Molecular Human Reproduction, Vol. 9, No. 3, 117-124,
March 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Expression of aromatase in human ejaculated spermatozoa: a putative marker of motility
Submitted on January 23, 2002; resubmitted on September 10, 2002. accepted on November 26, 2002
1 UPRES EA 2608—USC INRA, University-IRBA and 2 Department of Genetic and Reproduction, CHRU Clemenceau, Caen 14032, France
3 To whom correspondence should be addressed at: UPRES EA 2608—USC INRA, Université-IRBA, Esplanade de la Paix, 14032 Caen Cedex, France. e-mail: carreau{at}ibba.unicaen.fr
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ABSTRACT |
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Cytochrome P450 aromatase (P450arom) is a key enzyme responsible for the irreversible transformation of androgens into estrogens. In the present study, we have analysed the ability of human ejaculated spermatozoa to produce estrogens and for that purpose we have looked for the expression of specific aromatase transcript and protein. We have confirmed the presence of P450arom transcript in all normospermic purified samples by nested PCR. The sequence of PCR products from purified spermatozoa shares 98% identity with published human P450arom sequence. Using a semi-quantitative approach, we have observed in immotile sperm a significant decrease (28%) of the aromatase/glyceraldehyde-3-phosphate dehydrogenase ratio compared with the motile sperm fraction. On Western blot with a monoclonal antibody directed against aromatase, we have detected two bands (53 and 49 kDa) in microsome preparations from purified spermatozoa. In total protein extracts of purified spermatozoa (with and without cytoplasmic droplets), we have only found the aromatase as a 49 kDa band with a stronger intensity when cytoplasmic droplets are present. Moreover, the band seems to be weaker in immotile spermatozoa (with and without cytoplasmic droplets). Our data demonstrate the expression of aromatase both in terms of mRNA and protein in each sample of human purified spermatozoa and in addition, our results suggest that aromatase could be concerned with the acquisition of sperm motility.
Key words: aromatase/human spermatozoa/motility/mRNA/protein
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Introduction |
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The biosynthesis of estrogens from androgens is catalysed by a microsomal enzymatic complex named aromatase which is composed of a specific cytochrome P450 aromatase (P450arom) and a ubiquitous non-specific flavoprotein, the NADPH-cytochrome P450 reductase. The localization of P450arom within mammalian testicular cells has been a subject of interest and controversy during the last decades (for a review see Carreau et al., 1999). In addition, in meotic and post-meiotic germ cells of mouse (Nitta et al., 1993) and rat (Janulis et al., 1998; Levallet et al., 1998), a biologically active aromatase has been described. In the rat, the amount of P450arom mRNA decreases according to the stage of germ cell maturation (Janulis et al., 1998; Levallet et al., 1998). Conversely, aromatase activity is increased in sperm cells (Janulis et al., 1998; Levallet et al., 1998), then decreases during the epididymal transit (Janulis et al., 1998). Moreover, aromatase-deficient mice (ArKO mice) are infertile due to an impairment of spermiogenesis associated with a decrease in sperm motility and an inability to fertilize oocytes (Robertson et al., 1999, 2001).
In humans, Leydig cells have for a long time been considered a main source of estrogens (Payne et al., 1976; Inkster et al., 1995). In in-vitro studies, we have shown that both Leydig cells and Sertoli cells produce estrogens, and that Sertoli cell aromatase activity is under germ cell control (for a review see Carreau, 1996). It has also been claimed that spermatozoa are able to transform pregnenolone into androgens which would in turn be metabolized into estrogens (Chew et al., 1993; Gunasegaram et al., 1995). Durkee et al. (1998) have also demonstrated the existence of estrogen receptor (ER) in human sperm cells and in addition, it has been shown that the sperm membrane contains an ER-related protein able to bind steroids (for a review see Luconi et al., 2002).
Some cases of estrogen deficiency have been published in men. An inactivating mutation of the ER
gene has been reported by Smith et al. (1994): the number of spermatozoa was in the normal range although the viability was diminished. Five cases of estrogen deficiency due to an inactivating mutation of the CYP 19 gene have been described (Morishima et al., 1995; Carani et al., 1997; Deladoey et al., 1999; Murata et al., 2001; Kottler et al., 2002); the analysis of spermatic parameters in two patients showed decreased motility (Carani et al., 1997; Kottler et al., 2002).
Therefore, taking into account the possible role of estrogens in the male reproductive tract (Hess et al., 1997), we have looked for the expression of aromatase in human ejaculated spermatozoa. We then hypothesized that aromatase could be used as a marker of sperm quality (particularly in the acquisition of motility) and for that purpose we have analysed aromatase mRNA transcript and protein in motile and immotile spermatozoa from healthy donors.
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Materials and methods |
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Human sample preparation
Sperm samples (n = 37; mean age: 32.9 ± 5.1 years) were obtained from healthy donors by masturbation after 3 days of sexual abstinence and allowed to liquefy for 30–60 min at room temperature before processing. The selected specimens had normal semen parameters according to the WHO guidelines (World Health Organization, 1999). Infectious sperm and samples with >1x106 round cells/ml were excluded. A spermocytogramm was performed in order to eliminate sample with cytoplasmic droplets. To rule out the possibility of any contamination by residual cells (germ cells or polynuclear cells), human ejaculated spermatozoa were purified on a discontinuous Puresperm® gradient. The liquefied semen samples were fractionated on a discontinuous Puresperm gradient (JCD, Lyon, France) consisting of four successive layers with the following densities: 95, 76, 57 and 45%. After centrifugation (20 min at 400 g, 25°C), motile and immotile spermatozoa were isolated respectively from the 95% layer and from the interface 76–57%. The vitality of the spermatozoa isolated from the interface 76–57% was >80%. These two fractions were then washed twice with Earle’s medium (Eurobio, les Ullis, France) before being used. A microscopic examination of the enriched-sperm fractions obtained (95 and 76–57%) was performed to control the quality of the preparations (motility, survival and morphology) and no remaining round cells and cytoplasmic droplets were observed (Figure 1A–C). Moreover, we have been unable to amplify mRNA of STAT-4 in human spermatozoa in spite of its presence in crude preparations of germ cells or testicular cells, excluding therefore any contamination by these cells (data not shown). Three sperm samples with cytoplasmic droplets (>35%) were also selected for an additional experiment (Figure 1D). Human granulosa cells (positive aromatase control) were obtained by collection of human follicular fluid from preovulatory follicles in the IVF centre (CHRU Clémenceau).
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RNA extraction
Total RNAs from purified sperm fractions and granulosa cells were extracted using the guanidium thiocyanate-derived method (Chomczynski and Sacchi, 1987). Briefly, after centrifugation the cell pellets were homogenized on ice in a 1 mol/l Tris buffer containing guanidium thiocyanate (4 mol/l). The RNA was then isolated with a phenol-chloroform-isoamylic acid solution. It was precipitated twice from the aqueous phase with isopropanol, washed with 75% ethanol, dried on a speed-vac and dissolved in diethylpyrocarbonate-treated water and then stored at –80°C. Purity of the RNA samples was checked spectrophotometrically at 260 and 280 nm.
RT–PCR assay
Total RNA (750 ng) was reverse-transcribed to first-strand cDNA as follows: 1 h at 37°C with 200 IU M-MLV-RT (Promega, Charbonnières-France), 500 µmol/l dNTP, 0.2 µg of oligo-dT (12–18mer) and 24 IU RNasin in a final volume of 10 µl, then 5 min at 94°C. The cDNAs were further amplified by PCR using selected oligonucleotides. PCR was performed for 30 cycles (94°C for 1 min, 60°C for 1 min and 72°C for 2 min 30 s with a 2 s delay for each cycle) in the presence of 1.5 mmol/l MgCl2, 200 µmol/l dNTP, 1.5 IU Taq polymerase and 50 pmol of the forward and reverse primers (Life Technology, Eragny-France) in a final volume of 50 µl. The P450arom primers (Table I) were chosen to amplify a highly conserved sequence of 424 bp length including helical and aromatic regions between exons IX and X. We have used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts, already described in human spermatozoa (Goodwin et al., 2000a), to control for the integrity of the RNA. The PCR conditions for GAPDH were 95°C for 1 min, 60°C for 1 min, 72°C for 1 min 30 s for 30 cycles in the presence of 25 pmol of GAPDH primers (Table I), giving rise to a PCR product of 431 bp. All cDNA fragments were run on a 1.5% agarose gel stained with ethidium bromide and visualized under UV transillumination.
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Nested PCR for P450arom product
Amplified product (1 µl) was used as a template for the second-round PCR. Amplification was performed under the same conditions as those used in the first PCR in the presence of a new set of internal primers (Table I).
DNA sequence analysis
The RT–PCR products were extracted from the agarose gel by centrifugation in Microcon PCR (Millipore, Saint-Quentin en Yvelines, France). The DNAs were amplified and then sequenced using a DNA sequencing kit (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction®) and the protocol for fluorescence-based DNA sequencing with Taq polymerase (Applied Biosystems, Courtaboeuf-France).
Protein extraction
The 95% and the interface 76–57% Puresperm fractions were washed twice with Earle’s medium and the pellets were resuspended in lysis buffer [100 mmol/l Tris-HCl, pH 7.4, 20% glycerol, 150 mmol/l KCl, 1 mmol/l dithiothreitol, 1 mmol/l EDTA, containing one protease inhibitor tablet (Roche, Germany)/10 ml buffer]. Samples were homogenized and centrifuged (11 000 g, 15 min, 4°C) and the resulting supernatants were referred to as total proteins.
Microsomes prepared as described elsewhere (Turner et al., 2002), were obtained from a pool of purified spermatozoa (95% Puresperm fraction) and from human granulosa-lutein cells. The protein concentrations were determined with the Bradford assay (Bradford, 1976).
Western blot analysis
Denaturing loading buffer (5 µl) was added to aliquots of protein (10 µg) and boiled for 10 min. The samples (10 µg) were electrophoresed at 30 mA for 2 h on a 10% sodium dodecyl sulphate (SDS)–polyacrylamide gel using a running buffer consisting of 25 mmol/l Tris, 192 mmol/l glycine (pH 8.3), 0.1% SDS. Then the proteins were transferred onto a nitrocellulose ECL membrane (Amersham Biosciences, France). The membranes were blocked for 1 h at room temperature in 4% non-fat dried milk in Tween TBS or Tween Tris buffer saline (0.1% Tween 20, 10 mmol/l Tris, 15 mmol/l NaCl, pH 7.4), incubated overnight with aromatase antibody (kindly provided by Dr Saunders, Serotec, UK) diluted 1:500 in TTBS containing 1% non-fat dried milk. The antigen–antibody complexes were detected by incubation of the membranes for 90 min with peroxidase-coupled anti-mouse IgG (Amersham, France) and developed using the ECL Plus Western blotting detection system (Amersham, France).
Deglycosylation
Proteins were denatured in a buffer consisting of 50 mmol/l Na2HPO4 pH 7.5, 0.1% SDS and 50 mmol/l ß-mercaptoethanol, then heated at 100°C for 5 min. Triton X-100 (1%) was added and samples were incubated for 16 h with N-glycosidase F (Roche, Germany). Proteins were run on a 10% SDS–polyacrylamide gel.
Aromatase activity
The aromatase activity was assessed by measurement of 3H2O released from [1ß-3H]androst-4-ene-3,17-dione (New England Nuclear, Les Ulis, France) (Lephart and Simpson, 1991). Puresperm-purified spermatozoa were incubated with 0.5 µmol/l [1ß-3H]androst-4-ene-3,17-dione for 4 h at 37°C in a 5% CO2/95% air atmosphere. The reaction was stopped by the addition of 1 ml of chloroform. The aqueous phase was removed and an activated-charcoal (7%) suspension in K2HPO4 containing dextran (1.5%) was added. 3H2O released during aromatization was measured in a liquid scintillation counter.
Statistical analysis
Results are means ± SEM. Statistical analyses between groups were determined by two-way analysis of variance (ANOVA). P < 0.05 was considered significant.
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Results |
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Detection of aromatase transcript in individual semen samples
All samples were treated individually and the presence of specific P450arom mRNA transcript from the 95% Puresperm fraction was checked by nested PCR. A strong signal was observed in each semen sample (Figure 2A). The same observations were recorded for GAPDH transcript (Figure 2B). There was no detectable signal in samples without reverse transcriptase (data not shown). Sequence analysis of the PCR products obtained from human sperm cDNA and granulosa cells (Figure 3), showed >98% sequence identity to the published sequence of human P450arom (Corbin et al., 1988).
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Semi-quantitative nested PCR for P450arom transcript
In order to quantify the P450arom transcript in human sperm samples we determined the optimal conditions for the RT–PCR using 50 ng of total RNA. The PCR amplification of cDNA was carried out for 30 cycles for aromatase transcript. For the reamplification, the kinetic analysis showed a linear increase of the amount of P450arom transcript between 14 and 25 amplification cycles; therefore, we selected 19 cycles for further semi-quantitative experiments (Figure 4A). A similar procedure was used for the GAPDH products and according to the amplification kinetics, 15 and 21 cycles were selected respectively, for the first and the second amplification during the nested PCR (Figure 4B).The resulting PCR products of 189 bp for aromatase and 294 bp for GAPDH were analysed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Gels were photographed using Polaroid film under UV light and analysed using a AGFA Snap-Scan 1200U Scanner, PhotoExpress software and the NIH image computer program (htpp://rsb.info.nih.gov/nih-image). The intra- and inter-assay coefficients of variation were 3% (n = 6) and 5% (n = 3, repeated in three independent nested PCRs) respectively.
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Comparison of aromatase expression between immotile and motile spermatozoa
Motile and immotile spermatozoa from the same sample were isolated as described in Materials and methods. In order to compare P450arom expression in these two populations, the motility of each preparation was evaluated. The motility was
95% in the motile fraction and was <10% in the immotile fraction. After purification on a discontinuous Puresperm gradient, the amount of RNA per 106 spermatozoa was 2.5-fold higher (P < 0.001) in the immotile sperm fraction compared with the motile sperm fraction (346 ± 24 and 142 ± 14 ng per 106 spermatozoa respectively; n = 9). We performed semi-quantitative nested PCR of the samples obtained (n = 9). The staining intensities of the aromatase signals showed a statistical difference between immotile and motile populations (P < 0.001) but not for GAPDH (P = 0.37). Consequently, using the same amount of total RNA from the two populations of spermatozoa, we have observed a significant decrease (28%) of the aromatase/GAPDH ratio in immotile compared with motile sperm fractions (P < 0.001; Figure 5).
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Expression of P450arom protein in human ejaculated spermatozoa
To characterize the aromatase protein in human spermatozoa we have performed Western blot analysis with a monoclonal antibody directed against a highly conserved region of human aromatase developed by Turner et al. (2002).
First, we looked for the protein in a preparation of microsomes obtained from a pool of motile spermatozoa and from granulosa cells. Two bands were observed in both preparations: a strong band at 53 kDa and a weaker band at 49 kDa in granulosa-lutein cells. These two bands were also present in spermatozoa, but the intensities of the signals were reversed compared to granulosa cells (Figure 6A).
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In order to better characterize the 49 kDa protein, proteins obtained from granulosa cells and human spermatozoa were deglycosylated. After deglycosylation, the motility of the 49 kDa protein was not shifted in human spermatozoa, suggesting that the aromatase was not glycosylated, whereas it was in granulosa cells (Figure 6B).
We then compared the expression of aromatase in total protein extracts isolated from motile and immotile spermatozoa. The aromatase was present as a 49 kDa band (with weak intensity) in total proteins obtained from the motile spermatozoa fraction, whereas no signal was visible in the immotile fraction (Figure 6C).
In another experiment, we looked for the presence of aromatase in sperm with cytoplasmic droplets. As shown in Figure 7A, using total proteins extracted from the 95% Puresperm fraction (motile), a single band at 49 kDa was visible in sperm with cytoplasmic droplets, although a very weak band was observed in sperm without cytoplasmic droplets.
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The same observation was recorded when we compared the expression of aromatase in motile and immotile spermatozoa with cytoplasmic droplets. The intensity of the band seemed to be weaker in total protein isolated from immotile spermatozoa (Figure 7B).
Aromatase activity
Aromatase activity was evaluated in two different samples. The activity was greater in the motile compared to immotile fractions (147 ± 35 versus 98 ± 2 fmol/h/108 spermatozoa). Moreover, the activity was reduced to 93 ± 4 fmol/h/108 spermatozoa when motile spermatozoa were incubated with 4-hydroxyandrostenedione, a specific aromatase inhibitor.
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Discussion |
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This study provides evidence for the expression of aromatase both in terms of transcript and protein in human ejaculated spermatozoa from normal donors. Our data are in agreement with those of Aquila et al. (2002), who have used pooled human semen samples, but we have worked on individual human semen samples to amplify aromatase transcript, explaining why we have only found aromatase transcript in human spermatozoa by nested PCR. In addition, we have checked the identity of the sperm PCR products by sequencing and the signal corresponds to the DNA sequence of human aromatase (Corbin et al., 1988), at least for those sequenced nucleotides which encompass the aromatization region of the gene.
Moreover, we have shown a significant decrease in the amount of P450arom mRNA transcript in immotile spermatozoa compared with motile spermatozoa of the same sample. Our data showing that total RNA was significantly higher in immotile spermatozoa compared to motile spermatozoa are in agreement with those of Roudebush and Purnell (2001) who reported a 5-fold increase of total RNA in the immotile sperm population. Several explanations could be proposed: an increase of the transcriptional activity, a decrease at the translational level or a longer half-life of the RNAs (Genissel et al., 2001). Nevertheless for the same amount of RNA analysed, the level of specific P450arom transcript was significantly lower in the immotile cells, as also reported for the PAF-receptor mRNA (Roudebush et al., 2000).
Various transcripts in human ejaculated spermatozoa have already been described such as those encoding for ß1-integrins (Rohwedder et al., 1996), phosphodiesterase subtypes (Richter et al., 1999), calcium channel -1c subunit (Goodwin et al., 2000a), protamine 2 (Miller et al., 1994), N-cadherins (Goodwin et al., 2000b), and progesterone receptor (Sachdeva et al., 2000). The presence of mRNAs encoding for transition proteins or protamines has been shown in the human sperm nucleus (Siffroi and Dadoune, 2001); in addition, these authors suggest the presence of sperm-specific mRNAs such as cyclin B1 originating from a low transcriptional activity. Several explanations could be proposed for the existence of transcripts in spermatozoa: (i) they may be remnants of spermatogenesis or spermiogenesis and thus represent a marker of the spermatozoa quality; (ii) it has been shown that transcriptional and translational activities could occur during capacitation and acrosome reaction (Naz, 1998); (iii) the putative existence of translationally repressed mRNAs in spermatozoa has been reported (Siffroi and Dadoune, 2001).
On Western blot analysis using a monoclonal antibody directed against aromatase, we have shown the presence of two bands in microsome preparations, one at the expected size (53 kDa) and another at 
49 kDa in both granulosa-lutein cells and spermatozoa; however, the predominant form in human spermatozoa is the 49 kDa band. In total protein extracts of purified spermatozoa (with and without cytoplasmic droplets), we have visualized aromatase as a single band of 49 kDa with a stronger intensity when cytoplasmic droplets are present. Moreover, the intensity of the band seems to be weaker in protein extracts from immotile spermatozoa when compared to motile spermatozoa, whatever the samples used (with or without cytoplasmic droplets). These data are not in agreement with those published by Aquila et al. (2002) who have found a single band at 53 kDa in total protein extracts using a polyclonal antibody. The monoclonal antibody used in the present study recognizes a highly conserved region of aromatase (corresponding to amino acids 376–390) (Turner et al., 2002). The small variation observed between the aromatase expected size and the observed molecular weight in human spermatozoa seems to be due to the level of glycosylation. Shimozawa et al. (1993) have reported a difference of 3 kDa between the glycosylated and the non-glycosylated form of aromatase. We have not observed a different molecular weight between spermatozoa treated or not treated with N-glycosidase. Moreover, in granulosa cell microsomes treated with N-glycosidase, the mobility of aromatase was shifted by 4 kDa. However, as already described (Sethumadhavan et al., 1991; Moslemi et al., 1997), the level of glycosylation does not seem to be essential for aromatase activity.
It is now well established that aromatase plays a role in male reproduction. Aromatase has been detected in Sertoli and Leydig cells as well as in germ cells of various mammalian testes (for a review see Carreau et al., 1999). Indeed, it has been shown that the recrudescence of spermatogenesis in rodents and some steps in spermiogenesis are under estrogen control (for reviews see O’Donnell et al., 2001; Carreau et al., 2002). The administration of an aromatase inhibitor in rat (Tsutsumi et al., 1987) and monkey (Shetty et al., 1998) leads to the reduction of round and elongated spermatid numbers. In addition, in bank voles treated with estradiol during the resting season, a recrudescence of spermatogenesis has been demonstrated (Bilinska et al., 2002). In rat, whereas the amount of P450arom mRNA decreases according to the stage of germ cell maturation, the aromatase activity increases and is the highest in testicular spermatozoa (Janulis et al., 1998; Levallet et al., 1998). Therefore, in rodents, the aromatase activity decreases during the epididymal transit which could be due to the shedding of the cytoplasmic droplet (Janulis et al., 1995, 1998). The cytoplasmic droplet migrates along the sperm tail as the sperm crosses the epididymis (this does not however exclude a residual aromatase in mature sperm). In humans, it has been noted that the cytoplasmic droplet disappearance is achieved prior to the beginning of the sperm epididymal transport and the acquisition of sperm motility. In fact, the presence of cytoplasmic droplets seems to be associated with a default of spermiogenesis and/or a defective remodelling of the sperm plasma membrane (Huszar et al., 1998).
Our results, suggesting that the levels of aromatase are higher in spermatozoa with cytoplasmic droplets than in spermatozoa without, are in keeping with the above data.
In that context, Hess et al. (1997) have reported an alteration of fluid reabsorption in the proximal parts of the epididymis leading to an accumulation of fluid within seminiferous tubules, which in turn induces an atrophy of germ cells in the ERKO mice. Moreover, in the epididymis of monkey, aromatase activity has been demonstrated; this activity is increased in the proximal parts of the epididymis compared to corpus and cauda regions (Pereyra-Martinez et al., 2001).
A decrease of sperm motility has been reported in ArKO mice as well as in a man with an aromatase deficiency (Carani et al., 1997). We observed a significant decrease (28%) of aromatase mRNA transcript in immotile spermatozoa versus motile spermatozoa which seems to correlate with the amount of protein on Western blot analysis. In addition, we have been unable to amplify aromatase mRNA by nested PCR in one asthenospermic spermatozoa sample (data not shown). Together, these data suggest that aromatase could be involved in the acquisition of sperm motility.
It has also been claimed that spermatozoa contain estrogens (Chew et al., 1993; Galeraud-Denis et al., 2001) and are able to transform pregnenolone into androgens (Gunasegaram et al., 1995). Recently, Aquila et al. (2002) have shown that spermatozoa could synthesize estrogens. This steroid could also act as a survival factor for the reproductive cells and it has been shown that physiological concentrations of estradiol block in-vitro human germ cell apoptosis (Pentikaïnen et al., 2000).
In summary, we have demonstrated the presence of aromatase (mRNA and protein) in purified motile and immotile human ejaculated spermatozoa. The possible role of these P450arom transcripts encoding for aromatase are still not completely understood but they may represent a good marker of motility. Taking into account the presence of ER in sperm, further investigations on the expression of aromatase in both normal and pathological sperm will help to understand the physiological role of estrogens in capacitation and/or sperm survival.
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Acknowledgements |
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We are grateful to Dr Philippa Saunders for the generous gift of anti-aromatase and to Dr J.Levallet for the critical reading of the manuscript. S.L. is a recipient of a fellowship from the French Ministry of Education and Research; this work was in part supported by a grant from Organon/Akzo Nobel.
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