O objetivo do presente estudo foi comparar o efeito da idade de doadores de oócitos sobre os parâmetros reprodutivos, a taxa de recuperação e qualidade de oócitos após ICSI utilizando sêmen sexado. Éguas Pôneis de Polo (n = 79) e Puro-Sangue (n = 23) doadoras de oócitos (400-600kg e 3-29 anos) foram utilizados durante as estações reprodutivas de 2009/2010 e 2011 no hemisfério Sul (San Luis, Argentina) e norte (Kentucky, EUA), respectivamente. As éguas foram divididas em três categorias experimentais: Jovens (YM: 3-10 anos), Meia Idade (MA: 11-17a) e Idosas (OM≥18a). Um total de 326 oócitos recuperados in vivo a partir de folículos pré-ovulatórios (n=279) e folículos imaturos em crescimento (n=47). Desses, 224 oócitos foram recuperados, classificado e dirigido a um programa comercial ICSI com Pôneis de Polo (primeira estação). Durante a segunda estação, 57 oócitos foram recuperados a partir de folículos pré-ovulatórios e 47 oócitos de folículos em crescimento e congelados em nitrogênio líquido para ser usado posteriormente em outro estudo. Os pontos experimentais avaliados foram: intervalos (dias) entre a aspiração ou a ovulação (AS/OV) ao PGF, PGF ao GnRH (Dia 0=GnRH), AS/OV ao Dia 0, e AS/OV ao Dia+1; edema uterino (UE); tonus cervical (CT); diâmetro máximo do folículo dominante (MdF1) em D0 e D+1, e a taxa de crescimento folicular. O número total de aspirações, o número de folículos aspirados e oócitos por aspiração e/ou folículo, o grau de expansão das células do cúmulos, a presença e qualidade do corpo polar (PB), o volume ooplasma, o intervalo de GnRH a aspiração, a GnRH a ICSI, e a taxa de clivagem (CR) depois da ICSI também foram avaliados. Foram observadas diferenças significativas entre as três categorias experimentais (YM, MA e OM) para: intervalos (dias) entre PGF-GnRH, AS/OV-GnRH, AS/OV-Day 1, edema uterino no Dia 0, CT em Dia 1, MdF1 no D0 e D+1 e volume de ooplasma. A taxa de recuperação in vivo (RR) não foi afetada pela idade (média de 102%, 85% e 73,4% de oócitos recuperados por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as categorias experimentais. Em conclusão, um efeito de envelhecimento pode ser observado em vários parâmetros reprodutivos em éguas. Os ovócitos no presente estudo tiveram um menor volume de ooplasma para OM comparados com YM, mas isso não foi eficaz em predizer a viabilidade dos oócitos ou o potencial para o desenvolvimento para a avaliação da taxa de clivagem após ICSI utilizando sêmen sexado / The aim of the present study was to compare the effect of oocyte donors` age on reproductive parameters, recovery rate and quality of oocytes after ICSI using sexed semen. Polo Ponies (n=79) and Thoroughbred (n=23) oocyte donor mares (400-600kg and 3-29 years) were used during the 2009/2010 and 2011 reproductive seasons in the Southern (San Luis, Argentina) and in the Northern hemispheres (Kentucky, USA), respectively. Mares were divided into three experimental categories: Young Mares (YM: 3-10У), Middle Age Mares (MA: 11-17У) and Old Mares (OM18У). A total of 326 oocytes were recovered in vivo from pre-ovulatory follicles (n=279) and immature-growing follicles (n=47). From those, 224 oocytes were recovered, sorted and directed to a commercial ICSI program with Polo Ponies (first season). During the study`s second season, 57 oocytes were recovered from pre-ovulatory follicles and 47 oocytes from growing follicles and frozen in liquid nitrogen to be used in another study. The evaluated experimental end-points were: Intervals (days) between aspiration or ovulation (AS/OV) to PGF, PGF to GnRH (Day 0=GnRH), AS/OV to Day 0, and AS/OV to Day + 1; uterine edema (UE); cervical tone (CT); maximum diameter of dominant follicle (MdF1) on D0 and D+1, and follicular growth rate. Total number of aspirations, number of follicles aspirated and oocytes recovered per aspiration and/or follicle, the degree of cumulus cell expansion, the presence and quality of polar body (PB), the ooplasm volume, the interval from GnRH to aspiration, GnRH to ICSI, and the cleavage rate (CR) after ICSI were also evaluated. Significant differences between the three experimental categories (YM, MA and OM) were observed for: intervals (days) between PGF-GnRH, AS/OV-GnRH, AS/OV-Day +1, uterine edema on Day 0, CT on Day +1, MdF1 on D0 and D+1 and ooplasm volume. The in vivo recovery rate (RR) was not affected by age (average of 102%, 85% and 73.4% of oocyte recovered per cycle, per F1 and per follicle, respectively; P>0.05). The CR did not differ among experimental categories. In conclusion, an effect of aging could be observed in several reproductive parameters in mares. The oocytes in the present study had a smaller ooplasm volume for OM compared to YM, but this was not effective in predicting oocyte viability or the potential to development through evaluation of the cleavage rate after ICSI using sexed semen

A utilização de uréia na alimentação de fêmeas bovinas pode prejudicar o desempenho reprodutivo destes animais, provavelmente decorrente do aumento do teor de nitrogênio uréico plasmático (NUP). O objetivo deste estudo foi avaliar se alimentação com uréia por curto prazo oferecida a novilhas, e conseqüente aumento de NUP, exerce influência sobre a quantidade, qualidade e competência dos complexos cumulus-oócito (CCO). O experimento teve duração de 62 dias, dividido em oito semanas e dois períodos. Foram utilizadas 40 novilhas mestiças mantidas a pasto, alocadas a dois tratamentos, utilizando-se delineamento \"cross-over\": dieta sem uréia (SU): 5 kg (matéria original) de silagem de milho e 1 kg de concentrado/animal/dia ou dieta com uréia (U): 5 kg de silagem de milho e 1 kg de concentrado contendo 75 g de uréia/animal/dia. Os animais selecionados a cada semana receberam as dietas por seis dias, uma única vez ao dia. No sexto dia de oferecimento das dietas, foram realizadas aspiração folicular (OPU) e colheita de sangue, em jejum e 3 horas após a alimentação. Imediatamente após a OPU, foi realizada contagem total de CCO recuperados e classificação dos mesmos em viáveis e inviáveis. Apenas os viáveis foram destinados à produção In vitro de embriões. Em relação ao teor de NUP, houve efeito de interação entre tratamento e tempo de colheita (P=0,04) e dentro de cada tempo foi observado aumento significativo (P<0,01) para os animais do tratamento U. Não foi observado efeito de tratamento em relação ao número total de CCO recuperados por animal (média ± EPM: SU=9,15 ± 0,82 vs. U=8,82 ± 0,95), nem sobre a porcentagem de CCO viáveis sobre total de CCO recuperados por animal (SU=73,61% ± 2,47 vs. U=70,26% ± 2,31). A taxa de clivagem obtida no dia 3 após a inseminação In vitro (IIV) e as taxas de blastocisto nos dias 6, 7 e 9 após a IIV, não foram influenciadas pelo tratamento. Porém, no 11º pós IIV, houve diminuição (P=0,04) da taxa de blastocistos eclodidos para o tratamento U (SU=82,50% ± 0,05 vs. U=64,33% ± 0,07). Apesar do aumento do teor de NUP observado nas novilhas do tratamento U, a quantidade, qualidade e competência dos CCO durante as primeiras clivagens até atingirem o estádio de blastocisto In vitro não foram influenciadas pelo oferecimento de 75 g de uréia na dieta, durante seis dias. Porém, foi observada redução da taxa de eclosão dos blastocistos das novilhas do tratamento U. / Urea feeding offered to bovine dams may damage their reproductive performance, probably due to an increase in levels of plasmatic urea nitrogen (PUN). The aim of this study was evaluate if short-term urea feeding of heifers, following high PUN levels, would have an influence on the quantity, quality and competence of cumulus-oocyte complexes (COC). Trial lasted 62 days, divided into eight weeks and two periods. Forty crossbred grazing heifers were allocated to one of two treatments, using a cross-over design: diet without urea (WU): 5 kg (as fed) of corn silage and 1 kg of concentrated/animal/day, or diet with urea (U): 5 kg of corn silage and 1 kg of concentrated with 75 g of urea/animal/day. Heifers selected in each week received these diets once a day, for six days. On the sixth day of diets? offer, ovum pick-up (OPU) and blood sampling at fasting and 3 hours after feeding were carried out. Immediately after OPU, total COC recovered was counted and classified as either viable or unviable. Only viable were destined to In vitro embryo production. In relation to PUN level, there was a significant interaction between treatment and sampling time (P=0.04) and a significant (P<0.01) increase in animals undergoing U treatment for each of the test times. No significant effect was observed relative to either the total number of recovered COC per animal (mean ± SEM: WU=9.15 ± 0.82 vs. U=8.82 ± 0.95), or the viable COC as a percentage of total recovered COC per animal (WU=73.61% ± 2.47 vs. U=70.26% ± 2.31). Cleavage ratio assessed on day 3 post In vitro insemination (IVI) and blastocyst ratio on days 6, 7 and 9 post IVI were not influenced by treatments. However, there was a significant treatment effect (P=0.04) in relation to hatched blastocysts on day 11 after IVI (WU=83% ± 0.05 vs. U=64% ± 0.07). Even though higher PUN levels were observed in animals from treatment U, quantity, quality and competence of the COC during first cleavages until reaching the blastocyst stage In vitro were not influenced by adding 75 g of urea on the diet offered to heifers, during six days. Neverthless, a decline in hatched blastocysts rate was observed in heifers of treatment U.

The organisation of microtubule networks into a bipolar spindle is essential for reliable chromosome segregation during cell division. A pair of centrioles surrounded by pericentriolar material (PCM), define the canonical centrosome that acts as the main microtubule organising centre (MTOC) during mitosis. In mammalian meiosis, centrioles are eliminated early on during oogenesis. Despite the absence of centrosomes, a large number of centrosomal proteins are highly expressed in mouse oocytes. Here, I characterise the localisation and function of centrosomal proteins at a previously undescribed meiotic spindle pole domain (MSPD). An initial protein screen identified a group of pericentriolar satellite proteins that localised to a previously undescribed spindle pole domain throughout meiotic maturation in mouse oocytes, including Pericentriolar material 1 protein (PCM1). This domain was distinct from spindle microtubules and the acentrosomal microtubule organising centres (aMTOCs). Initial characterisation focused on PCM1, the main centriolar satellite scaffold protein in somatic cells. Depletion of PCM1 revealed interdependence with the essential aMTOC component, Pericentrin. In the absence of PCM1, aMTOCs could no longer assemble or maintain their structural integrity. PCM1 degradation and disassembly of aMTOCs disrupted spindle assembly and reduced the total amount of nucleated microtubules throughout meiosis. In the absence of the main microtubule nucleating aMTOCs, oocytes relied on the Ran GTPase activity to form a small bipolar spindle. A similar mechanism was previously reported in human oocytes that lack prominent MTOCs. The extended centrosomal protein screen identified additional components of the MSPD. TACC3, under the regulation of Aurora-A at aMTOCs, drive assembly of the MSPD. This domain was absent in MTOC free human oocytes but a second population of TACC3 (identified in mouse oocytes) localised to the meiotic spindle and K-fibres was essential for maintaining spindle pole integrity. Establishing the Lightsheet Z.1 system for live cell imaging of human oocytes enabled us to observe the dynamic distribution of TACC3 in these oocytes. In the absence of prominent MTOCs and the MSPD, human oocytes likely rely on other spindle assembly factors and motor proteins to organise their spindle. Future work to address if the absence of the MSPD could account (in part) for the observed spindle instability in human oocytes is an exciting outlook.

An estimated 1.6 million American couples struggle with infertility. Some causes for poor fertility can be clearly defined but in many instances, subfertility is unexplained. Poor oocyte quality is now considered to be a main contributing factor for many causes of infertility. Good oocyte quality is crucial for many processes including embryo development and maintaining pregnancy. There is a possibility that any alterations to the oocyte can have long lasting effects on embryo development and the health of the offspring. The oocyte is very sensitive to any perturbations to its surrounding environment. Transcripts for apoptosis inhibitors and epigenetic modifiers were found to be significantly more abundant in in vivo-matured oocytes compared to oocytes that were matured in vitro. RNA degradation and chromatin remodeling pathways may also be perturbed in in vitro-matured oocytes. While examining the effects of maternal age on the oocyte, there are age-related differences in gene expression in equine cumulus-oocyte complexes. Differences in gene expression may lead to a decrease in oocyte developmental competence. Age related alterations to gene expression in the equine cumulus-oocyte complexes might be caused by increased rates of oxidative stress and subsequent DNA damage. These alterations could directly impact many processes within the oocyte. Higher incidences of apoptosis may be possible in the cumulus cells from aged mares, which would directly impact the developmental competence of the oocyte. Lastly, oocyte quality may be impacted by western dietary consumption patterns, which could lead to many genes being differentially expressed in oocytes. Alterations to the abundance of these genes have been shown to lead to effects that are commonly seen with metabolic syndrome, such as glucose intolerance, insulin resistance, obesity and diabetes. The results of this work will ultimately provide insight into the effects environmental influences have on the oocyte at the molecular level.

It was hypothesized that technical factors associated with in vitro production (IVP) of embryos may influence rate of blastocyst development of oocytes matured at 38.5 or 41.0 C. To test this hypothesis, a retrospective meta-analysis was performed. Simple linear regression was performed to analyze continuous variables and ANOVA for categorical variables. Interactions among factors and maturation temperature on blastocyst development were analyzed using dummy regression for continuous variables, and using a factorial treatment design and ANOVA for categorical variables. Month of collection was the only variable that impacted responsiveness of ova to heat stress. Independent of maturation temperature, variables that explained most variation in blastocyst development included technician, total number of sliced ovaries per collection, ova number placed per well of oocyte maturation media, oocyte collection time, bull ID, sperm concentration added to ova, and ova age at IVF. The proportion of 8 to 16-cell embryos at time of cleavage assessment was the best predictor of blastocyst development. Results of model selection showed that development of ova matured at 38.5 C was associated with size of the collection, while development of ova matured at 41.0 C was mainly associated with ova age at fertilization. When data for ova matured at 38.5 and 41.0 C were combined, the effect of number of PZ per well on blastocyst development became evident. Use of these findings for optimizing efficiency of IVP procedures would effectively reduce experimental costs related to embryo production and increase laboratory productivity.

The key functions of the mammalian ovary are the production of fertilizable oocytes and thesecretion of steroid hormones. At the time of birth the human ovary is composed of basic unitstermed primordial follicles. Primordial follicles are long-lived structures in the ovary and some ofthem last until the woman reaches menopause. However, the intra-oocyte signaling pathways thatactivate primordial follicles and early follicular development are largely unknown. In this thesis, the functional roles that the phosphatidylinositol 3-kinase (PI3K) signaling pathwayplays in follicular development were investigated. In vivo approaches using genetically modifiedmouse models were used to determine the functions of several members of the PI3K signalingpathway in oocytes and in follicles. The function of Foxo3a, a substrate of Akt, was investigatedby expressing Foxo3a constitutively in oocytes of primary follicles. We found that continuouslyactive Foxo3a in mouse oocytes caused retardation of oocyte growth, resulting in arrest offollicular development. The functions of p27kip1 (p27) were studied using p27-deficient (p27-/-)mice. It was found that p27 suppresses follicle endowment/formation and activation, and that itinduces follicle atresia. The functions of PI3K signaling in oocytes during follicular activationwere also investigated using conditional mutant mice, by disrupting the Pten in oocytes ofprimordial follicles. We found that, all primordial follicles were prematurely activated due toovergrowth of oocytes and these follicles were depleted in young adulthood, causing prematureovarian failure (POF). At the same time, disruption of the Pten from oocytes of primary follicleshad no effect on activation of primordial follicles, and the follicles developed and maturednormally. The results clearly show that the PI3K pathway in the mammalian oocyte plays a keyrole in follicular activation through control of initiation of oocyte growth and folliculardevelopment. / Ovary development

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.

Posttranscriptional regulation plays a very important role in animal oocytes and embryos. Maternally synthesized mRNAs and proteins control early animal development up until the maternal-to-zygotic transition (MZT). This is the point when the zygotic genome takes control. The maternally deposited mRNAs are posttranscriptionally regulated right from the time they are produced during oogenesis, through egg activation, and in the embryo. microRNAs (miRNAs) are posttranscriptional regulators that have been shown to play a role in both RNA stability and translation. I examined miRNA abundance in Drosophila stage 14 oocytes, activated unfertilized eggs, and embryos and have grouped all the then known Drosophila miRNAs into four distinct temporal classes. Class I and III appear to be maternally deposited, while Class II appears to be both maternally and zygotically transcribed, and Class IV appears to be strictly zygotically transcribed. Follow-up experiments validated three of the four classes.

Early embryonic development in vertebrates is directed in part by maternal mRNAs expressed in oocytes and stored in cytoplasmic messenger ribonucleoprotein particles (mRNPs). Abundant evidence demonstrates the importance of mRNPs in embryonic development and in post-embryonic cellular function; however their characterization has been hampered by lack of suitable methodologies. The Xenopus oocyte has been the primary model system for studies of mRNPs. YY1 is a well-studied transcriptional regulatory factor that is sequestered in the oocyte cytoplasm and present entirely in cytoplasmic oocyte mRNPs. The objective of this thesis was to examine the biochemistry of YY1 association with maternal mRNA molecules in order to shed light on the role of YY1 in development and the poorly understood biology of oocyte mRNPs. The initial working hypotheses were that association of YY1 with mRNPs is dependent on sequence-specific RNA-binding activity and, therefore, that YY1 associates with a definite subset of maternal mRNA. A number of unique methods were developed in this study to address these hypotheses. RNA immunoprecipitation-DNA microarray (RIP-CHIP) analysis establishes that YY1 associates with a subset of mRNAs in the oocyte pool. A novel sequence-specific RNA-binding activity of the YY1 protein is demonstrated, and the RNA-binding activity of YY1 is shown to be required for its association with oocyte mRNPs in vivo. The functional roles of YY1 mRNA substrates are discussed in the context of embryological development and the biological function of YY1 in oocyte mRNPs. Extension of the experimental approaches developed in this thesis to the entire set of mRNP proteins would significantly advance our understanding of mRNP composition and heterogeneity, as well as the biological function of maternal mRNAs and mRNPs in development.

The main objective of this study was to investigate if natural honey can be used as a cryoprotecting agent (CP) in vitrification medium to improve the viability of vitrified-warmed bovine oocytes. The first study was conducted to investigate the dehydration capability of natural honey compared with sucrose, and to determine the proper concentration of honey-based medium and the optimum time for sufficiently safe dehydration of bovine oocytes. Matured cumulus-oocyte complexs (COCs) were denuded and introduced individually into different concentrations (0.25, 0.5, 1.0, 1.5 or 2.0 M) of honey and sucrose-based medium followed by rehydration in control media (TCM). Video images were recorded during dehydration and rehydration, and oocyte images were captured at 12 time intervals to calculate oocyte-volume changes during dehydration and rehydration. Results demonstrated that, in honey-based media, the maximum oocyte shrinkage was achieved after 60 sec exposure in 0.25M, 0.5M and 1.0M concentrations; while at higher concentrations 1.5M and 2.0M, the maximum dehydration occurred at 30 and 20 seconds respectively. In sucrose-based medium, the maximum oocyte shrinkage was achieved after 60 sec exposure in 0.25 or 0.5M concentrations. However, at higher concentrations (1M, 1.5M or 2M), the maximum dehydration occurred at 30, 20 and 10 sec. For rehydration, oocytes dehydrated in honey or sucrose-based medium were able to regain their original volume within 60-120 sec. However, oocytes dehydrated in higher concentrations (2M honey, and 1.5M and 2M sucrose) were rehydrated back to their original volume within 20 sec. This study concluded that natural honey and sucrose caused similar cell dehydration. Only oocytes dehydrated in 1M honey-based media reached maximal dehydration after 60 sec and equally regained original volume. Therefore, 1M of honey-based medium is suggested for sufficient and safe oocyte dehydration during vitrification. The second study was conducted to determine in vitro maturation (IVM), in vitro fertilization (IVF) and embryonic development of bovine oocytes vitrified in honey-based vitrifcation media. In Experiment 1, bovine COCs were randomly distributed in control group (non-vitrified; G1), 0.5M sucrose group (second control; G2), and 0.5M, 1M and 1.5M honey groups (G3, G4 and G5 respectively). The COCs were exposed to equilibration solution 1 (VS1) at ~ 22 oC for 5 min and to vitrification solution 2 (VS2) for 1 min, mounted on Cryotops and plunged into LN2. COCs were warmed in TCM and honey/sucrose medium at 38.5oC for 1 min, washed, matured in vitro (IVM), denuded, and immunostained to evaluate maturation. Maturation rate was significantly higher (80.7%) in control group (G1) than in vitrified groups (56, 52, 55 and 51% in G2, G3, G4 and G5, respectively) (P<.0001), whereas there was no significant difference among the vitrified groups (P>0.05). In Experiment 2, bovine COCs distributed in control (not vitrified, G1) and vitrified groups using 1M honey and 0.5M sucrose (G2 and G3 respectively), underwent for IVM, IVF and in vitro culture (IVC) for 9 days. Cleavage rate was significantly higher (P<.0001) in the control group (74%, G1, n=183) than rates of vitrified groups (51% in G2, n=137; and 42% in G3, n=131), whereas no differences among vitrified groups (P=0.0723). Rate of blastocyst formation was significantly higher (34%) in G1 than in the vitrified groups (P<.0001); however, blastocyst formation rates in the honey group were significantly higher (P=0.0026) than in the sucrose group (13% and 3% respectively). Addition of natural honey (1.0M; or 21.7%w/v) in vitrification medium can safely and sufficiently dehydrate bovine oocytes during vitrification procedure. The vitrification of bovine oocytes in 1M honey improved their post-warming maturation abtility and embryonic development.