Follicular guidance for oocyte developmental competence

The advancement of folliculogenesis is coincident with the sequential acquisition of oocyte developmental competence. In practical bovine/porcine ART, cumulus-oocyte complexes (COCs) aspirated from small antral follicles have low developmental competence relative to COCs from medium/large antral follicles, as evidenced by a poor capacity to support embryogenesis up to the blastocyst stage. This is in part because of incomplete differentiation of cumulus cells in small antral follicles, in particular under-developed functionality of EGF signalling. Gonadotrophins and oocyte-secreted paracrine factors cooperate to establish EGF receptor functionality in cumulus cells, which appears to be involved in the acquisition of oocyte developmental competence. Here we review the modification of follicular cumulus cells during antral folliculogenesis involved in oocyte developmental competence.

Follicular guidance for oocyte developmental competence

The advancement of folliculogenesis is coincident with the sequential acquisition of oocyte developmental competence. In practical bovine/porcine ART, cumulus-oocyte complexes (COCs) aspirated from small antral follicles have low developmental competence relative to COCs from medium/large antral follicles, as evidenced by a poor capacity to support embryogenesis up to the blastocyst stage. This is in part because of incomplete differentiation of cumulus cells in small antral follicles, in particular under-developed functionality of EGF signalling. Gonadotrophins and oocyte-secreted paracrine factors cooperate to establish EGF receptor functionality in cumulus cells, which appears to be involved in the acquisition of oocyte developmental competence. Here we review the modification of follicular cumulus cells during antral folliculogenesis involved in oocyte developmental competence.(AU)

Follicular environment and oocyte maturation: roles of local peptides and steroids

A large amount of data on the mechanisms regulating cumulus-oocyte maturation in mammals has been generated in the last 20 years. It has been made clear that oocyte-secreted factors play a central role in the control of cumulus differentiation and oocyte developmental competence. However, more recent data indicate that cumulus-derived factors are also involved. In this mini-review, we have compiled and discussed data produced in our laboratory about the involvement of oocyte and cumulus-derived peptides, including fibroblast growth factors, bone morphogenetic protein 15, Kit ligand and natriuretic peptide C, in the regulation of cumulus metabolism and oocyte nuclear maturation. In addition, we discuss the interaction of follicular steroids with natriuretic peptide C in the control of meiosis progression.

Follicular environment and oocyte maturation: roles of local peptides and steroids

A large amount of data on the mechanisms regulating cumulus-oocyte maturation in mammals has been generated in the last 20 years. It has been made clear that oocyte-secreted factors play a central role in the control of cumulus differentiation and oocyte developmental competence. However, more recent data indicate that cumulus-derived factors are also involved. In this mini-review, we have compiled and discussed data produced in our laboratory about the involvement of oocyte and cumulus-derived peptides, including fibroblast growth factors, bone morphogenetic protein 15, Kit ligand and natriuretic peptide C, in the regulation of cumulus metabolism and oocyte nuclear maturation. In addition, we discuss the interaction of follicular steroids with natriuretic peptide C in the control of meiosis progression.(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.

Effects of BMP-4 and FSH on growth, morphology and mRNA expression of oocyte-secreted factors in cultured bovine secondary follicles

This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.

Effects of BMP-4 and FSH on growth, morphology and mRNA expression of oocyte-secreted factors in cultured bovine secondary follicles

This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.(AU)

Melatonina e desenvolvimento embrionário / Melatonin and embryonic development

A melatonina é o principal hormônio sintetizado e secretado pela glândula pineal e desempenha umafunção importante na regulação dos ciclos reprodutivos sazonais. O seu papel na reprodução concentra-se,sobretudo, em suas ações diretas no ovário. Ela também é conhecida como uma eliminadora de espécie reativade oxigênio (ROS). Dessa forma, o estresse oxidativo é um dos fatores que prejudicam o desenvolvimento tantoin vitro quanto in vivo dos oócitos até a fase de blastocisto. Esta revisão aborda os diversos efeitos da melatoninasobre o desenvolvimento embrionário, assim como os mecanismos responsáveis pela diminuição do estresseoxidativo durante a maturação oocitária e o desenvolvimento embrionário.(AU)

Melatonin is the principal hormone synthesized and secreted by the pineal gland and plays animportant role in the regulation of seasonal breeding cycles. Your role in reproduction is focused on its directactions in the ovary and also be known as a scavenger of reactive oxygen species (ROS). Thus oxidative stress isone of the factors hindering the development in vitro and in vivo oocyte to the blastocyst stage. This reviewcovers the various effects of melatonin on embryonic development, as well as the mechanisms responsible for thereduction of oxidative stress during oocyte maturation and embryo development.(AU)

Melatonina e desenvolvimento embrionário / Melatonin and embryonic development

A melatonina é o principal hormônio sintetizado e secretado pela glândula pineal e desempenha umafunção importante na regulação dos ciclos reprodutivos sazonais. O seu papel na reprodução concentra-se,sobretudo, em suas ações diretas no ovário. Ela também é conhecida como uma eliminadora de espécie reativade oxigênio (ROS). Dessa forma, o estresse oxidativo é um dos fatores que prejudicam o desenvolvimento tantoin vitro quanto in vivo dos oócitos até a fase de blastocisto. Esta revisão aborda os diversos efeitos da melatoninasobre o desenvolvimento embrionário, assim como os mecanismos responsáveis pela diminuição do estresseoxidativo durante a maturação oocitária e o desenvolvimento embrionário.

Melatonin is the principal hormone synthesized and secreted by the pineal gland and plays animportant role in the regulation of seasonal breeding cycles. Your role in reproduction is focused on its directactions in the ovary and also be known as a scavenger of reactive oxygen species (ROS). Thus oxidative stress isone of the factors hindering the development in vitro and in vivo oocyte to the blastocyst stage. This reviewcovers the various effects of melatonin on embryonic development, as well as the mechanisms responsible for thereduction of oxidative stress during oocyte maturation and embryo development.

Paracrine and autocrine factors in the differentiation of the cumulus-oocyte complex

A better understanding of the paracrine and autocrine regulatory loops within the cumulus-oocyte complex (COC) is fundamental for the improvement of in vitro maturation (IVM) outcomes in humans and domestic species. This review presents the most important local regulators identified in the COC to date with special attention to th ose secreted by the oocyte and acting on cumulus cells, as well as their roles in different processes crucial for the successful maturation of the COC. An autocrine re gulatory loop mediated by epidermal growth factor-lik e (EGF-like) peptides in cumulus cells triggers COC maturation. During COC differentiation, oocyte s ecreted factors (OSFs), particularly members of the transforming growth factor-  (TGF  ) and fibroblast growth factor (FGF) families, regulate meiotic resumption, cumulus expansion, cumulus metabolism, apoptosis and steroidogenesis.

Paracrine and autocrine factors in the differentiation of the cumulus-oocyte complex

A better understanding of the paracrine and autocrine regulatory loops within the cumulus-oocyte complex (COC) is fundamental for the improvement of in vitro maturation (IVM) outcomes in humans and domestic species. This review presents the most important local regulators identified in the COC to date with special attention to th ose secreted by the oocyte and acting on cumulus cells, as well as their roles in different processes crucial for the successful maturation of the COC. An autocrine re gulatory loop mediated by epidermal growth factor-lik e (EGF-like) peptides in cumulus cells triggers COC maturation. During COC differentiation, oocyte s ecreted factors (OSFs), particularly members of the transforming growth factor-  (TGF  ) and fibroblast growth factor (FGF) families, regulate meiotic resumption, cumulus expansion, cumulus metabolism, apoptosis and steroidogenesis.(AU)

Efeito de um meio condicionado por células do cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos.

O principal desafio para a produção in vitro de embriões é a obtenção de oócitos com competência para o desenvolvimento Esta competência é adquirida através da maturação, processo complexo que envolve maturação nuclear, citoplasmática e o perfeito sincronismo entre ambas (GILCHRIST, ROBERT B.; LANE; THOMPSON, 2008). As células do cumulus oophorus são chave para a sinalização local e endócrina e para a maturação oocitária (SUTTON et al., 2003). Diferentes sistemas usam células homólogas e heterólogas para tirar proveito de fatores por elas secretados (GOOVAERTS et al., 2011). Neste sentido, o presente trabalho avaliou o efeito de um meio condicionado por células do cumulus-oophorus cultivadas previamente na maturação in vitro de complexos cumulus-oócitos e oócitos desnudados de bovinos e em seu potencial de desenvolvimento subsequente. A maturação oocitária in vitro foi avaliada com base nas taxas de maturação nuclear, maturação citoplasmática, expansão das células do cumulus, dissolução da zona pelúcida e por análise em microscopia eletrônica de transmissão. O potencial de desenvolvimento embrionário foi avaliado através da taxa de clivagem, taxa de blastocistos e taxa de eclosão.

One of the challenges of reproductive biology and medicine is to understand the nature of the molecular and cellular processes that control oocyte developmental competence. This competence, acquired through maturation, a complex process that comprises nuclear and cytoplasmic maturation, involves a deep interaction between the oocyte and Cumulus oophorus cells. Cumulus oophorus cells are key for paracrine and endocrine signaling and for oocyte maturation; however, in many applications, removal of these cells, or oocyte denudation, is required before maturation begins or prior to in vitro fertilization. Because denudation reduces developmental competence, an efficient in vitro maturation system for denuded oocytes is needed, as no system has been well established in mammalians. Different systems use co-cultures of homologous and heterologous cells or media conditioned by these cells, to take advantage of factors secreted by them and optimize results in in vitro production. The present study evaluates the effect of a medium conditioned by a culture of bovine Cumulus-oophorus cells in the in vitro maturation of bovine denuded oocytes and their embryonic development potential. The experiment was carried out in the Laboratory of in vitro Production of Embryos at the School of Veterinary Medicine, Federal University of Minas Gerais, Brazil. The oocytes were aspirated from follicles between 2 and 8 mm in diameter from slaughterhouse ovaries. Grade I and II oocytes, half of which had been denuded by successive pipetting prior to maturation, were matured in control medium and in the conditioned media previously obtained from the culture of Cumulus oophorus cells. 2927 oocytes were matured, of which 894 were used for evaluation of maturation and 2033 for evaluation of embryonic development. The evaluation of in vitro oocyte maturation was performed based on nuclear maturation, cytoplasmic maturation (mitochondrial distribution), enzymatic dissolution of zona pellucida with chymotrypsin 1% and pronase 0.5% and ultrastructure by transmission electron microscopy. The evaluation of embryonic development potential was based on embryo cleavage and blastocyst rates. There were no significant differences in nuclear maturation of oocytes in control medium and conditioned medium, as well as no differences between nuclear maturation of CCOs and denuded oocytes. Cytoplasmic maturation in conditioned medium was not significantly different from that in control medium. Denuded oocytes presented a predominantly heterogeneous-peripheral mitochondrial distribution pattern, whereas CCOs had a predominant heterogeneous-perinuclear pattern. Denuded oocytes matured in the conditioned medium showed a significant reduction of the dissolution time of the zona pellucida in enzymatic dissolution assay with chymotrypsin 1%, but there were inconsistencies between the results obtained in the assays with the two enzymes. There were no significant differences between cleavage and blastocysts rates between oocytes matured in control medium and conditioned medium. In conclusion, under these experimental conditions, the conditioned medium from bovine Cumulus oophorus cells did not prove to be superior to the control medium in relation to the in vitro oocyte maturation of the denuded oocytes nor with respect to its embryonic development and, therefore, its incorporation into in vitro embryo production routines is not recommended. Further studies are still needed to better understand the events related to the acquisition of oocyte competence and to develop more efficient in vitro maturation media for denuded oocytes.

Efeitos e regulação da expressão do kit ligante (KL) durante a maturação in vitro do complexo cumulus-oócito bovino

O desenvolvimento da competência oocitária ocorre de forma gradual durante a foliculogênese e é dependente da interação do oócito com as células do cumulus via fatores parácrinos. O KL é expresso pelas células do cumulus e estimula a maturação nuclear do oócito em roedores, mas suas funções no complexo cumulus-oócito (CCO) bovino não são conhecidas. Neste trabalho, avaliou-se o padrão de expressão do KL, os efeitos do KL sobre a progressão da meiose e a expansão do cumulus, bem como a regulação da expressão do KL por fatores secretados pelo oócito (FSOs; BMP15, FGF8, FGF10, FGF16 e FGF17) durante a maturação in vitro (MIV) de CCOs bovinos. Adicionalmente, os efeitos do KL sobre a expressão de BMP15, GDF9, FGF16 e c-KIT foi avaliada. As isoformas do KL (KL1 e KL2) apresentaram padrões de expressão semelhantes nas células do cumulus ao longo da MIV, com pico de expressão às 12 horas do cultivo. A adição de KL ao cultivo aumentou a porcentagem de oócitos em meiose II e diminuiu a de oócitos em meiose I ao final da MIV, mas não afetou a expansão do cumulus. O FGF8 aumentou a expressão dos RNAm do KL1 e do KL2, enquanto a BMP15 estimulou apenas a expressão do RNAm do KL1. Distintamente, a dose máxima do FGF10 diminuiu a expressão do KL2 às 22 horas do cultivo em estudo dose-resposta, embora o mesmo não tenha sido observado quando uma dose menor, porém efetiva em estimular a expansão do cumulus, foi testada ao longo da MIV (4, 12 e 22 horas). O tratamento com FGF10 e BMP15 associados aumentou a expressão do KL1 às 12 horas do cultivo em relação ao controle e aos fatores de crescimento sozinhos, sugerindo cooperação entre eles na regulação da expressão do KL. A adição de KL ao meio de cultivo não alterou a expressão dos FSOs. Em conjunto, os resultados deste estudo sugerem o...

The development of oocyte competence occurs gradually during folliculogenesis and is dependent on the interaction of the oocyte with cumulus cells via paracrine factors. The KL is expressed by cumulus cells and stimulates oocyte nuclear maturation in rodents, but its functions in cumulus-oocyte complex (COC) cattle are unknown. In this study, we evaluated the expression pattern of KL, the effects of KL on the progression of meiosis and cumulus expansion and regulation of KL expression by factors secreted by the oocyte (OSF; BMP15, FGF8, FGF10, FGF16 and FGF17) during in vitro maturation (IVM) of bovine COCs. Additionally, the effect of KL on the expression of BMP15, GDF9, FGF16 and c-KIT was evaluated. Isoforms of KL (KL1 and KL2) showed similar expression patterns in cumulus cells along the MIV, with higher expression at 12 hours of cultivation. The addition of KL increased the percentage of oocytes in meiosis II and decreased oocyte meiosis I in the end of IVM, but did not affect cumulus expansion. The FGF8 increased expression of KL1 and KL2 mRNA, while only BMP15 stimulated mRNA expression KL1. Distinctly, the maximum dose of FGF10 decreased expression of KL2 at 22 hours of cultivation in dose-response study, although the same was not observed when a lower dose, but effective to stimulate cumulus expansion was tested along MIV (4, 12 and 22 hours). Treatment with FGF10 and BMP15 associated with increased expression of KL1 at 12 hours of culture in relation to the control and growth factors alone, suggesting cooperation between them in the regulation of expression of the KL. The addition of KL to the culture medium did not alter the expression of the FSOs. Taken together, our results suggest the involvement of KL in controlling bovine oocyte nuclear maturation and expression of KL in bovine cumulus cells is...