Estrogen in the adult male reproductive tract: A review

Testosterone and estrogen are no longer considered male only and female only hormones. Both hormones are important in both sexes. It was known as early as the 1930's that developmental exposure to a high dose of estrogen causes malformation of the male reproductive tract, but the early formative years of reproductive biology as a discipline did not recognize the importance of estrogen in regulating the normal function of the adult male reproductive tract. In the adult testis, estrogen is synthesized by Leydig cells and the germ cells, producing a relatively high concentration in rete testis fluid. Estrogen receptors are present in the testis, efferent ductules and epididymis of most species. However, estrogen receptor-α is reported absent in the testis of a few species, including man. Estrogen receptors are abundant in the efferent ductule epithelium, where their primary function is to regulate the expression of proteins involved in fluid reabsorption. Disruption of the α-receptor, either in the knockout (αERKO) or by treatment with a pure antiestrogen, results in dilution of cauda epididymal sperm, disruption of sperm morphology, inhibition of sodium transport and subsequent water reabsorption, increased secretion of Cl(-), and eventual decreased fertility. In addition to this primary regulation of luminal fluid and ion transport, estrogen is also responsible for maintaining a differentiated epithelial morphology. Thus, we conclude that estrogen or its α-receptor is an absolute necessity for fertility in the male

E2f4 and E2f5 are essential for the development of the male reproductive system

The E2F transcription factors are primarily implicated in the regulation of entry and exit from the cell cycle. However, in vivo studies have established additional roles for E2Fs during organ development and homeostasis. With the goal of addressing the intestinal requirements of E2f4 and E2f5, we crossed mice carrying Vil-cre, E2f4 conditional and E2f5 germline alleles. E2f4 deletion had no detectable effect on intestinal development. However, E2f4f/f;E2f5+/−;Vil-cre males, but not E2f4f/f;Vil-cre littermates, were unexpectedly sterile. This defect was not due to defective spermatogenesis. Instead, the seminiferous tubules and rete testes showed significant dilation, and spermatozoa accumulated aberrantly in the rete testis and efferent ducts. Our data show that these problems result from defective efferent ducts, a tissue whose primary function is to concentrate sperm through fluid absorption. First, Vil-cre expression, and consequent E2F4 loss, was specific to the efferent ducts and not other reproductive tract tissues. Second, the E2f4f/f;E2f5+/−;Vil-cre efferent ducts had completely lost multiciliated cells and greatly reduced levels of critical absorptive cell proteins: aquaporin1, a water channel protein, and clusterin, an endocytic marker. Collectively, the observed testis phenotypes suggest a fluid flux defect. Remarkably, we observed rete testis dilation prior to the normal time of seminiferous fluid production, arguing that the efferent duct defects promote excessive secretory activity within the reproductive tract. Finally, we also detect key aspects of these testis defects in E2f5−/− mice. Thus, we conclude that E2f4 and E2f5 display overlapping roles in controlling the normal development of the male reproductive system.National Institutes of Health (U.S.) (Grant NIH-P01 CA42063

The antiestrogen ICI 182,780 induces early effects on the adult male mouse reproductive tract and long-term decreased fertility without testicular atrophy

BACKGROUND: Estrogen receptors (ER) have important physiological roles in both the female and male reproductive systems. Previous studies using the estrogen receptor-α knockout mouse (αERKO) or antiestrogen treatment in adult rodents have shown that ERα is essential for normal function of the male reproductive tract. In the present study, time-response effects of the antiestrogen ICI 182,780 were determined to better understand ERα function in the adult male. METHODS: Adult male mice, 30 days old, were injected subcutaneously with ICI 182,780 (5 mg) once per week for 17 weeks. Tissues were fixed by vascular perfusion to study the time responses from day 2 to 125 post treatment. RESULTS: No difference was seen in body weight due to treatment. Testis weight was decreased 18% on day 59 and 21.4% on day 125. Other significant treatment-related effects included the following: 1) dilation of rete testis and efferent ductule lumen; 2) decreased height of the rete testis and efferent ductule epithelium; 3) decreased height of the supranuclear epithelial cytoplasm in efferent ductules; 4) decreased height of the efferent ductule epithelial microvilli, particularly in the proximal ductules; 5) decrease in the PAS-positive granules and endocytotic vesicles in nonciliated epithelial cells of efferent ductules; 6) capping and vesiculation of narrow cells in the initial segment of the epididymis; 7) accumulation of PAS-positive granules in apical cells of the caput epididymis; 8) increase in lysosomal granules in clear cells of the corpus and cauda epididymis; 9) limited induction of atrophic seminiferous tubules and abnormal spermatogenesis; and 10) decreases in the concentration of cauda sperm, progressive sperm motility and decreased fertility. CONCLUSIONS: Antiestrogen treatment of the pubertal male mouse resulted in reproductive effects similar to those observed in the αERKO mouse as early as day 4; however, testis weight did not increase substantially and total atrophy was not observed with extended treatment

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules

BACKGROUND: The antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown. METHODS: In the present study, adult male rats were treated with ICI 182,780 for 7 to 150 days, to evaluate the time-response effects of the treatment on the pattern of ERα, ERβ and AR protein expression in the efferent ductules. The receptors were localized using immunohistochemistry. RESULTS: ERα, ERβ and AR have distinct cellular distribution in the testis and efferent ductules. Staining for ERα is nearly opposite of that for ERβ, as ERα shows an increase in staining intensity from proximal to distal efferent ductules, whereas ERβ shows the reverse. Androgen receptor follows that of ERα. ICI 182,780 caused a gradual but dramatic decrease in ERα expression in the testis and efferent ductules, but no change in ERβ and AR expression. CONCLUSIONS: The differential response of ERα and ERβ proteins to ICI 182,780 indicates that these receptors are regulated by different mechanisms in the male reproductive tract

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

BACKGROUND: Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite. METHODS: To test this hypothesis, adult male rats were submitted to surgical castration followed by estradiol, DHT or 3-beta-diol replacement. Changes in AQP9 expression in the efferent ductules were investigated by using immunohistochemistry and Western blotting assay. RESULTS: Data show that, after castration, AQP9 expression was significantly reduced in the efferent ductules. 3-beta-diol injections restored AQP9 expression, similar to DHT and estradiol. The results were confirmed by Western blotting assay. CONCLUSION: This is the first evidence that 3-beta-diol has biological activity in the male reproductive tract and that this androgen metabolite has estrogen-like activity in the efferent ductules, whose major function is the reabsorption of luminal fluid

Mechanistic insights into the regulation of the spermatogonial stem cell niche

Potential therapeutic use of stem cells in the treatment of human diseases depends on our ability to control the balance of their differentiation and self-renewal in vitro and in vivo. The stem cell “niche,” or specialized microenvironment, is now recognized as one of the major contributors to this regulation in many species. Our recent study, which was reported in Nature, was the first to demonstrate that expression of a vertebrate animal transcription factor is essential for the maintenance of a stem cell niche. In that letter, targeted disruption of ERM (ets-related molecule), which was localized only in the somatic support cell of the testis, the Sertoli cell, resulted in failure of self-renewal by spermatogonial stem cells, following the first wave of spermatogenesis. One of the more important conclusions drawn was the realization that regulation of the stem cell niche during the perinatal period, a phase characterized by rapid mitosis of both spermatogonial stem cells and Sertoli cells, differed from that in the adult. It appears that the ERM-regulated pathways are coincident with the termination of Sertoli cell proliferation and commencement of the cycle of spermatogenesis, which is sustained by the same cell that regulates the stem cell niche. Several likely targets for ERM regulation are discussed, as well as their potential implications for increasing our understanding of spermatogonial stem cell activity and the uniqueness of the Sertoli cell's immune privilege and possible utility for the protection of transplanted adult stem cells

Intraflagellar transport protein IFT20 is essential for male fertility and spermiogenesis in mice

Intraflagellar transport (IFT) is a conserved mechanism thought to be essential for the assembly and maintenance of cilia and flagella. However, little is known about its role in mammalian sperm flagella formation. To fill this gap, we disrupted the Ift20 gene in male germ cells. Homozygous mutant mice were infertile with significantly reduced sperm counts and motility. In addition, abnormally shaped elongating spermatid heads and bulbous round spermatids were found in the lumen of the seminiferous tubules. Electron microscopy revealed increased cytoplasmic vesicles, fiber-like structures, abnormal accumulation of mitochondria and a decrease in mature lysosomes. The few developed sperm had disrupted axonemes and some retained cytoplasmic lobe components on the flagella. ODF2 and SPAG16L, two sperm flagella proteins failed to be incorporated into sperm tails of the mutant mice, and in the germ cells, both were assembled into complexes with lighter density in the absence of IFT20. Disrupting IFT20 did not significantly change expression levels of IFT88, a component of IFT-B complex, and IFT140, a component of IFT-A complex. Even though the expression level of an autophagy core protein that associates with IFT20, ATG16, was reduced in the testis of the Ift20 mutant mice, expression levels of other major autophagy markers, including LC3 and ubiquitin were not changed. Our studies suggest that IFT20 is essential for male fertility and spermiogenesis in mice, and its major function is to transport cargo proteins for sperm flagella formation. It also appears to be involved in removing excess cytoplasmic components

Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the 9+2\u27\u27 axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required for ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27

Differential hormonal regulation of estrogen receptors ERalpha and ERbeta and androgen receptor expression in rat efferent ductules

Estrogen receptors, in addition to the androgen receptor (AR), are expressed at high levels in efferent ductules of the male reproductive tract and it is now well recognized that estrogen receptor (ER) α is required for the maintenance of normal structure and function of the ductules. However, little is known regarding the hormonal regulation of the receptors themselves in the male. In the present study, efferent ductule ligation and castration, followed by replacement with testosterone, dihydrotestosterone (DHT) or estradiol was used to investigate the relative importance of circulating and luminal sources of steroid for the modulation of ERα, ERβ and AR in rat efferent ductules. Uni- or bilateral castration and ligation did not affect the expression of ERα and ERβ, but bilateral castration caused down-regulation of AR. Replacement with DHT and testosterone alone or in combination with estradiol caused the recovery of AR expression to control levels. A slight recovery of AR was also observed after estrogen replacement. ERα expression was decreased to nearly undetectable levels after estrogen replacement. On the other hand, ERβ did not show evident effects following any of the treatments, suggesting a constitutive expression of this receptor. This differential modulation of the steroid hormone receptors highlights the importance of maintaining a physiological androgen-estrogen balance to regulate the structure and function of efferent ductules in the male