A protective role of cumulus cells after short-term exposure of rat cumulus cell-oocyte complexes to lifestyle or environmental contaminants

The developing acrosome in spermatids contains pituitary adenylate cyclase-activating polypeptide (PACAP). However, the role of the acrosomal PACAP remains unclear because it has not been detected in mature spermatids and sperm. We reinvestigated whether the sperm acrosome contains PACAP. An antiserum produced against PACAP reacted to the anterior acrosome in epididymal sperm fixed under mild conditions, suggesting that PACAP acts on oocytes and/or cumulus cells at the site of fertilization. Immunolabeling and RT-PCR demonstrated the presence of PACAP type I receptor, a PACAP-specific receptor, in postovulatory cumulus cells. To investigate the role of PACAP in fertilization, we pretreated cumulus–oocyte complexes with the polypeptide. At a low concentration of sperm, the fertilization rate was significantly enhanced by PACAP in a dose-dependent manner. Sperm penetration through the oocyte investment, cumulus layer, and zona pellucida was also enhanced by PACAP. The enhancement was probably due to an enhancement in sperm motility and the zona-induced acrosome reaction, which were stimulated by a cumulus cell-releasing factor. Indeed, PACAP treatment increased the secretion of progesterone from the cumulus–oocyte complexes. These results strongly suggest that in response to PACAP, cumulus cells release a soluble factor that probably stimulates sperm motility and the acrosome reaction, thereby promoting fertilization.

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

Reproduction ◽

10.1530/rep-20-0123 ◽

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.

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Roles of gene transcription and PKA subtype activation in maturation of murine oocytes

Reproduction ◽

10.1530/rep.0.1230799 ◽

2002 ◽

pp. 799-806 ◽

Cited By ~ 13

Author(s):

KF Rodriguez ◽

RM Petters ◽

AE Crosier ◽

CE Farin

Keyword(s):

Gene Transcription ◽

Cumulus Cell ◽

Cumulus Cells ◽

Type I ◽

Type Ii ◽

Germinal Vesicle Breakdown ◽

Series Of Experiments ◽

And Control ◽

Alpha Amanitin

The aims of this study were to examine the role of transcription and the coincident involvement of type I and type II protein kinase A (PKA) in the resumption of meiosis in murine cumulus-oocyte complexes (COCs) using the transcriptional inhibitors 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. The first series of experiments was designed to: (i) characterize the role of transcription in gonadotrophin-mediated and spontaneous maturation of murine oocytes; (ii) examine the roles of specific gonadotrophins (FSH versus hCG) and cumulus cells in transcriptionally mediated oocyte maturation; and (iii) determine the reversibility of the transcriptional arrest of meiosis. In the presence of FSH, transcriptional inhibitors arrested germinal vesicle breakdown (GVBD) (DRB: 2 +/- 2% and control: 76 +/- 2%; alpha-amanitin: 4 +/- 4% and control: 70 +/- 4%). Furthermore, cumulus cells were required for transcriptional inhibitors to arrest GVBD (DRB with cumulus cells: 0 +/- 15%; DRB without cumulus cells: 94 +/- 13%; alpha-amanitin with cumulus cells: 15 +/- 2%; alpha-amanitin without cumulus cells: 99 +/- 2%). Thus, in mice, FSH-mediated GVBD uses a transcriptional mechanism, which probably occurs within the cumulus cell compartment. In a second series of experiments, the role of transcription in mediating the resumption of meiosis after activation of either type I or type II PKA was examined. Activation of type I PKA in murine COCs resulted in an arrest of GVBD that was independent of a transcriptional event (with DRB: 7 +/- 9% GVBD; without DRB: 11 +/- 9% GVBD). In contrast, activation of type II PKA resulted in a resumption of meiosis, which required the occurrence of gene transcription (with DRB: 12 +/- 9% GVBD; without DRB: 80 +/- 9% GVBD). As FSH binding to cumulus cells activates the PKA second messenger system, our results indicate that, in cultured murine COCs, FSH binding to cumulus cells results in the activation of type II PKA, which, in turn, mediates a downstream transcriptional event required for the initiation of GVBD.

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Evidence of the role of short-term exposure to ozone on ischaemic cerebral and cardiac events: the Dijon Vascular Project (DIVA)

Heart ◽

10.1136/hrt.2010.200337 ◽

2010 ◽

Vol 96 (24) ◽

pp. 1990-1996 ◽

Cited By ~ 37

Author(s):

J.-B. Henrotin ◽

M. Zeller ◽

L. Lorgis ◽

Y. Cottin ◽

M. Giroud ◽

...

Keyword(s):

Cardiac Events ◽

Short Term ◽

Short Term Exposure

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69 REMOVAL OF CUMULUS CELLS AND PRE-INCUBATION IN CYTOCHALASIN B IMPROVE SURVIVAL OF IMMATURE CAT OOCYTES DURING VITRIFICATION

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab69 ◽

2008 ◽

Vol 20 (1) ◽

pp. 115 ◽

Cited By ~ 4

Author(s):

P. Comizzoli ◽

D. E. Wildt ◽

B. S. Pukazhenthi

Keyword(s):

Room Temperature ◽

Cytochalasin B ◽

Cumulus Cells ◽

Lower Percentage ◽

Short Term ◽

No Removal ◽

Short Term Exposure ◽

Highly Sensitive ◽

Rapid Transport ◽

Oocyte Membrane

Vitrification might be the best option to cryopreserve the immature cat oocyte because our recent studies have demonstrated that COCs are (1) highly sensitive to cryoprotectant (CPA) and require short-term exposure to prevent toxicity, and yet (2) resistant to extreme hyperosmotic conditions when pre-incubated in the cytoskeleton stabilizer cytochalasin B (CB). However, surrounding cumulus cells may inhibit the necessary rapid transport of water and CPA across the oocyte membrane during short-term exposure to vitrification solutions (VS) containing high CPA concentrations. The objective was to examine the influence of cumulus cells on oocyte survival during vitrification with or without pre-incubation in CB. Our metric of focus was the chromatin status after warming and IVM. Grade I immature oocytes (n = 420, 4 replicates) were equally allocated to one of four treatments: T1 (removal of cumulus cells in 0.2% hyaluronidase); T2 (pre-incubation in 7.5 µg mL–1 CB for 20 min); T3 (removal of cumulus cells and pre-incubation in CB (as above)); and T4 (control, no removal of cumulus cells and no pre-incubation in CB). After each treatment, half of the oocytes was washed and immediately cultured for IVM (28 h; denuded oocytes being co-cultured with an equal number of fresh COCs to circumvent the absence of cumulus cells). The other half was exposed to 10% (v/v) ethylene glycol (EG) + 10% (v/v) DMSO for 30 s, followed by exposure to VS (20% EG + 20% DMSO + 0.5 m sucrose) for 20 s at room temperature. Oocytes then were vitrified in <1 µL of VS at the tip of a plastic gutter (2 oocytes/gutter) by direct plunge into liquid nitrogen. After 1 day of storage, vitrified oocytes were warmed in 0.25 m sucrose, extensively washed, and cultured for IVM as described above. All oocytes were then fixed and Hoechst-stained to assess their chromatin status. Oocytes subjected to T1, T2, T3, and T4 without vitrification exhibited no degenerated chromatin (clumped or dispersed) after IVM. After vitrification, the incidence of degenerated chromatin was different (P < 0.05; ANOVA) among treatments, with a lower percentage (P < 0.05) when oocytes were subjected to T3 (15.5 � 3.9%; mean � SD) compared to T1 (26.6 � 3.1%), T2 (48.9 � 3.8%), or T4 (71.1 � 3.8%). The percentage of vitrified oocytes reaching the metaphase II stage (MII) also was different (P < 0.05) among treatments with a higher incidence (P < 0.05) in the T3 group (57.8 � 3.9%) compared to T1 (44.6 � 3.7%), T2 (31.1 � 7.7%), or T4 (15.5 � 3.9%) counterparts. Percentages of non-vitrified oocytes reaching the MII were not different (P > 0.05) among treatments (range, 84.7 to 89.0%) but were higher (P < 0.05) compared to vitrified oocytes. The combination of removal of cumulus cells and pre-incubation in CB prior to vitrification had a cumulative, beneficial influence on cat oocyte survival. Interestingly, removal of cumulus cells imparted a greater benefit on oocyte survival than CB pre-incubation alone.

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Effect of Short-Term Metal Exposure on Epididymal Antioxidant Status and Sperm Parameters and the Protective Role of Melatonin.

Biology of Reproduction ◽

10.1093/biolreprod/81.s1.648 ◽

2009 ◽

Vol 81 (Suppl_1) ◽

pp. 648-648

Author(s):

Niraj R. Joshi ◽

Sudip Banerjee ◽

Raktim Mukherjee ◽

Shail Chaube ◽

A.V. Ramachandran

Keyword(s):

Antioxidant Status ◽

Protective Role ◽

Sperm Parameters ◽

Metal Exposure ◽

Short Term

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

Biology of Reproduction ◽

10.1093/biolre/ioaa196 ◽

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.

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

Reproduction ◽

10.1530/rep.1.00761 ◽

2005 ◽

Vol 130 (6) ◽

pp. 857-868 ◽

Cited By ~ 29

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.

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