Maternal contributions to pregnancy success: from gamete quality to uterine environment

The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle.(AU)

Effects of increasing lipopolysaccharide concentrations on in vitro developmental competence of ovine oocytes

Although a considerable number of studies have investigated the effects of lipopolysaccharide (LPS) on the reproductive performance of dairy cows, the response of ovine oocytes to LPS during their in vitro maturation and development is not well defined yet. Ewe’s ovaries were obtained from a slaughterhouse, the oocytes were collected and matured in the presence of increasing concentrations (0, 0.01, 0.1, 1 and 10 µg/mL) of LPS in order to evaluate the meiotic maturation by measuring the proportion of oocytes reaching the MII stage. The concentration of intracellular glutathione (GSH) was measured in oocytes following maturation in vitro. In addition, concentrations of selected metabolites including glucose, pyruvate, lactate and glutamine were quantified in the medium following maturation. A number of treated matured oocytes along with the control group were subsequently fertilized using frozen semen and assessed for the rate of cleavage and for the proportion reaching the blastocyst stage. The number of oocytes in MII stage was significantly reduced in response to the increasing concentrations of LPS (77.83%, 70.64%, 68.86%, 66.32%, respectively, in case of 0.01, 0.1, 1 and 10 µg/mL LPS when compared to the control group, 76.34%; P<0.05). There were no differences neither in the intracellular concentration of GSH in the oocytes nor in case of the metabolites in the maturation medium. Although the rate of cleaved oocytes was not affected by increasing levels of LPS, the blastocyst rate was reduced in a dose dependent manner (36.69%, 34.21%, 30.35%, 17.27% and 14.03% for the control, 0.01, 0.1, 1 and 10 µg/mL LPS, respectively (P<0.05). These results demonstrate that the developmental competence of ovine oocytes may be affected detrimentally by LPS and such deleterious effects could be related to the maturation process.

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential in vitro

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P 0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.

Effects of increasing lipopolysaccharide concentrations on in vitro developmental competence of ovine oocytes

Although a considerable number of studies have investigated the effects of lipopolysaccharide (LPS) on the reproductive performance of dairy cows, the response of ovine oocytes to LPS during their in vitro maturation and development is not well defined yet. Ewes ovaries were obtained from a slaughterhouse, the oocytes were collected and matured in the presence of increasing concentrations (0, 0.01, 0.1, 1 and 10 µg/mL) of LPS in order to evaluate the meiotic maturation by measuring the proportion of oocytes reaching the MII stage. The concentration of intracellular glutathione (GSH) was measured in oocytes following maturation in vitro. In addition, concentrations of selected metabolites including glucose, pyruvate, lactate and glutamine were quantified in the medium following maturation. A number of treated matured oocytes along with the control group were subsequently fertilized using frozen semen and assessed for the rate of cleavage and for the proportion reaching the blastocyst stage. The number of oocytes in MII stage was significantly reduced in response to the increasing concentrations of LPS (77.83%, 70.64%, 68.86%, 66.32%, respectively, in case of 0.01, 0.1, 1 and 10 µg/mL LPS when compared to the control group, 76.34%; P<0.05). There were no differences neither in the intracellular concentration of GSH in the oocytes nor in case of the metabolites in the maturation medium. Although the rate of cleaved oocytes was not affected by increasing levels of LPS, the blastocyst rate was reduced in a dose dependent manner (36.69%, 34.21%, 30.35%, 17.27% and 14.03% for the control, 0.01, 0.1, 1 and 10 µg/mL LPS, respectively (P<0.05). These results demonstrate that the developmental competence of ovine oocytes may be affected detrimentally by LPS and such deleterious effects could be related to the maturation process.(AU)

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential in vitro

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P 0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.(AU)

Influência do hepes na maturação in vitro de oócitos caninos obtidos de cadelas em diestro e anestro / Influence of hepes on in vitro maturation of canine oocytes in different stages of the estrous cycle

Diferentemente de outras espécies domésticas, a maturação in vitro (MIV) de oócitos caninos apresenta sucesso limitado. O objetivo deste estudo foi avaliar o efeito do HEPES na maturação in vitro de oócitos caninos obtidos de cadelas em diestro e anestro. Os ovários foram coletados, isolados assepticamente e transportados refrigerados a uma temperatura de 4 ºC. Os complexos cumulus-oócito (CCOs), provenientes das duas fases do ciclo estral, foram submetidos a dois tratamentos: meio TCM-199 com adição de 25 mM de HEPES (GT) e meio sem suplementação (GC). Depois de 72 horas de maturação, os CCOs foram desnudados, fixados e corados com HOESCHT 33342 para avaliação da maturação nuclear. Os oócitos obtidos da fase de anestro e diestro do GT demonstraram, em relação ao grupo GC, maior frequência de oócitos nos estágios de M-II (p<0,01). Comparando-se os diferentes status reprodutivos, observou-se que os oócitos obtidos da fase de diestro apresentaram índices maiores de QVG e M-II. Nossos resultados demonstraram que o HEPES preserva a viabilidade e morfologia oocitária, indispensáveis para a aquisição da competência meiótica, potencializando as taxas de M-II, e que os oócitos obtidos da fase de diestro estão mais aptos a completarem a maturação oocitária.

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The aim of this study was to evaluate the effect of HEPES on the in vitro maturation of dog oocytes in anestrus and diestrus. Ovaries were collected, aseptically isolated and transported refrigerated at a temperature of 4ºC. Cumulus-oocyte complexe (COCs) from the two phases of estrous cycle were subjected to two treatments: medium TCM-199 with addition of 25 mM HEPES (TG) and medium without supplementation (CG). After 72 h of maturation, COCs were denuded, fixed and stained with Hoechst 33342 to assess nuclear maturation. The oocytes obtained from the anestrus and diestrus phases of the TG showed a higher frequency of oocytes in metaphase II (M-II) (p <0.01) stage.Comparing the different reproductive status, it was observed that the oocytes obtained from the diestrus phase presented higher rates of GVBD (germinal vesicle breakdown) and M-II. Our results showed that HEPES preserves the viability and oocyte morphology essential for the acquisition of meiotic competence, increasing the M-II rates and that the oocytes obtained from the diestrus phase are better able to complete oocyte maturation.

Influência do hepes na maturação in vitro de oócitos caninos obtidos de cadelas em diestro e anestro / Influence of hepes on in vitro maturation of canine oocytes in different stages of the estrous cycle

Diferentemente de outras espécies domésticas, a maturação in vitro (MIV) de oócitos caninos apresenta sucesso limitado. O objetivo deste estudo foi avaliar o efeito do HEPES na maturação in vitro de oócitos caninos obtidos de cadelas em diestro e anestro. Os ovários foram coletados, isolados assepticamente e transportados refrigerados a uma temperatura de 4 ºC. Os complexos cumulus-oócito (CCOs), provenientes das duas fases do ciclo estral, foram submetidos a dois tratamentos: meio TCM-199 com adição de 25 mM de HEPES (GT) e meio sem suplementação (GC). Depois de 72 horas de maturação, os CCOs foram desnudados, fixados e corados com HOESCHT 33342 para avaliação da maturação nuclear. Os oócitos obtidos da fase de anestro e diestro do GT demonstraram, em relação ao grupo GC, maior frequência de oócitos nos estágios de M-II (p<0,01). Comparando-se os diferentes status reprodutivos, observou-se que os oócitos obtidos da fase de diestro apresentaram índices maiores de QVG e M-II. Nossos resultados demonstraram que o HEPES preserva a viabilidade e morfologia oocitária, indispensáveis para a aquisição da competência meiótica, potencializando as taxas de M-II, e que os oócitos obtidos da fase de diestro estão mais aptos a completarem a maturação oocitária.(AU)

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The aim of this study was to evaluate the effect of HEPES on the in vitro maturation of dog oocytes in anestrus and diestrus. Ovaries were collected, aseptically isolated and transported refrigerated at a temperature of 4ºC. Cumulus-oocyte complexe (COCs) from the two phases of estrous cycle were subjected to two treatments: medium TCM-199 with addition of 25 mM HEPES (TG) and medium without supplementation (CG). After 72 h of maturation, COCs were denuded, fixed and stained with Hoechst 33342 to assess nuclear maturation. The oocytes obtained from the anestrus and diestrus phases of the TG showed a higher frequency of oocytes in metaphase II (M-II) (p <0.01) stage.Comparing the different reproductive status, it was observed that the oocytes obtained from the diestrus phase presented higher rates of GVBD (germinal vesicle breakdown) and M-II. Our results showed that HEPES preserves the viability and oocyte morphology essential for the acquisition of meiotic competence, increasing the M-II rates and that the oocytes obtained from the diestrus phase are better able to complete oocyte maturation.(AU)

In vitro maturation of collared peccary (Pecari tajacu) oocytes after different incubation times / Maturação in vitro de oócitos de cateto (Pecari tajacu) após diferentes períodos de incubação

Oocyte in vitro maturation (IVM) is the first step of the in vitro reproductive technologies that enables mature oocytes to be generated ex vivo and after used for embryo production. In this sense, the establishment of culture environment, as oocyte incubation time, is essential for the success of the IVM. Therefore, the study was carried out to investigate the relationship between the meiotic potential and the IVM times of collared peccary oocytes, wild mammals of great commercial and ecological interest. Thus, ovaries were collected of females derived from captivity and transported to the laboratory within 1 hour of slaughtering. The oocytes derived from follicles (3-6mm in diameter) were recovered by aspirated and sliced. Good quality oocytes (evenly granulated cytoplasm with a least one layer of surrounding cumulus cells) were selected and subjected to culture in TCM 199 supplemented with 10µg/mL FSH, 10% FBS and 100µM cysteamine at 38.5°C, 5% CO2 and maximum humidity for 24 or 48 hours. After the incubation period, the nuclear status, the presence of first polar body and the expansion of cumulus cells of oocytes were assessed. The data obtained were analyzed by Fisher exact test (P<0.05). A total of four sessions (2-3 females per session) were performed, resulting in eighteen aspirated and sliced ovaries with normal morphological characteristics. An oocyte recovery rate of about 83.1% (59/71) was obtained with 3.3 oocytes/ovary and 2.3 viable oocytes/ovary. After different incubation times, differences (P<0.05) were observed in 24 and 48 hours for expansion of the cumulus cells (38.1% vs. 100%), presence of first polar body (52.4% vs. 90.5%) and nuclear status in second metaphase (19.0% vs. 76.2%), respectively. In conclusion, 48 hours is suitable time for the in vitro maturation of oocytes derived from collared peccaries when compared to the time of 24 hours, according to the meiotic potential observed. Additional studies should be conducted to improve the quality of the oocyte culture environment, as medium composition, aiming to obtain viable mature oocytes for other in vitro biotechnologies.(AU)

A maturação in vitro (MIV) oocitária é a primeira etapa das tecnologias reprodutivas in vitro que permite que oócitos maturados sejam gerados ex vivo e depois usados para a produção de embriões. Nesse sentido, o estabelecimento do ambiente de cultivo, como o período de incubação de oócitos, é essencial para o sucesso da MIV. Portanto, o estudo foi realizado para investigar a relação entre o potencial meiótico e os períodos de MIV de oócitos derivados de catetos, mamíferos silvestres de grande interesse comercial e ecológico. Para tanto, os ovários foram coletados de fêmeas derivadas de cativeiro e transportados ao laboratório dentro de 1 h após o abate. Os oócitos derivados de folículos (3-6mm de diâmetro) foram recuperados por aspiração e fatiados. Oócitos de boa qualidade (citoplasma uniformemente granulado com pelo menos uma camada circundante de células cumulus) foram selecionados e submetidos ao cultivo em TCM 199 suplementado com 10µg/mL de FSH, 10% de SFB e 100μM de cisteamina a 38,5°C, 5% de CO2 e umidade máxima por 24 e 48 h. Após o período de incubação, o estado nuclear, a presença do primeiro corpúsculo polar e a expansão das células do cumulus dos oócitos foi avaliada. Os dados obtidos foram analisados pelo teste exato de Fisher (P<0,05). Um total de quatro sessões (2-3 fêmeas por sessão) foi realizado, resultando em dezoito ovários aspirados e fatiados com características morfológicas normais. Uma taxa de recuperação oocitária de aproximadamente 83,1% (59/71) foi obtida com 3,3 oócitos/ovário e 2,3 oócitos viáveis/ovário. Após diferentes períodos de incubação, diferenças (P<0,05) foram observadas entre 24 e 48 h para a expansão das células cumulus (38,1% vs. 100%), presença de primeiro corpúsculo polar (52,4% vs. 90,5%) e estado nuclear na segunda metáfase (19,0% vs. 76,2%), respectivamente. Em conclusão, 48 h é o período adequado para a maturação in vitro de oócitos derivados de catetos quando comparado ao tempo de 24 h, de acordo com o potencial meiótico observado. Estudos adicionais devem ser conduzidos para melhorar a qualidade do ambiente de cultivo oocitário, como a composição de meio, objetivando obter oócitos maturados viáveis para outras biotecnologias in vitro.(AU)

In vitro maturation of collared peccary (Pecari tajacu) oocytes after different incubation times / Maturação in vitro de oócitos de cateto (Pecari tajacu) após diferentes períodos de incubação

Oocyte in vitro maturation (IVM) is the first step of the in vitro reproductive technologies that enables mature oocytes to be generated ex vivo and after used for embryo production. In this sense, the establishment of culture environment, as oocyte incubation time, is essential for the success of the IVM. Therefore, the study was carried out to investigate the relationship between the meiotic potential and the IVM times of collared peccary oocytes, wild mammals of great commercial and ecological interest. Thus, ovaries were collected of females derived from captivity and transported to the laboratory within 1 hour of slaughtering. The oocytes derived from follicles (3-6mm in diameter) were recovered by aspirated and sliced. Good quality oocytes (evenly granulated cytoplasm with a least one layer of surrounding cumulus cells) were selected and subjected to culture in TCM 199 supplemented with 10µg/mL FSH, 10% FBS and 100µM cysteamine at 38.5°C, 5% CO2 and maximum humidity for 24 or 48 hours. After the incubation period, the nuclear status, the presence of first polar body and the expansion of cumulus cells of oocytes were assessed. The data obtained were analyzed by Fisher exact test (P<0.05). A total of four sessions (2-3 females per session) were performed, resulting in eighteen aspirated and sliced ovaries with normal morphological characteristics. An oocyte recovery rate of about 83.1% (59/71) was obtained with 3.3 oocytes/ovary and 2.3 viable oocytes/ovary. After different incubation times, differences (P<0.05) were observed in 24 and 48 hours for expansion of the cumulus cells (38.1% vs. 100%), presence of first polar body (52.4% vs. 90.5%) and nuclear status in second metaphase (19.0% vs. 76.2%), respectively. In conclusion, 48 hours is suitable time for the in vitro maturation of oocytes derived from collared peccaries when compared to the time of 24 hours, according to the meiotic potential observed. Additional studies should be conducted to improve the quality of the oocyte culture environment, as medium composition, aiming to obtain viable mature oocytes for other in vitro biotechnologies.(AU)

A maturação in vitro (MIV) oocitária é a primeira etapa das tecnologias reprodutivas in vitro que permite que oócitos maturados sejam gerados ex vivo e depois usados para a produção de embriões. Nesse sentido, o estabelecimento do ambiente de cultivo, como o período de incubação de oócitos, é essencial para o sucesso da MIV. Portanto, o estudo foi realizado para investigar a relação entre o potencial meiótico e os períodos de MIV de oócitos derivados de catetos, mamíferos silvestres de grande interesse comercial e ecológico. Para tanto, os ovários foram coletados de fêmeas derivadas de cativeiro e transportados ao laboratório dentro de 1 h após o abate. Os oócitos derivados de folículos (3-6mm de diâmetro) foram recuperados por aspiração e fatiados. Oócitos de boa qualidade (citoplasma uniformemente granulado com pelo menos uma camada circundante de células cumulus) foram selecionados e submetidos ao cultivo em TCM 199 suplementado com 10µg/mL de FSH, 10% de SFB e 100μM de cisteamina a 38,5°C, 5% de CO2 e umidade máxima por 24 e 48 h. Após o período de incubação, o estado nuclear, a presença do primeiro corpúsculo polar e a expansão das células do cumulus dos oócitos foi avaliada. Os dados obtidos foram analisados pelo teste exato de Fisher (P<0,05). Um total de quatro sessões (2-3 fêmeas por sessão) foi realizado, resultando em dezoito ovários aspirados e fatiados com características morfológicas normais. Uma taxa de recuperação oocitária de aproximadamente 83,1% (59/71) foi obtida com 3,3 oócitos/ovário e 2,3 oócitos viáveis/ovário. Após diferentes períodos de incubação, diferenças (P<0,05) foram observadas entre 24 e 48 h para a expansão das células cumulus (38,1% vs. 100%), presença de primeiro corpúsculo polar (52,4% vs. 90,5%) e estado nuclear na segunda metáfase (19,0% vs. 76,2%), respectivamente. Em conclusão, 48 h é o período adequado para a maturação in vitro de oócitos derivados de catetos quando comparado ao tempo de 24 h, de acordo com o potencial meiótico observado. Estudos adicionais devem ser conduzidos para melhorar a qualidade do ambiente de cultivo oocitário, como a composição de meio, objetivando obter oócitos maturados viáveis para outras biotecnologias in vitro.(AU)

The variable success of in vitro maturation: can we do better?

The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques

The variable success of in vitro maturation: can we do better?

The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques(AU)

Only VEGF is necessary to stimulate the meiotic competence of caprine oocytes during in vitro maturation / Apenas VEGF é necessário para estimular a competência meiótica de oócitos caprinos durante a maturação in vitro

Para o sucesso da maturação in vitro (MIV), faz-se necessário uma sincronização entre as maturação nuclear e citoplasmática. Desta forma, o presente estudo teve por objetivo comparar o efeito do fator de crescimento do endotélio vascular (VEGF) como fator de crescimento na maturação in vitro (MIV) de oócitos caprinos com os fatores de crescimento epidermal (EGF) e semelhante à insulina I (IGF-I) adicionados juntos. Complexos cúmulos oócito (CCOs) foram isolados por slicing de ovários caprinos (n=40) utilizando bisturi cirúrgico. Apenas oócitos ≥110 μm com citoplasma homogêneo circundado por pelo menos uma camada compacta e coesiva de células do cúmulus foram selecionados para MIV. O meio de cultivo de base foi o TCM199 suplementado com 1% de BSA, 5 g/mL de hormônio luteinizante (LH), 0,5 g/mL de hormônio folículo estimulante recombinante (rFSH®), 0,911 mMol/L de piruvato,1μg/mL de estradiol, e 100 μM de cisteamina, o qual foi denominado TCM199+. OsCCOs selecionados foram distribuídos aleatoriamente nos seguintes tratamentos: 1) EGF e IGF-I, TCM199+ Suplementado com 10 ng/mL de EGF e 50 ng/mL de IGF-I; 2) VEGF, TCM199+ suplementado com 100 ng/mL de VEGF. Após a MIV, foram utilizados marcadores fluorescentes para avaliar a configuração da cromatina (Hoechst) e a viabilidade oocitária (calceína-AM e etídio homodímero). De uma maneira geral, ambos os tratamentos VEGF (80.2 e 35.7%) e EGF e IGF-I (70.5 e 29.9%) apresentaram resultados similares de quebra da vesícula germinativa (GVBD) e metáfase II rates (MII), respectivamente, sendo o tratamento VEGF numericamente superior ao EGF e IGF-I. Desta forma, conclui-se que o VEGF atua de forma similar ao EGF e IGF-I na competência oocitária para retomar ameiose.

For successful IVM, oocytes must undergo synchronically nuclear and cytoplasmic maturation. Thus, the aim of the present study was to compare vascular endothelial growth factor (VEGF) as a growth factor to the in vitro maturarion (IVM) to caprine oocytes instead of epidermal growth factor (EGF) plus insulin like growth factor I (IGF-I).Then, cumulus oocyte complexes (COCs) were pooled from slicing of caprine ovaries (n=40) using a surgical blade. Only oocytes ≥110 μm with homogeneous cytoplasm and surrounded by at least one compact layer of shiny and cohesive cumulus cells were selected to the IVM. The basic culture medium consisted of TCM199 supplemented with 1% of BSA, 5 g/mL of luteinizing hormone (LH), 0.5 g/mL of recombinant follicle-stimulanting hormone (rFSH®), 0.911 mMol/L pyruvate,1 μg/mL estradiol, and 100 μM cysteamine named TCM199+. The selected COCs were randomly distributed in the following treatments: 1) EGF plus IGF-I, TCM199+supplemented with 10 ng/ml of EGF and 50 ng/mL of IGF-I; 2) VEGF, TCM199+supplemented only with 100ng/mL of recombinant VEGF-A. After the IVM, oocytes were stained with fluorescent markers for the assessment of chromatin configuration (Hoechst) and to evaluate oocyte viability (calcein-AM and ethidium homodimer). Overall, both VEGF (80.2 and 35.7%) and EGF plus IGF-I (70.5 and 29.9%) treatments presented similar results of germinal vesicle breakdown (GVBD) and metaphase II rates (MII), respectively, being VEGF numerically greater than EGF plus IGF-I. Thus, we can infer that VEGF may act similar to EGF plus IGF-I in in vitro oocyte competence to resumes meiosis.

Only VEGF is necessary to stimulate the meiotic competence of caprine oocytes during in vitro maturation / Apenas VEGF é necessário para estimular a competência meiótica de oócitos caprinos durante a maturação in vitro

Para o sucesso da maturação in vitro (MIV), faz-se necessário uma sincronização entre as maturação nuclear e citoplasmática. Desta forma, o presente estudo teve por objetivo comparar o efeito do fator de crescimento do endotélio vascular (VEGF) como fator de crescimento na maturação in vitro (MIV) de oócitos caprinos com os fatores de crescimento epidermal (EGF) e semelhante à insulina I (IGF-I) adicionados juntos. Complexos cúmulos oócito (CCOs) foram isolados por slicing de ovários caprinos (n=40) utilizando bisturi cirúrgico. Apenas oócitos ≥110 μm com citoplasma homogêneo circundado por pelo menos uma camada compacta e coesiva de células do cúmulus foram selecionados para MIV. O meio de cultivo de base foi o TCM199 suplementado com 1% de BSA, 5 g/mL de hormônio luteinizante (LH), 0,5 g/mL de hormônio folículo estimulante recombinante (rFSH®), 0,911 mMol/L de piruvato,1μg/mL de estradiol, e 100 μM de cisteamina, o qual foi denominado TCM199+. OsCCOs selecionados foram distribuídos aleatoriamente nos seguintes tratamentos: 1) EGF e IGF-I, TCM199+ Suplementado com 10 ng/mL de EGF e 50 ng/mL de IGF-I; 2) VEGF, TCM199+ suplementado com 100 ng/mL de VEGF. Após a MIV, foram utilizados marcadores fluorescentes para avaliar a configuração da cromatina (Hoechst) e a viabilidade oocitária (calceína-AM e etídio homodímero). De uma maneira geral, ambos os tratamentos VEGF (80.2 e 35.7%) e EGF e IGF-I (70.5 e 29.9%) apresentaram resultados similares de quebra da vesícula germinativa (GVBD) e metáfase II rates (MII), respectivamente, sendo o tratamento VEGF numericamente superior ao EGF e IGF-I. Desta forma, conclui-se que o VEGF atua de forma similar ao EGF e IGF-I na competência oocitária para retomar ameiose.(AU)

For successful IVM, oocytes must undergo synchronically nuclear and cytoplasmic maturation. Thus, the aim of the present study was to compare vascular endothelial growth factor (VEGF) as a growth factor to the in vitro maturarion (IVM) to caprine oocytes instead of epidermal growth factor (EGF) plus insulin like growth factor I (IGF-I).Then, cumulus oocyte complexes (COCs) were pooled from slicing of caprine ovaries (n=40) using a surgical blade. Only oocytes ≥110 μm with homogeneous cytoplasm and surrounded by at least one compact layer of shiny and cohesive cumulus cells were selected to the IVM. The basic culture medium consisted of TCM199 supplemented with 1% of BSA, 5 g/mL of luteinizing hormone (LH), 0.5 g/mL of recombinant follicle-stimulanting hormone (rFSH®), 0.911 mMol/L pyruvate,1 μg/mL estradiol, and 100 μM cysteamine named TCM199+. The selected COCs were randomly distributed in the following treatments: 1) EGF plus IGF-I, TCM199+supplemented with 10 ng/ml of EGF and 50 ng/mL of IGF-I; 2) VEGF, TCM199+supplemented only with 100ng/mL of recombinant VEGF-A. After the IVM, oocytes were stained with fluorescent markers for the assessment of chromatin configuration (Hoechst) and to evaluate oocyte viability (calcein-AM and ethidium homodimer). Overall, both VEGF (80.2 and 35.7%) and EGF plus IGF-I (70.5 and 29.9%) treatments presented similar results of germinal vesicle breakdown (GVBD) and metaphase II rates (MII), respectively, being VEGF numerically greater than EGF plus IGF-I. Thus, we can infer that VEGF may act similar to EGF plus IGF-I in in vitro oocyte competence to resumes meiosis.(AU)

Influence of epidermal growth factor (EGF) supplementation at different times of in vitro maturation of canine oocytes / Influência da suplementação do fator de crescimento epidermal (EGF) em diferentes momentos da maturação in vitro de oócitos caninos

The aim of this study was to evaluate the influence of epidermal growth factor (EGF) on in vitro maturation of canine oocytes at different times of the process. Ovaries were collected from 55 bitches considered healthy and aseptically isolated, immersed in physiological solution (0.9% NaCl) and transported under refrigeration. Grade 1 cumulus-oocyte complexes (COCs) were selected and divided into two groups: control group (CG) and treatment group (TG). In CG 698 grade I COCs were placed in 4-well plates containing TCM-199 medium supplemented with 25 mM HEPES, 100 IU/mL penicillin, 100 mg/mL streptomycin, 26 mM sodium bicarbonate, 1.5 mM sodium pyruvate, 2.9 mM sodium lactate pentahydrate, 0.6 mM cysteine, 0.03 IU/mL hCG, 0.5 µg/mL FSH, 20 µg/mL estrogen at 38.5ºC in a humidified atmosphere of 5% CO2 in times of 24 h, 48 h, and 72 h. In TG 547 COCs received the same maturation medium plus 10 ηg/mL EGF. Logistic regression models (SAS, 2011) were constructed in order to estimate the chances of oocytes being observed at nuclear maturation stages in different culture times (24 h, 48 h, and 72 h). Based on the results found EGF-supplemented medium showed 2.56 times more chances of having an oocyte at metaphase I (M-I) than medium without EGF (p < 0.0001). The results of this study demonstrated that the time of 72 h showed 5.88 times more chances of having an oocyte at metaphase II (M-II) compared to time of 24 h (p = 0.0001) and 7.69 times more chance than time of 48 h (p = 0.0001). The chances of finding an oocyte at M-II were also 9.09 times higher in medium supplemented with EGF than in medium without EGF (p = 0.0001). Thus, these results demonstrated the essential importance of EGF at different moments of oocyte maturation, being a key component for the acquisition of meiotic competence in bitches, increasing the M-I and M-II rates.(AU)

O objetivo deste estudo foi avaliar a influência do fator de crescimento epidermal (EGF) em diferentes momentos da maturação in vitro de oócitos caninos. Os ovários foram coletados de 55 cadelas consideradas sadias e isolados assepticamente, imersos em solução fisiológica e transportados refrigerados. Os complexos cumulus-oócito (COCs) grau 1 foram selecionados e divididos em dois grupos, denominados grupo controle (GC) e grupo tratamento (GT). No GC, 698 COCs grau I foram cultivados em placas de quatro poços contendo meio TCM-199 suplementado com 25 mM de HEPES, 100 UI/mL de penicilina, 100 mg/mL de estreptomicina, 26 mM de bicarbonato de sódio, 1,5 mM de piruvato de sódio, 2,9 mM de lactato de sódio penta hidratado, 0,6 mM de cisteína, 0,03 UI/mL de hCG, 0,5 µg/mL de FSH, 20 µg/mL de estrógeno em estufa úmida a 38ºC, 5% de CO2 nos períodos de 24h, 48 h e 72 h . Já no GT, 547 COCs receberam o mesmo meio de maturação acrescido de 10 ηg/mL do EGF. Modelos de regressão logística foram elaborados para estimar as chances do oócito ser observado nos estágios de maturação nuclear em diferentes tempos de cultivo. Com base nos resultados encontrados, o meio suplementado com EGF demonstrou 2,56 vezes mais chances de ter um oócito no estágio de metáfase I (M-I) do que o meio sem EGF (p < 0,0001). Os resultados desse estudo demonstraram também que o tempo de 72 h mostrou 5,88 vezes mais chances de ter um oócito no estágio de metáfase II (M-II) do que o tempo de 2 h (p = 0,0001) e 7,69 vezes mais chance do que o tempo de 48h (p = 0,0001). As chances de se encontrar um oócito em M-II também foram 9,09 vezes maiores no meio suplementado com EGF do que no meio sem EGF (p = 0,0001). Dessa forma, estes resultados demonstraram a importância essencial do EGF em diferentes momentos da maturação oocitária, sendo componente chave para a aquisição da competência meiótica nas cadelas, aumentando os índices de M-I e M-II.(AU)

Influence of epidermal growth factor (EGF) supplementation at different times of in vitro maturation of canine oocytes / Influência da suplementação do fator de crescimento epidermal (EGF) em diferentes momentos da maturação in vitro de oócitos caninos

The aim of this study was to evaluate the influence of epidermal growth factor (EGF) on in vitro maturation of canine oocytes at different times of the process. Ovaries were collected from 55 bitches considered healthy and aseptically isolated, immersed in physiological solution (0.9% NaCl) and transported under refrigeration. Grade 1 cumulus-oocyte complexes (COCs) were selected and divided into two groups: control group (CG) and treatment group (TG). In CG 698 grade I COCs were placed in 4-well plates containing TCM-199 medium supplemented with 25 mM HEPES, 100 IU/mL penicillin, 100 mg/mL streptomycin, 26 mM sodium bicarbonate, 1.5 mM sodium pyruvate, 2.9 mM sodium lactate pentahydrate, 0.6 mM cysteine, 0.03 IU/mL hCG, 0.5 µg/mL FSH, 20 µg/mL estrogen at 38.5ºC in a humidified atmosphere of 5% CO2 in times of 24 h, 48 h, and 72 h. In TG 547 COCs received the same maturation medium plus 10 ηg/mL EGF. Logistic regression models (SAS, 2011) were constructed in order to estimate the chances of oocytes being observed at nuclear maturation stages in different culture times (24 h, 48 h, and 72 h). Based on the results found EGF-supplemented medium showed 2.56 times more chances of having an oocyte at metaphase I (M-I) than medium without EGF (p < 0.0001). The results of this study demonstrated that the time of 72 h showed 5.88 times more chances of having an oocyte at metaphase II (M-II) compared to time of 24 h (p = 0.0001) and 7.69 times more chance than time of 48 h (p = 0.0001). The chances of finding an oocyte at M-II were also 9.09 times higher in medium supplemented with EGF than in medium without EGF (p = 0.0001). Thus, these results demonstrated the essential importance of EGF at different moments of oocyte maturation, being a key component for the acquisition of meiotic competence in bitches, increasing the M-I and M-II rates.(AU)

O objetivo deste estudo foi avaliar a influência do fator de crescimento epidermal (EGF) em diferentes momentos da maturação in vitro de oócitos caninos. Os ovários foram coletados de 55 cadelas consideradas sadias e isolados assepticamente, imersos em solução fisiológica e transportados refrigerados. Os complexos cumulus-oócito (COCs) grau 1 foram selecionados e divididos em dois grupos, denominados grupo controle (GC) e grupo tratamento (GT). No GC, 698 COCs grau I foram cultivados em placas de quatro poços contendo meio TCM-199 suplementado com 25 mM de HEPES, 100 UI/mL de penicilina, 100 mg/mL de estreptomicina, 26 mM de bicarbonato de sódio, 1,5 mM de piruvato de sódio, 2,9 mM de lactato de sódio penta hidratado, 0,6 mM de cisteína, 0,03 UI/mL de hCG, 0,5 µg/mL de FSH, 20 µg/mL de estrógeno em estufa úmida a 38ºC, 5% de CO2 nos períodos de 24h, 48 h e 72 h . Já no GT, 547 COCs receberam o mesmo meio de maturação acrescido de 10 ηg/mL do EGF. Modelos de regressão logística foram elaborados para estimar as chances do oócito ser observado nos estágios de maturação nuclear em diferentes tempos de cultivo. Com base nos resultados encontrados, o meio suplementado com EGF demonstrou 2,56 vezes mais chances de ter um oócito no estágio de metáfase I (M-I) do que o meio sem EGF (p < 0,0001). Os resultados desse estudo demonstraram também que o tempo de 72 h mostrou 5,88 vezes mais chances de ter um oócito no estágio de metáfase II (M-II) do que o tempo de 2 h (p = 0,0001) e 7,69 vezes mais chance do que o tempo de 48h (p = 0,0001). As chances de se encontrar um oócito em M-II também foram 9,09 vezes maiores no meio suplementado com EGF do que no meio sem EGF (p = 0,0001). Dessa forma, estes resultados demonstraram a importância essencial do EGF em diferentes momentos da maturação oocitária, sendo componente chave para a aquisição da competência meiótica nas cadelas, aumentando os índices de M-I e M-II.(AU)

Influence of anestrus and diestrus stages of oocyte on meiotic competence in bitches / Influência das fases de anestro e diestro na competência meiótica oocitária de cadelas

The bitch has reproductive peculiarities that differentiate it from other species. Several experiments have been conducted to establish efficient protocols for maturation; however, the results appear to be unsatisfactory. In this respect, the reproductive female donor should be considered, since it can be a factor of variability of findings in this species. The objective of this study was to evaluate the relationship between the estrous cycle phases phase diestrus and anestrus on canine oocyte in vitro maturation (IVM). The ovaries were transported in sodium chloride 0.9% solution, and were cut into phosphate-buffered saline (PBS) and the cumulus-oocyte complexes (COCs) selected in tissue culture medium (TCM), supplemented with 199 HEPES. A total of 469 grade 1 oocytes were collected from bitches in anestrus and diestrus. These selected oocytes were transferred to the maturation medium for a period of 72 hours, then subjected to hyaluronidase solution and stained with Hoechst 33342 to assess nuclear configuration. The comparison of anestrus and diestrus phase showed no differences (p > 0.05) between the nuclear maturation stages. Thus, the phase of the estrous cycle did not influence the in vitro maturation of canine oocytes, increasing the rates of M-II in this species.(AU)

A cadela apresenta particularidades reprodutivas que a diferencia de outras espécies. Diversos experimentos têm sido realizados visando estabelecer protocolos eficientes para a maturação, entretanto os resultados mostram-se insatisfatórios. Nesse aspecto, a fase reprodutiva da fêmea doadora deve ser considerada, já que pode ser um fator de variabilidade dos achados ate então presenciados nessa espécie. O objetivo deste estudo foi avaliar a influencia das fases do ciclo estral (anestro e diestro) na maturação in vitro (MIV) de cadelas. Os ovários foram transportados em solução de cloreto de sódio 0,9% e seccionados em solução salina fosfato tamponado (PBS) e os complexos cumulus-oócito (COCs) selecionados em meio de cultura de tecidos (TCM) 199 suplementado com HEPES. Foram obtidos 469 oócitos grau 1 de cadelas em anestro e diestro. Esses oócitos selecionados foram transferidos para o meio de maturação por um período de 72 horas, sendo posteriormente submetidos a solução de hialuronidase e corados com HOESCHT 33342 para avaliação da configuração nuclear. A comparação das fases de anestro e diestro não revelou diferença (p > 0,05) entre os estágios de maturação nuclear. Dessa maneira, a fase do ciclo estral não influenciou na maturação in vitro de oócitos caninos, incrementando os índices de M-II nesta espécie.(AU)

DINÂMICA DA MATURAÇÃO NUCLEAR E CITOPLASMÁTICA DE OÓCITOS BOVINOS CULTIVADOS IN VITRO EM MEIO SUPLEMENTADO COM FULEROL

A eficiência da maturação in vitro (MIV) de oócitos está intimamente relacionada com a competência bioquímica, intrínseca ao desenvolvimento do oócito e sua posterior fecundação. Os meios de MIV tem sido suplementados e testados afim de melhorar o potencial oocitário para a produção in vitro de embriões (PIVE). No presente estudo objetivou-se avaliar, in vitro, a dinâmica da maturação nuclear e citoplasmática de oócitos bovinos cultivados em meio MIV suplementado com fulerol. O fulerol é uma nanomolécula derivada da polihidroxilação do fulereno, é estável e formado exclusivamente por átomos de carbono, e tem sido utilizado em algumas áreas biológicas devido sua atividade antioxidante, quando em concentrações mais baixas. Nesse trabalho objetivou-se avaliar se o fulerol é capaz de bloquear a retomada da meiose de oócitos bovinos maturados in vitro. Foram utilizados dois meios de MIV: no tratamento controle (TC), meio utilizado foi o TCM 199 bicarbonato; e o segundo tratamento, com meio TCM 199 bicarbonato suplementado com 50nM de fulerol (MF50). Os oócitos foram maturados por 24 horas em estufa a 38,5ºC, 5% de CO2 e 95% de umidade. A avaliação da maturação nuclear do TC (n=300) e MF50 (n=270) foi realizada a cada 6 horas, durante 36 horas, por meio da coloração dos oócitos com Hoechst 33342. Foram identificados os seguintes estádios: vesícula germinativa (VG), quebra da vesícula germinativa (QVG), metáfase I (MI) e metáfase II (MII). Na maturação citoplasmática avaliou-se oócitos do TC (n=197) e MF50 (n=159) a cada 12 horas, durante 36 horas, corados com Mitotracker Orange (Life® Technologies, Carlsbad, CA, USA), de acordo com a distribuição citoplasmática das mitocôndrias. Durante a experimentação verificou-se dificuldade de desnudamento dos oócitos expostos ao fulerol 50nM como uma informação observacional. De maneira descritiva, a partir de 6 horas, observou-se retardo na maturação nuclear dos oócitos do grupo MF50. Às 6 horas, oócitos do TC (19%) se encontravam em MI, enquanto no MF50 estavam em VG ou QVG, o que também ocorreu com 12 horas. Já às 18 horas, enquanto 46,3% dos oócitos já estavam maturados no TC (oócito em estádio MII), em MF50 o percentual foi de 20%. Com 24 horas de maturação, verificou-se 43,9% de maturação no grupo MF50, quando comparado com 63,8% no controle. Às 30 e 36 horas, o padrão de maturação foi estável, contudo, foi identificado início de degeneração dos oócitos. Com relação à maturação citoplasmática, houve retardo da mesma com 36 horas de maturação (P<0,05) no grupo MF50 (53,9%), comparado ao tratamento controle (69,8% de gametas maduros). E em relação aos oócitos com citoplasma imaturo, foram encontrados 10,4% e 31,7% para o TC e MF50 (P<0,05), respectivamente. Conclui-se que a adição de 50nM de fulerol ao meio de maturação in vitro possivelmente interferiu no mecanismo de expansão das células do cumulus oophorus, bem como retardou a progressão meiótica e a maturação citoplasmática dos oócitos

The efficiency of in vitro maturation (IVM) of oocytes is closely related to biochemical competence, intrinsic to the development of the oocyte and subsequent fertilization. IVM medium is supplemented in order to test and improve the oocyte potential for in vitro embryo production (IVEP). This study aimed to evaluate, in vitro, the dynamics of nuclear and cytoplasmic maturation of bovine oocytes cultured in IVM medium supplemented with fullerol. Fullerol is a nanomolecule derived from fullerene polyhydroxylation, it is stable and formed exclusively by carbon atoms. It is being used in some biological areas due to its antioxidant activity at lower concentrations. This study aimed to evaluate whether fullerol is able to block the resumption of meiosis in bovine oocytes matured in vitro. Two MIV media were used: the control treatment (CT), TCM 199 bicarbonate medium; and treatment with TCM 199 bicarbonate medium supplemented with 50nM fullerol (MF50). The oocytes were matured for 24 hours in a incubator at 38.5ºC, 5% CO2 and 95% humidity. The evaluation of nuclear maturation of CT (n = 300) and MF50 (n = 270) was performed every 6 hours, for 36 hours, by staining the oocytes with Hoechst 33342 and identifying the following stages: germinal vesicle (GV), breakdown of the germinal vesicle (GVB), metaphase I (MI) and metaphase II (MII). At cytoplasmic maturation, oocytes from CT (n = 197) and MF50 (n = 159) were evaluated every 12 hours, for 36 hours, stained with Mitotracker Orange (Life® Technologies, Carlsbad, CA, USA), according to cytoplasmic distribution of mitochondria. During the experimentation, there was difficulty in stripping the oocytes exposed to 50nM fulerol, as an observational information. Descriptively, after 6 hours of incubation, a delay in the nuclear maturation of the oocytes of the MF50 group was observed. At 6 hours of maturation, oocytes of the CT (19%) were in MI, while the MF50 were in GV or GVB, which also occurred with 12 hours. At 18 hours, while 46.3% of oocytes were matured on CT (oocyte in stage MII), on MF50 the percentage was 20%. Within 24 hours of maturation, it was observed 43.9% and 63.8% of matured oocytes for MF50 and CT groups, respectively. At 30 and 36 hours, the pattern of maturation was stable, but degenerate oocytes were identified. Regarding cytoplasmic maturation, there was a delay of 36 hours of maturation (P<0.05) in the MF50 group (53.9%) compared to the control group (69.8% of mature gametes). In relation to cytoplasmic immature oocytes, they were 10.4% for CT and 31.7% for MF50 (P<0.05). It is concluded that the addition of 50nM fullerol to the in vitro maturation medium possibly interfered in the expansion mechanism of cumulus oophorus cells, as well as delayed meiotic progression and cytoplasmic maturation of oocytes.

Large-scale chromatin structure and function changes during oogenesis: theinterplay between oocyte and companioncumulus cell

The process of chromatin configuration remodeling within the mammalian oocyte nucleus or germinal vesicle (GV), which occurs towards the end of its differentiation phase before meiotic resumption, has received much attention and has been studied in several mammals. This review is aimed to highlight the relationship between changes in chromatin configurations and to both functional and structural modifications occurring in the oocyte nuclear compartment. During the extensive phase of meiotic arrest at the diplotene stage, the chromatin enclosed within the GV is subjected to several levels of regulation. Morphologically, the chromosomes lose their individuality and form a loose chromatin mass. Then the decondensed chromatin undergoes profound rearrangements during the final stages of oocyte growth in tight association with the acquisition of meiotic and developmental competence. Functionally, the discrete stages of chromatin condensation are characterized by different level of transcriptional activity, DNA methylation and covalent histone modifications. Interestingly, the program of chromatin rearrangement is not completely intrinsic to the oocyte, but follicular cells exert their regulatory actions through gap junction mediated communications and intracellular messenger dependent mechanism(s). With this in mind and since oocyte growth mostly relies on the bidirectional crosstalk with the follicular cells, experimental manipulation of large-scale chromatin configuration is discussed. Besides providing tools to determine the key cellular pathways involved in genome-wide chromatin modifications , the present findings will aid to the refinement of physiological culture systems that can have important implications in treating human infertility as well as managing breeding schemes in animal husband .

Large-scale chromatin structure and function changes during oogenesis: theinterplay between oocyte and companioncumulus cell

The process of chromatin configuration remodeling within the mammalian oocyte nucleus or germinal vesicle (GV), which occurs towards the end of its differentiation phase before meiotic resumption, has received much attention and has been studied in several mammals. This review is aimed to highlight the relationship between changes in chromatin configurations and to both functional and structural modifications occurring in the oocyte nuclear compartment. During the extensive phase of meiotic arrest at the diplotene stage, the chromatin enclosed within the GV is subjected to several levels of regulation. Morphologically, the chromosomes lose their individuality and form a loose chromatin mass. Then the decondensed chromatin undergoes profound rearrangements during the final stages of oocyte growth in tight association with the acquisition of meiotic and developmental competence. Functionally, the discrete stages of chromatin condensation are characterized by different level of transcriptional activity, DNA methylation and covalent histone modifications. Interestingly, the program of chromatin rearrangement is not completely intrinsic to the oocyte, but follicular cells exert their regulatory actions through gap junction mediated communications and intracellular messenger dependent mechanism(s). With this in mind and since oocyte growth mostly relies on the bidirectional crosstalk with the follicular cells, experimental manipulation of large-scale chromatin configuration is discussed. Besides providing tools to determine the key cellular pathways involved in genome-wide chromatin modifications , the present findings will aid to the refinement of physiological culture systems that can have important implications in treating human infertility as well as managing breeding schemes in animal husband .(AU)

Influência do ciclo estral no diâmetro oocitário em cadelas / Influence of the estrous cycle bitches in diameter oocyte / Influence del ciclo estral en diámetro de los ovocitos en hembras

A baixa competência meiótica de oócitos caninos quando submetidos às condições artificiais de cultivo é considerada um grande obstáculo para o desenvolvimento de biotecnologias reprodutivas nessa espécie. As baixas taxas de MIV podem estar associadas ao tamanho dos oócitos recuperados das diferentes fases do ciclo estral. Assim, o objetivo desse trabalho é avaliar a relação entre as fases do ciclo estral - fase luteal e anestro, com o diâmetro oocitário de cadelas. Foram utilizadas 41 fêmeas caninas, com idades entre 6 meses e 7 anos, submetidas à ovariosalpingohisterectomia eletiva no Ambulatório de Reprodução de Pequenos Animais do Departamento de Reprodução Animal e Radiologia Veterinária, da Faculdade de Medicina Veterinária e Zootecnia da UNESP, Botucatu. Os ovários foram fatiados e selecionados apenas os COCs grau I, sendo submetidos à solução de hialuronidase a 2% para a retirada das células do cumulus. Após esse processo, os oócitos foram corados com 10 ug/ml de Hoechst 33342 para avaliação do diâmetro oocitário, sem a zona pelúcida. Os resultados não demonstraram diferença estatisticamente significativa entre os oócitos obtidos das fases de anestro e luteal (77.62 µm x 78.64 µm). Dessa forma, há a necessidade de novos estudos para que seja possível uma compreensão adequada de quais os fatores que possam estar interferindo na competência oocitária, não devendo-se considerar apenas um fator determinante já que a ação coordenada de diversos fatores contribui para o crescimento e desenvolvimento do oócito.

The low meiotic competence of canine oocytes when subjected to conditions of artificial cultive is considered a major obstacle to the development of reproductive biotechnologies in this species. Low rates of IVM may be linked to the size of oocytes recovered from different phases of the estrous cycle. The objective of this study is to evaluate the relationship between the phases of the estrous cycle - luteal phase and anestrus, with oocyte diameter of bitches. A total of 41 female dogs, aged 6 months to 7 years, subject to ovariosalpingohisterectomy elective in the Clinic of Small Animal Reproduction, Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, UNESP, Botucatu. Ovaries were sliced and selected only those COCs grade I, being subjected to the, solution of 2% hyaluronidase to remove cumulus cells. After this process, the oocytes were stained with 10 ml of Hoechst 33342 for assessment of oocyte diameter without the zona pellucida. The results showed no statistically significant difference between the oocytes from anestrous and luteal phases (77.62 um x 78.64 µm). Thus, there is a need for further studies to be able to have a proper understanding of the factors that may be interfering with oocyte competence and should not be considered only a determining factor since the coordinated action of several factors contributing to the growth and developing oocyte.

La baja competencia meiótica de ovocitos caninos cuando se someten a condiciones de cultivo artificial se considera un obstáculo importante para el desarrollo de biotecnologías reproductivas de esta especie. Las bajas tasas de IVM puede estar relacionado con el tamaño de los ovocitos recuperados de las diferentes fases del ciclo estral. El objetivo de este estudio es evaluar la relación entre las fases del ciclo estral - fase lútea y anestro, con un diámetro de ovocitos de hembras. Un total de 41 hembras, con edades entre 6 meses a 7 años, conforme a ovariosalpingohisterectomy el trabajo electiva en la Clínica de Reproducción de Pequeños Animales, Departamento de Reproducción Animal y Radiología Veterinaria, Facultad de Medicina Veterinaria y Zootecnia, UNESP, Botucatu. Los ovarios fueron cortados y seleccionar sólo aquellos COCs grado I, se someten a la solución de 2% de hialuronidasa para eliminar las células del cumulus. Después de este proceso, los ovocitos se tiñeron con 10 g/ml de Hoechst 33342 para evaluar diámetro de los ovocitos sin zona de los ovarios pelúcida. Os rodajas y se selecciona sólo aquellos AOC grado I, siendo sometidos a solución de hialuronidasa de 2% para la retirada de los células del cumulus. Después de este proceso, los ovocitos se tiñeron con 10 g/ml de Hoechst 33342 para la evaluación del diámetro del ovocito sin la zona pelúcida. Los resultados no mostraron diferencias estadísticamente significativas entre los ovocitos de las fases lútea y en anestro (77,62 x 78,64 mm microM). Por lo tanto, hay una necesidad de más estudios para ser capaz de tener una adecuada comprensión de los factores que pueden interferir con la competencia de ovocitos y no debe considerarse simplemente un factor determinante ya que la acción coordinada de varios factores que contribuyen al crecimiento y desarrollo de los ovocitos.