Age-related changes in human testicular function

Main testicular functions are spermatogenesis and steroidogenesis. Androgen production, Testosterone (T) in particular, stimulates the differentiation of germ cells under the strict endocrine control of the pituitary gland via the two gonadotropins LH and FSH. Today we know that the reduction in serum T concentration (late onset hypogonadism) observed with age can be associated with an overall unhealthy status of the body. Moreover, the prevalence of obesity, metabolic syndrome and diabetes increases with age, and these conditions are significantly associated with hypogonadism, as assessed by sexual symptoms and low serum T levels in spite of normal LH. There is few information about mechanisms underlying androgen deficiency with age and little is known about human Leydig cell (LC) aging, in terms of number and function, mainly because of the scarce availability of the tissue. In this study, we evaluated the age-related changes in testicular function in healthy human testicular tissues of different ages (19-85 years) obtained from heart-beating organ donors and from patients referred to the andrology clinic having normal spermatogenesis and hormone levels. We demonstrated that aging is associated with changes in testicular morphology which are variable in different areas of the parenchyma. Changes included a significative reduction of the area occupied by seminiferous tubules coupled with an increased area occupied by interstitium, mainly composed by fibrotic tissue. In elderly donors, we noticed also peritubular thickening and the appearance of sclerotic tubules and empty tubules. Another morphological parameter evaluated was the LC micronodules frequency and size, since these aggregates composed by more than 15 LCs have been associated with a lower Testosterone/LH ratio. We did not find any correlation between age and micronodules distribution or size, pointing to a normal Hypothalamus-Pituitary-Gonad axis in our group of donors. We found a significative reduction of both LC and Sertoli cell (SC) number with age indicating that cellular senescence generally observed in many tissues with advancing age affects also the testis. Moreover, we evidenced a significative positive correlation between LC and SC number at all ages, which has not been described before in men. Concerning the function of LCs, we analysed gene expression of steroidogenic pathway enzymes and other LC markers by qRT-PCR. We did not find a significative correlation between enzyme gene expression and age but a negative trend was observed for 17β-HSD3, StAR and CYP11A1 genes. In contrast, INSL3 transcript, which also reflects LC number, significantly declined in aged men, consistent with our observation that LC population size changed during aging. Using the in vitro organ culture model previously developed in our laboratory, we demonstrated that three hours in vitro culture of both fresh and cryopreserved testis fragments allows to analyse the LC androgen production (testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA) and 17-Hydroxyprogesterone (17OHP)) secreted into the culture media and simultaneously measured by mass spectrometry both in basal conditions and under recombinant gonadotropin stimulation. We did not find any correlation between androgens secreted in basal conditions and age, indicating that LCs of the donors analysed were able to produce T, A, DHEA and 17OHP in an age- independent manner. The response to gonadotropin stimulation was found greatly variable among donors of different ages and not related to the age. The response to hCG was higher than that to LH when fresh tissue was used. Tissue cryopreservation did not alter interstitial compartment morphology but caused a significative reduction in T concentration, whereas A, DHEA and 17-OH-P levels increased, pointing to a particular vulnerability of specific enzymes to the freezing condition. These data are in line with the finding that the response to hCG was lost when the fragments were cultured after cryopreservation, reinforcing the idea that cryopreservation represents a stressor to the testicular tissue. In conclusion, our data point to a cellular senescence of the aging human testis which is not associated to the in vitro ability to produce androgens. Thus, our results support the idea that LC dysfunction is largely driven by aging of the whole testicular microenvironment rather than aging of LC population alone

Cytoskeleton Dynamics in Peripheral T Cell Lymphomas: An Intricate Network Sustaining Lymphomagenesis.

Defects in cytoskeleton functions support tumorigenesis fostering an aberrant proliferation and promoting inappropriate migratory and invasive features. The link between cytoskeleton and tumor features has been extensively investigated in solid tumors. However, the emerging genetic and molecular landscape of peripheral T cell lymphomas (PTCL) has unveiled several alterations targeting structure and function of the cytoskeleton, highlighting its role in cell shape changes and the aberrant cell division of malignant T cells. In this review, we summarize the most recent evidence about the role of cytoskeleton in PTCLs development and progression. We also discuss how aberrant signaling pathways, like JAK/STAT3, NPM-ALK, RhoGTPase, and Aurora Kinase, can contribute to lymphomagenesis by modifying the structure and the signaling properties of cytoskeleton

Involvement of sperm acetylated histones and the nuclear isoform of Glutathione peroxidase 4 in fertilization

We previously demonstrated that the nuclear form of Glutathione peroxidase 4 (nGPx4) has a peculiar distribution in sperm head, being localized to nuclear matrix and acrosome and that sperm lacking nGPx4 are more prone to decondensation in vitro. In this study we have hypothesized that sperm retained acetylated histones and nGPx4 are implicated in paternal chromatin decondensation and male pronucleus formation at fertilization. Indeed, significant higher amounts of acetylated histone H4 and acetylated histone H3 were observed by both immunofluorescence and western blotting in nGPx4-KO sperm vs WT ones. In vitro fertilization of zona pellucida- deprived oocytes by WT sperm in the presence of trichostatin (TSA) also demonstrated that paternal histone acetylation was inversely related to the timing of sperm nucleus decondensation at fertilization. In contrast, TSA had no effect on nGPx4-KO sperm, indicating they had a maximal level of histone acetylation. Moreover the paternally imprinted gene Igf2/H19 was hypomethylated in KO sperm compared to WT ones. The lack of nGPx4 negatively affected male fertility, causing a marked decrease in total pups and pregnancies with delivery, a significant reduction in pronuclei (PN) embryos in in vitro fertilization assays and an approximately 2 h delay in egg fertilization in vivo. Because the zona pellucida binding and fusion to oolemma of nGPx4-KO and WT sperm were similar, the subfertility of nGPx4 sperm reflected a decreased sperm progression through egg cumulus/zona pellucida, pinpointing a defective acrosome in line with acrosomal nGPx4 localization. We conclude that paternal acetylated histones and acrosomal nGPx4 are directly involved in fertilization

The DNA-helicase HELLS drives ALK - ALCL proliferation by the transcriptional control of a cytokinesis-related program.

Deregulation of chromatin modifiers, including DNA helicases, is emerging as one of the mechanisms underlying the transformation of anaplastic lymphoma kinase negative (ALK-) anaplastic large cell lymphoma (ALCL). We recently identified the DNA-helicase HELLS as central for proficient ALK-ALCL proliferation and progression. Here we assessed in detail its function by performing RNA-sequencing profiling coupled with bioinformatic prediction to identify HELLS targets and transcriptional cooperators. We demonstrated that HELLS, together with the transcription factor YY1, contributes to an appropriate cytokinesis via the transcriptional regulation of genes involved in cleavage furrow regulation. Binding target promoters, HELLS primes YY1 recruitment and transcriptional activation of cytoskeleton genes including the small GTPases RhoA and RhoU and their effector kinase Pak2. Single or multiple knockdowns of these genes reveal that RhoA and RhoU mediate HELLS effects on cell proliferation and cell division of ALK-ALCLs. Collectively, our work demonstrates the transcriptional role of HELLS in orchestrating a complex transcriptional program sustaining neoplastic features of ALK-ALCL

The interplay between NF-Y, AR and lipid metabolism regulates tumor aggressiveness in prostate cancer.

Prostate cancer (PCa) is the second most frequent cancer in Western men. Computational analyses linked the transcription factor NF-Y to progression from benign to localized PCa, aggressive signatures, response to androgen deprivation therapy (ADT) and metastasis. The NF-YA gene encodes two alternatively spliced transcripts, NF-YAs and NF-YAl. We demonstrated that PCa samples are characterized by increased NF-YA levels, as well as higher NF-YAs/NF-YAl transcriptional ratio. In vitro and in vivo, NF-YA depletion affects PCa tumorigenicity and changes in NF-YA isoforms expression are associated with key clinical and molecular features of aggressive PCa. NF-YAs enhances tumor growth and metastasis, while NF-YAl increases cell motility. Stratification of patients based on NF-YAs expression is predictive of clinical outcome, although a significant decrease in the NF-YAs/NF-YAl ratio characterizes PCa circulating cells. PCa cells depend on male sex hormones for growth and survival, which is the basis of ADT. While ADT is initially effective, patients eventually relapse with castration-resistant prostate cancer (CRPC). One of the cellular processes most affected by androgens is lipid metabolism, whose rewiring contributes to PCa development, progression, metastasis and recurrence. Androgens stimulate de novo lipogenesis and lipid uptake by activating SREBPs, the master transcription factors of cholesterol and fatty acid biosynthesis. SREBPs interact with target promoters in cooperation with NF-Y, which controls de novo lipid biosynthesis pathway. A feedforward mechanism between SREBP and the Androgen Receptor (AR) was also described. We modulated NF-YA in androgen-sensitive healthy and cancer cell lines, performing cellular and molecular studies. RNA-seq analysis highlighted the alteration of lipid and cholesterol metabolic pathways and of the unfolded protein response (UPR) in NF-YAl-overexpressing cells. Bioenergetics metabolic profiling of NF-YA transduced cells confirmed the key role of NF-YAl in PCa cell metabolism. We found a reciprocal regulation of AR and NF-Y that relies in particular on NF-YAl. Our data suggest that NF-Y, and specifically the longer NF-YA isoform, can participate in ADT responsiveness and resistance mediated by metabolic alterations. The characterization of these pathways could be useful in the stratification of PCa patients into sub-categories with different levels of aggressiveness and ADT sensitivity

The NF-YA Splicing Signature Controls Aggressiveness of Colon Cancer by Regulating Cell Metabolism and Different Types of Cell Migration

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-Y isoforms, NFYAs and NF-YAl, differentially control cell proliferation and differentiation. The analysis of patient’s transcriptome profiles from The Cancer Genome Atlas database highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA have a lower overall survival probability. We investigated the role of NF-YA in the metabolism of CRC cells, and the measurement of mitochondrial fuel usage in live cells shows that NF-YAl overexpression enhances the capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Moreover, we demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT, extracellular matrix and adhesion genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NFYAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patients’ stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities

The NF-YA splicing signature controls aggressiveness of colon cancer by regulating different modes of cell migration and cell metabolism.

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-YA isoforms, NF-YAs and NF-YAl, differentially control cell proliferation and differentiation. TCGA data highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA levels have a lower overall survival probability. We demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NF-YAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Since metabolic reprogramming is a hallmark of cancer and metastasis, we also investigated the role of NF-YAl in the metabolism of CRC cells. The measure of mitochondrial fuel usage in live cells showed that NF-YAl overexpression enhances the dependency and capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patient stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities