International Joiimal of Andrology, 1991, 14, pages 423-431

Induction of the acrosome reaction in sperm by exposure to low temperature increases their rate of fusion with zona-free hamster oocytes

R. SANCHEZ and W.-B. SCHILL Andrology, Justus Liebig University, Giessen, Federal Republic of Germany

Center of Dermatology and

Summary The fusion rate of human sperm with zona-free hamster ova was investigated after induction of the acrosome reaction by exposure to a low temperature (4°C). Sperm were collected from 14 patients, and selected by the ‘swim-up’ method. The sperm were incubated for 24 h at either room temperature (control group) or at 4°C (low temperature group), followed by additional incubation at 37°C for 3 h. The mean sperm penetration rate, number of swollen sperm heads as well as the number of sperm attached to the oocyte increased significantly after exposing sperm to low temperature. The sperm penetration rate showed a significant correlation (Spear- man test, r = 0.572, n = 28, P<0.0035) with the acrosome reaction in the low temperature group. These results were associated with an increase in the rate of penetration of hamster ova observed in this study, presumably due to the increase in induction of the acrosome reaction by low temperature. Incubation of sperm at low temperature might be useful in the evaluation of so-called false negative results in the zona-free hamster test.

Keywords: acrosome reaction, fertilization, HOP-test, human sperm, low temper- ature, zona-free hamster test.

Introduction The accurate prediction or evaluation of fertility and infertility of men by conven- tional semen analysis is very difficult except for such extreme cases as severe oligozoospermia or azoospermia, which obviously indicate infertility. Utilization of the zona-free hamster oocyte penetration (HOP) test permits evaluation of the function of human sperm in the process of adhesion and fusion to the oocyte plasma membrane. False positive results can occur because the HOP-test does not assess sperm binding and penetration of the human zona pellucida, but false negative results must be kept to a minimum to achieve fertility prognostic value.

The critical events which precede penetration of zona free hamster ova by human sperm are capacitation and the subsequent acrosome reaction (AR) (Yana- gimachi et al., 1976). Only acrosome-reacted sperm are capable of fusing with oocytes (Koehler et al., 1982). An increase- in the proportion of capacitated and

Correspondence: Professor W.-B. Schill, Center of Dermatology and Andrology, Justus Liebig University, Gaffkystrasse 14, D-6300, Giessen, Germany.

423

424 R. Sanchez and W.-B. Schill

acrosome-reacted sperm has been suggested to enhance sperm-egg fusion in the HOP-test (Van Kooij et al., 1985).

Sperm capacitation and the acrosome reaction can be induced artificially under a variety of conditions. These include prolonged incubation (Gould et al., 1983), the addition of albumin (Cohen etal . , 1982), TEST-yolk buffer (Yang etal . , 1988), zona pellucidae (Cross et a f . , 1988), follicular fluid (Suarez et al., 1986), human oocyte-cumulus complexes (Stock et al. , 1989), calcium ionophores (Aitken et al. , 1984) and low temperature (Sanchez et al., 1989). Some of these treatments increase the proportion of acrosome-reacted sperm and enhance penetration rates in the HOP-test, thus minimizing false negative results (Yee & Cummings, 1988; Van Kooij & te Velde, 1988).

In this communication we report on the results obtained with utilization of low temperature in assessing the ability of sperm from subfertile men to adhere to and penetrate zona-free hamster ova.

Materials and methods

Semen collection Semen specimens were obtained from 14 patients attending the andrology out- patient service. Samples were obtained by masturbation after 5 days of sexual abstinence. The semen was collected in a clean plastic jar, allowed to liquefy for 30 min at room temperature and analysed using the criteria suggested by the W.H.O. (1987). All samples were normal with a volume > 2 ml, >40 X 10' sperm ml-', >50% motility and 50% normal forms.

Preparation of sperm The medium used was HEPES-buffered Biggers, Whitten and Whittingham (H- BWW) (94.7 mM NaCI, 4.78 mM KCI, 1.71 mM CaCI2 X 2H20 , 1.19 mM KH2P04, 1.19 mM MgS04 X 7H20 , 25.07 mM NaHC03, 21.58 mM Na-Lactate, 0.5 mM Na-Pyruvate, 5.56 mM Glucose, 20 mM HEPES, pH 7.4 at 20°C) supplemented with 5 mg ml-' human serum albumin (Behring Institute, Germany), 25 pg ml-' streptomycin (Sigma, USA) and 15 pg m1-l penicillin (Sigma, USA).

A 'swim-up' method was applied to obtain a suspension of motile sperm. Briefly, 1-2 ml semen was pipetted into two sterile plastic tubes (Greiner, Art. 166101, W. Germany) with 3 ml H-BWW and centrifuged at 250 g for 10 min. The resulting pellets were resuspended in 2 ml H-BWW and centrifuged at 250 g for 5 min. The final sperm pellet was overlayed with 1 ml H-BWW and then incubated for 1 h at 37°C under 5% C 0 2 in air. After incubation, the supernatant containing 70-95% motile cells was recovered. The concentration of motile sperm in the final suspension was determined by counting in a haemocytometer and adjusted to give 2.5-3 X 10" motile sperm m1-I. Aliquots (300-500 ~ 1 ) of the sperm suspension were placed in sterile plastic tubes and sealed with plastic caps.

(i)

The sperm suspension was then treated in two different ways.

The control group was incubated for 24 h at room temperature, followed by an additional incubation at 37°C for 3 h,

Increased fusion rate in zona-free hamster oocytes 425

(ii) Low temperature group was incubated for 24 h at 4"C, followed by an addi- tional incubation at 37°C for 3 h.

At the end of each incubation period, sperm motility was evaluated and 50 yl medium containing sperm (concentration = 2.5-3 x 10' sperm ml-') was trans- ferred into a petri dish with paraffin oil, following the method described for the HOP-test. Triple staining of sperm was then performed to detect acrosome-reacted sperm as described below.

Determination of sperm motility The motility of each sperm sample was assessed by examining a 10 pl drop of the swim-up selected sperm on a microscope slide and by estimating the proportion of sperm with progressive motility (scores of 3-4 on a scale of 1-4 according to Amelar & Dubin, 1982). Sperm motility was analysed immediately after the 'swim-up' procedure, and at the end of the initial 24 h incubation period (at either room temperature or 4°C) followed by 3 h incubation at 37°C.

Triple-stain technique To each sperm suspension, an equal volume of Trypan blue (T-0887, Sigma, USA) (2% in H-BWW) was added and incubated for 15 rnin at 35°C in a water bath, followed by washing with 10 ml H-BWW and centrifugation at 600 g for 10 min. The resulting pellet was resuspended in 3 ml H-BWW and centrifuged at 750 g for 5 min. The final sperm pellet was fixed with 500 yl 3% glutaraldehyde in H-BWW at 35°C in a water bath. After 30 min, the fixative was removed by washing with distilled water and the sample centrifuged at 600 g for 10 min. The pellet was resuspended in 3 ml distilled water and centrifuged at 750 g for 5 min. The supernatant was then discarded and the remaining pellet resuspended by repeated pipetting and 20 yl then smeared onto two microslides and air-dried for 30 min.

The triple stain sequence consisted of incubation in 0.8% Bismarck Brown Y (B-2759, Sigma, USA) for 5 min at 40°C in a waterbath, three brief washings in distilled water, followed by incubation in 0.8% Rose Bengal (R-3877, Sigma, USA) for 40 rnin at room temperature. After three washings in distilled water, sperm were then dehydrated in graded alcohol (50, 70, 96%), cleared in Xylol and examined double-blind, under oil-immersion brightfield optics at 1000 x magnification by one investigator. For estimation of the acrosome reaction, 400 morphologically normal sperm were evaluated in randomly selected fields and the proportion of viable acrosome-reacted sperm was determined in and expressed as a percentage of the viable sperm.

Four staining patterns were obtained according to Talbot & Chacon (1980):

(i) dead sperm with completely intact acrosomes showing a dark blue post- acrosomal region and pink acrosomes;

(ii) degenerative acrosome reaction of dead sperm with a dark blue post- acrosomal region and a white acrosomal region;

(iii) viable sperm with intact acrosomes - a light brown post-acrosomal region and a pink acrosome;

(iv) viable sperm with normal reacted acrosomes - a light brown post-acrosomal region and a white acrosomal region.

426 R. Sanchez and W. - B. Schill

Zona-free hamster oocyte penetration ( H O P ) test

Collection of hamster oocytes. Mature oocytes were collected from the oviducts of superovulated adult (aged 8- 12 weeks) Golden Hamsters. Superovulation was induced by intraperitoneal administration of 30 IU pregnant-mare serum gonado- trophin (G-4877, Sigma, USA) on the morning of day 1 of the oestrus cycle, followed by 30 IU human chorionic gonadotrophin (CG-B, Sigma, USA) on day 3 (i.e. 56-58 h later). The animals were killed by cervical dislocation 15-17 h after the hCG injection. The oviducts were placed in H-BWW medium supplemented with human serum albumin. The cumuli were collected by tube washing into the medium and transferred to a 35 x 10 mm Petri dish (Greiner, Art. 627102, W. Germany) containing 1 mg ml-l hyaluronidase (H-3536, Sigma, USA) in H-BWW, in which dispersion of cumulus cells occurred during incubation for 5 min at room temperature. The oocytes were then aspirated by micropipette, washed twice in H-BWW medium, and then transferred into a Petri dish and incubated at 4°C. Afterwards, for the HOP-test, oocytes were transferred to 0.06% trypsin (T-8253, Sigma, USA) in H-BWW medium to remove the zona pellucida. Immediately after zona dissolution (approximately 1 min), the oocytes were removed and washed by rapid transfer into three consecutive Petri dishes containing fresh H-BWW.

inseminution and incubation of sperm and ova. Capacitated sperm were transferred in 50 pl drops to the centre of a petri dish and covered with warmed (37°C) paraffin oil. Ten-twenty zona-free ova were added to each drop of sperm and incubated at 37°C for 2 h . The concentration of motile sperm in the droplet was adjusted at 2 x 10" sperm r n 1 - I .

Evaluation of sperm penetration. At the end of the incubation period, the oocytes were washed in H-BWW medium. transferred to a clean microscope slide, and compressed to a depth of about 30 pm under a 22 X 22 mm cover slip mounted on two paraffin wax supports. All ova were fixed for 10 min with 1.5% glutaraldehyde, stained with 1% acetoorcein, and examined under oil-immersion at X 1000 using a phase contrast microscope. The presence of a swollen sperm head or male pronu- cleus with a visible tail in the cytoplasm was considered indicative of positive penetration. The penetration rate was defined as the number of ova with positive penetration, divided by the total number of ova examined, multiplied by 100.

Statistical ana fysis Comparison between two sets of data was by the paired Student's r-test. Correla- tion coefficients were determined using the non-parametric Spearman test.

Results

induction of the acrosome reaction by tow temperature Utilizing the triple staining technique, a significant induction of the acrosome reaction was observed in all sperm samples after exposure to low temperature. In controls ( n = 14), the acrosome reaction occurred in 5.9 k 0.6% (range 3.0-9.0%) of sperm, whereas it was found to be 15.9 k 1.0% (range 7.1-20.9%) in the low temperature group (P<O.0005).

Increased fusion rate in zona-free hamster oocytes 421

Sperm motility The percentage of swim-up sperm exhibiting progressive motility examined in suspensions obtained (1) after incubation for 3 h at 37°C with the suspension kept at room temperature (75% k 5) (control) as well as (2) after incubation for 3 h at 37°C with the suspension kept at 4°C (80% f 6) (low temperature) did not, show any statistically significant difference.

Sperm penetration in the HOP-test after incubation at low temperature (4°C) Fourteen patients were tested in the HOP-test after exposure of sperm to low temperature. Higher penetration rates were found in all cases when compared with the corresponding control (Table 1). Four of the 14 samples in the control group showed zero penetration, whereas exposure to low temperature yielded an incre- ment of > 10%.

Table 1. Enhancement of penetration rate in the HOP-test in sperm exposed to a low temperature (4°C)

Penetration rate (%)

Patient Control test (37°C) Low temperature (4°C) ~~ ~

I 2 3 4 5 6 I 8 9

10 11 12 13 14

58.8 16.6 55.5 42.9 25.0 27.3 0.0 0.0 0.0 0.0

46.2 10.0 16.7 75.0

100.0 100.0 100.0 100.0 62.5 60.0 12.5 62.5

100.0 54.6 70.6

100.0 100.0 100.0

The penetration index was calculated as the number of penetrated eggs divided by the total number of oocytes.

Effect of increasing the acrosome reaction Indirect evidence for an increase in the acrosome reaction in sperm exposed to low temperature were as follows.

(9

(ii)

(iii)

The number of sperm attaching to hamster oocyte membranes increased from 6. Uoocyte in the control test to 26.4/oocyte after low temperature exposure (P<O.O005; Table 2) . The total number of decondensing sperm heads per oocyte increased from 0.84/oocyte in the control test to 3.8/oocyte after low temperature exposure ( P < 0.0005). The sperm penetration rate increased from 26.7% in the control group to 80.2% in the low temperature group (P<0.0005).

428 R. Sanchez and W.-B. Schill

Table 2. Effect of expoaure of sperm to a low temperature performance in the HOP-test (means f SEM; n = 14)

Incubation condition

Parameter Control Low temperature (37°C) (4°C)

Number of sperm attached 6.1 f 1.3 26.4 f 4.7* per oocyte

per oocyte Penetration rate 26.7 f 6.7 x0.2 i 7.2*

* P < 0.0005, in comparison with respective control value.

Decondensing sperm heads 0.8 f 0.2 3.8 f 0.7";

Calculation of the correlation coefficient between the sperm penetration rate and the acrosome reaction revealed a significant positive correlation (Spearman r = 0.572, f z = 28, P<0.0035).

Discussion Capacitation and the acrosome reaction of sperm are necessary for in-vitro fertiliz- ation as well as for penetration of zona-free hamster oocytes. An increase in the proportion of capacitated and acrosome-reacted sperm has been postulated to enhance sperm fusion and penetration in the HOP-test. Aitken et a f . (1984) obtained a mean penetration rate of about 75% of oocytes using normozoospermic samples after addition of the Ionophore A23187 in concentrations of 50-100 yM. Similar results were reported after the addition of zona pellucida (ZP) in culture media (van Kooij & te Velde, 1988), which increased the percentage of penetrated oocytes in a dose-dependent manner (4.5% penetration without ZP; 5.1% with 2 ZP; 11.6% with 4 ZP; > 13% with 7 ZP). Similar results were obtained using follicular fluid (Yee & Cummings. 1988), showing an increase in the penetration rate by an average of 38% in three-quarters of samples from subfertile patients tested. Bolanos et al. (1983) and Chan et af. (1987) demonstrated a significant increase in both the sperm penetration rate and the index of fertilization in the HOP-test after 24 h storage of sperm at 4°C in TEST yolk-buffer, without assuming that the temperature per se could be responsible for the increase in penetration rate observed. Low temperature storage in the TEST-yolk buffer appeared to enhance the fertilizing capacity of the human sperm irz vitro.

Recent studies (Sanchez et al., 1989) have demonstrated that viable sperm incubated for 24 h at 4"C, followed by additional incubation at 37°C for 3 h, leads to an increase in the number of sperm undergoing the acrosome reaction in both fertile donors (23%) and a patient group (17%), when compared with the relevant control groups (incubation for 24 h at room temperature, followed by additional incubation at 37°C) which were 6.3 and 4.9% respectively for the fertile donors and the patient group. The triple stain technique was used to determine the acrosome reaction in each group. Using a similar protocol and the HOP-test, the present report demonstrates a significant increase in the percentage of sperm attached to

increased fusion rate in zona-free hamster oocytes 429

oocytes, the number of decondensing sperm heads per oocyte, as well as an increase in the penetration rate and the acrosome rection (15.9% determined by triple stain), after exposing sperm to low temperature. The incidence of the acrosome reaction in these samples is consistent with previous observations that only 10-20% of human sperm normally complete this reaction when incubated under capacitating conditions (Plachot et al . , 1984; Mallett et a l . , 1985). The enhancing effect of low temperature was evident from two important indices for assessment of the fertilizing capacity of human sperm in the HOP-test, namely the number of decondensing sperm heads (0.8 k 0.2 in the control group and 3.8 f 0.7 after exposure to low temperature) and the percentage of eggs penetrated. The penetration rate was found to be significantly higher in low temperature-treated sperm compared with the controls (80.2 -t 7.2 versus 26.7 _+ 6.7%). The sperm penetration rate showed a significant correlation (Spearman test, r = 0.572, n = 28, P<0.0035) with the acrosome reaction in the treated group. Interestingly. no penetration was observed in four patients of the control treatment group, although after exposure to low temperature, high penetration rates were observed, with values higher than lo%, the lowest limit accepted as normal for the HOP-test. These data are consistent with the increase in penetration of hamster oocytes observed in this study, due probably to the influence of low temperature on the capacitation process and on the influx of Ca'+ to induce the acrosome reaction (Canvin & Buhr, 1989).

From a clinical point of view, incubation of sperm at low temperature (4°C) might be particularly useful in the evaluation of so-called false negative results in the HOP-test. It is possible that insufficient sperm from these patients undergo spontaneous acrosome reactions in vitro to enable penetration of hamster oocytes. The utilization of low temperature stimulus for inducing the acrosome reaction provides a new possibility for the correct evaluation of the fertilizing capacity of human sperm using the HOP-test.

Acknowledgments This study was supported by Deutsche Forschungsgemeinschaft Sch 86/8-1. R. Sanchez is a recipient of a fellowship of the Alexander von Humboldt Foundation, Bonn-Bad Godesberg, Germany.

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Received 17 May 1991; accepted 26 June 1991