# Estrogens and male reproduction: a new concept

Serge Carreau, Dorothée Silandre, Sonia Bourguiba, Khaled Hamden, Lamia Said, Sophie Lambard, Isabelle Galeraud-Denis, Christelle Delalande

► To cite this version:

Serge Carreau, Dorothée Silandre, Sonia Bourguiba, Khaled Hamden, Lamia Said, et al.. Estrogens and male reproduction: a new concept. Brazilian Journal of Medical and Biological Research, 2007, 40, pp.761-768. 10.1590/S0100-879X2007000600003. hal-02657021

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<figure><img src="image_1.png" /></figure>
# Estrogens and male reproduction: a new concept

S. Carreau<fnref n="1" />, D. Silandre<fnref n="1" />,

S. Bourguiba<fnref n="2" />, K. Hamden<fnref n="1" />,

L. Said<fnref n="1" />, S. Lambard<fnref n="3" />,

I. Galeraud-Denis<fnref n="1" />

and C. Delalande<fnref n="1" />

## Abstract

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them estrogens are the end products obtained from the irreversible transformation of androgens by a microsomal enzymatic complex named aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rats, the aromatase activity is mainly located in Sertoli cells of immature rats and then in Leydig cells of adult rats. We have demonstrated that germ cells represent an important source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes compared to gonocytes or round spermatids; conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank voles, bears, and monkeys express aromatase. In humans, we have shown the presence of a biologically active aromatase and of estrogen receptors (α and β) in ejaculated spermatozoa and in immature germ cells in addition to Leydig cells. Moreover, we have demonstrated that the amount of P450arom transcripts is 30% lower in immotile than in motile spermatozoa. Alterations of spermatogenesis in terms of number and motility of spermatozoa have been described in men genetically deficient in aromatase. These last observations, together with our data showing a significant decrease of aromatase in immotile spermatotozoa, suggest that aromatase could be involved in the acquisition of sperm motility. Thus, taking into account the widespread localization of aromatase and estrogen receptors in testicular cells, it is obvious that, besides gonadotrophins and androgens, estrogens produced locally should be considered to be physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis.

## Key words

• Aromatase

• Estrogens

• Estrogen receptors

• Male germ cells

• Fertility

• Mammals

## Correspondence

S. Carreau

EA 2608-USC 2006 INRA

Université de Caen

Esplanade de la paix, 14032

Caen

France

Fax: +33-231-565-120

E-mail: serge.carreau@unicaen.fr

Research supported by the French Ministry of Education and Research, National Institute of Research in Agronomy and Région Basse-Normandie.

Received January 3, 2007

Accepted May 4, 2007

## Introduction

It is well known that normal testicular development and the maintenance of spermatogenesis are controlled by gonadotrophins and testosterone whose effects are modulated by a complex network of locally

produced factors, with estrogens being obviously involved. Estrogens have long been considered to be specific female hormones; however, the presence of estrogens in the male gonad has been well documented since the publication of Zondek (1) more than 70 years ago showing the presence of estrogen

<footnote n="1">¹Department of Biochemistry, University of Caen, Caen, France</footnote>

<footnote n="2">²Physiology Department, Turku University, Turku, Finland</footnote>

<footnote n="3">³Centre Hospitalier Universitaire St. Antoine, Paris, France</footnote>
in stallion urine (for a review, see Ref. 2). Indeed the androgen/estrogen balance is es- sential for normal sexual development and reproduction in mammals. In the mamma- lian testis, the maintenance of this balance is under a fine tuning via endocrine and para- crine factors, but is also related to aromatase activity. Aromatase, an enzymatic complex, ensures androgen conversion to estrogens and is localized in the endoplasmic reticu- lum of various tissues including placenta, gonads, brain, bones, and adipose tissue.

Besides the well-known negative effect exerted by these female hormones on the secretion of gonadotrophins, estrogens play a major role *in situ* and appear to be impor- tant not only in women but also in men, especially taking into account the data ob- tained from men genetically deficient in aromatase (for a review, see Ref. 3). More- over, in several epidemiological studies de- creased sperm counts and increased male reproductive tract disorders (cryptorchidism, hypospadia, testicular cancer) have been at- tributed to a deleterious effect of endocrine disruptors with either estrogenic or antian- drogenic actions (4,5). Therefore, the capac-

ity of the testis to synthesize estrogens has been extensively studied. Over the last two decades the role of estrogens in male repro- duction has been seriously revisited espe- cially in primates taking into account the existence of specific estrogen receptors (ERα and ERβ) which are distributed all along the genital tract (for reviews, see Refs. 2,6-9).

## Presence of aromatase in testicular cells of mammals and humans

### The aromatase gene

Aromatase is composed of two proteins: a ubiquitous NADPH-cytochrome P450 re- ductase and a cytochrome P450 aromatase (P450arom), which contains the heme and the steroid-binding pocket. In humans, P450arom is the product of a single gene located in region q21.1 of chromosome 15 and called *cyp19*, which belongs to the cyto- chrome P450 gene family. The *cyp19* gene is more than 123 kb in length with a coding region of 9 exons (II-X) and 9 nontranslated exons I (Figure 1). Expression of the *cyp19* gene is regulated by tissue-specific promot-

<figure><img src="image_2.png" /><figcaption>Figure 1. Schematic presentation of the human aromatase gene. P = promoter.</figcaption></figure>
much more intense under long-light cycle conditions which coincide with the breeding season (23). Identical data have been reported for the bear (24).

In the stallion, many estrogens are synthesized by Leydig cells (for a review, see Ref. 25). In contrast to the data of Hess and Roser (26), Hejmej et al. (27) have recently immunohistolocalized aromatase not only in Leydig cells, but also to epididymal cells and to the germ cells present in the lumen of the epididymis. In the boar testis the main source of estrogens is in the Leydig cells (28).

## Aromatase in primate testes

In humans, Leydig cells have long been considered to be the only source of estrogens (29); however, *in vitro* both Leydig cells and Sertoli cells produce estrogens (for a review, see Ref. 30). It has also been claimed that spermatozoa can convert pregnenolone into estrogens (31). After obtaining data on the expression of aromatase in male germ cells of the rat, we decided to check for the presence of aromatase in ejaculated spermatozoa from healthy donors. In a second approach, we hypothesized that aromatase could be used as a marker of sperm quality (especially motility) and thus we measured the amount of P450arom transcripts in motile and immotile spermatozoa. In addition, since no information was available on the source of estrogens in testicular germ cells, we examined by RT-PCR the putative expression of *cyp19* in immature germ cells (spermatozoa and spermatids) obtained from semen samples with large number of round cells (32). Briefly, the quality of the germ cell preparations was carefully analyzed in order to eliminate any sample contamination with leukocytes via the expression of CD45 transcript and with Sertoli cells via the presence of the Sertoli cell factor mRNA. All samples containing detectable levels of these transcripts were eliminated; conversely the presence of *c-kit* mRNA in round cells was used

as a positive control for the presence of germ cells. We detected the presence of P450arom transcripts in both immature germ cells and ejaculated spermatozoa. The sequence analysis of the PCR products obtained was identical and showed more than 98% identity to the published sequence of human P450arom (33). Using Western blots and a specific monoclonal antibody against a highly conserved region of aromatase we demonstrated the presence of aromatase in both immature germ cells and ejaculated sperm cells, with the staining intensity being more abundant in spermatozoa containing cytoplasmic droplets.

Moreover, we demonstrated that the amount of P450arom transcripts was 30% lower in immotile than in motile spermatozoa from several samples individually prepared by density gradient purification; in addition, the aromatase activity was 50% greater in the motile fraction compared to immotile spermatozoa. Our observations showing that aromatase was expressed both as a transcript and as a biologically active protein in spermatozoa from normal donors agree with other data (34). These latter observations are correlated with the immunolocalization of aromatase in cytoplasm surrounding elongated spermatids in man (35) and aromatase has also been revealed in cytoplasmic droplets of ejaculated human spermatozoa (36). Recently, Carpino et al. (37) have immunolocalized aromatase in the epithelial cells of human efferent ducts and to the proximal caput epididymis, suggesting an additional source of estrogens in the male genital tract. Similar observations have been made on rhesus monkeys, whose testis and, to a lesser extent, epididymis contained two P450arom transcripts, one of them being truncated (38).

## Regulation of aromatase gene expression

In order to obtain insights about the role of estrogens in male reproduction and espe-
cially within seminiferous tubules, it is nec-
essary to study the regulation of the *cyp19*
gene (for a review, see Ref. 39). Thus, using
RACE-PCR we demonstrated that promoter
II directs the expression of the aromatase
gene whatever the testicular cell type stud-
ied in the rat (40). In the testis, follicle-
stimulating hormone and luteinizing hor-
mone both act to increase the concentrations
of intracellular cyclic AMP (cAMP), there-
by inducing expression of P450arom which
in turn requires the transcription factors
cAMP response element-binding protein,
cAMP response element modulator and ste-
roidogenic factor-1 (SF-1). SF-1 belongs to
the nuclear orphan receptor superfamily and
regulates the transcription of steroidogenic
genes, among them P450arom via its inter-
action with numerous co-activators such as
cAMP response element-binding protein,
DAX-1, SOX-9, and WT1. In addition, we
have shown that liver receptor homologue-
1, an SF-1 homologue, which is present both
in Leydig cells and germ cells, but not in
Sertoli cells, increases the P450arom gene
expression in a mouse Leydig cell line (41).
Numerous functional motifs have been iden-
tified in P.II (11); in this context it is worth
noting that truncated transcripts of the
P450arom gene giving rise to putative inac-
tive proteins have been described in PS and
RS of adult rats (42). Moreover, it is now
clear that not only P.II drives the aromatase
gene in rat testis but two additional promot-
ers, P.I.f (brain promoter) and a new one that
we called P.I.Tr (testis rat; Silandre D,
Delalande C, Carreau S, unpublished re-
sults), are involved. We have also demon-
strated that the nutritional status of fetuses
(43) and aging (Hamden K, Silandre D,
Delalande C, El Feki A, Carreau S, unpub-
lished results) can modulate aromatase gene
expression in male rats. In mice it has been
shown that aromatase expression is con-
trolled by three different promoters, among
them a testis-specific promoter (44). In hu-
man gonads, only the P.II promoter has been

reported to control aromatase gene expres-
sion thus far (45).

Estrogen receptors in the male
genital tract

In order to exert a biological role, testicu-
lar estrogens should interact with ERs, which
in turn modulate the transcription of specific
genes. Until 1996, the only data on ERs
concerned ERα; however, with the discov-
ery of a novel ER denoted ERβ, the localiza-
tion of ERs has been reexamined, and it has
been shown that the α and β forms are not
always present in the same cells (or are
present in different amounts) within the male
genital tract. The distribution of the mRNAs
coding for the two types of ERs in the male
rat gonad has been studied (46) and Saun-
ders et al. (47) have shown the presence of
ERβ in pachytene spermatocytes and sper-
matids. ERβ has also been immunolocalized
in Leydig cells and in the seminiferous tu-
bules of the bank vole (23).

Mutembei et al. (48) have shown that
ERß is ubiquitously distributed in the tes-
ticular cells of the boar including Leydig
cells and is absent only in elongated sperma-
tids. Conversely ERα is very weakly ex-
pressed in Leydig cells and is much more
abundant in young germ cells (spermatogo-
nia and PS). Therefore, species differences
are obvious and the pig seems to have a
unique distribution of ERs consistent with the
large amounts of estrogen produced in boar
testes (for a review, see Ref. 25).

In immature germ cells of men we have
identified both in terms of transcripts and
proteins the two main isoforms of ERs, not
only the full-length one but also some vari-
ants (32). Besides the wild type of ERα, an
additional transcript related to the exon1-
deleted variant of ERα with a 46-kDa mo-
lecular mass has been reported in ejaculated
sperm. Concerning ERß, two isoforms (in
terms of transcript and protein) correspond-
ing to the expected sizes (full-length and
shorter one) were detected in germ cells, whereas in spermatozoa only the PCR product was found (49). Aquila et al. (50) have described the presence of $ER\alpha$ and $ER\beta$ in human ejaculated spermatozoa both as transcripts and as proteins corresponding to the well-characterized ER forms. These discrepancies with our studies could be due to the different methodologies used. Recently, Aschim et al. (51) reported the presence of several splice variants of $ER\beta$ in human testicular cells but the proteins have not been yet identified and thus their specific functions remain to be elucidated. Solakidi et al. (52) have also demonstrated by confocal analysis the presence of the two main ERs in ejaculated sperm. In primates, both ERs have been demonstrated but it is clear that Leydig cells mainly express $ER\alpha$ whereas in seminiferous tubules $ER\beta$ and $ER\alpha$ are present especially in PS and RS (for a review, see Ref. 53).

Thus, a general statement can be made: $ER\alpha$ is mainly localized in Leydig cells whereas $ER\beta$ is found in the seminiferous tubules and mainly in germ cells.

## Conclusions: estrogens and male fertility

In conclusion, the net biological effect of estrogen in the testis was long thought to be mainly negative. In view of the widespread distribution of ERs in male gonads and an additional expression of aromatase in germ cells, the role of estrogens in male reproduction is more complex than previously realized. Regarding the role of estrogens in male gamete maturation, we have shown not only the existence of estrogen sources in various germ cells but also the presence of $ER\alpha$ and

$ER\beta$ and, in addition, the presence of a truncated form of $ER\alpha$ (probably localized on the membrane) in ejaculated human sperm. Indeed, the motility and number of spermatozoa are reduced in diminished aromatase-deficient men (for a review, see Ref. 3). In general, an impairment of fertility is observed in men with a defect of the aromatase gene. These observations of decreased sperm motility in men with aromatase deficiency, together with our data showing a significant decrease of aromatase in immotile human spermatozoa, could suggest that aromatase is involved in the acquisition of sperm motility. In mouse as well as in man it has been shown that estrogens are positively involved in sperm capacitation and acrosome reaction (54). The existence of ERs on the sperm membrane (55) and in the mitochondria (52) is likely to be relevant for a role of estrogens in male gamete motility.

Consequently, the production of estrogens by germ cells and the existence of ERs represent good markers which should help to understand the physiological role of estrogens in human spermatogenesis. As also suggested by Ostermeier et al. (56), the genetic fingerprint of fertile men could include the analysis of mRNA profiles and thus aromatase and ERs may be helpful. Nevertheless, it is clear that further studies are necessary to elucidate the real impact of estrogen on human male reproduction, although it is obvious that several steps are involved (54). Even if numerous estrogen-targeted genes remain to be defined, there is now a body of evidence in favor of a positive role for estrogens in male reproduction via genomic and rapid membrane effects (for a review, see Ref. 57).
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