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
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Abstract |
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Key words: embryo/phosphatidylinositol-3 kinase/sperm motility
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Introduction |
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Materials and methods |
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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 spermMII 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 7080%, depending on egg quality. In vitro fertilized embryos were cultured in drops of Medium 16 (M16) (Whittingham, 1971
) 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).
SDSPAGE and western blot analysis
After different treatments, mouse sperm samples were processed for SDSPAGE for detection of AKAP3 phosphorylation as previously described (Luconi et al., 2004, 2005
). 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 Laemmlis 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% polyacrylamidebisacrylamide gels. After SDSPAGE, 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 -mercaptoethanol) and probed again with the appropriate primary antibody.
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Results |
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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., 2004, 2005
). 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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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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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 020 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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Discussion |
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In the present study, we report that the PI3K inhibitor LY294002, previously shown to increase human sperm motility in vitro (Luconi et al., 2001, 2004
), 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., 1999; Carr et al., 2001
), in both ejaculated human sperm (Luconi et al., 2004
) 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., 2004
; Liguori et al., 2005
) 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., 2002). 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., 2004), 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.
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Acknowledgements |
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References |
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Submitted on March 21, 2005; resubmitted on June 28, 2005; accepted on July 14, 2005.
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