Marta Santoro

Estrogen receptor beta (ERβ) produces autophagy and necroptosis in human seminoma cell line through the binding of the Sp1 on the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) promoter gene.

Testicular germ cell tumors are the most common tumor in male and the least studied. We focused on human seminoma using the TCAM2 cell line. Through ERβ, 10 nM estradiol (E2) was able to induce PTEN gene expression and promoter transactivation. Transient transfections, ChIP and EMSA assays evidenced the 5′-flanking region of PTEN gene promoter E2-responsive. The ERβ binding to the Sp1 on PTEN promoter decreased cell survival. The presence of ERβ or PTEN is necessary to induce the loss of cell survival upon E2, addressing their cooperation in this action. pAKT and AKT expression decreased under E2 and DPN, while known apoptotic markers appeared to be unchanged. The PI3K/AKT pathway inhibition also leads to autophagy: E2 and DPN enhanced the expression of autophagy-related markers such as PI3III, Beclin 1, AMBRA and UVRAG. MDC and TEM assays confirmed E2-induced autophagy. The absence of DNA fragmentation, caspase 9 and PARP1 cleavages suggested that necroptosis and/or parthanatos may occur. FACS analysis, LDH assay and RIP1 expression attested this hypothesis. Our study reveals a unique mechanism through which ERβ/PTEN signaling induces cell death in TCAM2 by autophagy and necroptosis. These data, supporting estrogen-dependency of human seminoma, propose ERβ ligands for therapeutic use in the treatment of this pathological condition.

In vitro mechanism for downregulation of ER-α expression by epigallocatechin gallate in ER+/PR+ human breast cancer cells

SCOPE Exposure of the breast to estrogens and other sex hormones is an important cancer risk factor and estrogen receptor downregulators are attracting significant clinical interest. Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, has gained considerable attention for its antitumor properties. Here we aimed to investigate the molecular mechanisms through which EGCG regulates ER-α expression in ER+ PR+ breast cancer cells. MATERIAL AND METHODS Western blotting analysis, real-time PCR, and transient transfections of deletion fragments of the ER-α gene promoter show that EGCG downregulates ER-α protein, mRNA, and gene promoter activity with a concomitant reduction of ER-α genomic and nongenomic signal. These events occur through p38(MAPK) /CK2 activation, causing the release from Hsp90 of progesterone receptor B (PR-B) and its consequent nuclear translocation as evidenced by immunofluorescence studies. EMSA, and ChIP assay reveal that, upon EGCG treatment, PR-B is recruited at the half-PRE site on ER-α promoter. This is concomitant with the formation of a corepressor complex containing NCoR and HDAC1 while RNA polymerase II is displaced. The events are crucially mediated by PR-B isoform, since they are abrogated with PR-B siRNA. CONCLUSION Our data provide evidence for a mechanism by which EGCG downregulates ER-α and explains the inhibitory action of EGCG on the proliferation of ER+ PR+ cancer cells tested. We suggest that the EGCG/PR-B signaling should be further exploited for clinical approach.

Human sperm physiology: Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) influence sperm metabolism and may be involved in the pathophysiology of varicocele-associated male infertility†

The mechanisms by which varicocele affects fertility remain undetermined. Estrogens play a key role in the human male reproduction and human sperm expresses the estrogen receptors (ERs) and aromatase. In this study, by Western blotting we evidenced the ERs content concomitantly in healthy sperm and in oligoastenoteratozoospermic (OAT) samples without and with varicocele. In varicocele a strong reduction of the ERβ was observed, while the ERα was almost absent. Besides, transmission electron microscopy (TEM) confirmed the reduction of ERs expression in “varicocele” sperm, indicating that varicocele has a detrimental effect on sperm structure at molecular level. To further define the estrogen significance in male gamete and the pathophysiology of varicocele we investigated both the expression of ERα and ERβ in normal and pathologic sperm samples as well as we evaluated estradiol (E2) action on lipid and glucose sperm metabolism. Responses to E2 treatments on cholesterol efflux, protein tyrosine phosphorylations, motility, and acrosin activity in varicocele sperm were reduced or absent. The evaluation of the triglycerides content, lipase and acyl‐CoA dehydrogenase activities, suggest that E2 exerts a lipolytic effect on human sperm metabolism. Concerning glucose metabolism, it appears that E2 induces G6PDH activity concomitantly to the insulin secretion. In “varicocele” sperm, the E2 did not induce energy expenditure. OAT sperm had E2‐responsiveness but in a lesser extent with respect healthy sperm. This study discovered a novel role for E2/ERs in human sperm physiology, since they modulate sperm metabolism and new detrimental effects related to the pathophysiology of the varicocele condition. J. Cell. Physiol. 226: 3403–3412, 2011.

The Mitochondrial Citrate Carrier (CIC) Is Present and Regulates Insulin Secretion by Human Male Gamete

The mechanisms through which sperm manage their energy metabolism are poorly understood. The present study provides biochemical and morphological evidence that mitochondrial citrate carrier (CIC) is present in ejaculated human sperm and is restricted to the midpiece. The inhibition of CIC with the specific substrate analog 1,2,3-benzenetricarboxylate resulted in the reduction of cholesterol efflux, protein tyrosine phosphorylation, phospho-AKT, phospho-p60src, hyperactivated motility and acrosome reaction, suggesting a role for this mitochondrial carrier in sperm physiology. Furthermore, inhibition of CIC by 1,2,3-benzenetricarboxylate resulted in a reduction of glucose-stimulated insulin secretion and autocrine insulin secretion by sperm. Remarkably, blocking CIC also reduced glucose-6-phosphate dehydrogenase activity, probably in accordance with its regulation on insulin secretion. Capacitation and glucose metabolism were stimulated by glucose as well as citrate, the specific substrate of CIC, implying a similar action because glucose and citrate both induced insulin secretion by sperm. In the present finding, we discovered a new site of action for CIC in the regulation of metabolism, and it may be assumed that CIC works with other factors in the regulation of sperm energy metabolism to sustain capacitation process and acrosome reaction.

Epigallocatechin gallate affects survival and metabolism of human sperm

SCOPE Green tea and its major constituent epigallocatechin gallate (EGCG) have been extensively studied as potential treatment for a variety of diseases. We assessed the influence of EGCG on male fertilizing potential by analyzing different features of human sperm involved in capacitation process. METHODS AND RESULTS Using human normozoospermic samples, we evaluated the effect of EGCG (2 μM, 20 μM, 60 μM) on sperm activities. Our results showed that lower doses of EGCG (from 2 to 20 μM) increased cholesterol efflux and tyrosine phosphorylation through the estrogen receptor (ER), since ICI 182,780, a specific ER antagonist, abrogated 20 μM EGCG effects. Besides, we evidenced that EGCG at similar concentrations, increased sperm motility, viability, and phosphorylation of proteins controlling cell survival such as Bcl2, Akt, and Src, via ER. Furthermore, we observed reduction of triglycerides content, induction of lipase, as well as the G6PDH activity. These results address to an increase in energy expenditure. On the contrary, treatment of 60 μM EGCG produced opposite effects that still appear after ICI cotreatment. CONCLUSION These results provide a novel mechanism involving ERs through which low doses of EGCG exerted benefits to sperm physiology, also detected data evidence the adverse action of high EGCG concentrations probably related to its prooxidant and antiestrogenic potential.

Expression of cyclooxygenase-1 (COX-1) and COX-2 in human male gametes from normal patients, and those with varicocele and diabetes: a potential molecular marker for diagnosing male infertility disorders

Rising rates of varicocele and diabetes mellitus (DM) pose a significant problem to human fertility. Recent studies have pointed out the impact of cyclooxygenase (COX) in the regulation of testicular function and male fertility. Prominent COX‐2 expression has been described recently in the testes of infertile patients, but little is known about the role and identity of COX isoforms in human sperm under certain disease states such as varicocele and DM. We therefore examined the expression profile and ultrastructural localization of COX‐1 and COX‐2 concomitantly in semen samples from healthy donors, and patients with varicocele and DM. Using Western blotting assay, ‘varicocele’ and ‘diabetic’ sperm showed enhanced COX isoforms expression with respect to the ‘healthy’ sperm. Immunogold labeling revealed human sperm anatomical regions containing COX‐1 and COX‐2, confirming their increased expression in pathological samples. Our data demonstrate that both COX isoforms are upregulated in the spermatozoa of varicocele and diabetic patients, suggesting the harmful effect of the diseases also at the sperm molecular level, going beyond the abnormal morphology described to date. In conclusion, COX enzymes may possess a biological relevance in the pathogenesis and/or maintenance of male factor infertility associated with varicocele and DM, and may be considered additional molecular markers for the diagnosis of male infertility disorders.

Bergapten drives autophagy through the up-regulation of PTEN expression in breast cancer cells.

BackgroundBergapten (5-methoxypsoralen), a natural psoralen derivative present in many fruits and vegetables, has shown antitumoral effects in a variety of cell types. In this study, it has been addressed how Bergapten in breast cancer cells induces autophagic process.ResultsIn MCF7 and ZR-75 breast cancer cells Bergapten exhibited anti-survival response by inducing the autophagic process increasing Beclin1, PI3KIII, UVRAG, AMBRA expression and conversion of LC3-I to LC3-II. LC3-GFP, Acridine orange assay and transmission electron microscopy even confirmed the increased autophagosome formations in treated cells. Bergapten-induced autophagy is dependent by PTEN up-regulation, since silencing this gene, the induction of Beclin1 and the p-AKT/p-mTOR signal down-regulation were reversed. PTEN is transcriptionally regulated by Bergapten through the involvement of p38MAPK/NF-Y, as evidenced by the use of p38MAPK inhibitor SB203580, site-direct mutagenesis of NF-Y element and NF-Y siRNA. Furthermore NF-Y knockdown prevented Bergapten-induced acid vesicular organelle accumulations (AVOs), strengthening the role of this element in mediating autophagy.ConclusionsOur data indicate PTEN as a key target of Bergapten action in breast cancer cells for the induction of autophagy. These findings add further details on the mechanism of action of Bergapten, therefore suggesting that phytochemical compounds may be implemented in the novel strategies for breast cancer treatment.

Sperm metabolism in pigs: a role for peroxisome proliferator-activated receptor gamma (PPARγ)

SUMMARY Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor expressed predominantly in adipose tissue, also implicated in energy homeostasis. In this study, we used western blotting and immunofluorescence techniques to demonstrate for the first time that pig spermatozoa express PPARγ. To investigate the functional role of PPARγ in pig sperm, we evaluated its action on different events that characterize the biology of sperm cells, i.e. motility, capacitation, viability and acrosome reaction, using the PPARγ-agonist 15-deoxy-12,14-prostaglandin J2 (PGJ2). In responses to PGJ2 treatment, motility, cholesterol efflux and tyrosine phosphorylation were increased, which broadens the role of PPARγ from that previously described in the literature, as it also acts to improve sperm functionality. To further our understanding of the significance of PPARγ in pig sperm, we focused its effects on lipid and glucose metabolism. Evaluation of triglyceride content and lipase, acyl-CoA dehydrogenase and G6PDH activities suggests that PPARγ induces energy expenditure in pig spermatozoa. These data represent a meaningful advance in the field of sperm energy metabolism. Taken together, our results demonstrate for the first time that PPARγ is expressed by pig sperm, thus improving its functionalities in terms of motility, capacitation, acrosome reaction, survival and metabolism.

Red wine consumption may affect sperm biology: the effects of different concentrations of the phytoestrogen myricetin on human male gamete function.

Myricetin is a natural flavonoid, particularly enriched in red wines, whose occurrence is widespread among plants. Despite extensive research, the beneficial effects of Myricetin on human health are still controversial. Here, we tested the estrogen‐like effect of the phytoestrogen Myricetin on human ejaculated sperm biology. To this aim, human normozoospermic samples were exposed to increasing concentrations (10 nM, 100 nM, and 1 µM) of Myricetin. Motility, viability, capacitation‐associated biochemical changes (i.e., cholesterol efflux and tyrosine phosphorylation), acrosin activity, as well as glucose utilization and fatty‐acid oxidation (i.e., glucose and lipid metabolism) were all significantly increased by low doses of Myricetin. Importantly, both estrogen receptors α and β (ERs) and phosphatidylinositol‐3‐OH kinase (PI3K)/AKT signaling are activated in the presence of Myricetin since these were both abrogated by specific inhibitors of each pathway. Our results show how Myricetin, through ERs and PI3K/AKT signalings, potentiates sperm function. This effect is dose‐dependent at low concentrations of Myricetin (up to 100 nM), whereas higher amounts do not seem to improve any further sperm motility, viability, or other tested features, and, in some cases, they reduced or even abrogated the efficacy exerted by lower doses. Further studies are needed to elucidate if high levels of Myricetin, which could be attained even with moderate wine consumption, could synergize with endogenous estrogens in the female reproductive tract, interfering with the physiological sperm fertilization process. Mol. Reprod. Dev.

Human sperm molecular anatomy: the enzyme 5α-reductase (SRD5A) is present in the sperm and may be involved in the varicocele-related infertility

Abstract The most common cause of male infertility is the testicular varicocele, a condition that impairs production and decreases quality of sperm. Male fertility also strictly depends on androgens acting through their own receptor. The enzyme 5α-reductase (SRD5A) is involved in the conversion of testosterone to 5α-dihydrotestosterone, both required for the development and maintenance of male reproductive function. Here, we evaluated, by western blotting analysis, the presence of SRD5A in human ejaculated spermatozoa and evidenced differences in sperm SRD5A content between healthy donors and varicocele-affected patients. Additionally, SRD5A sperm ultrastructural localization was also assessed by transmission electron microscopy and immunogold assay. We evidenced that SRD5A enzyme is present in the human spermatozoa and that its cellular content is lowered in sperm samples from varicocele patients compared to healthy subjects. The presence of SRD5A in human ejaculated spermatozoa highlights the potential role of this enzyme in sperm physiopathology suggesting that the decrease in its content, by affecting the conversion of testosterone into 5α-dihydrotestosterone, may be an important additional mechanism involved in the harmful effect of varicocele in male fertility.