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From the * Department of Veterinary Clinical
Medicine and the Department of Veterinary
Biosciences, University of Illinois, Urbana, Illinois; and the
Department of Clinical Studies, New Bolton
Center, University of Pennsylvania, Kennett Square, Pennsylvania.
| Correspondence to: Dr Gary Althouse, 382 W Street Rd, Department of Clinical Studies-NBC, University of Pennsylvania, Kennett Square, PA 19348 (e-mail: gca{at}vet.upenn.edu). |
| Received for publication April 2, 2003; accepted for publication December 8, 2003. |
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Abstract |
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Key words: Sperm, boar
The CD migrates from the proximal to the distal position during maturation in the distal caput and proximal corpus epididymis and is normally shed from boar spermatozoa during or shortly after ejaculation (Larsen et al, 1980; Kato et al, 1996). In contrast to rats, which dispose of the contents of the CD by epithelial phagocytosis following their loss in the distal region of the epididymis, as many as 70%–90% of cauda epididymal boar spermatozoa retain the distal droplet (DD) (Kaplan et al, 1984; Hermo et al, 1988; Kato et al, 1996). D-Fructose, a component of the seminal vesicular fluid, is thought to be the factor responsible for normal shedding of the DD from boar sperm (Harayama et al, 1996). In the bull and ram, phospholipid-binding protein, synthesized in the ampullary glands and seminal vesicles, is believed to induce the release of the CD (Matousek and Kysilka, 1984).
Proximal droplets (PDs) found on ejaculated spermatozoa are generally considered indicative of a defect of testicular origin and have been implicated in the depressed fertility of bulls and boars (Dausend, 1974; Amann et al, 2000; Thundathil et al, 2001). The effect of a retained DD on fertility is less well defined, although there is some evidence suggesting a negative impact for stored boar semen used in artificial insemination programs (Larsson et al, 1984; Zeuner, 1992; Waberski et al, 1994; Althouse, 1998). An elevated incidence of retained DDs has been described in conjunction with biochemically altered plasma membranes in heat-stressed boars (Althouse, 1992). Additionally, boar sperm with retained CDs have a reduced binding affinity for porcine oviductal epithelial explants in culture (Petrunkina et al, 2001).
It has long been recognized that a selective mechanism may exist for the removal of abnormal spermatozoa in the efferent ductules and epididymis (Crabo et al, 1971). Rao (1980) recognized that the incidence of abnormal sperm heads decreased as spermatozoa passed along the length of the excurrent duct system in dairy bulls and that further reduction occurred in the epididymis of bulls with pathological semen. Although the process whereby abnormal sperm cells were recognized was not known, it was proposed that epithelial phagocytosis was the primary route of clearance (Rao, 1980). Recently, a ubiquitin-dependent sperm quality control mechanism functioning in the bovine epididymis was described, showing that ubiquitin is secreted by epithelial cells in the epididymis and is reported to bind to the surface of defective bull sperm (Sutovsky et al, 2001a). Most of the ubiquitinated, defective sperm are subsequently phagocytized by the epididymal epithelial cells, but some escape phagocytosis and can be identified in the ejaculate (Sutovsky et al, 2001a). In the stallion, sperm ubiquitination may be associated with seasonal changes in sperm numbers and semen quality (Sutovsky et al, 2003).
Ubiquitin, a small peptide of 8.5 kd, is a universal marker for proteolysis found in all tissues and organisms (Hershko, 1998). Since its initial discovery, ubiquitin has been assumed to operate exclusively in the intracellular space, where it marks proteins for recycling and identifies misfolded or damaged proteins for degradation (Hershko, 1998). Ubiquitin was recently identified at relatively high levels in an extracellular fluid—human seminal plasma (Lippert et al, 1993). Shortly thereafter, intense ubiquitin immunoreactivity was detected in the principal cells of the distal caput and in the clear cells of the cauda epididymidis of the rat (Martin et al, 1995). Epithelial clear cells of the rat epididymis actively dispose of the contents of CDs released from spermatozoa (Hermo et al, 1988).
The preferential binding of ubiquitin to the surface of defective spermatozoa has been reported in bulls, stallions, men, rhesus monkeys, and mice (Sutovsky et al, 2001a,b, 2003) but not in the boar. Although speculation abounds as to the significance of retained DDs to fertility and some work has been done to elucidate the factors involved in the normal release and abnormal retention of the CD, more can be learned about the CD at the molecular level. A greater understanding of the CD will help discern the potential impact it has on the sperm that carry it and on the oocytes those sperm encounter. The authors propose that a ubiquitin-dependent mechanism, similar to that identified in other species to remove morphologically abnormal sperm from the epididymis, functions in the boar by marking defective sperm for disposal. In addition, since retained CDs on spermatozoa represent the majority of abnormalities in boar ejaculates, we hypothesize that ubiquitination of the CD in particular should be expected to occur and will reinforce the need for studies addressing the interaction of retained CDs with in vivo fertility. Therefore, the purpose of the present study was twofold: 1) to determine if antibodies raised against ubiquitin would recognize antigens associated with the porcine CD, and 2) to determine if the same antibody would identify ubiquitinated substrates on the surface of morphologically abnormal boar spermatozoa.
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Materials and Methods |
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Reagents
Anti-ubiquitin (whole antiserum) developed in rabbits using bovine red
blood cell ubiquitin conjugated to keyhole limpet hemocyanin as the immunogen
was purchased from Sigma Chemical Co (St Louis, Mo). Secondary antibodies were
obtained from Zymed Laboratories, Inc (South San Francisco, Calif). Normal
goat serum (NGS) was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz,
Calif). Vectashield Mounting Medium with propidium iodide (PI) was purchased
from Vector Laboratories (Burlingame, Calif). Beltsville Thaw Solution (BTS)
was supplied by IMV (Minneapolis Minn). Buffered formalin was purchased from
Fisher Scientific (Pittsburgh, Pa), and glutaraldehyde was obtained from
Electron Microscopy Sciences (Fort Washington, Pa). Unless otherwise noted,
all other reagents were purchased from Sigma-Aldrich (St Louis, Mo).
Immunofluorescence
Ten samples of fresh extended porcine semen were prepared for the ubiquitin
immunofluorescence assay (U-IFA) by an initial slide mounting technique
similar to that of Sutovsky (2001a), while 2 others were prepared by
conducting the fixation, incubation, and washing steps in microtubes rather
than on slides. For the slide-mounted technique, extended semen was initially
separated on an isotonic discontinuous Percoll gradient (72%/90%) to
concentrate the sperm cells, remove the extender, and isolate sperm cells from
bacteria, free CDs, and other debris
(Parrish et al, 1986; Grant et al, 1994). Following
Percoll separation, sperm cells were washed once in Tyrode albumin lactate
pyruvate (Bavister et al,
1983); then, 10 µL of concentrated cell suspension was added to
a 250-µL drop of warm (37°C) BTS
(Johnson et al, 1988) on a
charged slide (Superfrost/Plus, Fisher) and incubated at that temperature for
5 minutes. After attachment, excess fluid was shaken off, and the sperm were
fixed by first covering the attachment area with 200 µL of
phosphate-buffered saline (PBS) and then slowly adding 50 µL of 10% neutral
buffered formalin (NBF). After 40 minutes of fixation, the samples were
blocked and permeabilized for 30 minutes using 5% NGS in PBS containing 0.1%
Triton X-100 (TX-100) and then incubated for 40 minutes in a 1:100 dilution of
anti-ubiquitin in PBS. Following primary antibody incubation, the samples were
rinsed 3 times in PBS and incubated for 40 minutes with the secondary
antibody, fluorescein isothiocyante (FITC)-conjugated goat anti-rabbit diluted
1:100 in PBS. After 3 rinses in PBS, a coverslip was mounted using Vectashield
with PI for the DNA counterstain. Fluorescence was evaluated with an Olympus
IX70 microscope (NIB filter), and images were captured with a Spot RT digital
camera (Diagnostic Instruments Inc, Sterling Heights, Mich).
For the microtube technique, a volume of extended semen containing 3.5–5.0 x 106 sperm cells was added to a 1.5-mL microtube and centrifuged at 200 x g for 3 minutes (all subsequent centrifugations were at 200 x g for 4 minutes). The supernatant was removed, and the pellet was resuspended with 500 µL of 0.2% glutaraldehyde in PBS. After 45 minutes of fixation, the supernatant was removed by centrifugation, and the sperm cells were resuspended in a blocking solution containing 10% NGS and 0.05% TX-100 in PBS for 45 minutes. Blocking/permeabilization fluid was removed by centrifugation, and the sperm cells were incubated for 1 hour in 100 µL of anti-ubiquitin antibody diluted 1:100 in PBS. The samples were then centrifuged, the supernatant was discarded, and the sperm cells were washed once in 500 µL of PBS prior to a 45-minute incubation with 100 µL of FITC-conjugated goat anti-rabbit secondary antibody diluted 1:100 in PBS. The samples were then washed once with 500 µL of PBS and resuspended in 100 µL of fresh PBS. After gentle mixing, a 10-µL drop of the sperm suspension was placed on a Superfrost/Plus Slide, and the edge of a coverslip was used to make a smear that was allowed to air dry. A coverslip (22 x 50 mm) was mounted over the area of the smear using Vectashield medium with PI as described above.
Rat cauda epididymidis served as the positive controls. Negative controls included the following: 1) reagent control—exclusion of the primary antibody (pure PBS), 2) normal rabbit serum (NRS) control—incubation with NRS (1:100 dilution in PBS) in place of the primary antibody, and 3) peptide competition— preabsorption of the primary antibody with excess ubiquitin prepared from bovine erythrocytes.
Western Blot
To reduce contamination with free CDs and remove excess seminal plasma and
extender components, diluted boar semen was first separated through an
isotonic Percoll gradient (72%/90%). After washing once with PBS, the sperm
pellet was resuspended in precooled (4°C) PBS containing 1% TX-100 and
selected protease inhibitors (5 mM benzamidine HCl, 5 µg/mL leupeptin, 1
µg/mL pepstatin A, and 25 mg/mL phenylmethylsulfonyl fluoride); it was then
incubated on ice for 30 minutes with light vortexing at 5-minute intervals.
Following detergent extraction, the sample was clarified by centrifugation at
10 000 x g for 5 minutes, and the supernatant was drawn off and
stored at –20°C. Protein concentration was determined using the BCA
Protein Assay Reagent Kit from Pierce Biotechnology (Rockford, Ill).
Supernatant fluid containing 65 µg of sperm proteins was mixed 5:1 with
6x sample buffer and boiled for 3 minutes prior to 1-dimensional
discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) using a 5% stacking gel and a 15% resolving gel
(Laemmli, 1970). Proteins were
transferred to 0.2-µm-thick nitrocellulose membranes, with the transfer
efficiency evaluated using Ponceau S prior to immunostaining
(Towbin et al, 1979;
Salinovich and Montelaro,
1986). Membranes were blocked in Tris saline (TS 20) containing 5%
nonfat dried milk for 1 hour, incubated for 90 minutes with anti-ubiquitin
antibody (1:100 in TS 20 with 1% dried milk) at room temperature, washed 3
times in TS 20, and incubated for 45 minutes with horseradish
peroxidase–conjugated goat anti-rabbit immunoglobulin G (IgG) (1:100).
After 3 washes, blots were developed using the ImmunoPure Metal Enhanced DAB
(diaminobenzidine) Substrate Kit (Pierce). Specificity of labeling was
evaluated by including a negative control that was incubated with primary
antibody preabsorbed to excess bovine ubiquitin and 2 positive controls: 1
µg of bovine red blood cell ubiquitin (Sigma) and 1 µg of Ub (FL-76), a
35-kd tagged fusion protein produced in Escherichia coli representing
full-length ubiquitin of human origin (Santa Cruz Biotechnology).
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Results |
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Discussion |
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Surface ubiquitination was not apparent on nonpermeabilized morphologically abnormal porcine spermatozoa. Although this finding could be because of species differences in epididymal ubiquitination, it is more likely that the probe used in the present study was limited in its affinity for certain types of polyubiquitinated chains (Hershko and Ciechanover, 1998). The primary antibody (U5379) was generated against mono-ubiquitin purified from bovine erythrocytes, whereas ubiquitin potentially located on the surface of spermatozoa would be covalently bound to sperm surface proteins in the form of polyubiquitinated chains (Sutovsky et al, 2001a). That positive IFA results were obtained following permeabilization of the CD supports the belief that the antibody was effective at detecting ubiquitinated sperm proteins; however, further investigation may be elected to determine if using a broader range of epitope-specific ubiquitin probes and recombinant isopeptides will definitively verify the presence of ubiquitin-protein conjugates on the surface of the boar spermatozoa. The ubiquitinated proteins revealed within the boar CD after permeabilization suggest that the antibody used in the present study detects mainly intracellular ubiquitin that would most likely have originated from ubiquitin-dependent proteolysis during spermatid elongation (Sutovsky, 2003). As a remnant of the spermatid cytoplasm, the CD contains numerous vesicular elements and other small particles that may have originated from the Golgi apparatus, sperm centrosome, and sperm histones (Bloom and Nicander, 1961; Moreno et al, 2000; Sutovsky, 2003). The detection of intracellular ubiquitin does not rule out the possibility that the surface of the CD will also become ubiquitinated at a later stage, such as during epididymal passage and/or during postejaculatory sperm modifications.
Ubiquitination of the retained CDs has important implications when coupled with the knowledge that PDs and DDs have been associated with depressed fertility in vitro and in vivo. In mammals, some of the paternally derived organelles, such as mitochondria, are degraded in the lysosomes of the oocyte after fertilization, while others, such as the centrosome and male pronucleus, become vital zygotic components (Yanagimachi, 1994; Sutovsky et al, 1999). It has been theorized that following natural fertilization, the ubiquitin present on the surface of spermatozoa from subfertile ejaculates are carried over to the oocyte cytoplasm, where it could potentially target vital paternal organelles for destruction by the proteolytic machinery of the oocyte, effectively preventing further embyronic development (Sutovsky et al, 2001b). In support of this theory, a relatively high correlation coefficient (r = –0.432) was obtained by comparing Sperm Ubiquitin Tag Immunoassay (SUTI) to cleavage rate after in vitro fertilization (IVF) in human infertility patients, even though fertilization rates were poorly correlated (r = 0.046) (Sutovsky et al, 2001b).
Although ubiquitin was not simultaneously investigated, another study found that semen from yearling beef bulls containing more than 30% spermatozoa with PDs had severely compromised IVF fertility, which subsequently improved as the bulls matured and the incidence of PDs decreased (Amann et al, 2000). It has been reported that bull sperm with PDs do not bind to the zona pellucida at the same rate as morphologically normal sperm (Thundathil et al, 2001), and boar sperm with retained CDs have a reduced binding affinity to porcine oviductal epithelial explants in culture (Petrunkina et al, 2001); however, the ability of porcine sperm with retained DDs to bind to the zona and fuse to the oolema has not been ruled out.
A possible explanation for this reduced binding capacity of sperm with a CD relates to the maturity level achieved during spermiogenesis. In humans, the presence of a chaperone protein, HspA2, throughout the tail and on the surface of the tail plasma membrane has been associated with the level of sperm cellular maturity, function, and fertility (Huszar et al, 2000; Ergur et al, 2002). Expression of HspA2 in human sperm occurs during remodeling of the plasma membrane when zona pellucida–binding sites are formed (Huszar et al, 1994, 1997). Human sperm with a diminished HspA2 expression retain cytoplasm, are considered immature, and fail to bind to the zona pellucida (Ergur et al, 2002).
The boar sperm CD contains numerous membranous structures (Kaplan et al, 1984). One comparison finds the area of these internal CD membranes equivalent to 54% of the total surface area of the external sperm plasma membrane (Kaplan et al, 1984). The demonstrated presence of antigens recognized by anti-ubiquitin antibodies in the boar sperm CD, coupled with the possibility that superfluous ubiquitin species are detrimental to embryonic development through targeted destruction of critical paternally contributed zygotic organelles, raises concerns that retained DDs may be more detrimental to fertility than previously suspected. Further studies are warranted to investigate the binding, penetrating, and fertilizing ability of boar sperm with retained distal droplets and the fate of the CD and its contents during and after fertilization, as well as the developmental competence of any embryos produced from sperm with a CD.
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Acknowledgments |
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