The leaner mouse carries an autosomal recessive mutation in the α1A subunit of neuronal P/Q-type voltage gated calcium ion channels. Due to this mutation, the leaner mouse exhibits severe ataxia, absence seizures and paroxysmal dyskinesia. Mutations in this same gene in humans cause: episodic ataxia type 2, familial hemiplegic migraine, spinocerebellar ataxia type 6 and probably the newly recognized form of human inherited epilepsy. Decreased amplitude of calcium current in cerebellar Purkinje cells and decreased calcium buffering capacity suggest that failure of calcium homeostasis may lead to the neurodegeneration observed in these mutant mice. Both sexes are affected. Despite their neurological dysfunction, homozygous leaner mice are able to breed and produce viable offspring. The survival rate for these pups is highly correlated with early fostering to normal lactating dams. This thesis studies the reproductive dysfunction observed in female homozygous leaner mice and is divided into four parts: onset of puberty, estrous cycle, pregnancy and litter assessment, and hormone levels. We have discovered that the onset of puberty is precocious in leaner females compared to age-matched wild type females, and leaner mice spend more time in estrous than age-matched wild type females. Also, we have observed that leaner mice became pregnant less readily than wild type mice, but once pregnant, female leaner mice produced more pups per litter compared with wild type mice. The number of corpora lutea observed in leaner mice is greater than in wild type mice. In leaner mice, the number of corpora lutea in the ovary corresponding to the uterine horn with the highest number of offspring is larger than the number of corpora lutea found in the ovary corresponding to the other uterine horn. Radioimmunoassays of estradiol hormone levels at postnatal day 28 shows higher levels in leaner compared to age-matched wild type mice. However, at postnatal day 28, the luteinizing hormone levels are similar in both categories of mice. This study of reproductive dysfunction in leaner mice was performed to gain further understanding about the role of intracellular calcium ion signaling in neuronal regulation of reproductive processes in females.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A torção testicular (TT) é uma afecção de relativa frequência, alvo de muitos estudos clínicos e experimentais ao longo dos anos. Pode acometer qualquer idade, no entanto é mais incidente em pacientes com menos de 21 anos de idade. Sua distribuição ocorre em dois picos: um no período neonatal e outro ao redor dos 13 anos de idade. A taxa dessa morbidade em jovens com menos de 25 anos é na ordem de 1/4000. A TT resulta em rotação do cordão espermático, que gera um comprometimento do suprimento sanguíneo testicular, considerada uma urgência urológica. Depois da TT, a artéria testicular e o plexo pampiniforme sofrem oclusão, interrompem o suprimento arterial e a drenagem venosa do testículo, diminuindo o aporte de nutrientes e oxigênio aos tecidos e células, levando à isquemia testicular. Esta condição afeta a produção de espermatozóides do testículo torcido podendo comprometer a fertilidade do indivíduo. Este presente estudo avaliou a função reprodutiva, alguns parâmetros espermáticos e morfologia dos testículos de ratos adultos submetidos à torção testicular antes, durante e após a puberdade, e também o efeito do tratamento com resveratrol. Os testículos direitos dos ratos foram torcidos a 720 por quatro horas em todos os grupos, exceto no grupo controle. A TT foi realizada durante os períodos pré-púbere (4 semanas de idade), púbere (6 semanas de idade) e adulto jovem (9 semanas de idade). Para cada grupo, houve um outro grupo que os animais foram submetidos ao mesmo procedimento e foram tratados com resveratrol, 30 mg/kg resveratrol foi injetado por via intraperitonel, 30 minutos antes da destorção, seguido por gavage por 7 dias. Cada rato na idade adulta acasalou com três fêmeas adultas com 12 semanas de idade para avaliar parâmetros de fertilidade. Todos os animais foram mortos com 14 semanas de idade e os testículos foram removidos para análise de concentração, motilidade e viabilidade dos espermatozóides de amostras colhidas da cauda dos epidídimos. Os parâmetros morfológicos foram avaliados por métodos estereológicos macro e microscópicos. A função reprodutiva dos animais nas diferentes idades não foi afetada pela TT, no entanto, após o tratamento os ratos operados com 4 e 6 semanas de idades tiveram maior potência e os ratos operados com 9 semanas de idade apresentaram maior taxa de fecundidade na idade adulta.. Os parâmetros morfológicos foram afetados em todos os testículos torcidos em todas as idades. Alguns desses parâmetros melhoram após o tratamento com resveratrol, e o efeito adjuvante foi maior nos ratos submetidos à TT com 4 semanas de idade. Entretanto, o resveratrol recuperou o epitélio seminífero no grupo que sofreu TT com 9 semanas de idade, os quais não apresentaram nenhum epitélio após a TT. Os parâmetros espermáticos dos testículos torcidos decresceram em todas as idades, e o resveratrol não foi efetivo, embora tenha recuperado a produção de espermatozóides no grupo que foi submetido à TT com 9 semanas de idade, em que todos os animais foram aspermatogênicos após a TTNão houve nenhum dano nos testículos contralaterais. O resveratrol promoveu efeito protetor para TT, principalmente quando os animais foram submetidos à TT em idade prépúbere. / Testicular torsion (TT) is a disease relatively frequent, which was assessed in many experimental and clinical studies over the years. TT can occur in any age, but is more common in patients under 21 years old, mostly in neonatal period and around 13 years old. The rate of this disease in youths under 25 years is about 1/400. The TT also twists the spermatic cord, which generates an impairment of the testicular blood supply and is considered a urological emergency. After TT, the testicular artery and pampiniform plexus become occluded, disrupting arterial supply and venous drainage of the testis, decreasing provision of nutrients and oxygen for tissues and cells, leading to testicular ischemia. This condition affects sperm production of the twisted testicle and may impair the fertility. This study assessed reproductive function, some morphological and spermatic parameters in adult rats which underwent experimental TT before, during and after puberty and also the effect of the treatment with resveratrol. Right testicles of rats were twisted 720 for 4 hours in all groups except the control group. TT was done during the prepubertal period (4 weeks old), puberty (6 weeks old) and young adult period (9 weeks old). For each group, there was another group which animals underwent the same procedure and were treated with resveratrol, 30 mg/kg resveratrol was injected by an intraperitoneal route, 30 minutes before detorsion, followed by gavage for 7 days. Each rat in adulthood was mated with three adult females at 12 weeks old, in order to assess parameters of fertility. All animals were killed at 14 weeks old and testes were removed for analyses Concentration, motility and viability of spermatozoids were evaluated on samples taken from the tail of the epididymis. Morphological parameters were evaluated by macroscopic and microscopic stereological methods. Reproductive function of animals at different ages was not affected by TT, however, after treatment with resveratrol, rats operated at 4 and 6 weeks old presented greater potency and animals operated at 9 weeks old showed higher fertility rate in adulthood. Morphological parameters were affected in all twisted testes at all ages. Some of these parameters improved after treatment with resveratrol and the adjuvant effect was greater in rats which underwent TT at 4 weeks old. However, resveratrol recovered the seminiferous epithelium in the group that suffered TT at 9 weeks of age, which did not show any epithelium after TT. The spermatic parameters decreased in all ages and the resveratrol did not affect it, despite it has recovered sperm production in group that underwent TT with 9 weeks old, once all animals were azoospermic after the TT. There was almost no damage in the contralateral testis. Resveratrol promoted protective effect for TT, mainly when animals underwent TT at prepubertal age.

Reproductive function is essential to the survival of all species. In mammals and other vertebrates, the control of reproduction relies on the hypothalamic-pituitary-gonadal axis, with the primary driving force provided by hypothalamic GnRH neurons. In the median eminence, the decapeptide GnRH are released in a unique pattern from GnRH neuroterminals into the portal capillary system as part of reproductive cycle. During aging, the biological rhythms of GnRH release are altered in a species-specific manner, with a reduction of GnRH pulsatility and surge in aging female rats resulting in reproductive senescence, which happens much earlier than gonadal failure in rats. Relatively few studies have focused on regulation of GnRH release at the neuroterminal level in the median eminence during reproductive aging. Therefore, the aims of this dissertation are to 1) Study the regulation of GnRH secretion at the neuroterminal level, focusing on glutamate transmission; 2) Ascertain the interaction between GnRH neuroterminals and their surrounding microenvironment focused on glial cells and the portal capillary system in the median eminence; and 3) Analyze age and hormone effects on GnRH neuroterminals and their microenvironment. An aging ovariectomized female rat model was used to study the effects of age and hormones on GnRH neuroterminal system. Fluorescence microscopy, confocal microscopy and transmission electron microscopy were used in conjunction with several imaging analysis tools. I mastered the use of cryo-embedding multi-probe immunogold labeling electron microscopy, which was essential to visualize and quantify the ultrastructral changes in GnRH neuroterminals. I combined the serial electron microscopy with cryo-embedding immunogold electron microscopy preparation and developed a new technique to examine biological markers with a three-dimensional perspective at the cellular level. Results from a series of four research projects showed: 1) There is a novel glutermatergic pathway in GnRH neuroterminals, which may regulate GnRH secretion; 2) There are dramatic age related morphological changes in the GnRH neuroterminal /glia/ portal capillary system of the median eminence that may be involved in reproductive senescence and other neuroendocrine system impairments with age; 3) Serial electron microscopy combined with immunogold labeling technique is a useful method to study the regulation of neuronal signaling pathway. Although my studies were performed on a rat model, it seems reasonable to predict that some of these changes in the median eminence with age may apply to other species, including humans, relevant to some of the menopausal symptoms in women. / text

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A torção testicular (TT) é uma afecção de relativa frequência, alvo de muitos estudos clínicos e experimentais ao longo dos anos. Pode acometer qualquer idade, no entanto é mais incidente em pacientes com menos de 21 anos de idade. Sua distribuição ocorre em dois picos: um no período neonatal e outro ao redor dos 13 anos de idade. A taxa dessa morbidade em jovens com menos de 25 anos é na ordem de 1/4000. A TT resulta em rotação do cordão espermático, que gera um comprometimento do suprimento sanguíneo testicular, considerada uma urgência urológica. Depois da TT, a artéria testicular e o plexo pampiniforme sofrem oclusão, interrompem o suprimento arterial e a drenagem venosa do testículo, diminuindo o aporte de nutrientes e oxigênio aos tecidos e células, levando à isquemia testicular. Esta condição afeta a produção de espermatozóides do testículo torcido podendo comprometer a fertilidade do indivíduo. Este presente estudo avaliou a função reprodutiva, alguns parâmetros espermáticos e morfologia dos testículos de ratos adultos submetidos à torção testicular antes, durante e após a puberdade, e também o efeito do tratamento com resveratrol. Os testículos direitos dos ratos foram torcidos a 720 por quatro horas em todos os grupos, exceto no grupo controle. A TT foi realizada durante os períodos pré-púbere (4 semanas de idade), púbere (6 semanas de idade) e adulto jovem (9 semanas de idade). Para cada grupo, houve um outro grupo que os animais foram submetidos ao mesmo procedimento e foram tratados com resveratrol, 30 mg/kg resveratrol foi injetado por via intraperitonel, 30 minutos antes da destorção, seguido por gavage por 7 dias. Cada rato na idade adulta acasalou com três fêmeas adultas com 12 semanas de idade para avaliar parâmetros de fertilidade. Todos os animais foram mortos com 14 semanas de idade e os testículos foram removidos para análise de concentração, motilidade e viabilidade dos espermatozóides de amostras colhidas da cauda dos epidídimos. Os parâmetros morfológicos foram avaliados por métodos estereológicos macro e microscópicos. A função reprodutiva dos animais nas diferentes idades não foi afetada pela TT, no entanto, após o tratamento os ratos operados com 4 e 6 semanas de idades tiveram maior potência e os ratos operados com 9 semanas de idade apresentaram maior taxa de fecundidade na idade adulta.. Os parâmetros morfológicos foram afetados em todos os testículos torcidos em todas as idades. Alguns desses parâmetros melhoram após o tratamento com resveratrol, e o efeito adjuvante foi maior nos ratos submetidos à TT com 4 semanas de idade. Entretanto, o resveratrol recuperou o epitélio seminífero no grupo que sofreu TT com 9 semanas de idade, os quais não apresentaram nenhum epitélio após a TT. Os parâmetros espermáticos dos testículos torcidos decresceram em todas as idades, e o resveratrol não foi efetivo, embora tenha recuperado a produção de espermatozóides no grupo que foi submetido à TT com 9 semanas de idade, em que todos os animais foram aspermatogênicos após a TTNão houve nenhum dano nos testículos contralaterais. O resveratrol promoveu efeito protetor para TT, principalmente quando os animais foram submetidos à TT em idade prépúbere. / Testicular torsion (TT) is a disease relatively frequent, which was assessed in many experimental and clinical studies over the years. TT can occur in any age, but is more common in patients under 21 years old, mostly in neonatal period and around 13 years old. The rate of this disease in youths under 25 years is about 1/400. The TT also twists the spermatic cord, which generates an impairment of the testicular blood supply and is considered a urological emergency. After TT, the testicular artery and pampiniform plexus become occluded, disrupting arterial supply and venous drainage of the testis, decreasing provision of nutrients and oxygen for tissues and cells, leading to testicular ischemia. This condition affects sperm production of the twisted testicle and may impair the fertility. This study assessed reproductive function, some morphological and spermatic parameters in adult rats which underwent experimental TT before, during and after puberty and also the effect of the treatment with resveratrol. Right testicles of rats were twisted 720 for 4 hours in all groups except the control group. TT was done during the prepubertal period (4 weeks old), puberty (6 weeks old) and young adult period (9 weeks old). For each group, there was another group which animals underwent the same procedure and were treated with resveratrol, 30 mg/kg resveratrol was injected by an intraperitoneal route, 30 minutes before detorsion, followed by gavage for 7 days. Each rat in adulthood was mated with three adult females at 12 weeks old, in order to assess parameters of fertility. All animals were killed at 14 weeks old and testes were removed for analyses Concentration, motility and viability of spermatozoids were evaluated on samples taken from the tail of the epididymis. Morphological parameters were evaluated by macroscopic and microscopic stereological methods. Reproductive function of animals at different ages was not affected by TT, however, after treatment with resveratrol, rats operated at 4 and 6 weeks old presented greater potency and animals operated at 9 weeks old showed higher fertility rate in adulthood. Morphological parameters were affected in all twisted testes at all ages. Some of these parameters improved after treatment with resveratrol and the adjuvant effect was greater in rats which underwent TT at 4 weeks old. However, resveratrol recovered the seminiferous epithelium in the group that suffered TT at 9 weeks of age, which did not show any epithelium after TT. The spermatic parameters decreased in all ages and the resveratrol did not affect it, despite it has recovered sperm production in group that underwent TT with 9 weeks old, once all animals were azoospermic after the TT. There was almost no damage in the contralateral testis. Resveratrol promoted protective effect for TT, mainly when animals underwent TT at prepubertal age.

Philosophiae Doctor - PhD / Introduction: Typha capensis, commonly referred to as bulrush also called "love reed'' growing in Southern Africa's wetlands, is one of South Africa indigenous medicinal plants that are traditionally used to treat male fertility problems and various other ailments. Previous studies revealed that T. capensis has indeed a beneficial effect on male reproductive functions and aging male symptoms. The T.capensis rhizomes are used in traditional medicine during pregnancy to ensure easy delivery, for venereal diseases, dysmenorrhea, diarrhoea, dysentery, and to enhance the male potency and libido. Typha genuses contain flavones and other phenolic compounds, which exhibit anti-oxidative capacity. Materials and Methods: This study encompasses three parts (part 1: Exposure of different cell lines to crude aqueous extracts of T. capensis rhizomes; part 2: HPLC analysis of Typha capensis crude rhizome extract and exposure of different cell lines to the F1 fraction of the summer season; part 3: Compound identification by means of NMR spectrometric analysis and exposure of different cell lines to bioactive compounds (Quercetin and Naringenin) isolated from T. capensis rhizomes. Part 1: TM3-Leydig cells and LNCaP cells incubated with different concentrations of crude aqueous extract of T. capensis rhizomes (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) and control (without extract) for 24 and 96 hours, after incubation. The following parameters were evaluated: cell morphology and viability (determined by means of MTT assay). Part 2: The crude extract HPLC profiles were obtained by preparing the extracts for different seasons (Autumn, Winter, Spring, Summer). TM3-Leydig cells, LNCaP cells and PWR-1E cells incubated with different concentrations T. capensis rhizomes extract F1 fraction of the summer season (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) and control (without extract) for 24 and 96 hours, after incubation. The following parameters were evaluated: cell morphology was observed and recorded, viability (determined by means of MTT assay), testosterone production (testosterone ELISA test), cell early apoptosis (determined by means of Annexin V-Cy3 binding), DNA fragmentation (determined by means of the TUNEL assay). Part 3: NMR spectrometric analysis was performed on a 13C spectra were recorded at 400 MHz. TM3-Leydig cells and LNCaP cells incubated with different concentrations of bioactive compounds (Quercetin and Naringenin) isolated from T. capensis rhizomes, for acute exposure (24, 96 hours) and chronic exposure (96 hours), after incubation, the following parameters were evaluated: cell morphology and viability (determined by means of MTT assay), testosterone production (testosterone ELISA test), cell early apoptosis (determined by means of Annexin V-Cy3 binding) and DNA fragmentation (determined by means of the TUNEL assay). Results: Part 1: for TM3-Leydig cells the results reveal no observable morphological changes and no significant influence on cell viability except at highest concentration indicating cellular stress. However, LNCaP cells showed a decline in cell viability at the incubation period 96 hours (-82.4%) more than 24 hours (-64.7%) indicating more cell death. Part 2: HPLC data showed that the most effective fraction was the F1 fraction from the summer harvest. Results revealed that the T. capensis rhizome extract F1 fraction of the summer season significantly enhanced testosterone production in TM3 cells and was more toxic towards cancer cells (LNCaP cells ) compared to the normal cell lines (TM3-Leydig, PWR-1E cells). Part 3: NMR data showed 2 bioactive compounds which were identified as Quercetin and Naringenin. The assays showed that LNCaP cells are more sensitive to the cytotoxic effects and apoptosis induction of both compounds, whereas, the assays resulted in weak effects toward TM3-Leydig cells. However, testosterone production in TM3-Leydig cells was significantly enhanced at low concentrations of Quercetin and Naringenin at all exposure types (acute and chronic) testosterone beak significantly at around 0.100 and 0.125 μg/ml (P<0.0001), stimulatory activity in a dose-dependent manner. Conclusion: Typha capensis enhanced the production of testosterone and might be useful to treat male infertility and aging male problems. Results further reveal that the F1 fraction from the summer harvest had highest biological activity. This study, for the first time, investigated the effects of bioactive compounds (Quercetin and Naringenin) yeilded from aqueous extraction of Typha capensis rhizomes in cell lines investigating male reproductive functions. Active compounds present in the rhizomes have caused an increased production of testosterone level in TM3-Leydig cells. Furthermore, the active compounds of Typha capensis rhizomes in the high dose had a negative effect on the percentage of DNA fragmentation in LNCaP cells. When compared to the effect of the low dose, the two compounds induced significant apoptosis in cancer cell line (LNCaP) compared with the normal cell line (TM3-Leydig). The isolated compounds are significantly selective towards the cancer cells than the normal cell compared with the exposure of bioactive compounds used in this study.

No / Sexual dysfunction is a major, although poorly understood, side-effect of treatment with antipsychotic drugs. We have recently show marked disruption of reproductive function and weight gain in female rats treated subchronically with risperidone and haloperidol. The aim of the present study was to examine further the potential relationship between reproductive dysfunction and weight gain in female rats treated with olanzapine. The effects of olanzapine on weight gain, food and water intake, intra-abdominal fat, the oestrous cycle and uterine weight were assessed in group-housed adult female hooded-Lister rats. Olanzapine (0.5-4.0 mg/kg i.p.) or vehicle was administered once daily for 21 days and body weight, food and water intake measured, with histological examination of vaginal lavage to determine the stage of the oestrous cycle. On day 22, animals were sacrificed and intra-abdominal fat, wet and dry uterine weights measured. Olanzapine induced significant weight gain with concomitant increases in food and water intake and intra-abdominal fat without an effect on the oestrous cycle, wet and dry uterine weights or plasma prolactin levels. These results confirm the ability of olanzapine to induce weight gain in female rats on unrestricted normal diet with a concomitant increase in food and water intake and increased intra-abdominal fat. These effects of olanzapine were produced in the absence of any apparent impairment in reproductive function, in contrast to the substantial disruption of oestrous and uterine atrophy previously shown in rats treated with risperidone and haloperidol.

L’impact de l’utilisation des agropesticides sur la fonction de reproduction mâle est évalué chez des agriculteurs de Djutitsa (Ouest Cameroun). Le pesticide le plus utilisé par ces agriculteurs est testé in vivo sur la fonction de reproduction du rat mâle, ex vivo et/ou in vitro sur la stéroïdogenèse Leydigienne afin d’évaluer son implication dans les déséquilibres observés chez les agriculteurs. Ensuite, l’effet protecteur des extraits de Carpolobia alba et Basella alba contre les effets délétères dudit pesticide est évalué in vivo chez des rats mâles, ainsi que leur influence sur la production de la protéine de transport des stéroïdes (SHBG) par les cellules HepG2. Les résultats montrent que les agriculteurs utilisent 25 agropesticides et présentent des symptômes liés à une protection inadéquate. De plus, ils présentent une diminution de la testostérone avec une élévation de l’androstènedione sériques comparé au groupe témoin non exposé (P < 0,05) et ces déséquilibres androgéniques sont accompagnés de difficultés de reproduction. Le manèbe est l’agropesticide le plus utilisé et perturbe chez le rat mâle la stéroïdogenèse Leydigienne (à travers l’inhibition de la CYP11A1) et la fertilité. Cette fertilité est améliorée/rétablie lorsque le pesticide est concomitamment administré avec l’un des extraits, suggérant ainsi l’effet protecteur desdits extraits qui est imputable à leur activité androgénique également démontré chez les rats. Toutefois, ces extraits et le manèbe n’affectent pas la production de la SHBG in vitro. Ces résultats soulignent l’altération de la fonction de reproduction mâle par les agropesticides et l’effet bénéfique des plantes médicinales / The effect of agropesticides use on male reproductive function was evaluated in male farmers in Djutitsa (West Cameroon). The most frequently used agropesticide by farmers was selected and tested in vivo on the reproductive function of male rats, ex vivo and/or in vitro on Leydig cells steroidogenesis, in order to evaluate its implication in disorders observed in farmers. The ability of 2 Cameroonian medicinal plants (Carpolobia alba and Basella alba) to prevent pesticide-induced toxicity was also investigated in vivo in male rats. Furthermore, both extracts and selected pesticide were tested on the release of a steroid transporter protein (SHBG) by HepG2 cells. The results showed that farmers used 25 agropesticides and presented symptoms related to inadequate protection. Moreover, they had lower serum testosterone and higher androstenedione levels compared to a control group with no history of agropesticide exposure (P < 0.05), and these androgenic imbalances were accompanied by difficulties of reproduction. Maneb was the most common ingredient, and its administration to male rats resulted in decrease/alteration of Leydig cells steroidogenesis (through inhibition of CYP11A1) as well as fertility. The latter fertility was improved/restored when maneb was coadministrated to rats with any of the plant extracts, suggesting their protective effect that may be attributed to their proven androgenic activity. However maneb and the plant extracts did not affect SHBG release by HepG2 cells. These results highlight agropesticides deleterious effect on male reproductive function, which may be prevented by the investigated plant extracts