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Gamete Biology |
a Departments of Animal Sciences and Obstetrics and Gynecology, University of Missouri-Columbia, Columbia, Missouri 65211-5300
b Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square, Pennsylvania 19348
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, gamete biology, seasonal reproduction, sperm, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In humans and other mammalian species, male infertility has been associated with a higher content of sperm surface proteins that cross-react with anti-ubiquitin antibodies [8, 9]. These ubiquitin cross-reactive proteins appear to be secreted from the epididymal epithelium [10, 11] and bind predominantly to the surface of defective spermatozoa [9]. Although the fate of the ubiquitinated spermatozoa awaits further clarification, it appears that at least a portion of them are removed during epididymal passage. Similarly, sperm cytoplasmic droplets contain ubiquitin [9] and are removed during epididymal passage by a phagocytotic mechanism residing in the clear cells of the epididymal epithelium [12]. The presence of ubiquitin on ejaculated spermatozoa can be exploited as a sensitive, objective assay for infertility in men (SUTI, for sperm-ubiquitin tag immunoassay [8]). Furthermore, the ubiquitin system has been implicated in the seasonal control of spermatogenesis in the Japanese macaque [13] and in ascidian fertilization [14]. In addition, ubiquitin appears to be involved in cell cycle control, retroviral infection, endocytosis, gametogenesis, and embryonic development (reviewed in [15, 16]).
Ubiquitin has the highest degree of amino acid sequence conservation of any known protein [17]. Polyubiquitin chains of four or more molecules target substrate proteins for proteasomal degradation by binding to their lysine residues. This isopeptide-bond formation is catalyzed by ATP and the ubiquitin-conjugating factors E1–E4 (reviewed in [18, 19]).
Deubiquitinating enzymes, including ubiquitin C-terminal hydrolase, PGP 9.5 (for protein gene product 9.5), regulate the ubiquitin system and recycle ubiquitin molecules [20].
The variability in ubiquitin-protein and ubiquitin-ubiquitin conjugation somewhat complicates immunodetection of ubiquitin. In addition to the unconjugated, monomeric ubiquitin, available antiubiquitin antibodies recognize only certain types of polyubiquitin chains, ubiquitin-homology-proteins, and ubiquitin-protein conjugates [19]. To minimize this potential complication, in this study we used an antibody raised against recombinant human ubiquitin and an antibody raised against bovine erythrocyte ubiquitin to examine differential sperm ubiquitination in stallions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stallion tissue samples (Figs. 3 and 4) were collected postmortem from two adult thoroughbred stallions that were killed at the Widener Hospital for Large Animals, New Bolton Center because they had severe colic. Both had histories of being successful breeding stallions before their presentations and the testicles of both animals were palpably normal at the time of death. Tissue samples were obtained from each animal's testicles, epididymides, ampullae, seminal colliculi, seminal vesicles, prostate, and bulbourethral glands, and fixed in 4% paraformaldehyde.
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Semen samples for microscopy (Figs. 1 and 2) and for protein extraction (Fig. 6) were routinely collected from four additional systemically healthy stallions using a Missouri model artificial vagina (Nasco, Fort Atkinson, WI) with the stallion mounted either on an ovariectomized mount mare or on a dummy mount. Routine semen analysis, including measurement of semen volume, sperm concentration, sperm motility, and sperm morphology, was performed on each ejaculate according to the recommendations of the Society for Theriogenology [21].
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Stallions 1 and 2 (Fig. 6) were classified as fertile based on current histories of achieving at least 70% seasonal pregnancy rates in an average of less than two heat cycles per pregnancy and based on good semen quality (
60% progressively motile sperm; 60% morphologically normal sperm; 1 billion morphologically normal, progressively motile [MNPM] sperm per ejaculate; and good longevity of sperm motility). Stallion 3, although still fertile based on seasonal pregnancy rates and average heat cycles per pregnancy, had been experiencing an age-related decline in testicular size and semen quality over the past several years. This stallion's ejaculates intermittently contained 
60% progressively motile sperm and 60% morphologically normal sperm, although he did consistently ejaculate at least 1 billion MNPM sperm per ejaculate. Stallion 4 had a history of subfertility and marginal semen quality. This stallion's testicles were palpably and ultrasonographically abnormal. Histopathologic examination of testicular tissue confirmed a diagnosis of testicular degeneration and suspected testicular neoplasia.
To obtain samples for flow cytometric evaluation of seasonal changes in sperm ubiquitination, semen samples were collected quarterly (March, June, September, and December) from each of two fertile stallions (Fig. 5; stallions 1 and 2 described above) in residence at the Hofmann Center for Reproductive Studies, New Bolton Center.
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For all semen samples, four 1.5-ml aliquots were centrifuged at 16 000 x g in an Eppendorf 5415C centrifuge to pellet the sperm. The seminal plasma supernatants were removed and the sperm pellets were washed twice in PBS before being frozen at -20°C.
For extraction of sperm proteins, one sperm pellet from each stallion of interest was thawed, resuspended in SDS sample buffer containing 40 mM dithiothreitol and a 1x protease inhibitor cocktail (Complete Protease Inhibitor Cocktail tablets; Roche Molecular Biochemicals, Mannheim, Germany) and boiled for 5 min. The samples were centrifuged at 16 000 x g for 3 min and the supernatant was saved for analysis. Each protein sample was diluted in deionized water and the concentration of protein in each diluted sample was determined using the Protein Assay Kit (BioRad, Hercules, CA). Concentration was multiplied by the dilution factor to determine protein concentration in the original sample.
Immunological Reagents
Monoclonal mouse immunoglobulin (Ig) M raised against recombinant human ubiquitin (clone KM691) was obtained from Kamiya Biomedical Company (Seattle, WA [22]). This antibody has been used previously to identify ubiquitin-tagged proteins in human sperm [8]. Monoclonal mouse IgG raised against bovine erythrocyte ubiquitin (clone MK12-3) was obtained from MBL (Nagoya, Japan). This antibody has been used previously to detect ubiquitinated proteins in defective bull spermatozoa and in normal sperm mitochondria [9, 23, 24]. Polyclonal rabbit serum raised against ubiquitin conjugating enzyme E2 was obtained from Affinity Research Products, Inc. (Mamhead, U.K.). Polyclonal rabbit serum raised against ubiquitin C-terminal hydrolase, PGP 9.5 was obtained from Chemicon International (Temecula, CA). Fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Zymed, Inc. (San Francisco, CA) and Amersham Life Science (Arlington Heights, IL). Colloidal gold conjugates were obtained from Jackson Immunochemicals (West Grove, PA).
One-Dimensional Gel Electrophoresis and Immunoblotting
Ejaculated sperm proteins were separated under reducing conditions by SDS-PAGE on a 10% (w/v) gel. Ten micrograms of sperm protein from each stallion were loaded in each lane. After electrophoresis, proteins were electrophoretically transferred to polyvinylidene fluoride membranes (Immobilon-P Transfer Membranes; Millipore Corp., Bedford, MA). Following blocking in PBS containing 10% (w/v) fish gelatin, the blots were probed for 1 h with antiubiquitin (antibody KM691 or MK12-3; 1:5000 v/v) in PBS containing 0.1% (v/v) Tween-20 and 3% (w/v) BSA. The blots were washed three times for 10 min with PBS containing 0.2% (v/v) Tween-20 and then incubated for 1 h with goat anti-mouse IgM conjugated to HRP (Zymed) for KM691 or sheep anti-mouse IgG conjugated to HRP (Amersham Life Science) for MK12-3, each diluted 1:5000 (v/v) in PBS containing 0.2% (v/v) Tween-20 and 3% BSA. Blots then were washed five times in PBS, overlaid with enhanced chemiluminescence detection reagent (Amersham) and exposed to Kodak X-Omat Blue Scientific Imaging Film (NEN Life Science Products, Inc., Boston, MA). As a control, antibodies were removed from the membrane by submersion in 40% methanol, rinsing it twice in PBS, and then submerging in stripping buffer (100 mM 2-mercaptoethanol, 2% w/v SDS, and 62.4 mM Tris-HCl ph 6.7). The membrane then was washed for at least 30 min in two changes of PBS/Tween-20. After stripping and washing, the membrane was blocked and then reprobed as described above using nonimmune mouse serum (1:5000 w/v; Sigma Chemical Company, St. Louis, MO) instead of the primary antibody. To demonstrate equal protein loading, the same membrane was again stripped as above and then stained with a solution of Coomassie brilliant blue (Bio-Rad, Hercules, CA; data not shown).
Immunofluorescence
Spermatozoa were attached to poly-L-lysine coated coverslips in 37°C KMT medium [9], fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 (TX-100; TX 100 was not used for MK12-3 and KM691) and blocked for 30 min in 5% normal goat serum (NGS; Sigma) in PBS with 0.1% TX-100. Coverslips were incubated for 1 h at 37°C with the appropriate primary antibody, (KM691 diluted 1:100, MK12-3 diluted 1:10, and anti-E2 diluted 1:50) in PBS with 1% goat serum and 0.1% TX-100. Coverslips were washed three times in PBS before being incubated in a 1:80 dilution of the appropriate secondary antibody (FITC-conjugated goat anti-mouse IgM for KM691, FITC-conjugated goat anti-mouse IgG for MK12-3), or FITC-conjugated goat anti-rabbit IgG (for anti-E2) for 40 min at 37°C. DNA was counterstained with 4',6'-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR) added at 2.5 µg/ml DAPI to the solution containing the secondary antibody. Samples were washed in PBS and mounted on microscopy slides in VectaShield mounting medium. Negative control samples were incubated with the secondary antibody alone (Fig. 3L).
Immunohistochemistry on Paraffin Tissue Sections
Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and 4 µm-thick sections were placed on microscopy slides as described elsewhere [9]. Sections then were deparaffined with three xylene changes, rehydrated, permeabilized using 0.1% TX-100, and blocked in 5% NGS in PBS with TX-100. Slides were incubated for 1 h at 37°C with a primary antibody, (KM691, diluted 1:100; or PGP 9.5 diluted 1:50) in PBS with 1% goat serum and 0.1% TX-100. Sections were washed three times in PBS before being incubated with the appropriate secondary antibody (FITC-conjugated goat anti-mouse IgM (for KM691) or FITC-conjugated goat anti-rabbit IgG (for PGP9.5; both diluted 1:80; both from Zymed Laboratories, Inc., San Francisco, CA) for 1 h at 37°C. DNA was counterstained with DAPI (Molecular Probes) added at 2.5 µg/ml to the solution containing the secondary antibody. Samples were washed three times in PBS and covered with microscopy coverslips on VectaShield mounting medium. Negative control samples were incubated with the secondary antibody alone (Fig. 4G). Positive controls were performed by staining brain tissue sections from patients with Alzheimer disease with antiubiquitin antibodies (KM691 labeling of amyloid plaques in neurons is shown in Fig. 4H). Images of epididymal tissue were acquired at 100–150 msec exposure time, while the background fluorescence, necessary in order to visualize sections from testis and sex accessory glands, was acquired at 600 msec.
Electron Microscopy
Sperm samples in suspension were fixed in 2% formaldehyde, permeabilized with 0.1% TX-100, and processed with antibody KM691 as described for immunofluorescence. For immunoelectron microscopy, the fluorescent secondary antibody was replaced with goat anti-mouse IgG, conjugated to 12 nm colloidal gold (Jackson Immunochemicals, diluted 1:10). Where indicated, the samples were incubated with KM691 and GAM (goat anti-mouse) IgM/gold without fixation and permeabilization. Gold-labeled sperm were fixed again in a mixture of 2.5% glutaraldehyde and 0.6% paraformaldehyde in cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated, and embedded in PolyBed 812 as previously described [9]. Ultrathin sections were cut on a Reichert ultramicrotome, stained by uranyl acetate, and photographed in a Philips EM 300 electron microscope (Philips N.V., Eindhoven, The Netherlands). Negatives were scanned using a UMAX Powerlook 300 flatbed scanner with a transparency attachment (UMAX Technologies, Fremont, CA) and printed on an Epson Stylus 1280 photo printer (Epson American Inc., Long Beach, CA) using Adobe Photoshop software (Adobe Systems, Mountain View, CA).
Flow Cytometry
Ejaculates were collected and evaluated as described above for motility, morphology, and total sperm numbers. Each semen sample was washed in PBS and sperm pellets were frozen at -80°C. These samples were thawed and processed for labeling with the antiubiquitin antibody KM691 (SUTI assay [8]). Briefly, sperm pellets were fixed for 40 min in 2% formaldehyde in PBS and blocked for 30 min with 5% NGS in PBS (no TX-100 was used in this processing to ensure that only ubiquitinated substrates located on the sperm surface contributed to the signal). After a brief centrifugation wash in PBS, samples were processed with KM691 as described for immunofluorescence and the appropriate fluorescent secondary antibody (GAM IgM-FITC). Blank, negative control samples were generated by the omission of KM691. Ubiquitin-induced fluorescence was measured in 10 000 cells screened in each sample and each run using a FACS Vantage Analyzer (Becton Dickinson, Franklin Lakes, NJ). Median values of positive fluorescence (ubiquitin medians), histograms of fluorescent cell distribution, and light scatter plots were recorded. As a positive control, samples of the processed cells were mounted on microscopy slides and verified with an epifluorescence microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ejaculated stallion spermatozoa were examined by immunofluorescence using antibody KM691 (Fig. 1, A–D; anti-human recombinant ubiquitin). The results revealed that most ejaculated sperm had detectable ubiquitin signals on both the head and the tail. This was true whether sperm were processed unfixed (Fig. 1, A, B, and D), or fixed with formaldehyde (Fig. 1C). Of particular interest was the association of ubiquitin with defective sperm tails (Fig. 1B), defective sperm heads (Fig. 1C), and nuclear vacuoles (Fig. 1D).
Compared with the results for KM691, relatively few sperm had a detectable ubiquitin signal when probed with the antibody MK12-3 (Fig. 1, E and F; anti-bovine erythrocyte ubiquitin), and the staining was confined mainly to the sperm tail. These findings suggest that some stallion sperm surface proteins become ubiquitinated before ejaculation. Because the two different antibodies (KM691 and MK12-3, each recognizing a different subset of ubiquitin-tagged proteins) resulted in two different labeling patterns (Fig. 1; sperm tails are mainly recognized by MK12-3, whereas both tails and heads are labeled by KM691), it is possible that individual sperm surface proteins are differentially ubiquitinated. This is also supported by the differences in band patterns yielded by these two antibodies in Western blotting (Fig. 6).
Ubiquitin-conjugating enzyme E2 is involved in covalent ligation of ubiquitin molecules to target protein substrates [18]. Immunofluorescence using an antibody raised against this enzyme produced labeling in the tail and head-equatorial segment of most spermatozoa fixed with formaldehyde and permeabilized with TX-100 (Fig. 1G).
The results of our immunofluorescence experiments on nonpermeabilized sperm suggested that proteins on the surface of the sperm head and tail became ubiquitinated at some point before ejaculation. To confirm that this ubiquitin labeling was, in fact, on the sperm surface and not inside the cell, immunoelectron microscopy of fixed/permeabilized ejaculated sperm (Fig. 2A) and nonpermeabilized (Fig. 2, B and C), ejaculated sperm using the KM691 antibody was performed. The results confirmed that the ubiquitin cross-reactive substrates are largely confined to the sperm surface.
The Sperm Ubiquitin Tag Is Added in the Epididymis
We used immunocytochemistry to examine testicular and epididymal spermatozoa to confirm the site of sperm surface ubiquitination within the stallion's reproductive tract. Immunocytochemistry was performed with the KM691 antibody on paraffin sections of testicular and epididymal tissues. Cytosolic ubiquitin was detected in the Leydig cells of testicular stroma (Fig. 3A), but there was lesser accumulation of ubiquitin in the seminiferous tubules and testicular spermatozoa (Fig. 3B).
In contrast to the low levels of ubiquitination found on the surface of testicular spermatozoa, large amounts of surface ubiquitination were detected on spermatozoa located on the apical surface of the epididymal epithelium, spermatozoa intermingled with the apical stereocilia, and spermatozoa that were free in the epididymal lumen (Fig. 3, E and F). Some of the sperm heads appeared to be completely engulfed by the apical stereocilia (Fig. 3F). Defective spermatozoa and other cells (most likely somatic cells or immature spermatogenic cells) within the epididymal lumen appeared to be very highly ubiquitinated (Fig. 3G). In particular, isolated epididymal spermatozoa with morphologically abnormal tails (Fig. 3, H, I, and K) and heads (Fig. 3J) displayed strong ubiquitin cross-reactivity. The accumulation of ubiquitin in the epididymal lumen coincided with strong accumulation (Fig. 3, C and D) of the ubiquitin-recycling enzyme, ubiquitin C-terminal hydrolase PGP 9.5 (shown in Fig. 4F), within the stereocilia on the apical surface of epididymal epithelium. PGP 9.5 functions to regulate the ubiquitin system by recycling ubiquitin molecules [20]. Thus, multiple components of the ubiquitin system are present in the apical epididymal stereocilia, suggesting that the ubiquitin system is functional within the epididymis. In addition, these findings are consistent with the hypothesis that ubiquitin is secreted by the apical stereocilia of the epididymal epithelium [9, 11], thus allowing for the addition of sperm surface ubiquitin in the epididymis.
To determine whether reproductive tissues other than the epididymis also might be sources of sperm surface ubiquitin, we used immunocytochemistry and the KM691 antibody to examine other regions of the stallion reproductive tract. Relatively low levels of ubiquitin cross-reactive substrates were identified in tissues from the ampulla (Fig. 4A), bulbourethral gland (Fig. 4B), seminal colliculus (Fig. 4C), prostate (Fig. 4D), or seminal vesicle (Fig. 4E). The amount of ubiquitin cross-reactive substrates identified in each of these tissues was significantly less than that identified in the epididymis. Negative controls showed low or no cross-reactivity in epididymal tissue sections processed with the secondary antibody only (Fig. 4G). Strong accumulation of ubiquitin was revealed by antibody KM691 in the neuronal tangles of human brains with Alzheimer disease, used as a positive control (Fig. 4H).
Indications of Seasonal Variations in Sperm Surface Ubiquitination
The above studies were performed on sperm and tissue samples obtained during the nonbreeding season (winter). A recent report suggests a role for ubiquitin in the seasonal control of spermatogenesis in the Japanese macaque [13]. We hypothesized that seasonal changes in sperm surface ubiquitination also might be involved in the physiologic seasonal changes in sperm numbers and semen quality observed in stallions. As a first step in investigating this possibility, semen samples were collected from two fertile stallions in March (spring equinox), June (summer solstice), and September (fall equinox), and a sample from one of the stallions also was obtained in December (winter solstice). In both stallions, a flow cytometric SUTI assay [8] showed the lowest levels of surface ubiquitinated sperm in March, coinciding with the onset of the natural horse breading season (Fig. 5, A–C, and Table 1). Sperm ubiquitin levels progressively increased during summer and peaked in September. Based on these samples, there appears to be a seasonal variation in sperm surface ubiquitination, consistent with the hypothesis that increases in sperm surface ubiquitination could play a role in the seasonal decline in sperm numbers and semen quality seen in the nonbreeding season.
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Ubiquitinated Proteins Vary among Fertile and Subfertile Stallions
We have shown that some cell surface proteins in stallion sperm are ubiquitinated, possibly aiding their turnover and degradation during epididymal sperm maturation. However, the identity of these target proteins is not known. As an initial step in characterizing these proteins, and to determine whether the same proteins are ubiquitinated in all stallions, immunoblotting analysis using the KM691 antibody was performed on proteins extracted from ejaculated sperm from four stallions of varying fertility (Fig. 6B). In addition, ejaculated spermatozoa from each animal were probed with KM691 and subjected to flow cytometric analysis to determine whether or not different percentages of sperm were ubiquitinated in each animal (Fig. 6A). All semen samples were collected during the nonbreeding season. Stallions 1 and 2 were highly fertile and all measures of reproductive efficiency were normal. Stallion 3 was an aging stallion and, although he was still fertile, showed evidence of declining reproductive efficiency (lower sperm numbers, lower semen quality, and smaller testicle size). Stallion 4 was subfertile. This stallion experienced difficulty rendering mares pregnant, and histopathological examination of testicular tissue confirmed the presence of significant testicular pathology.
Flow cytometric analysis revealed differential levels of sperm surface ubiquitination in the four animals, with stallion 4 possessing the highest levels of ubiquitin (Fig. 6A). Immunoblotting analysis with antibody KM691 revealed that all four stallions shared four major ubiquitin cross-reactive bands. However, stallion 4 showed two unique ubiquitin cross-reactive bands (Fig. 6B, lane 4). In addition, stallion 3, the animal with declining reproductive efficiency, appeared to have a higher protein concentration in several of the prominent ubiquitin bands (Fig. 6B, lane 3).
The same membrane, stripped and reprobed with an unrelated antiubiquitin antibody, MK12-3, also revealed a major, unique ubiquitin cross-reactive band in stallion 4 (Fig. 6C), whereas some unique bands of lesser density were also detected in stallion 1. These results suggest that the identity of ubiquitinated proteins as well as the levels of sperm-surface ubiquitination vary among stallions, and may be correlated with reproductive efficiency. The same blot was again stripped and reprobed with nonimmune mouse serum as a negative control. No bands were identified, thus supporting the specificity of the antiubiquitin antibodies. Finally, the blot was stripped and stained with Coomassie brilliant blue to confirm equal protein loading in each lane. Except for the bands identified above, all remaining bands were of similar intensities in all four sample lanes (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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From where does this ubiquitin tag originate? Ubiquitin ([10, 11]; this study) and the ubiquitin-recycling enzyme, C-terminal hydrolase PGP 9.5 ([10]; this study), are present within the stereocilia on the apical surface of the lumenal epididymal epithelial cells (EECs). Fraile et al. [11] were the first to suggest that ubiquitin and PGP 9.5 are secreted into the epididymal lumen in humans and rats. Our work suggests that the same is true in stallions. Because the stereocilia and apical blebs of the epididymal epithelium are known sites of apocrine protein secretion [27], it is possible that ubiquitin is secreted from stallion EECs into the epididymal lumen where it binds to epididymal spermatozoa. Accordingly, pulse-chase studies of cultured bull EECs showed that newly synthesized, radiolabeled ubiquitin can be immunoprecipitated from culture medium conditioned by these cells [9]. A recent study of stallion seminal plasma proteins, separated by two-dimensional PAGE, showed that increased expression of some seminal plasma proteins correlated negatively with fertility [28].
In addition to the high levels of ubiquitin found in the epididymis, ICC experiments detected lower levels of ubiquitin in several other organs of the stallion reproductive tract, including the testis and sex accessory glands. Secretion of ubiquitin from these tissues could contribute to the protein content of seminal plasma, found to be rich in ubiquitin [29], but most likely is not the predominant source of sperm ubiquitin.
While this initial study does not comprise a statistically powerful group of stallions, our flow cytometric measurements of ubiquitin in ejaculated sperm provide preliminary evidence that the ubiquitin system could be involved in seasonal or pathological decreases (or both) in stallion sperm numbers. This is similar to our previous observations in breeding bulls [9, 23] and infertile men [8]. Further support for a role of the ubiquitin system in seasonal variations in sperm numbers is provided by studies showing an increased expression of ubiquitin C-terminal hydrolase, PGP 9.5, in the spermatogonia of Japanese macaque (Macaca fascicularis) during the nonbreeding, winter season [13]. Further studies comprising a significantly larger group of stallions will be necessary to address this issue in a satisfactory and conclusive manner. Because semen samples for this study were collected over the course of a year, we elected to freeze the sperm pellets and store them before performing cytometric analysis on all samples. Because these sperm were frozen and thawed, one must take into consideration the possibility that cryodamage to sperm surface epitopes could affect the cross-reactivity of some epitopes (e.g., ubiquitin).
The role of the hypothalamic-pituitary-gonadal axis in the control of stallion reproductive seasonality has been thoroughly investigated (reviewed in [6, 7]). However, little is known about how decreases in total sperm numbers, sperm quality, and sperm motility are achieved at the gonadal level. One possibility is that apoptosis, a mechanism by which cells initiate suicide, is induced in quiescent stallion spermatogenic cells. Once activated, apoptotic pathways result in DNA fragmentation and subsequent programmed cell death. In this way, apoptosis could be used to decrease cell numbers, prevent cell overgrowth, and eliminate defective cells from a population [30]. In humans and mice, it has been shown that apoptosis during spermatogenesis in the testis is involved in regulating sperm numbers [31–33]. Similarly, in humans it has been shown that evidence of apoptosis in ejaculated sperm may reflect an increase in apoptosis during spermatogenesis and a resultant decrease in ejaculated sperm numbers [34]. TUNEL assays for DNA fragmentation and sperm chromatin structure assays of chromatin integrity [35, 36] also suggest that apoptosis occurs in ejaculated human spermatozoa. Based on these findings, it is possible that apoptotic pathways might be involved in the seasonal changes in sperm numbers that are observed in stallions and other seasonal breeders. Several markers of apoptosis seem to correlate with fertility in men and animals [23, 37–39]. For example, the levels of annexin V, a marker of apoptotic plasma membrane rearrangement, are elevated in the sperm of infertile men [40]. Active caspase 3 [41] and Fas ligand [34] were detected in defective spermatozoa of mice and men, respectively. In stallions it has been shown that Fas ligand is intimately associated with midpiece mitochondria [16], thus raising the possibility of the existence of an active apoptotic pathway in equine spermatozoa.
How does this connect with ubiquitin? Ubiquitin accumulates in apoptotic tissues and may play a pivotal role in regulating the signaling molecules involved in executing programmed cell death (reviewed in [42]). Recently, it has been reported that a ubiquitin-like protein expressed in somatic cells was able to initiate apoptosis, thus providing a possible link between the two systems [43, 44]. Apoptosis has been implicated in mediating seasonal testicular regression in mammals [45]. In this regard, the accumulation of ubiquitin on TUNEL-positive (i.e., possibly apoptotic) bull spermatozoa, is correlated with sperm morphology and fertility in bulls with good but varied fertility [23]. Thus, surface ubiquitination of epididymal spermatozoa could initiate or accompany apoptosis of male germ cells, providing a novel mechanism for physiologic, seasonal reduction in sperm numbers. As has been shown in men, these systems also could be involved in some of the pathologic reductions in sperm numbers seen in subfertile stallions.
This and other studies [8] suggest a negative correlation between sperm surface ubiquitination and sperm count, raising the possibility that the ubiquitin system could be involved in physiological or pathological reductions in sperm numbers. Tagging of a spermatozoon with ubiquitin could reduce sperm numbers either by directly activating apoptotic pathways within the sperm cells, by making defective cells prone to phagocytosis or liquefaction in the epididymis, or both.
The issue of sperm phagocytosis remains controversial despite ultrastructural evidence of phagocytosis occurring in the normal epididymis of horses [46] and bulls [47]. Our data show that stallion sperm often become engulfed by the apical stereocilia of the epididymal epithelium, a possible first step toward phagocytosis. However, sperm nuclei were not observed deep within the basal cytoplasm of EECs and thus it is difficult to determine whether or not whole spermatozoa actually are phagocytosed in any significant quantities. On the other hand, a growing number of reports are showing that the percentage of primary sperm defects (mainly sperm head abnormalities) decreases during epididymal transit, thus suggesting that some spermatozoa are selectively removed by the epididymis (e.g., [48–50]). In contrast, some secondary sperm tail abnormalities could actually be induced in the epididymis [51]. We have documented in detail the ability of cultured, bovine epididymal epithelial cells to phagocytose bull and rhesus monkey spermatozoa in vitro [9, 16]. If sperm liquefaction or intraluminal phagocytosis occur within the normal epididymis as proposed [52, 53], they may lower the number of defective spermatozoa during epididymal transit. Taking into consideration that the major components of the ubiquitin-dependent proteolytic pathway, including ubiquitin C-terminal hydrolase PGP 9.5, ubiquitin-conjugating enzyme E2, and monomeric ubiquitin are present in epididymal sperm ([9–11]; this study) and in seminal plasma [29], one could envision that at least some percentage of defective sperm become liquefacted and their parts endocytosed during epididymal passage. A similar process has been previously described for sperm cytoplasmic droplets in the caput epididymis [9, 12]. From previous studies [9, 24] it appears that the ubiquitin-conjugating enzyme E2 is already present in spermatids and testicular spermatozoa, and that it may become activated, or unmasked in defective spermatozoa.
Thus far, ubiquitin appears to be a reliable, objective marker of sperm quality and perhaps infertility in men and bulls [8, 23]. In any andrological investigation, fertility evaluation relies heavily on highly subjective, light microscopic assessment of semen quality. The inherent variability in these evaluations could account for some of the inaccuracy of predicting potential fertility or infertility in clinical situations. Objective sperm quality markers, suitable for automated, objective semen analysis, are therefore sought after [54]. In horse breeding, the existing tests included in the Society for Theriogenology's stallion breeding soundness examination (BSE, [21]) fairly reliably identify stallions that can be categorized within the two extremes of outstanding fertility and complete sterility. However, these same tests have several shortcomings. First, they are not well suited for identifying the gradations of fertility that are observed in the majority of animals that fall somewhere between the two extremes. Second, several of the tests included in the BSE are highly subjective. Third, in some cases, stallions identified as satisfactory breeders fail to efficiently render mares pregnant. It is likely that some of these individuals possess genetic or molecular defects in their sperm that are not detectable with standard tests. For these reasons, stallion fertility evaluation could be greatly improved by the introduction of objective, molecular-based tests that could more sensitively separate gradations of stallion fertility. Our results suggest that differential ubiquitination of stallion spermatozoa occurs in the epididymis, thus raising the possibility that a sensitive, objective, ubiquitin-based assay could be developed as a test for fertility in the horse.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Peter Sutovsky, Assistant Professor, University of Missouri-Columbia, S141 ASRC, 920 East Campus Drive, Columbia, MO 65211-5300. FAX: 573 884 5540; e-mail: sutovskyp@missouri.edu
Received: 8 March 2002.
First decision: 4 April 2002.
Accepted: 16 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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