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From the * Division of Animal Sciences and the
Departments of Obstetrics & Gynecology,
University of Missouri-Columbia, Columbia, Missouri; the
Animal Science Department, California
Polytechnic State University, San Luis Obispo, California; and the
Department of Veterinary and Animal Sciences,
Clemson University, Clemson, South Carolina.
| Correspondence to: P. Sutovsky, Division of Animal Sciences, University of Missouri-Columbia, Columbia, MO 65211 (e-mail: SutovskyP{at}missouri.edu). |
| Received for publication April 7, 2006; accepted for publication August 21, 2006. |
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Abstract |
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Key words: Bull, sperm, PAF, fertility
The platelet activating factor is an important phospholipid mediator in reproduction, affecting sperm motility (Minhas et al, 1991), sperm-oocyte interaction (Angle et al, 1993), and pregnancy establishment (Tiemann et al, 2001; Yang et al, 2002). Recent data indicate that PAF is associated with sperm motility and fertility (Roudebush and Diehl, 2001). The pleiotropic functions of PAF are mediated through a membrane-bound receptor (PAFr). Characterization of the PAFr gene and its role in mediating PAF actions implicated in reproduction has been discussed (Honda et al, 1991). It is not surprising that PAF and PAFr are present in the mammalian spermatozoa (Kuzan et al, 1990; Reinhardt et al, 1999; Roudebush et al, 2000). The PAFr-encoding mRNA and PAFr protein are found in a variety of other cells and tissues, including oocytes (Stojanov and O'Neill 1999), preimplantation and peri-implantation embryos, endometrium (Tiemann et al, 2001; Yang et al, 2002); leukocytes, platelets, Kupffer cells, epithelial cells, and several internal organs (Ishii and Shimizu, 2000). The expression patterns of PAFr are tissue-dependent and species-specific (Ishii and Shimizu, 2000; Yang et al, 2002). Our hypothesis postulates that high levels of PAF and PAFr correlate positively with sperm motility and normal morphology, and thus PAF/PAFr can serve as a measurable fertility marker in bulls, boars and humans. It is expected that the highly fertile subpopulations of spermatozoa should have a higher level of PAFr expression which enhances utilization of PAF and is associated with specific motility parameters and subsequently higher fertility.
Ubiquitin (UBI) is a small protein involved in the degradation of either damaged or normal proteins that are targeted for turnover (reviewed by Hershko and Ciechanover, 1998; Pickart, 1998). A molecule of ubiquitin consists of 76 amino acids (AA), with a molecular mass of 8.5 kd. Ubiquitin attaches covalently to other proteins via its C-terminal, glycine residue (G-76) forming an isopeptide bond with an internal lysine residue on the substrate protein. Similarly, UBI molecules can bind to each other, forming multiubiquitin chains of various lengths linked through G-76 residue of one molecule and an internal lysine residue (K-7, 27, 29, 31, 33, 48, 63) of the other UBI molecule. A single UBI molecule, which can exist free or attached to a substrate protein, is referred to as monoubiquitin. Chains of higher orders are commonly referred to as polyubiquitin chains. Based on such nomenclature of UBI chains, the attachment of single or multiple ubiquitin molecules to the substrate is referred to as mono-, di-, tri-, tetra-, or polyubiquitination. The basic function of ubiquitination is the degradation of outlived or damaged proteins (Hershko and Ciechanover, 1998). The common signal for protein degradation is tetraubiquitination, that is, the formation of a 4-UBI molecule-long chain on a substrate protein. Tetra-ubiquitin and polyubiquitin chains target and dock the tetraubiquitinated substrate towards the 26S proteasome, a multisubunit protease common in most eukaryotic cells (Pickart, 1998). Although monoubiquitination does not target substrate protein towards 26S proteasome, it has been implicated in the endocytosis of membrane receptors and possibly other proteins (Strous and Govers, 1999) and degradation by a lysosome.
Ubiquitin has been implicated in the processing of defective epididymal spermatozoa (UBI-dependent sperm quality control; Sutovsky et al, 2001a), a process that may also involve specific epididymal secretory glycoproteins (Olson et al, 2004). The secretion of UBI by the epididymal epithelium was first suggested by Santamaria et al (1993) and supported by others (Sutovsky et al, 2001a; Hermo and Jacks, 2002; Jones, 2004). Our recent studies document that an active enzymatic system for UBI-substrate conjugation and recycling exists in the bovine epididymal fluid (Baska and Sutovsky, 2005). Lippert et al (1993) detected UBI in human seminal plasma, but found no correlation with fertility or sperm quality. In contrast, flow cytometric measurements of UBI associated with human sperm surface correlated with various measures of sperm quality in men with known and unexplained causes of infertility (reviewed by Baska and Sutovsky, 2005). Similar results were reported in bulls (Sutovsky et al, 2002), stallions (Sutovsky et al, 2003), and boars (Lovercamp et al, 2006). The flow cytometric sperm-ubiquitin tag immunoassay (SUTI; Sutovsky et al, 2001b) has been developed for objective, automated semen evaluation in infertility patients and farm animals. Immunocytochemical detection of UBI by SUTI reveals that cell surface ubiquitination occurs in ejaculated bull spermatozoa with gross morphological abnormalities (Sutovsky et al, 2001a), but also in the seemingly morphologically standard spermatozoa with intrinsic defects such as postapoptotic or necrotic DNA fragmentation (dual ubiquitin-TUNEL assay; Sutovsky et al, 2002). The present study explores the relationship between sperm ubiquitination and expression of fertility-associated sperm proteins, namely the PAFr, with particular focus on the application of objective flow cytometric assays in the breeding soundness evaluation in yearling sires.
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Materials and Methods |
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Reproductive Soundness Exam
Semen from yearling bulls was collected using an electroejaculator
(ElectroJac 5; Neogen Corp, Lexington, Ky). At the time of collection each
bull was given a reproductive soundness exam. Scrotal circumference was
measured; scrotum, sheath, and vesicular glands were palpated; and semen was
collected. Bulls were divided into 4 groups based on the outcome of vesicular
gland palpation (group 1 = excellent, group 2 = good, group 3 = minor
inflammation, group 4 = major inflammation), and into 5 groups based on
semen-WBC content (group 0 = less than 20 WBC per field in a Makler counting
chamber under 20x primary lens magnification, group 1 = more than 20 WBC
per field in light microscope at 20x lens magnification, group 2 =
paired WBC present, group 3 = clumped WBC present, group 4 = amorphous debris
and/or puss present). Bulls that produced samples with a high white blood cell
count were treated with antibiotics and were reevaluated every 3 days until
they were clean or had been through 5 treatments. The final evaluation was
based on BSE (above) and semen analysis (below). Bulls were divided into 3
groups based on overall satisfaction of evaluation: 1) satisfactory (WBC group
of 0, minimum 50% motility and less than 40% abnormal spermatozoa; 2) not
satisfactory (WBC group 1–4 and/or less than 50% motility and/or more
than 40% abnormal sperm); 3 = marginal (sexually immature; high % of secondary
sperm abnormalities).
Semen Analysis
Based on sample concentration or density, the semen was either evaluated
raw (not diluted) or diluted in 0.01 mol phosphate buffered saline (PBS with
BSA, Sigma P-3688, 0.138 mol NaCl, 0.0027 mol KCl, pH 7.4) to one of the
following dilutions: 1:1, 4:1, 8:1, 12:1, saline solution to semen. The
temperature of the saline was maintained at 37°C using a slide warmer.
Once the fresh samples were appropriately diluted, they were evaluated for
motility, morphology, and concentration related to fertility using an inverted
light microscope (Olympus Inc, Center Valley, Pa) at 20x and a Makler
counting chamber (Irvine Scientific, Santa Ana, Calif). Approximately 15 µL
of the semen sample was placed on the center of the Makler chamber and used to
evaluate sperm parameters. To estimate the sperm motility in a sample, the
number of progressively motile spermatozoa moving in a forward direction in a
3x3 grid on the Makler chamber was counted. Concentration was calculated
by counting the total number of sperm cells in 10 squares on the Makler
chamber, then counting the total number of sperm cells contained in a
different set of 10 squares, and an average concentration value was determined
from those respective values. Morphology was determined by evaluating 100
spermatozoa per sample and assigning to them either the normal
characteristics, primary abnormalities, or secondary abnormalities as defined
by the Society for Theriogenology
(Chenoweth et al, 1993). Based
on these numbers, the percent of normal sperm morphology was calculated. In
addition to these primary sperm parameters, concentration of sperm per
ejaculate and volume of semen were also calculated for each sample.
Sample Handling and Fixation
Raw semen was spun in a 15-mL Falcon tube at 400 x g for 4
minutes, then the supernatant was decanted. Sperm TL medium (1.84 mL lactic
acid, 2.920 g NaCl, 1.190 g HEPES, 1.050 g NaHCO3, 0.115 g KCl,
0.0174 g Na2HPO4, 0.005 g phenol red, 0.155 g
CaCl2, 0.40 g MgCl2 per 500 mL volume; pH 7.0;
osmolarity 290–300 mOsm) was added to bring volume to 8 mL, then spun at
400 x g for 4 minutes, and the supernatant was decanted. Three
milliliters of 2% formaldehyde (10% Ultra Pure EM Grade formaldehyde from
Polysciences, Warrington, Pa, was diluted in DPBS from Sigma, St Louis, Mo)
was added and the sample incubated at room temperature for a minimum of 40
minutes, then spun at 400 x g for 4 minutes, and the
supernatant was decanted. After adding 3 mL DPBS and spinning at 400 x
g for 4 minutes, the supernatant was decanted. Enough DPBS to bring
the volume to 2 mL was added and the sample split into two 1-mL NUNC tubes
(Fisher Scientific, Pittsburgh, Pa). Samples were labeled, wrapped in
parafilm, and stored in refrigerator. Fixed samples were shipped on wet ice to
University of Missouri, Columbia, Mo, for flow cytometric and microscopic
analyses completed within 4 weeks after collection.
Sample Processing for Flow Cytometry
Processing was performed in 15-mL Falcon tubes using a clinical bench-top
centrifuge with fixed rotor at 350 x g. All solutions were
filtered by using 0.22-µm syringe filters (Fisher Scientific) or, for large
volumes, 0.22-µm Stericub disposable filtration bottles (Millipore Corp,
Billerica, Mass). For flow cytometry processing, 200 µL of each fixed semen
sample in PBS were transferred to a 15-mL Falcon tube and collected by
centrifugation. Supernatants were removed by using a glass Pasteur pipette
attached to a small vacuum pump and samples were resuspended in 100 µL of
blocking and permeabilizing solution composed of 2% normal goat serum (NGS;
Sigma) and 0.1% Triton-X-100 (TX-100; Sigma) in PBS. After 1 hour of blocking,
sperm pellets were collected by centrifugation.
In trial 1 (n = 156), sperm pellets were incubated with 100 µL of a rabbit affinity purified serum raised against a synthetic peptide corresponding to amino acids 1–17 from the human PAF receptor (MEPHDSSHMDSEFRYTL; Nakamura et al, 1991; purchased from Cayman Chemicals, Ann Arbor, Mich; dilution 1/100 in PBS+1% NGS). After an overnight incubation at 4°C, a goat anti-rabbit IgG conjugated to FITC (GAR-FITC; Zymed) was added at dilution of 1/80 directly to 15-mL Falcon sample tubes containing a mixture of spermatozoa in PBS with anti-PAFr antibody and allowed to incubate for 40 minutes at room temperature under an aluminum foil wrap to reduce fluorochrome quenching. Omitting sample washing between first and second antibody incubation accelerated the procedure and reduced sample losses during centrifugation and supernatant removal without causing increased background fluorescence (comparison data not shown). Blank negative control samples were prepared by substituting a nonimmune rabbit serum (Sigma) for the anti-PAFr antibody. After the last incubation, 5 mL of filtered washing solution composed of filtered PBS with bovine serum albumin (BSA, 3 mg/mL; Sigma) were added to each sample. Sperm pellets were collected after 5 minutes washing by centrifugation and 500 µL of filtered PBS with no other additives was added to each tube. Samples were transferred into 5-mL cell strainer-cap flow cytometry tubes (#352235; Falcon brand; Becton Dickinson Labware, Franklin Lakes, NJ) and stored in dark at 4°C until the analysis, performed on the same day. The quality of processing was monitored randomly by epifluorescence microscopy of multiple processed samples.
In trial 2 (n = 88), samples of 200 µL of sperm-PBS suspension were pelleted by centrifugation, permeabilized, and blocked as described above. First, overnight incubation was with a mixture of rabbit polyclonal anti-PAFr antibody (dil 1/100), described above, and mouse monoclonal anti-UBI antibody MK-12-3 (dil 1/100; MBL, Nagoya, Japan; Sutovsky et al, 2001a). Secondary antibodies, added directly to sperm suspensions at a dilution of 1/80, included red fluorescent goat-anti-mouse-TRITC to detect ubiquitin and goat anti-rabbit-FITC to detect PAFr (both antibodies from Zymed Inc, San Francisco, Calif). Double-negative controls samples were generated by the replacement of anti-ubiquitin and anti-PAFr antibodies with a mixture of nonimmune rabbit and mouse sera (purchased from Sigma).
Flow Cytometry Data Acquisition and Analysis
Samples were analyzed by using FACS Scan Analyzers (Becton Dickinson) at
488 nm for FITC emission and at 568 nm for TRITC. In PAFr analysis (trial 1),
only the FITC channel was acquired. In dual analysis (trial 2), both channels
were acquired simultaneously. Relative levels of red (UBI) and green (PAFr)
fluorescence in 10 000 individual cells were recorded for each sample. A
sample of PBS buffer used for labeling and washing was used to adjust the
output to suppress nonspecific fluorescence and to calibrate the cytometer; a
blank sample from a corresponding sire, labeled with secondary antibody alone,
was measured before each antibody-labeled sample. Scatter diagrams of visible
light and histograms of relative fluorescence were generated for each channel
(red/TRITC and green/FITC). The median values of relative fluorescence
(UBI/PAFr median) were recorded for each sample. Median values are relative
(no units), represent the channel number about which half the cells are dimmer
and half the cells are brighter, and increase with the increase in the number
of brighter cells. Dual analysis was performed in a dual acquisition mode. In
the resultant dual flow cytometric diagram, the x-axis represented the green,
PAFr-induced fluorescence, and the y-axis was the red, ubiquitin-induced
fluorescence. The data were entered into Microsoft Excel tables and analyzed
by statistical analysis tools and the SAS (version 8.2) statistical package.
Pearson's correlation coefficients (r) and P values were
calculated for PAFr and UBI medians, % PAFr/UBI-positive cells, and semen
field analysis data. Scatter diagrams were drawn and supplemented with
regression lines. Single-factor analysis of variance (ANOVA) was used to
determine if there is a significant degree (P < .05) of variation
among individual sperm samples.
Immunofluorescence
Dual detection of PAFr and UBI was performed by using a described procedure
(Sutovsky, 2004). Sperm samples were mounted onto poly-L-lysine coated
coverslips and fixed in 2% formaldehyde in 0.1% phosphate buffered saline
(PBS) for 40 minutes. Samples were washed, permeabilized for 40 minutes with
0.1% TX-100, then blocked in 5% NGS in PBS for 25 minutes. Samples were then
incubated for 40 minutes with a mixture of mouse monoclonal anti-ubiquitin IgG
(dil 1/100; MK12-3; MBL, Nagoya, Japan) and rabbit-anti-PAFr serum (dil 1/100;
Cayman Chemicals), followed by a brief wash in PBS and incubation for 40
minutes with a mixture containing goat anti-rabbit IgG-TRITC (Zymed, dil
1:80), goat-anti mouse IgG-FITC (Zymed, #81-6114; dil 1:80) and DNA-stain DAPI
(#D1306; Molecular Probes, Eugene, Ore). In some experiments, the colors were
reversed (ie, PAFr-FITC and ubiquitin-TRITC detection). After second antibody
incubation, coverslips were washed in PBS and mounted on microscopy slides in
VectaShield mounting medium (#H-1000; Vector Laboratories, Inc, Burlingame,
Calif). Images were captured by using a Nikon Eclipse E800 microscope equipped
with a CoolSnap HQ CCD camera operated by the MetaMorph software.
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Results |
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Related to palpation results, the content of WBC was also evaluated and used to subdivide the bulls into 5 groups, ranging from those with no WBC (group 0) to those with large masses of amorphous debris and WBC clumps (group 4). Data were compared among all groups and between group 0 (no WBC) and combined groups 1–4 (WBC present). Both UBI and PAFr levels increased proportionally to the increased content of WBC (Tables 1B and 2B). The increases in PAFr (P = .03 in the first trial) and ubiquitin content (P = .03 in second trial) were statistically significant, while no significant differences between these groups were seen for sperm morphology. Consequently, there were few significant differences in PAFr and UBI levels among the 3 groups of bulls divided based on subjective sperm morphology (Tables 1C and 2C).
Based on motility division (70% motile sperm margin), a numerical increase in PAFr levels but a reduction in UBI levels was observed among semen samples with lesser sperm motility (Tables 1D and 2D). This reduced UBI measurement was attributable to the increased presence of a large proportion of UBI-negative WBC in samples with lesser motility, as discussed below. There was a statistically significant reduction in normal sperm morphology in samples with low motility in both trials (Tables 1D and 2D).
Measured scrotal circumference (SC) was compared with the expected SC based on body weight, and bulls were divided into 2 groups (acceptable SC and not acceptable SC). Both PAFr and UBI (Tables 1E and 2E) levels were increased in bulls with unacceptable SC. The increase in sperm ubiquitin levels was statistically significant (Table 2E). Surprisingly, normal morphology was slightly better in the bulls with unacceptable SC (Tables 1E and 2E).
The overall satisfaction of evaluation (Tables 1F and 2F; 1 = satisfactory, 2 = not satisfactory, 3 = marginal) favored bulls with high percentage of normal morphology, one of the major parameters impacting this categorization, while the PAFr and UBI measurements were numerically increased in the "marginal" group 3. Altogether, our data revealed that a measurable relationship exists between semen PAFr and UBI content in yearling bulls (Figure 1).
Correlation Analysis
Two trials were performed with some overlapping and some unique samples;
the high degree of correlation between PAFr measurements of the overlapping
subgroup (n = 84) in trial 1 and 2 (r = .71) illustrates the
repeatability of assay. Breed effects were not assessed, since 186/273 bulls
were Angus. The other 9 breeds were represented disproportionately (2–34
bulls/breed). Extensive statistical analysis was conducted to compare the
informative value of flow cytometric PAFr and UBI measurements with the value
of conventional semen and BSE workup. Analyses were conducted for all screened
animals divided by semen WBC content (Table
3), palpation results (Table
4), and satisfaction of evaluation
(Table 5). The most significant
correlations are depicted in Figure
2. We have found a close correlation between PAFr measurements
between Trials 1 and 2, demonstrating the repeatability of the assay
(Figure 2A and B). A positive
correlation between the percentage PAFr-positive cells and UBI-positive cells
in trial 2 indicated higher incidence of ubiquitinated, defective spermatozoa
in poor-quality semen samples with high PAFr content
(Figure 2C). Only a moderate
negative correlation was found between percentage normal sperm morphology by
conventional semen analysis and percentage PAFr/UBI positive cells by flow
cytometric evaluation (Figure 2D through
F). This can be explained by the known shortcoming of conventional
sperm morphology. Frequently, the ubiquitinated spermatozoa carry hidden
defects (eg, sperm DNA fragmentation;
Sutovsky et al, 2002) and
appear to be morphologically normal in conventional sperm morphology analysis.
Low correlation was also observed between sperm motility and PAFr/UBI values
(Figure 2G through I). Sperm
motility was the only semen characteristic that showed a substantial positive
correlation with percentage of morphologically normal spermatozoa in
conventional analysis (r = .53;
Table 4, all groups),
suggesting that conventional sperm morphology evaluation was confounded with
sperm damage introduced by sample processing. SC, thought to be related to
fertility, showed low correlation with sperm motility, conventional sperm
morphology, and flow cytometric PAFr/UBI levels
(Figure 2J through L). Sperm
concentration showed a negative correlation with percentage of
PAFr–positive cells in trial 1 but not in trial 2, where the cells with
medium-levels of PAFr and UBI seemed to show highest concentrations
(Figure 2M through O). Overall
a high degree of statistical correlation (r = .79 for all 88 bulls in
trial 2) was observed between semen content of PAFr and UBI
(Table 6).
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Immunofluorescence Analysis
Most morphologically normal spermatozoa displayed a distinct labeling with
anti-PAFr antibodies in the postacrosomal sheath of the sperm head and in the
principal piece of the sperm tail (Figure
3A–A''). A reduced immunoreactivity to anti-PAFr
antibodies was observed in the defective spermatozoa immunoreactive to
anti-UBI antibodies (Figure
3A–A''). Frequently, clusters of UBI-coated spermatozoa
were observed (Figure 3B and
C). While we initially expected that semen samples with high
leukocyte content and poor sperm morphology would display high UBI and low
PAFr levels, there was a strong positive correlation between ubiquitin and
PAFr in flow cytometric analysis. This discrepancy was reconciled by
immunofluorescence analysis revealing high expression of PAFr on the surface
of a subpopulation of leukocytes (Figure 3D
and E), which were common semen contaminants in bulls from
palpation groups 2–4 with poor sperm morphology and semen quality. This
is consistent with previous finding that platelets, monocytes, neuthrophils,
and B-cells, but not the resting T-cells and natural killer cells, express
PAFr (Muller et al, 1993). Neither PAFr nor UBI labeling were detected in
negative control samples processed with nonimmune rabbit and mouse sera and
appropriate secondary antibodies and fluorochromes
(Figure 3F). The presence of
ubiquitinated spermatozoa and PAFr-expressing WBC varied greatly among
individual sires (Figure 4).
Altogether, the findings obtained by immunofluorescence microscopy
corroborated the flow cytometric measurements (compare
Figure 1 and
Figure 3).
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Discussion |
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The present study identifies the WBC content as a major factor in the semen quality evaluation during BSE. While WBC content was reflected by the increased relative content of PAFr in semen, it had no effect on sperm morphology evaluated by conventional light microscopic analysis (Tables 1B and 2B). In contrast, semen samples with low WBC content showed a statistically significant reduction in semen UBI content, indicating the potential value of UBI-based semen evaluation for BSE. We attribute this discrepancy between light microscopic sperm morphology and sperm UBI measurements to the fact that anti-UBI antibodies bind to visibly anomalous spermatozoa as well as to spermatozoa that are seemingly normal by conventional light microscopic evaluation standards, but may carry intrinsic defects such as post-apoptotic or necrotic nuclear DNA-fragmentation (Sutovsky et al, 2002). In our dual immunofluorescence analysis, the ubiquitinated spermatozoa often lack visible morphological anomalies but display a reduced immunoreactivity to anti-PAFr antibodies (eg, Figure 3A-A").
The only parameter that showed substantial correlation with percentage of morphologically normal spermatozoa was sperm motility. Motility is perhaps the least informative semen characteristic with regard to semen quality and fertility in humans and farm animals, due mainly to a rapid motility decline after sample isolation and during sample processing for analysis (Drobnis, 1992; Eliasson, 1981). The fact that motility and morphology correlated positively in the present trial could be explained by high incidence of secondary sperm defects, such as sperm tail coiling, that are not necessarily reflective of primary sperm abnormalities generated by deviant spermatogenesis. Sperm tail coiling is rather an effect of cold shock and/or mixing of sperm with WBC-containing seminal plasma components contributed by sex accessory glands. In contrast, the levels of sperm surface expression of UBI are established in the individual cells during epididymal sperm maturation and thus are not altered by semen handling and processing technique.
In chute-side evaluation of trial 1 (Table 1F), only 21/156 bulls were identified as unsatisfactory, and 8/156 bulls were identified as marginal. In trial 2 (Table 2F), only 6/88 and 4/88 bulls were identified as unsatisfactory and marginal, respectively. While these bulls showed greater variability in the levels of PAFr, the average UBI values were not significantly different from the "satisfactory" group. This is most likely due to the fact that this category was greatly affected by the outcome of conventional light microscopic semen analysis, which will reliably account for the presence of WBC but likely miss many of the ubiquitinated spermatozoa without obvious morphological defects. We recorded a number of bulls with acceptable sperm morphology and field analysis criteria, but increased ubiquitin immunoreactivity.
Comparison of Figure 1C and D illustrates how antibiotic treatment reduced semen levels of both PAFr and UBI in a bull treated for inflammation without changing the percentage of morphologically normal spermatozoa that can be captured by conventional semen analysis. Bulls were further divided into 4 groups by scrotal palpation test, in which mean UBI and PAFR levels were elevated in groups 3 and 4 with pathological finding, as compared to groups 1 and 2 with excellent or good palpation result (Table 2A). This observation is not surprising, as the poor palpation result indicates inflammation and coincides with increased presence of WBC in semen. Inflammation will likely affect sperm maturation in the epididymis, a stage at which surface ubiquitination occurs in the defective spermatozoa (Sutovsky et al, 2001a). Ubiquitin indeed showed a robust negative correlation with sperm count and percentage normal morphology in groups 1 and 2 (Table 3).
Our data on expression of PAFr in both WBC and spermatozoa illustrate the complexity of objective semen evaluation based on protein markers not unique to male gametes. In this case, high expression of PAFr on WBC surface could have resulted in mistaken proclamation of high semen quality in young bulls which actually suffered from reproductive tract inflammation. Without the additional parameter of UBI expression, the value of PAFr as a fertility marker could also have been misinterpreted. It is thus likely that assays most useful for a large-scale objective semen analysis will have to screen concomitantly for more than 1 marker.
In summary, the present study shows that sperm WBC content in yearling bulls correlates with increased sperm PAFr and UBI content, but not with the results of a conventional sperm morphology analysis. It will be desirable to factor the WBC parameter into BSE determinations and to further develop objective semen quality assays that will distinguish between fertility marker expression in spermatozoa versus WBC. High UBI expression and poor sperm morphology correlate with reduced PAFr expression in bull spermatozoa alone, while high PAFr expression in semen contaminating WBC may reduce the value of PAFr as a lone fertility marker in young bulls. Dual PAFr-UBI semen analysis may provide more accurate fertility evaluation than the chute-side morphology-based system. We concluded that both the increased presence of ubiquitin and PAFr are indicators of poor semen quality and, if properly applied, could be useful as markers for automated bull semen analysis and breeding soundness evaluation. In general, UBI is a stronger indicator of sperm quality than PAFr, while PAFr is indicative of semen contamination with leukocytes. Leukocyte presence in semen of yearlings greatly affects the outcome of semen evaluation during BSE. Both conventional and automated semen evaluation could be more productive at a later age. Dual analysis could be further improved by combining the measurement of ubiquitin and PAFr fluorescence with DNA stains that will allow determining DNA content and ploidy of each measured cell, to assess separately the expression of PAFr and UBI in diploid contaminating WBC and haploid spermatozoa. Overall, sperm morphology and motility evaluation during BSE may be of limited value and could be replaced by semen marker-based analysis in the future.
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Acknowledgments |
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Footnotes |
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