in cases of human post-ICSI fertilization failures.
Bovine and human spermatozoa display two distinct proteasome populations, one located over the acrosome and the other
one as distinct punctuate densities on the connecting piece.
This is in accordance with the few previous reports available
(Wójcik et al., 2000; Sutovsky et al., 2004). Early in fertilization, most cytoplasmic proteasomes translocate to the newly
formed pronuclei where they possibly take part in active chromatin remodeling prior to syngamy. The present data show that
the inhibition of proteasomes by MG132 in early stage pronuclei results in PCC and incomplete pronuclear development.
These findings suggest that degradation mediated by proteasomes is required for the release of oocytes from MII block
and full pronuclear progression.
To explore the role of proteasomes in the release of the centriole from the complex structure of the sperm tail connecting
piece, we incubated bovine IVF zygotes with proteasomal inhibitor MG132 or with Chariot coupled to E446 anti-proteasome antibodies. Also, donated human oocytes were injected with SLO
permeabilized spermatozoa preloaded with function-blocking
anti-proteasome antibody p31. In all experiments, a common
pattern of fertilization failure emerged. Proteasomes accumulated around the sperm tail connecting piece in a dense cloud-like
configuration and there was absent or defective sperm aster formation and pronuclear development/apposition. Identical findings were present in the zygotes from couples with spontaneous
post-ICSI fertilization failure that also displayed dense proteasome aggregations overlying the sperm connecting piece and
lack or deficiencies in sperm asters and pronuclei. We propose
that the proteasome recruitments observed after experimental
inhibition of proteasomes and in spontaneous post-ICSI fertilization failures represent abortive attempts of the zygotes to overcome the impaired release of the sperm centriole caused by
experimental or spontaneous proteasome deficiencies.
Upon proteasome inhibition, proteasomes and polyubiquitinated proteins aggregate around the centrosomes forming an
inclusion body or ‘aggresome’ in the area of the proteolytic
center (Wójcik et al., 1996; Wójcik, 1997a,b; Johnston et al.,
1998; Wójcik and DeMartino, 2003). The proteasome clusters
reported by us are strikingly similar to aggresomes, suggesting
that their formation is a general response of cells when proteasomes are unable to adequately degrade ubiquitinated proteins
(Johnston et al., 1998). Thus, the sperm centrosome appears to
be a site of proteasome-regulated proteolytic activity just like
their counterparts in somatic cells. Altogether, these data
support our hypothesis that proteasomes are involved in proteolysis of the sperm tail connecting piece. Failure of fertilization
after microinjection of p31 antibody-loaded human spermatozoa
indicates that the sperm centriole-associated proteasomes could
function as a first-wave degradation mechanism of the structures
surrounding the sperm-borne proximal centriole. After this
Rawe et al.
priming of the sperm tail connecting piece by proteasomes, the
abundant set of oocyte proteasomes could contribute to degrading
and re-structuring the connecting piece to ensure the release of a
sperm functional centriole and its conversion to the zygotic centrosome, the organelle that nucleates a sperm aster responsible for
bringing the parental genomes together prior to first mitosis.
Acknowledgements
Funding
CEGyR Foundation and Grants from CONICET (PIP 2565);
ANPCyT (PICT 9591); Fondecyt 1040295.
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