Lic.-avh. (sammanfattning) Umeå : University. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 108 pages. Includes Vita. Includes bibliographical references.

Mammalian ovulation is a complex, hormone-dependent developmental program in which several events must take place in an ordered progression to ensure that the oocyte is competent for fertilization. This process requires the coordinated expression of many genes which must be turned on and off in the right place at the right time for proper development of the follicle. While the hormone signals from the brain that initiate ovulation are known, the master control genes which regulate this process are not well known. Homeobox proteins are potential candidates to perform as master regulators. Homeobox proteins are DNA-binding proteins that regulate the transcription of downstream genes and thereby control biological events. We recently identified a new homeobox gene cluster on the mouse X chromosome that are only expressed in reproductive tissues. These reproductive homeobox (Rhox) homeobox genes are expressed in the ovary, placenta, testis, and epididymis, and thus are good candidates to regulate both male and female reproductive tissue development and physiology. Rhox gene expression fell into three categories: Class I exhibited peak expression prior to ovulation (0-8 hours after hCG), Class II were predominantly expressed during ovulation (8-16 hours after hCG), and Class III peaked after ovulation. The slightly overlapping windows of peak Rhox gene expression suggest that these genes may regulate specific events during the ovulatory cycle. The founding member of the cluster, Rhox5, is highly expressed in granulosa cells of pre-ovulatory follicles. We previously reported that Rhox5-null female mice are viable and fertile, suggesting that RHOX5 is either not essential for ovulation, or that one of the other RHOX factors may compensate functionally in granulosa cells. In order to identify potentially redundant RHOX factors, we examined the expression patterns of all 32 Rhox genes using an eCG primed, hCG induced superovulation model, in wild-type, Rhox5-null, and heterozygous littermate mice. Expression levels of Rhox1, exhibited peak expression prior to being hormonal primed, was reduced in the Rhox5-null animal. However, Rhox8 mRNA and protein were reduced at 2h and 4h post hCG, but recovered once the follicles passed the antral stage of development. Conversely, in progesterone receptor knockout mice (PRKO), Rhox8 exhibited normal stimulation by eCG, but failed to reach its peak mRNA level at 8h post-hCG found in WT mice. This suggests a model in which Rhox8 transcription is dependent upon RHOX5 during early folliculogenesis and progesterone during the periovulatory window when RHOX5 normally wanes. Subsequent promoter analysis in granulosa cells revealed essential homeobox binding and progesterone response elements within Rhox8's 5'-flanking region. Transfection of RHOX5 and PGR expression plasmids stimulated, whereas dominant negative and mutant constructs inhibited, Rhox8 promoter activity. At present, the specific impact of misregulation of Rhox5 and Rhox8 during early folliculogenesis is not known. However, follicle counts from serially sectioned ovaries, extirpated from normal cycling animals, indicated that Rhox5-null mice possess ~50% fewer follicles than heterozygous littermates. Loss of RHOX5 in Sertoli cells results in male subfertility characterized by poor germ cell survival due in part to the misregulation of metabolism promoting genes. One of these genes, Ins2, is also stimulated by RHOX5 and RHOX8 in granulosa cells, suggesting impaired insulin signaling may contribute reduced follicle development in Rhox5-null ovaries.

Programmed cell death (PCD) and removal of cell corpses are important processes in animal development and homeostasis. Typically, engulfment of cell corpses is performed by professional phagocytes, such as macrophages. In tissues with limited accessibility to circulating cells, engulfment is carried out by neighboring non-professional phagocytes, such as epithelial cells. Compared to professional phagocytosis, the mechanisms that govern non-professional phagocytosis are not well characterized. The Drosophila ovary provides a powerful in vivo model for the study of PCD and engulfment by non-professional phagocytes. This dissertation identifies genetic pathways that govern non-professional phagocytosis during starvation-induced PCD and elucidates the major mechanism promoting non-apoptotic developmental PCD. During mid-oogenesis, germline nurse cells can be induced to die by starvation and their remnants are engulfed by surrounding epithelial follicle cells. We show that the engulfment receptor Draper is enriched and required in engulfing follicle cells. Additionally, we demonstrate that the JNK pathway is activated by Draper and required in engulfing follicle cells. Overexpression of Draper or the JNK pathway is sufficient to induce death of the germline, suggesting that there is coordination between the germline and follicular epithelium to promote cell death. Furthermore, activation of JNK bypasses the need for Draper in engulfment. These data demonstrate that JNK and Draper are crucial regulators of engulfment by non-professional phagocytes. During late oogenesis, nurse cells transfer their contents into the oocyte and undergo developmental PCD. Disruption of apoptosis or autophagy only partially inhibits PCD, indicating that other mechanisms contribute to the process. We demonstrate that the large-scale non-apoptotic developmental PCD in the Drosophila ovary occurs by a novel cell death program where follicle cells non-autonomously promote death of the germline. The phagocytic machinery of follicle cells, including Draper, JNK, and Ced-12, is essential for death and removal of nurse cells. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation are impaired when phagocytosis genes are inhibited. Moreover, elimination of a subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of non-apoptotic, non-autonomous PCD, providing insight on the diversity of cell death mechanisms.

Polycystic ovary syndrome (PCOS) is one of the most common and complex endocrinopathies among reproductive-aged women. Symptom heterogeneity among clinical cases and in sibling studies suggests that PCOS is a multifactorial disease with environmental components. Investigators hypothesize that endocrine disruptors may contribute to the pathophysiological origins of PCOS in utero. The goal of this literature review is to present and compare the available research investigating the developmental origins of PCOS deriving from prenatal exposure to three major classes of endocrine disruptors: bisphenol A (BPA), phthalates, and androgenic endocrine disruptors. Considerable evidence has been found to suggest links between fetal exposure to endocrine disruptors and PCOS. Rodent studies reported that maternal BPA exposure dysregulates postnatal development and sexual maturation. In rats, gestational exposure to dibutyl phthalate (DBP) and di(2-ethylhexyl)phthalate (DEHP) resulted in polycystic ovaries among first and third generation offspring. Additionally, serum DBP concentrations are higher among women with PCOS. Regarding DEHP, animal studies have shown that prenatal exposure results in an endocrinological profile similar to PCOS. Nicotine and 3,4,4'-trichlorocarbanilide (TCC) are known androgenic endocrine disruptors. Inducing prenatal exposure to excess androgens in animal models has successfully recreated a PCOS-like phenotype that includes abnormal ovarian function, anovulatory cycling, impaired fertility, and increased visceral fat distribution. Based on the literature, endocrine disruptors appear to have an etiological role in PCOS development through prenatal exposure and thus may pose one of the greatest hazards to fetal health and development.

Polycystic ovary syndrome is a complex, heterogeneous disease that affects 5-10% of reproductive-aged women. It is characterized by clinical or biochemical hyperandrogenism, oligo-anovulation, and polycystic ovary morphology. Instigating endocrine findings include aberrantly rapid gonadotropin-releasing hormone pulsatile secretion, elevated luteinizing hormone, sub-optimal levels of follicle-stimulating hormone, and hyperandrogenism. Metabolic symptoms are also present including hyperinsulinemia, insulin resistance, and dyslipidemia. These endocrine and metabolic findings are also accompanied by ovarian dysfunction and improper folliculogenesis. Because aberrant functioning of the hypothalamic-pituitary-gonadal axis is central to the pathophysiology of polycystic ovary syndrome, it is beneficial to examine it for abnormalities. Polycystic ovary syndrome has been shown to have both genetic and environmental components. Its strong genetic component has been demonstrated in twin studies, family association studies, candidate gene studies, and genome-wide association studies. Previous genome-wide association studies have found many candidate genes including those for DENND1A (differentially expressed in normal and neoplastic cells domain containing 1A), THADA (thyroid adenoma-associated protein), FSHR (follicle-stimulating hormone receptor), and LHR (luteinizing hormone receptor). This, together with the central endocrine abnormalities, prompted this review on polymorphisms of receptors of the hypothalamic-pituitary-gonadal axis, including those for gonadotropin-releasing hormone, luteinzing hormone, follicle-stimulating hormone, estrogen, and progesterone, as well as anti-müllerian hormone. Studies on single-nucleotide polymorphisms of these receptors were found on PubMed, Web of Science, and Google Scholar and subsequently analyzed. Many different single-nucleotide polymorphisms were studied, but only a handful of them have been subjected to repeated studies. Only rs2293275 of the luteinizing hormone receptor and rs2349415 of the follicle-stimulating hormone receptor, both at 2p16.3, were found to have a possible role in the etiology of polycystic ovary syndrome, but all eight were found to have a possible phenotypic role: rs13405728, rs2293275, and rs4539842 of the luteinizing hormone receptor; rs6165, rs6166, rs2268361, and rs2349415 of the follicle-stimulating hormone receptor; and rs2002555 of the anti-müllerian hormone receptor. The limitations most affecting the results of these studies include small sample sizes, heterogeneous study populations, lack of generalizability due to ethnicity, and lack of control or adjustment for confounders. It is necessary to develop a standardized study protocol and separate polycystic ovary syndrome patients based on phenotype in order to obtain stronger results in the future.

Cell death is an integral part of oogenesis in the fruit fly, Drosophila melanogaster. When the fly is starved of protein, some pre-vitellogenic egg chambers die apoptotically. As egg chambers mature, excess germline cells die via a non-apoptotic, developmentally programmed death. Overexpression of the transcription factor escargot was found to block both death events in the ovary, which is very unusual. escargot overexpression blocked starvation-dependent death upstream of caspases, but still needed a death signal to produce undead egg chambers. In maturing egg chambers, escargot overexpression blocked death more effectively than disrupting both apoptosis and autophagy, indicating that it must affect non-apoptotic, non-autophagic death mechanisms. RNA-Seq and a genetic modifier screen were used to identify potential escargot targets that inhibit cell death. Studies were also undertaken to characterize the loss-of-function phenotype of escargot in the ovary. escargot is a member of the Snail family of transcription factors that play integral roles in development and gene regulation throughout Bilaterian organisms. In Drosophila melanogaster, the genes snail, escargot, and worniu are critical for stem cells in neuroblasts, gut, and testis, but a role in the ovary had not been shown. To analyze Snail family function in the ovary, I made a triple deficiency that removed the three Snail family members, called ΔSF. Surprisingly, ΔSF homozygous follicle stem cells are rapidly lost. Follicle stem cell loss was rescued by the expression of escargot or worniu but not snail, indicating that there is shared capability between genes. Moreover, follicle stem cells did not linger in the germarium, and their loss was not prevented by blocking apoptosis, indicating that the ΔSF defect is a failure of stem cell maintenance. Together, the results described in this dissertation show that Snail genes are needed for the normal function of the Drosophila ovary, and that escargot can regulate multiple kinds of cell death. Understanding Snail family genes is particularly important for the study of cancer, as they are implicated in mechanisms underlying the cancer stem cell state. Analysis of the highly conserved Snail family genes in Drosophila illuminates their function and dysfunction in human health and disease.

Objetivo: Investigar a influência do tamanho dos fragmentos ovarianos na preservação dos folículos primordiais e primários pós-congelamento. Desenho: Experimento laboratorial controlado. Foram realizadas análises histológicas comparativas em grupos controle e pós-congelamento de fragmentos de ovários bovinos. No grupo dos fragmentos menores, a menor das medidas era de 2mm e no grupo dos fragmentos maiores, todos as medidas eram maiores de 2mm. Local de Realização: Universidade Federal do Rio Grande do Sul (UFRGS), Faculdade de Veterinária da UFRGS e Centro de Reprodução Humana Nilo Frantz, Porto Alegre, RS, Brasil. Animais: Foi utilizado um ovário de vaca raça Hereford. Resultados Principais: Nos fragmentos congelados com medida de 2mm, e com medidas maiores de 2mm, a percentagem de folículos normais foi de 56% e 34%, respectivamente. O risco relativo para a manutenção dos folículos normais póscongelamento nos fragmentos com de 2mm e maiores de 2mm foi de 0,63 e de 0,47, respectivamente. A comparação desse dois riscos relativos mostrou, de forma significativa (qui-quadrado de heterogeneidade p=0,022), que o impacto do congelamento na redução das taxas de sucesso foi maior nos fragmentos com espessuras maiores de 2mm. Conclusões principais: Este estudo mostrou que há um comprometimento maior na preservação folicular em fragmentos ovarianos criopreservados com espessuras maiores de 2mm. / Aims of the study: To assess the effect of different tissue sizes on the morphology of primordial and primary follicles after cryopreservation. Desing: Laboratorial controlled experiment. Comparative histological evaluations were performed of fresh control and cryopreserved tissue strips. Two groups were analyzed. In the group of the smaller fragments, the major face measure 2mm and in the group of the larger fragments, all the faces measure more than 2mm. Setting: Veterinary School of the Federal University of Rio Grande do Sul and Nilo Frantz Assisted Reproduction Clinic, Porto Alegre, RS, Brazil. Animal (s): One ovary from a Hereford cow. Main results: The percentages of normal follicles in large ovarian fragments cut into 2mm and >2mm slices were 56% and 34%, respectively. The relative risks to obtain normal follicles in the 2mm and >2mm strips after cryopreservation were 0.63 and 0.47, respectively. The comparison of the two relative risks shows significantly (heterogeneity qui-square p=0.022) that the effect of freezing on reducing success rate was higher in large tissue fragments that are cut >2mm. Conclusion(s): The present study shows that there is an increased risk of damage to primary and primordial follicles when the cryopreserved fragment has all its measures larger than 2mm.