Relationship between sperm mitochondrial membrane potential, sperm motility, and fertility potential

Tsuyoshi Kasai, Keigo Ogawa, Kaoruko Mizuno, Seiichiro Nagai, Yuzo Uchida, Shouji Ohta, Michiko Fujie, Kohta Suzuki, Shuji Hirata, Kazuhiko Hoshi

Department of Obstetrics and Gynecology, Yamanashi Medical University, Yamanashi 409-3898, Japan

Asian J Androl  2002 Jun; 4:  97-103             

Keywords: fertility; mitochondria; membrane potential; in vitro fertilization; sperm motility; computer assisted sperm analysis; fluorescence; hyperactivation; sperm function

Abstract

Aim: To analyze the relationship between sperm mitochondrial membrane potential and sperm motility parameters by means of a computer-assisted sperm analyzer (CASA) and in-vitro fertilization rate (%FR). Methods: Semen samples were obtained from 26 men undergoing in vitro fertilization-embryo transfer (IVF-ET). Informed consent was obtained from all men prior to the study. Samples were prepared using wash and swim-up method in HEPES-HTF medium. The sperm motility (%MOT), progressive motility (%PMOT), average path velocity (VAP) (mm/s), straight line velocity (VSL) (mm/s), curvilinear velocity (VCL) (mm/s) and %hyperactivated sperm (%HA), and the %FR were assessed. The samples were incubated in the presence of 2.0 mmol/L of 5,5',6,6' -tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) for 30 min at 37 in air and washed in PBS before flow cytometry (FACSCalibur: Becton Dickinson) analysis. The mitochondrial probe JC-1 was used to identify the mitochondrial membrane potential. The sperm was divided into three populations according to the fluorescence pattern as follows: the high mitochondrial membrane potential group (n=8), the moderate group (n=5), and the low group (n=13). Statistical analysis was performed using unpaired t-test. Results: Significant differences were found between the high and the low groups in %MOT (91.18.5 vs 63.032.7, meanSD), VAP (73.014.2 vs 52.112.5), VCL (127.028.1 vs 87.022.6), %HA (27.323.6 vs 7.29.0) and %FR [73.2 (48/56) vs 59.0 (69/117)]. No significant differences were found in other CASA parameters. Conclusion: When the sperm mitochondrial membrane potential increases, sperm motility parameters and fertility potential will also increase. The JC-1 dye method is useful to predict sperm fertility potential.

1 Introduction

In vitro fertilization-embryo transfer (IVF-ET) is an important assisted reproductive technique (ART) for many couples suffering from long-standing unexplained infertility. The in vitro fertilization rate is significantly lower in patients with unexplained infertility in comparison with tubal patients [1]. Now, intracytoplasmic sperm injection (ICSI) is a technique that is widely used in unexplained infertility [2] and previous IVF failures [3]. Therefore, predicting the sperm fertilization ability prior to the IVF-ET procedure is very important in choosing the appropriate ART for couples with unexplained infertility. The standard semen analysis using a microscope provides the initial estimate of semen quality. However, it provides very little information on the sperm fertilizing capacity [4]. Many sperm function tests [4], such as morphology assessment (Kruger's strict criteria) [3], computer-aided sperm motion analysis (CASA) [6], nuclear maturity test using acridine orange [7], hypo-osmotic swelling test [8], induced acrosome reaction test [9], human sperm-hamster oocyte penetration assay (SPA) [10] and sperm-zona pellucida binding assay [11], have been proposed.

Some studies [12-16] suggested that the characteristics of progressive forward motility of the spermatozoa was related to their fertilizing capacity and the sperm motility was dependent on mitochondrial function. The aim of this study was to analyze the relationship between the sperm mitochondrial membrane potential and sperm motility parameters and in vitro fertilization rate, so as to assist determining the sperm fertilizing potential of patients with unexplained infertility.

2 Materials and methods

2.1 Patients

Subjects were 26 couples recruited between June and December 1999 with unexplained infertility undergoing conventional IVF treatment at Yamanashi Medical University Hospital. Unexplained infertility was diagnosed: (1) 3 or more years of infertility, (2) the woman had a menstrual cycle length of 25-35 days with a midluteal progesterone level in the ovulatory range and normal tubal patency, and (3) repeated semen analyses in the man were normal according to the World Health Organization (WHO) criteria [17]. The couples provided written consent after being given detailed explanations of the proposed study.

Fresh semen samples were collected by masturbation into sterile plastic containers after 2-5 days of sexual abstinence on the day of oocyte collection and were allowed to liquefy for 30 minutes at room temperature in the dark. Semen analysis was performed according to WHO protocols [17]. Samples were processed according to the routine wash and swim-up procedure in HEPES-HTF (Irvine Scientific, Santa Ana, CA, USA) medium supplemented with 10 % serum substitute supplement (SSS) (Irvine Scientific, Santa Ana, CA, USA).

Following incubation for 1 hour at 37 in air, 10 mL of sperm suspension was placed on a Makler chamber and sperm motility parameters were analyzed by CASA. The semen analyzer used was the Hamilton Thorne Research semen analyzer (IVOS, Version 10.8x. Hamilton Thorne, Beverly, USA). The standard parameter settings employed for analysis were as follows: frame acquired, 30; frame rate, 60Hz; minimum contrast, 80; minimum cell size, 3; static head size limits, 1.00~2.90; static head intensity limits, 0.60~1.40; static elongation limits, 0-80 and temperature, 37. At least 10 fields were examined.

The post swim-up sperm suspensions were used to assess the sperm motility, as it was proposed that the post swim-up motility is a better predictor of fertilizing ability of spermatozoa than fresh semen [18]. The following motility parameters were measured: concentration (CON, Million/mL), rate of motility (%MOT) and rate of rapid progression (%PMOT). For those spermatozoa that exhibit an average path velocity (VAP) greater than 25 mm/s, we determined the following parameters: average path velocity (VAP, mm/s), straight line velocity (VSL, mm/s), curvilinear velocity (VCL, mm/s), amplitude of lateral head displacement (ALH, mm), beat-cross frequency (BCF, Hz), Straightness (STR, %); VSL/VAP, Linearity (LIN, %); CSL/VCL, and rate of hyperactivated spermatozoa (%HA). Hyperactivated spermatozoa was measured according to Burkmans criteria [19] : LIN less than or equal to 65 %, VCL greater than or equal to 100 mm/s and ALH greater than or equal to 7.5 mm.

2.4 Flow cytometric analysis of mitochondria staining

5,5',6,6' -tetra-chloro-1,1',3,3'-tetraethylben-zimidazolyl-carbocyanine iodide (JC-1, T3168, Molecular Probes, Eugene, USA). Stock solution was prepared at 1 mg/mL in dimethylsulfoxide (DMSO, D8779, Sigma, USA). Stock solution was divided into small aliquots and stored at -20 until use.

After motility measurement, the sperm suspension was divided into two aliquots: one for subsequent oocytes insemination and the other for fluorescence measurements. Sperm suspension was diluted to a concentration of 210⁶ sperm/mL in HEPES-HTF. One milliliter of the sperm suspension was incubated with 2.0 mL of JC-1 stock solution (final concentration 2.0 mmol/L) for 30 min at 37 in air shielded from light. The samples were washed in PBS. The staining properties of JC-1 in each sample were visually confirmed by epifluorescence microscopy at 400 before flow cytometric analysis.

The samples were analyzed by FACSCalibur (Becton Dickinson, Mountain View, USA) and CELLQuest (Becton Dickinson). In order to determine the forward scatter (FSC) and side scatter (SSC) of the population of human spermatozoa to be analyzed, normal motile spermatozoa stained with JC-1 acquired from two volunteers with proven fertility were used to backgate. The FSC and SSC region corresponding to the JC-1 stained spermatozoa was determined for acquisition of normal spermatozoa (Figure 1).

Figure 1. Dot plots of Forward scatter (FSC) versus side scatter (SSC) of normal motile spermatozoa from a fertile man. Events occurring in this region were backgated from normal motile spermatozoa stained with JC-1.

The flow cytometer was equipped with a 488 nm excitation filter and a 530 nm emission filter (the fluorescence 1:FL1) and a 585 nm emission filter (the fluorescence 2:FL2). The values of photomultiplier were logarithmically set. Green fluorescence (FL1) represents the monomeric form of JC-1 corresponding to the mitochondrial mass. Red-orange fluorescence (FL2) corresponds to the J-aggregate form of JC-1. As positive control, normal motile spermatozoa stained with JC-1 acquired from two volunteers with proven fertility were used (Figure 2). Spermatozoa staining red-orange appear in the upper half of the four decades of the red -orange fluorescence parameter in cytogram of FL1 and FL2.

Figure 2. Cytogram of FL1 (green fluorescence) and FL2 (red-orange fluorescence) of normal motile spermatozoa from a fertile man, corresponding to the events based on the size gate of FSC and SSC. Spermatozoa staining red-orange appearing in the upper side serve as the positive control.

As a negative control, immotile spermatozoa were treated with 0.1% (w/v) Triton X-100 for 30 minutes and stained with JC-1 (Figure 3). Spermatozoa staining red-orange shift in the lower half of the four decades of the red-orange fluorescence parameter in the cytogram.

Figure 3. Cytogram of FL1 (green fluorescence) and FL2 (red-orange fluorescence) of immotile spermatozoa treated with 0.1% (w/v) Triton X-100 corresponding to the events based on the size gate of FSC and SSC. Spermatozoa staining red-orange appearing in the upper side serve as the negative control.

A total of 10,000 events were analyzed for each sample for both green and red-orange fluorescence. The samples acquired from patients with unexplained infertility were divided into three groups according to the red-orange fluorescence patterns of spermatozoa stained with JC-1 (Figure 4). High, moderate, and low mitochondrial membrane potential groups contained 8, 5, and 13 cases, respectively. Each sperm population was dominant in the upper half, the middle and the lower half of the four decades of the red-orange fluorescence parameter, respectively.

Figure 4. Cytograms of FL1 (green fluorescence) and FL2 (red-orange fluorescence) of spermatozoa from patients with unexplained infertility (upper lane). Histograms of FL2 (red-orange fluorescence, lower lane). Sperm populations of high, moderate and low mitochondrial membrane potential group were dominant in the upper half, the middle and the lower half of the four decades of the red-orange fluorescence parameter, respectively.

2.5 Procedure for conventional IVF-ET

The procedures for IVF-ET have been described previously [20]. A brief description is given here. Pituitary down-regulation was achieved by using a gonadotropin-releasing hormone agonist (GnRHa) (buserelin acetate, Suprecur; Aventis Pharma, Tokyo, Japan), starting from the 21st day of the preceding menstrual cycle. Ovarian stimulation was done by using human menopausal gonadotropin (hMG) (Pergonal, TEIKOKU HORMONE Co., Tokyo, Japan) at a dose of 3-4 ampoules (75 U/ampoule) daily. Ovulation was induced with 10,000 U of human chorionic gonadotropin (hCG) (Gonatropin, TEIKOKU HORMONE Co.). Oocytes were retrieved 36 h later. In each of the patients, more than 5 oocytes were retrieved. Standard oocyte insemination was performed in a standard fashion in HTF medium (Irvine Scientific, Santa Ana, CA) supplemented with 10 % SSS.

Fertilization was confirmed 16 to 18 h after insemination by visualization of two pronuclei and two polar bodies. Immature oocytes, such as germinal vesicle stage, metaphase I stage and degenerative oocytes were excluded from calculation of the fertilization rate. After verification of fertilization, pronuclear embryos were transferred to growth medium until they were transferred on Day 3 in the morning. Embryos were transferred to the uterine cavity with the use of a Wallace catheter.

The patients were given progesterone tablets 200 mg vaginally two times a day beginning the day after oocyte retrieval until the day of serum b-hCG assay, a total of 14 days after oocyte retrieval. Pregnancy was defined clinically as the presence of the fetal heartbeat.

2.6 Data processing

Results are expressed as meanSD. Statistical analysis was performed using unpaired t-test and Fisher's exact test where appropriate. P< 0.05 was considered statistically significant. This research was approved by the institutional review board of Yamanashi Medical University.

The post swim-up sperm motility parameters are shown in Table 1. There are no significant differences between the high and the moderate potential groups. The values of %MOT, VAP, VCL, and %HA are significantly greater in the high potential compared with the low potential group. The value of VAP is significantly higher in the moderate potential compared with the low potential group. No significant differences between the moderate and low potential groups were seen in other motility parameters.

Table1. Comparative results of post swim-up sperm motility parameters corresponding to three mitochondrial membrane potentials. MeanSD. a: High vs. Low, P<0.05, b: High vs. Low, P<0.01, c: Moderate vs. Low, P<0.05.

The clinical characteristics of patients and the comparative outcomes of IVF-ET are shown in Table 2. There were no significant differences in the demographic characteristics among the three groups. The fertilization rates and the proportions of fertilized oocytes are significantly higher in the high potential compared with the low potential group. Total fertilization failure occurred only in the low potential group. However, pregnancy rates were the same in the three groups.

Table2. Demographic characteristics and outcomes of IVF-ET corresponding to three mitochondrial membrane potentials. a: High vs. Low, P<0.05, b: Moderate vs. Low, P <0.01.

The importance of sperm motility during the fertilization process has received considerable attention over the past decades. Several researchers have reported the relationship of fertility potential in vitro and sperm motility parameters measured with CASA [21]. Objective analysis of sperm motility parameters resulted in significant correlations between the value of ALH [22], VCL [23-25], VAP [26], LIN [24] and the in vitro fertilization rates. In addition to VCL and VAP, sperm hyperactivation has been shown to be an important marker of fertilizing ability in the in vitro situation [27-30].

Energy is stored in the mitochondria as a proton concentration gradient and an electric potential gradient across the membrane. These gradients are generated by electron transport maintained by the inner mitochondrial membrane and drive the synthesis of ATP. Membrane permeable lipophilic cations accumulate in the mitochondria and exhibit a negative interior membrane potential. Recently, the lipophilic cationic fluorescent carbocyanine dye, JC-1, has been used to differentially label mitochondria with high and low membrane potential. When JC-1 forms monomers in mitochondria with low potential, the JC-1 stain emits a green fluorescence (510-520 nm), while the JC-1 form multimers known as J-aggregates after accumulation in mitochondria with high membrane potential, the JC-1 stain emits a bright red-orange fluorescence at 590 nm. Any changes in mitochondrial membrane potential could be a good indicator of sperm motility. Mitochondria membrane potential of spermatozoa has been evaluated with JC-1 in a variety of species [31-33], including human [34,35]. However, evaluation of the relationship between the mitochondria membrane potential and the sperm fertility potential has not yet been reported for human spermatozoa.

In the present study, JC-1 was used with flow cytometry to assess the sperm mitochondrial functional status. The use of flow cytomery allows the analysis of large populations of spermatozoa from a given sample. Our results show that there are significant differences in %MOT (P<0.05), VAP (P<0.01) and VCL (P<0.01) between the high and low mitochondrial membrane potential group. These results suggest that there might be significant correlations between the value of %MOT, VAP and VCL reflecting the fertility potential in vitro, and the mitochondrial membrane potential. Our results also show that there are significant correlation between the percentage of %HA and the mitochondrial membrane potential.

In conclusion, the results indicate that as the sperm mitochondrial membrane potential increases, sperm motility parameters, such as %MOT, VAP, VCL and %HA, also increase together with their fertility potential. When the sperm mitochondrial membrane potential is low, application of ICSI to couples with unexplained infertility should be considered. It is suggested that the JC-1 staining method is useful to predict of sperm fertility potential.

We thank Ms. K.L.K. Tamashiro, Department of Psychiatry, University of Cincinnati, for her assistance in the preparation of the manuscript.

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Correspondence to: Prof. Kazuhiko HOSHI, Department of Obstetrics and Gynecology, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi 409-3898, Japan.
Tel: +81-55-273-1111 Ext. 2360, Fax: +81-55-273-3746
E-mail: kazuho@res.yamanashi-med.ac.jp
Received 2002-04-29      Accepted 2002-05-08