Orientador: José Buratini Junior / Banca: Fernanda da Cruz Landim e Alvarenga / Banca: José Antonio Visintin / Banca: Mario Binelli / Banca: Marcelo Marcondes Seneda / Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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) / Doutor

Orientador: José Gonçalves Franco Júnior / Banca: Anagloria Pontes / Banca: José Carlos Peraçoli / Banca: Valdemar Ortiz / Banca: Mario Cavagna Neto / Resumo: Desde o primeiro relato de sucesso em humanos, a Injeção Intracitoplasmática de Espermatozóide (ICSI) tem sido particularmente indicada para casos de alterações seminais graves, sendo já demonstrada uma relação diretamente proporcional entre os resultados deste procedimento e a qualidade seminal. Tem sido sugerido que a incapacidade de o espermatozóide iniciar a ativação oocitária seja uma das principais causas de falha de fertilização após a ICSI. Estudos prévios identificaram o cálcio como um importante segundo mensageiro durante a ativação oocitária e que o tratamento com ionoforo do calcio e capaz de favorecer a ativação oocitária, resultando em fertilização, desenvolvimento embrionário e gestações normais. O objetivo deste estudo foi avaliar o efeito da Ativação Oocitária Artificial (AOA) com ionoforo do calcio A23187 em ciclos de ICSI quando utilizados espermatozóides de diferentes origens. Foram avaliados 314 ciclos de ICSI divididos em tres Grupos: EJACULADO (n = 92), EPIDIDIMARIO (n = 82) e TESTICULAR (n = 140), sendo cada um deles dividido em subgrupos, dependendo da realizacao (Subgrupo AOA) ou nao de AOA (Subgrupo Controle . CT). Foram avaliados tambem separadamente os ciclos de mulheres com idade inferior a 36 anos, objetivando minimizar o efeito da idade sobre os resultados dos tratamentos. Para a ativação oocitária, os oócitos foram mantidos por 30 minutos apos a ICSI em meio de cultivo para AOA, contendo 5ÊM de ionoforo de calcio A23187 e em aproximadamente 20 horas a fertilizacao foi confirmada pela presenca de 2 Pro-Nucleos. Para cada grupo experimental foram comparados, entre os Subgrupos AOA e CT, os seguintes parametros: taxa de fertilizacao normal, taxa de falha parcial de fertilizacao, porcentagem de bons embrioes no dia da transferencia, taxa de gestacao clinica, taxa de implantacao e taxa de abortamento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Since the first report of a birth after ICSI, it has been specially used for severe male factor of infertility and it was demonstrated that the semen quality influences the ICSI outcomes. Previous studies suggested that failure of fertilization after ICSI may be due to a spermatozoa inability in trigger the oocyte activation. Calcium has long been identified as a universally important second messenger during oocyte activation and many studies demonstrated that treatment with calcium ionophore may increase the free intracellular calcium, promoting oocyte activation, resulting in fertilization, embryo development and pregnancies. The aim of this study was to evaluate the effect of artificial oocyte activation (AOA) with calcium ionophore A23187 in ICSI cycles using sperm from different origins. It was evaluated 314 ICSI cycles divided in groups according to the origin of spermatozoa: EJACULATED group (n = 92), EPIDIDYMAL group (n = 82) and TESTICULAR group (n = 140). Each group was split into sub-groups depending on the AOA treatment: sub-group AOA, when it was performed AOA and sub-group Control-CT, when it was not performed AOA. Furthermore, it was evaluated separately only cycles in each woman were younger then 36 years old. After ICSI, oocytes were incubated in culture medium containing calcium ionophore A23187 (5ìM concentration) for 30 minutes and in approximately 20 hours oocytes were checked for normal fertilization, defined as observation of two distinct pronucleous. For each experimental group the following parameters were compared between the sub-groups AOA and CT: normal fertilization rate; partial fertilization fail rate; percentage of high quality embryos on the transfer day; implantation rate; pregnancy rate and miscarriage rate. For all the evaluated parameters, it was not observed any significant difference between the subgroups for the three spermatozoa origin groups.... (Complete abstract click electronic access below) / Doutor

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Na produÃÃo in vitro (PIV) de embriÃes bovinos vÃrios fatores contribuem para as variÃveis na produÃÃo e qualidade dos oÃcitos e embriÃes. Avaliou-se o uso parenteral de vitamina A (VA) e vitamina E (VE) na produÃÃo de oÃcitos colhidos por aspiraÃÃo folicular (OPU) e embriÃes por produÃÃo in vitro (PIV) em de vacas (n=22), sendo: Simental (S) (n=2); Nelore (N) (n=4); Brahma (B) (n=5) e Gir (G) (n=11). Todos os animais foram alocados na fase prÃ-tratamento (F1) (n=22) (nÃo receberam vitaminas) e os mesmos animais utilizados para a fase pÃs-tratamento (F2) (receberam 1.000.000 UI de vitamina A e 1g de vitamina E). A primeira OPU foi na F1, logo em seguida foi aplicado 1.000.000 UI de VA e 1g de VE, e apÃs 12 dias realizou-se nova OPU para fazer a F2. Os oÃcitos (CCO) foram maturados, fecundados e cultivados in vitro. As 44 OPU produziram 520 oÃcitos, 217 (F1) e 303 (F2), havendo efeito significativo, com acrÃscimo de 86 oÃcitos, obtendo mÃdia e desvio padrÃo 9,86Â5,53 F1 e 13,77Â2,0 F2, (*p<0,0219). Quando separada as raÃas NBG (Nelore, Brahma e Gir) (n=20) houve acrÃscimo de 95 oÃcitos, obtendo mÃdia e desvio padrÃo 9,90Â5,81 F1-NBG e 14,65Â9,44 F2-NBG, (*p<0,0085). As 44 PIV produziram 224 embriÃes, sendo 93 F1 e 131 F2, obtendo mÃdia e desvio padrÃo 4,23Â3,09 F1 e 5,95Â4,05 F2, (*p<0,0228). Quando separada as NBG a produÃÃo foi de 214 embriÃes, havendo acrÃscimo de 38 embriÃes, obtendo valores de 4,45Â3,15 F1 e 6,25Â4,09 F2, (*p<0,0285). Houve um efeito significativo na quantidade produzida de oÃcitos (n=22) e oÃcitos NBG (n=20). Houve efeito na produÃÃo de embriÃes de todas as raÃas (n=22) e embriÃes NBG (n=20). A suplementaÃÃo com VA e VE aumentou o nÃmero de oÃcitos totais (1,7Â0,7); oÃcitos NBG (1,8Â0,8); embriÃes totais (3,9Â1,6) e embriÃes NBG (4,7Â1,6). A resposta da F2 comparado com a F1 na produÃÃo de oÃcitos e embriÃes foi significativa quando todas as raÃas estavam agrupadas e tambÃm quando foi agrupado apenas as Bos taurus indicus (NBG). O uso das vitaminas A e E pode ser usada para maior recuperaÃÃo oÃcitÃria e embrionÃria em raÃas ZebuÃnas. / The in vitro (IVP) bovine embryos production has several factors that contribute to the variables in the production and quality of oocytes and embryos. We evaluated the parenteral use of vitamin A (VA) and vitamin E (VE) in the production of oocytes collected by follicular aspiration (OPU) and embryos by in vitro production (IVP) in cows (n = 22), where: Simmental (S) (n = 2), Nelore (N) (n = 4), Brahma (B) (n = 5) and Gir (G) (n = 11). All animals were allocated in the pre-treatment (F1) (n = 22) (not receiving vitamins) and the same animals used for post-treatment (F2) (received 1,000,000 IU of vitamin A and vitamin 1g E). The first OPU was in F1, soon after 1,000,000 IU was administered 1 g of VE and VA, and after 12 days was held to make the new OPU F2. Oocytes (COC) were matured, fertilized and cultured in vitro. The OPU 44 yielded 520 oocytes, 217 (F1) and 303 (F2), with significant effect, an increase of 86 oocytes, obtaining mean and standard deviation 9.86 Â 5.53 13.77 Â 2.0 F1 and F2, (*p <0.0219). When separate races NBG (Nelore, Brahman and Gir) (n = 20) there was an increase of 95 oocytes, obtaining mean and standard deviation 9.90 Â 5.81 and 14.65 NBG F1-F2-NBG Â 9.44, (*p <0.0085). The 44 IVP embryos produced 224, 93 F1 and 131 F2, getting mean and standard deviation 4.23 Â 3.09 5.95 Â 4.05 F1 and F2, (*p <0.0228). When separated from the NBG production was 214 embryos, with an increase of 38 embryos, obtaining values of 4.45 Â 3.15 6.25 Â 4.09 F1 and F2, (*p <0.0285). There was a significant effect on the quantity produced of oocytes (n = 22) and NBG oocytes (n = 20). It was an increased in all breeds embryos production (n = 22) and NBG embryos (n = 20). Supplementation with VE and VA increased the total number of oocytes (1.7 Â 0.7); NBG oocytes (1.8 Â 0.8); total embryos (3.9 Â 1.6) and embryos NBG (4 7 Â 1.6). The response of the F2 compared to F1 in the production of oocytes and embryos was significant when all races were grouped together and also when it was grouped only Bos taurus indicus (NBG). The use of vitamins A and E can be used to greater oocyte recovery and embryo in Zebu breeds.

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&ge;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&#43;1; edema uterino (UE); tonus cervical (CT); diâmetro máximo do folículo dominante (MdF1) em D0 e D&#43;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&#43;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: 3-10&#1059;), Middle Age Mares (MA: 11-17&#1059;) and Old Mares (OM18&#1059;). 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 &#43; 1; uterine edema (UE); cervical tone (CT); maximum diameter of dominant follicle (MdF1) on D0 and D&#43;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 &#43;1, uterine edema on Day 0, CT on Day &#43;1, MdF1 on D0 and D&#43;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

With meiosis spanning from embryonic development to the end of reproductive life in females, scientists have faced considerable limitations in studying female meiosis and the effects of toxicants on the developing oocyte. Over the last half century, various culture methods have been developed with the aim of studying the mechanisms of early ovary development, as well as for use in reproductive toxicology. However, very few of the established embryonic ovary culture systems have been used to investigate potential reproductive toxicants on the embryonic ovary, in particular when compared with the vast number of in vitro reproductive toxicity studies on the post-natal ovary. Here, a novel test compound, a topoisomerase II inhibitor: AstraZeneca Test Compound (AZTC), was used to investigate the efficacy and validity of ovarian culture methods when compared with in vivo reprotoxicity studies. AZTC was selected due to preliminary in vivo studies demonstrating its detrimental effects on spermatogenesis in male rats. AZTC targets bacterial type II topoisomerases that might have mammalian homologues involved in meiosis. Topoisomerase-II α was expressed within the female germ cells pre-natally, but became localised to the granulosa and stroma cells post-natally. This occurred both in vivo and in vitro. Ovaries from female rats exposed pre-natally to AZTC in vivo were analysed histologically and a significant increase in the number of primordial follicles was observed within the ovaries, as well as an increase in the number of unhealthy follicles. A novel mouse embryonic ovary culture system was developed by adapting, improving and bridging existing available culture techniques. The culture system supported growth of pre-meiotic mouse germ cells through prophase I of meiosis, the formation of primordial follicles and initiation of follicle growth. Cultured ovaries contained follicles at stages in comparable ratios to those in vivo and appeared morphologically normal and healthy. The culture also supported meiotic progression of oocytes to the pachytene stage, albeit with a slight delay. AZTC was used to validate the novel embryonic ovary culture by comparing the results with those from the in vivo study, where AZTC exposure had also occurred during embryonic development. Similar results were consistently observed between the in vivo and in vitro studies. In vitro effects of AZTC on the post-natal mouse ovary were also investigated, where neonatal mouse ovaries cultured with AZTC had fewer primordial follicles and more unhealthy follicles than did control ovaries. AZTC therefore demonstrated different effects when exposure occurred pre-natally vs. post-natally. The embryonic ovary culture was then used to examine the effects of another topoisomerase II inhibitor, etoposide, on the pre-natal ovary. Etoposide is a chemotherapy agent and has previously been prescribed to pregnant women. A significant reduction in the size of the follicle pool was observed in exposed cultured embryonic ovaries, where primordial and transitional follicles were targeted. Overall, establishment of post-natal culture systems have become a useful addition to in vivo reproductive toxicology studies. The embryonic ovary culture system developed here could become a valuable and powerful tool to screen potential reproductive toxicants, as well as to study the dynamics and regulation of early ovary development.

Abstract Apoptosis is a genetically programmed mechanism for a multicellular organism to remove cells that are unnecessary, or potentially harmful. The female reproductive system is characterised by a high rate of cellular proliferation. At the same time, apoptosis is also abundant during the normal physiological function of the ovary and endometrium. More than half of the 7 million oocytes that are produced during human ovarian development are deleted before birth and only about 400 oocytes reach the stage of ovulation during the female fertile lifespan. The fate of the non-ovulatory follicles is atresia, occurring through the mechanism of apoptosis. The endometrium goes through radical renewal processes during each menstrual cycle. Apoptosis has been suggested to participate in the regulation of endometrial cellular homeostasis. Errors in this mechanism can result in endometrial diseases such as hyperplasia and cancer. In this work, apoptosis and its regulation were studied in the human fetal and adult ovary, normal endometrium and endometrial pathologies. In fetal ovaries, apoptosis was already abundantly present in oocytes at 13 weeks of gestation. The maximum rate of apoptosis was seen between the 14th and 20th weeks, after which apoptosis decreased towards term. Ovarian Bcl-2 expression was detected in early fetal life during weeks 13 and 14. Bax expression was observed throughout the studied period, from week 13 to 40. The expression of transcription factor GATA-4, which is linked to follicular survival, was localised to the granulosa cells and was high in early fetal life and decreased somewhat towards term. In adult life apoptosis was located in the granulosa cells of the growing follicles. In ovarian biopsies from women homozygous for the inactivating C566T mutation of the FSH receptor, apoptosis or GATA-4 expression was not detected. During corpus luteum regression a peak in apoptosis was detected 10 - 12 days after the LH surge, and was preceded by an increase in 17HSD type 1 and TNF-α expression. During normal menstrual cycles, the highest rate of apoptosis was observed in the menstrual endometrium. This increase in apoptosis was preceded by a decreased Bcl-2/Bax ratio. In endometrial hyperplasia, the rate of apoptosis was similar to that seen during normal proliferation of the endometrium, but an apparent increase was observed in grade II endometrial carcinoma. In grade III carcinoma, the rate of apoptosis was lower than in grade II carcinoma but higher than in hyperplasia. These results indicate that apoptosis is the mechanism behind the substantial oocyte demise during ovarian development. During adult life, apoptosis was mainly localised to the granulosa cells of the growing follicles which do not reach the stage of a dominant follicle. In ovaries where FSH action is abolished, folliculogenesis was impaired and ovarian apoptosis was negligible. Apoptosis is also the underlying mechanism of corpus luteum regression. In the endometrium, apoptosis has a role in rejuvenating the endometrium for growth during the next endometrial cycle and in regulating cellular homeostasis.

Master of Science / Department of Clinical Sciences / James W. Carpenter / Current in vitro maturation (IVM) systems for domestic dog oocytes are inefficient, largely due to the species' unique reproductive physiology. The size of donor follicle influences developmental competence of dog ovarian oocytes. Specifically, oocytes from follicles > 2 mm in diameter complete in vitro nuclear maturation at a higher rate than those from smaller follicles. The objective was to determine the influences of follicular size, maturation time, and meiotic status on oocyte metabolism. We hypothesized that metabolic patterns differed between oocytes from small versus large follicles. Oocytes (n = 531) from adult ovaries were collected and grouped based on follicular size (small, < 1 mm, n = 252; medium, 1 to 2 mm, n = 231; and large, > 2 mm, n = 48). Oocytes were cultured for 0, 24, or 48 hours at 38.5°C in 5% CO[subscript]2 in 80 [Mu]L of TCM 199 + 25[Mu]M [Beta]- mercaptoethanol + 10 ng/ml epidermal growth factor + 0.25 mM pyruvate + 2.0 mM glutamine + 0.1% polyvinyl alcohol + 0.03 mg/ml streptomycin + 0.03 mg/ml penicillin G sodium (IVM medium), assessed for metabolism and evaluated for nuclear status. For metabolic assessments, oocytes were incubated for 3 h in 3 [Mu]l of IVM medium containing (1) 0.005 mM [0.064 [Mu]Ci/[Mu]l] D-53H-glucose (glycolysis) + 1 mM D-614C [0.053 [Mu]Ci/[Mu]l] glucose (glucose oxidation) or (2) 0.001 mM [0.041 [Mu]Ci/[Mu]l] L-G-3H-glutamine + 1 mM [0.027 [Mu]Ci/[Mu}l] 1-14C pyruvate, placed on the lid of a centrifuge tube containing 25 mM NaHCO3 and trapped radioactivity was measured using a [Beta]-counter. Only oocytes at an appropriate meiotic stage for each culture period (n = 380) were included in data analysis (e.g., germinal vesicle stage at 24 and 48 h culture were excluded). Differences in metabolism among groups were analyzed by ANOVA (main effects being follicular class, culture interval, and meiotic status). Oocytes recovered from large follicles metabolized significantly more pyruvate, glutamine, and glucose (via glycolysis) than those from small ones (p < 0.05). Across meiotic stages and follicular sizes, glycolytic rate was lowest in oocytes cultured for 24 hours (p < 0.05) compared to 0 or 48 hours. Metaphase II oocytes had a significantly higher glycolytic rate than those at other meiotic stages (p < 0.05). At culture onset (0 h), oocytes from small follicles predominately used pyruvate (p < 0.05), while oocytes from larger follicles (p < 0.05) predominately metabolized glucose. The present data suggests that dog oocytes preferentially use glucose as an energy substrate and that increasing glycolytic rate correlates with meiotic maturation. In addition, oocytes collected from large follicles exhibit increased metabolic capabilities that may be responsible for their increased developmental competence during IVM.

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.

La méiose ovocytaire comprend une succession de deux divisions cellulaires, sans phase intermédiaire de réplication de l'ADN, permettant l'haploïdisation du gamète femelle en vue de la fusion des génomes parentaux lors de la fécondation. Le caractère fortement asymétrique de ces divisions permet l'expulsion du matériel génétique surnuméraire, dans de petits globules polaires, tout en conservant l'essentiel des ressources cytoplasmiques qui seront nécessaires au développement précoce de l'embryon. De nombreuses études réalisées sur l'ovocyte de souris ont mis en évidence les capacités intrinsèques du gamète à rompre sa symétrie en positionnant son fuseau de manière excentrée à proximité du cortex. En se positionnant de la sorte le fuseau induit, via un gradient de Ran-GTP porté par les chromosomes, une polarisation du cortex ovocytaire qui permettra de restreindre le site d'émission des futurs globules polaires. Cette polarisation se caractérise notamment par une forte accumulation de filaments d'actines dépendante du facteur de nucléation Arp2/3. Nos travaux nous ont permis de mettre en évidence le rôle de Cdc42-GTP, via l'activation de N-WASP, comme intermédiaire entre le gradient de Ran-GTP et la polymérisation polarisée des filaments d'actine. Nous nous sommes également intéressés à la localisation des protéines ERM (Ezrin Radixin Moesin), connues pour favoriser la formation des microvillosités membranaires. Dans l'ovocyte, les microvillosités et les ERM sont toutes deux exclues du cortex polarisé et nous avons pu démontrer le rôle de Ran-GTP dans ce processus. Enfin, nous avons étudié la localisation du réseau d'acto-myosine cortical lors de la deuxième division méiotique qui nécessite la rotation du fuseau de l'ovocyte de souris. Nos résultats révèlent l'existence de deux sous-populations de myosine 2 corticale, l'une dépendante de la chromatine (Ran-GTP/Cdc42-GTP) et l'autre dépendante du fuseau central (Ect2/RhoA). / Oocyte meiosis is accomplished through two successive rounds of cellular divisions, without DNA replication, allowing for gamete haploidization necessary for parental genome fusion after fertilization. These divisions are highly asymmetric and allow extra-DNA expulsion, in small polar bodies, while retaining most of the cytoplasmic resources needed for early embryo development. Studies in mouse oocyte have demonstrated the capabilities of the gamete to autonomously break his symmetry by positioning the spindle near the cortex. By doing so, the spindle is able to induce a cortical polarization that is dependent on a Ran-GTP gradient emanating from the chromosomes. This polarization will be necessary for delimiting extrusion sites of the future polar bodies. A polarized accumulation of Arp2/3 actin filaments is one of the most evident features of oocyte polarization. We have shown that polarization of Cdc42-GTP, trough N-WASP activation, is an essential intermediate between Ran-GTP and the polarized polymerization of actin filaments. We also investigated ERM (Ezrin Radixin Moesin) proteins localization that are known to promote microvilli assembly. According to our data, microvilli and ERM are excluded from the polarized cortex in a Ran-GTP dependent manner. Finally, we studied cortical acto-myosin dynamics during the second meiotic division which requires spindle rotation. We demonstrated the existence of two cortical myosin 2 sub-populations which depend either on chromosomes (Ran-GTP/Cdc42-GTP) or on the central spindle (Ect2/RhoA).

This randomized, double-blind, placebo-controlled study was conducted to compare the effectiveness of intravenous acetaminophen vs. oral acetaminophen or placebo as an adjunct to opioids on lowering post-operative pain scores, discharge time, need for opioids, and opioid-related side effects, as well as assessing for any effects pain treatment has on embryological and pregnancy outcomes. Secondary analysis included identifying risk factors in patients that cause them to have worsening pain or minor relief from traditional pain management. This study was conducted at a single academic fertility center at Massachusetts General Hospital in Boston with a patient population of 159 English-speaking women between 18-43 years old, undergoing oocyte retrieval as a part of In Vitro Fertilization procedure. Participants were randomly placed in one of three treatment groups to receive either 1000mg IV acetaminophen and PO placebo (Group A), IV placebo, and 1000mg PO acetaminophen (Group B), or IV and PO placebo (Group C) as pain control before oocyte retrieval procedure. The primary outcomes measured were patient-reported post-operative visual analogue scale pain scores in the recovery room at 10 minutes, 30 minutes, and discharge time. Using these values to measure the effectiveness of each treatment at improving post-operative pain. To assess the relationship, if any, between demographical or clinical factors and pain, we analyzed what factors were common in those experiencing high or low pain. We used the Visual Analog Scale (VAS) which has patients rank pain from 1-10. For this analysis, low pain is defined as those whose 10-minute post-operative pain score was less than 5 (VAS score <5/10) and high pain as those whose 10-minute postoperative pain score is 5 or greater (VAS score 5+/10). Results showed that mean post-operative pain scores were similar between the study groups at 10 minutes (A:2.3, B: 2.6, C:2.8, p=0.51). Timing of discharge was also similar (A:60.1 mins, B: 58.8 mins, C:57.6mins; p=0.76). Although not statistically significant, the mean post-operative opioid dose for patients in group A was less than half of that in Group B and C (0.24mg vs. 0.59mg vs. 0.58mg; p=0.34) and fewer required rescue pain medication in the recovery room (4% vs. 19% vs. 15% respectively; p=0.24). There was a trend towards decreased side effects of constipation in Group A compared to Groups B and C (15% vs. 31% vs. 33%, respectively; p=0.07). There were no differences in embryological or early pregnancy outcomes between study groups. An analysis of predictors of pain, patients with BMI >/=30kg/m2 (obese) were more likely to report high post-operative pain (p=0.009). Prior abdominal surgeries, including pelvic laparoscopy and laparotomy, were associated with low post-operative pain (p=0.069 and p=0.025, respectively). Those who reported having pre-operative pain greater than zero were more likely to report lower postoperative pain (p=0.002). There was no significant relationship between race/ethnicity, infertility diagnosis, and procedure length and pain. This study's findings showed no significant difference between post-operative pain scores or discharge times in women undergoing oocyte retrieval when given IV acetaminophen, PO acetaminophen, or a placebo. There were severe findings that suggested that IV acetaminophen may reduce the need for post-operative narcotics and lead to fewer opioid-related side effects, however these findings while large were statistically insignificant. The predictors of higher post-operative pain we found, including high BMI, no prior history of abdominal procedures, and lack of pre-operative pain, indicate that further investigation into these predictors could be beneficial. This information may allow physicians and anesthesiologists to optimize their pain control.