Estrogens In Male Reproduction Research Articles

Seasonal variations of aromatase and estrogen receptors expression in the testis of free-ranging sand rats

An increasing number of studies revealed the importance of estrogen in male reproduction. However, most research was conducted in laboratory rodents subjected to standardized environmental conditions. Therefore, seasonal regulations of estrogen pathways remain poorly understood under natural conditions. Using immunohistochemistry, the expression of several molecules involved in the functioning of testis (i.e. 17-β estradiol [E2], P450 aromatase, estrogen receptors ESR1, ESR2, and GPER1 [also known as GPR30]) were investigated in free-ranging fat sand rats, Psammomys obesus, during the breeding and resting seasons. Leydig cells showed a strong immunoreactivity for aromatase in the testis sampled during the breeding season only; however, E2, ESR1, ESR2 and GPER1 were present during both seasons. Sertoli cells showed a positive signal for E2 and ESR2 during the breeding season; though, all molecules, except GPER1, were present during the resting season. Spermatogonia were reactive for E2, ESR2 and GPER1 during the breeding season and for ESR1 and GPER1 during the resting season. During both seasons, spermatocytes-I presented a moderate reactivity for E2, ESR1, ESR2 and a strong reactivity for GPER1; aromatase was detected during the resting season only. Spermatids and spermatozoa were present exclusively during breeding season and were reactive for all molecules; except round spermatids that were negative for aromatase. The functioning of the testis depends on finely tuned stimulation and inhibition systems. Our results suggest that differential expression of aromatase, ESR1, ESR2, and GPER1 across cells types is involved in the seasonal activation/inactivation cycle of spermatogenesis in a free-ranging species.

The Effects of Exogenous Estrogens on Estrogen Receptors in Male Reproductive Organs

There is an essential physiological role for estrogen in male reproduction. Conversely, exposure to exogenous sources of estrogen has negative effects on reproductive physiology and fertility in men. Infertility, affecting nearly 15% of couples, is defined as the inability to conceive after one year of unprotected sexual intercourse. In at least 20% of cases, male reproductive pathology is the major cause for a couple’s infertility. Thus, it is essential to investigate potential causes of infertility in adult males. Evidence shows that exposure to certain endocrine disruptors is associated with reduced semen quality and impaired fertility in men. Bisphenol A and Diethylstilbestrol are endocrine disruptors that act as exogenous sources of estrogen and have been associated with male reproductive pathology. This review will examine the role of exogenous estrogens on changes in gene expression of estrogen receptors ERα, ERβ, and GPR30. Previous studies have had conflictive results, suggesting that the effects of exogenous estrogens on male reproduction are multi-faceted. Future studies should focus on determining whether exogenous estrogens have a stimulatory and/or inhibitory effect on gene expression and whether this relationship is dose-dependent or if it follows a more complex dosage pattern.

Identification of a Membrane Estrogen Receptor in Zebrafish with Homology to Mammalian GPER and Its High Expression in Early Germ Cells of the Testis1

To study the rapid action of estrogen on the male reproductive system in teleost, a full-length cDNA homologous to the seven-transmembrane receptor GPER of humans and rodents was cloned from the testis of zebrafish. Biological characterization of this cloned zebrafish gper was performed based on its functional expression in cultured eukaryotic cells. Saturation analysis and Scatchard plotting of [(3)H]-estradiol binding to plasma membranes of gper-transfected COS-7 cells and cAMP response element transactivation assay demonstrated the biological function of the cloned gper as an estrogen receptor. In addition, treatment of gper-transfected COS-7 cells with 17beta-estradiol increased the phosphorylation of MAPK3/MAPK1. However, the inactivity of Gper in the FOS promoter transactivation study indicated some functional difference between the zebrafish and human receptors. We found gper to be highly expressed in the brain and testis by RT-PCR analysis. Results of in situ hybridization demonstrated the localization of gper in specific brain regions and in early germ cells of the testis, including the spermatogonia, spermatocytes, and somatic cells such as Sertoli cells in adult male zebrafish. Subsequent RT-PCR analysis in cells derived from laser capture microdissection microscopy further confirmed the high expression of gper in early germ cells of the testis. The present study demonstrates the existence of a functionally active Gper in zebrafish and suggests a putative role in mediating the rapid action of estrogen in male reproduction.

A Therapeutic Scheme For Oligospermia Based On Serum Levels Of FSH And Estradiol

Oligospermia is a major cause of infertility in couples. It is usually managed by assisted reproductive techniques. In these techniques the female partners are treated, while the male partners having this medical problem are left untreated! Though several empirical therapies are available to treat oligospermia, it is difficult to identify a therapy that is most likely to benefit a man having oligospermia. This leads to trial of different therapies resulting in varied success rates. These empirical therapies put emphasis on the stimulatory role of testosterone in the process of spermatogenesis while estrogens are considered inhibitory in this respect. In the last decade, however, positive role of estrogens in male reproduction has been reported by some authors. In view of this evidence, a therapeutic scheme for oligospermia based on serum levels of FSH and estradiol, is presented. Its aim is to develop a system by which a clinician may possibly identify a suitable therapy before starting the treatment of an infertile man. Thus it may reduce unnecessary trial of treatments and save on costs and time, and it may save some women from being treated for a problem of male infertility.

Estrogen receptor α promoter polymorphism: stronger estrogen action is coupled with lower sperm count

Although the importance of estrogens in male reproduction is indisputable, little attention has been paid to the role of estrogen receptor (ER) gene mutations in male infertility. Significant correlation between (TA)n repeat allelic variants and lumbar bone mineral density was previously observed in the promoter region of the ERalpha gene, indicating that allelic combinations with higher number of (TA)n repeats are functionally more active genetic variants. We studied the (TA)n repeat polymorphism situated in the promoter region of the ERalpha gene in a large group of infertile and normospermic men (n = 347). Although the (TA)n polymorphism failed to show a significant association with male infertility, we found a significant effect of this polymorphism on sperm count. In the group of infertile men, the mean TA repeat number and sperm concentration (P = 0.022) and total sperm number (P = 0.043) were inversely correlated, showing an association between higher TA repeat number (genotype A) and lower sperm production. In line with this observation, normospermic subjects with genotype A had a significantly lower mean sperm concentration with respect to men bearing genotype B with shorter TA alleles (P < 0.05) and a lower total sperm count (P < 0.01). Our data indicate that specific allelic combinations of the ERalpha, which confer a stronger estrogen effect, may negatively influence human spermatogenesis.

The penis: a new target and source of estrogen in male reproduction.

In the past decade, interest and knowledge in the role of estrogen in male reproduction and fertility has gained significant momentum. More recently, the cellular distribution and activity of estrogen receptors (alpha and beta)(ER) and aromatase (estrogen synthesis) has been reported in the penis, making the penis the latest "frontier" in the study of estrogen in male reproduction. ER and aromatase are broadly and abundantly expressed in various penile compartments and cell types (erectile tissues, urethral epithelia, vascular and neuronal cells), suggesting the complexity and significance of the estrogen-ER system in penile events. Unraveling this complexity is important and will require utilization of the various resources that are now at our disposal including, animal models and human lacking or deficient in ER and aromatase and the use of advanced and sensitive techniques. Some of the obvious areas that require our attention include: 1) a comprehensive mapping of ER-alpha and -beta cellular expression in the different penile compartments and subpopulations of cells, 2) delineation of the specific roles of estrogen in the different subpopulations of cells, 3) establishing the relationship of the estrogen-ER system with the androgen-androgen receptor system, if any, and 4) characterizing the specific penile phenotypes in human and animals lacking or deficient in estrogen and ER. Some data generated thus far, although preliminary, appear to challenge the long held dogma that, overall, androgens have a regulatory monopoly of penile development and function.

Human ejaculated spermatozoa contain active P450 aromatase.

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 (3)H(2)O 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.

Human Ejaculated Spermatozoa Contain Active P450 Aromatase

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.

Estradiol acts as a germ cell survival factor in the human testis in vitro.

The necessity of estrogens for male fertility was recently discovered in studies on both estrogen receptor alpha knockout and aromatase (cyp 19 gene) knockout mice. However, direct testicular effects of estrogens in male reproduction have remained unclear. Here we studied the protein expression of ERalpha and the recently described estrogen receptor beta in the human seminiferous epithelium and evaluated the role of 17beta-estradiol, the main physiological estrogen, in male germ cell survival. Interestingly, both estrogen receptors alpha and beta were found in early meiotic spermatocytes and elongating spermatids of the human testis. Furthermore, low concentrations of 17beta-estradiol (10(-9) and 10(-10) mol/L) effectively inhibited male germ cell apoptosis, which was induced in vitro by incubating segments of human seminiferous tubules without survival factors (i.e. serum and hormones). Dihydrotestosterone, which, in addition to estradiol, is an end metabolite of testosterone, was also capable of inhibiting testicular apoptosis, but at a far higher concentration (10(-7) mol/L) than estradiol. Thus, estradiol appears to be a potent germ cell survival factor in the human testis. The novel findings of the present study together with the previously reported indirect effects of estrogens on male germ cells indicate the importance of estrogens for the normal function of the testis.

Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene.

It is well established that spermatogenesis is controlled by gonadotrophins and testosterone. However, a role for estrogens in male reproduction recently was suggested in adult mice deficient in estrogen receptor alpha. These mice became infertile primarily because of an interruption of fluid reabsorption by the efferent ductules of the epididymis, thus leading to a disruption of the seminiferous epithelium [Hess, R. A., Bunick, D., Lee, K. H., Bahr, J., Taylor, J. A., Korach, K. S., and Lubahn, D. B. (1997) Nature (London) 390, 509-512]. Despite the demonstration of the aromatase enzyme, which converts androgens to estrogens, and estrogen receptors within the rodent seminiferous epithelium, the role of aromatase and estrogen in germ cell development is unknown. We have investigated spermatogenesis in mice that lack aromatase because of the targeted disruption of the cyp19 gene (ArKO). Male mice deficient in aromatase were initially fertile but developed progressive infertility, until their ability to sire pups was severely impaired. The mice deficient in aromatase developed disruptions to spermatogenesis between 4.5 months and 1 year, despite no decreases in gonadotrophins or androgens. Spermatogenesis primarily was arrested at early spermiogenic stages, as characterized by an increase in apoptosis and the appearance of multinucleated cells, and there was a significant reduction in round and elongated spermatids, but no changes in Sertoli cells and earlier germ cells. In addition, Leydig cell hyperplasia/hypertrophy was evident, presumably as a consequence of increased circulating luteinizing hormone. Our findings indicate that local expression of aromatase is essential for spermatogenesis and provide evidence for a direct action of estrogen on male germ cell development and thus fertility.

Characterization of an estrogen receptor in the testis of the urodele amphibian Necturus maculosus.

The exceptionally high rate of aromatization previously demonstrated in the testis of the urodele amphibian Necturus maculosus prompted us to investigate estrogen binding activity in this tissue. When crude cytosolic extracts were incubated with [3H] estradiol prior to DNA-cellulose affinity chromatography or sucrose gradient centrifugation, no estrogen binding was detected. Under these conditions, the presence of an endogenous estrogen-conjugating system in the crude cytosol resulted in extensive conversion of radiolabeled tracer to sulfoconjugates which presumably were poor receptor ligands; however, when this enzyme was first removed from the crude cytosol by fractionation through DNA-cellulose columns, DNA-adhering components could then be labeled with radioactive estradiol. This post-labeling method revealed an estrogen binding component with the physicochemical properties of a classical estrogen receptor: high affinity (Kd=10−10); low capacity (2–5 fmol/mg protein); and affinity for estrogenic steroids only. On sucrose gradients the estrogen receptor complex eluted from DNA-cellulose had a sedimentation coefficient of 4.7–5S. Estrogen receptors having properties similar to the cytosolic form were also demonstrated in nuclear extracts prepared from testes of untreated animals, suggesting that translocation of estrogen synthesized from endogenous precursors is a normal in vivo occurrence in this species. Preliminary measurements indicate that both cytoplasmic and nuclear estrogen receptors may show seasonal changes keyed to the annual reproductive cycle. We reported earlier that estrogen binding activity in the crude cytosol of Necturus testis is located mainly in regions comprised of differentiating or fully differentiated Leydig cells. Further studies of aromatization, the estrogen receptor system, and the estrogen-conjugating enzyme in Necturus testis may provide new insights into the role of intratesticular estrogen in male reproduction.