Dissertations / Theses on the topic 'Female reproductive function'

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.

Chronic alcohol (ethanol) consumption has been known to affect the major organs of the body and particularly the liver. However, the effects of chronic ethanol consumption on the female reproductive system remain relatively unstudied. A convenient way to study these effects is by analyzing laboratory mice that have been fed an ethanol diet for an extended period of time and comparing them to control mice. In this study, female mice were separated into control and ethanol fed groups. The mice were placed on their specified diets and observed over the course of six weeks. The mice were fed and weighed daily throughout the duration of the experiment. Once a week, vaginal washes were performed on both groups of mice to determine the stage of the estrous cycle for each mouse. At the end of the six weeks, the mice were sacrificed and the ovaries were harvested and fixed in 4% paraformaldehyde. The ovaries were then paraffin embedded and sectioned. Glass microscope slides were then stained using Hematoxylin and Eosin staining procedures for evaluation using standard light microscopy. The tissue’s morphology, follicle development, presence of corpora lutea, and overall appearance were analyzed. Due to the premature deaths of several mice in first group of ethanol fed mice, the experiment was repeated with three more groups of mice to obtain a better representation of data. The data from the control group was compared to that of the ethanol fed group. The mice that received the ethanol fed diet ceased to cycle and arrested in the diestrous phase of the estrous cycle. Our data indicates that the ovarian follicles within the ethanol fed mice show signs of degeneration in the 4b, 5a, 5b, 6, and 7 levels of development. There are also no notable corpora lutea present within the ovaries of the ethanol fed mice. Our findings indicate that chronic alcohol consumption has deleterious effects on ovarian morphology in mice.

The orphan nuclear receptor NR5A2 is implicated in a multitude of biological processes including cholesterol homeostasis and development. Its role in cholesterol metabolism and cell proliferation is now well established in vitro and in vivo. Both in vitro and gene expression studies have suggested a role for NR5A2 in ovarian function. In this study, we provide in vivo evidence for its involvement in reproductive function by demonstrating that heterozygosity for a null mutation of NR5A2 leads to a reduction in female fertility. Furthermore, we showed that NR5A2+/- females display a severe reduction in ovarian progesterone production and that progesterone supplementation can rescue the NR5A2+/- subfertility phenotype. We also provide evidence that one of the mechanisms by which NR5A2 regulates ovarian progesterone production is through modulating the expression of SCAR, which controls one of the rate-limiting steps of progesterone synthesis.
A targeted disruption of the NR5A2 gene in the mouse leads to early lethality in utero between embryonic days 6.0 and 7.5, showing that NR5A2 plays a crucial role during early embryogenesis. The molecular mechanisms underlying this early lethality, however, are poorly understood. In this study, we used a morphological and marker gene analysis to characterize the NR5A2-/- embryonic phenotype and showed that although initial axis specification occurs in NR5A2-/- embryos, primitive streak and mesoderm fail to form. Using a chimeric approach, we demonstrated a requirement for NR5A2 function in the visceral endoderm (VE), an extra-embryonic tissue, for proper primitive streak morphogenesis and gastrulation. Our results also indicate a reduction in the expression of VE marker genes involved in the nutritive function of this tissue, suggesting that NR5A2 play a dual role in the VE, being implicated in the mediation of both its patterning and nutritive activity.
Taking advantage of the LacZ knock-in approach used to inactivate the NR5A2 gene, we also demonstrated that NR5A2 is expressed during craniofacial and nervous system development, suggesting a novel role for NR5A2 in head formation and neural development.

Philosophiae Doctor - PhD
In different parts of the world, medicinal plants have demonstrated a lot of health benefits to mankind and remains an important source for the discovery of new bio-active compounds. Libya is a typical example of a country where medicinal plants are widely used. Plant extracts of five Libyan medicinal plants were used in this study to investigate their in vivo effects on spermatogenesis and steroidogenesis in male rats and on ovulation and fertility in female rats. The In vitro effects of these plant extracts were also investigated on TM3 Leydig cells and MCF 7 breast cancer cells. A phyto-chemical analysis of the five Libyan medicinal plants (flaxseed, black seeds, radish seed, date palm pollen and nutmeg) was done. The results showed that date palm pollen had a higher antioxidant activity than all of the above mentioned plants. In addition to this, Nigella sativa was observed to possess high flavonol content as well as high antioxidant activity. Male rats exposed to flaxseed, radish seeds and date palm pollen showed no significant alterations in body weight gain, whereas date palm pollen (240 mg/kg, p < 0.05) promoted an increase in body gain. This study also revealed a significant increase in the relative testicular weight of animals exposed to either flaxseed (300mg/kg) or date palm pollen (120mg/kg). In addition, the relative weights of the seminal vesicles of all treated groups showed significant increased values. The level of serum testosterone showed a significant increase after exposure to radish seed (80mg/kg) and a significant dose- dependent increase for date palm pollen when compared to control (P< 0.05). In contrast, flaxseed caused a dose-dependent significant (p <0.01) decrease in testosterone level at radish seed (300mg/Kg). All plant extracts caused a significant increase in sperm concentration. Sperm vitality significantly (p < 0.05) increased by radish seed (80mg/kg), flaxseed (300mg/kg) and date palm pollen (120, 240mg/kg) respectively. Total progressive motility improved significantly at flaxseed (300 mg/kg) (p < 0.001) as well as date palm pollen (p < 0.01). Histological examination of the cross sections of the testis showed clear presence of all stages of spermatogenesis in all the treated groups. Rat epididymides showed normal morphological appearance and their lumen were filled with spermatozoa. The diameter of seminiferous tubules in male rats exposed to date palm pollen (120 and 240 mg/kg) was significantly higher (p < 0.001). The heights of the germ cell epithelia within the eminiferous tubules were also significantly increased in all treated groups. Liver and renal functions tests showed a significant decrease in Alanine transaminase (ALT) and creatinine in all treated groups (p < 0.05), and this demonstrates the lack of cytotoxic effects of date palm pollen, radish seed and flaxseed on the rats. However, these plant extracts produced a non-significant (p > 0.05) increase in Aspartate transaminase (AST) levels. Besides this, superoxide dismutase activity (SOD) in testis was increased significantly by radish seed (160 mg/kg), flaxseed (200 mg/kg) and date palm pollen (120 mg/kg). There was also improved catalase activity in testis of male rats exposed to radish seed and date palm pollen. Regarding male sexual behavior, the time to reach the female and the mount frequency decreased significantly in male rats exposed to flaxseed (300 mg/kg) and date palm pollen (120 and 240 mg/kg; p > 0.05) thus, these plant extracts exhibit aphrodisiac properties. In addition, exposure of male rats to date palm pollen (120 mg/kg) produced a significant (p < 0.01) increase in the number of embryos in untreated female rats. In the female rats, the body weight gain was not affected (p > 0.05). However, the relative uterus weights exposed to nutmeg (200 mg/kg) and date palm pollen (120 and 240 mg/kg) were significantly decreased (p < 0.05). In addition, the relative weights of ovaries after treatment with nutmeg (400 mg/kg) and black seed (400 mg/kg) showed significantly increased values (p < 0.01). Serum FSH was significantly increased (p > 0.05 or 0.01) when the female rats have been exposed to black seed (200 mg/kg), nutmeg (200 mg/kg) or date palm pollen (120 mg/kg). The LH level significantly (p < 0.01) decreased following exposure to black seed (200 mg/kg), date palm pollen (120 mg/kg). On the other hand, serum LH concentration was significantly increased in female rats exposed nutmeg (400 mg/kg; p > 0.05). The creatinine activity in female rat serum in all treated groups was significantly decreased (p < 0.05). Whereas the higher dose of date palm pollen (240 mg/kg) caused only a non-significant decrease. ALT activity in serum of female rat exposed to either black seed (400 mg/kg) or date palm pollen (120 and 240 mg/kg) was shown to decrease significantly (p < 0.05). Histology of the reproductive organs, kidney and liver in the female rats showed no obvious alterations in any of the treated groups. In addition, the number of embryos in female rats significantly increased (p < 0.01; p < 0.001) following exposure of female rats to black seeds 400 and date palm pollen 240 mg/kg, respectively. Incubation of TM3 Leydig cells with radish seeds for 24, 48 or 72 hours caused a significant (p < 0.01) decrease in mitochondrial dehydrogenase activity. Besides that, date palm pollen and flaxseed increased the mitochondrial dehydrogenases activity of TM3 Leydig cells. In addition, higher concentration of date palm pollen, nutmeg and black seed were cytotoxic to MCF7 breast cells. In testis slices testosterone secretion in vitro was significantly increased by flaxseed (500 μg/ml; p > 0·05) and date palm pollen (500 μg/ml; p > 0·01). MCf-7 cells treated with BS 10-50 μg/ml black seed and nutmeg 10-50μg/ml significantly increased cell proliferation. However, the treatment with date palm pollen produced only a weak estrogenic effect, which resulted in a concentration dependent significant increase as observed between 50-1000 μg/ml date palm pollen. In conclusion, in this study, we observed that date palm pollen, radish seed and flaxseed increased libido as well as steroidogenesis and spermatogenesis, improved hepato and nephron-protective effects. In female rats, the plant extracts NM, BS and date palm pollen potentiated the production of gonadotropic hormones. In addition to this, at lower concentrations these medicinal plants promoted cell growth, whereas at higher concentrations they inhibited cell proliferation of MCF- 7 breast cancer cells. The anti-oxidant effects of these plant extracts have been implicated for the above mention effects.

Mestrado em Biologia Molecular e Celular
The impact of the Fragile Mental Retardation-1 (FMR1) gene CGG repeat number in the female reproductive function is well established. Carriers of a CGG repeat number between 55 and 200, designated a premutation, are prone to develop primary ovarian insufficiency or early menopause. Yet, an impact on the reproductive function in carriers of “normal” genotypes and sub-genotypes (CGG<54) is controversial. The presence of AGG in normal-sized alleles confers stability, hampering the expansion of the repeat number in future generations. To the best of our knowledge testing the influence of the AGG number and pattern on the female reproductive function has never been endeavored. Herein, the ovarian reserve markers were correlated with CGG number as well as AGG number and pattern, in female carriers of FMR1 normal-sized alleles. Our cohort comprised 50 healthy young females, candidates for oocyte donation. Considering AGG number and pattern are not routinely determined different methodologies were implemented: 1) Triplet-Primed Polymerase Chain Reaction; 2) Sanger sequencing; and 3) Restriction Fragment-Length Analysis. A projection of the association between the CGG repeat values and the hormonal levels, by multivariate analysis, was performed, considering the FMR1 new “normal” sub-genotypes previously defined. The hormonal levels associated with the different samples were not sufficient to discriminate the sub-genotypes, indicating that the individualization of the samples classified by sub-genotype was not possible. Resorting to a mathematical formula that determines the allelic score, taking into account total allele size, and AGG number and pattern. After statistical analysis, it was possible to divide the samples into two groups: a first called an equivalent group and a second called an opposite group. The equivalent group is composed mainly of samples carrying alleles in the normal FMR1 sub-genotype and the opposite, where most of the samples have an FMR1 low/normal sub-genotype. In the equivalent group, a positive and significant correlation was observed between the number of antral follicles and the hormonal levels: prolactin and luteinizing hormone (LH). Thus, it is possible to predict the largest number of antral follicles produced combining the levels of prolactin and LH. These results actually confirm prior publications as the low/normal sub-genotype has been previously associated with a diminished ovarian reserve. Overall, this study confirms the association of the FMR1 CGG repetitive region in the female reproductive function and suggests that the stability of the alleles is a determining factor for the ovarian response success.
A relação entre o número de repetições CGG do gene Fragile Mental Retardation-1 (FMR1) e a função reprodutiva em mulheres não é uma novidade. Está descrito que as portadoras de alelos com um número de repetições CGG entre 55 e 200, designados por pré-mutação, têm uma predisposição para desenvolver insuficiência ovárica primária ou menopausa precoce. Porém, a existência de risco de diminuição da função reprodutiva nas mulheres, com genótipos considerados “normais” (CGG<54), e respetivos subgenótipos ainda não é clara. Sabe-se que a presença de interrupções AGG confere a esses alelos uma maior estabilidade, impedindo a expansão do número de repetições CGG para um tamanho considerado patogénico. A forma como o número e o padrão de interrupções AGG poderá influenciar a função reprodutiva feminina, nunca foi estudada. No presente trabalho, os marcadores de reserva ovárica foram correlacionados com o número de repetições CGG e perfil das interrupções AGG. A população em estudo incluiu 50 mulheres jovens e saudáveis, candidatas à doação de oócitos. Dado que o número e o padrão das interrupções AGG não são determinados por rotina, foi então necessário implementar a sua análise, recorrendo a diferentes metodologias: 1) Triplet-Primed Polymerase Chain Reaction; 2) Sanger sequencing; and 3) Restriction Fragment-Length Analysis. Foi realizada uma projeção da associação entre o número de repetições CGG e os níveis hormonais, através de uma análise multivariável, considerando os novos subgenótipos "normais" FMR1 previamente definidos. Os níveis hormonais associados às diferentes amostras não foram suficientes para discriminar subgenótipos, indicando que a individualização das amostras classificadas por sub-genótipos não era possível. Recorrendo a uma fórmula matemática que determina a pontuação alélica, tendo em consideração o tamanho total do alelo, e o número e o padrão de AGG. Após análise estatística foi possível dividir as amostras em dois grupos: um primeiro designado por grupo equivalente e um segundo designado por grupo oposto. O grupo equivalente, que é composto principalmente por amostras que possuem alelos do subgenótipo “normal” FMR1, e o oposto, onde a maioria das amostras possui subgenótipo “normal/baixo” FMR1. No grupo equivalente, observou-se correlação positiva e significativa entre número de folículos antrais e os níveis hormonais: prolactina e hormona luteinizante (LH). Assim, é possível prever o número de folículos antrais produzidos combinando os níveis de prolactina e LH. Estes resultados confirmam publicações anteriores, já que o sub-genótipo “normal/baixo” foi anteriormente associado a uma diminuição da reserva ovárica. No geral, este estudo confirma a associação da região repetitiva CGG do FMR1 na função reprodutiva feminina e sugere que a estabilidade dos alelos é um fator determinante para o sucesso da resposta ovárica.

Thesis (DMed (Obstetrics and Gynaecology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Chapter 1 is a literature review investigating the incidence of cancer in children and young adults. It describes the most important treatment options including chemotherapy, radiotherapy and surgery and the effect of treatment on future endocrine development and fertility. Different primary cancer sites are discussed in more detail. Chapter 2 is a literature review on the effects of cancer surgery in women and the options for fertility sparing. Cervical cancer and pre-cancer are discussed in detail with options for more conservative surgery in selected patients. A summary of the available published cases of trachelectomy with pregnancy outcomes is included. Other gynaecological cancers requiring surgery are also discussed with reference to conservative options. Chapter 3 is a literature review about the medical (pharmacological) options for protection of ovarian function in patients undergoing oncotherapy. The role of gonadotrophin releasing hormone analogues and hormonal contraceptives in ovarian suppression is discussed in detail. Chapter 4 This chapter examines germ cell physiology with reference to cryopreservation. It includes two major parts. Part 1 is the description of germ cell- and follicle physiology, the principles of cryobiology followed by a review of oocyte cryopreservation and ovarian tissue preservation. Both slow freezing and vitrification techniques are described. The second part of chapter 4 is a report on a randomised controlled evaluation of two different slow freezing cryopreservation protocols. This experimental study compared ultrastructural changes in fresh and previously cryopreserved ovarian cortical tissue after equilibration and thawing using two different cryoprotectants. This is the first randomised investigation into DMSO and PROH as cryoprotectants. Chapter 5 is an investigation into cryopreservation of ovarian tissue as a strategy to protect hormonal function and fertility against gonadotoxic treatment. This chapter consists of two parts. The first part is a thorough literature review of all the published work about grafting of previously cryopreserved ovarian tissue. The largest case series found from a single institution was five patients. Another report of six patients included patients from various sites in Denmark. Part 2 is a description of a cohort of patients followed up after re-implantation of previously cryopreserved ovarian cortical tissue. Follow-up hormone levels of 13 individual cases are described in detail. This is the largest case series ever reported. The experimental study described in Chapter 4 and the clinical study described in Chapter 5 was approved by the ethical research committee of the Faculty of Health Sciences, Stellenbosch University, project number N05/10/182. Chapter 6 provides an integrated overview of the incidence and treatment of cancer in young women and how its negative effects may be prevented or mitigated. Aspects of chemotherapy, radiotherapy and surgery are evaluated where it may affect future reproductive health. The role of oocyte and ovarian tissue cryopreservation is discussed. Guidelines are provided for clinicians.
AFRIKAANSE OPSOMMING: Hoofstuk 1 Hierdie is ‘n literatuuroorsig wat die insidensie van kanker in kinders en jong volwassenes ondersoek. Dit sluit die mees belangrike behandelingsopsies in, naamlik chemoterapie, radioterapie en chirurgie en die effek wat behandeling mag hê op toekomstige endokriene ontwikkeling en fertiliteit. ‘n Verskeidenheid kanker tipes word in meer detail beskryf. Hoofstuk 2 Hoofstuk 2 is ‘n literatuuroorsig oor die effekte van kankerchirurgie in vroue en die geleenthede tot beskerming van fertiliteit. Servikale kanker en voorlopers van servikale kanker word bespreek en die opsies vir konserwatiewe chirurgie in uitgesoekte pasiënte word gegee. ‘n Opsomming van die inligting wat beskikbaar is oor tragelektomie en swangerskap uitkomste word ingesluit. Ander ginekologiese kankers wat chirurgie mag benodig, word ook bespreek met verwysing na konserwatiewe hantering. Hoofstuk 3 ‘n Literatuuroorsig oor die mediese (farmakologiese) opsies vir die beskerming van ovariële funksie in pasiënte wat behandeling ontvang vir kanker. Die rol van gonadotropien-vrystellingshormoon-analoë en hormonale kontrasepsie vir ovariële onderdrukking word in detail bespreek. Hoofstuk 4 Hierdie hoofstuk ondersoek kiemselfisiologie met verwysing na vriesbewaring. Dit is verdeel in twee dele. Deel 1 is ‘n beskrywing van kiemsel- en follikelfisiologie en die beginsels van vriesbiologie. Dit word gevolg deur ‘n oorsig van oösiet vriesbewaring en ovariële weefselbewaring. Stadige bevriesing en vitrifikasie- metodes word bespreek. Die tweede deel van hoofstuk 4 is ‘n verslag oor ‘n gerandomiseerde, gekontroleerde evaluasie van twee stadige bevriesingsmetodes. Hierdie eksperimentele studie het die ultrastrukturele veranderinge vergelyk in vars en voorheen bevrore ovariële kortikale weefsel na ekwilibrasie en ontdooiing met twee verskillende vriesbeskermers. Dit is die eerste gerandomiseerde studie oor DMSO en PROH as vriesbeskermers. Hoofstuk 5 Hierdie hoofstuk handel oor ‘n ondersoek na vriesbewaring van ovariële weefsel as ‘n benadering tot beskerming van hormonale funksie en fertiliteit teen gonadotoksiese behandeling. Die hoofstuk bestaan uit twee dele. Die eerste deel is ‘n deeglike oorsig van die literatuur oor al die beskikbare werk wat handel oor terugplasing van voorheen bevrore ovariële weefsel. Die grootste pasiëntreeks van ‘n enkel instelling was slegs vyf pasiënte. ‘n Ander beskrywing van ses pasiënte het pasiënte van verskeie eenhede in Denemarke ingesluit. Deel 2 is ‘n beskrywing van ‘n groep pasiënte wat opgevolg is na oorplanting van voorheen bevrore ovariële kortikale weefsel. Opvolg hormoonvlakke van 13 gevalle word in detail bespreek. Hierdie is die grootste pasiëntreeks wat tot nog toe beskryf is. Die eksperimentele studie wat in hoofstuk 4 beskryf word en die kliniese studie wat in hoofstuk 5 beskryf word, is goedgekeur deur die etiese navorsingskomitee van die Fakulteit Gesondheidswetenskappe van die Universiteit Stellenbosch met die projeknommer N05/10/182 Hoofstuk 6 Hierdie is ‘n geïntegreerde oorsig van die voorkoms en behandeling van kanker in jong vroue en hoe die negatiewe effekte daarvan voorkom of verminder kan word. Aspekte van chemoterapie, radioterapie en chirurgie word geëvalueer ten opsigte van die effek op toekomstige reproduktiewe gesondheid. Die rol van oösiet- en ovariële weefselvriesbewaring word bespreek. Riglyne vir klinici word gegee.

Experiments were designed to determine: a) The effect of undernutrition in utero and in early post-natal life on subsequent activity of the hypothalamic-pituitary-ovarian axis in prepubertal and peripubertal female sheep; b) The effect of undernutrition in utero from the time of mating on development of the ovaries of foetal female sheep at days 47 and 62 of gestation. It is concluded that undernutrition in early life significantly retards pituitary development as demonstrated by the selective hypersensitivity of prepubertal L group pituitary glands but that this is compensated for around 18 months of age, by which time peripubertal L group pituitary glands are hyposensitive to positive GnRH stimulation. As there were no effects of treatment on metabolic hormone profiles or glucose clearance rate, it can be concluded that undernutrition acts directly at the pituitary level and not through differences in L group metabolism. In experiment 2, ewes were fed 150% (High; H) or 50% (Low; L) of maintenance energy requirements from mating until 47 or 62d of gestation in separate studies. At 47d, L lambs exhibited normal foetal weight, foetal ovarian weight and foetal ovary steroidogenic capacity but had significantly retarded germ cell degeneration as indicated by higher concentrations of oogonia in L group foetal ovaries (p<0.001). At 62d, L lambs exhibited significantly retarded germ cell degeneration, as indicated by higher oocyte concentrations (p<0.01), and delayed arrest of meiosis, as indicated by higher meiotic activity (p<0.001). It is concluded that undernutrition of the ewe from the time of mating significantly retards ovarian development in foetal ovaries. This retardation may be responsible for the known reduction in lifetime reproductive performance of ewes undernourished in early life.

Antimicrobial peptides (AMPs) are small proteins produced by epithelial surfaces, which have broad-spectrum antimicrobial and immunomodulatory activities. In the lung, skin and alimentary tract AMPs are known to be important in infectious and inflammatory conditions. Far less is known regarding the role of AMPs within the female reproductive tract, but as infection and inflammation are causes of preterm labour, AMPs may have a key function in maintain and protecting pregnancy. The major groups of human AMPs include the human beta defensins (HBDs), two antileukoproteinases (secretory leukocyte protease inhibitor (SLPI) and Trappin-2/Elafin), and the human cathelicidin hCAP18/LL-37, with several studies identifying their presence at sites throughout the reproductive tract. The cervix in pregnancy is positioned between the upper genital tract containing the developing fetus and the lower tract where infections usually arise. I hypothesise that AMPs are fundamental to mucosal immune defence of the cervix in pregnancy, preventing ascending infection and excessive inflammation that can cause preterm labour. This thesis focused on the human cathelicidin hCAP18/LL-37 and its role within the cervix and vagina. The aims of this thesis were to; investigate the inflammatory effects of LL-37 from cervical and vaginal derived epithelial cells and determine the pathways and receptors in which LL-37 may elicit its effects and how production may be regulated; investigate the role of CRAMP in a mouse model of preterm birth; and determine the production of AMPs by the pregnant cervix whilst investigating the relationship between AMP concentrations in cervicovaginal secretions and preterm labour. The inflammatory effect of LL-37 was investigated using cell lines derived from endocervical, ectocervical and vaginal epithelium. The study of these cell lines suggests divergent responses of cervical and vaginal epithelial cells. LL-37 mediated induction of IL-8 and IL-6 production from endocervical epithelial cells was observed in a dose-dependent and time-dependent manner, whilst ectocervical and vaginal cells also respond to treatment with LL-37 through IL-8 and IL-6 production. To determine a possible mechanism of action of LL-37 on IL-8 and IL-6 in the three cell lines, inhibitors against MAPK cascades, ERK, p38 MAPK and JNK, and known LL-37 receptors were investigated. In endocervical cells LL-37 mediated IL-8 occurs via activation of unidentified GPCRs, whilst in ectocervical cells this effect on IL‐8 and IL-6 is via the activation of ERK and p38 MAPK cascades. The mechanism by which LL-37 induces IL-8 secretion in vaginal epithelial cells remains unknown. Expression of LL-37 was shown to be mediated by vitamin D3 in vitro in cervical and vaginal epithelial cells. However when this relationship was investigated in vivo, using matched serum and cervicovaginal secretions from woman at early pregnancy, no correlation was observed between circulating vitamin D and cervicovaginal or circulating hCAP18/LL-37. However, the majority of women in this study reported with insufficient levels of vitamin D, which may effect the relationship observed with hCAP18/LL-37. Using a mouse model of LPS-induced preterm labour, to mimic the presence of intrauterine infection bacterial infection, I aimed to characterise the role of CRAMP, the mouse orthologue of hCAP18/LL-37, in the lower inflammatory and immune response that results in preterm labour. Wild type C57Bl/6J mice receiving an intrauterine injection of LPS deliver prematurely, within 24 hours of injection. However mice deficient in CRAMP (Camp -/-) receiving an intrauterine injection of LPS deliver significantly later and have a non-significant increase in pup survival compared to wild type C57Bl/6J mice. Cervical tissue collected post partum showed no difference in inflammatory markers between wild type C57Bl/6J and Camp -/- mice, however there was increased expression of the neutrophil chemoattractant marker, Cxcl5, and the neutrophil marker, Ngp in Camp -/- mice. In the lower genital tract, levels of antimicrobial peptides were determined in samples of cervicovaginal secretions collected from pregnant women. AMPs, hCAP18/LL-37, HBD-2 and SLPI were found in cervicovaginal secretions, and levels of hCAP18/LL-37 were increased in women with the common vaginal infection bacterial vaginosis. However no relationship was identified between the concentration of AMPs and preterm birth in this study. This work has shown that the lower genital tract, where infections that are associated with preterm labour originate, expresses the human cathelicidin hCAP18/LL-37. It may play an important role in modulating the immune response to invading infection associated with preterm labour. Further investigation of these responses may increase understanding of the physiology and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

Chez les mammifères femelles, une fertilité optimale repose sur la synchronisation des évènements neuroendocriniens et comportementaux régulant la fonction de reproduction. Pour cela, l’horloge circadienne principale, entrainée par l’alternance lumière/obscurité, rythme l’axe hypothalamo-hypophyso-ovarien. Ainsi, des cycles jour/nuit irréguliers, comme lors du travail en horaires décalés, peuvent altérer la fonction de reproduction et diminuer la fertilité, notamment chez les femmes. Ce travail de recherche visait à évaluer les effets de la perturbation du rythme circadien sur la fonction de reproduction féminine et à étudier les mécanismes neuroendocriniens sous-jacents. Chez la souris femelles, l’exposition à un décalage horaire chronique a entrainé une désynchronisation majeure du pic préovulatoire de LH, persistant plusieurs semaines. Cette altération était associée à une modification de la transmission de l’information journalière de l’horloge principale aux neurones à kisspeptine qui régulent la sécrétion de LH. De plus, la capacité reproductive des souris était diminuée, mais sans effet majeur sur le développement de leur descendance
In female mammals, optimal fertility relies on the synchronization of neuroendocrine and behavioral events regulating reproductive function. To this end, the circadian timing system, entrained by the light-dark cycle, sets the pace for the hypothalamic-pituitary-ovarian axis. Therefore, irregular light-dark cycles, such as those experienced in shift work, can disrupt reproductive function and compromise fertility, especially in women. This research aimed to assess the effects of circadian disruption on female reproductive function and investigate the underlying neuroendocrine mechanisms. In female mice, exposure to a light-based shift work model led to a major desynchronization of the preovulatory LH surge, which persisted for several weeks. This disruption was associated with altered transmission of daily signals from the master circadian clock to kisspeptin neurons, which regulate LH secretion. Additionally, reproductive outcomes in mice were affected, though without any major impact on offspring development

The plant life cycle alternates the diploid sporophyte and the haploid gametophyte. The female gametophyte of flowering plants develops within the ovule, a specialized structure within the ovary, which gives rise to the seed after fertilization. Sexual reproduction in plants entails a series of developmental steps that culminate in the formation of the seed. The developing ovule protects the haploid female gametophyte, which is formed as the result of the megasporogenesis and megagametogenesis. Inside the female gametophyte, the two female gametic cells, the central and the egg cells, upon fertilization give rise to the seed endosperm and embryo respectively. During my PhD, I dissect the genetic and molecular networks controlling female gametophyte formation and differentiation. I employed a yeast one-hybrid approach to identify EC1.1 regulators; the EC1 genes are specifically expressed in the female gamete and they are required for gamete fusion, therefore they are good candidates for clarify how gamete differentiation occurs in Arabidopsis thaliana. Among the transcription factors isolated, we focused on SUPPRESSOR OF FRIGIDA4 (SUF4). In vivo and in vitro evidences support SUF4 capacity to regulate AtEC1.1, furthermore suf4 mutants show also a mild ec1 phenotype. Plant can produce progeny without sexual reproduction. One example is apomixis, where meiosis and fertilization of the egg by male gametes are by passed to result in the production of clonal progeny without a parental contribution. Apomixis is due to modifications of the sexual reproduction and it does not occur in the major crop species, but is found in many wild species like Poa pratensis and Brachiaria brizantha. The idea of this work is to study genes involved in apomixis in apomictic plants, and then studies the function in the model organism Arabidopsis thaliana. In Poa pratensis by the cDNA-AFLP technique several genes differentially expressed in apomictic and sexual genotypes have been isolated. During my PhD I characterized the Arabidopsis homologue of PpAPO1 (Poa pratensis APOSTART 1) that has been renamed AtAPO1. Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction.

The menopause is associated with a deficiency of reproductive hormones, and accompanied by a significant loss of bone mass. This bone loss is accelerated within the first five years post-menopause. Muscle strength at this time would have important clinical implications for correcting imbalance and preventing falls. The aim of the studies within this thesis were to 1) determine the rate and time course of force loss of the quadriceps muscle group over 12 months in three groups of women with varying hormonal status 2) establish the role of oestrogen in this weakness and 3) investigate the effectiveness of hormone replacement therapy (HRT) in maintaining muscle function. The reliability of an isokinetic dynamometer and a strain gauge assembly was examined initially to determine the inherent variability of muscle function assessment. Strength of the knee extensors measured on the isokinetic dynamometer was deemed reliable in middle-aged women, although at 1.05 rad/s more practice trials were needed to attain peak torque. Measurements of the knee flexors were highly variable. Maximal voluntary isometric contractions were repeatable using the strain gauge system, for both the knee extensors and first dorsal interosseus (FOI) muscle. There was greater variability in force production generated from electrically stimulated contractions. Maximal strength of the knee extensors declined by 9.3-4.6 and I0.3?3.1% (mean?SE) for dynamic (1.05 radls) and isometric strength respectively over 9 months in hypoestrogenic post-menopausal women. There were no changes at higher angular velocities, or for handgrip strength. These results support the role of reproductive hormones in influencing force production, which is further endorsed by the observation that females on HRT did not experience a reduction in strength over this time. The force loss was significant only when the post-menopausal and HRT group were compared (p < 0.05). The postmenopausal group were within I to 3 years past the menopause, the time period in which bone loss is rapid. This rapid loss of strength would therefore be expected to level out, similarly to bone. The menopause is an oestrogen-deficient and progesterone-deficient endocrinopathy. It is not possible to identify which hormone, if not both, is responsible for these observed changes in strength. To explore the relationship between acute changes in oestrogen and progesterone and strength, maximal force production of the quadriceps and first dorsal interosseus (FOI) was measured across the menstrual cycle. Maximal strength of the quadriceps was lowest prior to the surge in luteinizing hormone (LH) and reached its peak mid-luteal, a difference of 12.6?4.3% (mean?SE). These changes were significantly different (p < O.OS). From these results, there does not appear to be a role of unopposed oestrogen influencing force production but the pattern of strength changes implicates progesterone. There were no corresponding fluctuations in strength of the FOI, which remained relatively stable across the menstrual cycle. The contractility and fatigue resistance of the quadriceps did not differ significantly between any phase (p > O.OS).The difficulty in isolating oestrogen during the menstrual cycle does not render this a good model to assess its effects upon force production. Maximal strength and fatiguability of the FDI were examined in young women undergoing in vitro fertilisation (IVF) treatment when acute, massive changes in oestrogen are induced. There were no differences in muscle function of the FDI when assessed under very low or high oestrogen changes (p > O.05). The independent effects of oestrogen upon muscle function were not demonstrated here. Hormone replacement therapy is the most efficacious treatment for preventing menopausally-related bone loss. The results from the longitudinal study suggest that HRT confers protection against muscle weakness as a consequence of ovarian failure. Whether HRT maintains or restores strength was examined in the FDI of post-menopausal women (n=9). The oestrogen only and oestrogen-progestogen phases were compared with baseline measurements. A positive change in strength was observed, although this did not reach significance (p < O.1). The increase in strength (15.2±20.6%) between baseline and the oestrogen-progestogen phase of HRT corroborates the involvement of progesterone in determining muscle function. The findings suggest that the menopause is associated with a loss of strength, prevented by the administration of HRT. Oestrogen alone does not influence force production, although progesterone is implicated. This has important ramifications in hysterectomised women who are prescribed preparations containing oestrogen only.

Women are twice as likely as men to suffer from depression and anxiety disorders and have an increased risk of onset during periods associated with hormonal changes, such as the postpartum period and the menopausal transition. Furthermore, some women seem more sensitive to normal hormone fluctuations across the menstrual cycle, since approximately 3-5% suffers from premenstrual dysphoric disorder (PMDD). Why these disorders are so common in women has not been established but there is a probable involvement of the ovarian hormones. The aim of this thesis was to investigate the effect of the ovarian hormones on the female brain during different reproductive states using psychological tests known to affect brain activity in different ways. Paper one examined the effect of the ovarian hormones on prepulse inhibition (PPI) on the acoustic startle response (ASR) and comprised cycling women and postmenopausal women. The cycling women had lower levels of PPI compared to postmenopausal women and postmenopausal women with moderate estradiol levels had lower PPI compared to postmenopausal women with low estradiol levels. Paper two examined the effect of anticipation and affective modulation on the ASR in women with PMDD and healthy controls. Women with PMDD have an increased modulation during anticipation of affective pictures compared to healthy controls during the luteal phase of the menstrual cycle. Paper three examined brain activity during response inhibition among women with PMDD and healthy controls by the use of a Go/NoGo task and fMRI. Women with PMDD displayed a decreased activity in the left insula during follicular phase and an increased activity during the luteal phase compared to controls. Paper four comprised women in the postpartum period and non-pregnant controls to examine brain activity during response inhibition. While this study revealed decreased activity at 4 weeks postpartum compared to 48 hours postpartum we cannot ascertain the role of the ovarian steroids, since none of the significant brain areas correlated with ovarian steroid or neurosteroid serum concentrations. The results of this thesis demonstrate that the ovarian hormones, or at least various hormonal states, have a probable impact on how the female brain works.

Introduction Endocannabinoid system (ECS) includes lipid messengers termed endocannabinoids (eCBs), their receptor (CBRs) targets type-1 (CB1) and type-2 (CB2) cannabinoid receptors, G-protein coupled receptor 55 (GPR55), transient receptor potential vanilloid type 1 channel (TRPV1) and a number of metabolic enzymes. ECS has a key-role in virtually all steps of female reproduction. To date, among the 4 main receptors, only 2 receptors have been extensively studied in the mammalian oocytes, i.e. CB1 and CB2, both modulated during meiotic maturation. The aim of this thesis was (a) to determine expression levels of all these CBRs and (b) to investigate their role in the process of mouse oocyte meiotic maturation. Methods Adult CD1 female mice were primed with 5 IU PMSG and sacrificed: (a) 44 h later to obtain GV oocytes, (b) 8 or 12h after hCG (5 IU) injection to obtain MI or MII oocytes. CBRs mRNA and protein contents were assessed by qRT-PCR and Western blot; CBRs localization by confocal microscopy. CB1, CB2 and GPR55 roles during meiotic resumption (germinal vesicle breakdown, GVBD) or maturation up to MI and MII stages were assessed by incubating oocytes in the presence/absence of receptor antagonists (SR1, SR2, and ML193, respectively). cAMP concentration was assessed by EIA kit; MI/MII spindle morphology by appropriate immunofluorescent antibodies. Experiments were repeated at least 3 times. Results Despite a significant decrease of CB1, CB2 and GPR55 mRNAs occurring after GVBD, CB2 and GPR55 protein contents increased significantly from GV to MI and MII. At GV, only CB1 was localized in the oolemma, although it disappeared at MI. TRPV1 (mRNA/protein) was always undetectable. When oocytes were in vitro matured with CB1 and CB2 antagonists, a significant delay of GVBD was recorded, sustained by higher intraoocyte cAMP concentration. Although CBRs antagonists did not affect polar body I emission nor chromosome alignment at metaphase I or II plates, ML193 impaired the formation of normal MI or MII spindles in about 70% of oocytes. Indeed, ML193-incubated oocytes showed a significant reduction of spindle length as compared with control. Conclusion In mouse oocytes, all the major eCB-receptors are differentially expressed during meiotic maturation. CB1 and CB2 have a prominent role in the control of meiosis resumption, while GPR55 could be involved in the assembly of MI and MII spindles. These findings offer potentially novel biomarkers involved in the physiological maturation of mouse oocytes and could be a starting point for investigating female fertility issues also in women.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Introduction: Infertility, besides being a medical condition that deserves medical attention and treatment, is a disturbing development, with implications on various aspects of life of infertile couples and individuals (personal, relational, social and sexual). The impact of infertility on women's sexuality is not entirely clear. Studies that have investigated the topic reported contradictory results and methodological limitation. Objectives: • Review important aspects of female sexual function, including, in Brazil the prevalence, diagnosis and treatment. • Establish the risk of female sexual dysfunction in infertile couples. • Determine the prevalence of sexual dysfunction among infertile and fertile women and among women undergoing the techniques of low and high complexity. • Compare the dysfunctions in fertile and infertile women and in women subjected to low and high technical complexity. Methods: A literature review article, constructed from research on PubMed/Medline and SciELO databases between 1985 and 2012 was drafted. Then an original article where a study of the case-control was developed with 278 infertile participants met at the Laboratory of Huma n Reproduction, Hospital das Clínicas and fertile patients recruited at the Clinic of Gynecology in the same hospital, from March 2012 to September 2013. The case group consisted of 92 women with sexual dysfunction and a control group of 186 women without sexual dysfunction. The questionnaire Female Sexual Function Index (FSFI) Portuguese version, which assesses the domains desire, arousal, lubrication, orgasm, satisfaction and pain, was used. Data were collected through interviews after signing the WIC. Two controls per case were randomly selected. The odds ratio (OR) was calculated for chance of female sexual dysfunction in infertile couples (p ≤ 0.05). Results: In the literature, it is observed that female sexual dysfunction have a multifactorial etiology, prevalence ranged from 35.9 % to 49.0 % and is rarely studied in the Brazilian population. Infertile and fertile women have the same chance for sexual dysfunction (OR = 1.45, 95% CI 0.86 to 2.44, p = 0.20). The prevalence of sexual dysfunction in infertile women was 36.31 %, and the fertile women was 28.18 %. In women undergoing low technical complexity prevalence was 38.88 %, 34.37 % and high complexity. Desire and arousal were significantly lower in infertile women. No significant differences were observed in relation to sexual dysfunction in women subjected to the techniques of low and high complexity. Conclusions: The risk of infertile women experiencing sexual dysfunction is the same fertile women. There was no statistical difference regarding the prevalence in infertile women compared to fertile, and women undergoing fertilization of low complexity when compared to high complexity. The desire and arousal domains were the most affected in infertile women. No differences were observed in the areas in relation to the techniques of low and high complexity.
Introdução: A infertilidade, além de ser uma condição clínica que merece atenção médica e tratamento, é um acontecimento perturbador, com implicações em diversas dimensões da vida dos casais e indivíduos inférteis (pessoal, relacional, social e sexual). O impacto da infertilidade na sexualidade da mulher não está inteiramente claro. Os estudos que investigaram o tema apresentam resultados contraditórios e limitações metodológicas. Objetivos: • Revisar aspectos importantes sobre a função sexual feminina, incluindo, prevalência no Brasil, diagnóstico e tratamento. • Estabelecer o risco de disfunções sexuais femininas em casais inférteis. • Determinar a prevalência de disfunção sexual entre mulheres inférteis e férteis e entre mulheres submetidas às técnicas de baixa e alta complexidade. • Comparar as disfunções em mulheres férteis e inférteis e em mulheres submetidas às técnicas baixa e alta complexidade. Métodos: Foi redigido um artigo de revisão da literatura, construído a partir de pesquisa nas bases de dados PubMed/Medline e SciELO entre 1985 e 2012. Em seguida um artigo original onde um estudo do tipo caso-controle foi desenvolvido com 278 participantes inférteis atendidas no Laboratório de Reprodução Humana do Hospital das Clínicas e pacientes férteis recrutadas no Ambulatório de Ginecologia do mesmo hospital, no período de março de 2012 a setembro de 2013. O grupo caso foi composto por 92 mulheres com disfunção sexual e o grupo controle por 186 mulheres sem disfunção sexual. Foi utilizado o questionário Female Sexual Function Index (FSFI) versão em português, que avalia os domínios desejo, excitação, lubrificação, orgasmo, satisfação e dor. Os dados foram colhidos por entrevista após assinatura do TCLE. Dois controles por caso foram selecionados aleatoriamente. Foi calculado o odds ratio (OR) para chance de disfunção sexual feminina em casais inférteis (p ≤0,05). Resultados: Na revisão da literatura, observa-se que as disfunções sexuais femininas apresentam etiologia multifatorial, a prevalência pode variar de 35,9% a 49,0% e é pouco estudada na população brasileira. Mulheres inférteis e férteis apresentam a mesma chance para disfunção sexual (OR= 1,45; IC 95% 0,86–2,44; p= 0,20). A prevalência de disfunção sexual em mulheres inférteis foi de 36,31%, e nas mulheres férteis foi de 28,18%. Em mulheres submetidas à técnica de baixa complexidade a prevalência foi de 38,88%, e alta complexidade 34,37%. Desejo e excitação foram significativamente inferiores em mulheres inférteis. Não foram observadas diferenças significativas em relação às disfunções sexuais em mulheres submetidas às técnicas de baixa e alta complexidade. Conclusões: O risco de mulheres inférteis apresentarem disfunção sexual é o mesmo de mulheres férteis. Não houve diferença estatística em relação à prevalência em mulheres inférteis quando comparadas às férteis, e em mulheres submetidas à fertilização de baixa complexidade quando comparadas a alta complexidade. Os domínios desejo e excitação foram os mais comprometidos em mulheres inférteis. Não foram observadas diferenças nos domínios em relação às técnicas de baixa e alta complexidade.

Pendant de nombreuses années, les femelles ont été décrites comme passives dans les interactions mâles-femelles et ont parfois été négligées dans les études sur les comportements reproducteurs et la communication animale. Pourtant, il est de plus en plus évident que les comportements des femelles influencent ceux des mâles et que les mâles peuvent ajuster leurs comportements de parades aux comportements des femelles. L’objectif principal de cette thèse est de mieux comprendre les signaux que les femelles émettent lors d’interactions avec un mâle dans un contexte de reproduction chez le canari domestique. L’ensemble de mes résultats montre tout d’abord que les femelles utilisent des signaux de parades visuels et acoustiques, c’est-à-dire des postures de sollicitation à l’accouplement et des trilles-spécifiques de femelles, comme une invitation à s’accoupler, mais qu’elles peuvent aussi les utiliser pour inciter le mâle à parader et l’aider à échantillonner la qualité de partenaires potentiels. D’autre part, ces deux signaux n’auraient pas la même efficacité selon le contexte d’émission. Ensuite, il semblerait que les signaux véhiculés par la modalité visuelle pourraient jouer un rôle plus important qu’on ne le pensait jusque-là dans les interactions intersexuelles chez cette espèce. Enfin, cette thèse a aussi pu apporter de nouveaux éléments concernant les préférences des femelles pour les chants de mâles et a montré que les méthodes utilisées en laboratoire pour tester les préférences des femelles étaient fiables et congruentes. Cette thèse permet de contribuer aux recherches grandissantes mettant en évidence le rôle important de la femelle dans les interactions intersexuelles
Much studies on reproduction and animal communication have considered the female as the passive sex; the role of the female during male-female interactions have often been overlooked. However, there is growing evidence that female behaviours can affect those of the males and that males can adjust their courtships to female behaviours. The aim of this thesis is to understand the signals produced by females during interactions with a male in a reproductive context in the domestic canary. Overall, my results show that females not only use their visual and acoustic signals, the copulation solicitation display and the female-specific trills, as an invitation to copulate but also to incite male to sing as an aid to sample potential mates. Then, these two signals could not have the same efficacy in different contexts of transmission. Moreover, the visual components of the communication seem to be more important than previously thought during intersexual interactions in this species. Finally, this thesis provides new elements about the female preferences for male songs and shows that methods used to test female preferences in laboratory are reliable and congruent. This thesis contributes to the growing number of researches showing that females play an active role in intersexual interactions

博士
國立中山大學
生物科學系研究所
106
Sleep plays an important role in many physiological and psychological aspects of human endocrinology, carbohydrate metabolism, cardiovascular diseases, immune function and mental status. Sleep deprivation (SD) adversely affects female reproductive function. In this study, we explored the effect of lack of sleep on reproductive function using rat model of total sleep deprivation (TSD), and the in vitro cell culture of intact ovarian follicles of juvenile rats. The concentrations of serum estradiol, corticosterone and serotonin in rats were systemically measured. Mechanically dissected intact rat follicles were cultured in vitro for the evaluation of follicular development and steroidogenic functions. TSD led to a significant difference in serum estradiol concentrations between the treatment and the control groups. The serum serotonin and corticosterone concentrations were significantly elevated in groups with more than 2 days of SD. FSH induced an increase in both follicle size and follicular cell number, while follicular cell differentiation was accompanied by enhanced inhibin-α and connexin 43 expression. Preantral follicular growth was dose-dependently inhibited by betamethasone (BET; 0.001–1 µg/ml). Such inhibition led to a decrease in follicular cell numbers, the suppressed expression of a proliferating cell nuclear antigen, inhibin-α, and connexin 43. Increasing doses of serotonin could reduce the estradiol production from the large follicles cultured in a fixed FSH level (50 mIU/ml) by as much as 20%. Serotonin also attenuated the expression of FSH-stimulated follicular steroidogenic acute regulatory protein (StAR) but did not affect the follicular cell proliferation. These findings supported the notion that TSD retards the follicular development by overstimulating the ovaries with elevated corticosterone. The reduced serum level of estradiol in TSD rats is likely caused by serotonin-mediated inhibition of estradiol production and the expression of StAR protein in the follicles.

Due to their phylogenetic position as an outgroup to humans and the other African apes, empirical data from orangutans are an especially valuable comparative tool with which questions about the evolution of human life history and reproductive characteristics can be addressed. Yet few such data are available. In this dissertation, I use endocrinological and behavioral data from 7 female and 3 male orangutans housed at the Woodland Park Zoo in Washington and the Great Ape Trust in Iowa to characterize the ovarian function and reproductive behaviors of captive female orangutans at different points in the life cycle. Ovarian hormone measurements were achieved through the use of non-invasive urine sampling, and assays reveal both intra- and inter-individual variation in hormone production. Results indicate that (1) adolescent females in captivity do not experience a marked period of subfecund estrogen and progesterone levels in association with reproductive maturation, (2) individual females exhibit both "high quality" and "low quality" cycles, including instances of anovulation, in the absence of fluctuating dietary and environmental conditions, (3) mating behaviors vary between individuals and with cycle phase, but are not strongly influenced by absolute ovarian hormone concentrations, and (4) reproductive senescence does not significantly impact the ovarian function and mating behaviors of aging female orangutans. These results demonstrate that many aspects of human reproductive biology and behavior, such as an extended period of mating receptivity, are evolutionarily conserved. They suggest, however, that the decline in human ovarian function in mid-life may be derived and of possible adaptive significance. The potential significance of differences between captive and wild ape populations, and the character, history, and familial relationships of the particular individuals discussed are considered in the interpretation of all data.

The goal of the current research was to examine the expression, signaling and function of the membrane progestin receptors (mPRs) in the ovarian follicular cells of the Atlantic croaker (Micropogonias undulatus) and in human breast cancer cells. Multiple studies have examined the role of mPRs in the germ cells of several vertebrate classes, yet few studies have examined the role of the mPRs in the somatic cells of reproductive tissues. Therefore this research examines the mechanism of mPR action and its function in somatic cells of female reproductive tissues. Results from studies on the expression, localization and signaling of the mPR[alpha] in co-cultures of granulosa and theca cells from the croaker suggest that the mPR[alpha] is localized to the plasma membrane of both cell types and that the mPR[alpha] is associated with and signals via pertussis toxin-sensitive inhibitory G proteins to decrease intracellular cAMP and activate ERK. In addition, exposure of follicular co-cultures to progestins that activate the mPR[alpha] results in a decrease in serum starvation-induced cell death which is not replicated by progestins which activate the nuclear progestin receptor (nPR), indicating mPR mediation. Similar studies in two immortalized human breast cancer cell lines, MDA-MB-468 and SKBR3, suggest that the mPR[alpha] is also present in the membranes of these cells and signals in human breast cancer cell lines via activation of a pertussis toxin-sensitive G protein to significantly decrease in intracellular cAMP and activate ERK. Progesterone exposure also decreased serum starvation-induced cell death in SKBR3 cells which are nPR positive and in MDA-MB-468 cells which are nPR negative. Synthetic progestins which activate the nPR but not the mPR were ineffective in inhibiting death in either cell type suggesting that the mPR is the mediator of this progestin action. mPR[alpha], mPR[beta] and mPR[gamma] expression analysis of paired normal and malignant breast tissue biopsies from thirteen women revealed that at least one mPR isoform was upregulated in the malignant tissue of 70% of the women. In addition the expression of mPR[gamma] was positively correlated with the expression of the nPR and CK19, a breast epithelial cell marker.
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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.

No
Rationale: Weight gain caused by some antipsychotics is not only confined to adults but can also adversely affect both children and adolescents. Indeed, olanzapine and risperidone have been associated with extreme weight gain in adolescents even greater than that reported in adults. We have recently shown substantial weight gain in adult female rats following treatment with olanzapine and risperidone but not ziprasidone. Objectives: The aim of the present study was to compare the effects of several antipsychotics on weight gain and reproductive function in juvenile (aged 7 weeks) female hooded Lister rats. Methods: Olanzapine (4 mg/kg), risperidone (0.5 mg/kg), ziprasidone (2.5 mg/kg), sulpiride (10 mg/kg), haloperidol (0.5 mg/kg) or vehicle was administered i.p. once per day for 21 days. Body weight, food and water intake were measured daily, in addition to the determination of stage of the oestrous cycle. Results: Sub-chronic administration of olanzapine, risperidone, sulpiride and haloperidol, but not ziprasidone, significantly increased body weight compared to vehicle-treated animals during weeks 1-3. Sulpiride significantly increased food and water intake. Significantly increased percentage intra-abdominal fat weight was observed in olanzapine, risperidone, sulpiride and haloperidol, but not ziprasidone-treated animals. Marked disruption of the oestrous cycle was observed in all but the ziprasidone-treated group, which continued to have regular 4-day oestrous cycles. Conclusions: Weight gain observed in these juvenile animals was 1.5-2 times greater than that previously observed in adult rats. These findings have important implications for the use of antipsychotics in children and adolescent patients.

博士
國立屏東科技大學
環境工程與科學系所
106
This study is composed of two parts which are related to the residues of persistent organic pollutants (POPs) in human breast milk in Southern Taiwan and their possible associations with female reproductive function (i.e. infertility, gynecological diseases, and menstruation characteristics) and their correlation with sociodemographic parameters (i.e. age, pre-pregnant body mass index (BMI), annual incomes, population, birth year, and parity) and dietary habits. In the Part I of this study, the congener-specific concentrations of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polybrominated dibenzo-p-dioxins/furans (PBDD/Fs), and polybrominated diphenyl ethers (PBDEs) in 25 breast milk samples from southern Taiwan were investigated. It was found that most PCDD/F and PBDE congeners in the 25 samples were detectable. However, the concentrations of PBDD/F congeners were below method detection limits (MDLs). The geometric means of PCDD/Fs and PBDEs in the breast milk are 2.44 pg WHO2005-TEQ/g lipid and 2810 pg/g lipid, respectively. Several PCDD/F and PBDE congeners were highly correlated to each other, like 1,2,3,7,8-PeCDD and 2,3,4,7,8-PeCDF (r = 0.919, p < 0.001). Moreover, the longest duration of menstruation could be predicted by the congeners BDE-153 (β = 0.252) and 1,2,3,4,6,7,8-HpCDF (β = 0.345) with adjustment of confounders using a multiple stepwise linear regression model (r = 0.963, p < 0.001). In the Part II of this study, 68 breast milk samples from Southern Taiwan were analyzed for organochlorine pesticides (OCPs). It was found that the most abundant OCP residues in the breast milk were ƩDDT followed by ƩHCH, whose geometric means ± standard deviations were 9.81 ± 7.52 ng/g lipid and 0.539 ± 0.557 ng/g lipid, respectively. Additionally, cis-chlordane (cis-CHL) and γ-HCH were related to participants who received medical treatment for infertility, while 4,4’-DDT was associated with those who received gynecological surgery. The logistic regression showed that the odds ratio (OR) of log γ-hexachlorocyclohexane (γ-HCH) was higher for mothers who had received medical treatment for infertility than for the normal group (OR = 25.6, p = 0.035) after adjustments for age, pre-pregnant BMI, annual income, population (i.e., native-born Taiwanese), birth year, and parity. Cow milk and beef consumption as well as menstruation characteristics such as average menstrual period (>5 days), shortest menstrual period (<3 days), and women who had taken hormonal drugs were significantly associated with several OCP residues in the breast milk. In addition, ƩHCH including β-HCH and γ-HCH was correlated with annual family income, gravidity, and cow milk and beef consumptions. γ-HCH exhibited a probable association with the infertility diseases of Taiwanese women, and dietary habit might play an important role in the female Taiwanese exposure to OCPs. Overall, the findings in the present study support that PBDEs, PCDD/Fs, and OCPs have adverse health effects on female reproductive function.

Preterm birth is the leading cause of newborn mortality, with 15 million babies born premature worldwide every year. Children that do survive early delivery are more likely to develop cognitive abnormalities, motor deficits, heart disease, cerebral palsy, and more. While little is known about the pathophysiology of preterm birth, several pregnancy-related complications are related to preterm birth, namely cervical insufficiency and preeclampsia. In the former, premature cervical remodeling and softening can result in the shortening of the cervix, increasing a woman’s risk of preterm birth; this condition is called cervical insufficiency (CI), which is the inability of the cervix to remain closed as a result of weakened tissues. CI is currently measured by a one-dimensional sonographic cervical length, where < 25 mm indicates shortening. Preeclampsia is a disorder that can be explained through the Page kidney phenomenon: compression of the left renal vein (LRV) causes renal venous outflow obstruction, leading to elevated intrarenal pressure and hypertension. The supine pressor test (SPT) is a diagnostic tool for preeclampsia where a positive test is defined by an increase of 20 mmHg in diastolic blood pressure (BP) when shifting from the left lateral recumbent to the supine position. Due to the intense risk of morbidity and mortality for both the mother and the fetus, the need to monitor BP changes is critical. Currently, there is an unmet clinical need to characterize the hemodynamic and geometric properties of the female reproductive organs throughout gestation. Utilizing ultrasound imaging can increase our knowledge about the 3D anatomy and systemic changes during pregnancy, unravel risk factors, establish preventative methods, and standardize treatment plans. In this thesis research, we developed a murine model to 1) examine the pathophysiology of renal vein stenosis, and 2) investigate the effects of stenosis on various cervical dimensions. Renal vein stenosis was found to greatly impact blood flow velocities, as well as cervical width (p<0.05). LRV and cervical area and height also trend towards significance, and there is negative damage to the left kidney and placentae within the stenosed cohort. We also conducted a human study that showed reduced change in postural BP in patients with higher body mass index (BMI). Systolic and diastolic BP in the supine position was significantly greater than in the lateral position for all BMIs with a baseline increase in BP of approximately 9-14 mmHg. These findings suggest that therapeutic positioning and close monitoring of BP could mitigate the risk of developing related disorders in pregnancy.

碩士
臺北醫學大學
醫學科學研究所
96
Guanylyl cyclase(GC) is known to transmit signaling by synthesizing of intracellular cyclic GMP. We have previously demonstrated an orphan GC receptor on mouse sperm (mouse GC-G, mGC-G) which is able to regulate sperm motility and capacitation-associated protein tyrosine phosphorylation(2006 Endocrinology). To further examine the role of mGC-G in female reproductive system, we detected the expression and localization of mGC-G in female reproductive organs. Reverse transcriptase(RT)-PCR and immunostaining analysis revealed that mouse GC-G is expressed in ovary (granulosa cells and ooplasm), oviduct, and endometrium. In granulosa cells, the mGC-G receptor was expressed on the cell surface by confocal image analysis. Besides, the mGC-G receptor was immunostained at oviductal epithelial cells, and endometrium in all estrus stages, including proestrus, estrus, metestrus, and diestrus. The expression of mGC-G in endometrium is estrogen-dependent by utilizing ovariectomized female mouse model. This result hints the upstream regulation of mGC-G on the function of granulosa cells. Given the specific homo-extracellular domain (ECD) interaction of mGC-G receptor has been demonstrated(unpublished data), the mGC-G-ECD-Fc recombinant protein is used to interact with the granulosa cells. cGMP assay demonstrated the activity of mGC-G-ECD-Fc recombinant protein to elevate the intracellular cGMP level and decrease aromatase (Cyp19) gene expression in granulosa cells. In summary, here we demonstrated the expression of mGC-G in female reproductive system, and the mGC-G may play a role in estrogen production through its ECD-homophilic interaction and cGMP elevation in granulosa cells.

Human tissue kallikrein-related peptidases (KLK) are fifteen genes located on chromosome 19q13.4, encoding hormonally regulated, secreted serine proteases with trypsin/chymotrypsin-like activity. I identified expression of many KLKs in tissues throughout the female reproductive system and in cervico-vaginal fluid (CVF). The female reproductive system is hormonally regulated during the menstrual cycle, suggesting KLKs may also be regulated by these hormones. Measurement of KLKs levels in CVF and saliva samples throughout the menstrual cycle revealed a peak in expression following ovulation in both fluids. Progesterone levels rise during this period suggesting KLK regulation by progesterone during the menstrual cycle. Using proteomic techniques, I resolved the CVF proteome to identify potential KLK substrates. Among 685 proteins identified, several cell-cell adhesion molecules, cervical mucins and defense-related proteins were found. KLKs play a role in the desquamation of skin corneocytes through cleavage of cell-cell adhesion proteins. The vaginal epithelium undergoes cyclical changes during the menstrual cycle involving desquamation of cells upon rising progesterone levels. The post-ovulatory peak in KLK expression suggests that KLKs may contribute to cell desquamation during the menstrual cycle. Cervical mucus acts to block the uterus from vaginal microorganisms. Around ovulation, cervical mucus loses viscosity to facilitate sperm passage through the cervix. Proteolytic enzymes are thought to aid in this mucus remodelling. Our immunohistochemical studies localized KLK expression to the mucus secreting cervical epithelium and I investigated KLK processing of cervical mucin proteins in vitro. KLKs 5 and 12 were found to cleave mucins, suggesting their potential involvement in cervical mucus remodelling. CVF plays a role in host defense. KLKs are known to process the antimicrobial cathelicidin protein in skin and I investigated whether KLKs may also process antimicrobial proteins found in CVF. KLK5 was found to cleave defensin-1 alpha, in vitro, suggesting KLKs may aid in defense of the female reproductive system. Here I provide evidence of potential physiological roles for KLKs in the female reproductive system: in desquamation of vaginal epithelial cells, remodelling of cervical mucus and processing of antimicrobial proteins. These findings suggest KLKs may function in female fertility, in pathological conditions such as vaginitis and in host defense.