<p>Eurycoma longifolia (Tongkat Ali / TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is also used to improve male fertility and no report&nbsp / regarding its safety has been published, this study investigated the effects of a patented, aqueous TA extract on various sperm and testicular functions. Materials and Methods This study&nbsp / encompasses two parts (part 1: on spermatozoa / part 2: on TM3-Leydig and TM4-Sertoli cells). Part 1: Semen samples of 27 patients and 13 fertile donors were divided into two groups,&nbsp / washed and swim-up prepared spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 &mu / g/ml) for 1 hour at 37&deg / C. A sample without addition of TA served as control. After incubation with TA,&nbsp / the following parameters were evaluated: viability (Eosin-Nigrosin test), total and progressive motility (CASA), acrosome reaction (triple stain technique), sperm production of reactive oxygen&nbsp / species (ROS / dihydroethidium test / DHE), sperm DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (&Delta / &psi / m) (Depsipher kit). Part 2: TM3-Leydig and TM4-Sertoli cells&nbsp / incubated with different concentrations of TA (0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 &mu / g/ml) and control (without extract) for 48 and 96 hours. After incubation with TA, the following parameters were&nbsp / evaluated: viability (XTT), cell proliferation (protein assay), testosterone (testosterone ELISA test) and pyruvate (pyruvate assay). Results Part 1: For washed spermatozoa, significant&nbsp / dose-dependent trends were found&nbsp / for viability, total motility, acrosome reaction and sperm ROS production. However, these trends were only significant if the highest concentrations were included in the calculation. In the swim-up spermatozoa, ROS production of spermatozoa showed a biphasic relationship with its lowest percentage at 10 &mu / g/ml, yet, no significance could be&nbsp / observed (P=0.9505). No influence of TA could be observed for sperm DNA fragmentation nor &Delta / &psi / m.</p>

<p>Eurycoma longifolia (Tongkat Ali / TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is also used to improve male fertility and no report&nbsp / regarding its safety has been published, this study investigated the effects of a patented, aqueous TA extract on various sperm and testicular functions. Materials and Methods This study&nbsp / encompasses two parts (part 1: on spermatozoa / part 2: on TM3-Leydig and TM4-Sertoli cells). Part 1: Semen samples of 27 patients and 13 fertile donors were divided into two groups,&nbsp / washed and swim-up prepared spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 &mu / g/ml) for 1 hour at 37&deg / C. A sample without addition of TA served as control. After incubation with TA,&nbsp / the following parameters were evaluated: viability (Eosin-Nigrosin test), total and progressive motility (CASA), acrosome reaction (triple stain technique), sperm production of reactive oxygen&nbsp / species (ROS / dihydroethidium test / DHE), sperm DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (&Delta / &psi / m) (Depsipher kit). Part 2: TM3-Leydig and TM4-Sertoli cells&nbsp / incubated with different concentrations of TA (0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 &mu / g/ml) and control (without extract) for 48 and 96 hours. After incubation with TA, the following parameters were&nbsp / evaluated: viability (XTT), cell proliferation (protein assay), testosterone (testosterone ELISA test) and pyruvate (pyruvate assay). Results Part 1: For washed spermatozoa, significant&nbsp / dose-dependent trends were found&nbsp / for viability, total motility, acrosome reaction and sperm ROS production. However, these trends were only significant if the highest concentrations were included in the calculation. In the swim-up spermatozoa, ROS production of spermatozoa showed a biphasic relationship with its lowest percentage at 10 &mu / g/ml, yet, no significance could be&nbsp / observed (P=0.9505). No influence of TA could be observed for sperm DNA fragmentation nor &Delta / &psi / m.</p>

Orientador: Mary Anne Heidi Dolder / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T22:42:21Z (GMT). No. of bitstreams: 1 Gomes_MarcosdeLuccaMoreira_D.pdf: 17022356 bytes, checksum: 8f52bd976bb7a3f25e260b1c7c06dc69 (MD5) Previous issue date: 2011 / Resumo: A espécie Heteropterys aphrodisiaca é amplamente utilizada como afrodisíaca. Estudos mostram o potencial efeito estimulante sobre o tamanho das vesículas seminais e de populações de células de Leydig em ratos, ambos andrógenos dependentes. Neste trabalho foi proposto o tratamento com a infusão de H. aphrodisiaca aliado ao exercício de resistência física com o intuito de observar seus efeitos nos testículos de ratos. Foram utilizados ratos Wistar (90 dias) divididos em 4 grupos (n=10, cada): controles - água, sedentário e com treinamento físico; tratados com H. aphrodisiaca (104mg/kg/dia) sedentário e com treinamento físico. O experimento teve duração total de 56 dias. Foi realizada dosagem de testosterona plasmática e avaliados parâmetros morfométricos e estereológicos do parênquima testicular. No epitélio seminífero, foram contados os números de células germinativas e somáticas, calculando-se o rendimento da espermatogênese, índices mitótico e meiótico e eficiência e capacidade de células de Sertoli. Foi realizado Western Blotting para quantificação da concentração de receptores de andrógeno, e TUNEL para determinação de porcentagem de células apoptóticas. A concentração de testosterona circulante foi significativamente maior nos animais tratados e sedentários. Os diâmetros médios tubulares dos animais treinados foram maiores quando comparados aos dos animais sedentários. Não ocorreram variações significativas quanto ao volume do tecido intersticial, na proporção de macrófagos, espaço linfático e vasos sanguíneos. Entretanto, ocorreram diminuições significativas na proporção de tecido conjuntivo em ambos os grupos que receberam a infusão vegetal. Houve aumentos significativos do diâmetro e volume nucleares das células de Leydig dos animais treinados. Houve diminuição significativa do número de células de Leydig por grama de parênquima testicular nos animais controle/treinados quando comparados aos outros grupos experimentais. O grupo tratado/treinado apresentou o maior índice de mitoses espermatogoniais, sendo significativamente maior que a média do grupo sedentário/tratado. As curvas de rendimento e índice mitótico possuem mesmo padrão de distribuição, entretanto, para o índice meiótico, o grupo sedentário/tratado mostrou as maiores médias quando comparado aos dois grupos controle, sedentário e treinado. Não houve alteração de índices apoptóticos tampouco na concentração de receptores androgênicos entre os grupos experimentais. O protocolo de treinamento aplicado no presente experimento não acarretou em danos no parênquima testicular, como tem sido afirmado em outros experimentos envolvendo exercício aeróbico. Contudo, a infusão de nó-de-cachorro parece influenciar tanto no estímulo de células de Leydig, com aumento do volume celular, quanto no comportamento espermatogonial, induzindo mitoses e aumentando o rendimento geral do processo espermatogênico / Abstract: The species Heteropterys aphrodisiaca is traditionally used as an aphrodisiac. Some studies showed its stimulating potential of seminal vesicle and Leydig cell population growth, both androgen dependent. In this study, we proposed to observe the effects of H. aphrodisiaca infusion treatment combined with endurance exercise protocol. For this investigation, 40 adult Wistar rats (90 days) were used (4 groups, n = 10 each): two controls receiving water, sedentary and trained, and two treated with H. aphrodisiaca (104mg/kg/dia), sedentary and trained. The experiment lasted for 56 days. Plasma testosterone concentration was measured by radioimmunoassay. Morphometrical and stereological parameters were evaluated from the testicular parenchyma. The numbers of somatic and germ cells were estimated by counting the seminiferous epithelium cell population. Also, the spermatogenic yield, meiotic and mitotic index, efficiency and capacity of Sertoli cells were estimated. Western blotting was performed to quantify the concentration of androgen receptors, and TUNEL technique to determine the percentage of apoptotic cells within the seminiferous tubules. Control/trained animals showed a significant decrease in body mass gain compared to sedentary animals. The testosterone concentrations were significantly higher in treated/sedentary animals. The mean tubular diameters of trained animals were higher when compared to sedentary animals. There were no significant differences regarding the volume of interstitial tissue, the proportion and volume of macrophages, lymphatic space and blood vessels. However, the treated/sedentary group showed lower connective tissue content compared with the control/sedentary, as well as the diminished volume of Leydig cells, compared with the treated/trained group. There was a significant increase of both nuclear diameter and volume of Leydig cells in trained animals. On the other hand, there was a significant decrease of Leydig cell number per gram of testicular parenchyma in control/trained animals. Considering the spermatogenesis dynamics, both treated groups showed the highest spermatogenic yield, in relation to the control/trained group. The treated/trained group showed the highest index of spermatogonial mitosis - significantly higher than the sedentary/treated one. Spermatogenic yield and mitotic index showed the same distribution pattern when plotted on graphs; however, the meiotic index was significantly higher only in the sedentary/treated group. The apoptotic indexes, as well as the androgen receptor concentrations were not affected by the protocols employed. Treatment with the plant infusion did not lead to an increase of the parameters analyzed. However, the infusion seems to stimulate either the Leydig cell (increase of cell volume) or the spermatogonial behavior, inducing more mitosis and increasing the spermatogenic yield / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Introduction: Eurycoma longifolia (Tongkat Ali; TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is also used to improve male fertility and no report regarding its safety has been published, this study investigated the effects of a patented, aqueous TA extract on various sperm and testicular functions. Materials and Methods: This study encompasses two parts (part 1: on spermatozoa; part 2: on TM3-Leydig and TM4-Sertoli cells). Part 1: Semen samples of 27 patients and 13 fertile donors were divided into two groups, washed and swim-up prepared spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 μg/ml) for 1 hour at 37°C. A sample without addition of TA served as control. After incubation with TA, the following parameters were evaluated: viability (Eosin-Nigrosin test), total and progressive motility (CASA), acrosome reaction (triple stain technique), sperm production of reactive oxygen species (ROS; dihydroethidium test; DHE), sperm DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (Δψm) (Depsipher kit). Part 2: TM3-Leydig and TM4-Sertoli cells incubated with different concentrations of TA (0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 μg/ml) and control (without extract) for 48 and 96 hours. After incubation with TA, the following parameters were evaluated: viability (XTT), cell proliferation (protein assay), testosterone (testosterone ELISA test) and pyruvate (pyruvate assay). Results Part 1: For washed spermatozoa, significant dose-dependent trends were found for viability, total motility, acrosome reaction and sperm ROS production. However, these trends were only significant if the highest concentrations were included in the calculation. In the swim-up spermatozoa, ROS production of spermatozoa showed a biphasic relationship with its lowest percentage at 10 μg/ml, yet, no significance could be observed (P=0.9505). No influence of TA could be observed for sperm DNA fragmentation nor Δψm. Part 2: The viability rates and protein production of TM3-Leydig and TM4-Sertoli cells at 48-hour exposure to TA showed increases whereas at 96-hour incubation periods viability and protein production declined especially as from concentration 25 μg/ml TA. Similar results could be seen for TM4-Sertoli cells pyruvate production. The testosterone production at 48-hour exposure marginally increased (P=0.0580) at the highest (50 μg/ml) concentration of TA. However, at 96-hour exposure to TA the testosterone production significantly (P=0.0065) increased. It is also apparent that after 96 hours the concentration of testosterone has increased [12 x 10-4 ng/ml] when compared to 48-hour exposure [6 x 10-7ng/ml] of Tongkat Ali. Conclusion: Part 1: Results indicate that the Tongkat Ali extract has no deleterious effects on sperm functions at therapeutically used concentrations (<2.5 μg/ml). Part 2: The cytotoxic effect of TA are only presented at higher concentration from 25 μg/ml. TM3-Leydig cells appears to be more resilient than TM4-Sertoli cells in viability and protein production yet at prolonged periods of exposure it is detrimental. Testosterone production only increases after 96 hours exposure to TA.