Androgen Receptor In Sertoli Cells Research Articles

Sertoli Cell-Androgen Receptor DNA-Binding Domain Is Essential for Estradiol-Induction of Spermatogenesis.

Androgen receptor (AR) actions are vital for spermatogenesis. Paradoxically, estradiol (E2) induces spermatogenesis and increases circulating FSH levels in androgen-deficient hypogonadal (Gnrh1-/−) but not global AR−/Y mice. We have determined the role of Sertoli cell AR in E2-induced spermatogenic development using our transgenic model with Sertoli cell-specific disruption (in frame Ar exon 3 deletion) of a crucial zinc finger in the AR DNA binding domain; denoted SARΔZF. Mature (10 week old) SARΔZF males were infertile and displayed testes 30% of normal size, which were found to have cytoplasmic Sertoli cell ARΔZF localisation (confocal microscopy). Detailed stereological (optical disector) analysis showed normal total Sertoli and spermatogonial cell numbers, but 50% of normal spermatocyte numbers and limited spermatid development in SARΔZF versus wildtype testes. Serum LH and FSH levels were significantly elevated in SARΔZF males, and total intratesticular testosterone (T), DHT, 3a-diol and 3β-diol levels (ng/ testis) were all significantly elevated in SARΔZF relative to wildtype testes, whereas E2 remained undetectable (LC-MS). Disruption of Sertoli cell AR function had differential effects on expression of steroidogenic factors (qPCR), with elevated Hsb3b6, Hsd17b3 and Lhr mRNA levels contrasting with normal Cyp11a1 and reduced Cyp17a1 and Star mRNA expression in SARΔZF compared to wildtype testes. To determine Sertoli cell AR requirement for E2-induced spermatogenesis, SARΔZF males were treated with E2 (1 cm sc implants) for 6 weeks. Circulating FSH levels were reduced to normal levels in E2-treated SARΔZF males. Total intratesticular T remained elevated, whereas total DHT, 3a-diol and 3β-diol were all reduced to normal levels in E2-treated versus untreated SARΔZF testes. E2 had no effect on Sertoli cell numbers and spermatogenic development in SARΔZF males, indicating that Sertoli AR DNA binding is indispensable for E2-induced meiotic competence and postmeiotic development. Our SARΔZF paradigm represents a valuable model to differentiate Sertoli cell AR-regulation of Leydig cell steroidogenesis and the steroidal induction of spermatogenesis.

Effect of Temporal Sertoli Cell Androgen Receptor Expression on Leydig Cell Development and Function in Mice.

Effect of Temporal Sertoli Cell Androgen Receptor Expression on Leydig Cell Development and Function in Mice.

Identification of testosterone-/androgen receptor-regulated genes in mouse Sertoli cells

Androgen and androgen receptor (AR) play important roles in male spermatogenesis and fertility, yet detailed androgen/AR signals in Sertoli cells remain unclear. To identify AR target genes in Sertoli cells, we analyzed the gene expression profiles of testis between mice lacking AR in Sertoli cells (S-AR(-/y)) and their littermate wild-type (WT) mice. Digital gene expression analysis identified 2276 genes downregulated and 2865 genes upregulated in the S-AR(-/y) mice testis compared to WT ones. To further nail down the difference within Sertoli cells, we first constructed Sertoli cell line TM4 with stably transfected AR (named as TM4/AR) and found androgens failed to transactivate AR in Sertoli TM4 and TM4/AR cells. Interestingly, additional transient transfection of AR-cDNA resulted in significant androgen responsiveness with TM4/AR cells showing 10 times more androgen sensitivity than TM4 cells. In the condition where maximal androgen response was demonstrated, we then analyzed gene expression and found the expression levels of 2313 genes were changed more than twofold by transient transfection of AR-cDNA in the presence of testosterone. Among these genes, 603 androgen-/AR-regulated genes, including 164 upregulated and 439 downregulated, were found in both S-AR(-/y) mice testis and TM4/AR cells. Using informatics analysis, the gene ontology was applied to analyze these androgen-/AR-regulated genes to predict the potential roles of androgen/AR in the process of spermatogenesis. Together, using gene analysis in both S-AR(-/y) mice testis and TM4/AR cells may help us to better understand the androgen/AR signals in Sertoli cells and their influences in spermatogenesis.

Expression of Tubb3, a Beta-Tubulin Isotype, Is Regulated by Androgens in Mouse and Rat Sertoli Cells1

Our previous analysis of Sertoli cell androgen receptor (AR) knockout (SCARKO) mice revealed that several cytoskeletal components are a potential target of androgen action. Here, we found that one of these components, the beta-tubulin isotype Tubb3, is differentially regulated in testes from SCARKO mice (relative to littermate controls) from Postnatal Day 10 to adulthood. The Tubb3 gene is unique in this respect, as at Day 10, no other beta-tubulin genes are significantly regulated by AR. We further characterized androgen regulation of Tubb3 in vivo and in vitro and demonstrated that it is a conserved feature in both mice and rats. To investigate whether androgens directly regulate Tubb3 expression, we screened for androgen response elements (AREs) in the Tubb3 gene. In silico analysis revealed the presence of four ARE motifs in Tubb3 intron 1, two of which bind to AR in vitro. Mutation of one of these (ARE1) strongly reduced androgen-dependent reporter gene expression. These results, coupled with the finding that the AR binds to the Tubb3 ARE region in vivo, suggest that Tubb3 is a direct target of AR. Our data strengthen the contention that androgens exert their effects on spermatogenesis, in part, through modulation of the Sertoli cell cytoskeleton. Androgen regulation of beta-tubulin has also been described in neurons, fortifying the already known similarity in microtubule organization in Sertoli cell processes and neurons, the only other cell type in which Tubb3 is known to be expressed.

Selective ablation of the androgen receptor in mouse sertoli cells affects sertoli cell maturation, barrier formation and cytoskeletal development.

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Early Expression of the Androgen Receptor in the Sertoli Cells of a Marsupial Coincides with Downregulation of Anti-Müllerian Hormone at the Time of Urogenital Virilization

Anti-Müllerian hormone (AMH), responsible for the regression of Müllerian ducts, is strongly expressed by eutherian fetal and postnatal Sertoli cells. Both AMH and testosterone levels are high during the period of fetal reproductive tract virilization which occurs largely in utero in eutherian mammals. Taking advantage of the fact that differentiation of the urogenital tract occurs after birth in marsupials, we studied the ontogeny and regulation of AMH in the tammar wallaby testis and related it to the expression of the androgen receptor in Sertoli cells. Testicular AMH expression was high between days 10–30 post partum, then fell to basal levels by day 60 and remained low until day 90, the oldest age examined. AMH expression was repressed by treatment of male pouch young with the potent androgen androstanediol. Thus, in the tammar, AMH expression decreases in response to androgen at the time of initial urogenital masculinization, in contrast to the situation in humans in which AMH is repressed by testosterone only at the time of puberty. The difference might be explained by the timing of androgen receptor expression which appears in tammar Sertoli cells at around day 40 of pouch life but only at a later developmental stage in eutherians.

Age Dependent Expressiones of Androgen Receptores in Testes and Epididymes of Mandarin Voles（<I>Lasiopodomys mandarinus</I>）

In order to investigate the effects of age on androgen receptor (AR) in testes and epididymides of mandarin voles（Lasiopodomys mandarinus）, the expression of AR from five age groups [postnatal 1(neonatal), 10, 25, 45 and 60 days (adult) of age] was examined using immunohistochemistry method. The results were as follows: ① There was AR expression in leydig cells in neonatal voles and the expression of AR decreased at postnatal 10 days and 25days. AR expression reached its peak at postnatal 45 days and then decreased in adults（P＜0.05）. ② The positive expression of AR in myoid cells was found from neonatal to adult. The positive expression of AR was unchanged at postnatal 1 day and postnatal 10 days, but decreased at postnatal 25 days, reaching its highest level at postnatal 45 days and then decreased in adults (P＜0.05). ③ The positive expression of AR in prospermatogonia was weak in neonatal voles. There was no expression of AR in spermatogonia at postnatal 10 days. There was AR expression in spermatoon at postnatal 25 days, while in spermatocyte and some spermatoon at postnatal 45days. The positive expression of AR in spermatogonia, spermatoon and sperm was also found in adults. ④ The positive expression of AR in sertoli cells was found in adults, but there were few expressions of ARs at other ages. ⑤ The positive cells of ARs were both detected in epithelium cells and stroma cells of epididymides. These results suggest that AR expressiones in leydig cells, myoid cells and spermatogenic cells change significantly with mandarin vole’s individual development. Androgen facilitates leydig cells differentiation during puberty, and myoid cells play an important role during spermatogenesis. Androgen may regulate function of epididymises.

Sertoli Cell Androgen Receptor DNA Binding Domain Is Essential for the Completion of Spermatogenesis

We examined the biological importance of Sertoli cell androgen receptor (AR) genomic interaction, using a Cre-loxP approach to selectively disrupt the AR DNA-binding domain (AR-DBD). Sertoli cell (SC)-specific transgenic Abpa or AMH promoters targeted Cre-mediated inframe excision of mouse Ar exon-3, encoding the AR-DBD second zinc-finger (ZF2), generating SC-specific mutant AR(DeltaZF2) lines designated Abp.SCAR(DeltaZF2) and AMH.SCAR(DeltaZF2), respectively. Both SCAR(DeltaZF2) lines produced infertile males exhibiting spermatogenic arrest, despite normal SC numbers and immunolocalized SC nuclear AR. Adult homozygous TgCre((+/+)) SCAR(DeltaZF2) or double-TgCre((+/-)) Abp/AMH.SCAR(DeltaZF2) males displayed equivalent small testes 30% of normal size, representing maximal Cre-loxP-disruption of Sertoli AR function. Hemizygous TgCre((+/-)) vs. homozygous TgCre((+/+)) Abp.SCAR(DeltaZF2) testes were larger (47% normal size) with more postmeiotic development, indicating dose-dependent Cre-mediated disruption of SC-specific AR-DBD activity. SCAR(DeltaZF2) males exhibited adult Leydig cell hypertrophy but normal serum testosterone levels. Sertoli cell-specific Rhox5 and Spinlw1 transcription, regulated by divergent or classical androgen-response elements, respectively, were both decreased in postnatal SCAR(DeltaZF2) vs. control testes, demonstrating SC-specific AR-DBD function as early as postnatal d 5. However, Rhox5 expression declined dose-dependently, whereas Spinlw1 expression increased, in adult TgCre((+/-)) and TgCre((+/+)) SCAR(DeltaZF2) testes, revealing differential temporal control for distinct AR-regulated transcripts. Androgen-repressed Ngfr was not up-regulated in SCAR(DeltaZF2) testes, suggesting maintenance of a nonclassical mechanism independent of AR-DBD. Thus, our unique SCAR(DeltaZF2) paradigm provided dose-dependent Cre-mediated disruption of testicular development and gene expression revealing that the AR-DBD is essential for SC function and postmeiotic spermatogenesis. Nongenomic or AR-DBD-independent pathways appear secondary or play no major independent role in SC function.

Androgen Receptor Roles in Spermatogenesis and Fertility: Lessons from Testicular Cell-Specific Androgen Receptor Knockout Mice

Androgens are critical steroid hormones that determine the expression of the male phenotype, including the outward development of secondary sex characteristics as well as the initiation and maintenance of spermatogenesis. Their actions are mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily. AR functions as a ligand-dependent transcription factor, regulating expression of an array of androgen-responsive genes. Androgen and the AR play important roles in male spermatogenesis and fertility. The recent generation and characterization of male total and conditional AR knockout mice from different laboratories demonstrated the necessity of AR signaling for both external and internal male phenotype development. As expected, the male total AR knockout mice exhibited female-typical external appearance (including a vagina with a blind end and a clitoris-like phallus), the testis was located abdominally, and germ cell development was severely disrupted, which was similar to a human complete androgen insensitivity syndrome or testicular feminization mouse. However, the process of spermatogenesis is highly dependent on autocrine and paracrine communication among testicular cell types, and the disruption of AR throughout an experimental animal cannot answer the question about how AR in each type of testicular cell can play roles in the process of spermatogenesis. In this review, we provide new insights by comparing the results of cell-specific AR knockout in germ cells, peritubular myoid cells, Leydig cells, and Sertoli cells mouse models that were generated by different laboratories to see the consequent defects in spermatogenesis due to AR loss in different testicular cell types in spermatogenesis. Briefly, this review summarizes these results as follows: 1) the impact of lacking AR in Sertoli cells mainly affects Sertoli cell functions to support and nurture germ cells, leading to spermatogenesis arrest at the diplotene primary spermatocyte stage prior to the accomplishment of first meiotic division; 2) the impact of lacking AR in Leydig cells mainly affects steroidogenic functions leading to arrest of spermatogenesis at the round spermatid stage; 3) the impact of lacking AR in the smooth muscle cells and peritubular myoid cells in mice results in similar fertility despite decreased sperm output as compared to wild-type controls; and 4) the deletion of AR gene in mouse germ cells does not affect spermatogenesis and male fertility. This review tries to clarify the useful information regarding how androgen/AR functions in individual cells of the testis. The future studies of detailed molecular mechanisms in these in vivo animals with cell-specific AR knockout could possibly lead to useful insights for improvements in the treatment of male infertility, hypogonadism, and testicular dysgenesis syndrome, and in attempts to create safe as well as effective male contraceptive methods.

Androgen Receptor in Sertoli Cells Is Not Required for Testosterone-Induced Suppression of Spermatogenesis, but Contributes to Sertoli Cell Organization in Utp14bjsd Mice

Testosterone acting through the androgen receptor (AR) maintains the arrest of spermatogonial differentiation in juvenile spermatogonial depletion (jsd mutation in the Utp14b gene) mutant adult male mice. It is not known which of the somatic cell types expressing AR mediates this inhibition. To determine whether Sertoli cells are responsible, we selectively eliminated AR in Sertoli cells in jsd mice containing a floxed-Ar gene and an anti-Müllerian hormone-Cre transgene. In these Sertoli AR-knockout (SCARKO)-jsd mice, spermatogonial differentiation did not recover. However, the normal organization of Sertoli cell nuclei was drastically disrupted in SCARKO-jsd mice compared with SCARKO or jsd mice. In addition, the extent of ectoplasmic specializations was reduced; tight junctions were not found; vinculin, an anchoring protein found in ectoplasmic specializations, became uniformly distributed in the cytoplasm; and the adult Sertoli cells showed excess heterochromatin subjacent to their nuclear envelope. Despite the abnormalities in Sertoli cells in SCARKO-jsd mice, global suppression of testosterone action and levels was still effective in restoring the differentiated germ cells, and this was accompanied by an improved arrangement of Sertoli cell nuclei. We conclude that Sertoli cells are not targets for the testosterone-mediated inhibition of spermatogonial differentiation in jsd mice, and that both AR in Sertoli cells and the presence of differentiated germ cells contribute to maintaining the organization of Sertoli cells within the seminiferous tubules.

Sertoli Cell-Specific Modification of The Androgen Receptor DNA-Binding Domain Disrupts Sertoli Cell Function and Completion of Spermatogenesis.

Androgen receptor (AR) actions are vital for spermatogenesis, but male germ cells do not express AR, highlighting its key role in testicular somatic cells. Recent genetic models with targeted Sertoli cell (SC) AR disruption confirmed the vital role for Sertoli AR-activated pathways in spermatogenesis. However, complete loss of functional AR in these SC AR knockout (SCARKO) models excludes selective analysis of classical (via direct AR DNA binding) versus potential indirect non-classical or non-genomic AR actions. We have used a Cre-loxP strategy to selectively study the in vivo requirement of AR DNA-binding in SC function. Transgenic (Tg) mice with SC-specific Cre were crossed with mice carrying a floxed-AR gene (Arflox) to target exon 3. Cre-mediated inframe excision of exon 3, encoding a zinc finger essential for DNA-binding, was predicted to generate mutant ARΔex3 to selectively disrupt AR-regulated gene transcription via direct DNA interaction. We established two unique SC-specific ARΔex3 (SCARΔex3) models: i) AMH.SCARΔex3 derived from [Tg antimullerian hormone (AMH) promoter-driven Cre mice] X [Arflox mice], and ii) Abp.SCARΔex3 derived from [Tg androgen-binding protein (Abp) promoter-driven Cre mice] X [Arflox mice]. ROSA-LacZ reporter mice showed that the Abp promoter directs SC-specific Tg expression consistent with our past work, with testis Abp.Cre expressed as early as day 17 postcoitum. RT-PCR detected testis ArΔex3 RNA in both SCARΔex3 models. Using hemizygous TgCre mice, Abp.SCARΔex3 (49.3 ± 1.8 mg) testes were significantly larger (p < 0.001) than AMH.SCARΔex3 testes (29.9 ± 0.9 mg), and both were smaller (p < 0.001) than age-matched 9 week old controls (105.4 ± 4.4 mg). Homozygous TgCre mice were used to increase Cre expression levels. Testis size was further reduced in homozygous Abp.SCARΔex3 (31.6 ± 1.9 mg, p < 0.001) but not homozygous AMH.SCARΔex3 (32.1 ± 1.5 mg) mice relative to corresponding hemizygous TgCre SCARΔex3 testes, suggesting a maximal effect for SC Ar exon 3 disruption was achieved using Tg homozygous Abp.Cre or hemizygous AMH.Cre mice. Immunohistochemistry revealed SC nuclear AR expression was retained in SCARΔex3 testes. Despite histological detection of Leydig cell hypertrophy in SCARΔex3 testes, serum testosterone levels were equivalent to normal (22.8 ± 5.0 nM) in Abp.SCARΔex3 (15.7 ± 8.0 nM) and AMH.SCARΔex3 (16.6 ± 7.0 nM) 9 week old males, unlike the elevated or reduced androgen levels reported in SCARKO models with complete SC AR loss. Stereology revealed total SC numbers were normal in SCARΔex3 testes, whereas spermatogenesis in both SCARΔex3 models showed predominant meiotic arrest, noting larger Abp.SCARΔex3 testes using hemizygous Tg Abp.Cre mice exhibited more post-meiotic development. The testicular phenotype of these unique SCARΔex3 models demonstrates that despite nuclear ARΔex3 localisation, a functional AR DNA-binding domain is vital for Sertoli cell function and full spermatogenic development. The SCARΔex3 testes resemble SCARKO testes with complete SC AR loss, suggesting that non-genomic or pathways independent of direct AR-DNA binding play no major role in SC function. In combination, our novel SCARΔex3 models provide unique opportunity to further determine dose-dependent effects of AR activity in Sertoli cells. This research was supported by NHMRC funding.

Androgen Receptor in Sertoli Cell Is Essential for Germ Cell Nursery and Junctional Complex Formation in Mouse Testes

To examine the role of androgen receptor (AR) in Sertoli cells (SC), we used a SC-specific AR knockout (S-AR-/y) mouse to further evaluate the chronological changes of seminiferous tubules and the molecular mechanisms of SC androgen/AR signals on spermatogenesis. Testes morphology began changing as early as postnatal day (PD) 10.5 in wild-type (WT), but not in S-AR-/y mice. After puberty (PD 50), the SC nuclei of WT testes migrated to the basal area of the seminiferous epithelium; however, in S-AR-/y testes, SC nuclei were disarranged and dislocated. Results from electron microscopy further showed an obvious duplication of basal lamina of the seminiferous epithelium in S-AR-/y testes at PD 50 compared with WT testes. Using quantitative RT-PCR analyses, the expression of SC gene profiles were compared in PD 10.5 testes. In S-AR-/y testes, the expression levels of 1) vimentin were significantly increased and laminin alpha5 was significantly decreased in PD 10.5, which contributed to functional defects in cytoskeletons and production of the basement membrane component of SC leading to cell morphology deterioration and thus affecting the integrity of seminiferous epithelium; 2) claudin-11, occludin, and gelsolin were significantly decreased in PD 10.5, which contributed to defects in intact junctional complex formation of SC leading to impairment of the integrity of the blood-testis barrier; 3) calcium channel, voltage-dependent, P/Q-type, alpha1A subunit; tissue-type plasminogen activator; transferrin; and epidermal fatty-acid-binding protein were significantly decreased in PD 10.5, which contributed to functional defects in production and/or secretion of specific proteases, transport proteins, and paracrine factors of SC, leading to impairment of its germ cells' nursery functions.

Ontogeny of androgen and estrogen receptor expression in porcine testis: Effect of reducing testicular estrogen synthesis

Reducing endogenous estrogen leads to increased proliferation of porcine Sertoli cells during the first 2 months of life. The resulting increase in porcine Sertoli cell numbers is maintained through puberty. The reduced estrogen appears to be the direct hormonal mediator because essentially no changes are observed in other hormones. However, the mechanism for this effect on Sertoli cell proliferation is unknown. The objective of these studies was to evaluate estrogen receptors α and β (ESR1 and ESR2) in conjunction with androgen receptor (AR) on Sertoli cells and other testicular cell types, as an initial step toward understanding how reduced estrogen leads to increased Sertoli cell numbers. Testis sections from treated animals (aromatase inhibition to decrease endogenous estrogen beginning at 1 week of age) and from littermate controls treated with vehicle were subjected to immunocytochemical labeling for ESR1, ESR2, and AR. Three observers scored Sertoli cells, interstitial cells, peritubular myoid cells, and germ cells for intensity of labeling (0: absent; 1+: weak; 2+: moderate; or 3+: strong labeling). AR in Sertoli cells was readily detected at 1 week of age, was very faint in 2-month vehicle controls, and labeling appeared to increase in 3-month vehicle controls. AR in Sertoli cells, interstitial cells, and apparently germ cells was increased in treated animals at 2 months of age compared with the vehicle controls. This increase was confirmed in western blots. ESR1 and ESR 2 were clearly present in Sertoli cells from 1-week-old animals; ESR in Sertoli cells generally decreased with age with the decrease more apparent for ESR2. ESR1 in Sertoli cells and peritubular myoid cells exhibited some treatment-related effects but reduction of endogenous estrogen did not appear to affect ESR2 in the boar testis. The observed alterations in AR and ESR1 may mediate the increases in Sertoli cell proliferation following inhibition of endogenous estrogen production or may reflect the altered function of the Sertoli cells and peritubular myoid cells.

Expression of Aromatase, Estrogen Receptor α and β, Androgen Receptor, and Cytochrome P-450scc in the Human Early Prepubertal Testis

The expression of aromatase, estrogen receptor alpha (ERalpha) and beta (ERbeta), androgen receptor (AR), and cytochrome P-450 side chain cleavage enzyme (cP450scc) was studied in prepubertal testis. Samples were divided in three age groups (GRs): GR1, newborns (1- to 21-d-old neonates, n = 5); GR2, postnatal activation stage (1- to 7-mo-old infants, n = 6); GR3, childhood (12- to 60-mo-old boys, n = 4). Absent or very poor detection of ERalpha by immunohistochemistry in all cells and by mRNA expression was observed. Leydig cells (LCs) of GR1 and GR2 showed strong immunostaining of aromatase and cP450scc but weak staining of ERbeta and AR. Interstitial cells (ICs) and Sertoli cells (SCs) expressed ERbeta, particularly in GR1 and GR2. Strong expression of AR was found in peritubular cells (PCs). For all markers, expression in GR3 was the weakest. In germ cells (GCs), i.e. gonocytes and spermatogonia, aromatase and ERbeta were immunoexpressed strongly whereas no expression of ERalpha, AR, or cP450scc was detected. It is proposed that in newborn and infantile testis, testosterone acting on PCs might modulate infant LC differentiation, whereas the absence of AR in SCs prevents development of spermatogenesis. The role of estrogen is less clear, but it could modulate the preservation of an adequate pool of precursor LCs and GCs.

Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells.

Androgens and the androgen receptor (AR) play important roles in male fertility, although the detailed mechanisms, particularly how androgen/AR influences spermatogenesis in particular cell types, remain unclear. Using a Cre-Lox conditional knockout strategy, we generated a tissue-specific knockout mouse with the AR gene deleted only in Sertoli cells (S-AR(-/y)). Phenotype analyses show the S-AR(-/y) mice were indistinguishable from WT AR mice (B6 AR(+/y)) with the exception of testes, which were significantly atrophied. S-AR(-/y) mice were infertile, with spermatogenic arrest predominately at the diplotene premeiotic stage and almost no sperm detected in the epididymides. S-AR(-/y) mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone concentrations than B6 AR(+/y) mice. Further mechanistic studies demonstrated that S-AR(-/y) mice have defects in the expression of anti-Müllerian hormone, androgen-binding protein, cyclin A1, and sperm-1, which play important roles in the control of spermatogenesis and/or steroidogenesis. Together, our Sertoli cell-specific AR knockout mice provide in vivo evidence of the need for functional AR in Sertoli cells to maintain normal spermatogenesis and testosterone production, and ensure normal male fertility.

A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis.

Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre/loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Müllerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte/spermatid development/survival critically depends on androgens.

Expression of the E3 SUMO-1 ligases PIASx and PIAS1 during spermatogenesis in the rat

PIASx and PIAS1, two members of the conserved protein inhibitor of activated STAT (PIAS) family, are able to interact with and modulate activities of several distinct nuclear proteins, including androgen receptor (AR). PIASx and PIAS1 also function as E3-type ligases in small ubiquitin-related modifier 1 modifications. To gain better insight into the physiological roles of PIASx and PIAS1 in vivo, their cell-type specific expression and regulation were analyzed during testicular development and spermatogenesis in the rat. The expression of both PIASx and PIAS1 was low or undetectable in newborn rats. PIASx mRNA started to accumulate after day 20 of postnatal life, whereas expression of PIAS1 mRNA increased around day 30 after birth. In the adult rat testis, both PIASx and PIAS1 mRNA were present in Sertoli cells and in germ cells in the seminiferous epithelium at all stages. However, PIASx mRNA was more abundant in spermatocytes than in other cell types, whereas higher levels of PIAS1 mRNA were detected in late spermatocytes and round spermatids than in early spermatocytes. Since PIASx and PIAS1 accumulate in developing male germ cells, their regulatory functions are not only restricted to AR in Sertoli cells, but they also participate in molecular processes during meiosis.

Androgen receptor expression in sertoli cells as a function of seminiferous tubule maturation in the human cryptorchid testis.

Androgen receptor (AR) immunohistochemistry was performed in an archival collection of adult human cryptorchid testes to determine whether AR cellular distribution and intensity of immunostaining were functions of the severity of cellular dysgenesis. The seminiferous tubule histology of cryptorchid testes collected from adults is marked by three specific patterns. 1) Seminiferous tubules are characterized as maintaining focal areas of germinal cell differentiation (albeit incomplete) that are interspersed with 2) tubules composed of Sertoli cells only, these latter cells being principally of the adult type, although dysgenetic and immature Sertoli cells may also be detected. 3) In contrast, there is a class of tubule that is characterized as being composed exclusively of Sertoli cells that are extremely dysgenetic in appearance. The majority of adult-type Sertoli cells found in the first types of tubules exhibited either robust or moderate AR staining intensity. Peritubular cells of these tubules also expressed a similar AR staining intensity. In contrast, in the more dysgenetic and immature type Sertoli cells found in the second type of tubules, the intensity of AR staining was significantly less, if not missing altogether. Finally, in the most dysgenetic tubules, Sertoli cell AR staining was never detected. To our knowledge, this is the first report in the literature that addresses the intensity of AR immunostaining in Sertoli cells of cryptorchid testes. The results presented herein are consistent with the interpretation that the intensity of AR staining in Sertoli cells diminishes as a function of the severity to which the cells are afflicted within a cryptorchid testis and that focal absence of AR expression in Sertoli cells correlates with a lack of local spermatogenesis in the tubules.

Protein inhibitor of activated STAT-1 (signal transducer and activator of transcription-1) is a nuclear receptor coregulator expressed in human testis.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquid beta-galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent. [35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544-634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25-30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.

Protein Inhibitor of Activated STAT-1 (Signal Transducer and Activator of Transcription-1) Is a Nuclear Receptor Coregulator Expressed in Human Testis

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquidβ -galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent.[ 35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544–634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25–30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.