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The strict connections/interactions between microbial infections and cancer are nowadays widely accepted. Hence, the dual (or multiple) targeting of microbial infections and cancer is an essential issue to be addressed. In this context, metal complexes have gained considerable importance and effectiveness in medicinal chemistry. Particularly, N-heterocyclic carbene (NHC) complexes with transition metals have emerged as very promising compounds. Among the myriad of NHC–metal complexes, those bearing silver will be the subject of this review. Numerous Ag(I)-NHC complexes have revealed high antibacterial and/or anticancer properties, even higher than those of reference drugs. Herein, we summarize the most recent studies while also discussing the proposed mechanism of action and offering an interesting remark about the research in this field. Literature databases (PubMed/MEDLINE, Scopus, and Google Scholar) were used as sources to search the literature, referring to the last five years. [ABSTRACT FROM AUTHOR]

Glucocorticoids play a physiologic role in the adult male reproductive functions, modulating gonadal steroid synthesis and spermatogenesis, through the glucocorticoid receptor (GR). The expression of GR has been described in several key testicular cell types, including somatic cells and early germ cell populations. Nothing is known on GR in human spermatozoa. Herein, we explored the GR expression and its possible role in normal and testicular varicocele semen samples from volunteer donors. After semen parameter evaluation by macro- and microscopic analysis, samples were centrifuged; then spermatozoa and culture media were recovered for further investigations. By western blotting and immunofluorescence analyses we evidenced for the first time in spermatozoa the presence of GR-D3 isoform which was reduced in sperm from varicocele patients. By treating sperm with the synthetic glucocorticoid dexamethasone (DEXA), we found that survival, motility, capacitation, and acrosome reaction were increased in both healthy and varicocele samples. GR involvement in mediating DEXA effects, was confirmed by using the GR inhibitor mifepristone (M2F). Worthy, we also discovered that sperm secretes different cortisol amounts depending on its physio-pathological status, suggesting a defence mechanism to escape the immune system attach in the female genital tract thus maintaining the immune-privilege as in the testis. Collectively, our data suggests a role for glucocorticoids in determining semen quality and function, as well as in participating on sperm immune defensive mechanisms. The novelty of this study may be beneficial and needs to take into account in artificial insemination/drug discovery aimed to enhancing sperm quality. [ABSTRACT FROM AUTHOR]

Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that the male gonad is a potential target of glucagon-like peptide-1 (GLP-1). We investigated the effects of glucagon-like peptide-1 (GLP-1) on sperm function and the molecular mechanisms through which it may act. Semen samples of healthy men were incubated in the presence or absence of a GLP-1 mimetic analog, exendin-4 (Exe). In a different analysis, sperm were exposed to tumor necrosis factor (TNF-α) alone and, in some tubes, TNF-α was added after previous exposure to exendin-4 (Exe). Sperm parameters and protein-kinase B (p-Akt), insulin receptor substrate-1 (p-IRS-1 Ser312), and c Jun N-terminal protein kinase (p-JNK Thr183/Tyr185) were considered and evaluated. Sperm parameters, when incubated for 4 h in a simple defined balanced salt solution lacking protein, declined progressively with incubation time. The maximum decline was associated with a significant decrease in phosphorylated protein kinase B (p-Akt), concomitantly to an increase in insulin receptor substrate-1 (p-IRS-1 Ser312) and c Jun N-terminal protein kinase (p-JNK Thr183/Tyr185). Preincubation with exendin-4 (Exe) prevented this decline and maintained sperm motility (progressive—PM and total—TM). TNF-α exposure resulted in decreased sperm motility (PM and TM) and viability (V) in a concentration-dependent manner. Exe addition attenuated this TNF-α negative effect on sperm parameters. Glucagon-like peptide-1 (GLP-1) also acts by reducing levels of the "negative" kinases p-IRS-1Ser312 and p-JNK. An imbalance involving these three kinases in sperm, as it occurs in somatic cells, is a novel scenario that may participate in sperm physiopathology. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming is an emerging hallmark of cancer, involving the overexpression of metabolism-related proteins, such as glucose and monocarboxylate transporters and intracellular glycolytic enzymes. The biology of testicular germ cell tumors (TGCTs) is very complex, and although their metabolic profile has been scantily explored, some authors have recently reported that the metabolic rewiring of cancer cells resulted in an association with aggressive clinicopathological characteristics. In this study we have investigated, by immunohistochemical analysis, the expression of key proteins sustaining the hyperglycolytic phenotype in pure seminoma (SE, nr. 35), pure embryonal carcinoma (EC, nr. 17) tissues samples, and normal testes (nr. 5). GLUT1, CD44, PFK-1, MCT1, MCT4, LDH-A, and PDH resulted in more expression in EC cells compared to SE cells. TOM20 was more expressed in SE than in EC. GLUT1, MCT1, and MCT4 expression showed a statistically significant association with SE histology, while for EC, the association resulted in being significant only for GLUT1 and MCT4. Finally, we observed that EC resulted as negative for p53, suggesting that the GLUT1 and MTC overexpression observed in EC could be also attributed to p53 downregulation. In conclusion, our findings evidenced that EC exhibits a higher expression of markers of active aerobic glycolysis compared to SE, suggesting that the aggressive phenotype is associated with a higher glycolytic rate. These data corroborate the emerging evidence on the involvement of metabolic reprogramming in testicular malignancies as well, highlighting that the metabolic players should be explored in the future as promising therapeutic targets. [ABSTRACT FROM AUTHOR]

Abstract Background Nitric oxide (NO) is a signaling molecule produced by intracellular nitric oxide synthase (NOS) enzymes. This free radical appears to affect sperm capacitation, a maturation step preceding acrosome reaction. Recent studies have reported leptin ability to promote capacitation and acrosome reaction in pig male gametes. Methods This study has investigated nitric oxide production in leptin-treated pig spermatozoa by fluorescence-activated cell sorting, while the intracellular NOS isoforms were assessed by Western blot analysis. In addition, acrosome status of treated-spermatozoa was evaluated by FITC-PNA staining. Results Significant increases of nitric oxide levels and acrosome reaction extent were detected in leptin-treated spermatozoa, but both the effects were reversed in presence of L-NAME. Furthermore, the immunoblots of sperm extracts have evidenced three bands of ~160 Kd(bNOS), ~130 Kd (iNOS) and ~135 Kd (eNOS). Conclusions The identification of the three intracellular NOS isoforms suggests that pig spermatozoa could produce NO, while the augmented nitric oxide levels in leptin-treated male gametes indicates the capacity of the hormone to induce nitric oxide production. Furthermore, the inhibitory effect of L-NAME and of Ab-ObR on the promotion of acrosome reaction triggered by leptin suggests a possible involvement of NO in the hormone action.

Abstract Background A wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified. Methods and Results By RT-PCR, Western blot and Immunofluorescence techniques, we demonstrated that human sperm expresses the 1,25(OH)2D3 receptor (VDR). Besides, 25(OH)D3-1 alpha-hydroxylase, evidenced by Western blot analysis, indicated that in sperm 1,25(OH)2D3 is locally produced, highlighting the potential for autocrine-paracrine responses. 1,25(OH)2D3 through VDR, increased intracellular Ca2+ levels, motility and acrosin activity revealing an unexpected significance of this hormone in the acquisition of fertilizing ability. In sperm, 1,25(OH)2D3 through VDR, reduces triglycerides content concomitantly to the increase of lipase activity. Rapid responses stimulated by 1,25(OH)2D3 have been observed on Akt, MAPK and GSK3 implying that this secosteroid is involved in different sperm signalling pathways. Conclusion Our data extended the role of 1,25(OH)2D3 beyond its conventional physiological actions, paving the way for novel therapeutic opportunities in the treatment of the male reproduction disorders.

Abstract Background Androgens and estrogens are crucial for mammalian sperm differentiation but their role in biology of mature male gamete is not still defined. The expression of proteins involved in the biosynthesis and action of these steroid hormones has been demonstrated in human spermatozoa, but very few data have been reported in mature sperm from non human species. The purpose of the current study was to investigate the expression of aromatase (P450arom), estrogen (ERalpha/ERbeta) and androgen (AR) receptors in ejaculated spermatozoa of pig. Methods The immunfluorescence experiments were carried out treating pig sperm with anti-P450arom, anti-ERalpha, anti-ERbeta and anti-AR as primary antibodies, while Texas-Red/FITC conjugated IgG were applied as secondary antibodies. Furthermore, Western blot analysis was performed on sperm lysates. Results Aromatase was immunolocalized in the sperm tail, ERalpha and AR were localised in the sperm midpiece, while ERbeta was confined in the acrosomal region of the male gamete. Immunoblots detected a ~52 kDa aromatase band, a ~110 kDa AR band, a ~67 kDa ERalpha and two ERbeta bands, at ~50 kDa and ~59 kDa. Conclusion This is the first report demonstrating that pig ejaculated spermatozoa express aromatase, estrogen and androgen receptors with a differential intra-cellular localization revealing a specie-specific expression pattern. Therefore, pig sperm could be considered as a potential estrogen source while the different hormone cellular sites suggest distinct roles of androgens and estrogens in pig sperm physiology.

Abstract Background A key role of estrogens in human sperm biology has been recently suggested by aromatase and estrogen receptor detection in human testicular germ cells and ejaculated spermatozoa. However, the involvement of these hormones in the sperm maturation process is still not defined. The aim of this work was to investigate the expression of estrogen receptors, ER-alpha and ER-beta, in human ejaculated immature spermatozoa with excess residual cytoplasm. Methods Immunofluorescence labelling has been used to localize ER-alpha and ER-beta proteins in immature spermatozoa isolated by Percoll gradient, while Western blot analysis was carried out on sperm protein extracts. Results Both estrogen receptors were localized in excess residual cytoplasm of immature sperm, while sperm tails showed only ER-beta. Furthermore, in the same cells, immunoblots detected the presence of the full-length (~67 kDa) ER-alpha and (~59 kDa) ER-beta proteins, together with a ~50 kDa ER-beta species, lacking in mature sperm. Conclusion The present investigation demonstrated ER-alpha and ER-beta presence in excess residual cytoplasm of human abnormal sperm cells, suggesting the hypothesis that both the 'classical' ERs could be able to mediate estrogen action in spermatogenetic cells. Furthermore, the presence of the short ER-beta form in abnormal germ cells and its disappearance in mature sperm, support estrogen modulation via different ER forms during sperm maturation.

The action of myo-inositol (MI), belonging to the inositol family, has been shown to improve sperm quality. To further elucidate the efficacy of this substance in male fertility, we investigated in vivo the effects of a nutraceuticals mix, containing mainly myo-inositol (MI) and in vitro the action of the MI on human male gamete performance. Sperm samples were evaluated from 51 men: 21 healthy normozoospermic and 30 oligoasthenoteratozoospermic (OAT). In the latter group, 15 patients were orally treated with the nutraceutical mix and in the remaining 15 patients only MI was used directly on their ejaculated sperm. Comparing the pathologic samples with respect to normal samples we observed that motility, viability, Bcl-2 phosphorylation, and cholesterol efflux increased after in vitro and in vivo treatments. Glucose-6-phosphate dehydrogenase activity as well as triglycerides level and lipase activity highlighted an enhancement of energy expenditure upon the treatment. Uncapacitated sperm is characterized by an anabolic metabolism, to generate an energy reservoir which will be spent during the capacitation, an energy-consuming process needed to acquire the competence for the fertilization. Intriguingly, our finding highlights that the treatment with these substances facilitated the switch from uncapacitated to capacitated sperm, promoting the acquisition of the male gamete fertilizing capacity. Our data suggested that these substances act both directly on sperm and on spermatogenesis, improving the performance of OAT sperm invitro and invivo. The positive effects of these treatments could be of great help for men and couples who have difficulty to conceive achild in anatural way and/or during medical-assisted reproduction. Abbreviations: 30 OAT-untreated patients; B: 15 OAT patients treated in vivo; Bovine serum albumin (BSA); C: 15 OAT patients treated in vitro; cholesterol oxidase–peroxidase (CHOD–POD); H: Normozoospermic samples; HM: sperm from normospermic patients treated in vitro with MI; MI: Myoinositol: IM: Immobile motility; NP: Non-progressive motility; OAT: Oligoasthenoteratozoospermic; PPP: Pentose Phosphate Pathway; PR: Progressive motility; WHO: World Health Organization. [ABSTRACT FROM AUTHOR]

Abstract Background The enzyme cytochrome P450 aromatase, catalysing the conversion of androgens into estrogens, has been detected in normal human testicular cells suggesting a physiological role of local estrogen biosynthesis on spermatogenesis control. Estrogens, regulating cell growth and apoptosis, can also be involved in tumorigenesis process, but the possible link between estrogens and testicular neoplastic process is, up to now, scarcely known. This study examined aromatase expression in human seminoma, which is the most common germ cell tumour of the testis. Methods The tumour-bearing testes were obtained from 20 patients with classic seminoma undergoing to therapeutic orchidectomy. Paraffin embedded tissues were processed for immunohistochemistry using a mouse monoclonal antibody generated against human placental cytochrome P450 arom, as primary antibody, and a biotinylated goat-anti-mouse IgG, as secondary antibody. Furthermore, Western blot analysis of seminoma extracts was carried out. Results Intense P450 arom immunoreactivity was observed in the seminoma cells and Western blot analysis confirmed the immunodetection. A strong immunostaining was also detected in cells of intratubular germ cell neoplasia (IGCN), adjacent to seminoma. Conclusion The present study demonstrated, for the first time in human, aromatase expression in neoplastic cells of seminoma suggesting a relation between local estrogen biosynthesis and germ cell tumorigenesis. The P450 arom immunolocalization in the cells of IGCN, representing the common precursor of most germ cell tumors, seems to support these findings.

Background: The incretin hormone glucagon‐like peptide‐l (GLP‐1) is an important regulator of post‐prandial insulin secretion, acting through a G protein‐coupled cell surface receptor (GLP‐1R). In addition to its expression in pancreatic β‐cells, several studies suggested that GLP‐1R is located in extra‐pancreatic tissues. Objectives: In this study, we examined for the first time the testicular distribution of the GLP‐1R, both in normal human and neoplastic testicular tissues as well as in rodent testis and rodent testicular cell lines. Methods and Methods: The GLP‐1R distribution in testicular section has been evaluated by immunohistochemistry, the specificity of IHC was validated by demonstrating a positive staining for GLP‐1RmRNA by RISH technology. While GLP‐1R expression in terms of protein was detected by western blot analysis, Moreover, mRNA levels were determined in human testis, in rodent Leydig, and Sertoli cell lines. Results: Using immunohistochemistrya specific staining for GLP‐1R was detected in Leydig cells. The specificity of IHC was validated by demonstrating a positive staining for GLP‐1RmRNA only in these cell types. Species differences in the GLP‐1R expression between humans and rodents were observed. Interestingly, a decreased expression of the receptor in rodent tumor Leydig cell line and an absence in human Leydig tumor samples was detected. Discussion: It may be hypothesized that GLP‐1R acts like an oncosuppressor in Leydig tumors. A role in regulation of hormone secretion by GLP‐1 has been shown in other endocrine cells, therefore we hypothesized that GLP‐1R is able to modulate somehow the Leydig cell function. Conclusion: In our findings, a careful evaluation of human testicular tissues and rodent testis revealed Leydig cells as a potential target for GLP‐1. Collectively, an effect of GLP‐1R in Leydig cell function may be presumed although future studies are needed to ascertain the GLP‐1R's role both in normal and tumor Leydig cells. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor gamma (PPARγ) acts as a ligand activated transcription factor and regulates processes, such as energy homeostasis, cell proliferation and differentiation. PPARγ binds to DNA as a heterodimer with retinoid X receptor and it is activated by polyunsaturated fatty acids and fatty acid derivatives, such as prostaglandins. In addition, the insulin-sensitizing thiazolidinediones, such as rosiglitazone, are potent and specific activators of PPARγ. PPARγ is present along the hypothalamic-pituitary-testis axis and in the testis, where low levels in Leydig cells and higher levels in Sertoli cells as well as in germ cells have been found. High amounts of PPARγ were reported in the normal epididymis and in the prostate, but the receptor was almost undetectable in the seminal vesicles. Interestingly, in the human and in pig, PPARγ protein is highly expressed in ejaculated spermatozoa, suggesting a possible role of PPARγ signaling in the regulation of sperm biology. This implies that both natural and synthetic PPARγ ligands may act directly on sperm improving its performance. Given the close link between energy balance and reproduction, activation of PPARγ may have promising metabolic implications in male reproductive functions. In this review, we first describe PPARγ expression in different compartments of the male reproductive axis. Subsequently, we discuss the role of PPARγ in both physiological and several pathological conditions related to the male fertility. [ABSTRACT FROM AUTHOR]

The mechanisms by which varicocele affects fertility remain undetermined. Vitamin A (all‐trans retinoic acid [ATRA]) is required for fertility and normal spermatogenesis; however, the mechanisms driving its action are not defined yet. Previously, we demonstrated in varicocele sperm a reduced RARα expression and that ATRA influence sperm performance. To further define vitamin A significance in male gamete and in the physiopathology of varicocele, we tested for the first time ATRA action on human sperm metabolism and antioxidant defense systems. Evaluating triglycerides content and lipase activity, in normal sperm ATRA had a lipid lowering effect, which was not observed in varicocele sperm. The modulation of the glucose‐6‐phosphate dehydrogenase activity, concomitantly with a reduction of the glucose content, highlight an ATRA role on glucose metabolism. ATRA induced the superoxide dismutase (SOD) and glutathione transferase activities, while it reduced the malondialdehyde and reactive oxygen species (ROS) production both in healthy and varicocele sperm. Interestingly, SOD1 and SOD2 have been localized in the acrosome and midpiece, glutathione‐ S‐transferase omega 2 (GSTO2) in the acrosome, equatorial, and subacrosomial regions. SOD1, SOD2, and GSTO2 levels were significantly lower in varicocele with respect to healthy sperm. Herein, we discovered that ATRA treatment was able to reprogram sperm metabolism toward that of the capacitation status. The retinol protected human sperm from ROS damage enhancing the antioxidant enzymes activity, providing evidence toward the efficacy of vitamin A as therapeutic tool in improving sperm quality. These novel findings further confirm the importance of vitamin A in male fertility adding new insights into the retinoids complex biological framework. Vitamin A significance in human male gamete and in the physiopathology of varicocele. Vitamin A action on metabolism and antioxidant defense systems in human sperm SOD1, SOD2, and GSTO2 expression and activities were significantly lower in varicocele with respect to healthy sperm. [ABSTRACT FROM AUTHOR]

Human testis, gonocytes, and adult germ cells mainly express estrogen receptor beta, and estrogen receptor beta loss is associated with advanced tumor stage; however, the molecular mechanisms of estrogen receptor beta-protective effects are still to be defined. Herein, we provide evidence that in human seminoma TCam-2 cells, E2 through estrogen receptor beta upregulates the mitochondrial deacetylase sirtuin-3 at protein and messenger RNA levels. Specifically, E2 increases sirtuin-3 expression through a transcriptional mechanism due to the occupancy of sirtuin-3 promoter by estrogen receptor beta, together with the transcription factor Sp1 as evidenced by Chip reChIp assay. This complex binds to a GC cluster located between -128 bp/+1 bp and is fundamental for E2 effects, as demonstrated by Sp1 small interfering RNA studies. Beside, after 24 h, E2 stimulus significantly increased activities of superoxide dismutase and catalase to scavenge reactive oxygen species produced by 30 min of E2 stimulus. In summary, this article indicates a novel functional interplay between estrogen receptor beta and sirtuin-3 counteracting reactive oxygen species production in TCam-2 cells. Our findings thus show that an important tumor-suppressive pathway through estrogen receptor beta is target of E2, actually proposing a distinctive protecting action against seminoma. Future studies may lead to additional strategies for the current therapy of seminoma. [ABSTRACT FROM AUTHOR]