J Med Dent Sci 2004; 51: 99–104

Original Article

Impaired hyperactivation of human sperm in patients with infertility

Mirihasimu Munire, Yasufumi Shimizu, Yu Sakata, Reiko Minaguchi and Takeshi Aso

Department of Comprehensive Reproductive Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Hyperactivation and are pre- Introduction requisite steps for sperm to be able to fertilize an oocyte. In mammals, hyperactivation is defined as Hyperactivation is a movement pattern observed in a movement pattern seen in spermatozoa at the site spermatozoa at the site and time of fertilization in mam- and time of fertilization. The objectives of the pre- mals and is critical to its success. Hyperactivation is sent experiments were to analyze the process of considered as a part of the complex process of hyperactivation and to investigate its relationship capacitation1. However, little is known about the with progesterone evoked intracellular calcium mechanisms which lead the sperm to hyperactivation. 2+ concentration ([Ca ]i) increase and their implica- Cumulus cells surrounding the oocyte may provide sig- tions with infertility. nals for hyperactivation, since the supernatant from the After capacitation the sperm from patients, culture of cumulus oophorus cells increased curvilinear when compared with donor’s sperm, showed velocity and side-to-side movement of the heads of decreased percentage of hyperactivated sperm, human sperm2. molitily, progressive , and curvilinear Changes in intracellular ion concentrations are velocity (VCL). On the other hand, the linearity associated with different aspects of sperm function (LIN) was increased. Amplitude of lateral head such as sperm motility, capacitation and the acrosome 2+ displacement (ALH) and [Ca ]i increase (peak reaction, and the external ion composition and the and plateau from baseline) showed good correla- effect of channel blockers can therefore influence tion in patients with infertility. them. These ion channels thus play an important role in These data suggest that impaired hyperactivation the regulation of sperm function. might be involved in the pathophysiology of infer- It is known that extracellular calcium is required to tility. maintain hyperactivation in hamster sperm3 and that 2+ intracellular calcium concentration ([Ca ]i) within the Key words: progesterone, hyperactivation, calci- flagella of hyperactivated sperm is increased. The cal- um, sperm, infertility cium influx occurring with capacitation might switch on hyperactivation by directly affecting the flagellar axoneme4,5. But little is known about the precise mechanisms by which calcium affects hyperactivation in human sperm. Corresponding Author: Yasufumi Shimizu Two different amino acid neurotransmitter recep- Department of Comprehensive Reproductive Medicine, Graduate tor/Cl- channels, a gamma-aminobutyric acid (GABA) School, Tokyo Medical and Dental University, 1-5-45, Yushima, - - Bunkyo-ku, Tokyo 113-8519, Japan. receptor-like/Cl channel and a glycine receptor/Cl Tel: 81-3-5803-5318 Fax: 81-3-5803-0148 channel, have recently been found in mammalian E-mail address: [email protected] sperm and shown to be involved in the sperm acro- Received November 7; Accepted December 19, 2003 some reaction, an exocytotic event essential to mam- 100 M. MUNIRE et al. J Med Dent Sci malian fertilization6. They reported that progesterone hyperactivation were measured using computer- induced acrosome reaction involved the sperm assisted sperm analyzer (Hamilton-Thorn Motility - GABAA receptor-like/Cl channel, thus causing an out- Analyzer, Hamilton-Thorn Research, Danvers, MA, flow of the Cl- ion from the cell6. In reverse, this will facil- USA). 2+ 7 itate the influx of [Ca ]i through the calcium channel . The Burkman’s criteria were used to identify hyper- Acrosome reaction assays demonstrated a clear dif- activated sperm 60 minutes after swim up and ference between progesterone- and A23187- induced overnight incubation. The Burkman’s criteria was as fol- acrosome reaction. A23187 can cause a rapid influx of lows: VCL (curvilinear velocity)＞100 Òm/s, LIN (lin- 2+ [Ca ]i and thus inducing the acrosome reaction, earity)＜65%, and ALH (amplitude of lateral head dis- which is independent of the capacitation time8. placement)＞7.5 Òm.11 We reported that progesterone induced increase in Curvilinear velocity (VCL) is calculated from the 2+ sperm [Ca ]i is significantly correlated with the egg- sum of the straight lines joining the sequential positions penetrating ability of spermatozoa from fertile men, and of the sperm head along the ’s track. sperm from infertile men with abnormal morphology Linearity (LIN) is defined as straight line velocity exhibited lower egg penetrating ability and lower peak (VSL)/VCL×100. The amplitude of the lateral dis- 2+ progesterone-evoked [Ca ]i than morphologically nor- placement of the sperm head (ALH) is calculated from mal sperm9. the amplitudes of its lateral deviations about the cell’s In the present experiments we analyzed hyperacti- axis of progression. vation in infertile and healthy humans and investigated 2+ its correlation with progesterone-evoked [Ca ]i. Measurement of changes in intracellular calcium concentration After overnight incubation intracellular calcium con- Materials and Methods centration of the sperm obtained from patients were monitored fluorometrically using the Ca2+-sensitive Preparation of human spermatozoa indicator fura-2AM as previously described.9,12,13 Semen samples were obtained from ejaculates of Fura-2 and its Ca2+ complex fluoresce strongly, but their donors, which were selected after analyzes of sperm excitation peaks differ in wavelength. Alternation following WHO guidelines, and from those of infertile between the two preferred wavelength allows assess- men submitted for semen analysis to the Department of ment of the ratio of Ca2+-bound dye to free dye and Obstetrics and Gynecology, Tokyo Medical and Dental hence the intracellular calcium can be measured.14 In University Hospital. Semen was evaluated following the brief, capacitated sperm (7.5x106/ml) were loaded World Health Organization criteria10. All specimens with 2 ÒM fura-2AM for 45 minutes at 37°C. Loaded were allowed to liquefy at room temperature for at least sperm were rinsed and assayed in BWW/HSA 30 minutes. A highly motile sperm population was 5 mg/ml. Fluorescence measurement were performed recovered after a 60-minute swim-up in Biggers, on a Hitachi spectrofluorometer F-2000 (Tokyo, Whitten and Whittingham (BWW) medium supple- Japan) at excitation wavelength 340/380 nm and mented with 5 mg/ml human serum albumin (HSA). For emission wavelength 510 nm with slit widths set at swim up, 1 ml aliquots of semen were injected under 5 nm. An aliquot of fura-2 loaded sperm was introduced 2ml BWW/HSA 5 mg/ml in 15 ml loosely capped coni- into a 4 ml cuvette, and continually stirred in a ther- cal tubes. After a 60-minute incubation at 37°C in mostated cuvette holder (37°C). Solutions containing

5%CO2 in air, the upper 1 to 1.5 ml of medium was col- test compounds were added to the cuvette in a 1:100 lected, and spermatozoa were washed by centrifuga- dilution. Progesterone was dissolved in dimethly- tion for 5 minutes at 1500 g. Thereafter spermatozoa sulphoxide (DMSO) at a concentration of 100 Òg/ml and were resuspended in BWW/HSA 30 mg/ml, and incu- 15 Òl was added to the cuvette containing 1.5ml bated at 37°C in 5%CO2 for overnight (18 to 22 hours) sperm suspension to make a final concentration of 1 2+ for capacitation. These spermatozoa were used within Òg/ml. Ratio 340:380 was converted to [Ca ]i using the 10 minutes of their removal from the incubator. formula as described by Gynkiewicz et al.15 Rmax and Rmin were determined in the presence of 0.01% Assessment of sperm motility, velocity and hyper- (wt/vol) digitonin and 15mM ethylene-bis (oxy-ethyle- activation nenitriolo) tetraacetic acid (EGTA), respectively.12,13 Motile percent, progressive sperm percentage and Rmax was the ratio340:380 when intracellular calcium IMPAIRED HYPERACTIVATION OF HUMAN SPERM 101 was saturated, and Rmin was the ratio 340:380 when (Fig. 1). intracellular calcium was depleted. The percentage of motile sperm was decreased after overnight incubation in both infertile patients Reagents (p＜0.0001) and donors (p＜0.0001). After overnight Fura-2AM was purchased from Molecular Probes incubation, the percentage of motile sperm of infertile (Junction City, OR, USA) and dissolved in DMSO and patients (33.9±4.4%) was smaller than that of donors stored at 0°C until use. Human serum albumin, BSA, (52.6± 3.4%) (p=0.001). There was no difference penicillin G, streptomycin sulfate and progesterone between both groups when compared after 60 minutes were from Sigma Chemical Company (St.Louis, MO, swim up (Fig. 2a). USA). The progesterone was dissolved in DMSO, The percentage of progressive motility was digitonin was dissolved in distilled water and stored decreased after overnight incubation in both infertile frozen at –20°C. patients (p = 0.0008) and donors (p＜0.0001). After overnight incubation, the percentage of progressive Statistical analysis motility of infertile patients (19.9±3.7%) was smaller Results were expressed as the mean ± SE. than that of donors (30.5±2.7%) (p = 0.021). There Statistical comparisons were made using Student’s t was no difference between both groups when com- test for paired or un-paired samples. Correlations pared after 60 minutes swim up (Fig. 2b). were calculated using Pearson’s correlation coeffi- cient.

Results

Sixty sperm samples obtained from 18 different donors and 35 samples from 35 patients were used for analyses. After overnight incubation, the percentage of hyperactivated sperm of infertile patients (7.1±1.2%) was smaller than that of donors (11.4±0.9%) (p = 0.006). There was no difference between both groups when compared after 60 minutes swim up. The per- centage of hyperactivated sperm was increased after overnight incubation in donors (p＜0.0001), whereas this increase was not observed in infertile patients Fig. 2a.

Fig. 2b. Fig. 1. The percentage of hyperactivated sperm in donors (dark The percentages of motile sperm (2a) and progressive movement bars) and infertile patients (dotted bars) after 60 minutes swim up and (2b) in donors (dark bars) and infertile patients (dotted bars) after 60 overnight incubation. minutes swim up and overnight incubation. 102 M. MUNIRE et al. J Med Dent Sci

Fig. 4a.

Fig. 3a.

Fig. 4b. 2+ Correlation between [Ca ]i increase from baseline to peak (4a) or from baseline to plateau (4b), after the administration of 1 Òg/ml of progesterone, and amplitude of lateral head displacement (ALH) of sperm in infertile patients.

Fig. 3b. Curvilinear velocity (VCL) (3a) and linearity (LIN) (3b) in donors (dark In infertile patients, there was a significant positive bars) and infertile patients (dotted bars) after 60 minutes swim up and 2+ correlation between [Ca ]i increase from baseline to overnight incubation. peak after the administration of 1 Òg/ml of progesterone and the ALH (r = 0.49, n = 35, p = 0.002) (Fig. 4a). 2+ [Ca ]i increase from baseline to plateau after the After overnight incubation, the curvilinear velocity administration of 1 Òg/ml of progesterone was also pos- (VCL) of infertile patients (87.9± 3.2 Òm/sec) was itively correlated with the ALH in infertile patients (r = smaller than that of donors (99.3±2.6 Òm/sec) (p = 0.54, n = 35, p = 0.0006) (Fig. 4b). 0.008). There was no difference between both groups Regarding ALH, there was no significant difference when compared after 60 minutes swim up (Fig. 3a). between both groups when compared after 60 minutes On the other hand, the linearity (LIN) of infertile swim up (donors; 4.0±0.4 Òm, patients; 4.2±0.1 patients (79.2±1.3%) was greater than that of donors Òm). Such an insignificant difference was also (46.3±8.1%) after overnight incubation (p＜0.0001). observed when the ALH was compared between the There was no difference between both groups when both groups after overnight incubation (donors; 4.7± compared after 60 minutes swim up. After overnight 0.6 Òm, patients; 4.5±0.2 Òm). incubation the linearity was increased in infertile patients (p＜ 0.01), whereas it was decreased in donors (p = 0.015) (Fig. 3b). IMPAIRED HYPERACTIVATION OF HUMAN SPERM 103

that extracellular calcium is required to maintain 2+ Discussion hyperactivation in hamster and that [Ca ]i within the fla- gella of hyperactivated sperm is increased3. Hyperactivated motility of sperm was first reported by In summary, our data showed that hyperactivation Yanagimachi et al16. Although fertilization still represents and calcium concentration are closely correlated and the endpoint of sperm capacitation, the ability of the that the sperm of patients with infertility has a sperm to undergo a regulated acrosome reaction (e.g. decreased capability to be hyperactivated, stressing the in response to the or progesterone) can importance of hyperactivation in human fertility. be taken as an earlier, upstream endpoint of capacita- tion17. Capacitation is also correlated with changes in sperm motility patterns, designated as hyperactivation, Acknowledgements in a number of species18. Hyperactivated motility, a swimming pattern of mammalian sperm in the oviduct, This work was supported by Grant-in-Aid is essential for fertilization in vivo. (12671538) from the Ministry of Education, Science In the present experiment we showed that the per- and Culture of Japan. centage of hyperactivated sperm increased signifi- cantly in healthy donors after overnight incubation. This change might be important in the preparing the sperm References for further fertilization, since this increase was not 1. Kay VJ, Robertson L. Hyperactivated motility of human sper- observed in the sperm from infertile patients. matozoa : a review of physiological function and application in Congruently the VCL was decreased in the sperm from assisted reproduction. Hum Reprod 1998;4:776-786. 2. Fetterolf PM, Jurisicova A, Tyson JE, et al. Conditioned infertile patients after 18 hours incubation whereas the medium from human cumulus oophorus cells stimulates linearity was decreased in the same group. 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