Enhancement of mouse sperm motility by the PI3-kinase inhibitor LY294002 does not result in toxic effects on preimplantation embryo development

Michaela Luconi1, Simona Torcia2, Domenico Grillo2, Maria Teresa Fiorenza2, Gianni Forti1, Franco Mangia2 and Elisabetta Baldi1,3

1 Department of Clinical Physiopathology, Center of Research, Transfer and High Education, ‘DENOthe’ Andrology Unit, University of Florence and 2 Department of Psychology, Section of Neuroscience, University of Rome La Sapienza, Rome, Italy M.Luconi and S.Torcia contributed equally to this work and they should be regarded as joint First Authors

3 Corresponding author. E-mail: e.baldi{at}dfc.unifi.it


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: A reduced number of progressively motile sperm (as may occur in cases of asthenozoospermia or when cryopreserved spermatozoa are used for fertilization) limits the possibility of applying various assisted reproductive techniques (ARTs). We previously showed that incubation of sperm with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 increases sperm progressive motility and enhances the number of sperm recovered by capacitation protocols used in ART. METHODS AND RESULTS: In the present study, we investigate the motility-enhancing effects of this compound in epididymal mouse sperm, and examine the use of the mouse system to investigate the effect of LY294002 on oocyte fertilization and preimplantation embryo development. Our results show that neither pre-incubation of mouse spermatozoa with the inhibitor during in vitro capacitation nor the direct addition of LY294002 to the sperm–oocyte mixture significantly affects the process of fertilization and preimplantation development of embryos produced even when they developed in the presence of LY294002. CONCLUSIONS: The present data encourage the design of new drugs based on the molecular structure of LY294002, which may open up new options for the in vitro treatment of human/animal asthenozoospermia.

Key words: embryo/phosphatidylinositol-3 kinase/sperm motility


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Motility is a characteristic function of the male gamete, allowing sperm to reach and penetrate the oocyte. Due to this fundamental role, sperm motility is under the control of intrinsic and extrinsic factors (reviewed by Luconi and Baldi, 2003Go) that mediate its acquisition and regulate its maintenance. We have demonstrated recently that the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 is able to increase human sperm motility (Luconi et al., 2001Go) by increasing tyrosine phosphorylation of the protein kinase A (PKA)-anchoring protein AKAP3, as well as the AKAP3 interaction with PKA in sperm flagellum (Luconi et al., 2004Go). Treatment of spermatozoa from asthenozoospermic subjects with LY294002 not only increases motility (Luconi et al., 2001Go; du Plessis et al., 2004Go), but also enhances sperm–oocyte binding in the hemizona assay (du Plessis et al., 2004Go), opening up the possibility that this compound, or other PI3K inhibitors, may have a therapeutic use in ART procedures to treat spermatozoa having reduced motility, such as those recovered from asthenozoospermic patients. However, the role of PI3K-dependent signals in promoting embryonic cell survival and cell cycle progression (Burdon et al., 2002Go) and in participating in oocyte meiotic maturation (Hoshino et al., 2004Go) is well documented. Therefore, it is possible that sperm treatment with a PI3K inhibitor results in detrimental effects on fertilization and preimplantation embryo development. In this study, we have exploited the mouse system to investigate the effect of LY294002 on oocyte fertilization and preimplantation embryo development. To this aim, we have also determined the effect of LY294002 on epididymal mouse sperm motility and the involvement of AKAP3 phosphorylation in such an effect.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Mouse sperm preparation and IVF
Epididymal spermatozoa were routinely obtained from 2- to 3-month-old B6D2F1 male mice by squeezing the cauda epididymis in HTF medium (Quinn et al., 1985Go) supplemented with 30% bovine serum albumin (BSA). Sperm were then capacitated for 30 min at 37°C in a 1 ml drop of HTF medium under paraffin oil with an atmosphere of 5% CO2 in air. During this procedure, spermatozoa move away from each other and capacitate spontaneously, acquiring the ability to fertilize cumulus-enclosed metaphase II (MII) oocytes in vitro. MII oocytes were obtained from 1.5- to 2-month-old B6D2F1 female mice following priming with pregnant mare serum gonadotrophin (PMSG) and HCG, according to standard procedures (Nagy et al., 2003Go).

For IVF, an aliquot of capacitated sperm suspension was added to MII oocytes in a 1 ml drop of HTF medium under oil, resulting in ~4 x 106/ml and 1 x 104/ml final sperm concentrations. The sperm–MII oocyte mixture was incubated further for 3 h at 37°C and then scored for the presence of fertilized eggs at the pronuclei stage of embryo development. Under these conditions, the fertilization rate consistently approximated 70–80%, depending on egg quality. In vitro fertilized embryos were cultured in drops of Medium 16 (M16) (Whittingham, 1971Go) under paraffin oil and an atmosphere of 5% CO2 in air at 37°C.

Mouse sperm motility assay
Aliquots (10 µl) of sperm capacitated for 30 min as described above were collected near the border of the sperm-containing drop of medium. Even though this procedure resulted in the selection of spermatozoa having a high motility, it was necessary to obtain cells dispersed enough to be individually analysed for their motility. Sperm aliquots were rapidly transferred to pre-warmed glass slides, covered with a coverslip and eventually assayed for motility at 37°C, using a Nikon Eclipse inverted microscope equipped with a warming table, Nomarsky optics and a CCD camera. Sperm motility was determined using the Metamorph software equipped with the ‘cell track’ function (Universal Imaging Co., Downingtown, PA).

SDS–PAGE and western blot analysis
After different treatments, mouse sperm samples were processed for SDS–PAGE for detection of AKAP3 phosphorylation as previously described (Luconi et al., 2004Go, 2005Go). Briefly, sperm samples were washed with saline and eventually resuspended in lysis buffer [20 mmol/l Tris, pH 7.4, 150 mmol/l NaCl, 0.25% NP-40, 1 mmol/l Na3VO4, 1 mmol/l phenylmethylsulfonyl fluoride (PMSF)]. After protein measurement (Coomassie kit, Bio-Rad Laboratories, Hercules, CA), sperm extracts, containing ~30 µg of protein, were diluted by an equal volume of 2x Laemmli’s reducing sample buffer (62.5 mmol/l Tris pH 6.8, 10% glycerol, 2% SDS, 2.5% pyronin and 200 mmol/l dithiothreitol), incubated at 95°C for 5 min and loaded on 8 or 10% polyacrylamide–bisacrylamide gels. After SDS–PAGE, proteins were transferred to nitrocellulose (Sigma, St Louis, MO). In some experiments, the amount of protein loaded was determined by Coomassie staining of a parallel gel. After a 2 h incubation in 1% BM blocking solution (Roche, Milan, Italy) in Tris-buffered saline containing 0.1% Tween-20, pH 7.4 (TTBS), nitrocellulose membranes were washed and then immunostained with peroxidase-conjugated anti-phosphotyrosine (PY20-HRP) or anti-AKAP3 FSP95 antibody (kindly provided by Dr J.Herr, University of Charlottesville, VA), followed by the peroxidase-conjugated secondary antibody.

The antibody-reacted proteins were revealed by an enhanced chemiluminescence system (BM, Roche). For conjugation with a different antibody, as needed, the nitrocellulose membrane was washed for 30 min at 50°C in stripping buffer (10 mM Tris, pH 6.8, 2% SDS, 100 mmol/l {beta}-mercaptoethanol) and probed again with the appropriate primary antibody.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Effect of LY294002 on mouse sperm motility and AKAP3 phosphorylation
To determine if LY294002 positively affects sperm motility in the mouse as it does on human sperm (Luconi et al., 2001Go), mouse epididymal sperm were treated with increasing LY294002 concentrations ranging from 2.0 to 10.0 µmol/l during the 30 min capacitation period at 37°C in vitro. The motility of individual spermatozoa was then determined for each LY294002 concentration tested, as described in Materials and methods. The linear velocity of untreated sperm approximated sperm speed values previously reported in the mouse (Quill et al., 2003Go). Minor differences were probably related to the different velocity detection methods used. The results obtained with different LY294002 concentrations are summarized in Figure 1. The frequency of immotile sperm (data not shown) did not vary between LY294002 treatments (P > 0.3, calculated by {chi}2 analysis). In contrast, the frequency of circularly moving (in situ) sperm significantly decreased with 5.0 and 10.0 µmol/l, but not 2.0 µmol/, LY294002. In agreement with this result, the 5.0 and 10.0 µmol/l LY294002 treatments elicited a significant increase in mean sperm velocity, with no apparent effect on maximal sperm velocity (Figure 1). It therefore appears that, in the mouse, LY294002 mediates the recruitment of sperm having in situ/low motility to the compartment of those having intermediate/high and progressive motility. This conclusion is fully consistent with previous observations obtained in humans showing that LY294002 increases the velocity of spermatozoa having a low/intermediate motility (Luconi et al., 2001Go, 2004Go).



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Figure 1. Effect of LY294002 on the motility of mouse epididymal sperm. Diagrams represent frequencies of arbitrary sperm motility classes including the velocity of 100–400 individual spermatozoa in the range 2–200 µm/s, determined on 20–25 time-lapse frames of at least nine randomly collected microscopic fields obtained in three independent experiments. See Materials and methods for experimental details. Immotile sperm (velocity <2 µm/s) are not shown. Grey areas indicate the frequency of in situ moving sperm (velocity 2–15 µm/s). White areas indicate the frequency of progressive motile sperm (velocity 16–200 µm/s). The vertical dashed line indicates the mean progressive velocity of control sperm, as a visual reference for LY294002-treated sperm. The mean ± SEM (µm/s) velocities of the progressive motile sperm following the different concentrations of LY294002 are indicated in the respective panels. Differences in the mean progressive velocity following LY294002 treatment with respect to control motile sperm were calculated by ANOVA: 2.0 µmol/l versus control, P > 0.18; 5.0 µmol/l versus control, **P < 0.001; 10.0 µmol/l versus control, *P < 0.05.

 

In human sperm, the stimulatory effect of LY294002 on sperm motility is exerted through an increased phosphorylation of the PKA-anchoring protein AKAP3, which, in turn, recruits PKA at the sperm tail level (Luconi et al., 2004Go, 2005Go). To verify whether a similar mechanism underlies the positive effect of LY294002 on sperm motility in the mouse (Figure 1), we evaluated the level of AKAP3 phosphorylation following sperm exposure to increasing concentrations of the PI3K inhibitor (Figure 2). LY294002 concentrations ranging from 2 to 10 µmol/l induced an increase in the phosphorylation in a 110 kDa protein band (left panel) which was revealed by anti-AKAP3 antibody after stripping and reprobing of the same blot (right panel), suggesting that human and mouse sperm share a common and AKAP3-dependent mechanism(s) of motility enhancement by LY294002. Similarities between the LY294002 effects on mouse and human sperm validated the use of the mouse system to investigate the effect of this drug on preimplantation embryo development.



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Figure 2. Western blot analysis of mouse epididymal sperm lysates treated or not with the indicated concentrations of LY294002. The blot had been first probed with PY20 antibody (left panel), stripped and re-probed with anti AKAP3 antibody (right panel). Human sperm were run as a positive control for AKAP3. Representative of three similar experiments.

 

Effect of LY294002 on fertilizing ability of mouse sperm
The issue of whether sperm treatment with LY294002 affected the fertilizing ability in the mouse was addressed by two different protocols. In the first set of experiments (Figure 3A), epididymal sperm were exposed to increasing LY294002 concentrations during the 30 min capacitation period in vitro. An aliquot of treated sperm was then added to the MII oocyte-containing drop of medium, giving a final sperm concentration of ~4 x 106/ml and a 20-fold dilution of initial LY294002 concentration during sperm capacitation. As an example, when spermatozoa were capacitated in the presence of 20.0 µmol/l LY294002, the final inhibitor concentration during fertilization was 1.0 µmol/l. As for the second protocol (Figure 3B), sperm were capacitated with plain medium and then added to eggs for the 3 h fertilization period in the presence of increasing LY294002 concentrations. Fertilization rates were eventually determined by scoring eggs for the presence of pronuclei at the end of the 3 h incubation. With both protocols, the rate of egg fertilization (assessed by emission of the second polar body and the presence of two expanded pronuclei) did not differ from that obtained in the absence of LY294002, showing that the inhibitor had no effect on egg fertilization per se.Similar results (data not shown) were also obtained in a full series of experiments performed with the same initial and final LY294002 concentrations used previously, but a final sperm concentration of 1 x 104/ml. These findings ruled out the possibility that a high sperm concentration during fertilization somehow masked a putative toxic effect of LY294002 by increasing the absolute number of drug-resistant sperm in the fertilization drop.



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Figure 3. Effect of LY294002 on mouse sperm fertilizing ability. Histograms represent frequencies (mean ± SEM of at least 120–170 embryos obtained in at least three independent experiments) of eggs having both male and female pronuclei after an incubation in the presence of capacitated sperm for 3 h, as described in the text. Results were analysed by {chi}2analysis. (A) LY294002 was administered during sperm capacitation. (B) LY294002 was administered during fertilization. See text for details.

 

Effect of sperm treatment with LY294002 on preimplantation embryo development
To determine whether sperm treatment with LY294002 during capacitation affected the preimplantation development of resulting embryos, epididymal sperm were capacitated in vitro for 30 min in the presence of 0–20 µmol/l LY294002. A sperm aliquot was then added to the MII oocyte-containing drop of fertilization medium (resulting in a 20-fold dilution of the drug, as described above). Following the 3 h incubation, fertilized eggs at the pronuclei stage were carefully washed of surrounding spermatozoa and fertilization medium by sequential transfer through drops of fresh M16 supplemented with 3% BSA under paraffin oil and then cultured in vitro for 4 days. Developing embryos were scored daily for the developmental stage(s) they had attained. Results obtained (Figure 4) showed that sperm treatment with LY294002 before/during fertilization did not significantly affect preimplantation embryo development at all drug concentrations tested, ruling out the possibility that sperm exposure to LY294002 was toxic to preimplantation embryo development.



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Figure 4. Preimplantation development of embryos derived from sperm treated with LY294002 during capacitation. Histograms represent frequencies (mean ± SEM of at least 120–170 embryos obtained in at least three independent experiments) of embryos at the 1-cell (empty bars), 2-cell (large striped bars), 4-cell (narrow striped bars), 8- to 16-cell (grey bars) and blastocyst (full bars) stages of development. Results were analysed by ANOVA. LY294002 concentrations: (A) medium only; (B) 5 µmol/l; (C) 10 µmol/l; (D), 20 µmol/l.

 


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The development and maintenance of sperm motility is one of the most characteristic reproductive functions of the human male. Men characterized by absence of sperm motility (asthenozoospermia) are infertile and the only curative option they have is ICSI in the oocyte. In patients with reduced sperm motility, however, the possibility of ART applications other than ICSI is restricted by the low number of progressively motile sperm recovered by current sperm selection techniques. Although clinical pregnancy rates do not differ between ICSI and IVF, according to a recent multicentre study that evaluated 5-year-old children conceived with those techniques (ESHRE Report, 2005), the odds of a major malformation were almost doubled in the ICSI versus IVF children (Bonduelle et al., 2005Go). These results pointed out the necessity to develop new strategies aimed at increasing sperm motility in vitro and recovering highly motile spermatozoa in large enough concentrations to apply less invasive ARTs, such as IVF.

In the present study, we report that the PI3K inhibitor LY294002, previously shown to increase human sperm motility in vitro (Luconi et al., 2001Go, 2004Go), also has a similar stimulatory effect on mouse epididymal sperm, validating the mouse as a useful model to study the effect of LY294002 on fertilization and embryo preimplantation development in mammals. In this respect, the present experiments have shown that mouse sperm treatment with LY294002 during either in vitro capacitation or fertilization does not affect the sperm fertilizing ability, nor does it alter the preimplantation development of resulting embryos, at least within the drug concentration range tested. Although unlikely in light of the present results, the possibility that sperm treatment with LY294002 affects mouse post-implantation development still remains to be addressed by further experiments.

The finding that LY294002 enhances epididymal sperm motility in the mouse as it does in ejaculated human sperm, as well as in rat and rabbit epididymal sperm (Luconi M and Baldi E., unpublished data), supports the idea that such an effect is a general property of mammalian sperm and that the signalling pathway(s) that mediate such LY29402 effects is(are) already active before ejaculation. In agreement with this conclusion, the present data have shown that LY29402 increased the tyrosine phosphorylation of AKAP3, a fibrous sheath protein involved in sperm capacitation and motility (Vijayaraghavan et al., 1999Go; Carr et al., 2001Go), in both ejaculated human sperm (Luconi et al., 2004Go) and epidydimal mouse sperm. Tyrosine phosphorylation of sperm AKAP3 was evident at all LY294002 concentrations used, with a maximal response between 2 and 5 µmol/l, although 2 µmol/l was not effective on mouse sperm motility. It is possible that AKAP phosphorylation is required not only to enhance sperm motility, but also to mediate other effects of the compound as described recently (Nauc et al., 2004Go; Liguori et al., 2005Go) and thus the AKAP3 phosphorylation induced by 2 µmol/l LY294002 may affect a signalling pathway other than motility.

Overall, these results open up the possibility of using PI3K inhibitors as sperm motility enhancers for veterinarian purposes.

It is well established that the PI3K/AKT pathway is involved in embryonic cell survival and development (Burdon et al., 2002Go). In the light of this, the lack of detrimental effects caused by the presence of LY294002 on the processes of sperm capacitation and fertilization, as well as the apparently normal preimplantation development of embryos derived from LY294002-treated sperm, is intriguing, because it indirectly indicates that PI3K is dispensable during fertilization, even though it is relevant to early embryo development from the one-cell stage (Torcia S and Mangia F, unpublished data). The molecular reason for the dispensability of PI3K during fertilization still remains to be investigated by further study. In any case, we conclude that stimulation of sperm motility by LY294002 during capacitation and/or fertilization does not significantly impair preimplantation embryo development, provided the drug is carefully washed out from sperm before/soon after fertilization. This is further evidence that LY294002 is a candidate as a potentially useful tool for improving the efficiency of IVF performed with low-quality sperm in human and/or domestic animals.

Taken together with the recent finding that LY294002 increases capacitation and phosphorylation of several sperm proteins in ejaculated human sperm (Nauc et al., 2004Go), the present data encourage the design of new drugs based on the molecular structure of LY294002, which may open upnew options for the treatment of human/animal asthenozoospermia in vitro.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was partially supported by grants from Serono International S.A. (Geneva, Switzerland), Istituto Pasteur-Fondazione Cenci Bolognetti (to F.M.) and the Italian Ministry of Education and Research (MIUR-Cofin, to G.F.).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Luconi M, Porazzi I, Ferruzzi P, Marchiani S, Forti G and Baldi E (2005) Tyrosine phosphorylation of the a kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate. Biol Reprod 72,22–32.[Abstract/Free Full Text]

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Submitted on March 21, 2005; resubmitted on June 28, 2005; accepted on July 14, 2005.





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