Characterization of proteins in cryopreserved and non-cryopreserved seminal plasma of dairy bulls of dif-fering fertility

doi:10.4236/ojas.2011.12005

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Vol.1, No.2, 33-40 (2011) Open Journal of Animal Sciences

doi:10.4236/ojas.2011.12005

Characterization of proteins in cryopreserved and

non-cryopreserved seminal plasma of dairy bulls of

differing fertility

——Seminal plasma proteomics

J. F. Odhiambo1,2, R. A. Dailey1*

1Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, USA; *Corresponding Author: rdailey@wvu.edu

2Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada;

Received 6 May 2011, revised 25 May 2011, accepted 10 June 2011.

ABSTRACT

Seminal plasma is composed of secretions from

accessory sex glands, which are mixed with

sperm at ejaculation and contribute the majority

of semen volume. Seminal plasma is considered

a transport and support medium for sperm in

the female reproductive tract. Because seminal

plasma is not required for fertilization, the im-

portance of its constituents to the establishment

of normal pregnancy has been overlooked. Four

seminal plasma proteins, Osteopontin, Sper-

madhesin Z13, BSP 30 kDa and Phospholipase

A2, have been identified as markers of fertility in

dairy bulls [1-3]. The objective of the present

study was to characterize the expression pat-

terns of these proteins and other proteins found

to be of interest in seminal plasma of cryopre-

served and non-cryopreserved bull semen.

Seminal plasma samples were obtained from 16

mature Holstein- Friesian bulls at Select Sires

Inc. Samples were divided into two groups

based on assigned fertility score expressed as

the percentage point deviation (PD) of the bull’s

non-return rate (NRR) from the average NRR of

all bulls in the Select Sires Inc. reproductive

management program. Group 1 (high fertility

bulls, n = 8) 1.9% ≤ PD ≤ 2.7%, and group 2 (low

fertility bulls, n = 8) - 6.5% ≤ PD ≤ 1.8%. Addi-

tionally, the samples were categorized as proc-

essed (cryopreserved) or unprocessed (non-

cryopreserved) for protein analysis. Protein ex-

pression was analyzed by 2 - D fluorescence

difference gel electrophoresis (2D-DIGETM). Pro-

tein spots were picked from a reference gel,

analyzed by mass spectrometry and, subse-

quently identified by MS/MS ion searches per-

formed on the SwissProt database. Protein ex-

pression did not differ (P > 0.05) with fertility

grouping but displayed two distinct pa- tterns

among the processing groups: majority of the

functional proteins were highly expressed in

seminal plasma of non-cryopreserved semen

while the cryopreserved semen contained mainly

structural/ extender derived proteins. Functional

proteins identified included Spermadhesin Z13,

BSP A1/2, BSP 30 kDa, Nucleobindin-1 and

metalloproteinase inhibitor 2. Some of these

proteins have been identified as anti-fertility or

fertility enhancing agents in males. Whether this

alteration in protein expression after processing

might affect semen fertility is worthy of further

evaluation.

Keywords: Seminal Plasma; Proteomics;

Bull Fertility

1. INTRODUCTION

Secretions from the accessory sex glands are mixed

with sperm at ejaculation and contribute to the majority

of semen volume and components. However, during

cryopreservation, most seminal plasma is replaced with

semen extenders, mainly egg yolk or milk proteins. Se-

minal fluid contains signaling agents that influence fe-

male reproductive physiology to improve chances of

conception and pregnancy success. These factors include

cytokines, sex hormones, and prostaglandins [4]. Ex-

periments in rodents and pigs showed that seminal

plasma is the second most vital component of the ejacu-

late, absence of which at mating reduced fertilization

and increased fetal loss after implantation [5]. Artificial

inseminations with adjunctive seminal plasma consis-

tently tended to have little or no effect on pregnancy

outcome [6]. The precise nature of active constituents

J. F. Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40

that might influence pregnancy and their relative

amounts remain relatively unknown.

High concentrations of TGF-β cytokines were de-

tected in boar seminal fluid [7], and their characteristic

immunosuppressive activity was associated with protein

fractions of appropriate size in boar seminal fluid [4].

Four proteins (osteopontin, spermadhesin Z13, bovine

seminal plasma protein (BSP) 30 kDa, and phospholi-

pase A2) were identified as markers of fertility in dairy

bulls [1-3]. In those studies, proteins were visualized

with Coomassie brilliant blue staining after 2-D gel

separation, a process that might not reveal some of the

low abundance proteins in seminal plasma that might be

of biologic importance.

Therefore, a more sensitive protein detection tech-

nique that would give a broader approach was used in

the present study to identify seminal plasma proteins.

The multiplexing capability of 2 - D fluorescence dif-

ference gel electrophoresis (2 - D DIGE) technology was

expected to broaden the number of markers included in

the assay and yield more robust predictions of bio-

markers for bull fertility. The objectives of this study

included: 1) large scale identification and differential

expression of seminal plasma proteins between high and

low fertility bulls, 2) correlation of expression of spe-

cific proteins to fertility phenotype, and 3) comparison of

the expression patterns pre- and post-cryopreservation.

2. MATERIALS AND METHODS

Semen samples from 16 dairy bulls were obtained

from Select Sires Inc. (Plain City, Ohio). All samples

used (cryopreserved and non-cryopreserved) were ob-

tained from the same ejaculate of each bull and proc-

essed at Select Sires Inc. At each collection time, ejacu-

lates from the same bull were pooled and an aliquot was

obtained for the non-cryopreserved sample. The rest of

the semen was processed and extended for cryopreserva-

tion in 0.5 mL semen straws.

Semen was extended in egg-yolk-citrate (EYC) com-

posed of a 2.9% sodium citrate buffer supplemented

with 20% egg yolk (vol/vol) prepared with a glycer-

olated and non-glycerolated fractions containing 14 or

0% glycerol (vol/vol), respectively. Samples were di-

luted 1:3 (vol/vol) with non-glycerolated semen extender,

placed in a 200-mL water bath and allowed to equilibrate

to 5˚C over a 2-h period. Samples were extended to half

volume (80 × 106/ml) in non-glycerolated extender and

then an equal volume of glycerolated extender was

slowly titrated over a 10 min period. The final dilution

yielded a sperm concentration of 20 × 106 per 0.5-mL

French straw. Straws were then frozen in liquid nitrogen.

To obtain seminal plasma, semen was centrifuged at

3,000 g for 10 min and then aspirated into Eppendorf

tubes for volumes < 1 mL, or 15 mL centrifuge tubes for

larger volumes. Samples were then further clarified in

0.45 micron syringe filter and finally frozen in 0.5 mL

straws.

Samples were assigned to high or low fertility groups

based on fertility score expressed as the percentage point

deviation (PD) of the bull’s non-return rate (NRR) from

the average NRR of all bulls in the Select Sires Inc. re-

productive management program. These scores were

based on inseminations with frozen semen from the bulls

under this program. Range of scores for high fertility

bulls (n = 8) was 1.9% ≤ PD ≤ 2.7% and for low fertility

bulls (n = 8) was –6.5% ≤ PD ≤ 1.8%.

Following delivery, semen samples were thawed at

room temperature and centrifuged at 10,000 × g for 15

min at 4˚C to remove sperm and associated debris. Ali-

quots of 0.5 mL seminal samples were obtained from the

supernatant and stored frozen at –80˚C until further use.

2.1. Electrophoresis

Frozen samples of seminal plasma were thawed at

room temperature and centrifuged at 10,000 × g for 60

min at 4˚C. The supernatant was processed by 2-D

Clean-UP protocol (GE Healthcare, Piscataway, NJ) to

remove impurities such as nucleic acids, lipids and salts.

Samples were then assayed for protein content [8] using

BSA as standard, and aliquots were frozen at –800˚C.

Samples for electrophoresis were thawed at room tem-

perature, concentrated to 1 - 10 mg protein/mL and la-

beled with cyanine dye (CyDye) DIGE Fluor Cy3/5 (GE

Healthcare, Piscataway, NJ) at a ratio of 50 µg protein to

400 pmol fluor. A pooled internal standard was created

from equal aliquots of each sample and labeled with Cy2

dye. Samples were separated by isoelectric focusing on

an Ettan™ IPGphor™ apparatus (GE Healthcare, Pis-

cataway, NJ) using 24 cm Immobiline DryStrip gels (GE

Healthcare, Piscataway, NJ) containing a mixture of

ampholytes with pH ranging from 3 to 10.

Following isoelectric focusing, strip gels were trans-

ferred to 24 cm Tris-Tricine gradient gels (Bio-Rad La-

boratories, Hercules, CA) mounted on low-fluorescence

glass plates. Thereafter, proteins were separated by mo-

lecular mass in the second dimension using Ettan™ Dalt

II Electrophoresis System (GE Healthcare, Piscataway,

NJ). Dalt gels were scanned using a Typhoon 9400 Va-

riable Mode imaging densitometer (GE Healthcare, Pis-

cataway, NJ) at 100 µm resolution. A fully automated

image analysis software, Progenesis SameSpots™ (Non-

linear Dynamics, Durham, NC), was used to analyze the

protein expression data.

2.2. Statistical Analysis

In 2D-DIGE experiments, the pooled internal stan-

J. F.Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40

3535

dards were essential for assessing biological and ex-

perimental (between gels) variations and increasing the

robustness of statistical analysis. Individual protein data

from sample groups (Cy3 or Cy5) were normalized

against the Cy2 labeled internal standard. Scanned im-

ages of the labeled proteins were sequentially analyzed

by differential in-gel analysis (DIA) that performed

Cy3/Cy5:Cy2 normalization, and then by biological var-

iation analysis (BVA) that performed inter-gel statistical

analysis to provide relative abundance in various groups.

Log abundance ratios were then compared between

sample groups using ANOVA and t-test in Progenesis

SameSpots. The analyzed spots were ranked by their

probability values and then grouped into experimental

groups for further evaluation. Principal component

analysis (PCA) was used to determine the presence of

outliers in the data and also to compare how well the

samples fitted to the experimental groups. The expres-

sion profiles of the selected proteins were then examined

by correlation analysis.

2.3. Protein Identification

A list of protein spots of interest (pick-list) was gener-

ated using the image analysis software and exported di-

rectly into a Spot Picking Ettan™ Spot Handling Work-

station (GE Healthcare, Piscataway, NJ) equipped to

automatically pick spots from the Dalt gels. Selected

protein spots were washed by 50 mM ammonium bicar-

bonate/50% (vol/vol) methanol in water, dried by vac-

uum centrifugation, and incubated overnight at 37 0C in

140 ng of sequencing grade trypsin [9]. Tryptic digests

were analyzed by capillary liquid chromatography-

nanoelectrospray ionization-tandem mass spectrometry

(CapLC-MS/MS, Thermo Finnigan, San Jose, CA). Pro-

teins were identified by MS/MS ion searches performed

on the processed spectra against the SwissProt and NCBI

protein databases using a Bioworks Browser 3.1

(Thermo Finnigan, San Jose, CA) search engine. The

identification of a protein was confirmed when the Bio-

works confidence interval was greater than 95%. The

protein mass and pI accuracy on the 2D gel was used as

a guide to confirm protein identification.

3. RESULTS

3.1. Distribution of Protein Spots in Seminal

Plasma of Dairy Bulls

Three patterns of protein expression were observed

consistently in seminal plasma samples of cryopreserved

semen (Figure 1). Spot volume analysis and peptide

identification indicated a higher expression of proteins

from semen extender at the molecular weight range be-

tween 30 to 60 kDa (Group 1) accounting for 55% of

protein spots by Coomassie staining. A “train” of spots

was visible within the 20 to 25 kDa range (Group 2)

accounting for about 35% of the spot volume, while the

remaining spots (10%) were expressed below the 20 kDa

range (Group 3).

There was a two-fold difference (P < 0.01) in total

protein content between extended and non-extended se-

minal plasma (41.6 ± 2.3 vs. 19.5 ± 2.1 ng/mL, respec-

tively). However, the expression patterns of proteins in

seminal plasma of high and low fertility bulls did not

differ (P > 0.05). Therefore, subsequent analyses were

done between cryopreserved (processed) and non-cryopre-

served (unprocessed) seminal plasma. A total of 54 spots

differed (P < 0.001) in expression pattern between proc-

essed and unprocessed seminal plasma (Figure 2(a)).

The spots were then examined by principal component

analysis and clustered into two groups: those 31 spots

(57.4%) that were highly expressed in processed seminal

plasma but not in unprocessed seminal plasma and those

23 spots (42.6%) that were highly expressed in unproc-

essed seminal plasma but not in processed seminal

plasma (Figure 2(b)).

3.2. Protein Identification

A reference image (Figure 3) was generated from the

expression data and used to pick spots for protein identi-

fication (Table 1). Extender derived proteins, mainly

chicken vitellogenin-2 (MW 20.5 kDa), fibrinogen β

chain (MW 52.70) and chicken albumin, were predomi-

nant proteins identified from spots above 20 kDa in se-

minal plasma of processed semen. In unprocessed semi-

nal plasma, which lacked extender proteins, several

spots above 20 kDa were identified. Notable among

these proteins were nucleobindin-1, clusterin, phosphol-

ipase A2 isoforms, seminal plasma protein BSP-30 kDa,

metalloproteinase inhibitor-2 and cathepsins (B and D).

Below 20 kDa range, greater amounts of spermadhesin

(SPAD1 and Z13) isoforms were expressed than the ma-

jor bovine seminal plasma proteins (PDC - 109 and

BSP-A3) in seminal plasma of processed semen. How-

ever, in unprocessed seminal plasma, PDC - 109 and

BSP-A3 predominated over the spermadhesins below the

20 kDa range.

4. DISCUSSION

The main finding of this study was that expression

pattern of seminal plasma proteins differed between,

cryopreserved and non-cryopreserved dairy bull semen.

Indeed, during cryopreservation, major seminal plasma

proteins are replaced with extender proteins. Most artifi-

cial inseminations in cattle involve use of processed

(cryopreserved) semen, and as a consequence, most fer-

tility data have been derived from inseminations with

J. F. Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40

Table 1. Proteins identified from corresponding spots in Figure 3.

Gel ID Fold increase* Protein ID Accession number Coverage % Molecular

weight pI

Membrane stabilizing proteins

1 95.4 Spermadhesin Z13 P82292 44.0 15.2 6.3

19 30.2 Spermadhesin Z13‡ P82292 43.3 15.2 6.3

3 54.4 Spermadhesin 1 P29392 78.4 15 5.1

20 30.1

Epididymal secretory pro-

tein E1 P79345 55.0 16.6 8.4

ECM interacting proteins

6 38.8 Cathepsin B P07688 16.1 36.7 --

26 27.3 Cathepsin D P80209 5.1 42.5 --

43 20.4 Clusterin P17697 31.4 51.1 6.0

12 33.9

Metalloproteinase inhibitor

2 P16368 42.7 24.3 7.8

Capacitation/acrosome reaction proteins

9 37.3 BSP-A1/A2 P02784 44.8 15.5 4.9

19 30.2 BSP A1/A2† P02784 44.8 15.5 4.9

34 24.6 BSP-30kDa P81019 27.9 21.3 5.9

51 17.1 Phospholipase A2 IPI00760435.1 47.1 50.1 6.5

90 7.9

Platelet-activating factor

acetylhydrolase Q28017 53.2 50.1 6.5

100 5.5 Nucleobindin-1 Q0P569 63.1 54.9 5.2

Ubiquitination proteins

14 33.3 Kelch-like protein 9 Q2T9Z7 24.5 153.6 8.8

Motility associated proteins

30 26.4

Fast myosin heavy chain

extraocular IPI00829549.2 30.6 186.1 5.7

*Spot volumes differed in relative amounts compared to internal standard；

†Protein was identified at this spot only in unprocessed semen.；

‡Identification at this

spot was enhanced by semen processing.

processed semen. Therefore, it was prudent to utilize

cryopreserved semen in this study to examine changes in

its protein profile between low and high fertile bulls, and

also between processed and unprocessed semen.

Abundant low molecular weight bovine seminal plasma

proteins were replaced during cryopreservation by high

molecular weight extender-derived chicken structural

proteins. Major bovine seminal plasma proteins

PDC-109 (BSP-A1/A2), BSP-A3 and BSP-30 kDa play

important roles in fertility by maintaining sperm in an

appropriate state in the female tract until the oocyte

reaches the site of fertilization [10-13]. It is unclear

whether the effect of processing that decreased the ab-

undance of these BSP proteins by more than half would

render them ineffective in preventing premature capaci-

tation and acrosome reaction of sperm from normal fer-

tility bulls in the female tract. Spot volume data indicated

that removal of the major seminal plasma proteins ex-

posed less abundant low molecular weight proteins like

spermadhesins, especially spermadhesin Z 13 which has

been identified as an antifertility factor [2,14] in bovine

semi-nal plasma.

Killian et al. [14] suggested that four bovine seminal

plasma proteins were associated with fertility. These pro-

teins were later characterized as osteopontin and BSP-30

kDa in high fertility bulls and spermadhesin Z

J. F.Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40

3737

Figure 1. Reference 2D gel depicting distribution of protein spots in seminal plasma of dairy bulls. Protein spots were

characterized as: 1) probable extender-derived proteins, 2) probable medium and high molecular weight seminal

plasma proteins, and 3) major bovine seminal plasma proteins.

(a)

J. F. Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40

(b)

Figure 2. Cluster analysis of protein expression in seminal plasma of dairy bulls. Reference 2D gel (a) depicting

protein spots that differed (P < 0.001) and their standard expression profiles (b) in processed (Red outlines) and

unprocessed (Green outlines) semen.

Figure 3. Pick list for protein spots on a reference gel of a pooled internal standard of seminal plasma from processed

and unprocessed dairy bull semen. Thirty spots from this list were picked and digested with trypsin for protein identi-

fication by CapLC-MS/MS mass spectrometry.

J. F.Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40

3939

13 in low fertility bulls [2]. In the present study, seminal

plasma protein expression did not differ between high

and low fertility dairy bulls. A difference in protein ex-

pression between fertility groups had been demonstrated

in a previous study [2]. It was anticipated that by utiliz-

ing the multiplexing capability of the 2-DIGE technol-

ogy, experimental errors would be minimized, and a

more robust analysis would be achieved as opposed to

the densitometric analysis utilized in the former study.

The discrepancy in outcome between the two studies can

be attributed to sample type utilized or fertility grouping,

or both. Samples in the present study were from proc-

essed insemination straws and unprocessed seminal

plasma from the same ejaculates as opposed to fresh

seminal plasma samples utilized in the previous study

that avoided secretions from epididymis and vas diferens.

Therefore, differential expression of proteins in samples

examined in this study might have been affected by

sample type as well as semen processing as evidenced

by the comparison of processed and unprocessed semi-

nal plasma protein profile. The narrow range of fertility

differences in the bulls used in this trial also impeded the

classification of semen samples into distinct high and

low fertile groups as was done in the previous study [2].

Bulls used in the present study had percentage point de-

viations (PDs) from the average of +2.7% to –6.5%. In

contrast, the previous study had PDs from +7.7% to

–18.1%. Consequently, low and high fertility groups in

the present study corresponded to the intermediate fertil-

ity groups in that study. Because the previous authors

reported no differences in expression levels of osteopon-

tin, spermadhesin Z13, phospholipase A2 and BSP

30kDa among these two groups, it was not surprising

that the groups did not differ in their seminal plasma

protein profile in the present study. This is a major lim-

iting factor in fertility studies in farm animals because it

is rare to find sires at the lower extremes of fertility due

selection pressure.

Seminal plasma proteins have been characterized by

other investigators and their association with male fertil-

ity continues to be explored [2,3,15-17]. Functions of

sperm that may be affected by seminal plasma proteins

include capacitation, acrosome reaction, motility, DNA

integrity and interaction with the oocyte [3,18].

Major BSP proteins (BSP-A1/A2, A3 and BSP-30

kDa) are known to influence capacitation by their ability

to modulate membrane cholesterol [18]. Phospholipase

A2 (PLA2) and osteopontin are involved in acrosome

reaction and sperm-oocyte interaction and possibly early

embryonic development [3]. Proteins that might be asso-

ciated with interaction and modulation of extracellular

matrix (ECM) components are TIMP-2, clusterin and

cathepsins. These functions may be important during

fertilization when the sperm is required to interact with

and cross barriers established by the cumulus cells, zona

pellucida and oocyte membrane. Albumin, aSFP and

clusterin are involved either directly or indirectly in

mechanisms aimed at preventing damage to sperm

membrane, oxidative stress, and immune attack. Proteins

associated with sperm motility in the female reproduc-

tive tract include BSP A1/A2, aSFP, PLA2 and ecto

5'-nucleotidase (5'-NT). Spermadhesin Z13 might also

be included in the motility associated group because it

shares 50% homology with aSFP. However, expression

of spermadhesin Z13 in seminal plasma of dairy bulls

was inversely related to fertility [2].

5. CONCLUSIONS

Although expression pattern of seminal proteins was

not associated with fertility ranking in this study, altera-

tion of protein expression might affect fertility in bulls

that show more divergence in fertility ranking. In addi-

tion, expression patterns of seminal plasma proteins

might be altered during cryopreservation as has been

demonstrated in this study. Whether this alteration might

affect fertility remains to be explored.

6. ACKNOWLEDGEMENTS

The authors acknowledge the support of Select Sires Inc for provi-

sion of fertility data and semen samples. We greatly acknowledge the

support of Ms. Linda Corrum, Mr. Steve Wolf and Dr. Peter Perotta of

the Core Proteomics facility at West Virginia University. This work is

published with the approval of the director of the WV Agriculture and

Forestry Experiment Station as a scientific paper from the Division of

Animal and Nutritional Sciences and was supported by Hatch Project

421, NE1007 and Select Sires grant.

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