Oxidative state and zona‐binding ability in mouse spermatozoa treated with reduced glutathione

Abstract

Background and Aims: Nuclear proteins in mature mammalian spermatozoa nuclei are oxidized to form numerous disulfide bonds. Reduced glutathione (GSH) in the oocyte has been linked to spermatozoan nuclear decondensation after fertilization. In this study, we analyzed whether GSH reduced protamines in sperm nuclei in vitro, and examined the zona‐binding ability of treated nuclei.

Methods:  Three groups of mouse cauda epididymal spermatozoa were prepared. The first group was cultured in Chatot–Tasca–Ziomek (CZB; control group), the second in 10 mmol/L GSH (GSH group), and the third group was the GSH group re‐cultured in CZB (re‐cultured group). Each sperm was stained with acridine orange, and the oxidative and reductive state of nuclei was analyzed by using fluorescence microscopy. Furthermore, we examined the zona‐binding ability for each group by insemination to mouse oocytes after exposure to hyaluronidase.

Results:  All sperm nuclei from the control group displayed an oxidized pattern (green), and those from the GSH group displayed a reduced pattern (red), attributable to reduced protamines. Sperm nuclei from the re‐cultured group displayed the oxidized pattern. Although the zona‐binding ability of the GSH group was deteriorated compared with the control group sperm, no significant differences were observed between the control and re‐cultured groups.

Conclusion:  From these results, in vitro reduced sperm were shown to be oxidized in CZB. A reduction of mouse spermatozoa in vitro by GSH is, therefore, reversible, and the oxidative state of sperm nuclei exerts an effect on zona‐binding ability. (Reprod Med Biol 2002; 1: 55–58)

INTRODUCTION

DURING EARLY SPERMATOGENESIS, the nuclear chromatin are the same histones present in somatic cell nuclear chromatin. In the sperm nucleus, however, the histones are gradually replaced with protamines to form filamentous structures. The nucleus then condenses to form a highly dense packed structure. ¹ , ² , ³ The protamines in the sperm nucleus form disulfide bonds, changing from a reduced to an oxidized state. ⁴ However, after the sperm nucleus enters the oocyte, the disulfide bonds break and the nucleus decondenses. Evidence now strongly suggests that reduced glutathione (GSH) in the oocyte plays a role in the mechanisms of sperm nuclear decondensation. ⁵ In vitro studies have demonstrated that decondensation does not occur in the absence of both GSH and heparin. ⁶ , ⁷ In this study, we used acridine orange staining to analyze in vitro oxidation and reduction of spermatozoa. We also evaluated the effects on the zona pellucida binding ability of spermatozoa.

MATERIALS AND METHODS

CAUDA EPIDIDYMAL SPERMATOZOA from 8‐week‐old B6D2F I mice were used. The cauda epididymides were severed by using scissors, and a dense sperm mass was squeezed out into a Chatot–Tasca–Ziomek (CZB) medium. ⁸ , ⁹ The suspended sperm were divided into three groups. The control group sperm were stained with acridine orange, as shown in Fig. 1. The remaining suspended sperm were cultured in 10 mmol/L GSH (pH 7.4) at 37°C in 5% CO₂ for 30 min to prepare in vitro reduced sperm (GSH group). Some of the latter sperm were then re‐cultured in CZB for 30 min (re‐cultured group). The oxidative and reductive state of the nuclear proteins in each sperm group was then analyzed by using acridine orange staining. ¹⁰

Figure 1.

Acridine orange staining method. ¹⁰

We also evaluated the effects of GSH on the zona pellucida binding ability of the sperm. We carefully dissected the cauda epididymides in CZB or 10 mmol/L GSH to prepare 1 h swim‐up samples. Some of the sperm cultured in GSH were again re‐cultured in CZB. The swim‐up sperm of the three groups were carefully prepared so that no change in the oxidation or reduction of the sperm was made. In the meantime, 8‐week‐old female mice were induced to superovulate by using an intraperitoneal injection of 10 IU pregnant mare serum gonadotrophin (PMS) followed by 10 IU human chorionic gonadotrophin (HCG) 48 h later, and oocytes were collected from the fallopian tubes after another 14 h. Oocytes were freed from cumulus cells by exposure to 0.1% hyaluronidase. They were inseminated by placement into each sperm group suspension. The oocytes were retrieved 20 min later and washed three times with a pipette to determine the number of spermatozoa bound to the zona.

RESULTS

ALL SPERM NUCLEI from the control group (100%) were stained green under acridine orange. This confirmed that the mice cauda epididymal spermatozoa displayed the oxidized pattern, with disulfide bond formation.

The sperm nuclei of the GSH group displayed red staining, indicating in vitro reduction of the sperm nucleus protamines. To investigate the relationship between GSH concentration and degree of reduction, we prepared GSH at concentrations of 2.5, 5 and 10 mmol/L. The results of culturing the epididymal spermatozoa in GSH at each concentration are shown in Table 1. As the GSH concentration increased, the proportion of the oxidised pattern sperm decreased. No significant changes in motility were observed, even at a concentration of 20 mmol/L, however, more detailed evaluation could not be performed because of mechanical damage to the sperm heads during smear preparation.

Table 1.

Reduction and sperm motility of mouse epididymal spermatozoa treated with GSH

To evaluate the potential for auto‐oxidation of sperm reduced by treatment with 10 mmol/L GSH, we re‐cultured some of the sperm in CZB. All sperm nuclei in this re‐cultured group (100%) displayed green staining. This confirmed that the in vitro reduced sperm protamines were able to readily auto‐oxidize in the presence of CZB without GSH. The series of treatment steps caused no significant changes to sperm motility (Table 2).

Table 2.

Motility rates in each sperm group (mean value for three mice)

In addition, to evaluate the zona‐binding ability of the in vitro reduced sperm, we determined the mean number of sperm in each group bound to the zona pellucida. Sperm motility after swim‐up for insemination did not significantly differ among the three groups. Although reduction of sperm nuclei with GSH significantly decreased the number of bound sperm, the auto‐oxidized sperm in the re‐cultured group again regained this binding ability. No significant differences were observed between the control and re‐cultured groups (Table 3).

Table 3.

Zona‐binding ability in each sperm group

DISCUSSION

Reduced glutathione IN the presence of heparin ¹¹ is reported to cause decondensation of sperm nuclei. ¹² In our study, mouse cauda epididymal sperm nuclei displayed 100% green staining under acridine orange, indicating an oxidized type. When treated with 10 mmol/L GSH, almost all sperm nuclei changed from yellow to red staining, indicating a reduced pattern. Our findings are the first to demonstrate that even in the absence of heparin, disulfide bond formation of intranuclear protamines can be altered by treatment with GSH. An interesting finding was that these structural changes were different from the change with reducing agents such as dithiothreitol (DTT), and could be produced while retaining sperm viability and causing no significant effect on motility.

The proportion of reduced sperm varied with GSH concentration. Less reduction was observed at lower concentrations, and sperm nuclei tended to display yellow staining rather than red with acridine orange. At concentration ≥20 mmol/L, sperm heads tended to be easily destroyed during smear preparation, suggesting a change to rather weak intranuclear bonding. In addition, at least 30 min was required for a sufficient reaction with GSH.

Recent studies report that phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium‐containing protein, plays a role in the formation of disulfide bonds in sperm nuclei and mitochondria. During sperm maturation, the two known different structures of PHGPx each play a role in sperm nuclei or mitochondria. In nuclei, PHGPx enables nuclear condensation by disulfide bond formation with nuclear protamine. ¹³ , ¹⁴ In mitochondria, PHGPx is thought to be involved in the formation of disulfide bonds with mitochondrial capsule proteins, resulting in a more tightly packed capsule structure. ¹⁵ , ¹⁶ The rapid oxidation of GSH‐reduced sperm protamines in the absence of GSH probably involves PHGPx.

A reduction of sperm protamines by GSH was associated with a significant decrease in the number of spermatozoa bound to the zona pellucida. This is probably caused by changes in the stereostructure of zona receptors present in the sperm membrane where the nuclear disulfide bonds were broken. Our findings suggest an association between nuclear immaturity and membrane‐binding ability. In human ejaculated sperm, unlike in mice, acridine orange often produces yellow or red fluorescent staining of immature sperm. In patients with a high percentage of these immature sperm, the fertilization rate with in vitro fertilization is generally low. ¹⁷ The results of the present study suggest a mechanism leading to these differences in human fertilization rates. In addition, zona‐binding ability was recovered in the re‐cultured sperm group, with auto‐oxidation at a level similar to that in the control group. This finding indicates reversible changes in the membrane.

In conclusion, the reduction of mouse spermatozoa in vitro by GSH is reversible, and the oxidative state of sperm nuclei affects zona‐binding ability. Further research is needed to elucidate factors such as the interaction of the sperm nucleus and membrane.