ANDROLOGIA

ACCEPTED: AUGUST 28, 1991

24, 77-81 (1992)

Association of human sperm nuclear decondensation and in vitro penetration ability P. J. Chan and D. R. Tredway Key words. Spermatozoa

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nuclear decondensation

- penetration

Summary. Sperm nuclear decondensation is an integral step in fertilization which leads to the formation of the male pronucleus. The association between the in vitro spontaneous nuclear decondensation of human sperm and its fertilizing ability was studied in infertile male patients. The ability of sperm to fertilize a n egg using the discontinuous two-layer Percoll method was significantly correlated to the percentage of decondensed swollen head ( r = 0.43; P < 0.005). The fertilizing ability of sperm processed with TestYolk buffer was correlated with the percentage of sperm a t the fully decondensed stalk stage ( r = 0.5 1; P < 0.05). There were insignificant correlations for the whole-wash centrifugation, cryopreserved-thawed and swim-up methods. Samples of sperm that were positive ( > 0% fertilization) in the sperm penetration assay had a higher percentage of decondensed sperm heads (66.7% vs. 20.6%) after Percoll wash or wholewash centrifugation (60.5% vs. 44.3%) treatments compared with samples with no fertilization. Treatments that included Test-Yolk resulted in high percentages of decondensed swollen heads. The results suggest a positive association between sperm nuclear decondensation and the fertilizing ability of sperm, and affirm the importance of nuclear decondensation to the study of fertilization events.

ability - human

Introduction An interesting phenomenon that we observed in sperm in 1 d-old glass slides, which had been used for the sperm penetration assay (SPA; Rogers, 1985), was the spontaneous ballooning or decondensation of the sperm heads. The decondensation process could easily be divided through microscopic examination into intact (unreacted), swollen (balloon-shaped) or stalk (midpiece and tail remaining with dispersed nuclear material) stages. Bronson & Rogers' article (1988) on the SPA procedure notes that human sperm which undergo the acrosome reaction in human serum albumin (HSA) exhibited a ballooning of the plasma membrane. In vitro sperm nuclear decondensation after treatment with reducing agents, e.g. dithiothreitol (DTT), sodium dodecyl sulphate, Triton X- 100 and thioglycolate has been reported (Zirkin el al., 1985). It is proposed in this study that the percentage of patient sperm that exhibits spontaneous sperm nuclear decondensation after each SPA procedure is correlated with the sperm's ability to fertilize the egg. The objective was to determine whether there was an association between the two sperm functions: sperm penetration ability and sperm nuclear decondensation.

Material and methods Semen samples Andrology/Male Reproduction Laboratory, Department of Gynecology and Obstetrics and Department of Physiology and Pharmacology, Loma Linda University Medical Center, Loma Linda, CA 92350, USA. Correspondence: Dr Philip J. Chan, Andrology/Male Reproduction Laboratory, Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, Loma Linda, CA 92350, USA.

Semen samples were obtained from 21 patients for SPA processing. The samples were allowed to liquify for 20-30 min and processing was initiated within 1 h. The sperm count ranged from 2.8 to 172 x lo6 ml-' with an average of 55.2 x lo6 ml-'. Basic semen analysis was performed on all samples in accordance with the

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P. J. CHAN& R. TREDWAY

standards of the World Health Organization (WHO, 1987). Sperm processing SPA procedure. Th e culture medium used to wash the sperm and for the SPA procedure was Biggers, Whitten, Whittingham (BWW) medium (Biggers et al., 1971) supplemented with 3.5% HSA. The centrifuge washing procedure or whole-cell wash procedure consisted of an equal volume of the liquified semen with the BWW medium which was centrifuged at 300 G for 10 min in 15 ml centrifuge tubes. The resultant pellets were resuspended in 2 ml of BWW medium and centrifuged again at 300 G for 10 min. The final pellets were each resuspended at a concentration of 10 x lo6 sperm ml-l and preincubated in a 5% CO, in air mixture at 37 "C for 3 h prior to sperm zona-free oocyte interaction. Test- Yolk Buffer procedure. The Test-Yolk Buffer (TYB) procedure (Bolanos et al., 1983) consisted of an equal volume of semen in a centrifuge tube with Refrigeration Medium (Irvine Scientific, Irvine, CA, USA), which contained fresh egg yolk and TES Tris buffer. The tube was then placed in a cup of water at room temperature (25 "C) and stored at 4 "C for 48 h. At the end of the storage period, the mixture was added to 37°C pre-warmed BWW medium and kept at 37 "C for 30 min followed by centrifugation at 300 G for 10 min. The pellet was resuspended at a concentration of 10 x lo6 sperm ml-' and preincubated in a 5% CO, in air mixture at 37°C for 3 h prior to sperm zona-free oocyte interaction. Swim-Up Wash procedure. The Swim-Up sperm wash procedure (Berger et al., 1985) involved mixing an equal volume of semen with the BWW medium in a 15 ml centrifuge tube, centrifuging at 300 G for 10 min, discarding the supernatant and gently layering 0.5-1.0 ml BWW medium over the pellet. The tube was placed at a 45" angle in an incubator at 37°C in a 5% CO, in air mixture for 1 h. At the end of the incubation period, the motile sperm in the top layer of the medium were pipetted out and BWW medium added to adjust to a final concentration of 10 x lo6 sperm mlThe swim-up sample was then preincubated in a 5% CO, in air mixture at 37°C for 3 h prior to sperm zona-free oocyte interaction.

'.

Percoll wash procedure. The Percoll wash procedure (McClure et al., 1989) consisted of layering the

semen on top of a discontinuous two-layer Percoll gradient in a 15 ml centrifuge tube. The bottom layer consisted of 1.5 ml of 95% isotonic Percoll while the next, uppermost layer was 1.5 ml of 47.5% isotonic Percoll. The gradients were centrifuged at 300 G for 20 min and the bottom layer (1.5 ml) and the pellet were added to an equal volume of BWW medium and recentrifuged. The final pellet was resuspended at a concentration of 10 x 1O6 sperm ml - and preincubated in a 5 % CO, in air mixture at 37 "C for 3 h prior to sperm zona-free oocyte interaction. Cryopreseruation procedure. The cryopreservation procedure (Weidel & Prins, 1987) involved adding an equal volume of Freezing Medium (Irvine Scientific, Irvine, CA, USA) drop-bydrop to the semen in 1.8 ml cryovials, refrigerating the mixture for about 1 h and storing the cryovials in liquid nitrogen for at least 1 month. The semen in the cryovials was thawed by placing the cryovials in a 37°C waterbath for 8 min, adding an equal volume of BWW medium to the thawed sample, followed by centrifugation at 300 G for 10 min. The pellet was resuspended and recentrifuged, and the final pellet resuspended at a concentration of 10 x lo6 sperm ml- ' and preincubated in a 5% CO, in air mixture at 37 "C for 3 h prior to sperm zona-free oocyte interaction. The fertile control sperm samples were processed using the cryopreserved thawed procedure. Sperm penetration assay ( S P A ) The hamster oocytes were obtained from commercially available Cryotech straws (Charles River, Wilmington, MA, USA). The oocytes were treated with 0.1% trypsin to remove the zona pellucida. Each group of zona-free oocytes ( n = 15) was then incubated in 1 0 0 ~ 1sperm preparations for 3 h (Margolioth et al., 1989). At the end of the 3 h interaction period, wet preparations were made which consisted of gently squashing the oocytes between the glass slide and cover slip. The number of penetrated sperm (pronuclei with visible sperm tail) in the oocytes was determined using an inverted phase-contrast, dark-field microscope. Controls were carried out for each SPA run using cryopreserved fertile donor sperm. All SPA controls in this study had over 80% penetration. Sperm nuclear decondensation After each determination of sperm penetration in the zona-free oocytes, the slides were individually ANDROLOGIA 24, 77-81 (1992)

SPONTANEOUS SPERM

kept in large petri dishes a t room temperature (25 "C) for 24-48 h. The number of sperm in the wet area of the slide showing different stages of sperm nuclear decondensation was determined by means of the inverted phase-contrast microscope. Evaluations carried out in the dried areas of the slides were not possible due to scattering of the microscope light. Sperm that were fully decondensed and with just the midpiece and tail remnants without heads were placed in the stalk (S) category. Swollen sperm heads that resembled balloons with grey coloration were grouped into the decondensed (D) category while sperm that showed no changes and usually had a dark head were grouped into the intact ( I ) category. T h e SDI index was calculated from the percentage of decondensed sperm divided by the product of the percentages of stalk and intact sperm. Statistical methods Linear regression statistics with regression correlation coefficients were used to analyse the data. Significance was determined using the Student's t-test analysis of the regression slope. Means were tested using Student's t-test. Outliers were tested according to the residuals method of Grubbs & Beck (1972).

Results The data presented in Table 1 showed significant positive correlation between the percentage of penetration and the percentage of decondensed sperm in the Percoll wash group. There was a significant negative correlation between the percentage of penetration and the percentage of intact sperm in the Percoll wash treatment and also for the percentage of stalk sperm in the TYB

NUCLEAR DECONDENSATION

processed group. There were no other significant correlations. When the data were categorized, according to the outcome of the SPA results, into negative (percentage penetration = 0%) and positive (percentage penetration> 0%) SPA groups (Table 2), the data indicated a significantly higher percentage of decondensed balloon-type sperm heads in the positive SPA groups for both the centrifugewash and Percoll-wash procedures. A significantly lower percentage of intact sperm heads was observed in the Percoll-wash procedure in the positive SPA group compared with the group with no penetration. The Percoll treatment of sperm yielded the highest percentage of intact or unchanged sperm heads while the TYB treatment of the sperm produced the highest percentage of decondensed balloon-type sperm heads. There were no negative SPA results in the TYB and cryopreserved groups in this study. The intra-assay coefficients of variation were 36.4, 12.6%, and 14.5% for the intact, decondensed, and stalk sperm, respectively.

Discussion During fertilization, the sperm which penetrates the ooplasm undergoes nuclear chromatin decondensation followed by the formation of the male pronucleus. The decondensation of the sperm nucleus is critical, particularly for successful in vitro fertilization or sperm microinjection techniques. Studies have indicated that the mechanism of decondensation probably involves both a loss of zinc from the sperm (Kvist, 1980) and a reduction in the disulphide bonds of the sperm nucleus by glutathione reductase localized in the ooplasm (Gall & Ohsumi, 1976; Huret, 1983). Sperm have been shown to undergo in vitro

Table 1. Linear correlation coefficients (Y) for the in-vitro sperm nuclear decondensation stages and the sperm penetration assay results Type of wash

Intact (I) sperm ehad

Decondensed (D) swollen head

Fully decondensed stalk (S) stage

Centrifuged wash (n=51)

0.19 (P=NS)* 0.13 (P=NS) 0.10 (P=NS) 0.49 (P< 0.0005) (P=NS)

0.10 (P=NS) 0.29 (P=NS) 0.15 (P=NS) 0.43 (P

Association of human sperm nuclear decondensation and in vitro penetration ability.

Sperm nuclear decondensation is an integral step in fertilization which leads to the formation of the male pronucleus. The association between the in ...
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