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Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (ARLmon/Y). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in ARLmon/Y mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation in vivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition. ispartof: Embo Reports vol:22 issue:12 pages:1-19 ispartof: location:England status: published

N-methyl pyrrolidone (NMP) is an FDA approved molecule used as an excipient in pharmaceutical industry. Besides having a central role in formulation of drugs, the most important function of any excipient is to guarantee the safety of the medicine during and after its administration. Several studies have shown that exposure to NMP and especially in rats produce a gonadotoxic effect leading to infertility. However, the mechanisms underlying the effect of NMP on male reproduction are unknown. The aim of this study was to assess the reproductive toxicity of NMP in male rats and to elucidate the underlying mechanism. Male Sprague Dawley rats were injected intraperitoneally, twice/ week, at a dose of 108 mg/ 100 g of body weight with NMP. Analysis of reproductive parameters revealed testicular atrophy in NMP treated animals compared to control animals. Germ cell composition within the seminiferous tubules was disturbed and manifested in an increase in number of cells with fragmented DNA. A subsequent decrease in number of spermatocytes and spermatids was observed. Alpha screen assay shows that NMP acts at the concentrations we applied in vivo as a low affinity inhibitor for BRDT (testis specific bromodomain protein). BRDT inhibition is mirrored by a significant decrease in the expression of early stage spermatocyte markers (lmna, aurkc and ccna1), during which BRDT expression predominates. A significant decrease in testosterone levels was also observed. Since NMP interferes with spermatogenesis on various levels, its use in humans must be carefully monitored.

Introduction: The testis is an immune privileged organ that provides a specific environment for germ cell development. Various factors responsible for inflammatory changes can lead to deterioration of the immune tolerant model found in the testis. As a result, the thickness of the proper membrane of seminiferous tubules changes and the process of spermatogenesis is disturbed. Aim: The purpose of the present study was to find the connection between the changes in the level of testis-specific isoform of angiotensin-converting enzyme (tACE) expression and the morphological changes of the seminiferous tubule wall of the testis in patients with infertility. Materials and methods: The study included 24 infertile men who underwent a testicular biopsy. Routine histological techniques, immunohistochemical reactions for tACE, α-smooth muscle actin, and morphometric analysis were performed to examine the biopsy preparations. Results: By using testicular biopsy to diagnose patients with infertility, a stage-specific pattern of the processes associated with thickened proper membrane of seminiferous tubules was established and a decreased or absent spermatogenic activity was observed. Conclusions: A significant increase in the proper membrane thickness of the seminiferous tubules in the testis was found in patients with infertility. This finding shows that the processes take place gradually over time, correlating with the degree of pathology, and that changes do not depend on the factors causing them. We also found that the degree of proper membrane thickening correlated with disturbances of spermatogenesis, using tACE expression as a marker for spermatogenic epithelium differentiation.&nbsp

N, N-Dimethylacetamide is an FDA approved solvent widely used in pharmaceutical industry to facilitate the solubility of lipophilic, high molecular weight drugs with poor water solubility. However, the cytotoxic effects of DMA raises the concern about its use in clinical applications. In the present study, we address the effect of DMA on spermatogenesis. Male Sprague Dawley rats were injected intra-peritoneally for 8 weeks, once a week at a dose of 862 mg/kg. Analysis of reproductive parameters revealed that DMA treated animals exhibit spermatid formation defects within the testis describing the characteristics of oligozoospermia. A subsequent decrease in epididymal sperm concentration along with distortion of sperm morphology was observed. The mitochondrial and microtubule organization in the sperm is considerably modified by DMA. This disrupts the sperm kinetics thus decreasing the total and progressive sperm motility. Finally, DMA treatment resulted in loss of fertility. Our results indicate that exposure to DMA has a negative impact on spermatogenesis and leads to infertility in male rats by inhibiting the post meiotic stages of sperm development. Therefore, the use of DMA in humans must be closely monitored.

The IGFs are the major intratesticular factors regulating immature Sertoli cell proliferation and are, therefore, critical to establish the magnitude of sperm production. However, the intratesticular source of IGF production and the downstream signaling pathway mediating IGF-dependent Sertoli cell proliferation remain unclear. Single-cell RNA sequencing on mouse embryonic testis revealed a robust expression of Igf1 and Igf2 in interstitial steroidogenic progenitors, suggesting that IGFs exert paracrine actions on immature Sertoli cells. To elucidate the intracellular signaling mechanism that underlies the proliferative effects of IGFs on immature Sertoli cells, we have generated mice with Sertoli cell-specific deletion of the Pten gene, a negative regulator of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway, alone or together with the insulin receptor (Insr) and the IGF1 receptor (Igf1r). Although ablation of Pten appears dispensable for Sertoli cell proliferation and spermatogenesis, inactivation of Pten in the absence of Insr and Igf1r rescued the Sertoli cell proliferation rate during late fetal development, testis size, and sperm production. Overall, these findings suggest that IGFs secreted by interstitial progenitor cells act in a paracrine fashion to promote the proliferation of immature Sertoli cells through the IGF/PTEN/PI3K pathway.

Aims Experimental and clinical studies have demonstrated that components of renin-angiotensin system are elevated in the hippocampus in epileptogenic conditions. In the present work, we explored the changes in the expression of angiotensin II receptor, type 1 (AT1 receptor) in limbic structures, as well as the effect of the AT1 receptor antagonist losartan in a model of comorbid hypertension and epilepsy. Main methods The expression of AT1 receptors was compared between spontaneously hypertensive rats (SHRs) and Wistar rats by using immunohistochemistry in the kainate (KA) model of temporal lobe epilepsy (TLE). The effect of losartan was studied on AT1 receptor expression in epileptic rats that were treated for a period of 4 weeks after status epilepticus. Key findings The naive and epileptic SHRs were characterized by stronger protein expression of AT1 receptor than normotensive Wistar rats in the CA1, CA3a, CA3b, CA3c field and the hilus of the dentate gyrus of the dorsal hippocampus but fewer cells were immunostained in the piriform cortex. Increased AT1 immunostaining was observed in the basolateral amygdala of epileptic SHRs but not of epileptic Wistar rats. Losartan exerted stronger and structure-dependent suppression of AT1 receptor expression in SHRs compared to Wistar rats. Significance Our results confirm the important role of AT1 receptor in epilepsy and suggest that the AT1receptor antagonists could be used as a therapeutic strategy for treatment of comorbid hypertension and epilepsy.

Leukemia inhibitory factor (LIF), a pleiotropic cytokine belonging to the interleukin-6 family, is most often noted for its role in maintaining the balance between stem cell proliferation and differentiation. In rodents, LIF is expressed in both the fetal and adult testis; with the peritubular myoid (PTM) cells thought to be the main site of production. Given their anatomical location, LIF produced by PTM cells may act both on intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, the leukemia inhibitory factor receptor (LIFR) is expressed in germ cells, Sertoli cells, Leydig cells, PTM cells and testicular macrophages, suggesting that LIF signalling via LIFR may be a key paracrine regulator of testicular function. However, a precise role(s) for testicular LIFR-signalling in vivo has not been established. To this end, we generated and characterised the testicular phenotype of mice lacking LIFR either in germ cells, Sertoli cells or both, to identify a role for LIFR-signalling in testicular development/function. Our analyses reveal that LIFR is dispensable in germ cells for normal spermatogenesis. However, Sertoli cell LIFR ablation results in a degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules.

Experimental and clinical studies have demonstrated that components of renin-angiotensin system are elevated in the hippocampus in epileptogenic conditions. In the present work, we explored the changes in the expression of angiotensin II receptor, type 1 (ATThe expression of ATThe naive and epileptic SHRs were characterized by stronger protein expression of ATOur results confirm the important role of AT

Cobalt is a transition metal and an essential trace element that is required for vitamin B12 biosynthesis, enzyme activation, and so on but is toxic in high concentrations. It was shown that the content of different elements in the plasma of2-month-old BALB/c mice (control group) decreased inthe following order: Ca>Mg>Si>Fe>Zn>Cu ≥Al ≥ B. The treatment of mice with CoCl2 did not appreciably change the relative content of Ca, Cu, and Zn, but a significant increase in the content of B (2.3-fold), Mg (1.5-fold), Al and Fe (2.1-fold), and Si (3.4-fold) was found. The treatment of mice led to a 2.2-fold decrease in the concentration of the total blood protein and a 1.70.2-fold decrease of total immunoglobulin Gs (IgGs). Deoxyribonuclease IgGs corresponding to mice treated (t-IgGs) and non-treated (nt-IgGs) with CoCl2 contained intrinsically bound metal ions; these IgGs hydrolyzed DNA with very low activity but were not active in the presence of ethylenediaminetetraacetic acid or after Ab dialysis against ethylenediaminetetraacetic acid. The average RAs of deoxyribonuclease nt-IgGs increased after addition of external metal ions in the following order: Zn 2+ Co 2+ (5.6-fold) Zn 2+ (5.1-fold)>Mg 2+ (4.1-fold)>Ca 2+ (3.0-fold)>Fe 2+ (1.3-fold). However, the RAs of t-IgGs were remarkably more active than nt-IgGs in the presence of best activators of t-IgGs Ni 2+ (1.4-fold) and especially Mn 2+ (2.2-fold). The data may be useful for an understanding of Co toxicity, its effect on the concentration of other metal ions, and a change of metal-dependent specificity of Abzs. Copyright © 2012 John Wiley & Sons, Ltd.

Vitamin A together with testosterone (T) and FSH play an essential role in regulating spermatogenesis. Androgens and Vitamin A act through specific nuclear receptors: AR, retinoic acid receptors (R...

Cobalt is an essential oligoelement for mammals. It is not a cumulative toxin but chronic exposure induces negative effects on the organism. Data from the literature evidenced that in experimental animals cobalt impaired male reproductive organs and fertility when applied chronically. The aim of our study is to follow the effect cobalt on pubertal male progeny of female mice treated with cobalt in late pregnancy and during suckling period. Macroscopic parameters as weight of male reproductive organs and organ/body weight ratio were established. Significant reduction in body weight and 20% decrease (non significant) of testicular and epididymal weight as well as in testis/body weight index was found. The impact of cobalt on male progeny could be explained with transplacental route of exposure and with possible transfer of cobalt into mothers' milk. The negative effect of cobalt was not seen in mid puberty (day 25) with the exception of epididymal weight which was not compensated suggesting that epi...

Recent studies demonstrated that one isoform of angiotensin-converting enzyme named testicular or germinal (tACE) is localized in postmeiotic male germ cells and is essential for fertilizing ability of spermatozoa. Hypertension in spontaneously hypertensive rats (SHR) is androgen-dependent and reduction in male gametes is reported in this experimental conditions. Expression of tACE was not studied under conditions of spontaneous hypertension. The aim of this work is to characterize immuno-expression of tACE in the testis of adult (16-week-old) SHR rats in relation to the changes in blood pressure and serum testosterone level. In 82% of adult SHR, the immuno-expression of tACE followed the normal stage-specific pattern. Destructive testicular changes, germ cells depletion have been observed in 18% of 16-week-old SHR and stronger expression of tACE in stages 8-11 compared to controls was detected. As a result stage specificity in SHR was not as evident as in control. No reaction was found in germ cell depleted tubules in which elongated spermatids were absent. Degenerating germ cells exhibited strong immunostaining comparable to that in residual bodies. The blood pressure was significantly higher in SHR and testosterone levels were more than twice but non-significantly elevated. There was no clear correlation between testicular structural changes, blood pressure level values or serum testosterone levels. Expression of tACE in postmeiotic germ cells, specifically altered by SHR, suggested possible involvement of components of renin-angiotensin system in the process of spermiogenesis. Loss of enzyme expression we found in germ cell depleted tubules in SHR is due to absence of corresponding stages of spermatid differentiation. Therefore, tACE can be used as a marker for germ cell depletion due to hypertension and other pathological conditions.

The human being is exposed to a great variety of endocrine disruptors (EDs), widely spread in environment and they are potential risk for male reproductive health and fertility. The present study aimed to investigate the influence of EDs with estrogenic activity on testis development and function. Seven experimental groups were used and rats were treated neonatally with different oestrogen active compounds (diethylstilbestrol/DES/, Bisphenol-A/Bis-A/, octylphenol/OP/and genistein). The oestrogen effect was evaluated by using a complex system of functional and morphological criteria. Data showed dose-dependent effect of DES (inhibitory for medium and high doses and stimulatory for low dose) on the testis during puberty and in adulthood. Genistein affects negatively but temporary spermatogenesis in puberty, while Bis-A and OP has slight stimulatory effect. No significant effect was observed in the adulthood neither for genistein treated group nor for animals injected with Bis-A and OP. In conclusion...

Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction.

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in tissues during aging in animals and humans and are the basis for mitochondrial diseases. Testosterone synthesis occurs in the mitochondria of Leydig cells. Mitochondrial dysfunction (as induced here experimentally in mtDNA mutator mice that carry a proofreading-deficient form of mtDNA polymerase γ, leading to mitochondrial dysfunction in all cells types so far studied) would therefore be expected to lead to low testosterone levels. Although mtDNA mutator mice showed a dramatic reduction in testicle weight (only 15% remaining) and similar decreases in number of spermatozoa, testosterone levels in mtDNA mutator mice were unexpectedly fully unchanged. Leydig cell did not escape mitochondrial damage (only 20% of complex I and complex IV remaining) and did show high levels of reactive oxygen species (ROS) production (>5-fold increased), and permeabilized cells demonstrated absence of normal mitochondrial function. Nevertheless, within intact cells, mitochondrial membrane potential remained high, and testosterone production was maintained. This implies development of a compensatory mechanism. A rescuing mechanism involving electrons from the pentose phosphate pathway transferred via a 3-fold up-regulated cytochrome b5 to cytochrome c, allowing for mitochondrial energization, is suggested. Thus, the Leydig cells escape mitochondrial dysfunction via a unique rescue pathway. Such a pathway, bypassing respiratory chain dysfunction, may be of relevance with regard to mitochondrial disease therapy and to managing ageing in general.

The role of androgens in the proliferation and maturation of Sertoli cells (SC) and the development of their capacity to support spermatogenesis remains poorly understood. We evaluated these functions in complete androgen receptor knockout (ARKO) and SC-selective androgen receptor knockout (SCARKO) mice. Compared with controls, ARKO mice exhibited a progressive reduction in SC number/testis, whereas SCARKOs showed minor changes, suggesting that androgen effects on SC number are not mediated via direct action on SCs. Immunoexpression of anti-Mullerian hormone (AMH), p27(kip1), GATA-1, and sulfated glycoprotein-2, which changes according to SC maturational status, occurred normally in ARKOs and SCARKOs. Functional capacity of SCs to support spermatogonia was similar in SCARKOs and controls, whereas ARKOs showed reduced capacity with age. SC capacity to support total germ cells revealed major deficits in ARKO and SCARKO adults, particularly with respect to postmeiotic germ cells. Using quantitative RT-PCR, the expression of SC markers was compared in d 50 testes. In ARKOs, expression of Pem, fatty acid binding protein, platelet-derived growth factor-A, and transferrin were all significantly reduced, whereas FSH receptor and AMH were increased. In SCARKOs, there were modest reductions in expression of cystatin-related gene highly expressed in testis and epididymis (cystatin-TE) and claudin-11, whereas expression of Pem, fatty acid binding protein, and platelet-derived growth factor-A was markedly reduced, highlighting these as potentially androgen-regulated SC genes that merit further study. In conclusion, androgen action is not required for maturation-dependent changes in immunoexpression of the SC markers AMH, p27(kip1), GATA-1, and sulfated glycoprotein-2 but is essential for expression of other SC genes, the attainment of normal SC number, and the support of meiotic and postmeiotic germ cell development.

This study investigated whether transient, neonatal (days 2–12) treatment of rats with the potent oestrogen, diethylstilboestrol (DES), altered the structure of the cauda epididymis/vas deferens in adulthood, and if the changes observed related to altered development of basal cells in early puberty. Neonatal treatment with 10 μg DES resulted in the following during adulthood: (a) coiling of the normally straight initial vas deferens, (b) gross epithelial abnormalities, (c) 4-fold widening of the periductal non-muscle layer, (d) infiltration of immune cells across the epithelium into the lumen, and (e) reduction/absence of sperm from the vas deferens lumen. Amongst affected animals >75% exhibited reduced epithelial immunoexpression of androgen receptor and aberrant oestrogen receptor-α immunoexpression and 63% exhibited multi-layering of basal cells coincident with increased epithelial cell proliferation. None of the aforementioned changes occurred in rats treated neonatally with 0.1 μg DES.As basal cells play a key role in the development of epithelia such as that in the epididymis and vas deferens, we went on to investigate if neonatal DES treatment affected basal cell development. In controls, basal cells were first evident at day 10 (vas deferens) or day 18 (cauda). Rats treated with 10 μg, but not those treated with 0.1 μg, DES, showed ~90% reduction (P< 0.001) in basal cell numbers at day 15 and day 18. This decrease coincided with gross suppression of testosterone levels; co-treatment of rats with 10 μg DES + testosterone maintained basal cell numbers at control levels at day 18. However, suppression of testosterone production (GnRH antagonist treatment) or action (flutamide treatment) did not alter basal cell numbers. It is concluded that neonatal exposure to high oestrogen levels coincident with reduced testosterone action results in abnormal changes in the adult cauda/vas deferens that are preceded by delayed differentiation of basal cells. These findings imply a role for androgens and oestrogens in basal cell development and suggest that this may be pivotal in determining normal epithelial (and stromal) development of the cauda/vas deferens.

Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI–VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubul...

Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.

Oestrogen exposure of the male during fetal/neonatal life can fundamentally alter the structure and function of the reproductive system, though how is unknown. This study examined whether such treatment was able to induce a 'female' characteristic, namely immunoexpression of progesterone receptor (PR), in the reproductive system of the male. Rats were treated on postnatal days 2, 4, 6, 8, 10 and 12 with either 10, 1 or 0.1 microg diethystilbestrol (DES) or with the vehicle (20 microl corn oil). Groups of control and treated rats were killed on days 18, 25, 35 and 90 (= adults) and tissues fixed in Bouins for immunolocalisation studies using antisera to PR (recognises A and B forms) and oestrogen receptor-beta (ER beta). PR immunoexpression was absent from all tissues studied in control rats at all ages with the exception of the parasympathetic ganglia of the prostate. In rats treated with 10 microg DES, intense immunoexpression of PR was detected in the nuclei of stromal, but not epithelial, cells of the caput and cauda epididymis, the vas deferens, seminal vesicles and at the base of the dorsolateral prostatic complex (DLPC) at day 18, but was absent from the ventral prostate and from the testis. DES induction of PR immunoexpression was evident after a single injection (on day 3) and at 18-35 days the intensity of immunoexpression was DES dose-dependent; rats treated neonatally with 0.1 microg DES showed no detectable PR immunoexpression at any age. These findings were confirmed by Western analysis which indicated that most of the PR induced was probably the B form. Co-localisation studies, using confocal microscopy, demonstrated that PR and ER beta frequently co-localised to the same stromal cells in the DLPC, epididymis and seminal vesicles of DES-treated rats at day 18, whereas epithelial cells, which also expressed ER beta, did not express PR. In the tissues studied, only occasional stromal cells expressed ER alpha in comparison to the more widespread expression of ER beta, although epithelial cell expression of ER alpha was also detected in the epididymis on day 18 (but not on day 10). In DES-treated rats, immunoexpression of PR in the reproductive tract decreased progressively in intensity from days 18-35 and was non-detectable in adulthood. In conclusion, these findings are interpreted as evidence that neonatal oestrogen treatment exerts pervasive 'reprogramming' effects throughout the reproductive system of the developing male as indicated by the induction of PR immunoexpression. This induction was restricted to stromal tissue even though both stromal and epithelial cells at most sites expressed ER beta and/or ER alpha.

The primary purpose of this study was to establish whether Sertoli cells proliferate in the neonatal period in the marmoset monkey (Callithrix jacchus) and whether administration of a long-acting GnRH antagonist (GnRHa) during this phase induced any transient or permanent effects on Sertoli cell number or on any other aspect of testicular development. Male marmoset co-twins (n = 9) were treated during Weeks 1–14 with either vehicle or GnRHa. Four sets of co-twins were examined at Weeks 18–22 (start of infancy) and 5 sets in adulthood (92+ wk), and Sertoli cell number was determined using either the nucleator or optical disector methods; other testicular morphometric analyses (e.g., germ cell volume, Leydig cell volume) used standard point-counting. Data for the marmoset were compared with that obtained in similarly treated rats. Sertoli cell number in marmosets treated neonatally with GnRHa was reduced by 35% compared with that of controls at Weeks 18–22 but was comparable to control values in ad...

A germ cell nuclear antigen with approximately 44-kDa molecular weight was identified by a novel monoclonal antibody designated as Mab 2F2 from the library we have accumulated against rat testicular cells. In immature 20-day-old and adult rat testis the recognized antigen was expressed in the nuclei of early meiotic cells from preleptotene to early pachytene spermatocytes exhibiting a stage-specific appearance in the cycle of the seminiferous epithelium. The immunoreactivity was clearly associated with the meiotic chromosomes. The antigen was not detected in the late pachytene spermatocytes and more advanced stages of spermatogenesis. No labeling was observed in spermatogonia and somatic Sertoli and Leydig cells. The pattern of expression of the recognized antigen during early meiotic stages of spermatogenesis but not in mitotically dividing spermatogonia could strengthen its possible role in meiotic division.

This study aimed to identify the mechanism(s) for impairment of spermatogenesis in adulthood in rats treated neonatally with estrogens. Rats were treated (days 2-12) with 10, 1, or 0.1 microg diethylstilbestrol (DES), 10 microg ethinyl estradiol (EE), 10 mg/kg of a GnRH antagonist (GnRHa), or vehicle and killed in adulthood. DES/EE caused dose-dependent reductions in testis weight, total germ cell volume per testis, and Sertoli cell volume per testis. Sertoli cell number at 18 days of age in DES-treated rats was reduced dose dependently. GnRHa treatment caused changes in these parameters similar to those in rats treated with 10 microg DES. Plasma FSH levels were elevated (P < 0.001) to similar levels in all treatment groups regardless of differences in Sertoli cell number and levels of inhibin B; the latter reflected Sertoli cell number, but levels were disproportionately reduced in animals treated with high doses of DES/EE. Neonatal estrogen treatment, but not GnRHa, caused dose-dependent reductions (40-80%) in plasma testosterone levels in adulthood, but did not alter LH levels. Preliminary evidence suggests that the decrease in testosterone levels in estrogen-treated rats is not due to reduced Leydig cell volume per testis. GnRHa-treated rats exhibited a significant increase in germ cell volume per Sertoli cell and a reduction in germ cell apoptosis, probably because of the raised FSH levels. Despite similar raised FSH levels, rats treated with DES (10 or 1 microg) or EE (10 microg) had reduced germ cell volume/Sertoli cell and increased germ cell apoptosis, especially when compared with GnRHa-treated animals. The latter changes were associated with an increase in lumen size per testis, indicative of impaired fluid resorption from the efferent ducts, resulting in fluid accumulation in the testis. Rats treated neonatally with 0.1 microg DES showed reduced germ cell apoptosis comparable to that in GnRHa-treated animals. The changes in apoptotic rate among treatment groups occurred across all stages of the spermatogenic cycle. It is concluded that 1) neonatal estrogen treatment results in dose-dependent alterations in Sertoli cell numbers, germ cell volume, efficiency of spermatogenesis, and germ cell apoptosis in adulthood; 2) the relatively poor spermatogenesis in estrogen-treated animals is most likely due to altered testis fluid dynamics and/or altered Sertoli cell function; 3) as indicated by FSH (LH) and testosterone levels, the hypothalamic-pituitary axis and Leydig cells are probably more sensitive than the Sertoli cells to reprogramming by estrogens neonatally; and 4) elevated FSH levels in adulthood may improve the efficiency of spermatogenesis.

The effect of bradykinin on germ cell proliferation was studied by using in vitro organ cultures of testicular fragments from 3.5- and 4.5-day-old rats in the presence of 3H-thymidine. Bradykinin in tested concentrations of 10(-8), 10(-6) and 10(-4) M manifested a significant stimulation of rat prespermatogonial cell proliferation. The percentage of labeled germ cells increased up to 3-fold in comparison with the control value especially in 3.5-day-old rat testicular explants. Two kininase inhibitors: phosphoramidon and captopril were also used in combination with bradykinin. The percentage of labeled germ cells was slightly increased as compared to the samples treated with bradykinin only. Experiments with two agonists and two antagonists of B1 and B2 receptors for kinins showed that the rat prespermatogonial cell proliferation response to bradykinin is probably mediated via B2 receptors. The present findings suggest that bradykinin may be an important local testicular factor with paracrine or autocrine role in the regulation of spermatogonial cell proliferation and germ cell number.

Diethylstilbestrol (DES) was administered neonatally (Days 2-12; 10 microg on alternate days) to rats, and developmental changes in Sertoli cell function were evaluated at 18, 25, and 35 days of age and compared to those observed in rats administered a GnRH antagonist (GnRHa; Days 2 and 5; 10 mg/kg) or a vehicle (controls). DES and GnRHa treatments resulted in similar reductions in both Sertoli cell numbers (40% for DES, 48% for GnRHa) and suppression of testicular growth at 18 and 25 days, though by 35 days the suppression was more pronounced (p 60% reduction in testis weight with many Sertoli cell-only tubules and very low daily sperm production. Taken together, these data are interpreted as providing evidence for direct modulation of Sertoli cell (maturational) development by DES.

The Leydig cells situated in the interstitial compartment of the mammalian testis are responsible for most of the testosterone produced in males. Previous studies in rats have shown that the ability of Leydig cells to produce testosterone significantly declines with age. The present study was focused on the description of some morphological alterations in rat Leydig cells that could be associated with the decreased testicular steroidogenic capacity during aging. Ultrastructural study of aging rat testes revealed the presence of Leydig cells with intact morphology as well as Leydig cells with different degree of degeneration. The decrease of both smooth endoplasmic reticulum and mitochondria, together with an accumulation of lipid droplets and residual bodies in aging Leydig cells, were observed. Our electron microscopical analysis revealed that the majority of the mitochondria in aged Leydig cells (24-month-old) exhibited significant features of degeneration: increasing in size, swelling, lighten of the matrix, reduction of cristae number, and alterations in the structural integrity of the membranes. During aging progressive increase was established in the number of Leydig cells exhibiting nuclear changes such as chromatin compaction and fragmentation and nuclear shrinkage that could be a sign of elevated apoptotic tendency. The observed morphological alterations in rat Leydig cells reflect the decreased testicular steroidogenic capacity during aging, resulting in decrease of testosterone production.

Hydroxysteroid dehydrogenases (HSDs) belong to the NADPH/NAD+-dependent oxidoreductases, which interconvert ketones and the corresponding secondary alcohols. As their names imply, they catalyze the oxidoreduction in different positions of steroidal substrates (3α-, 3β-, 11β-, 17β-, 20αand 20β-position). The steroid-converting HSDs play central roles in the biosynthesis and inactivation of steroid hormones, but some of them are also involved in the metabolism of diverse non-steroidal compounds [1]. The HSDs are integral parts of systemic (endocrine) and local (intracrine) mechanisms. In target tissues they convert inactive steroid hormones to their corresponding active forms and viceversa, thus modulating the transactivation of steroid hormone receptors or other elements of the non-genomic signal transduction pathways. Therefore, HSDs act as molecular switches allowing pre-receptor modulation of steroid hormone action [2].

Cobalt (Co) is a transition metal and an essential trace element, required for vitamin B 12 biosynthesis, enzyme activation and other biological processes, but toxic in high concentrations. There is lack of data for the effect of long-term Co(II) treatment on the concentrations of other trace elements. We estimate the influence of cobalt chloride (CoCl 2 ) on the relative content of different metals in mouse plasma using two-jet arc plasmatron atomic emission and on the total protein content. On average, the content of different elements in the plasma of 2-month-old balb/c mice (control group) decreased in the order: Ca > Mg > Si > Fe > Zn > Cu ≥ Al ≥ B. The treatment of mice for 60 days with CoCl 2 (daily dose 125 mg/kg) did not appreciably change the relative content of Ca, Cu, and Zn, while a 2.4-fold statistically significant decrease in the content of B and significant increase in the content of Mg (1.4-fold), Al and Fe (2.0-fold) and Si (3.2-fold) was found. A detectable amount of Mo was observed only for two control mice, while the plasma of 9 out of 16 mice of the treated group contained this metal. The administration of Co made its concentration detectable in the plasma of all mice of the treated group, but the relative content varied significantly. The treatment led to a 2.2-fold decrease in the concentration of the total plasma protein. Chronic exposure to CoCl 2 affects homeostasis as well as the concentrations and metabolism of other essential elements, probably due to competition of Co ions for similar binding sites within cells, altered signal transduction and protein biosynthesis. Long-term treatment also leads to significant weight changes and reduces the total protein concentration. The data may be useful for an understanding of Co toxicity, its effect on the concentration of other metal ions and different physiological processes.

Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be important for normal testis function as it regulates interstitial fluid (IF) dynamics which is an important intra-testicular transport medium. Androgens control vasomotion, but how they exert these effects remains unclear. One possibility is by signalling via androgen receptors (AR) expressed in testicular arteriole smooth muscle cells. To investigate this and determine the overall importance of this mechanism in testis function, we generated a blood vessel smooth muscle cell-specific AR knockout mouse (SMARKO). Gross reproductive development was normal in SMARKO mice but testis weight was reduced in adulthood compared to control littermates; this reduction was not due to any changes in germ cell volume or to deficits in testosterone, LH or FSH concentrations and did not cause infertility. However, seminiferous tubule lumen volume was reduced in adult SMARKO males while interstitial volume was increased, perhaps indicating altered fluid dynamics; this was associated with compensated Leydig cell failure. Vasomotion was impaired in adult SMARKO males, though overall testis blood flow was normal and there was an increase in the overall blood vessel volume per testis in adult SMARKOs. In conclusion, these results indicate that ablating arteriole smooth muscle AR does not grossly alter spermatogenesis or affect male fertility but does subtly impair Leydig cell function and testicular fluid exchange, possibly by locally regulating microvascular blood flow within the testis.

Aim 11beta hydroxysteroid dehydrogenase (11beta HSD) catalyzes the interconversion of glucocorticoids to inert metabolites in man and rodents and plays a crucial role in regulating corticosteroid hormone action. The physiological role and regulation of 11beta HSD type 2 in the adrenal gland remains obscure. Therefore, the aim of the present study was to establish the pattern of 11beta HSD type 2 expression in rat adrenal gland under conditions of testosterone withdrawal. Material and methods We performed immunohistochemical analyses of adrenal gland sections of ethane dimethanesulphonate (EDS)-treated adult rats. Results In controls, strong positive 11beta HSD type 2 signals were detected in the adrenal cortex cells, but not in the medulla. We observed the lowest 11beta HSD type 2 expression intensity 7 days after initial treatment with ethane dimethanesulphonate (EDS) followed by progressive increase in the immunoreactivity toward days 14 and 21. Maximal staining intensity of 11beta HSD type 2 in the adrenocorticocytes was found by day 35 after EDS treatment. Conclusions By using the EDS model the present study provides new data about 11beta HSD type 2 expression in the adrenal gland under conditions of testosterone withdrawal of adult rats. Our results elucidate further the functional significance of 11beta HSD system in rat adrenal gland and the regulatory role of testosterone in its activity.

Previous studies in rats have shown that the ability of Leydig cells (LCs) to produce testosterone significantly declines with age. To address the possible mechanisms by which aging LCs lose their steroidogenic function, we determined the effect of aging on the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2. The enzyme plays a protective role in blunting the suppressive effects of glucocorticoids on LCs steroidogenesis. Our immunohistochemical analysis revealed progressive decline in 11beta-HDS type 2 expression in LCs of the 18 months of age rats and the most significant reduction in 11beta-HSD2 immunoreactivity was evident in the testicular interstitium of 24- month-old rats. The decrease in the 11beta-HDS type 2 immunostaining in LCs during aging coincided with decline in insulin-like 3/relaxin-like factor (INSL3/RLF) expression, an independent marker for LCs differentiation status. Concomitant with the age-related decrease of 11beta-HDS type 2 immunoreactivity in the LCs population, the immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), marker for LCs steroidogenic activity, was greatly reduced at 24 months compared to 3-month-old control. Similar pattern of expression exhibited also androgen receptor (AR) which is localized in the nuclei of Sertoli cells (SCs), LCs, and peritubular cells. During ages we observed progressive decrease in the immunoreactivity for AR in the testicular types and there was a loss of stage specificity in SCs at age of 24 months. It now seems evident that a variety of factors are likely to be involved in age-related decreases in LCs steroidogenesis, including 11beta-HSD type 2. The observed reduction in 11beta-HSD type 2 expression in aging LCs reflects the decline in their protection ability, opposing the suppressive effect of glucocorticoids on testosterone production.

Cluster analysis at Postnatal Day 8-20 of putative androgen-regulated genes in mice with Sertoli cell-selective knockout of the androgen receptor (SCARKO) has pinpointed three genes (Spinlw1, Gpd1, Drd4) with an expression pattern strongly resembling that of Rhox5, the definitive Sertoli cell (SC) androgen-regulated gene. We used organotypic testis cultures from Day 8 mice to study control of these genes by (anti)androgens and follicle-stimulating hormone (FSH). Testis morphology and androgen induction of the studied genes were preserved for 48 h. Preincubation with ketoconazole for 24 h to block endogenous androgen production, followed by 24-h incubation with the synthetic androgen R1881, resulted in 45-, 5-, 19-, and 6-fold induction of mRNA levels of Rhox5, Spinlw1, Gpd1, and Drd4, respectively. However, noticeable differences in control of the studied genes were observed. Rhox5 and Spinlw1 were fully induced by R1881 in the continuous (48 h) presence of ketoconazole, whereas only marginal effects were observed on expression of Gpd1 and Drd4. Similarly, FSH only marginally affected expression of Rhox5 and Spinlw1, whereas it markedly increased Gpd1 and Drd4 expression. Explant cultures of SCARKO testes confirmed the differential effects of FSH on the studied genes and, for Gpd1, showed that the effect did not depend on a functional androgen receptor in SC, whereas this was essential for the effects of FSH on Drd4. In conclusion, organotypic cultures represent the first in vitro approach to preserving androgen responsiveness of putative SC-expressed genes. This approach facilitates detailed analysis of their regulation in ways not possible in vivo.

Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert this effect remains uncertain. Androgen receptors (ARs) are expressed in several testicular cell types, but continuing uncertainty exists over which cell type mediates androgen control of spermatogenesis. Androgen signaling via Sertoli cells (SCs) is essential for complete spermatogenesis, but the role for androgen signaling via peritubular myoid (PTM) cells is contentious. To address this controversy, we generated PTM-specific AR-knockout (PTM-ARKO) mice in which gross reproductive development was normal, but all PTM-ARKO males were azoospermic and infertile. Testis weight was reduced beyond puberty, and in adulthood there was an 86% reduction in germ cells, compared with wild-type littermates. These changes were not explained by any deficits in testosterone, luteinizing hormone, or follicle-stimulating hormone concentrations. SC function was impaired in PTM-ARKO males, indicated by reduced seminiferous tubule fluid production and reduced expression of some androgen-dependent SC genes. Androgen action via PTM cells is therefore essential for normal testis function, spermatogenesis, and fertility in males. This study also provides the first direct evidence for the importance of androgen-driven stromal-epithelial interactions underpinning the regulation of spermatogenesis; PTM-ARKO mice will enable identification of the new molecular pathways involved.—Welsh, M., Saunders, P. T. K., Atanassova, N., Sharpe, R. M., Smith, L. B. Androgen action via testicular peritubular myoid cells is essential for male fertility.

It is established that androgens and unidentified Sertoli cell (SC)-derived factors can influence the development of adult Leydig cells (LC) in rodents, but the mechanisms are unclear. We evaluated adult LC development and function in SC-selective androgen receptor (AR) knockout (SCARKO) and complete AR knockout (ARKO) mice. In controls, LC number increased 26-fold and LC size increased by approximately 2-fold between 12 and 140 d of age. LC number in SCARKOs was normal on d 12, but was reduced by more than 40% at later ages, although LC were larger and contained more lipid droplets and mitochondria than control LC by adulthood. ARKO LC number was reduced by up to 83% at all ages compared with controls, and LC size did not increase beyond d 12. Serum LH and testosterone levels and seminal vesicle weights were comparable in adult SCARKOs and controls, whereas LH levels were elevated 8-fold in ARKOs, although testosterone levels appeared normal. Immunohistochemistry and quantitative PCR for LC-specific markers indicated steroidogenic function per LC was probably increased in SCARKOs and reduced in ARKOs. In SCARKOs, insulin-like factor-3 and estrogen sulfotransferase (EST) mRNA expression were unchanged and increased 3-fold, respectively, compared with controls, whereas the expression of both was reduced more than 90% in ARKOs. Changes in EST expression, coupled with reduced platelet-derived growth factor-A expression, are potential causes of altered LC number and function in SCARKOs. These results show that loss of androgen action on SC has major consequences for LC development, and this could be mediated indirectly via platelet-derived growth factor-A and/or estrogens/EST.