322 LEPTIN TREATMENT DURING BOVINE OOCYTE MATURATION AFFECTS mRNA LEVELS OF APOPTOSIS-RELATED GENES

2006 ◽  
Vol 18 (2) ◽  
pp. 268
Author(s):  
M. Boelhauve ◽  
F. F. Paula-Lopes ◽  
F. A. Habermann ◽  
F. Sinowatz ◽  
E. Wolf
Keyword(s):  
Quantitative Pcr ◽  
Fas Ligand ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Inhibitor Of Apoptosis ◽  
Mrna Levels ◽  
Oocyte Growth ◽  
Transcript Levels ◽  
Apoptosis Protein ◽  
Bovine Oocytes

The series of events associated with oocyte growth and maturation determines its ability to undergo successful fertilization, cleavage, and embryonic development. Among the molecules involved in these events, leptin has been identified as a modulator of oocyte function. The latest studies have focused on long-term effects of leptin during maturation of bovine oocytes on apoptosis and gene expression in in vitro-produced blastocysts. Briefly, blastocysts originating from leptin-treated oocytes exhibited decreased transcript levels of BCL2 associated X-protein (BAX), but increased mRNA concentrations for leptin receptor (LEPR), signal transducer and activator of transcription 3 (STAT3), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP) (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). In the present study, we analyzed single oocytes and their surrounding cumulus cells matured in the presence of 0, 1, and 10 ng/mL leptin. Transcript levels of LEPR, STAT3, BAX, BIRC4, FASLG (encoding Fas ligand), and FAS (encoding Fas receptor) were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of matured oocytes and cumulus cells. Four IVM replicates with four individual samples were collected 22 h after the start of IVM. Total RNA was isolated using a modified TriZol protocol, and reverse transcribed using the enzyme Superscript II-RT and random hexamer primers. Quantitative PCR was conducted with SYBR-Green as a double-stranded DNA-specific fluorescent dye in an ABI 7000 SDS apparatus. Treatment of COCs with 1 or 10 ng/mL leptin increased transcript levels of LEPR (2-fold; P < 0.001), STAT3 (2-fold; P < 0.001), BAX (2-fold; P < 0.001) and FAS (2-fold; P < 0.001) in cumulus cells. Interestingly, the transcript level of the well-known inhibitor of apoptosis, BIRC4, was increased about 4-fold (P < 0.001). In oocytes, leptin treatment increased the mRNA abundance of STAT3 (10 ng/mL; P < 0.05), FAS (1 ng/mL; P < 0.05 and 10 ng/mL; P < 0.001), and FASLG (10 ng/mL; P < 0.05). In conclusion, physiological doses of leptin during maturation of COCs have effects on the expression of developmentally important and apoptosis-associated genes in the oocyte and surrounding cumulus cells. The higher level of BIRC4 mRNA in leptin-treated cumulus cells was associated with a reduced proportion of cumulus cell apoptosis, which might explain the positive and long-lasting effects of leptin on the developmental competence of bovine oocytes.

scholarly journals Assessment of transcript and protein levels contributing to cell cycle control and gap junction connections in morphologically variable groups of porcine cumulus-oocyte complexes

2010 ◽  
Vol 55 (No. 10) ◽  
pp. 512-521 ◽  
Author(s):  
P. Antosik ◽  
B. Kempisty ◽  
M. Jackowska ◽  
H. Piotrowska ◽  
D. Bukowska ◽  
...  
Keyword(s):  
Connexin 43 ◽  
Protein Localization ◽  
Cell Cycle Control ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Dapi Staining ◽  
Transcript Levels ◽  
Grade Iii

Oocytes and somatic cumulus cells are connected by an extensive network of gap junctions. These connections contribute in a major way to oocyte maturation and developmental competence. Cumulus-oocyte complexes (COCs) were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The morphological classification of COCs is based on the number of cumulus cell layers and the degree of their compaction, as well as on cytoplasm composition (homogenous, heterogeneous). The obtained COCs were divided into four grades according to this classification system. By assessing the activity of glucose-6-phosphate dehydrogenase (G6PDH) using the brilliant cresyl blue (BCB) test, real-time quantitative PCR (RQ-PCR) reaction methods and confocal microscopic observations, we determined the transcript levels of connexins 43 and 45, cyclin dependent kinases (cdk5 and cdk5r), and cdk inhibitors 1 and 3 (p27kip1 and cdkn3) as well as cdk4 protein in morphologically different groups of porcine oocytes isolated from puberal gilts. To assess their nuclear status the completely denuded oocytes were subjected to DAPI staining. We found statistically increased cdkn3, cdk5 and connexin 45 mRNA levels in oocytes graded as I as compared to II, III, and IV. The cdkn1, cdk5r and connexin 43 transcript contents were higher only when comparing between oocytes graded as I, III and IV. The cdk4 protein in oocytes graded I and II is localized mainly in the zona pellucida, although in grade III COCs the expression of this protein is decreased and observed only in the cytoplasm. Grade IV COCs do not demonstrate a significant presence of cdk4 protein. With regards to nuclear maturation, the percentage of MII stage oocytes was significantly (P &lt; 0.05) higher in grade I and II oocytes as compared to grade III and IV oocytes. Our results demonstrate for the first time that cdk4 protein localization and all of the investigated transcript levels are associated with COC morphology and may be related to further maturation ability as well as developmental competence of oocytes.

192 The effect of invitro maturation on the PI3K-Akt pathway in bovine cumulus cells

2020 ◽  
Vol 32 (2) ◽  
pp. 224
Author(s):  
G. Andrade ◽  
M. Del Collado ◽  
R. Nociti ◽  
W. J. Da Silva ◽  
F. Meirelles ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Extraction ◽  
Cumulus Cell ◽  
Principal Component ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Signalling Pathway ◽  
Differentially Expressed ◽  
Akt Pathway ◽  
Apoptosis Protein

Oocyte quality is influenced by invitro oocyte maturation (IVM) because the culture conditions can alter the metabolism and gene expression of cumulus cells. Proper oocyte development requires fine regulation of signalling pathways involved with cell proliferation and survival, such as the PI3K-Akt (phosphatidylinositol-3-kinase/protein kinase B) signalling pathway. The aim of this study was to determine the effect of IVM on the expression of PI3K-Akt-related genes in bovine cumulus cells. To this aim, cumulus cells associated with immature oocytes, associated with oocytes invitro-matured for 24h, or associated with oocytes invivo-matured were compared in terms of gene expression. Pools (n=4) of cumulus cells from 20 cumulus-oocyte complexes (COCs) per group were submitted to total RNA extraction using the TRizol protocol, libraries were prepared with TruSeq stranded mRNA sample prep kit (Illumina Inc.), and the sequencing was performed in the HisEqn 2500 V4 (Illumina Inc.). After quality check with FastQC and filtering with Trim Galore, read alignment was performed with STAR and analysis of differential gene expression was done using DESEqn 2 in R considering the Benjamini-Hochberg method for adjusted P-values&lt;0.10, and absolute value of log2-fold change &gt;0.5. Principal component analysis was able to separate, with 94% cumulative variance (81% and 13% for PC1 and PC2, respectively), the cumulus cells groups, especially the immature from the matured counterparts. Gene ontology and enrichment analysis showed that the PI3K-Akt signalling pathway was affected in immature cumulus cells compared with cumulus cells from invitro- or invivo-matured oocytes, with 77 and 88 genes from PI3K-Akt pathway being differentially expressed, respectively. A total of 51 genes were common in invivo- and invitro-matured oocytes cumulus cells groups compared with immature group. Regarding cumulus cells after the maturation process, 48 genes from the PI3K-Akt pathway were differentially expressed; of those, 26 genes were upregulated in cumulus cells from invitro-matured oocytes and 22 genes were upregulated in cumulus cells from invivo-matured oocytes. Comparing the invitro and invivo cumulus cells, the main genes of the pathway (AKT, PI3K, and PTEN) were not differentially expressed. The differences in expression between invitro and invivo cumulus cells were in genes responsible for different cellular functions controlled by the PI3K-Akt pathway, such as apoptosis, protein synthesis, and DNA repair, among others, which, in general, were increased in cumulus cells from invitro-matured oocytes. These results demonstrated the effect of culture conditions on cumulus cell gene expression modulating important pathways involved in oocyte competence acquisition, such as PI3K-Akt signalling. Financial support was provided by FAPESP grants 2014/22887-0, 2018/01431-9, and 2018/13155-6.

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 X-linked inhibitor of apoptosis protein and active caspase-3 expression patterns in antral follicles in the sheep ovary

Reproduction ◽  
2011 ◽  
Vol 142 (6) ◽  
pp. 855-867 ◽  
Author(s):  
Hollian R Phillipps ◽  
Ilona C Kokay ◽  
David R Grattan ◽  
Peter R Hurst
Keyword(s):  
Mrna Expression ◽  
Caspase 3 ◽  
Expression Patterns ◽  
Inhibitor Of Apoptosis ◽  
Mrna Levels ◽  
Follicular Atresia ◽  
Inhibitor Of Apoptosis Protein ◽  
Antral Follicles ◽  
Apoptosis Protein ◽  
Active Caspase

X-linked inhibitor of apoptosis protein (XIAP) interacts with caspases to inhibit their activity, thereby providing a potential mechanism for regulation of granulosa cell apoptosis occurring during follicular atresia. The aim of this study was to determine the presence and localization of XIAP mRNA and protein content in the sheep ovary and compare these expression patterns with active caspase-3 protein in the same antral follicles. Romney ewe estrous cycles (n=25) were synchronized with 2–3 Estrumate injections and ovarian tissue collected during the luteal and follicular phases of the cycle. The presence ofXIAPmRNA was confirmed by RT-PCR using laser capture microdissected ovarian cell samples.XIAPmRNA was subsequently localized byin situhybridization histochemistry and XIAP and active caspase-3 protein visualized by immunohistochemistry. In antral follicles extensive XIAP localization was evident in both granulosa and thecal cells. In contrast, mRNA expression was widespread in granulosa cells and only detected in thecal tissue from a small proportion of antral follicles. Active caspase-3 and XIAP comparative expression analysis showed positiveXIAPmRNA expression in all late luteal phase (day 14) follicles, despite varying levels of active caspase-3 protein. A proportion of follicular phase (days 15 and 16) follicles, however, showed an inverse expression relationship at the protein and mRNA levels in both granulosa and thecal tissue, as did XIAP protein in day 14 follicles. These results suggest high XIAP may prevent activation of caspase-3, thereby regulating follicular atresia in antral follicles and could potentially be utilized as a marker of follicular health.

scholarly journals 325RETINOID-DEPENDENT POLY(A) MRNA CONTENTS IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 282
Author(s):  
L.J. Royo ◽  
A. Rodriguez ◽  
A. Gutierrez-Adan ◽  
C. Diez ◽  
E. Moran ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Defined Medium ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Atp Production ◽  
Beneficial Effects ◽  
Oocyte Developmental Competence ◽  
Grant Support ◽  
Bovine Oocytes

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP&lt;0.01; a,bP&lt;0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

236 NUCLEAR AND CYTOPLASMIC MODIFICATIONS OF GERMINAL VESICLE BOVINE OOCYTES IN RELATION TO CHROMATIN REMODELING

2006 ◽  
Vol 18 (2) ◽  
pp. 226
Author(s):  
V. Lodde ◽  
P. Maddox-Hyttel ◽  
S. Modina ◽  
A. M. Luciano
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Condensation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Cortical Granules ◽  
Oocyte Growth ◽  
Structural Modifications ◽  
Chromatin Configuration ◽  
Hoechst Staining ◽  
Bovine Oocytes

We previously reported that germinal vesicle (GV) bovine oocytes can be classified on the basis of their chromatin organization and that increased chromatin condensation is accompanied by a major incidence of gap junction-mediated coupling interruption between germ and cumulus cells and by an increase in oocyte developmental competence (Lodde et al. 2005 Reprod. Fertil. Dev. 17(2), 294-295). The aim of this study was to characterize, at the ultrastructural level, both nuclear and cytoplasmic compartments of bovine oocytes classified according to their chromatin configuration because key structural modifications, such as nucleolar inactivation and remodeling of specific ooplasmic structures, take place during the later phases of oocyte growth. Cumulus-oocyte complexes collected from 0.5-2-mm early antral (EA) and 2-6-mm mid-antral (MA) follicles were freed of cumulus cells. Denuded oocytes were stained with Hoechst 33342, classified according to the degree of chromatin condensation, and processed for light microscopy of semi-thin sections (LM; n = 10 in each class) and transmission electron microscopy (TEM; n = 5 in each class). Four classes of oocytes were identified by the Hoechst staining: GV0 with filamentous chromatin diffused in the nuclear area, GV1 with few foci of condensed chromatin, GV2 with chromatin further condensed into distinct clumps, and GV3 with chromatin condensed into a single clump. Almost all oocytes collected from EA follicles were classified as GV0. Oocytes of this class were absent in MA follicles, whereas class GV1, GV2, and GV3 oocytes occurred at similar frequency. LM confirmed the chromatin condensation found by the Hoechst staining and revealed that in class GV2 and GV3 oocytes the chromatin was mainly located close to the nucleolus. Ultrastructurally, the nucleolus was fibrillo-granular in GV0 oocytes; the oocytes in the other classes displayed an electron dense fibrillar sphere with the remnant of a fibrillar center on the surface. Organelles were dispersed in the cytoplasm at GV0 while at GV1 and GV2 most organelles were homogenously distributed in the oocyte cortex. At GV3 most organelles were found in clusters in the oocyte cortex. Typical features of completion of the oocyte growth phase, like undulation of the nuclear envelope and reduction of the size of Golgi complex, were found at GV2 and GV3. Moreover, GV3 oocytes presented cortical granules that displayed varying degrees of degeneration. Our findings indicate that the process of chromatin remodeling is strictly related to structural modifications that characterize the later stages of the oocyte growth phase. Because the highest degree of chromatin condensation was combined with degenerative features of cortical granules, we hypothesize that this class of oocytes (GV3) originated from early atretic follicles, as also suggested in other species. The evaluation of oocytes on the basis of chromatin configuration may be useful for the development of new strategies for manipulating fertility in mammals. This work was supported by a COFIN Grant.

256 SYNERGISTIC EFFECT ON EMBRYO DEVELOPMENT AND EMBRYO QUALITY WHEN BOVINE DENUDED OOCYTES ARE CO-CULTURED WITH CUMULUS - OOCYTE COMPLEXES DURING IN VITRO MATURATION

2011 ◽  
Vol 23 (1) ◽  
pp. 226
Author(s):  
S. R. Dey ◽  
G. K. Deb ◽  
J. I. Bang ◽  
S. J. Cho ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Tunel Staining ◽  
Oocyte Growth ◽  
Respective Control ◽  
Bovine Oocytes

The oocyte and its surrounding somatic cells are metabolically coupled to each other through gap junctions. This phenomenon allows intercellular communication and transfer of different low-molecular-weight substrates between the cells necessary for oocyte growth. The oocyte itself regulates the cumulus cell microenvironment through oocyte-secreted factors. The development competence of the bovine oocytes is increased when denuded oocytes (DO) are co-cultured with cumulus–oocyte complexes (COC) during in vitro maturation (IVM). However, the fate of the DO, which are usually discarded after IVM, has not been determined. The present study aimed to investigate whether there is a synergistic effect of co-culturing COC and DO during IVM. We performed 3 IVM schemes: 1) COC and DO co-culture, with 12 COC and 60 DO; 2) COC control, with 12 COC; and 3) DO control, with 60 DO in 120-μL drop of TCM-199 for 22 to 24 h. Following IVM, IVF and in vitro culture were separately performed for the COC (COC co-culture) and DO (DO co-culture) from the IVM co-culture group. In vitro fertilization and in vitro culture (modified CR1aa) were done in 60-μL drops. Embryos were cultured at 38.5°C and 5% CO2 in air. Cleavage and blastocyst rates were checked at Day 3 and 8 from IVF on total COC/DO placed in IVM drop. Day 8 blastocysts were used for TUNEL staining using In Situ Cell Death Detection Kit (Roche, Budapest, Hungary). Data were analysed by one-way ANOVA, and significant differences among groups were tested by DMRT. Compared with the respective control treatments, co-culture has no effect on cleavage rates of COC and DO (see Table 1). However, blastocyst rates and total cell numbers of blastocysts were increased in COC co-culture and DO co-culture group compared with their respective control groups (see Table 1). Co-culture had no effect on apoptosis of blastocysts. These data show that co-culture of COC and DO improved developmental competence and quality of embryos from the COC co-culture and DO co-culture group. Table 1.Development competence and blastocyst quality of intact and denuded bovine oocytes This work was partly supported by the BK21 program, the KRF (KRF-2008-211-F00011), the IPET (108068-03-1-SB010), and the KOSEF (10525010001-05N2501-00110).

186 EFFECT OF POLYVINYLPYRROLIDONE SUPPLEMENTATION IN CULTURE MEDIUM ON THE GROWTH OF MOUSE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 242
Author(s):  
S. Mizumachi ◽  
K. Sasaki ◽  
K. Matsubara ◽  
Y. Hirao
Keyword(s):  
Granulosa Cell ◽  
Culture Medium ◽  
Polar Body ◽  
Cumulus Cell ◽  
Mouse Oocyte ◽  
Oocyte Diameter ◽  
Oocyte Growth ◽  
Cell Complexes ◽  
Bovine Oocytes

A high volume of polyvinylpyrrolidone (PVP) supplementation in culture medium has a significant impact on the growth of bovine oocytes. The objective of the present study was to determine whether or not PVP affects oocyte growth in the mouse. Oocyte–granulosa cell complexes were isolated from 11- or 12-day-old mice (ICR) by mechanical isolation of follicles, followed by a collagenase treatment (0.1%; 10 min). Twenty complexes were placed on each insert fit in the 24-well culture plate and cultured for 10 days in an atmosphere of 5% CO2 in air at 37°C. The culture medium was a modified α-MEM supplemented with 5% fetal bovine serum and 1 ng mL–1 FSH. The concentration of PVP (molecular weight of 360 000) was 0%, 1%, 2%, or 3% (w/v). During the first 2 days, only medium with 0% PVP was used. The oocytes recovered on Day 10 were subjected to in vitro maturation, IVF, and embryo culture. In 12 replications, the total numbers of oocytes cultured in medium with 0%, 1%, 2%, and 3% PVP were 235, 233, 233, and 231, respectively. In some additional experiments, oocytes were fixed on Day 10 and processed for transmission electron microscopy (TEM). The oocytes in medium with 0% PVP became located within an enlarged dome-like structure. In medium with 2% PVP and 3% PVP, no such domes were formed, and the oocytes within several granulosa cell layers were exposed to medium; however, the cumulus cell mass specifically became larger than that in medium with 0% PVP. The viabilities of oocytes recovered from medium with 0%, 1%, 2%, and 3% PVP were 83%, 81%, 91%, and 93%, respectively. The survival rate was significantly higher in medium with 3% PVP than in medium with 0% PVP or 1% PVP (P < 0.05). The mean oocyte diameter increased from 59 µm (Day 0) to 72, 71, 71, and 72 µm in medium with 0, 1, 2, and 3% PVP, respectively, but they continued to be smaller than in vivo grown oocytes (81.0 µm; P < 0.01). When maturation was induced, cumulus cell mucification occurred irrespective of PVP concentration during the growth. No significant differences were found between the groups in the percentage of polar body extrusion (ranging from 78 to 88%). Developmental outcomes based on oocytes used for in vitro fertilization were the following: cleavage rates were 67, 78, 74, and 76%; and blastocyst rates were 37, 44, 47, and 36% of oocytes that had been grown in medium with 0, 1, 2, and 3% PVP, respectively. The numbers of oocytes included were 60, 59, 68, and 66, respectively. The TEM observation suggests that more intimate contacts were maintained between the oocyte and cumulus cells in medium with 2% PVP than in medium with 0% PVP. Taken together, PVP supplementation in medium has a considerable influence on the morphology of mouse oocyte–granulosa cell complexes and close contacts within the complexes in the long-term culture, as having been observed with bovine oocytes.

scholarly journals 288 RELATIONSHIP BETWEEN CHROMATIN ORGANIZATION AND OOCYTE - CUMULUS CELL COMMUNICATION IN GERMINAL VESICLE STAGE BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 294
Author(s):  
V. Lodde ◽  
C. Galbusera ◽  
S. Modina ◽  
M.S. Beretta ◽  
A. Lauria ◽  
...  
Keyword(s):  
Chromatin Condensation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Hoechst 33342 ◽  
Oocyte Growth ◽  
The Status ◽  
Chromatin Configuration ◽  
Bovine Oocytes

Chromatin configuration in the germinal vesicle (GV) undergoes dynamic changes during oocyte growth, and the progressive chromatin condensation has been related to the acquisition of embryonic developmental potential. However, little is known about the mechanisms that regulate chromatin remodeling. In immature mouse oocytes, chromatin condensation and redistribution around the nucleolus are associated with transcriptional repression in both in vivo-derived and in vitro-cultured oocytes in the presence of an intact cumulus oophorus (de la Fuente et al. 2001 Dev. Biol. 229, 224). It is widely accepted that oocyte communication with the somatic cell compartment is essential for both oocyte growth and acquisition of meiotic competence (Eppig et al. 1997 Hum. Reprod. 12, 127). In particular, cumulus cells play an active role in modulating the levels of transcription in the nucleoplasm and in perinuclear domains as well as in chromatin configuration of GV stage oocytes. In cattle, a heterogeneous population of cumulus-oocyte complexes (COCs) has been found after isolation from the follicle, and this is characterized by a different functional degree of gap junction-mediated communication (Luciano et al. 2004 Biol. Reprod. 70, 465). This study was aimed at investigating the possible correlation between the chromatin configuration of immature bovine oocytes and the status of communication between the oocyte and cumulus cells, and oocyte developmental competence. In the first experiment, 138 COCs, isolated from follicles 2–6 mm in diameter, were injected with a 3% solution of Lucifer Yellow to assess the communication status between oocytes and cumulus cells. Successively, COCs were freed of cells, and denuded oocytes (DOs) were stained with Hoechst 33342 to determine the chromatin configuration. In a second experiment, 330 COCs were denuded and stained with Hoechst 33342 in order to assess chromatin configuration and then matured in vitro according to their GV stage. After IVM, DOs were fertilized, and presumptive zygotes were cultured for 7 days at which time blastocyst rate was assessed. Data were analyzed by ANOVA and Fisher's PLSD test. Three stages of GV oocytes were identified: GVI, with filamentous chromatin distributed in the nucleoplasm; GVII, with chromatin condensed into thick clumps; and GVIII, with chromatin condensed into a single clump. The GVIII stage showed a lower proportion of functional open communication than the GVI and GVII groups (8.5 vs. 45.7 and 46.1, respectively, P < 0.05). However, when compared with each other, the GVI stage oocytes showed lower embryonic developmental competence (12.9 in GVI vs. 22.1 and 24.2 in GVII and GVIII, respectively, P < 0.05). Our findings indicate that the status of communication between oocytes and cumulus cells could be related to the chromatin organization in immature bovine oocytes. A direct correlation between the communications grade, the modulation of oocyte transcriptional activity, and the acquisition of oocyte developmental competence remain to be confirmed. This work was supported by a 2003 UniMi Grant.

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.

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