An increasing number of women delay childbearing and consequently face infertility and pregnancy complications associated with age. The central contributor to compromised reproductive performance is poor oocyte quality. Despite advances in assisted reproductive technologies, a strategy to overcome the damage that oocytes receive with age is yet to be identified. This work focuses on the influence of TAp73, a protein that decreases in mouse and human eggs with age, on the developmental capacity of mouse oocytes. TAp73 deficient mice were found to have fewer active mitochondria and compromised clearance of damaged material in their oocytes, possibly due to reduced mTOR-TAp73 axis signaling. These qualities were shown to contribute to low oocyte maturation rates. Additionally, TAp73 likely mediates the action of coenzyme Q10, which restores oocyte TAp73 levels and mitochondrial quality in aged mice. Together these findings suggest that TAp73 is a promising therapeutic target for improving oocyte function.

An increasing number of women delay childbearing and consequently face infertility and pregnancy complications associated with age. The central contributor to compromised reproductive performance is poor oocyte quality. Despite advances in assisted reproductive technologies, a strategy to overcome the damage that oocytes receive with age is yet to be identified. This work focuses on the influence of TAp73, a protein that decreases in mouse and human eggs with age, on the developmental capacity of mouse oocytes. TAp73 deficient mice were found to have fewer active mitochondria and compromised clearance of damaged material in their oocytes, possibly due to reduced mTOR-TAp73 axis signaling. These qualities were shown to contribute to low oocyte maturation rates. Additionally, TAp73 likely mediates the action of coenzyme Q10, which restores oocyte TAp73 levels and mitochondrial quality in aged mice. Together these findings suggest that TAp73 is a promising therapeutic target for improving oocyte function.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

Research Doctorate - Doctor of Philosophy (PhD ) / In Australia almost 40% of the carnivorous marsupials, or dasyurids, are threatened. Assisted reproductive techniques (ART), especially genome resource banking, have the potential to contribute to the conservation of these species by reducing the loss of genetic diversity. This project aimed to advance the knowledge of ART in dasyurids by focusing on the long term preservation of male and female gametes and establishing protocols for the production of mature oocytes for use in future ART. These studies used the fat tailed dunnart (Sminthopsis crassicaudata) as a model dasyurid and replicated many of the findings on threatened dasyurids. Dasyurid spermatozoa had a relatively unstable acrosome which lacked acrosomal membrane disulphide stabilisation. There was no evidence that S. crassicaudata spermatozoa were susceptible to high concentrations of cryoprotectants, but spermatozoa frozen with up to 40% glycerol using a rapid freezing protocol were not viable. Nonetheless the morphology and acrosomal integrity of frozen spermatozoa was normal and there was no evidence of DNA damage. The lack of success with cryopreservation is likely to be an artifact of cold shock, which was observed in S. crassicaudata and had not previously been described in any other marsupial. This susceptibility to low temperature can be overcome by slow cooling spermatozoa to 0 ºC at 0.5 ºC minute -1 with up to 20% egg yolk, and it is likely that this finding will result in successful sperm cryopreservation in the near future. Freeze drying spermatozoa represents an additional strategy for long term sperm preservation and freeze dried S. crassicaudata spermatozoa had normal morphology and nuclear integrity. In this study preserved dasyurid spermatozoa were immotile and non-viable but had no nuclear damage, suggesting that fertilisation may be achieved with intracytoplasmic sperm injection (ICSI). As ICSI requires a large number of mature oocytes to be collected, a reliable timed ovarian stimulation protocol was established in S. crassicaudata. This protocol enabled the collection of up to 28 oocytes which were either mature, or able to be cultured to the first polar body stage within 48 hours. Despite the success of induced ovulation, methods for preservation of the female gamete are essential to genome resource banking. This study also described a protocol for the enzymatic dissociation of dasyurid ovarian tissue allowing collection of high quality individual preantral follicles. The oocytes inside these follicles were able to be vitrified without any loss of viability and short term in vitro culture of immature follicles repaired the small amount of vitrification-induced damage to the surrounding granulosa cells. This collection of studies describes progress in genome resource banking for spermatozoa and oocytes from dasyurids and the development of protocols allowing the collection of a large number of oocytes for use in fertilisation experiments. These advances provide a solid and comprehensive framework for continuing the study of dasyurid ART which is timely due to the urgent need for genome resource banking in several threatened dasyurid marsupials.

No description available.

In the developing mouse oocyte, as well as in the preimplantation embryo, betaine (N,N,N-trimethylglycine) plays an important role first as a mechanism for cell volume regulation and second as a major methyl donor. Thus, the presence of betaine has implications both during development, and throughout the lifespan. It has previously been observed that betaine accumulates in the mouse oocyte as it matures, however its origin in the egg is unknown. Here I explore the enzyme choline dehydrogenase (CHDH; EC 1.199.1) as a method by which the mouse oocyte synthesizes the betaine that we observe prior to initiation transport activity in the preimplantation embryo. I carefully monitored betaine transport throughout meiotic maturation to confirm that no other previously unobserved membrane transport existed in the maturing oocyte. However, no betaine transport into oocytes was detected during meiotic maturation suggesting de novo synthesis. Previous data suggests that the enzyme is expressed (at the transcript level) in the developing oocyte, and becomes active during meiotic maturation. I demonstrated the presence of CHDH protein in the oocyte and preimplantation embryo. I then examined whether the mouse oocyte synthesizes betaine autonomously and addressed whether CHDH is a requirement for this process. Chdh knockout oocytes did not accumulate betaine in vivo, while normal betaine levels were observed in Chdh wildtype oocytes. CHDH-mediated synthesis of betaine was directly confirmed by detection of increased betaine in oocytes matured in vitro in the presence of choline. Chdh-/- oocytes failed to produce betaine when similarly cultured in choline. This establishes the production of betaine as an autonomous process in maturing oocytes. Overall, I have built upon previous data to demonstrate that betaine accumulation is a feature of meiotic maturation that occurs by de novo synthesis of the molecule, a process that requires transient activation of the enzyme choline dehydrogenase.

Cryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future.

Made available in DSpace on 2014-06-11T19:32:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-06Bitstream added on 2014-06-13T20:42:44Z : No. of bitstreams: 1 caixeta_es_dr_botib.pdf: 1185802 bytes, checksum: ef647e66df4e5f5d7f8ca9a758116412 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O oócito participa ativamente dos mecanismos reguladores da maturação do complexo cumulus-oócito (COC) via secreção de fatores parácrinos. A proteína morfogênica óssea 15 (BMP15) e o fator de crescimento e diferenciação 9 (GDF9) têm concentrado a maior parte da atenção direcionada aos fatores secretados pelo oócito (FSO) e têm sido associados com a melhora na competência para o desenvolvimento do COC. Em adição, recentemente, detectamos a expressão de fatores de crescimento fibroblástico (FGFs) no oócito e seus receptores nas células do cumulus (FGF10 e seus receptores FGFR1B e 2B; FGF8 e 17 e seus receptores FGFR2C e 3C), sugerindo o envolvimento do sistema FGF na regulação da diferenciação das células do cumulus. O presente trabalho investigou a regulação da expressão do RNAm de FSOs (BMP15, GDF9, FGF8, FGF10 e FGF17) e seus receptores, bem como de membros da família de fatores de crescimento epidermal (EGF)-like [ampiregulina (AREG), epiregulina (EREG) e betacelulina (BTC)] durante a maturação in vitro (MIV) bovina estimulada pelo FSH. O FSH estimulou a expressão do FGFR2C, FGFR3C, FGFR1B, ALK6, AREG e EREG nas células do cumulus durante a MIV. A expressão do RNAm do FGF8 e FGF17, mas não da BMP15, GDF9 e FGF10 diminuiu no oócito durante a MIV. Em adição foram investigadas especificamente as ações da BMP15 e do FGF10 sobre a expansão do cumulus e expressão gênica de membros da família desintegrina e metaloproteinases (ADAM10 e ADAM17), membros da família dos fatores EGF-like (AREG, EREG e BTC) e de genes sabidamente envolvidos no controle da expansão do cumulus [ciclooxigenase 2 (COX2), hialurona sintetase 2 (HAS2), proteína indutora do fator de necrose tumoral 6 (TSG6) e pentraxina 3 (PTX3)]. A BMP15 e o FGF10 aumentaram a porcentagem de COCs completamente... / The oocyte actively participates in the regulatory mechanisms of cumulus-oocyte complex (COC) maturation via secretion of paracrine factors. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) have concentrated most of the attention directed to oocyte secreted factors (OSFs) and have been shown to enhance developmental competence of the COC. In addition, fibroblast growth factors (FGFs) have also been recognized as important OSFs. Recently, we detected the expression of FGFs in the oocytes and their receptors in cumulus cells (FGF10 and its receptors FGFR1B and 2B; FGF8 and 17 and their receptors FGFR2C and 3C), suggesting the involvement of the FGF system in the regulation of cumulus cells differentiation. The present study investigated the mRNA expression pattern for FSOs (BMP15, GDF9, FGF8, FGF10 and FGF17) and their receptors, as well as of epidermal growth factor (EGF)-like family members [ampiregulina (AREG), epiregulina (EREG) and betacelulina (BTC)] during bovine COC in vitro maturation (IVM) stimulated by FSH. The FSH stimulated mRNA expression of FGFR2C, FGFR3C, FGFR1B, ALK6, AREG and EREG in cumulus cells during IVM. Messenger RNA expression of FGF8 and FGF17, but not of BMP15, GDF9 and FGF10, decreased in the oocyte during IVM. In addition were specifically investigated the actions of BMP15 and FGF10 on cumulus expansion and gene expression of disintegrin and metalloproteinase family members (ADAM10 and ADAM17), of EGF-like family members (AREG, EREG and BTC) and the major expansion-inducing genes genes [cyclooxygenase 2 (COX2), hyaluronan synthase 2 (HAS2), pentraxin 3 (PTX3), tumor necrosis factor-stimulated gene-6 protein (TSG6)]. BMP15 and FGF10 increased the percentage of fully expanded cumulus-oocyte... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:23:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-27Bitstream added on 2014-06-13T19:30:09Z : No. of bitstreams: 1 pires_ea_me_jabo.pdf: 426419 bytes, checksum: b57d15a1404fbc5b5fdc2c88bcefb224 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo desta pesquisa foi avaliar os efeitos da suplementação de cisteína e cisteamina no desenvolvimento meiótico de oócitos caninos durante o processo de maturação ín vítro. Os oócitos foram coletados de sete cadelas hígidas em fase pré-ovulatória imediata, submetidas à ovario-histerectomia. Os COC's selecionados foram cultivados por um período de 72 horas em quatro meios diferentes: A (controle) - TCM199 suplementado com BSA (3 mg/mL) + FSH (5 J.Lg/mL) + LH (10 f.Lg/mL) + progesterona (2 f.Lg/mL) + estradiol (2 f.Lglml); 8 - controle + 0,1mM de cisteína; C - controle + 100J.1M de cisteamina; D - controle + 0,1 mM de cisteína + 100J.1M de cisteamina. Os resultados demonstraram que não houve diferença significativa entre os tratamentos (p<0,05), ou seja, a suplementação de compostos antioxidantes no meio de maturação não favoreceu a competência meiótica. Além disso, neste estudo pode-se inferir que para cada fase do ciclo estral, talvez seja necessário um período de maturação diferenciado. / The aim of this research was to evaluate the effects of the cysteine and cysteamine supplementation on meiotic deveropment of canine oocytes dunng the process of in vitro maturation. The oocytes were collected atter ovanohysterectomy from seven healthy bitches in immediate preovulatory stage. The selected COC's were cultured by a period of 72 hours in four different media: A (control) - TCM199 supplemented with BSA (3 mg/mL) + FSH (5 J-Lg/mL) + LH (10 J-Lg/mL) + progesterone (2 f.Lg/mL) + estradiol (2 f.LglmL); 8 - control + 0,1mM of cysteine; C - control + 100JlM of cysteamine; O - control + 0,1 mM of cysteine + 100J,lM of cysteamine. The present study demonstrated that there was not significant difference among the treatments (p<0,05), in other words, the supplementation of antioxidant in the medium of maturation didn't favor the meiotic competence. Besides, in this study it can be inferred that for each stage of the oestrus cycle, perhaps it is necessary a different maturation penod.

Submitted by LORENA SANCHES (losanches25@yahoo.com.br) on 2016-09-05T23:48:16Z No. of bitstreams: 1 Dissertação Lorena Sanches.pdf: 910469 bytes, checksum: c0365e51eb8f6cf8b8d23bb263de599e (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-09-06T12:34:18Z (GMT) No. of bitstreams: 1 sanches_l_me_bot.pdf: 910469 bytes, checksum: c0365e51eb8f6cf8b8d23bb263de599e (MD5) / Made available in DSpace on 2016-09-06T12:34:22Z (GMT). No. of bitstreams: 1 sanches_l_me_bot.pdf: 910469 bytes, checksum: c0365e51eb8f6cf8b8d23bb263de599e (MD5) Previous issue date: 2016-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A maturação in vitro (MIV) é uma etapa fundamental da produção in vitro de embriões bovinos (PIV). A conclusão prematura da maturação nuclear durante a MIV é considerada como uma das principais causas da eficiência limitada da PIV. Em camundongos, o fator de crescimento dos fibroblastos 8 (FGF8) regula a atividade do peptÌdeo natriurético tipo C (NPPC) aumentando a expressão de seu receptor (NPR2) e contribuindo assim para o bloqueio meiótico. O principal objetivo deste trabalho foi testar os efeitos do FGF8 sobre a dinâmica da quebra da vesícula germinativa e progresso da meiose durante a MIV induzida com FSH ou ampirregulina (AREG) em bovinos. Paralelamente, os efeitos do FGF8 sobre a expansão do cumulus e metabolismo da glicose também foram testados. Na MIV induzida com AREG, mas não com FSH, o FGF8 diminuiu a porcentagem de oócitos com quebra da vesícula germinativa às 6 e 9 horas do cultivo, enquanto aumentou a expressão do RNAm do NPPC, mas não do NPR2, nas células do cumulus. Distintamente, na MIV com FSH, o FGF8 diminuiu a porcentagem de oócitos em Meiose I às 9 horas de cultivo. O FGF8 não afetou a porcentagem de oócitos em Meiose II às 22 horas da MIV, nem a expansão do cumulus, embora tenha aumentado a expressão do RNAm de genes da cascata ovulatória [prostaglandina endoperoxido sintase 2 (PTGS2) e desintegrina e metaloproteinase de domínio contendo proteÌna 10 (ADAM 10)]. Além disso, o FGF8 diminuiu o consumo de glicose e a produção de lactato na MIV com FSH, mas não com AREG. Em conclusão, o FGF8 desacelera a dinâmica da maturação nuclear enquanto aumento a expressão do NPPC nas células do cumulus durante MIV induzida com AREG e, portanto, apresenta-se como fator potencialmente útil para melhorar a eficiÍncia da MIV. / FAPESP: 2012/06417-8