High Trypanosoma cruzi prevalence in armadillo (Zaedyus pichiy; Xenarthra: Chlamyphoridae) populations from Mendoza, Argentina.

Armadillos are considered important reservoir hosts for Trypanosoma cruzi, the causative agent of Chagas disease. The first report of T. cruzi infection in pichis (Zaedyus pichiy), a small armadillo species endemic to central Argentina and Chile, dates back to 1935. However, more recent reports on T. cruzi in this species are scarce. The objective of this study was to assess T. cruzi infection and parasite load in Z. pichiy from Mendoza Province, an area endemic to human Chagas disease. Blood samples were obtained in 2014-2016 from pichis from Lavalle (low Monte), Malargüe (Patagonian steppe), and San Carlos (ecotone) departments, Mendoza Province, Argentina. The detection and quantification of T. cruzi was performed through qPCR amplification using satellite primers. Of the 265 analyzed samples, 201 (76%) were positive for T. cruzi. Parasite loads varied between < 0.1-55.8 parasite-equivalents/mL (par-eq/mL), with a median of 1.1 par-eq/mL in quantifiable samples. The prevalence was similar in Malargüe and Lavalle (85-94%), but significantly lower in pichis from San Carlos (50%). Animals from Lavalle captured after hibernation had significantly higher parasite loads (median 2.0 par-eq/mL). In Malargüe, T. cruzi infection and parasite loads were significantly lower before than after hibernation in 2016. The high prevalence and low median parasite load suggest a chronic and persistent infection of T. cruzi in pichis. Regional differences and a marked increase in precipitation during 2015-2016 could have influenced annual and seasonal infection rates of this vector-borne disease.

Effect of peripheral neural stimulation with allopregnanolone on ovarian physiology.

Neuroactive steroids can rapidly regulate multiple physiological functions in the central and peripheral nervous systems. The aims of the present study were to determine whether allopregnanolone (ALLO), administered in low nanomolar and high micromolar concentrations, can: (i) induce changes in the ovarian progesterone (P4) and estradiol (E2) release; (ii) modify the ovarian mRNA expression of Hsd3b1 (3ß-hydroxysteroid dehydrogenase, 3ß-HSD)3ß-, Akr1c3 (20α-hydroxysteroid dehydrogenase, 20α-HSD), and Akr1c14 (3α-hydroxy steroid oxidoreductase, 3α-HSOR)); and (iii) modulate the ovarian expression of progesterone receptors A and B, α and ß estrogenic receptors, luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). To further characterize ALLO peripheral actions, the effects were evaluated using a superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) and a denervated ovary (DO) systems. ALLO SMG administration increased P4 concentration in the incubation liquid by decreasing ovarian 20α-HSD mRNA, and it also increased ovarian 3α-HSOR mRNA expression. In addition, ALLO neural peripheral modulation induced an increase in the expression of ovarian LHR, PRA, PRB, and ERα. Direct ALLO administration to the DO decreased E2 and increased P4 concentration in the incubation liquid. The mRNA expression of 3ß-HSD decreased and 20α-HSD increased. Further, ALLO in the OD significantly changed ovarian FSHR and PRA expression. This is the first evidence of ALLO's direct effect on ovarian steroidogenesis. Our results provide important insights about how this neuroactive steroid interacts both with the PNS and the ovary, and these findings might help devise some of the pleiotropic effects of neuroactive steroids on female reproduction. Moreover, ALLO modulation of ovarian physiology might help uncover novel treatment approaches for reproductive diseases.

Intrauterine androgen exposure impairs gonadal adipose tissue functions of adult female rats.

Prenatal androgen exposure induces fetal programming leading to alterations in offspring health and phenotypes that resemble those seen in women with Polycystic Ovary Syndrome. It has been described that prenatal androgenization affects the reproductive axis and leads to metabolic and endocrine disorders. Adipose tissue plays a crucial role in all these functions and is susceptible to programming effects. Particularly, gonadal adipose tissue is involved in reproductive functions, so dysfunctions in this tissue could be related to fertility alterations. We aimed to investigate the extent to which prenatal hyperandrogenization is able to alter the functionality of gonadal adipose tissue in female adult rats, including lipid metabolism, adipokines expression, and de novo synthesis of steroids. Pregnant rats were treated with 1 mg of testosterone from day 16 to day 19 of pregnancy, and female offspring were followed until 90 days of age, when they were euthanized. The prenatally hyperandrogenized (PH) female offspring displayed two phenotypes: irregular ovulatory (PHiov) and anovulatory (PHanov). Regarding lipid metabolism, both PH groups displayed disruptions in the main lipid pathways with altered levels of triglyceride and increased lipid peroxidation levels. In addition, we found that Peroxisome Proliferator-Activated Receptors (PPARs) alpha protein expression was decreased in both PH phenotypes (p < 0.05), but no changes were found in PPARγ protein levels. Furthermore, regarding adipokines, no changes were found in Leptin and Adiponectin protein levels, but Chemerin protein levels were decreased in the PHiov group (p < 0.05). Regarding de novo synthesis of steroids, the PHanov group showed increased protein levels of Cyp17a1 and Cyp19, while the PHiov group only showed decreased protein levels of Cyp19 (p < 0.05). These results suggest that prenatal androgen exposure affects females' gonadal adipose tissue in adulthood, disturbing different lipid pathways, Chemerin expression, and de novo synthesis of steroids.

Alterations in Hypothalamic Mechanisms Associated with Reduced Suckling-Induced Prolactin Secretion in the Lactation-Deficient OFA hr/hr Rat.

INTRODUCTION: OFA hr/hr rats have deficient lactation with impaired suckling-induced PRL release. Unlike their background strain, Sprague-Dawley (SD) rats, OFA rats display abnormal mediobasal hypothalamus (MBH) dopaminergic tone during late pregnancy and lactation. We explored if the expression of MBH components, including various receptors (R) and proteins that regulate the dopaminergic system, is altered in mid-lactating OFA compared to SD rats, which may be associated with the abnormality. METHODS: Four groups of mid-lactating rats were used: continuous lactation; pups separated overnight; 30-min suckling (S); and 2 h or 4 h S after separation. Mothers were sacrificed to obtain serum for PRL RIA and MBHs to determine tyrosine hydroxylase (TH), PRL-R, PRL signaling molecules (activator: STAT5b; inhibitors: SOCS1, SOCS3, CIS), opioids (PENK, PDYN), and µ- and κ-opioid R (MOR, KOR) mRNA expression by qPCR and phospho-TH (p-TH) and TH proteins by Western blot. RESULTS: Suckling-induced PRL was lower in OFA and p-TH expression diminished in both strains. Separation increased TH mRNA and protein in SD, which decreased after 4 h S, but OFA protein levels remained unchanged. Separation of pups also resulted in decreased PRL-R and CIS expression in SD but increased PRL-R and SOCS3 in OFA. Despite the lower PRL-R, STAT5b, SOCS1, and SOCS3 levels in OFA compared to SD, suckling diminished them further. We observed subtle changes in SD opioids and their R, but in OFA, suckling decreased PENK, KOR, and MOR. CONCLUSION: The different patterns of TH, opioids, their R, and PRL signaling inhibitor expression with conserved TH activation by suckling may disturb the balance between stimulation and inhibition of PRL release resulting in impaired suckling-induced PRL secretion in OFA rats.

Superior mesenteric ganglion neural modulation of ovarian angiogenesis, apoptosis and proliferation by the neuroactive steroid allopregnanolone.

Allopregnanolone (ALLO), a potent neuroactive steroid, is synthesized and active in the peripheral nervous system. Previous studies have shown that ALLO participates in the central regulation of reproduction with effects on ovarian physiology, although there is little evidence for its ability to modulate peripheral tissues. The present study aimed to determine whether ALLO, administered to an ex vivo system that comprises the superior mesenteric ganglion (SMG), the ovarian nervous plexus (ONP) and the ovary (O), or to the denervated ovary (DO), was able to modify ovarian apoptosis, proliferation and angiogenesis. For this purpose, the SMG-ONP-O system and DO were incubated during 120 min at 37°C, in the presence of two ALLO doses (0.06 µm and 6 µm). The intrinsic and extrinsic pathways of apoptosis were analyzed. Incubation of the SMG-ONP-O system with ALLO 0.06 µm led to an increase in the BAX/BCL-2 ratio and a reduction of FAS-L mRNA levels. ALLO 6 µm induced a decrease of FAS-L levels. Incubation of DO with ALLO 0.06 µm reduced FAS-L, whereas ALLO 6 µm significantly increased it. Cyclin D1 mRNA was measured to evaluate proliferation. Treatment with ALLO 6 µm increased proliferation in both SMG-ONP-O and DO. ALLO 0.06 µm produced an increase of Cyclin D1 in DO only. Administration of either ALLO dose led to a higher ovarian expression of vascular endothelial growth factor in the SMG-ONP-O system, but a lower one in the DO system. ALLO 6 µm induced ovarian sensitization to GABA by increasing GABAA receptor expression. In conclusion, ALLO participates in the peripheral neural modulation of ovarian physiology. It can also interact directly with the ovarian tissue, modulating key mechanisms involved in normal and pathological processes in a dose-dependent manner.

The epigenetic role of breastfeeding in mammary differentiation.

Maternal milk consumption can cause changes in the mammary epithelium of the offspring that result in the expression of molecules involved in the induction of differentiation, reducing the risk of developing mammary cancer later in life. We previously showed that animals that maintained a higher intake of maternal milk had a lower incidence of mammary cancer. In the present study, we evaluated one of the possible mechanisms by which the consumption of maternal milk could modify the susceptibility to mammary carcinogenesis. We used Sprague Dawley rats reared in litters of 3 (L3), 8 (L8), or 12 (L12) pups per mother in order to generate a differential consumption of milk. Whole mounts of mammary glands were performed to analyze the changes in morphology. Using real-time polymerase chain reaction (PCR), we analyzed the expression of mammary Pinc, Tbx3, Stat6, and Gata3 genes. We use the real-time methylation-specific polymerase chain reaction method to assess the methylation status of Stat6 and Gata3 CpG sites. Our findings show an increase in the size of the epithelial tree and a smaller number of ducts called terminal end buds in L3 vs. L12. We observed an increased expression of mRNA of Stat6, Gata3, Tbx3, and a lower expression of Pinc in L3 with respect to L12. Stat6 and Gata3 are more methylated in the CpG islands of the promoter analyzed in L12 vs. L3. In conclusion, the increased consumption of maternal milk during the postnatal stage generates epigenetic and morphological changes associated with the differentiation of the mammary gland.

High rates of Trypanosoma cruzi infection in goats from Mendoza province, Argentina: Parasite loads in blood and seasonal variation.

Mendoza province, in central west Argentina, is considered among the high-risk provinces for vector transmission of Trypanosoma cruzi, the causative agent of Chagas disease. Extensive goat farming is common in large parts of rural Mendoza, and goats may act as a reservoir for this parasite. The objective of this study was to determine infection rates, parasite loads, and seasonal variation of these parameters in T. cruzi infection in goats from rural areas of three departments of Mendoza. A total of 349 peripheral blood samples with EDTA / guanidine were analyzed from goats on 11 farms (three in Lavalle, three in San Carlos, and five in Malargüe department) in spring of 2014, 2015, and 2016; and in fall of 2015 and 2016 (only Malargüe). DNA was extracted using a Phenol: Chloroform: Isoamyl protocol. The detection and quantification of T. cruzi was performed through qPCR amplification using satellite oligonucleotides. Of the 349 blood samples, 267 (77%) were positive, with parasite loads ranging between <0.10 and 10.90 par-eq/mL (median 0.10). In spring, frequencies of infection in the three departments ranged between 86% and 95%, but differences were not significant. Median parasite loads were higher in Lavalle than in the other departments, while those in goats from San Carlos were consistently low. The frequency of infection and parasite loads in Malargüe were significantly higher in spring than in fall. This seasonal variation may have been related to a reduced nutritional status and impaired immune response of goats in spring. In conclusion, the high proportion of positive goats confirms the persistence of T. cruzi in rural Mendoza.

Human renal adipose tissue from normal and tumor kidney: its influence on renal cell carcinoma.

Tumor cells can interact with neighboring adipose tissue. We evaluated components present in human adipose explants from normal (hRAN) and kidney cancer (hRAT) tissue, and we evaluated the effects of conditioned media (CMs) from hRAN and hRAT on proliferation, adhesion and migration of tumor and non-tumor human renal epithelial cell lines. In addition, we evaluated the expression of AdipoR1, ObR, CD44, vimentin, pERK and pPI3K on cell lines incubated with CMs. hRAN were obtained from healthy operated donors, and hRAT from patients who underwent a nephrectomy. hRAT showed increased levels of versican, leptin and ObR; and decreased levels of perilipin, adiponectin and AdipoR1, compared to hRAN. Cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRAT-CMs vs. hRAN- or control-CMs. Surprisingly, HK-2, 786-O and ACHN cells showed a significant decrease in cell migration after incubation with hRAN-CMs vs. control-CMs. No difference in proliferation of cell lines was found after 24 or 48 h of treatment with CMs. AdipoR1 in ACHN and Caki-1 cells decreased significantly after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. ObR and CD44 increased in tumor line cells, and vimentin increased in non-tumor cells, after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. We observed an increase in the expression of pERK and pPI3K in HK-2, 786-O and ACHN, incubated with hRAT-CMs. In conclusion, results showed that adipose microenvironment can regulate the behavior of tumor and non tumor human renal epithelial cells.

Fetal programming by androgen excess in rats affects ovarian fuel sensors and steroidogenesis.

Fetal programming by androgen excess is hypothesized as one of the main factors contributing to the development of polycystic ovary syndrome (PCOS). PCOS is more than a reproductive disorder, as women with PCOS also show metabolic and other endocrine alterations. Since both ovarian and reproductive functions depend on energy balance, the alterations in metabolism may be related to reproductive alterations. The present study aimed to evaluate the effect of androgen excess during prenatal life on ovarian fuel sensors and its consequences on steroidogenesis. To this end, pregnant rats were hyperandrogenized with testosterone and the following parameters were evaluated in their female offspring: follicular development, PPARG levels, adipokines (including leptin, adiponectin, and chemerin as ovarian fuel sensors), serum gonadotropins (LH and FSH), the mRNA of their ovarian receptors, and the expression of steroidogenic mediators. At 60 days of age, the prenatally hyperandrogenized (PH) female offspring displayed both an irregular ovulatory phenotype and an anovulatory phenotype with altered follicular development and the presence of cysts. Both PH groups showed altered levels of both proteins and mRNA of PPARG and a different expression pattern of the adipokines studied. Although serum gonadotropins were not impaired, there were alterations in the mRNA levels of their ovarian receptors. The steroidogenic mediators Star, Cyp11a1, Cyp17a1, and Cyp19a1 were altered differently in each of the PH groups. We concluded that androgen excess during prenatal life leads to developmental programming effects that affect ovarian fuel sensors and steroidogenesis in a phenotype-specific way.

Estradiol and progesterone regulate proliferation and apoptosis in colon cancer.

Epidemiological studies describe estrogens as protectors in the development of colon cancer in postmenopausal women treated with hormone replacement therapy. However, the role of progesterone in colon cancer has been minimally studied and the results are controversial. For the above, the objective of this work was to determine the hormonal regulation exerted by natural ovarian steroids on proliferation and apoptosis in an experimental model of colon cancer in ovariectomized rats treated with 17-beta estradiol and progesterone. Sprague-Dawley rats were exposed to the carcinogen 1,2-dimethylhydrazine to induce colon tumors. Thirty days later, the rats were ovariectomized and treated with estradiol (60 µg/kg), progesterone (10 mg/kg), estradiol plus progesterone (60 µg/kg and 10 mg/kg) or vehicle. We observed no significant differences in colon cancer incidence and tumor multiplicity between the groups. Nevertheless, we observed a decrease in PCNA expression and a greater number of apoptotic index, higher expression of caspase 3, cleaved PARP and cleaved caspase 8 in tumors, confirming the activation of the extrinsic pathway of apoptosis by the combined treatment. In addition, we observed a higher expression of estrogen receptor beta in these tumors. We conclude that the action of both hormones, estradiol and progesterone, is necessary to reduce proliferation and increase apoptosis in colon tumors, probably through estrogen receptor beta activation.

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma.

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease.

Hypothyroidism decreases JAK/STAT signaling pathway in lactating rat mammary gland.

Thyroid pathologies have deleterious effects on lactation. Especially hypothyroidism (HypoT) induces premature mammary involution at the end of lactation and decreases milk production and quality in mid lactation. Milk synthesis is controlled by JAK2/STAT5 signaling pathway and prolactin (PRL), which activates the pathway. In this work we analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT on PRL signaling pathway on mammary glands from rats on lactation (L) days 2, 7 and 14. HypoT decreased prolactin receptor expression, and expression and activation of Stat5a/b protein. Expression of members of the SOCS-CIS family, inhibitors of the JAK-STAT pathway, decreased in L2 and L7, possibly as a compensatory response of the mammary cells to maintain PRL responsiveness. However, on L14, the level of these inhibitors was normal and the transcription of α-lactoalbumin (lalba), a target gene of the PRL pathway, decreased by half. HypoT altered the transcriptional capacity of the cell and decreased mRNA levels of Prlr and Stat5b on L14. Stat5b gene has functional thyroid hormone response elements in the regulatory regions, that bind thyroid hormone receptor ß (TRß) differentially and in a thyroid hormone dependent manner. The overall decrease in the PRL signaling pathway and consequently in target gene (lalba) mRNA transcription explain the profound negative impact of HypoT on mammary function through lactation.

Hypothyroidism advances mammary involution in lactating rats through inhibition of PRL signaling and induction of LIF/STAT3 mRNAs.

Thyroid diseases have deleterious effects on lactation, litter growth and survival, and hinder the suckling-induced hormone release, leading in the case of hyperthyroidism, to premature mammary involution. To determine the effects of hypothyroidism (HypoT) on late lactation, we analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT on mammary histology and the expression of members of the JAK/STAT/SOCS signaling pathway, milk proteins, prolactin (PRLR), estrogen (ER), progesterone (PR) and thyroid hormone (TR) receptors, markers of involution (such as stat3, lif, bcl2, BAX and PARP) on lactation (L) day 21. HypoT mothers showed increased histological markers of involution compared with control rats, such as adipose/epithelial ratio, inactive alveoli, picnotic nuclei and numerous detached apoptotic cells within the alveolar lumina. We also found decreased PRLR, ß-casein and α-lactoalbumin mRNAs, but increased SOCS1, SOCS3, STAT3 and LIF mRNAs, suggesting a decrease in PRL signaling and induction of involution markers. Furthermore, Caspase-3 and 8 and PARP labeled cells and the expression of structural proteins such as ß-Actin, α-Tubulin and Lamin B were increased, indicating the activation of apoptotic pathways and tissue remodelation. HypoT also increased PRA (mRNA and protein) and erß and decreased erα mRNAs, and increased strongly TRα1, TRß1, PRA and ERα protein levels. These results show that lactating HypoT rats have premature mammary involution, most probably induced by the inhibition of prolactin signaling along with the activation of the LIF-STAT3 pathway.

Effect of hypothyroidism on the expression of nuclear receptors and their co-regulators in mammary gland during lactation in the rat.

Thyroid hormones (TH) regulate mammary function. Hypothyroidism (HypoT) has deleterious effects on lactation, litter growth and survival. We analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT in the expression of nuclear receptors, co-regulators and oxytocin receptor (OTR) on lactation (L) days 2, 7 and 14. TH receptors (TRs) were increased on L7 at mRNA and protein levels, except TRα protein, that fell on L14. HypoT decreased TRα2 mRNA on L7 and TRα1 protein on L2, while TRß1 protein increased on L14. HypoT increased estrogen receptor ß (ERß) mRNA on L7 but decreased its protein levels on L14. Progesterone receptor A (PRA) mRNA decreased from L2 to L14 while PRB increased, and at protein levels PRA levels showed a nadir on L7, while PRB peaked. HypoT decreased PRA mRNA and protein and increased PRB mRNA at L14. Nuclear receptor co-activator (NCOA) 1 and RXRα mRNA showed an opposite pattern to the TRs, while NCOA2 increased at L14; HypoT blocked the variations in NCOA1 and NCOA2. HypoT increased NCOR1 on L2 and decreased OTR at L2 and circulating estradiol and NCOR2 at L14. In controls the most notable changes occurred on L7, suggesting it is a key inflection point in mammary metabolism. The low levels of TRα1, NCOA1 and OTR, and increased NCOR1 produced by HypoT on L2 may hinder the mammary ability to achieve normal milk synthesis and ejection, leading to defective lactation. Later on, altered ER and PR expression may impair further mammary function.

Estradiol promotes luteal regression through a direct effect on the ovary and an indirect effect from the celiac ganglion via the superior ovarian nerve.

There is evidence suggesting that estradiol (E(2)) regulates the physiology of the ovary and the sympathetic neurons associated with the reproductive function. The objective of this study was to investigate the effect of E(2) on the function of late pregnant rat ovaries, acting either directly on the ovarian tissue or indirectly via the superior ovarian nerve (SON) from the celiac ganglion (CG). We used in vitro ovary (OV) or ex vivo CG-SON-OV incubation systems from day 21 pregnant rats. Various concentrations of E(2 )were added to the incubation media of either the OV alone or the ganglion compartment of the CG-SON-OV system. In both experimental schemes, we measured the concentration of progesterone in the OV incubation media by radioimmunoassay at different times. Luteal messenger RNA (mRNA) expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD) enzymes, respectively, involved in progesterone synthesis and catabolism, and of antiapoptotic B-cell lymphoma 2 (Bcl-2) and proapoptotic Bcl-2-associated X protein (Bax), were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) at the end of the incubation period. Estradiol added directly to the OV incubation or to the CG of the CG-SON-OV system caused a decline in the concentration of progesterone accumulated in the incubation media. In addition, E(2), when added to the OV incubation, decreased the expression of 3ß-HSD and the ratio of Bcl-2/Bax. We conclude that through a direct effect on the OV, E(2) favors luteal regression at the end of pregnancy in rats, in association with neural modulation from the CG via the SON.