Gestational Low Protein Diet Modulation on miRNA Transcriptome and Its Target During Fetal and Breastfeeding Nephrogenesis

BackgroundThe kidney ontogenesis is the most structurally affected by gestational protein restriction, reducing 28% of their functional units. The reduced nephron number is predictive of hypertension and cardiovascular dysfunctions that are generally observed in the adult age of most fetal programming models. We demonstrate miRNAs and predict molecular pathway changes associated with reduced reciprocal interaction between metanephros cap (CM) and ureter bud (UB) and a 28% decreased nephron stem cells in the 17 gestational days (17GD) low protein (LP) intake male fetal kidney. Here, we evaluated the same miRNAs and predicted targets in the kidneys of 21GD and at 7 days of life (7DL) LP offspring to elucidate the molecular modulations during nephrogenesis.MethodsPregnant Wistar rats were allocated into two groups: NP (regular protein diet- 17%) or LP (diet-6%). miRNA transcriptome sequencing (miRNA-Seq) was performed on the MiSeq platform from 21GD and 7DL male offspring kidneys using previously described methods. Among the top 10 dysfunctional regulated miRNAs, we validated 7 related to proliferation, differentiation, and apoptosis processes and investigated predicted target genes and proteins by RT-qPCR and immunohistochemistry.ResultsIn 21GD, LP fetuses were identified alongside 21 differently expressed miRNAs, of which 12 were upregulated and 9 downregulated compared to age-matched NP offspring. In 7-DL LP offspring, the differentially expressed miRNAs were counted to be 74, of which 46 were upregulated and 28 downregulated. The curve from 17-GD to 7-DL shows that mTOR was fundamental in reducing the number of nephrons in fetal kidneys where the mothers were subjected to a protein restriction. IGF1 and TGFβ curves also seemed to present the same mTOR pattern and were modulated by miRNAs 181a-5p, 181a-3p, and 199a-5p. The miRNA 181c-3p modulated SIX2 and Notch1 reduction in 7-DL but not in terms of the enhanced expression of both in the 21-GD, suggesting the participation of an additional regulator. We found enhanced Bax in 21-GD; it was regulated by miRNA 298-5p, and Bcl2 and Caspase-3 were controlled by miRNA (by 7a-5p and not by the predicted 181a-5p). The miRNA 144-3p regulated BCL6, which was enhanced, as well as Zeb 1 and 2 induced by BCL6. These results revealed that in 21GD, the compensatory mechanisms in LP kidneys led to the activation of UB ramification. Besides, an increase of 32% in the CM stem cells and a possible cell cycle halt of renal progenitor cells, which remaining undifferentiated, were observed. In the 7DL, much more altered miRNA expression was found in LP kidneys, and this was probably due to an increased maternal diet content. Additionally, we verified the activation of pathways related to differentiation and consumption of progenitor cells

Prostate telocytes change their phenotype in response to castration or testosterone replacement

Telocytes are CD34-positive cells with a fusiform cell body and long, thin cytoplasmic projections called telopodes. These cells were detected in the stroma of various organs, including the prostate. The prostate is a complex gland capable of undergoing involution due to low testosterone levels; and this condition can be reversed with testosterone replacement. Telocyte function in the mature prostate remains to be dermined, and it is not known whether telocytes can take place in tissue remodeling during prostate involution and regrowth. The present study employed structural, ultrastructural and immunohistochemical methods to investigate the telocyte's phenotypes in the ventral prostate (VP) from control (CT), castrated (CS) and testosterone replacement (TR) groups of adult male Wistar rats. Telocytes were found in the subepithelial, perimuscular and interstitical regions around glandular acini. Telocytes from CT animals have condensed chromatin and long and thin telopodes. In CS group, telocytes appeared quiescent and exhibited layers of folded up telopodes. After TR, telocytes presented loose chromatin, abundant rough endoplasmic reticulum and enlarged telopodes, closely associated with bundles of collagen fibrils. We called these cells "telocytes with a synthetic phenotype". As testosterone levels and glandular morphology returned toward to the CT group parameters, after 10 days ofTR, these telocytes progressively switched to the normal phenotype. Our results demonstrate that telocytes exhibit phenotypic plasticity upon androgen manipulation and interact with fibroblast and smooth muscle cells to maintain glandular architecture in control animals and during tissue remodeling after hormonal manipulation9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305391/2014-3; 306900/2016-5; 305840/2015-0; 310663/2018-02002/11102-4; 2014/26660-0; 2017/01063-

Efeito do estradiol sobre a prostata da cobaia Cavia porcellus em diferentes fases do desenvolvimento pos-natal

Orientador : Sebastião Roberto TabogaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A próstata é uma glândula anexa do sistema reprodutor masculino que tem despertado grande interesse na área biomédica devido às inúmeras patologias que a acometem. Frente ao desbalanço hormonal, ocasionado principalmente pelo processo de senescência, o tecido prostático desenvolve dentre outras doenças, a hiperplasia benígna prostática (BPH), metaplasias do epitélio, prostatites e o câncer prostático. O estrógeno parece ter papel fundamental na gênese da hiperplasia benígna prostática e do câncer da próstata, porém, os aspectos que envolvem a relação estroma-epitélio não são completamente esclarecidos. O objetivo do presente estudo foi avaliar os efeitos da administração crônica do estradiol sobre os componentes epitelial e estromal da próstata de cobaias machos Cavia porceUus ao longo do desenvolvimento pós-natal, utilizando técnicas histológicas, histoquímicas e ultra-estruturais. Foram utilizados 10 animais de cada idade: pré-púbere, púbere, pós-púbere e adultos, sendo que 5 foram destinados ao grupo controle e 5 ao grupo tratado, que recebeu injeção subcutânea semanal de Benzoato de Estradiol durante 4 semanas. Após o tratamento as próstatas foram fixadas e incluídas para a microscopia de luz e microscopia eletrônica de transmissão. Foram feitos testes histoquímicos para fibras colágenas (Tricrômico de Masson, Picrossírius-Hematoxilina), fibras elásticas (Resorcina Fucsina de Weigert) e fibras reticulares (Reticulina de Gõmõn), além das colorações usuais pela Hematoxilina-eosina e pelo Azul de Toluidina. Para a caracterização quantitativa dos componentes prostáticos foi realizada uma análise morfométrica-estereológica, sendo que os dados obtidos foram comparados aplicandose o Teste T (p::;;O.OS). Na microscopia eletrônica de transmissão foram feitos procedimentos rotineiros para a caracterização ultra-estrutural de elementos epiteliais e do estroma prostático. A análise histopatológica do material revelou regiões onde há ocorrência da Neoplasia Intraepitelial Prostática (PIN) característica do padrão epitelial denominado cribriforme. Além disso, nota-se nos animais experimentais, um estreitamento da região clara supranuclear, descrita como sendo a região do complexo de Golgi. Observa-se perda da polaridade celular, núcleos de diversas formas e tamanhos, em diferentes alturas e com padrões de distribuição cromatínica irregular. Tais alterações foram encontradas, principalmente, nos animais púberes, pós-púberes e adultos. A análise histoquímica , juntamente com a análise morfométrica estereológica, revelaram nos animais tratados hiperplasia estromal com aumento da camada fibromuscular que circunda os ácinos glandulares nas idades pré-púbere, pós-púbere e adulta. Além disso, observou-se um aumento na quantidade dos elementos fibrilares da matriz estromal, como o colágeno, as fibras reticulares e as fibras do sistema elástico, entremeando células musculares lisas que se arranjam, às vezes, de forma não concêntrica. A ultta-estrutura mostrou que nos animais tratados ocorre um certo desarranjo no sistema de membranas internas nas células epiteliais que assumem fenótipos variados, hipertrofia de células basais, presença de feixes fibrilares colagênicos espessos e de fibras do sistema elástico em maior quantidade quando comparado aos animais do grupo controle, além de reforçar os achados histológicos como o padrão epitelial cribriforme e a hiperplasia estromal. Estes dados sugerem que o estrógeno pode provocar modificações celulares e teciduais em qualquer etapa do desenvolvimento, principalmente nas fases em que os níveis de testosterona estão aumentados (após a puberdade), e com isso provocar alterações fisiopatológicas relevantes para o funcionamento da glândula prostáticaAbstract: The prostate is an attached gland of the male reproductive system that has developed great interest in the biomedical area because to the innumerable pathologies that attacks it. Front to hormonal disorder, caused mainly for the aging, the prostatic tis sue develops amongst other diseases, the benign prostatic hyperplasia (BPH), metaplasias of the epithelium, prostatitis and the prostate cancer. The estrogen hormone seems to have essential role in genesis of the benign prostatic hyperplasia and prostate cancer however the aspects that involve the stroma-epithelium relation completely are not clarified. The objective of the present study was to evaluate the effect of the chronic administration of estrogen on epithelial and stromal components of the male Guinea pig prostate to long of developmen_ being used the hystological and structural techniques. For this experiment were utilized 10 animals of each age: pre-puberal age, puberal age, post-puberal age and adult age, which 5 animaIs were destined to control group whereas 5 animals were destined to treated group that received weekly subcutaneous injections of Benzoate of estradiol during 4 weeks. After the treatment the prostates were fixed and embedded for light and transmission electron microscopy. Histochemical tests were carried out for collagen fibers (Masson's trichrome, Picrossirius Hematoxylin), elastic fibers (Weigert's Resorcin Fucsin) and reticular fibers (Gõmõri's reticulin), as well as the usual staining methods with Hematoxilin-eosin and Toluidine blue. To quantitative characterization of the prostatic components were carried out using the morphometric-stereological analyze, where the data were compared using statistical test T (p_0.05). At the transmission electron microscopy the routine procedures were followed for the ultrastructural characterization of the epithelium and prostatic stroma elements. The hystopatological analysis of the material disclosed regions where it has occurrence of Prostatic Intraepithelial Neoplasia (pIN), characteristic of the cribriform epithelial pattem. Moreover, it is noticed in the experimental animaIs, a narrowing of the clear up-nuclear region, described as being the region of the Golgi complexo One observes loss of the cellular polarity, nuclei of diverse forms, sizes, in different heights and with pattem of irregular chromatin distribution. Such alterations had been found, mainly, in the puberal , post-puberal and adult animals. The hystochemical analysis, together with the morphometric-stereological analysis, had disclosed in the treated animals stromal hyperplasia with increased to the layer to fibromuscular that surrounds the glandular acini in the pre-puberal, post-puberal and adult ages. Moreover, there were an increased in the amount of the fibrilar elements from the stromal matrix, as the collagen and reticular fibers and the elastic system fibers, intermixing smooth muscular cells that if they arrange, sometimes, of disorganized formo The structural analysis showed that in the treated animals occurs a certain disarrangement in the system of internal membranes in the epithelial cells that assume varied phenotypes, hypertrophy of basal cells, presence of thick collagen fibers and the elastic system fibers in bigger amount than control group, besides strengthening the hysthologic findings as the cribriform epithelial pattem and the stromal hyperplasia. These data suggest that estrogen can provoke cellular and tecidual modifications in any stage of the development, mainly in the phases where the testosterone levels are increased (after the puberty), and with this to cause important physiopathological alterations for the functioning of the prostate glandMestradoBiologia CelularMestre em Biologia Celular e Estrutura

Tissue Evidence Of The Testosterone Role On The Abnormal Growth And Aging Effects Reversion In The Gerbil (meriones Unguiculatus) Prostate.

Prostate differentiation during embryogenesis and its further homeostatic state maintenance during adult life depend on androgens. Abundant biological data suggest that androgens play an important role in the development of the prostate cancer and other prostatic diseases. The objective of this work was to evaluate the effects of the testosterone supplementation in gerbil (a new experimental model) at different ages. Tissues from experimental animals were studied by histological and histochemistry procedures, androgen receptor immunohistochemistry assay, morphometric-stereological analysis, and transmission electron microscopy (TEM). After the treatment were observed increase of prostate weight and epithelium height in all ages studied. In some adult and aged treated animals, hyperplasic and dysplastic process were observed, including prostatic intraepithelial neoplasias and adenocarcinomas. Increase of the thickness of the smooth muscle cell (SMC) layer was observed in pubescent and adult animals and TEM revealed apparent SMC hypertrophy. An apparent increase in the frequency of blood vessels distributed by the subepithelial stroma in the treated animals was noticed. Reversion of the natural effects of aging on the prostate was observed in the aged treated animals in some acini of the gland. These data demonstrate that the gerbil prostate is susceptible to androgenic action at the studied ages and it can serve, for example, as experimental model to studies of prostate neoplastic process induction and hormonal therapy in aged animals.2881190-20

Sexual maturation of the Mongolian gerbil (Meriones unguiculatus): a histological, hormonal and spermatic evaluation

This study determined the phases of sexual development of the male Mongolian gerbil (Meriones unguiculatus) based on an integrative analysis of testicular morphology, hormonal data and sperm parameters. Male gerbils were analysed at 1, 7, 14, 21, 28, 35, 42, 50, 60, 70, 90, 100 and 120 days of age. Body, testicular and epididymal weights increased up to Day 70, 60 and 90, respectively. The impuberal phase, characterised by the presence of gonocytes, extended until Day 14. The prepubertal period lasted until Day 42, when puberty was achieved and a drastic increase in serum testosterone levels, mature adult Leydig cells and elongated spermatids was observed. Gerbils at 60 days of age showed a remarkable number of spermatozoa in the testis, epididymidis caput/corpus and cauda, and at Day 70 the maximum daily sperm production was reached. However, the gerbil may be considered sexually mature only from Day 90 onward, when sperm reserves become stable. The total transit time of spermatozoa along the epididymis of sexually mature gerbils was 11 days, with 1 day in the caput/corpus and 10 days in the cauda. These data cover a lacuna regarding the reproductive parameters of this rodent and provide foundations for its use in testicular toxicology studies.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Gestational protein malnutrition impairs c-myc and p63 protein expression, increases prostatic intraepithelial neoplasia incidence and prostatitis aggressiveness in adult male offspring subjected to hormonal handling

Background: Prostate cancer is the second most common cancer diagnosed in men; however its etiology remains unknown. Previous studies have shown that environmental adverse factors, such as maternal nutritional status during pregnancy, can influence fetal development and predispose people to diseases in adult life. The feeding of low-protein diets to pregnant rats result in fetal growth disturbance, androgen/estrogen unbalance and changes in the expression and sensibility of hormone receptors in male offspring. These alterations can promote permanent changes in androgen dependent organs, such as in the prostate. In this sense, we hypothesized that the hormonal unbalance that occurs during aging can lead to an increase in the susceptibility to prostatic disorders. Aim: To evaluate our hypothesis, malnourished male rat offspring were submitted to simultaneous estrogen and testosterone exposure in adulthood, to drive lesions in the rat ventral prostate gland (VP). Methods: 17 week-old Wistar rats (n=48) that received in utero normal protein diet (NP group, AIN93G=17% protein) or low protein diet (RP group, AIN93G modified=6% protein) were given implants with 17β-estradiol plus testosterone administration (NPH and RPH groups) for 17 weeks. The animals were killed at the age of 34 weeks and the VP were excised, weighted and processed for histopathological, immunohistochemical (Ki67, AR, p63, e-caderin, laminin, c-myc and GSTP), biochemical and ultrastructural analysis. Results: Both absolute and relative VP weight from NPH animals were about 30% higher than RPH. Serological data showed that estradiol levels were similar in both groups, but testosterone levels were lower in the RPH male offspring. The steroid hormone exposure in adult life promoted prostate lesions in both RPH and NPH offspring associated with reactive stroma. VP from RPH group exhibited heightened susceptibility to prostatic intraepithelial neoplasia (mainly cribriform and signet ring-cell patterns) and increased the incidence and aggressiveness of prostatitis. In this group, a higher proportion of basal cells, increased proliferation index, lower expression ofthe androgen receptor and increased focus of collagenous micronodules closely associated to epithelial neoplasias were also observed. Conclusion:These observations suggest that maternal protein restriction alters adult prostate response to androgen/estrogen handling and increases susceptibility to prostate diseases. Ethical protocol:CEEA,476/2013 IBB-UNESP; Funding Support: 2009/50204-6 and 2013/09649-0

Genistein reduces the noxious effects of in utero bisphenol A exposure on the rat prostate gland at weaning and in adulthood

Bisphenol A (BPA) is one hormonally active chemical with potential deleterious effects on reproductive organs, including breast and prostate. In contrast, genistein (GEN) is the major phytoestrogen of soy that presents potential protective effects against hormone-dependent cancers, including that of the prostate. Thus, pregnant Sprague-Dawley rats were treated with BPA at 25 or 250 μg/kg/day by gavage from gestational day (GD) 10-21 with or without dietary GEN at 250 mg/kg/chow (∼5.5 mg/kg/day). Then, male offspring from different litters were euthanized on post-natal day (PND) 21 and 180. At PND21, BPA 25 exposure induced early prostatic changes while dietary GEN attenuated some deleterious actions this xenoestrogen on epithelial cell proliferation levels, androgen receptor expression and prostatic architecture in male offspring. At PND180, a significant increase in incidence of prostatic multifocal inflammation/reactive hyperplasia and atypical hyperplasia were observed in male offspring from dams that received BPA 25. On the other hand, maternal GEN feeding attenuated some the adverse effects of BPA 25 on prostate disease at late-in-life. This way, the present findings point to preventive action of dietary GEN on deleterious effects of gestational BPA exposure in both early and late prostate development in offspring F1

Testosterone Therapy Differently Regulates the Anti- and Pro-Inflammatory Cytokines in the Plasma and Prostate of Rats Submitted to Chronic Ethanol Consumption (UChB)

ProblemEthanol consumption damages the prostate, and testosterone is known by anti-inflammatory role.Methods of StudyThe cytokines were investigated in the plasma and ventral prostate of UChB rats submitted or not to testosterone therapy by ELISA and Western blot, respectively. Additionally, inflammatory foci and mast cells were identified in the ventral prostate slides stained by hematoxylin and eosin and toluidine blue, respectively.ResultsInflammatory foci were found in the ethanol-treated animals and absent after testosterone therapy. Plasma levels of IL-6 and IL-10 were not changed while TNF alpha and TFG-beta 1 were increased in the animals submitted testosterone therapy. Regarding to ventral prostate, IL-6 did not alter, while IL-10, TNF alpha, and TFG-b1 were increased after testosterone therapy. Ethanol increases NFR2 in addition to high number of intact and degranulated mast cell which were reduced after testosterone therapy.ConclusionsSo, ethanol and testosterone differentially modulates the cytokines in the plasma and prostate.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES