Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism

Asian J Androl 2003 Mar; 5: 43-46             

Keywords: human spermatozoa; seminal plasma; heat-shock proteins 90; western blotting; sperm preservation; image analysis

Abstract

Aim: To study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism. Methods: In 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis. Results: The sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.13.2 and 243.021.6, respectively, while those after thawing were 23.22.5 and 105.728.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation. Conclusion: HSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.

1 Introduction

Semen cryopreservation plays an important role in helping those who suffer from irreparable infertility problems. The cryopreservation process, however, results in reduced fertility compared with the fresh semen. One obvious characteristic of cryopreserved spermatozoa is the decline in motility. Recently, Huang et al. [1] found that there was a substantial decrease of HSP90 in cooling the boar sperm, that preceded the decline of sperm motility. In another paper, it was indicated that an HSP90 specific inhibitor, geldanamycin (GA), significantly reduced the sperm motility in a dose- and time-dependent manner [2]. The above findings suggested the possibility of a cause and effect relationship between the decrease in HSP90 and the decline in semen characteristics during the cooling process. The present study was designed to help clarify whether a similar relationship exists in human spermatozoa and the cause of HSP90 reduction.

2 Materials and methods

Samples of semen were obtained from 18 donors referred to the Sperm Bank of this Institute/Hospital. Semen was collected by masturbation and allowed to liquefy for 15~30 min at room temperature before evaluation. The sperm concentration and progressive motility were determined by computer-aided sperm analysis. Only ejaculates with progressive motility higher than 70 % were used for this study.

Semen sample was diluted to a sperm concentration of 510⁷/mL with PBS (0.01 mol/L, pH 7.4) and transferred 1 mL each into 2, one for cryopreservation and the other placed in a 37 water bath as the control. The egg-yolk citrate buffer, containing 15 % glycerol, 20 % egg yolk, 1 % glycine, 4 % glucose, 4 % fructose and 4 % sodium citrate in distilled water prepared according to Zelder [3], was used as the cryopreservative medium (CPM). It was stored at 4 until use.

The buffer was gradually added to the semen sample to a ratio of 1:1 at room temperature and the diluted semen were decanted into plastic vials (Nunc CryoTubeTM, Denmark). The vials were placed in a controlled rate freezer (Planer, Kryo 360-1.7, UK), cooled down from room temperature to -5 at a rate of -2 /min, then from -5 to -70 at -50 /min and subsequently to -130 at -30 /min. Finally they were plunged into a liquid nitrogen tank for half an hour. Vials were then allowed to thaw for 10 min in a 37 water bath. After that, samples in both the control and the thawed vials were separately transferred into 6 mL tubes and centrifuged at 800 g for 10 min. The supernatant was used for protein analysis and the pellets, for further processing after washed with PBS and centrifuged for 10 min at 800 g for 2 times.

For spermatozoal protein profile analysis, the pellets were lysed in 0.5 mL sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2 % SDS, 5 % b-mercaptoethanol, 10 % glycerol and 0.002 % bromophenyl blue) for SDS-PAGE separation. For seminal plasma protein profile analysis, the supernatants were diluted 1:1 with the sample buffer, boiled for 5 min and stored at -20 until use.

Proteins were separated by 9 % SDS-PAGE according to the method of Laemmli [4]. The molecular standards employed were phosphorylase B (97 KDa), bovine serum albumin (66 KDa), rabbit actin (43 KDa), bovine carbonic anhydrase (31 KDa), trypsin inhibitor (20 KDa) and hen egg white lysozyme (14 KDa). After electro-phoresis, the gels were stained with 0.1 % Coomassie brilliant blue R-250 in 50 % methanol and 10 % acetic acid. After being stained for 60 min, the gels were destained with destaining solution (30 % methanol and 10 % acetic acid) until the background was clear.

After electrophoresis, the gels were soaked in a transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, pH 8.3) for 20 min. The proteins on the gels were then transferred to polyvinylidine diluoride (PVDF) membrane by electrophoretic transfer technique (Trans-Blot^® Cell, Bio-Rad, USA). The membranes were blocked with Tween-20 Tris-buffered saline solution (TTBS: 20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 0.05 % Tween-20) containing 1 % bovine serum albumin for 2 h and rinsed 15 min with TTBS for 3 times. The membranes were incubated with rabbit polyclonal antibodies against human HSP90 (diluted 1:200 with TTBS). After washing with TTBS, the membranes were incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (diluted 1:50 with TTBS) for 2 h at room temperature. The membranes were then rinsed with TTBS and developed for 3-5 min at room temperature with the developing buffer [5 µg diamino benzidine tetrahydrochloride (DAB), 10 mL 0.1 mol/L PBS, PH7.4, 5 µL 30 % H₂O₂]. The color of the immuno-complex was developed to a proper intensity.

After western blotting, the average and integrated grey levels of HSP90 bands on PVDF membrane were determined by computerized image analysis (KS400 image analysis systems, ZEISS, Germany). Proteins from both fresh and frozen-thawed seminal plasma were subjected to SDS-PAGE and Western blotting to see whether HSP90 would leak into seminal plasma

Data were expressed in meanSD and the comparisons of means were made with the paired t test. HSP90 data were analyzed with SAS 6.12 software.

3 Results

Figure 1 shows that there are no significant changes in sperm protein during the cooling process, except for proteins with molecular weights of 35, 90, 110 and 130 kDa (designated as P35, P90, P110 and P130, respectively). P35, P110 and P130 were increased and P90, decreased. The former 3 could be found in the CPM, but the latter could not (lane 8). The remains of these three proteins in the CPM, which could not be removed completely during sample preparation, resulted in the increase of these three proteins, whereas P90 did change after cryopreservation.

Figure 1. Protein profiles of human spermatozoa before and after cryopreservation. Molecular weight standards (lane1), sample before cooling (lane 2, 4 and 6), sample after thawing (lane 3, 5 and 7), cryopreservative medium (lane 8). hshows the protein band changed during cryopreservation.

Western blot analysis was performed using rabbit polyclonal antibodies against HSP90 to study P90 (Figure 2). The result demonstrated that P90 was HSP90. Quantitative results of HSP90 and semen characteristics obtained by computerized image analysis during cryopreser-vation are shown in Figure 2 and Table 1. The results suggested sperm motility and HSP90 in frozen-thawed spermatozoa had significantly decreased (P<0.01), compared with fresh spermatozoa before cryopreservation. Figures 3 and 4 showed that HSP90 was not found in the seminal plasma before or after cryopreservation.

Table 1. Sperm motility and HSP90 level before and after cryopre-servation. Data analysis using MEANS procedure of SAS. ^cP< 0.01, compared with fresh sperm.

Figure 2. Immunoblot analysis of spermatozoal proteins with antibody against human HSP90. Lane1 and lane2 are from the same subject, lane3 and lane4 are from the same subject. Sample before cooling (lane1 and lane3), sample after thawing (lane2 and lane4). The figure shows significant decrease of HSP90 after cryopre-servation.

Figure 3. Protein profiles of human seminal plasma before and after cryopreservation. Molecular weight standards (lane1), sample before cooling (lane 2), sample after thawing (lane 3), cryopre-servative medium (lane 4), sample 2 and 3 are from the same subject.

Figure 4. Immunoblot analysis of seminal plasma proteins with antibody against human HSP90. Lane1 and lane5 are spermatozoal proteins as a positive control, lane2 and lane3 are from the same subject. cryopreservative medium (lane 4). Sample before cooling (lane2), sample after thawing (lane3).

4 Discussion

Sperm motility impairment was found in the majority of cryopreserved spermatozoa, which has a negative influence on assisted reproductive technology. Since good sperm motility is helpful for clinical therapy of male infertility [5], the mechanism of motility impairment needs to be further elucidated.

Using boars as the study subject, Huang et al.[1] concluded that HSP90 might play a crucial rule in regulating porcine sperm motility, which decreased substantially during the cooling process. Using SDS-PAGE and Western blotting, we also confirmed this phenomenon in human spermatozoa. There may exist several mechanisms leading to the loss of HSP90. First, more than one protein was found leaking from spermatozoa to the extracellular medium, including membrane and cytosolic proteins [6]. Second, the decrease in HSP90 may be due to degradation of HSP90 itself. According to our results, no HSP90 was found in seminal plasma before and after cryopreservation.

Although HSP90 has been regarded as a cytosolic protein, its exact function still remains unclear. As a member of the heat shock protein family and regarded as a molecular chaperone, HSP90 plays an essential role in stress tolerance, protein folding, signal transduction, etc. HSP90 has been shown to possess an inherent ATPase, that is essential for the activation of authentic 'client' proteins in vivo [7]. Certain studies suggested that HSP90 might also be associated with sperm motility. Garcia-Cardena et al.[8] reported that HSP90 could activate nitric oxide synthase (NOS), which is beneficial to sperm motility. During the cooling process, reactive oxygen species (ROS) increased significantly and ROS can greatly impair the sperm motility. Fukuda et al. [9] reported that HSP90 protected cells from ROS. It was suggested that the ATP level was diminished after cold shock and would not restore later [10]. Recent studies have also demonstrated that HSP90 is involved in ATP metabolism [11].

In summary, HSP90 was decreased significantly after cryopreservation. It may be the result of protein degradation. Further research is worthwhile to elucidate its mechanism.

References

[1] Huang SY, Kuo YH, Lee WC, Tsou HL, Lee YP, Chang HL, et al. Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa. Theriogenology 1999; 51: 1007-16.
[2] Huang SY, Kuo YH, Tsou HL, Lee YP, King YT, Huang HC, et al. The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (HSP90). Theriogenology 2000; 53: 1117-84.
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[8] Garcia-Gardena G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, et al. Dynamic activation of endothelial nitric oxide synthase by HSP90. Nature 1998; 392: 821-4.
[9] Fukuda A, Osawa T, Oda H, Tanaka T, Toyokuni S, Uchida K. Oxidative stress response in iron-induced acute nephrotoxicity: enhanced expression of heat shock protein 90. Biochem Biophys Res Commun 1996; 219: 76-81.
[10] Watson PF. The effect of cold shock on sperm cell membranes. In: Morris GJ, Clarke A, editors. Effects of low temperature on biological membranes. London: Academic Press; 1981. p189-218.
[11] Prodromou C, Roe SM, O'Brien R, Ladbury JE, Piper PW, Pearl LH. Identification and structural characterization of the ATP/ADP-binding site in the HSP90 molecular chaperone. Cell 1997; 90: 65-75.

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Correspondence to: Dr. Wen-Lei CAO, Shanghai Institute of Andrology, Renji Hospital, Shanghai Second Medical University, Shanghai 200001, China.
Tel: +86-21-6326 1981, Fax: +86-21-6373 0455
E-mail: caowenlei@hotmail.com
Received 2002-09-02      Accepted 2003-01-17