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Health Centre (S.Aq., D.S., M.G., E.M.), Department of Cell Biology (L.S.), and Faculty of Pharmacy (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: . sebando@tin.it or aquisav{at}tin.it
Abstract
The generation of cytochrome P450 aromatase (P450arom) and estrogen receptor (ER) knockout mice has raised new interest in the physiological role of estrogens in male reproduction. Testicular expression of P450arom, the enzyme that converts androgens into estrogens, has been shown in both somatic and germ cell types in several species, whereas in humans, testicular expression is confined to the somatic cells. The aim of this study was to determine whether P450arom is present in human ejaculated spermatozoa. Using RT-PCR and specific primers, we amplified the highly conserved helical, aromatic, and heme-binding sequences of the conventional human P450arom from RNA isolated from human spermatozoa. Employing a rabbit polyclonal antiserum directed against human placental P450arom, immunoblotting analysis demonstrated aromatase protein expression, which was localized primarily to the tail and midpiece of spermatozoa. Measurement of enzymatic activity using a sensitive 3H2O aromatase assay revealed that activity was enhanced by the 2'-O-dibutyryl cAMP and completely inhibited in the presence of the specific aromatase inhibitor, letrozole. These results represent the first demonstration that human spermatozoa are a potential site of estrogen biosynthesis. The physiological relevance of estrogen synthesis in spermatozoa remains to be elucidated and opens a new area of investigation in male fertility.
DURING THE LAST decade, the role of estrogens in male reproduction has been extensively investigated (1, 2). Generation of knockout mice for estrogen receptors (ER) 
and ß and the cytochrome P450 aromatase (P450arom) genes has provided direct evidence for a crucial role of estrogens in the maintenance of normal spermatogenesis. Male ER knockout (ERKO) mice are completely infertile (3, 4, 5). From the onset of puberty, they show atrophy of the testes and dysmorphogenesis of the seminiferous tubules that results in decreased spermatogenesis and inactive sperm. The infertility of male ERKO mice is due to a lack of fluid reabsorption in the efferent ductules, resulting from an altered regulation of ion transport and fluid movement (6, 7). ERß knockout (ERßKO) male mice apparently exhibit normal fertility, whereas mice with double knockouts in ER and ERß (ERßKO) seem to have a similar phenotype to ERKO mice. These findings suggest that estrogen action through ER is required for normal male mouse reproduction (8, 9).
Male aromatase knockout (ArKO) mice are initially fertile, and histologic examination of the testes reveals no gross morphological abnormalities. Testis weight and size are within the normal range and exhibit normal testicular development; however, these mice have a progressive long-term deterioration of spermatogenesis. In older ArKO mice, approximately 4–5 months of age, the progressive disruption of spermatogenesis results in a decrease of round and elongated spermatids. This is associated with an increase of apoptosis and reduced or absent sperm maturation in the cauda epididymis (10, 11). Thus, in contrast to the ERKO animals, which exhibit alterations of spermatogenesis primarily related to the mechanical effects of altered fluid dynamics, the ArKO mice exhibit alterations within the adult seminiferous epithelium itself.
In various species, P450arom has been found in meiotic and postmeiotic germ cells of the testis (12, 13, 14). In bear (15), rooster (16), rat (17), and mouse (12), a biologically active P450arom was immunolocalized in pachytene spermatocytes, round spermatids, elongated spermatids, flagella of late spermatids, and spermatozoa in the epididymus (16). Aromatase displays an age-dependent pattern of expression in the rat testis with mainly Sertoli cell expression in prepubertal animals and Leydig cell expression in mature animals (18, 19). Moreover, in mouse germ cells the aromatase transcript presents a pattern of expression that does not parallel that of aromatase protein content. P450arom mRNA is more abundant in less differentiated germ cells (12), whereas P450arom protein and activity are higher in elongated spermatids and testicular spermatozoa. P450arom expression decreases as sperm traverses from the efferent ductules and moves along the epididymal tract from the caput to the cauda (12, 20, 21).
It remains to be clarified whether aromatase expression in human gametes is lost during transit through the genital tracts or whether ejaculated spermatozoa continue P450arom expression. In addition, the expression of P450arom in human spermatozoa has not been studied. In the present study, we demonstrate for the first time that ejaculated spermatozoa of normal men contain functional P450arom.
Materials and Methods
Chemicals
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 Corp. (Milan, Italy); Moloney murine leukemia virus (M-MLV) was from Life Technologies-Italia (Milan, Italy). Oligonucleotide primers were made by Invitrogen (Milan, Italy). DMEM-F10 medium, 2'-O-dibutyryl cAMP [(Bu)2cAMP], BSA protein standard, activated charcoal, Laemmli sample buffer, prestained molecular weight markers, Percoll (colloidal polyvinyl pyrrolidone-coated silica for cell separation), Earle’s balanced salt solution, and all other chemicals were purchased from Sigma Chemical (Milan, Italy). Acrylamide bisacrylamide was obtained from Labtek Eurobio (Milan, Italy). ECL Plus Western blotting detection system, Hybond ECL, and Dextran T70 were from Amersham Pharmacia Biotech (Buckinghamshire, UK). The rabbit polyclonal antiserum directed against human placental P450arom was provided by Dr. Yoshio Osawa (Hauptman-Woodward Medical Research Institute, Buffalo, NY). Secondary antibody and peroxidase-coupled antirabbit IgG was from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). [1ß-3H]androst-4-ene-3,17-dione was obtained from NEN Life Science Products (Boston, MA). Bradford protein assay was performed using a kit from Bio-Rad Laboratories, Inc. (Milan, Italy).
Semen samples
Human semen samples were obtained from normozoospermic men, who were otherwise undergoing semen analysis for couple infertility, by masturbation after 3 d of sexual abstinence. All samples were obtained according to the World Health Organization (WHO)-recommended procedure (22). Samples were allowed to liquefy for 30–60 min and were examined for seminal parameters according to the WHO Laboratory Manual (22). Samples with a concentration of less than 20 x 106 sperm/ml and/or the presence of leukocytes and/or immature germ cells at a concentration greater than 2 x 105 cells/ml were not included in the study. Ejaculates with abnormal viscosity were also excluded.
Spermatozoa purification
Spermatozoa were isolated from pooled normal semen samples by centrifugation on a discontinuous Percoll density gradient (80:40, vol/vol) (22). After centrifugation for 20 min at 800 x g, the 80% Percoll fraction was examined using an optical microscope equipped with a 100x oil objective to ensure that a pure sample of spermatozoa was obtained. An independent observer, who observed several fields for each slide, inspected the cells. The pellet was then washed twice with BSA-free Ham’s-F10 medium.
After purification, the cells were used immediately for RNA isolation or protein extraction. For the assessment of aromatase activity, the final pellet was resuspended in Ham’s-F10 medium supplemented with 0.2% BSA, and sperm concentration was reevaluated.
Assessment of purity for spermatozoa samples
To demonstrate the purity of the spermatozoa population, RT-PCR analysis of myelo peroxidase (Myelo Pox) transcript, a gene restricted to granulocytes, was performed on mRNA from the 80% Percoll fraction. As positive control, mRNA from leukocytes was used, and separation of leukocytes from whole blood was performed with a PMN Separation Medium Kit, according to the manufacturer’s protocol.
Isolation of sperm heads and tails
Purified sperm were decapitated (23) by an incubation with trypsin (100 µmol/liter), and then the samples were homogenized in a loose-fitting Dounce homogenizer for 3–4 min until the heads and tails were separated. A 10-fold excess of soybean trypsin inhibitor was added immediately, and the samples were placed on ice to inhibit further tryptic activity. The head and the tail-midpiece were washed three times in PBS containing protease inhibitors (1 mmol/liter phenylmethylsulfonylfluoride, 1 mmol/liter aprotinin) and in 50 mmol/liter Tris-HCl (pH 7.4) at 4 C.
This sperm preparation was subjected to centrifugation on a gradient of 40–80% metrizamide (24, 25). After centrifugation (at 800 x g for 4 h), the heads and tails were recovered in 60–64% and 45–47% fractions, respectively, as expected. When examined under microscopy, the head and the tail-midpiece were found to have separated. An independent observer, who characterized several fields on each slide, verified head/tail separation. The two fractions were washed with Ham’s-F10 medium, and the pellets were resuspended in lysis buffer for Western blot analysis.
RNA isolation
Total RNA was extracted from human spermatozoa with Total RNA Isolation System kit (Promega Corp.) according to the manufacturer’s protocol. The purity and integrity of the RNA was assessed spectroscopically before carrying out the analytical procedures.
mRNA analysis
The P450arom mRNA present in the total RNA samples was analyzed by the RT-PCR. Reverse-transcriptase generation of cDNA was performed on 3 µg of total RNA for both P450arom and Myelo Pox, in a final volume of 10 µl, by incubation for 30 min at 37 C with 200 IU M-MLV reverse transcriptase, 0.4 µg oligo-dT, 0.5 mmol/liter deoxy-nucleotide-triphosphate, and 24 IU RNasin, followed by heat denaturation for 5 min at 95 C. Subsequent PCR analysis was performed on 4 µl of the cDNA in a final volume of 20 µl.
The following pairs of primers were used to amplify 659 bp of human P450arom, which included the helical aromatic and heme-binding region: 5'-CTGGAAGAATGTATGGACTT-3' located in exon 7, and 5'-GATCATTTCCAGCATGTTTT-3' located in exon 10. The primer pairs used to amplify 349 bp of Myelo Pox were: 5'-CCTTGCTGGGCTGGGGGTCTCAC-3' located in exon 1, and 5'-GGGCGGGCGTCAGCACATCAG-3' located in exon 3.
The PCR mixtures (each reaction tube final concentrations) consisted of 10 mmol/liter Tris-HCl, 50 mmol/liter KCl, 3 mmol/liter MgCl2, 0.2 mmol/liter of each deoxy-nucleotide-triphosphate, 20 pmol/liter of each primer, and 2 U of Taq DNA polymerase. The temperature program used was 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min for 35 cycles. To control for false-positive PCR amplification by contaminating genomic DNA, some samples did not include M-MLV reverse transcriptase (data not shown). For all amplifications, negative controls were included that consisted of water added instead of sample to test for contamination with extraneous DNA. For each sample, a 6-µl aliquot of PCR amplification products was analyzed on 2% agarose gels, stained with ethidium bromide, visualized under UV light, and photographed. Standard DNA markers (1000-bp DNA ladder) were also used to determine the size of amplified products. As a positive control for each reaction, P450arom was amplified from a plasmid-containing human CYP19 (pcDNA3.1-CYP19), the gene encoding P450arom (25). Amplification using cDNA prepared from leukocytes RNA served as positive control for Myelo Pox.
Western blot analysis of spermatozoa proteins
Percoll-purified spermatozoa were washed twice with Ham’s-F10 medium and centrifuged for 5 min at 5000 x g; the pellet was resuspended in lysis buffer [62.5 mmol/liter Tris-HCl (pH 6.8), 50 mmol/liter dithiothreitol, 2% SDS, 10% glycerol, 1 mmol/liter Na3VO4, 1 mmol/liter phenylmethylsulfonylfluoride]. The suspension was shaken for 15 min, and protein concentration was estimated using Bradford’s assay (26). After protein measurement, the sperm extracts, each containing the same protein amount (10 µg), were adjusted with lysis buffer to the same volume and diluted in an equal volume of reducing 2x standard Laemmli SDS buffer. Samples were boiled for 5 min and electrophoresed on 10% SDS-polyacrylamide gels.
After SDS-PAGE, proteins were transferred to nitrocellulose membranes. Nitrocellulose filters with transferred proteins were blocked in TTBS (0.1% Tween 20, 20 mmol/liter Tris, and 150 mmol/liter NaCl) containing 5% BSA, then washed three times in TTBS, and incubated for 1 h in 5% BSA-TTBS containing rabbit polyclonal antiserum directed against the human placental P450arom (1:6000). The antigen-antibody complexes were detected by incubation of the membranes for 1 h with peroxidase-coupled antirabbit IgG and developed using the ECL Plus Western blotting detection system.
Aromatase activity assay
Aromatase activity was detected in human spermatozoa by the tritiated water (3H2O) release assay (27). Percoll-purified spermatozoa were incubated with 0.5 µmol/liter [1ß-3H]androst-4-ene-3,17-dione. Because a functional cAMP-signaling system has been documented in human spermatozoa (28, 29), some samples were pretreated for 1 h with different concentrations (0.5, 1, and 2 mmol/liter) of (Bu)2cAMP, followed by assay of aromatase activity. In addition, spermatozoa were pretreated with 10 µmol/liter letrozole (30) for different times (30 min, 1 h, and 2 h), followed by aromatase activity assay. Enzyme activity assays were carried out for 4 h at 37 C in a 5% CO2/95% air atmosphere and were terminated by the addition of 3 ml of chloroform. The aqueous phase was separated, treated with 2.5% charcoal suspension to eliminate residual steroids, and centrifuged. An aliquot was removed, and 3H2O was released during aromatization of [1ß-3H]androstenedione to estrogens was measured in a liquid scintillation counter. The protein concentration of the lysates was measured by Bradford’s assay. Aromatase activity was expressed as picomoles of 3H2O released per hour per milligram of protein.
Statistical analysis
The experiments for RT-PCR were repeated on at least three independent occasions, whereas Western blot analysis was performed in at least 20 independent experiments. The data obtained from replicate experiments of aromatase activity assay (nine replicate experiments) are presented as the mean ± SEM, and differences in mean values were calculated using a paired t test with a P value less than 0.05 significance level. Regression analysis was performed using the SPSS program (SPSS, Inc., Richmond, CA).
Results
Assessment of the purity for spermatozoa samples
Contamination by other cellular types was excluded by the spermatozoa purification protocol using a Percoll gradient and by microscopic examination. In addition, to ensure purity of spermatozoa preparations, we examined mRNA expression of Myelo Pox, a marker used to examine granulocyte contamination. RT-PCR of Myelo Pox in the sperm samples was not detectable, providing evidence that spermatozoa preparations are not contaminated by granulocytes (Fig. 1
).
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P450arom mRNA expression was studied using RT-PCR. Results of the amplification of the highly conserved sequence of the human P450arom cDNA sequence are shown in Fig 2. cDNA amplified from human spermatozoa revealed the same mobility product as that amplified from a plasmid containing the full coding region of P450arom, which was used as a positive control. No amplification was observed in samples that lacked RNA or from samples that were amplified without M-MLV reverse transcriptase (data not shown).
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Using a rabbit polyclonal antiserum directed against human P450arom, a single band corresponding to a molecular mass of 55 kDa, the correct size of human P450arom protein, was present in all spermatozoa samples tested (Fig. 3A). The band comigrated at the same mobility as purified human placental P450arom protein that was used as positive control. To better define the location of aromatase expression in spermatozoa, sperm heads were separated from the tail-midpieces. Immunoblot analysis demonstrated that the immunoreactive 55-kDa protein was expressed at higher levels in the tail-midpiece region than in the sperm head (Fig. 3B).
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Human ejaculated spermatozoa contain active P450arom protein
To determine whether the P450arom in ejaculated spermatozoa was biologically active, enzymatic activity was studied using the incorporation of tritium from [1-ß3H] androstenedione into 3H2O. The 3H2O assay for aromatase activity was validated for spermatozoa and exhibited a concentration-dependent increase in P450arom activity (Fig. 4A). The aromatase activity in human ejaculated spermatozoa was 0.62 ± 0.14 pmol/h·mg protein. This value represents the mean ± SEM of nine independent determinations. The activities from these experiments ranged from 0.181–2.25 pmol/h·mg protein. The rat R2C, Leydig tumor cell line that constitutively expresses elevated levels of P450arom, was used as a positive control.
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). The addition of (Bu)2cAMP, which stimulates the cAMP-dependent signaling pathways, increased aromatase activity in a concentration-dependent manner (Fig. 4C). This effect was completely reversed in the presence of letrozole (10 µmol/liter) (Fig. 5).
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Expression of P450arom has been extensively studied in the male genital tract of several species. Expression within the testis has been shown for both somatic and germ cells from pachytene spermatocytes through elongated spermatids (12, 13, 14). To date, expression of P450arom in the human male genital tract has been limited to prostate (31), Leydig cells (32), and elongate spermatids (33). In this study, the presence of P450arom in human ejaculated spermatozoa was demonstrated at three different functional levels: mRNA expression, protein content, and enzymatic activity.
New reports firmly establish that the presence of mRNA in mammalian ejaculated spermatozoa is a general phenomenon. About 12 transcripts have been identified (34, 35), and the analysis and significance of mRNA presence in these cells are currently under investigation. The aromatase mRNA in ejaculated spermatozoa is most likely carried over from earlier stages of spermatogenesis, because the P450arom mRNA at various stages of germ cells development in the testis of several species has been well documented. In rat and mouse, aromatase content as well as enzymatic activity decrease as spermatozoa traverse along the epididymal tract from the caput to the cauda. However, although the ability to synthesize estrogen by spermatozoa gradually decreases, estrogen synthesis is never completely lost (21).
Recent studies have demonstrated that estrogens are involved in the function of mature spermatozoa. In particular, the effects of estradiol on human spermatozoa have been associated with an enhanced motility, oocyte penetration, longevity, oxygen intake, lactate production, and metabolization of several substrates (36). These aspects may support a role for endogenous estradiol production in sperm survival, because low estradiol concentrations have been demonstrated to be more effective than androgens in the prevention of germ cell apoptosis in human adult seminiferous tubules (37). The effects of estradiol on spermatozoa are too rapid to be mediated by genomic activation (38, 39), and this supports the evidence that the stimulatory action of the estrogen on spermatozoa is mediated by membrane-associated ER (39). Autoradiographic data, on the other hand, confirm that the plasma membrane is the site of a receptor with specificity for 17ß-estradiol and it is mainly concentrated in the central part of the spermatozoa tail (40).
Estradiol induces a rapid increase of intracellular cAMP and calcium concentrations and an enhanced tyrosine phosphorylation of proteins (39, 28) in sperm. It is worth noting that these effects of estradiol recapitulate the events that characterize sperm capacitation (41, 42). During capacitation, the increase in intracellular cAMP concentration leads to an activation of protein kinase A (PKA) pathway, and the role of the cAMP/PKA pathway in spermatozoa has been extensively investigated (43). The PKA pathway involves, as final targets, dynein and PKA-anchoring proteins that are located in the tailpiece of spermatozoa and are important for the modulation of sperm motility (44).
The present study demonstrates an enhancement of aromatase activity upon (Bu)2cAMP exposure. Thus, the cAMP/PKA signaling pathway may play an important role in the regulation of aromatase activity in ejaculated spermatozoa. By Western blot analysis, aromatase was localized primarily to the tail and mid-piece regions rather than in the head of ejaculated spermatozoa. Given the coincident location of cAMP/PKA pathway effectors (i.e. dynein, A kinase-anchoring proteins), ER (39, 45), and aromatase in the tail of spermatozoa, it is reasonable to assume that the estradiol produced locally might be able to stimulate sperm motility in a paracrine and/or autocrine fashion through a nongenomic pathway.
A physiological role for enzymatically active P450arom in human ejaculated spermatozoa is supported by its potential use of readily available aromatizable androgens (e.g. testosterone) that are present in seminal plasma. The possible link between locally produced estradiol by ejaculated spermatozoa and effects on sperm motility and sperm capacitation, although attractive, currently relies on indirect evidence. For example, it has been demonstrated that testosterone in seminal plasma prevents premature capacitation (46). Thus, it is possible that aromatization of testosterone would remove this inhibition. Whether the ability of spermatozoa to synthesize estrogens influences their fertilizing capacity remains as an intriguing issue to be clarified, and this opens a new area of investigation in male reproduction.
Acknowledgments
We express our sincere thanks to E. R. Simpson, Ph.D. (Prince Henry’s Institute of Medical Research, Monash Medical Center, Clayton, Australia) for the gift of the pcDNA3.1 vector containing human CYP19. We also thank W. E. Rainey, Ph.D., and M. Young, Ph.D., for editorial assistance.
Footnotes
This work was supported by Grant 2001063981 from the COFIN MIUR.
Abbreviations: ArKO, Aromatase knockout; ER, estrogen receptor; ERKO, ER knockout; ERßKO, ERß knockout; M-MLV, Moloney murine leukemia virus; Myelo Pox, myelo peroxidase; P450arom, cytochrome P450 aromatase; PKA, protein kinase A.
Received September 7, 2001.
Accepted March 17, 2002.
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