Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes

Warzych, Ewelina; Peippo, Jaana; Szydłowski, M.; Lechniak, D.

doi:10.1016/j.anireprosci.2006.01.011

Cited by 49 publications

(38 citation statements)

References 41 publications

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“…In spite of this method being frequently used to evaluate the genotoxicity of test substances there is evidence that comet pictures might be associated with apoptotic nuclei (Choucroun et al, 2001). Additionally, a significant relationship between supplementation of maturation medium and apoptotic index of resulting embryos has been reported (Warzych et al, 2007). This confirms the effect of the composition of maturation media on embryo developmental competence.…”

Section: Resultssupporting

confidence: 70%

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Takada

Martins

Mingoti

et al. 2010

Sci. agric. (Piracicaba, Braz.)

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Melatonin (MEL) acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM) on bovine embryos. This study tested the addition of MEL to maturation medium (MM) with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum -FCS) at 39°C and 5% CO 2 in air. After 24 hours of culture in MM with 0.5 μg mL -1 FSH and 5.0 μg mL -1 LH; 10 -9 M MEL) or 10 -9 M MEL, 0.5 μg mL -1 FSH and 5.0 μg mL -1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10 -9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM. Key words: comet assay, embryos, gonadotropins, in vitro fertilization, melatonin Melatonina no meio de maturação não melhorou as taxas de maturação dos ovócitos, de desenvolvimento embrionário e a fragmentação do DNA dos embriões bovinos RESUMO: Melatonin (MEL) atua como um potente redutor de radicais livres. Efeito direto da MEL na função gonadal também foi observado. Existem poucos estudos relacionados ao efeito da MEL durante a maturação no desenvolvimento embrionário in vitro. Avaliou-se a adição de MEL no meio de maturação (sem gonadotrofinas) nas taxas de maturação nuclear e de desenvolvimento embrionário e na incidência de fragmentação do DNA nos embriões produzidos in vitro. Complexos cumulus-ovócitos foram aspirados de ovários provenientes de matadouros e cultivados em meio de maturação (Meio TCM-199 suplementado com 10% de soro fetal bovino) a 39°C e 5% CO 2 em ar. Após 24 horas de cultivo em meio de maturação com 0,5 μg mL -1 de FSH e 5,0 μg mL -1 de LH; 10 -9 M de MEL ou 10 -9 M de MEL, 0,5 μg mL -1 de FSH e 5,0 μg mL -1 de LH, os ovócitos foram corados com Hoechst 33342 para avaliação da taxa de maturação nuclear. A fragmentação do DNA dos blastocistos foi avaliada pela técnica do Cometa. Não houve efeito da MEL adicionada ao meio de maturação, com ou sem gonadotrofinas, nas taxas de maturação nuclear, clivagem e blastocistos. Os percentuais variaram entre 88% a 90%, 85% a 88% e 42% a 46%, respectivamente. O grau de fragmentação do DNA também não foi afetado pela adição de MEL ao meio de matur...

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Section: Resultssupporting

confidence: 70%

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Takada

Martins

Mingoti

et al. 2010

Sci. agric. (Piracicaba, Braz.)

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“…In our study, when EGF (10 ng/mL), FSH, LH was used together with or without FBS, the proportion of good quality oocytes showing grade 3 cumulus expansion was higher than in other treatment groups but this effect was not evident in nuclear maturation status of oocytes. Similar results were also reported Warzych et al 17 . In the study of Lonergan et al 3 contrary to the present study, addition of EGF to culture media, irrespective of concentration, or 10% FCS to TCM-199 stimulated cumulus expansion as well as significantly increasing the proportion of oocytes attaining M II.…”

Section: Discussionsupporting

confidence: 82%

“…Stimulatory effect of these hormones on CCE may not necessarily be reflected on nuclear maturation, if one assumes that these hormones act only at the cumulus cell level but not at on the oocytes directly or indirectly. It may also be possible that culture systems in vitro may differ in their response to hormonal effects due to effects of hormonal supplementation to culture media 8,[16][17][18] .…”

Section: Discussionmentioning

confidence: 99%

“…Some systems require hormonal supplementation while some studies report no beneficial or adverse effects of hormonal supplementation 16,17 in addition to develop serum-free culture systems 18 . This study was designed to evaluate the effects of hormones (FSH, LH and E2) and EGF alone or in combination, in serum-free and serum supplemented TCM-199 medium on cumulus expansion and nuclear maturation in vitro of bovine cumulus-oocyte complexes (COCs) with a view to develop a valid maturation systems responsive to the known factors.…”

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confidence: 99%

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Sığır Oositlerinin Kumulus Ekspansiyonu ve Nükleer Maturasyon Üzerine Serum, Gonadotropinler, Epidermal Büyüme Faktörü ve Östradiol 17-Beta’nın Etkileri

Çevik¹,

Şen²,

Koçyiğit³

et al. 2009

Kafkas Univ Vet Fak Derg

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SummaryThe aim of the study was to evaluate the effects of follicle stimulating hormone (FSH), luteinizing hormone (LH), epidermal growth factor (EGF) and estradiol 17-β (E2) in maturation medium on nuclear maturation and cumulus expansion of cumulus-oocyte complexes (COCs) obtained from bovine ovaries. COCs were classified as good and poor quality grades based on cumulus investment. COCs were then subjected to in vitro maturation in TCM-199 in a humidified atmosphere of 5% CO2 in air at 38.5°C for a period of 24 h. The combination of the hormones added to the medium was as follows: T1=10% (v/v) heat inactivated Fetal Bovine Serum (FBS); T2=5 µg/ml bLH, 0.5 µg/ml bFSH and 10 ng/ml EGF; T3=FBS, bLH, bFSH and EGF; T4=1 µg/ml E2; T5=FBS and E2. Supplementation of maturation medium with the FSH, LH, EGF irrespective to supplementation with FBS stimulated expansion of cumulus around the oocytes compared to oocytes in other treatment groups (P<0.05). However there were no significant differences between treatment groups in terms of proportion of oocytes reached to metaphase II (M II) stage determined by nuclear staining. In general cumulus expansion degree increased with the quality of COCs in all treatment groups (P<0.05). In conclusion, hormone and growth factor supplementation (FSH, LH and EGF) stimulated cumulus cell expansion and maturation rate. Keywords: Bovine oocyte, IVM, EGF, Estradiol 17-β, Gonadotropins Sığır Oositlerinin Kumulus Ekspansiyonu ve Nükleer Maturasyon Üzerine Serum, Gonadotropinler, Epidermal Büyüme Faktörü ve Östradiol 17-Beta'nın Etkileri ÖzetÇalışmanın amacı, sığır ovaryumlarından elde edilen kumulus oosit komplekslerinin (KOK) nükleer olgunlaşma ve kumulus ekspansiyonu üzerine olgunlaşma medyumuna katılan folikül stimüle edici hormon (FSH), luteinleştirici hormon (LH), epidermal büyüme faktörü (EGF) ve östradiol 17-β (E2)'nin etkisini değerlendirmektir. KOK'lar oositlerin kumulus hücreleri ile kuşatılma derecesine göre iyi ve zayıf kaliteli olarak sınıflandırılmıştır. Sonrasında KOK'lar 24 saat süreyle TCM-199 medyumunda 38,5°C ve %5 CO2'li ortamda olgunlaşmaya bırakılmıştır. Medyuma eklenen hormonların kompozisyonu şu şekildedir; T1=10% (v/v) inaktif Fötal Sığır Serumu (FBS); T2=5 µg/ml bLH, 0.5 µg/ml bFSH ve 10 ng/ml EGF; T3=FBS, bLH, bFSH ve EGF; T4=1 µg/ml E2; T5=FBS ve E2. Olgunlaşma medyumuna FBS hariç FSH, LH ve EGF eklenmesinin diğer muamele grupları ile karşılaştırıldığında kumulus ekspansiyonunu artırdığı görülmüştür (P<0.05). Fakat nükleer boyama ile metafaz II (MII) safhasına ulaştığı belirlen oositlerin oranı muamele grupları arasında önemli bir fark göstermemiştir. Genel olarak tüm muamele gruplarında kumulus ekspansiyon derecesi KOK'ların kalitesiyle birlikte artış göstermiştir (P<0.05). Sonuç olarak hormon ve büyüme faktörü ilavesi kumulus ekspansiyonu ve olgunlaşma oranını stimüle etmiştir.

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“…Many factors have been shown to affect the oocyte's developmental potential, including follicle size (Lonergan et al 1994), health of the follicle (Blondin & Sirard 1995, Vassena et al 2003, phase of follicular wave (Hagemann 1999, Machatková et al 2004, hormonal stimulation (Blondin et al 2002; for review Sirard et al 2006), maturation environment (Warzych et al 2007; for review Sutton et al 2003), season (Al-Katanani et al 2002, Sartori et al 2002, nutrition (Fouladi-Nashta et al 2007) and age (Rizos et al 2005). Although previous studies support the notion that oocyte competence depends on multiple factors, it remains difficult to draw clear and reliable criteria for oocyte selection.…”

Section: Introductionmentioning

confidence: 86%

Molecular and subcellular characterisation of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity

Torner¹,

Ghanem²,

Ambros³

et al. 2008

Reproduction

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Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here, we aim to identify molecular and functional markers associated with oocyte developmental potential when selected based on G6PDH activity. Immature compact cumulus-oocyte complexes were stained with brilliant cresyl blue (BCB) for 90 min. Based on their colouration, oocytes were divided into BCB K (colourless cytoplasm, high G6PDH activity) and BCB C (coloured cytoplasm, low G6PDH activity). The chromatin configuration of the nucleus and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement. The abundance and phosphorylation pattern of protein kinases Akt and MAP were estimated by Western blot analysis. A bovine cDNA microarray was used to analyse the gene expression profiles of BCB C and BCB K oocytes. Consequently, marked differences were found in blastocyst rate at day 8 between BCB C (33.1G3.1%) and BCB K (12.1G1.5%) oocytes. Moreover, BCB C oocytes were found to show higher phosphorylation levels of Akt and MAP kinases and are enriched with genes regulating transcription (SMARCA5), cell cycle (nuclear autoantigenic sperm protein, NASP ) and protein biosynthesis (RPS274A and mRNA for elongation factor 1a, EF1A). BCB K oocytes, which revealed higher mitochondrial activity and still nucleoli in their germinal vesicles, were enriched with genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10) and growth factor activity (bone morphogenetic protein 15, BMP15). This study has evidenced molecular and subcellular organisational differences of oocytes with different G6PDH activity.

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Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes

Cited by 49 publications

References 41 publications

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Sığır Oositlerinin Kumulus Ekspansiyonu ve Nükleer Maturasyon Üzerine Serum, Gonadotropinler, Epidermal Büyüme Faktörü ve Östradiol 17-Beta’nın Etkileri

Molecular and subcellular characterisation of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity

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