Eugen Plas

Effects of aging on male fertility

The increase in male life expectancy has raised issues concerning the impact of aging on the endocrine system and male fertility. This review focuses on the relationship of spermatogenesis to changes with age in androgen production and testicular morphology, the influence of age on semen parameters and chromosomal quality, and the impact of paternal age and pregnancy outcome. While age-related endocrine changes are well documented, those concerning semen parameters and consequent fertility are based on cross-sectional studies alone. Nevertheless, characteristic age-related morphological testicular alternations have been described, such as decreased numbers of Leydig cells paralleling decreased testosterone production, arteriosclerotic lesions, thickening and hernia-like protrusions of the basal membrane of the seminiferi tubules, and fibrotic thickening of the tunica albuginea. Surprisingly, these alterations do not lead to significant differences in sperm-morphology, time of spermatozoa development or sperm function between young and elderly males. Reports on decreased sperm motility, semen volume and changes in sperm count are contradictory. Although numerical chromosomal abnormalities of spermatozoa are not higher in aging males, an increase in structural aberrations can be observed. Consequently, children of elderly fathers show a 20% higher risk for autosomal dominant diseases, presumably due to increasing numbers of germ cell meioses and mitoses. Thus, the American Fertility Society recommends an age limit for semen donors of 50 years or less.

ROS Signaling by NOX4 Drives Fibroblast-to-Myofibroblast Differentiation in the Diseased Prostatic Stroma

Stromal remodeling, in particular fibroblast-to-myofibroblast differentiation, is a hallmark of benign prostatic hyperplasia (BPH) and solid tumors, including prostate cancer (PCa). Increased local production of TGFβ1 is considered the inducing stimulus. Given that stromal remodeling actively promotes BPH/PCa development, there is considerable interest in developing stromal-targeted therapies. Microarray and quantitative PCR analysis of primary human prostatic stromal cells induced to undergo fibroblast-to-myofibroblast differentiation with TGFβ1 revealed up-regulation of the reactive oxygen species (ROS) producer reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and down-regulation of the selenium-containing ROS-scavenging enzymes glutathione peroxidase 3, thioredoxin reductase 1 (TXNRD1), and the selenium transporter selenoprotein P plasma 1. Consistently, NOX4 expression correlated specifically with the myofibroblast phenotype in vivo, and loss of selenoprotein P plasma 1 was observed in tumor-associated stroma of human PCa biopsies. Using lentiviral NOX4 short hairpin RNA-mediated knockdown, pharmacological inhibitors, antioxidants, and selenium, we demonstrate that TGFβ1 induction of NOX4-derived ROS is required for TGFβ1-mediated phosphorylation of c-jun N-terminal kinase, which in turn is essential for subsequent downstream cytoskeletal remodeling. Significantly, selenium supplementation inhibited differentiation by increasing ROS-scavenging selenoenzyme biosynthesis because glutathione peroxidase 3 and TXNRD1 expression and TXNRD1 enzyme activity were restored. Consistently, selenium depleted ROS levels downstream of NOX4 induction. Collectively, this work demonstrates that dysregulated redox homeostasis driven by elevated NOX4-derived ROS signaling underlies fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Further, these data indicate the potential clinical value of selenium and/or NOX4 inhibitors in preventing the functional pathogenic changes of stromal cells in BPH and PCa.

TGF-β cytokines increase senescence-associated beta-galactosidase activity in human prostate basal cells by supporting differentiation processes, but not cellular senescence

The family of transforming growth factors betas (TGF-betas) comprises molecules involved in growth inhibition, stress-induced premature senescence, epithelial mesenchymal transition and differentiation processes. The aim of this study was to clarify the effect of long term exposure of human prostate basal cells to TGF-betas, which are found in high concentrations in prostatic fluid and areas of benign prostatic hyperplasia (BPH). Basal cell cultures established from prostate explants (n=3) were either grown into cellular senescence, or stimulated with TGF-beta1, beta2 and beta3. Similar to cellular senescence, TGF-beta stimulation resulted in an increase of SA-beta galactosidase (SA-beta-gal) activity, flattened and enlarged cell morphology, and down-regulation of the inhibitor of differentiation Id-1. TGF-beta-treated prostate epithelial cells neither showed terminal growth arrest nor induction of important senescence-relevant genes, such as p16(INK4A), IFI-6-16, IGFBP-3 or Dkk-3. Cells stained positive for cytokeratins 8/18, but did not express other lumenal markers, such as prostate-specific antigen and androgen-receptors. TGF-betas increased also the expression of the mesenchymal marker vimentin, indicating that basal epithelial cells underwent differentiation with lumenal and mesenchymal features. In contrast, in vitro-differentiated neuroendocrine-like cells from prostate organoide cultures, expressing chromogranin A and cytokeratin 18, strongly stained positive for SA-beta-gal. Thus, SA-beta-gal activity is not only a marker for senescence, but also for differentiation of human prostate epithelial cells. With regard to the in vivo situation, in addition to cellular senescence, TGF-beta could contribute to the increased number of SA-beta-gal positive epithelial cells in BPH.

A low-molecular-weight fraction of human seminal plasma activates adenylyl cyclase and induces caspase 3-independent apoptosis in prostatic epithelial cells by decreasing mitochondrial potential and Bcl-2/Bax ratio

The majority of elderly men are affected by benign and malign diseases of the prostate that are governed by endocrine factors and local stromal/epi‐thelial and luminal/epithelial interactions. Prostate epithelial cells secrete numerous factors into the seminal plasma (SMP) that are thought to be responsible for nutrition, accurate pH, and ionic environment of sperm. Our hypothesis assumes that prostatic factors responsible for optimal fertility might have retrograde influences on epithelial cell growth, differentiation, and function. SMP was analyzed for proteins and other biologically active substances by size exclusion high‐performance liquid chromatography. Each fraction was investigated for its effect on cell growth and death. A low molecular mass fraction (2–4 kDa) was responsible for inducing apoptosis in proliferating prostate epithelial cells. Signal transduction was mediated by the production of cAMP; no significant changes in tyrosine phosphorylation of membrane receptors were observed. Mechanisms of apoptosis, i. e., caspase‐ and mitochondria‐dependent pathways, were investigated in prostate epithelial cells by caspase activity assays, an‐nexin/propidium iodide staining, changes in mitochondrial potential, p53, Par‐4, Bax, and Bcl‐2 protein levels. SMP induced p53‐ and Bcl‐2‐dependent apoptosis without activation of caspase‐3. Obviously, SMP contains protective factors that help eliminate degenerated cells and control epithelial renewal. Age‐related changes in the composition of SMP or the susceptibility of epithelial cells might, therefore, contribute to proliferative prostatic diseases.—Untergasser, G., Rumpold, H., Plas, E., Madersbacher, S., Berger, P. A low molecular weight fraction of human seminal plasma activates adenylyl cyclase and induces caspase 3‐independent apoptosis in prostatic epithelial cells by decreasing mitochondrial potential and Bcl‐2/Bax ratio. FASEB J. 15, 673‐683 (2001)

GAGEC1, a cancer/testis associated antigen family member, is a target of TGF-β1 in age-related prostatic disease

Transforming growth factor beta (TGF-beta) is a multi-functional cytokine that plays a fundamental role during embryonic development and tissue homeostasis in metazoans. Changes in TGF-beta signalling are implicated in prostate cancer (PCa) and benign prostatic hyperplasia (BPH), two of the most common diseases affecting ageing males. GAGEC1 belongs to the GAGE-related family of cancer/testis associated antigens and in males is expressed only in prostate and testis. Previous reports demonstrate that GAGEC1 is up-regulated in symptomatic BPH and PCa. We demonstrate GAGEC1 up-regulation by TGF-beta1 in primary prostatic stromal and epithelial cells. Our data suggest that disease-associated increases in TGF-beta1 may account for the increase in GAGEC1 expression in BPH and PCa. Given its restricted spatial expression in males, GAGEC1 represents a promising target for therapeutic intervention of BPH and PCa.

Activators and stimulators of soluble guanylate cyclase counteract myofibroblast differentiation of prostatic and dermal stromal cells.

BACKGROUND Fibrotic diseases encompass numerous systemic and organ-specific disorders characterized by the development and persistence of myofibroblasts. TGFβ1 is considered the key inducer of fibrosis and drives myofibroblast differentiation in cells of diverse histological origin by a pro-oxidant shift in redox homeostasis associated with decreased nitric oxide (NO)/cGMP signaling. Thus, enhancement of NO/cGMP represents a potential therapeutic strategy to target myofibroblast activation and therefore fibrosis. METHODS Myofibroblast differentiation was induced by TGFβ1 in human primary prostatic (PrSCs) and normal dermal stromal cells (NDSCs) and monitored by α smooth muscle cell actin (SMA) and IGF binding protein 3 (IGFBP3) mRNA and protein levels. The potential of enhanced cGMP production by the sGC stimulator BAY 41-2272 or the sGC activator BAY 60-2770 to inhibit and revert myofibroblast differentiation in vitro was analyzed. Moreover, potential synergisms of BAY 41-2272 or BAY 60-2770 and inhibition of cGMP degradation by the PDE5 inhibitor vardenafil were investigated. RESULTS BAY 41-2272 and BAY 60-2770 at doses of 30µM significantly inhibited induction of SMA and IGFBP3 levels in PrSCs and reduced myofibroblast marker levels in TGFβ1-predifferentiated cells. At lower concentrations (3 and 10µM) only BAY 41-2272 but not BAY 60-2770 significantly inhibited and reverted myofibroblast differentiation. In NDSCs both substances significantly inhibited differentiation at all concentrations tested. Attenuation of SMA expression was more pronounced in NDSCs whereas reduction of IGFBP3 levels by BAY 41-2272 appeared more efficient in PrSCs. Moreover, administration of BAY 41-2272 or BAY 60-2770 enhanced the efficiency of the PDE5 inhibitor vardenafil to inhibit and revert myofibroblast differentiation in vitro. CONCLUSIONS Increase of cGMP by sGC stimulation/activation significantly inhibited and reverted myofibroblast differentiation. This effect was even more pronounced when a combination treatment with a PDE5 inhibitor was applied. Thus, enhancement of NO/cGMP-signaling by sGC stimulation/activation is a promising strategy for the treatment of fibrotic diseases. Whereas, in NDSCs BAY 60-2770 and BAY 41-2272 exerted similar effects on myofibroblast differentiation, higher potency of BAY 41-2272 was observed in PrSCs, indicating phenotypical differences between fibroblasts form different organs that should be taken into account in the search for antifibrotic therapies.

Hormonal Changes in Ageing Men

ABSTRACT The hypothalamic-pituitary-gonadal hormone axis undergoes profound changes with ageing in both sexes. While there is a rather abrupt cessation of sex steroid production in women with age (menopause), alterations in men are more subtle. Recent cross-sectional and longitudinal studies have identified a number of age-related changes in men that can ultimately lead to androgen deficiency. A number of symptoms and disorders frequently seen in elderly men (e.g. loss of body mass, sexual dysfunction, osteoporosis, depression) have been linked to these endocrine changes. Up to one third of men beyond the age of 60 have low serum testosterone levels. The role of androgen supplementation for these men is controversial due to the lack of long-term, randomised clinical trials with defined clinical endpoints. The aim of this review is to summarize age-related changes of the hypothalamic-pituitary-axis in men, determine their clinical relevance with particular reference to androgens, and discuss advantages and disadvantages of androgen supplementation.