The objective of this study was to uncover molecular and cellular dynamics in spermatozoa from Holstein bulls with Good (11 bulls) and Poor (5 bulls) cryotolerance. Post-thaw sperm kinetics, membrane integrity, and DNA fragmentation were assessed. Data was analyzed using principal component analysis. The spermatozoa from Good bulls had a higher number of cells with intact membranes (P = 0.029), nonragmented DNA (P = 0.018), post-thaw viability (P < 0.001) in comparison to those of the Poor bulls. The Good bulls also had a faster average path velocity (VAP; P = 0.017), straight-line velocity (VSL; P = 0.036), a greater distance average path (DAP; P = 0.006) and distance straight line (DSL; P = 0.011). No differences were found in total antioxidant capacity (TAC), number of live cells or other kinetic parameters between spermatozoa from Good and Poor groups, showing that no one characteristic can be used to determine cryotolerance.

Orientador: Rejane Maira Góes / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T19:26:40Z (GMT). No. of bitstreams: 1 Negrin_AnaCarolina_M.pdf: 3573186 bytes, checksum: 84ff9397d827717b776f6497a21bc510 (MD5) Previous issue date: 2014 / Resumo: Desreguladores endócrinos (DE), como o di-n-butil ftalato (DBP), podem alterar o panorama dos hormônios esteroides ou sua ação, comprometendo o desenvolvimento testicular e a capacidade reprodutiva na vida adulta. Estudos toxicológicos mostram que ratos adultos, quando expostos a altas doses de ftalatos durante a fase de diferenciação sexual, exibem diversas anomalias reprodutivas, como agenesia do epidídimo e redução na produção diária de espermatozoides. Dados mostram que exposições a 2mg/kg/dia de DBP durante a gestação e a lactação são suficientes para prejudicar o desenvolvimento das células germinativas. Além disso, há relatos de que DE contrinuem para o aumento da adipogênese por causar alterações na sinalização celular de adipócitos. É conhecido que a obesidade masculina pode afetar a qualidade do sêmen e aumenta as taxas de infertilidade. Considerando que os ftalatos podem se acumular no tecido adiposo, ficamos interessados em avaliar os efeitos da exposição prolongada a baixas doses de DBP a as possíveis interferências do excesso de lipídeos na dieta para a função testicular e os parâmetros espermáticos de gerbilos adultos. Gerbilos fêmeas adultas, alimentadas com dieta balanceada (4% de lipídeos) ou hiperlipídica (20% de lipídeos) por oito semanas, foram acasaladas com machos normais. A prole masculina foi dividida em grupos controle (C), di-n-butil ftalato (Ph), dieta hiperlipídica (HF) e hieta hiperlipídica mais di-n-butil ftalato (HFPh). DBP (5 mg/kg/dia) foi administrado na água de beber às mães durante a gravidez e lactação e aos filhotes do desmame até a idade adulta (14 semanas). A resposta do testículo foi avaliada por meio de análises microscópicas e esterológicas, da sensibilidade de suas principais populações celulares a andrógenos e estrógenos, e da produção espermática. Também foram examinados os efeitos sobre a reserva espermática, o tempo de trânsito dos espermatozoides pelo epidídimo e a motilidade espermática. Isoladamente, baixas doses de DBP resultaram em obesidade e dislipidemia nos animais adultos. Nenhuma alteração foi observada na estrutura testicular, mas a síntese de estrógeno pelo testículo foi reduzida, resultando em tendência a diminuição na eficiência espermática. A redução no estrógeno intratesticular, após exposição ao DBP, foi acompanhada de um aumento de ~70% na expressão de ER? nos testículos, que pode ser uma resposta adaptativa aos baixos níveis deste hormônio. A ingestão crônica de dieta hiperlipídica não induziu os gerbilos adultos à obesidade, mas causou uma leve queda na eficiência espermática. Esta redução não está relacionada a alterações na estrutura testicular ou na sua capacidade esteroidogênica, mas podem estar ligadas ao comprometimento da sinalização testicular, já que foi verificada uma redução no conteúdo de AR. A exposição combinada à dieta hiperlipídica e a baixas doses de DBP atuaram sinérgica e negativamente na síntese de testosterona intratesticular, prejudicando a eficiência espermática e aumentando o tempo de trânsito dos espermatozoides pelo epidídimo. A motilidade espermática não sofreu alteração frente as exposições isoladas ou combinadas. Este estudo demonstrou que o ambiente nutricional pode interferir na resposta dos testículos frente aos ftalatos e proporciona novas informações para o entendimento das consequências da exposição aos DE para a redução na reverva espermática e fertlidade humana / Abstract: Endocrine disrupting chemicals (EDC), as di-n-butyl phthalate (DBP), can alter the scenary of steroid hormones or their action, impairing the testicular development and reproductive capacity in adulthood. Toxicological studies show that adult rats, when exposed to high doses of phthalates during sexual differentiation, exhibit several reproductive anomalies, such as agenesis of the epididymis and reduction in daily sperm production. Data show that exposures to 2mg/kg/day of DBP during gestation and lactation periods are enough to impair the development of the male germ cells. Furthermore, there are reports that EDC contribute to the increase of the adipogenesis by altering cell signaling of adipocytes and lipid metabolism. It is known that the male obesity can affect semen quality and increases infertility rates. Whereas that phthalates can accumulate in adipose tissue, we were interested in evaluating the effects of prolonged exposure to low doses of DBP and possible interferences of excess dietary fat for testicular function and spermatic parameters of adult gerbils. Adult female gerbils, fed high-fat (20% fat) or balanced diet (4% fat) for eight weeks, were mated with normal males. The male offspring was divided into control (C), di-n-butyl phthalate (Ph), high-fat diet (HF) and high-fat diet plus di-n-butyl phthalate (HFPh) groups. DBP (5 mg/kg/day) was administered in drinking water to pregnant and breastfeeding mothers and to offspring from weaning up to adulthood (14-week-old). Testis response was evaluated by means of microscopic and stereological analyses, sensitivity of its major cell populations to androgens and estrogens, steroidogenic capacity and spermatic efficiency. We also examined the effects on sperm reserves, sperm transit time through the epididymis and sperm motility. Alone, low doses of DBP resulted in obesity and dislipidemy at adulthood. No histological change was observed in testicular structure of these animals, but there was reduction in intratesticular estrogen synthtesis, resulting in a tendency to decrease in sperm production. The decrease in intratesticular estrogen after lifetime DBP exposure was accompanied by a ~70% increment in ER? content in the testis, that might be an adaptative response to low estrogen levels. Chronic intake of high-fat diet did not induce obesity in aldult gerbils, but led to a slight decrease in spermatic efficiency. This decrease was not associated with changes in testicular structure or steroidogenic capacity, because serum or intratesticular testosterone and estrogen concentrations were not altered, but it can be linked to an unbalanced in androgen receptor signaling, since it was observed a decrease in AR content. The combined exposure to high-fat diet and to low doses of DBP acted synergical and negatively on intratesticular testosterone synthesis, impairing the spermatic efficiency and increasing the sperm transit time. The sperm motility was not changed in isolated or combined exposures. This study demonstrated that fat nutritional environment may adversely affect the response of testes to phthalates, and provide new informations for understanding the consequences of exposures to ECD in decreasing human sperm counts and fertility / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Au cours des dernières décennies, un contexte alarmant de déclin des paramètres du sperme etd’accroissement constant des pathologies génitales masculines a été décrit dans de nombreuxpays industrialisés.Notre travail évalue dans un premier temps l’évolution des paramètres spermatiques chez11 330 hommes ayant consulté pour infertilité conjugale au Centre de ProcréationMédicalement Assistée du CHU de Marseille entre 1988 et 2007. Les données ont étérecueillies de manière rétrospective et analysées par régression linéaire multi-variée afin detenir compte de l’effet lié à l’âge. Nous montrons une diminution significative des principauxparamètres du sperme: concentration en spermatozoïdes (-1.4% par an), numération totale enspermatozoïdes (-1.5% par an), mobilité progressive rapide (-5.6% par an) et morphologienormale (-1.9% par an). Les possibles biais de selection ont été discutés. L’effet del’environnement sur notre fonction de reproduction est l’une des hypothèses principalesévoquées pour expliquer ce phénomène.Nous montrons ensuite comment un modèle de culture de tubes séminifères de rat en chambrebicamérale, couplé à des techniques d’étude de la méiose en immunocytochimie peut êtreutilisé comme nouvel outil de reprotoxicologie. Nous décrivons tout d’abord avec l’anticorpsanti-SCP3 la prophase de première division méiotique chez le rat in-vivo et nous validons lemodèle de culture par comparaison entre les résultats des cellules obtenues in vivo et ceux descellules obtenues ex-vivo. Nous montrons ensuite grâce à ce système l’impact du ChromeHexavalent et du Cadmium sur la méiose du rat. Ces 2 métaux lourds, largement présent dansnotre environnement, peuvent être responsables d’atteintes importantes des cellulesméiotiques, y compris à de faibles doses pouvant correspondre à celles retrouvées chez desindividus exposés dans leur vie quotidienne et/ou professionnelle. / In recent decades, an alarming decline of sperm parameters and a constant increase in malegenital diseases has been described in many industrialized countries.In a first time, our study evaluates the evolution of sperm parameters in 11 330 menconsulting for infertility of their couple in the Reproduction Biology Laboratory of anUniversity Hospital in Marseilles between 1988 and 2007. Data were collected retrospectivelyand analyzed by multivariate linear regression to take into account the effect related to age.We show a significant decrease of the main sperm parameters: sperm concentration (-1.4%per year), total sperm count (-1.5% per year), rapid progressive motility (-5.6% per year) andnormal morphology (-1.9 % per year). Possible selection bias were discussed. The effect ofenvironment on our reproductive function is one of the main hypothesis to explain thisphenomenon.Secondly, we show how a rat seminiferous tubule culture in a bicameral chamber system,coupled with study of meiosis by immunocytochemistry can be used as a new tool ofreprotoxicology. We first describe with an anti-SCP3 antibody the first prophase of ratmeiosis and we validate the model of culture by comparing the results obtained on cellsobtained after in vivo spermatogenesis and those of cells obtained ex-vivo. Then, we showusing this system, the impact of hexavalent chromium and cadmium on rat meiosis. These twoheavy metals, widely present in our environment, may cause substantial impairment ofmeiotic cells, including low doses which can fit with those found in individuals exposed intheir personnal life and / or occupational training.

Orientador: Wilma De Grava Kempinas / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T02:46:53Z (GMT). No. of bitstreams: 1 Fernandez_CarlaDalBianco_D.pdf: 4399629 bytes, checksum: 5e3c4d594df1ba1dd65fdfd541b7fa14 (MD5) Previous issue date: 2011 / Resumo: O sobrepeso e a obesidade estão aumentando rapidamente, tornando-se uma epidemia mundial. Estas duas condições podem ser definidas simplesmente como o acúmulo excessivo de gordura nos adipócitos, células do tecido adiposo responsáveis pela síntese e liberação de leptina, um hormônio protéico cuja principal ação é inibir o apetite e estimular o gasto energético, mas que também atua na regulação do eixo hipotalâmico-hipofisáriogonadal. As concentrações séricas de leptina apresentam-se elevadas, proporcionalmente à quantidade de gordura, em muitos modelos animais de obesidade e na obesidade humana. Recentemente alguns estudos têm mostrado a relação entre obesidade e infertilidade masculina, mas até o momento os resultados são controversos. Da mesma forma, o papel da leptina no sistema reprodutor masculino não está muito esclarecido, principalmente quando este hormônio está em excesso, como na obesidade. Assim, os objetivos do presente estudo foram investigar os efeitos da obesidade induzida por consumo de dieta rica em gordura em parâmetros reprodutivos de ratos machos. Também foi investigado o papel da leptina na fertilidade e em alguns parâmetros espermáticos, em ratos machos adultos. Para isso, ratos machos da variedade Wistar foram alimentados com dieta hipercalórica rica em gordura (DH) ou com dieta padrão (DP) durante 15, 30 ou 45 semanas, e foram avaliados quanto ao índice de obesidade, níveis séricos de leptina, peso dos órgãos reprodutores e contagens espermáticas. Em um segundo experimento, os animais receberam as diferentes dietas apenas por 15 semanas, tempo suficiente para provocar obesidade. Esses animais foram avaliados quanto às dosagens dos hormônios sexuais, comportamento sexual, motilidade e morfologia espermática, fertilidade após cruzamento natural e inseminação artificial in utero. Além disso, ratos machos adultos não-obesos foram tratados com leptina exógena, ou com salina, ip, por 42 dias e avaliados quanto ao peso dos órgãos reprodutores, contagens espermáticas, motilidade dos espermatozóides e fertilidade após inseminação artificial. Após 15, 30 ou 45 semanas, os animais alimentados com DH apresentaram um aumento significativo no índice de obesidade e nos níveis de leptina. O peso dos órgãos reprodutores e as contagens espermáticas foram similares entre os dois grupos, em todos os períodos estudados. Nos animais que receberam DH por 15 semanas, o comportamento sexual e a fertilidade após acasalamento natural foram semelhantes aos do grupo controle. Da mesma forma, os níveis de testosterona não foram alterados, mas os de estradiol aumentaram nos animais que ingeriram DH. Além disso, a qualidade espermática foi reduzida nestes animais, como evidenciado pela baixa porcentagem de espermatozóides com movimento progressivo. A alteração da motilidade espermática foi seguida por uma redução quantitativa do potencial de fertilidade após inseminação artificial in utero. Os animais tratados com leptina não apresentaram diferenças nas contagens espermáticas nem na motilidade quando comparados aos animais controle. No entanto, o potencial de fertilidade reduziu cerca de 40% e a taxa de perdas pré-implantação foi duas vezes maior nos animais tratados com leptina. Juntos, esses resultados indicam que a obesidade diminuiu a qualidade espermática, prejudicando a fertilidade dos ratos machos, e que, provavelmente a leptina está relacionada a este prejuízo / Abstract: Overweight and obesity are rapidly becoming a worldwide epidemic that affects children and adults. These two statuses are often defined simply as a condition of abnormal or excessive fat accumulation in adipose tissue, whose cells are responsible for synthesis and release of leptin. This is a protein hormone whose main action is to decrease appetite and increase energy expenditure, but it also act as regulatory signal for gonadal axis, among other functions. Serum leptin concentration is augmented in several obesity animal models and in obese human, proportionately to the amount of fat. Some studies have shown a relationship between obesity and male infertility, but until now it remains controversial. In the same way, the role of leptin on male reproductive function is not clear, mainly when the hormone is in excess as occur in obesity. Thus, the aims of the present study were to investigate the effects of high-fat diet-induced obesity on male rats' reproductive parameters. Furthermore it was evaluated the possible role of the excess of leptin on fertility and some sperm parameters in adult male rats. To achieve these aims, male Wistar rats were fed a high-fat diet (HFD) or standard chow (SD) for 15, 30 or 45 and were evaluated by adiposity index, serum leptin levels, reproductive organ weights and sperm counts. In a second experiment rats received HFD or SD only for 15 weeks, long enough to cause obesity. Sexual hormones, sexual behavior, sperm morphology and motility, fertility after natural mating and after artificial in utero insemination were evaluated. Moreover, non-obese adult male rats were treated with exogenous leptin ip (30?g/kg/day) or vehicle for 42 days and were evaluated for sperm count in testis and epididymis, sperm motility and fertility after in utero artificial insemination. After 15, 30 or 45 weeks, HFD-fed animals presented significant increases in obesity index and serum leptin levels. Reproductive organ weights and sperm counts in the testis and epididymis were similar between the two groups at all timepoints studied. In HFD-fed animals only for 15 weeks, sexual behavior and fertility after natural mating were not altered by the diet regimen. In these animals, intergroup testosterone levels were also comparable, but estradiol levels were increased in HDF rats. Furthermore, sperm quality was reduced in 15 weeks HFD-fed animals as evidenced by their decreased percentage of sperm with progressive movement. This altered motility parameter was followed by a quantitative reduction in fertility potential after artificial in utero insemination. Leptin treated animals did not show any differences in sperm counts either in the testis or epididymis. In the same way, the percentage of progressive, non-progressive and immotile sperm was similar between the two groups. However, the fertility potential showed a reduction of about 40% whereas the pre-implantation loss rate increased more than two fold in leptin treated animals. These results indicate that obesity lead to a decrease in sperm quality, impairing male fertility and probably the excess of leptin is related with this alteration of fertility / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

The presence of pathogens in semen can compromise the outcome of assisted reproductive treatment, together with the possibility of the female partner or offspring becoming infected. This is cause for concern, especially in South Africa with a high prevalence of HIV-1. Most of these infected individuals are in their reproductive years with the desire to have their own genetically related children. Therefore, assisted reproductive treatment with effective risk reduction procedures, such as semen processing for the elimination of these pathogens is crucial. However, during sperm preparation by standard discontinuous density gradient centrifugation, the supernatant is aspirated to allow access to the purified sperm pellet. Pathogens from the upper layers can adhere to the inside surface of the test tube and flow down to re-infect the purified sperm sample. The use of a centrifuge tube insert may prevent the re-contamination of sperm samples after discontinuous density gradient centrifugation. Furthermore, seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective. Serine proteases, such as trypsin, have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could have a negative impact on sperm parameters. This research was therefore aimed towards the determination of: i) The effect of semen processing with trypsin and trypsin inhibitor on sperm parameters. ii) The prevalence of various bacteria in semen samples from men attending the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. iii) The effectiveness of semen processing by discontinuous density gradient centrifugation with a centrifuge tube insert, for the elimination of some of the most prevalent bacteria, white blood cells and in vivo derived HIV-1. Evaluation of sperm parameters after semen processing indicated that trypsin and trypsin inhibitor did not have an impact on sperm mitochondrial membrane potential, vitality, motility and zona binding potential, or acrosin activity, respectively. Seminal bacteria were highly prevalent in patients wishing to participate in the Unit’s assisted reproductive program, with 49.5% of semen samples presenting with positive bacterial cultures. Semen processing by means of discontinuous density gradient centrifugation with the tube insert, eliminated significantly more in vitro derived (spiked) bacteria and white blood cells from semen compared to processing without the insert. Furthermore, the semen decontamination procedure was effective in removing HIV-1 RNA from 100% of samples and proviral DNA from 98.1% of semen samples from HIV-1 sero-positive patients. The effectiveness of discontinuous density gradient centrifugation for the elimination of seminal pathogens could, therefore, be improved by the addition of trypsin to the upper density layer, without supplementing the bottom layer with trypsin inhibitor. Additionally, semen decontamination efficiency could also be improved by the prevention of re-contamination of processed sperm samples by the utilization of a tube insert during discontinuous density gradient centrifugation. / Thesis (PhD)--University of Pretoria, 2014. / gm2014 / Obstetrics and Gynaecology / unrestricted