Vitrification leads to transcriptomic modifications of mice ovaries that do not affect folliculogenesis progression.

Ovarian tissue cryopreservation is emerging as a promising alternative for fertility preservation of cancer survivors. To date, more than a hundred couples have successfully had babies using this procedure, although it is still considered experimental and demands further investigation. In this work, we evaluated the effects of vitrification, warming and autotransplantation procedures on the morphology and gene expression of murine ovaries. Ovaries were removed from adult female C57BL6 mice (n = 15), vitrified, warmed and autotransplanted (vitrified group), additionally, ovaries were autotransplanted without vitrification (control group, n = 15). After twenty days, grafted ovaries were harvested and used for histological and ultrastructural analysis, germinal vesicle (GV) oocyte collection, RNA sequencing, and Transmission Electron Microscopy (TEM). All classes of follicles and GV were observed in both control and vitrified/warmed transplanted ovaries, and the numbers of primordial, antral and atretic follicles were not different (p > 0.05). Using RNA-seq, we detected 16,602 vs 13,527 expressed genes in vitrified and control ovaries, respectively; and 623 significantly dysregulated genes (fold change >1.5; 332 up-regulated and 291 down-regulated). Cellular membranes, cytoskeletons, and extracellular matrices were found as the main functions of the differentially expressed genes. Moreover, vitrified samples also presented ultrastructural alterations in the cytoskeleton, cell junctions, and endoplasmic reticulum. Taken together, this work showed for the first time that ovarian cells might trigger a compensatory gene regulation mechanism to maintain cellular structure and folliculogenesis progression after vitrification and autotransplantation.

Effects of anabolic steroid treatment associated with physical training in adipose tissue of male Wistar rats / Efeito do tratamento com anabolizante associado com atividade física no tecido adiposo de ratos machos Wistar

Anabolic androgenic-steroids (AAS) include a broad class of synthetic derivatives of testosterone, being nandrolone decanoate the most widely used in sports environment. The aim of this study was to evaluate the metabolic effects of nandrolone decanoate in sedentary and trained adult male rats. We established four experimental groups: sedentary control, sedentary treated, trained control and trained treated. The training had consisted of running on a treadmill for nine weeks. Treated animals received intramuscular injections of nandrolone decanoate (0.5 mg kg-1) during the last four weeks of physical training. The training time as the drug used were not sufficient to significantly reduce body weight gain, but caused a significative decrease on diameter of adipocytes and in the amount of adipose tissue stored, as well as decreased the plasma levels of glucose and total cholesterol.

Os esteróides anabólicos androgênios incluem uma ampla classe de derivados sintéticos da testosterona, sendo o decanoato de nandrolona um dos mais utilizados no meio esportivo. Este trabalho teve como objetivo avaliar os efeitos metabólicos desse anabolizante esteróide em ratos machos adultos sedentários e treinados. Foram estabelecidos quatro grupos experimentais: sedentário controle, sedentário tratado, treinado controle e treinado tratado. O treinamento consistiu de corrida em esteira ergométrica durante nove semanas. Os animais tratados receberam injeção intramuscular de decanoato de nandrolona (0.5 mg kg-1) durante as quatro últimas semanas de treinamento físico. Tanto o tempo de treinamento, quanto a dose de anabolizante utilizado não foi eficiente para reduzir significativamente o ganho de peso corporal, mas causou reduções significativas no diâmetro dos adipócitos e na quantidade de tecido adiposo armazenado, assim como diminuiu os valores plasmáticos de glicose e de colesterol total.

Morphometric evaluation of the spermatogonial stem cell distribution and niche in vertebrates.

Morphometry is a classical quantitative method often used in biology to provide a data basis for functional interpretations/interactions of a particular organ or system. Herein we took advantage of this valuable approach to evaluate the spermatogonial stem cell niche using the horse testis and immunocytochemical localization of GFRA1 [glial cell line-derived neurotrophic factor receptor produced by Sertoli cells)] as an example. Using the NIH ImageJ free software, we describe in detail all the necessary steps to investigate this specific and crucial microenvironment. Based on several recently published papers from our research group, this approach has proved to be fast, simple, and adaptable to a wide range of species and has the potential to be easily reproducible in different laboratories.

Testis morphophysiology of rats treated with nandrolone decanoate and submitted to physical training / Morfofisiologia testicular de ratos tratados com decanoato de nandrolona e submetidos ao treinamento físico

The mammalian testis is a complex organ with endocrine and exocrine functions. It consists of seminiferous tubules where the production of the male gametes called spermatogenesis occurs. This process is influenced by a number of factors including the use of physical performance-enhancing drugs. The objective of this study was to evaluate the effects of the anabolic steroid nandrolone decanoate on the morphofunctional structure of testes in sedentary rats and rats subjected to moderate aerobic exercise training. Twenty-four male rats were divided into four experimental groups: sedentary control, sedentary treated, trained control and trained treated.The training lasted eight weeks and consisted of running on a programmable ergometer treadmill, tailored to train eight rats simultaneously. Treated animals received intramuscular injections of nandrolone decanoate (0.5 mg kg-1 body weight) during the last four weeks of physical training, while the control groups received intramuscular injections of vehicle (vegetable oil).The male reproductive system morphology showed that treatment with nandrolone decanoate, in both sedentary and trained rats, promoted morphological and functional changes that result in reduced efficiency of spermatogenesis.

O testículo de mamíferos é um órgão complexo com funções endócrinas e exócrinas. É composto por túbulos seminíferos, local onde ocorre a espermatogênese, processo de produção dos gametas masculinos. Este processo é influenciado por uma série de drogas potencializadoras do desempenho físico, que são utilizadas para melhorar a performance. O objetivo deste estudo foi avaliar os efeitos do anabolizante esteróide decanoato de nandrolona sobre a estrutura morfofuncional de testículos de ratos sedentários e submetidos ao treinamento físico aeróbico moderado. Foram utilizados vinte e quatro ratos machos divididos em quatro grupos experimentais: sedentário controle, sedentário tratado, treinado controle e treinado tratado. O treinamento teve duração de oito semanas e consistiu de corrida em esteira ergométrica programável, adaptada para treinar oito ratos simultaneamente. Os grupos tratados receberam injeção intramuscular de decanoato de nandrolona (0,5 mg kg -1) durante as quatro últimas semanas de treinamento físico, enquanto os grupos controles receberam injeção intramuscular do veículo (óleo vegetal). Os resultados mostraram que o tratamento com o decanoato de nandrolona, tanto em ratos sedentários como submetidos ao exercício físico, promove alterações morfofuncionais que resultam na redução da eficiência da espermatogênese.

Phenotypic characterization and in vitro propagation and transplantation of the Nile tilapia (Oreochromis niloticus) spermatogonial stem cells.

In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.

Morfologia testicular de ratos wistar obesos sedentários e submetidos a treinamento físico / Testicular morphology in obese and sedentary wistar rats submitted to physical training

O objetivo deste trabalho foi avaliar morfologicamente os efeitos da dieta de cafeteria e o treinamento físico em esteira sobre o testículo de ratos Wistar. Ratos machos adultos foram divididos em grupos (sedentário-controle; sedentário-cafeteria; treinado-controle; e treinadocafeteria). Para comprovar a instalação da obesidade calculou-se o índice de Lee e o peso dos tecidos adiposos periepididimal e retroperitoneal. A análise testicular envolveu o peso da gônada e após processamento histológico e coloração por Hematoxilina-Eosina, os parâmetros de diâmetro tubular, altura do epitélio seminífero, identificação dos tipos celulares presentes nos túbulos seminíferos, contagem de células e rendimento geral da espermatogênese. O aumento significativo do Índice de Lee e do peso dos tecidos adiposos, nos grupos que receberam dieta de cafeteria, comprovou a instalação da obesidade e indicou ser este um modelo adequado para induzir obesidade experimental. Não houve efeito da dieta ou do treinamento sobre o peso testicular, diâmetro tubular e altura do epitélio seminífero não havendo também diferenças na organização histológica dos testículos e túbulos seminíferos. Após quantificação celular e cálculo dos índices mitótico e meiótico e da capacidade total de suporte das células de Sertoli, verificamos efeito positivo do treinamento físico, independente da dieta recebida, sobre o rendimento geral da espermatogênese.

The aim of this study was to morphologically evaluate the effects of the cafeteria diet and physical training on the testicles of adult Wistar rats. Adult male rats were divided into groups (sedentary-control, sedentary-cafeteria, trained-control, and trainedcafeteria) In order to state the obesity condition both the Lee index and the weight of retroperitoneal and periepididymal adipose tissues were calculated. The testicular analysis involved the gonad weight and after the histological processing the Hematoxylin-Eosin staining parameters of tubular diameter, height of seminiferous epithelium, identification of the cellular sorts in the seminiferous tubules, counting of cells and overall output of spermatogenesis were done. The significant increase in the Lee index and the adipose tissue weight, in groups which received the cafeteria diet, stated the obesity condition indicating that this model is adequate to induce experimental obesity. There was no effect either from diet or training on the testicular weight, tubular diameter and height of seminiferous epithelium and also no difference in the histological organization of testicles and semineferous tubules. After cellular quantification and being calculated both mitotic and meiotic index and total capacity of Sertoli cells, it was verified a positive effect of the physical training, regardless of diet, on the overall output of spermatogenesis.

Effect of the nandrolone decanoate on the efficiency of spermatogenesis of sedentary rats and rats subjected to physical training / Efeito do decanoato de nandrolona sobre a eficiência da espermatogênese de ratos sedentários e ratos submetidos a treinamento físico

Spermatogenesis is a complex, highly organized and coordinated process that results in the production of male gametes. This process is influenced by a number of drugs that enhance physical performance, which have been used mainly by young people, athletes or not, seeking to gain athletic performance or only a prominent social position. The objective of this study was to evaluate the effects of the anabolic steroid nandrolone decanoate on the efficiency of spermatogenesis in sedentary rats and rats subjected to physical training. Twenty-four male rats were divided into four experimental groups: sedentary control, sedentary treated, trained control and trained treated. Treated animals received intramuscular injection of nandrolone decanoate (0.5 mg kg-1 body weight) during the last four weeks of physical training. The training program consisted of running on a programmable ergometer treadmill, tailored to train eight rats simultaneously, for nine weeks. We evaluated the morphological and morphoquantitative profiles of the male reproductive system. The results showed that the treatment causes a reduction in the efficiency of spermatogenesis; however, when associated with physical training, this compensates for the anabolic action, keeping the process of spermatogenesis within normality.

A espermatogênese é um processo complexo, altamente organizado e coordenado, que resulta na produção dos gametas masculinos. Este processo é influenciado por uma série de drogas potencializadoras do desempenho físico, as quais têm sido utilizadas principalmente, por jovens, atletas ou não, que buscam adquirir maior desempenho esportivo ou mesmo apenas uma posição de destaque na sociedade. O objetivo deste estudo foi avaliar os efeitos do anabolizante esteroide decanoato de nandrolona na eficiência da espermatogênese de ratos sedentários e submetidos ao treinamento físico. Foram utilizados 24 ratos machos divididos em quatro grupos experimentais: sedentário-controle, sedentário tratado, treinado-controle e treinado tratado. Os animais tratados receberam injeção intramuscular de decanoato de nandrolona (0,5 mg kg-1) durante as quatro últimas semanas de treinamento físico. O programa de treinamento consistiu de corrida em esteira ergométrica programável, adaptada para treinar oito ratos simultaneamente, durante nove semanas. Foi avaliado o perfil morfológico e morfoquantitativo do sistema reprodutor masculino. Os resultados demonstraram que o tratamento causa redução da eficiência da espermatogênese, no entanto, o treinamento físico quando associado compensa a ação do anabolizante, mantendo o processo de espermatogênese normal.

Estrous cycle, anatomy and histology of the uterine tube of the mongolian gerbil meriones unguiculatus

Fue clasificado el ciclo estral del gerbo de Mongolia y descritas las estructuras y relaciones de la tuba uterina en la cavidad abdomino-pélvica, así como la histología de su epitelio de revestimiento. Las tubas uterinas de 19 hembras adultas del gerbo de Mongolia fueron investigadas a través de técnicas de anatomía e histología. Fue observado el ciclo estral poliéstrico e irregular, con cinco fases: proestro, estro I, estro II, metaestro y diestro. El estro I se caracterizaba por presentar células queratinizadas, esparcidas y en menor número con respecto al estro II. La tuba uterina presentaba relaciones con la extremidad distal del cuerno uterino y con el ovario. La tuba presenta serosa de revestimiento externo (lámina visceral del peritoneo), túnica media muscular de fibras circulares lisas y túnica mucosa con epitelio de revestimiento simple, constituido de células cilíndricas ciliadas y secretoras. Anatómicamente las tubas uterinas, se dividen en cuatro regiones: intramural, istmo, ampolla e infundíbulo. El gerbo no constituye modelo ideal para estudios del ciclo estral debido a su irregularidad. La anatomía e histología de la tuba uterina son semejantes a la presentada por los roedores usados como animales de laboratorio

Scanning electron microscopic study of the tongue of chinchila

A través de la microscopía electrónica de barrido se observó la estructura tridimensional de las papilas linguales y el tejido conectivo de la lengua de chinchilla. Se utilizó la técnica de maceración celular con NaOH para remover la capa epitelial y visualizar la arquitectura del tejido conectivo. El dorso de la lengua está cubierto por epitelio escamoso estratificado querantizado y posee cuatro tipos de papilas: filiformes, fungiformes, foliadas y valadas. Las imágenes de microscopía de barrido mostraron superficies de las células epiteliales con micropuentes distintos de los bordes intercelulares y poros linguales. El tejido conectivo central de las papilas filiformes de las partes anterior e intermedia de la lengua, presentaban dos o tres protrusiones semejantes a vástagos, mientras que en la parte posterior se observó una amplia base con varias protrusiones apicales. La lámina propia de las papilas fungiformes era columnar y las papilas valadas presentaron una muesca central rodeada por numeroso tejido conectivo papilar, las papilas foliadas presentaban orificios elípticos rodeados por escaso tejido conectivo papilar