scholarly journals Apoptosis in bovine cumulus–oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation

Reproduction ◽  
2005 ◽  
Vol 130 (6) ◽  
pp. 857-868 ◽  
Author(s):  
Paola Pocar ◽  
Daniela Nestler ◽  
Michaela Risch ◽  
Bernd Fischer
Keyword(s):  
Cell Apoptosis ◽  
Polychlorinated Biphenyl ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Coplanar Pcbs ◽  
Apoptotic Gene ◽  
Commercial Mixture ◽  
Bovine Oocytes

Aroclor-1254 (A-1254) is a commercial mixture of coplanar (dioxin-like) and non-coplanar (non dioxin-like) polychlorinated biphenyls (PCBs) affecting bovine oocytein vitromaturation (IVM) and developmental competence. In the present study, the role of cumulus cell apoptosis in mediating the toxic effects of PCBs duringin vitromaturation has been investigated. Results indicate that exposure of cumulus–oocyte complexes (COCs) to A-1254 significantly induced apoptosis of cumulus cells. Furthermore, A-1254 significantly increased the expression of the pro-apoptotic gene, Bax, concomitantly reducing the level of the anti-apoptotic gene, Bcl-2, in the cumulus cell compartment. The effects of pure mixtures of coplanar (PCB 77, 126 and 169) or non-coplanar (PCB 52, 101 and 153) PCBs were examined. Exposure of COCs to coplanar PCBs affected maturation at doses as low as 100.6 pg/ml. Furthermore, a significant increase in apoptosis and in Bax mRNA expression was observed. No variations in maturation or apoptosis were observed in the non-coplanar PCB group. To further analyze the role of cumulus cells, COCs and denuded oocytes (DOs) have been exposed to A-1254 or coplanar PCBs during IVM. Exposure of COCs significantly reduced the percentage of matured oocytes after 24 h of culture in both treatments. In contrast, exposure of DOs significantly decreased the maturation rate only at the highest dose investigated (100-fold greater than that affecting COCs). Taken together, the results indicate a direct role of cumulus cell apoptosis in mediating PCB toxicity on bovine oocytes, and a direct relationship between congener planarity and toxicity in bovine oocytes is suggested.

scholarly journals Sperm binding to ZP2-coated beads improve the efficiency of porcine in vitro fertilisation

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. 725-735
Author(s):  
Julieta Gabriela Hamze ◽  
María Jiménez-Movilla ◽  
Raquel Romar
Keyword(s):  
Acrosome Reaction ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
In Vitro Fertilisation ◽  
Sperm Binding ◽  
Fertilisation Rate ◽  
Gamete Recognition ◽  
Boar Spermatozoa

The role of specific zona pellucida (ZP) glycoproteins in gamete interaction has not yet been elucidated in many species. A recently developed 3D model based on magnetic sepharose beads (B) conjugated to recombinant ZP glycoproteins (BZP) and cumulus cells (CBZP) allows the study of isolated ZP proteins in gamete recognition studies. The objective of this work was to study the role of porcine ZP2, ZP3 and ZP4 proteins in sperm binding, cumulus cell adhesion and acrosome reaction triggering. ZP protein-bound beads were incubated with fresh ejaculated boar spermatozoa and isolated cumulus cells for 24 h. The number of sperm bound to the beads, the acrosomal shrouds (presence of acrosomal content) on the bead’s surface, and the acrosome integrity (by means of PNA-FITC lectin) in bound and unbound sperm were studied. Finally, in vitro matured porcine oocytes mixed with BZP2 were inseminated in vitro using fresh sperm and fertilisation results evaluated. Over 60% of beads had at least one sperm bound after 2 h of coincubation. ZP2-beads (BZP2) and cumulus-ZP2-bead complexes (CBZP2) reached the highest number of sperm per bead, whereas BZP3 and BZP4 models showed the highest number of unbound reacted sperm cells and acrosomal shrouds. Fertilisation efficiency and monospermy rate increased when oocytes were fertilised in the presence of BZP2. We, therefore, conclude that in pigs, it is mainly ZP2 that is involved in sperm-ZP binding whereas ZP3 and ZP4 induce acrosome reaction. Using magnetic sepharose ZP2-bound beads might be a valuable tool to improve the fertilisation rate in pigs.

scholarly journals Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†

2020 ◽  
Author(s):  
Aslihan Turhan ◽  
Miguel Tavares Pereira ◽  
Gerhard Schuler ◽  
Ulrich Bleul ◽  
Mariusz P Kowalewski
Keyword(s):  
Oocyte Maturation ◽  
Cell Function ◽  
Hypoxia Inducible Factor ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Negative Effects ◽  
Protein Levels ◽  
Maturation In Vitro

Abstract Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus–oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

230 ROLE OF PITUITARY HORMONES AND CUMULUS CELLS IN MODULATING THE DEVELOPMENTAL CAPACITY OF AGING BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 204
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
N. Zinovieva
Keyword(s):  
Embryonic Development ◽  
Tyrosine Kinases ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Prolonged Culture ◽  
Bovine Oocytes

The competence for embryonic development acquired during the oocyte maturation attenuates during the subsequent oocyte aging both in vivo and in vitro. Thus, the successful control of the female fertility requires information regarding factors responsible for the oocyte protection from early aging. The aim of the present research was to study the pattern and pathways of actions of two closely related pituitary hormones, prolactin (PRL), and growth hormone (GH), on the developmental potential of bovine oocytes during their aging in vitro. Therefore, we analysed (1) effects of PRL and GH during the prolonged culture of bovine oocytes on their subsequent development up to the blastocyst stage and (2) the role of cumulus cells (CC) and tyrosine kinases, the well-known mediators of PRL and GH signalling, in these effects. Bovine cumulus-enclosed oocytes (CEO) were cultured for 22 h in the following maturation medium: TCM 199 containing 10% fetal calf serum (FCS), 10 μg mL–1 of porcine FSH, and 10 μg mL–1 of ovine LH. After IVM, CEO or denuded oocytes (DO) were transferred to the aging medium consisting of TCM 199 supplemented with 10% FCS and cultured for 10 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL or 10 ng mL–1 recombinant bovine GH and/or 10 μg mL–1 genistein (a non-selective inhibitor of tyrosine kinases). Genistein was not applied in the case of aging DO, since their developmental potential was not affected by both hormones. Following the prolonged culture, oocytes underwent IVF and IVC. Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 8. The embryo development was evaluated at Days 2 and 8 for cleavage and blastocyst formation. The data from 5 to 6 replicates using 135–184 oocytes per treatment were analysed by ANOVA. Aging of oocytes in the control medium had no effect on the cleavage rate, but caused the blastocyst yield to decline (P < 0.001) from 31.1 ± 2.3% (CEO fertilized immediately after maturation) to 10.5 ± 2.4% (aged CEO) and 7.9 ± 1.9% (aged DO). Cleavage rates of aging CEO and DO were unaffected by both PRL and GH. In the case of CEO, the addition of PRL (but not GH) to the aging medium raised the blastocyst yield from 8.2 ± 0.9% to 15.2 ± 2.1% (P < 0.05), whereas the removal of CC abolished this effect, reducing the yield up to 9.1 ± 2.7% (P < 0.05). At the same time, genistein did not influence the blastocyst yield in the PRL-treated group. The findings demonstrate that PRL can inhibit the attenuation of the developmental competence of bovine oocytes aging in vitro, with this effect being achieved via cumulus cells. Tyrosine kinases are unlikely to mediate the beneficial action of PRL on the CEO capacity for embryonic development. Meanwhile, closely related GH does not affect the developmental competence of aging bovine oocytes.This research was supported by RFBR (project No. 13-04-01888).

Role of EGF, IGF-I, sera and cumulus cells on maturation in vitro of bovine oocytes

1995 ◽  
Vol 44 (1) ◽  
pp. 109-118 ◽  
Author(s):  
P.L. Lorenzo ◽  
M.J. Illera ◽  
J.C. Illera ◽  
M. Illera
Keyword(s):  
Cumulus Cells ◽  
Maturation In Vitro ◽  
Igf I ◽  
Bovine Oocytes

scholarly journals Peroxiredoxin 6 Is Upregulated in Bovine Oocytes and Cumulus Cells During In Vitro Maturation: Role of Intercellular Communication1

2004 ◽  
Vol 71 (5) ◽  
pp. 1646-1651 ◽  
Author(s):  
Gregory Leyens ◽  
Benjamin Verhaeghe ◽  
Marie Landtmeters ◽  
Joëlle Marchandise ◽  
Bernard Knoops ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Peroxiredoxin 6 ◽  
Bovine Oocytes

scholarly journals The role of follicular-secreted factors in the maintenance of meiotic arrest in rats

2021 ◽  
Author(s):  
◽  
Zaramasina Lena Clark
Keyword(s):  
Gap Junction ◽  
Cumulus Cell ◽  
Indirect Evidence ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Regulatory Pathway ◽  
Meiotic Arrest ◽  
Intracellular Cgmp

<p>Meiosis is the process by which diploid germ cells develop into competent haploid gametes. In female mammals, meiosis is characterised by two periods of arrest, the duration of which is species-specific. This study investigated the first period of meiotic arrest which occurs at the diplotene stage of prophase I. This period of arrest has important implications for artificial reproductive technologies as the maintenance of meiotic arrest in the in vitro situation has been correlated with improved embryological outcomes. Despite there being extensive evidence that the somatic cells of the follicle (granulosa and cumulus cells) produce meiosis-inhibiting factors, the factors themselves and the mechanisms through which they act are unclear. Recent evidence implicates C-type natriuretic peptide (CNP) and oestradiol in the regulation of meiotic arrest in mouse oocytes. In this proposed hypothesis, CNP is produced by the granulosa cells and activates its cognate receptor, NPR2, on cumulus cells. This results in the production of cyclic guanosine monophosphate (cGMP) in cumulus cells which is transferred to the oocyte via gap junctions. In the oocyte, cGMP slows the rate of hydrolysis of cyclic adenosine monophosphate (cAMP) by phosphodiesterase 3A resulting in elevated intra-oocyte cAMP levels. By maintaining high levels of cAMP in the oocyte, maturation-promoting factor (MPF) activity is inhibited, preventing re-entry into the cell cycle, thus maintaining meiotic arrest. The overall objective of this study was to investigate the validity of this aforementioned hypothesised regulatory pathway in another mammalian species, the rat. Four fundamental components of this pathway were chosen to be investigated and these framed the four aims of this study.  The aims of this study were to investigate in cultured rat cumulus cell-oocyte complexes (COCs) the short and long-term effects of CNP and oestradiol, both alone and in combination on (1) gap junction permeability using a validated gap junction assay, (2) intracellular cGMP levels using a direct competitive immunoassay, (3) mRNA expression levels of key cumulus cell-derived genes (Npr2, the receptor for CNP; and Pde4b and Pde4d, phosphodiesterases) using an optimised multiplex TaqMan qPCR reaction, and (4) duration of meiotic arrest.  Overall, the results of this study indicated that the assessed treatments did not alter gap junction permeability in rat COCs in vitro. Whilst treatment with CNP and oestradiol appeared to increase the intracellular levels of cGMP in COCs, this requires further investigation. Notably, this study confirmed the role of steroid hormones in upregulating Npr2 expression. Indirect evidence suggests that PDE4D in particular, is a major regulator of cyclic nucleotide levels in the cumulus cells. Finally, treatment of rat COCs with CNP and oestradiol increased the duration of meiotic arrest in oocytes incubated in vitro.  The results of this study provide the first evidence that the hypothesised regulatory pathway proposed above is also relevant in the rat. Nonetheless, further investigation of the effects of CNP and oestradiol on the modulation of intracellular cGMP levels are required to fully validate the model.</p>

scholarly journals ID: 1064 Effects of FSH, cumulus cell morphology and follicular fluid from different follicular sizes on the in vitro maturation of bovine oocytes

2017 ◽  
Vol 4 (S) ◽  
pp. 146
Author(s):  
Nguyen Hoang-Kieu Linh ◽  
Phung Ngoc Minh Doan ◽  
Pham Truong Duy ◽  
Bui Hong Thuy ◽  
Nguyen Van Thuan
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mature Oocyte ◽  
Vital Role ◽  
Oocyte Maturity ◽  
Maturation Rate ◽  
Bovine Oocytes

The quality of mature oocyte plays a vital role in assisted reproductive technology, as well as animal cloning. Therefore, optimization of the in vitro maturation procedure for oocytes has long been of interests for researchers in the fields of reproduction. In this study, we investigated the effect of different supplement culture factors on in vitro maturation of bovine oocytes such as follicular-stimulating hormone (FSH) (experiment 1), different layers of cumulus cells (CCs) (experiment 2), and follicular fluid (FF) collected from different follicle sizes (experiment 3). With result from experiment 1, bovine oocytes cultured in in vitro maturation (IVM) medium supplemented with FSH reached to higher maturation rate than cultured in the basic one (85.9% and 69.3% respectively). In addition, experiment 2 suggested that, the groups of 3-4 layers and 2-3 layers achieve higher rate of oocyte maturity than group of <1 layers (84.38%; 82.46%; 47.83% respectively). However, the result of experiment 3 show that FF collected from different follicle size did not affect to the maturation rate. In conclusion, FSH and layers of CCs affect to the maturation of bovine oocytes.

scholarly journals The role of follicular-secreted factors in the maintenance of meiotic arrest in rats

2021 ◽  
Author(s):  
◽  
Zaramasina Lena Clark
Keyword(s):  
Gap Junction ◽  
Cumulus Cell ◽  
Indirect Evidence ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Regulatory Pathway ◽  
Meiotic Arrest ◽  
Intracellular Cgmp

<p>Meiosis is the process by which diploid germ cells develop into competent haploid gametes. In female mammals, meiosis is characterised by two periods of arrest, the duration of which is species-specific. This study investigated the first period of meiotic arrest which occurs at the diplotene stage of prophase I. This period of arrest has important implications for artificial reproductive technologies as the maintenance of meiotic arrest in the in vitro situation has been correlated with improved embryological outcomes. Despite there being extensive evidence that the somatic cells of the follicle (granulosa and cumulus cells) produce meiosis-inhibiting factors, the factors themselves and the mechanisms through which they act are unclear. Recent evidence implicates C-type natriuretic peptide (CNP) and oestradiol in the regulation of meiotic arrest in mouse oocytes. In this proposed hypothesis, CNP is produced by the granulosa cells and activates its cognate receptor, NPR2, on cumulus cells. This results in the production of cyclic guanosine monophosphate (cGMP) in cumulus cells which is transferred to the oocyte via gap junctions. In the oocyte, cGMP slows the rate of hydrolysis of cyclic adenosine monophosphate (cAMP) by phosphodiesterase 3A resulting in elevated intra-oocyte cAMP levels. By maintaining high levels of cAMP in the oocyte, maturation-promoting factor (MPF) activity is inhibited, preventing re-entry into the cell cycle, thus maintaining meiotic arrest. The overall objective of this study was to investigate the validity of this aforementioned hypothesised regulatory pathway in another mammalian species, the rat. Four fundamental components of this pathway were chosen to be investigated and these framed the four aims of this study.  The aims of this study were to investigate in cultured rat cumulus cell-oocyte complexes (COCs) the short and long-term effects of CNP and oestradiol, both alone and in combination on (1) gap junction permeability using a validated gap junction assay, (2) intracellular cGMP levels using a direct competitive immunoassay, (3) mRNA expression levels of key cumulus cell-derived genes (Npr2, the receptor for CNP; and Pde4b and Pde4d, phosphodiesterases) using an optimised multiplex TaqMan qPCR reaction, and (4) duration of meiotic arrest.  Overall, the results of this study indicated that the assessed treatments did not alter gap junction permeability in rat COCs in vitro. Whilst treatment with CNP and oestradiol appeared to increase the intracellular levels of cGMP in COCs, this requires further investigation. Notably, this study confirmed the role of steroid hormones in upregulating Npr2 expression. Indirect evidence suggests that PDE4D in particular, is a major regulator of cyclic nucleotide levels in the cumulus cells. Finally, treatment of rat COCs with CNP and oestradiol increased the duration of meiotic arrest in oocytes incubated in vitro.  The results of this study provide the first evidence that the hypothesised regulatory pathway proposed above is also relevant in the rat. Nonetheless, further investigation of the effects of CNP and oestradiol on the modulation of intracellular cGMP levels are required to fully validate the model.</p>

182 EFFECTS OF IGF-1 OR IGF-1-LongR3 ON CELLULAR AND MOLECULAR ASPECTS OF CUMULUS-OOCYTE COMPLEXES DURING IN VITRO OOCYTE MATURATION IN CATTLE

2016 ◽  
Vol 28 (2) ◽  
pp. 222
Author(s):  
M. S. Araujo ◽  
M. D. Guastali ◽  
A. C. S. Castilho ◽  
F. Landim-Alvarenga
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Synthetic Analogue ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Metaphase Stage ◽  
Bovine Oocytes

The insulin-like growth factor-1 recombinant -3 (IGF-1-LongR3), a synthetic analogue of IGF-1 with increased bioavailability has not yet been used in vitro maturation (IVM) medium of bovine oocytes. Therefore, the aim of this study was to evaluate and compare the addition effects of IGF-1-LongR3 or IGF-1 in IVM bovine oocytes on meiotic progression, apoptosis, and profile of oocytes genes (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGFBP4 and IGFBP5) and genes in cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). Bovine ovaries were collected in slaughterhouses, and 739 oocytes with grades 1 or 2 were selected after aspiration of 2- to 8-mm follicles. IVM was carried out in TCM199 with FSH, LH, and antibiotics (BM) supplemented with 100 ng mL–1 IGF-1 or 100 ng mL–1 LongR3-IGF-1. Control oocytes were matured in BM supplemented with 0.1% polyvinyl alcohol (PVA) or 10% FCS. For all groups, maturation was performed during 22–24 h in an incubator at 38.5°C and 5% CO2 in air. Subsequently oocytes were denuded and analysed for apoptosis, nuclear maturation, and gene expression by TUNEL assay, staining Hoechst 33342, and RT-qPCR, respectively. Statistical analysis was performed using a linear mixed effects model, which correlated the change in metaphase stage 1 to 2 and the absence of apoptosis among the experimental groups. ANOVA and Tukey tests were used to analyse the results obtained by RT-qPCR. After 10 replicates of IVM, 339 oocytes were evaluated for meiotic progression and apoptosis and 400 oocytes for gene expression. There was no statistical difference between the experimental groups with respect to meiotic progression and apoptosis. BCL2 and IGFBP4 gene were less expressed in oocytes matured with IGF-1 and LongR3-IGF-1 compared with control groups. GFBP4 was also less expressed in cumulus cell of oocytes from the experimental groups. Moreover COX2 expression was statistically elevated in cumulus cells matured in the presence of IGF-1 and LongR3-IGF-1 It was possible to perform IVM of bovine oocytes in the presence of LongR3-IGF-1, allowing its use in replacement of IGF-1 and FCS. The results of this study will provide more information on the interaction of IGF with the IGFBP and its importance for oocyte maturation.

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