Cryopreservation of human sperm in the presence of Zn²⁺ increases the motility rate

Introduction

The practice of human sperm freezing using liquid nitrogen was first introduced in the 1960s [1] and is currently routinely used in the assisted reproductive laboratory. Recovery of motile sperm after thawing is essential for further fertility treatment. However, risks following thawing including thermal shock, formation of intracellular ice crystals and osmotic shock can cause cell membrane damage and cell death that might lead to a reduced percentage of motile sperm [2, 3]. Indeed, the most common detrimental effects of the freeze-thaw sperm process include reduced motility, membrane damage, ultrastructural changes [4], DNA damage [5] and loss of mitochondrial function [6]. Various substances have been used for enhancing human sperm resistance to the stress of cryopreservation-thawing. Of these progesterone and/or acetyl carnitine were found not to be adequately effective in preventing human sperm stress after thawing [7]. Brugnon et al. reported that density gradient centrifugation prior to cryopreservation and hypotaurine supplementation improve post-thaw quality of sperm. However, total motility and progressive motility were in the low range of 18% and 11%, respectively [8]. A different method for sperm freezing by vitrification (without the use of conventional cryoprotectants) which requires 1% human serum albumin and 0.25 M of sucrose was compared to the conventional cryoprotectant freezing protocol. Compared to the initial sperm motility prior to freezing (74%), no statistically significant differences between the two methods were observed after thawing, in total motility and rapid progressive motility (20% and 11%, respectively) [9].
The aim of the present study was to assess the improvement in sperm cryopreservation conditions by adding Zn²⁺ to the freezing medium. Cryopreservation causes oxidative stress to the spermatozoa [2, 3] while it is known that Zn²⁺ acts as an antioxidant [10]. In human sperm, Zn²⁺ has been shown to improve membrane integrity and reduce DNA damage [11, 12]. Moreover, the addition of Zn²⁺ to human semen before freezing prevents DNA damage and preserves sperm function [13].
It is known that protein kinase A(PKA) mediates sperm motility [14]. In a recent study we demonstrated that Zn²⁺ activates PKA in sperm, which is mediated by the G-protein-coupled receptor GPR39 [15]. Zn²⁺ activates adenylyl-cyclase in sperm [15], and the increase in cAMP leads to actin polymerization [16, 17], a process necessary for the regulation of sperm motility [18]. The increase in flagellar beat required for the development of hyper-activated motility is also mediated by cAMP [19, 20]. Zn²⁺ is required for normal physiological function and development [21, 22]. The testes contain a high concentration of Zn²⁺ [23], which is essential in both the earlier and late stages of spermatogenesis [24, 25]. It has been suggested that extracellular Zn²⁺ activates a signal transduction pathway by several mechanisms, including the activation of ZnR, a member of the G-protein-coupled receptor (GPCR) family [26].
A high concentration of Zn²⁺ is needed for the earlier and the late stages of spermatogenesis in the testes [24, 25]. However, after penetration into the female reproductive tract, sperm are exposed to 1.0–1.5 µM Zn²⁺ [27], which is about 1000 times lower compared to its concentration in the seminal plasma. Thus, under physiological conditions in the female reproductive tract Zn²⁺ may enhance sperm capacitation and fertilization ability. Sperm motility is known to be extremely reduced after the freeze-thaw process, thus further highlighting the importance in improving the cryopreservation protocol, in order to maintain a higher motility rate after freezing. The aim of the present study was to verify the enhancement of sperm motility by Zn²⁺ after thawing.

Material and methods

Sperm preparation

After obtaining informed consent, the left over spermatozoa after IVF or ICSI procedures were transferred for freezing evaluation to the andrology laboratory of Bar-Ilan University. It should be emphasized that since our research was performed with left over spermatozoa, the length of time between ejaculation and freezing was approximately 4 to 6 h, in all the samples.
Human semen was liquefied by incubation for 30 min at room temperature; afterwards, the semen was loaded on a gradient (PureCeption Lower and Upper Phase Gradient 80% and 40%) and centrifuged for 10 min at 3000 g at room temperature. The lower layer containing the sperm was collected and washed in Ham’s F-10 medium. The motile cells were collected and resuspended in Ham’s F-10 medium containing 21 mM HEPES, 4 mM sodium bicarbonate, 0.6% human serum albumin, and 3.6 ml sodium lactate (60% syrup). The lower layer containing the sperm was collected and washed twice in Ham’s F-10 medium, then centrifuged again, and the sperm were allowed to “swim up” after the last wash at 37°C. The motile cells were collected without the pellet and resuspended in capacitation medium and supplemented with 0.1% human serum albumin (HSA, Sigma-Aldrich, USA, Cat. No. A1653).
This procedure allowed us to obtain motile sperm without leukocyte contamination.
Statement about IRB: An informed consent form was signed to allow further evaluation of the remaining spermatozoa. No further statement of Institutional Review Board is required.

Cryopreservation and thawing

The liquefied ejaculates were divided into two aliquots; ZnCl2 was added to one aliquot and the other aliquot served as a control. The samples were kept at room temperature for 10 min, after which they were mixed with equal volumes of cryoprotective medium (sperm freezing medium kit, Quinn’s Advantage, SAGE). The aliquots were transferred to screw-top cryovials and stored in liquid nitrogen for several days, as previously described. The samples were removed from liquid nitrogen, subjected to rapid thawing and washed in HamF-10 medium.

Sperm motility determinations

Sperm cells (1 × 107 cells/ml) were incubated in capacitation medium HamF-10 for 1, 2, 3 or 4 h. Samples (5 µl) were taken out at the indicated time and placed in a pre-warmed standard counter four-chamber slide (20 l m-depth) (Leja, Nieuw-Vennet, Netherlands) at 37°C, and analyzed using a CASA (computer-aided sperm analysis) device with IVOS software (version 12, Hamilton-Thorne Biosciences). Up to 10 sequences, each 30 s long, were acquired for each sample. At least 700 cells were analyzed in each sample, according to parameters identifying human sperm motility.

Results

Freezing of human sperm in the presence of 50 µM Zn²⁺ revealed a 26%-184% increase in the number of motile sperm in 10 out of 13 semen samples (Table 1). In 3 semen samples a decrease in total motility was observed after freezing with Zn²⁺. Freezing in the presence of Zn²⁺ also showed a 130% increase in sperm presenting progressive motility (Figure 1). Similar results were obtained when motility determination was performed at zero time or after 1 h of incubation (Figure 1). These data clearly indicate that the presence of Zn²⁺ in the freezing medium significantly improved sperm motility after freezing-thawing. Incubation of frozen sperm after thawing revealed a gradual reduction in the number of motile cells, reaching zero motility after 4 h of incubation; however, this reduction did not occur when sperm were frozen in medium containing Zn²⁺ (Figure 2). Thus, we conclude that the presence of Zn²⁺ in the freezing medium enhances the percentage of motile sperm and preserves motility for a longer period of time.
The percentage of cells presenting progressive motility was also significantly increased depending on the fluid volume used for freezing (Figure 3). Freezing in 100 µl resulted in a two-fold improvement compared to freezing in 50 µl when both total and progressive motility were determined (Figure 3). The presence of Zn²⁺ in the freezing medium revealed about a 2-fold increase in the number of motile cells and cells presenting progressive motility when 50 µl of freezing medium was used, whereas in 100 µl there was about a 3-fold increase in total and progressive motility (Figure 3). We can conclude that the sample volume used for freezing the cells is critical for achieving a higher proportion of motile cells, especially in medium containing Zn²⁺.
The presence of Zn²⁺ also protects sperm motility after a second freezing. When frozen cells were thawed and then refrozen in the presence of Zn²⁺, there was a 48% and 93% increase in total and progressive motility, respectively (Figure 4). After the second freezing and thawing 2% and 1% of the cells were motile or showed progressive motility, respectively, compared to slightly higher rates of 3% and 2% (total and progressive motility, respectively) when the second freezing was performed with Zn²⁺. These data clearly indicate that Zn²⁺ protects the cells against loss of motility even after a second freezing.
We also tested the effect of glucose and pentoxifylline on sperm motility after freezing with Zn²⁺. Glucose increases intracellular ATP levels by operating the glycolysis pathway. Pentoxifylline, an inhibitor of the enzyme phosphodiesterase which hydrolyzes cAMP, will cause an increase in intracellular levels of cAMP. It is well known that both ATP and cAMP are necessary molecules for functional sperm motility. The data in Figure 5 show that glucose plus pentoxifylline caused 93% stimulation of progressive motility after the second freezing and thawing, whereas when cells were frozen with Zn²⁺ there was a 154% increase in progressive motility. No significant differences were observed by adding glucose or pentoxifylline when cells were frozen in the presence or absence of Zn²⁺ in the freezing medium. Addition of Zn²⁺ plus glucose caused 43% and 71% increases in progressive motility when cells were frozen in the absence or presence of Zn²⁺, respectively (Figure 5). Thus, we conclude that the best conditions to achieve good motility are to freeze the sperm in 100 µl of medium containing 50 µM Zn²⁺ and to add glucose and pentoxifylline to the cell suspension after thawing.

Discussion

The data described here clearly indicate that the presence of Zn²⁺ in the freezing medium significantly improved sperm motility after freezing-thawing. It was previously shown that the presence of 50 µM Zn²⁺ in human sperm cryopreservation increases sperm total motility by 12.6% and progressive motility by 8.4% [13], while under the conditions of our study a considerably larger increase of motility was demonstrated (Table 1).
The presence of Zn²⁺ in the freezing medium increases the percentage of motile sperm and preserves motility for a longer period of time (Figure 2). There are contradictory reports regarding the effect of Zn²⁺ on sperm motility. Several studies have suggested that Zn²⁺ inhibits motility [28, 29], whereas other reports show that Zn²⁺ enhances sperm motility [30, 31]. The higher percentage of motile sperm after freezing in the presence of Zn²⁺ observed in the present study can be attributed to the stabilizing effect of Zn²⁺ on microfilaments in the outer dense fibers [32]. We found that Zn²⁺ stimulates production of actin filaments in human sperm (data not shown), a process found to enhance human sperm motility [18]. In addition, Zn²⁺ may stabilize the sperm plasma membrane during the freeze-thaw process [33, 34], thus protecting the sperm from motility damage.
The data also clearly indicate that Zn²⁺ protects the cells against loss of motility even after a second freezing (Figure 4).
One of the main problems in semen cryopreservation concerns DNA damage [5] and loss of mitochondrial function [6]. It was previously shown that semen samples cryopreserved in the presence of Zn²⁺ had a higher percentage of sperm with intact DNA and better mitochondrial function compared to freezing without Zn²⁺ [13]. These data provide further support to our suggestion to add Zn²⁺ to the cryopreserved medium.
We also tested the effect of glucose and pentoxifylline on sperm motility after freezing with Zn²⁺. The data in Figure 5 show that glucose plus pentoxifylline caused 93% stimulation of progressive motility after the second freezing and thawing, whereas when cells were frozen with Zn²⁺ there was a 154% increase in progressive motility. Thus, we conclude that the best conditions to achieve good motility are to freeze the sperm in 100 µl of medium containing 50 µM Zn²⁺ and to add glucose and pentoxifylline to the cell suspension after thawing.
Several reports have suggested that Zn²⁺ inhibits sperm capacitation and the acrosome reaction, two processes essential for successful fertilization [29, 35, 36]. In our recent study we showed that Zn²⁺ in micromolar concentration stimulates bovine sperm capacitation and the acrosome reaction [15]. In human sperm we also found that Zn²⁺ stimulates sperm capacitation including stimulation of protein tyrosine phosphorylation and hyper-activated motility (submitted for publication). It was also demonstrated elsewhere that survival, the rate of capacitation and the acrosome reaction are significantly enhanced in human semen samples cryopreserved in the presence of Zn.

Conclusions

Our data clearly demonstrated that freezing sperm in the presence of Zn²⁺ significantly enhanced sperm total and progressive motility.

Conflict of interest

The authors declare no conflict of interest.

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