The Crosstalk between Melatonin and Sex Steroid Hormones.

Melatonin, an indolamine mainly released from the pineal gland, is associated with many biological functions, namely, the modulation of circadian and seasonal rhythms, sleep inducer, regulator of energy metabolism, antioxidant, and anticarcinogenic. Although several pieces of evidence also recognize the influence of melatonin in the reproductive physiology, the crosstalk between melatonin and sex hormones is not clear. Here, we review the effects of sex differences in the circulating levels of melatonin and update the current knowledge on the link between sex hormones and melatonin. Furthermore, we explore the effects of melatonin on gonadal steroidogenesis and hormonal control in females. The literature review shows that despite the strong evidence that sex differences impact on the circadian profiles of melatonin, reports are still considerably ambiguous, and these differences may arise from several factors, like the use of contraceptive pills, hormonal status, and sleep deprivation. Furthermore, there has been an inconclusive debate about the characteristics of the reciprocal relationship between melatonin and reproductive hormones. In this regard, there is evidence for the role of melatonin in gonadal steroidogenesis brought about by research that shows that melatonin affects multiple transduction pathways that modulate Sertoli cell physiology and consequently spermatogenesis, and also estrogen and progesterone production. From the outcome of our research, it is possible to conclude that understanding the correlation between melatonin and reproductive hormones is crucial for the correction of several complications occurring during pregnancy, like preeclampsia, and for the control of climacteric symptoms.

The effect of premature termination codon mutations on CFTR mRNA abundance in human nasal epithelium and intestinal organoids: a basis for read-through therapies in cystic fibrosis.

A major challenge in cystic fibrosis (CF) research is applying mutation-specific therapy to individual patients with diverse and rare CF transmembrane conductance regulator (CFTR) genotypes. Read-through agents are currently the most promising approach for Class I mutations that introduce premature termination codons (PTCs) into CFTR mRNA. However, variations in degradation of PTC containing transcripts by nonsense mediated decay (NMD) might lower read-through efficacy. Allele specific quantitative real time (qRT)-PCR was used to measure variations in CFTR mRNA abundance for several PTC mutations in respiratory cells and intestinal organoids. The majority of PTC mutations were associated with reduced levels of relative mRNA transcript abundance (∼33% and 26% of total CFTR mRNA in respiratory cells and intestinal organoids, respectively, compared to >50% for non-PTC causing mutations). These levels were generally not affected by PTC mutation type or position, but there could be twofold variations between individuals bearing the same genotype. Most PTC mutations in CFTR are subject to similar levels of NMD, which reduce but do not abolish PTC bearing mRNAs. Measurement of individual NMD levels in intestinal organoids and HNE cells might, therefore, be useful in predicting efficacy of PTC read-through in the context of personalized CFTR modulator therapy.

The effects of the obesogen tributyltin on the metabolism of Sertoli cells cultured ex vivo.

Human exposure to environmental contaminants is widespread. Some of these contaminants have the ability to interfere with adipogenesis, being thus considered as obesogens. Recently, obesogens have been singled out as a cause of male infertility. Sertoli cells (SCs) are essential for male fertility and their metabolic performance, especially glucose metabolism, is under a tight endocrine control, being essential for the success of spermatogenesis. Herein, we studied the impact of the model obesogen tributyltin in the metabolic profile of SCs. For that, ex vivo-cultured rat SCs were exposed to increasing doses of tributyltin. SCs proliferation was evaluated by the sulforhodamine B assay and the maturation state of the cells was assessed by the expression of specific markers (inhibin B and the androgen receptor) by quantitative polymerase chain reaction. The metabolic profile of SCs was established by studying metabolites consumption/production by nuclear magnetic resonance spectroscopy and by analyzing the expression of key transporters and enzymes involved in glycolysis by Western blot. The proliferation of SCs was only affected in the cells exposed to the highest dose (1000 nM) of tributyltin. Notably, SCs exposed to 10 nM tributyltin decreased the consumption of glucose and pyruvate, as well as the production of lactate. The decreased lactate production hampers the development of germ cells. Intriguingly, the lowest levels of tributyltin were more prone to modulate the expression of key players of the glycolytic pathway. This is the first study showing that tributyltin reprograms glucose metabolism of SCs under ex vivo conditions, suggesting new targets and mechanisms through which obesogens modulate the metabolism of SCs and thus male (in)fertility.

Regucalcin counteracts tert-butyl hydroperoxide and cadmium-induced oxidative stress in rat testis.

Regucalcin (RGN) is a calcium (Ca2+ )-binding protein with multiple physiological roles and has also been linked to the suppression of oxidative stress. It is widely known that oxidative stress adversely affects spermatogenesis, disrupting the development of germ cells, and interfering with sperm function. The present study aims to analyze the role of RGN modulating testicular oxidative stress. To address this issue, seminiferous tubules (SeT) from transgenic rats overexpressing RGN (Tg-RGN) and wild-type (WT) were cultured ex vivo for 24 h in the presence/absence of pro-oxidant stimuli, tert-butyl hydroperoxide (TBHP, 250 and 500 µM) and cadmium chloride (Cd, 10 and 20 µM). Noteworthy, SeT from Tg-RGN animals displayed a significantly higher antioxidant capacity and diminished levels of thiobarbituric acid reactive substances relatively to their WT counterparts, both in control and experimental conditions. Regarding the antioxidant defense systems, a significant increase in the activity of glutathione-S-transferase was found in the SeT of Tg-RGN whereas no differences were observed in superoxide dismutase activity throughout experimental conditions. The activity of apoptosis executioner caspase-3 was significantly increased in the SeT of WT rats treated with 250 µM of TBHP or 10 µM of Cd, an effect not seen in Tg-RGN animals. These results showed that the SeT of Tg-RGN animals displayed lower levels of oxidative stress and increased antioxidant defenses, exhibiting protection against oxidative damage and apoptosis. Moreover, the present findings support the antioxidant role of RGN in spermatogenesis, which may be an important issue of further research in the context of male infertility. Copyright © 2016 John Wiley & Sons, Ltd.

Pre-diabetes alters testicular PGC1-α/SIRT3 axis modulating mitochondrial bioenergetics and oxidative stress.

Pre-diabetes, a risk factor for type 2 diabetes development, leads to metabolic changes at testicular level. Peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) and Sirtuin 3 (Sirt3) are pivotal in mitochondrial function. We hypothesized that pre-diabetes disrupts testicular PGC-1α/Sirt3 axis, compromising testicular mitochondrial function. Using a high-energy-diet induced pre-diabetic rat model, we evaluated testicular levels of PGC-1α and its downstream targets, nuclear respiratory factors 1 (NRF-1) and 2 (NRF-2), mitochondrial transcription factor A (TFAM) and Sirt3. We also assessed mitochondrial DNA (mtDNA) content, mitochondrial function, energy levels and oxidative stress parameters. Protein levels were quantified by Western Blot, mtDNA content was determined by qPCR. Mitochondrial complex activity and oxidative stress parameters were spectrophotometrically evaluated. Adenine nucleotide levels, adenosine and its metabolites (inosine and hypoxanthine) were determined by reverse-phase HPLC. Pre-diabetic rats showed increased blood glucose levels and impaired glucose tolerance. Both testicular PGC-1α and Sirt3 levels were decreased. NRF-1, NRF-2 and TFAM were not altered. Testicular mtDNA content was decreased. Mitochondrial complex I activity was increased, whereas mitochondrial complex III activity was decreased. Adenylate energy charge was decreased in pre-diabetic rats, as were ATP and ADP levels. Conversely, AMP levels were increased, evidencing a decreased ATP/AMP ratio. Concerning to oxidative stress pre-diabetes decreased testicular antioxidant capacity and increased lipid and protein oxidation. In sum, pre-diabetes compromises testicular mitochondrial function by repressing PGC-1α/Sirt3 axis and mtDNA copy number, declining respiratory capacity and increasing oxidative stress. This study gives new insights into overall testicular bioenergetics at this prodromal stage of disease.

Oestrogens as apoptosis regulators in mammalian testis: angels or devils?

In the mammalian testis, spermatogenesis is a highly coordinated process of germ cell development, which ends with the release of 'mature' spermatozoa. The fine regulation of spermatogenesis is strictly dependent on sex steroid hormones, which orchestrate the cellular and molecular events underlying normal development of germ cells. Sex steroids actions also rely on the control of germ cell survival, and the programmed cell death by apoptosis has been indicated as a critical process in regulating the size and quality of the germ line. Recently, oestrogens have emerged as important regulators of germ cell fate. However, the beneficial or detrimental effects of oestrogens in spermatogenesis are controversial, with independent reports arguing for their role as cell survival factors or as apoptosis-inducers. The dual behaviour of oestrogens, shifting from 'angels to devils' is supported by the clinical findings of increased oestrogens levels in serum and intratesticular milieu of idiopathic infertile men. This review aims to discuss the available information concerning the role of oestrogens in the control of germ cell death and summarises the signalling mechanisms driven oestrogen-induced apoptosis. The present data represent a valuable basis for the clinical management of hyperoestrogenism-related infertility and provide a rationale for the use of oestrogen-target therapies in male infertility.

Regucalcin is an androgen-target gene in the rat prostate modulating cell-cycle and apoptotic pathways.

BACKGROUND: Regucalcin (RGN) is a calcium (Ca(2+) )-binding protein underexpressed in prostate adenocarcinoma comparatively to non-neoplastic prostate or benign prostate hyperplasia cases. Moreover, RGN expression is negatively associated with the cellular differentiation of prostate adenocarcinoma, suggesting that loss of RGN may be associated with tumor onset and progression. However, the RGN actions over the control of prostate cell growth have not been investigated. METHODS: Androgens are implicated in the promotion of prostate cell proliferation, thus we studied the in vivo effect of androgens on RGN expression in rat prostate. The role of RGN modulating cell proliferation and apoptotic pathways in rat prostate was investigated using transgenic animals (Tg-RGN) overexpressing the protein. RESULTS: In vivo stimulation with 5α-dihydrotestosterone (DHT) down-regulated RGN expression in rat prostate. Cell proliferation index and prostate weight were reduced in Tg-RGN, which was concomitant with altered expression of cell-cycle regulators. Tg-RGN presented diminished expression of the oncogene H-ras and increased expression of cell-cycle inhibitor p21. Levels of anti-apoptotic Bcl-2, as well as the Bcl-2/Bax protein ratio were increased in prostates overexpressing RGN. Both caspase-3 expression and enzyme activity were decreased in the prostates of Tg-RGN. CONCLUSIONS: Overexpression of RGN resulted in inhibition of cell proliferation and apoptotic pathways, which demonstrated its role maintaining prostate growth balance. Thus, deregulation of RGN expression may be an important event favoring the development of prostate cancer. Moreover, the DHT effect down-regulating RGN expression in rat prostate highlighted for the importance of this protein in prostatic physiology.

Expression pattern of G protein-coupled receptor 30 in human seminiferous tubular cells.

The role of estrogens in male reproductive physiology has been intensively studied over the last few years. Yet, the involvement of their specific receptors has long been a matter of debate. The selective testicular expression of the classic nuclear estrogen receptors (ERα and ERß) argues in favor of ER-specific functions in the spermatogenic event. Recently, the existence of a G protein-coupled estrogen receptor (GPR30) mediating non-genomic effects of estrogens has also been described. However, little is known about the specific testicular expression pattern of GPR30, as well as on its participation in the control of male reproductive function. Herein, by means of immunohistochemical and molecular biology techniques (RT-PCR and Western blot), we aimed to present the first exhaustive evaluation of GPR30 expression in non-neoplastic human testicular cells. Indeed, we were able to demonstrate that GPR30 was expressed in human testicular tissue and that the staining pattern was consistent with its cytoplasmic localization. Additionally, by using cultured human Sertoli cells (SCs) and isolated haploid and diploid germ cells fractions, we confirmed that GPR30 is expressed in SCs and diploid germ cells but not in haploid germ cells. This specific expression pattern suggests a role for GPR30 in spermatogenesis.

In vitro cultured human Sertoli cells secrete high amounts of acetate that is stimulated by 17ß-estradiol and suppressed by insulin deprivation.

BACKGROUND: Several important functions for a successful spermatogenesis are dependent on Sertoli cells (SCs). Besides their unique characteristics as support cells, they produce essential cofactors and metabolites, and are responsible for nurturing the developing germ cells. The continuous production of lipids, phospholipids and proteins by germ cells must require high amounts of metabolic precursors. Thus, we hypothesized that hSCs could produce acetate in a hormonally-regulated manner. METHODS: hSC-enriched primary cultures were maintained in the absence of insulin or in the presence of 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT). Acetate production was determined by 1H-NMR. mRNA gene expression levels of Acetyl CoA hydrolase (ACoA Hyd) and Acetyl CoA synthase (ACoA Synt) were determined by RT-PCR. RESULTS: hSCs produced high amounts of acetate suggesting that this metabolite should play a key role on the progression of spermatogenesis, namely as a metabolic precursor for the synthesis of cellular constituents. In addition, acetate metabolism proved to be under strict hormonal regulation. In the presence of E2 or DHT, hSCs produced different amounts of acetate. While E2 treatment increased acetate production, increasing ACoA Hyd gene transcript levels, DHT-treated cells showed decreased acetate production, differently modulating the ratio ACoA Hyd/ACoA Synt. Surprisingly, insulin-deprivation completely suppressed acetate production/export and significantly decreased the ACoA Hyd gene transcript levels. GENERAL SIGNIFICANCE: Taken together, these results suggest that, although hSCs are primarily described as lactate producers, the elevated production of acetate deserves special attention, in order to clarify the mechanisms behind its hormonal regulation and its role on a successful spermatogenesis.

Control of Sertoli cell metabolism by sex steroid hormones is mediated through modulation in glycolysis-related transporters and enzymes.

Sertoli cells (SCs) glucose metabolism is crucial for spermatogenesis since developing germ cells consume lactate produced by SCs as their main energy source. Recently, androgens and estrogens have been implicated in SCs energy metabolism modulation, although the molecular mechanisms remained undisclosed. Here, we report the effect of sex steroid hormones on key points of cultured rat SCs glycolytic pathway. We used primary cultures of immature rat SCs treated with 17ß-estradiol (E2) or 5α-dihydrotestosterone (DHT). The transcript levels of glucose transporters (GLUTs), phosphofructokinase 1 (PFK-1) and lactate dehydrogenase C (LDH C) were analyzed after 25 and 50 h of culture by qPCR. Protein levels of GLUTs, PFK-1, LDH and monocarboxylate transporter 4 (MCT4) after 25 and 50 h were determined by western blot and LDH activity was also assessed. Our results show that both E2 and DHT downregulated the transcript levels of PFK-1, GLUT1 and GLUT3 after 50 h. However, only DHT-treated cells presented a downregulation of LDH C transcript levels. Interestingly, the protein levels of these enzymes and transporters remained unaltered except in DHT-treated cells that presented a significant decrease on GLUT1 protein levels evidencing a possible site for the regulation of SCs glucose metabolism by androgens. Taken together, our results provide evidence that sex steroid hormones action in SCs energy metabolism is mediated through modulation in glycolysis-related transporters and enzymes, particularly at the transcriptional level. DHT decreased GLUT1 protein levels and increased LDH activity after 25 h, evidencing key points for this hormone action in the regulation of SCs metabolism.

Regulation of ABC transporters by sex steroids may explain differences in drug resistance between sexes.

Drug efficacy is dependent on the pharmacokinetics and pharmacodynamics of therapeutic agents. Tight junctions, detoxification enzymes, and drug transporters, due to their localization on epithelial barriers, modulate the absorption, distribution, and the elimination of a drug. The epithelial barriers which control the pharmacokinetic processes are sex steroid hormone targets, and in this way, sex hormones may also control the drug transport across these barriers. Thus, sex steroids contribute to sex differences in drug resistance and have a relevant impact on the sex-related efficacy of many therapeutic drugs. As a consequence, for the further development and optimization of therapeutic strategies, the sex of the individuals must be taken into consideration. Here, we gather and discuss the evidence about the regulation of ATP-binding cassette transporters by sex steroids, and we also describe the signaling pathways by which sex steroids modulate ATP-binding cassette transporters expression, with a focus in the most important ATP-binding cassette transporters involved in multidrug resistance.

Regucalcin, a calcium-binding protein with a role in male reproduction?

Regucalcin (RGN) is a calcium (Ca(2+))-binding protein which plays an important role in the regulation of Ca(2+) homeostasis and has been shown to catalyse an important step in L-ascorbic acid biosynthesis. It is encoded by an X-linked gene and differs from other Ca(2+)-binding proteins by lacking the typical EF-hand Ca(2+)-binding domain. RGN controls intracellular Ca(2+) concentration by regulating the activity of membrane Ca(2+) pumps. Moreover, RGN has been indicated to regulate the activity of numerous enzymes and to act in the regulation of cell proliferation and apoptosis. The importance of Ca(2+) homeostasis in spermatogenesis has been demonstrated by several studies, and its disruption has been shown to cause reversible male infertility. Recently, the expression of RGN in male reproductive tissues has been described and its localization in all testicular cell types was demonstrated. In addition, RGN expression is regulated by androgens, a class of steroid hormones recognized as male germ cell survival factors and of uttermost importance for spermatogenesis. Altogether, available information suggests the hypothesis that RGN might play a role in spermatogenesis, directly or as a mediator of androgen action. This review discusses this hypothesis presenting novel data about RGN expression in human testis.

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy.

BACKGROUND AND AIM: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. METHODS: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. RESULTS: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. CONCLUSIONS: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization.

The Choroid Plexus Is an Alternative Source of Prolactin to the Rat Brain.

Among the more than 300 functions attributed to prolactin (PRL), this hormone has been associated with the induction of neurogenesis and differentiation of olfactory neurons especially during pregnancy, which are essential for maternal behavior. Despite the original hypothesis that PRL enters the central nervous system through a process mediated by PRL receptors (PRLR) at the choroid plexus (CP), recent data suggested that PRL transport into the brain is independent of its receptors. Based on transcriptomic data suggesting that PRL could be expressed in the CP, this work aimed to confirm PRL synthesis and secretion by CP epithelial cells (CPEC). The secretion of PRL and the distribution of PRLR in CPEC were further characterized using an in vitro model of the rat blood-cerebrospinal fluid barrier. RT-PCR analysis of PRL transcripts showed its presence in pregnant rat CP, in CPEC, and in the rat immortalized CP cell line, Z310. These observations were reinforced by immunocytochemistry staining of PRL in CPEC and Z310 cell cytoplasm. A 63-kDa immunoreactive PRL protein was detected by Western blot in CP protein extracts as well as in culture medium incubated with rat pituitary and samples of rat cerebrospinal fluid and serum. Positive immunocytochemistry staining of PRLR was present throughout the CPEC cytoplasm and in the apical and basal membrane of these cells. Altogether, our evidences suggest that CP is an alternative source of PRL to the brain, which might impact neurogenesis of olfactory neurons at the subventricular zone, given its proximity to the CP.

Attritional evaluation of lipophilic and hydrophilic metallated phthalocyanines for oncological photodynamic therapy.

BACKGROUND AND AIM: Oncological photodynamic therapy (PDT) relies on photosensitizers (PSs) to photo-oxidatively destroy tumor cells. Currently approved PSs yield satisfactory results in superficial and easy-to-access tumors but are less suited for solid cancers in internal organs such as the biliary system and the pancreas. For these malignancies, second-generation PSs such as metallated phthalocyanines are more appropriate. Presently it is not known which of the commonly employed metallated phtahlocyanines, namely aluminum phthalocyanine (AlPC) and zinc phthalocyanine (ZnPC) as well as their tetrasulfonated derivatives AlPCS4 and ZnPCS4, is most cytotoxic to tumor cells. This study therefore employed an attritional approach to ascertain the best metallated phthalocyanine for oncological PDT in a head-to-head comparative analysis and standardized experimental design. METHODS: ZnPC and AlPC were encapsulated in PEGylated liposomes. Analyses were performed in cultured A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and EGFR overexpression. First, dark toxicity was assessed as a function of PS concentration using the WST-1 and sulforhodamine B assay. Second, time-dependent uptake and intracellular distribution were determined by flow cytometry and confocal microscopy, respectively, using the intrinsic fluorescence of the PSs. Third, the LC50 values were established for each PS at 671 nm and a radiant exposure of 15 J/cm2 following 1-h PS exposure. Finally, the mode of cell death as a function of post-PDT time and cell cycle arrest at 24 h after PDT were analyzed. RESULTS: In the absence of illumination, AlPC and ZnPC were not toxic to cells up to a 1.5-µM PS concentration and exposure for up to 72 h. Dark toxicity was noted for AlPCS4 at 5 µM and ZnPCS4 at 2.5 µM. Uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. After 60 min, the entire non-nuclear space of the cell was photosensitized, with PS accumulation in multiple subcellular structures, especially in case of AlPC and AlPCS4. PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 µM, 0.04 µM, and 0.81 µM, respectively, 24 h post-PDT (based on sulforhodamine B assay). ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. At 4 h post-PDT, the mode of cell death comprised mainly apoptosis for ZnPC and AlPC, the extent of which was gradually exacerbated in AlPC-photosensitized cells during 8 h. ZnPC-treated cells seemed to recover at 8 h post-PDT compared to 4 h post-PDT, which had been observed before in another cell line. AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested at 2 h after PDT. During the course of 8 h, necrotic cell death transitioned into mainly late apoptotic cell death. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). Cell cycle arrest was most profound in cells that had been photosensitized with AlPC and subjected to PDT. CONCLUSIONS: Liposomal AlPC is the most potent PS for oncological PDT, whereas ZnPCS4 was photodynamically inert in A431 cells. AlPC did not induce dark toxicity at PS concentrations of up to 1.5 µM, i.e., > 37 times the LC50 value, which is favorable in terms of clinical phototoxicity issues. AlPC photosensitized multiple intracellular loci, which was associated with extensive, irreversible cell death signaling that is expected to benefit treatment efficacy and possibly immunological long-term tumor control, granted that sufficient AlPC will reach the tumor in vivo. Given the differential pharmacokinetics, intracellular distribution, and cell death dynamics, liposomal AlPC may be combined with AlPCS4 in a PS cocktail to further improve PDT efficacy.

Tubular fluid secretion in the seminiferous epithelium: ion transporters and aquaporins in Sertoli cells.

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K(+) than the blood and express various membrane and water transporters (Na(+)/K(+)-ATPase; Ca(2+)-ATPase; V-type ATPase; Cl(-) channels; CFTR Cl(-) channels; K(+) channels; L-, T- and N-type Ca(2+) channels; Na(+)/H(+) exchangers; Na(+)-driven HCO(3) (-)/Cl(-) exchangers (NDCBEs); Na(+)/HCO(3) (-) cotransporters (NBCes); Na(+)-K(+)-2Cl(-) cotransporter; Na(+)/Ca(2+) exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.

[Primary Pulmonary Tumor in an Adolescent]. / Lesão Tumoral Primária do Pulmão em Adolescente.

Primary lung tumors in the pediatric age group are rare, histologically diverse and have different therapeutic approaches. The inflammatory myofibroblastic tumor of the lung accounts for 0.04% - 1.2% of all lung tumors, is more common in children and young adults and its etiology is unknown. The diagnosis is difficult as clinical and radiological findings are highly variable. We report a case of a 15-year-old adolescent who presented with a single pulmonary nodule on a chest radiograph, in the context of a respiratory infection, and whose etiological investigation revealed an inflammatory myofibroblastic tumor of the lung. Atypical resection was performed by video-assisted thoracoscopic surgery, with full recovery. We highlight the rarity of this entity, the need for a high suspicion index and the diagnostic investigation undertaken to reach a definitive diagnosis and a successful outcome.

Os tumores pulmonares primários em idade pediátrica são raros, existindo, contudo, uma multiplicidade de tipos histológicos, com diferentes abordagens terapêuticas. O tumor miofibroblástico inflamatório do pulmão representa 0,04% - 1,2% de todos os tumores pulmonares, é mais frequente em crianças e adultos jovens e a sua etiologia é desconhecida. A apresentação clínica e radiológica é muito variável, pelo que o diagnóstico é difícil. Descrevemos o caso de um adolescente de 15 anos, com achado de nódulo pulmonar em radiografia de tórax realizada no contexto de infeção respiratória e cujo estudo etiológico revelou tratar-se de um tumor miofibroblástico inflamatório do pulmão. Foi realizada resseção atípica por toracoscopia videoassistida, verificando-se evolução favorável. Salientamos a raridade desta entidade, a necessidade de elevado índice de suspeição clínica e a marcha diagnóstica que levou ao esclarecimento definitivo e à sua resolução.

Regulation of ABC transporters by sex steroids may explain differences in drug resistance between sexes

Drug efficacy is dependent on the pharmacokinetics and pharmacodynamics of therapeutic agents. Tight junctions, detoxification enzymes, and drug transporters, due to their localization on epithelial barriers, modulate the absorption, distribution, and the elimination of a drug. The epithelial barriers which control the pharmacokinetic processes are sex steroid hormone targets, and in this way, sex hormones may also control the drug transport across these barriers. Thus, sex steroids contribute to sex differences in drug resistance and have a relevant impact on the sex-related efficacy of many therapeutic drugs. As a consequence, for the further development and optimization of therapeutic strategies, the sex of the individuals must be taken into consideration. Here, we gather and discuss the evidence about the regulation of ATP-binding cassette transporters by sex steroids, and we also describe the signaling pathways by which sex steroids modulate ATP-binding cassette transporters expression, with a focus in the most important ATP-binding cassette transporters involved in multidrug resistance. (AU)

Cystic Fibrosis Newborn Screening in Portugal: PAP Value in Populations with Stringent Rules for Genetic Studies.

Newborn screening (NBS) for cystic fibrosis (CF) has been shown to be advantageous for children with CF, and has thus been included in most NBS programs using various algorithms. With this study, we intend to establish the most appropriate algorithm for CF-NBS in the Portuguese population, to determine the incidence, and to contribute to elucidating the genetic epidemiology of CF in Portugal. This was a nationwide three-year pilot study including 255,000 newborns (NB) that were also screened for congenital hypothyroidism (CH) and 24 other metabolic disorders included in the Portuguese screening program. Most samples were collected in local health centers spread all over the country, between the 3rd and 6th days of life. The algorithm tested includes immunoreactive trypsinogen (IRT) determination, pancreatitis associated protein (PAP) as a second tier, and genetic study for cases referred to specialized clinical centers. Thirty-four CF cases were confirmed positive, thus indicating an incidence of 1:7500 NB. The p.F508del mutation was found in 79% of the alleles. According to the results presented here, CF-NBS is recommended to be included in the Portuguese NBS panel with a small adjustment regarding the PAP cut-off, which we expect to contribute to the improvement of the CF-NBS performance. According to our results, this algorithm is a valuable alternative for CF-NBS in populations with stringent rules for genetic studies.

The protective effect of regucalcin against radiation-induced damage in testicular cells.

AIMS: Regucalcin (RGN), a protein broadly expressed in the male reproductive tract, has shown to have beneficial effects on spermatogenesis suppressing chemical-induced apoptosis. This study aimed to evaluate whether RGN overexpression ameliorates the spermatogenic phenotype after radiation treatment. MAIN METHODS: Transgenic rats overexpressing RGN (Tg-RGN) and their wild-type (Wt) counterparts were exposed to a single dose of X-rays (6Gy), and at ten weeks after irradiation, the testicular status and the epididymal sperm parameters were evaluated. The expression of RGN and several cell cycle and apoptosis regulators, the enzymatic activity of caspase-3, and RGN immunostaining were also assessed. KEY FINDINGS: Tg-RGN animals displayed higher gonadosomatic index, and augmented sperm viability and motility relatively to their Wt counterparts after irradiation, as well as higher frequency of normal sperm morphology and a diminished incidence of head-defects. The differences in reproductive parameters were underpinned by a lower rate of apoptosis, as evidenced by the reduced activity of caspase-3, lower levels of caspase-8, and increased Bcl-2/Bax ratio in the testis of Tg-RGN animals. Supporting the involvement of RGN in the anti-apoptotic response, an enhanced expression of RGN was observed in irradiated rats. SIGNIFICANCE: Transgenic-overexpression of RGN protected against radiation-induced testicular damage, which strengthens the role of this protein protecting cells from the damage of external agents. These findings also indicated that the modulation of RGN testicular levels would be a mechanism for fertility preservation in men undergoing oncological treatment.