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Thymosin alpha-1 enhances the fertilizing capacity of human sperm cell: implication in diagnosis and treatment of male infertility



The effects of synthetic thymosin peptides (T alpha 1 and T beta 4) and their antibodies on the fertilizing capacity of human sperm cells were investigated. T alpha 1, but not the T beta 4, significantly (p < 0.001) increased the human sperm penetration rates in sperm penetration assay (SPA). Antibodies to both T alpha 1 and TB4, which predominantly bound to the acrosomal region of human sperm cell in the indirect immunofluorescence technique (IFT), also significantly (p < 0.001) increased (up to 4.7-fold) the human sperm penetration rates in SPA. The T alpha 1 and antibodies to both T alpha 1 and T beta 4 enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of T alpha 1 and antibodies to T alpha 1 and T beta 4 on percent sperm motility, although they significantly affected various motility characteristics such as velocity, amplitude of lateral head displacement (ALH), and beta frequency--the motility parameters involved in hyperactivation phenomenon of sperm cells. Both T alpha 1 and T beta 4 were detected in the seminal plasma of fertile men, and the levels of T alpha 1 were significantly (p = 0.002) lower in the seminal plasma of infertile men having defective sperm function. These results indicate that the thymosin molecules, especially T alpha 1, may have a role in human sperm capacitation leading to acrosome reaction. These findings also suggest that the T alpha 1 may find clinical applications in the specific diagnosis and treatment of male infertility in humans.
BIOLOGY OF REPRODUCTION 47, 1064-1072 (1992)
Thymosin Alpha-i Enhances the Fertilizing Capacity of Human Sperm Cell: Implication
in Diagnosis and Treatment of Male Infertility1
Reproductive Immunology and Molecular Biology Laboratories,3 Department of Obstetrics and Gynecology
The Albert Einstein College of Medicine, Bronx, New York 10461
Department of Obstetrics, Gynecology and Reproductive Science,4 The Mount Sinai School of Medicine
New York, New York 10029
Department of Biochemisby and Molecular Biology,5 The George Washington University School of Medicine
Washington, District of Columbia 20037
The effects of synthetic thymosin peptides (Tot 1 and T4) and their antibodies on the fertilizing capacity of human sperm cells were
investigated. Totl, but not the T4, significantly (p <0.001) increased the human sperm penetration rates in sperm penetration as-
say (SPA). Antibodies to both Tal and Th4, which predominantly bound to the acrosomal region of human sperm cell in the indirect
immunofluorescence technique (1FF), also significantly (p <0.001) increased (up to 4.7-fold) the human sperm penetration rates
in SPA. The Tal and antibodies to both Tal and T134 enhanced spontaneous as well as calcium ionophore-induced acrosome reac-
tion and release of acrosin from the human sperm cells. There was no effect of Ta 1 and antibodies to Tot 1 and T4 on percent sperm
motility, although they significantly affected various motility characteristics such as velocity, amplitude of lateral head displacement
(ALH), and beat frequency-the motility parameters involved in hyperactivation phenomenon of sperm cells. Both Tat and T134 were
detected in the seminal plasma of fertile men, and the levels of Tot! were significantly (p =0.002) lower in the seminal plasma of in-
fertile men having defective sperm function. These results indicate that the thymosin molecules, especially Tot 1, may have a role in
human sperm capacitation leading to acrosome reaction. These findings also suggest that the Tot 1 may find clinical applications in the
specific diagnosis and treatment of male infertility in humans.
The thymus gland is involved in immunologic and en-
docrinologic homeostasis of the body [1]. There have been
reports indicating the correlation of gonadal activity with
thymic function. Both estrogen and androgen receptors have
been identified in thymic epithelial cells, and various fac-
tors of the thymus gland can modulate the levels of sex
steroids, which in turn can cause spontaneous and experi-
mentally induced autoimmune diseases and alter the im-
mune response during pregnancy [2]. Several humoral fac-
tors processing hormone-like activity have been isolated and
characterized from the thymus or its extracts [3,4]. Some
of the most important factors among them include prothy-
mosin-a (ProTa; 12.6 kDa), thymosin-al (Tal; 3.108 kDa),
and thymosin-34 (TB4; 4.982 kDa); Tal and T134 have been
sequenced and synthesized. Tal is thought to be derived
from ProTa, since its 28-amino acid sequence is identical
to that of the latter’s N-terminus [3, 4]. Tal has been shown
to induce growth factor-like activity on cell proliferation in
the 1713-estradiol-treated breast carcinoma cell line (MCF-
7) [5], and synergizes with epidermal growth factor in the
stimulation of rat fibroblast cell proliferation by modulating
Accepted August 5, 1992.
Received May 22, 1992.
‘This work was supported by NIH grant HD 24425 to RK.N.
2Correspondence: Rajesh K. Naz, Ph.D., obstetrics/Gynecology, Ullmann #123,
The Albert Einstein College of Medicine. 1300 Morris Park Avenue, Bronx, NY 10461.
FAX: (212) 863-0599.
the expression of c-fos proto-oncogene [6]. It was further
found that the thymosin-immunoreactive peptides are as-
sociated with proliferation, particularly during G1 phase of
the proliferative cycle of rat small intestinal cell line (IEC-
6) [7]. T134 was originally proposed solely as a thymic hor-
mone, on the basis of its ability to stimulate the T-lympho-
cyte terminal deoxynucleotidyl transferase and to inhibit
macrophage migration [2,4]. Recently, it was shown that TfM
may be a potent regulator of actin polymerization in living
cells [8]. Thus, Ted and possibly Tf34 may be of marked
importance in cell growth and differentiation.
Fertilization is a complex process requiring the sper-
matozoon to undergo a cascade of events before it can fuse
with the egg plasma membrane. This chain of events in-
clude capacitation, binding to the zona pellucida, acrosome
reaction, and penetration through the zona pellucida [9, 10].
Capacitation is a physiological process that the mammalian
sperm cell must undergo before it can fertilize the oocyte
[9, 10]. Although this phenomenon was discovered in 1951
and has been extensively studied since then, the molecules
and exact mechanisms involved in sperm cell capacitation
leading to acrosome reaction are not clearly understood at
The aims of the present study were (1) to investigate the
effects of synthetic thymosin peptides (Tal and Tt34) and
their antibodies on human sperm cell capacitation and/or
acrosome reaction, and (2) to determine the presence and
concentration of these thymosin peptides in the seminal
plasma from fertile and infertile men. The overall objective
was to investigate the role of thymosin molecules in human
sperm cell function and investigate their utility in diagnosis
and treatment of human male infertility.
Thyinosin Peptides (Tal and T(34) and Their Antibodies
Tal, a synthetic 28 amino acid (a.a) peptide (3.108 kDa)
of thymic origin, and T134, a synthetic peptide (4.982 kDa)
of thymic origin, were obtained from Alpha 1 Biomedicals,
Inc., (Washington, DC); both were dissolved in sterile PBS
(pH 7.4) at a concentration of 1 mg/mI, aliquoted, and stored
at -20#{176}C.The endotoxin level in these thymosin peptides
was less than 0.03 pg/mg of Tad and T134 as measured by
standard limulus lysate assay.
Antibodies to Tal and T134 were raised in rabbits as de-
scribed elsewhere [11]. Briefly, Tal and T34 were coupled
to keyhole limpet hemocyanin (KLH) as a carrier, and rab-
bits were injected at intramuscular and subcutaneous sites
with Tal-K1.H or T134-KLH emulsified with Freund’s com-
plete adjuvant. After 2 wk, the rabbits were immunized,
weekly for 3 wk, with the respective antigen preparation
(Tal-KLH or T34-KLH) emulsified with the incomplete ad-
juvant. One week after the last injection, the animals were
bled by retro-orbital puncture and the antisera were ti-
trated using the radioimmunoassay, as described later.
The sera collected from rabbits injected with the same
amount of KLH (without the thymosin peptides) or with an-
other 30 a.a synthetic control peptide, termed human p17 gag
peptide (HGP), conjugated with KLH served as controls. The
HGP cross-reacts with human immunodeficiency virus (HIV)
p17 glycosaminoglycan peptide and shares a 9 a.a homology
with Tad [11, 12]. However, the antibodies to HGP do not
recognize Tal and also the antibodies to Tal do not recog-
nize HGP [11, 12]. These antibodies will henceforth be re-
ferred to as anti-KU-I alone and anti-HGP-30 antibodies re-
spectively. All these antisera were heat-inactivated (56#{176}C,30
mm), aliquoted, and stored at -20#{176}Cuntil used.
These antibodies were analyzed for their binding with
methanol-fixed and unfixed viable non-capacitated as well
as capacitated human sperm in an indirect immunofluo-
rescence technique (IFT) as described elsewhere [13, 14].
Briefly, the ejaculated sperm from fertile men were washed
in PBS, and a swim-up population of highly motile sperm
was collected, washed in PBS, air-dried on slide, fixed with
methanol (30 mm at room temperature), air-dried again,
and then treated with antiserum (1:50 dilution) at room
temperature for 1.5 h in a humid chamber. The antibody-
binding was localized by incubating with fluorescein iso-
thiocyanate-labeled goat anti-rabbit antibodies (1:50 dilu-
tion; Cappel Labs., Malvern, PA) [13, 14]. The slides were
washed with PBS (three times), mounted in 90% glycerol
in PBS containing sodium azide (0.1%) and 1,4-diazabicyclo
(2,2,2) octane (10 mg/mI) to reduce photobleaching dur-
ing observation. Samples were kept at 4#{176}Cin a humid
chamber until examined. The capacitation was induced by
incubating (8 h, 37#{176}C,in 5% CO2 and 95% air mixture) the
swim-up sperm (10 X10 sperm/mi) in Ham’s F-b me-
dium containing 5% BSA The capacitated sperm were
washed twice with PBS, methanol-fixed, and studied for an-
tibody reactivity as described above. For IFT on unfixed vi-
able cells, the swim-up sperm were washed twice with PBS
and incubated (37#{176}Cfor 2 h) with the thymosin antibodies
(4 x 106 sperm/mI, 1:20 final dilution of the antibody),
washed twice in PBS, and then incubated (37#{176}Cfor 1.5 h)
with fluorescein isocthiocyanate-labeled goat anti-rabbit im-
munoglobulin (1:40 dilution containing 0.02% sodium azide
to avoid capping). The cells were washed, mounted, and
examined as described above.
Both Tad and T134 and their antibodies were further in-
vestigated for their effects on fertilizing capacity of human
sperm cell using the human sperm penetration assay of zona-
free hamster oocytes (SPA), and the assays for acrosome
reaction and human sperm motility.
Sperm Penetration Assay
SPA was performed by the method of Yanagimachi et al.
[15] as described elsewhere [16, 17]. Briefly, superovulation
was induced in adult female golden hamsters by i.p. injec-
tion of 30 IU eCG (Sigma Chemical Co., St. Louis, MO) on
Day 1 of the cycle. After 5 5-72 h, 20 IU hCG (Sigma Chem-
ical Co.) was administered i.p. The animals were killed 15-
17 hafter hCG injection, and the mature unfertilized ova
were collected and separated from the surrounding cu-
mulus cells and from the zonae pellucidae.
Semen from fertile men (n =3) was liquilhed for 15-
30 mm at 37#{176}Cand the swim-up sperm population was col-
lected as described elsewhere [16, 17]. The sperm cells in
the swim-up were washed with Biggers, Whitten, and Whit-
tingham medium (B’W) supplemented with 1% BSA (frac-
tion V, No. A-7906; Sigma Chemical Co.), adjusted to 5-10
X10 motile sperm/ml and then allowed to incubate for
5-6 h at 37#{176}C(in 5% CO2 and 95% air mixture) with the
Tal and T134 (0.01-10 p.g/b00 p.l of the sperm suspen-
sion), or their antibodies (10-20 p.l of antiserum/100 l
of sperm suspension), or the same amount of control an-
tibodies (normal rabbit serum/anti-KLH alone antibodies/
anti-HGP-30 antibodies), or the equivalent volume of the
PBS containing 0.5% BSA (PBS-BSA). After incubation, the
sperm were washed to remove the unreacted antibody and
co-incubated with zona-denuded hamster oocytes (20-50
eggs/treatment in each assay) for 3-4 h. The oocytes were
removed, washed thoroughly, fixed with 3% giutaralde-
hyde, and stained with acetocarmine solution. Penetration
was determined by the presence of a swollen sperm head
with discernible tail in the cytoplasm of the ovum. Motility
of sperm before and after incubation with ova was re-
corded. The assays were repeated at least two to eight times
using three different fertile donors, and each sample was
tested with at least 41-480 oocytes.
1066 NAZ ET AL.
The percentage of ova penetrated was calculated ac-
cording to the following formula:
%ova penetrated =
Totalnumber of sperm penetrated
Total number of ova incubated
Using this assay [15-17], we obtain approximately 93-100%
ova penetrated with sperm from fertile men, with an av-
erage of one sperm penetrated per oocyte.
The SPA was also performed in infertile (n =18) and
fertile men (n =5), described in Table 5, by the modified
method of Syms et al. [18], using test yolk buffer, as de-
scribed in detail elsewhere [19]. In this assay, liquified se-
men was combined with an equal volume of prewarmed
(37#{176}C)test yolk buffer and allowed to capacitate for 24 h at
4#{176}C.The sperm were then washed and added to the zona-free
hamster oocytes; the number of sperm penetrated per ovum
was determined by the procedure described above. Using this
assay [18, 19], we obtain 100% ova penetrated with sperm
from fertile men, with an average of 26.2-34.9 sperm pene-
trated per oocyte (Table 5). If the sperm function is abnor-
mal, there is a decrease in number of sperm penetrated per
oocyte and also in the percentage of ova penetrated if the
sperm abnormality is severe. In these infertile men (28-37
yr old), infertility was attributed to the male factor or idio-
pathic etiology; they demonstrated abnormal semen analy-
sis and/or defective sperm function in SPA These men were
unable to impregnate their female partners after more than
1 yr of unprotected sexual intercourse. The fertilemen (27-
34 yr old) used in this study were those who had sired a
healthy baby within previous 3 yr.
For immunoabsorption experiments, the anti-Tab and
anti-T134 antibodies (20 .tl each) were reacted with Tab or
T134 or PBS-BSA (10 J.g each) in PBS overnight at 4#{176}C;the
reaction mixture was centrifuged and the supernatants were
tested as described above.
Assess,ne’nt of Acrosome Reaction
The effect of thymosmn peptides and their antibodies (with
appropriate controls) was also investigated on the human
sperm acrosome reaction. The effect on acrosome reaction
was assessed by determining the acrosomal status of the
sperm after antibody incubation as well as by studying the
acrosin distribution after the acrosome reaction.
Assessment of acrosomal status. The motile sperm were
collected from fertile men by use of the swim-up proce-
dure [16, 17]. The good quality sperm samples (5 x 10 mo-
tile sperm/mI, >75% motility, +3 to +4 forward progres-
sion on a scale of 0 to +5) were incubated with Tab or T134
or anti-Ta 1 antibodies or anti-T34 antibodies. The controls
were treated with the same amount of normal rabbit serum
or anti-KLH alone antibodies or anti-HGP-30 antibodies or
PBS-BSA. After incubation, the spermatozoa were centri-
fuged, washed with PBS, and divided into 2 aliquots. One
aliquot was induced to acrosome react with calcium iono-
phore (10 p.M final concentration of calcium ionophore
A23 187 [Sigma] was incubated with sperm suspension for 1
h) [20], and the other aliquot was studied for the sponta-
neous acrosome reaction without treatment with the cal-
cium ionophore. The acrosomal status was assessed by us-
ing the triple stain procedure [21] as described elsewhere
[22]. Briefly, the ionophore-treated sperm or non-iono-
phore-treated sperm (spontaneous acrosome-reacted sperm)
were washed (twice) in PBS and then incubated for 37#{176}Cfor
15 mm with 2% Tiypan blue (Sigma) (1:1, v/v) in PBS. The
sperm were washed (twice), fixed in 3% glutaraldehyde in
0.1 M cacodylate buffer for 60 mm, and then washed (twice)
again in PBS. A drop of suspension was placed on a glass slide
and allowed to air-dry overnight [21, 22]. The slides were
stained in 0.8% Bismarck Brown (Sigma) in deionized water
(pH 1.8 with 2N HC1), rinsed in deionized water, and stained
for 45 mm (at room temperature) in 0.8% Rose Bengal
(Sigma) in 0.1 M cacodylate buffer (pH 6.0). The slides were
then washed in deionized water, dehydrated in an alcohol
series, cleared in xylene, mounted with permount, and cov-
ered with a coverslip [22]. A total of 200-500 sperm per
sample were evaluated and recorded as either live or dead,
and the live sperm were further evaluated as acrosome-in-
tact or non-acrosome-mntact (reacted) sperm. The experi-
ments were repeated three to five times using sperm of at
least three different fertile donors.
Assessment of acrosin distribution. After incubation with
the thymosin peptides or their antibodies or control anti-
bodies or PBS-BSA, and then with or without treatment with
calcium ionophore A23b87 for 1 h as described above, the
sperm cells were centrifuged and the acrosin activity was
determined in the supernatant and cells by the method of
Kennedy et al. [23] as described elsewhere [22]. Briefly, 90
p.1 of the supernatant or the pelleted sperm cells sus-
pended in 90 p.1 of PBS were mixed with 1 ml of the re-
action mixture (1 mg/mI of N-benzoyl-d/-arginine-p-ni-
troanilide [BAPNA] hydrochloride in 0.055 M HEPES buffer,
0.055 M NaC1, 10% [v/v] dimethyl sulfoxide, 0.1% [v/v] Tri-
ton X-100) and incubated for 3 h at room temperature. The
reaction was stopped by addition of 100 p.l of 0.5 M ben-
zamidine and the absorbance was measured at 405 nm. Ac-
rosin activity was expressed at p.IU/106 spermatozoa. The
daily variability of the assay was normalized using a cryo-
preserved, partially purified human acrosin extract pre-
pared as described elsewhere [22].
Sperm Motion Analysis
After incubation with the thymosin peptides or their an-
tibodies or control antibodies or PBS-BSA for 5-6 h as de-
scribed above, an aliquot (7 p.1) of the sperm suspension
was placed into a Malder chamber (Sefi-Medical Instru-
ments, Israel) and the sperm motion characteristics were
determined using a computerized semen analyzer (Cell Soft,
Cyro Resources Ltd., New York, NY) as described elsewhere
[16, 17, 22]. The following parameter settings were used
throughout the study: 30 frames analyzed at an image fre-
quency of 30 Hz, 3 frames minimum sampling for motility,
15 frames minimum sampling for both velocity and ampli-
tude of the lateral head movement (ALH) measurements,
10 p.m/sec threshold velocity, minimum linearity of 2.5 for
AU-I measurement, and cell size range of 4-40 pixels with
a magnification calibration of 0.688 p.m/pixel.
The levels of thymosmn peptides (Tal and T134) were as-
sayed in the seminal plasma of fertile and infertile men by
radioimmunoassay. The seminal plasma were collected from
the liquefied semen of fertile (n =5) and infertile (n =
18) men by centrifugation (Table 5).
The radioimmunoassay was performed as described
elsewhere [11,24]. Briefly, 25-50 p.1 of seminal plasma was
diluted to 400 p.1 with radioimmunoassay buffer (PBS, pH
7.4, supplemented with azide and normal rabbit serum).
Standards using synthetic Tal or T134 were prepared and
diluted to 400 p.1 with radioimmunoassay buffer. Tab tracer
(1251-Tyr-Tal) or T134 tracer (125I-Tyr-C13-T34) was added
in 50 pA, as was the anti-Tab or the anti-TIM antiserum. The
samples were vortexed and incubated for 24 h at 4#{176}C.A
second antibody (goat anti-rabbit IgG) was added, and the
samples were incubated for an additional 12-18 h at 4#{176}C.
The complexes were precipitated by centriftigation, the su-
pernatants were aspirated, and the pellets were counted.
Alternatively, goat anti-rabbit IgG bound to a polymer (Kynar,
Roche Diagnostics, Raritan, NJ) was added to precipitate the
The thymosmn levels (Tab and T4) were correlated with
the sperm concentration, sperm motility, and the penetra-
tion rates.
Statistical Analysis
Significance of differences between treated (thymosmn
peptides or their antibodies) and control (normal rabbit
serum or anti-KLH alone antibodies or anti-HGP-30 anti-
bodies or PBS-BSA) was calculated using paired as well as
unpaired Student’s t-test. The correlation between the Tad
and T[34 levels in seminal plasma, semen parameters (total
sperm, % motility, and % normal sperm), and the penetra-
tion rates (Table 5) was determined by analyzing for linear
regression. Correlation coefficients (-y) with p<0.05 were
considered signfficant.
Effects on SPA
The thymosmn al (Tal) significantly (p <0.001) in-
creased (up to 2.6-fold) the percentage of ova penetrated
compared to PBS-BSA-treated control sperm (Table 1), tested
at a concentration of 0.05-0.5 p.g Tal /100 p.1 sperm sus-
pension. At higher concentrations (2-10 p.g/bOO p.1 solu-
TABLE 1. Effects of Tel and T4 on human sperm penetration rates.
(g/100 l) Ova tested
(No.) Ova penetrated, %
(Mean ±SD)
Tel 0.01 41 120.3 ±11.6
0.05 82 268.2 ±26.4
0.1 124 251.0 ±36.3
0.5 167 204.0 ±13.9
2.0 82 100.5 ±0.7
10.0 261 102.2 ±18.9
T4 0.1 122 102.7 ±6.5
0.5 128 99.7 ±10.1
2.0 173 61.8 ±279b
10.0 179 31.5 ±12.2
PBS-BSA 302 97.6 ±8.4
#{149}bavs. control, p <0.001; b vs. control, p =0.00 9; others were insignificant
vs. control.
tion), there was no increase in the penetration rates as
compared to control. However, these concentrations did not
decrease the percentage of ova penetrated. In contrast to
Tal, incubation of sperm with similar doses of TIM did not
significantly increase the penetration rates; in fact, at con-
centration of 2-10 p.g/100 p.1, there was a significant de-
crease in the percentage of ova penetrated.
In contrast to thymosmn molecules (Tab and TIM), the
antibodies to both Tad and TIM significantly (p <0.001)
increased (up to 4.7-fold) the penetration rates in a con-
centration-dependant manner (Table 2). Antibodies to KLH
carrier, or to HGP-30 a.a. peptide-KLH conjugate or normal
rabbit serum, tested at the same concentration (20 p.1/100
p.!) as the anti-Tal and anti-T4, did not affect penetration
rates as compared to the PBS-BSA-treated control sperm.
Immunoabsorption of the antibodies (anti-Tal and anti-TIM)
with the respective peptide caused a significant (p <0.001)
reduction in the fertilization-enhancing activity of the an-
tibodies (Table 2).
There were no visible effects of the thymosin molecules
or their antibodies on sperm motility (percent and pro-
The anti-Tad antibody demonstrated binding, predomi-
nantly with acrosomal regions of methanol-fixed non-ca-
pacitated human sperm cells in the 1FF (Fig. la). The anti-
TIM antibody also reacted strongly with the acrosomal
regions, besides reacting weakly with the tail regions, of
methanol-fixed human sperm cells (Fig. lb). The unfixed
viable non-capacitated human sperm cells also demon-
strated the same binding pattern, with the antibodies pre-
dominantly binding with the plasma membrane localized
on the acrosomal region of the sperm cell. Though the
binding pattern remained the same after capacitation, the
antibody binding slightly decreased on the capacitated sperm
compared to non-capacitated sperm, resulting in a de-
creased fluorescence intensity. The absorption of these an-
tibodies with the respective synthetic thymosmn molecules
1068 NAZ ET AL.
TABLE 2. Effects of thymosin antibodies on human sperm penetration rates.
(pi/lOO l)
Antibody absorbed
(p.g peptide) Ova tested
(No.) Ova penetrated. %
(Mean ±SD)
10 -83 121.0 ±5.7
20 -164 376.5 ± 557b
20 10 171 208.5 ±595bi
20 -166 457.8 ±
20 10 170 25001b
Anti-KLH alone
20 -83 108.0 ±2.8
20 - 84 83.5± 9.19
Normal rabbit serum
20 -80 109.5 ±12.0
PBS-BSA -480 98.0 ±6.9
#{149}bavs. control, p =0.004; b and b1 vs. control, p <0.001; others were insignificant vs. control.
FIG. 1. Epifluorescent photomicrographs indicating the indirect immunofluorescent reaction patterns of the an-
tibodies to Tel and T134. The antibody to Tal reacted with acrosomal regions of unfixed viable or methanol-fixed
capacitated as well as non-capacitated human sperm cells (a). The antibody to T4 demonstrated reaction with ac-
rosomal (stronger) and tail regions (weaker) of human sperm cells (b). The control antibodies to KLH alone or to HGP-
30 a.a. peptide-KLH conjugate did not react with the human sperm cell (d). The phase-contrast picture of d has been
included for comparison (ci. a-d, x850.
TABLE 3. Effects of thymosins and their antibodies on the acrosome reaction of human sperm.
Without ionop hore treatment With ionoph ore treatment
Acrosome-reacted sperm (%)
Acrosin activity”
Acrosome-reacted sperm (%)
Acrosin activity”
Cells Supernatant
Cells Supernatant
Treatment (Mean ±SD) (Mean ±SD) (Mean ±SD) (Mean ±SD) (Mean ±SD) (Mean ±SD)
Thymosins (0.5 g/l00 .i.l)
Tel 18.5 ±3.5’ 64.5 ±6.4’ 27.5 ±13.4’ 69.2 ±3.8#{176} 30.6 ±7.2C 68.2 ±16.6#{176}
Tf34 14.5 ±2.1 76.5 ±9.1 21.0 ±14.4 61.5 ±4.8 38.5 ±7.1 52.8 ±16.2
Antibodies (20 l/100 l)
Anti-Tel 16.4 ±3.2#{176} 69.6 ±11.0#{176} 26.3 ±13.6#{176} 67.3 ±4.2#{176} 31.0 ±2.6#{176} 70.0 ±6.5#{176}
Anti-TI34 17.0 ±2.6#{176} 66.3 ±8.5#{176} 25.0 ±13.1#{176} 68.0 ±3.5#{176} 32.6 ±3.8#{176} 67.3 ±8.0#{176}
Anti-KLH alone 12.7 ±1.2 74.5 ±7.8 19.0 ±15.5 60.7 ±4.03 41.2 ±13.6 56.4 ±8.9
Anti-HGP-30 13.0 ±1.4 73.0 ±11.3 19.5 ±13.4 61.5 ±3.5 - -
PBS-BSA 12.6 ±2.5 77.4 ±10.0 15.0 ±12.1 59.5 ±3.9 43.2 ±7.5 53.8 ±10.4
Assays (3-5) were performed on various days using sperm collected from at least 3 different fertile men.
bACrOsin activity was expressed as lU of acrosin/lO” sperm cells.
CC vs. control, p =0.04 to p <0.001; others were insignificant vs. control.
(20 p.1 of antibody +10 p.g of Tal or TIM) significantly
absorbed out the immunoreactivity of the antibody when
tested using methanol-fixed sperm cells. The normal rabbit
serum or the anti-KLH alone antibodies or the anti-HGP-30
a.a peptide-KLH conjugate antibodies did not react with the
methanol-fixed or unfixed viable non-capacitated or capa-
citated human sperm cells (Fig. id).
Effects on Acrosomal Reaction of Human Sperm
The Tab, but not the TIM, tested at a concentration of
0.5 p.g/100 pA (dose at which the Tal significantly in-
creased the penetration rates and T134 did not reduce the
penetration rates) significantly enhanced capacitation and
acrosome reaction of the human sperm cells (Table 3). Upon
incubation with the Tal for 5-6 h, the samples demon-
strated significantly higher percentages of acrosome-re-
acted sperm compared to sperm samples treated with PBS-
BSA (Table 3). The sperm samples treated with Tab showed
a higher percentage of acrosome-reacted sperm whether
they were tested for spontaneous acrosome reaction (with-
out ionophore treatment) or investigated after ionophore-
induced acrosome reaction. However, the effects were more
prominent after the ionophore treatment.
In contrast, the antibodies to both Tad and T134 caused
a significant increase in the percentage of acrosome-re-
acted sperm (Table 3). Again the effects were apparent
whether checked for spontaneous acrosome reaction or in-
vestigated after ionophore-induced acrosome reaction of the
antibody-treated sperm, although the effects were more
prominent after the ionophore treatment.
The antibodies to neither KLH alone nor to HGP-30 a.a
peptide-KLH conjugate showed any significant effect on the
percentage of acrosome-reacted sperm as compared to PBS-
BSA control.
Effects on Acrosin Distribution
Again, on incubation with Tal (0.5 p.g/100 p.!) (and not
with T134) or with anti-Tal (20 p.1/100 p.1) or anti-T134 an-
tibody (20 p.1/100 p.1), the sperm samples demonstrated a
significantly (p =0.04 to p<0.001) higher quantity of
acrosin concentration released in the supematant and a lower
quantity of acrosin concentration in the sperm cells, com-
pared to PBS-BSA-treated sperm (Table 3). The effects were
apparent whether investigated with or without the iono-
phore treatment, although the effects were more promi-
nent after the ionophore-treatment.
The antibodies to neither KLH alone nor to HGP-30 a.a.
peptide-KLH conjugate demonstrated any affect on the ac-
rosin distribution of sperm cells.
Effects on Sperm Motility Characteristics
Neither thymosin molecules (Tab or T134; 0.5 p.g/100
p.1) nor their antibodies (anti-Tal or anti-T134; 20 p.1/100
p.! each) significantly affected the percentage of motile sperm
cells in the sample, up to 5-6 h of the observation period
(Table 4). However, Tal and antibodies to both Tal and
T4 significantly (p =0.02 top <0.001) affected velocity,
ALH, and beat frequency of sperm cells; effects were more
apparent on the increase in ALH and beat frequency of sperm
cells.In contrast, neither T134 nor antibodies to KLH alone or
HGP-30 a.a. peptide-KLH conjugate affected any motility
characteristics of the sperm cells compared to PBS-BSA-
treated control (Table 4).
Presence of Thymosin Molecules in Seminal Plasma of
Infertile and Fertile Men
Tab was detected in seminal plasma of both infertile and
fertile men (Table 5). However, the Tal levels were sig-
nificantly (p =0.002) lower in the seminal plasma of in-
fertile men as compared to those of fertile men (mean ±
SD: infertile men, 1321.89 ±516.63; fertile men 2962.20 ±
1811.25). The levels of Tal correlated significantly (r =0.566,
p<0.01) with the number of sperm penetrated per ovum
in SPA. Three of these infertile patients [13, 16, 17], who had
‘SPA was performed according to the method of Syms et al. 1181, as described elsewhere 1191.
TABLE 4. Effects of thymosins and their antibodies on human sperm motility parameters.
Motility characteristics (Mean ±SD)
Treatment Percent motility Velocity Linearity ALHb Beat frequency
Thymosins (0.5 g/100 pM
Tel 80.3 ±6.2 61.1 ±3.0C 3.9 ±0.6 4.0 ±0.3#{176} 13.7 ±0.6#{176}
T4 76.0 ±4.3 58.5 ±2.6 4.0 ±0.6 3.7 ±0.4 13.1 ±0.3
Antibodies (20 p.1!lOO l)
Anti-Tal 75.0 ±5.0 60.9 ±1.1#{176} 3.9 ±0.7 3.8 ±0.1#{176} 14.0 ±0.2#{176}
Anti-T134 75.0 ±4.6 60.8 ±1.8#{176} 4.0 ±0.7 3.7 ±0.2#{176} 13.3 ±0.9#{176}
Anti-KLH alone 71.0 ±7.5 56.8 ±1.7 3.9 ±0.2 3.6 ±0.3 12.8 ±0.3
Anti-HGP-30 71.0 ±5.6 57.1 ±2.0 3.8 ±0.3 3.7 ±0.3 12.7 ±0.3
PBS-BSA 78.0 ±9.1 57.2 ±2.7 3.8 ±0.4 3.5 ±0.4 12.6 ±0.5
Assays (n =3-5) were performed on various days using sperm collected from 5 different fertile donors.
“ALH means amplitude of lateral head displacement.
CC vs. control, p =0.02 to p <0.001; others were insignificant vs. control.
most severe impairment of sperm motility and reduced DISCUSSION
penetration rates and percentage of ova penetrated, also Our results demonstrate that incubation with synthetic
had the least concentration (<1000 pg/mi) of Tal in sem- Tal increases the penetration rates of human sperm cells
inal plasma (616, 942, and 938 pg/mI). in zona-free hamster oocytes as measured by SPA, the assay
Again, T134 was also detected in seminal plasma of both routinely used in various clinical laboratories to evaluate
infertile and fertile men. However, T134 levels were slightly the fertilizing capacity of human sperm cells. In contrast, a
higher in seminal plasma of infertile men as compared to similar concentration of TIM, another thymosin peptide, did
fertile men (mean ±SD: infertile men, 527.75 ±689.83; not affect the penetration rates. The effects of Tab reached
fertile men, 219.00 ±92.98), although the differences were a plateau at a concentration of 0.5 p.g/100 p.1. TIM dem-
statistically insignificant. The levels of T134 did not correlate onstrated either no effect (0.1 p.g/100 pA) or a decrease (at
significantly (r =-0.283, p>0.05) with the number of concentrations of 2 and 10 p.g/bOO p.1, with 10 p.g showing
sperm penetrated per ovum in SPA. the most drastic effects) in the penetration rates. At the
TABLE 5. Ta1 and TB4 levels in seminal plasma of infertile and fertile men.
Semen analysis Penetration rate?
Total Sperm Ova TB4
sperm Motility Normal sperm penetrated/ovum penetrated Ta, level level
Patient (#1 (xlO6) (%) (%) (No.) (%) (pg/mI) (pg/mI)
Infertile men 476 70 70 5.9 100 1115 381
2 165 82 76 24.1 100 1370 -
3 216 64 61 -- 1222 352
4 41 68 56 - - 1271 156
5246 69 53 7.1 100 1403 369
6 19 62 69 18.1 100 1169 282
7 128 60 58 14.6 100 2171 318
8 73 61 77 14.2 100 1435 178
9 43 60 59 13.4 100 1007 2643
10 140 55 65 18.8 100 1330 805
11 81 47 53 22.3 100 2965 452
12 295 60 70 - - 1436 294
13 46 32 24 6.5 40 616 -
14 230 50 64 22.14 100 1206 -
15 245 79 61 21.55 100 1123 -
16 28 21 19 3.2 62 942 -
17 41 18 27 0.55 42 938 -
18 241 59 56 17.2 100 1075 103
Fertile men
1632 82 84 34.9 100 1438 102
2385 65 72 26.2 100 2175 197
31430 65 74 32.1 100 1931 354
4383 76 83 29.7 100 5962 188
5 1336 68 76 31.3 100 3305 254
Sperm extract
LIS-sperm extract (6.94 mg/mI) - - - - 1626 66
present time, we do not know the reasons for the detri-
mental effects of T134 on penetration rates. However, these
decreasing effects were not due to effects on sperm motil-
Antibodies to both Tal and TI34 showed a highly sig-
nificant increase in the penetration rates of human sperm
cells. The effects were specific since (1) antibodies to KLH
(carrier used for conjugating the thymosin peptides to raise
antibodies) or to another 30 a.a. HGP peptide conjugated
to KLH did not affect the penetration rates, and (2) ab-
sorption of the antibodies with the respective peptide sig-
nificantly reduced the fertilization-enhancing activity of both
these antibodies. Both these antibodies predominantly
demonstrated specific binding with the acrosomal regions
(TIM also showed binding with the tail regions, albeit weakly)
of unfixed viable as well as capacitated or non-capacitated
human sperm cells in 1FF, indicating the presence of Tal
and TIM on the head regions of sperm cells. Also, the LIS-
solubilized human sperm preparations showed the pres-
ence of Tab as well as TIM by radioimmunoassay (Table
5). Thus, the anti-Tal antibody simulated the effects of pu-
rifled Tal peptide in stimulating the penetration rates, rather
than inactivating or inhibiting the receptor molecules and!
or mechanisms involved in increasing the fertilizing capac-
ity of human sperm cells. These types of stimulating (rather
than blocking) effects have also been noted with other an-
tibodies. In another study carried out in our laboratory, it
was found that the binding of monoclonal antibody to epi-
dermal growth factor (EGF) receptor activates the receptor
to induce phosphorylation (as does the EGF molecule),
rather than blocking it [25].
It was intriguing to find that the antibody to T4 in-
creased the penetration rates, while T134 itself did not sig-
nificantly increase the fertilizing capacity of human sperm
cells. These effects may be due to the higher affinity and/
or valency of the antibody as compared to T[34. It is also
possible that TIM has negative effects on the fertilizing ca-
pacity of human sperm cells in a normal, healthy, fertile
individual, and that the antibodies to it neutralize the TI34
on the sperm cells, thus down-regulating the negative effect
of T134, which results in an increase in the fertilization po-
tential of sperm cells. This suggestion is supported by the
observations that (1) T134, at a higher concentration, de-
creases the penetration rates, and (2) the seminal plasma
of fertile men demonstrate a lower concentration of TI34
as compared to those of infertile men (Table 5). Interest-
ingly, actin, a major cytoskeletal protein of many cell types,
has been localized by immunological and biochemical
methods in the spermatozoa of several mammalian species
including humans [26,27], and TIM has been shown to be
a potent regulator of actin polymerization in living cells [8].
Although a unifying hypothesis for the role of actin in sperm
cell function is still lacking, actin polymerization has been
shown to be involved in many important cellular processes
such as cell locomotion, chemotaxis, phagocytoxis, and cy-
tokinesis. The exact mechanism of action needs further study,
but it is possible that the T134 and its antibodies may act
through modulation of actin polymerization in the sperm
In our study, Tal and anti-Tal antibodies increased the
sperm penetration rates in SPA, and the seminal plasma from
normal fertile men demonstrated a significantly (p = 0.002)
higher concentration of Tal compared to seminal plasma
from infertile men. These results agree with our previous
report showing the presence of Tal in the seminal plasma
of men and in the follicular fluids of women [24]. These
findings suggest that the thymosin molecules, especially Tab,
may have a role in sperm cell function-especially in sperm
capacitation and/or in the acrosome reaction-since the
SPA has been reported to be a measure of ‘capacitation”
of human sperm cells [28]. Indeed, the sperm sample treated
with the Tal or anti-Tab antibody demonstrated an in-
crease in the percentage of acrosome-reacted sperm when
allowed to spontaneously acrosome-react or induced to ac-
rosome-react in the presence of calcium ionophore. These
results were further confirmed by an increase of the ac-
rosin activity in the supernatant and decrease of the acrosin
activity in the sperm cells in both spontaneously acrosome-
reacting or ionophore-induced acrosome-reacting sperm.
These results indicate that both Tad and its antibody may
enhance the acrosome reaction. Since capacitation is a pre-
requisite for the sperm cell to undergo acrosome reaction,
Tab and its antibody may be involved in the capacitation
process. Neither Tab nor its antibody affected the percent
motility of sperm cells, although both significantly affected
the various motility characteristics, especially the velocity,
ALH, and beat frequency. The ALH and beat frequency have
been reported as important indicators in estimating hyper-
activation of sperm cells, which is considered an integral
part of capacitation preceding acrosome reaction and sperm
binding to zona pellucida [29, 30].
The exact mechanism of induction of capacitation by Tab
or its antibody or anti-TIM antibody needs further investi-
gation. These observations provide new insights into the
relationship between Tal and non-lymphoid non-somatic
cell differentiation. The role of Tab in sperm differentiation
is substantiated by several considerations. First, the thymic
hormones are multifunctional peptides, since expression of
prothymosin and thymosin is found in almost all tissues
and is not restricted to lymphoid cells, as could be ex-
pected for a T-cell-specific growth factor [5,31]. Second,
prothymosin and thymosin expression and function are re-
stricted to proliferating cells and are induced by application
of mitogenic stimuli to quiescent cells, as has been dem-
onstrated by activation of the a-prothymosin gene by myc
proteins [32]. Interestingly, our laboratory recently dem-
onstrated that the c-myc protein, c-ras protein, and EGF re-
ceptor are present on the human sperm cell and may be
involved in the signal transduction pathway leading to sperm
capacitation and/or the acrosome reaction [16,22, 25]. The
1072 NAZ El AL.
exact mode of interaction between c-rnyc protein, c-ras
protein, EGF-receptor, fertilization antigen (FA-1) [33], Tab,
T34, and various other unidentified growth factors and their
receptors, and their participation in the physiologic cascade
leading to sperm capacitation and the acrosome reaction
needs further study.
In conclusion, our data suggest that the human sperm
cell has Tad- and T134-like proteins that are recognized by
their respective antibodies, and that these molecules may
be directly or indirectly involved in some step vital for sperm
capacitation and/or the acrosome reaction. This investiga-
tion is first to report that the interaction of thymosin mol-
ecules with sperm can result in enhancement of fertilizing
capacity of human sperm cells. Further studies are needed
to investigate the exact signal transduction pathway by which
thymosin molecules and their antibodies are involved in
sperm cell capacitation and subsequent acrosome reaction.
These studies may also have clinical applications in the di-
agnosis and treatment of idiopathic and male factor-me-
diated human infertility especially since (1) Tal and anti-
bodies to Tal and T4 enhanced the fertilizing capacity of
human sperm cells, and (2) infertile men had significantly
lower concentration of Tad in their seminal plasma. At
present, we are extending this study to investigate the pres-
ence of Tad and T134 in serum, seminal plasma, and sperm
of infertile men with idiopathic infertility or defective sem-
inal parameters and/or who are incapable of fertilizing the
oocytes in a human in vitro fertilization-embryo transfer (1W-
ET) program. We are also studying the modulation of sperm
motility and function in infertile men by thymosins and their
We sincerely thank Laura Keller for providing some of the seminal plasma from
the Andrologv Laboratory. The superb secretarial assistance provided h’ Ms. Daniela
Pulitano is gratefully acknowledged.
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... The differential effects of the cyclin A and cyclin B. antibodies may be due to the differences in nature of the antibodies. We have seen that some antibodies, e.g., antibodies to epidermal growth factor (EGF) receptors, antibodies to p185 c-neu proto-oncogene product, and antibodies to thymosin o,, have enhancing (rather than neutralizing) effects after binding to the receptors ( [25,26]; our unpublished data]). Alternatively, it is possible that cyclin A and cyclin B. may have a differential roles in the sperm cell differentiation process. ...
The presence and roles of mitotic cyclins (cyclin A and cyclin B1), and cdc2 and related (having an N-terminal PSTAIRE conserved sequence) serine/threonine protein kinases were investigated by use of specific antibodies. The cyclin A and cyclin B1 antibodies reacted specifically with the acrosomal regions of human sperm cells in the indirect immunofluorescence technique (IFT) and recognized the specific band of p60 (cyclin A) and p62 (cyclin B1) on the Western blot of sodium deoxycholate (DOC)-solubilized noncapacitated human sperm preparation. Both antibodies reacted more strongly with the specific cell region/band of capacitated sperm than with that of noncapacitated sperm. The cdc2 and PSTAIRE antibodies also reacted predominantly with the acrosomal regions of human sperm cells in IFT and recognized the specific band of 34 kDa corresponding to p34 cdc2 protein on the Western blot of DOC-solubilized noncapacitated human sperm preparation. Again, both antibodies reacted more strongly with the specific cell region/band of capacitated sperm than with that of noncapacitated sperm. The cyclin A antibodies (but not the cyclin B1 antibodies) and cdc2 antibodies as well as the PSTAIRE antibodies significantly (p = 0.02 to p < 0.001) increased (rather than decreased) the human sperm penetration rates of zona-free hamster ova; the cyclin A and cdc2 antibodies showed the strongest enhancing effects. These three antibodies significantly increased the acrosome reaction and release of acrosin activity from the sperm cells.(ABSTRACT TRUNCATED AT 250 WORDS)
... 42 Despite enormous advances in sperm phosphoproteomic research, 44,56 the mechanisms of phosphorylation, especially tyrosine phosphorylation, in mammalian spermatozoa remain poorly understood. Capacitating human spermatozoa reportedly shed their sperm cytoplasm, which contains several growth factors/cytokines, including interleukin-2 (IL-2), 57,58 interleukin-6 (IL-6), 59 interleukin-8 (IL-8), 60 colony stimulating factor-1 (CSF-1), 61 interferon-g (IFN-g), 62 tumor necrosis factor-a (TNF-a), 63 epidermal growth factor (EGF), 64 Ta1, 65 and thymosin β4 (Tβ4), 66 in addition to several unidentified factors. Several of these factors are also present in the cervical mucus of the female reproductive tract. ...
Male infertility refers to the inability of a man to achieve a pregnancy in a fertile female. In more than one-third of case, infertility arises due to male factor. Therefore, developing strategies for the diagnosis and prognosis of male infertility is critical. Simultaneously, a satisfactory model for the cellular mechanisms that regulate normal sperm function must be established. In this regard, tyrosine phosphorylation is one of the most common mechanisms through which several signal transduction pathways are adjusted in spermatozoa. It regulates the various aspects of sperm function for example, motility, hyperactivation, capacitation, the acrosome reaction, fertilization and beyond. Several recent large-scale studies have identified the proteins that are phosphorylated in spermatozoa to acquire fertilization competence. However, most of these studies are basal and have not presented an overall mechanism through which tyrosine phosphorylation regulates male infertility. In this review, we focus of this mechanism, discussing most of the tyrosine-phosphorylated proteins in spermatozoa that have been identified to date. We categorized tyrosine-phosphorylated proteins in spermatozoa that regulate male infertility using MedScan Reader (v5.0) and Pathway Studio (v9.0).
... Thymosin has been reported to be localized at the acrosomal region of human spermatozoa and to increase its penetration rate and fertilizing capacity by enhancing the acrosome reaction (Naz et al., 1992). ...
Full-text available
Proteins of the signal crayfish Pacifastacus leniusculus egg and spermatophore were identified using in-gel digestion, mass spectrometry, and Mascot search. Forty-one and one-hundred-fifty proteins were identified in egg and spermatophore, respectively. The proteins were classified into nine categories including cell defence, cell signalling, cytoskeleton, DNA related activity, metabolism and energy production, protease and protease inhibitor, respiration, transportation, and others and unknown. Twenty-two proteins were found in both egg and spermatophore. The respiration and cytoskeleton groups are the most diverse categories in the protein profiles of the egg and spermatophore, respectively. No protein was assigned to DNA related activity and cell defence categories in the protein profile of the crayfish egg. Differences between protein profiles of the crayfish egg and spermatophore show different functional priorities for each of gametes. Several proteins having possible roles in gametogenesis, capacitation, acrosome reaction, and fertilization were identified. This proteomic profile of signal crayfish gametes provides a basis for further investigation of functional roles of the identified proteins in aspects of reproduction such as capacitation and fertilization.
... Because AT III is a serine proteinase inhibitor, we believe that AT III-immunoreactive molecules on the zona pelucida may act as sperm acceptor and may induce acrosome reaction of the capacitated sperm after binding of sperm to the egg. Although the identification of AT III as a sperm motility stimulator and chemoattractant was unexpected, there are many other examples of multifunctional proteins and the number of such proteins is increasing with time ( Morin et al., 1990;D'Alessio et al., 1991;Jenne and Tschopp, 1992;Marx, 1992;Naz et al., 1992;Singh and Green, 1993). In this study, we clearly demonstrated that porcine AT III stimulates the motility of boar sperm and possesses chemotactic activity for sperm and that AT III-immunoreactive molecules are distributed on the zona pellucida of ovulated eggs (cf. ...
Purified porcine antithrombin III stimulates motility and acts as a chemoattractant of washed boar sperm. Porcine follicular fluid and blood plasma both possess chemotactic activity. Porcine antithrombin III-immunoreactive molecules are distributed on the zona pellucida of ovulated eggs flushed from the swine oviducts. We propose that antithrombin III plays important roles in the mammalian fertilization process in vivo.
Thymosin β4 (TMSB4X) belongs to a class of highly conserved small proteins that are present in a high abundance in immune tissues, where it participates in various biological activities, including anti-inflammation, wound healing, apoptosis, and cell survival. However, little is known about the expression and regulation of TMSB4X in reproductive tissues. The aim of this study was to examine the expression of rat Tmsb4x and chicken TMSB4X genes during testicular and epididymal development. Rat Tmsb4x was strongly detected in the spermatogonia and spermatocytes of testis at early postnatal development and transmitted to Leydig cells at sexual maturation. Also, rat Tmsb4x was detected at an increased level in the epididymis during postnatal development. When compared to the rat, expression of the chicken TMSB4X gene was low in the testis and epididymal region, and the mRNA localization was also unexpected. Three experiments were performed to examine the regulation of rat Tmsb4x in the epididymis: after elimination of Leydig cells using ethylene dimethane sulfonate (EDS); after destruction of the testis by cryptorchidism; and after castration. EDS-treated and castrated rats were injected with testosterone propionate. The expression of Tmsb4x was significantly reduced in the epididymis of EDS-treated, and castrated rats. In contrast, Tmsb4x was significantly enhanced in the epididymis after testosterone treatment. The expression of rat Tmsb4x was also regulated in the epididymis after cryptorchidism. Collectively, the expression of Tmsb4x was strongly detected in the testis and epididymis of rats, and was highly regulated in the epididymis by testosterone. J. Exp. Zool. 9999A: 1-12, 2013. © 2013 Wiley Periodicals, Inc.
The effects of thymosin α1 (Tα1) and FA-1 monoclonal antibody (anti-FA-1 mAb) on murine preimplantation embryonic development were investigated by performing 2-cell embryo bioassay and by studying ova/embryos protein phosphorylation pattern (by 32P metabolic labeling and by in vitro kinase assay) and protein synthesis (by in vitro [35S]methionine labeling). Tα1 treatment (0.1, 0.5 and 5 ng/100 μl) significantly increased blastulation rates (P < 0.01), blastocyst hatching rate (P < 0.0001), blastocyst diameter (P < 0.001) and number of cells per blastocyst (P < 0.0001) of the in vitro cultured 2-cell stage embryos. Anti-FA-1 mAb reduced blastulation rates (P < 0.001) primarily due to an arrest of development at morula stage. In vitro metabolic labeling of murine ova/embryos showed 32P incorporation into 4 major protein bands of murine ova (Mr 125, 90, 68 and 31 kDa, respectively), 7 protein bands of 2-cell (Mr 90, 68 and 31; and 145, 52, 38 and 32 kDa, respectively), 10 protein bands of morula (Mr 150, 110, 92, 82, 70, 54, 39, 34, 30 and 29 kDa, respectively), and 15 protein bands of blastocyst (150, 110, 92, 70, 68, 54, 39, 34 and 30; and 131, 105, 52, 44, 43 and 33 kDa, respectively) stage embryos. Tα1 treatment (0.1–0.5 ng/100 μl) resulted in a general increase in 32P labeling in all proteins of 2-cell, morula and blastocyst stage embryos. Anti-FA-1 mAb completely blocked 32P labeling of various proteins of murine ova, 2-cell, morula and blastocyst stage embryos, whereas control mouse myeloma IgG did not affect phosphorylation of these proteins. In vitro kinase assay performed directly on various ova/embryos extracts revealed 6 phosphoproteins (Mr 105, 82, 55, 38, 34 and 33 kDa, respectively) that were common to ova and 2-cell embryos, besides a 43 kDa protein detected only in the ova extract. Of these phosphoproteins, Tα1 treatment specifically enhanced whereas anti-FA-1 mAb inhibited autophosphorylation of a 55 kDa protein of 2-cell embryos. Similarly, Tα1 treatment enhanced 6 phosphoproteins of morula (Mr 102, 60, 58, 49, 35 and 32 kDa, respectively) and 4 phosphoproteins of blastocyst (Mr 102, 60, 49 and 35 kDa, respectively) stage embryo extracts in the kinase assay. In [35S]methionine labeling experiments, Tα1 treatment did not affect labeling of any of the 5 proteins (Mr 170, 95, 86, 62 and 42 kDa, respectively) of 2-cell embryos, however, it enhanced labeling of 1 protein band of ova (46 kDa), 7 proteins of morula (Mr 95, 86, 70, 43, 42, 39 and 35 kDa, respectively) and 9 proteins of blastocyst (Mr 95, 86, 70, 43, 42, 39 and 35; and 51 and 33 kDa, respectively) stage embryos. Anti-FA-1 mAb did not affect 35S labeling of murine ova and 2-cell, morula or blastocyst proteins compared to control myeloma IgG. These results suggest that Tα1 enhance murine preimplantation embryonic development by modulating protein phosphorylation pattern as well as protein synthesis. On the other hand, anti-FA-1 mAb inhibit embryonic development by modulating protein phosphorylation pattern without affecting protein synthesis.
This investigation was carried out to examine the levels of CSF-1 in sera and seminal plasma of fertile, immunoinfertile, and male factor-infertile men in order to study the role of CSF-1 in human infertility. Sera, sperm, and seminal plasma were analyzed for antisperm antibodies by the sperm immobilization technique, tray agglutination technique, and immunobead binding technique. Sperm motility and fertilizing capacity were analyzed by sperm motion analysis and sperm penetration assay, respectively. CSF-1 concentration was estimated by the competitive radioimmunoassay. CSF-1 was detected in sera of both fertile and immunoinfertile men (mean ± SD; fertile men: 52.9 ± 10.3 pg/mg protein; immunoinfertile: 58.2 ± 19.5 pg/mg proteinic p ±.05). CSF-1 was also detected in the seminal plasma of fertile, immunoinfertile, and male factor-infertile men (mean ± SD; normal fertile men: 1.7 ± 3.5 ng/mg; immunoinfertile men: 10.9 ± 15.6 ng/mg; male factor-infertile men: 0.85 ± 0.25 ng/mg). The levels of CSF-1 were highest in the immunoinfertile men, followed by those in fertile and male factor-infertile men. The average level in immunoinfertile men was significantly different from the average level in fertile or male factor-infertile men (p <. 0001). Interestingly, the concentrations of CSF-1 per milliliter of seminal plasma was in general approximately 5 times greater than those in serum. These findings indicate that elevated seminal plasma CSF-1 concentration is correlated with male infertility.
The population explosion, unintended pregnancies, sexually transmitted diseases, and cancer (cervical and breast) continue to cause major public health issues worldwide. Curcumin, diferuloyl methane, the yellow pigment component of the curry spice turmeric (Curcuma longa), has immense biological effects and has recently drawn considerable attention. Curcumin has antibacterial, antiviral, antiinflammatory, and anticancer properties. It has shown a lack of toxicity in animals and human clinical trials. Yet, its effect on reproduction has not been examined. The present study was conducted to examine if curcumin affects sperm function in vitro and fertility in vivo. Sperm (human and murine) were collected and incubated with curcumin to examine the effect on motility, capacitation/acrosome reaction, and in vitro fertilization. The effect on in vivo fertility using the mouse model was also examined. Incubation of sperm with curcumin caused a concentration-dependent decrease in sperm forward motility, capacitation/acrosome reaction, and murine fertilization in vitro. At higher concentrations, there was a complete block of sperm motility and function within 5-15 min. Administration of curcumin, especially intravaginally, caused a significant (P<0.001) reduction in fertility. The antifertility effect of curcumin was reversible. This is the first study to report the inhibitory effect of curcumin on sperm function, fertilization, and fertility. The findings suggest that curcumin may constitute a double-edged sword to block conception, infection, and cancer, thus providing an ideal contraceptive.
To investigate the protein phosphorylation pattern and role of products of c-erbB-1 and c-abl proto-oncogenes with known tyrosine kinase activity in preimplantation embryonic development in mice. The protein phosphorylation pattern was studied by in vitro 32P metabolic labeling of murine ova/embryos as well as by in vitro kinase assay performed directly on various ova/embryos extracts. The role of products of c-erbB-1 (170 kDa, receptor for epidermal growth factor [EGF]) and c-abl proto-oncogenes (150 kDa) was examined by in vitro culturing murine embryos in the presence of monoclonal antibodies to respective protein products and by co-culturing with EGF, the ligand for EGF receptor (EGF-R). In vitro metabolic labeling of murine ova/embryos showed 32P incorporation into at least two protein bands of murine ova (M(r) 81 and 36 kDa), six protein bands of two-cell (M(r) 81, 36; and 97, 52, 22 and 19 kDa, respectively), six protein bands of morula (M(r) 81, 36; 97, 22, and 19; and 33 kDa, respectively), and eight protein bands of blastocyst (81, 36; 97, 22, 19; and 115, 58, and 15 kDa, respectively), stage embryos; there were some specific bands in each stage. Prolonged labeling from 2 to 4 h not only resulted in a relative increase in 32P incorporation into these proteins but also revealed additional bands in morula (M(r) 133 and 115 kD) and blastocyst (M(r) 49, 33, and 31 kD) stage embryos. In vitro kinase assays performed directly on various ova/embryos extracts revealed at least three phosphoproteins (M(r) 58, 36 and 33, respectively) that were common to ova, two-cell, morula, and early/late blastocyst stage embryos. Additionally, three protein bands each in murine ova and two-cell embryos (M(r) 108, 81, 73 kDa, respectively), and four protein bands of late blastocyst (M(r) 108, 73; 133 and 18 kDa, respectively) stage embryos were also revealed. Culture of two-cell embryos in the presence of EGF, the ligand for EGF-receptor, resulted in a concentration dependent increase (P < .001) in the number of cells per blastocyst. Monoclonal antibody to c-erbB-1 170 kDa protein (receptor for EGF) did not affect development of in vitro cultured murine embryos from two-cell to morula, but significantly (P < .001) inhibited the in vitro development of morula to late blastocyst stage. Monoclonal antibody to c-abl protein inhibited the development of murine embryos from two-cell to morula (P < .017), as well as, from morula to late blastocyst stage (P < .002 to .01). These results suggest that the stage-specific protein phosphorylation pattern and specific products of c-erB-1 and c-abl proto-oncogenes may have a role in preimplantation embryonic development in mice.
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Evaluation of the immune response of individuals exposed to human immunodeficiency virus (HIV) is an important component of any plan designed to lead toward the development of an AIDS vaccine. Since the levels of antibodies to HIV p17 and the synthetic p17 peptide HGP-30 correlate with stages of progression to AIDS, studies were initiated to determine whether cytotoxic lymphocytes directed toward target cells pulsed with HGP-30 and radioactive chromium were present in seropositive individuals. The significance of such cells in controlling HIV viral infection has recently been enhanced by reports that HIV p17 is on the surface of infected cells and that an inactivated virus vaccine depleted of viral envelope appears to be effective in controlling expression. The selection of HGP-30 as the p17 peptide to be evaluated in early studies is based on the presence of both T-cell and B-cell epitopes as predicted by computer modeling and mouse studies and the demonstration of in vitro neutralization activity by antibodies to the epitope. By using B-lymphoblastoid cells pulsed with HGP-30 and radioactive chromium as autologous targets and mixed leukocyte culture-expanded peripheral blood lymphocytes as effectors, CD8+ cytotoxic T lymphocytes against HGP-30-coated targets were identified in seropositive individuals. In this report we demonstrate that a synthetic p17 epitope can be a target for major histocompatibility complex-restricted cytotoxic T lymphocytes in HIV-infected individuals.
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An antigen was isolated from deoxycholate-solubilized rabbit testis and sperm by using an immunosorbent column containing IgG from a monoclonal antibody (8C10.5) that inhibits fertility. Elution was by stepwise increases in pH (8.0, 10.0, and 11.4), with the pH 11.4 fraction after recycling through the column showing a single band at 63 kilodaltons in slab NaDodSO4/PAGE with a silver stain. The antigen molecule was composed of two subunits, which on two-dimensional PAGE showed many spots within the same molecular size range (50-70 kilodaltons) but differing in charge. The antigen isolated either from testis or sperm showed mainly the same spots. The antigen is periodic acid/Schiff positive and contained 21% carbohydrate. An asialo-derivative of the antigen did not change its characteristics on NaDodSO4/PAGE. This glycoprotein resolved into two types of polypeptides, those binding and those not binding to a lens culinaris lectin column; some of the polypeptides appeared common to both fractions. Murine antiserum against the antigen neither agglutinated nor immobilized rabbit sperm but in immunofluorescence reacted with the plasma membrane of viable rabbit sperm as well as with murine and human sperm. Fertilization of female rabbits inseminated with treated sperm was not affected, but, by 9 days, fertility was significantly reduced (21% of controls). The postfertilization antifertility effect was not due to parthenogenic activation or to polyspermy. The antiserum reacted with one specific band in the one- and two-dimensional gel electrophoretic transfer blot procedure and was unaffected by absorption with different somatic tissues.
While studying ovum sperm interactions among various animals it was found that human spermatozoa, which were initially incapable of penetrating zona pellucida free animal (hamster) ova, acquired the ability to do so when incubated several hr in vitro. Experimental evidence suggests that this ability is associated with the completion of sperm capacitation and the acrosome reaction. Thus it appears that zona free animal ova can be substituted for human ova in the preliminary assessment of the fertilizing capacity of human spermatozoa when human ova are not readily available.
The monoclonal anti-phosphotyrosine antibody (PTA) recognized proteins related to relative molecular mass regions of 94,000 +/- 3000 and 46,000 +/- 3000 Mr on Western blots of detergent-solubilized non-capacitated human sperm extract (HSE). The pattern of phosphorylation at tyrosine residues depended upon the physiological state of the sperm cells. At least six protein bands corresponding to four molecular regions of 94,000 +/- 3000, 46,000 +/- 3000, 25,000 +/- 7000 and 12,000 +/- 2000 Mr, respectively, were labeled with 32P when human sperm were capacitated in vitro; the proteins belonging to the former three regions were phosphotyrosine proteins as they were precipitable by PTA. In vitro kinase assay performed directly on HSE indicated autophosphorylation of proteins of the same four molecular regions, with the capacitated sperm preparations having 30% higher 32P incorporation into 94,000 +/- 3000 Mr proteins and 17% less incorporation into 12,000 +/- 2000 Mr proteins as compared to the non-capacitated sperm preparations. Both of these protein regions were also autophosphorylated at tyrosine residues when immunoprecipitated phosphotyrosine proteins were used for the kinase assay. Phosphorylation of tyrosine residues of 94,000 +/- 3000 Mr proteins was further stimulated by 1.38- to 1.46-fold in response to exposure to zona pellucida proteins, namely the porcine ZP3 and human zona proteins (HZP); the HZP induced the highest response. Immunofluorescence observations on fixed human sperm demonstrated that capacitation as well as exposure to zona proteins increased the degree of tyrosine-specific fluorescence per sperm cell as well as the number of sperm cells that showed fluorescence at the acrosomal region of the spermhead.(ABSTRACT TRUNCATED AT 250 WORDS)
The presence and role of c-myc protein was investigated in mature sperm cells of the human, mouse, and rabbit. The monoclonal antibodies against c-myc protein (c-myc) reacted with the acrosomal region of the sperm of these mammalian species in the indirect immunofluorescence technique. The c-myc monoclonal antibody (MCA) recognized c-myc protein of 62 and 64 kDa on Western blots of lithium diiodosalicylate-solubilized sperm preparations of these species. The c-myc MCA showed a dose-dependent inhibition of human sperm penetration of zona-free hamster eggs, inhibition of murine in vitro fertilization, and reduced in vivo fertilization in rabbits. There was no effect of the antibody on percent sperm motility, though the antibody significantly affected various motility characteristics such as mean and maximum amplitude of lateral head displacement and curvilinear velocity involved in hyperactivation phenomenon of human sperm cells. These results suggest that c-myc or c-myc-like protein is present in mature sperm cells and may have a role in sperm cell function especially in capacitation and/or acrosome reaction.
The proto-oncogene MYC encodes a nuclear protein whose biochemical and physiological functions remain uncertain. We used an estrogen-regulated version of the MYC protein to explore these functions. Activation of MYC in quiescent rat and mouse fibroblasts elicited re-entry into and progression through the cell cycle, bypassing early events that would follow stimulation of the cells with serum. Activation of MYC led to a rapid increase in transcription of the alpha-prothymosin gene, even in the absence of protein synthesis. We conclude that the product of MYC acts directly on transcription, in accord with inferences based on the structure of the MYC protein. The function of alpha-prothymosin is not known, but our results suggest that the protein may play a role in the proliferation of mammalian cells.
Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.
High levels of thymosin alpha 1 (T alpha 1) were detected in human seminal plasma and follicular fluid. In the seminal plasma of 19 males studies, T alpha 1 levels varied from 614 to 2,604 pg/mL (mean +/- SD, 1,682.4 +/- 453.9 pg/mL). There was a correlation between the T alpha 1 levels and the total number of sperm in the ejaculate (r = .18) and seminal volume (r = .26). The infertile males, who had low levels of T alpha 1 also demonstrated fewer sperm, reduced motility, and lower semen volume. In follicular fluid collected from 24 follicles of 10 infertile females, T alpha 1 levels varied from 1,019 to 6,384 pg/mL (mean +/- SD, 3,572.8 +/- 1,599.7 pg/mL), which were higher when compared with the corresponding serum levels (mean +/- SD, 1,666.9 +/- 1,378.9 pg/mL). T alpha 1 levels present in follicular fluids which had "immature" oocytes were lower when compared with follicular fluids which had "intermediate" or "mature" oocytes. The immunoreactive T alpha 1 present in seminal plasma of males and in the follicular fluids of females may be involved in some aspect of germ cell maturation and function. The measurement of T alpha 1 levels may be useful in the diagnosis and treatment of male and female infertility, and also as a novel marker for the assessment of maturity of oocytes required for in vitro fertilization-embryo transfer.
The penetration of zona pellucida-free hamster ova by human spermatozoa has been used to quantitate sperm penetration potential. However, since mammalian eggs in vitro have limited viability, the effect of in vitro aging on the ability of hamster ova to be penetrated by human spermatozoa was examined. Zona-free ova maintained at room temperature (25 degrees C) lost their ability to be subsequently penetrated with a half-life of 50.1 +/- 8.8 minutes. This was partly the result of removing the zona pellucida by trypsin digestion, since zona-free oocytes in the presence of trypsin inhibitor or zona pellucida-intact oocytes had half-lives of 99.1 +/- 15.2 and 120.5 +/- 17.4 minutes, respectively. Reduction in penetration rates associated with ovum aging did not appear to be due to loss of viability and could be completely prevented by maintaining the ova on ice (4 degrees C). In the presence of TEST-yolk buffer at 4 degrees C, ova retained (100%) their ability to be penetrated for up to 24 hours and were morphologically indistinguishable from fresh ova. These observations show that ovum aging in vitro at 25 degrees C is much greater than previously anticipated. This may result in artifactually low and variable scores in the penetration bioassay.
The triple-stain technique according to Talbot and Chacon was adapted for use in routine semen analysis. This staining technique allows evaluation of spermatozoal morphology and determination of the percentage of dead and "live" spermatozoa and of the percentages of spermatozoa with and without intact acrosome. Optimal results were obtained if centrifugation prior to fixation was avoided, the semen was diluted before fixation, and the spermatozoa were fixed in suspension. This modification of the triple-stain technique produces preparations with low background and excellent visibility of all spermatozoal structures. Use of Trypan Blue leads to underestimation of the percentage of dead spermatozoa, but use of other supravital stains was even less satisfactory. Round cells could not be evaluated in the triple-stain technique.