Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation'

Radiotherapy causes destruction of tumor cells, but also threatens the integrity and survival of surrounding normal cells. Then, woman submitted to irradiation for cancer treatment may present permanent ovary damage, resulting in impaired fertility. The objective of this study was to investigate the effects of therapeutic doses of ionizing radiation (IR), used for ovarian cancer treatment in humans, on bovine cumulus-oocyte complexes (COCs) as experimental model. Bovine ovaries were exposed to 0.9 Gy, 1.8 Gy, 3.6 Gy or 18.6 Gy IR, and then COCs were collected and used to evaluate: (a) oocyte nuclear maturation; (b) presence of phosphorylated H2A.X (γH2AX), as an indicator of DNA double-strand breaks (DSBs); and (c) expression of genes involved in DNA repair (TP53BP1, RAD52, ATM, XRCC6 and XRCC5) and apoptosis (BAX). The radiation doses tested in this study had no detrimental effects on nuclear maturation and did not increase γH2AX in the oocytes. However, IR treatment altered the mRNA abundance of RAD52 (RAD52 homolog, DNA repair protein) and BAX (BCL2-associated X protein). We conclude that although IR doses had no apparent effect on oocyte nuclear maturation and DNA damage, molecular pathways involved in DNA repair and apoptosis were affected by IR exposure in cumulus cells.(AU)

Diet crude protein reduction on follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows

This study evaluated the effect of crude protein (CP) reduction in four diets (156, 139, 132, and 127 g Kg-1 DM) maintaining constant metabolizable protein (188 g/day) on the follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows. Twenty-two Girolando cows with average of 21.55 ±3.19 L daily milk yield, 105.30 ±22.62 days in lactation and 3.22 ±0.03 body condition score were selected. To reduce CP in diets and maintain constant metabolizable protein, urea and soybean meal were gradually replaced by lignosulfonate-treated soybean meal (SoyPass®, Cargill), resulting in an increase in rumen-undegradable protein and a reduction in rumen degradable protein. A linear and quadratic reduction was observed in the plasma and follicular fluid urea nitrogen concentration following CP reduction, with the most intense reduction occurring in the 127 g Kg-1 DM group (p<0.001). As CP reduced, there was a tendency for a linear increase in the follicular growth rate (P=0.0696), on the number and proportion of viable oocytes (P<0.09), and also a linear increase for the number (P=0.0397) and proportion (P<0.09) of grade I viable oocytes. Plus, there was a linear effect for the number of cumulus oophorus cells. Cows fed with the lowest amount of CP had cumulus-oocyte complexes with higher numbers of cumulus oophorus cells (P=0.0238). Also, the reduction of diet crude protein was followed by a decrease in the probability of oocytes' DNA degradation. In conclusion, the reduction of CP in the diet of mid-lactating Girolando cows, reduces urea nitrogen concentration in both blood plasma and follicular fluid, and, as a consequence, increases the viability of oocytes and the number of cumulus oophorus cells while reducing oocytes' DNA degradation of follicular included cumulus-oocyte complex. The reduction on dietary CP may improve in vivo oocytes' embryo development impacting fertility of lactating dairy cows.(AU)

Relative transcript level of BMP15, BAX and CASP3 with qRT-PCR in vitrified equine immature cumulus-oocytes complexes / Relative transcript level of BMP15, BAX and CASP3 with qRT-PCR in vitrified equine immature cumulus-oocytes complexes

Research focused on female gamete vitrification has increased attention to develop a reliable cryopreservation method to preserve immature equine oocytes. Despite the intensive implementation of biotechnological procedures for horse breeding, vitrification of immature equine cumulus-oocyte complexes (COCs) remain to be clearly elucidated. We aimed to determine the relative transcript level of target genes Bone morphogenetic protein 15 (BMP15); Bcl-2-associated X protein (BAX); and Caspase 3 (CASP3) in equine COCs prior to and after vitrification. Ovarian follicles were aspirated from ovaries collected from an abattoir. A total of 240 COCs were collected and distributed into vitrified COCs (VIT, n=120) and nonvitrified (Non-VIT, n=120) groups. Then, COCs were preserved and relative transcript expressions of BMP15, BAX, CASP3 were measured and normalized against GAPDH performed by qRT-PCR. In addition, 38 COCs were evaluated to assess chromatin configuration of germinal vesicl e stage prior and after vitrification by exposure to 10 µg/ml of bisbenzimide. A difference was observed in the COCs' mRNA level of abundance for the BAX gene between the VIT (2.05 ± 0.47) and (0.85 ± 0.08) Non-VIT groups. There was no difference in mRNA relative transcript level of CASP3 and BMP15 in Non-VIT (0.63 ± 0.20 and 1.55 ± 0.73, respectively) compared to VIT (0.64 ± 0.01 and 2.84 ± 2.20, respectively) equine COCs. All COCs where considered at immature stage of development even though COCs in Non-VIT group showed higher condensed chromatin configuration compared to VIT (100% vs 60.7%, respectively). We demonstrate that BMP15 and CASP3 are detected in VIT and Non-VIT immature COCs. In conclusion, BAX is expressed highly in vitrified immature equine COCs and indicates that activation of apoptosis signaling cascades in cells exposed to vitrification.(AU)

Pesquisas sobre a vitrificação de gametas femininos estão sendo realizadas para o desenvolvimento de um método confiável de criopreservação dos complexos cumulus-oócitos (CCOs) na espécie equina. Apesar da implementação intensiva de biotecnologias reprodutivas em equinos, a vitrificação dos CCOs imaturos permanece em estágio experimental em relação à competência celular. O objetivo do estudo foi determinar o nível de transcrição relativo dos genes Proteína morfogenética óssea 15 (BMP15); Proteína X associada a Bcl-2 (BAX); e Caspase 3 (CASP3) em CCOs equinos antes e após a vitrificação. Folículos ovarianos foram aspirados de ovários coletados em matadouro. O total de 240 CCOs foi coletado e distribuído em grupos vitrificados (VIT, n=120) e não vitrificados (N-VIT, n=120). Os CCOs foram preservados e as expressões transcritas relativas de BMP15, BAX, CASP3 foram determinadas pela técnica de qRT-PCR sendo normalizadas em relação ao GAPDH. Além disso, 38 CCOs foram avaliados para determinar a configuração da cromatina no estágio de vesícula germinativa antes e após a vitrificação pela exposição a 10 µg/ml de bisbenzimida. Os resultados mostraram uma diferença no nível de abundância de mRNA dos CCOs para o gene BAX entre os grupos VIT (2,05 ± 0,47) e N-VIT (0,85 ± 0,08). Não houve diferença no nível de transcrição relativa do mRNA de CASP3 e BMP15 nos CCOs do grupo N-VIT (0,63 ± 0,20 e 1,55 ± 0,73, respectivamente) em comparação com VIT (0,64 ± 0,01 e 2,84 ± 2,20, respectivamente). Todos os CCOs foram considerados em estágio imaturo de desenvolvimento, embora os CCOs no grupo N-VIT apresentaram a configuração de cromatina condensada em maior número de células avaliadas em comparação com VIT (100% vs 60,7%, respectivamente). Demonstramos que BMP15 e CASP3 são detectados em CCOs imaturos em VIT e N-VIT. Conclui-se que o BAX é altamente expresso em CCOs equinos imaturos vitrificados sendo relacionado à sinalização de apoptose em células expostas ao processo de vitrificação.(AU)

Dynamics of nuclear and cytoplasm maturation of bovine oocytes cultivated in vitro in medium supplemented with fulerol / Dinâmica da maturação nuclear e citoplasmática de oócitos bovinos cultivados in vitro em meio suplementado com fulerol

The aim of this work was to evaluate, in vitro, the dynamics of nuclear and cytoplasmic maturation of bovine oocytes in traditional IVM medium (CT) and supplemented with fullerol (MF50), for 36 hours. The nuclear maturation of CT (n=300) and MF50 (n=270) every 6 hours, stained with Hoechst33342 and cytoplasmic, the mitochondrial distribution of CT (n=197) and MF50 (n=159) at every 12 hours, stained with Mitotracker Orange. At 6 hours, CT oocytes (19%) were in MI (metaphase I), while in MF50 they were in GV (germ vesicle) or GVB (GV breakeage), repeating at 12 hours. At 18 hours, 46.3% were matured in CT, and 20% in MF50. At 24 hours, 43.9% of maturation was observed in the MF50 group, and 63.8% in the CT. At 30 and 36 hours, the maturation pattern was stable, but with the onset of oocyte degeneration. There was a delay in cytoplasmic maturation with 36 hours (P < 0.05) in MF50 (53.9% of mature gametes), compared to CT (69.8%). With immature cytoplasm, they were 10.4% and 31.7% for CT and MF50 (P< 0.05), respectively. It was concluded that fullerol possibly interfered in the expansion of cumulus oophorus cells, as well as delayed the meiotic progression and cytoplasmic maturation.

O objetivo deste estudo foi avaliar, in vitro, a dinâmica da maturação nuclear e citoplasmática de oócitos bovinos em meio MIV tradicional (TC) e suplementado com fulerol (MF50), durante 36 horas. Na maturação nuclear do TC (n=300) e do MF50 (n=270) a cada seis horas, corados com Hoechst 33342, e na citoplasmática, avaliou-se a distribuição mitocondrial do TC (n=197) e do MF50 (n=159) a cada 12 horas, corados com Mitotracker Orange (Life® Technologies). Às seis horas, oócitos do TC (19%) se encontravam em MI (metáfase I), enquanto no MF50 estavam em VG (vesícula germinativa) ou QVG (quebra VG), repetindo com 12 horas. Às 18 horas, 46,3% estavam maturados no TC, e 20% no MF50. Com 24 horas, verificaram-se 43,9% de maturação no grupo MF50, e 63,8% no TC. Às 30 e 36 horas, o padrão de maturação foi estável, mas com início de degeneração oócitária. Houve retardo na maturação citoplasmática com 36 horas (P<0,05) no MF50 (53,9% de gametas maduros), comparado ao TC (69,8%). Com citoplasma imaturo, foram 10,4% e 31,7% para TC e MF50 (P<0,05), respectivamente. Conclui-se que o fulerol possivelmente interferiu na expansão das células do cumulus oophorus, bem como retardou a progressão meiótica e a maturação citoplasmática dos oócitos.

Influência das espécies reativas de oxigênio durante o cultivo in vitro de oócitos e folículos ovarianos de mamíferos domésticos / Influence of reactive oxygen species during in vitro culture of ovarian oocytes and follicles of domestic mammals

Sistemas de cultivo in vitro de folículos pré-antrais e de complexos cumulus-oócitos (CCOs) se tornaram uma poderosa ferramenta capaz de subsidiar diversas biotécnicas reprodutivas, bem como possibilitar estudos do efeito de substâncias sobre a dinâmica folicular. No entanto, esses sistemas podem resultar em estresse oxidativo e na diminuição da proteção antioxidante das células, distúrbio associado a altas taxas de morte celular durante o cultivo in vitro. Para contornar esses efeitos adversos, a adição de substâncias antioxidantes aos meios de cultivo tem sido proposta. Abrangendo diferentes classes e mecanismos de ação, compostos antioxidantes têm por função interferir no processo de oxidação para inibir ou retardar o dano oxidativo causado pelos radicais livres às biomoléculas. Além de antioxidantes que têm sido rotineiramente utilizados com esse propósito, recentemente, substâncias alternativas de origem natural como extratos vegetais e óleos essenciais têm ganhado destaque. Dessa forma, diante da influência do estresse oxidativo e da importância do uso de antioxidantes no cultivo celular, a presente revisão objetiva abordar os principais mecanismos de síntese e atuação dos radicais livres bem como o papel dos antioxidantes em protocolos de cultivo in vitro de folículos ovarianos e de CCOs de animais domésticos.(AU)

In vitro culture systems for preantral follicles and cumulus-oocyte complexes (COCs) have become a powerful tool capable of subsidizing several reproductive biotechniques, as well as enabling studies of the effect of substances on follicular dynamics. However, these systems can favor oxidative stress and decrease the antioxidant protection of cells, a disorder associated with high rates of cell death during in vitro culture. To overcome these adverse effects, the addition of antioxidant substances to the culture media has been proposed. Covering different classes and mechanisms of action, these antioxidant compounds have the function of interfering in the oxidation process to inhibit or delay the oxidative damage caused by free radicals to biomolecules. In addition to antioxidants that have been commonly used for this purpose, recently, alternative substances of natural origin such as plant extracts and essential oils have gained prominence. Thus, given the influence of oxidative stress and the importance of using antioxidants in cell culture, this review aims to address the main mechanisms of synthesis and action of free radicals as well as the role of antioxidants in in vitro protocols for ovarian follicles and COCs in domestic animals.(AU)

The effect of adding L-ergothioneine in culture medium on blastocyst development rates

Background: Oocytes and embryos produce energy through mitochondrial oxidative phosphorylation by using oxygen. The membrane structure of the embryo is mostly composed of unsaturated fatty acids, for this reason DNA fragmentation, apoptosis, and abnormal gene expression are shaped as a result of the lipid peroxidation during culture. Oxidative stress (OS) is one of the most important problems affecting the in vitro embryo development. Antioxidant supplementation to the culture medium has been an alternative way to reduce cell damage caused by oxidative stress in in vitro embryo production systems. In this study, it was aimed to determine the effect of L-ergothioneine on blastocyst development when added to the culture medium. Materials, Methods & Results: The material of the study consisted of oocytes aspirated from the ovaries of Holstein cows which were collected from the local slaughterhouse. The ovaries were delivered to the laboratory within 2-3 h in a thermos which provided a constant temperature of 25-30o C with physiological saline (0.9%) containing antibiotics. All follicles in the 3-8 mm range on the ovaries were aspirated using 20 G needle. The collected follicle fluid was filtered through filters with a pore diameter of 70 micrometers. Cells remaining in the filter were washed with OPU medium and transferred to the petri dishes. Fluids were examined under a stereomicroscope. The cumulus-oocyte complexes were classified, and A and B quality oocytes were included to the study (A, B, C, and D quality COC). Oocytes aspirated from the ovaries and collected later on were incubated in IVM medium for 22 h. After maturation, it was taken into IVF medium, semen was added and incubated for 20-22 h. Possible zygotes to be taken to the culture stage were transferred to culture (IVC) drops with (L-ergothioneine 100 µL/mL (n:121) added and without antioxidant (control (n:124)), and kept in the incubator for 6-7 days. Evaluated on the 7th day differences in in vitro embryo production stages were evaluated with the Chi-square test. The study was run in 5 replicates each time, with at least 20 possible zygotes for per group being cultured. It was determined that 262 (87.33%) of a total of 300 oocytes undergoing in vitro maturation were matured. It was determined that 245 of the mature oocytes were fertilized (93.51%). The cleavage rates of the groups were determined as 87.60% and 86.29%, respectively. Eighty-two (33.47%) blastocysts were obtained from 245 zygotes taken into the culture stage, and the blastocyst rates in the groups were found to be 40.50% and 26.61%, respectively. After the study, it was determined that the statistical difference between L-ergothioneine and control in cleavage rates was insignificant (P > 0.05) and blastocyst rates was significant (P < 0.05) Discussion: Oxygen content above normal ratios can increase the formation of reactive oxygen species (ROS), particularly hydrogen peroxide (H2 O2 ), hydroxyl radical (HO·), and peroxyl radicals (ROO·). The increased rate of ROS negatively affects the success of IVP in mammalian embryos. It was observed that L-ergothioneine, which has high antioxidant activity, improved blastocyst development rates, and higher blastocyst rates could be achieved compared to the control group. By investigating the use of L-ergothioneine in different doses, it was thought that the dose with the highest antioxidant activity could be added to the culture medium in in vitro embryo production and more blastocysts could be produced.

Quantification of multi-oocyte follicles in ovaries of bitches / [Quantificação de folículos multiócitos em ovários de cadelas]

The objective of this study was to quantify the number and frequency of monocyte (MnOF) and multi-oocyte (MtOF) follicles in ovaries of bitches subjected to ovary salpingohysterectomy (OSH). Right and left ovaries of 38 bitches were collected after OSH, prepared, and a histological analysis was carried out. The ovaries were subjected to surface and deep histological cuts; the follicles were classified, and the number of follicles and cumulus oophorus complexes (COC) per follicle were quantified for each histological cut. MnOF and MtOF were found in all ovaries, at different developmental stages; primary follicles were grouped in the ovarian cortex, and follicles at other follicular stages presented a random distribution. MtOF containing two, three, four, or more COC were found in the ovaries of bitches, with a decreasing frequency trend, according to the number of COC in the MtOF. The effect of the age, number of estrus, estrus interval, and number of progenies per delivery was not significant for the number and frequency of MtOF in the ovaries of the bitches, whereas the size, number of pregnancies, use and number of contraceptive applications had some effect on the number and frequency of MtOF in the ovaries of the bitches.(AU)

Objetivou-se, com este estudo, quantificar o número e a frequência de folículos monocitários (MOF) e polioocitários (POF) provenientes de ovários de cadelas submetidas à ovariossalpingo-histerectomia (OSH). Para tanto, coletaram-se os ovários (direito e esquerdo) de 38 cadelas após OSH, com posterior preparação e análise histológica. Cada ovário foi submetido a dois cortes histológicos (superficial e profundo) onde se quantificou o número e a classificação dos folículos, bem como o número de complexos cumulus oophorus (COCs) por folículo em cada corte histológico. Observaram-se MOF e POF em todos os ovários estudados, em diferentes estádios de desenvolvimento, sendo os folículos primários agrupados no córtex ovariano, frente a uma distribuição aleatória dos outros estádios foliculares. FOPs contendo dois, três, quatro ou mais COCs foram observados nos ovários de todas as fêmeas estudadas, e sua frequência tendeu a diminuir de acordo com o número de COC presente no POF. Não se observou influência da idade, do número e do intervalo de estros, assim como do número de filhotes por gestação sobre o número/frequência de FOP nos ovários das cadelas estudadas, enquanto o porte, o número de gestações, o uso e o número de contraceptivo apresentaram algum grau de influência sobre o número/frequência de FOP nos ovários das cadelas estudadas.(AU)

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Abstract Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.(AU)

Influence of long-term thermal stress on the in vitro maturation on embryo development and Heat Shock Protein abundance in zebu cattle

The objective of this study was to investigate the influence of long-term temperature stress during the in vitro maturation (IVM) of oocytes on the in vitro embryo production (IVP) and the abundance of HSP70 and HSP90 in zebu cattle. Viable cumulus-oocyte complexes (COCs) were incubated for 24 h at 37 °C, 38.5 °C, or 40 °C for the low-, physiological, and high-temperature stress treatments, respectively. Thereafter, they were subjected to in vitro fertilization and culture. Temperature did not affect the polar body extrusion. However, IVP was adversely affected when IVM took place at 37 °C and 40 °C. The highest abundance of HSP70 was observed in cumulus cells after maturation of COCs at 40 °C. In contrast, HSP70 was more abundant in oocytes at both 37 °C and 40 °C; however, at 40 °C, the difference to the control group (38.5 °C) was not significant. In contrast, the highest abundance of HSP90 was observed in oocytes and cumulus cells at 37 °C. It appears that HSP70 and HSP90 respond to cold and heat stress in different ways. In conclusion, moderately high (40 °C) and low (37 °C) thermal stress for 24 h during IVM is detrimental to the developmental competence of oocyte and is accompanied by changes in the abundances of HSP70 and HSP90, especially in cumulus cells.

Influence of long-term thermal stress on the in vitro maturation on embryo development and Heat Shock Protein abundance in zebu cattle

The objective of this study was to investigate the influence of long-term temperature stress during the in vitro maturation (IVM) of oocytes on the in vitro embryo production (IVP) and the abundance of HSP70 and HSP90 in zebu cattle. Viable cumulus-oocyte complexes (COCs) were incubated for 24 h at 37 °C, 38.5 °C, or 40 °C for the low-, physiological, and high-temperature stress treatments, respectively. Thereafter, they were subjected to in vitro fertilization and culture. Temperature did not affect the polar body extrusion. However, IVP was adversely affected when IVM took place at 37 °C and 40 °C. The highest abundance of HSP70 was observed in cumulus cells after maturation of COCs at 40 °C. In contrast, HSP70 was more abundant in oocytes at both 37 °C and 40 °C; however, at 40 °C, the difference to the control group (38.5 °C) was not significant. In contrast, the highest abundance of HSP90 was observed in oocytes and cumulus cells at 37 °C. It appears that HSP70 and HSP90 respond to cold and heat stress in different ways. In conclusion, moderately high (40 °C) and low (37 °C) thermal stress for 24 h during IVM is detrimental to the developmental competence of oocyte and is accompanied by changes in the abundances of HSP70 and HSP90, especially in cumulus cells.(AU)

Produção de embriões partenogenéticos de cutias usando Cloreto de Estrôncio e Citocalasina B / Production of parthenogenetic embryos from agoutis using strontium chloride and cytochalasin B

The study aimed to evaluate the effect of strontium chloride (SrCl2) with cytochalasin B (CB) on the activation of agouti oocytes matured in vitro for embryo production. Thus, ovaries were used for oocyte recovery by slicing. Subsequently, viable oocytes were destined for in vitro maturation (IVM) and after 24 h evaluated for the expansion and viability of the cumulus cells and presence of the first polar body (1PB). After IVM, oocytes were activated with a combination of 10 mM SrCl2 and 5 μg/mL CB for 6 h and evaluated for embryo development kinetics. Hence, 93.3% of cumulus cell expansion was observed, with 91.2% viability and 37.3% of oocytes with the presence of 1PB. Regarding embryonic development, 43.2% (19/44) of cleaved structures and 6.8% (3/44) of morulae were observed in relation to the number of oocytes and 18.8% (3/16) morulae in relation to the number of cleaved structures. Thus, the combination of SrCl2 with CB promoted the activation of oocytes matured in vitro from agouti resulting in morulae. Finally, with this study, fundamental steps for in vitro conservation through reproductive biotechniques were developed in agoutis.

Produção de embriões partenogenéticos de cutias usando Cloreto de Estrôncio e Citocalasina B / Production of parthenogenetic embryos from agoutis using strontium chloride and cytochalasin B

The study aimed to evaluate the effect of strontium chloride (SrCl2) with cytochalasin B (CB) on the activation of agouti oocytes matured in vitro for embryo production. Thus, ovaries were used for oocyte recovery by slicing. Subsequently, viable oocytes were destined for in vitro maturation (IVM) and after 24 h evaluated for the expansion and viability of the cumulus cells and presence of the first polar body (1PB). After IVM, oocytes were activated with a combination of 10 mM SrCl2 and 5 μg/mL CB for 6 h and evaluated for embryo development kinetics. Hence, 93.3% of cumulus cell expansion was observed, with 91.2% viability and 37.3% of oocytes with the presence of 1PB. Regarding embryonic development, 43.2% (19/44) of cleaved structures and 6.8% (3/44) of morulae were observed in relation to the number of oocytes and 18.8% (3/16) morulae in relation to the number of cleaved structures. Thus, the combination of SrCl2 with CB promoted the activation of oocytes matured in vitro from agouti resulting in morulae. Finally, with this study, fundamental steps for in vitro conservation through reproductive biotechniques were developed in agoutis.(AU)

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation

Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation

Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs(AU)

Intrafollicular barriers and cellular interactions during ovarian follicle development

Follicles are composed of different interdependent cell types including oocytes, cumulus, granulosa, and theca cells. Follicular cells and oocytes exchange signaling molecules from the beginning of the development of the primordial follicles until the moment of ovulation. The follicular structure transforms during folliculogenesis; barriers form between the germ and the somatic follicular cells, and between the somatic follicular cells. As such, communication systems need to adapt to maintain the exchange of signaling molecules. Two critical barriers are established at different stages of development: the zona pellucida, separating the oocyte and the cumulus cells limiting the communication through specific connections, and the antrum, separating subpopulations of follicular cells. In both situations, communication is maintained either by the development of specialized connections as transzonal projections or by paracrine signaling and trafficking of extracellular vesicles through the follicular fluid. The bidirectional communication between the oocytes and the follicle cells is vital for driving folliculogenesis and oogenesis. These communication systems are associated with essential functions related to follicular development, oocyte competence, and embryonic quality. Here, we discuss the formation of the zona pellucida and antrum during folliculogenesis, and their importance in follicle and oocyte development. Moreover, this review discusses the current knowledge on the cellular mechanisms such as the movement of molecules via transzonal projections, and the exchange of extracellular vesicles by follicular cells to overcome these barriers to support female gamete development. Finally, we highlight the undiscovered aspects related to intrafollicular communication among the germ and somatic cells, and between the somatic follicular cells and give our perspective on manipulating the above-mentioned cellular communication to improve reproductive technologies.

Intrafollicular barriers and cellular interactions during ovarian follicle development

Follicles are composed of different interdependent cell types including oocytes, cumulus, granulosa, and theca cells. Follicular cells and oocytes exchange signaling molecules from the beginning of the development of the primordial follicles until the moment of ovulation. The follicular structure transforms during folliculogenesis; barriers form between the germ and the somatic follicular cells, and between the somatic follicular cells. As such, communication systems need to adapt to maintain the exchange of signaling molecules. Two critical barriers are established at different stages of development: the zona pellucida, separating the oocyte and the cumulus cells limiting the communication through specific connections, and the antrum, separating subpopulations of follicular cells. In both situations, communication is maintained either by the development of specialized connections as transzonal projections or by paracrine signaling and trafficking of extracellular vesicles through the follicular fluid. The bidirectional communication between the oocytes and the follicle cells is vital for driving folliculogenesis and oogenesis. These communication systems are associated with essential functions related to follicular development, oocyte competence, and embryonic quality. Here, we discuss the formation of the zona pellucida and antrum during folliculogenesis, and their importance in follicle and oocyte development. Moreover, this review discusses the current knowledge on the cellular mechanisms such as the movement of molecules via transzonal projections, and the exchange of extracellular vesicles by follicular cells to overcome these barriers to support female gamete development. Finally, we highlight the undiscovered aspects related to intrafollicular communication among the germ and somatic cells, and between the somatic follicular cells and give our perspective on manipulating the above-mentioned cellular communication to improve reproductive technologies.(AU)

Efeito de diferentes sistemas de maturação e cultivo sobre a produção in vitro de embriões bovinos / Effect of different maturation and growth systems on in vitro production of bovine embryos

O objetivo desse estudo foi comparar dois diferentes sistemas de incubação (incubadora convencional – CONV e minibancada – MINI) sobre a produção in vitro de embriões (PIVE) de bovinos. Para tanto, complexos cumulusoócito (CCOs) foram submetidos a maturação in vitro e posterior fecundação e cultivo in vitro em ambos os sistemas. As estruturas foram avaliadas pós-maturação e pós-cultivo, para avaliação de maturação nuclear e quantidade de células/blastocisto, respectivamente. Não foram observadas diferenças estatísticas (p>0,05) nos diferentes parâmetros estudados. Concluímos que ambos os sistemas testados demonstraram ser eficientes para PIVE de bovinos.

The objective of this study was to compare two different incubation systems (conventional incubator - CONV and minibank - MINI) on the in vitro production of bovine embryos (PIVE). For this purpose, cumulus cytotoxic complexes (CCOs) were submitted to in vitro maturation and subsequent fertilization and in vitro culture in both systems. The structures were evaluated post-maturation and post-culture, for evaluation of nuclear maturation and amount of cells / blastocyst, respectively. No statistical differences (p>0.05) were observed in the different parameters studied. We conclude that both tested systems proved to be efficient for bovine PIVE.

Efeito de diferentes sistemas de maturação e cultivo sobre a produção in vitro de embriões bovinos / Effect of different maturation and growth systems on in vitro production of bovine embryos

O objetivo desse estudo foi comparar dois diferentes sistemas de incubação (incubadora convencional CONV e minibancada MINI) sobre a produção in vitro de embriões (PIVE) de bovinos. Para tanto, complexos cumulusoócito (CCOs) foram submetidos a maturação in vitro e posterior fecundação e cultivo in vitro em ambos os sistemas. As estruturas foram avaliadas pós-maturação e pós-cultivo, para avaliação de maturação nuclear e quantidade de células/blastocisto, respectivamente. Não foram observadas diferenças estatísticas (p>0,05) nos diferentes parâmetros estudados. Concluímos que ambos os sistemas testados demonstraram ser eficientes para PIVE de bovinos.(AU)

The objective of this study was to compare two different incubation systems (conventional incubator - CONV and minibank - MINI) on the in vitro production of bovine embryos (PIVE). For this purpose, cumulus cytotoxic complexes (CCOs) were submitted to in vitro maturation and subsequent fertilization and in vitro culture in both systems. The structures were evaluated post-maturation and post-culture, for evaluation of nuclear maturation and amount of cells / blastocyst, respectively. No statistical differences (p>0.05) were observed in the different parameters studied. We conclude that both tested systems proved to be efficient for bovine PIVE.(AU)

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.