Abstract Although the genetic sex of an embryo is determined at conception by the presence or absence of the Y chromosome, both females and males have bipotential, undifferentiated gonads early in their development. Genes and testicular hormones direct differentiation into either testes or ovaries. The first relevant gene to be identified was the Y-linked master regulatory gene, SRY, since when several other genes have been found to be of importance for sex determination. The primary aim here was to identify the role of Wnt-4 in the development of the gonad and adrenal gland. Wnt-4 was found to be expressed in the developing gonad, the Müllerian duct and the adrenal gland, in addition to the kidney, pituitary gland and mammary gland as observed earlier. Expression in the gonad was found to be regulated in a sex-specific manner. After sex determination Wnt-4 was downregulated in the testis, but the expression persisted until birth in the ovary. Wnt-4-deficient female mice demonstrated a partial female-to-male sex reversal and a reduction in the number of oocytes, while the Müllerian duct was absent from both sexes. Lack of Wnt-4 in the adrenal gland led to reduced aldosterone production, indicating abnormal development of the zona glomerulosa. Flutamide administration to pregnant Wnt-4 heterozygote females was shown to partially restore the sex reversal. The results suggest that female development is not a default pathway but needs active signalling, in which Wnt-4 plays an essential role.

Ovarian changes, patterns of reproduction, and age-specific productivity of female Columbian black-tailed deer (Odocoileus hemionus columbianus) on Vancouver Island were elucidated largely by examination of serial, stained sections of ovaries from 444 females. Well-defined, 8 or 9-day, follicular cycles occur during the breeding season in November and early December. Many follicles rupturing at first ovulation are asynchronous and of extreme sizes. Relatively small (4-5 mm³), short-lived (8-day) corpora lutea develop after first ovulation; these rapidly regress after second ovulation, while the corpora lutea of pregnancy grow to 100 mm³, within 5-8 days. The changes in ovarian structures during the breeding season were ascertained in 12 females, whose follicles and corpora lutea were dated by the cleavage stage of fertilized ova. These provided criteria for estimating ovulation dates in other females. In each cycle, about 50% of the adult females ovulated within an 8-day period. The mean date of first ovulation in each of the 5 years was approximately November 16; the second ovulation followed after a remarkably short period of 8-9 days. About 96% of the females conceived at second ovulation and 4% on subsequent ovulations. A high proportion of 'silent heats' accompany first ovulation, as indicated by lack of sperm on four of six ova. Growth curves, based on fetuses conceived at second ovulation, provided the conception dates of two late conceivers—those containing more than one generation of regressed corpora lutea of non-pregnancy. Females may cycle at least five times if pregnancy does not occur. Accessory corpora lutea develop in large and small unruptured follicles, in small ruptured follicles, and in regressing corpora lutea. They occurred in 47% of females between first and second ovulation and in 36% of pregnant females. Minimum loss of ova was 8.3% in all females that produced corpora lutea and 4.3% in females that became pregnant, of which 3.1% was moribund fetuses. Corpora lutea of pregnancy, corresponding to the number of fetuses, develop into distinctive scars, which persist for the life of the doe. Pregnancy rates of the previous season, as well as the long term productivity of individuals and the population, were estimated from these. The average number of viable fetuses per doe increased progressively from 0.91 in yearlings to 1.81 in the 5-5 to 6.5 age-classes, and thereafter decreased. These changes in fertility with age were mirrored by changes in weight and girth. The relative contribution of fawns by each age-class in the population progressively decreased with age. One hundred females of reproductive age produced about 137 fawns. / Science, Faculty of / Zoology, Department of / Graduate

No description available.

Menopause is associated with disorders such as osteoporosis and ovarian cancer. It is unclear whether the postmenopausal ovary retains steroidogenic capacity and how it can impact the development of these disorders. The present studies used the VCD-treated follicle-depleted mouse model of menopause to test the hypothesis that residual ovarian tissue retains steroidogenic capacity following ovarian failure and, thus, affects the development of these disorders. Microarray technology was used to evaluate gene expression in residual ovarian tissue of follicle-depleted mice compared to that in ovaries from cycling animals. Among the genes identified were those encoding proteins for synthesis of androgens. Steroidogenic capacity of residual ovarian tissue was further evaluated by determining the expression of genes and proteins involved in ovarian steroidogenesis, and by measuring levels of circulating and rostenedione and gonadotropins. Follicle-depleted ovaries were enriched in mRNAs for androgenic enzymes, receptors involved in the internalization of cholesterol, and luteinizing hormone receptor. Increased circulating levels of FSH and LH and detectable and rostenedione were measured throughout the study. Protein for 3β-hydroxysteroid dehydrogenase, 17α- hydroxylase/17,20-lyase and luteinizing hormone receptor was detected in follicledepleted ovaries by Western blot analysis and localized by immunofluorescence staining. The contribution of retaining residual ovarian tissue to accelerated bone loss following ovarian failure was evaluated by comparing bone mineral density from young and aged VCD-treated mice to that in age-matched ovariectomized (OVX) animals. Retaining residual ovarian tissue resulted in protection against accelerated bone loss in young but not aged VCD-treated mice. Whether residual ovarian tissue is more susceptible to development of ovarian neoplasms compared to ovaries from cycling animals was addressed by combining the VCD-treated mouse with the DMBA model of ovarian carcinogenesis. VCD-treated follicle-depleted mice that received DMBA developed Sertoli-Leydig cell tumors while no tumors were observed in cycling animals. Residual ovarian tissue following ovarian failure appears to have a protective effect against loss of bone integrity, but a detrimental effect on development of ovarian neoplasms. Findings from these studies: provided evidence of a physiological role for residual ovarian tissue following ovarian failure, and furthered the use of the VCD-treated mouse as a relevant model for menopause and associated disorders.

No description available.

Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, the principal angiogenic factor, vascular endothelial growth factor A (VEGFA), may influence follicle growth via regulation of angiogenesis. However, VEGFA may also influence follicular development through nonangiogenic mechanisms since its expression has been localized to nonvascular follicles and cells. Alternative mRNA splicing of 8 exons from the VEGFA gene results in the formation of different VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms are encoded by exon 8a while a sister set of isoforms with antiangiogenic properties are encoded by exon 8b. The antiangiogenic isoforms comprise the majority of VEGFA expressed in most tissues while expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. The Vegfa angiogenic isoforms (Vegfa_120, Vegfa_164, and Vegfa_188) were detected in developing rat ovaries, and quantitative RT-PCR determined that Vegfa_120 and Vegfa_164 mRNA was more abundant after birth, while Vegfa_188 mRNA was highest at embryonic day 16. The antiangiogenic isoforms, Vegfa_165b and Vegfa_189b, were amplified and sequenced from rat ovaries and quantitative RT-PCR determined that Vegfa_165b mRNA was more abundant around embryonic day 18, but Vegfa_189b lacked a distinct pattern of abundance. VEGFA and its receptors were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. Antiangiogenic VEGFA isoforms were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. To determine the role of VEGFA in developing ovaries, postnatal day 3/4 rat ovaries were cultured with VEGFR-TKI, a tyrosine kinase inhibitor that blocks signaling through the VEGFA receptors, FLT1 and KDR. Ovaries treated with VEGFR-TKI had vascular development reduced by 94%. In addition, treated ovaries had more primordial follicles, fewer early primary, transitional, and secondary follicles, and greater total follicle numbers compared with control ovaries. This suggests that VEGFA promotes follicle recruitment and early follicular development. These effects may be dependent upon increased ovarian vascularization or they may be mediated by nonvascular mechanisms.

Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies (MAbs), due to their ability to perform correct post-translational modifications. A major issue for use of CHO cells lines for the production of recombinant proteins is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval. Protein production is complex and is influenced by cell growth, transcription, translation, protein folding and post-translational processing and secretory events, which may interact to determine stability of expression during prolonged culture. This thesis aims to identify features associated with stability/instability of recombinant protein expression and methods to improve protein production, with the addition of chemically defined (CD) feed and chemicals. Two exemplar CHO cell lines, which secrete the same recombinant antibody were characterised in response to LTC, feed and DMSO addition. Both cell lines (3.90 and 51.69) exhibited unstable protein production over LTC, with a loss in final antibody titres and specific productivity (Qp). The instability observed within the exemplar cell lines was not due to decreased recombinant gene copy numbers or mRNA expression but was associated with lower viable cell densities, increased ER stress (GADD153 and spliced XBP-1 [XBP-1(s)]) and enhanced rates of lactate utilisation (observed during the decline phase of batch culture). Improvement of recombinant protein expression in response to feed or DMSO addition was associated with lower expression of ER stress markers (ATF4, XBP-1(s) and GADD153 at mRNA level and GADD153 at protein level) and alterations to the metabolic activity of the cultures (prevention of alanine and lactate re-utilisation, and greater glucose utilisation between the stationary and decline phase of batch culture).Although feed or DMSO addition improved recombinant protein production, these additions did not reverse the appearance or progression of instability for cells after LTC. ER stress expression was not abolished as a consequence of feed or DMSO addition. Expression of stress markers at earlier time points may be the factor that limits antibody production and secretion. The consequences of the presence of feed and DMSO addition on ER stress markers and antibody production serves to highlight approaches that may be utilised for engineering more productive or stable protein production phenotypes in parental cell lines.

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-03-16Bitstream added on 2014-06-13T19:40:31Z : No. of bitstreams: 1 oliveira_vtp_dr_rcla.pdf: 3167477 bytes, checksum: 7c78f3049a399eb9b91205136ee7ba11 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Nas abelhas eussociais há duas castas femininas, as rainhas que são responsáveis pela produção dos indivíduos que mantêm a população da colônia e fazem sua multiplicação e as operárias que compartilham entre si todas as tarefas de manutenção da colônia e das próprias atividades da rainha. Na espécie estudada, Melipona quadrifasciata anthidioides, uma abelha sem ferrão, as operárias normalmente produzem ovos em seus ovários na fase em que se ocupam do aprovisionamento das células de cria (operárias nutridoras), diferentemente de Apis mellifera em que feromônios da rainha são capazes de inibir a vitelogênese nos ovários das operárias. As diferenças funcionais entre rainhas e operárias em ambas espécies são controladas hormonalmente. O corpo gorduroso (CG) é um tecido constituído basicamente por um único tipo de célula designado trofócito, cuja função se assemelha em vários aspectos à dos hepatócitos. Os trofócitos ao mesmo tempo em que retiram substâncias da hemolinfa e as armazenam ou metabolizam, sintetizam outras que nela descarregam. Entre estas últimas está a vitelogenina, uma proteína precursora do vitelo acumulado pelos ovócitos durante a vitelogênese, como reserva para o futuro desenvolvimento do embrião. Neste trabalho a citoquímica dos trofócitos de rainhas virgens, fisogástricas e de operárias nutridoras foi estudada com microscopia eletrônica de transmissão (MET) e comparadas com a morfologia do desenvolvimento dos ovócitos no ovário com a finalidade de comparar a atividade dos trofócitos com a absorção de substâncias da hemolinfa pelo ovário. Em outra vertente a presença da vitelogenina foi pesquisada no CG e no ovário de ambas as castas, na tentativa de verificar se havia concordância quanto à produção da proteína nos trofócitos e sua presença nos ovários... / Eussocial bees have two female castes, the queens that are responsible for producing individuals that will keep the colony population and workers that share among them the tasks of colony maintenance and of the queen care. In the studied species Melipona quadrifasciata anthidioides, a stingless bee, workers normally produce eggs in their ovaries when they are provisioning the brood cells (nurse workers), differently from Apis mellifera, where pheromones from the queen are able of prevent the vitellogenesis in the workers ovaries. The functional differences between queens and workers are, in both castes controlled by hormones. The fat body (FB) tissue is constituted basically by a single kind of cell designated throphocyte whose function is similar in several aspects to the hepatocytes. The throphocytes at the same time that take substances from the hemolymph and storage or metabolize them also synthesize others that are discharged in the body fluid. Among these is the vitellogenin, a precursor protein of the yolk accumulated by oocytes during the vitellogenesis as a reserve for further development of the embryo. In this work the cytochemistry of trophocytes of virgin and physogastric queens and nurse workers was studied with transmission electron microscopy (TEM) and compared to the morphology of oocytes development in the ovary with the purpose of correlate morphologically the activity of trophocytes to the absorption of substances from hemolymph by the ovaries. The presence of vitellogenin was searched in the FB and ovaries extracts of both castes by electrophoresis, as an attempt to see if there was an agreement between production of the protein in the trophocytes and their presence in the ovaries. Finally, taking into account the action of the morphogenetics hormones over the trophocytes, these cells of both castes were incubated into medium containing juvenile hormones III... (Complete abstract, click electronic address below)

Paper III reprinted with kind permission of Elsevier, sciencedirect.com

Paper III reprinted with kind permission of Elsevier, sciencedirect.com