1 National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, Laboratory of Molecular Endocrinology, 48 Vas Constantinou Ave, 11635 Athens, 2 Foundation for Biomedical Research of the Academy of Athens, Center for Basic Research, Laboratory of Biochemistry, 4 Soranou Efesiou, 11527 Athens and 3 IVF and Genetics, 296 Kifisias Ave and 40 Navarinou St, 15232 Athens, Greece
4 To whom correspondence should be addressed. E-mail: csekeris{at}eie.gr
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Abstract |
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Key words:
androgen receptor/estrogen receptor /estrogen receptor
/mitochondrion/sperm
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Introduction |
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Androgens are steroid hormones, necessary for normal male phenotype expression, including the outward development of secondary sex characteristics and the initiation and maintenance of spermatogenesis (McLachlan et al., 2002). Many physiological actions of androgens are mediated by the androgen receptor (AR), whose function is essential in males for proper sexual differentiation, pubertal development and the regulation of normal spermatogenesis. AR activity is regulated by the steroid ligand testosterone and its metabolite dihydrotestosterone, the binding of which on the receptor initiates nuclear translocation and the transcriptional regulatory function of AR (Lindzey et al., 1994
). AR itself also plays an important role in the feedback regulation of testosterone levels. Several studies suggest that high testosterone concentrations inhibit spermatogonial proliferation in models in which spermatogenesis has been damaged by processes such as irradiation (Meistrich and Kanganiemi, 1997
). During meiosis, testosterone stimulates the progression of spermatocytes through this process and inhibits apoptosis (Singh et al., 1995
). The site of action of androgens in spermatogenesis has been extensively studied (McLachlan et al., 2002
). Non-genomic actions mediated by androgens have been described in several cell types, including Sertoli cells. Some of these cells transduce androgen signals using surface receptors that await final characterization, whereas other cells employ the classical AR (Walker et al., 2003
).
The central role of estrogens and androgens in the male reproductive system has therefore focused attention on the presence and distribution of their cognate receptors in male reproductive tissues and cells. ER has been detected in the nuclei of spermatogonia, spermatocytes and early developing spermatids. Elongating spermatids, mature sperm, Sertoli and Leydig cells were negative for ER
, indicating that ER
is likely to be the isoform mediating estrogen effects in human testis (Makinen et al., 2001
). Nevertheless, other studies (Pelletier and El-Alfy, 2000
; Pelletier et al., 2000
) have presented conflicting data: ER
has been localized in the nuclei of Leydig cells, round spermatocytes and spermatids, while ER
has been found in the nuclei of Sertoli cells. Sperm has been increasingly targeted, particularly in respect to the involvement of the sex hormone receptors in sperm metabolism, flagellar activity and motility and in the acrosome reaction. The first research group that provided evidence for the expression of ER by human sperm had reported the localization of ER on the tailpiece (Durkee et al., 1998
). Recent data from immunolocalization experiments have demonstrated the presence of ER
and ER
in human and rat sperm and their differential localization in these cells, with ER
being detected in the midpiece and ER
in the sperm cell tail (Aquila et al., 2004
). It is therefore questionable whether the two ER isoforms have redundant or distinct roles in estrogen signalling in sperm. The expression of AR has been exclusively restricted in spermatogonia, suggesting a direct effect of androgens on germ cells during the stages of pre-spermatogenesis and early spermatogenesis (Zhou et al., 1996
; Arenas et al., 2001
; Weber et al., 2002
). With the onset of chromatin condensation, no AR can be observed in these cells (Holdcraft and Braun, 2004
; Vornberger et al., 1994
).
The presence of steroid and thyroid hormone receptors in mitochondria of mammalian cells has been well documented (Demonacos et al., 1993; Wrutniak et al., 1998
; Monje and Boland, 2001
; Scheller et al., 2000
, 2003
; Koufali et al., 2003
; Psarra et al., 2003
; Cammarata et al., 2004
; Chen et al., 2004a
; Yang et al., 2004
). The possibility of mitochondrial localization of sex steroid hormone receptor not only in somatic cells, but also in sperm, as well as the conflicting findings concerning the expression pattern of ER
, ER
and AR in sperm cells, have motivated us to explore the distribution of these steroid hormone receptors in human sperm.
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Materials and methods |
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Sperm were isolated by centrifugation on a discontinuous Percoll density gradient (90:70:50 v/v) according to the method of Ord et al.(1990), with modifications. After centrifugation at 300 g for 30 min, the 90% Percoll fraction was examined using an optical microscope to ensure that a pure sample of sperm was obtained. The 90% Percoll fraction was then washed twice with protein-free Hams F-10 medium (Gibco). Sperm were pooled and subjected to experimental procedures.
Cells and culture conditions
Human prostate carcinoma LnCap cells, known to be positive for androgen receptor were used as a positive control for AR staining. They were maintained in Roswell Park Memorial Institute medium (Gibco BRL, Germany) supplemented with 10% fetal bovine serum. Cells were grown at 37°C in a humidified atmosphere with 5% CO2 and expanded to 75 cm2 cell culture flasks. For immunostaining, cells were grown on coverslips.
Antibodies
Rabbit polyclonal anti-ER, anti-ER
and anti-AR antibodies were commercially provided by Santa Cruz Biotechnology (CA, USA) (G20 and MC20 recognize an epitope mapping at the C-terminus and hinge region of ER
respectively, H150 and L20 recognize epitopes mapping at the N-terminus and C-terminus of ER
respectively, and H280 and C19 recognize epitopes mapping at the N-terminus and C-terminus of AR respectively). Secondary fluorescein isothiocyanate (FITC)- and horseradish peroxidase-conjugated antibodies were also purchased from Santa Cruz Biotechnology.
Immunostaining
Sperm were placed on coverslips, incubated for 30 min at 37°C with 400 nmol/l MitoTracker Red CMXRos (CMX; Invitrogen CA, USA-Molecular Probes), washed 3 x 5 min with phosphate-buffered saline (PBS), fixed for 7 min in ice-cold methanol and transferred to ice-cold acetone for 2 min. After rinsing in PBS, cells were blocked at room temperature with serum from a non-immunized animal (5% in PBS) for 1 h, to reduce non-specific binding. For immunofluorescence microscopy, specimens were incubated with primary antibodies (1:20 dilution for anti-ER and and anti-ER
and 1:10 dilution for anti-AR antibodiessimilar results were obtained with up to 1:100 dilution for all antibodies) for 2 h in a moist chamber, at room temperature. Following three washing steps with PBS, anti-rabbit FITC-conjugated secondary antibody (Jackson Immunoresearch Europe Cambridge shire, UK), diluted 1:50, was added for 1 h. The specimens were washed 3 x 3 min in PBS and mounted in a 50% glycerol solution.
The specificity of ER, ER
and AR was tested by preincubation of the antibodies with the respective blocking peptides purchased from Santa Cruz Biotechnology. According to the instructions, the antibodies were incubated with a 5-fold (by weight) excess of blocking peptide, for 2 h, at room temperature and were then added to fixed cell preparations. Sperm cells incubated without the primary antibodies were also used as negative controls.
Confocal laser scanning microscopy
Cell specimens were observed with a Nikon confocal laser scan fluorescence-inverted microscope (EZ 2000, Nikon), equipped with two lasers used simultaneously: a helium-neon laser (excitation wavelength at 543 nm) and an argon laser (excitation wavelength at 488 nm). The excitation spectra were separated by a dichroic beam splitter of 475/505 nm, and the emission spectra of the two fluorochromes were separated by a 565 nm dichroic beam splitter. Two detectors were used in parallel and were preceded with a 560 to 620 nm (rhodamine channel) or a 500 to 530 nm (fluorescein channel) narrow-band barrier filter. The partial superposition of the emission spectra of the two fluorochromes was negligible. Specimens were observed through an oil immersion x60/1.4 objective.
Preparation of sperm lysates
Pooled sperm were washed twice in Hams F-10 protein-free medium (centrifugation at 300 g for 10 min) and lysed in lysis buffer (20 mmol/l Tris pH 7.6, 0.5% Triton X-100, 250 mmol/l NaCl, 3 mmol/l EDTA, 3 mmol/l EGTA, 10 µg/ml pefabloc, 2 mmol/l sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mmol/l dithiotreitol). The supernatant was aliquoted and stored at 70°C.
Protein (western blot) analysis
Sperm lysates (810 x106 per western blot lane) were electrophoresed on a 10% sodium dodecyl sulphatepolyacrylamide gel. Reducing agent was added in a 1:10 ratio. Proteins were then transferred to nitrocellulose membranes, which were blocked for 2 h, at room temperature, in 5% non-fat dry milk with Tris-buffered saline (TBS)0.1% Tween 20. The blots were subsequently incubated overnight at 4°C, either with anti-ER, anti-ER
or AR polyclonal antibodies (1:333 dilution in 3% non-fat dry milk with TBS0.1% Tween 20). After incubation, the membranes were washed twice in TBS0.1% Tween 20, for 15 min. Horseradish peroxidase-conjugated secondary antibody (1:1000 dilution in 1% non-fat dry milk with TBS0.1% Tween 20) was then added for 90 min at room temperature, and after washing twice, detection of protein bands was carried out using an enhanced chemiluminescence system (Amersham Biosciences, UK).
The specificity of ER, ER
and AR was tested by preincubation of the antibodies with the respective blocking peptides purchased from Santa Cruz Biotechnology. According to the instructions, the antibodies were incubated with a 5-fold (by weight) excess of blocking peptide for 2 h at room temperature, and were then used for incubation of membranes.
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Results |
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Preincubation of the primary antibodies with blocking peptides before immunolocalization, reduced fluorescence to background levels. As another control for the specificity of the staining procedure, sperm were incubated with CMX, without the use of primary antibody (Figure 2A).
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Immunofluorescence localization of AR
Immunolocalization of androgen receptor in sperm treated as mentioned above, demonstrated an intense staining of the sperm midpiece (Figure 2Bb and e), corresponding to the site of concentration of the mitochondria (Figure 2Ba and d). The merged images of 2Bc and 2Bf show an excellent overlap of the mitochondria-specific dye CMX with the anti-AR staining. In addition to the mitochondrial fluorescence the AR antibody also reacted diffusely in the tail region. Preincubation of the C19 primary antibody with the respective blocking peptide before immunolocalization reduced fluorescence to background levels.
Immunolocalization of AR in LnCap human prostate cancer cells was also performed (Figure 2C). AR was present not only in the cell nucleus (Figure 2Cb), but was also enriched in the sites of mitochondria (Figure 2Cc).
Western blotting of extracts with ER , ER
and AR antibodies
Sperm were lysed and the lysates were subjected to western blotting using ER, ER
and AR antibodies (Figure 3). As shown in Figure 3Aa and Ba, the presence of a 66 kDa band staining with antibody against ER
was observed. A second ER
band with a mol. wt of 45 kDa was also detected (see Discussion). The reaction of antibody against ER
(Figure 3Ab and Bc) with the sperm lysate yielded a 64 kDa band, consistent with the size of ER
. Western blotting with anti-AR antibody demonstrated the presence of a prominent 110 kDa band, corresponding to the AR (Figure 3Ac) and an
90 kDa band, which could either represent an AR isoform or an AR cleavage product (see Discussion).
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Similar results were obtained after submitting intact sperm to western blotting. The specificity of the antibody reactions was verified by preincubation experiments with respective blocking peptides, which abolished the appearance of the protein bands in the three respective blots (Figure 3Ad and Bb and d).
Computer analysis of AR for the presence of mitochondria-targeting peptide signals
Most proteins that target to mitochondria contain an aminoterminal-targeting peptide signal (mTPS; Bauer et al., 2000). The localization of AR in mitochondria led us to search for such an mTPS in this receptor. The primary sequence of human AR (accession number AR P10275) was analysed using a Target P program (http://www.ebs.dk/services/Target p ) (Emanuelsson et al., 2000
). No aminoterminal classical mTPS was found in AR. However, the receptor contains in two regionsone at the aminoterminal part (amino acids 1100) and one at the carboxyterminal part (amino acids 841900)sequences with properties similar to those of the internal mTPS of two proteins, BSC1 and Tim 23p (Foelsch et al., 1996
; Davis et al., 1998
), located at the mitochondrial inner membrane.
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Discussion |
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The seminal role of androgens in the differentiation process of spermatogonia to sperm also led to the search and detection of the respective receptors in germinal cells in various phases of their differentiation, with somewhat conflicting data as to the presence of AR in fully differentiated sperm.
Due to our interest in the role of mitochondria in the context of hormonal regulation of energy production and apoptosis and the relevant demonstration of steroid and thyroid receptors in mitochondria of a variety of cells (Demonacos et al., 1993; Wrutniak et al., 1998
; Scheller et al., 2000
; Monje and Boland, 2001; Psarra et al., 2003
; Koufali et al., 2003
), we proceeded to study the presence and distribution of ER and AR in human sperm and their possible localization and function in the mitochondria of these cells. By applying immunofluorescence confocal microscopy with antibodies specific for ER
and ER
, we demonstrated the presence of both estrogen receptor isoforms in human sperm, differentially distributed within the cells. ER
staining was concentrated in the midpiece region. By labelling the mitochondria with the specific mitochondrial marker CMX, we could demonstrate the dense packaging of the mitochondria in the midpiece and, importantly, the co-localization of ER
with the mitochondria. Whereas ER
was mainly localized in mitochondria, ER
was found in the cytoplasmic tail and in the head region of the sperm, as a discrete, highly fluorescent, compact band. There are some discrepancies between our findings and the results of Aquila et al.(2004)
. According to the latter group, ER
was prevalently localized in the midpiece region, whereas ER
was detected in all of the tail region, with an overlapping distribution of ER
and ER
in the proximal region of the tail. The discrepancies observed, which focus mainly on the ER
localization, could be due to the differences in processing methods, the condition of the sperm samples (i.e. degradation), the use of different fixationpermeabilization methods and the use of a different anti-ER
antibody. As regards the ER
localization, we used the same anti-ER
antibody as Aquila et al. and unlike them we did not detect a strong signal in the tail region, a finding we verified with the use of a second anti-ER
antibody detecting a different ER
epitope. Western blot confirmed the presence of proteins of 66 and 64 kDa, corresponding to ER
and ER
respectively. A second ER
species of 45 kDa was also detected, probably representing the 46 kDa ER
described by Denger et al. (2001)
in human primary osteoblasts and Lambard et al. (2004)
in human sperm, acting as a strong ER
inhibitor.
Using the same immunofluorescence labelling technique, we also detected the presence of AR in sperm. The pattern of labelling was similar to that of ER, with an intense co-localization of AR with mitochondria in the midpiece of the cells. Western blot analysis with an anti-AR antibody revealed the presence of a prominent 110 kDa band, corresponding to the intact AR and a faint 90 kDa protein, which could represent an AR isoform or an AR degradation product, due to proteases present in the sperm. The possibility that such cleavage products represent functionally active receptor species must be explored. We have verified the presence of AR in sperm with the use of two different methods (immunofluorescence and western blot) and two anti-AR antibodies recognizing epitopes mapping at the C-terminus and N-terminus of human AR respectively. Although, to our knowledge, no mitochondrial localization of AR has been yet reported, we were able to detect AR not only in the nucleus (as already known) but also in mitochondria of the LnCap human prostate cancer cell line, a finding implying that the mitochondrial localization of AR is not restricted to sperm, but could be a characteristic of different eukaryotic cell types.
Indeed, the presence of steroid and thyroid receptors in mitochondria of a variety of cell types has been well documented (Demonacos et al., 1993; Scheller et al., 2000
; Koufali et al., 2003
; Psarra et al., 2003
). In respect to ER, recent papers refer to its presence in a variety of cells, most reports pointing to a mitochondrial localization of ER
(Chen et al., 2004a
,b
; Cammarata et al., 2004
; Yang et al., 2004
; Solakidi S et al., unpublished data). The central role of mitochondria in energy production and cell apoptosis and the known effects of steroid and thyroid hormones on these processes (Hughes et al., 1996
; Scheller et al., 2003
) led to the hypothesis (Sekeris, 1990
) that the hormone receptors could be involved in these important regulatory processes by acting directly on the mitochondria, a hypothesis which has found experimental support (Demonacos et al., 1993
, 1995
, 1996
; Wrutniak et al., 1998
; Enriquez et al., 1999
).
The presence of ER and AR in mitochondria of sperm could be related to the energy requirements of these cells. It must be stressed that the path which the released sperm need to travel in order to reach the ovum is quite long and that the sperm cell has to consume enormous quantities of energy to maintain flagellar movement. Nevertheless, the findings of Diez-Sanchez et al.(2003)
demonstrated that human ejaculated sperm were not capable of synthesizing proteins, being biogenetically inactive and unable to modify their capacity for oxidative phosphorylation by de novo synthesis of respiratory complexes once differentiated. As a consequence, when motility is activated after maturation, increasing the need for a high energy supply, a compensatory biogenetic response is not allowed. As demonstrated by Williams and Ford (2001)
, the glycolytic production of ATP is required for sperm motility and hyperactivation. The presence of ER and AR in the mitochondria of sperm may therefore be only indirectly involved in the regulation of their motility.
The localization of ER in the equatorial segment could imply its involvement in the fertilization process, since this segment is supposed to have a crucial role in the fusion of sperm with oocytes (Ramalho-Santos et al., 2002
). In the equatorial segment, proteins were detected, such as equatorin and oscillin, which are closely related to this process: the successful activation of the oocyte, as an initial element of mammalian fertilization, is limitated by the sperm cell via its ability to induce intracellular calcium oscillations. Oscillin may be a key component of the sperm cytosolic activation factor, which seems to be necessary for proper oocyte activation (Montag et al., 1998
).
Luconis group (Luconi et al., 1999, 2002
; Baldi et al., 2000
) in a series of papers hypothesized an ER
isoform in the plasma membrane of sperm, conserving the hormone-binding domain only, through which rapid, non-genomic effects of estradiol on cell motility could be mediated. The possibility that some rapid effects of sex hormones in sperm could be mediated by ER
and AR action on mitochondria should be explored.
The localization of ER and AR in mitochondria necessitates the presence of mitochondrial localization signals in these receptors. No classical aminoterminal amphipathic, basic,
-helical signals have been detected in ER
or in AR. However, in publications reporting the presence of ER
in mitochondria of MCF-7 cells, Chen et al.(2004a,b)
described internal sequences in ER
having characteristics similar to those observed in some mitochondrial inner membrane proteins, targeted to mitochondria by such internal protein signals. As shown in our computer search of AR, similar sequences are present in this receptor and the role of these sequences is now being explored. It should be added that in another receptor of the nuclear receptor family, the glucocorticoid receptor, such sequences have also been detected (A-M.G.Psarra et al., unpublished data).
Our findings suggest that the differential localization of androgen and estrogen receptor isoforms in human sperm reveals distinct roles for these receptors in the physiology of sperm cells and perhaps also in the process of fertilization.
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Acknowledgements |
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Submitted on March 2, 2005; resubmitted on July 8, 2005; accepted on July 12, 2005.
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