The Endocrine Disruptor Compound Bisphenol-A (BPA) Regulates the Intra-Tumoral Immune Microenvironment and Increases Lung Metastasis in an Experimental Model of Breast Cancer.

Breast cancer (BC) metastasis represents the main physiopathology leading to poor prognosis and death. Bisphenol A (BPA) is a pollutant, classified as an endocrine-disrupting chemical compound with estrogenic properties, their exposure in the early stages of neonatal life leads to an increase in the size and weight of breast tumors and induces cellular changes in the tumoral immune microenvironment where cytokines play a key role. Thus, we used female BALB/c mice exposed neonatally to a single dose of BPA. Once mice reached sexual maturity, a mammary tumor was induced, injecting 4T1 cells in situ. After 25 days of injection, we evaluated endocrine alterations, cytokine expression, tissue alterations denoted by macro or micro-metastasis in the lung, and cell infiltration induced by metastasis. We found that BPA neonatal treatment did not show significant endocrine alterations. Noteworthy, BPA led to an augmented rate of metastasis to the lung associated with higher intratumoral expression of IL-1ß, IL-6, IFN-γ, TNF-α, and VEGF. Our data suggest that cytokines are key players in the induction of BC metastasis and that BPA (an environmental pollutant) should be considered as a risk factor in the clinical history of patients as a possible inductor of BC metastasis.

Characterization of the myoepithelial cells in the major salivary glands of the fruit bat Artibeus jamaicensis.

Bats constitute one of the most numerous mammalian species. Bats have a wide range of dietary habits and include carnivorous, haematophagous, insectivorous, frugivorous and nectivorous species. The salivary glands of these species have been of particular research interest due to their structural variability among chiropterans with different types of diets. Myoepithelial cells (MECs), which support and facilitate the expulsion of saliva from the secretory portions of salivary glands, are very important for their function; however, this cell type has not been extensively studied in the salivary glands of bats. In this study, we characterized the MECs in the major salivary glands of the fruit bat Artibeus jamaicensis. Herein, we describe the morphology of the parotid, submandibular and sublingual glands of A. jamaicensis at the light- and electro-microscopic level and the distribution of MECs in these glands, as defined by their expression of smooth-muscle markers such as α-smooth muscle actin (SMAα) and desmin, and of epithelial cell markers, such as KRT14. We found that the anatomical locations of the major salivary glands in this bat species are similar to those of humans, except that the bat sublingual gland appears to be unique, extending to join the contralateral homologous gland. Morphologically, the parotid gland has the characteristics of a mixed-secretory gland, whereas the submandibular and sublingual glands were identified as mucous-secretory glands. MECs positive for SMAα, KRT14 and desmin were found in all of the structural components of the three glands, except in their excretory ducts. Desmin is expressed at a lower level in the parotid gland than in the other glands. Our results suggest that the major salivary glands of A. jamaicensis, although anatomically and structurally similar to those of humans, play different physiological roles that can be attributed to the dietary habits of this species.

In vitro capacitation and acrosome reaction in sperm of the phyllostomid bat Artibeus jamaicensis.

Sperm capacitation occurs during the passage of sperm through the female reproductive tract. Once the sperm binds to the pellucid zone, the acrosome reaction to enable penetration of the oocyte is completed. In this study, sperm of Artibeus jamaicensis bat was used to evaluate both capacitation status and the acrosome reaction under in vitro conditions, incubating sperm at 32 and 37°C with and without progesterone. Sperm was incubated at different times to assess sperm cells' functionality in terms of capacitation and acrosome reaction, using the chlortetracycline staining, lectin fluoresceinisocyanate conjugate-Pisum sativum agglutinin (FITC-PSA), and transmission electron microscopy. Sperm cells that presented uniform fluorescence throughout the head and mid-piece were classified as non-capacitated. Subsequently, sperm cells, which were observed with fluorescence only in the anterior portion of the head and mid-piece, were classified as capacitated. Sperm cells with no fluorescence in the head, but fluorescence in the mid-piece, were categorized as sperm cells that have carried out the acrosome reaction. During the acrosome reaction, sperm cells showed changes in their morphology, so it was not possible to distinguish the plasma and acrosomal membranes. Around the entire head, it was not possible to distinguish the fusion points between these membranes that made it possible for the acrosomal reaction to take place and thus to release the enzymes necessary to penetrate the pellucid zone. In conclusion, under appropriate in vitro conditions and by supplementing the culture medium with progesterone, A. jamaicensis bat sperm cells are able to be capacitated in a period from 6 to 8 h and to carry out the acrosome reaction.

The endocrine-immune network during taeniosis by Taenia solium: The role of the pituitary gland.

It is well known that sex hormones play an important role during Taenia solium infection; however, to our knowledge no studies exist concerning the immune response following complete or lobe-specific removal of the pituitary gland during T. solium infection. Thus, the aim of this work was to analyze in hamsters, the effects of lack of pituitary hormones on the duodenal immune response, and their impact on T. solium establishment and development. Thus, in order to achieve this goal, we perform anterior pituitary lobectomy (AL, n = 9), neurointermediate pituitary lobectomy (NIL, n = 9) and total hypophysectomy (HYPOX, n = 8), and related to the gut establishment and growth of T. solium, hematoxylin-eosin staining of duodenal tissue and immunofluorescence of duodenal cytokine expression and compared these results to the control intact (n = 8) and control infected group (n = 8). Our results indicate that 15 days post-infection, HYPOX reduces the number and size of intestinally recovered T. solium adults. Using semiquantitative immunofluorescent laser confocal microscopy, we observed that the mean intensity of duodenal IFN-γ and IL-12 Th1 cytokines was mildly expressed in the infected controls, in contrast with the high level of expression of these cytokines in the NIL infected hamsters. Likewise, the duodenum of HYPOX animals showed an increase in the expression of Th2 cytokines IL-5 and IL-6, when compared to control hamsters. Histological analysis of duodenal mucosa from HYPOX hamsters revealed an exacerbated inflammatory infiltrate located along the lamina propria and related to the presence of the parasite. We conclude that lobe-specific pituitary hormones affect differentially the T. solium development and the gut immune response.

Transient transgenesis of the tapeworm Taenia crassiceps.

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). Infestation of the human brain, also known as neurocysticercosis, is the most common parasite disease of the central nervous system worldwide. Significant advances in the understanding of the disease have been achieved using the Taenia crassiceps murine model. We describe here a successful transfection protocol of T. crassiceps cysticerci as the first step to approach a number of currently inaccessible biological questions on cysticercosis. T. crassiceps cysticerci (ORF strain) were microinjected with the plasmid pcDNA3.1/NT-GFP-TOPO, encoding the green fluorescent protein (GFP) driven by a cytomegalovirus promoter (CMV). Twelve hours after the microinjection, GFP fluorescence gradually developed in patches associated to bud structures in the bladder wall of cysts. Fluorescence reached a peak at 24-48 h and lasted up to 72 h after the microinjection. Immunohistochemical studies on tissue sections of transfected cysts using an anti-GFP antibody, demonstrated co-localization of the antibody and the GFP fluorescence in the tegumentary cytoplasm and subtegumentary cytons. To validate at the mRNA level the expression of GFP, we carried out RT-PCR using two pairs of nested primers. Results showed expression of GFP-mRNA at 24 h post-transfection. Moreover, western blot assays of crude extracts of transfected cysts, carried out using the anti-GFP specific antibody, showed the expected protein band of 27 kDa, demonstrating that the GFP expression started at 24 after plasmid microinjection and was maintained up to 72 h. These findings will facilitate the development of functional genomics approaches applied to this model of cysticercosis.

Regulation of intestinal immune response by selective removal of the anterior, posterior, or entire pituitary gland in Trichinella spiralis infected golden hamsters.

The influence of anterior pituitary hormones on the gastrointestinal tract of humans and animals has been previously reported. Hypophysectomy (HYPOX) in the rat causes atrophy of the intestinal mucosa, and reduction of gastric secretion and intestinal absorption, as well as increased susceptibility to bacterial and viral infections. However, to our knowledge, no findings have been published concerning the immune response following HYPOX during worm infection, particularly that which is caused by the nematode Trichinella spiralis. The aim of this work was to analyze the effects of total or partial HYPOX on colonization of T. spiralis in the intestinal lumen, together with duodenal and splenic cytokine expression. Our results indicate that 5 days post infection, only neurointermediate pituitary lobectomy (NIL) reduces the number of intestinally recovered T. spiralis larvae. Using semiquantitative inmunofluorescent laser confocal microscopy, we observed that the mean intensity of all tested Th1 cytokines was markedly diminished, even in the duodenum of infected controls. In contrast, a high level of expression of these cytokines was noted in the NIL infected hamsters. Likewise, a significant decrease in the fluorescence intensity of Th2 cytokines (with the exception of IL-4) was apparent in the duodenum of control and sham infected hamsters, compared to animals with NIL surgeries, which showed an increase in the expression of IL-5 and IL-13. Histology of duodenal mucosa from NIL hamsters showed an exacerbated inflammatory infiltrate located along the lamina propria, which was related to the presence of the parasite. We conclude that hormones from each pituitary lobe affect the gastrointestinal immune responses to T. spiralis through various mechanisms.

Different levels of testicular organization during gonadal differentiation in B6.Y(Tir) mice manifesting sex reversal.

B6.Y(Tir) (mice with Y chromosome from a strain in Tirano, Italy, and autosomes and X-chromosomes from the B6 strain) mice provide an excellent model for analysing sex development that occurs during gonadal differentiation; however, the molecular mechanisms that contribute to sex reversal are unclear. Our aim has been to establish which molecular events participate in this sex reversal. The pattern of gene expression related to testicular [Sry (sex-determining region of the Y chromosome), Sox9 (Sry-related high-mobility group box gene 9) and Mis (Müllerian-inhibiting substance)] and ovarian [Wnt4 (Wingless-type MMTV (murine-mammary-tumour virus) integration site family, member 4), Rspo1 (cysteine-rich secretory protein containing a thrombospondin type 1 repeat) and Stra8 (stimulated by retinoic acid gene 8)] differentiation was analysed by applying immunofluorescence and real-time RT-PCR (reverse transcription-PCR), focusing on XY gonads from the B6.Y(Tir) mouse, but also analysing the normal strains CD-1 and C57BL/6J (B6). The expression of genes related to the process of sexual differentiation was altered in the case of the B6.Y(Tir) strain, both at the transcript and protein level, inducing differentiation of ovaries and ovotestes, but not the formation of the testes, which were normal. Our results indicate that the expression of testicular genes is inhibited at various levels, permitting the expression of ovarian genes such as Wnt4, Stra8 and Rspo1. However, their activity was not clear when the data were averaged. Correlation analysis indicated that an ovary differentiation pathway is activated when the testicular differentiation pathway is inhibited.

Effect of allopurinol on damage caused by free radicals to cryptorchid testes.

Cryptorchidism causes apoptosis of germ cells. It has been suggested that the redox regulatory system is involved in this process. The free radicals produced are thought to be generated during the production of uric acid, a reaction catalyzed by xanthine oxidase. This enzyme is inhibited by allopurinol; however, the role of allopurinol in neonate rats with inguinal cryptorchidism has not been assessed yet. Sixty male Wistar rats were used and five groups were formed: a control, a sham, a sham group with allopurinol administration and two groups with surgical unilateral cryptorchidism, which either did not receive, or received, allopurinol. The rats were assessed at 40 days post-partum. Reactive oxygen species concentration and epithelial area were measured and the histopathological, apoptotic and cellular proliferation indexes were determined. We found a decrease in reactive oxygen species, histopathological and apoptotic indexes and an increase in proliferation index and epithelial area in rats with cryptorchidism treated with allopurinol in comparison with rats with untreated cryptorchidism. We suggest that the over-production of reactive oxygen species plays an important role in the damage of the cryptorchid testes. Allopurinol administration decreases reactive oxygen species concentrations as well as the damage to the germ epithelium.

The effect of estrogen on testicular gonocyte maturation.

During the first days of postnatal life in rats the male germ cells (gonocytes) proliferate and move towards the seminiferous tubule basal lamina maturing into spermatogonia. This process is necessary for spermatogenesis and can be affected by estrogen (E); therefore, it is important to determine whether the damaging mechanism induced by E administration during the postnatal period impairs gonocyte maturation. One-day-old rat pups were given 1 microg 17-beta-estradiol daily and studied at 3, 5, 8, 10 and 16 days of age, corresponding to the critical gonocyte differentiation period in the rat. Testicles were isolated and the number of gonocytes in contact with the basal lamina of the seminiferous tubule was estimated, as well as the proliferation rate and apoptosis of the gonocytes. We observed that the administration of E changed the migration of gonocytes towards the basal lamina, decreased cell proliferation and increased apoptosis, resulting in a decrease in spermatogonia and spermatocytes. The migration of gonocytes and subsequent proliferation is required for survival of this germ cell type. The lack of maturation and the death of gonocytes could be one of the causes of infertility following exogenous E treatment.

Gubernacular fibroblasts express the androgen receptor during testis descent in cryptorchid rats treated with human chorionic gonadotrophin.

Cryptorchidism was provoked in 3 day old rats treated with 17-beta-estradiol over 30 days to identify the cells that express the androgen receptor (AR) during experimental testis descent in the gubernaculum. In one group of animals, testis descent was induced with human chorionic gonadotrophin (hCG) applied daily for 5 or 10 days. A correlative study using a testosterone radioimmunoassay with electron microscopy and immunocytochemical detection of AR was performed in gubernacula of hCG treated and untreated control animals. The gubernaculum of rats undergoing testes descent showed a dramatic increase in the number of AR-positive cells. These were located in the connective tissue among smooth muscle cells in the gubernacular cord and between striated muscle fibers in the bulb. In both regions, the AR-positive cells were identified as fibroblasts. Several clusters of amorphous material appeared in the extracellular matrix of the connective tissue in hCG treated rats. Our results suggest that testosterone induces the expression of AR in gubernacular fibroblasts which seem to degrade the extracellular matrix during gubernacular involution.

Androgen receptor and calcitonin gene-related peptide in neurons of the genitofemoral nerve during testicular descent induced with human chorionic gonadotropin.

BACKGROUND: Low levels of circulating testosterone during testis descent cause cryptorchidism in humans and rats. Treatment with human chorionic gonadotrophin (hCG) induces testis descent by stimulating production of testosterone (T). Neurons of genitofemoral nerve (GFN), which innervate testicular gubernaculum, may play a role in testis descent. METHODS: In the current study, putative correlations were made between T and GFN motor and sensory neuron activity during inguinoscrotal testis descent. Cryptorchidism was provoked in prepuberal rats with estradiol. Rats with testicular descent induced with hCG and cryptorchid controls were used. Cells of spinal cord and dorsal root ganglia were labeled by retrograde staining with fast-blue. Expression of androgen receptor (AR) and calcitonin gene-related peptide (CGRP) were detected with indirect immunofluorescence. RESULTS: Neurons labeled with fast-blue were found in the center of motor horn and dorsal root ganglia at levels L1 and L2. While number of motor neurons expressing AR was significantly higher in the group treated with hCG, number expressing CGRP was higher in controls. In dorsal root ganglion, number of cells immunostained with CGRP antibody was similar in both groups but AR was not detected. CONCLUSIONS: Present results support the hypothesis that motor nucleus of the GFN is a direct target of testosterone and that regulation of CGRP in sensory nucleus may be involved in testicular descent.

Progesterone receptor gene and protein expression in the anterior preoptic area and hypothalamus of defeminized rats.

Progesterone receptor (PR) plays an important role during sexual differentiation of the rat brain. The objective of the present study was to determine PR protein and gene expression pattern in preoptic-anterior hypothalamic area (POA-AHA) and hypothalamus (HYP), after estradiol or testosterone treatment during the postnatal critical period of sexual differentiation of the rat brain (defeminized animals). Three-day-old female rats were subcutaneously (s.c.) injected with a single dose of 17beta-estradiol (200 microg), or testosterone enanthate (200 microg), or vehicle (corn oil). POA-AHA and HYP were dissected 3 h, 24 h, and 14 days, as well as on the day of vaginal opening (VO) after treatments. Other animals, previously treated as above, were acutely injected with 17beta-estradiol (5 microg) on the day of VO; POA-AHA and HYP were obtained 3 h later. Total RNA was extracted and processed for semiquantitative RT-PCR and tissue slices were prepared for protein detection by immunohistochemistry. We observed that PR mRNA expression was increased in POA-AHA and HYP of the animals treated with estradiol or testosterone 3 hours after treatments, compared with the vehicle-treated control group. We also found a significant increase in PR mRNA and protein expression in POA-AHA and HYP on the day of VO in both estradiol and testosterone defeminized rats. Interestingly, the acute administration of estradiol on the day of VO (VO + E(2)) did not increase PR mRNA or protein expression in POA-AHA and HYP of either estradiol or testosterone defeminized animals, as opposed to the marked induction observed in the intact animals of the control group. The overall results suggest that estradiol and testosterone treatment during the postnatal critical period of sexual differentiation of the brain modifies the regulation of the PR mRNA and protein expression during early onset of maturity.

c-fos and estrogen receptor gene expression pattern in the rat uterine epithelium during the estrous cycle.

Different studies in ovariectomized estrogen treated animals support the idea that c-fos plays a role in the proliferation of uterine epithelial cells. However, these studies invite us to reassess the role played by c-fos in epithelial cell types of the endometrium during the estrous cycle. The present study was undertaken to determine the c-fos and estrogen receptor (ER) gene expression pattern in the rat uterine epithelium during the estrous cycle in which natural and cyclic changes of steroid hormones occur, and correlate these changes with the proliferation status of this cellular types. Proliferation was assessed during the estrous cycle using bromodeoxyuridine incorporation to DNA. ERalpha and beta proteins were assessed by immunohistochemistry. The regulation of c-fos gene expression in the uterus of intact animals during the estrous cycle was evaluated using both in situ hybridization and immunohistochemistry. Estradiol (E(2)) and progesterone (P(4)) plasma levels were assessed by radioimmunoassay. The results indicated that luminal (LE) and glandular epithelia (GE) presented maximal proliferation during the metestrus (M) and the diestrus (D) days. However, during the proestrus (P) day only LE presented proliferation, and during the estrus (E) day only the stromal cells proliferated. A marked immunostaining for ERalpha was detected in both LE and GE cells during the early phases of the cycle but diminished on the P and the E day. In contrast, ERbeta was undetectable in both epithelia during all stages of the cycle. The highest c-fos mRNA level was detected in both epithelia on the M day, followed by a significant reduction during the other days of the cycle. The highest protein content was observed on the M and D days, and the minimal value was detected on the E day. The c-Fos protein level in LE was increased during M and D days, presenting a high correlation with the cellular proliferation pattern of this cell type. In conclusion, the overall results indicate that c-Fos protein presented a good correlation with uterine epithelial cell proliferation of LE. In the case of GE, the same tendency was observed, although no significant correlation was found. Both in LE and GE, c-fos mRNA did not strictly correlate with its protein levels. c-fos seems to have a postranscriptional regulation in uterine epithelial cells during the rat's estrous cycle.

Expression of p53 in luminal and glandular epithelium during the growth and regression of rat uterus during the estrous cycle.

It has been well recognized that epithelial cells of the rat endometrium cyclically proliferate and die during the estrous cycle. The aim of the present study was to determine p53 expression pattern and correlate it with the the apoptotic pattern of epithelial cells of the rat uterus during the estrous cycle. The p53 mRNA and protein expression pattern was assessed by in situ hybridization and immunohistochemistry. The apoptotic index was determined by using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and electron microscopy. The highest p53 mRNA content, detected by in situ hybridization, was observed on the metestrus day both in the luminal and the glandular epithelia. During this period both epithelia presented high proliferation. The content of p53 mRNA markedly decreased in the following days, presenting its minimal values on the estrus day. The highest number of p53 immunopositive nuclei, in both the luminal and the glandular epithelia, was also detected on the metestrus day, while the lowest one was found on estrus day. On the proestrus day, p53 protein was predominantly detected in the glandular epithelium. However, on the estrus day, p53 protein was detected both in the nuclei and in the cytoplasm of luminal epithelial cells, predominantly in the cytoplasm. The highest apoptotic index in both the luminal and the glandular epithelia was observed on the estrus day whereas the lowest one was observed on the proestrus day. The apoptotic index values were higher in the luminal than in the glandular epithelia. The overall results indicate that p53 expression at both mRNA and protein levels is higher on the metestrus day when the apoptotic index is low. This suggests that p53 should play an important physiological role during proliferative phases of the estrous cycle in the rat uterus.