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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
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].

Several studies have investigated the effect of cryopreservation on the sperm membrane and have found a decline in the membrane integrity after thawing[4-8]. However, there have been few attempts to evaluate the cryodamage done to head and tail membranes of a single spermatozoon, and to assess the effect of cryodamage on the sperm function. Recently, the World Health Organization (WHO) has recommended the eosin Y (EY) exclusion and the hypoosmotic swelling (HOS) tests as integral parts of sperm assay[9,10]. As the EY method determines the head plasma membrane's integrity and the HOS test, the tail's[11,12], we integrated the two methods into a single test to make the whole assay more convenient. The present work is designed to investigate the effect of cryopreservation on plasma membrane integrity in the head and tail regions of individual spermatozoon, and the relationship between intact cryopreserved spermatozoa and their motility and zona-free hamster oocyte penetration rate.

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

The Student's t-test was used to compare pre- and post-thaw samples for HOS-EY test scores. A difference with P<0.05 was considered significant. The correlation between the values of Type IV integrity and the values of other tests were evaluated by the linear regression analysis and correlation coefficient determinations.

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.


I (%)

II (%)

III (%)

IV (%)





















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 

The work was supported by the Natural Science Foundation of Guangdong Province, China.


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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