Aromatase is an enzymatic complex localized in the
endoplasmic reticulum of numerous tissues which ensures the
irreversible conversion of androgens into estrogens. Aromatase
is involved in sexual differenciation, in lipid metabolism
and bone structures but also in cancer development which
therefore suggest a major role for that enzyme in humans.
It is well known that the normal testicular development
and the maintenance of spermatogenesis are controlled by
gonadotrophins and testosterone whose effects are modulated
by a complex network of factors produced locally and among
them, estrogens are concerned. Aromatase is composed of two
proteins: a ubiquitous NADPH-cytochrome P450 reductase
and a specific cytochrome P450 aromatase (P450arom),
which contains the heme and the steroid binding pocket. In
humans the P450arom is the product of a single gene located
on chromosome 15 and called cyp19, which belongs to the cytochrome P450 gene family. The cyp19 gene lies on more
than 123 kb length with a coding region of 9 exons (II-X) and
9 untranslated exons I. The cyp19 gene expression is
regulated by tissue-specific promoters producing alternate 5’-
untranslated exons I that are then spliced onto a common 3’-
splice acceptor site in the exon II, upstream of the translation
starting site (for reviews [1-3]. Therefore cyp19 variants are
numerous but the coding sequences are identical in humans
giving rise to a unique protein of 55 kDa [4].
The estrogens role in male reproduction has been reexamined
especially after the clinical and biological analysis
of aromatase-deficient men [5]. Moreover from several
epidemiological studies, decrease sperm counts and increase
male reproductive tract disorders (cryptorchidism, hypospadia,
testicular cancers) have been attributed to a deleterious effect of
endocrine disruptors with either estrogenic or antiandrogenic
actions [6,7]. An other interesting feature about estrogens
in male is provided by studies showing that the prenatal exposure to diethylstilbestrol induces abnormalities of the
genital tract in the newborn male mice and in men [8]. All
together the capacity of the testis to synthesize estrogens has
been extensively studied since the last two decades and there
is a growing body of evidence suggesting that estrogens play
a role via their specific receptors (ERα and ERβ), which are
present throughout the genital tract [9,10].
Aromatase in human germ cells
In humans, during several decades Leydig cells have been
considered as the only source of estrogens [11,12]. However
in vitro both Leydig and Sertoli cells produce estrogens [13].
Therefore taking into account the possible role of estrogens
in the mammalian testes [10,14] we have looked for the
expression of aromatase in motile and immotile spermatozoa
from healthy donors. Using “nested PCR”, P450arom
transcripts have been detected in ejaculated spermatozoa from
healthy men and the PCR products showed more than 98%
identity compared to the human placenta aromatase sequence
[15-16]. On Western blots we have evidenced the presence of
aromatase in both immature germ cells and ejaculated sperm
cells [15] and the intensity of staining was more abundant
in spermatozoa containing cytoplasmic droplets as reported
elsewhere [17]. Moreover we have observed that the amount
of P450arom transcripts was 30% lower in immotile than in
motile spermatozoa from several samples prepared individually
by density gradient purification; in addition the aromatase
activity was 50% greater in motile fraction compared to
immotile spermatozoa [18]. Our observations showing that
aromatase was expressed both in terms of transcript and of
biologically active protein in spermatozoa from normal donors
are in fitting with other reports [19]. According to our recent
data collected from infertile patients and using Real-Time
PCR, we have measured the amounts of aromatase mRNA in asthenospermic, teratospermic and asthenoteratospermic
patients: they were decreased respectively by 44%, 52% and
67% as compared to controls. A high degree of correlation
between the percentage of abnormal forms of spermatozoa
and the aromatase/GAPDH transcripts ratio have revealed
a high degree of correlation (especially head defects) (Saïd,
Galeraud-Denis, Carreau, unpublished data). Moreover
Carpino et al. [20] have immunolocalized aromatase in the
epithelial cells of human efferent ducts and in the proximal
caput epididymis suggesting an additional source of estrogens
in the male genital tract. Similar observations have been
published in the Rhesus monkey, in which it has been reported
that testis contained two P450arom transcripts [21]. As to the
regulation of the aromatase gene expression in human testis til
now only the promoter PII has been reported [1].
Our recent data about the implication of aromatase in
human spermatozoa have permitted to discuss new aspects of
the estrogens role in male reproduction either as a diagnostic
tool for evaluating male infertility or as a possible marker of
spermiogenesis.
Signification of aromatase transcripts and Putative
translational activity in spermatozoa
In spermatozoa the transcripts are believed to be remnants
from post-meiotically active genes found mainly in spermatids
[22-23]. However spermatozoa functions such as motility
or sperm morphology could also be related to the mRNA
profile of aromatase. We have also described differential
mRNAs distribution between motile and immotile fractions
of normospermic patients [18]. Analysis of spermatozoal
mRNA including aromatase transcripts could represent the
fingerprint for monitoring past events such as spermatogenesis
and/or spermiogenesis. The relationship between .the active
synthesis of estrogens in mature spermatozoa and the amount
of aromatase transcripts must be further investigated.
The potential involvement of mRNAs in functional
spermatozoal activities is actually subject to debate [24-27].
However a sperm endogenous reverse transcriptase activity
[28] and the incorporation of foreign DNA sequences into
sperm issuied from reverse transcription of RNA molecules
suggests that reverse transcribed products are fully active
in spermatozoa [29]. Moreover Pittoggi et al. [30] have
demonstrated the correct outsplice of an intronic sequence
incorporated into a DNA construct and Shaman et al. [31] have
reported the existence of an active topoisomerase (TOP2B)
associated with nucleases which thus are in favor of a less inert
activity of sperm chromatin than previously evoked [24].
Estrogen roles in spermatogenesis
In order to exert a biological role, testicular estrogens should
interact with estrogen receptors (ER) which in turn modulate
the transcription of specific genes. Therefore considering
the presence of at least two ERs (ERα and ERβ) in most of
the testicular cells and in the other parts of the genital tract,
the physiological role of estrogens in mammalian testes and
especially in human reproduction has been extensively reevaluated
[8,32-33]. In sperm cells, we have found specific
transcripts for ERs but only an ERα protein with a mol wt
of 46 kDa was revealed [16]. That variant could be located on
the membrane as reported elsewhere [34]. Aquila et al. [35],
Solakidi et al. [36] have described the presence of ERα and
ERβ proteins in human ejaculated spermatozoa with some
discrepancies on their respective localisation. Recently Aschim
et al. [37] reported the presence of several splice variants of
ERβ in the human testicular cells and a putative relationship
between two ERβ polymorphisms and man infertility has
been suggested [38]. Indeed the effects of estrogens in human
ejaculated spermatozoa are more and more obvious: besides
the classical genomic effects, membrane estrogen receptors
connected with numerous signal transduction pathways
involving quick answers have been described [39]. In that
respect Fraser et al. [40] have demonstrated that genistein
improves the capacitation and acrosome loss of human
spermatozoa. In addition, the existence of estrogen receptors in
mitochondria a cytoplasmic organelle very concentrated in the
mildpiece of spermatozoa [41] could be additionally relevant for a significant role of estrogens in male gamete motility.
Recently the presence of androgen receptor has been
demonstrated in human spermatozoa [42] which thus could
provide andogens for the aromatase activity since the human
spermatozoa express a functional P450arom. Together with
the presence of ERs new considerations about the role of
estrogens all along the male genital tract and likely in sperm
mobility and fertilizing ability should be taken into account
[43]. The observations of decreased sperm motility in men
with aromatase deficiency [5] which is a feature in common
with the knockout models of mice [8,44] together with our
data showing a significant decrease of aromatase expression in
spermatozoa from infertile men likely suggest that aromatase
is involved in the acquisition of sperm motility.
Besides the positive role for estrogens in male gamete
quality we should also keep in mind the adverse effects of
these female hormones in man testis. As a matter of fact, in
leydigoma [45] and in seminoma [46-47] estrogens (and/
or xenoestrogens) are responsible for the abnormal cell
proliferation and thus play a major role in these deseases.