Increase in the concentration of cytosolic‐free calcium induced by human follicular fluid was decreased in single human spermatozoon with abnormal morphology

Abstract

Aim:  The increase in the concentration of cytosolic‐free calcium ([Ca²⁺]i) induced by follicular fluid or progesterone has been reported to promote an acrosome reaction and alternation in several motion parameters in human sperm (hyperactivation). We previously reported that populations of sperm in cell suspension obtained from infertile men with abnormal morphology exhibited lower mean peak progesterone‐evoked [Ca²⁺]i compared with morphologically normal sperm using cell‐suspension methods. In the present study, the change in [Ca²⁺]i in individual normally and abnormally shaped spermatozoa was compared.

Methods:  The change in [Ca²⁺]i induced by human follicular fluid in individual spermatozoa with normal and abnormal morphology was compared using the fluorescent calcium‐sensitive dye fluo‐3/AM. The spatial distribution of the increase in [Ca²⁺]i in single sperm was also investigated.

Results:  The [Ca²⁺]i of normally shaped spermatozoa increased rapidly after the administration of human follicular fluid. The response reached a peak within 2–3 s and then slowly declined to a plateau phase. The baseline and peak fluorescence in spermatozoa with abnormal morphology was lower when compared with normal spermatozoa. The follicular‐fluid‐induced increase in [Ca²⁺]i (expressed as a percentage increase in [Ca²⁺]i over basal) in morphologically abnormal sperm was 39.2 ± 5.3% (n = 107, mean ± standard error), which was smaller than that of morphologically normal sperm (61.6 ± 5.7%, n = 100, P < 0.005) from seven healthy donors. The follicular‐fluid‐induced [Ca²⁺]i increases observed in sperm with morphologically abnormal mid‐pieces (20.9 ± 4.3%, n = 12, P < 0.05) or tails (40.7 ± 6.0%, n = 92, P < 0.05) were lower than those of morphologically normal spermatozoa (61.6 ± 5.3%, n = 101). The follicular‐fluid‐induced [Ca²⁺]i increase of morphologically normal spermatozoa from infertile couples (35.1 ± 6.3%, n = 25, P < 0.05) was also found to be lower than that of morphologically normal spermatozoa from healthy donors.

Conclusion:  The present study shows that spermatozoa with abnormal morphology in healthy donors have disorders of signal transduction, as do normally shaped sperm in men from infertile couples. (Reprod Med Biol 2008; 7: 143–149)

INTRODUCTION

TRADITIONAL PARAMETERS OF semen analysis, such as concentration, motility and viability of spermatozoa have little or no predictive value for sperm‐fertilizing ability. ¹ As physiological stimuli of the acrosome reaction in human spermatozoa, progesterone ² , ³ , ⁴ , ⁵ , ⁶ and follicular fluid ⁷ , ⁸ are reported to promote an acrosome reaction and enhance the fertilizing ability of sperm. Morales et al. stated that the progesterone in follicular fluid is the molecule responsible for inducing the acrosome reaction in human spermatozoa. ⁷ A correlation between changes in the concentration of cytosolic‐free calcium [Ca²⁺]i in sperm induced by progesterone and fertilizing ability has been reported by several laboratories.

Decreased sperm responsiveness to progesterone has been shown in oligozoospermic men, ⁹ as well as in infertile patients. ¹⁰ Prostaglandins, which exist in high concentrations in human seminal plasma and follicular fluid, also increase sperm [Ca²⁺]i. ¹¹ , ¹² , ¹³ Assessments of morphology using strict criteria can also be predictive of in vitro fertilization (IVF) outcome. ¹⁴ In another report, the average percentage [Ca²⁺]i and the rate of increase in the frequency of acrosome reaction following progesterone challenge were significantly lower in a group of patients with a fertilization rate of less than 50%. ¹⁵

We have previously reported that sperm from infertile men with abnormal morphology show low egg‐penetrating ability, and the increase in sperm [Ca²⁺]i induced by progesterone was lower in cell suspensions from these teratozoospermic men compared with the sperm obtained from morphologically normal ejaculates. ¹⁶ In the present study, changes in [Ca²⁺]i induced by human follicular fluid in individual normally and abnormally shaped spermatozoa were compared. In addition, the spatial distribution of the increase in [Ca²⁺]i in single sperm was also investigated.

METHODS

Preparations of human spermatozoa

SEMEN SAMPLES WERE obtained after 2–7 days of sexual abstinence from 13 ejaculates from seven healthy volunteers (who were not proven fertile) and from five ejaculates from five infertile men submitted for semen analysis to the Department of Obstetrics and Gynecology and the Department of Urology, Tokyo Medical and Dental University Hospital. Informed consent for using sperm in the present study was obtained from the donors and also from the infertile patients. Semen was evaluated following the World Health Organization criteria. ¹⁷

After the specimens were allowed to liquefy, a highly motile population was recovered after a 60‐min swim‐up in Minimum Essential Medium (MEM) supplemented with 5 mg/mL human serum albumin (HSA) (Sigma Chemical Company, St Louis, MO, USA). For swim‐up, 500 µL aliquots of semen were injected under 2 mL Biggers–Whitten–Whittingham (BWW)/HSA 5 mg/mL in 15 mL loosely capped conical tubes. After a 60‐min incubation at 37°C in 5% CO₂ in air, the upper 1–1.5 mL of medium was collected, and spermatozoa were washed by centrifugation for 5 min at 1500 g. Human follicular fluid was obtained at the time of oocyte retrieval with the patient's permission, as part of the in vitro fertilization‐embryo transfer (IVF‐ET) program at Tokyo Medical and Dental University Hospital, from two women who became pregnant after IVF‐ET treatment. The human follicular fluid was pooled for the present study. Follicular fluid was centrifuged at 1000 g for 30 min and the supernatant was collected and stored (for up to 6 months) at –20°C until use.

Measurement of changes in the concentration of cytosolic‐free Ca²⁺

The swim‐up fractions were incubated with 10 µmol/L fluo‐3/AM (Molecular Probes, Eugene, OR, USA) suspended in MEM without HSA for 30 min at 37°C in 5% CO₂ in air, and then centrifuged at 600 g for 5 min and resuspended in MEM. Then, 2.5 µL droplets of the sperm cell suspension were fixed onto a glass coverslip coated with Cell‐Tak (BD, Franklin Lakes, NJ, USA). ¹⁸ The coverslip was then placed onto a microscope stage. Minimum Essential Medium supplemented with 5 mg/mL HSA was perfused at a rate of 3 mL/min (at 37°C) for 2–3 minutes to wash out the motile, non‐adherent sperm. Changes in [Ca²⁺]i within a single spermatozoon were measured after exposure to 1% follicular fluid. A glass micropipette with a tip opening of 4–5 µm was filled with 10% follicular fluid diluted with MEM and applied to the droplet on the coverslip and 10 µL of the 10% follicular fluid was injected.

The relative value of intracellular calcium within individual spermatozoa was measured in specific areas using fluo‐3/AM, as described by Tesarik et al. ¹⁹ Fluorescence measurements of individual spermatozoon were carried out using a confocal laser scanning microscope (Meridian Insight plus‐IQ, Meridian Instruments Far East, Tokyo, Japan) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. A drop of follicular fluid was added to the sperm droplet as a stimulus. After incubation with fluo‐3/AM, all sperm within the field of view were first assessed at 400× for morphology and then they were re‐assessed for the measurement of cytosolic calcium after the follicular fluid was added to the sperm droplet. Measurements were carried out with a ×40 objective, which allowed examination of an optical field comprising 5–10 living spermatozoa at one time. The fluorescence signal emitted from the fluo‐3‐loaded cells was recorded at 2 s intervals. Abnormal‐shaped sperm was scored using the World Health Organization criteria ¹⁷ with regard to head abnormality (tapering, amorphous, pin head, large oval, small oval, double head), mid‐piece abnormality (mid‐piece anomalies), and tail abnormality (coiled tail, bent tail).

The increase in [Ca²⁺]i evoked by human follicular fluid in individual spermatozoa with abnormal morphology from healthy donors was compared with that observed in normal sperm from healthy donors. We also compared the increase in [Ca²⁺]i evoked by human follicular fluid in normally shaped spermatozoa from healthy donors with that from infertile patients.

Statistical analysis

The results are expressed as the mean ± standard error (SE). Mann–Whitney U‐tests and Student's t‐tests were used for the statistical analyses.

RESULTS

THE PARAMETERS OF the 13 semen samples from the seven healthy donors were as follows: semen volume 3.5 ± 0.3 mL (1.1–4.0), sperm concentration 119 ± 19 × 10⁶/mL (24–266), motility 61.9 ± 3.4% (43.9–87.9), and the percentage of sperm with abnormal morphology 15.5 ± 2.6% (4–32.7). The parameters of the five semen samples from the five infertile patients were as follows: semen volume 3.0 ± 0.6 mL (1.1–4.0), sperm concentration 86 ± 14 × 10⁶/mL (38–117), motility 55.4 ± 8.8% (23.7–73.0), and the percentage of sperm with abnormal morphology 20.0 ± 2.7% (10.3–25.0). There was no significant difference between each parameter when comparing healthy donors and infertile patients (Mann–Whitney U‐test).

The [Ca²⁺]i of morphologically normal spermatozoa from healthy donors increased rapidly after the administration of human follicular fluid (Fig. 1). The response reached a peak within 2–3 s and then slowly declined to a plateau phase. The increase in [Ca²⁺]i induced by follicular fluid was observed only at the sperm head, and no increase was observed at the tail of normally shaped sperm.

Figure 1.

Representative tracing of the concentration of cytosolic‐free calcium ([Ca²⁺]i) in a single spermatozoon. [Basal] indicates the fluorescence value before the addition of the follicular fluid. After the administration of the follicular fluid, the response reached a peak [peak] within 2–3 s and then slowly declined to a plateau phase.

The baseline and peak fluorescence of the spermatozoa with abnormal morphology from healthy donors was lower compared with the morphologically normal sperm from healthy donors. One percent follicular fluid induced a [Ca²⁺]i increase (expressed as the percentage increase of [Ca²⁺]i over basal) in morphologically abnormal sperm of 39.2 ± 5.3% (n = 107), which was smaller than the increase in morphologically normal sperm (61.6 ± 5.3%, n = 101, P < 0.05) from healthy donors (Fig. 2). Follicular‐fluid‐induced [Ca²⁺]i increases of spermatozoa with abnormal morphology of the mid‐piece (20.9 ± 4.3%, n = 12, P < 0.05) or tail (40.7 ± 6.0%, n = 92, P < 0.05) were lower than the increases in spermatozoa with normal morphology (61.6 ± 5.3%, n = 101), but there was no significant difference between abnormal head and normal morphology from healthy donors (Fig. 3).

Figure 2.

Comparison of the percentage increase in the concentration of cytosolic‐free calcium ([Ca²⁺]i) between morphologically normal and abnormal sperm from healthy donors. One percent follicular fluid induced a [Ca²⁺]i increase (expressed as the percentage increase of [Ca²⁺]i over basal) in morphologically abnormal sperm of 39.2 ± 5.3% (n = 107), which was smaller than the increase in morphologically normal sperm (61.6 ± 5.3%, n = 101, P < 0.05) from healthy donors.

Figure 3.

Increase in the concentration of cytosolic‐free calcium ([Ca²⁺]i) induced by follicular fluid in morphologically normal and abnormal sperm from healthy donors. Follicular‐fluid‐induced [Ca²⁺]i increases in spermatozoa with abnormal morphology of the mid‐piece (20.9 ± 4.3%, n = 12, P < 0.05) or tail (40.7 ± 6.0%, n = 92, P < 0.05) were lower than those in spermatozoa with normal morphology (61.6 ± 5.3%, n = 101), but there was no significant difference between abnormal head and normal morphology from healthy donors.

The follicular‐fluid‐induced [Ca²⁺]i increase in spermatozoa with normal morphology from infertile patients (35.1 ± 6.3%, n = 24) was significantly lower than the increase in the spermatozoa with normal morphology from healthy donors (61.6 ± 5.3%, n = 10) (P < 0.05) (Fig. 4).

Figure 4.

Comparison of the percentage increase in the concentration of cytosolic‐free calcium ([Ca²⁺]i) in morphologically normal spermatozoa between healthy donors and infertile patients. Follicular‐fluid‐induced [Ca²⁺]i increases in spermatozoa with normal morphology from infertile patients (35.1 ± 6.3%, n = 24) were significantly lower than those from spermatozoa with normal morphology from healthy donors (61.6 ± 5.3%, n = 101) (P < 0.05).

DISCUSSION

IN COUPLES WITH reproductive failure, it is important to be able to predict the egg‐penetrating ability of sperm so that appropriate treatments can be selected. An alternation in the ability of sperm to undergo an acrosome reaction in response to calcium ionophores has been shown in human spermatozoa with low‐fertilizing ability. ²⁰ , ²¹ Progesterone has been shown to be a physiological stimulator of the acrosome reaction in human spermatozoa. ² , ³ , ⁴ , ⁵ Progesterone appears to be primarily involved in the action of follicular fluid on spermatozoa, as the ability of the follicular fluid to stimulate acrosome reaction is highly correlated with its progesterone content. ⁷ , ⁸ Once human spermatozoa have been exposed to progesterone, they rapidly develop an insensitivity to further stimulation that lasts for 0.5–1 h. ²² It has been proposed that: (i) the acrosome reaction and flagellar beat are regulated by separate Ca²⁺ stores; (ii) these stores are mobilized through different mechanisms by different agonists; and (iii) progesterone in vivo acts as a switch for the oscillator that regulates the flagellar beat mode. ²² A progesterone concentration ramp (0–3 µmol/L) is reported to induce [Ca²⁺]i oscillations (repetitive store mobilization), which modify flagellar beating, whereas bolus application of micromolar progesterone causes a single large transient (causing acrosome reaction), which is apparently dependent on Ca²⁺ influx. ²³

Downregulation also occurs when human spermatozoa are exposed repeatedly to prostaglandins, but does not occur between progesterone and prostaglandin. ¹³ This suggests that there are different progesterone‐induced and prostaglandin‐induced pathways leading to transients of [Ca²⁺]i within spermatozoa. The mechanism by which progesterone induces calcium transient does not involve significant mediation by gamma‐aminobutyric acid (GABA)‐A receptors, voltage or second messenger operated calcium channels or pathways involving phospholipase C or G proteins. ²⁴

There have been several reports describing a correlation between changes in [Ca²⁺]i in sperm induced by progesterone and the fertilizing‐ability of the sperm. Infertile patients with a high incidence of abnormal sperm forms, as diagnosed by strict criteria, also have a low incidence of spontaneous acrosome reaction, and a diminished progesterone‐stimulated acrosome reaction and parallel abnormalities of [Ca²⁺]i have been observed. ²⁵ Significant correlations between the fertilization rate, the progesterone‐stimulated [Ca²⁺]i and acrosome reaction increases (r = 0.78 and r = 0.79, respectively) have been observed. In a subsequent study of cases of fertilization failure, no increases in [Ca²⁺]i or acrosome reaction were observed in response to progesterone, with the exception of one case. However, the correlation coefficient between morphology and fertilization in this study was only 0.46. ¹⁵

In the present study, changes in [Ca²⁺]i induced by human follicular fluid were compared between individual morphologically normal and abnormal sperm, at the level of a single cell. Image analysis of the calcium transients in a normally formed spermatozoon revealed a rapid increase in [Ca²⁺]i, although individual differences in patterns of their increasing and declining curves were noted. At the level of individual cells, the generation of calcium transients in human spermatozoa appears to be a ubiquitous response, localized to the acrosomal domain of these cells. An increase in [Ca²⁺]i induced by follicular fluid was observed only at the sperm head, and no increase was observed at the tail of normally shaped sperm. This is in accord with other reports by Shirakawa and Miyazaki ²⁶ and Kirkman‐Brown et al. ²⁷ The peak in [Ca²⁺]i of sperm with abnormal morphology was lower than that of normally shaped sperm in healthy donors. This might be one of the reasons that morphologically abnormal sperm have lower penetrating ability. In our study, the percentage increase in [Ca²⁺]i was lower in spermatozoa with mid‐piece and tail abnormalities in healthy donors compared with sperm that demonstrated head abnormalities. Further work is needed to elucidate the relationship between signal transduction and abnormally shaped lesions of the spermatozoon.

Only a few spermatozoa with head abnormalities could be measured by the perfusion system used in the present study because it was difficult to immobilize the sperm on the glass coverslip with Cell‐Tak. In addition, we could not measure the calcium response of sperm with high velocity.

There have been several reports concerning the spatial distribution of [Ca²⁺]i in response to progesterone or follicular fluid. In ram spermatozoa, calcium was initially evident on the outer acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. ²⁸ Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly (observed by electron microscopy). Video image processing enhanced fluorescence microscopy was used to monitor mammalian sperm [Ca²⁺]i response to the stimulatory agonist in the egg's zona pellucida during exocytosis. In the mouse, two types of agonist‐dependent [Ca²⁺]i transport pathways were defined: one has the characteristics of a poorly selective cation channel and the other is the sperm L channel. ²⁹

Defects in [Ca²⁺]i transients in response to progesterone or follicular fluid have been reported by several laboratories. The selective binding of progesterone–bovine serum albumin (BSA) conjugates to human spermatozoa has been confirmed. ³⁰ Progesterone has been reported to induce changes in [Ca²⁺]i of sperm from infertile men, but with normal basic parameters, while there were no increases or lower increases compared with fertile donors. ³⁰ However, this non‐genomic progesterone receptor could be observed only in 10% of spermatozoon, even from fertile donors. In another report, the mean percentage of cells binding the progesterone–BSA ligand was 3.01 ± 0.29%. ²² In contrast, when the responses of the same sperm populations to progesterone stimulation were imaged, 91.79 ± 1.8% of cells were found to exhibit a definite calcium transient. There was no significant correlation between the small percentage of spermatozoa binding the fluoresceined probe and the proportion subsequently generating calcium transients on exposure to progesterone.

Spermatozoa with morphologically abnormal heads might have defects in the plasma membrane that result in an alteration in progesterone or prostaglandin receptor density or function so that the increases in Ca²⁺ induced by follicular fluid are smaller. Evidence has previously been presented that a threshold Ca²⁺ concentration is required to initiate the acrosome reaction, which is not achieved by abnormally shaped spermatozoa. This in turn would lead to a lower ability to penetrate eggs, which is suggested by the observation of Liu and Baker. ³¹ They showed that morphologically abnormal sperm bind the zona pellucida, but exhibit an impaired ability to penetrate the zona, and that this resulted from a disordered acrosome reaction. These observations suggest that the zona‐induced acrosome reaction might be impaired in these spermatozoa, in addition to that observed in response to human follicular fluid, prostaglandins and progesterone. Whether this effect is mediated by alterations in calcium transients or further downstream events is unclear. Some patients with disordered zona‐pellucida‐induced acrosome reaction failed to respond to protein kinase C promoters, suggesting defects of pathways downstream of protein kinase C. ³²

The present study shows that spermatozoa with abnormal morphology have disorders of signal transduction in healthy donors, and normal‐shaped sperm from infertile patients have disorders of signal transduction when compared with sperm from healthy donors.