Impact of an acute heat shock during in vitro maturation on interleukin 6 and its associated receptor component transcripts in bovine cumulus-oocyte complexes

An acute heat stress event after the LH surge increased interleukin 6 (IL6) levels in the follicular fluid of the ovulatory follicle in hyperthermic cows. To examine direct consequences of a physiologically-relevant elevated temperature (41.0°C) on the cumulus-oocyte complex (COC), IL6 transcript abundance and related receptor components were evaluated throughout in vitro maturation. Heat-induced increases in IL6 were first noted at 4 hours of in vitro maturation (hIVM); peak levels occurred at 4.67 versus 6.44 hIVM for 41.0 and 38.5°C COCs, respectively (SEM = 0.23; P < 0.001). Peak IL6ST levels occurred at 6.95 versus 8.29 hIVM for 41.0 and 38.5°C, respectively (SEM = 0.23; P < 0.01). Transcript for LIF differed over time (P < 0.0001) but was not affected by 41.0°C exposure. Blastocyst development after performing IVF was not affected by 41.0°C exposure for 4 or 6 h. When limiting analysis to when IL6 was temporally produced, progesterone levels were only impacted by time and temperature (no interaction). Heat-induced shift in the temporal production of IL6 and IL6ST along with its impact on progesterone likely cooperate in heat-induced hastening of meiotic progression described by others.

Impact of an acute heat shock during in vitro maturation on interleukin 6 and its associated receptor component transcripts in bovine cumulus-oocyte complexes

An acute heat stress event after the LH surge increased interleukin 6 (IL6) levels in the follicular fluid of the ovulatory follicle in hyperthermic cows. To examine direct consequences of a physiologically-relevant elevated temperature (41.0°C) on the cumulus-oocyte complex (COC), IL6 transcript abundance and related receptor components were evaluated throughout in vitro maturation. Heat-induced increases in IL6 were first noted at 4 hours of in vitro maturation (hIVM); peak levels occurred at 4.67 versus 6.44 hIVM for 41.0 and 38.5°C COCs, respectively (SEM = 0.23; P < 0.001). Peak IL6ST levels occurred at 6.95 versus 8.29 hIVM for 41.0 and 38.5°C, respectively (SEM = 0.23; P < 0.01). Transcript for LIF differed over time (P < 0.0001) but was not affected by 41.0°C exposure. Blastocyst development after performing IVF was not affected by 41.0°C exposure for 4 or 6 h. When limiting analysis to when IL6 was temporally produced, progesterone levels were only impacted by time and temperature (no interaction). Heat-induced shift in the temporal production of IL6 and IL6ST along with its impact on progesterone likely cooperate in heat-induced hastening of meiotic progression described by others.(AU)

Avaliação morfológica e nuclear de oócitos bovinos imaturos, obtidos de ovários com e sem a presença de corpo lúteo / Morphological evaluation and nuclear oocytes immature, obtained from ovarian with and without the body presence luteum

A produção in vitro de embriões (PIVE) é uma biotecnologia que possibilita principalmente o rápido aumento no número de descendentes com mérito genético superior. Atualmente o Brasil é líder mundial na PIVE e grande parte desse sucesso deve-se às inúmeras pesquisas realizadas nessa área. A seleção de oócitos de boa qualidade para essa técnica é determinante para obtenção de embriões de qualidade e satisfatórios índices de prenhez. Desta forma, esse trabalho teve como objetivo avaliar oócitos, obtidos de ovários com e sem a presença de corpo lúteo (CL), de acordo com a classificação morfológica e nuclear, afim de identificar a possível influência do CL na viabilidade dos oócitos. Foram coletados ovários de abatedouro e separados em dois grupos, com e sem CL. Os folículos foram aspirados, e os oócitos obtidos foram selecionados, classificados e em seguida corados com Hoechst 33342 e avaliados sob microscópio de epifluorescência, para avaliação nuclear. Realizou-se análise descritiva de cada tratamento com relação a classificação morfológica e nuclear. Os resultados obtidos foram analisados através de testes não-paramétricos, sendo o efeito do corpo lúteo verificado por meio do teste Qui-quadrado (2) (R core Team, 2013). Concluiu-se que a presença de CL não influencia na qualidade de oócitos imaturos, do ponto de vista quantitativo, morfológico e nuclear.

The in vitro embryo production (IVP) is a biotechnology that primarily enables the rapid increase in the number of offspring with superior genetic merit . Currently, Brazil is the world leader in IVP and much of this success is due to the extensive research carried out in that area. The selection of good quality oocytes for this technique is critical to obtaining quality embryos and satisfactory pregnancy rates. Thus, this study aimed to assess oocytes obtained from ovaries with and without the presence of corpus luteum (CL), according to the morphological and nuclear classification, in order to identify the possible influence of CL on the viability of oocytes. They were collected from slaughterhouse ovaries and separated into two groups, with and without CL. Follicles were aspirated and the oocytes were selected, ranked, and then stained with Hoechst 33342 and evaluated by epifluorescence microscope for nuclear evaluation. A descriptive analysis of each treatment with respect to morphological and nuclear classification. The results were analyzed using non-parametric tests, with the effect of the corpus luteum verified using the chisquare test (χ2) (R Core Team, 2013). It was concluded that the presence of CL does not influence the quality of immature oocytes, from the quantitative and morphological nuclear standpoint.

Avaliação morfológica e nuclear de oócitos bovinos imaturos, obtidos de ovários com e sem a presença de corpo lúteo / Morphological evaluation and nuclear oocytes immature, obtained from ovarian with and without the body presence luteum

A produção in vitro de embriões (PIVE) é uma biotecnologia que possibilita principalmente o rápido aumento no número de descendentes com mérito genético superior. Atualmente o Brasil é líder mundial na PIVE e grande parte desse sucesso deve-se às inúmeras pesquisas realizadas nessa área. A seleção de oócitos de boa qualidade para essa técnica é determinante para obtenção de embriões de qualidade e satisfatórios índices de prenhez. Desta forma, esse trabalho teve como objetivo avaliar oócitos, obtidos de ovários com e sem a presença de corpo lúteo (CL), de acordo com a classificação morfológica e nuclear, afim de identificar a possível influência do CL na viabilidade dos oócitos. Foram coletados ovários de abatedouro e separados em dois grupos, com e sem CL. Os folículos foram aspirados, e os oócitos obtidos foram selecionados, classificados e em seguida corados com Hoechst 33342 e avaliados sob microscópio de epifluorescência, para avaliação nuclear. Realizou-se análise descritiva de cada tratamento com relação a classificação morfológica e nuclear. Os resultados obtidos foram analisados através de testes não-paramétricos, sendo o efeito do corpo lúteo verificado por meio do teste Qui-quadrado (2) (R core Team, 2013). Concluiu-se que a presença de CL não influencia na qualidade de oócitos imaturos, do ponto de vista quantitativo, morfológico e nuclear.(AU)

The in vitro embryo production (IVP) is a biotechnology that primarily enables the rapid increase in the number of offspring with superior genetic merit . Currently, Brazil is the world leader in IVP and much of this success is due to the extensive research carried out in that area. The selection of good quality oocytes for this technique is critical to obtaining quality embryos and satisfactory pregnancy rates. Thus, this study aimed to assess oocytes obtained from ovaries with and without the presence of corpus luteum (CL), according to the morphological and nuclear classification, in order to identify the possible influence of CL on the viability of oocytes. They were collected from slaughterhouse ovaries and separated into two groups, with and without CL. Follicles were aspirated and the oocytes were selected, ranked, and then stained with Hoechst 33342 and evaluated by epifluorescence microscope for nuclear evaluation. A descriptive analysis of each treatment with respect to morphological and nuclear classification. The results were analyzed using non-parametric tests, with the effect of the corpus luteum verified using the chisquare test (χ2) (R Core Team, 2013). It was concluded that the presence of CL does not influence the quality of immature oocytes, from the quantitative and morphological nuclear standpoint.(AU)

Natural infection in ovarian structures by bovine herpesvirus 1: molecular and serological detection / Infecção natural em estruturas ovarianas pelo Herpesvírus bovino 1: detecção molecular e sorológica

In this study, the polymerase chain reaction (PCR) was used to evaluate the presence of viral DNA in ovarian tissue, in the cumulus-oocyte complex (COC), follicular liquid, and blood of animals naturally infected with bovine herpesvirus-1 (BoHV-1). The serum profile of the sampled animals was also evaluated. Samples of serum, blood, ovarian tissue, follicular liquid, and COC were collected from 147 slaughterhouse animals that were not vaccinated against BoHV-1. Contaminated or insufficient samples were disregarded. Serological tests allowed the identification of serum-positive animals with neutralizing antibodies against BoHV-1. Analysis of samples by PCR revealed the presence of viral DNA in 0.9% (1/115) of the COC samples, in 4.3% (5/117) of the ovarian tissue samples, and in 2.8% (3/108) of the blood samples. Viral DNA was not detected in any of the follicular liquid samples. In serological samples, a positivity of 83.6% (117/140) was observed for BoHV-1. All PCR-positive animals, regardless of the samples analyzed, showed positivity in the serum neutralization test for the detection of BoHV-1-specific antibodies. According to these results, a high prevalence of antibodies against BoHV-1 was detected in naturally infected animals from different herds, and the molecular tests revealed the presence of viral DNA in bovine ovarian tissue, providing evidence that this might be...(AU)

Neste trabalho foi avaliada a presença do DNA viral, por meio da Reação em Cadeia da Polimerase (PCR), no tecido ovariano, nos oócitos, líquido folicular e sangue de vacas naturalmente infectadas. Também foi avaliado o perfil sorológico dos animais amostrados. Foram coletadas amostras de soro, sangue, tecido ovariano, líquido folicular e complexo cumulus-oócitos de 147 animais abatidos em frigorífico não vacinados contra o herpesvirus bovino 1 (BoHV-1). Amostras tóxicas ou insuficientes foram descartadas. Os testes sorológicos foram realizados permitindo a identificação dos animais soropositivos para anticorpos neutralizantes contra o BoHV-1. Foram realizadas as PCRs onde foi observada a presença do DNA viral em 0,9% (1/115) dos oócitos, em 4,3% (5/117) do tecido ovariano e em 2,8% (3/108) do sangue. Em nenhuma das amostras de líquido folicular foi detectado o DNA viral. Nas amostras sorológicas observou-se 83,6% (117/140) de positividade para o BoHV-1. Dentre os animais positivos na PCR, independente das amostras, todos apresentavam positividade no teste de soroneutralização para detecção de anticorpos para BoHV-1. Conclui-se que em animais de diferentes rebanhos analisados foi detectada alta prevalência de anticorpos contra o BoHV-1 e que nos testes moleculares houve a presença do DNA viral em amostras de tecidos ovarianos de bovinos, evidenciando que estas estruturas poderia...(AU)

INTERAÇÃO YAP1-TEAD REGULA A CASCATA DE SINALIZAÇÃO DO EGF EM CÉLULAS DO CUMULUS EM BOVINOS

O mecanismo de expansão das células do cumulus (CC) e ovulação in vivo é desencadeado pelo pico de LH nas células da granulosa e CC, culminando com uma série de comunicações parácrinas e autócrinas entre estas células ovarianas. Está estabelecido que fatores secretados pela granulosa atuam sobre as CC estimulando a transcrição de genes importantes para a expansão, maturação oocitária e ovulação. Embora já sejam conhecidas diversas vias de sinalização envolvidas com esse processo, ainda não se tem o completo entendimento. Recentemente, a via de sinalização Hippo e seus efetores (YAP1 e TAZ) vêm sendo relacionados com diferentes contextos da fisiologia ovariana, inclusive com o processo ovulatório, em camundongos e humanos. Porém, ainda são escassos os estudos relacionando essa via com espécies de produção. O objetivo deste estudo foi demonstrar a função da ligação YAP1 com fatores de transcrição TEAD na modulação de genes-chave para a expansão e mecanismo ovulatório nas células do cumulus de bovinos e a sua regulação. Para isso, complexos cumulus-oócito (CCO) foram cultivados in vitro durante 0 e 12h e submetidos a imunofluorescência para demonstrar a localização intracelular de YAP1 e pYAP1. Os resultados sugerem que YAP1 transloca do citoplasma para o núcleo das CC durante o processo de maturação in vitro (MIV). Para elucidar a regulação de YAP1, receptores de EGF (EGFR; com AG 1478) foram inibidos e então, os CCOs foram estimulados com EGF ou FSH. Com isso, foi demonstrado que a inibição de EGFR resulta na redução da expressão de YAP1 e CTGF (um dos principais genes-alvo de YAP1-TEAD) nas CC. Então, os CCOs foram tratados com diferentes concentrações de verteporfina, um inibidor da interação YAP1-TEAD e obteve-se a redução de maneira concentração-dependente de genes-chave para a expansão e ovulação nas CC. A interação YAP1-TEAD está envolvida na expressão de genes-chave para a expansão e mecanismo ovulatório em células do cumulus de bovinos e a regulação de YAP1 é mediada através da estimulação de EGFR, por ação direta de EGF, ou indireta de FSH.

The mechanism of cumulus cell (CC) expansion and ovulation in vivo is triggered by LH surge in granulosa cells and CC, which culminating in a series of paracrine and autocrine communications between these ovarian cells. It is well established that granulosa-secreted factors act on CC, stimulating important genes involved in cumulus expansion, oocyte maturation and ovulation. Although several signaling pathways involved in this process are already known, there is no complete understanding in this physiological process. Recently, the Hippo signaling pathway and its effectors (YAP1 and TAZ) have been related to different contexts of ovarian physiology, including the ovulatory process, in mice and humans. However, few studies relating this pathway are available in economic relevant species. The aim of this study was to demonstrate the function of YAP1-TEAD interaction in the modulation of key genes for expansion and ovulatory mechanism, as well as, their regulation in bovine cumulus cells. For this, cumulus-oocyte complexes (COCs) were in vitro cultured during 0 or 12h and subjected to immunofluorescence to demonstrate YAP1 and pYAP1 intracellular localization. Our results suggest that YAP1 translocates from the cytoplasm to the nucleus during in vitro maturation (IVM) time. To elucidate YAP1 regulation, we inhibited EGF receptor (EGFR, with AG 1478) and stimulated COCs with EGF or FSH. Thus, EGFR inhibition results in reduction of YAP1 and CTGF (one of the major YAP1-TEAD target genes) expression. Then, COCs were subjected to different concentrations of verteporfin, an YAP1-TEAD inhibitor, and it was obtained dose-dependent reduction of cumulus expansion (HAS2 and PTX3) and ovulation (ADAM17, EREG and PTGS2) related genes. Based on that, it is suggested that YAP1-TEAD interaction is involved in the expression of key genes for cumulus expansion and ovulation in bovine cumulus cells. Also, YAP1 regulation is mediated through EGFR stimulation, by EGF direct action, or FSH indirect action.

Fatores anatomofisiologicos que afetam a qualidade oocitária em bovinos / Anatomophysiologic factors affecting oocyte quality in cattle

Para estudar os fatores anatomofisiológicos que interferem na qualidade de complexos cumulus-oócitos (CCOs) bovinos, foram obtidas 396 ovários após abate de 198 fêmeas Bos indicus em frigorífico. Os ovários foram separados por categorias, sendo distribuídos em nulípara vs multípara e com progesterona (P4 - presença de corpo lúteo em um dos ovários) vs sem progesterona (NP4 - ausência de corpo lúteo). Todos os folículos foram mensurados e categorizados em pequenos (<6mm), médios (6 a 9mm) ou grandes (>9mm). Em seguida todos os folículos foram puncionados e os CCOs recuperados e avaliados morfologicamente. Não houve diferença na taxa de recuperação nem na qualidade dos CCOs de fêmeas nulíparas vs multíparas. O percentual de CCOs desnudos/degenerados foi maior no grupo NP4 e os CCOs expandidos foram superiores no grupo P4. A taxa de recuperação e o percentual de CCOs selecionados para PIV (graus I e II) foram similares nos grupos P4 vs NP4. Folículos pequenos apresentam menor taxa de recuperação em comparação aos de tamanho médio e grande, porém o percentual de CCOs de grau I foi superior em folículos pequenos e médios. Diante dos resultados aqui encontrados conclui-se que a categoria da doadora e a progesterona não influenciaram a qualidade de CCOs selecionados para PIV e que folículos menores apresentam de CCOs de melhor qualidade.(AU)

To study the anatomical and physiological factors that affect the bovine cumulus-oocyte complexes (COCs) quality, were examined 396 ovaries obtained after slaughter of 198 Bos indicus females. Ovaries were categorized in nulliparous vs multiparous and progesterone (P4 - a corpus luteum at least one ovary) vs without progesterone (NP4 - absence of corpus luteum). All follicles were measured and categorized into small (<6mm), medium (6 to 9mm) or large (>9mm). Then all follicles were punctured and COCs recovered and assessed morphologically. There was no difference in COCs recovery rate or quality from nulliparous vs multiparous females. The rate of denuded or degenerated oocytes was higher in NP4 and expanded COCs were higher in P4. The COCs recovery and grades I and II rates were similar in P4 vs NP4 groups. Small follicles have lower COCs recovery rate than medium or large follicles, but the grade I oocytes rates was higher in small and medium follicles. In conclusion, the category of donor female and progesterone did not affect the quality of COCs selected for PIV and smaller follicles present best quality of COCs.(AU)

Fatores anatomofisiologicos que afetam a qualidade oocitária em bovinos

Abstract: To study the anatomical and physiological factors that affect the bovine cumulus-oocyte complexes (COCs) quality, were examined 396 ovaries obtained after slaughter of 198 Bos indicus females. Ovaries were categorized in nulliparous vs multiparous and progesterone (P4 - a corpus luteum at least one ovary) vs without progesterone (NP4 - absence of corpus luteum). All follicles were measured and categorized into small ( 6mm), medium (6 to 9mm) or large (>9mm). Then all follicles were punctured and COCs recovered and assessed morphologically. There was no difference in COCs recovery rate or quality from nulliparous vs multiparous females. The rate of denuded or degenerated oocytes was higher in NP4 and expanded COCs were higher in P4. The COCs recovery and grades I and II rates were similar in P4 vs NP4 groups. Small follicles have lower COCs recovery rate than medium or large follicles, but the grade I oocytes rates was higher in small and medium follicles. In conclusion, the category of donor female and progesterone did not affect the quality of COCs selected for PIV and smaller follicles present best quality of COCs.

Resumo: Para estudar os fatores anatomofisiológicos que interferem na qualidade de complexos cumulus-oócitos (CCOs) bovinos, foram obtidas 396 ovários após abate de 198 fêmeas Bos indicus em frigorífico. Os ovários foram separados por categorias, sendo distribuídos em nulípara vs multípara e com progesterona (P4 - presença de corpo lúteo em um dos ovários) vs sem progesterona (NP4 - ausência de corpo lúteo). Todos os folículos foram mensurados e categorizados em pequenos ( 6mm), médios (6 a 9mm) ou grandes (>9mm). Em seguida todos os folículos foram puncionados e os CCOs recuperados e avaliados morfologicamente. Não houve diferença na taxa de recuperação nem na qualidade dos CCOs de fêmeas nulíparas vs multíparas. O percentual de CCOs desnudos/degenerados foi maior no grupo NP4 e os CCOs expandidos foram superiores no grupo P4. A taxa de recuperação e o percentual de CCOs selecionados para PIV (graus I e II) foram similares nos grupos P4 vs NP4. Folículos pequenos apresentam menor taxa de recuperação em comparação aos de tamanho médio e grande, porém o percentual de CCOs de grau I foi superior em folículos pequenos e médios. Diante dos resultados aqui encontrados conclui-se que a categoria da doadora e a progesterona não influenciaram a qualidade de CCOs selecionados para PIV e que folículos menores apresentam de CCOs de melhor qualidade.

Origins and effects of oocyte quality in cattle

Oocyte quality is the resultant of multifactor interactions that should be carefully taken into account either for in vitro embryo production technologies or for studying follicle dynamics. Different approaches may be used to perform an analysis of variables related to oocyte quality which may focus on the ovary, the follicle, the cumulus-oocyte complex and, finally, the oocyte. The information obtained may answer key questions, such as what does the oocyte need to acquire meiotic competence and whether follicle activity can be manipulated to improve the in vitro embryo production efficiency. Although morphological evaluation represents the most common procedure used to discriminate the developmental potential of the oocyte and in spite of a good relationship that has been found between the quality of the oocyte and the atresia grade of the follicle that comprises it, there is not yet a clear correspondence between th e visual criteria and the developmental competence of oocytes submitted to in vitro embryo production. New technologies have b ecome available, including emerging ‘omics’ sciences that, through analysis of the cumulus cells, offer the opportunity, by a non-invasive method, to indirectly predict the developmental potential of the oocyte.

Origins and effects of oocyte quality in cattle

Oocyte quality is the resultant of multifactor interactions that should be carefully taken into account either for in vitro embryo production technologies or for studying follicle dynamics. Different approaches may be used to perform an analysis of variables related to oocyte quality which may focus on the ovary, the follicle, the cumulus-oocyte complex and, finally, the oocyte. The information obtained may answer key questions, such as what does the oocyte need to acquire meiotic competence and whether follicle activity can be manipulated to improve the in vitro embryo production efficiency. Although morphological evaluation represents the most common procedure used to discriminate the developmental potential of the oocyte and in spite of a good relationship that has been found between the quality of the oocyte and the atresia grade of the follicle that comprises it, there is not yet a clear correspondence between th e visual criteria and the developmental competence of oocytes submitted to in vitro embryo production. New technologies have b ecome available, including emerging ‘omics sciences that, through analysis of the cumulus cells, offer the opportunity, by a non-invasive method, to indirectly predict the developmental potential of the oocyte.(AU)