Developmental capability of denuded bovine oocyte in a Co-culture system with intact cumulus-oocyte complexes: Role of cumulus cells, cyclic adenosine 3?,5?-monophosphate, and glutathione

2005 ◽  
Vol 71 (3) ◽  
pp. 389-397 ◽  
Author(s):  
Alberto M. Luciano ◽  
Valentina Lodde ◽  
Matteo S. Beretta ◽  
Silvia Colleoni ◽  
Antonio Lauria ◽  
...  
Keyword(s):  
Culture System ◽  
Cumulus Cells ◽  
Bovine Oocyte

176 REGULATORY ROLE OF miR-20a DURING BOVINE OOCYTE MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 196 ◽  
Author(s):  
E. Andreas ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
E. Held ◽  
M. Hoelker ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Expression Analysis ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Fine Tuning ◽  
Bovine Oocyte ◽  
Total Rna ◽  
Progesterone Synthesis ◽  
Spent Media

The role of microRNA in oocyte maturation is mostly associated with optimal turnover of the accumulated maternal transcripts during their growth to allow maturation. MiR-20a is a member of the miR-17–92 cluster, which has been found to be differentially expressed in bovine granulosa cells derived from preovulatory dominant and subordinate follicles. Our recent study showed that miR-20a is involved in the regulation of granulosa cell proliferation, differentiation, and progesterone synthesis by targeting PTEN and BMPR2 genes. Here, we aimed to investigate the role of miR-20a in the bovine oocyte maturation processes. For this, cumulus-oocyte complexes (COC) were aspirated from small antral follicles (2–8 mm in diameter) and cultured in groups of 50 in 400 µL of maturation media (TCM-199 media supplemented with 12% oestrus cow serum and 10 µg/ml Follitropin®) at 39°C in a humidified atmosphere with 5% (vol/vol) CO2 in the air for 22 h. The cumulus cells and oocytes before (germinal vesicle) and after maturation (metaphase II) were mechanically separated in 0.1% hyaluronidase (in TCM-199 media). To study whether the presence of cumulus cells or oocyte has an impact on the miR-20a expression, we cultured oocytectomized cumulus cells and oocytes with and without their companion cells. Moreover, COC were co-cultured with miR-20a mimic, inhibitor, or corresponding controls to investigate the role of this miRNA in oocyte maturation. The total RNA from cumulus cells and oocytes was extracted using miRNeasy® mini kit (Qiagen GmbH, Hilden, Germany). Total RNA from respective samples was reverse transcribed for mRNA and microRNA expression analysis. Quantitative expression analysis was performed using StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) and subsequent data were analysed using a comparative cycle threshold method. The progesterone released in the spent media was measured using progesterone enzyme-linked immunosorbent assay kit (ENZO Life Sciences GmbH, Loerrach, Germany). Here, we found that miR-20a expression in cumulus cells increased (P < 0.05) during oocyte maturation. Conversely, miR-20a expression in metaphase II stage oocytes was significantly lower (P < 0.001) compared with the germinal vesicle stage. The absence of oocyte cytoplasm resulted in reduced miR-20a expression in cumulus cells. On the other hand, the absent of cumulus cells increased miR-20a expression in oocytes. The miR-20a expression revealed that the microRNA transduction is restricted in the cumulus cells. The overexpression of miR-20a increased oocyte maturation rate (P < 0.05) by 4.8% (as determined by extrusion of the polar body) and the expression of oocyte maturation-related genes (INHBA, MAPK1, PTGS2, PTX3, and EGFR). The progesterone released in spent media of COC co-cultured with miR-20a mimic and inhibitor showed increasing (P = 0.0936) and decreasing (P = 0.0993) trends, respectively. In this study, we also found that miR-20a modulation altered the expression of PTEN and BMPR2 in cumulus cells. In conclusion, the modulation of miR-20a expression in cumulus cells regulates the oocyte maturation and partially involved in the progesterone synthesis by fine-tuning the expression of PTEN and BMPR2 genes.

scholarly journals PACAP-mediated sperm–cumulus cell interaction promotes fertilization

Reproduction ◽  
2011 ◽  
Vol 141 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Ichiro Tanii ◽  
Tadashi Aradate ◽  
Kouhei Matsuda ◽  
Akira Komiya ◽  
Hideki Fuse
Keyword(s):  
Sperm Motility ◽  
Acrosome Reaction ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cell Interaction ◽  
Fertilization Rate ◽  
Type I ◽  
Dependent Manner ◽  
Sperm Penetration

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.

Contrasting effects of the Toll-like receptor 4 in determining ovarian follicle endowment and fertility in female adult mice

Zygote ◽  
2021 ◽  
pp. 1-7
Author(s):  
Júlio Panzera Gonçalves ◽  
Breno Augusto Magalhães ◽  
Paulo Henrique Almeida Campos-Junior
Keyword(s):  
Ovarian Follicle ◽  
Cumulus Cells ◽  
Toll Like Receptor ◽  
Female Reproduction ◽  
Toll Like Receptor 4 ◽  
Genetic Ablation ◽  
Adult Mice ◽  
Cumulus Oocyte Complex ◽  
Estrous Cyclicity

Abstract Toll-like receptor 4 (TLR4) is best known for its role in bacteria-produced lipopolysaccharide recognition. Regarding female reproduction, TLR4 is expressed by murine cumulus cells and participates in ovulation and in cumulus–oocyte complex (COC) expansion, maternal–fetal interaction and preterm labour. Despite these facts, the role of TLR4 in ovarian physiology is not fully understood. Therefore, the aim of the present study was to investigate the effects of TLR4 genetic ablation on mice folliculogenesis and female fertility, through analysis of reproductive crosses, ovarian responsiveness and follicular quantification in TLR4−/− (n = 94) and C57BL/6 mice [wild type (WT), n = 102]. TLR4-deficient pairs showed a reduced number of pups per litter (P = 0.037) compared with WT. TLR4−/− mice presented more primordial, primary, secondary and antral follicles (P < 0.001), however there was no difference in estrous cyclicity (P > 0.05). A lower (P = 0.006) number of COC was recovered from TLR4−/− mice oviducts after superovulation, and in heterozygous pairs, TLR4−/− females also showed a reduction in the pregnancy rate and in the number of fetuses per uterus (P = 0.007) when compared with WT. Altogether, these data suggest that TLR4 plays a role in the regulation of murine folliculogenesis and in determining ovarian endowment. TLR4 deficiency may affect ovulation and pregnancy rates, potentially decreasing fertility, therefore the potential side effects of its blockade have to be carefully investigated.

Role of Cumulus Cells in Oocyte Maturation

1982 ◽  
pp. 175-188 ◽  
Author(s):  
Torbjörn Hillensjö ◽  
Claes Magnusson ◽  
Carl Ekholm ◽  
Håkan Billig ◽  
Lars Hedin
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells

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 276 THE EFFECT OF CUMULUS CELLS DURING MATURATION ON THE RISE IN THE CONCENTRATION OF INTRACELLULAR Ca2+ ([Ca2+]i) OF PORCINE OOCYTES INDUCEDBY INOSITOL 1,4,5-TRISPHOSPHATE

2005 ◽  
Vol 17 (2) ◽  
pp. 288
Author(s):  
T. Amano ◽  
T. Mori ◽  
K. Matsumoto ◽  
T. Watanabe ◽  
A. Iritani
Keyword(s):  
Cumulus Cells ◽  
Immature Oocytes ◽  
R Ratio ◽  
Transient Rise ◽  
Activation Rate ◽  
Sperm Penetration ◽  
Cytoplasmic Maturation ◽  
Student’S T ◽  
Rate Of Activation

Increase of inositol 1,4,5-triphosphate (IP3) in the cytoplasm of mammalian oocytes is said to be responsible for [Ca2+]i oscillation observed in the oocytes immediately after sperm penetration, and the [Ca2+]i oscillation is known to be essential for the development of embryos. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of oocytes and affecting the embryonic development. To obtain more information about the role of cumulus cells in cytoplasmic maturation, the effects of cumulus cells during maturation on the rise in [Ca2+]i and on the rate of activation of porcine mature oocytes induced by IP3 injection were investigated. The immature porcine oocytes were divided into three groups: COCs (intact cumulus-oocyte complexes), DOs (oocytes denuded of their cumulus cells), Co-culture (DOs attached to separated cumulus cells). These groups of immature oocytes were cultured in NCSU23 46 h for maturation. To examine the function of cumulus cells, two groups of immature oocytes were also prepared: DOs + pyruvate (DOs put into NCSU23 with pyruvate) and COCs-glucose free (COCs put into NCSU23 without glucose). The mature oocytes from each group were loaded with Ca2+ indicator fluorescent dye Fura2-AM, and then were irradiated by 340 nm and 360 nm of ultraviolet immediately after the injection of IP3. The intensities of emission light caused by the irradiation of 340 nm and 360 nm ultraviolet were recorded as E340 and E360. Since coupling of Ca2+ and the dye intensifies E340, but does not change E360, the level of [Ca2+]i was shown as R (ratio = E340/E360) in this study. Activation rate was calculated by counting the number of the oocytes that formed pronuclei by injection of IP3. ANOVA and Student's t-test were used in this study. Transient rise in [Ca2+]i was observed in the mature oocytes from every group. The peak R of the rise in [Ca2+]i of the mature oocytes derived from COCs, Dos, and Co-culture and induced by IP3 were 7.2, 4.0, and 6.9, respectively. The R of DOs was significantly lower than those of the others (P < 0.05). Also, the activation rate of the mature oocytes from DOs was significantly lower than those from COCs and Co-culture (31, 66, and 66%). The mature oocytes from DOs + pyruvate showed the same level of peak R compared with those from COCs (7.4 and 6.3), but COCs-glucose free showed a slight but significantly lower peak R compared with the mature oocytes from COCs (6.0 and 7.4, P < 0.05). In conclusion, cumulus cells appeared to support the rise in [Ca2+]i of porcine oocytes induced by IP3 during maturation and the following activation. Moreover, a function of cumulus cells supposedly produces pyruvate by metabolizing glucose and provides it to oocytes during maturation for promoting the cytoplasmic maturation. A part of this study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

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

Reproduction ◽  
2002 ◽  
pp. 799-806 ◽  
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.

P–142 Comparison of the IVF outcome in the course of ICSI vs PICSI treatment continuation in exactly the same group of HBA abnormal patients

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Rusin ◽  
W Szecel ◽  
M Jagiello ◽  
J Liss ◽  
K Lukaszuk
Keyword(s):  
Binding Sites ◽  
Poor Response ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Retrospective Case ◽  
Plasmatic Membrane ◽  
Registration Number ◽  
Main Component ◽  
Control Study

Abstract Study question Does the PICSI have a beneficial effect for men with abnormal HBA on the fertilization rate, blastocysts number and clinical pregnancies in the next attempt? Summary answer Patients with HBA &lt;80% choosing to undergo PICSI after ICSI failure see an increase in blastocyst and pregnancy rates. What is known already Hyaluronic acid (HA) is a main component of cervical mucus and the extracellular matrix of cumulus cells. The formation of HA-binding sites in sperm cell membranes is one of the markers of sperm maturation indicating completion the spermatogenic process of remodelling the plasmatic membrane, cytoplasmic extrusion and nuclear maturity. Spermatozoa selected by the HA-binding technique (the physiologically selected intracytoplasmic sperm injection – PICSI) have a potentially reduced risk of chromosomal aneuploidy or DNA fragmentation. Recent evidence do not show significant benefits in using PICSI. However, it has not been analysed in the course of treatment continuation in the same patients. Study design, size, duration This was a retrospective case-control study. It included exactly the same 58 patients with abnormal HBA, who underwent IVF treatment with ICSI initially and later with PICSI, between January 2014 and October 2020 at INVICTA Fertility Centre, Poland. Median female partner age in PICSI group was 36,2±5,34, without PICSI 35,8 ±5,28. Participants/materials, setting, methods 275 cycles (130 ICSI and 145 PICSI) resulted in 793 and 897 MII respectively. Patients were also divided into two groups &lt;80% and ≥80% depending on the obtained HBA score expressed as the percentage of sperm bound with hyaluronan. The analysis covered the fertilization rate (FR), TQ and total blastocyst rate on day 5 and clinical pregnancy rate. Patients with poor response to stimulation were excluded from the study. Main results and the role of chance FR in ICSI and PICSI groups was not significantly difference (57.00%±31.2 vs 59.87%±30.8) even when taking into account the division of patients according to the obtained HBA score. In the &lt;80% group the FR was 57.04%±29.3 vs 59.54%±30.8 in ICSI vs PICSI group respectively. There were no significant differences when comparing the under HBA ≥80% subgroups for all analysed outcomes. Fertilization rate ​​was 56.88% in the ICSI group vs 61.03% in the PICSI group. The percentage of blastocysts was 28.61% vs 34.45% and the percentage of TQ blastocysts on day 5 was 15.32% vs 16.81% with ICSI and PICSI respectively, in the group consisting of the same patients. In the HBA &lt;80% group significant differences were observed in the percentage of obtained blastocysts 37.81% vs 47.61% by comparing the ICSI and PICSI approaches (p &lt; 0.05). Also, percentage of TQ blastocyst on day 5 also was higher in patients with &lt;80% HBA score after PICSI and was statistically significant (17.07% ICSI vs 23.92% PICSI, p &lt; 0.05). We saw statistically significant (p &lt; 0.01) increase in percentage of clinical pregnancies from 29.03% without PICSI to 69.44% in patient’s subsequent procedures involving PICSI. Limitations, reasons for caution More data is required to confirm that improved results of PICSI procedure are consistent and possible to reproduce in a larger group – and as a result could be included as part of the standard treatment process. Wider implications of the findings: The presented results show that in patients with normal HBA score, PICSI does not bring a measurable benefit and this may be important factor to consider in decision-making for couples seeking assistance. Trial registration number Not applicable

scholarly journals Reply to Comment on “Kadomoto, S. et al. Tumor-Associated Macrophages Induce Migration of Renal Cell Carcinoma Cells via Activation of the CCL20-CCR6 Axis” Cancers 2020 12, 89

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 354
Author(s):  
Kadomoto ◽  
Izumi ◽  
Hiratsuka ◽  
Nakano ◽  
Naito ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Tumor Microenvironment ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Culture System ◽  
Staining Intensity ◽  
Carcinoma Cells ◽  
Definite Difference ◽  
M1 And M2 Macrophages

We appreciate Zins and Abraham [1] commenting on our paper studying the role of the CCL20-CCR6 axis on renal cell carcinoma (RCC) cells [2]. As they pointed out, our study has certain limitations. Although M1- and M2-types cannot be separated clearly and a consecutive change of character might exist between them, it has been reported that plural specific markers express on M1- and M2-types. Unfortunately, a definite difference between M1 and M2 macrophages was not confirmed in our study. For more differentiation, multiple stimulations, such as suggested in the comments of Zins and Abraham, might be needed. Hence, we needed to expediently use “M1-like” and “M2-like” to mention specific status of these macrophage-like cells. Meanwhile, CCL20 expression levels of M2-like-THP-1 cells co-cultured with RCC cells were dramatically increased compared with parental THP-1 cells, indicating that certain stimulations within the tumor microenvironment rather than theoretical stimulations make macrophages differentiated; however, further studies are needed to clarify this mechanism using a more appropriate co-culture system mimicking the tumor microenvironment. Immunohistochemistry of CCL20 and M2 markers will help to better understand the role of tissue infiltrating macrophages, even tissue CD68 staining intensity itself was reported to correlate with prognosis of RCC patients [3]. [...]

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