Este trabalho caracteriza os efeitos das condições reais de exposição ao material particulado urbano na espermatogênese por meio de análises histológicas de testículos de duas gerações de camundongos BALB/c durante o período gestacional, pósgestacional ou em ambos os momentos combinados. A geração parental foi exposta à poluição do ar em câmaras com ou sem filtros para PM2,5 (câmaras filtrada e não filtrada, respectivamente) por 4 meses, formando dois grupos: não exposto; e exposto. Estes animais foram acasalados e a freqüência do plug vaginal apresentou tendência de queda nas fêmeas expostas (p>0,05). O número de fêmeas prenhes também foi reduzido (p=0,007) e o número de nativivos foi menor na câmara não filtrada (186) quando comparada com a filtrada (268), contudo, o número de animais por ninhada não foi alterado (p>0,05). Após o acasalamento os machos foram eutanasiados, seus testículos pesados, fixados em solução de Bouin ou paraformaldeído 4% e corados com HE, PCNA, Ki67 ou TUNEL. Metade dos animais produzidos na primeira geração, constituída de animais de 1 dia de vida, foi transferida de uma câmara para outra formando os grupos pré-natal e pós-natal. Os animais remanescentes das câmaras filtrada e não filtrada constituíram os grupos não exposto e pré+pós-natal, respectivamente. Após 90 dias, os animais da primeira geração foram eutanasiados e seus testículos foram retirados, pesados, fixados e corados da mesma forma que sua geração parental. Os animais expostos ao PM2,5 da geração parental apresentaram aumento do peso dos testículos (p=0,002), dos epidídimos (p<0,001) e do peso relativo testículo/corpo (p=0,003), epidídimo/corpo (p=0,001). Não houve alteração no número de células germinativas e somáticas e nem na proliferação celular (p>0,05). A apoptose pela coloração de HE foi reduzida no estádio IV (p=0,046) e aumentada no estádio VIII (p=0,019) da espermatogênese. Pela técnica de TUNEL os estádios IV (p=0,017), V (p=0,035) e VIII (p=0,024) mostraram apoptose menor nos animais do grupo exposto. O estádio IV foi identificado como o de maior apoptose espontânea nas duas técnicas empregadas, HE (p<0,001); e TUNEL (p<0,001), entre os animais não expostos. O ciclo do epitélio seminífero foi alterado com freqüência reduzida do estádio IV entre os animais expostos (p=0,005). Os animais da primeira geração expostos no período pré-natal apresentaram redução de peso corpóreo (p<0,001) e dos testículos (p=0,012), bem como, aumento do peso relativo testículos/corpo (p=0,013). O número de células somáticas não foi alterado, mas o de espermatócitos nos grupos pós-natal (p=0,011) e pré+pós-natal foi aumentado (p=0,035) enquanto nos grupos pré-natal e pós-natal houve redução no número de espermátides alongadas (p<0,001). Não houve diferença significativa na taxa de proliferação, apoptose e freqüência dos estádios do ciclo do epitélio seminífero entre os grupos de exposição (p>0,05). O estádio IV mostrou-se o mais sensível para a ocorrência de apoptose espontânea nas duas técnicas empregadas: HE (p<0,001); e TUNEL (p<0,001). A freqüência normal dos estádios entre os animais não expostos mostrou que os estádios finais são os mais freqüentes (VI, VIII e VII, nesta ordem) e os iniciais os menos freqüentes (II e I, nesta ordem) para ambas as gerações. Estes resultados fornecem dados que sugerem que o PM2.5 da poluição atmosférica urbana é capaz de alterar o sistema reprodutivo masculino e a espermatogênese não dependendo do período da vida (durante ou após a gestação) em que os animais são expostos. / The present paper describes the effects of real exposure to urban PM2.5 on spermatogenesis by histological analysis of testes of mice (BALB/c) from two generations during fetal or postnatal phases of development and of mice exposed in both phases of development. Parental generations (BALB/c mice) were exposed to air pollution in chambers with or without filters for PM2.5 for 4 months (filtered and non-filtered chambers, respectively), forming two groups, namely non-exposed and exposed. These animals were mated and a frequency of decrease on vaginal plug in the exposed females was observed (p>0.05). The number of pregnant females was reduced as well (p=0.007) and the number of born alive decreased in the non-filtered chamber (186) when compared to the filtered chamber (268); however, the litter size was not altered (p>0.05). After mating, the male were killed, their testes were weighed and fixed in Bouins solution or 4% paraformaldehyde and stained in H&E, PCNA, Ki67 or TUNEL. Half of 1-day old offspring was crossed over between chambers forming the prenatal and postnatal groups; remaining offspring from filtered and non-filtered chambers comprised the non-exposed and pre+postnatally exposed groups, respectively. After 90 days, the animals from first generation were killed and their testes were removed, weighed, fixed and stained like the parental generation. The animals exposed to PM2.5 from the parental generation showed increased testis weight (p=0.002), epididymis weight (p<0.001), relative testis weight (p=0.003), and relative epididymis weight (p=0.001). The germ and somatic cells number was not reduced, and neither was cell proliferation (p>0.05). The apoptosis labeled by H&E was reduced in stage IV (p=0.046) and increased in stage VIII (p=0.019) of spermatogenesis. By using the TUNEL technique, stages IV (p=0.017), V (p=0.035) and VIII (p=0.024) showed fewer apoptosis in the exposed animal group. Stage IV was identified as the most spontaneous apoptosis in both methods: HE (p<0.001) and TUNEL (p<0.001), among the non-exposed animals. The cycle of the seminiferous epithelium was altered with reduced frequency of stage IV between the exposed animals (p=0.005). The animals from the first generation exposed during the prenatal period had a reduced body (p<0.001) and testis weight (p=0.012) and an increased relative testis weight (p=0.013). Differences in germ cell proliferation, apoptosis, and staging were not significantly different among treatment groups (p>0.05). Nevertheless, germ cell populations of post- (p=0.011) and pre+postnatally (p=0.035) PM-exposed animals contained an increased percentage of spermatocytes, while pre- and postnatal groups (p<0.001) had a reduced number of elongated spermatids. Stage IV was shown to be the most sensitive for the occurrence of spontaneous apoptosis in both methods used: H&E (p<0.001); and TUNEL (p<0.001). The normal frequency of the stages between non-exposed animals showed that the final stages are more frequent (VI, VIII e VII) and the beginning stages less frequent (II e I) to both generations. These results suggest that PM2.5 from urban air pollution is capable of altering the male reproductive system and spermatogenesis independently of the period of life when the animals are exposed to it (during or after pregnancy).

Neste estudo foram investigados os efeitos da inalação crônica de cocaína crack na espermatogênese de camundongos púberes e maduros. Camundongos Balb/c machos de duas diferentes idades, jovem e adulta (n=20), foram expostos à fumaça de 5g de cocaína crack em uma câmara de inalação, 5 dias por semana, durante 2 meses. Animais controle (n=10) foram mantidos em biotério durante o período de experimentação. Análises morfométricas quantitativas de cortes histológicos de testículos foram feitas em microscopia óptica. A qualidade da espermatogênese foi avaliada durante a fase VII de maturação do epitélio seminífero, através da quantificação dos tipos celulares normais e degenerados presentes nos espaços intra e intertubular (células germinativas, células de Sertoli e células de Leydig). As diferenças foram consideradas significativas quando p<0,05. O número de túbulos seminíferos em fase VII por testículo mostrou significante redução (p=0,023) em animais jovens expostos. Houve redução significativa observada no número de células de Sertoli (p=0,000) e espermátides alongadas (p=0,005) de animais jovens expostos. A degeneração celular é aumentada em todos os grupos expostos, com maior severidade no grupo jovem (p=0,000). A inalação de fumaça de cocaína crack induz a alterações na espermatogênese, sendo sua toxicidade maior em animais jovens expostos durante a fase de maturação gonadal . Estes achados são de interesse na saúde pública e mais investigações devem ser feitas focando efeitos similares em homens. / In the present study, the effects of chronic inhalation of crack cocaine on the spermatogenesis of pubertal and mature mice were investigated. Balb/c mice of two different ages, young and adult (n=20), were exposed to the smoke of 5g of crack in an inhalation chamber for 5 days a week during 2 months. Correspondents control animals (n=10) were kept in animal house during experimentation. Morphological quantitative analyses of testis were made in optical microscope. The spermatogenesis was evaluated during phase VII of maturation of the seminiferous epithelium. The number of spermatogenesis cell types (germ cells and Sertoli cells), the germ cell degenerations and the intertubular Leydig cells population was scored. Differences were considered significant when p<0.05. The number of tubular phase VII per testis showed significant (p= 0,023) reduction in young exposed animals. Significant reductions (p=0,000) were observed in Sertoli cells and spermatids elongated (p=0,005) in young intoxicated animals. Apoptosis is also increased in all intoxicated groups being more severe in young groups (p=0,000). Inhalation of crack cocaine smoke induced spermatogenesis disruption of chronic exposed mice. Crack toxicity was more severe in pubertal mice when sexual gonad undergoes maturation. We think that our findings should be of public health concern and that further investigations focusing similar effects on human males are warranted.

O objetivo, deste estudo foi investigar o comportamento dos hormônios esteróides 17β-Estradiol (E2), 17α-hidroxiprogesterona (17α-OHP), Testosterona (T) e 11-Ketotestosterona (11-KT), em piracanjuba Brycon orbignyanus de diferentes sexos e idades, na estação reprodutiva, e nas fêmeas submetidas à reprodução induzida. Os animais utilizados no trabalho, eram criados em piscicultura comercial, mantidos em 3 viveiros, separados por lotes de diferentes idades. A coleta dos animais, consistiu de quatro machos e cinco fêmeas (48 meses), identificados através do dimorfismo sexual da espécie, e as demais idades, (12 e 24 meses), coletaram-se, 20 peixes de cada idade, para identificação do sexo através de histologia. Já o experimento de caracterização dos esteróides sexuais na reprodução induzida, foram coletadas cinco fêmeas, selecionadas através das características com: abdome abaulado, papila urogenital, saliente e avermelhada. Após captura, os peixes foram transportados ao laboratório, onde houve coleta de sangue, para quantificação do perfil plasmático de E2, 17α-OHP, T e 11-KT. Posteriormente, os animais foram abatidos e suas gônadas coletadas e fixadas, a fim de que fosse realizada análise histológica para identificação do sexo. Na reprodução induzida, foi coletado sangue em dois momentos: pré-indução (PI) e pós-extrusão (PE). O nível plasmático de E2 nos machos de 12 meses destaca sua ação no processo de proliferação e renovação das espermatogônia observado em machos imaturos. Nas fêmeas o E2 apresentou os maiores níveis (P<0,05) nos animais de 48 meses, confirmando assim, sua principal função na estimulação do processo de vitelogênese, e maturação final do oócito. Quanto aos andrógenos T e 11-KT, os maiores níveis (p<0,05) foram observados nos peixes adultos (48 meses), permitindo afirmar que estes atuam como feedback negativo, do FSH e feedback positivo do LH, fundamental no processo de maturação final e liberação dos gametas, além de regular o comportamento reprodutivo. O resultado da 17α-OHP, sugere que, nas idades estudadas, é indispensável por participar como precursor dos principais esteróides (T, E2 e 11-KT), além da 17α,20β dihydroxy-4-pregnen-3-one (17α,20β-DHP), essencial no estágio final de maturação, e desova na reprodução induzida. / The objective of this study was to investigate the physiological behavior of steroid hormones 17β-Estradiol (E2), 17α-hydroxyprogesterone (17α-OHP), testosterone (T) and 11-Ketotestosterone (11-KT) in Brycon orbignyanus with different sex and ages on the reproductive season and in females submitted to induced reproduction. The animals used in the study were kept in three ponds on a commercial fish farming, separated by lots with different ages. The sampling of animals consisted of the collection of four males and five females (48 months) identified by the sexual dimorphism of the specie. In the other groups (12 and 24 months), 20 fish of each age were collected for identification of sex through histology. In the experiment with characterization of the sexual steroids in the induced reproduction, were collected five females selected through the following characteristics: bulging abdomen and prominent reddish genital papilla. After capture, the fish were transported to the laboratory, where blood was collected for quantification of the plasma profile of E2, 17α-OHP, T and 11-KT Subsequently, the animals were slaughtered and their gonads were collected and fixed for histological analysis. In the induced females, blood was collected at two moments: pre-induction (PI) and post-extrusion (PE). The plasma profile of E2 is fundamental in immature males, highlighting its action in the process of proliferation and renewal of spermatogonia, observed in males of 12 months. In females E2 presented the highest levels (P <0.05) in animals at 48 months, thus confirming its main function in the stimulation of the vitellogenesis process and final oocyte maturation. The highest levels (p <0.05) of T and 11-KT androgens were observed in adult fish (48 months), allowing to affirm that they are acting as FSH negative feedback and LH positive feedback, fundamental in the final maturation and release of the gametes, besides regulating the reproductive behavior of the fish. The results of 17α-OHP suggest this hormon is fundamental in the studied ages because it is a precursor of the main steroids (T, E2 and 11-KT) and 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DHP), essential in the final stage of maturation and spawning in induced reproduction.

Made available in DSpace on 2017-07-10T14:57:32Z (GMT). No. of bitstreams: 1 camila_ machado.pdf: 1503244 bytes, checksum: 361dda1eae6076e04495b3c6a4455f6e (MD5) Previous issue date: 2016-03-14 / The triclosan (TCS) is a bactericidal agent widely used in personal hygiene products, in the context of odontology, this substance has been showing itself efficient in reducing the dental plaque and gingivitis, besides controlling the progression of periodontal disease. However, it's questionable the real benefits of using the TCS in large scale in different products, once this compound has been included on the list of endocrine disrupters. The growing observation of the prevalence of environmental contaminant with properties for the endocrine disrupting has been generating a considerable debate between the scientists, regulatory agencies and general public, about the potential risks which these substances represent to the man's and animal's productive health. Many of these compounds are present in our daily routine. The objective of this study was to evaluate the effects resulted from the exposure to TCS bactericidal, during pregnancy and lactation of mother rats, on the physical development, initial sexual and testicular function of the male son on the following phase of the development: puberty and sexual maturity. For this, 16 rats Wistarprenhes were used, separated in four experimental groups, with 4 animals each one: GI- received corn oil daily by gavage; GII - received TCS, at a dose of 75 mg/kg/day. GIII- received TCS, at the dose of 150 mg/kg/day and GIV - received TCS, at the dose 300 mg/kg/day. The rats where weighed in alternate days throughout the experimental period, to the adjustment of the dose and monitored about the birth of the offspring. At birth, the offspring was weighed and evaluated about the initial physical development: age of the ears' detachment, hair onset, and eruption of incisors and eyes opening. The male offspring were kept and monitored about the external physical parameter of initial sexual development (descended testicles age and prepuce separation). When they're 60 (puberty) days and 90 (sexual maturity) years old, they were weighed and sacrificed to the organs collecting and weighing and histological analysis of the spermatogenesis stages and Sertoli cells counting. The results showed that the gain of body weight of the female rats during the pregnancy was similar to the experimental groups. However, there was a body weight-loss in the end of the experimental period, of the rats of group GIV in comparison to 14 group GI. The pregnancy average time and size of the offspring were similar between the groups. The average weigh of the offspring treated with TCS, in different doses was smaller (p<0,05), in comparison to group GI. The initial physical development and the descended testiscles were similar between the experimental groups. The offspring exposed to TCS, during pregnancy and lactation, it was observed a delay (days) on the prepucial separation. In puberty (60 days), it was observed a meaningful weight-loss of the seminal gland on groups GIII and GIV, in comparison to group GI. When they were 90 days old, it was observed liver weight-loss and a weight gain of the prostate of the animals of group GIV, in comparison to the animals of group GI. The analysis of the stages of the seminiferous epithelium cycle of the 60 days animals showed an increase of the stages I-VI on the animals treated with TCS 300mg/kg/day in comparison to group GI (p<0,01). The 90 days animals showed an increase of the stages VII-VIII, IX-XIII and decrease on the frequency of the stage XIV of the animal spermatogenesis treated with different doses of TCS when compared to group GI (p<0,01). There was no difference on the number counting of Sertoli cells between the animals of the different experimental groups. We concluded that the maternal exposition to TCS during the gestation period and lactation causes on the male offspring, intrauterine development restriction, delay on the puberty installation, change the weight of the seminal gland in animals at 60 days, as well, changes in liver weight and prostate in animals with 90 days, and disrupting the seminiferous epithelial cycle in both age. / O triclosan (TCS) é um agente bactericida amplamente utilizado em produtos de higiene pessoal. No contexto da odontologia, esta substância tem se mostrado eficaz em reduzir a placa dentária e gengivite, além de controlar a progressão da doença periodontal crônica. Entretanto, questiona-se o real benefício da utilização em larga escala do TCS em diferentes produtos, uma vez que este composto tem sido incluído na lista dos desreguladores endócrinos (DE). A crescente observação da prevalência de contaminantes ambientais com propriedades para a desregulação endócrina tem gerado considerável debate entre os cientistas, agências regulatórias e público em geral, sobre os potenciais riscos que estas substâncias representam para a saúde reprodutiva do homem e dos animais e muitos destes compostos estão presentes em nosso cotidiano. O objetivo deste estudo foi avaliar os efeitos decorrentes da exposição ao TCS, durante a prenhez e lactação das ratas mães, no desenvolvimento físico, sexual inicial e função testicular da prole masculina na puberdade e vida adulta. Para tanto, foram utilizadas 16 ratas Wistar prenhes separadas em quatro grupos experimentais, com 4 animais em cada: GI- receberam óleo de milho diariamente por gavage; GII - receberam TCS, na dose de 75 mg/kg/dia; GIII- receberam TCS, na dose de 150 mg/kg/dia e GIV- receberam TCS, na dose de 300 mg/kg/dia. As ratas foram pesadas em dias alternados ao longo de todo o período experimental, para ajuste da dose, e monitoradas quanto ao nascimento dos filhotes. Ao nascimento, a ninhada foi pesada e avaliada quanto ao desenvolvimento físico inicial: idades de descolamento das orelhas, nascimento de pêlos, erupção dos incisivos e abertura dos olhos. Os filhotes machos foram mantidos e monitorados quanto aos parâmetros físicos externos do desenvolvimento sexual inicial (idades da descida testicular e separação prepucial). Aos 60 dias de idade (puberdade) e 90 dias de idade (maturidade sexual), foram pesados e sacrificados para a coleta e pesagem de órgãos e análise histológica dos estágios da espermatogênese e contagem de células de Sertoli. Os resultados indicaram que o ganho de peso corporal das ratas ao longo da gestação foi semelhante entre os grupos experimentais. Entretanto, houve diminuição do peso corporal, ao final do período experimental, das ratas do grupo GIV, 12 quando comparado ao grupo GI. O tempo médio da gestação e tamanho da ninhada foi semelhante entre os grupos. O peso médio das ninhadas das ratas tratadas com TCS, nas diferentes doses, foi menor (p<0,05), quando comparado ao grupo GI. O desenvolvimento físico inicial e as idades de descida testicular foram semelhantes entre os grupos experimentais. Na prole exposta ao TCS, durante a gestação e lactação, foi observado um atraso no tempo (dias) da separação prepucial. Na puberdade (60 dias), foi observada uma diminuição significativa no peso da glândula seminal nos grupos GIII e GIV, quando comparado ao grupo GI. Aos 90 dias de idade, foi observada diminuição do peso do fígado e um aumento no peso da próstata dos animais do grupo GIV, quando comparado aos animais do grupo GI. A análise dos estágios do ciclo do epitélio seminífero dos animais de 60 dias mostrou aumento dos estágios I-VI nos animais tratados com TCS 300mg/Kg/dia quando comparado ao grupo GI (p<0,01). Aos 90 dias de idade os animais mostraram aumento dos estágios VII-VIII, IX-XIII e diminuição na frequência do estágio XIV da espermatogênese nos animais tratados com diferentes doses de TCS quando comparado ao grupo GI (p<0,01). Não houve diferença na contagem do número de células de Sertoli entre os animais dos diferentes grupos experimentais. Foi possível concluir que, a exposição materna ao TCS durante a gestação e lactação causa efeitos na prole masculina, tais como: Restrição de desenvolvimento intrauterino, atraso na instalação da puberdade, alteração do peso da glândula seminal nos animais com 60 dias, como também, alteração no peso do fígado e da próstata nos animais com 90 dias e desregulação no ciclo do epitélio seminífero em ambas as idades.

The Prdm9 gene (PR domain containing 9, Meisetz, Hybrid sterility 1) encodes enzyme that trimethylates histone 3 on lysines 4 and 36. These methylation marks determine the positions of DNA double-strand breaks that are repaired by meiotic homologous recombination. In this study, we assayed genetic interactions of Prdm9 with two genes important for spermatogenesis - Mili (Piwil2) involved in piRNA biogenesis and Mybl1 encoding transcription factor that regulates many genes important for prophase I, including piRNA precursors. We crossed laboratory mice carrying mutation in Prdm9 with heterozygotes for mutation in Mybl1 or Mili, and created compound heterozygotes and, in case of Mybl1, also double homozygotes. We assessed body weight and male fertility parameters (weight of testes, sperm count, malformed sperm, percentage of tubules containing spermatocytes and of abnormal nuclei of pachytene spermatocytes) of these mice and compared them to controls. We also investigated the effect of Mybl1 and Mili mutations on fecundity of F1 intersubspecific hybrids. Our results revealed possible interactions of Prdm9 and Mybl1 in the laboratory mouse. Decreased gene dosage of Mybl1 reduced fertility of intersubspecific F1 hybrids. Interaction between Prdm9 and Mili in both laboratory mouse and F1 hybrids remain...

Depot intramuscularly administered testosterone cypionate (TC) is indicated for treatment of hypogonadism in males. However, illegal use of TC and other anabolic steroids in athletic competition has been occurring for over 50 years. A randomized three-arm clinical trial was conducted to investigate side effects of long-term abuse of testosterone cypionate. The objective of the thesis is to apply modeling approach to characterize pharmacokinetics of long-term TC injections as well as identify its side effects on healthy male volunteers. A linear one-compartment model with first-order absorption best described the concentration-time profile of testosterone obtained from 31 healthy males. The population clearance estimates for total and free testosterone were 2.42*103 and 6.03*105 L/day, respectively. Weight and albumin were identified as significant covariates for total testosterone. Given the known inhibitory effect of testosterone on HPG axis, an indirect effect model was applied to describe the suppression of luteinizing hormone and spermatogenesis. The estimated potency of total testosterone with respect to LH suppression was 9.38ng/ml. Model simulation showed that suppression of luteinizing hormone and spermatogenesis after TC injection was more severe and of greater duration in the highest dose level. A polynomial change point mixed effects model was successfully built to describe the change in weight and lipid profiles after weekly injection of testosterone cypionate. Model simulation showed that both 250mg and 500mg would incur an average increase of body weight of 3.5kg at 8 weeks after dosing. A polynomial change point model also identifies that there is a tendency for lipid decrease after TC administration. However, no difference was found in the lipid change between three dose groups, which precludes any definite conclusion on the effect of long-term TC administration on lipid profiles.

Iron has a dualistic character. On the one hand it is essential for the life of most organisms, on the other hand it is involved in the generation of reactive oxygen species that are implicated in diseases and aging. During evolution efficient mechanisms for uptake, handling and storage of iron in a safe way have developed to keep the balance between iron availability and minimizing the hazards. In eukaryotes, mitochondria are the central organelle for “metabolizing” iron and consequently play an important role in cellular iron homeostasis. Mitoferrins are mitochondrial carrier proteins, which are involved in iron transport into mitochondria. In vertebrates two mitoferrins exist, one (mitoferrin1) of which is essential for heme synthesis during erythropoiesis, while the function of the other (mitoferrin2) is not well defined. In the fruit fly we found only one mitoferrin gene (dmfrn), which codes most likely for a functional homologueof vertebrate mitoferrin2. In Drosophila cell culture, dmfrn overexpression resulted in an overestimation of cell sensed iron levels. The signal responsible for this, is most likely a yet unidentified compound of ISC synthesis. In the cell culture system we also showed that iron chelation blocks the progression of the cell cycle in a reversible and therefore most likely controlled way. Study of different dmfrn mutants indicates a role of dmfrn during spermatogenesis and development to adulthood. As dmfrn deletion mutants are not lethal, it is likely that other lower affinity iron transporters exist. A similar conclusion has been drawn by others from the study of yeast mitoferrin homologuemutants. Rim2p/Mrs12p has recently been implicated in mitochondrial iron transport, and might be an alternative metal carrier. We identified a putative homologuein the fruit fly and found a possible link between mutants in this gene and iron. Our results emphasize the importance of the mitochondrial iron metabolism in cellular iron homeostasis. We also show for the first time, a direct connection between the mitochondrial iron metabolism and spermatogenesis. Mutants characterized and developed by us will help to study these processes in further detail and reveal the underlying mechanisms.

<p>PIWI proteins belong to an evolutionary conserved protein family as the sister sub-family of ARGONAUTE (AGO) proteins. While AGO proteins are functionally well-characterized and shown to mediate small-RNA guided gene regulation, the function of PIWI proteins remain elusive. Here we pursued functional characterization of PIWI proteins by studying MILI and MIWI, two PIWI proteins in the mouse.</p><p>We first show that both MIWI and MILI co-immunoprecipitate with a novel class of non-coding small RNAs from the post-natal mouse testis extract, which are named Piwi-interacting RNAs (piRNAs). Our cloning efforts identified thousands of different piRNA sequences, mostly derived from intergenic regions. Interestingly, both MILI and MIWI piRNAs correspond to the same regions on the genome and differ primarily in length. We propose piRNAs in the adult testis are produced by the processing of long, single stranded RNA precursors, based on the observation that piRNAs originate in clusters from a number of sites on the genome in a head-to-tail homology. In support, we bioinformatically predicted putative promoters, and yeast one hybrid analysis on two such regions found out that they interact with Krueppel C2H2 type zinc finger transcription factors. We did not observe the features of the "ping-pong" mechanism in their biogenesis: Both MILI and MIWI piRNAs are biased for 5` Uracil without an Adenine bias on the 10th nucleotide position, and do not significantly consist of sequences complementary to each other along their first 10nt. Moreover, MILI piRNAs are not down-regulated in Miwi-/- testis. These results indicate that the post-natal testicular piRNAs are produced independent of the ping-pong mechanism. </p><p>Although piRNAs are highly complex, PAGE and in situ analyses showed that piRNAs are germ cell-specific with predominant expression in spermatocytes and round spermatids, suggestive of a meiotic function. Correspondingly, we found that Miwi-/-; Mili-/- mice undergo only male infertility with terminal spermatogenic arrest during meiosis. piRNAs show a nucleo-cytoplasmic distribution, with enrichment in the chromatoid and dense bodies, two male germ cell-specific structures. The dense body has been implicated in synapsis and in the heterochromatinization of the sex chromosomes during male meiosis, a process known as meiotic sex chromosome inactivation (MSCI). Our histological analysis on Miwi-/-; Mili-/- testes showed that, while the overall synapsis is not affected, the sex chromosomes retain the euchromatin marker acetyl-H4K16 and lacks the heterochromatin marker H3K9-dimethyl. These observations indicate that murine PIWI proteins are necessary for MSCI. Moreover, we identified piRNA production from the X chromosome before MSCI, and propose PIWI proteins utilize piRNAs to target and silence unpaired chromosomal regions during meiosis.</p> / Dissertation

Expression of the Tudor domain containing protein 6 (TDRD6), which is restricted to the male germ line, starts at day 16 of spermatogenesis, i.e. in pachytene spermatocytes. TDRD6 is a 250 kDa protein, which we recently found to be cleaved at the C-terminal end during germ cell development, resulting in a 230 kDa product. Neither is the process of cleavage itself nor are the functions of the two different forms known. The 230 kDa isoform is the most prominent form in round spermatids, where it localizes to the chromatoid body (CB), i.e. a single filamentous, perinuclear granule. One characteristic component of the CB is the RNA helicase MVH. CBs contain components of the microRNA (miRNA) pathway, including Piwi-interacting RNAs (piRNAs), as well as MIWI, MIWI2, and MILI, the mouse homologs of the Piwi proteins, which bind piRNAs and also act in transposon regulation. We showed that TDRD6 interacts with MIWI and MILI in vitro, and a direct interaction with MVH was shown before. To reveal the function of TDRD6, we generated Tdrd6-/- mice, which lack the protein. These mice are generally healthy but the males are sterile, due to the absence of mature spermatozoa. The most striking intracellular phenotype of Tdrd6-/- mice is the highly aberrant architecture of chromatoid bodies in round spermatids. Tdrd6-/- CBs appear as diffuse, disrupted, and less condensed structures. Their interior is largely missing, and only a “ghost”-like structure remains, expected to be significantly impaired in function. Other CB components like MVH, MIWI and MILI are expressed in Tdrd6-/- testes, but they cannot localize to the disrupted CBs. This suggests a role for TDRD6 in assembling the chromatoid body complex by recruiting other proteins. The CB is important for storage and translational regulation of mRNA, through interaction with miRNAs. In Tdrd6-deficient testes 10 % of all known murine miRNAs are differently expressed, whereas most of the mature miRNAs are up-regulated, indicating less turnover, and thus, accumulation of mature miRNAs. Since some precursor miRNAs are up-regulated as well, we assume, that TDRD6 affects miRNA transcription most likely by indirectly influencing transcriptional regulation of miRNA genes. In Tdrd6-/- mice an overall abnormal mRNA gene expression pattern was observed by microarray analyses. Of all mis-regulated genes 36 % are located to the centromer-proximal region of Chr 8, and 11 % are located to the distal end of Chr 1. This mis-regulation might be due to a common transcriptional regulation. The orthologous regions on the human chromosomes show altered chromosomal structures in many different carcinomas. If TDRD6 plays a role in carcinogenesis has to be investigated.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "August 2000." Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.