Androgens and the ovary

Between 10-15% of women suffer from polycystic ovary syndrome (PCOS), making it the most common cause of female infertility. Clinical features of PCOS include high circulating levels of ovarian androgens (T and A4), anovulation and obesity. The aetiology of this reproductive endocrinopathy is likely to be multifactorial, through the interplay of genetics, epigenetics and environmental factors. Primate research into sexual behaviour development noted that fetally androgenised monkeys developed symptoms like those of PCOS. There are now multiple animal models of PCOS using primates, sheep, rats and transgenic mice. The investigations described in this thesis use rodent models to examine the role of androgens in the pathogenesis of female infertility. An attempt to generate a granulosa cell specific androgen receptor knockout mouse model will first be described, followed by several studies into the developmental programming of female Wistar rat infertility and metabolism by steroid hormones. Initial investigations showed that testosterone proprionate (TP) administered to female rats during different windows of fetal and neonatal life alters the reproductive and metabolic axes of the adult animals. Fetal plus neonatal TP exposure led to complete ovarian dysgenesis, while postnatal exposure produced a PCOS-like phenotype. Animals which received TP postnatally were heavier and had an increased proportion of primordial follicles in their ovaries by postnatal day (pnd) 90 of life. Evaluation of this PCOS model showed that neonatally androgenised rats had ovarian follicles with larger antra and a greater ovarian stromal compartment. In addition, these animals were heavier when compared to controls. However, unlike human studies, neonatally androgenised rats showed no differences in circulating gonadotrophin or ovarian androgen levels. Nor did they show any programming effect of neonatal TP upon the theca interna by pnd 90. Further investigations to narrow the windows and dose of TP required to produce a PCOS phenotype showed that TP administered in an early window of neonatal life, between postnatal days (pnd) 1-6 not only led to anovulation, but potentially reprogrammed the hypothalamic-pituitary axis, as there was minimal gonadotrophin response to reduced ovarian negative feedback (inhibin B and estradiol) in these rats. Neonatal TP also affected the rat metabolic axis with adult animals becoming heavier after weaning without any change in food intake. Animals developed mesenteric and retroperitoneal obesity along with insulin resistance (IR). Increased hepatic glucocorticoid turnover and altered adipokine expression were also noted in neonatally androgenised females, possibly contributing to the pathogenesis of obesity. No effect of TP dose upon the severity of infertility or metabolic abnormalities in adult animals was observed. To delineate which features of the rat PCOS model resulted from androgenic, estrogenic or corticosteroid action, a final study used administration of different steroids during the early window of postnatal life: TP, estradiol valerate (EV), dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA) and dexamethasone (DEX). The anovulatory PCO-like phenotype observed with TP was also seen in animals which received EV, but not those which received DHT, DHEA or DEX. TP and EV treatment also resulted in a reduction of ovarian follicle numbers and activated follicle proportions, with an increase in primordial follicle proportions. Although glucose tolerant, animals treated with TP and EV were highly IR. Unlike dexamethasone, DHT and DHEA also produced IR in adult animals, to a lesser extent than TP and EV. Taken collectively, the results described in this thesis demonstrate that the PCOS-like phenotype observed in the neonatally androgenised female rat is likely to be due to the estrogenic actions of testosterone, potentially through as yet unknown epigenetic mechanisms. The programming of the metabolic components described may additionally be due to the actions of androgens. Furthermore, these studies demonstrate a novel estrogenic effect of neonatal steroids upon primordial follicle populations and show that the neonatally androgenised rat may be a rational PCOS model in a poly-ovulatory species

An analysis of the proliferative response of oral keratinocytes during the re-epithelialization of a chemically induced wound

The wound healing response requires the interaction of a series of events, culminating in the restoration of tissue integrity. In the present study, the proliferative phase of this process was examined with particular reference to re-epithelialization of the wound defect and the manner in which epithelial keratinocytes respond. PC-lO (Dako), a mouse monoclonal antibody against proliferating cell nuclear antigen, was employed as a marker of cell division. This 36kDa auxilliary protein 2 for DNA polymerase-delta is essential for DNA replication at S-phase and has been employed with varying success as a marker of cell proliferation. The majority of work utilizing PCNA to evaluate proliferative activity has been performed on histopathological tissue. Studies examining PCNA in non-neoplastic tissue, and in particular, oral epithelium, are limited. The proliferative status of tongue for instance, one of the more readily accessible regions of the oral cavity, has been determined using analagous techniques, however there is a paucity of information regarding the application of PCNA. In fact, research by Warnakulasuriya and Johnson (1993) investigating cell proliferation in hamster tongue appears to be the only equivalent study. Consequently, in the present investigation the performance of PCNA was examined in both normal and wounded rat tongue. An animal model, adapted from unpublished work by Shaw and Young (1983), was established and involved the subepithelial injection of 35ul of the sclerosant sodium tetradecyl sulphate (STS) into the right ventrolateral aspect of the tongue of anaesthetised animals using a Hamilton microsyringe. The resulting wound was assessed at two, four, seven and fourteen days subsequent to the STS injection. The excised tongues were microwave irradiated in Kryofix (Merck) and submitted for immunostaining with PC-lO (Dako). Detection of the incorporated antibody was performed using an indirect PAP technique. Stained sections were quantified using a "chromatic" image analysis system to determine the proliferative activity in the healing epithelium . A PCNA labelling index, expressed as the number of PCNA-stained cells per 100 epithelial basal cells, was calculated for the dorsal, ventral and total epithelial surface. Each experimental wound was mapped to indicate the spatial distribution of proliferating cells, as determined by PCNA with regard to the wound margin. The results of this study showed that epithelial regeneration of the STS induced wound began within forty eight hours and confluence was achieved fourteen days following the injection. PCNA labelling indices for the dorsal, ventral and total basal epithelial surfaces of normal rat tongue, expressed as the mean :- SD, were 23: 6.3, 21 t 16.7 and 22 i 9.1 respectively. These findings reflect the more complex proliferative pattern that occurs in dorsal epithelium to maintain the undulating rete ridge pattern. Comparison of the PCNA indices for the four experimental groups with those obtained for normal rat tongue demonstrated that maximal epithelial activity occurred at seven days post-wounding in the dorsal surface and four days post-wounding in the ventral surface. Quantitative analysis of the PCNA labelling combined with microscopic evaluation also enabled a general sequence of healing to be established, based on proliferative activity. From these data, a number of events known to occur during the reparative process could be identified, namely the lag phase, a period of peak proliferative activity and an interval of maximal migratory activity. lmmunohistochemical detection of PCNA also revealed that following wounding, oral keratinocytes undergo cell division in a regular temporal distribution rather than in random order. The findings of this investigation suggest that the STS animal model is reproducible and results in a slow healing wound that allows various aspects of the reparative process to be considered. The STS model, when used in conjunction with the methods employed for the quantitative assessment of PCNA, may have utility in wound healing studies. In particular, this procedure would be of benefit in evaluating the contribution of proliferation to the reparative process

Histološka različitost u koštanoj biopsiji postmenopauzalnih i osteoporotičnih žena

Many investigations have been carried out on osteoporosis and showed this disorder in a new light. The reduction in bone structure and dynamics points to the etiology that may be of therapeutic significance. Today we still do not know everything about the histomorphometric characteristics of each metabolic disturbance and bone disease. Bone biopsy and histomorphometry are not available for most patients and that is the reason why a broad classification of these reductions in osteoporosis and osteomalacia are widely accepted in clinical practice. New findings from this complex domain are necessary to design a strategy in the evolution of new therapeutic devices and drugs with the intention to decrease the high disability and mortality rate in the vulnerable population of postmenopausal women.Brojna istraživanja provedena posljednjih godina pokazala su osteoporozu u novom svjetlu. Promjene u koštanoj strukturi i dinamici upućuju na etiologiju poremećaja, što je od presudnog značenja za liječenje bolesti. S obzirom na činjenicu da još uvijek malo znamo o histomorfometrijskim značajkama pojedinih metaboličnih bolesti te da je biopsija kosti i histomorfometrija još uvijek teže dostupna većem broju bolesnika, danas se u kliničkoj praksi uglavnom rabi klinička klasifikacija po kojoj se one dijele u dvije široke kategorije: osteoporozu i osteomalaciju. Nova saznanja iz ovoga područja su od presudne važnosti za planiranje buduće strategije razvoja novih terapijskih sredstava i lijekova kako bi se smanjio visok postotak invaliditeta i smrtnosti u osjetljivoj populaciji postmenopauzalnih žena

Utjecaj morfološkog tipa stanica na ekspresiju bcl-2, Ki-67 i p-53 u uvealnim melanomima

Uveal melanomas are known to be less aggressive malignant tumors compared with melanoma of other localizations but the same morphological characteristics. We hypothesized that immunohistochemical determination of expression of the known proliferation markers bcl-2 and ki-67, and p-53 as a suppressor gene marker, could better explain the biological behavior of uveal melanoma vs. melanoma of other localizations. Thirty cases of primary uveal melanoma of two levels of invasion were tested retrospectively for the presence of bcl-2, Ki-67 and p-53 proteins with the indirect peroxidase-antiperoxidase immunoenzyme method and three step ABC/AP method. The intensity of reaction was assessed by semiquantiative method.Usprkos morfološkoj građi sličnoj drugim melanomima, poznato je da uvealni melanomi imaju daleko bolju prognozu od melanoma drugih lokalizacija. Upravo stoga pretpostavili smo da bi određivanje ekspresije bcl-2, Ki-67 kao poznatih čimbenika proliferacije, te p-53 proteina u stanicama uvealnih melanoma moglo protumačiti ovakvo biološko ponašanje uvealnih melanoma. U tu svrhu ispitano je retrogradno 30 primarnih uvealnih melanoma stadija pT2 i pT3 na prisutnost bcl-2, Ki-67 i p-53 proteina indirektnom metodom peroksidaze-anti-peroksidaze i trofaznom metodom ABC/AP. Stupanj reaktivnosti određen je semikvantitativnom metodom