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and ERß Are Both Expressed in Human Ejaculated Spermatozoa: Evidence of Their Direct Interaction with Phosphatidylinositol-3-OH Kinase/Akt Pathway
Centro Sanitario (S.Aq., M.G., E.M., S.C.), Department of Cell Biology (A.C., V.R.), Faculty of Pharmacy (D.S., S.An.), University of Calabria 87030 Arcavacata di Rende (Cosenza - Italy)
Address all correspondence and requests for reprints to: Prof. Sebastiano Andò, Faculty of Pharmacy, University of Calabria, Arcavacata di Rende (CS) 87030, Italy. E-mail: sebastiano.ando{at}unical.it. Alternate E-mail: aquisav{at}libero.it.
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Abstract |
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and ERß. In the present study, we have first demonstrated the concomitant expression of ERß and ER in human ejaculated spermatozoa. By RT-PCR and Southern blot, transcripts of both ERs were detected. Western blot analysis showed ER and ERß proteins at the same size as the "classical" ERs. The localization of ER and ERß with the immunocytochemistry shows a differential distribution of the two ER subtypes, the former being prevalently located in the midpiece, but the latter being in the tail. Estradiol has been associated with sperm longevity; however, the mechanism through which estradiol acts in sperm survival was never investigated. Upon estradiol exposure, we observed an enhanced phosphorylation of the proteins involved in the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway like PDK1, Akt, GSK-3, Bcl-2, together with ERK1/2, which was also involved in cell survival signals. Moreover, such phosphorylations were reduced in the presence of ICI 182, 780, addressing the role of estradiol and ERs in sperm survival. For instance we have provided, for the first time, a different interaction of the two ERs with the PI3K/Akt pathway, because ER interacts with the p55 regulatory subunit of PI3K, whereas ERß interacts with Akt1. However, it still remains to be elucidated whether the functional role of each of the ER subtypes in sperm survival signaling is redundant or distinct. |
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and ERß, each encoded by a unique gene, differing in the C-terminal ligand-binding domain and in the N-terminal trans-activation domain (3). Both receptors seem to be expressed in germ cells in various stages of development from spermatogonia to elongated spermatids (Ref. 3 and references therein). ERß expression appears to be the predominant ER in germ cells (Ref. 3 and references therein), whereas ER was found in early meiotic spermatocytes and elongated spermatids only in one study (4) and in human ejaculated spermatozoa (5, 6).
The human and animal phenotypes related to aromatase deficiency and estrogen resistance have been previously detailed (7). For instance, ER knockout (ERKO) mouse was infertile from the onset of puberty (8, 9), whereas P450arom knockout (ArKO) mouse displayed a progressive long-term deterioration of spermatogenesis consisting of abnormalities in postmeiotic early cells associated with an increase in apoptosis that suggests a role for estrogen in germ cell survival (10, 11).
A stimulatory role for estrogen in germ cell differentiation was demonstrated; moreover, during development, germ cells are able to synthesize estrogen, directly modulating via paracrine and/or intracrine actions their own maturation (Ref. 3 and references therein). Recent data from our laboratory have demonstrated that a biologically active P450arom is present in ejaculated spermatozoa (12); in addition, its physiological role is associated either with capacitation or acrosome reaction (13). This raises the possibility that sperm not only would be exposed to estrogens in female genital tract but can provide itself a persisting local source of estrogen that may target on its own receptors modulating sperm extratesticular maturation. The biochemical changes during capacitation induced by estrogens occur rapidly, addressing the nongenomic action of ERs as demonstrated in other cell types (14). On the other hand, the fast ERs responses, instead to their classic genomic action, represent the exclusive modality of ERs action in spermatozoa because they are considered transcriptionally inactive. However, the mechanism by which ERs mediate the rapid effects of estrogen is still not well understood. The identification of ERß has indicated that the cellular responses to estrogen are far more complex (15). It was suggested that the two receptors may play redundant roles in estrogen signaling. On the other hand, this appears questionable, on the basis of localization studies that have revealed distinct expression patterns for each receptor.
In the present study, we demonstrated that both ER and ERß are expressed in human ejaculated spermatozoa. Focusing on the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway involved in cell survival, we have evidenced, together with its up-regulation upon estradiol exposure, that ER coprecipitates with the p55 regulatory subunit of PI3K, whereas ERß coprecipitates with the downstream protein Akt1. This makes it extremely intriguing to investigate a potential distinct role of each ER in controlling sperm survival.
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PMN cell isolation medium was from BIOSPA (Milan, Italy). Total RNA Isolation System kit, enzymes, buffers, nucleotides, and 100-bp ladder used for RT-PCR were purchased from Promega (Milan, Italy). Moloney murine leukemia virus (M-MLV) was from Life Technologies, Inc. - Life Technologies Italia (Milan, Italy). Oligonucleotide primers were made by Invitrogen (Milan, Italy). DMEM-F12 medium, BSA protein standard, activated charcoal, Laemmli sample buffer, prestained molecular weight markers, Percoll (colloidal PVP-coated silica for cell separation), sodium bicarbonate, sodium lactate, sodium pyruvate, estradiol (E2), dimethylsulfoxide, IgG Texas-red conjugated, antirabbit IgG fluorescein isothiocyanate (FITC) conjugated, Earle’s balanced salt solution, and all other chemicals were purchased from Sigma Chemical (Milan, Italy). Acrylamide bisacrylamide was from Labtek Eurobio (Milan, Italy). Triton X-100, Eosin Y was from Farmitalia Carlo Erba (Milan, Italy). Enhanced chemiluminescence (ECL) Plus Western blotting detection system, Hybond ECL, [
-32P]ATP, and HEPES sodium salt were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). ICI 182, 780 (ICI) was purchased from Zeneca Pharmaceuticals (Cheshire, UK). Monoclonal mouse antibody to human ER (F-10), rabbit polyclonal antibody to human ERß (H-150), mouse anti-p85 regulatory subunit monoclonal antibody, goat policlonal actin (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), peroxidase-coupled antirabbit, antigoat, antimouse IgG, and protein A/G-agarose plus were from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit policlonal antibody to human ERß was from Upstate (Lake Placid,). Rabbit anti-p-ERK (p-ERK1/2) policlonal antibody, rabbit anti-p-Bcl-2 antibody, rabbit anti-p-Akt1/Akt2/Akt3 S473 or anti-p-Akt1/Akt2/Akt3 T308 antibodies, rabbit anti-p-PDK1 antibody, rabbit anti-p-GSK-3 antibody, and mouse anti-ERK1/2 were from Cell Signaling (Milan, Italy). Nylon membranes were provided by Roche diagnostics Corporation (Indianapolis, IN). Thin-layer chromatography aluminum sheets were from MERK (Milan, Italy).
Semen samples and spermatozoa preparations
Semen specimens from normozoospermic men were obtained by masturbation (approved by the University of Virginia Human Investigation Committee) after 3 d of sexual abstinence. The samples were produced into sterile containers and left for at least 30 min to completely liquefy before being processed. Sperm samples with normal parameters of semen volume, sperm count, motility, vitality, and morphology, according to the WHO Laboratory Manual (16), were pooled and included in this study. Ejaculates with observed spermatozoa agglutination or abnormal viscosity were discarded. Spermatozoa preparations were performed as previously described (12).
RNA isolation
Total RNA was isolated from human ejaculated spermatozoa using a Total RNA Isolation System kit as described by the manufacturer. The purity and quantity of the RNA was assessed spectroscopically before carrying out the analytical procedures. All RNA preparations were evaluated by denaturating formaldehyde-agarose gel electrophoresis.
RT-PCR
RNA was amplified by the technique of RT-PCR. Before RT-PCR, RNA was incubated with ribonuclease-free deoxyribonuclease (Dnase) I in single-strength reaction buffer at 37 C for 15 min. This was followed by heat inactivation of Dnase I at 65 C for 10 min. Five micrograms of Dnase-treated RNA samples were reverse transcribed by 200 IU M-MLV reverse transcriptase in a reaction vol of 20 µl (0.4 µg oligo-dT, 0.5 mM deoxy-NTP and 24 IU Rnasin) for 30 min at 37 C, followed by heat denaturation for 5 min at 95 C. PCR amplification of cDNA was performed with 2 U of Taq DNA polymerase, 50 pmol primer pair for ER (forward, 5'-GTG TAC AAC TAC CCC GAGG-3'; reverse, 5'-CAG ATT CAT CAT GCG GAA CCG AGATG-3') or 50 pmol primer pair for ERß (forward, 5'-CCA TGA TGA TGT CCC TGA CCA-3'; reverse, 5'-GCC CTC TTT GCT TTT ACT GTCC-3') in 10 mM Tris-HCL (pH 9.0) containing 0.1% Triton X-100, 50 mM KCl, 1.5 mM MgCl2, and 0.25 mM of each deoxy-NTP. The reactions were carried out for 35 cycles, with each consisting of denaturation for 1 min at 95 C, annealing for 1 min at 59 C, and extension for 2 min at 72 C. Standard DNA marker (100-bp DNA ladder) was also run to determine the size of amplified products. To check for the presence of DNA contamination, a RT-PCR was performed without M-MLV reverse transcriptase (negative control). In each reaction, HEG0 (containing human ER gene) and pCMV5-hERß (containing human ERß gene) served as positive control for ER and ERß, respectively.
Southern blotting
The identity of the PCR-amplified cDNA fragment of ER and ERß transcripts from human spermatozoa was verified using Southern hybridization. The PCR-amplified products were subjected to electrophoresis in 2% agarose gels and transferred on nylon membranes with the use of a capillary method and lasted for 16 h. The DNA, covalently linked to the nylon membrane by exposure to UV light (254 nm) at 1.5 J/cm2, were prehybridized for 4 h at room temperature in 6x saline sodium citrate (SSC). A total of 1–2 ng cDNA probes (5'-GTGCAATGACTATG-CTTCAGGCTACCAT-3' for ER and 5'-TCGAGAGTTAAAACTCCAACACAAAGAATA-3' for ERß) were labeled with [32P] ATP using polynucleotide kinase and were added to a second solution identical to the prehybridization solution. The hybridization was carried out overnight at room temperature. The membranes were washed first for 45 min at 50 C with 2x SSC containing 0.1% sodium dodecyl sulfate (SDS), then with 1x SSC, and finally with 0.1x SSC for 30 min at the same temperature. Washed blots were exposed to Kodak XAR-2 film (Sigma, Milan, Italy) with intensifying screens.
Western blot analysis
Sperm samples were washed twice with Earle’s balanced salt solution (uncapacitating medium), incubated with the various compounds as indicated in Results, and then centrifuged for 5 min at 5000 x g. The pellet was resuspended in lysis buffer as previously described (13). Briefly, the pellet was resuspended in lysis buffer (62.5 mmol/liter Tris-HCl, pH 6.8; 150 mM NaCl; 2% SDS; 1% Triton X100; 10% glycerol; 1 mM phenylmethylsulfonylfluoride; 10 µg/ml leupeptin; 10 µg/ml aprotinin; 2 µg/ml pepstatin). Lysates were quantified using Bradford protein assay reagent (17). Equal amounts of protein (20 µg) were boiled for 5 min, separated under denaturing conditions, by SDS-PAGE on 10% polyacrylamide Tris-glycine gels, and electroblotted to nitrocellulose membrane. Nonspecific sites were blocked with 5% nonfat dry milk in 0.2% Tween-20 in Tris-buffered saline (TBS-T) for 1 h at room temperature and then probed with an appropriate dilution of the various antibodies as indicated in the figure legends. After extensive washings (three times for 15 min each time in TBS-T), the secondary antirabbit or antimouse horseradish peroxidase-conjugated antibody was added for 1 h at 22 C. Blots were again washed three times for 15 min in TBS-T, and the bound of secondary antibody was located with the ECL Plus Western blotting detection system according to the manufacturer’s instructions. Each membrane was exposed to the film for 2 min. As internal control, all membranes in which the phosphorylation levels were determined were subsequently stripped (glycine, 0.2 M, pH 2.6, for 30 min at room temperature) of the first antibody and reprobed with the antibody recognizing the nonphosphorylated form of the proteins or with actin.
Immunoprecipitation of spermatozoa proteins
Spermatozoa were washed twice with unsupplemented Earle’s medium and were incubated in the unsupplemented Earle’s medium for 1 h at 37 C and 5% CO2, without (control) or in the presence of 10 nM E2. Besides, some samples were washed and incubated in capacitating medium (Earle’s balanced salt solution medium supplemented with 266 mg/100 ml CaCl2, 600 mg/100 ml BSA, 3 mg/100 ml sodium pyruvate, 360 µl/100 ml sodium lactate, and 200 mg/100 ml sodium bicarbonate). To avoid aspecific binding, the sperm lysates were incubated for 2 h with protein A/G-agarose beads at 4 C and centrifuged at 12,000 x g for 5 min. The supernatants (each containing 500 µg total protein) were then incubated overnight with 10 µl anti-ER or 10 µl anti-ERß or 5 µl anti-p-Akt1/2/3 and 500 µl HNTG (IP) buffer (50 mM HEPES, pH 7.4; 50 mM NaCl; 0.1% Triton X-100; 10% glycerol; 1 mM phenylmethylsulfonylfluoride; 10 µg/ml leupeptin; 10 µg/ml aprotinin; 2 µg/ml pepstatin) for each. Immune complexes were recovered by incubation with protein A/G-agarose. The beads containing bound proteins were washed three times by centrifugation in IP buffer, then denaturated by boiling in Laemmli sample buffer and analyzed by Western blot to identify the coprecipitating effector proteins. Immunoprecipitation with protein A/G alone was used as negative control. Membranes were stripped of bound antibodies by incubation in glycine (0.2 M, pH 2.6) for 30 min at room temperature. Before reprobing with different primary antibodies, stripped membranes were washed extensively in TBS-T and placed in blocking buffer (TBS-T containing 5% milk) overnight.
Immunofluorescence assay
Sperm cells, recovered from Percoll gradient, were rinsed three times with 0.5 mM Tris-HCl buffer (pH 7.5) and were allowed to settle onto slides in a humid chamber. The overlying solution was carefully pipetted off and replaced by absolute methanol for 7 min at -20 C. After methanol removal, sperm cells were washed in TBS, containing 0.1% Triton X-100, and were treated for immunocytochemistry. ER staining was carried out, after blocking with normal horse serum (10%), using a mouse antihuman ER IgG as primary antibody and a Texas-red-conjugated antimouse IgG (1:50) as secondary antibody. ERß immunocytochemical staining was performed, after blocking with normal goat serum (10%), using a rabbit antihuman ERß (1:100) followed by an FITC-conjugated antirabbit IgG (1:50). Sperm cells incubated without the primary antibodies were used as the negative controls. The slides were examined under a fluorescence microscope (Olympus BX41, Milan, Italy) with a suitable filter for FITC and Texas-red, scoring a minimum of 200 spermatozoa per slide.
ER-associated PI-3K activity
Purified spermatozoa were washed in Earle’s balanced salt solution (uncapacitating medium) and centrifuged at 800 x g for 20 min. Sperm were resuspended in the same uncapacitating medium and in different tubes containing no E2 (control) or 100 nM E2. Some samples were resuspended in capacitating medium. Some samples were treated with ICI (100 nM) or LY294002 (LY) (10 µM alone or with 100 nM E2) after a preincubation of 30 min. The negative control was performed using a sperm lysate, where p110 catalyzing subunit of PI3K was previously removed by preincubation with the respective antibody (1 h at room temperature) and subsequently immunoprecipitated with protein A/G-agarose. As a positive control, MCF-7 samples were treated with 100 nM insulin for 10 min before lysis and immunoprecipitated with anti-IRS-1 from 500 µg of cell lysates. ER was precipitated from 500 µg of sperm lysates. The immunoprecipitates were washed once with cold PBS, twice with 0.5 M LiCl, 0.1 M Tris (pH 7.4), and finally with 10 mM Tris, 100 mM NaCl, 1 mM EDTA. The presence of PI3K activity in immunoprecipitates was determined by incubating the beads with reaction buffer containing 10 mM HEPES (pH 7.4), 10 mM MgCl2, 50 µM ATP, 20 µCi [-32P] ATP, and 10 µg L--phosphatidylinositol-4,5-bis phosphate (PI-4,5-P2) for 20 min at 37 C. The reactions were stopped by adding 100 µl of 1 M HCl. Phospholipids were extracted with 200 µl CHCl3/methanol. For extraction of lipids, 200 µl chloroform:methanol (1:1, vol/vol) were added to the samples and vortexed for 20 sec. Phase separation was facilitated by centrifugation at 5000 rpm for 2 min in a tabletop centrifuge. The upper phase was removed, and the lower chloroform phase was washed once more with clear upper phase. The washed chloroform phase was dried under a stream of nitrogen gas and redissolved in 30 µl chloroform. The labeled products of the kinase reaction, the PI phosphates, then were spotted onto trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid–treated silica gel 60 thin-layer chromatography plates. Radioactive spots were visualized by autoradiography.
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Results |
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To determine whether mRNAs for both ERs are present in human ejaculated spermatozoa, RNA isolated from pooled purified sample of normal men was subjected to reverse PCR and then to Southern blot analysis. The products were not a result of any contamination in semen samples because the RNA was extracted from a pooled sperm population selected by Percoll procedure. Moreover, sperm samples were checked under x100 magnification under a bright-field light microscope to rule out the possibility of any contamination with other cells. Furthermore, the samples were also subjected to RT-PCR using primers specific for Myelo Pox to rule out the possibility of granulocyte contamination, and no product was detected for Myelo Pox in sperm RNA (12). The RT-PCR products for ER and ERß in sperm were also not a result of any DNA contamination because the RNA samples were subjected to Dnase treatment before RT-PCR. Therefore, sequences of the two ERs were deduced from the cDNA sequence of the human conventional ER and ERß. Further, we did not obtain any product when RT was omitted from the amplification reaction carried out in the presence of Taq polimerase alone. RT-PCR amplification of ER and ERß in human ejaculated spermatozoa revealed the expected PCR product size of 1170 bp for ER and of 692 bp ERß. To verify the identity of the amplified products, we performed Southern blot analysis. The results depicted in Fig. 1
show the hybridized bands (S) with similar mobility in the positive controls (+).
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and ERß
To demonstrate the presence of the two ERs proteins, we performed Western blot analysis. We observed that human ejaculated spermatozoa showed the expression of both ER and ERß as a single band corresponding to the molecular mass values of 67 kDa for ER (Fig. 2A) and of 55 kDa for ERß (Fig. 2B) noted in other tissues. ER was detected with a monoclonal ER antibody (epitope mapping at the carboxy terminus of ER of human origin), whereas ERß was detected with two different polyclonal antibodies raised against the ammino terminus of ERß, tested separately. The negative controls (lane 2) were performed using a sperm lysate, where ER and ERß were previously removed by preincubation with the respective antibodies (1 h at room temperature) and subsequently immunoprecipitated with protein A/G-agarose. As positive controls, MCF-7 (breast cancer cell line) was used for ER (lane 1), whereas LnCap (prostate cancer cell line) was used for ERß (lane 1).
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and ERß in the human spermatozoa
To investigate the cellular localization of the two ERs, we did perform immunofluorescence assay. Positive stainings for both ER proteins were observed (Fig. 3). Furthermore, a different localization of the two ERs was detected in sperm cells. In fact, ER was prevalently localized in the midpiece region (A1), according to a previous report (5), whereas ERß was detected in all the tail region (B1), with an overlapping distribution of ER and ERß in the proximal region of the tail. No immunoreaction was detected in the negative controls (A2, B2), thus demonstrating the immunostaining specificity.
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To determine the potential role of estrogen/ERs in sperm survival signaling, E2 was added to spermatozoa at the increasing concentrations of 10 nM, 100 nM, and 1 µM for 30 min. To test whether the effects of E2 were mediated by ERs, ICI was added at a final concentration of 100 nM alone or with E2 (100 nM) after a preincubation of 30 min. An increase on the activation of PDK-1, Akt S473, Akt T308, GSK-3, and Bcl-2 was observed in sperm lysates in a dose-dependent manner, whereas ICI reduces E2-induced activation (Fig. 4A). Because ERK1/2 (18) also play an important role in survival signaling, we evaluated whether they were activated by E2 in human ejaculated spermatozoa. E2 induced the activation of the kinases in a dose-dependent manner, whereas ICI is able to reduce this effect, suggesting an involvement of ERs (Fig. 4B). A total of 10 µM wortmannin or 10 µM LY reduced estrogen-induced Akt S473 and Akt T308 phosphorylations, suggesting how this occurs through a PI3K activation (Fig. 4C).
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coprecipitates with the p55 regulatory subunit of PI3K
Recent studies demonstrated that ER binds to the p85 regulatory subunit of PI3K in endotelial cells (19, 20), so we asked whether the association would occur in human ejaculated spermatozoa. As shown in Fig. 5A, ER constitutively associated with the p55 regulatory subunit of PI3K. Our antibody anti-p85 regulatory subunit of PI3K recognized in spermatozoa a single band of 55 kDa; whereas in other cellular types (i.e. TM4 and MCF-7), it recognized both 85-kDa and 55-kDa bands (Fig. 5A). This result fits well with previous findings reporting the 55 kDa as the predominant regulatory subunit of PI3K in human testis (21). As can be noted, the interaction is specific for the subtype of the ER, because we were unable to detect any association between ERß and the p55 regulatory subunit of PI3K in any experimental condition. The ER/p55 complex exhibits an intrinsic PI3K activity that is enhanced in the presence of E2, and it is reduced by ICI as well as by LY (Fig. 5B).
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To further investigate the effects of the two ERs in the PI3K pathway, sperm lysates were immunoprecipitated with anti-ER or two different antibodies raised against ERß and detected with anti-Akt1 or anti-pAkt1/2/3Ser or anti-Akt1Threo or vice versa (Fig. 6A). As shown, the interaction is specific for the subtype ß of the ER, because we were unable to detect association between ER and Akt in our experimental conditions. Besides, ERß constitutively associated with Akt1 or p-Akt1/Akt2/Akt3 S473 or p-Akt1/Akt2/Akt3 T308 or vice versa, and this interaction was unaffected by 10 nM E2 as well as by 100 nM ICI or 10 µM LY (Fig. 6B). We were unable to assign Akt phosphorylation to different Akt isoforms, because the commercially available antibodies recognize sites analogous to S473 and T308 in Akt1, Akt2, and Akt3.
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Discussion |
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and ERß can heterodimerize in vitro (23) have complicated the analysis of the molecular mechanisms of estrogen action. ER expression has already been reported in human ejaculated spermatozoa (5), whereas the concomitant presence of ERs subtype has never been investigated. In the present study, we have first demonstrated the presence of both ER and ERß in human ejaculated spermatozoa at different levels: mRNA, protein content, and immunolocalization.
The presence and the significance of several mRNAs shown in mammalian spermatozoa are currently under investigation (24). New findings suggest that some of these transcripts code for proteins essential in early embryo development (25). Expression of ERß protein in the male gamete is a novel finding. In Western blot analysis, ER and ERß were recognized at the same sizes as reported for the human native ER and ERß. It is now quite clear that ERs, in addition to their classic genomic action, also regulate cellular processes through their nongenomic mechanism (26). For instance, we demonstrated how the rapid effect of either estrogens or aromatizable steroids in ejaculated spermatozoa may trigger both capacitation and acrosome reaction (13). Because here we provide evidence that estrogens may also modulate an important pathway related to survival in different cell types, it remains to be clarified the specific role of each receptor in mediating the sperm survival.
To date, most of the localization studies for both ERs have been performed in rodents (27, 28). In human testis, conflicting data are present with regard to the distribution of the different ER types; both receptors are expressed in germ cells at various stages of development from spermatogonia to elongated spermatids (29, 30). By using specific antibodies to the ER subtypes, we have immunolocalized ER prevalently in the midpiece region as previously demonstrated (5), whereas ERß is uniformly distributed along the tail. An overlap of the two ERs occurs in the proximal region of the tail. On the basis of these observations, it is reasonable to presume how each receptor subtype exerts different functions potentially linked to its specific cellular localization. Indeed, the highly polarized structure and function of spermatozoa do compartmentalize specific metabolic and signaling pathways to regions where they are needed.
Recent data show how estradiol acts as a germ cell survival factor in the human testis (4), even though its mechanism of action remains to be elucidated. We have explored the most important pathways involved in cell survival and previously investigated in other cell types, PI3K/Akt (31) and ERK1/2 (32, 33). In various cell types, estradiol stimulates the cascade that activates the ERK1/2 to inhibit apoptosis (18). This kinase is recognized to mediate cell survival in response to a variety of growth factors targeting a myriad of cell types and has also been proposed to act as a survival protein (18). In human ejaculated spermatozoa, both PI3K/Akt and ERK1/2 pathways appear activated by E2, and ICI reduces E2-induced activation of some protein, addressing the involvement of estradiol and ERs in these events. In the same vein, Bcl-2 (34), a key protein in survival signaling, phosphorylated at Serine 70, the physiologically relevant phosphorylation site, necessary for its full and potent antiapoptotic function, is enhanced upon E2 exposure and inhibited in the presence of ICI. A recent described nongenomic estrogen-signaling pathway exhibits the direct interaction of ER with the p85 regulatory subunit of PI3K (20, 21). So, we questioned whether the two ERs were able to interact with the PI3K/Akt pathway in human ejaculated spermatozoa. It is worth noting that ER coprecipitates with the p55 kDa regulatory subunit of PI3K, reported to be the prevalent regulatory subunit expressed in the testis (21), whereas ERß coprecipitates with Akt1. The complex ER/p55 contains an intrinsic PI3K activity enhanced in the presence of E2. The fact that the above described pathway, upon E2 exposure, is inhibited in a similar extent in the presence of either ICI or the specific PI3K inhibitor led us to postulate that ER per se enhances PI3K activity and consequently also the activity of the downstream effector proteins: PDK1, Akt, and GSK-3. Further, we observed that in the presence of ICI, the ERK1/2, PDK1, and Akt phosphorylations are somehow activated. This unexpected effect could be argued by the fact that the ICI-induced conformational changes of ER able to abrogate ER transcriptional activity do not necessary interfere in the ER nongenomic signaling in the absence of its natural ligand.
Even though Akt phosphorylation either in S473 or in T308 residues appears to be not influenced by E2 in ERß coprecipitates, it appears clearly up-regulated upon E2 exposure in cell lysate. Such apparent discrepancy could be due to the fact that the Akt fraction, much more phosphorylated upon E2 exposure, may not be necessarily involved in the coimmunoprecipitation with ERß, or that the conformational changes induced by coprecipitation make the phosphorylated Akt not discriminated by our antibody. The selective coimmunoprecipitation of ER and of ERß with the p55 regulatory subunit of PI3K and Akt, respectively, discloses a potential separate action of the two ERs on the same pathway. At present, we are unable to distinguish whether the functional role of each ER subtype in sperm survival signaling is redundant or distinct.
Normal male fertility relies on normal spermatogenesis, and the importance of ERs and aromatase in the process was shown at all stages of testicular development, both in somatic cells (35) and in the germ cells (Ref. 3 and references therein). A specific role of estrogen in maintaining sperm fertilizing capability (36, 37, 38) appears to be questioned by a recent study demonstrating that mice germ cells do not require ER (39) for their terminal morphofunctional differentiation when implanted in normal seminiferous tubules of a wild-type recipient. However, in these circumstances, we should consider that the production and secretion of many proteins/factors by Sertoli cells, involved in germ cells development, may overcome the lack of ER, adapting themselves to the changed needs of transplanted germ cells. Besides, the potential action of estrogens in germ cells via nonclassical receptors cannot be ruled out.
On the other hand, several findings have demonstrated how estradiol may influence sperm fertilizing capability (36, 37, 38) as well as sperm survival (Ref. 5 and references therein). However, the molecular mechanisms related to these events did remain to be disclosed. In the present study, we provided evidence that estrogens in ejaculated spermatozoa activate the PI3K/Akt pathway: ER and ERß seem to influence this pathway at different levels and may co-work in controlling sperm survival.
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Acknowledgments |
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Footnotes |
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S.A. and D.S. contributed equally to this work.
Abbreviations: Dnase, Deoxyribonuclease; E2, estradiol; ER, estrogen receptor; FITC, fluorescein isothiocyanate; ICI, ICI 182, 780; KO, knockout; LY, LY294002; M-MLV, Moloney murine leukemia virus; PI3K, phosphatidylinositol-3-OH kinase; SDS, sodium dodecyl sulfate; SSC, saline sodium citrate; TBS-T, Tween-20 in Tris-buffered saline.
Received September 24, 2003.
Accepted November 26, 2003.
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References |
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has a functional role in the mouse rete testis and efferent ductules. Biol Reprod 63:1873–1880(ER) via interaction between ER and PI3K. Cancer Res 61:5985–5991 in vivo and in vitro. Biochem Biophys Res Commun 243:122–126[CrossRef][Medline]
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and ß in human testis. Mol Hum Reprod 7:497–503 and ß in the human reproductive organs. J Clin Endocrinol Metab 85:4835–4840 is required by the supporting somatic cells for spermatogenesis. Mol Cell Endocrinol 178:57–63[CrossRef][Medline]
for development or function. Endocrinology 141:1273–1276This article has been cited by other articles:
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 E. Agirregoitia, A. Valdivia, A. Carracedo, L. Casis, J. Gil, N. Subiran, C. Ochoa, and J. Irazusta Expression and Localization of {delta}-, {kappa}-, and {micro}-Opioid Receptors in Human Spermatozoa and Implications for Sperm Motility J. Clin. Endocrinol. Metab., December 1, 2006; 91(12): 4969 - 4975. [Abstract] [Full Text] [PDF]
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 R. K. Naz and R. Sellamuthu Receptors in Spermatozoa: Are They Real? J Androl, September 1, 2006; 27(5): 627 - 636. [Full Text] [PDF]
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Y. S. Devi, K. Sarda, B. Stephen, P. Nagarajan, and S. S. Majumdar Follicle-Stimulating Hormone-Independent Functions of Primate Sertoli Cells: Potential Implications in the Diagnosis and Management of Male Infertility J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 1062 - 1068. [Abstract] [Full Text] [PDF]
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S. Solakidi, A-M.G. Psarra, S. Nikolaropoulos, and C.E. Sekeris Estrogen receptors {alpha} and {beta} (ER{alpha} and ER{beta}) and androgen receptor (AR) in human sperm: localization of ER{beta} and AR in mitochondria of the midpiece Hum. Reprod., December 1, 2005; 20(12): 3481 - 3487. [Abstract] [Full Text] [PDF]
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H. Schuel and L. J. Burkman A Tale of Two Cells: Endocannabinoid-Signaling Regulates Functions of Neurons and Sperm Biol Reprod, December 1, 2005; 73(6): 1078 - 1086. [Abstract] [Full Text] [PDF]
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S. Aquila, M. Gentile, E. Middea, S. Catalano, C. Morelli, V. Pezzi, and S. Ando Leptin Secretion by Human Ejaculated Spermatozoa J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4753 - 4761. [Abstract] [Full Text] [PDF]
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 L. Liguori, E. de Lamirande, A. Minelli, and C. Gagnon Various protein kinases regulate human sperm acrosome reaction and the associated phosphorylation of Tyr residues and of the Thr-Glu-Tyr motif Mol. Hum. Reprod., March 1, 2005; 11(3): 211 - 221. [Abstract] [Full Text] [PDF]
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 S. Aquila, M. Gentile, E. Middea, S. Catalano, and S. Ando Autocrine Regulation of Insulin Secretion in Human Ejaculated Spermatozoa Endocrinology, February 1, 2005; 146(2): 552 - 557. [Abstract] [Full Text] [PDF]
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Z. Huang, P. R. Somanath, R. Chakrabarti, E. M. Eddy, and S. Vijayaraghavan Changes in Intracellular Distribution and Activity of Protein Phosphatase PP1{gamma}2 and Its Regulating Proteins in Spermatozoa Lacking AKAP4 Biol Reprod, February 1, 2005; 72(2): 384 - 392. [Abstract] [Full Text] [PDF]
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 L. Moro, S. Reineri, D. Piranda, D. Pietrapiana, P. Lova, A. Bertoni, A. Graziani, P. Defilippi, I. Canobbio, M. Torti, et al. Nongenomic effects of 17{beta}-estradiol in human platelets: potentiation of thrombin-induced aggregation through estrogen receptor {beta} and Src kinase Blood, January 1, 2005; 105(1): 115 - 121. [Abstract] [Full Text] [PDF]
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