JBRA Assist. Reprod. 1998;02(02):74-82
ARTIGO ORIGINAL
doi: 10.5935/1518-0557.1998.2.2.02
The effects of sperm preparation by swim-up technique and capacitation on acrosome status of normozoospermic men
Os efeitos da seleção espermática pela técnica de swim-up e da capacitação sobre o perfil acrossômico de homens normozoospérmicos
Abstract
Introduction: We studied whether sperm preparation by swim-up technique can select a better intact acrosome sperm population and analyzed how subsequent in vitro capacitation influenced the acrosomal status of these selected spermatozoa.
Material and Methods: Semen specimens from normal donors (n = 15) were divided into three equal aliquots: the first received no treatment (raw; control) and the others were processed by swim-up technique. After swim-up, the second aliquot received no further treatment (swim-up) and the third was capacitated in air for 3 hours by incubation in a modified-BWW with 3% HSA at 37°C under 5% C02 (capacitated). Sperm motion parameters in raw, swim-up and capacitated specimens were analyzed by CASA. The acrosome status was assessed by FITC-PNA lectin. Sperm viability was measured by Hoechst-33258 stain.
Results: Percent sperm motility, percent viability and all motion parameters, except linearity, were significantly increase in both swim-up and capacitated specimens. The frequency of spermatozoa exhibiting intact acrosomes was not different between raw and swim-up specimens (p=0.63). However, the frequency of acrosome reacted spermatozoa was significantly higher in the capacitated group (20.5%) compared to both raw and swim-up groups (p<0.001). Acrosome reaction was observed in 13.1% and 13.0% of raw and swim-up spermatozoa, respectively, and this has occurred independent of capacitation.
Conclusions: We conclude that sperm preparation by swim-up technique does not select a better acrosome intact sperm population in spite of yielding a highly motile and viable one. In addition, in vitro sperm capacitation significantly optimizes the acrosome reaction rate. However, a small proportion of sperm do not require capacitation to undergo in vitro spontaneous acrosome reaction.
Keywords: swim-up, sperm capacitation, FITC PNA labeling, Hoechst-33258 stain
Resumo
Introdução: Avaliamos se a técnica de "swim-up" permite selecionar uma subpopulação espermática com acrossomos intactos superior às amostras não selecionadas. Além disso, analisamos os efeitos da capacitação espermática subseqüente, "in vitro", sobre o perfil acrossômico das amostras selecionadas pela técnica de "swim-up".
Material e Métodos: Amostras de sêmen de doadores normais (n=15) foram divididas em três alíquotas iguais: a primeira não recebeu qualquer tratamento (sêmen nãó processado; controle) e as outras foram processadas pela técnica de "swim-up". Após swim-up, a segunda alíquota não recebeu tratamento subseqüente ("swim-up"), enquanto que a terceira foi capacitada durante 3 horas através de incubação no meio de BWW modificado, suplementado com 3% de albumina humana, em atmosfera de 5% de C02 à 3 7°C (capacitada). Os parâmetros da motilidade espermática foram avaliados em todas as alíquotas através de análise computadorizada. 0 perfil acrossômico foi analisado através da técnica de fluorescência envolvendo a lecitina FITC-PNA. A vitalidade espermática foi mensurada através da coloração de Hoechst-33258.
Resultados: Tanto as amostras selecionadas através da técnica de "swim-up" quanto as capacitadas apresentaram motilidade percentual, vitalidade e parâmetros da motilidade espermática, exceto linearidade, significativamente superiores às amostras controle. A freqüência de espermatozóides exibindo acrossomos intactos não diferiu significativamente entre as amostras controle e aquelas selecionadas pela técnica de "swim-up" (p=0,63). Entretanto, a freqüência de reação acrossômica foi significativamente superior (20.5%) nas amostras submetidas à capacitação espermática (p<0,001). Taxas de reação acrossômica de 13,1 % e 13,0% foram observadas nas amostras controle e nas selecionadas pela técnica de "swim-up", independentemente de capacitação.
Conclusão: Embora o preparo do sêmen através da técnica de "swim-up" propicie a seleção de espermatozóides com melhor motilidade e vitalidade, tal técnica não se mostrou eficaz para selecionar uma subpopulação espermática exibindo melhor perfil acrossômico. A capacitação espermática "in vitro" optimiza significativamente a taxa de reação acrossômica. Entretanto, uma pequena fração de espermatozóides não requer capacitação para sofrer reação acrossômica espontânea, em condições laboratoriais.
Unitermos: swim-up, capacitação espermática, coloração por FITC-PNA, corante Hoechst-33258
Introduction
Under normal physiological conditions, ejaculated spermatozoa need to be washed free from seminal plasma to undergo capacitation before they are capable of acrosome reaction and of penetrating the zona pellucida (Henkel et al., 1993). Seminal plasma is removed as the spermatozoa transverse the cervical mucus, and capacitation occurs as they are transported across the cervix, uterus, and Fallopian tubes (Mortimer et al., 1985). Previous studies have found that spermatozoa have to be capacitated before they can undergo acrosome reaction and that a normal acrosome reaction is essential for the sperm to penetrate through the zona pellucida and to prepare for the fusion with the oolema (Zaneveld et al., 1991; Wang et al., 1993).
Prolonged exposure to seminal plasma results in marked decline in both sperm motility and viability, which is not the case if sperm is incubated in synthetic culture medium free of seminal plasma contamination. By virtue of constituents that inhibit or reverse capacitation, the presence of seminal plasma in in vitro fertilization systems inhibits the occurrence of acrosome reaction and hence the fertilizing ability of human spermatozoa. It is clearly essential, therefore, that spermatozoa for clinical procedures such as IUI, IVF or GIFT, must be separated from the seminal environment as soon as possible after ejaculation.
The swim-up procedure remains the simplest means of obtaining populations of highly motile human spermatozoa, and it has been utilized by many laboratories as the main sperm preparation technique for assisted reproduction procedures (Brandeis et al., 1993). However, not only good motility butalso intact acrosomes that can undergo normal acrosome reaction is critical for successful sperm fertilization (Polanski et al., 1988), and the latter is one of the important factors that correlates with fertilization rate in vitro and pregnancy outcome (Calvo et al., 1994; Parinaud et al., 1995; Henkel et al., 1993; Liu et al, 1988).
The goals of this study were to verify whether sperm preparation by swim-up can select a better intact acrosome sperm population and to study the influence of subsequent in vitro capacitation on acrosome reaction of these selected spermatozoa.
Materials and methods
Chemicals
Sperm preparation media (modified Biggers-Whitten-Whittingam [BWW]) was purchased from Irvine Scientific (Santa Ana, CA). Fluoresceine isothiocyanate-conjugated peanut agglutinin (FITCPNA) and bis-benzimide (Hoechst-33258) were obtained from Sigma (Sigma Chemical Co., St. Louis, MO).
Semen collection and assessment of quality
Semen samples were obtained from 15 normal healthy volunteers of proven fertility. All subjects were asked to abstain from ejaculation for at least 48 hours before their appointments. Semen was collected at the appointment by masturbation into sterile specimen cups. The ejaculates were allowed to liquefy for 30 minutes at 37°C. A small aliquot was removed from each liquefied raw specimen and was analyzed within I hour from collection on a computer-assisted semen analyzer (CASA; Motion Analysis, Cell-Trak, model VP 110, Santa Rosa, CA)to assess sperm concentration, motility and motion characteristics. All subjects included in the study had normal semen volume, sperm count and motility as defined by the World Health Organization criteria (WHO, 1992).
Sperm preparation and capacitation procedures
After the initial semen analysis, each semen specimen was divided into three equal aliquots. The first aliquot received no subsequent treatment (raw). The highly motile sperm population from the second and third aliquots was isolated by the swim-up method (Berger et al., 1985, Russell et al., 1987). Briefly, the liquefied semen sample was diluted with an equal volume of BWW. After centrifugation at 300 g for 10 minutes, the supernatant was removed and the pellet resuspended in 2 mLof BWW. A second centrifugation was followed by resuspension to a final volume of 600 μL of BWW supplemented with 0.3% bovine serum albumin factor V (BSA) (Irvine Scientific, Santa Ana, CA). This small volume, containing a large number of spermatozoa, was divided in three equal aliquots of 200 μL. Each of them was underlayered beneath 800 μL of BWW supplemented with 0.3% BSA in Falcon tubes. The tubes were loosely capped and placed in a 37°C incubator, under 5% C02 in air, at a 45 degree angle for 1 hour. During this period, motile spermatozoa migrated from the underlayered sperm suspension to the upper layer. Subsequently, the top 600-700 uL was removed with extreme care to avoid disturbing the interface of the two layered fluids. The removed fluid contained the actively motile spermatozoa and was termed swim-up. After swim-up, the second aliquot received no further treatment (swim-up) and the third aliquot was capacitated for 3 hours by incubation in a BWW medium with 3% human serum albumin (HSA) at 37°C, under 5% C02 in air (capacitated). Raw, swim-up and capacitated specimens were analyzed by CASA for percent motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH).
Calibration setup of the motion analyzer
The CASA calibration setup was as follows: 2-well, 20 microns, duration of data capture (frames): 15 (raw) and 30 (prepared); maximum motile speed (µ/sec): 600 (raw) and 800 (prepared); distance scale factor (μ/pixel): 0.9457; centroid cell size minimum (pixels): 2; centroid cell size maximum (pixels): 8; number of cells to find per well : 200; minimum number of fields per sample: 3. High degree of correlation seen between the CASA and manual sperm counts (r2 =1, slope 1 ) and motility (r2 = 0.97, slope 0.97) established the accuracy of CASA measurements. Reproducibility of the analyzer was determined by using a calibration video tape recording. Rejection criteria for results was values greater than 2 SD. For raw specimens, sperm count was: 38.3 to 42.5 X 106/mL (30 frame/sec) and for prepared specimens 33.1 to 34.7 X 106 /mL (60 frame/sec); sperm motility: 60.6 to 75.0 % (30 frame/ sec) and for prepared specimens 78.1 to 81.5 % (60 frame see), respectively.
Assessment of acrosome status
The acrosome status in raw, swim-up and capacitated specimens was evaluated by fluoresceine isothiocyanate-conjugated peanut agglutinin (FITCPNA, Sigma Co., St. Louis, MO) in conjunction with a supra-vital dye, bis-benzimide (Hoechst-33258, Sigma Co., St. Louis, MO), as a viability test (Cross et al., 1986; Mortimer et al., 1990; Aitken et al., 1993). Simultaneous assessment of viability by Hoechst-33258 and acrosome status by FITC-PNA was done by mixing 100 μL of semen specimen to 100 μL of 2 μg/mL Hoechst-33258 solution and incubating for 10 minutes in the dark. Spermatozoa were then washed in phosphate-buffered saline (PBS) solution by centrifugation at 1200 rpm for 5 minutes to remove excess stain, and the pellet was resuspended in 100 uL of BWW. Twenty microliters of this solution were subsequently smeared on a microscope slide and allowed to dry. At least three slides of each sample were prepared, in case of problems with labeling or scoring. The slides were then immersed in ice-cold methanol for 30 seconds to permeabilize the sperm membranes and allowed to air dry. The fixed smears were immersed in a 40-μg/mL FITC-PNA solution, incubated at room temperature for 20 minutes in foilcovered Coplin jar, and washed gently in PBS to remove the excess label. Scoring was completed within 48 hours of staining.
A Leitz Orthoplan fluorescence microscope (Leitz, Germany) equipped with a Ploemopak epiillumination module and a mercury ultraviolet source was used to examine the slides at 1000x magnification in the presence of an anti-quenching agent (Cargille immersion oil, type DF, Fisher Scientific, Pittsburgh, PA) to minimize the loss of fluorescence. Filter cube 1.2 was used for FITC-PNA, which fluoresces "apple-green," and cube A.2 for Hoechst-33258, which fluoresces a bright medium blue. Hoechst-33258 stains the nuclei of damaged cells (dead spermatozoa), which show a bright blue-white fluorescence and is excluded from viable cells (live spermatozoa), which show a pale blue fluorescence (Cross et al., 1986). The same slide was examined for FITC-PNA labeling and for Hoechst-33258 staining by interchanging the two filters.
Categorization of staining patterns
Acrosome staining on FITC-PNA labeling was classified as follows. In an intact acrosome, the acrosomal region of the sperm head exhibited a uniform apple-green fluorescence. In a reacted acrosome, only the equatorial segment of the acrosome was stained (figure Ia). Viability staining on Hoechst-33258 was classified as follows. In a viable spermatozoa, the sperm head showed a pale-blue fluorescence, and in a dead spermatozoa, the sperm head showed a bright blue-white fluorescence (figure I b). A total of 200 spermatozoa per sample were scored. The acrosome scores were based on viable cells only.
Figura I A. Photomicrograph of spermatozoa labeled with FITC-PNA. Labeled spermatozoa were viewed under epifluorescence using cube 1.2 at 1000X, and photographed on 400 ASA film with exposure time of 30 seconds. Acrosome intact (small arrow) and acrosome-reacted spermatozoa (large arrow).
Figura I B. Photomicrograph of spermatozoa labeled with Hoechst-33258 stain. Labeled spermatozoa were viewed under epifluorescence using cube A.2 at 1000X and photographed on 400 ASA film with exposure time of 60 seconds. Live (small arrow) and dead cells (large arrow).
Statistical analysis
Repeated measures analysis of variance (ANOVA) was used to test for statistical differences in sperm motion characteristics in raw, swim-up and capacitated specimens. For multiple pairwise comparisons the Bonferroni correction factor was used and a P value of </= 0.02 was considered to be significant. Due to non-normally distributed data for the acrosome scores, pairwise comparisons between groups were performed using the Wilcoxon rank sum test with P </= 0.02 (with Bonferroni correction) considered statistically significant. Statistical analysis was performed using the SAS statistical software package (Cary, NC).
Results
Sperm characteristics in the raw, swim-up and capacitated specimens are compared in Table I. There was a significant increase in percent motility and in all motion parameters, except linearity, in both swim-up and capacitated groups compared to the raw specimens. There was no statistically significant increase in the motion parameters in the capacitated group compared to the swim-up group, although the VCL, VAP, and ALH values were higher in the former. Viability scores in swim-up specimens were significantly higher than in non-selected ones (raw) (P = 0.03).
TABLE I. Characteristics of raw, swim-up and capacitated sperm*
The frequency of spermatozoa exhibiting intact acrosomes was not different between raw and swim-up specimens (P = 0.63) (Table II). However, the frequency of acrosome reacted spermatozoa was significantly higher (20.5%) in the capacitated group compared to both raw and swim-up groups (P < 0.001 ) (Table III). Acrosome reaction was observed in 13.1% and 13.0% of raw and swim-up spematozoa, respectively, and this has occurred independent of capacitation.
TABLE II. Frequency of spermatozoa exhibiting intact acrosomes in raw and swim-up non capacitated specimens*
TABLE III. Frequency of acrosome reaction in raw, swim-up and capacitated specimens*
Discussion
Sperm processing is an integral step in assisted reproductive technologies. The most common methods are the swim-up technique and the separation through a discontinuous gradient. The advantage of these methods is that a highly motile sperm population is selected, rather than the mere nonselective concentration o f spermatozoa obtained through a simple wash (Brandeis et al., 1993). Although motility is an important aspect of sperm fertilizing ability in vivo and in vitro, efforts have been currently directed to the understanding of sperm function and to developing assays to evaluate and to accurately predict the fertilization potential of a given sperm population (Jeyendran et al., 1984; Kruger et al., 1988; Calvo et al., 1989).
Many studies have shown that normal morphology using strict criteria (Kruger et al., 1988), sperm-zona pellucida (ZP) binding (Gould et al., 1983) and normal intact acrosomes are the factors that most important correlates with fertilization rate in vitro and pregnancy outcome (Calvo et al., 1994; Parinaud et al., 1995; Henkel et al., 1993; Liu et al., 1988). It is reasonable to assume that techniques which select high numbers of spermatozoa with normal morphology, forward progression, high zona-binding capacity and normal intact acrosomes may improve the results of IVF and others procedures in assisted reproduction. In the present study, in spite of selecting a highly motile sperm population, the swim-up procedure was not effective in yielding higher numbers of spermatozoa with intact acrosomes. The reason for this is unclear, but it is possible that prepared spermatozoa lack substances that inhibit the acrosome reaction, or that some spermatozoa may undergo spontaneous acrosome reaction dependent on the concentration of protein used in culture medium and time of incubation (Mortimer et al., 1989; Tesarik, 1989; Van Kooij et al., 1985).
The capacity for fertilization in vivo develops during the passage of the spermatozoa through the female genital tract and is termed "capacitation". Spermatozoa of all eutherian mammals must undergo capacitation before they can fertilize (Henkel et al., 1993). Sperm transport through the female genital tract can occur quite rapidly (times as short as 15 to 30 minutes have been reported in humans), where as capacitation time can vary from 3 to 24 hours (Mortimer, 1985). Therefore, it can be speculated that capacitation is not completed until after the spermatozoa has entered the cumulus oophorus. This delay is physiologically beneficial because the spermatozoa do not respond to acrosome-reaction-inducing signals until they approach the zona pellucida, preventing premature acrosome reactions that lead ultimately to the sperm inability to penetrate the egg vestments (Fraser, 1984; Mortimer, 1985).
The mechanism of capacitation is complexand poorly understood. Inhibiting factors in or on the sperm surface block the acrosome reaction or prevent spermatozoa from binding to the zona (Zaneveld et al., 1991). The removal of these factors is necessary before the acrosome reaction can occur and is an essential aspect of the capacitation process. A variety of techniques can induce capacitation in vitro. Albumin, a frequently used agent for in vitro capacitation, removes glycoconjugates from the sperm surface (Focarelli et al., 1990). The standard incubation medium for capacitation purposes used in the present study was BWW (Wang et al., 1993) supplemented with 3% human serum albumin. This medium supports in vitro functional capacitation and the acrosome reaction of human spermatozoa (Henkel et al., 1993; Bielfeld et al., 1994; Calvo et al., 1993, Esteves et al., in press).
Under experimental conditions, capacitation often is said to have taken place when spermatozoa are able to acrosome react in response to an agonist. Because human spermatozoa in an albumin-containing medium usually do not respond to an agonist but will respond after a 3-hour incubation period or longer, capacitation has been defined as the changes that occur when spermatozoa are incubated in an albumin-containing medium for 3 hours or longer (Bielfeld et al., 1994). Furthermore, it has been shown that capacitated spermatozoa exhibit a change in the type of flagellar motility expressed, from progressive, linear motility to less progressive, less linear, more vigorous, so-called hyperactivated motility (Robertson et al., 1988). Robertson et al. (1988) and Burkman (1991) demonstrated that the most fertile specimens reach their maximum incidence of hyperactivation within 3 hours, and that after this period of time the percentage of spermatozoa that undergo acrosome reaction is significantly higher.
In the present study, the population of viable spermatozoa was considered as the total sperm population. The reasons for this were two-fold. First, non-viable spermatozoa may show intact acrosomes or different patterns of reacted acrosomes on fluorescence labeling. However, it is difficult to differentiate between the dead cells that have lost their acrosomal contents due to membrane rupture and those that have undergone acrosome reaction prior to death. Second, only viable, acrosome-intact spermatozoa can undergo acrosome reaction and potentially fertilize the oocyte.
Acrosome reaction independent of capacitation has occurred in a small number of raw and swim-up selected spermatozoa in our study. This study supports the idea that in vitro capacitation and acrosome reaction may occur independently, as proposed by others (Bielfeld et al., 1994, Esteves et al., 1997), since the occurrence of the latter is not always preceded by the former. On the other hand, we found that in vitro incubation under capacitating conditions significantly optimized the acrosome reaction rate in the swim-up selected population. Based on our results and on previous studies (Tesarik, 1989, Zaneveld et al., 1991; Wang et al., 1993), we emphasize that sperm capacitation must be considered an extremely important aspect in the normal acrosome reaction process.
Tesarik (1989) has showed that the acrosome reaction must be precisely timed to ensure fertilization, in vivo. A premature acrosome reaction leads to the loss of zona pellucida recognition sites from the sperm surface and thus compromises sperm-zona pellucida binding (Liu et al., 1994). The term acrosome reaction prematurity is used for the cases in which the frequency of spontaneous acrosome reaction (not related to capacitation) is > 20%. This condition is seen in males with unexplained infertility (Tesarik et al., 1995). The cause of the premature acrosome reaction is not known, but the premature stimulus-independent initiation of acrosomal exocytosis may be related to a perturbation of the plasma membrane. The exocytosis process decreases membrane stability, thereby increasing the propensity to membrane fusion. In this situation, the acrosome reaction may not involve a premature activation of the receptor-mediated process, but rather reflects an inherent fragility of the sperm membrane, leading to a receptor-independent loss of acrosome. Consequently, spontaneous or capacitation-induced acrosome reaction prior to therapeutic procedures, such as intrauterine insemination, may lead to the sperm inability to fertilize the egg. On the other hand, the swim-up preparation does not capacitate the sperm population. This method is effective to remove the seminal plasma, which is the first step on sperm capacitation process. We believe that therapeutic procedures such as intrauterine insemination must be done in a time frame variying from immediately after the swim-up up to 3 hours culture at 37°C, because spermatozoa can complete capacitation inside the female reproductive tract. Although these considerations seems to be true for normozoospermic men, some infertile individuals appear to benefit for prolonged in vitro incubation under capacitating conditions prior to insemination.
Acknowledgments
The authors wish to thank Mrs. Fabíola Couceiro Bento for the editorial assistance and language correction.