1 Centres for Assisted Reproduction (CARE), The Park Hospital, Sherwood Lodge Drive, Arnold, Nottingham, NG5 8RX and 2 School of Pharmacy and Chemistry, John Moores University, Bysom Street, Liverpool L3 3AF, UK
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Abstract |
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Key words: acrosome reaction/capacitation/hyperactivation
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Introduction |
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The objective of this report was to investigate whether capacitating human spermatozoa that reach hyperactivation in vitro in synthetic media, collected individually using the Computer Image Sperm Selection (CISS) technique (Green, 1995; Green et al., 1995
; Green and Fishel, 1999
), have an increased risk of spontaneous acrosome loss.
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Materials and methods |
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Because the HST is not commonly used, the computer assisted sperm analysis (CASA) system for the study of HA motility in human spermatozoa and the computer image sperm selection (CISS) technique itself incorporates a unique application for CASA, the HST was validated for the accuracy and reproducibility for measurement of amplitude, frequency and velocity and hyperactivation thresholds were independently established for the HST. Both procedures have been described previously (Green and Fishel, 1999).
The thresholds for HA spermatozoa trajectories obtained from the HST operating at 25 Hz, which was the European standard at the time these data were collected (Sukcharoen et al., 1995), were VCL
70 µm/s, VSL
30 µm/s, LIN
30% and ALH
7 µm. More recent ESHRE guidelines, however, now recommend using a minimum of 50 Hz for the reliable measurement of HA motility in human spermatozoa (ESHRE Andrology Special Interest Group, 1998
).
Criteria for non-hyperactivating spermatozoa (non-HA)
Our criteria for non-HA spermatozoa were those displaying uniformly progressive trajectories which were highlighted with yellow computer generated trails, visually confirming that hyperactivation parameters for these spermatozoa had not been reached.
Computer image sperm selection (CISS)
The details of the CISS technique has been published previously (Green, 1995; Green et al., 1995
; Green and Fishel, 1999
). Briefly, the selection of spermatozoa was performed from a 200 µl volume of medium under light mineral oil held within a temperature conserving stage (Research Instruments Ltd, Penryn, Cornwall) on an IMT2 inverted microscope (Olympus UK Ltd, London) equipped with micromanipulation tools (Research Instruments Ltd, Penryn, Cornwall).
Reproducible identification of HA spermatozoa was achieved by a computer program, specially developed for the HST, that changed the colour of the computer generated overlay on the spermatozoon, seen on a tracking monitor, from yellow to white for those trajectories that reached all the minimum centroid derived criteria defining hyperactivation. This ensured that only computer verified HA spermatozoa were recovered for analysis. Spermatozoa were removed individually with a 12 µm diameter microneedle using micromanipulating tools under the visual control of the tracking monitor.
Manual reconstruction of these trajectories corresponded to those of the starspin and thrashing classification previously reported (Burkman, 1991).
Sperm preparation
Semen samples were obtained from the male partners of couples attending the Nottingham University fertility clinic who consented for their spermatozoa to be used for research. The semen samples were produced by masturbation and the liquefied ejaculate carefully layered over a 45% and 90% discontinuous Percoll (Sigma UK, Poole) gradient obtained by volumetric dilution of the supplier's `100%' Percoll. Semen samples were centrifuged through this gradient at 500 g for 10 min. The spermatozoa at the bottom of the 90% fraction were removed with a sterile glass pipette and mixed with Earle's Balanced Salt Solution (EBSS) containing 10% maternal serum (EBSS 10%) warmed to 37°C. The spermatozoa were centrifuged at 250 g for 5 min after which the supernatant was removed. The spermatozoa were resuspended in fresh EBSS 10% and centrifuged at 250 g for 5 min. After the second washing step the spermatozoa were diluted to a concentration of ~200 000 motile spermatozoa per ml, a concentration that would facilitate CISS, and divided into aliquots. All samples were incubated at 37°C in 5% CO2 in air under relative humidity of 95%.
Preparation of samples
The evaluation of the spontaneous acrosome reaction in selected HA and selected non-HA spermatozoa was reported at time intervals of 0, 1, 2.5, 3.5 and 6 h to allow for capacitation and samples were divided into aliquots to ensure subsequent incubation time points would not be disturbed.
The time points for CISS were arrived at because the sampling had to be performed around the routine morning use of the IMT2 inverted microscope equipped for CISS. A 24 h time point was initially incorporated but subsequently abandoned, since all samples examined showed no hyperactivation.
At each designated time point, an aliquot was taken out of the incubator and placed on the heated stage of the inverted IMT2 microscope. At each time point CISS was performed for HA spermatozoa and these were transferred with the microneedle under microscopic visualization into microwells on PH 133 glass slides (C.A. Hendley Ltd, Loughton, Essex, UK). Only after all the available spermatozoa satisfying hyperactivation thresholds had been collected and transferred to microwell slides was sampling for non-HA spermatozoa performed. These were collected in the same manner and transferred to separate microwell glass slides. The location of HA and non-HA spermatozoa was labelled using a code and the slides were fixed and stained. The slides were subsequently scored blind by technicians in the Seminology department who had no knowledge of the code.
Slide preparation and evaluation
All the microwell slides were rinsed and wiped in alcohol before use to remove traces of grease. Once the selected spermatozoa were transferred, the specimen was air dried for 10 min. The slide was then fixed in absolute alcohol for 15 min followed by dual staining with a 1:1 dilution of fluorescein isothiocyanate (FITC) conjugated Pisum sativum lectin (1 mg/ml) and propidium iodide (PI) (20 µg/ml) in a humidified chamber at room temperature for 20 min. The slide was rinsed in distilled water for 10 min, air dried and mounted in glycerol.
Acrosomal assessment was performed by epifluorescence microscopy utilizing a dichroic mirror and filter system, where one filter was used specifically for PI with an emission wavelength of 615 nm and another filter specifically for FITC with an emission wavelength of 520 nm, using a BH2 fluorescence microscope with a x40 oil immersion objective (Olympus UK Ltd, London, UK).
PI was included in order to use the bright orange staining of the nucleus to locate the individually collected spermatozoa fixed on the microwell. Once the spermatozoa where located, it was a simple matter to move to the FITC filter for acrosomal assessment of the fixed spermatozoa, determined by the pattern of binding of the FITC-conjugated P.sativum.
Acrosome reacted spermatozoa (R) were characterized by FITC emitting at the equatorial segment only, partially reacted spermatozoa (PR), by a patchy emission over the acrosome cap and non-reacted spermatozoa as a bright fluorescence over the acrosomal cap (Cross et al., 1986).
The number of spermatozoa in each staining category at each time point for HA and non-HA were recorded.
Statistical analysis
The data for the three end points, the proportion of selected spermatozoa fully acrosome reacted (R), the proportion partially acrosome reacted (PR) and the proportion of spermatozoa showing any acrosome reaction (R+PR), were analysed using two way analysis of variance with spermatozoa type (HA and non-HA), and time as the two factors. The data were also broken down into two separate sets, one for HA and the other for non-HA spermatozoa. These were each subjected to one-way analysis of variance with time as the factor. All analyses were carried out using the SPSS statistical package for Windows version 6.0.1 (SPSS Inc., Chicago, Illinois, USA)
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Results |
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The one-way analysis showed that among the selected HA spermatozoa there were marked differences among the proportion spontaneously acrosome reacted at the various different incubation time points (for R+PR, P = 0.005). In contrast, there was no significant evidence of change with time for the selected non-HA spermatozoa using the same end point.
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Discussion |
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Of the 9540 selected spermatozoa examined, the results demonstrated a significant difference between HA and non-HA spermatozoa in the propensity to undergo the spontaneous acrosome reaction, regardless of whether complete (R), partially (PR) or any (R+PR) acrosome reaction was used as the end point (P < 0.001). Although the occurrence of spontaneous acrosome reaction was significantly higher in HA spermatozoa, the proportion undergoing any acrosome reaction, showed marked differences at the various different time points (P = 0.005). In contrast, there was no significant evidence of change with time for the non-HA spermatozoa using the same end point.
These data suggest that HA human spermatozoa may have a greater propensity for spontaneous acrosomal loss than non-HA spermatozoa during an incubation time course.
Capacitation has been reported to be a prerequisite for both partially and complete acrosome reaction (Jaiswal et al., 1998) and since the onset of HA motility is accepted to be a concomitant feature of capacitation (Mortimer et al., 1998
), it is probable that the HA spermatozoa consisted of capacitated cells more susceptible to undergo a spontaneous acrosome reaction.
Peak values of spontaneous acrosome loss reached 25% of all HA spermatozoa after only 2.5 h incubation when the combined R+PR were considered, compared to a maximum of 15% of all non-HA spermatozoa after 6 h incubation (Table I). In this report we have found that potential levels of spontaneous acrosome reaction in HA spermatozoa suggest `acrosome reaction prematurity' (Tesarik and Mendoza, 1995
).
We recently reported that hyperactivation in vitro occurs more frequently in spermatozoa with normal morphology (Green and Fishel, 1999). It is therefore possible that the fertilizing potential of a suspension of spermatozoa incubated over time at 37°C may decline (Fishel et al., 1985
), as a consequence of acrosome reaction prematurity within this subpopulation of morphologically normal spermatozoa, particularly if levels of hyperactivation within the suspension are high.
It is routine practice in embryology laboratories performing in-vitro fertilization to incubate suspensions of spermatozoa at 37°C for variable lengths of time prior to mixing with the oocytes. In light of this report, we propose that these samples are incubated for a short period of <2 h, prior to insemination of oocytes to minimize the risk of loss of fertilizing potential as a consequence of acrosome reaction prematurity within a functionally important subpopulation of morphologically normal spermatozoa (Kruger et al., 1988).
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Acknowledgments |
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Notes |
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References |
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Submitted on January 18, 1999; accepted on April 8, 1999.