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

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Abstract

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)
1064
Thymosin Alpha-i Enhances the Fertilizing Capacity of Human Sperm Cell: Implication
in Diagnosis and Treatment of Male Infertility1
RAJESH K. NAZ,2’ PAUL KAPLAN,4 and ALLAN L. GOLDSTEIN5
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
ABSTRACT
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.
INTRODUCTION
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
present.
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
ROLE OF THYMOSINS IN SPERM CELL FUNCTION 1065
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.
MATERIALS AND METHODS
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.
X100
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
ROLE OF THYMOSINS IN SPERM CELL FUNCTION 1067
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.
Radioimmunoassay
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
complexes.
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
RESULTS
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.
Treatment
(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
Control
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-
gressive).
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.
Antibody
(pi/lOO l)
Antibody absorbed
with
(p.g peptide) Ova tested
(No.) Ova penetrated. %
(Mean ±SD)
Anti-Tel
10 -83 121.0 ±5.7
20 -164 376.5 ± 557b
20 10 171 208.5 ±595bi
Anti-T4
20 -166 457.8 ±
20 10 170 25001b
Anti-KLH alone
20 -83 108.0 ±2.8
Anti-HGP-30
20 - 84 83.5± 9.19
Normal rabbit serum
20 -80 109.5 ±12.0
Control
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.
ROLE OF THYMOSINS IN SPERM CELL FUNCTION 1069
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 - -
Control
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.
1070 NAZETAL.
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
Control
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
ROLE OF THYMOSINS IN SPERM CELL FUNCTION 1071
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-
ity.
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
cell.
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
antibodies.
ACKNOWLEDGMENTS
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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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.
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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.
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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.
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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.
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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.
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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.
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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.