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Cryodamage
to plasma membrane integrity in
head and tail regions of human sperm
Wei-Jie
ZHU, Xue-Gao LIU
Center
for Reproductive Immunology Research, Jinan University, Guangzhou 510632,
China
Asian J Androl 2000 Jun; 2: 135-138
Keywords:
spermatozoa;
cryopreservation; cell membrane; supravital staining; hypoosmotic swelling
test
Abstract
Aim: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions of individual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-free hamster oocyte penetration rate. Methods: The eosin Y exclusion and the hypoosmotic swelling tests were combined to form a single test (HOS-EY test) to identify the spermatozoa with four types of membrane integrity. Results: After cryopreservation, there was a marked decline in the percentage of spermatozoa with Type IV membrane integrity (head membrane intact/tail membrane intact), and a significant increase in those with Type I (head membrane damaged/tail membrane damaged) and Type III (head membrane damaged/tail membrane intact) membrane integrity (n=50, P<0.01). The value of Type III integrity had a wide range of variability, whereas Type II (head membrane intact/tail membrane damaged) was uncommon after thawing. A high correlation was observed between the percentage of Type IV integrity and sperm motility (n=50, r=0.74, P<0.01). However, the values of Type IV integrity were usually lower than those of post-thaw motility in most cryopreserved samples. The value of Type IV integrity did not correlate with the sperm penetration rate (n=25, r=0.22, P>0.05). Conclusion: (1) The HOS-EY test has the advantage of showing four patterns of membrane integrity in individual spermatozoon; (2) Cryopreservation causes a significant membrane rupture in the head and tail regions of spermatozoa; Type III is the main transitional state of membrane cryodamage; (3) Cryodamage to head and tail membrane may occur independently; the presence of an intact tail membrane does not necessarily indicate the intactness of head membrane. (4) Intact membranes are closely related to post-thaw motility, but do not reflect the fertilizing potential.1 Introduction
The
sperm plasma membrane is an outer cell structure that acts as a physiological
barrier and its integrity is required for their normal activities[1].
During cryopreservation sperm undergo dramatic
changes in their intracellular and extracellular environment due to exposure
to cryoprotectives, cooling, freezing, storage in liquid nitrogen, and
thawing. The chemical and physical effects of these reagents/processes
may cause extensive cryodamage to plasma membranes with resultant changes
in their normal functioning[2,3].
2 Materials and methods
2.1
Semen samples
Semen
samples were obtained by masturbation after 3-6 days of sexual abstinence
from 50 adult, healthy and fertile men with a minimum sperm concentration
of 60106/mL
and a minimum motility of 40%.
Samples
were laid aside at room temperature for 30 min to allow complete liquefaction.
After that, a routine semen analysis was performed according to the WHO
laboratory manual[9].
2.2
Semen cryopreservation
Semen
samples were cryopreserved by using a method previously described[13,14].
Briefly, semen was mixed with an equal volume of yolk-free cryoprotective
medium containing 10% glycerol (v/v). The diluted semen was aspirated
into a 1-mL polypropylene syringe, and after placing in a 4 water bath
for 30 min, the syringe was plunged directly into liquid nitrogen for
storage. Thawing was accomplished by keeping the syringe at room temperature
for 30 and then in a 37
water bath for 5 min.
2.3
The HOS-EY test
The
HOS-EY test introduced in the present paper was a combination of the HOS
test (Jeyendran et al[12]) and the EY method (Eliasson
& Treichl[11]) with minor modifications. A hypoosmotic
solution consisting of sodium citrate 7.35 g/L and fructose 13.51 g/L
was employed. A 1.5% eosin Y solution was prepared by dissolving 0.15
g eosin Y in 10 mL hypoosmotic solution.
The
HOS-EY test was performed as follows: 0.1 mL of liquefied or cryopreserved
semen was mixed with 1.0 mL hypoosmotic solution and incubated at 37
for 30 min. During the last 2 min of incubation, 0.1 mL eosin solution
was added. After incubation, this suspension was immediately examined
under a light microscope at 400 magnification. The sperm head stained
red (EY positive) or unstained (negative) was observed. The swelling phenomena
of sperm tail irrespective of the types of tail coiling, type b to g described
by Jeyendran et al[12] was
determined. One hundred spermatozoa were counted and classified into the
four categories described below according to morphological changes of
membranes at both the head and the tail regions.
Type
I: head-red (EY+)
and tail-non-swollen (HOS-)
Type II: head-white
(EY-) and tail-non-swollen (HOS-)
Type III: head-red
(EY+) and tail-swollen (HOS+)
Type IV: head-white
(EY-) and tail-swollen (HOS+)
2.4
Sperm function tests
Sperm
motility was evaluated using a conventional microscopic method. Sperm
zona-free
hamster oocyte penetration assay (SPA) was performed as described by WHO[9,10]
with the following modifications. The washed spermatozoa were incubated
for 6 hours, the final insemination concentration was (7-10)106
motile sperm/mL, and the sperm-oocyte co-incubation time was 4 h.
2.5
Data analyses
3
Results
Table
1 showed that there were significant differences in HOS-EY test scores
between pre- and post-thaw specimens (P<0.01). With the cryopreserved
sperm, the percentage range
of Type III was 5-29, whereas Type II occurred
sparingly.
Table
1. Membrane integrity (Type
I-IV) before and after cryopreservation. Data in means; Values within
brackets: percentage range. cP<0.01, compared with
the fresh sperm.
|
(n=50) |
I
(%) |
II
(%) |
III
(%) |
IV
(%) |
|
Fresh |
24.511.3 |
0.91.3 |
6.24.4 |
68.611.6 |
|
sperm |
(4-56) |
(0-6) |
(1-14) |
(41-92) |
|
Frozen |
51.59.7c |
0.020.1 |
16.56.1c |
32.28.5c |
|
sperm |
(26-76) |
(0-1) |
(5-29) |
(21-61) |
In
the cryopreserved sperm, there was a high correlation between the percentage
of Type IV integrity and the motility (n=50, r=0.70, P<0.01)
(Figure 1). But in most cryopreserved
specimens, the values of Type IV were lower than those of post-thaw motility.
Besides, in cryopreserved sperm, the percentage of Type IV integrity did
not correlate with the percentage of SPA (n=25, r=0.22,
P>0.05) (Figure 2).
Figure
1. Relationship between post-thaw motility and Type IV integrity
in cryopreserved sperm.
Figure 2. Relationship between
zona-free hamster oocyte penetration rate and Type IV integrity in cryopreserved
sperm.
4
Conclusions
The
HOS-EY test is a combination of eosin Y staining and hypoosmotic effect
to assess the plasma membrane integrity in the head and tail regions of
a single spermatozoon. If the tail membrane is disrupted, it will lose
its osmoregulatory capacity and the swelling phenomenon of the sperm tail
will not occur when exposed to hypoosmotic conditions. If the head membrane
is damaged, it will lose its resistance to eosin penetration and the sperm
nuclei would be stained red. Type I is the group of non-viable sperm with
membrane defects both at the head and tail, Types II and III are transitional
states showing membrane defects either at the tail or the head, and Type
IV is the group of viable spermatozoa with intact membrane.
If
only the HOS test is used, sperm with swollen tail will include two groups
of cells: Types III and Type
IV. Meanwhile, a EY method alone can only identify live (Types II and
IV) and dead (Types I and
III) spermatozoa according to their staining characteristics, but can
not distinguish Type II from Type IV. With the combined HOSEY method
employed in the present study, four patterns of sperm membrane integrity
could be clearly differentiated in both fresh and cryopreserved spermatozoa.
The HOS-EY test has the advantage to discern in an individual spermatozoon
whether the damage is at the head, the tail, or both.
After
cryopreservation, the percentages of Types I and III were significantly
increased, which indicated that the procedure may impair membrane integrity
in both the head and tail regions. In the cryopreserved sperm, Type II
was rare and Type III was considered the main transitional state of membrane
cryodamage. An increase in the proportion of Type III after thawing indicated
that cryodamage to the sperm head and the tail membrane took place independently.
Thus, a functionally intact tail membrane does not necessarily indicate
that the head membrane is also
intact. In the freezing-thawing process, disruption of sperm head membrane can
occur more easily than the tail. The values of Type III may be highly
varied, as they seem to be related to both the freezing methodology and
the quality of spermatozoa. Disruption to sperm head or tail membrane
may lead to disturbed sperm function, therefore, only Type IV can be considered
functionally normal.
The
present study showed a high correlation between Type IV and motility in
cryopreserved sperm, which was in agreement with the results of Chan and
colleagues[7]. We indicated that in most cryopreserved sperm
specimens, the values of Type IV were usually lower than those of post-thaw
motility. However, theoretically, the former should be higher than the
latter. The discrepancy can be further investigated. Probably several
factors may be involved in this apparently paradoxical phenomenon: (1)
when cryopreserved sperm with a slight membrane damage were subjected
to a hypoosmotic solution for 30 minutes at 37, their membrane permeability
might have been enhanced; (2) the presence of glycerol might have promoted
eosin permeation into the slightly damaged sperm[16]; and (3when
the hyperosmotic medium of cryoprotective sperm was diluted into hypoosmotic, the
huge osmotic difference might have exerted certain stress on the sperm
membranes[17]. All these factors could aggravate the membrane
permeability and sperm with a slight membrane damage might have been stained
with eosin.
Obvious
efforts have been made to find out additional sperm functional tests to
predict their fertilizing capacity. Jeyendran et al[12]
found that the results of the HOS test correlated with SPA strongly for
non-cryopreserved human sperm. Others also have correlated the HOS test
and sperm fertilizing capacity[18,19]. In the present study,
the percentage of Type IV did not correlate with the percentage SPA for
the cryopreserved sperm. It suggests that SPA may not be associated with
membrane integrity. Thus, the results of these two tests appear to reflect
different aspects of cryopreserved sperm function.
The
HOS-EY test described in this paper has demonstrated its applicability
to identify the membrane integrity
of cryopreserved spermatozoa. The combined test makes possible the differentiation
of sperm with intact or defective membranes at either the head or the
tail; it is more useful for assessing membrane cryodamage than
the HOS test and the EY method. Besides, eosin added into the testing
medium can act as a background stain and allows the swelling phenomenon
of sperm tail to be clearly observed under a light microscope. This point
has a practical value for laboratories that are not equipped with a phase-contrast
microscope. In conclusion, cryopreservation causes a pronounced membrane
impairment in the head and
tail regions of the sperm. Cryodamage to the head and tail membrane can
occur independently. The HOS-EY test combines the advantages of the two
methods and may provide important
information to the cryodamage status in a single spermatozoon. It is a
useful adjunct to the existing sperm assays in determining the quality
of cryopreserved human sperm.
5
Acknowledgments
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Correspondence
to: Prof. Wei-Jie Zhu, PhD. Center for Reproductive Immunology Research,
Jinan University, Guangzhou 510632, China
Tel: +86-20-8522 0235 Fax:
+86-20-8522 1941
e-mail: tzhuwj@jnu.edu.cn
Received
2000-02-02 Accepted 2000-04-11
