Signals of seminal vesicle autoantigen suppresses bovine serum albumin-induced capacitation in mouse sperm

Signals of seminal vesicle autoantigen suppresses bovine serum

albumin-induced capacitation in mouse sperm

Yen Hua Huang

a,*

, Shin Peih Kuo

a

, Mei Hsiang Lin

a

, Chwen Ming Shih

a

,

Sin Tak Chu

b,c

, Chih Chun Wei

a

, Tasi Jung Wu

a

, Yee Hsiung Chen

b,c,* a

Department of Biochemistry and Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, Taipei, Taiwan b

Institute of Biochemical Sciences, College of Science, National Taiwan University, Taiwan c

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Received 14 September 2005

Available online 2 November 2005

Abstract

Capacitation is the prerequisite process for sperm to gain the ability for successful fertilization. Unregulated capacitation will cause

sperm to undergo a spontaneous acrosome reaction and then fail to fertilize an egg. Seminal plasma is thought to have the ability to

suppress sperm capacitation. However, the mechanisms by which seminal proteins suppress capacitation have not been well understood.

Recently, we demonstrated that a major seminal vesicle secretory protein, seminal vesicle autoantigen (SVA), is able to suppress bovine

serum albumin (BSA)-induced mouse sperm capacitation. To further identify the mechanism of SVA action, we determine the molecular

events associated with SVA suppression of BSA
s activity. In this communication, we demonstrate that SVA suppresses the BSA-induced

increase of intracellular calcium concentration ([Ca

2+

]

i

), intracellular pH (pH

i

), the cAMP level, PKA activity, protein tyrosine

phos-phorylation, and capacitation in mouse sperm. Besides, we also found that the suppression ability of SVA against BSA-induced protein

tyrosine phosphorylation and capacitation could be reversed by dbcAMP (a cAMP agonist).

2005 Elsevier Inc. All rights reserved.

Keywords: Capacitation; Seminal vesicle; Ca2+_{; Protein tyrosine phosphorylation; cAMP-PKA pathway}

During epididymal transit, sperm progressively acquire

the ability to move, but they are still fertilization

incompe-tent. Fertilization capacity is gained after residence in the

female reproductive tract for a finite period of time, and

the physiological changes in sperm during this period are

collectively called ‘‘capacitation.’’ Capacitation is a

com-plex process first described and defined independently by

Chang

[1,2]

and Austin

[3,4]

. The capacitation processes

involve changes in membrane properties and dynamics,

enzyme activities, elevation of [Ca

2+

]

i

, pH

i

, and the cAMP

level. It leads to energy consumption and hypermotility,

and eventually an acrosome reaction by sperm

[5,6]

.

Sperm capacitation occurs in the oviduct or uterus,

depending on the species

[6]

. The process of sperm

capacita-tion is tightly regulated by suppression factors (in the

epidid-ymis and seminal vesicles) and capacitation factors (in the

female reproductive tract). Serum albumin is abundant in

the female reproductive tract. It is thought to serve as a

cho-lesterol-binding protein to remove sperm membrane

choles-terol, by which to destabilize the sperm membrane and

induce sperm capacitation

[7–9]

. Serum albumin has also

been demonstrated to regulate the T-type Ca

2+

channel of

sperm, induce extracellular Ca

2+

and bicarbonate ion influx

[10]

, and elevate [Ca

2+

]

i

and pH

i

. The increase of [Ca

2+

]

i

and

pH

i

upregulates the cAMP-dependent signaling and

enhanc-es the protein tyrosine phosphorylation, ultimately inducing

hyperactivation and capacitation of sperm

[5,11]

.

The suppressive effect of capacitation by suppression

factors is referred to as ‘‘decapacitation’’

[12]

. Without

sup-pression regulation, most of the sperm would undergo a

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.120

* _{Corresponding authors. Fax: +886 2 2736166x3150 (Y.H. Huang),} +886 2 23635038 (Y.H. Chen).

E-mail addresses:rita1204@tmu.edu.tw (Y.H. Huang), bc304@gate. sinica.edu.tw(Y.H. Chen).

www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 338 (2005) 1564–1571

spontaneous acrosome reaction. An acrosome-reacted

sperm lose its acrosome cap which is required for sperm

binding to the zona pellucida of the egg. Thus, sperm

ulti-mately lose their fertilization ability even though they still

have hypermotility

[6]

. It has been reported that the

epidid-ymis and seminal plasma contain decapacitation activity

[12–23]

; the presence of suppression factors (decapacitation

factors) may prevent the unfruitful capacitation of sperm

and allow effective fertilization of an egg at the right time

and place

[17]

. Currently, several decapacitation factors

are known. Fraser et al.

[18]

suggested that the

decapacita-tion mechanism involves fucose residues and a

GPI-an-chored receptor on sperm in the epididymis. A low

molecular weight N-glycosidically linked oligomannosidic

glycopeptide (MGp) isolated from the autoproteolysis

products of human seminal plasma was reported to prevent

premature sperm exocytosis

[19]

. Studies by Villemure et al.

[21]

revealed the gelatin-binding proteins from goat seminal

plasma play a role in sperm decapacitation. A sperm

adhe-sin family of boar accessory sex gland fluids is also

sup-posed to consist of decapacitation factors

[22]

. In

addition, the platelet-activation factor, acetylhydrolase

(PAF-AH), was also suggested to play a role in

decapacita-tion by hydrolysis of PAF to lyso-PAF

[23]

. However, the

mechanisms of these potential factors in decapacitation

have not been well defined.

Recently, we demonstrated that serum obtained from

male and female mice immunized with seminal vesicle

secretion (SVS) fluid is immunoreactive to an

androgen-re-sponsive glycoprotein

[24,25]

, and it was designated

semi-nal vesicle autoantigen (SVA). SVA is a 19-kDa protein

secreted from luminal epithelium cells of seminal vesicles

and contribute to the dominant component of seminal

plasma (
300 lM)

[26]

. SVA binds Zn

2+

[27]

and

cho-line-containing phospholipids, such as phosphatidylcholine

and sphingomyelin

[26]

. SVA has been demonstrated to

suppress BSA-induced zinc ion removal from the sperm

membrane, sperm hypermotility, protein tyrosine

phos-phorylation, and capacitation

[28]

. In this communication,

we further demonstrate that SVA suppresses BSA-induced

[Ca

2+

]

i

, pH

i

, the cAMP level, and PKA activity in mouse

sperm. In addition, the suppressive effects of SVA on

BSA-induced protein tyrosine phosphorylation and

capac-itation in mouse sperm can be reversed by a cAMP agonist.

Materials and methods

Materials. Fatty acid-free BSA, polyvinylalcohol, and Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) were from Sigma (St. Louis, MO). Antiphos-photyrosine monoclonal antibody (clone 4G10) was from UBI (Lake Placid, NJ), horseradish peroxidase (HRP)-conjugated anti-mouse IgG was from Jackson ImmunoResearch Lab (West Grove, PA), Percoll, chemiluminescence detection ECL plus, [c-32P]ATP, and the cAMP assay kit (RPN 255) were from Amersham–Pharmacia Biotech (Buckingham-shire, UK), Fluo-3-AM and BCECF-AM were from Molecular Probes (Eugene, OR), dituylryl cAMP (dbcAMP), Rp-cAMPS, and IBMX were from Research Biochemicals International (Natick, MA), and H-89 was from LC Laboratories (Woburn, MA). Phosphocellulose Units SpinZyme Format for the radioactive kinase assay was from Pierce (Rockford, IL),

and the scintillation counting cocktail was from Merck (Darmstadt, Germany). All other chemicals were of reagent grade.

Sperm preparation and cytological observations. Outbred CD-1 mice purchased from Charles River Laboratories (Wilmington, MA) were bred in the Animal Center at Taipei Medical University School of Medicine. Animals were handled in accordance with institutional guidelines on animal experimentations.

The culture medium used throughout these studies was modified Krebs–Ringer bicarbonate HEPES medium (HM) as described previously

[28]. In brief, modified HM contains 120.0 mM NaCl, 2.0 mM KCl, 1.20 mM MgSO4Æ7H2O, 0.36 mM NaH2PO4, 15 mM NaHCO3, 10 mM Hepes, 5.60 mM glucose, 1.1 mM sodium pyruvate, and 1.7 mM CaCl2. The pH of the medium was adjusted to 7.3–7.4 with humidified air/CO2 (95:5) in an incubator at 37C for 48 h before use. Polyvinylalcohol (1 mg/ ml) was added to serve as a sperm protectant. Mature mouse sperm were harvested by a swim-up procedure from the caudal epididymides and isolated with a 20–80% Percoll gradient. The viability and progressive motility of the sperm fraction used in the present study were more than 95%. The population of the capacitated stage in sperm was analyzed by the CTC staining method as described previously[29].

Flow cytometry. [Ca2+_]

iof sperm was determined using fluo-3 AM by flow cytometry (FACScan, BD). In brief, Percoll-separated sperm were loaded with fluo-3 AM (10 lM) for 10 min. After 10 min incubation, sperm were washed twice with modified HM to remove any free fluo-3 AM. Fluo-3 AM-loaded sperm (106_{cells/ml) were treated with SVA (0–} 66 lM) in the presence or absence of BSA (0.3%) at 37C for 90 min and then analyzed by epifluorescence microscope and flow cytometry.

pHiof sperm was determined using BCECF-AM. In brief, Percoll-sep-arated sperm were loaded with BCECF-AM (2 lM) for 10 min and then sperm were washed twice with modified HM to remove any free fluore. The fluore-loaded sperm were treated with BSA (0.3%) ± SVA (2, 20, and 66 lM) and then analyzed by flow cytometry. For pHicalibration, a nigericin/high K+calibration protocol was used to derive the pHivalues as described previously[30]. The fluorescence of fluo-3 was excited at 488 nm and mea-sured via a 515–540 nm filter, and the fluorescence of BCECF was excited at 510 nm and measured via a 564–606 nm filter. PMT voltages and gains were set to optimize the dynamic range of the signal. The fluorescence intensity of sperm was quantified for 10,000 individual cells.

cAMP assay. The amount of cAMP produced in living, intact sperm was determined using a nonradioactive enzyme immunoassay kit according to the manufacturer
s instructions.

Assay of protein kinase A activity. Protein kinase A activity was measured using Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as the specific substrate. Sperm (107_{cells/ml) were incubated under different experimental} condi-tions, such as BSA (0.3%), SVA (66 lM) or BSA supplemented with SVA. DbcAMP (a cAMP agonist, 1 mM) plus IBMX (a phosphodiesterase inhibitor, 100 lM) were used to be a positive control, and H-89 (a PKA inhibitor, 30 lM) served as a negative control. After incubation at 37C for 90 min, the sperm suspension (10 ll) was mixed with an equal volume of 2· assay cocktail (10 ll) and incubated at 37C for additional 15 min. The final concentration of the assay components was 100 lM kemptide, [c-32P]ATP (6000 Ci/mmol) (2· 106

cpm/assay), 100 lM ATP, 1% (v/v) Triton X-100, 1 mg/ml BSA, 10 mM MgCl2, 40 mM b-glycerophosphate, 5 mM p-nitro-phenyl phosphate, 10 mM Tris–HCl (pH 7.4), 10 lM aprotinin, and 10 lM leupeptin. The reactions were stopped by an equal volume of 20% TCA, and reaction mixtures were cooled on ice for 20 min and followed by centrifu-gation at 10,000g at room temperature for 3 min. Twenty-five microliter of the resultant mixture was applied onto an affinity support of the phospho-cellulose unit and washed with 500 ll of 75 mM phosphoric acid for four times (10 min/each time). The washed-sample bucket was then transferred into a scintillation vial for counting.

Detection of protein tyrosine phosphorylation. Sperm (5· 106_cells/ml) were incubated with BSA (0.3%) in the absence or presence of SVA (66 lM). In some experiments, BSA and SVA supplemented with dbcAMP (a cAMP agonist, 1 mM) plus IBMX (a phosphodiesterase inhibitor, 100 lM), or BSA and H-89 (a PKA inhibitor, 30 lM) or Rp-cAMP (a Rp-cAMP antagonist, 1 mM) were added. The reactions were incubated at 37C. After 90 min incubation, the cell lysate was prepared

according to Visconti et al.[31], subjected to a 10% SDS–PAGE, and then transferred to a PVDF membrane for Western blot analysis. The mono-clonal anti-phosphotyrosine IgG (clone 4G10) (1 lg/ml) was used as the primary antibody and HRP-conjugated anti-mouse IgG (1:2000) served as the secondary antibody. The enzyme activity of HRP was detected by the ECL system according to the manufacturer
s instructions.

Statistical analysis. All experiments were repeated at least three times with three different pooled sperm samples from four or five male mice. The data were expressed as means ± SD. Difference in means was assessed by one-way analysis of variance (ANOVA), followed by the Tukey–Kramer multiple comparisons test.

Results

SVA suppresses BSA-stimulated capacitation in mouse

sperm

BSA, like serum albumin, is a well-known putative

capacitation factor used for stimulating mouse sperm

capacitation in vitro. Its related capacitation events involve

the removal of membrane-bound zinc ions, elevation of

[Ca

2+

]

i

, pH

i

, the cAMP level, sperm hypermotility, and

protein tyrosine phosphorylation

[5]

. To determine the

molecular events of SVA suppression of sperm

capacita-tion, we detected the SVA effect on BSA (0.3%)-induced

capacitation signaling. The capacitation states of mouse

caudal epididymal sperm under different experimental

con-ditions were determined by the CTC assay

[29]

. As shown

in

Fig. 1

, fewer than 15% of sperm underwent capacitation

in the absence of BSA with or without SVA. BSA (0.3%)

induced sperm capacitation by 53 ± 8%. SVA (66 lM)

suppressed BSA-induced capacitation in mouse sperm

from 53 ± 8% to 30 ± 4%. This effective concentration of

SVA on BSA activity is well below the physiological

con-centration in semen (
300 lM). These observations

fur-ther confirmed our previous report that SVA suppressed

BSA-induced mouse sperm capacitation

[28]

. In addition,

adding dbcAMP plus IBMX reversed the suppressive effect

of SVA on BSA-induced mouse sperm capacitation,

sug-gesting a role of cAMP in SVA suppression signaling.

SVA suppresses BSA-induced elevation of [Ca

2+

]

i

and pH

i

in mouse sperm

Elevation of [Ca

2+

]

i

and pH

i

has been shown to be

asso-ciated with mouse sperm capacitation induced by BSA

[5,32]

. [Ca

2+

]

i

and pH

i

have also been demonstrated to

upregulate membrane/soluble adenylate cyclase activity,

by which to produce cAMP, and activate PKA activity,

inducing protein tyrosine phosphorylation and

capacita-tion in mouse sperm

[32]

. Since SVA is capable of

suppress-ing BSA-induced capacitation, we decided to determine the

effect of SVA on [Ca

2+

]

i

and pH

i

in sperm stimulated by

BSA. [Ca

2+

]

i

was determined by epifluorescence

microsco-py and flow cytometry using fluo-3-AM. As shown in

Fig. 2

A, BSA (0.3%) increased [Ca

2+

]

i

(

Fig. 2

A, panel b

vs. panel a). SVA not only suppressed the BSA-induced

ele-vation of [Ca

2+

]

i

(

Fig. 2

A, panel d vs. panel b), but also

decreased the basal [Ca

2+

]

i

in mouse sperm (

Fig. 2

A, panel

c vs. panel a). Besides, the relative [Ca

2+

]

i

of sperm was also

detected by flow cytometry and expressed as percentages in

comparison with that of control cells. As shown in

Fig. 2

B,

BSA elevated [Ca

2+

]

i

to 153 ± 6%. SVA suppressed [Ca

2+

]

i

with or without BSA in a dose-dependent manner.

Elevation of pH

i

has been correlated with BSA-induced

sperm capacitation

[33]

. To determine the SVA effect on

pH

i

in mouse sperm, sperm pre-loaded with BCECF-AM

were incubated with SVA and analyzed by flow cytometry.

The pH

i

of sperm was estimated by a pH standard curve

which was calibrated according to a pH 6–7 standard

solu-tion (

Fig. 3

A) as described previously

[30]

. As shown in

Fig. 3

B, BSA elevated the pH

i

from 6.5 to 6.9, and SVA

suppressed the BSA-induced elevation of pH

i

. At 20 lM,

SVA significantly suppressed the BSA-induced elevation

of pH

i

to basal level of mouse sperm. In addition, SVA

was also shown to decrease the basal level of pH

i

of mouse

sperm (our unpublished results).

SVA suppresses BSA-induced elevation of the cAMP level in

mouse sperm

Since [Ca

2+

]

i

and pH

i

have been shown to be the

upstream regulators of adenylate cyclase activity, we then

determined the effect of SVA on the BSA-induced elevation

0 25 50 75 Co ntro l SVA BS A BS A + SV A BSA + S VA + dbc AM P

Capacitation,

%

*

† #

Fig. 1. cAMP agonist reverses SVA suppression of BSA-induced capacitation in mouse sperm. Capacitation stage of sperm (5· 106

cells/ml) under different experimental conditions was determined using the CTC fluorescence method. Each data point is the mean ± SD of three independent determinations. Data obtained from sperm treated with BSA alone were compared with that of control sperm, or data obtained from sperm treated with BSA and SVA were compared with that of sperm treated with BSA but without SVA, or data obtained from sperm treated with BSA and SVA in the presence of dbcAMP plus IBMX were compared with that of sperm treated with BSA and SVA but in the absence of dbcAMP plus IBMX, respectively, by one-way ANOVA, Tukey-Kramer multiple comparison test. (#_{p < 0.001,} *p < 0.001,_{p < 0.001).}

of the cAMP level in mouse sperm. As shown in

Fig. 4

,

BSA (0.3%) increased the cAMP level to 153 ± 11% as

compared with that of control sperm. SVA suppressed

the BSA-induced elevation of the cAMP level in a

dose-de-pendent manner with the maximum effect at 66 lM (from

153 ± 11% to 93 ± 10%). Besides, SVA (66 lM) decreased

the basal level of cAMP to 43 ± 8% in mouse sperm. This

observation is in coincidence with our previous results that

SVA suppressed the basal [Ca

2+

]

i

and pH

i

.

SVA suppresses BSA-induced the PKA activity in mouse

sperm

cAMP has been demonstrated to affect PKA activity

and regulate protein tyrosine phosphorylation of mouse

sperm

[34]

. Since SVA is able to suppress the BSA-induced

elevation of the cAMP level in mouse sperm, we suggested

that SVA may suppress the BSA-induced PKA activity. A

PKA-specific substrate, Kemptide

(Leu-Arg-Arg-Ala-Ser-Leu-Gly), was used to detect the PKA activity of sperm.

As shown in

Fig. 5

, dbcAMP plus IBMX served as a

posi-tive control, it enhanced the PKA activity to near 400% as

compared with that of the control sperm. A PKA inhibitor

H-89 was used to be a negative control; it suppressed the

PKA activity to

75%. BSA (0.3%) significantly enhanced

the PKA activity to 155 ± 6% and SVA both suppressed

the BSA-induced and the basal level of PKA activity of

sperm. These results support our previous observations

that SVA suppresses both the BSA-induced and the basal

[Ca

2+

]

i

and the cAMP levels (

Figs. 2–4

). Furthermore,

the suppressive effect of SVA (200 lM) on the PKA activity

of mouse sperm could be overcome by 10% BSA. This

observation further supports our previous report that

10% BSA reversed the SVA
s suppressive effect on 0.3%

BSA-induced protein tyrosine phosphorylation and

capac-itation in mouse sperm

[28]

.

cAMP agonist reverses SVA
s suppression of

BSA-stimulated protein tyrosine phosphorylation in mouse sperm

Protein tyrosine phosphorylation has been

demonstrat-ed to the molecular evidence of capacitation, and it is

cor-related with the cAMP level and PKA activity in mouse

sperm

[34]

. Since SVA decreased the cAMP level in sperm,

we therefore detected the effect of a cAMP agonist on

SVA
s suppression of BSA-induced protein tyrosine

phos-phorylation. As shown in

Fig. 6

A, two proteins of M.W.

120 and
50 kDa were tyrosine-phosphorylated in

con-trol sperm which showed normal motility. The M.W.

120-kDa protein did not respond to the BSA stimulation

and was similar to p95/106 hexokinase identified by Kalab

et al.

[35]

(

Fig. 6

A, indicated by an arrowhead). BSA

(0.3%) treatment of sperm resulted in significant

enhance-ment of protein tyrosine phosphorylation in the range of

MW 50–100 kDa (

Fig. 6

A, lane 3, indicated by arrows),

and this phenomenon was suppressed by Rp-cAMP and

H-89 (

Fig. 6

A, lanes 4 and 5). These results further confirm

the previous report that the cAMP-PKA pathway is

involved in BSA
s activity of mouse sperm

[5,34]

.

Treat-ment of sperm with SVA reduced motility

[26]

and the

tyro-sine phosphorylation of the MW 50 kDa protein of control

sperm (

Fig. 6

B, lane 2). Furthermore, the BSA-induced

protein tyrosine phosphorylation of sperm was also

signif-icantly suppressed by SVA (

Fig. 6

B, lane 4).

DbcAMP and IBMX have been reported to induce

pro-tein tyrosine phosphorylation and capacitation in mouse

sperm

[34]

. To define the role of cAMP in SVA
s

suppres-sive effect, we determined the effect of dbcAMP plus IBMX

on SVA suppression of BSA-induced protein tyrosine

phosphorylation. As shown in

Fig. 6

B, the suppressive

effects of SVA on BSA-induced protein tyrosine

phosphor-ylation were overcome in the presence of dbcAMP plus

0 25 50 75 100 125 150 175 0 10 20 30 40 50 60 70

SVA (

µ

M)

R

e

la

ti

v

e

[Ca

2+

]

I,

%

Control 0.3 % BSA #

**

**

† †

*

A

B

a

b

d

c

Fig. 2. SVA suppresses BSA-induced elevation of [Ca2+]iin mouse sperm. Sperm (5· 106cells/ml) were loaded with fluo-3 AM to detect [Ca2+]i. Fluorescence images of sperm under different experimental conditions were shown in (A): (a) control (HM only), (b) BSA (0.3%), (c) SVA (66 lM), and (d) BSA + SVA. Fluo-3-loaded sperm were incubated with increasing concentration of SVA (0–66 lM) in the presence (closed circles) or absence (open circles) of BSA (B). After 90 min incubation, sperm were subjected to a flow cytometry to analyze [Ca2+]i. [Ca2+]iof sperm was shown as percentages in comparison with that of control sperm. Each data point is the mean ± SD of five independent determinations. Data obtained from sperm treated with BSA or SVA alone were compared with that of control sperm (_{p < 0.001,} #_{p < 0.001), or data obtained from sperm} treated with BSA and SVA were compared with that of sperm treated with BSA but without SVA (*p < 0.01, **p < 0.001), respectively, by one-way ANOVA, Tukey–Kramer multiple comparison test. Scale bar, 10 lm.

IBMX (

Fig. 6

B, lanes 5–7). This result further supports our

previous observation that a cAMP agonist reversed SVA
s

suppression on capacitation of mouse sperm (

Fig. 1

).

Together with the fact that SVA decrease the cAMP level

and PKA activity, these observations strongly suggest that

cAMP not only participates in BSA-induced protein

tyro-sine phosphorylation and capacitation but also involves

in SVA
s suppressive mechanism.

Discussion

Successful fertilization is tightly regulated by

capacita-tion and decapacitacapacita-tion processes. Sperm interact with

sup-pression factors in the seminal plasma (e.g., seminal plasma

proteins) and capacitation factors in the female

reproduc-tive tract (e.g., serum albumin) while they are ejaculated

from the caudal epididymis to move to the female

repro-ductive tract. Serum albumin is thought to stimulate sperm

capacitation in the female genital tract. In vitro, [Ca

2+

]

i

and pH

i

have been demonstrated to be the upstream

medi-ators of BSA-induced mouse sperm capacitation. [Ca

2+

]

i

and pH

i

regulate the membrane and/or cytosolic adenylate

cyclase

[5,36–39]

, modulate cAMP metabolism and PKA

activity

[36]

, and ultimately affect capacitation-associated

Fluorescence Intensity

Cell Number

6 6.2 6.4 6.6 6.8 7 7.2 Con trol 0.3 % BS A 0.3 % BSA + 2 µM SVA 0.3 % BS A + 20 µ M S VA 0.3 % BS A + 66 µ M S VA p H V a lu e * * # Control medium 0.3 % BSA 0.3 % BSA + 2 mM SVA 0.3 % BSA + 20 mM SVA y = 32.3x - 92.516 R2 = 0.9905 90 100 110 120 130 140 150 160 6 6.25 6.5 6.75 7 7.25 pH Value Per c e n ta g e , %

Fluorescence Intensity

Cell Number

A

B

Fig. 3. SVA suppresses BSA-induced elevation of pHiin mouse sperm. Sperm (5· 10 6

cells/ml) pre-loaded with BCECF AM were calibrated with a pH 6–7 standard solution (A), or incubated with BSA (0.3%) in the presence or absence of SVA (2, 20, and 66 lM) (B). pHiof sperm under different experimental conditions are shown as percentages of that of control sperm. Each data point is the mean ± SD of three independent determinations. Data obtained from sperm treated with BSA alone were compared with that of control sperm, or data obtained from sperm treated with BSA and SVA were compared with that of sperm treated with BSA but without SVA, respectively, by one-way ANOVA, Tukey–Kramer multiple comparison test. (#p < 0.001, *p < 0.001). 0 25 50 75 100 125 150 175 0 10 20 30 40 50 60 70

SVA (

µ

M)

Le

vel of cAMP

Control 0.3 % BSA ** * # † †

Fig. 4. SVA suppresses BSA-induced elevation of the cAMP level in mouse sperm. Sperm (5· 106

cells/ml) were incubated with increasing concentration of SVA (0–66 lM) in the presence (closed circles) or absence (open circles) of BSA (0.3%). After 90 min incubation, the total cell lysate was extracted and the total intracellular cAMP amount was detected. Each data point is the mean ± SD of five independent determinations. Data obtained from sperm treated with BSA or SVA alone were compared with that of control sperm (#_{p < 0.001,}_{p < 0.001), or data obtained from} sperm treated with BSA and SVA were compared with that of sperm treated with BSA but without SVA (*p < 0.01, **p < 0.001), respectively, by one-way ANOVA, Tukey–Kramer multiple comparison test.

protein tyrosine phosphorylation of sperm

[32,40]

.

Stimula-tion of tyrosine phosphorylaStimula-tion is an important event

dur-ing mammalian sperm capacitation

[34,41]

. In somatic

cells, the tyrosine-phosphorylated proteins mediate a

vari-ety of cellular functions such as growth regulation, cell

cycle control, cytoskeletal assembly, ionic current

regula-tion, and receptor regulation

[42]

. In human sperm, the

capacitation-associated tyrosine-phosphorylated proteins

have been demonstrated to involve the valosin-containing

protein (VCP), SNARE-interacting protein and protein

kinas A-anchoring protein family, etc., suggesting the

changes in regulatory enzymes of acrosomes and

cytoskel-etal elements during the sperm capacitation process

[43]

.

An unregulated capacitation process causes sperm to

undergo unfruitful capacitation prior to reaching the egg

for fertilization. Seminal plasma is thought to contain some

suppressive factors to regulate the capacitation processes

[12,14–17,19–23,44–46]

. The accessory sexual organ is also

thought to play a role in maintaining an optimal calcium

environment in seminal plasma, by which to regulate sperm

function

[45]

. In supporting this hypothesis, Coronel et al.

[46]

reported that a calcium transport inhibitor, caltrin, is

secreted by bovine seminal vesicles and binds with sperm

to maintain sperm in a low cytosolic calcium concentration

during ejaculation. In our results, as a major component of

seminal plasma, SVA significantly suppresses BSA-induced

mouse sperm capacitation and its related signaling. The

SVA-affected molecular events include [Ca

2+

]

i

, pH

i

, the

cAMP level, PKA activity, and protein tyrosine

phosphor-ylation. The observations that SVA decreases [Ca

2+

]

i

and

the cAMP level in sperm are in coincidence with our

previ-ous studies that SVA suppresses sperm motility

[26]

. The

IC

50

of SVA suppression on BSA-induced [Ca

2+

]

i

and the

cAMP level was less than 20 lM, a concentration which

is well below the physiological concentration of SVA in

semen (
300 lM).

Since Ca

2+

is the upstream regulator of sperm

capacita-tion, the fact that [Ca

2+

]

i

decreases apparently plays an

important role in SVA
s activity. We hypothesized that there

are two possibilities for SVA suppression of [Ca

2+

]

i

of mouse

sperm. SVA may block the calcium influx or SVA may play a

role in calcium clearance of mouse sperm. The calcium influx

may not be affected by SVA because SVA is effective in

low-ering [Ca

2+

]

i

in Ca

2+

-free medium as well as in Ca

2+

-contain-ing medium (unpublished results). Besides, in view of

∆ ∆ 1 2 3 4 5 kDa 120 __ __ __ __ __ __ __ __ 50 Contr ol

Rp-cAMP BSA BSA + Rp-cAMP BSA + H-89 _{dbcAMP + IBMX}

_{+ + +}

Contr ol SV A BSA BSA + SV A Contr ol SV A BSA + SV A 1 2 3 4 5 6 7 kDa 120 50

A

B

_ _ _ _ _ _ _ _

Fig. 6. cAMP agonist reverses the SVA inhibition of BSA-induced protein tyrosine phosphorylation in mouse sperm. The pattern of protein tyrosine phosphorylation in mouse sperm (5· 106_{cells/ml) incubated in different experimental conditions was detected (A): (1) control (HM only), (2) Rp-cAMP} (1 mM), (3) BSA (0.3%), (4) BSA + Rp-cAMP, and (5) BSA + H-89 (30 lM). The suppressive effect of SVA on BSA-induced protein tyrosine phosphorylation was detected and is shown in (B): (1) control (HM only), (2) SVA (66 lM), (3) BSA (0.3%), (4) BSA + SVA, and (5–7) BSA + SVA + cAMP agonist (dbcAMP plus IBMX). The arrows indicate the location of capacitation-related tyrosine-phosphorylated proteins, and the arrowhead denotes the location of a capacitation-unrelated 120 kDa tyrosine-phosphorylated protein.

0 25 50 75 100 125 150 175 200 Cont rol dbcAM P H-89 0.3 % BSA 66 µ M S VA 200 µM SV A 0.3 % BSA + 6 6 µM SVA 0.3 % BS A + 20 0 µM SV A 10 % BS A + 2 00 µ M SV A R e la tiv e PK A Ac tiv it y , % † † # _* *

Fig. 5. SVA suppresses BSA-induced elevation of the PKA activity in mouse sperm. The PKA activities of sperm under different experimental conditions were determined, such as (1) control (HM only), (2) dbcAMP (1 mM) + IBMX (100 lM), (3) H-89 (30 lM), (4) BSA (0.3%), (5) SVA (66 lM), (6) SVA (200 lM), (7) BSA (0.3%) + SVA (66 lM), (8) BSA (0.3%) + SVA (200 lM), and (9) BSA (10%) + SVA (200 lM). Each data point is the mean ± SD of three-independent determinations. Data obtained from sperm treated with BSA or SVA alone were compared with that of control sperm, or data obtained from sperm treated with BSA and SVA were compared with that of sperm treated with BSA but without SVA, respectively, by one-way ANOVA, Tukey–Kramer multiple com-parison test. (#_{p < 0.001, *p < 0.001,}_{p < 0.001).}

intracellular Ca

2+

clearance, there are four major

mecha-nisms that have been reported in most cell types

[47]

. Two

are on the plasma membrane: the plasma membrane Ca

2+

-ATPase (PMCA, which exports a cytoplasmic Ca

2+

ion

while importing one or two extracellular protons), and the

plasma membrane Na

2+

-Ca

2+

exchanger (NCX, which

exports an intracellular Ca

2+

ion and imports approximately

three Na

+

ions). The other two clearance sites are in the

intracellular organelles: sarcoplasmic endoplasmic

reticu-lum Ca

2+

-ATPase (SERCA pumps) and mitochondrial

Ca

2+

uniporter (MCU). In mouse sperm, PMCA and

NCX are considered to be the most important molecules

for maintaining low [Ca

2+

]

i

, but MCU just play a minor role

and the SERCA pumps are not thought to be essential in

mediating Ca

2+

clearance in sperm

[47]

. In consideration

of that SVA decreases both the BSA-induced and basal

[Ca

2+

]

i

and pH

i

(

Figs. 2 and 3

) and the PMCA
s dominant

role in exporting Ca

2+

and importing extracellular protons,

we suggest PMCA is likely the site where SVA exerts its

action. However, this hypothesis needs to be further

examined.

BSA and SVA are both phospholipid binding proteins.

The interactions of BSA and SVA with sperm membrane

lip-id components may play roles in regulating capacitation

sig-naling. Recent studies by Sleight et al.

[48]

conclude that

BSA-induced cholesterol efflux alters the lipid raft domain

on sperm membrane, by which to initiate the capacitation

signaling. Lipid rafts are highly enriched in cholesterol,

gan-gliosides, and sphingolipids, and are thought to recruit

spe-cific types of proteins to serve as the cholesterol traffic

centers for signal transduction pathway originating at the

plasma membrane

[49]

. PMCA has also been shown to be

concentrated in the caveolae/raft and mediated by

sphingo-lipids

[50]

. As SVA is capable of binding choline-containing

phospholipids and sphingolipid

[26]

, the possible interaction

of SVA with these specific phospholipids/sphingolipids on

sperm membrane lipid-rafts may mediate the Ca

2+

signal

to suppress the BSA-induced capacitation in mouse sperm.

This suppressive effect of SVA (in semen) on capacitation

may enable sperm to avoid unfruitful capacitation before

encountering the egg. Sperm may undergo capacitation at

or near the site where the egg resides, and the concentration

of SVA is low in the uterus and oviduct.

Acknowledgments

This work was supported by grants from National

Sci-ence

Council,

Taiwan

NSC91-2320-B-038-029

and

NSC93-2311-B-038-006 (to Y.H.H.), and

NSC91-2311-B001-076 and NSC91-2311-B002-049 (to Y.H.C.), and

from Taipei Medical University, TMU92-AE1-B24 and

94TMU-TMUH-03 (to Y.H.H.).

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