1 Department of Obstetrics and Gynaecology, University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong, China
2 Present address: Department of Obstetrics and Gynaecology, Second Affiliated Hospital, Sun Yat-sen University, 107 West Yan Jiang Road, Guangzhou 510120, Guangdong, China
3 Present address: Innovation and Technology Commission, 14/F, Ocean Centre, 5 Canton Road, Kowloon, Hong Kong, China
4 To whom correspondence should be addressed. e-mail: wsbyeung{at}hkucc.hku.hk
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Abstract |
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Key words: cumulus oophorus/human spermatozoa
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Introduction |
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Various in vitro models had been used to study the effects of the cumulus oophorus on sperm function. These models include cumulusoocyte complex (COC) co-incubation (Suarez et al., 1983; Chen and Sathananthan, 1986
; Cherr et al., 1986
; Cummins and Yanagimachi, 1986
; Corselli and Talbot, 1987
; Stock et al., 1989
; Carrell et al., 1993
; Schroer et al., 2000
), cumulus oophorus co-incubation with spermatozoa (Magier et al., 1990
; Tesarik et al., 1990
; White et al., 1990
), cumulus cell monolayer co-culture (Mansour et al., 1995
) and culture in cumulus cell-conditioned medium (Sullivan et al., 1990
; Fetterolf et al., 1994
). All these models have their drawbacks and may not represent the in vivo condition.
The use of the COC theoretically is the best model. By the use of videomicroscopy, co-incubation with the COC had been applied to study sperm motility and acrosome reaction within the cumulus oophorus in golden hamster and guinea pig (Suarez et al., 1983; Cherr et al., 1986
; Cummins and Yanagimachi, 1986
; Corselli and Talbot, 1987
; Schroer et al., 2000
). However, this model cannot differentiate the effect of the cumulus oophorus from that of the oocyte. In addition, there are some ethical concerns on the use of living oocytes in this model. Also there would be restriction on the amount of work that can be done with this model when the experiments are incorporated in routine assisted reproduction services, e.g. one may only be able to study the effect of the COC on sperm functions 1620 h post-insemination when the fertilization check has been performed.
Co-culture with a cumulus cell monolayer disrupts the three-dimensional architecture of the cumulus oophorus and might not give the true picture of the physiology of the cumulus cells. This is manifested by the change in morphology of the cumulus cells from a spherical shape suspended in the matrix to flattened cells when they are attached to the culture dish (Bar-Ami et al., 1989). Such deficiency of the monolayer cumulus culture also questions the suitability of using its conditioned medium for physiological study of the cells. Moreover, this model would not allow us to study the impact of cumulus matrix on sperm function.
A few studies have used co-incubation of spermatozoa with the cumulus oophorus (Magier et al., 1990; Tesarik et al., 1990
; White et al., 1990
) to compare the function of spermatozoa inside and outside the cumulus oophorus. While this model has the advantage of maintaining the three-dimensional architecture of the cell mass and the morphology of the cumulus cells, it is uncertain whether the spermatozoa inside the cumulus oophorus would ever be able to penetrate through the whole thickness of the cell mass. The sperm population outside the cumulus oophorus is likely to represent a mixture of spermatozoa that have and have not interacted with the cumulus oophorus, and there currently is no method to separate these two sperm subpopulations. In addition, the number of spermatozoa in the cumulus oophorus that can be recovered in this model is limited, and further functional analyses with these spermatozoa are difficult.
We recently have developed a novel capillary-cumulus oophorus model to tackle the deficiencies in the above models. In this model, the cumulus oophorus is placed within a capillary and spermatozoa are allowed to traverse the cumulus oophorus from one end of the capillary. As such, the penetrated spermatozoa can be collected from the other end of the capillary for analysis. We have attempted to demonstrate the feasibility of this model in this report. This model will then be used to reappraise the effect of the cumulus oophorus on the functions of human spermatozoa passing through it.
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Materials and methods |
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Collection of cumulus oophorus
COCs were obtained from women who underwent treatment with ICSI due to male infertility. HMG (Serono, Sweden) was used for ovarian stimulation after downregulation with buserelin (Ng et al., 2000). After oocyte retrieval, cumulus oophorus were dissected mechanically from COCs using a sterile syringe needle and a glass pipette. The cumulus oophorus from each patient were pooled in EBSS/0.3% bovine serum albumin (BSA) before experimentation.
In vitro cumulus oophorus model
A sterile glass capillary (Microcaps, Drummund, USA) with an inner diameter of 0.7 mm was attached onto a 1 ml disposal syringe (Terumo, Tokyo, Japan) with a limited amount of nail polish and was pre-warmed in a mobile Hoffman IVF Chamber (Air-shields Vickers, Hatboro, USA) at 37°C with humidified air containing 5% CO2. EBSS/0.3% BSA was aspirated to a length 3 cm from the end of the capillary (Figure 1). This was followed by aspiration of one or two cumulus oophorus, forming a cumulus oophorus column of length determined by the experimental design. This end of the capillary was then dipped into a 100 µl droplet of sperm suspension containing 10 x 106 motile spermatozoa/ml overlaid with mineral oil. Another capillary containing only EBSS/0.3% BSA served as a control. The whole set-up was kept in the mobile IVF chamber for a time period determined by the experimental design. After incubation, the capillary was cut with a diamond pen at the interface between the cumulus oophorus column and the medium column (Figure 1). The medium column contained spermatozoa that had passed through the cumulus oophorus (penetrated spermatozoa). These spermatozoa were expelled into a 0.5 ml Eppendorf tube, and the spermatozoa from several capillaries having undergone the same treatment were pooled. The control capillary was cut at a level that was at the same distance from the end of the capillary as those capillaries containing the cumulus oophorus. Spermatozoa that had swum above the cutting level of the control capillary were collected as control spermatozoa.
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Determination of sperm capacitation
The capacitation status of spermatozoa was determined by chlortetracycline (CTC; Sigma) staining as previously described (Yao et al., 2000). The fixation solution consisted of 1 mol/l Tris buffer pH 7.8. Briefly, 10 µl of sperm suspension was stained with an equal volume of CTC staining solution (750 µM CTC in 20 mM Tris buffer supplemented with 130 mmol/l NaCl and 5 mmol/l cysteine) in a light-protected tube. Glutaraldehyde (12.5%) fixation solution (1 µl) was then added. The capacitation status and acrosomal status of 200 spermatozoa were evaluated under a fluorescence microscope (Zesis) at x630 magnification with a filter set consisting of an excitation filter BP 450490, a chromatic beam splitter FT510, and a barrier filter LP520. Five CTC staining patterns of the sperm head were identified according to the method of Perry et al. (1995
). They were: (i) CTC1, a fluorescent band in the post-acrosomal region; (ii) CTC2, a bright fluorescent head with a non-fluorescent post-acrosomal region; (iii) CTC3, a bright fluorescent head with a non-fluorescent thin band in the post-acrosomal region; (iv) CTC4, uniform head fluorescence; and (v) CTC5, a decrease in or loss of uniform fluorescence over the head. CTC1, CTC2 and CTC3 were the uncapacitated patterns; CTC4 was the capacitated pattern; and CTC5 was the acrosome-reacted pattern.
Determination of acrosomal status and motility of sperms
Fluorescein isothiocyanate-labelled Pisum sativum lectin (FITC-PSA; Sigma, St Louis, MO) was used to evaluate the acrosomal status of the sperm as described (Chiu et al., 2002). The fluorescence patterns of 300 spermatozoa in randomly selected fields were determined under a microscope (Zeiss) with x1000 magnification. The acrosomal status of spermatozoa was classified according to the lectin staining into (i) intact acrosome, complete staining of the acrosome; (ii) reacting acrosome, partial or patchy staining of the acrosome; and (iii) reacted acrosome, complete staining of the equatorial segment only or no staining of the whole sperm head. The proportions of intact, reacting and reacted acrosome were expressed as percentages of the respective patterns in the total number of spermatozoa counted. The Hobson Sperm Tracker System (HST; Hobson Tracking Systems Ltd, Sheffield, UK) was used to determine the motility of spermatozoa. A total of 500 spermatozoa were assessed for each sample. The set-up parameters of the system and the procedures have been described elsewhere (Chiu et al., 2003
).
Determination of spermatozoa-zona binding capacity
The hemizona binding test (HZA) was applied to assess the effect of the cumulus oophorus on the zona binding of the penetrated spermatozoa. As the number of penetrated spermatozoa collected was small, the protocol of HZA described previously (Chiu et al., 2002) was miniaturized, with the volume of incubation reduced from 100 to 30 µl. Briefly, hemizonae were prepared by micromanipulation as described (Chiu et al., 2002
). The hemizonae were placed in 30 µl droplet of EBSS/0.3% BSA containing penetrated or control spermatozoa at a concentration of 0.2 x 106 spermatozoa/ml. After 4 h of incubation at 37°C under 5% CO2 in air, the tightly bound spermatozoa on the outer surface of the hemizonae were counted. The coefficient of variation in the counting of the tightly bound spermatozoa was 10%. The index of HZA (HZI) was calculated as: HZI = number of spermatozoa bound in test droplet x 100%/number of spermatozoa bound in control droplet.
Determination of sperm count
Preliminary experiments showed that the concentration of penetrated spermatozoa collected in the medium was <0.5 x 106 spermatozoa/ml. In order to increase the accuracy in counting the sperm at such a low sperm count, spermatozoa in the central set of squares and the eight peripheral sets of squares in a haemocytometer were counted. The intra-assay variance of the counting at concentrations from 0.125 to 0.5 x 106 spermatozoa/ml was <5% and the inter-assay variance ranged from 3 to 7.5%.
Effect of the length of the cumulus oophorus column and incubation time on the number of penetrated spermatozoa
For the in vitro model to be useful, the model had to allow collection of a sufficient number of spermatozoa for subsequent functional analyses. This depended on two factors, namely the length of the cumulus oophorus column and the duration of incubation. Therefore, an experiment was performed to study the effects of these two factors on the number of penetrated spermatozoa collected. In this experiment, two lengths of the cumulus oophorus column (1 and 2 cm) and two incubation periods (1 and 2 h) were studied. Spermatozoa from semen samples of six men were tested with the cumulus oophorus from six female patients. As the results showed that a sufficient number of spermatozoa could be collected when the cumulus oophorus column was 2 cm in length and the incubation duration was 1 h (see below), these conditions were used in all subsequent experiments.
Effect of the cumulus oophorus on various sperm parameters
After semen processing, the spermatozoa were allowed to penetrate the cumulus oophorus in the capillary model. In each experiment, 46 identical capillaries with a cumulus oophorus were prepared, while four other capillaries containing medium only served as the control. After incubation, the above functional parameters of the penetrated and the control spermatozoa were determined.
Statistical analysis
SigmaStat statistical software (Jandel Scientific, San Rafael, CA) was used to analyse the data. Sperm counts and concentration were expressed as mean and SD. Results for the HZA were expressed as means ± SD. Paired Students t-test was used to compare the number of spermatozoa tightly bound to the hemizonae. Results of other sperm function parameters were expressed as medians and were compared with the MannWhitney U-test.
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Results |
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Effect of the cumulus oophorus on sperm motility
The effects of the cumulus oophorus on sperm motility parameters are shown in Table II. The velocity parameter, Curvilinear velocity (VCL), did not differ between the control and the penetrated spermatozoa. Penetrated spermatozoa had significantly higher (P < 0.05) Average path velocity (VAP), Straight line velocity (VSL), Linearity (LIN) and Beat cross frequency (BCF), but lower Amplitude of lateral head displacement (ALH) and hyperactivation than the control spermatozoa. These data suggested that the penetrated spermatozoa swam in a more linear and progressive manner.
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Effect of the cumulus oophorus on spermatozoa-zona binding
The effect of the cumulus oophorus on spermatozoazona binding capacity was assessed with HZA. Eight semen samples were used. In comparison with spermatozoa which had swum up in the control capillary, the penetrated spermatozoa had a higher zona-binding capacity with significantly more (P < 0.01) tightly bound spermatozoa on the hemizona (88.3 ± 7.6) than control spermatozoa (68.3 ± 8.8), and the HZI (129.7 x 7.3) was increased by 30%.
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Discussion |
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There are two criteria for a successful cumulus oophorus model. First, the natural structure of the cumulus oophorus has to be maintained. Secondly, the sperm population passing through the cumulus oophorus can be collected in sufficient quantity for functional study. In the present model, the three-dimensional architecture of the cumulus oophorus is maintained in glass capillaries. The elasticity of the cumulus matrix enables the cell mass to completely occupy the lumen of the cumulus oophorus column in the capillary. This set-up ensures that spermatozoa accumulated in the medium column above the cumulus oophorus column represent only those that have penetrated the cumulus oophorus. The amount of spermatozoa that can be recovered in this model depends on the length of the cumulus oophorus column and the duration of incubation. As expected, increasing the length of the cumulus oophorus column (1 versus 2 cm) and decreasing the incubation time (1 versus 2 h) decreased the number of penetrated spermatozoa collected. A 1 h incubation and a 2 cm long cumulus column were selected as standard conditions for subsequent experiments because the former was the shortest time reported for the spermatozoa to traverse the cumulus oophorus (see below), and the latter was used to ensure that all penetrated spermatozoa had sufficient interaction with the cumulus oophorus. With the conditions selected, an average of 2500 spermatozoa in 10 µl of medium were recovered from a capillary. This accounts for <1% of the spermatozoa in the droplet. In order to perform a sperm function assay with the penetrated spermatozoa, spermatozoa from several capillaries in an experiment had to be pooled together. The amount of penetrated spermatozoa recovered per capillary was much lower than that in the control capillary. This is in line with the observation that the cumulus oophorus is a barrier to the penetrating spermatozoa (Lavy et al., 1988; Inagaki et al., 1994
; Hourani et al., 1995
), supporting the idea that the cumulus oophorus may be involved in modifying/selecting good spermatozoa for fertilization.
A recent randomized study showed that a 1 h incubation of spermatozoa with the COC was sufficient to achieve fertilization (Gianaroli et al., 1996; Dirnfeld et al., 1999
). Incubation of the COC with Hoechst 30322-labelled spermatozoa showed that spermatozoa entered the complex within 15 min, traversed the cumulus layer within 3 h, and first appeared in the oocyte cortex at 4 h post-insemination (Gianaroli et al., 1996
). By serial sectioning of the COC, Chen and Sathananthan (1986
) reported that acrosome-intact, partial or fully reacted spermatozoa were found in the zona pellucida 13 h post-insemination. The discrepancy in the time required for the spermatozoa to pass through the cumulus oophorus in these two studies could be due to the reduced performance of spermatozoa after fluorescent labelling in the former study. In agreement with the latter study, our present results demonstrated that 1 h was sufficient for the spermatozoa to pass through the cumulus oophorus.
Sperm capacitation is a prerequisite of fertilization. Cumulus cells had been shown to affect sperm capacitation in several in vitro experiments (Hartmann et al., 1972; Gwatkin et al., 1974
; Bastias et al., 1993
) in both human and non-human eutherian mammals. In the present study, a significantly higher percentage of capacitated spermatozoa was found in the penetrated sperm population when compared with the control population. There are two possible mechanisms for this observation. First, the cumulus oophorus may stimulate capacitation of the spermatozoa passing through it. Secondly, capacitated spermatozoa penetrate the cumulus oophorus at a faster rate or uncapacitated spermatozoa are selectively retained in the cumulus oophorus (Cherr et al., 1986
). As capacitation is not a well-defined phenomenon with no clear starting point and end point, the present data cannot distinguish between these two possibilities.
The percentage of spermatozoa with normal morphology was significantly higher in the penetrated spermatozoa than in the control spermatozoa. In fact, no penetrated spermatozoa with severe morphological defects were found. This is in line with the previous electron microscopic observation that all the 36 human spermatozoa in the cumulus oophorus of a pronuclear egg recovered from a woman after natural intercourse had normal morphology (Pereda and Coppo, 1985). It has been suggested that the cumulus oophorus has a role in selecting morphologically normal human spermatozoa (Sundstrom, 1984
; Carrell et al., 1993
). How this is accomplished is unknown. Human sperm morphology is related to its fertilizing capacity (Rogers et al., 1983
; Schuffner et al., 2002
). In clinical studies, IVF and intrauterine insemination outcome are highly related to sperm morphology as evaluated with strict criteria (Enginsu et al., 1992
; Toner et al., 1995
; Burr et al., 1996
; Eggert-Kruse et al., 1996
; Vawda et al., 1996
; Van Waart et al., 2001
).
Data from this study showed that the penetrated spermatozoa moved in a more rapid, linear and progressive fashion, with higher VAP, VSL and BCF but lower ALH when compared with the control spermatozoa. A similar motility pattern of spermatozoa in the cumulus oophorus described as cumulus-related motility has been reported (Tesarik et al., 1990). Such a motility pattern was suggested to be beneficial for penetrating the cumulus oophorus. This is in great contrast to the hyperactivated motion with high VCL, ALH and low VSL. Therefore, the percentage of hyperactivated spermatozoa is lower in the penetrated spermatozoa (this study) and in spermatozoa within the cumulus oophorus (Tesarik et al., 1990
). It has been suggested that the change in motility pattern of spermatozoa in viscous medium, such as in a cumulus matrix, could be due to local heterogeneity in the mechanical resistance of the matrix (Drobnis et al., 1988
). However, the present observation that the penetrated spermatozoa exhibited cumulus-related motility even when they were swimming freely in culture medium suggests that the mechanical property of the cumulus matrix is not responsible for inducing this specific motility pattern. This is in agreement with the suggestion of the presence of unknown matrix components responsible for such induction (Tesarik et al., 1990
). The exact identity of this factor(s) needs further investigation.
Spent medium after culturing cumulus cells for 37 h does not affect sperm motility (Hong et al., 2003). However, specific cumulus-related motility is induced in the penetrated spermatozoa after traversing the cumulus oophorus. Cumulus cell monolayer co-culture (Mansour et al., 1995
) and cumulus cell-conditioned medium collected from IVF insemination dishes 2024 h after insemination or primary cumulus cell culture (Fetterolf et al., 1994
) also affect the motility of human spermatozoa in vitro. These studies demonstrate that secretory product(s) from the cumulus cells affect sperm motility, and that >7 h is required for the product(s) to accumulate in a monolayer culture to a level sufficient to induce a detectable change in motility. However, the threshold level of secretory product(s) is reached in the cumulus oophorus probably as a result of trapping of the product(s) in the matrix. It is likely that more than one component of the cumulus oophorus is involved in modifying sperm motility. While the cumulus oophorus induces the cumulus-related motility (high VAP, VSL and BCF but low ALH), cumulus cell-conditioned medium stimulates VCL and ALH but reduces BCF and LIN (Fetterolf et al., 1994
). Cumulus cells produce progesterone (Dirnfeld et al., 1993
; Bar-Ami and Khoury, 1994
; Chian et al., 1999
). The stimulatory activity of the conditioned medium is not correlated with the concentration of progesterone in the medium, but progesterone levels are inversely correlated with a decrease in BCF and LIN (Fetterolf et al., 1994
).
More penetrated spermatozoa had undergone the acrosome reaction when compared with the controls. This is consistent with other studies. Stock et al. (1989) showed that human COC increased the percentage of acrosome-reacted spermatozoa of the co-incubated spermatozoa 1418 h post-insemination from 15 to 31% when compared with spermatozoa that had been incubated without a COC. Others reached similar conclusions (Tesarik, 1985
; Carrell et al., 1993
). Cumulus oophorus had also been shown to induce capacitation and acrosome reaction of spermatozoa in cattle (Fukui, 1990
; Cox and Hormazabal, 1993
; Chian et al., 1995
). The mechanism responsible for this activity of cumulus cells is not yet fully understood.
Human cumulus cell-conditioned medium collected from IVF insemination dishes 1618 h post-insemination induced the acrosome reaction of spermatozoa (Siiteri et al., 1988), suggesting that secretory product(s) of the cells are involved. One possible candidate is progesterone, which stimulates the acrosome reaction of human spermatozoa (Osman et al., 1989
; Foresta et al., 1992
; Kay et al., 1994
). Another component in the cumulus oophorus that may affect the acrosome reaction is hyaluronic acid in the cumulus matrix, which can increase the intracellular calcium concentration of spermatozoa (Sabeur et al., 1998
). Calcium plays a critical role in sperm acrosomal exocytosis. The action of hyaluronic acid makes the spermatozoa more susceptible to undergo the acrosome reaction upon induction by constituents of the cumulus and/or zona pellucida (Sabeur et al., 1998
).
The zona-binding ability of spermatozoa decreases after the acrosome reaction (Liu and Baker, 1990). The physiological implication of the acrosome-inducing activity of the cumulus oophorus is unknown. Capacitation in mammals is considered as a dynamic and coordinated phenomenon in the female genital tract, and should be completed with the occurrence of the acrosome reaction in the vicinity of the oocyte (Tesarik, 1989
; Hunter, 2002
; Hunter and Rodriguez-Martinez, 2004
). Compared with the control spermatozoa, the increased incidence of the acrosome reaction of the penetrated spermatozoa might indicate that more of these spermatozoa had completed their capacitation and more readily underwent the acrosome reaction upon stimulation by components in the cumulus oophorus, as discussed above. It has been suggested that spermatozoa with better fertilizing potential should display elevated levels of acrosome-reacted spermatozoa with the appropriate stimulus (Tesarik, 1989
). Moreover, glycodelin-F in the cumulus matrix suppresses the progesterone-induced acrosome reaction (Chiu et al., 2003
), and may serve to protect against a premature acrosome reaction. The concentration of glycodelin-F in the cumulus oophorus used in this study is likely to be less than that in vivo as the cumulus oophorus were available for experimentation 46 h after their retrieval from patients due to logistic reasons. During this period, some of the glycodelin-F might have diffused out of the cumulus oophorus, making the spermatozoa in the cumulus matrix more susceptible to an induced acrosome reaction. In animals, it has been suggested that the cumulus oophorus retains non-capacitated and acrosome-reacted spermatozoa by sticking them to the cumulus cells (Cherr et al., 1986
; Cummins and Yanagimachi, 1986
). The present study showed that this retention system, if it exists in human, is leaky, with acrosome-reacted spermatozoa also capable of penetrating the cumulus oophorus.
In spite of having a higher incidence of the acrosome reaction, the zona-binding capacity of the penetrated spermatozoa is higher than that of the control spermatozoa. This does not contradict the current belief that acrosome-reacted spermatozoa do not bind to the zona pellucida, as there were still a substantial number of penetrated spermatozoa with intact acrosomes. Apart from the percentage of acrosome reaction, the number of tightly bound spermatozoa in HZA also depends on other parameters. The cumulus oophorus seems to have selected spermatozoa with better fertilizing potential that outweighed the adverse effect of the increased proportion of acrosome-reacted spermatozoa on zona binding. Compared with the control spermatozoa, the fertilization potential of the penetrated spermatozoa is likely to be better as they have a higher percentage of spermatozoa with normal morphology and capacitated pattern and have higher forward motility. Various studies have demonstrated that spermatozoa bound to the zona pellucida are usually of normal morphology (Kot and Handel, 1987; Menkveld et al., 1991
; Liu and Baker, 1992
). The binding of spermatozoa to the zona pellucida increases during capacitation by uncovering (Dostalova et al., 1995
; Iborra et al., 1996
; Flesch et al., 2001
) or by changing the binding properties of the putative zona receptors (Franken, 1998
). The chance of spermatozoa meeting with the hemizona in HZA increases with the motility of the spermatozoa, and more spermatozoa would be expected to bind to the hemizona in a sperm population having higher motility. Indeed, it has been demonstrated in rhesus monkeys that spermatozoa with higher motility have significantly higher zona binding ability in HZA (Pu et al., 1994
). It is also possible that certain components of the cumulus oophorus directly stimulate the binding of the penetrated spermatozoa to the zona pellucida. The exact mechanism for this to take place awaits further study.
In conclusion, a novel model has been developed to study the interaction of spermatozoa with the cumulus oophorus. Data from this model demonstrate that spermatozoa that have penetrated the cumulus oophorus have better morphology and higher zona-binding capacity, are more likely to be capacitated and exhibit specific motility patterns.
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Acknowledgements |
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References |
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Submitted on January 15, 2004; accepted on April 1, 2004.