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From the * J. O. Almquist Research Center,
Department of Dairy and Animal Science, and
The Huck Institute of Life Sciences, The
Pennsylvania State University, University Park, Pennsylvania.
| Correspondence to: Dr Gary J. Killian, J. O. Almquist Research Center, Department of Dairy and Animal Science, The Pennsylvania State University, University Park, PA 16802 (e-mail: lwj{at}psu.edu). |
| Received for publication December 1, 2005; accepted for publication March 21, 2006. |
 |
Abstract |
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 Top Abstract Material and MethodsResultsDiscussionReferences |
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0) and low-fertility
sires (n = 8; PD < 0) and were also used as independent variables in
regression analysis. Proteins were identified by capillary liquid
chromatography–nanoelectrospray ionization–tandem mass
spectrometry. An average of 118 spots was detected in 2-D maps of the CEF, but
we were unable to distinguish any protein that was expressed only in
high-fertility or in low-fertility bulls. However, the amount of
-L-fucosidase 2 and cathepsin D was 2.3- and 2.4-fold greater
(P < .05) in high-fertility than in low-fertility bulls,
respectively. Conversely, the intensities of 3 isoforms (24–27 kd; pl
6.3–5.8) of prostaglandin D-synthase (PGDS) were from 3.2- to 2.2-fold
greater in low-fertility sires (P < .05). An empirical regression
model established that a significant proportion (R2 =
0.72; P < .0001) of the variation in fertility scores (PD values)
was explained by the intensities of cathepsin D and 1 isoform of PGDS (24 kd;
pl 6.3). Thus, multiple proteins present in the CEF are potential biomarkers
of fertility in high-use, mature Holstein bulls.
Key words: -L-fucosidase, cathepsin D, epididymis, mass spectrometry, prostaglandin D-synthase, sperm
Previously, we utilized catheterization of the vasa deferentia to recover secretions from accessory sex glands and cauda epididymis of mature Holstein bulls (Henault et al, 1995). These surgically altered bulls had documented fertility based on artificial insemination (AI) of large numbers of cows and therefore provide a unique resource to study molecular indicators of fertility of the normal male. Using such a model, we have recently shown that proteins of the accessory sex gland secretions identified as spermadhesin Z13, osteopontin, BSP 30 kd, and phospholipase A2 were related to fertility indexes of sires (Moura et al, 2006). Thus, given the important events that take place in the epididymis and their potential effects on sperm function, we tested the hypothesis in the present study that proteins of the cauda epididymal fluid (CEF) are associated with fertility scores of mature dairy bulls.
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Material and Methods |
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TopAbstractMaterial and MethodsResultsDiscussionReferences |
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Information about nonreturn rates (NRRs) of Holstein bulls was provided by AI cooperatives in the northeastern United States and was based on the number of cows that did not return to service 60 days after the first insemination. Compensation for small variations among data sets obtained from different AI centers was achieved by expressing the fertility index of each bull as the percentage point deviation (PD) of its NRR from the average NRR of all bulls in a given AI unit (Killian et al, 1993). In this case, a bull with a PD of 0 had average fertility, relative to the average for the population it came from. The number of services using frozen semen from each bull ranged from 1074 to 52 820, and sires had PDs ranging from +7.7% to -6.6%. Bulls with PD values of 0 or higher were considered as high fertility (n = 12), and bulls with PD values lower than 0 were considered as low fertility (n = 8).
Eletroctrophoresis
Samples of CEF pulled from liquid nitrogen were thawed at room temperature
and centrifuged at 10 000 x g (60 minutes at 5°C). The
supernatant was then assayed for protein content
(Lowry et al, 1951) by using
bovine serum albumin as standards and aliquots frozen at -80°C. For
electrophoresis, samples were thawed at room temperature and subjected to 2-D
electrophoresis according to a previously described procedure
(Killian et al, 1993).
Briefly, isoelectric focusing was carried out in tube gels (Bio Rad)
containing a mixture of ampholytes with pH ranging from 3 to 7 (0.4 mL) and 3
to 10 (0.1 mL; Serva, Heidelberg, Germany). Samples of CEF containing 500
µg of protein were brought to a volume of 100 µL with a solution of
ß-mercaptoethanol, urea, and the same ampholytes used in the gels. Gels
were then subjected to 200 V for 15 minutes, 300 V for 30 minutes, 400 V for
30 minutes, 375 V for 16 to 18 hours, and 800 V for 1 hour. After focusing,
gels were removed from the tubes and placed on stacking gels that had been
prepared on the top of gels containing a linear gradient of acrylamide
(10%–17.5%). Standards from 66 to 14 kd were also used (Sigma Chemical
Co, St Louis, Mo). Gels were stained with Coomassie brilliant blue R-250;
destained in a solution of methanol, acetic acid, and deionized distilled
water; and scanned with a GS-670 imaging densitometer (Bio Rad). Images saved
as TIFII files were analyzed by PDQuest software (Bio Rad) as previously
described (Moura et al, 2006).
Briefly, for the set of 20 images of CEF gels, a single master gel was
generated by the software representing the best pattern of spots in the
samples. Additional spots consistently present in some gels were also added to
the master so that they could be matched to all samples. Proteins in key
regions of the gels were used as landmarks, and final matching of spots was
achieved after several rounds of extensive comparisons. Control of spot
matches was checked in each gel with the respective pattern in the master.
Protein quantities were given as parts per million (ppm) of the total
integrated optical density of the spots, according to PDQuest.
Protein Identification
Proteins separated by 2-D SDS-PAGE and selected by PDQuest were subjected
to in-gel trypsin digestion (Koc et al,
2001). Excised gel pieces were washed 3 times with 100 µL of
ammonium bicarbonate (25 mM) and dehydrated with 100 µL of acetonitrile
(50%) and dried in a speed vacuum. They were then incubated overnight at
37°C with trypsin (12.5 ng/µL in 25 mM ammonium bicarbonate). Peptides
were then extracted twice with 25 µL of formic acid (5%) for 20 minutes.
The extracts were dried in a speed vacuum again and resuspended in 10 µL of
5% acetonitrile with formic acid (0.1%). Tryptic digests were analyzed by
capillary liquid chromatography–nanoelectrospray ionization–tandem
mass spectrometry (CapLC-MS/MS). A Micromass Q-Tof API US mass spectrometer
coupled with a Waters CapLC high-performance liquid chromatography (HPLC) unit
(Waters Co, Milford, Mass) was used for the analysis
(Abbas et al, 2005). The
proteolytic digests (1–5 µL) were injected into solvent A
(acetonitrile–water–formic acid mixture of 5:95:0.1) supplied by
the auxiliary pump of the capillary HPLC unit and trapped in a Waters Symmetry
300 column (C-18, 5-µm film; 0.3 x 5 µm) for on-line desalting and
preconcentration (Abbas et al,
2005). After washing for 3 minutes with solvent A at 20 µL/min,
trapped peptides were then back flushed with the gradient solvent flow onto
the analytical column—a Dionex PepMap fused silica capillary column
(C-18 5 µm, 0.075 x 150 mm)—with a 10-port switching valve. The
analytical column was run with a gradient (5%–42% solvent B,
acetonitrile–water–formic acid mixture of 95:5:0.2, for 44
minutes). The mass spectrometry was calibrated with Glu-Fib product ion
fragments as needed to maintain mass accuracy within 10 ppm. The Q-Tof mass
spectrometer was operated to acquire MS/MS of tryptic peptides in
data-dependent acquisition mode for precursor ion selection by using
charge-state recognition and intensity threshold as selection criteria with
MassLynx 4.0 SP1. To carry out the tandem mass spectrometric data acquisition,
a survey scan (2 seconds) over the m/z of 400 to 1500 was performed.
From each survey scan, up to 4 most intense precursor ions based on the
selection criteria were selected for tandem mass spectrometry to obtain the
production spectra resulting from collision-induced dissociation in the
presence of argon. The product ion spectra (6–8 seconds) collected were
processed with Protein Lynx Global Server 2.1 and were converted to peak list
text files for database searching. To identify the proteins, MS/MS ion
searches were performed on the processed spectra against a locally maintained
copy of the National Center for Biotechnology Information nonredundant (NCBI
NR) database by using MASCOT Daemon and search engine (Matrix Science Inc,
Boston, Mass). The searches were made with the assumption that there was 1
maximum missed trypsin cleavage and that peptides were monoisotropic and
oxidized at methionine residues (variable modifications) and
carbamidomethylated at cysteine residues (fixed modifications). Peptide mass
tolerance and fragment mass tolerance were initially set to 1.2 and 0.6
daltons, respectively, for MS/MS ion searching; however, peptide mass values
were ensured to be within 0.1 dalton (typically less than 0.05 dalton) when
manually reviewing MASCOT search results.
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0) and low-fertility bulls (n = 8; PD < 0) were
evaluated by t test (Statistical
Analysis Systems, 2003). Protein quantities that significantly
differed among these bulls were used as independent variables to determine the
extent by which the percentage PD of bull NRRs (PD values) was explained by
regression equations. Criteria used to evaluate the regression models were
R2, Mallow's C(p) value, and multicollinearity
(Statistical Analysis Systems,
2003). |
Results |
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TopAbstractMaterial and MethodsResultsDiscussionReferences |
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-L-fucosidase 2,
cathpesin D, and isoforms of prostaglandin D-synthase (PGDS) in the CEF. The
intensity of -L-fucosidase and cathepsin D was 2.3- and 2.4-fold
greater in high-fertility bulls than in low-fertility bulls, respectively
(P < .05; Figure 1;
Table). Conversely, the average intensity of 3 isoforms of PGDS was from
3.2-fold (spot 1) to 2.2-fold (spot 2) greater in low-fertility bulls (P, .05;
Figure 2; Table). Spot
"A" of the PGDS train was not associated with fertility indexes.
Analysis of tryptic peptides from that particular spot generated 2 matches
with significant scores, though with different magnitudes: PGDS, with score
53, and apolipoprotein A-I, with score 318 (Table).
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A regression model including cathepsin D and isoform # 3 of PGDS (24 kd; pI 6.3) as independent variables generated the equation PD = 44.79 + 0.0016 x (cathepsin D) - 4.6 x log(PGDS_spot 3), where R2 = 0.72 and P < .0001. PD represents the percentage PD from the average NRR of bulls, and variables in parentheses are the integrated optical density of the respective spots in the CEF gels, as calculated by PDQuest.
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Discussion |
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TopAbstractMaterial and MethodsResultsDiscussionReferences |
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-L-fucosidase 2, cathepsin D, and PGDS were expressed at different
levels in bulls of high and low fertility. Moreover, an empirical regression
model established that a significant proportion (R2 =
0.72) of the variation in fertility scores was explained by cathepsin D and 1
isoform of PGDS. Holstein bulls of this study represent a population of
tested, mature sires that have been extensively used for AI with frozen semen.
Inseminations are performed with similar sperm numbers, and differences in
fertility scores among bulls were not correlated with results of routine semen
analysis conducted at AI centers. Animals were reproductively normal, and
although selected primarily for their ability to transmit genetic traits
linked to milk yield, these sires have also been screened for fertility merit.
To our knowledge, this is the first study to report that certain cauda
epididymal proteins are related to fertility of bulls with these
characteristics.
We have previously reported the existence of associations between bull
fertility and accessory sex gland components
(Moura et al, 2006). This
previous report and the findings of the present study suggest that molecular
markers of male fertility are associated with both epididymal sperm physiology
and postejaculation events regulated by accessory sex gland components.
Studies have reported correlations between NRRs of bulls and seminal plasma
proteins that are originally synthesized by the accessory sex glands and
epididymis (Killian et al,
1993; Gerena et al,
1998; McCauley et al,
2001). However, fluid produced by the epididymis is diluted about
8- to 10-fold when mixed with accessory sex gland secretions at ejaculation
(Gerena et al, 1998). This
makes it difficult to accurately identify epididymal proteins in the seminal
plasma milieu, particularly those secreted in low abundance or if they are
also secreted by other organs, such as the accessory sex glands. Unpublished
results from our laboratory show that most of the minor spots seen in the CEF
2-D maps are greatly diminished or undetectable in Coomassie-stained seminal
plasma gels or when the CEF is mixed 1:8 with the accessory sex gland fluid
from the same bulls.
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From 118 spots detected in the CEF maps, 26.3% were present in all gels. Despite the relatively small number of spots present in all members of the match set constructed by PDQuest, there was no protein expressed either in only high- or low-fertility bulls. These results for CEF are similar to those obtained with accessory sex gland fluid 2-D maps (Moura et al, 2006), when it was shown that no proteins were expressed solely in bulls with the highest fertility scores and absent in those with the lowest scores, or vice-versa.
Because most proteins from the rete testis are not present in the milieu of the epididymis lumen (Olson and Hinton, 1985; Dacheux et al, 1989), there is a general assumption that proteins of the epididymal fluid are mainly the product of the epididymis itself. Numerous proteins have been detected in the epididymal milieu of mammalian species (Cornwall et al, 2002; Dacheux and Dacheux, 2002) but the exact roles of most of them in sperm maturation are yet to be determined (Gatti et al, 2004). The cases of fertility-related proteins identified in the present study are not exceptions; therefore, the explanation for the links between their expression and fertility is still a matter of hypothesis.
As mentioned, both -L-fucosidase and cathepsin D were more
predominant in high-fertility bulls. -L-fucosidase catalyzes the
hydrolysis of -L-fucose, which is part of oligosaccharide moieties of
glycoproteins, glycolipids, and glycosaminoglycans. The enzyme detected in the
CEF by tandem mass spectrometry (54.4 kd; pI 6.6) appears similar to a soluble
form that has been found in seminal plasma (56 kd) of humans
(Alhadeff et al, 1999;
Khunsook et al, 2002) and
epididymal fluid of bulls (Jauhiainen and
Vanha-Perttula, 1986). -Fucosidase secreted in the
epididymal fluid may participate in the modification of carbohydrate moieties
of sperm membrane proteins during epididymal transit. This protein has been
found suppressed in the seminal plasma of bulls with elevated percentage of
abnormal sperm (Jauhiainen and
Vanha-Perttula, 1986), and dogs with insufficiency of
-fucosidase also have impaired sperm maturation
(Veeramachaneni et al,
1998).
Cathepsin D is a cysteine peptidase that degrades proteins commonly found
as part of the extracellular matrix and is involved in tissue growth and
remodeling (Dickinson, 2002).
A study has reported that seminal plasma of oligo- and azoospermic men has
less cathepsin D than their normal counterpart
(Dandekar and Harikumar, 1997).
In the stallion, cathepsin D is synthesized mainly in caput and corpus
epididymis (Fouchécourt et al,
2000), and in this regard it may participate in proteolytic
remodeling of membrane components of sperm during epididymal transit. Such
modifications, like those suggested to be mediated by -fucosidase, may
contribute to the fertilizing capacity of epididymal sperm
(Chapman and Killian, 1984;
Cuasnicú et al, 2002;
Sullivan et al, 2005).
In contrast to what was found with -L-fucosidase and cathepsin D, 3
isoforms of PGDS were significantly higher in bulls with low fertility scores.
An additional isoform of PGDS apparently comigrated with another protein
identified as apolipoprotein A-I, and, coincidentally, the intensity of that
particular spot did not show any association with bull fertility.
Apolipoprotein A-I is a component of high-density lipoproteins
(Sparrow et al, 1992) and is
suggested to be involved in sperm cholesterol efflux and capacitation
(Manjunath et al, 1989;
Thérien et al, 1997).
Previous studies have reported that PGDS detected in the seminal plasma was
positively related to sperm quality in men
(Olsson, 1975;
Diamandis et al, 1999;
Leone et al, 2001) and field
fertility of dairy bulls (Killian et al,
1993; Gerena et al,
1998; Fouchécourt et
al, 2002). The apparent contrast between the results presented
here (with CEF) and those previously reported by our own laboratory and other
authors (with seminal plasma) are interesting but not necessarily in conflict.
PGDS appears in the CEF as a series of spots with 24 to 27 kd and pI from 6.3
to 5.8, based on the analysis from tandem mass spectrometry and Western blots
(data not shown). These results are similar to what was detected in the rete
testis fluid of bulls (Gerena et al,
1998) and epididymis of rams and stallions
(Fouchécourt et al,
1999). Concentration of PGDS is much lower (eightfold) in the
seminal plasma than in the CEF (Gerena et
al, 1998), and the protein that was originally related to
fertility scores of bulls by Killian et al
(1993) was detected as a
single 26-kd (pI 6.2) spot in the seminal plasma 2-D maps. These facts
undoubtedly emphasize that PGDS spots from seminal plasma and CEF gels are
different variables and that their relative importance in explaining
variations in fertility scores may be different as well. Changes occur in both
molecular weight and pI of PGDS isoforms during epididymal transit in the ram
and stallion, and such changes may alter the attributes of this protein
(Fouchécourt et al,
1999). It is also possible that modifications occur in epididymal
PGDS when it is mixed with accessory sex gland secretions at ejaculation.
PGDS acts as a lipophilic ligand-binding protein after secretion (Urade and Hayaishi, 2000). It has the ability to bind molecules such as testosterone, thyroid hormones, and retinoids (Urade and Hayaishi, 2000; Leone et al, 2002), but its functions in the epididymis are unclear. As we have previously suggested (Gerena et al, 2000), if one considers PGDS as a retinoid-carrying protein, it is important to mention that retinoids can be detrimental to phospholipid membranes and their permeability to ions (Stillwell and Wassall, 1990). Also, PGDS binds to docosahexanoic acid, a major polyunsaturated fatty acid of human sperm (Alvarez and Storey, 1995) that regulates membrane fluidity and permeability (Stillwell and Wassall, 2003). The net concentration of docosahexanoic acid in sperm during epididymal transit is important because its loss prevents peroxidative damage to sperm, but a minimal amount is also required to maintain membrane fluidity and sperm mobility (Ollero et al, 2000). Thus, PGDS could influence male fertility by mediating the action of hydrophobic molecules on sperm during epididymal transit or cauda epididymal storage.
In conclusion, we have presented empirical evidence that certain cauda epididymal proteins are significant molecular indicators of bull fertility. It is known that secretions of the epididymis contain biologically active molecules (Dacheux and Dacheux, 2002; Gatti et al, 2004), and the present study confirms that only a select group of those molecules is linked to a fertility phenotype of proven, high-use dairy bulls. The distinct expression of these proteins may have been favored by the type of selection applied to dairy sires.
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Footnotes |
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